MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via regulation of HOXD10. This report was soon challenged by another group who reported that miR-10b expression in a large patient cohort correlated inversely and significantly with tumor size, grade, and vascular invasion, but did not correlate with development of distant metastases or survival. These latter data suggest that miR-10b may impede specific functions associated with breast cancer progression. In this thesis, I present my analysis of miR-10b function in breast carcinoma cells, which revealed that it suppresses their migration and invasion. To define a mechanism that accounts for this suppressive function, I identified T-lymphoma invasion and metastasis 1 (TIAM1), a guanine nucleotide exchange factor for Rac1, as a miR-10b target and demonstrated that miR-10b inhibits TIAM1-dependent Rac1 activation, migration, and invasion. In addition, I identified the VEGF receptor fms-related tyrosine kinase 1 (FLT-1) as a second target of miR-10b and discovered a novel function for FLT-1 in promoting breast carcinoma cell migration and invasion. My results show, for the first time, that Rac activation can be regulated by a specific miRNA and provide a novel mechanism for the regulation of TIAM1 and FLT-1 in breast cancer. These data support the conclusion from clinical data that miR-10b expression correlates inversely with breast cancer progression, and suggest that miR-10b functions to impede breast carcinoma progression by regulating key target genes involved in cell motility.

The focus of experiments presented in this dissertation is to determine how signals created by exposure to environmental stimuli are integrated at the level of transcription, resulting in the generation of specific patterns of gene expression. The model system used was expression of the neurotensinl neuromedin N (NT/N) neuropeptide gene in the neuroendocrine PC12 cell line. This gene is synergistically activated in PC12 cells in response to nerve growth factor, lithium, glucocorticoids, and activators of adenylate cyclase. Several cis-regulatory elements were identified within a 200 bp regulatory region, including AP-1, CRE, and GRE-like elements. Mutational analysis confirmed the importance of these elements for responses to inducer combinations. The primary objective was to identify proteins that interact with NT/N promoter sequences and determine if they are important in mediating responses to inducer combinations. The first set of experiments was designed to investigate changes in AP-1 binding activity. Previous analysis had shown that mutation of the AP-1 site severely curtails responses to all inducer combinations indicating that AP-1 plays a pivotal role in NT/N gene activation. DNA binding studies using in vitro synthesized AP-1 proteins revealed that all heterodimeric combinations could bind both the AP-1 and JARE sites; however, these complexes displayed a higher affinity for the AP-1 site. c-Jun homodimers were also found to bind both these sites albeit with a lower affinity and with a preference for the JARE site. These studies revealed that specificity is probably not at the level of DNA binding. Therefore, it was possible that only a subset of AP-1 proteins were activated upon stimulation. DNase I footprint analysis using nuclear extracts from PC12 cells showed changes in protection at the consensus AP-1 site upon treatment with inducers suggesting changes in AP-1 binding activity. It was found that AP-1 binding activity was increased upon stimulation, with the major component being Jun B. However, substantial levels of c-Fos and c-Jun were also detected at some time points. These results coupled with transfection data demonstrating that forced expression of c-Jun and c-Fos result in potent synergistic activation of the NT/N promoter support the hypothesis that c-Jun and c-Fos are also involved in NT/N gene activation. DNase I footprinting studies using PC12 nuclear extracts also revealed substantial areas of protection surrounding the CRE element. This result, along with the high degree of conservation of these sequences between human and rat, suggested they play a role in the regulation of the NT/N gene in PC12 cells. Mutational analysis of this region showed that sequences upstream of the CRE were important for full activation of the NT/N promoter. Specific mutation of the CRE resulted in a 75% decrease in activity upon induction, a level similar to that observed previously with less precise linker scanner mutations. This site had also been shown to be critical for c-Jun mediated NT/N activation, even though c-Jun homodimers do not bind this site in vitro. Therefore, nuclear extracts from PC12 cells were tested for the presence of proteins which could bind this site. Complexes composed of both c-Jun and ATF-2 were found in extracts from both uninduced and induced PC12 cells. ATF-2 could mediate both the recruitment of c-Jun to this site as well as mediate the effect of activators of adenylate cyclase, since ATF-2 has been shown to be a target for protein kinase A in vitro. Expression of ATF-2 in PC12 cells resulted in a modest increase in NT/N promoter activation. The significant levels of endogenous ATF-2 protein in PC12 cells most likely accounts for the relatively small magnitude of this effect. Experiments with the closely related protein, ATF-a2, revealed that it potently antagonizes c-Jun activation while forced expression of ATF-2 did not affect c-Jun activation under the conditions analyzed. Therefore, ATF proteins could be involved in both activation and repression of the NT/N gene. Both c-Jun and ATF-2 have been shown to be activated by c-Jun N-terminal kinase (JNK) in response to environmental stress or cytokine activation. Therefore, the ability of inducers to activate the previously described N-terminal ATF-2 activation domain was investigated using a GAL4-ATF-2 (1-109) chimer construct. This construct was not significantly activated by inducer combinations that result in high level NT/N gene expression, indicating that activation of ATF-2 through this pathway is not involved in NT/N gene activation. Also activation of JNK, a MAPK which activates both c-Jun and ATF-2, only partially substituted for NGF indicating that NGF activates an additional pathway. The data presented here support a model involving synergistic transcriptional activation of the NT/N promoter by c-Jun/c-Fos, ATF-2, ATF-2/c-Jun and the GR. ATF-2 was found to enhance NT/N promoter activation while a splice variant (ATF-2 195) lacking a central portion of ATF-2 that is rich in Ser/Thr residues had no effect suggesting that this region could be important for ATF-2 activation in PC12 cells. The identification of the signaling pathways that mediate the effects of inducer combinations on NT/N gene activation will be an important future goal and should provide insights into the control of neuronal gene expression.

