Os tumores odontogênicos são um grupo heterogéneo de lesões formadas a partir de tecidos que dão origem ao dente. A metilação do ADN, uma adição covalente de um grupo metilo na posição 5 de carbono de um nucleótideo de citosina, é considerado um importante regulador da expressão génica. A adição do radical metil é catalisada por ADN metiltransferases (DNMTs). Embora alguns estudos epigenéticos tenham sido realizados em tumores odontogênicos, um estudo com os três tipos de DNMTs em vários membros desse grupo está em falta. Este estudo analisa a expressão de DNMTs em tumores odontogênicos. Amostras de vinte ameloblastomas, dez Calcificante tumores odontogênicos císticos, dez calcificados tumores epiteliais, dez tumor odontogênico adenomatóide, dez tumores odontogênicos queratocísticos, quatro fibromas ameloblásticos, dois fibro-odontoma ameloblástico, quatro fibroma centrais odontogênicos, sete tecidos de fibromas odontogênicos periféricos e dez mixomas odontogênicos foram incluídos. DNMT1, 3A e 3B foram expressas no núcleo e / ou citoplasma de todos os tumores odontogênicos. A alta expressão de DNMTs em células de tumor odontogênico sugere metilação como um mecanismo importante para este grupo de tumores. / Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalyzed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyzes the expression of DNMTs in odontogenic tumours. Formalin-fixed and paraffin-embedded tissue samples of twenty ameloblastomas, ten calcifying cystic odontogenic tumors, ten calcifying epithelial tumors, ten adenomatoid odontogenic tumors, ten keratocystic odontogenic tumors, five ameloblastic fibromas, two ameloblastic fibro-odontoma, four central odontogenic fibroma, seven peripheral odontogenic fibroma and ten odontogenic mixoma were included. DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

Os meningiomas são os tumores primários do Sistema Nervoso Central (SNC) mais frequentes e representam 35,5% dos casos considerando-se todas as faixas etárias. Apesar dos progressos ocorridos nas últimas décadas, a tumorigênese dos meningiomas ainda permanece como um desafio. Há um consenso da necessidade de ferramentas moleculares para ajudar tanto no diagnóstico quanto no prognóstico dos meningiomas. Neste contexto, alguns trabalhos demonstram a importância do papel dos receptores de estrógeno e progesterona, assim como o entendimento das alterações nos níveis de expressão dos microRNAs (miRNAs) na tumorigênese dos meningiomas. Alguns estudos demonstram que o perfil de expressão sérico dos miRNAs tem correlação com a classificação e evolução clínica, sendo de grande interesse o uso desse material por se tratar de um procedimento não-invasivo, ou seja, como biomarcadores. Objetivos: avaliar o perfil de expressão tecidual e sérica de microRNAs associados as vias dos receptores de estrógeno e progesterona em meningiomas grau I, II e III. Pacientes e métodos: foram utilizadas amostras de tecido e plasma de 40 pacientes com meningiomas grau I, II e III. Para a análise da expressão dos miRNAs miR-34a, miR-143, miR-145 e miR-335 foi utillizada a técnica de PCR em tempo real. Resultados: os miRNAs: miR-34a e miR-145 apresentaram diferença estatística significativa nas amostras de tecido tumoral entre os grupos estudados com menor expressão nas amostras de meningiomas grau II quando comparadas as amostras grau I e III. Não observamos diferença estatística estatística significativa na expressão dos miRNAs nas amostras de plasma. Conclusão: os miRNAs selecionados não apresentaram correlação com a progressão tumoral em meningiomas. / Meningiomas are the most common primary Central Nervous System (CNS) tumors, accounting for 35.5% of the cases, considering all age groups. Despite the progress made in recent decades, the tumorigenesis of meningiomas still remains a challenge. There is a consensus of the need for molecular tools to assist both diagnosis and prognosis of meningiomas. In this context, some studies demonstrate the importance of the role of estrogen and progesterone receptors, as well as the understanding of alterations in microRNA (miRNAs) expression levels in the tumorigenesis of meningiomas. Some studies have shown that the serum expression profile of the miRNAs correlates with the classification and clinical evolution, being of great interest the use of this material because it is a non-invasive procedure, i.e., as biomarkers. Objectives: To evaluate the tissue and serum expression profile of microRNAs associated with the estrogen and progesterone receptor pathways in meningiomas grade I, II and III. Patients and methods: tissue and blood samples from 40 patients with grade I, II and III meningiomas were used. For analysis of miRNA expression miR-34a, miR-143, miR-145 and miR-335 was used the real-time PCR technique. Results: miRNAs: miR-34a and miR-145 presented a significant statistical difference in the tumor tissue samples between the groups with lower expression in the samples of grade II meningiomas when compared to samples I and III. We did not observe statistically significant statistical difference in miRNA expression in blood samples. Conclusion: the selected miRNAs showed no correlation with tumor progression in meningiomas.

