Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

RNA interference (RNAi) is a natural mechanism of cellular defence against exogenous double stranded RNA (dsRNA). The discovery of small dsRNA molecules which can be processed by the RNAi pathway in mammalian cells was one of the key advances in the study of functional genomics. These molecules can be designed to downregulate the expression of specific genes. Collections or libraries of dsRNA molecules targeting an extensive number of genes are now available. Using these libraries, numerous studies have implemented high-throughput screens for the study of molecular effectors of numerous phenotypes. The process of designing an RNAi screen requires the consideration of several critical factors during both the experimental and analysis phases. The experimental screen should aim to reproduce the biological phenomenon studied as closely as possible by choosing an adequate model and screening conditions. Phenotype evaluation and assessment of knockdown effects need careful consideration. The results obtained from large-scale RNAi screens are often complex. An analysis pipeline should be implemented which integrates the biological basis of the phenomenon and facilitates the interpretation of the data. This project designed and implemented an unbiased shRNA screen in two in vitro models relevant to carcinogenesis and diabetes. The first screen implemented used a model of neighbour suppression to study the molecular effectors of the response in tumorigenic cells to growth suppression cues from the surrounding tissue, a cellular interaction relevant in early tumorigenesis. The second screen studied two phenotypes relevant to diabetes: proliferation and resistance to lipotoxicity of β-cells in a reversibly immortalised  cell line. An integrative analysis pipeline was also developed to apply network biology and functional enrichment analysis methods for the interpretation of the data obtained from both screens.

Human pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has been made in elucidating the signalling pathways regulating pluripotency and differentiation, our mechanistic understanding of the downstream regulations is still incomplete. Moreover, studies aimed at clarifying these aspects are severely impeded by the lack of efficient methods to conditionally modulate gene expression in hPSCs and hPSC-derived cells. In this dissertation I provide new insights into the molecular mechanisms controlled by the Activin/Nodal-SMAD2/3 signalling pathway, whose activity dictates the balance between hPSC pluripotency and differentiation. First, I show that SMAD2/3 modulates the chromatin epigenetic landscape of hPSCs by cooperating with the pluripotency factor NANOG to recruit the DPY30-COMPASS complex and promote histone 3 lysine 4 trimethylation (H3K4me3). This regulation promotes expression of pluripotency genes, while poising developmental regulators for activation during differentiation. Secondly, I describe a novel efficient approach for inducible gene knockdown in hPSCs and hPSC-derived cells. By taking advantage of this technology, I demonstrate that DPY30 is required for early differentiation of hPSCs into certain mesoderm and endoderm derivatives. Finally, I report the first large-scale proteomic identification of SMAD2/3 interacting proteins in both undifferentiated and differentiating hPSCs. This analysis not only confirms that SMAD2/3 interacts with multiple epigenetic modifiers involved in hPSC fate choices, but also implicates SMAD2/3 in several functions other than transcriptional regulation. In particular, I describe how SMAD2/3 physically and functionally interacts with the METTL3-METTL14-WTAP complex to promote the formation of N6-methyladenosine (m6A). This epitranscriptional modification antagonizes the expression of selected mRNAs, including pluripotency factors whose transcription is promoted by SMAD2/3. Therefore, this provides a negative feedback that facilitates rapid exit from pluripotency upon inhibition of Activin/Nodal signalling. Overall, the work presented in this dissertation advances the stem cell field in two ways. First, it demonstrates that the Activin/Nodal-SMAD2/3 pathway finely orchestrates the balance between pluripotency and differentiation by shaping both the epigenome and the epitranscriptome of hPSCs. Secondly, it provides a novel powerful technology to facilitate further studies of the mechanisms that regulate cell fate decisions.

