The quality of life term is gaining importance day by day. However, its meaning has evolved throughout history from when it was first mentioned by Aristotle until it was given a central role in the UK national sustainable development strategy, launched in 1999. In this context, quality of life is about giving the opportunity to existing and future generations to achieve their potential through education, participation, access to information, good health and full employment. The various public institutions, private organisations and voluntary associations, collectively called the ‘well-being delivery chain’, share the responsibility to create, promote and foster high quality of life conditions. The drive to improve quality of life conditions has always been at the heart of what English local authorities do. However, up to now, local authorities’ activities are assessed through the performance on delivering their own local services, not on assuring quality of life among their citizens. This is going to change radically in the coming years, since this public assessment will evolve further to look at the well-being of local areas rather than the performance of services. So, local authorities will have to learn how to transform good public services into good quality of life conditions. In fact, it represents the shift of the local authorities’ role from local administration to local government. The present study aims to uncover what has been the impact of the local authorities’ performance on delivering services on local quality of life issues. It is believed that local authorities have the power to create and modify quality of life conditions. The study explores empirically the relationship between local quality of life and local authorities’ performance on delivering services at the level of English district in 2005, in the search for correlations between excellence of local public services and outstanding quality of life conditions, or contrarily. The in-depth study of the 2005 quality of life dataset and the 2003/04 report on local authorities’ performance on delivering services form the core of the present research study. Intensive quantitative techniques —ranging from statistics to data mining— have been used to explore data, pinpoint the possible different clusters, correlate indicators, associate phenomena and analyse both datasets. The study has revealed that behind the lower quality of life enjoyed by citizens in inner London boroughs, metropolitan districts and northern unitary authorities —compared to the rest of English districts— there could be a failure in the responsibility of the local authorities to manage their local schools, since a strong association between both phenomena exists.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

La O-GlcNAcylation est une glycosylation dynamique et réversible sous le contrôle de la O-GlcNAc Transférase (OGT) qui transfère un résidu de GlcNAc sur les Ser/Thr de protéines intracellulaires, et de la O-GlcNAcase (OGA). Plusieurs travaux dont ceux de notre équipe ont montré l'importance de la dynamique de O-GlcNAcylation pour la progression normale du cycle cellulaire, et plus particulièrement de la mitose. L’objectif de mes travaux de thèse était de comprendre comment la balance O-GlcNAc participe au contrôle des étapes précoces du cycle cellulaire. J’ai d’abord montré dans différentes lignées cellulaires que l’entrée en phase S s’accompagne d’une baisse marquée du niveau de O-GlcNAc, corrélée à une augmentation de l’expression et de l’activité de l’OGA endogène. Par protéomique, 58 protéines cytosoliques et nucléaires différentiellement O-GlcNAcylées à la transition G1/S ont ensuite été identifiées dans les cellules MCF7 synchronisées. Ces protéines interviennent dans des processus cellulaires essentiels à la phase G1 dont la régulation de la transcription, de la traduction et de la mise en conformation des protéines, et de la réplication de l’ADN. Par immunoprécipitation, les variations O-GlcNAc dépendantes du cycle cellulaire ont été confirmées sur les protéines cytosoliques CK8, hnRNP K et Caprine 1, et sur les protéines nucléaires du complexe de pré-réplication, MCM-3, -4, -6, et -7. Ces travaux montrent donc que la transition G1/S est étroitement liée à la dynamique de O-GlcNAcylation et soulignent un rôle potentiel de cette glycosylation dans le contrôle de l’initiation de la réplication de l’ADN et par là même, dans le maintien de l'intégrité génomique. / O-GlcNAcylation is a highly dynamic and reversible glycosylation which is governed by O-GlcNAc Transferase (OGT) that transfers the N-acetylglucosamine (GlcNAc) residue onto Ser/Thr of intracellular proteins, and O-GlcNAcase (OGA). Over the last decade, we and others have shown that dynamics of O-GlcNAcylation was important in regulating the cell cycle progression, and more particularly the mitosis events. The aim of my work was to explore how O-GlcNAc balance is implicated in the control of cellular proliferation by focusing on the early steps in the cell cycle. We highlighted in several cell lines that S-phase entry is associated with a marked decrease in the overall level of O-GlcNAcylated proteins, concordant with an increase in both the expression and activity of endogenous OGA. Then, using a proteomic approach we identified 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated between G0, G1 and S phases. These proteins are involved in key cellular functions that are essential for G1 and S progression, such as protein folding and translation, transcription or DNA replication. By immunoprecipitation, we further confirmed the cell cycle-dependent O-GlcNAc variations of CK8, hnRNP K, Caprin-1, and MCM -3, -4, -6, and -7 proteins which are part of the pre-replicative complex. To conclude, this study shows that there is a close link between the dynamics of O-GlcNAc and G1/S transition and provides a descriptive overview of differentially O-GlcNAcylated proteins at the G1/S transition, highlighting a potential role of O-GlcNAcylation in the initiation of DNA synthesis and therefore, in the maintenance of genome integrity.

