The robust regenerative capacity of muscle stem cells (MuSCs) and their progenitors depends on their ability to undergo rapid and vast changes to their transcriptome during cell state changes. While transcription factors and epigenetic remodelling proteins are critical to render genes permissive for transcription, often these genes are found to have paused promoter proximal RNA Polymerase II (Pol II) which remains in a rate-limiting poised state. Indeed, while prior studies have shown poised Pol II is often regulated by the Negative Elongation Factor (NELF) to induce rapid changes in gene expression, the specific need for NELF in somatic stem cell populations has not been previously examined. In this thesis, we identify a specific requirement for NELF-dependent promoter proximal Pol II pausing in proliferating myogenic progenitors. Here, NELF stabilizes nascent transcripts associated with the paused RNA Pol II at genes required to maintain muscle progenitors in cell cycle. This promotes expansion of the pool of myogenic progenitors required to adequately repair damaged skeletal muscle. Our molecular analysis suggests that in proliferating progenitors, NELF-bound Pol II ensures the stabilization of transcripts, and continued expression of genes that prevent p53-mediated cell cycle withdrawal and terminal differentiation. Unexpectedly, this work revealed a previously unappreciated contribution of proliferating myogenic progenitors to replenish the stem cell niche in support of MuSC self-renewal during skeletal muscle regeneration. Based on our results, new therapeutic avenues which could treat muscle wasting disease are also discussed.

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No description available.

No description available.

PEA3, ERM, and ER81 comprise a subfamily of ETS transcription factors that upregulate genes correlated with an increased metastastic potential of tumors. In mouse embryo fibroblast (MEF) cells, PEA3 is required for transformation by activated Ras or Neu, but the means by which PEA3 mediates Ras-transformation is not clear. Osteopontin (OPN) expression is induced upon B-ras-transformation and purified PEA3 can bind the OPN promoter by gel-shift analysis. In this study, OPN expressed higher transcript levels in the wildtype MEF 4 cell line than the PEA3 null MEF 1 cell line and was further characterized as a potential PEA3 target gene by Northern blot analyses and transient transfection studies. Northern blot analyses of 4 wildtype MEF (4, 100, 101, 104), 5 FEA3 null MEF (1, 115, B5, B10, B12), and 5 MEF 1 retransformant cell lines that stably reexpress PEA3 showed a good correlation between OPN and ERM transcript levels in 9/11 cell lines although at least 2 PEA3 subfamily members were coexpressed in 8/11 cell lines that expressed high OPN transcript levels. This suggested that the PEA3 subfamily additively regulated OPN and that ERM protein was more abundant than PEA3 and ER81 protein levels in the MEF cell lines. The relative PEA3 subfamily protein levels remain to be clarified. Transient transfection assays in the HEK 293-1 C cell line indicated that the OPN promoter was responsive to PEA3 and that the promoter region between -258 to -88 was required for maximal OPN promoter activity. There are 16 candidate core ETS binding sites in the -777 /+79 OPN promoter which could be responsible for PEA3 subfamily transactivation. The OPN promoter was more active in the MEF 4 cell line than the MEF 1 cell line, corresponding to their relative number of expressed PEA3 subfamily members. Ectopic expression of dominant negative PEA3 suppress ed OPN promoter activity in the MEF 4 cell line. Furthermore, ectopic expression of PEA3, ERM, or ER81 increased OPN promoter activity in the MEF 1 or COS-1 cell line. Thus OPN is transcriptionally regulated by the PEA3 subfamily and represents a target gene that can mediate the progression of tumor cells. / Thesis / Master of Science (MSc)