Filip Lhota: Study of alternatively spliced variants of estrogen receptor alpha in breast cancer cell lines Abstract: Estrogen receptor α (ER-α) is a transcription factor responsible for mediation of the activities of its natural ligand 17-β-estradiol (E2), the hormone that together with progesterone belongs to the key regulators of mammary epithelial as well as breast cancer cells proliferation. Except to the major gene product consisting of all eight coding exons of ER-α, numerous qualitatively and quantitatively different spliced variants originated from primary transcript by activity of alternative splicing is expressed. Despite that some of these spliced variants have been functionally characterized, their precise role on final ER-α cellular activity remains to be elucidated. The functional characterization of individual alternative forms of ER-α and description of its participation on the overall ER-α activity is important for our understanding of their biogenesis and is also critical for the delineation of molecular bases for ER-α regulation during anti cancer chemotherapy. This work aimed to study the influence of alternatively spliced ER-α variants on the growth characteristics of clones constructed from stable mammary tissue cell lines in regulation to cultivation conditions and cellular...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20662号 / 医博第4272号 / 新制||医||1024(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 松田 道行, 教授 原田 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

P-Glykoprotein als Produkt des MDR1-Gens stellt einen gut untersuchten Mediator der Multidrugresistenz (MDR) in humanen Malignomen dar. Die Überexpression dieses ABC-Transporters steht in Korrelation zu einer erniedrigten Tumorremission und einer kürzeren Überlebensrate der Patienten. Bisherige Versuche, das Protein über niedermolekulare Substanzen (MDR-Modulatoren) zu inhibieren, vermochten in allen bisherigen klinischen Studien nicht zu überzeugen, so daß diese bis heute keinen Eingang in Standardtherapieschemata gefunden haben. Ziel dieser Arbeit war es, mittels RNA-Interferenz Strategien die Expression von MDR1 zu hemmen und eine Reversion der zellulären Chemoresistenz sowohl im Zellkultur- als auch im Tiermodell zu erreichen. Für die in vitro Untersuchungen an drei humanen multidrug-resistenten Karzinomzellinien wurden verschiedene siRNA (short interfering) Duplexe und shRNA (short hairpin)-exprimierende Vektoren gegen die MDR1 mRNA entwickelt. Die Behandlung der Zellen mit siRNAs führte zu einer bis zu 91 %igen Inhibition der MDR1 mRNA-Expression und zu einer Sensitivierung der Zellen gegenüber dem Anthrazyklin um 89 %. Diese Effekte konnte über einen Zeitraum von drei bis fünf Tagen aufrechterhalten werden. Die stabile Expression von anti-MDR1 shRNAs führte in zwei der untersuchten Zellmodelle zu einer dauerhaften und kompletten Überwindung des MDR1-abhängigen Resistenzphänotyps. Im Mausmodell konnte durch intratumorale Applikation des anti-MDR1 shRNA-kodierenden Vektors mittels low-volume Jet-Injektion eine komplette Reversion der MDR1-Überexpression sowie eine Wiederherstellung der Chemosensitivität gegenüber Doxorubicin in dem resistenten Tumormodell erreicht werden. Die Effizienz der kombinierten Gen- und Chemotherapie wird durch die Verminderung des in vivo Tumorwachstums auf das Volumen des von der sensiblen Zellinien-abgeleiteten Tumors reflektiert. / Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp). For stable reversal of "classical" MDR in three human cancer cell lines by RNA interference (RNAi) technology, two small interfering RNA (siRNA) constructs and four H1-RNA gene promoter-driven expression vectors encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules were constructed. In all cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to a maximum of 89%. The introduction of anti-MDR1/P-gp shRNA expression vectors leads in two of the three human cancer cell lines to a complete reversion of the MDR phenotype. The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells. In a mouse xenograft model a complete in vivo restoration of MDR1 overexpression and chemosensitivity to doxorubicin could be obtained by intratumorally jet-injected anti-MDR1 shRNA in a multidrug resistant human cancer tumor model.

