Maspina, proteína da família das serpinas, tem um único gene descrito, porém diversas funções biológicas foram observadas: modulação da adesão; inibição do crescimento, da angiogênese e da invasão tumoral; efeito pró-apoptótico entre outras. Está no núcleo, no citoplasma e na membrana plasmática. Tantas funções e localizações não podem ser justificadas apenas por sua estrutura primária. Maspina pode sofrer modificações pós-traducionais controlando sua atividade, localização subcelular e interações proteicas. Propôs-se assim, caracterizar a modificação de maspina pela adição de SUMO. Dois prováveis sítios de sumoilação, nas lisinas 47 e 277 e um provável motivo de interação com SUMO entre aminoácidos 156 e 159 foram encontrados pelos programas de predição SUMOplot, SUMOsp e GPS-SBM. A estrutura 3D de maspina dessas regiões mostra que são compatíveis e coerentes com sumoilação. Ensaio de imunoprecipitação sugere que maspina endógena é sumoilada na linhagem MCF-10A. Esses dados sugerem que sumoilação pode ser importante na regulação das funções biológicas de maspina. / Maspin, a protein from the serpin family, has only one gene described, but diverse biological functions observed: adhesion modulation; inhibition of growth, of angiogenesis and of tumoral invasion; pro-apoptotic effect and others. It is in the nucleus, the cytoplasm and the plasma membrane. Those many functions and localizations cant be justified only by its primary structure. Maspin could suffer posttranslational modifications controlling its activity, subcellular localization and protein interaction. So, we propose to characterize maspin modification by the addition of SUMO. Two possible sumoylation sites in lisines 47 and 277 and a possible SUMO interacting motif between amino acids 156 and 159 were found by prediction programs SUMOplot, SUMOsp and GPS-SBM. The 3D structure of maspin in those regions shows that they are compatible and coehrent with sumoylation. Immunoprecipitation assay suggests that endogenous maspin is sumoylated in MCF-10A cell line. This data suggest that sumoylation could be important in the regulation of maspin biological functions.

人類乳頭瘤病毒 (HPV) 是引起宮頸癌的必要條件。在高危型HPV中，以HPV16在癌症樣本中最為常見，其全球盛行率達50%以上。近年來，用以辨認HPV16變異子譜系的序列特徵已經建立。雖然這個系統建基於全球的HPV16變異株，但是它只包含了四個亞洲地區。為了改善這個系統於亞洲樣本的準確性，是次研究收集了更多亞洲地區的序列。 / 是次研究提供了在香港和韓國收集的HPV16樣本的系統發生史及序列變異 (LCR、E6 和 E7)。此外，是次研究也檢測了HPV16變異株的在兩地的分佈和致癌風險。 / 是次研究從香港和韓國收集了329個HPV16呈陽性的宮頸樣本。利用LCR、E6、E7 和整合的LCR-E6基因序列以極大似然法來構建HPV16變異株的系統發生樹。序列變異會按照系統發生樹之拓撲結構來分類並詳細描述。卡方檢驗或費雪精確性檢定用於分析HPV16變異株在兩地的分佈和致癌風險。 / 是次研究結果顯示用以辨認HPV16變異子譜系的序列特徵需加以改善。我們建議採用A7287C/T作為亞洲子譜系的序列特徵，以替代原有的A7287C。有關HPV16變異株的地理分佈，亞洲和歐洲的變異株在香港 (亞洲變異株: 70%，歐洲變異株: 25.3%) 和韓國 (亞洲變異株: 61.2%，歐洲變異株: 20.2%) 均十分普遍。另外，1和2型亞美變異株在香港和韓國的分佈有著明顯差別 (1型亞美變異株: 2% 與12.4%，P < 0.001; 2型亞美變異株: 0% 與2.8%，P = 0.04)。 / 另外，是次研究發現亞洲子譜系於韓國民族中呈較高致癌風險 [比值比 (95% 置信區間) 2.02 (1.03-3.99)]。在進化支中，E6的第五進化支[2.44 (1.27-4.74)]和E7的第三進化支[2.02 (1.03-3.99)]也於韓國民族中呈較高致癌風險。在SNP中，E6 T178G [2.17 (1.11-4.23)]、兩個E7的SNPs (A647G [1.73 (0.88-3.42)]、T846C [2.27 (1.16-4.49)]) 和9個LCR SNPs (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] 和 C24T [2.36 (1.20-4.66)])於韓國民族中也呈較高致癌風險。這些進化支和SNPs都與亞洲子譜系有關聯。在香港方面，兩個LCR SNPs (A7289C [1.89 (0.92-3.87)] 和 T7781C [2.07 (0.92-4.71)])呈較高致癌風險。 / 是次研究發現的高危SNPs和進化支需要進一步的大型流行病學研究和生物化學實驗來核實。這些序列特徵可作為生物標誌物以檢測出與HPV有關的早期宮頸病變。 / Human papillomavirus (HPV) is necessary for the development of cervical cancer. Of those high-risk HPV types, HPV16 is the most common type detected in cervical cancer and accounts for a prevalence of greater than 50% worldwide. Recently, a sequence signature-based system for identifying the sub-lineages of HPV16 variants has been established. Although this system was developed from HPV16 variants collected worldwide, only four Asian regions were included. To improve the accuracy of this sub-lineage classification system for Asian samples, more sequence data from Asian regions were included in the current study. / The