Dong Zhiqian. / "June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / 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.

子宮内膜癌是最常见的妇科恶性肿瘤，其中有80%属于子宮内膜腺样癌，这一癌症发病的分子机制尚未清楚。研究表明，癌基因表达异常或者功能异常在肿瘤的发生发展过程中具有重要的作用。另一方面，抑癌基因在肿瘤细胞中特异性甲基化失活通常导致细胞恶性转变和肿瘤的发生。本实验将阐明癌基因阴阳1和抑癌基因PCDH10在人类子宮内膜腺样癌发病中的作用。 / 本实验第一部分研究多功能转录因子阴阳1（YY1）在子宮内膜腺样癌发病过程中的作用。首先本实验证实YY1在子宮内膜腺样癌临床标本和癌细胞系中均明显表达上调，并且上调的程度与肿瘤的FIGO分期相关。接着体外细胞培养和裸鼠荷瘤模型的实验均提示抑制YY1 表达可抑制癌细胞增殖和体外迁移，而过表达YY1则促进癌细胞增殖。这些结果表明YY1在子宮内膜腺样癌发病中具有促进作用。进一步全细胞基因组转录谱分析提示YY1 介入子宮内膜腺样发病的各个方面，并通过抑制抑癌基因APC的表达发挥发挥重要作用。深入的分子机制研究发现一个新的表观抑制作用模型：YY1可募集EZH2等多梳蛋白到APC启动子区并导致后者组蛋白3赖氨酸27上三甲基化，从而抑制APC基因转录。此外，本实验还发现YY1在子宮内膜腺样癌的表达增高是由于微小RNA，miR-193a-5p，在此癌中表达下降所导致的。所以，本实验第一部分的结果揭示了miR-193a-5p-YY1-APC这条全新的信号通路在子宮内膜腺样癌发病中发挥重要作用，并可作为潜在的治疗靶点。 / 本实验第二部分鉴定出PCDH10作为子宮内膜腺样癌一个新的抑癌基因。通过5-氮杂-2'-氧胞嘧啶处理和亚硫酸氢钠测序的方法，我们证实抑癌基因PCDH10在子宮内膜腺样癌中失活是由于其启动子区DNA甲基化所致，并且这种DNA甲基化介导的PCDH10表达沉默在子宮内膜腺样癌临床标本和癌细胞系中很常见，但不存在于正常子宮内膜组织。另外，在子宮内膜腺样癌细胞系体外实验中恢复PCDH10的表达可抑制细胞增殖、单细胞克隆形成，促进细胞凋亡。 同时在体实验荷瘤模型中恢复PCDH10的表达也可抑制肿瘤细胞增殖，这些结果与其肿瘤抑制功能相符。 / 总之，本实验结果阐明了YY1和PCDH10在子宮内膜腺样癌发病过程中新的作用，拓展了子宮内膜腺样癌发病分子机制的研究并为其药物治疗提供了潜在的靶点。 / Endometrial cancer is the most common gynecologic malignancy and about 80% of these cancers are endometrial Endometrioid carcinoma (EEC). The molecular mechanisms underlying EEC tumorigenesis are under-explored. Aberrant expression and function of oncogenes promote tumor development by modulating many aspects of tumor cell growth. On the other hand, tumor specific promoter methylation on tumor suppressor genes (TSG), which are generally unmethylated in normal cells, usually initiate and promote malignant transformation and cancer initiation. Our study aims to characterize the functions of an oncogenic transcription factor Yin Yang 1 (YY1) and a novel tumor suppressor gene PCDH10 in Human Endometrioid Endometrial Adenocarcinoma. / In the first part of our study, we investigated the function of a multifunctional TF, YY1 in EEC tumorigenesis. We demonstrated YY1 is up-regulated in EEC cell lines and primary tumors and its expression is associated with FIGO stages. Depletion of YY1 inhibits EEC cell proliferation and migration both in vitro and in vivo whereas over-expression of YY1 promotes EEC cell growth. These results suggest that YY1 functions as an onocogenic factor in EEC. Transcriptome analysis revealed a significant effect of YY1 on critical aspects of EEC tumorigenesis and its down-regulation of APC transcripts. Further mechanistic investigation uncovered a new epigenetic silencing mode of Adenomatosis Polyposis Coli (APC) by YY1 through recruitment of EZH2 and trimethylation of histone 3 lysine 27 in its promoter region. Additionally, YY1 over-expression was found to be a consequence of miR-193a-5p down-regulation through direct miR-193a-5p-YY1 interplay. Our results therefore established a novel miR-193a-5p-YY1-APC regulatory axis contributing to EEC development, which may serve as future intervention target. / In the second part of our study, we identified PCDH10 as a novel tumor suppressor gene in EEC. By using bisulfate genomic sequencing combined with pharmacologic demethylation drug treatment, we elucidated that PCDH10 inactivation in EEC is a consequence of DNA hypermethylation on its promoter region. Further study suggested that hypermethylation-mediated PCDH10 silencing was a common event in EEC cell lines and clinical samples, but not in normal endometrial tissues. Restoration of PCDH10 expression in EEC cells suppressed cell proliferation, inhibited single cell colony formation and induced cell apoptosis; moreover, overexpression of PCDH10 inhibited EEC xenograft tumor growth in vivo.These results suggest PCDH10 acts as a tumor suppressor. / Together, our results reveal the novel functions of YY1 and PCDH10 in EEC. These findings add novel insights into the molecular mechanisms of EEC development and progression, which may serve as potential therapeutic targets for this disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yang, Yihua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 170-186). / Abstracts also in Chinese. / TITLE --- p.I / ABSTRACT --- p.III / ACKNOWLEDGEMENTS --- p.VII / PUBLICATION --- p.IX / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XIII / LIST OF TABLES --- p.XVI / TABLES