Thesis advisor: Marc Jan Gubbels / Thesis advisor: Thomas Chiles / The length of the tachyzoite cell cycle, in particular G1, is an important virulence factor in Toxoplasma gondii. Cdk and Cyclin activities ultimately control the cell cycle; however, the checkpoint control mechanisms diverge from higher eukaryotes and are poorly understood. In order to elucidate these mechanisms, temperature sensitive (ts) mutants were generated by chemical mutagenesis. One of these mutants, called FV-P6, dies within one cell cycle in the G1 phase upon transfer from the permissive (35°C) to the restrictive temperature (40°C). Cosmid complementation identified the gene responsible for this G1 arrest as a `Cactin' ortholog. A single point mutation in this gene that resulted in an amino acid substitution from Tyrosine to Histidine at position 661 in the highly conserved C-terminus was shown to underlay the temperature sensitive effect. Cactin is highly conserved across eukaryotes and plays a role in embryonic development of metazoa although its mechanism of action is poorly understood. In agreement with the predicted nuclear localization signal in the N-terminus, expression of a fluorescent reporter gene fusion resulted in nuclear localization. Genome-wide expression profiling analysis of mutant and wild type at the permissive and restrictive temperatures confirmed the G1 arrest and furthermore demonstrated up-regulation of bradyzoite and Toxoplasma cat life cycle stage genes, hinting at TgCactin's role as a repressor. Since DNA binding domains or enzymatic domains are absent in TgCactin, TgCactin must act in a complex. Native blue gel electrophoresis demonstrated that TgCactin is present in large complexes of 720 and 800 kDa. A yeast two-hybrid screen (YTH) identified 40 potential TgCactin-interacting proteins of which 10 were selected for further validation. Eight out of these ten candidates are involved in DNA/RNA processes pertaining to transcription and translation, respectively. One-on-one YTH interactions between mutated and N-terminal deletion mutants of TgCactin and the above 10 interactors were abolished except for a single RNA helicase. Studies in Toxoplasma of four of these interactors demonstrated that only the RNA helicase localized to the nucleus; however, co-immunoprecipitation experiments to demonstrate that this protein is present in a complex with TgCactin were inconclusive. Furthermore, TgCactin self interactions identified domains necessary for TgCactin-TgCactin binding. Taken together, these findings indicate that TgCactin likely functions as a repressor of gene expression, possibly through an epigenetic mechanism reminiscent of an RNA/DNA helicase- based system in plants. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Much of the work on how the cell cycle is regulated has focused on Cyclin-Dependent Kinase (CDK)-mediated regulation of factors that control the coordinate expression of genes required for entry into the cell cycle. In Saccharomyces cerevisiae, SBF and MBF are related transcription factors that co-ordinately activate a large group of genes at the G1/S transition, and their activation depends on the Cln3-Cdk1 form of the cyclin-dependent kinase. However, cells are viable in the absence of Cln3, or SBF and MBF, indicating that other regulatory pathways must exist that activate the budding yeast cell cycle. The known CDK-independent pathways are made up of various phosphatases and plasma membrane transporters that control ion homeostasis in early G1 phase, a time when cells assess environmental growth conditions in order to commit to cell cycle entry. The enigmatic Bck2 protein is thought to act within these CDK-independent pathways, but the means by which it activates G1/S-regulated genes is not known. Bck2 contains little sequence homology to any known protein. In order to understand how CDK-independent pathways operate, I have studied the Bck2 protein using multiple approaches. In one approach, I have screened for novel SBF/MBF-binding proteins in order to determine if other non-CDK proteins, such as Bck2, might activate SBF and MBF. I have also investigated which region of Bck2 is required for its activity in order to determine if Bck2’s transcriptional activation region is essential. Using one of the iii truncation derivatives from this analysis, I have screened for proteins that interact with Bck2. One of these novel proteins is Mcm1, a global transcriptional activator of genes involved in cell cycle progression, mating gene transcription and metabolism. My studies suggest that Bck2 regulates the activity of Mcm1 in early G1 phase to activate the expression of SWI4, CLN3, and others. My evidence suggests that Bck2 competes for binding to a specific pocket on Mcm1 that is also bound by an Mcm1 repressor called Yox1. My findings suggest that CDK-independent pathways function through Bck2, in order to induce the initial suite of genes required for entry into the cell cycle.