Mastocitoma é uma das principais neoplasias cutâneas em cães, caracterizada por uma multiplicação anormal de mastócitos, com comportamento biológico muito variável. Os principais fatores prognósticos incluem grau histopatológico, marcadores de proliferação, como índice mitótico, Ki67 e AgNOR, além de estadiamento clínico. Diversos estudos concentram-se na avaliação da relação da apoptose com a oncogênese e seu papel no prognóstico. Em condições fisiológicas, a apoptose ocorre na maturação e senescência celular, mantendo a homeostasia dos diferentes tecidos, removendo do organismo células que tenham sofrido alguma mutação. A genética da apoptose pode ser interrompida frente à ocorrência de mutações, levando à perda do controle na proliferação celular, o que resulta no desenvolvimento de uma neoplasia. O presente estudo avaliou a ocorrência de apoptose por meio de ensaio TUNEL em mastocitomas cutâneos caninos, com o objetivo de testar sua relação com as graduações histopatológicas e o valor prognóstico quanto à sobrevida pós-cirúrgica, assim como compará-lo à expressão imuno-histoquímica de caspase 3 e Ki67. Quarenta e quatro mastocitomas cutâneos caninos, provenientes de 36 cães, foram submetidos à avaliação histopatológica para graduação quanto à diferenciação tumoral, à análise imuno-histoquímica para avaliação das expressões de Ki67 e caspase 3. A marcação positiva para TUNEL não mostrou relação com grau histopatológico, nem foi um bom indicador para sobrevida ou mortalidade em função da doença. Apesar disso, houve correlação positiva entre os índices apoptóticos. / Mast cell tumor is a very common neoplasm in dogs and characterized by an abnormal proliferation of mast cells, with variable biological behavior. The main prognostic factors include histological grade, proliferation markers, such as mitotic index, Ki67 and AgNOR, and clinical staging. Several studies focus in the relation of apoptosis and oncogenesis and its role in prognostication. In physiological conditions, apoptosis occurs due to aging and cell senescence, maintaining the homeostasis of different tissues by removing mutated cells. The genetics of apoptosis can be interrupted by mutations, leading to loss of control in cellular proliferation, and resulting in cancer development. This study evaluated the occurrence of apoptosis by TUNEL assay in canine cutaneous mast cell tumors, compared it with histopathological grading and the immunohistochemical expressions of caspase-3 and Ki67, as well as tested its prognostic value for post-surgical survival. Forty-four canine cutaneous mast cell tumors, from 36 dogs were submitted to histopathologic and immunohistochemical analyses. Positive staining for TUNEL showed no relation with histological grade, and was not considered a good indicator for survival or mortality. Nevertheless, a positive correlation between the apoptotic indexes was found.

by Xin-xia Tian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 195-212). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Kwan, Leo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 108-116). / Abstract --- p.i / Achnowledgment --- p.ii / List of abbreviations --- p.iii / List of table and figures --- p.v / Table of contents --- p.vi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- THE DISCOVERY OF TUMOUR NECROSIS FACTOR (TNF) --- p.1 / Chapter 1.2 --- THE MOLECULE AND ITS RECEPTORS --- p.1 / Chapter 1.3 --- THE BIOLOGICAL ACTIVITIES OF TNF --- p.3 / Chapter 1.4 --- STUDIES ON THE CYTOTOXIC MECHANISM OF TNF --- p.4 / Chapter 1.5 --- A TENTATIVE MECHANISM OF TNF CYTOTOXICITY --- p.11 / Chapter 1.6 --- THE GLUTATHIONE SYSTEM : A CELLULAR PROTECTIVE MECHANISM AGAINST OXIDATIVE STRESS …… --- p.12 / Chapter 1.7 --- OBJECTIVE AND STRATEGY OF THIS STUDY --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND APPARATI / Chapter 2.1 --- CELL LINES --- p.19 / Chapter 2.2 --- "ISOLATION, MAINTENANCE AND SUBCULTURE OF CELL LINES" --- p.19 / Chapter 1. --- Plain RPMI-1640 medium / Chapter 2. --- Penicillin-streptomycin solution / Chapter 3. --- Foetal bovine serum / Chapter 4. --- Complete RPMI-1640 medium / Chapter 5. --- Trypsin-ethylenediaminetetraacetate solution / Chapter 6. --- Phosphate buffered saline / Chapter 7. --- Cycloheximide / Chapter 8. --- Actinomycin D / Chapter 9. --- Trypan blue stain / Chapter 10. --- Neutral red stain / Chapter 11. --- Recombinant human tumour necrosis factor / Chapter 12. --- Cell culture plates and flasks / Chapter 2.3 --- GROWTH CHARACTERISTIC --- p.22 / Chapter 1. --- Tritiated Thymidine / Chapter 2. --- Tritiated Leucine / Chapter 3. --- Trichloroacetic