Host cell factor-1 (HCF-1) is an evolutionarily conserved protein first discovered through its interaction with the herpes simplex viral transactivator VP16. Both the amino-terminal VP 16 interaction domain and the adjacent, less studied basic domain of HCF-1 are essential in cell cycle regulation. However, the mechanism(s) of this regulation is unknown. Our objective was to provide insight into the importance of the basic domain by studying proteins that interact with this region. Using the yeast two-hybrid system with the HCF-1 basic region as bait, we identified a novel protein named HCF-1 interacting zinc finger protein (HIZ). The purpose of this research was to characterize HIZ and determine if its interaction with HCF-1 could reveal a novel cellular target for HCF-1. A putative HIZ full-length sequence was determined and its primary structure was studied in detail. HIZ contains 16 C₂H₂ zinc fingers in its amino terminal. HIZ also contains a novel glutamine-rich motif adjacent to a potent autonomous transactivation domain. The presence of a DNA-bincing domain structure and a strong, functional transactivation domain indicate that HIZ is a novel transcriptional factor. Northern blot analysis showed tissue specific expression of HIZ with highest levels in the testis, skeletal muscle, liver and pancreas, suggesting possible roles in spermatogenesis, differentiation and metabolism. The HIZ-HCF-1 interaction was verified 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 using glutathione-s-transferase (GST) pull-down assays and the minimal HIZ domain for HCF-1 interaction was mapped to the same region critical for transactivation. Recent studies have identified the transcription factors Miz-1, GABP, LZIP, Zhangfei and PGC-1β as proteins that interact with the two domains of HCF-1 important in cell cycle control. Also, these studies have shown that interaction with HCF -1 is important in regulation of their transactivation potential. Thus, the effect of HCF-1 on HIZ activation was studied using transient transfection assays. Similar to its effect on the known cell cycle inhibitor, Miz-1, our studies showed that cotransfection with HCF-1 significantly inhibited HIZ transactivation. The expression pattern of HIZ in a matched tumour/normal tissue array showed that HIZ is expressed at a significantly lower level in tumours of the lung and uterus in comparison to normal tissues from the same individual. This finding suggests a correlation between HIZ expression and tumour formation, possibly in conjunction with the known cell cycle regulator HCF -1. / Thesis / Master of Science (MSc)

Infection with Human papillomaviruses has been suggested to play an important role in the etiology of a number of human malignancies. The most investigated of HPVs is HPV-16. Infection with HPV-16, in association with other unknown factors, is implicated in the development of cervical cancer. Genetic analysis of HPVs has been hampered by the lack of an in vitro system in which the complete replicative cycle and the transcription of the various genes is possible. Replication and transcription of HPVs seem to be in part regulated by cellular factors expressed at different stages in the maturation of epithelial cells which constitute the normal hosts of those viruses. This study was primarily designed to address this difficulty. HPV-16 genome was introduced into a viral system, HSV-1, hoping that virally encoded factors can substitute for cellular factors required for the expression of HPV genes. This hypothesis was tested by analyzing the activity of the HPV-16 early promoter P₉₇ , in the constructed recombinant, in cells which are unsusceptible to infection with HPV-16. In Vero cells infected with the recombinant, P₉₇ was shown to be active. This suggests that HSV-1 encoded factors can influence transcription from endogenous papilloma promoters. / Thesis / Master of Science (MS)

Alexander Scriabin (1872-1915) composed primarily for his instrument, the piano. He did, however, compose five major works for orchestra and a piano concerto. Scriabin's last work for orchestra, Prometheus: Poem of Fire, Op. 60 (1910), exemplifies his mature compositional style. The purpose of this dissertation is to contribute a solo transcription of Prometheus to the piano's rich literature in that genre. Furthermore, the dissertation aims to identify and examine the problems encountered in transcribing this work for solo piano and the decision-making that led to musically acceptable solutions. Throughout the process of arrangement, one major question became apparent: What informs the transcription? In turn, this question and its numerous answers served as a guide during the transcription's realization and are the focus of the project.