Much of the work on how the cell cycle is regulated has focused on Cyclin-Dependent Kinase (CDK)-mediated regulation of factors that control the coordinate expression of genes required for entry into the cell cycle. In Saccharomyces cerevisiae, SBF and MBF are related transcription factors that co-ordinately activate a large group of genes at the G1/S transition, and their activation depends on the Cln3-Cdk1 form of the cyclin-dependent kinase. However, cells are viable in the absence of Cln3, or SBF and MBF, indicating that other regulatory pathways must exist that activate the budding yeast cell cycle. The known CDK-independent pathways are made up of various phosphatases and plasma membrane transporters that control ion homeostasis in early G1 phase, a time when cells assess environmental growth conditions in order to commit to cell cycle entry. The enigmatic Bck2 protein is thought to act within these CDK-independent pathways, but the means by which it activates G1/S-regulated genes is not known. Bck2 contains little sequence homology to any known protein. In order to understand how CDK-independent pathways operate, I have studied the Bck2 protein using multiple approaches. In one approach, I have screened for novel SBF/MBF-binding proteins in order to determine if other non-CDK proteins, such as Bck2, might activate SBF and MBF. I have also investigated which region of Bck2 is required for its activity in order to determine if Bck2’s transcriptional activation region is essential. Using one of the iii truncation derivatives from this analysis, I have screened for proteins that interact with Bck2. One of these novel proteins is Mcm1, a global transcriptional activator of genes involved in cell cycle progression, mating gene transcription and metabolism. My studies suggest that Bck2 regulates the activity of Mcm1 in early G1 phase to activate the expression of SWI4, CLN3, and others. My evidence suggests that Bck2 competes for binding to a specific pocket on Mcm1 that is also bound by an Mcm1 repressor called Yox1. My findings suggest that CDK-independent pathways function through Bck2, in order to induce the initial suite of genes required for entry into the cell cycle.

>Magister Scientiae - MSc / In this work, we describe the synthesis of ferrocenylimine and G1 dendrimeric N,N′ ligands, and their palladium(II) complexes. The ligands L1 – L4 were synthesized following Schiff base condensation reactions. The ferrocenylimine ligands L1 and L2 were prepared from ferrocenecarboxaldehyde and 1,3-diaminopropane or DAB-4-(NH2)4, while the G1 dendrimeric N,N′ ligands L3 and L4 were prepared from DAB-4-amine and 2- pyridinecarboxaldehyde or 2-quinolinecarboxaldehyde. The air and moisture stable ligands were obtained in fairly good yields of over 65 %. All the ligands were observed to be soluble in common organic solvents. They were characterized by FTIR spectroscopy, 1H-NMR spectroscopy, 13C-NMR spectroscopy, micro-analysis and ESImass spectrometry.A reaction of the ligands with PdCl2(COD) or PdCl2(MeCN)2 gave palladium(II) complexes C1 – C4 in good yields of over 70%. The complexes were observed to be stable. However,the complexes did not dissolve in common organic solvents. These were characterized by FTIR spectroscopy and 1H-NMR and 13C-NMR spectroscopy.All the complexes were screened for activity towards Heck coupling model reaction of iodobenzene with styrene. The complexes catalyzed the coupling of styrene with iodobenzene, giving over 60 % conversions. However, complex C3 was found to be the most active catalyst, yielding 75% conversion without formation of palladium black.