No description available.

Cette étude porte sur le contrôle moléculaire de l'expansion cellulaire dans l'apex de la racine du peuplier. L'étude a été focalisée sur la régulation de l'expression des aquaporines TIP1s sous deux niveaux de déficit hydrique. Un cadre conceptuel, combinant analyse de la densité de transcrits à haute résolution spatiale et un formalisme de la mécanique des fluides, a été établi pour décrire la régulation de l'expression des gènes dans le temps et l'espace, le long de l'apex. Deux états de croissance contrastés ont été étudiés : un où la croissance de l'apex de la racine est rétablie (après 3 jours de stress modéré, 80 mosmol kg-1, 100 g L-1 de polyéthylène glycol PEG à 3500 g mol-1) et un où elle est réduite (après 3 jours de stress fort, 250 mosmol kg-1, 200 g L-1 de PEG). L'analyse cinématique a révélé une sensibilité différentielle de la vitesse d'allongement relatif (REGR) selon le niveau de stress et la position le long de l'apex. Au niveau moléculaire, nous montrons que lorsque la croissance est réduite, les pics de densité de transcrits des TIP1s sont plus forts et décalés dans les premiers millimètres de l'apex, zone où la croissance des cellules est maintenue. Cependant les niveaux d'induction/répression n'étaient jamais plus élevés que dans la condition témoin, soulignant qu'une forte densité de transcrits n'est pas forcément synonyme d'une forte induction de l'expression. Focalisé sur la combinaison de deux processus dynamiques, l'expansion cellulaire et l'expression des gènes, cette étude a mis en évidence que les conclusions tirées de l'analyse de ces processus sont influencées par la façon dont le temps, l'espace sont considérés / This study consideres the molecular control of cell expansion in poplar root apex. The study was focused on the regulation of the TIP1 aquaporins expression under two levels of water deficit. A conceptual framework combining transcript density analysis (quantitative PCR) at a high spatial resolution and a fluid mechanics formalism was established to describe the regulation of gene expression in time and space along the root apex. Two contrasting growth status were both studied: root growth rate is either restored (after three days of moderate stress, 80 mmol kg-1, 100 g L-1 polyethylene glycol PEG 3500 g mol-1) or root growth rate is reduced (after 3 days of high stress, 250 mosmol kg-1, 200 g L-1 PEG). Kinematic analysis revealed a differential sensitivity of the relative elemental growth rate (REGR) according to the stress level and to the coordinate along the apex. At the molecular level, we showed that growth reduction was associated with a shift of maximal densities of transcripts towards the first millimeters of the apex, where cell expansion was maintained. Meanwhile the induction/repression levels were never stronger than in the control condition underlying that a high transcript density does not mean a high transcriptional induction. Focused on the combination of two dynamic processes, cell expansion and gene expression, my thesis showed that the conclusions issued from the analyses of these processes are influenced by the way time, space and age are considered

Orientador: Claudia Marcia Aparecida Carareto / Banca: Ricardo De Marco / Banca: André Luís Laforga Vanzela / Resumo: A resistência aos inseticidas é um modelo de processo evolutivo onde o inseticida atua como agente seletivo e, como resposta à seleção, ocorre a evolução da resistência nas populações de insetos. As enzimas citocromo P450 monooxigenases (CYP) formam uma família responsável pela resistência aos inseticidas. Tem sido proposto que a inserção de elementos transponíveis (TEs) em regiões reguladoras ou codificadoras dos genes da família Cyp pode alterar a expressão gênica e induzir a resistência aos inseticidas. No presente estudo foram realizadas análises in silico que permitiram identificar a ocorrência de inserções de fragmentos de TEs em 35 genes Cyps com diferentes funções, e em seus genes flanqueadores, em Drosophila melanogaster e D. simulans, além de 13 genes Cyps de seis espécies do grupo melanogaster de Drosophila. As inserções de TEs ocorreram principalmente nas regiões flanqueadoras 5' dos Cyps associados à resistência aos inseticidas e à função monooxigenase geral. Os resultados não indicaram qualquer relação entre a distância em relação ao gene e o número de inserções. As análises mostraram que a maioria das inserções pertence