acid / Chapter 4. --- Scintillation cocktail / Chapter 2.4 --- "RESPONSE TOWARDS ANTICANCER DRUGS, CYTOTOXIC AGENTS, AND ENZYME MODULATORS" --- p.23 / Chapter 1. --- N-acetyl-DL-homocysteinethiolactone / Chapter 2. --- Diethyldithiocarbamic acid / Chapter 3. --- Doxorubicin / Chapter 4. --- Acivicin / Chapter 5. --- Ethacrynic acid / Chapter 6. --- "L'Buthionine-[S,R]-sulfoximine" / Chapter 7. --- Hydrogen peroxide / Chapter 8. --- Methotrexate / Chapter 9. --- Menadione / Chapter 2.5 --- CULTURE OF BACTERIAL CELLS --- p.27 / Chapter 1. --- Ampicillin stock solution / Chapter 2. --- Chloramphenicol stock solution / Chapter 3. --- Tetracycline stock solution / Chapter 4. --- Luria-Bertani medium / Chapter 5. --- LB with ampicillin / Chapter 6. --- SOB medium / Chapter 7. --- SOB medium with ampicillin / Chapter 8. --- SOC medium / Chapter 9. --- SB medium / Chapter 10. --- SB medium with ampicillin / Chapter 11. --- Agar plates / Chapter 2.6 --- PREPARATION OF DNA PROBES FROM BACTERIAL CLONES --- p.29 / Chapter 1. --- FlexiPrep Kit / Chapter 2. --- Restriction endonucleases / Chapter 3. --- GeneClean® II Kit / Chapter 4. --- cDNA clones for making DNA probes / Chapter 5. --- TrisHCl EDTA buffer / Chapter 2.7. --- ELECTROPHORESIS OF DNA --- p.31 / Chapter 1. --- EDTA stock solution / Chapter 2. --- Tris acetate EDTA electrophoresis buffer / Chapter 3. --- Tris borate EDTA electrophoresis buffer / Chapter 4. --- Ethidium bromide / Chapter 5. --- DNA molecular size markers / Chapter 6. --- TAE/TBE agarose gel slab / Chapter 2.8 --- CONSTRUCTION OF MURINE TNFR1 PARTIAL cDNA CLONE --- p.33 / Chapter 1. --- Frist strand cDNA synthesis Kit / Chapter 2. --- Murine TNFR1 forward and reverse primers / Chapter 3. --- Polymerase chain reaction reagents / Chapter 4. --- Cloning vector / Chapter 5. --- Modifing enzymes / Chapter 6. --- T7 SequencingTM Kit / Chapter 7. --- Acrylamide/bis gel stock solution / Chapter 8. --- Urea / Chapter 9. --- TEMED and ammonium persulphate / Chapter 10. --- β-Galactosidase colour test reagents / Chapter 11. --- TFB solution / Chapter 12. --- DnD solution / Chapter 2.9. --- RADIOLABELLING OF DNA PROBES --- p.35 / Chapter 1. --- Oligolabelling kit / Chapter 2. --- Redivue [α-32P] dCTP / Chapter 3. --- PUSH column / Chapter 2.10 --- EXTRACTION OF TOTAL RNA FROM CELL LINES --- p.36 / Chapter 1. --- N-Lauroylsarcosine / Chapter 2. --- 2M sodium acetate (pH48) / Chapter 3. --- Phenol / Chapter 4. --- Isopropanol / Chapter 5. --- Ethanol / Chapter 6. --- Extraction buffer / Chapter 7. --- Chloroform / Chapter 8. --- Isoamyl alcohol / Chapter 2.11 --- HYBRIDIZATION AND NORTHERN ANALYSIS --- p.37 / Chapter 1. --- 20XSSC / Chapter 2. --- 5X formaldehyde running buffer / Chapter 3. --- RNA sample buffer / Chapter 4. --- 10X RNA loading buffer / Chapter 5. --- Formaldehyde slab gel / Chapter 6. --- Hybond®-N membrane / Chapter 7. --- Immobilon®-N membrane / Chapter 8. --- Salmon sperm DNA / Chapter 9. --- Sodium dodecyl sulphate / Chapter 10. --- Dextran sulphate / Chapter 11. --- Kodak Biomax MR and X-OMAT films and developing kits / Chapter 2.12 --- APPARATI USED --- p.39 / Chapter CHAPTER 3 --- METHODS / Chapter 3.1 --- ISOLATION AND MAINTENANCE OF TNF RESISTANT L929 CELLS --- p.40 / Chapter 3.1.1 --- Culture of L929 cells / Chapter 3.1.2 --- Trypan blue exclusion test / Chapter 3.1.3 --- Isolation of TNF-resistant variants of L929 / Chapter 3.1.4 --- Verification of the TNF-resistant trait of rL929 / Chapter 3.1.5 --- Neutral red uptake assay / Chapter 3.2 --- COMPARING L929 AND rL929 CELLS IN TERMS OF GROWTH CHARACTERISTICS --- p.43 / Chapter 3.2.1 --- Doubling time / Chapter 3.2.2 --- Rate of protein synthesis / Chapter 3.2.3 --- Rate of DNA synthesis / Chapter 3.3 --- COMPARING L929 AND rL929 CELLS IN TERMS OF RESPONSE TOWARDS DIFFERENT ENZYME INHIBITORS AND CYTOTOXIC AGENTS --- p.44 / Chapter 3.3.1 --- TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.2 --- Effects of inhibitors of gene transcription and protein synthesis on TNF cytotoxicity on L929 and rL929 cells --- p.44 / Chapter 3.3.3 --- Cytotoxic effect of hydrogen peroxide and menadione on L929 and rL929 cells --- p.44 / Chapter 3.3.4 --- TNF cytotoxicity on L929 and rL929 cells: effect of N-acetyl homocysteine thiolatone --- p.45 / Chapter 3.3.4.1 --- The tolerant limit of AHCT / Chapter 3.3.4.2 --- Effect of AHCT on TNF cytotoxicity / Chapter 3.3.5 --- TNF cytotoxicity on L929 and rL929 cells: effect of diethyldithiocarbamate --- p.46 / Chapter 3.3.5.1 --- The tolerant limit of DEDTC / Chapter 3.3.5.2 --- Effect of DEDTC on TNF cytotoxicity / Chapter 