OF CONTENT --- p.XVII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Endometrioid Endometrial Adenocarcinoma (EEC) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Etiology and risk factors --- p.3 / Chapter 1.1.3 --- Treatment and prognosis --- p.8 / Chapter 1.1.4 --- Molecular Mechanisms --- p.9 / Chapter 1.1.5 --- APC and Wnt/β-catenin signaling pathway --- p.12 / Chapter 1.1.6 --- Summary --- p.18 / Chapter 1.2 --- Epigenetic modifications and EEC --- p.20 / Chapter 1.2.1 --- Epigenetic modifications --- p.20 / Chapter 1.2.2 --- Epigenetic and cancer --- p.21 / Chapter 1.2.5 --- Summary --- p.30 / Chapter Chapter 2 --- Material and Method --- p.31 / Chapter 2.1 --- Tissue samples --- p.31 / Chapter 2.2 --- Cell culture --- p.32 / Chapter 2.3 --- Cell proliferation assays --- p.33 / Chapter 2.4 --- Cell migration assay --- p.34 / Chapter 2.5 --- 3-deazaneplanocin A (Dznep) or 5-aza-2'-deoxycytidine (5-aza) treatment --- p.34 / Chapter 2.6 --- Computational prediction --- p.35 / Chapter 2.7 --- Cell cycle assay --- p.35 / Chapter 2.8 --- Apoptosis assay --- p.36 / Chapter 2.9 --- Total RNAs, Total proteins and Genomic DNA extraction --- p.36 / Chapter 2.10 --- Bisulfite Genomic Sequencing --- p.38 / Chapter 2.11 --- Oligonucleotides --- p.39 / Chapter 2.12 --- RT-PCR, Semi-quantitative PCR and Real-time PCR --- p.41 / Chapter 2.13 --- microRNA validation --- p.43 / Chapter 2.14 --- Plasmid construction --- p.43 / Chapter 2.15 --- Transfection --- p.45 / Chapter 2.16 --- Luciferase reporter assay --- p.45 / Chapter 2.17 --- Western blotting --- p.46 / Chapter 2.18 --- Immunofluorescence ( IF ) --- p.48 / Chapter 2.19 --- Immunohistochemistry (IHC) --- p.50 / Chapter 2.20 --- ChIP assay --- p.53 / Chapter 2.21 --- Sequencing and base calling --- p.55 / Chapter 2.22 --- Read mapping to genome with splice-aware aligner sequenced --- p.55 / Chapter 2.23 --- Xenograft mouse model --- p.55 / Chapter 2.24 --- Statistical analysis --- p.57 / Chapter Chapter 3 --- Yin Yang 1 Plays an Oncogenic Role in Human Endometrioid Endometrial Adenocarcinoma --- p.58 / Chapter 3.1 --- YIN YANG 1(YY1) --- p.58 / Chapter 3.1.1 --- YY1 structure --- p.58 / Chapter 3.1.2 --- YY1 function --- p.59 / Chapter 3.1.3 --- YY1 and epigenetic --- p.61 / Chapter 3.1.4 --- YY1 and cancer --- p.62 / Chapter 3.1.5 --- Regulation of YY1 expression and activity --- p.66 / Chapter 3.2 --- Results --- p.68 / Chapter 3.2.1 --- YY1 is up-regulated in EEC lines and localizes in nuclei of EEC cells --- p.68 / Chapter 3.2.2 --- YY1 expression level is associated with EEC clinicopathological features --- p.72 / Chapter 3.2.3 --- Knock-down of YY1 by RNAi inhibits EEC cell proliferation and migration --- p.77 / Chapter 3.2.4 --- Ectopic expression of YY1 promotes EEC cell proliferation --- p.84 / Chapter 3.2.5 --- YY1 does not affect EEC cell cycle and cell apoptosis --- p.91 / Chapter 3.2.6 --- Genome-wide characterization of YY1-mediated transcriptome changes --- p.94 / Chapter 3.2.7 --- Gene Ontology analysis of YY1 targets on EEC tumorigenesis --- p.98 / Chapter 3.2.8 --- YY1 inhibits APC gene expression and functions --- p.101 / Chapter 3.2.9 --- YY1 inhibits APC expression through recruiting EZH2 and causing H3K27me3. --- p.105 / Chapter 3.2.10 --- Knock-down of YY1 does not change DNA methylation status of CpG island of APC gene --- p.117 / Chapter 3.2.11 --- SiYY1 oligo injection inhibits tumor grows in vivo --- p.119 / Chapter 3.2.12 --- miR-193a-5p is down-regulated in EEC cell lines and clinical samples --- p.126 / Chapter 3.2.13 --- miR-193a-5p targets YY1 3’UTR and inhibits YY1 expression --- p.128 / Chapter 3.2.14 --- miR-193a-5p inhibits tumor grow in vivo --- p.133 / Chapter 3.3 --- Discussion --- p.136 / Chapter 3.3.1 --- YY1 oncogenic functions in EEC --- p.136 / Chapter 3.3.2 --- YY1 epigenetically silences APC --- p.138 / Chapter 3.3.3 --- miR-193a-5p down-regulates YY1 in EEC --- p.139 / Chapter 3.4 --- Conclusion --- p.141 / Chapter Chapter 4 --- The tumor suppressive functions of PCDH10 in Human Endometrioid Endometrial Adenocarcinoma --- p.143 / Chapter 4.1 --- Introduction --- p.143 / Chapter 4.1.1 --- PCDH10 structure and function --- p.143 / Chapter 4.1.2 --- PCDH10 and tumor --- p.146 / Chapter 4.1 --- Results --- p.149 / Chapter 4.2.1 --- PCDH10 is down-regulated in EEC cell lines and clinical samples --- p.149 / Chapter 4.2.2 --- PCDH10 is hypermethylated in EEC cell lines and clinical samples --- p.150 / Chapter 4.2.3 --- Pharmacologic demethylation restores PCDH10 expression in EEC cell lines --- p.152 / Chapter 4.2.4 --- Ectopic over-expression of PCDH10 inhibits EEC cell proliferation --- p.154 / Chapter 4.2.5 --- PCDH10 over-expression induces EEC cell apoptosis --- p.161 / Chapter 4.2.6 --- PCDH10 over-expression inhibits tumor grows in vivo --- p.166 / Chapter 4.3 --- Discussion and future plan --- p.169 / REFERENCE --- p.170