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Nous avons identifié une nouvelle forme de phospho-S27-S33-Ku70 constitutivement surexprimée dans des cellules issues de la leucémie lymphocytaire chronique résistante à la chimiothérapie basée sur des agents alkylants de l’ADN et/ou analogues nucléotidiques. La protéine Ku70 est une protéine essentielle du maintien de la stabilité génomique par son rôle dans la réparation non-homologue (système NHEJ) des cassures double brin de l’ADN (CDB) et par sa fonction télomérique. Le laboratoire d’accueil a déjà démontré, in vitro et in vivo, dans les cellules LLC résistantes une altération de la réparation par le système NHEJ et un dysfonctionnement télomérique. Le travail de thèse a porté sur la caractérisation fonctionnelle de cette nouvelle forme phospho-S27-S33-Ku70. Pour ceci, nous avons utilisé des vecteurs d’expression permettant simultanément d’inhiber l’expression du Ku70 endogène (shRNA) et d’exprimer de façon épisomale différentes formes de Ku70 exogène. Ainsi, nous avons démontré : i) une stricte colocalisation de pS27-pS33-Ku70 avec les foyers γ-H2AX; ii) des cassures double brin (DSB) induisent la phosphorylation de S27-S33-Ku70 sous forme hétérodimère avec Ku80. Cette phosphorylation a lieu quelques minutes après le stress génotoxique et implique l'activité et l'interaction physique avec pS2056-DNA-PKcs, reliant ainsi pS27-pS33-Ku70 au système NHEJ ; iii) les cellules exprimant la forme sauvage exogène S27-S33-Ku70 ou la forme phosphomimétique E27-E33-Ku70 présentent une cinétique de réparation de l’ADN plus rapide que celle des cellules exprimant la forme mutée A27-A33-Ku70. Cependant, iv) la forme sauvage de Ku70 contribue à un niveau plus élevé d'aberrations structurales chromosomiques après la première division cellulaire suite à un stress génotoxique indiquant une infidélité lors de la réparation des dommages de l’ADN. En outre, les cellules exprimant A27-A33-Ku70 possèdent un index cellulaire plus élevé qui est corrélé avec une activation de la voie β-caténine. En adéquation avec sa surexpression dans la forme résistante de la LLC, l’ensemble de ces résultats suggère un rôle oncogénique de la forme phosphorylée de Ku70. Nous avons ensuite testé l’effet des nanodiamants hydrogénés (ND-H) dans des lignées exprimant différentes formes de Ku70. Grâce à leurs propriétés physico-chimiques les ND-H sont capables de potentialiser sous irradiation la production intracellulaire des espèces réactives de l’oxygène (ROS) et ainsi augmenter le taux des cassures (simple et double brin de l’ADN) et solliciter d’avantage le système de réparation de l’ADN. Nous observons que indépendamment de la forme exprimée de Ku70, ce double traitement induisait la sénescence cellulaire ; une découverte d’un intérêt à la fois fondamental (compréhension des voies apoptotiques vs senescence) et d’utilité pharmacologique potentielle. / We have identified a new form of phospho-S27-S33-Ku70 constitutively overexpressed in a subset of chronic lymphocytic leukemia (CLL) B cells resistant to apoptosis induced by DNA double strand breaks (DSB). Ku70 is one of the essential proteins involved in the maintenance of genomic stability through its role in DNA double strand break repair (non-homologous end-joining, NHEJ) and in telomeric protection.Laboratory previously established that resistant CLL cells disclose an upregulated NHEJ DNA repair and an impaired structure of telomeres. The goal of this thesis was to characterize the biological function(s) of this new form of Ku70. For this purpose we have constructed specific EBV-based vectors (siRNA / cDNA) enabling a simultaneous inhibition of endogenous Ku70 and an expression of different forms (mutated, wild, phosphomimetic at ser27-33) of Ku70 resistant to siRNA. Thus, we showed: i) a strict colocalisation of phospho-Ku70 with γ-H2AX foci; ii) that DSB induces the phosphorylation of Ku70 within minutes after genotoxic stress in heterodimer complex Ku70/Ku80. This phosphorylation necessitates both the physical interaction and the activity of pS2056-DNA-PKcs and/or ATM, linking phospho-Ku70 to NHEJ-mediated DNA DSB repair; iii) cells expressing mutated A27-A33-Ku70 exhibit a delayed G2/M cell cycle arrest, slower kinetic of DNA repair, lower level of genotoxic stress-induced chromosomal aberrations, and a higher cellular impedance correlated with translocation of transcriptional factor β-catenin from cytoplasmic membrane to the nucleus. Together, these data unveil an involvement of phospho-Ku70 in fast and inaccurate DNA repair; new paradigm for NHEJ regulation and to the control of resistance and maintenance of malignant cells.In parallel, we have initiated experimental approaches to explore other potential roles of phospho-Ku70. Especially, we were interested to determine whether it could play a role in an initiation of cell senescence induced by combined cells’ treatment by hydrogenated nanodiamonds (H-NDs) particles and ionizing irradiation. H-NDs exhibit positive surface charge in aqueous solutions allowing, when irradiated by photons, electrons’ emission and the release of reactive oxygen species (ROS) causing DNA damage. Effectively, we have established an intracellular increase of ROS that drive cell cycle arrest in G1/S in addition to the G2 arrest activated by irradiation alone. Finally, cells underwent the senescence process characterized byγ-galactosidaze activity, persistent large γ-H2AX foci and senescence-associated heterochromatinisation. Noteworthy, the senescence induced in this way occurred independently of Ku70 (ser27-ser33) status and irrespectively of cell resistance to genotoxic agents administrated alone; a finding of potential use in clinical trials.