current study provided data on the phylogeny and the sequence variation of Long control region (LCR), E6 and E7 open reading frames (ORFs) of HPV16 isolates collected in Hong Kong and Korea. The distribution of HPV16 variants between two regions and the risk association of HPV16 variants with cervical cancer development were also examined. / A total of 329 HPV16-positive cervical samples were collected from Hong Kong and Korea. The phylogenetic trees were constructed for the LCR, E6, E7 and concatenated LCR-E6 sequences using the maximum likelihood method. The sequence variation of each region was delineated and grouped according to the tree topology. The distribution and risk association of HPV16 variants were examined using the chi-square test or Fisher’s exact test as appropriate. / The results showed that the previously described sequence signatures for classifying sub-lineages of HPV16 variants required further improvement, especially for the Asian sub-lineage. We proposed A7287C/T as a signature SNP of the Asian sub-lineage rather than A7287C as suggested by Cornet et al. In regard to the distribution of HPV16 variants, the Asian (As) and European (Eur) variants were commonly found in Hong Kong (As: 70%, Eur: 25.3%) and Korea (As: 61.2%, Eur: 20.2%). Furthermore, Asian American-1 and 2 (AA1 and AA2) variants were found to distribute significantly different between Hong Kong and Korea (AA1: 2% versus 12.4%, P < 0.001; AA2: 0% versus 2.8%, P = 0.04). / A key finding was that variants of the Asian sub-lineage carried a higher oncogenicity among Korean population [odds ratio (95% confidence interval) = 2.02 (1.03-3.99)]. In clade level, E6 clade 5 [2.44 (1.27-4.74)] and E7 clade 3 [2.02 (1.03-3.99)] were found to carry a higher oncogenicity among Korean population. In SNP level, E6 T178G [2.17 (1.11-4.23)], two SNPs of E7 ORF (A647G [1.73 (0.88-3.42)] and T846C [2.27 (1.16-4.49)]) and nine SNPs of LCR (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] and C24T [2.36 (1.20-4.66)]) were also found to carry a higher oncogenicity among Korean population. Those clades and SNPs were linked to the Asian sub-lineage. In contrast, only two SNPs of LCR (A7289C [1.89 (0.92-3.87)] and T7781C [2.07 (0.92-4.71)]) were found to associate with a higher oncogenicity among Hong Kong population. / The risk associations of SNPs, clades of the HPV16 Asian sub-lineage revealed by the current study should be verified by large-scale epidemiological studies and biochemical experiments. These signatures may serve as biomarkers for early detection of HPV-related cervical neoplasia. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ma, Tsz Ue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 148-156). / Abstracts also in Chinese. / Abstract of Thesis --- p.I / 論文摘要 --- p.V / Acknowledgements --- p.VIII / Contents --- p.X / Figures --- p.XIII / Tables --- p.XIV / Abbreviations --- p.XVI / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- History of Human Papillomavirus --- p.2 / Chapter 1.2 --- Biology of Human Papillomavirus --- p.4 / Chapter 1.2.1 --- Genome Organization and Protein Functions --- p.4 / Chapter 1.2.1.1 --- E5 Protein --- p.7 / Chapter 1.2.1.2 --- E6 Protein --- p.8 / Chapter 1.2.1.3 --- E7 Protein --- p.9 / Chapter 1.2.2 --- Life Cycle of Human Papillomavirus --- p.10 / Chapter 1.2.3 --- Taxonomy of Human Papillomavirus --- p.12 / Chapter 1.3 --- Cervical Cancer --- p.16 / Chapter 1.3.1 --- Natural History --- p.16 / Chapter 1.3.2 --- Risk Factors --- p.17 / Chapter 1.4 --- Epidemiology of Cervical Cancer --- p.19 / Chapter 1.4.1 --- Global Disease Burden --- p.19 / Chapter 1.4.2 --- Disease Burden in Hong Kong --- p.21 / Chapter 1.4.3 --- Disease Burden in South Korea --- p.22 / Chapter 1.5 --- Human Papillomavirus Type 16 --- p.23 / Chapter 1.6 --- Background and Objectives --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Study Design --- p.31 / Chapter 2.2 --- Study Samples --- p.35 / Chapter 2.2.1 --- HPV16-Positive Samples --- p.35 / Chapter 2.2.2 --- Samples with Unknown HPV Status --- p.36 / Chapter 2.3 --- Laboratory Methods --- p.39 / Chapter 2.3.1 --- DNA Extraction --- p.39 / Chapter 2.3.2 --- Polymerase Chain Reaction --- p.40 / Chapter 2.3.2.1 --- PGMY09/11 PCR --- p.40 / Chapter 2.3.2.2 --- HPV16-Specific PCR --- p.42 / Chapter 2.3.3 --- Genotyping of HPV --- p.48 / Chapter 2.3.4 --- Purification of PCR Products --- p.51 / Chapter 2.3.5 --- Sequencing Reaction --- p.52 / Chapter 2.4 --- Data Analysis --- p.54 / Chapter 2.4.1 --- Sequence Edit and Alignment --- p.54 / Chapter 2.4.2 --- Sequence Variation of HPV16 Variants --- p.56 / Chapter 2.4.3 --- Construction of Phylogenetic Tree --- p.56 / Chapter 2.4.4 --- Distribution and Comparison of HPV16 Variants in Hong Kong and Korea --- p.57 / Chapter 2.4.5 --- Distribution of HPV16 Variants in Normal and Cancer Samples and Risk Association Study --- p.58 / Chapter Chapter 3 --- Results --- p.59 / Chapter 3.1 --- Study Samples --- p.60 / Chapter 3.1.1 --- HPV16-Positive Samples --- p.60 / Chapter 3.1.2 --- Samples with Unknown HPV Status --- p.61 / Chapter 3.2 --- Sub-lineage Identification of HPV16 Variants --- p.63 / Chapter 3.2.1 --- Based on the Phylogenetic Analysis in the Current Study --- p.63 / Chapter 3.2.1.1 --- Concatenated LCR-E6 Phylogenetic Tree --- p.63 / Chapter 3.2.1.2 --- LCR Phylogenetic Tree --- p.66 / Chapter 3.2.1.3 --- E6 Phylogenetic Tree --- p.69 / Chapter 3.2.2 --- Based on the Single Nucleotide Polymorphisms Proposed by Cornet et al. --- p.74 / Chapter 3.2.2.1 --- Single Nucleotide Polymorphisms of LCR Sequence --- p.74 / Chapter 3.2.2.2 --- Single Nucleotide Polymorphisms of E6 Open Reading Frame --- p.78 / Chapter 3.3 --- Sequence Variation of HPV16 Variants --- p.82 / Chapter 3.3.1 --- LCR Sequence --- p.82 / Chapter 3.3.2 --- E6 Open Reading Frame --- p.91 / Chapter 3.3.3 --- E7 Open Reading Frame --- p.95 / Chapter 3.4 --- Distribution of HPV16 Variants in Hong Kong and Korea --- p.100 / Chapter 3.4.1 --- Sub-lineage Level --- p.100 / Chapter 3.4.2 --- Clade Level of E6 Open Reading Frame --- p.101 / Chapter 3.4.3 --- Clade Level of E7 Open Reading Frame --- p.102 / Chapter 3.4.4 --- Single Nucleotide Polymorphisms Level --- p.105 / Chapter 3.4.4.1 --- LCR Sequence --- p.105 / Chapter 3.4.4.2 --- E6 Open Reading Frame --- p.107 / Chapter 3.4.4.3 --- E7 Open Reading Frame --- p.108 / Chapter 3.5 --- Risk Association and distribution of HPV16 Variants in normal and Cancer samples --- p.112 / Chapter 3.5.1 --- Sub-lineage Level --- p.112 / Chapter 3.5.2 --- Clade Level of E6 Open Reading Frame --- p.114 / Chapter 3.5.3 --- Clade Level of E7 Open Reading Frame --- p.115 / Chapter 3.5.4 --- Single Nucleotide Polymorphisms Level --- p.122 / Chapter 3.5.4.1 --- LCR Sequence --- p.122 / Chapter 3.5.4.2 --- E6 Open Reading Frame --- p.125 / Chapter 3.5.4.3 --- E7 Open Reading Frame --- p.126 / Chapter Chapter 4 --- Discussion --- p.132 / Chapter 4.1 --- HPV16 Variant Sub-lineages --- p.133 / Chapter 4.2 --- Comparison of HPV16 variants between Hong Kong and Korea --- p.137 / Chapter 4.3 --- Risk Association of HPV16 Variants --- p.138 / Chapter 4.4 --- Strength and Weakness --- p.144 / Chapter 4.5 --- Implications for Future Work --- p.146 / References --- p.148

by Lai Wing Hong Kevin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 63-74). / Acknowledgments --- p.i / Abbreviations --- p.ii / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction --- p.3 / Chapter 1.1 --- Advantages of Antisense Oligonucleotides Inhibition --- p.4 / Chapter 1.2 --- The Uses of Antisense Oligonucleotide in Leukemic Therapy --- p.5 / Chapter 1.3 --- Oncogenes in the Pathogenesis of Leukemia --- p.6 / Chapter 1.4 --- Apoptosis and Apoptosis-Related Genes --- p.9 / Chapter 1.5 --- Protooncogene bcl-2 --- p.10 / Chapter 1.6 --- Bcl-2 Homologues --- p.11 / Chapter 1.7 --- Regulation of Apoptosis by