à classe de transposons de DNA, sendo o transposon DNAREP1_DM o que apresentou o maior número de cópias. O fato de essas seqüências apresentarem putativos sítios de ligação de fatores de transcrição sugere que possam desempenhar algum papel na regulação dos genes Cyps. Também foi analisada a ocorrência de polimorfismos de inserção de TEs em regiões flanqueadoras de genes da família Cyp, em diferentes linhagens geográficas resistentes e suscetíveis, de D. melanogaster e D. simulans. Análises evidenciaram a presença de polimorfismo interpopulacional de tamanho das regiões flanqueadoras dos genes Cyp6w1, Cyp6a2 e Cyp12d1, porém, não indicaram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Insecticide resistance is a model of evolutionary process where the insecticide acts as the selective agent and resistance in the insect populations evolves as an answer to selection. Cytochrome monooxygenases (CYP) is family of enzymes responsible for the insecticide resistance. It has been proposed that insertion of transposable elements (TEs) in regulatory or coding regions of the Cyp genes can alter gene expression and induce insecticide resistance. In the present study in silico analyses allowed identifying the insertion of TE fragments in 35 Cyp genes with different functions, in Drosophila melanogaster and D. simulans, as well as in 13 Cyps of six species of the melanogaster group of Drosophila. The TE insertions occurred mainly in the 5' flanking regions of Cyp genes associated to resistance and to those with a general monooxygenase function. The results did not indicate any relationship between the number of insertions and the distance in relation to the gene. The analyses showed that most of the insertions belong to the DNA transposon class, being DNAREP1_DM the most numerous. Since this element carry putative biding sites of transcription factors it can be suggested they play same role in gene regulation. The polymorphism of TE insertions in the flanking regions of Cyp6w1, Cyp6a2 and Cyp12d1, genes associated to resistance, found in resistant and as well as in susceptible geographical strains of D. melanogaster and D. simulans, does not indicate any relationship between the presence of TEs in those regions and the insecticide resistance. The results also showed that the insertions of TEs in the proximities of the Cyps associated to resistance is differential among six species of the melanogaster group, not following the genomic proportion of TEs in each species. These results also suggest that TEs inserted in the Cyp flanking regions can carry out an adaptive... (Complete abstract click electronic access below) / Mestre

O ferro (Fe) é um oligoelemento capaz de aceitar e doar elétrons. Tal propriedade o torna extremamente útil em diversos componentes importantes ao bom funcionamento do organismo e da célula. O Fe está associado a algumas proteínas, está presente em citocromos, em moléculas que se ligam ao oxigênio (hemoglobina e mioglobina) e em uma grande variedade de enzimas. O aumento e a diminuição da sua oferta levam a alterações na expressão de RNAs mensageiros e proteínas responsáveis pela sua própria homeostase. Sabe-se que a expressão de vários genes envolvidos no metabolismo do Fe é regulada pós-transcricionalmente, por meio de mecanismo que é desencadeado por sua ligação em regiões não traduzíveis presentes em mRNAs específicos, o que interfere no seu grau de poliadenilação, e por conseguinte, na estabilidade e na tradução do transcrito. A Mb é uma heme-proteína de 18,8 kDa, altamente expressa no tecido muscular esquelético e cardíaco, e que pertence a mesma família da hemoglobina. Sabendo-se que cerca de 15% do Fe existente no organismo está presente nos músculos, no presente trabalho avaliamos se a suplementação e restrição de Fe, a curto e longo prazo, alteram a expressão gênica da Mb no músculo oxidativo Soleus (S), glicolítico Extensor Digital Longo (EDL) e no cardíaco. Observamos que a restrição de Fe, a longo prazo, provocou um aumento na expressão gênica e protéica da Mb, apenas no músculo Soleus, sem alterar o grau de poliadenilação do transcrito, enquanto a suplementação não alterou os parâmetros avaliados em nenhum dos tecidos. A administração aguda de Fe não alterou a expressão gênica e protéica da Mb, nem o grau de poliadenilação do transcrito em nenhum dos tecidos estudados. Estes resultados sugerem que a regulação da expressão da Mb pelo Fe se dá apenas