Poon Kin-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-147). / Abstracts in English and Chinese. / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.iii / ABBREVIATIONS --- p.viii / TABLE OF CONTENTS --- p.x / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- Cervical Intraepithelial Neoplasia (CIN) and Cervical Cancer --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.3 / Chapter 1.1.1.1 --- Descriptive Epidemiology --- p.4 / Chapter 1.1.1.2 --- Risk Factors --- p.7 / Chapter 1.1.2 --- Pathology --- p.22 / Chapter 1.1.2.1 --- Macroscopic Appearance --- p.22 / Chapter 1.1.2.2 --- Symptoms and Diagnosis --- p.23 / Chapter 1.1.2.3 --- Staging Classification --- p.25 / Chapter 1.1.2.4 --- Histopathology --- p.29 / Chapter 1.2 --- Microsatellite Instability (MSI) --- p.35 / Chapter 1.2.1 --- Microsatellite --- p.35 / Chapter 1.2.2 --- Mismatch Repair --- p.37 / Chapter 1.2.3 --- Microsatellite Instability (MSI) --- p.38 / Chapter 1.2.4 --- MSI in Various Cancers --- p.42 / Chapter 1.2.5 --- The Role of MSI in Carcinogenesis --- p.49 / Chapter 1.2.6 --- MSI as a Diagnostic / Prognostic Tool --- p.50 / Chapter CHAPTER II --- AIMS OF THE STUDY --- p.53 / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.56 / Chapter 3.1 --- Materials --- p.56 / Chapter 3.1.1 --- Patients and Specimens --- p.56 / Chapter 3.1.2 --- Microsatellite Markers --- p.57 / Chapter 3.2 --- Methods --- p.59 / Chapter 3.2.1 --- Preparation of OCT-embedded Specimen Sections --- p.59 / Chapter 3.2.2 --- Microdissection of Epithelial Cells and Neoplastic Cells from Specimen Sections --- p.60 / Chapter 3.2.3 --- DNA Extraction --- p.60 / Chapter 3.2.3.1 --- Normal Blood --- p.61 / Chapter 3.2.3.2 --- Dissected Cells --- p.62 / Chapter 3.2.4 --- DNA Amplification --- p.64 / Chapter 3.2.4.1 --- End-labeling of Primers --- p.64 / Chapter 3.2.4.2 --- Polymerase Chain Reaction --- p.65 / Chapter 3.2.5 --- Denaturing Polyacrylamide Gel Electrophoresis --- p.66 / Chapter 3.2.6 --- Autoradiography --- p.67 / Chapter 3.2.7 --- Determination of MSI --- p.67 / Chapter 3.2.8 --- HPV Detection --- p.68 / Chapter 3.2.9 --- Statistical Analysis --- p.69 / Chapter CHAPTER IV --- RESULTS --- p.70 / Chapter 4.1 --- Incidence of MSI in Cervix --- p.70 / Chapter 4.1.1 --- Incidence of MSI in Normal Cervix --- p.70 / Chapter 4.1.2 --- Incidence of MSI in CIN --- p.70 / Chapter 4.1.3 --- Incidence of MSI in Cervical Carcinoma --- p.71 / Chapter 4.1.4 --- Correlation of MSI-positive with the Evolution of Cervical Neoplasm --- p.77 / Chapter 4.2 --- Correlation of MSI-positive with Clinicopathological Characteristics in Cervical Carcinoma --- p.77 / Chapter 4.2.1 --- MSI and Age --- p.80 / Chapter 4.2.2 --- MSI and Clinical Stage --- p.80 / Chapter 4.2.3 --- MSI and Histological Grade --- p.80 / Chapter 4.2.4 --- MSI and Clinical Status --- p.81 / Chapter 4.3 --- Comparison between Two Panels of Microsatellite Markers used in MSI Detection --- p.84 / Chapter 4.4 --- Human Papilloma Virus (HPV) Infection in Cervical Neoplasm --- p.89 / Chapter 4.4.1 --- HPV Infection and Typing in CIN and Cervical Carcinoma --- p.89 / Chapter 4.4.2 --- Correlation of MSI-positive with HPV Infection in Cervical Carcinoma --- p.94 / Chapter CHAPTER V --- DISCUSSION --- p.96 / Chapter 5.1 --- MSI Detection --- p.96 / Chapter 5.1.1 --- Techniques in MSI Assays --- p.98 / Chapter 5.1.2 --- Choice of Microsatellite Markers --- p.101 / Chapter 5.1.3 --- Diagnostic Criteria of MSI --- p.105 / Chapter 5.2 --- The Role of MSI in the Carcinogenesis of Cervical Neoplasm --- p.107 / Chapter 5.3 --- The Clinical Significant of MSI in Cervical Carcinoma --- p.111 / Chapter 5.4 --- The Interaction between HPV Infection and MSI in Cervical Carcinoma --- p.113 / Chapter CHAPTER VI --- CONCLUSION --- p.116 / REFERENCES --- p.119