3.3.6 --- TNF cytotoxicity on L929 and rL929 cells: effect of buthionice sulfoximine --- p.47 / Chapter 3.3.6.1 --- The tolerant limit of BSO / Chapter 3.3.6.2 --- Effect of BSO on TNF cytotoxicity / Chapter 3.3.7 --- TNF cytotoxicity on L929 and rL929 cells: effect of Acivicin --- p.47 / Chapter 3.3.7.1 --- The tolerant limit of acivicin / Chapter 3.3.7.2 --- Effect of acivicin on TNF cytotoxicity / Chapter 3.3.8 --- TNF cytotoxicity on L929 and rL929 cells: effect of ethacrynic acid --- p.48 / Chapter 3.3.8.1 --- The tolerant limit of ethacrynic acid / Chapter 3.3.8.2 --- Effect of ethacrynic acid on TNF cytotoxicity / Chapter 3.3.9 --- Cytotoxic effect of doxorubicin on L929 and rL929 cells --- p.49 / Chapter 3.3.10 --- TNF cytotoxicity of L929 cells: effect of N-acetyl cysteine --- p.49 / Chapter 3.3.11 --- Cytotoxic effect of methotrexate on L929 and rL929 cells --- p.50 / Chapter 3.3.12 --- Cytotoxic effect of hyperthermia on L929 and rL929 cells --- p.50 / Chapter 3.4 --- NORTHERN ANALYSIS AND HYBRIDIZATION --- p.51 / Chapter 3.4.1. --- Preparing RNA blots --- p.51 / Chapter 3.4.1.1 --- Extraction of total RNA from cells / Chapter 3.4.1.2 --- Making equal loading of RNA samples in formaldehyde gel electrophoresis / Chapter 3.4.1.3 --- Northern blotting of RNA / Chapter 3.4.2. --- Preparation of cDNA probes --- p.53 / Chapter 3.4.2.1 --- Preparing plasmids from A TCC clones / Chapter 3.4.2.2 --- Preparing TNFR1 probe from first-strand cDNA of L929 cells / Chapter 1. --- Construction of recombinant clone from the PCR product of TNFRl fragment / Chapter 2. --- Transforming the recombinant vector into JM109 host / Chapter 3. --- Sequencing of PCR product for identity confirmation / Chapter 3.4.2.3 --- Preparing DNA inserts from plasmids / Chapter 3.4.3 --- Radiolabelling of cDNA probes --- p.56 / Chapter 3.4.4 --- Hybridization of radioactive probes to RNA blots --- p.57 / Chapter CHAPTER 4 --- RESULTS AND DISCUSSIONS / Chapter 4.1 --- ISOLATION OF TNF-RESISTANT VARIANTS OF L929 CELLS --- p.58 / Chapter 4.1.1 --- Single cell subcloning of TNF-resistant L929 variants / Chapter 4.1.2 --- Growth rates of L929 and rL929 cells / Chapter 4.1.3 --- Rate of protein synthesis in L929 and rL929 cells / Chapter 4.1.4 --- Rate of DNA synthesis in L929and rL929 cells / Chapter 4.2 --- EFFECT OF INHIBITORS OF GENE TRANSCRIPTION AND PROTEIN SYNTHESIS ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.67 / Chapter 4.3 --- RESPONSE OF L929 AND rL929 CELLS TOWARDS VARIOUS CYTOTOXIC AGENTS --- p.70 / Chapter 4.3.1 --- "Response towards methotrexate, an anti-metabolite used in cancer treatment" / Chapter 4.3.2 --- "Response towards doxorubicin, an chemotherapeutic agent used in cancer treatment" / Chapter 4.3.3 --- "Response towards menadione, a cytotoxic agent known to generate free radicals inside cells" / Chapter 4.3.4 --- Response towards hydrogen peroxide: a highly oxidative agent / Chapter 4.3.5 --- "Response towards hyperthermia, a treatment known to exert oxidative stress on cells" / Chapter 4.4 --- EFFECTS OF MODULATORS OF CYTOSOLIC SUPEROXIDE DISMUTASE ON TNF CYTOTOXICITY ON L929 and rL929 CELLS --- p.77 / Chapter 4.5 --- EFFECT OF MODULATORS OF GLUTATHIONE METABOLISM ON TNF CYTOTOXICITY ON L929 AND rL929 CELLS --- p.82 / Chapter 4.5.1 --- "Effects of L-buthionine [S,R] sulfoximine, an inhibitor of glutathione synthesis" --- p.82 / Chapter 4.5.2 --- "Effect of N-acetyl cysteine, a cysteine derivative" --- p.84 / Chapter 4.5.3 --- "Effects of acivicin , an inhibitor of GSH reuptake and recycle" --- p.85 / Chapter 4.5.4 --- "Effect of ethacrynic acid, an inhibitor of glutathione S- transferase" --- p.87 / Chapter 4.6 --- GENE EXPRESSION IN L929 AND rL929 CELLS IN THE COURSE OF TNF CHALLENGE --- p.89 / Chapter 4.6.1 --- Isolation of total RNA from L929 and rL929 cells --- p.89 / Chapter 4.6.2 --- Preparation of DNA probes for hybridization --- p.89 / Chapter 4.6.3 --- Hybridization of specific probes on RNA blots --- p.90 / Chapter 4.6.3.1 --- Expression of heat shock protein --- p.70 / Chapter 4.6.3.2 --- Expression of the p55 TNF receptor / Chapter 4.6.3.3 --- Expression of glutathione reductase / Chapter 4.6.3.4 --- Expression of glutathione S-transferase pi / Chapter 4.7 --- DISCUSSIONS OF THE EXPERIMENTAL RESULTS --- p.97 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.104 / APPENDIX / Generation of the TNF receptor 1 cDNA probe --- p.106 / REFERENCES --- p.108