transcricionalmente, e de maneira tecido específica. / Iron is a trace element that can accept and donate electrons. This property makes iron extremely important to several components involved with the proper functioning of the organism and cells. Iron is associated with some proteins, is present in cytochromes, molecules that bind to oxygen (hemoglobin and myoglobin) and a variety of enzymes. The increase and decrease of its offer lead to changes in the expression of mRNAs and proteins responsible for their own homeostasis. It is known that the expression of several genes involved in the metabolism of iron is regulated post-transcriptionally through a mechanism that is triggered by its binding in non-translatable regions of specific mRNAs, which interferes with their polyadenylation, and as a consequence, with the stability and translation of the transcripts. Mb is a heme-protein with 18,8 kDa, highly expressed in skeletal and cardiac muscle, and it belongs to the same family of hemoglobin. About 15% of iron in the body is present in muscle tissue. Thus, this study aimed to investigate if long- and short-term Fe supplementation and restriction affect Mb gene expression in the oxidative Soleus (S), glycolitic Extensorum Digitalis Longus (EDL), and cardiac muscles. It was shown that long- term Fe restriction increased Mb mRNA and protein expression only in S muscle, without interfering in the transcript polyadenylation, whereas Fe supplementation did not alter any parameter evaluated in the three tissues. The short-term iron administration did not change the Mb mRNA, polyadenylation and protein expression in any of the tissues studied. The present results indicate that the regulation of Mb gene expression by iron occurs only at transcriptional level and in a tissue specific manner.

No final do século passado, avanços ocorridos no campo da Biologia Molecular levantaram questionamentos sobre como organismos complexos, com poucos genes a mais que organismos relativamente mais simples, regulariam seu desenvolvimento e funções celulares tão mais intrincadas. A descoberta dos RNAs não codificadores de proteínas e suas funções lançou nova luz ao entendimento da regulação da expressão gênica em organismos superiores. Apesar do conhecimento adquirido nos últimos anos, pouco ainda é sabido sobre a regulação destes RNAs. MicroRNAs, por outro lado, são uma espécie bem estudada de pequenos RNAs preditos como possíveis reguladores de mais de 60 % dos genes codificadores de proteínas no genoma humano, considerados importantes reguladores da expressão gênica em nível pós-transcricional. O presente projeto estudou a possível regulação do gene não codificador de proteínas TUG1, envolvido em proliferação celular e apoptose, por miRNAs e o papel desta regulação em processos tumorigênicos. Para isto foram utilizadas técnicas que superexpressaram e silenciaram miRNAs e técnicas de PCR quantitativo em tempo real para medir o nível de TUG1 nas amostras tratadas. Verificou-se a possibilidade de regulação do TUG1 por microRNAs em diferentes linhagens celulares sendo que, no entanto, esta regulação não parece ser importante em nível fisiológico / At the end of the last century, advances occurred in the Molecular Biology field raised questions about how complexes organisms, with few more genes than relatively simpler organisms, regulate it so intricate development and cellular functions. The discovery of long non-protein coding RNAs and it functions gave light to the understanding of gene expression regulation in superior organisms. Despite the knowledge acquired in the last years, few is yet known about the regulation of these RNAs. MicroRNAs, other way, are a well-studied tiny RNAs specie. They are predicted as possible regulators of more than 60 % of protein-coding genes in the human genome and considered important regulators of gene expression regulation at post-transcriptional level. This project studied the possible regulation of the non protein-coding gene TUG1, involved in cell proliferation and apoptosis, by microRNAs and the role of this regulation in tumorigenic processes. In order to do this we used techniques that superexpressed and silenced miRNAs and techniques of real time quantitative PCR to measure the TUG1 levels in the treated samples. We find the possibility of regulation of TUG1 by microRNAs in different cell lineages but this regulation does not seems to be important in a physiologic context.