Li Chung Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 110-123). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.iv / TABLE OF CONTENTS --- p.v / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.xi / Chapter I. --- INTRODUCTION --- p.1 / Chapter I.1 --- Gastric Carcinoma --- p.1 / Chapter I.2 --- Etiology of Gastric Carcinoma --- p.2 / Chapter I.2.1 --- Environmental Factors: --- p.2 / Chapter I.2.2 --- Helicobacter Pylori Infection: --- p.2 / Chapter I.2.3 --- Genetic Factors: --- p.3 / Chapter I.2.4 --- Other Factors: --- p.4 / Chapter I.3 --- The Lauren Classification of Gastric carcinoma --- p.5 / Chapter I.3.1 --- Histolopathology of Intestinal Type of Gastric Carcinoma --- p.5 / Chapter I.3.2 --- Histopathology of Diffuse Type of Gastric Carcinoma --- p.8 / Chapter I.4 --- Cytogenetics Studies in Gastric Carcinoma --- p.10 / Chapter I.4.1 --- Cytogenetic Studies of Gastric carcinoma --- p.10 / Chapter I.5 --- Molecular Studies of Gastric Carcinoma --- p.14 / Chapter I.5.1 --- Genetic Instability --- p.14 / Chapter I.5.2 --- Amplification/ Mutation of Oncogenes --- p.15 / Chapter I.5.3 --- Alterations of Tumor Suppressor Genes --- p.20 / Chapter I.5.4 --- Cell Adhesion Molecules --- p.23 / Chapter I.5.5 --- Molecular Studies on Intestinal Metaplasia --- p.27 / Chapter II. --- LONG ARM OF CHROMOSOME 6 --- p.28 / Chapter III. --- BRIEF REVIEWS OF THE TECHNIQUES USED IN THIS STUDY --- p.34 / Chapter III.1 --- Comparative Genomic Hybridization (CGH) --- p.34 / Chapter III.2 --- Loss of Heterozygosity (LOH) --- p.37 / Chapter III.3 --- Methylation-Specific PCR (MSP) --- p.38 / Chapter IV. --- OBJECTIVES OF STUDY --- p.40 / Chapter V. --- MATERIALS AND METHODS --- p.41 / Chapter V.l --- Sample Collections --- p.41 / Chapter V.1.1 --- Patients Information for CGH Studies --- p.42 / Chapter V. 1.2 --- Patients Information for LOH Studies --- p.42 / Chapter V.2 --- Extraction of Genomic DNA for Tumor and Normal Tissues --- p.47 / Chapter V.2.1 --- Extraction of Genomic DNA from Frozen Tissues or Paraffin Embedded Sections --- p.47 / Chapter V.2.2 --- Extraction of Genomic DNA from Blood --- p.48 / Chapter V.3 --- Comparative Genomic Hybridization (CGH) of Gastric Carcinoma --- p.49 / Chapter V.3.1 --- Preparation of Normal Metaphase Slides --- p.49 / Chapter V.3.2 --- Metaphase Slide Denaturation --- p.49 / Chapter V.3.3 --- Nick Translation for DNA Labeling --- p.50 / Chapter V.3.4 --- Dot Blot Assay for Biotin and Digoxigenin-Labeled DNA --- p.51 / Chapter V.3.5 --- "Probe Preparation, Denaturation and Hybridization" --- p.51 / Chapter V.3.6 --- Post hybridization Washing and Detection --- p.52 / Chapter V.3.7 --- Image Acquisition and Analysis of CGH Images --- p.53 / Chapter V.4 --- Loss of Heterozygosity (LOH) Analysis on Chromosome 6q --- p.55 / Chapter V.4.1 --- Microsatellite Markers --- p.55 / Chapter V.4.2 --- Polymerase Chain Reaction (PCR) --- p.57 / Chapter V.4.3 --- Denaturing Polyacrylamide Gel Electrophoresis --- p.58 / Chapter V.4.4 --- SYBR Gold Nucleic Acid Gel Staining and Image Viewing --- p.58 / Chapter V.4.5 --- Assessment of Loss of Heterozygosity (LOH) --- p.59 / Chapter V.4.6 --- Statistical Analysis --- p.61 / Chapter V.5 --- Methylation Specific Polymerase Chain Reaction (MSP) --- p.62 / Chapter V.5.1 --- Bisulfite Modification of DNA --- p.62 / Chapter V.5.2 --- Mehtylation Specific PCR --- p.63 / Chapter VI. --- RESULTS --- p.66 / Chapter VI.1 --- Results of CGH --- p.66 / Chapter VI. 1.1 --- Chromosomal Copy Number Aberrations in Gastric Carcinoma --- p.66 / Chapter VI. 1.2 --- Comparison of CGH Results with Intestinal and Diffuse Type of Gastric Carcinoma --- p.67 / Chapter VI.2 --- LOH Analysis of Chromosome 6q --- p.73 / Chapter VII. --- DISCUSSIONS --- p.83 / Chapter VII.l --- Discussions on CGH --- p.83 / Chapter VII.2 --- Discussions on LOH Study --- p.89 / Chapter VII.2.1 --- Two Distinct Deletion Regions --- p.89 / Chapter VII.2.2 --- Possible Candidate Suppressor Genes in Two Deletion Regions --- p.93 / Chapter VII.2.3 --- Infrequent Loss of IGF2R Gene --- p.95 / Chapter VII.3 --- Relationship Between Intestinal Metaplasia and Gastric Carcinoma --- p.99 / Chapter VII.4 --- Microsatellite Instability --- p.100 / Chapter VII.5 --- Correlations --- p.103 / Chapter VII.6 --- Comparison Between CGH and LOH Results on Chromosome 6 --- p.104 / Chapter VII.7 --- Conclusions --- p.106 / Chapter VII.8 --- Limitations of the Study --- p.107 / Chapter VII.8.1 --- Limitation of CGH --- p.107 / Chapter VII.8.2 --- Limited Information Supply by LOH Analysis --- p.107 / Chapter VII.8.3 --- Small Sample Size --- p.107 / Chapter VII.9 --- Future Studies --- p.108 / Chapter VIII. --- REFERENCES --- p.110