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Computer and Applied Sciences) Vaal University of Technology / This study seeks to design and test specific short hairpin RNA (pshRNA) for their efficiency in knocking down the GALT gene RNA products thereby limiting the resultant enzyme activity. The following objectives were followed in designing the current study: 1. Designing a shorthairpin RNA (pshRNA) to target different regions of the coding sequence of the target GALT gene. 2. Propagating the pshRNAs in Escherichia coli (E.coli) and subsequently isolation of the respective plasmids for transfection. 3. Transfection of HeLa cells to test the efficiency of relevant pshRNAs in knocking down the GALT gene expression. 4. Transfection was followed by extraction of total mRNA, purification and quantification of total mRNA. 5. The GALT gene expression was qualitatively quantified against a house-keeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) to evaluate efficiency of knockdown using real time PCR. The three newly designed pshRNA (pshRNA2, pshRNA3 and pshRNA4) targeting the GALT gene expression showed a knockdown efficiency of 171 %, 48 % and 200 %, respectively. The results of this study will be useful for future evaluation of the possible long term glycosylation patterns under proper UDP glucose/UDP galactose levels compared with variable defective GALT gene levels.

Cystic Fibrosis (CF) is a degenerative disorder that is often associated with chronic lung disease. CF is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel, which lead to defective chloride and sodium ion movement across epithelia. Subsequent dehydration of the airway surface liquid (ASL) on airway epithelia, is associated with poor mucociliary clearance and chronic lung infection. The monogenic nature of CF, along with the accessibility of the lung, makes the disease amenable to gene replacement therapy. Gene therapy clinical trials have focused on replacing the mutated CFTR with a functional copy, which has led to improved chloride transport, but has shown no significant effects on sodium transport. An alternative strategy for CF gene therapy therefore, could be to reduce the expression of the epithelial sodium channel (ENaC) in the lung, using RNA interference (RNAi), combined with CFTR delivery. Developing a dual-function gene transfer vector could potentially restore chloride and sodium levels in the ASL and help alleviate CF lung disease. The aim of this thesis was to develop a recombinant lentivirus delivery system capable of simultaneously delivering CFTR expression and knocking down ENaC expression in the airways. A modular HIV vector genome plasmid was developed to allow simple insertion of various promoter elements, transgenes and knockdown sequences, for subsequent virus production. Insertion of the CFTR transgene and a short-hairpin RNA (shRNA) sequence targeting the ENaC alpha subunit (ENaCα) resulted in significant knockdown of human ENaCα and simultaneous expression of CFTR in A549 (human lung carcinoma) cell culture. Replacement of the ENaCα shRNA with an shRNA targeting the transcription factor BACH1 resulted in target gene knockdown and concomitant HMOX1 up-regulation, confirming specific knockdown effects, and demonstrating that the dual-function rLV vector could mediate target gene knockdown irrespective of the target. Attempts were made to knock down BACH1 in primary cultures of human bronchial epithelial cells grown at the air-liquid interface (ALI), but improved transduction efficiencies from the apical surface will be required to generate successful knockdown in this experimental model. These studies provide proof-of-principle for the utility of this versatile dual-function prototype virus. The dual function vector not only has the potential for treatment of CF lung disease, but could be readily altered to target other lung diseases where combinations of prolonged target gene knockdown and gene expression/up-regulation could collectively provide an appropriate therapy. In conclusion, the focus on the rational design of gene transfer vectors for specific therapeutic effects will aid the development and translation of gene therapy approaches to human studies.