Other Genes --- p.13 / Chapter 1.8 --- Promyelocytic Leukemia HL-60 Cell Line --- p.15 / Chapter 1.9 --- Aim of Project --- p.16 / Chapter Chapter 2 --- Chemical Synthesis of DNA Oligonucleotides / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.3 --- Results --- p.24 / Chapter 2.4 --- Discussion --- p.26 / Chapter Chapter 3 --- The Apoptotic Effects of TPA and Ouabain on the Promyelocytic Leukemic HL-60 cell line / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and methods --- p.33 / Chapter 3.3 --- Results --- p.40 / Chapter 3.4 --- Discussion --- p.44 / Chapter Chapter 4 --- Effect of Antisense Oligonucleotides on TPA-Induced Apoptosisin Leukemic HL-60 cells / Chapter 4.1 --- Introduction --- p.48 / Chapter 4.2 --- Materials and Methods --- p.49 / Chapter 4.3 --- Results --- p.52 / Chapter 4.4 --- Discussion --- p.54 / Chapter Chapter 5 --- General Discussion --- p.57 / References --- p.63

La tyrosine kinase cytoplasmique Src est un régulateur essentiel de la croissance et de l’adhésion cellulaires induites par de nombreux stimuli extracellulaires, dont les facteurs de croissance et certains composants de la matrice extracellulaire. La dérégulation de son activité catalytique lui confère des propriétés oncogéniques importantes conduisant à la formation de tumeurs agressives chez l’Homme. La plupart des connaissances actuelles sur sa régulation catalytique repose sur des données structurales de cristallographie qui ont révélé l’importance des interactions intramoléculaires SH2 et SH3-dépendantes dans le contrôle des conformations ouvertes et actives de Src. Les domaines SH3, SH2 et kinase de Src sont associés au domaine SH4 N-terminal d'ancrage à la membrane plasmique via un domaine unique (DU) flexible. Le rôle du DU dans la régulation de Src reste obscur car il est intrinsèquement désordonné et par conséquent, cette région n'a pas été incluse dans les analyses structurales originelles. L'analyse par RMN de l'extrémité N-terminale de Src a révélé une conformation partiellement structurée du DU par le contact de séquences peptidiques spécifiques avec les lipides membranaires et le domaine SH3 (Perez et al, 2013; Maffei et al, 2015). De plus, cette interaction est régulée par phosphorylation des Ser69 et Ser75 localisées à proximité de ce domaine. L’objectif de ma thèse était d’adresser la relevance biologique de ces données structurales en me focalisant sur le rôle d’une partie du DU identifiée comme importante par RMN. J’ai montré que l’inactivation de cette région par des mutations perte de fonction, diminue fortement la signalisation de Src dans les cellules non transformées humaines ainsi que ses propriétés tumorales dans les cellules cancéreuses colorectales métastatiques révélant la pertinence biomédicale du système. Des analyses de protéomique m’ont permis de révéler un mécanisme original par lequel cette région contrôle la capacité de Src à phosphoryler ses substrats. En conclusion, ces données confirment un rôle important du DU sur l'activité et la signalisation de Src et révèlent un rôle critique des domaines désordonnés dans les tumeurs chez l’Homme. L’inhibition de cette région unique par de petites molécules devrait conduire à de nouvelles stratégies thérapeutiques dans le cancer. / The membrane-anchored non-receptor tyrosine kinase Src is involved in numerous signal transduction pathways and hyperactive Src is a potent oncogene and driver of human metastasis. Most of our knowledge on Src kinase regulation relies on structural data, which revealed SH2 and SH3-dependent intramolecular interactions to control active conformations of the protein. The kinase, SH3 and SH2 domains of Src are attached to the membrane-anchored SH4 domain through the flexible unique domain (UD). The role of this UD remains obscure due to its intrinsically disordered properties for which reason it was not included in original structural analyses. Interestingly, membrane-associated intrinsic