Wu-shun, Felix Wong. / Thesis (M.D.)--Chinese University of Hong Kong, 1991. / Includes bibliographies. / Title page --- p.i / Table of contents --- p.ii / Acknowledgements --- p.vi / Abstract --- p.viii / Declaration --- p.xiii / List of abbreviation --- p.xiv / Chapter Chapter one： --- Introduction --- p.1 / Chapter 1.1 --- Overview --- p.2 / Chapter 1.2 --- Aims of the study --- p.5 / Chapter Chapter two： --- Literature review --- p.8 / Chapter 2.1 --- Cell cycle and tumour growth --- p.9 / Chapter 2.1.1 --- Cell cycle --- p.9 / Chapter 2.1.2 --- Tumour growth --- p.14 / Chapter 2.2 --- Kinetic studies --- p.21 / Chapter 2.2.1 --- Radioisotopic studies --- p.21 / Chapter 2.2.2 --- Flow cytometry --- p.28 / Chapter 2.2.3 --- Monoclonal antibody --- p.32 / Chapter 2.3 --- Monoclonal antibody Ki-67 --- p.39 / Chapter 2.3.1 --- Development of Ki-67 --- p.39 / Chapter 2.3.2 --- The nature of the Ki-67 antigen --- p.42 / Chapter 2.3.3 --- Comparison with other kinetic methods --- p.45 / Chapter 2.3.4 --- Reported studies --- p.50 / Chapter 2.4 --- immunocytochemical staining --- p.63 / Chapter 2.4.1 --- Principle of immunostaining --- p.63 / Chapter 2.4.2 --- Fixation and processing methods --- p.69 / Chapter Chapter three： --- Materials and methods --- p.75 / Chapter 3.1 --- Cell culture --- p.76 / Chapter 3.1.1 --- Culture medium --- p.76 / Chapter 3.1.2 --- Origin and maintenance of cell line --- p.76 / Chapter 3.1.3 --- Coversip monolayer culture --- p.80 / Chapter 3.1.4 --- Multicellular spheroid culture --- p.80 / Chapter 3.1.5 --- Growth curve study --- p.81 / Chapter 3.1.6 --- Cytocentrifuge slide preparation --- p.81 / Chapter 3.2 --- immunoperoxidase staining --- p.83 / Chapter 3.2.1 --- Materials of immunoperoxidase staining --- p.83 / Chapter 3.2.2 --- Immunoperoxidase staining method --- p.86 / Chapter 3.3 --- Cell counting method --- p.92 / Chapter 3.3.1 --- Interactive cell counting system --- p.92 / Chapter 3.3.2 --- Cell counting methods --- p.95 / Chapter Chapter four： --- Proliferative activities of tumour cells IN VITRO --- p.104 / Chapter 4.1 --- Identification of cell proliferation of B16 melanoma cellsin VITRO --- p.105 / Chapter 4.1.1. --- Materials and methods --- p.106 / Chapter 4.1.2. --- Results --- p.107 / Chapter 4.1.3 --- Discussion --- p.110 / Chapter 4.2 --- Staining patterns of proliferating OCC1 cells in vitro --- p.117 / Chapter 4.2.1 --- Materials and methods --- p.117 / Chapter 4.2.2 --- Results --- p.118 / Chapter 4.2.3 --- Discussion --- p.121 / Chapter 4.3 --- "Comparative in vitro studies of cell proliferation using AgNOR counts, anti-BrdU, AD203 and Ki-67" --- p.130 / Chapter 4.3.1. --- Materials and methods --- p.130 / Chapter 4.3.2. --- Results --- p.131 / Chapter 4.3.3 --- Discussion --- p.134 / Chapter 4.4 --- Proliferative activities of tumor cells in vitro --- p.139 / Chapter 4.4.1. --- Materials and methods --- p.140 / Chapter 4.4.2. --- Results --- p.141 / Chapter 4.4.3 --- Discussion --- p.146 / Chapter Chapter five： --- Growth fraction in human genital tissues --- p.156 / Chapter 5.1 --- Cell proliferation in normal and neoplastic cervical tissues --- p.157 / Chapter 5.1.1. --- Materials and methods --- p.158 / Chapter 5.1.2. --- Results --- p.161 / Chapter 5.1.3 --- Discussion --- p.154 / Chapter 5.2 --- Tumour growth fraction in cervical carcinoma --- p.172 / Chapter 5.2.1. --- Materials and methods --- p.172 / Chapter 5.2.2. --- Results --- p.173 / Chapter 5.2.3 --- Discussion --- p.177 / Chapter 5.3 --- Tumour growth fraction in ovarian carcinoma --- p.185 / Chapter 5.3.1. --- Materials and methods --- p.185 / Chapter 5.3.2. --- Results --- p.186 / Chapter 5.3.3 --- Discussion --- p.190 / Chapter Chapter six： --- Conclusion --- p.198 / Chapter 6.1 --- Overview and future work --- p.199 / Chapter 6.2 --- Conclusion --- p.211 / references --- p.213 / Appendix： --- p.246 / Chapter (A) --- Additional Experiments / Chapter Experiment 1 --- Highest selection counting method --- p.246 / Chapter Experiment 2 --- Double staining of B16 melanoma cells --- p.248 / Chapter Experiment 3 --- Trypan blue exclusion test for viability --- p.250 / Chapter (B) --- Selected publications by the author / Chapter Publication 1 --- Characteristics of a cell line established from a Chinese patient with a squamous carcinoma of the uterine cervix --- p.252 / Chapter Publication 2 --- Establishment and characterization of a new human cell line derived from ovarian clear cell carcinoma --- p.258 / Chapter Publication 3 --- "Identification of ""non-proliferating"" B16 melanoma cells using monoclonal antibody (AD203) against the Ml subunit of ribonucleotide reductase" --- p.267 / Chapter Publication 4 --- The correlation of agyrophilic nucleolar organiser regions (AgNORs) count to bromodeoxyuridine incorporation and Ki-67 scores in an ovarian carcinoma cell line --- p.275 / Chapter Publication 5 --- Immunohistochemical determination of tumour growth fraction in human ovarian carcinoma --- p.278 / Chapter Publication 6 --- Tumor growth fraction in cervical carcinoma --- p.283