O osteossarcoma é um tumor ósseo maligno comum em cães, com preferência por raças de grande porte, tem alto potencial metastático e ocorre frequentemente no esqueleto apendicular. O diagnóstico é baseado na história clínica, exame físico, achados radiológicos e histopatológicos. O tratamento consiste na ressecção cirúrgica do tumor e o protocolo de tratamento de escolha é a amputação associada à quimioterapia. O osteossarcoma apresenta semelhanças em cães e humanos, portanto o osteossarcoma canino pode ser um modelo útil para estudar esta doença em humanos. O objetivo deste trabalho foi estabelecer e caracterizar a linhagem celular primária de osteossarcoma canino. Os fragmentos tumorais foram obtidos por meio de biópsias e excereses cirúrgicas do osteosarcoma, a confirmação diagnóstica realizada pelo exame histopatológico. Os fragmentos foram cultivados em meios de cultura DMEM-H, DMEM-Low, RPMI-1640 e MEM para obtenção das linhagens. Para análise da morfologia celular foi realizada a fotodocumentação das garrafas em microscopia invertida, microscopia eletrônica de transmissão e varredura. A caracterização dos marcadores de superfície, citoplasmáticos e nucleares, análise das fases do ciclo celular e potencial elétrico mitocondrial foram realizados por citometria de fluxo. Foram obtidos 6 tumores, sendo 5 osteossarcomas e um condrossarcoma, dos quais foram obtidas 3 linhagens OST-1, OST-3, e CDS1. Os meios de cultivo DMEM-H e MEM foram eficientes para obtenção e manutenção das linhagens. Morfologicamente as células OST-1, apresentaram aspecto fusiforme enquanto as células OST-3 apresentaram pleomorfismo celular. Os marcadores de superfície, citoplasmáticos e nucleares nas células de osteossarcoma canino OST-3 apresentaram-se diferencialmente expressos, como os marcadores de origem mesenquimal, o marcador de reparo de DNA, P53, não foi expresso como também é encontrado em inúmeras linhagens de osteossarcoma. / Osteosarcoma is a common malignant bone tumor in dogs, with a preference for large breeds, has a high metastatic potential and occurs often in the appendicular skeleton. Diagnosis is based on clinical history, physical examination, radiological and histopatological findings. The treatment consists in surgical tumor resection, and treatment protocol choice is amputation associated with chemotherapy. Osteosarcoma has many similarities in dogs and humans, thus the canine osteosarcoma can be a usefull model to study this disease in humans. The aim of this work was to establish and characterize the canine osteosarcoma primary cell line. Tumor samples were obtained by surgical biopsy and the osteosarcoma confirmation was made by histopathological examination. Tumor fragments were cultured in DMEM-H, DMEM- LOW, RPMI-1640 and MEM culture media to establish the cell lines. Cell morphology analysis was carried out by photodocumentation in inverted microscopy and in transmission electron microscopy. The characterization of surface, cytoplasmic and nuclear markers and cell cycle phases, and mitochondrial electric potential analysis were performed by flow cytometry and analyzed with the Win MDI 2.8. Five osteosarcomas, 1 chondrosarcoma, and three cancer cell lines, OST-1, OST-3, and CDS1, were obtained. DMEM-H and MEM culture medium were efficient in cell lines establishing and maintenance. OST-1 cell line showed spindle shaped and OST-3 cell line showed pleomorphism. Surface, cytoplasmic and nuclear markers in the OST-3 canine osteosarcoma cell line were differentially expressed as mesenchimal origin markers, the DNA repair marker, P53, it was not expressed, it is also found in other osteossarcoma lines.