L’hypertension artérielle pulmonaire (HTAP) est une pathologie vasculaire résultant de l’obstruction des artères pulmonaires distales. L’obstruction de ces artères conduit à une augmentation drastique de la pression artérielle pulmonaire au-delà du niveau physiologique (≥ 25 mmHg), entrainant une adaptation pathologique du cœur droit. En effet, le cœur droit deviendra incapable de compenser cette augmentation de pression, ce qui conduira à l’arrêt cardiaque et la mort du patient. Jusqu’à présent, les traitements ont permis une amélioration de la qualité de vie des patients, mais aucun ne permet de soigner cette maladie. L’obstruction des artères pulmonaires observées chez les patients atteints d’hypertension artérielle pulmonaire s’explique, notamment, par l’acquisition d’un phénotype prolifératif des cellules musculaires lisses (CML) de ces artères. Précédemment, le laboratoire a démontré le rôle majeur de la diminution du micro-ARN 204 dans l’acquisition de ce phénotype des CML. Cette même diminution est observée dans les maladies vasculaires systémiques où elle conduit à l’acquisition d’un phénotype calcifiant des CML en permettant l’expression du facteur de transcription ostéogénique RUNX2. Durant ma thèse, j’ai investigué le rôle de la surexpression du facteur de transcription RUNX2 régulé par le micro-ARN 204 dans les CML des artères pulmonaires de patients atteints d’HTAP. Ce projet a permis de démontrer le rôle central de RUNX2 dans l’HTAP non seulement en participant à l’acquisition du phénotype prolifératif des CML, mais également en favorisant la transition phénotypique des CML vers un phénotype calcifiant conduisant à la formation de lésions calcifiantes. De plus, la diminution de l’expression de RUNX2 dans un modèle murin sugen/hypoxie d’HTAP permet l’amélioration des paramètres histologiques et hémodynamiques de l’HTAP, faisant de RUNX2 une cible thérapeutique prometteuse. / Pulmonary arterial hypertension (PAH) is a pulmonary vascular disease that drives the narrowing of distal pulmonary arteries. Pulmonary artery narrowing leads to an increase in the pulmonary arterial pressure above 25 mmHg. In order to compensate this increased pressure, the right ventricle undergoes a pathological adaptation. Shortly, the right ventricle becomes unable to compensate this increase leading to right heart failure and patient’s death. So far, PAH treatment has allowed significant improvement for patient’s life quality but none of them can cure the disease. Distal pulmonary artery narrowing is due, at least in part, to the acquisition of a proliferative phenotype of the smooth muscle cell (SMC). Previously, the laboratory has demonstrated the down-regulation of the micro-RNA 204 in the SMC and its major role in the acquisition of this proliferative phenotype. Interestingly, the same down-regulation is observed in systemic vascular disease where it leads to the acquisition of a calcifying phenotype of the SMC by allowing the expression of the osteogenic transcription factor RUNX2. During my thesis, I investigated the role of RUNX2 up-regulation in pulmonary artery SMC allowed by the down-regulation of the micro-RNA 204. This project uncovered a critical role for RUNX2 in PAH, not only by participating to the acquisition of SMC proliferative phenotype but also by allowing the acquisition of a calcifying phenotype of the SMC. Furthermore, targeting RUNX2 in a Sugen/hypoxia rat model of PAH reverse histologic and heomodynamic PAH phenotype, making RUNX2 an attractive therapeutic target.

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2006-2007 / Le facteur de transcription GATA4 joue un rôle majeur dans le développement du cœur, du système digestif et des gonades. Bien qu'un grand nombre de gènes cibles de GATA4 soit connu, les mécanismes qui régulent sa propre expression restent à définir. Afin de mieux comprendre la régulation de G AT A4 dans les gonades, un fragment de 5 kb situé en amont de l'exon 1 du gène Gata4 de rat a été clone et inséré devant le gène rapporteur luciferase. Des expériences de transfections transitoires dans des lignées gonadiques (MA-10, MSC-1, DC3) et hétérologue (CV-1) ont révélé que l'activité basale du promoteur Gata4 était assurée par une courte région d'ADN de 118 pb en amont de l'exon 1. Une étude in silico comparant les séquences orthologues de cette région a permis d'identifier plusieurs éléments « cis » conservés. Une mutagenèse de ces sites a démontré qu'un élément E-box était crucial pour l'activité basale du promoteur Gata4. Des essais de retardement sur gel ont mis en évidence la liaison de la E-box par les facteurs de transcription ubiquitaires USF1 et USF2. Cette étude décrit pour la première fois les éléments de régulation basale requis pour la transcription du gène G AT A4. / The GATA4 transcription factor plays a major role in the development of the heart, the gut and gonads. Although many target genes regulated by GATA4 have been identified, surprisingly little is known about the factors and the mechanisms implicated in the regulation of the GATA4 gene itself. In order to better understand how GATA4 expression is controlled in the gonads, 5 kb of the region immediately upstream of exon 1 of the rat Gata4 gene have been cloned and placed upstream of a firefly luciferase reporter. Transient transfection assays in both homologous (MA-10, MSC-1 and DC3) and heterologous (CV-1) cell lines revealed that the first 118 bp were sufficient to drive full promoter activity. Comparative sequence analysis revealed the presence of multiple conserved cisregulatory elements located within this minimal promoter region. Site-directed mutation of these sites identified an E-box as the crucial element required for the basal activity of the Gata4 promoter. Subsequent gelshift assays showed that the E-box was bound by the ubiquitous transcription factors USF1 and USF2. Therefore, this study provides the first description of the regulatory sequences required for GATA4 transcription.