When released extracellularly, the purine nucleoside guanosine (Guo) can exert a wide range of physiological effects in vitro and in vivo. Guo can induce the release of neurotrophic factors such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and can initiate the differentiation, growth and proliferation of neurons and glia. While structural and pharmacological evidence support the existence of a putative Guo binding site in the rat brain, there is a paucity of information on the mechanism through which Guo exerts these effects. Through bioinformatic research, our lab has identified an orphan G-protein coupled receptor as the first Guo receptor (termed G1R). The aim of this dissertation is to determine the mechanism of action of Guo using radioligand binding assays. It is hypothesized here that G1R is a distinct purinergic receptor for Guo. Using the calcium phosphate (CaP) co-precipitation (co-i.p.) method, Drosophila Schneider 2 (S2) cells were stably and transiently transfected with G1R recombinant cDNA. A series of binding assays using tritiated Guo ([3H]-Guo) showed no difference in binding between CaP transfection groups and wild S2 controls that do not endogenously express G1R, suggesting that the [3H]-Guo may not have a high binding affinity for the G1R binding site. Preliminary experiments using the Lipofectamine® 3000 to transfect S2 cells showed higher G1R mRNA expression as well as increased binding affinity to Guo when compared to the CaP transfected groups. This suggests that the results in the CaP mediated groups may be due to low transfection efficiency. In conclusion, transfections using the CaP method resulted in too low of a transfection efficiency to see a difference in binding affinity between wild S2 and transfected S2 cells. Findings from this work can be used to further examine the binding relationship of Guo to the G1R and optimize transfections using S2 cells and radioligand binding assays using purine based compounds. / Thesis / Master of Science (MSc)

Les cellules souches embryonnaires (cellules ES) sont capables de se multiplier de façon autonome en l’absence de facteurs de croissance et de cytokines, un état appelé “état fondamental de pluripotence”. Le cycle cellulaire des cellules ES se caractérise : (i) par une expression élevée et uniforme de la cycline E et des complexes Cycline E-CDK2 au cours de la progression dans le cycle cellulaire et (ii) par une phase G1 très courte (1 heure) dont la traversée ne dépend ni des MAPK ni des points de contrôles régulés par la protéine du rétinoblastome (RB) et p53. Ces observations soulèvent la question de l’existence d’un lien de cause à effet entre ce phénomène de réplication autonome et la pluripotence. Mon projet de thèse se construit autour de trois axes qui montrent que : 1/ la phase G1 des cellules ES de souris est une phase de sensibilité accrue aux inducteurs de différenciation. 2/ la balance entre autorenouvellement et différenciation est perturbée, (i) quand l’expression de la cycline E est altérée, ou (ii) quand l’association de la cycline E avec la kinase CDK2 et le centrosome est bloquée. 3/ La signalisation par le LIF contrôle la formation et l’activation des complexes Cycline E/CDK2. Dans les cellules ES naïves Rex1+, l’allongement de la durée de la phase G1 induit par la privation de LIF précède, ou est concomitante, à la diminution de l’expression de marqueurs de pluripotence et à l’activation des marqueurs les plus précoces de la différenciation. Finalement, nous proposons un modèle dans lequel la signalisation par le LIF régule la transition G1/S et permet le maintien de l’autorenouvellement des cellules ES murines / Pluripotency can be captured and propagated in vitro from the epiblast of the pre-implantation blastocysts in the form of embryonic stem cells (ESCs). ESCs are capable of unlimited proliferation in an undifferentiated state while maintain the potential to differentiate into cells of all three germ layers in the embryo, including the germline. Two key features the ES cell mitotic cycle are (i) a vastly elevated and uniform expression of Cyclin E and Cyclin E/CDK2 complexes throughout the cell cycle and (ii) a short G1 phase characterized by the lack of RB- and p53-dependent checkpoints, and reduced dependency on MAPK signalling. During my PhD project, we explored whether and how the regulation of the cell cycle actively sustains self-renewal of mouse ESCs (mESCs). We demonstrated that: 1/ the G1 phase of mESCs is a phase of increased susceptibility to differentiation inducers. Thus shortening of G1 might shield undifferentiated cells from differentiation inducers and help ESCs to self-renew in the pluripotent state. 2/ Cyclin E opposes differentiation and supports self-renewal of mESCs by two independent mechanisms, one of which being independent of CDK2 activation. 3/ LIF signalling regulates Cyclin E/CDK2 kinase activity therefore accelerating the G1 to S phase transition. Finally, we propose a model in which LIF signalling stimulates the G1 to S phase transition to shield mESCs from undesired differentiation signals and help them to self-renew in the pluripotent state

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.