Orientador : Valeria Valente / Banca: Cleslei Fernando Zanelli / Banca: Ana Lúcia Fachin Saltoratto / Resumo: Os tumores cerebrais primários mais comuns são denominados gliomas. Eles são definidos patologicamente pela presença de características histológicas e imuno-histoquímicas que evidenciam diferenciação glial. De acordo com a suposta linhagem de origem, eles são classificados como astrocitomas, oligodendrogliomas ou ependimomas. Dentre eles, os astrocitomas são os mais comuns e agressivos. O tratamento atualmente utilizado inclui remoção cirúrgica seguida de quimioterapia com temozolamida (TMZ) e radioterapia, porém sua eficácia é muito baixa devido à alta resistência das células tumorais. Buscando encontrar genes associados com a elevada resistência dos astrocitomas, realizamos um estudo anterior de expressão gênica diferencial utilizando uma coleção de genes de reparo de DNA. Nesta análise foram identificados sete genes significantemente superexpressos em glioblastoma multiforme (GBM), o tipo mais agressivo de astrocitoma. Estes genes são: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L e XRCC2. Através de RT-PCR quantitativo, avaliamos os níveis de expressão destes genes em um painel expandido de 54 casos clínicos de astrocitomas de diferentes graus de malignidade e em 5 linhagens celulares de GBM. Todos os genes analisados mostraram-se mais expressos nos astrocitomas, com exceção de RAD54L em amostras de astrocitoma de grau II. Além disso, a superexpressão dos 7 genes avaliada isoladamente não exerce influência direta na sobrevida dos pacientes. Evidenciou-se ainda a superexpressão mais acentuada de EXO1 e NEIL3, que foram selecionados para realização de ensaios funcionais de silenciamento, e avaliação do ciclo celular e taxas de apoptose/morte efetiva das células. Estes ensaios foram realizados com as linhagens celulares T98G e U138MG, que apresentaram maiores níveis de expressão destes genes. Nos ensaios funcionais, observamos que o silenciamento... / Abstract: Gliomas are the most common type of primary brain cancers. They are pathologically defined by the presence of histological and immunehistochemical characteristics that evidence glial differentiation. According to the hypothetical cell of origin they are classified in: astrocytomas, oligodendrogliomas and ependimomas. Among them, astrocytomas are the more common and aggressive type. The treatment currently used for GBM includes surgical resection of tumor followed by chemotherapy with temozolamide (TMZ) and radiotherapy, but this protocol is still insufficient due to the high resistance of cancer cells. Searching for repair genes associated with the high resistance of astrocytomas, we developed a previous study of differential gene expression using a collection of DNA repair genes. In this analysis, we identified seven genes significantly overexpressed in glioblastoma multiforme (GBM), namely: APEX1, BRCA2, BRIP1, EXO1, NEIL3, RAD54L and XRCC2. Using quantitative RT-PCR, we evaluated the expression of these genes in an expanded panel of samples with 54 clinical cases of different grade astrocytomas and five GBM cell lines. All genes showed expression significantly higher in astrocytomas, except RAD54L in grade II astrocytomas. Moreover, the overexpression of this 7 genes evaluated individually doesn't exert direct influence upon patient's survival rate. Remarkably, EXO1 and NEIL3 showed the higher fold changes and were chosen for functional silencing assays. This experiments were performed with T98G and U138MG cell lines that showed the higher expression levels among the GBM cell lines analyzed. In the functional assays, we observed that the silencing of EXO1 or NEIL3 doesn't induce changes in the apoptosis and cell death rates and doesn't change the distribution of cells in cycle. Beyond this, the silencing of this two genes doesn't sentisizes cells to ionizing radiation. / Mestre

During apoptosis, caspase cleavage of ROCK1 removes an auto-inhibitory region yielding a constitutively active kinase fragment. This results in phosphorylation of downstream targets that promote contractile force generation leading to cell shrinkage, membrane blebbing and nuclear disintegration. To address fundamental questions regarding the purpose of ROCK1 cleavage and consequent apoptotic morphological features, a novel mouse model was generated that carries a single amino acid substitution in the caspase cleavage site (D1113A) that converts ROCK1 to a caspase-resistant non-cleavable (ROCK1nc) form. When apoptosis was induced in ROCK1nc cells, morphological features were significantly impaired, although the biochemical apoptotic program itself was unaffected. To understand the biological role of apoptotic morphological features in the maintenance of tissue homeostasis, acute liver damage was induced in mice with the liver-selective genotoxic compound diethylnitrosamine (DEN). Following DEN treatment, there were increased TUNEL-positive apoptotic cells and increased neutrophil infiltration in ROCK1nc mice. Histologically, ROCK1nc livers were more damaged, paralleled by higher serum alanine transaminase levels. We hypothesized that uncleared apoptotic debris undergoing secondary necrosis may release damage associated molecular patterns (DAMPs) that may aggravate liver damage by recruiting neutrophils to the liver. Indeed, inhibiting the cytokine activities of HMGB1 reduced neutrophil infiltration as well as liver damage in ROCK1nc mice. Furthermore, to determine whether defects in tissue damage responses in ROCK1nc mice would affect tumour development, the ROCK1nc mutation was introduced into two different cancer models, specifically the DEN-induced hepatocellular carcinoma and Eµ-myc lymphoma mouse models. Defective caspase cleavage of ROCK1 promoted increased infiltration of CD8+ T-cells in ROCK1nc liver tumours and had a protective effect against tumour development in both tumour models. Taken together, our results indicate that apoptotic morphological features suppress inflammation which helps to maintain tissue homeostasis but enables tumourigenesis.