Yam Hin-Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 138-157. / ABSTRACT --- p.2 / ACKNOWLEDGEMENT --- p.5 / CONTENT --- p.6 / Chapter I. --- INTRODUCTION --- p.8 / Chapter II. --- LITERATURE REVIEWS / Chapter 1. --- Esophagus and Esophageal Carcinoma --- p.11 / Chapter 2. --- Characterization of Cell Line --- p.23 / Chapter 3. --- Membrane Surface --- p.26 / Chapter 4. --- Differentiation and Cancer --- p.36 / Chapter 5. --- Calcium Ion --- p.42 / Chapter III. --- MATERIALS AND METHODS / Chapter 1. --- Characterizations of EC/CUHK2 Cell Line --- p.48 / Chapter 2. --- SQM1 Localization on EC/CUHK2 Cells --- p.57 / Chapter 3. --- SQM1 Localization on Other Cells and Cell Lines --- p.62 / Chapter 4. --- Characterizations of EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.65 / Chapter 5. --- SQM1 Localization on EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.71 / Chapter 6. --- SQM1 Localization on EC/CUHK2 Cells with Changes of Extracellular Calcium Ion Concentrations --- p.73 / Chapter IV. --- RESULTS / Chapter 1. --- Characterizations of EC/CUHK2 Cell Line --- p.74 / Chapter 2. --- SQM1 Localization on EC/CUHK2 Cells --- p.81 / Chapter 3. --- SQM1 Localization on Other Cells and Cell Lines --- p.83 / Chapter 4. --- Characterization of EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.87 / Chapter 5. --- SQM1 Localization on EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.96 / Chapter 6. --- SQM1 Localization on EC/CUHK2 Cells with Changes of Extracellular Calcium Ion Concentrations --- p.105 / Chapter V. --- DISCUSSIONS / Chapter 1. --- Characterizations of Carcinoma Cell Line --- p.107 / Chapter 2. --- SQM1 Distribution on Esophageal Cancer Cells --- p.118 / Chapter 3. --- SQM1 Distribution on Other Cells --- p.122 / Chapter 4. --- Calcium-Induced Differentiation of Esophageal Carcinoma Cells --- p.125 / Chapter 5. --- SQM1 Distribution on Calcium-Induced Esophageal Carcinoma Cells 6 --- p.132 / Chapter VI. --- CONCLUSION --- p.136 / Chapter VII. --- REFERENCES --- p.138 / Chapter VIII. --- ILLUSTRATIONS --- p.158

by Suk-Fung Tsang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 101-112). / Abstract --- p.i / Acknowledgement --- p.iii / Abbreviation --- p.iv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- TNF molecule / Chapter 1.2 --- Molecular biosynthesis of TNF / Chapter 1.3 --- Antitumor activity of TNF / Chapter 1.4 --- Macrophage-mediated immunity / Chapter 1.5 --- Endogenous production of TNF / Chapter 1.6 --- LPS : the potent inducer for TNF release / Chapter 1.7 --- Natural product: as primer or inducer / Chapter 1.8 --- Aim of this project / Chapter Chapter 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Materials / Chapter 2.2 --- Animals / Chapter 2.3 --- Cell line / Chapter 2.4 --- Transformed cell line : EAT cells invivo / Chapter 2.5 --- Reagents / Chapter 2.6 --- Methods / Chapter Chapter 3 --- Preparation of sample --- p.33 / Chapter 3.1 --- Alcohol precipitaion of Bupleuri radix / Chapter 3.2 --- Endogenous TNF production by BR fractions / Chapter Chapter 4 --- Purification of BRI --- p.38 / Chapter 4.1 --- Gel filtration chromatography of BRI / Chapter 4.2 --- Anion exchange chromatography / Chapter Chapter 5 --- Purification of PQI --- p.52 / Chapter 5.1 --- Gel filtration chromatography of PQI / Chapter 5.2 --- Anion exchange chromatography / Chapter Chapter 6 --- Capacity of BR and PQ as eliciting agent for endogenous TNF production --- p.62 / Chapter 6.1 --- Time course of endogenous TNF production by BRI subfractions / Chapter 6.2 --- Time course of endogenous TNF production by PQI subfractions / Chapter 6.3 --- BRI subfractions as eliciting agents / Chapter 6.4 --- PQI subfractions as eliciting agents / Chapter Chapter 7 --- Are BR and PQ priming agents in endogenous TNF production ？ --- p.71 / Chapter 7.1 --- Priming by intraperitoneal route / Chapter 7.2 --- Priming by intravenous route / Chapter Chapter 8 --- Removal of LPS by acetic acid treatment --- p.79 / Chapter Chapter 9 --- Antitumor activities of BRI subfractionsin relationship with TNF production --- p.86 / Chapter 9.1 --- BRI subfraction as eliciting agent / Chapter 9.2 --- Pretreatment with BRIA subfractions followed by LPS treatment / Chapter Chapter 10 --- Conclusion --- p.95 / Bibliography --- p.101