INTRODUÇÃO: O aracnídeo Phoneutria nigriventer, também conhecido por aranha-armadeira, possui um veneno complexo, contendo vários peptídeos que ativam canais iônicos nas células. Dentre estes, só dois neuropeptídeos, Tx2-5 e Tx2-6, destacam-se por relaxar o músculo liso trabecular do corpo cavernoso, induzindo ereção peniana em camundongos e ratos. Este efeito tem sido associado à produção de oxido nítrico pela ativação de óxido nítrico sintases. No entanto, faltam estudos mais amplos para determinar o papel de Tx2-6 na indução da ereção. OBJETIVOS: Identificar os genes diferencialmente expressos no tecido erétil de camundongos após indução da ereção pela Tx2-6 utilizando microarranjos de oligonucleotídeos. Validação dos resultados obtidos nos microarranjos por PCR quantitativa e imuno-histoquímica. MATERIAIS E MÉTODOS: Camundongos machos e adultos da linhagem Swiss foram divididos em dois grupos: controle (n=10), inoculados pela via intracavernosa com 20 l de solução salina; e tratado (n=10), os quais receberam 0,006gg/animal do peptídeo Tx2-6 diluído em 20 l de salina pela via intracavernosa. Uma hora após o início da ereção no grupo tratado todos os animais foram sacrificados e retirou-se o pênis. Este último foi dividido em dois fragmentos, uma parte do material foi congelada em nitrogênio líquido e mantida a 80°C até a extração do RNA para os experimentos de microarranjos e PCR quantitativa; outra parte foi utilizada para avaliação imuno-histoquímica. RESULTADOS: No grupo tratado a ereção foi observada 30-45 minutos após aplicação de Tx2-6 e mantida durante 120 minutos. Os camundongos de grupo controle não apresentaram nenhum indício de ereção. Nos experimentos de microarranjos, onde foram analisados 34.000 genes representando o genoma total do camundongo, identificou-se 3.803 (12,3%) genes com expressão diferencial de pelo menos ±1,5 vez entre os grupos (1.823 genes superexpressos e 1.980 genes subexpressos no grupo tratado comparado ao controle). Os genes ednrb, sparc, fn1, sstr2, pdgfr foram selecionados para validação dos microarranjos por PCR quantitativa e confirmaram a superexpressão em relação aos controles. As proteínas Fn1, Sstr2 e Pdgfr resultaram aumentadas no grupo tratado após avaliação imuno-histoquímica. CONCLUSÕES: A inoculação de Tx2-6 pela via intracavernosa alterou o perfil de expressão gênica no tecido erétil de camundongos. O número de genes superexpressos foi similar ao de genes subexpressos. Serão necessários outros estudos para entender melhor as vias moleculares que Tx2-6 afeta na indução da ereção peniana. / INTRODUCTION: The Phoneutria nigriventer arachnid, also known as armed-spider, has a complex venom, composed by several peptides that affect cellular ionic channels. Among these, only two neuropeptides, Tx2-5 and Tx2-6 induce penile erection in mice and rats and this effect has been associated with the production of nitric oxide by the activation of nitric oxide synthases. Moreover, there is a scarcity of studies focusing on the role of Tx2-6 in the induction of erection. OBJECTIVES: To identify the differently expressed genes in the erectile tissue of mice after erection induction by Tx2-6 using oligonucleotide microarrays. To validate microarray results by quantitative PCR and immunohistochemistry. MATERIALS AND METHODS: Swiss adult male mice were divided in two groups: control (n=10) were injected intracavernously with 20 gl of saline solution; and treated (n=10) were injected intracavernously with 0.006gg/mouse of the Tx2-6 peptide diluted in 20 gl of saline solution. After checking the penile erection in the treated group, all mice were sacrificed one hour after the beginning of erection for the removal of the penis. Penile organ was divided into two fragments, one piece was immediately frozen in liquid-nitrogen and stored at -80°C until RNA extraction to make the microarray and quantitative PCR experiments; the other was reserved for immunohistochemistry analysis. RESULTS: In the treated group, erection was noticed 30-45 minutes after Tx2-6 inoculation and lasted for 120 minutes. Control mice did not present any sign of erection. Considering as differentially expressed genes with a ±1.5 fold expression difference, of the 34,000 genes on the microarray we identified 3,803 (12.3%) genes differentially expressed between the groups (1,823 genes up-regulated and 1,980 genes down-regulated in the treated group compared to controls). The ednrb, sparc, fn1, sstr2, pdgfr genes were selected for validation of microarray results by using quantitative PCR and confirmed the up-regulation when compared to controls. After immunohistochemistry analysis the Fn1, Sstr2 and Pdgfr proteins were found increased in the treated group. CONCLUSIONS: The intracavernous inoculation of Tx2-6 modified the gene expression profile of erectile tissue of mice. The number of upregulated and down-regulated genes was similar. Further studies are needed to understand the molecular pathways that Tx2-6 affect to induce penile erection.