Prostate cancer is a significant health problem for men in the western world. Of particular concern are patients who present with aggressive, invasive and metastatic disease, and develop lethal castration-resistant prostate cancer (CRPC) following androgen deprivation therapy. The activation of Wnt/β-catenin signalling is a common event in patients with the poorest prognosis, and frequently associated with the loss of PTEN and activation of the PI3K/Akt signalling pathway. However, the molecular basis for the significant impact of these aberrations in prostate cancer remains unclear. By using pre-clinical transgenic in vivo models, we have demonstrated that β-catenin is a potent proto-oncogene that drives prostate cancer tumourigenesis. Concurrent heterozygous loss of Pten exacerbates β-catenin-driven tumour progression and decreases host survival, while tumours are most aggressive when Pten is deleted. By investigating differential gene and protein expression, we have characterised co-operation between β-catenin activation and Pten loss through a complex network of intrinsic and extrinsic molecular events. These drive survival, growth and proliferation signals, and modulate tumour-immune response interactions to evade anti-tumourigenic processes, resulting in aggressive prostate cancer. Furthermore, by examining novel in vivo models of β-catenin-driven CRPC, we have indicated that β-catenin may promote treatment-resistance through androgen receptor (AR) reprogramming. We propose a mechanism for β-catenin-driven CRPC that is independent of classical AR signalling, and mediated through significant upregulation of canonical and non-canonical Wnt pathway components, which may be effectively targeted by Wnt inhibition. In summary, this thesis highlights a number of potential biomarkers and molecular targets that may be exploited to develop new strategies to manage patients with aggressive prostate cancer, to improve prognosis and avoid progression to lethal castration-resistant disease.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Estudos de acurÃcia diagnÃstica tÃm como finalidade apresentar indicadores clÃnicos que possam predizer as respostas humanas, gerando conhecimentos para melhorar o processo de inferÃncia diagnÃstica. Dessa forma, este estudo teve como objetivo analisar a acurÃcia dos indicadores clÃnicos do diagnÃstico de enfermagem Enfrentamento familiar comprometido no contexto de cÃncer na adolescÃncia. Trata-se de um estudo de acurÃcia diagnÃstica, com corte transversal. Participaram do estudo 236 cuidadores e adolescentes entre 10 e 19 anos completos, com diagnÃstico de cÃncer, internados ou em consulta ambulatorial ou no hospital dia de uma instituiÃÃo terciÃria de referÃncia em oncologia infatojuvenil, nos meses de marÃo a maio de 2015. A estratÃgia de amostragem foi nÃo probabilÃstica por conveniÃncia. O instrumento de coleta dados foi composto com base nos indicadores clÃnicos do diagnÃstico em estudo e foi aplicado por entrevista realizada com o adolescente e seu cuidador. Os dados foram reunidos no software Excel (2007), analisados com o apoio do pacote estatÃstico SPSS versÃo 19.0 for Windows e do software R versÃo 2.12.1 e sintetizados em 10 tabelas. A anÃlise descritiva dos dados incluiu o cÃlculo de frequÃncias absolutas, percentuais, medidas de tendÃncia central e de dispersÃo. Para as proporÃÃes de variÃveis categÃricas, foram calculados intervalos de confianÃa de 95%. Aplicaram-se os testes de Lilliefors, Qui-quadrado e Mann-Whitney. Para verificar a sensibilidade e especificidade de cada indicador clÃnico, foi utilizado o mÃtodo de anÃlise de Classes Latentes. Os resultados mostram que a maior parte dos adolescentes avaliados eram do sexo masculino, acompanhados em hospital dia. Metade dos adolescentes possuÃa idade de atà 14 anos, renda de R$ 780,00 reais e mais que cinco membros na famÃlia. Os tumores hematolÃgicos foram os mais frequentes. Os adolescentes consideraram a famÃlia como um todo importante na sua vida, mas quem mais ajuda no tratamento e mais o acompanha à a figura materna. Os indicadores clÃnicos de maior prevalÃncia foram: Pessoa significante relata entendimento inadequado, que interfere na eficÃcia dos comportamentos de apoio e Pessoa significante relata preocupaÃÃo com a reaÃÃo pessoal (p.ex. medo, pesar, culpa, ansiedade) à necessidade do cliente. O diagnÃstico Enfrentamento familiar comprometido apresentou baixa prevalÃncia tanto na populaÃÃo geral, quanto nos subgrupos amostrais de sexo, idade e tempo de diagnÃstico. Para adolescente com tempo de diagnÃstico igual ou inferior a 24 meses, foram encontradas: trÃs indicadores sensÃveis e um especÃfico. Para o sexo masculino, dois indicadores foram sensÃveis e dois especÃficos. Para o sexo feminino, foram encontrados quatro indicadores sensÃveis. Dentre estes, dois indicadores tambÃm se mostraram especÃficos. Para indivÃduos com idade menor que 14, dois indicadores revelaram-se sensÃveis e um mostrou-se especÃfico. Para indivÃduos com idade superior ou igual a 14 anos, trÃs indicadores clÃnicos obtiveram valores vÃlidos e elevados de sensibilidade e especificidade. A identificaÃÃo de indicadores clÃnicos preditores de um diagnÃstico de enfermagem à importante para a conclusÃo diagnÃstica. E este estudo contribuirà para a inferÃncia diagnÃstica de Enfrentamento familiar comprometido no contexto de cuidado de adolescentes com cÃncer. / Studies of accuracy diagnostic in family are intended to provide clinical indicators that can predict human