Os meningiomas são os tumores primários do Sistema Nervoso Central (SNC) mais frequentes e representam 35,5% dos casos considerando-se todas as faixas etárias. Apesar dos progressos ocorridos nas últimas décadas, a tumorigênese dos meningiomas ainda permanece como um desafio. Há um consenso da necessidade de ferramentas moleculares para ajudar tanto no diagnóstico quanto no prognóstico dos meningiomas. Neste contexto, alguns trabalhos demonstram a importância do papel dos receptores de estrógeno e progesterona, assim como o entendimento das alterações nos níveis de expressão dos microRNAs (miRNAs) na tumorigênese dos meningiomas. Alguns estudos demonstram que o perfil de expressão sérico dos miRNAs tem correlação com a classificação e evolução clínica, sendo de grande interesse o uso desse material por se tratar de um procedimento não-invasivo, ou seja, como biomarcadores. Objetivos: avaliar o perfil de expressão tecidual e sérica de microRNAs associados as vias dos receptores de estrógeno e progesterona em meningiomas grau I, II e III. Pacientes e métodos: foram utilizadas amostras de tecido e plasma de 40 pacientes com meningiomas grau I, II e III. Para a análise da expressão dos miRNAs miR-34a, miR-143, miR-145 e miR-335 foi utillizada a técnica de PCR em tempo real. Resultados: os miRNAs: miR-34a e miR-145 apresentaram diferença estatística significativa nas amostras de tecido tumoral entre os grupos estudados com menor expressão nas amostras de meningiomas grau II quando comparadas as amostras grau I e III. Não observamos diferença estatística estatística significativa na expressão dos miRNAs nas amostras de plasma. Conclusão: os miRNAs selecionados não apresentaram correlação com a progressão tumoral em meningiomas. / Meningiomas are the most common primary Central Nervous System (CNS) tumors, accounting for 35.5% of the cases, considering all age groups. Despite the progress made in recent decades, the tumorigenesis of meningiomas still remains a challenge. There is a consensus of the need for molecular tools to assist both diagnosis and prognosis of meningiomas. In this context, some studies demonstrate the importance of the role of estrogen and progesterone receptors, as well as the understanding of alterations in microRNA (miRNAs) expression levels in the tumorigenesis of meningiomas. Some studies have shown that the serum expression profile of the miRNAs correlates with the classification and clinical evolution, being of great interest the use of this material because it is a non-invasive procedure, i.e., as biomarkers. Objectives: To evaluate the tissue and serum expression profile of microRNAs associated with the estrogen and progesterone receptor pathways in meningiomas grade I, II and III. Patients and methods: tissue and blood samples from 40 patients with grade I, II and III meningiomas were used. For analysis of miRNA expression miR-34a, miR-143, miR-145 and miR-335 was used the real-time PCR technique. Results: miRNAs: miR-34a and miR-145 presented a significant statistical difference in the tumor tissue samples between the groups with lower expression in the samples of grade II meningiomas when compared to samples I and III. We did not observe statistically significant statistical difference in miRNA expression in blood samples. Conclusion: the selected miRNAs showed no correlation with tumor progression in meningiomas.

by Nellie Yuk Fei Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 111-120). / Abstract also in Chinese. / Abstract --- p.i / List of Abbreviations --- p.iii / Acknowledgements --- p.iv / Publication of this study --- p.vi / Table of Contents --- p.vii / Chapter Chapter1: --- Introduction --- p.1 / Chapter 1.1 --- Multiple Myeloma --- p.2 / Chapter 1.2 --- The Problem --- p.2 / Chapter Chapter2: --- Literature Review --- p.5 / Chapter 2.1 --- Molecular Genetics of Multiple Myeloma --- p.6 / Chapter 2.1.1 --- Cytogenetics --- p.6 / Chapter 2.2 --- Alterations of Proto-Oncogenes --- p.9 / Chapter 2.2.1 --- c-myc --- p.9 / Chapter 2.2.2 --- Ras --- p.10 / Chapter 2.2.3 --- Bcl-2 and Related Protein --- p.10 / Chapter 2.3 --- Alteration of Tumor-Suppressor genes --- p.11 / Chapter 2.3.1 --- p53 Gene Mutations --- p.11 / Chapter 2.3.2 --- Retinoblastoma (Rb) Gene --- p.11 / Chapter 2.3.3 --- p16 and p15 Genes --- p.13 / Chapter Chapter3: --- DNA Methylation and Cancers --- p.14 / Chapter 3.1 --- Role of DNA Methylation --- p.15 / Chapter 3.2 --- CpG Islands --- p.15 / Chapter 3.3 --- Abnormalities of DNA Methylation in Neoplasia --- p.16 / Chapter 3.3.1 --- DNA Hypomethylation in Cancer --- p.16 / Chapter 3.3.2 --- DNA Methyltransferase