Cheung Hoi Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 151-166). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / LIST OF FIGURES --- p.VII / LIST OF TABLES --- p.IX / CONTENTS --- p.X / Chapter CHAPTER ONE: --- GENERAL INTRODUCTION / Chapter 1.1 --- Non-coding RNAs in transcriptional output --- p.2 / Chapter 1.2 --- Diverse functions of non-coding RNAs --- p.5 / Chapter 1.3 --- HI9: imprinted non-coding RNA --- p.6 / Chapter 1.4 --- Objective --- p.7 / Chapter CHAPTER TWO: --- The ROLE OF H19 RNA IN MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- H19-Igf2 locus as a model for genomic imprinting --- p.10 / Chapter 2.1.2 --- HI9 as a non-protein coding regulatory RNA --- p.12 / Chapter 2.1.3 --- Controversial roles of H19 RNA --- p.13 / Chapter 2.1.4 --- Novel role of H19 RNA in drug resistance --- p.15 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.17 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Cell culture --- p.19 / Chapter 2.2.2.2 --- Plasmid construction and stable cell transfection --- p.19 / Chapter 2.2.2.3 --- Transient gene transfection --- p.20 / Chapter 2.2.2.4 --- RNA isolation and RT-PCR --- p.21 / Chapter 2.2.2.5 --- MTT drug sensitivity assay --- p.22 / Chapter 2.2.2.6 --- Western blot analysis --- p.22 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Differential expression of H19 RNA in different human cancer cell lines --- p.24 / Chapter 2.3.2 --- R-HepG2 cells over-expressed P-glycoprotein and H19 RNA --- p.24 / Chapter 2.3.3 --- Development of H19-silenced cell lines in HepG2 cells by RNA interference --- p.26 / Chapter 2.3.4 --- Altered drug sensitivity in H19-silenced cells --- p.28 / Chapter 2.3.5 --- Expression of P-glycoprotein in H19-silenced cells --- p.31 / Chapter 2.3.6 --- Overexpression of H19 RNA in HepG2 cells --- p.34 / Chapter 2.3.7 --- Induction of H19 RNA and MDR1 in HepG2 cells --- p.34 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- H19 regulation of MDR1 associated drug resistance --- p.38 / Chapter 2.4.2 --- The puzzle of riboregulation in drug resistance --- p.40 / Chapter CHAPTER THREE: --- The ROLES OF PTB AND IMP1 IN H19-RELATED MDR1 EXPRESSION OF HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- H19 RNA binding proteins --- p.43 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.46 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell culture --- p.48 / Chapter 3.2.2.2 --- Plasmid construction and stable cell transfection --- p.48 / Chapter 3.2.2.3 --- RNA extraction and RT-PCR --- p.48 / Chapter 3.2.2.4 --- MTT drug sensitivity assay --- p.48 / Chapter 3.2.2.5 --- Western blot analysis --- p.48 / Chapter 3.2.2.6 --- Real-time PCR analysis of gene expression --- p.49 / Chapter 3.2.2.7 --- DOX efflux assay --- p.49 / Chapter 3.3 --- Results / Chapter 3.3.1 --- PTB knockdown increased P-glycoprotein expression --- p.51 / Chapter 3.3.2 --- IMP1 knockdown decreased MDR1 /P-glycoprotein expression --- p.54 / Chapter 3.3.3 --- Altered drug sensitivity in IMP 1 -knockdown cells --- p.60 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Antagonistic effect of PTB and IMP1 on H19/MDR1 expressions --- p.64 / Chapter 3.4.2 --- Complexity of riboregulation --- p.65 / Chapter CHAPTER FOUR: --- IDENTIFICATION OF H19 RNA BINDING PROTEINS FROM HUMAN HEPATOCELLULAR CARCINOMA HepG2 CELLS / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Overview of RNA-protein interactions --- p.69 / Chapter 4.1.2 --- Methodology in the study of RNA-protein interactions --- p.71 / Chapter 4.1.3 --- Identification of RNA-binding proteins --- p.72 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.75 / Chapter 4.2.2 --- Methods / Chapter 4.2.2.1 --- Screening of H19 cDNA from human placenta cDNA library --- p.78 / Chapter 4.2.2.2 --- Preparation of nuclear and cytoplasmic extracts from HepG2 cells / Chapter 4.2.2.3 --- In vitro RNA transcription and RNA labeling --- p.80 / Chapter 4.2.2.4 --- RNA electrophoretic mobility