responses, generating knowledge to improve the diagnostic inference process. Thus, this study aimed to analyze the accuracy of clinical indicators of nursing diagnosis of Compromited family coping in the context of cancer in adolescence. It is a study of diagnostic accuracy, cross-sectional. Study participants were 236 caregivers and adolescents between 10 and 19 full years, diagnosed with cancer, hospitalized or outpatient visit or hospital day of a tertiary institution of reference in infatojuvenil oncology, in the months from March to May 2015. The strategy sample was not probabilistic for convenience and the collection held by the author and academic nursing. The data collection instrument was made based on clinical indicators of diagnosis under study and was administered by interview with the teen and his caregiver. Data were gathered on Excel software (2007), analyzed with the support of statistical package SPSS version 19.0 for Windows and the R version 2.12.1 software and synthesized in 10 tables. The descriptive analysis included the calculation of absolute frequencies, percentages, measures of central tendency and dispersion. For categorical variables the proportions of 95% confidence intervals were calculated. They applied to the Lilliefors tests, chi-square and Mann-Whitney. To check the sensitivity and specificity of each clinical indicator, we used the method of analysis of Latent Classes. Most of the adolescents evaluated were male, accompanied on hospital day. Sample teen half had aged under 14 years, real income of R$ 780.00 and more than 5 members in the family. Hematological tumors were the most frequent. Teenagers consider the family as a whole important in your life, but who else helps to treat and more accompanies is the mother figure. Clinical indicators of higher prevalence were: significant person reports inadequate understanding, which interferes with the effectiveness of behavior support and significant person reports preoccupation with personal reaction (eg fear, grief, guilt, anxiety) to customer needs. Compromited family coping diagnosis showed low prevalence both in the general population, as the sample subgroups of gender, age and time of diagnosis. For teen with diagnostic time equal to or less than 24 months were found: three sensitive and specific indicators. For males, two indicators were sensitive and two specific. For females, were found four sensitive indicators. Among these, also shown two indicators are specific. For individuals younger than 14, two indicators were receptive and proved to be specific. For individuals older than or equal to 14 years, three clinical indicators obtained valid and high levels of sensitivity and specificity. Identifying predictors of clinical indicators of a nursing diagnosis is important. And this study contributed to the diagnostic inference compromised family coping in the context of adolescent care with cancer.

O objetivo deste trabalho foi comparar o prognóstico de sobrevida da graduação histológica após efeito da quimioterapia (graus I versus II de Huvos), visando também identificar fatores prognósticos no que diz respeito à sobrevida livre de recidiva local (SLRL), sobrevida livre de metástase (SLM) e sobrevida global (SG), em pacientes portadores de osteossarcoma primário não metastático ao diagnóstico. Vinte e quatro entre 45 pacientes admitidos no Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo IOT/HC/FMUSP, entre 2000 e 2004, foram eleitos para o estudo, segundo os critérios de inclusão e exclusão utilizados. As probabilidades de sobrevida acumuladas foram feitas pela técnica de Kaplan-Meier e os índices I e II de HUVOS comparados pelos testes de Log Rank. A análise multivariada foi feita pela técnica de regressão logística com modelo de risco proporcional de COX e a validade estatística estabelecida para valores de p<0,05. Os graus I e II de Huvos, quando comparados, não foram considerados de valor prognóstico em nenhuma das sobrevidas estudadas (SLRL, SLM e SG). Os fatores adversos que influenciaram o risco de recidiva local e a sobrevida global, na análise univariada foram: subtipo histológico diferente do osteoblástico (p=0,017) e o tamanho tumoral maior que 15 cm (p=0,048). Em relação à SLM o subtipo não osteoblástico (p=0,007) teve um pior prognóstico. O subtipo histológico manteve sua significância na análise multivariada em todas as sobrevidas estudadas / The purpose of this study was to compare the prognostic of survivor of histologic graduation post chemotherapy (Huvos´s grade I versus II), aiming to identify prognostic factors concerning to local recurrence free survival (LRFS), metastases free survival (MFS) and overall survival (OS) in patients with nonmetastatic primary osteosarcoma. This study included 24 patients registred in the Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo - Brazil, from 2000 to 2004. Survivor rates were calculed using Kaplan-Meier method. Huvos´s grade (I e II) were compared using the Log Rank test. Cox proportional hazards model was used for multifatorial analysis. Statistical significance was defined as a p value less than 0, 05. The Huvos´s grade I versus II was not significant factor for LRFS, MFS or OS. The adverse factors for LRFS and OS in univariate analysis were nonosteoblastic histologic subtypes (p=0,017) and large tumor (p=0,048). For MFS nonosteoblastic histologic subtypes (p=0,007) had worse prognostic. The histologic subtypes maintained their significance in multivariate testing on all studied survivor