disordered domains are more prevalent among signaling and cancer-related proteins and they are thought to play critical roles in human disease. NMR analysis of Src N-terminus in the presence of lipids revealed a partially structured conformation of the UD through the contact of a small peptide sequence with membrane lipids and the SH3 domain of the protein (Perez et al, 2013; Maffei et al, 2015). This interaction was regulated by phosphorylation of Ser69 and Ser75 surrounded this central region. The aim of my thesis project was to assess the biological relevance of this structural data. Interestingly, I showed that expression of Src mutants with UD loss of function drastically affected Src activity and signaling in human non-transformed cells as well as Src oncogenic properties in metastatic colorectal cancer cells. This highlights the biomedical relevance of the system. Further proteomic analysis revealed an unsuspected mechanism by which this region controls the Src capacity to phosphorylate specific substrates involved in cancer cell activity. These data support a previously unrecognized important role of the UD on Src activity and signaling and reveals a critical role of intrinsic disordered domains in the regulation of kinase signaling in human disease. Targeting this unique region by small molecules may be of therapeutic value in human cancer.

by Yam Hin Fai. / "June 1998." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 118-151). / 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. / Abstract in Chinese.

O fator de indução de proteólise (proteolysis-inducing factor) ou PIF é uma glicoproteína de 24 kDa encontrada no plasma de pacientes com câncer e responsável pelo catabolismo de proteínas musculares associado a caquexia neoplásica. O presente trabalho investigou pelas técnicas de RT-PCR, RACE-PCR e Real-time PCR a presença de formas de expressão de mensagens do PIF em vários tipos celulares derivados de tecido normal e tumoral. A expressão de mRNA do gene foi testada em 37 amostras de tumores e 4 tecidos normais, sendo detectada em 7 de 20 linhagens tumorais de mama, uma linhagem tumoral de cólon e no tecido normal da mama, placenta e testículo. A expressão significativa do gene PIF entre as linhagens metastáticas da mama foi confirmada pela técnica de Real-time PCR. Uma nova variante da mensagem (PIF-SV), contendo um novo éxon de 64 bp, inserido entre os éxons 4 e 5, foi identificada em tecido normal de mama pela técnica de RACE-PCR. Esta forma variante de PIF é expressa concomitante com a forma principal de PIF em tumores primários da mama, linhagens tumorais de mama e nos tecidos normais da mama e placenta. Parece que o PIF exerce funções fisiológicas nos tecidos normais. / Proteolysis-inducing factor (PIF) is a 24 kDa glycoprotein identified in plasma of cancer patients and responsible for muscle catabolism associated with the process of cancer cachexia. The present study has investigated, using the RT-PCR, RACE-PCR and Real-time PCR techniques, the presence of PIF messages in different cell types derived from normal and tumor tissues. The PIF mRNA expression was examined in 37 tumor and 4 normal tissue samples and detected in 7 of 20 breast tumor cell lines, one colon tumor cell line and in normal breast, placenta and testis tissues. The differential expression of PIF message in metastatic breast cell lines was confirmed by the Real-time PCR technique. A new splicing form of the message, containing one new exon of 64bp, inserted between the exons 4 and 5, was identified in normal breast tissue. This splicing form of PIF is expressed concomitantly with a main form of PIF in breast tumor cell lines and also in normal breast and placenta tissue. These data suggest that PIF exhibits physiological functions in normal tissues. An over-expression and a production of a new splicing form (PIF-SV), seem to contribute in some event leading normal cell to a malignant phenotype.