shift assay --- p.81 / Chapter 4.2.2.5 --- In vitro UV-crosslinking assay --- p.82 / Chapter 4.2.2.6 --- Preparation of RNA-affinity column and isolation of RNA binding proteins --- p.83 / Chapter 4.2.2.7 --- In-gel digestion and MALDI-TOF mass spectrometry --- p.84 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Screening of H19 cDNA and preparation ofH19 RNA --- p.86 / Chapter 4.3.2 --- Electrophoretic mobility shift analysis of H19 RNA with HepG2 cytoplasmic extract --- p.87 / Chapter 4.3.3 --- UV-crosslinking of H19 RNA with HepG2 nuclear and cytoplasmic extract --- p.90 / Chapter 4.3.4 --- Isolation of H19 RNA binding proteins by RNA-affmity chromatography --- p.94 / Chapter 4.3.5 --- Confirmation of PTB and IMP1 as H19 RNA binding protein --- p.96 / Chapter 4.3.6 --- MALDI-TOF mass spectrometric analysis of isolated H19 RNA binding proteins --- p.96 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- RNA-protein interactions: an initial step for mechanistic study --- p.99 / Chapter 4.4.2 --- In vitro and in vivo methods for isolation of RNA binding proteins --- p.101 / Chapter 4.4.3 --- Novel role of hnRNP M protein in H19 RNA binding --- p.103 / Chapter CHAPTER FIVE: --- THE ROLE OF PTB IN APOPTOSIS / Chapter 5.1 --- Introduction / Chapter 5.1.1 --- Overview of polypyrimidine tract-binding protein in RNA processing and post-transcriptional gene regulation --- p.106 / Chapter 5.1.2 --- Evidences of polyrimidine-tract binding protein in the regulation of apoptosis --- p.108 / Chapter 5.2 --- Materials and methods / Chapter 5.2.1 --- Materials --- p.111 / Chapter 5.2.2 --- Methods / Chapter 5.2.2.1 --- Cell culture --- p.114 / Chapter 5.2.2.2 --- Stable cell transfection in A431 cells --- p.114 / Chapter 5.2.2.3 --- Western Blot analysis --- p.114 / Chapter 5.2.2.4 --- MTT drug sensitivity assay --- p.114 / Chapter 5.2.2.5 --- DNA fragmentation assay --- p.115 / Chapter 5.2.2.6 --- Flow cytometry analysis of apoptosis --- p.115 / Chapter 5.2.2.7 --- Caspase activity assay --- p.116 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Taxol as an apoptosis inducer in HepG2 cells --- p.117 / Chapter 5.3.2 --- PTB was cleaved during Taxol-induced apoptosis --- p.118 / Chapter 5.3.3 --- PTB knockdown increased Taxol cytotoxicity and apoptosis --- p.118 / Chapter 5.3.4 --- Effect of PTB knockdown on drug sensitivity of cells --- p.121 / Chapter 5.3.5 --- Effect of PTB knockdown on other drug-induced apoptosis --- p.121 / Chapter 5.3.6 --- Effect of PTB knockdown on the basal expressions of genes in apoptosis pathway --- p.126 / Chapter 5.3.7 --- The role of caspase-9 activation in PTB-regulated apoptosis --- p.129 / Chapter 5.3.8 --- The effect of PTB knockdown on pro-caspase-9 expression and Taxol-induced apoptosis in A431 cells --- p.133 / Chapter 5.3.9 --- The role of PTB in the regulation of intrinsic apoptosis pathway --- p.136 / Chapter 5.4 --- Discussion / Chapter 5.4.1 --- The role of PTB in intrinsic apoptosis pathway --- p.138 / Chapter 5.4.2 --- PTB in regulation of pro-caspase-9 expression --- p.139 / Chapter CHAPTER SIX: --- GENERAL DISCUSSION AND CONCLUSION / Chapter 6.1 --- H19 as a potential target in anti-cancer gene therapy --- p.143 / Chapter 6.2 --- Conclusion --- p.144 / Chapter 6.3 --- Unanswered questions and future work --- p.145 / Chapter 6.4 --- A proposed model for H19 pathway --- p.148 / REFERENCES --- p.151