Activity in Cancer --- p.17 / Chapter 3.4 --- Regional DNA Hypermethylation in Cancer --- p.17 / Chapter 3.4.1 --- p16 and p15 Genes in Solid Tumors --- p.18 / Chapter 3.4.2 --- The p16 and p15 Genes in Leukemia and other Hematopoietic Malignancies --- p.19 / Chapter 3.4.3 --- Retinoblastoma Gene --- p.20 / Chapter 3.5 --- Mechanism Underlying the DNA Methylation Changes --- p.21 / Chapter Chapter4: --- Background of Study --- p.23 / Chapter 4.1 --- Background of Study --- p.24 / Chapter 4.2 --- Project Objectives --- p.27 / Chapter Chapter5: --- Materials and Methods --- p.29 / Chapter 5.1 --- Patients Samples --- p.30 / Chapter 5.2 --- Normal Controls --- p.30 / Chapter 5.3 --- Storage of the Samples --- p.32 / Chapter 5.4 --- Materials --- p.32 / Chapter 5.4.1 --- Chemicals --- p.32 / Chapter 5.4.2 --- Primers --- p.33 / Chapter 5.4.3 --- Enzymes --- p.35 / Chapter 5.5 --- Methods --- p.35 / Chapter 5.5.1 --- Cloning of p16 and p15 Exon 1 Probes for Southern Analysis --- p.35 / Chapter 5.5.1.1 --- PCR Amplification of p16 and p15 exon1 Probes from Normal Blood DNA --- p.35 / Chapter 5.5.1.2 --- Recovery and Purification of p16 and p15 Exon 1 DNA Fragment --- p.36 / Chapter 5.5.1.3 --- Ligation --- p.37 / Chapter 5.5.1.4 --- Transformation --- p.37 / Chapter 5.5.1.5 --- Plating --- p.38 / Chapter 5.5.1.6 --- Screening of Recombinant Plasmid --- p.38 / Chapter 5.5.1.7 --- Confirmation of Cloned DNA by Sequencing --- p.42 / Chapter 5.5.2 --- DNA Extraction and Purification --- p.45 / Chapter 5.5.2.1 --- DNA Extraction from Bone Marrow Aspirate and Peripheral Blood --- p.45 / Chapter 5.5.2.2 --- Isolation of Plasmid DNA from Transformant Cutures --- p.46 / Chapter 5.5.2.3 --- Qualification and Quantification of DNA --- p.49 / Chapter 5.5.3 --- Detection of Hypermethylation by Southern Analysis --- p.50 / Chapter 5.5.3.1 --- Restriction Enzyme Digestion --- p.50 / Chapter 5.5.3.2 --- Agarose Gel Electrophoresis --- p.51 / Chapter 5.5.3.3 --- Southern Transfer --- p.51 / Chapter 5.5.3.4 --- Membrane Fixation --- p.51 / Chapter 5.5.3.5 --- Recovery and Purification of p16 and p15 Exon 1 Probes from Plasmid --- p.52 / Chapter 5.5.3.6 --- Probe Labeling --- p.54 / Chapter 5.5.3.7 --- Purification of Radioactive labeled DNA --- p.54 / Chapter 5.5.3.8 --- Southern Hybridization --- p.55 / Chapter 5.5.3.9 --- Post Hybridization --- p.55 / Chapter 5.5.3.10 --- Autoradiography --- p.56 / Chapter 5.5.4 --- Polymerase Chain Reaction-Single Strand Conformational Polymorphism Analysis (PCR-SSCP) --- p.56 / Chapter 5.5.4.1 --- 5'- end Radioactive Labeling of Primer --- p.56 / Chapter 5.5.4.2 --- Amplification of Target Sequence by PCR --- p.57 / Chapter 5.5.4.3 --- Non-denaturing Polyacrylamide Gel Electrophresis --- p.57 / Chapter 5.5.4.4 --- Direct DNA Sequence of PCR Products --- p.58 / Chapter 5.5.5 --- Prevention of Overall Contamination in PCR --- p.60 / Chapter 5.5.6 --- "Sensitivity, Specificity Controls" --- p.62 / Chapter Chapter6: --- Results --- p.64 / Chapter 6.1 --- Patient Characteristics --- p.65 / Chapter 6.1.1 --- General Patient Characteristics --- p.65 / Chapter 6.1.2 --- Clinical and Laboratory Features --- p.65 / Chapter 6.2 --- Southern Blot Analysis of p16/p15 and Rb --- p.79 / Chapter 6.2.1 --- Absence of Deletions or hypermethylationin Normal Controls --- p.79 / Chapter 6.2.2 --- Absence of Homozygous Deletions or Mutationsin p16/15 and Rb among all MM Patients --- p.79 / Chapter 6.2.3 --- Hypermethylation of p16 --- p.89 / Chapter 6.2.4 --- Hypermethylation of p15 --- p.92 / Chapter 6.3 --- Hypermethylation of p16/p15 and Clinico-pathologic Correlation --- p.94 / Chapter Chapter7: --- Discussion --- p.97 / Chapter 7.1 --- "Absence of Homozygous Deletions, Gene Rearrangements and Mutations in p16/p15 and Rb" --- p.98 / Chapter 7.2 --- Hypermethylation of p16/p15-An Alternative Way for Gene Inactivation --- p.100 / Chapter 7.2.1 --- Methylation of p15 Gene --- p.101 / Chapter 7.2.2 --- Methylation of 5'-CpG Island of p16/p15 and Lack of Gene Expression --- p.102 / Chapter 7.2.3 --- Comparison of Methylation Status of Primary Samples and Cell Lines in MM --- p.103 / Chapter 7.2.4 --- Progressive Gene Inactivation by Random Methylation Errors --- p.104 / Chapter 7.2.5 --- The Lack of Correlation of Tumor Contents Revealed by the Southern Analysis and Morphologic Assessment --- p.105 / Chapter 7.3 --- Knudson's Two-hit Model of Tumorigenesis --- p.106 / Chapter 7.4 --- Inverse Relationship of p16 and Rb --- p.107 / Chapter 7.5 --- Implications of Our Findings --- p.109 / Chapter 7.6 --- Future Studies --- p.109 / References --- p.111