Du Jing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 70-89). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.IV / PUBLICATIONS --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATIONS --- p.VIII / CONTENTS --- p.X / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- Laryngeal carcinoma and HPV --- p.1 / Chapter 1.2 --- HPV --- p.2 / Chapter 1.3 --- Human papillomavirus E6 protein --- p.6 / Chapter 1.3.1 --- Transformation by HPV E6 --- p.7 / Chapter 1.3.2 --- Inhibition of apoptosis by E6 --- p.8 / Chapter 1.3.3 --- Alteration of gene transcription --- p.11 / Chapter 1.3.4 --- E6 interation with other proteins --- p.12 / Chapter 1.3.5 --- E6 as a therapeutic target --- p.14 / Chapter 1.4 --- HPV E7 protein --- p.15 / Chapter 1.4.1 --- Regulation of viral life cycle by HPV E7 --- p.16 / Chapter 1.4.2 --- Degradation of retinoblastoma tumor suppressor by HPV E7 --- p.18 / Chapter 1.4.3 --- Inhibition of p53 by HPV E7 --- p.22 / Chapter 1.4.4 --- Interaction with other proteins by HPV E7 --- p.24 / Chapter 1.5 --- Objective --- p.26 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS --- p.28 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Materials for cDNA and RNA manipulation --- p.28 / Chapter 2.1.2 --- Culture media and transfection reagents --- p.28 / Chapter 2.1.3 --- Antibodies --- p.29 / Chapter 2.1.4 --- Materials for protein manipulation --- p.29 / Chapter 2.1.5 --- Kits --- p.30 / Chapter 2.1.6 --- Instrumentation --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Plasmid construction --- p.32 / Chapter 2.2.1.1 --- DNA preparation --- p.34 / Chapter 2.2.1.2 --- DNA ligation --- p.34 / Chapter 2.2.1.3 --- Transformation of competent E. coli --- p.35 / Chapter 2.2.2 --- Mini preparation --- p.35 / Chapter 2.2.3 --- Clone selection and confirmation --- p.37 / Chapter 2.2.4 --- Sequencing gel electrophoresis --- p.37 / Chapter 2.2.5 --- Cell culture and cytokine treatment --- p.39 / Chapter 2.2.6 --- Plasmid transfection --- p.39 / Chapter 2.2.7 --- Confirming construction of stable cell lines by RT-PCR --- p.40 / Chapter 2.2.7.1 --- Total cellular RNA extraction --- p.40 / Chapter 2.2.7.2 --- First strand cDNA synthesis --- p.41 / Chapter 2.2.7.3 --- Polymerase chain reaction (PCR) --- p.41 / Chapter 2.2.8 --- Fluorescence microscopy and imaging --- p.43 / Chapter 2.2.9 --- DNA fragmentation assay --- p.44 / Chapter 2.2.10 --- Protein detection --- p.46 / Chapter 2.2.10.1 --- Preparation of protein extract --- p.46 / Chapter 2.2.10.2 --- SDS-PAGE electrophoresis and protein transfer --- p.47 / Chapter 2.2.10.3 --- Immunoblotting analysis --- p.47 / Chapter 2.2.11 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE: --- RESULTS --- p.49 / Chapter 3.1 --- Plasmid construction --- p.49 / Chapter 3.2 --- Expression of HPV16 viral oncogenes in transfected UMSCC12 --- p.51 / Chapter 3.3 --- HPV16 E6 and E7 protect apoptosis induced by TNF-alpha and CHX --- p.53 / Chapter 3.4 --- Detection of apoptosis with fluorescence staining --- p.55 / Chapter 3.5 --- Regulation of the expression of apoptosis-associated proteins by E6 and E7 oncoproteins --- p.57 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.59 / Chapter CHAPTER FIVE: --- CONCLUSION AND FUTURE PERSPECTIVE --- p.68 / REFERENCES --- p.70 / APPENDIX DNA SEQUENCING RESULTS --- p.90

Les récepteurs de la famille HER jouent un rôle majeur dans le développement du cancer. Alors qu’EGFR, HER2 et HER3 sont très bien étudiés et ciblés par des anticorps thérapeutiques, le dernier récepteur de cette famille, HER4, n’est que peu étudié et son implication dans la cancérogénèse est controversée. Il n’existe à ce jour pas d’anticorps thérapeutique anti-HER4 sur le marché ou en phase clinique. Ce récepteur est présent à la surface en quatre isoformes (JMa/CYT1 ; JMa/CYT2 ; JMb/CYT1 ; JMb/CYT2). Les isoformes JMa sont activées par clivage du récepteur, contrairement aux deux isoformes JMb. Le clivage de ces isoformes conduit à la libération de la partie intracellulaire du récepteur, appelée 4ICD. Ce fragment peut être dirigée au noyau ou dans d’autres compartiments cellulaires, impliquant HER4 dans des signalisations oncogéniques ou suppresseurs de tumeur. La littérature décrivant une activité pro-apoptotique de 4ICD et de la NRG1, le principal ligand de HER4, nous avons étudié la signalisation de ces isoformes afin de déterminer leurs rôles au niveau tumoral. Nos résultats indiquent que la NRG1 induit une signalisation suppresseur de tumeur via JMa/CYT1 et une signalisation oncogénique via JMa/CYT2. Sur la base de ces résultats, nous avons développé un criblage original d’anticorps anti-HER4 par phage display, sur des cellules exprimant l’isoforme JMa/CYT1 