Gho Wai-Man. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-123). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENT --- p.VII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / ABBREVIATIONS --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Epidemiology --- p.2 / Chapter 1.3 --- Etiological factors --- p.6 / Chapter 1.3.1 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3.1.1 --- Hepatitis B Virus (HBV) --- p.7 / Chapter 1.3.1.2 --- Hepatitis C Virus (HCV) --- p.9 / Chapter 1.3.2 --- Aflatoxin Exposure --- p.10 / Chapter 1.3.3 --- Alcohol Abuse --- p.11 / Chapter 1.3.4 --- Liver Cirrhosis --- p.12 / Chapter 1.4 --- Genetic alterations in hcc --- p.16 / Chapter 1.4.1 --- Chromosomal Gain --- p.16 / Chapter 1.4.2 --- Chromosomal Loss --- p.17 / Chapter 1.5 --- Discovery of common activating transcription factor 5 (atf5) down-regulations in hcc --- p.19 / Chapter 1.5.1 --- Chromosome 19 Aberration in HCC --- p.19 / Chapter 1.5.2 --- Discovery of High Frequency of　ATF5 Down-regulations --- p.19 / Chapter 1.5.3 --- Activating Transcription Factor Family --- p.20 / Chapter 1.6 --- Aim of thesis --- p.28 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.29 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Buffers --- p.31 / Chapter 2.1.3 --- Cell culture --- p.31 / Chapter 2.1.4 --- Nucleic acids --- p.32 / Chapter 2.1.5 --- Enzymes --- p.32 / Chapter 2.1.6 --- Equipment --- p.32 / Chapter 2.1.7 --- Kits --- p.33 / Chapter 2.1.8 --- Software and Web Resource --- p.33 / Chapter 2.2 --- Dna extraction --- p.34 / Chapter 2.2.1 --- Cell Lines --- p.34 / Chapter 2.2.2 --- Primary HCC --- p.34 / Chapter 2.2.3 --- Lymphocytic DNA --- p.35 / Chapter 2.3 --- Rna extraction --- p.36 / Chapter 2.4 --- Dna sequencing --- p.38 / Chapter 2.4.1 --- Polymerase Chain Reaction (PCR) --- p.38 / Chapter 2.4.2 --- Cycle Sequencing --- p.39 / Chapter 2.5 --- Dual-labeled fluirescence in situ hybridization (fish) --- p.41 / Chapter 2.5.1 --- FISH Probe Preparation --- p.41 / Chapter 2.5.1.1 --- Preparation of Human Bacterial Artificial Chromosome (BAC) --- p.41 / Chapter 2.5.1.2 --- Nick Translation --- p.41 / Chapter 2.5.2 --- FISH --- p.42 / Chapter 2.6 --- 5-aza-2'-deoxycytidine & trichostatin a treatment on cell lines --- p.43 / Chapter 2.7 --- Bisulfite modificaiton of dna --- p.43 / Chapter 2.8 --- Methylation-specific pcr (msp) --- p.44 / Chapter 2.9 --- Bisulfite dna sequencing --- p.44 / Chapter 2.10 --- Quantitative reverse transcription pcr (qrt-pcr) --- p.46 / Chapter 2.11 --- In-vitro and in-vivo functinal examination --- p.49 / Chapter 2.11.1 --- ATF5 Transfection --- p.49 / Chapter 2.11.2 --- Cell Growth Assay --- p.50 / Chapter 2.11.3 --- Xenograft Development --- p.51 / Chapter 2.12 --- codelink expression microarray --- p.51 / Chapter 2.13 --- Statistical analysis --- p.53 / Chapter CHAPTER 3 --- INACTIVATION OF MECHANISMS UNDERLYING ATF5 DOWN-REGULATION --- p.54 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and methods --- p.58 / Chapter 3.2.1 --- Cell Lines --- p.58 / Chapter 3.2.2 --- Mutational Analysis --- p.58 / Chapter 3.2.3 --- Copy Number Loss --- p.59 / Chapter 3.2.4 --- Epigenetic Control --- p.59 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Sequencing Analysis of A TF5 Gene --- p.67 / Chapter 3.3.2 --- FISH Analysis of ATF5 Copy Number --- p.73 / Chapter 3.3.3 --- Epigenetic Control of A TF5 Expression --- p.73 / Chapter 3.4 --- Discussion --- p.82 / Chapter CHAPTER 4 --- FUNCTIONAL EXAMINATION AND INVESTIGATION OF DOWNSTREAM TARGETS MODULATED BY ATF5 --- p.85 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Cell Lines --- p.88 / Chapter 4.2.2 --- Plasmids and Transfection --- p.88 / Chapter 4.2.3 --- Cell Growth Assay --- p.88 / Chapter 4.2.4 --- Xenograft Development --- p.88 / Chapter 4.2.5 --- CodeLink Expression Microarray --- p.89 / Chapter 4.2.6 --- Quantitative RT-PCR --- p.90 / Chapter 4.2.7 --- Statistical analysis --- p.90 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Cell Proliferation --- p.91 / Chapter 4.3.1.1 --- In-Vitro Examination --- p.91 / Chapter 4.3.1.2 --- In-Vivo Examination --- p.91 / Chapter 4.3.2 --- Microarray A nalysis --- p.91 / Chapter 4.3.3 --- Correlation of A TF5 with Id-1 Expression --- p.103 / Chapter 4.4 --- Discussion --- p.106 / Chapter CHAPTER 5 --- PROPOSED FUTURE INVESTIGATIONS --- p.110 / Chapter 5.1 --- inactivation mechanisms of atf5 gene --- p.111 / Chapter 5.2 --- Molecular pathways modulated by atf5 --- p.112 / Chapter CHAPTER 6 --- REFERENCES --- p.114