et stimulées par la NRG1. Nous avons caractérisés quatre anticorps anti-HER4, dont l’activité et les signalisations de certains sont modulées par la NRG1. Deux de ces anticorps, caractérisés comme étant des agonistes du récepteur HER4, induisent la mort des cellules tumorales par des mécanismes que nous sommes en train d’élucider. De manière similaire a la NRG1, un des anticorps induit la relocalisation de 4ICD-CYT1 a la mitochondrie pour induire la mort cellulaire. Ces résultats prometteurs ouvrent la voie à un ciblage thérapeutique du récepteur HER4 a l’aide d’anticorps agonistes pour le traitement des cancers / HER family is composed by four members which play a major role in cancer development. EGFR, HER2 and HER3 are well described and targeted with therapeutic monoclonal antibodies. HER4, the last one, is poorly described with a contentious role in cancerogenesis. Nowadays, there is no therapeutic antibody targeting HER4 in clinic. Four isoforms of the receptor are addressed to the plasma membrane and are called JMa/CYT1; JMa/CYT2; JMb/CYT1 and JMb/CYT2. JMa isoforms are activated by cleavage, but not JMb isoforms. Following their activation, JMa isoform cleavage releases the intracellular part of the receptor called 4ICD. This part can be directed to the nucleus or others subcellular compartments, involving HER4 in oncogenic or tumor suppressor signalling. Because a pro-apoptotic activity of 4ICD and its main ligand NRG1 have been described, we studied JMa isoforms signaling to determine their roles in cancer. We demonstrated that NRG1 induce a tumor suppressor signalling from JMa/CYT1 and an oncogenic signalling from JMa/CYT2. Based on these results, we developed an innovative screening for anti-HER4 antibodies by whole cell panning with phage display. To this end, we used NRG1- stimulated cells expressing JMa/CYT1 isoforms. We characterized four anti-HER4 antibodies and functions of some of them are affected and modulated by NRG1. Two antibodies were characterized as agonistic anti-HER4 antibodies and induce cell death of cancer cells by different mechanisms. Like NRG1, one of them induce mitochondrial localization of 4ICD-CYT1 to induce cell death. These promising results pave the way to a therapeutic targeting of HER4 receptor with agonistic antibodies to treat cancer

Background. B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) acts as an oncogene in various cancer such as non-small cell lung cancer, colon cancer, gastric cancer, bladder cancer and nasopharyngeal cancer (NPC). / Methods. Immunohistochemistry was performed to evaluate Bmi-1 expression in 252 breast cancer samples. The correlations were analyzed between Bmi-1 expression and clinicopathologic parameters, including age, tumor size, lymph nodal involvement, distant metastasis, clinical stages, hormone receptor (ER, PR) and Human Epidermal Growth Factor Receptor 2 (HER-2). The overall survivals were compared by Kaplan-Meier analysis based on Bmi-1 expression. / Results. Bmi-1 expression was significantly increased in primary cancer tissues than in matched adjacent non-cancerous tissues ( P&lt;0.001). Only 35.9% (14 of 39) of adjacent non-cancerous tissues displayed high expression compared with 72.2% (182 of 252) in primary cancer tissues. Among adjacent non-cancerous tissues, no Bmi-1 staining signal was detected in 30.8% (12 in 39) samples. Only 28.2% (11 in 39) samples showed nucleus staining and the remaining 41.0% (16 in 39) samples exhibited cytoplasm staining. Of those cancer tissues, however, 75.4% (190 in 252) was stained in the nucleus and 24.6% (62 in 252) located in the cytoplasm. The elevated Bmi-1 expression was correlated with advanced clinicopathologic classifications (T, N, M) and clinical stages (P&lt;0.001, respectively). A high level of Bmi-1 expression displayed unfavorable overall survival ( P&lt;0.001). The overall survival rate, assessed by the Kaplan-Meier method, was 85.1% (57 in 67) in low Bmi-1 expression group, whereas it was only 59.9% (103 in 172) in high Bmi-1 expression group. In addition, Bmi-1 serves as a high risk for breast cancer and the relative risk increased almost four fold in patients with high Bmi-1 expression compared with that with low Bmi-1 expression by univariate Cox regression analyses. After the adjustment of the confounding factors, Bmi-1 was still found to predict the poor survival (P=0.042), which indicated Bmi-1 was an independent prognostic factor. The overexpression of Bmi-1 increased the mobility and invasiveness in 76N-TERT and MCF-10A, concurrent EMT-like molecular changes, the stabilization of Snail protein and the activation of Akt/GSK3beta pathway. Consistent with these observations, the repression of Bmi-1 in MDA-MB-435S remarkably attenuated the cellular mobility, invasiveness and transformation, as well as tumorigenesis and spontaneous lung metastases in nude mice. In addition, the repression of Bmi-1 reversed the EMT markers and inhibited the Akt/GSK3beta/Snail pathway. However, ectopic Bmi-1 alone was not able to lead to the phenotype of HMECs. Additionally, discordant mRNA expression levels of Bmi-1 and E-cadherin were detected between primary cancer tissues and matched adjacent non-cancerous tissues. The mRNA level of Bmi-1 was strongly up-regulated in breast cancer tissues compared with paired non-cancerous tissues ( P=0.001), whereas the mRNA level of E-cadherin was markedly down-regulated (P=0.042). Furthermore, there was a converse correlation between Bmi-1 and E-cadherin expression at the transcriptional level ( P=0.041). (Abstract shortened by UMI.) / Guo, Baohong. / Adviser: Kung, Hsiang Fu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 161-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Oncogene-induced senescence (OIS) is a fail-safe mechanism activated to halt the proliferation of cells at risk of malignant transformation. It is a cell cycle arrest program of biological changes to the cell comprising of the activation of tumour suppressor pathways, altered cellular metabolism, extensive chromatin remodelling and the activation of a senescence-associated secretory phenotype (SASP). The vast array of proteins secreted by the cells not only play a cell-autonomous role in reinforcing the senescence phenotype, but also modulate the cell's micro-environment by inducing senescence in neighbouring cells, promoting angiogenesis, and initiating an immune response through the recruitment of immune cells. To this end, senescence is a complex phenotype that has countless pathophysiological implications and understanding its molecular mechanisms of activation could prove to be fruitful for understanding its diverse functions. Components of the innate immune system have been shown to play an essential role in the development of the SASP through its processing and activation of Caspase 1 that in turn leads to activation of IL-1B. A gene set enrichment analysis of OIS cells showed significant upregulation in the Pattern Recognition Receptor (PRR) family, from the innate immune response. Hence, we explored the role of innate immune receptors in OIS. Methods and Materials IMR90 human diploid fibroblast cells, stably transfected with an oncogenic ER:RAS fusion protein undergo OIS upon treatment with 4-hydroxytamoxifen. A loss of function siRNA screen was conducted targeting components of the innate immune systems, including pattern recognition receptors. This served as a proof-of-principle screen for a larger screen of proteases and ubiquitin conjugation enzymes. Potential regulators of OIS were identified through siRNA that bypassed the proliferative arrest associated with OIS. We chose to focus on studying the role of TLR2 and TLR10 in senescence. A transcriptome analysis was carried out to identify genes regulated by these TLRs and further biological manipulation was used to confirm the mechanism through which these receptors control senescence. Results Toll-like receptor 2 (TLR2) and TLR10 have been identified as regulators of OIS. Their overexpression in IMR90 cells induces a premature form of senescence where the cells have significantly reduced proliferative activity and display senescence-associated β galactosidase activity. Moreover, the knockdown of TLR2 and TLR10 results in suppression of tumour suppressor pathway genes, reduced signaling through the pathway and blunting of the SASP. High TLR2 expression in patients with lung adenocarcinoma is associated with a higher survival rate. Concomitantly, the screening also identified Caspase 4, a critical component of non-canonical inflammasome signaling, to be regulated by TLR2 and TLR10 in OIS. A full transcriptome analysis of cells with TLR2 and TLR10 knockdown revealed serum amyloid amylase 1 (SAA1) and SAA2 are upregulated in OIS and were also confirmed to be activating ligands of TLR2. The activation of TLR2 by SAA, followed by the engagement of the non-canonical inflammasome by LPS electroporation induced senescence in proliferating IMR90 cells. Conclusion Our results suggest that the TLR2 and TLR10 act as potential tumour suppressor genes, signaling upstream of the inflammasome to initiate the production of inflammatory cytokines, and thereby the SASP. The production of the SASP develops a positive feedback loop, generating the damage-associated molecular pattern (DAMP) A-SAA that initiates an immune response signal cascade and subsequently activates senescence.