Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser.

No description available.

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

As we age, our ability to cope with a variety of stresses significantly decreases. One of the features of an ageing organism is the dramatic increase in the number of cells arrested in the G1 phase, a process known as senescence. It is well established that the senescence phenotype leads to a change in the way cells respond to stress. However, the molecular mechanisms by which these cells cope and/or respond to a variety of environmental challenges remain unknown. In general, cells respond to stress by engaging a variety of mechanisms; one of them is the assembly of cytoplasmic foci known as stress granules (SGs). These entities are considered as part of the survival pathways that are activated at the beginning of any stress to protect key cellular elements which allow a quick recovery if the stress is rapidly removed. However, we do not know whether SGs formation is activated during senescence. In this study, we investigated the formation and the role of SGs in senescent cells exposed to various stresses. We demonstrated that while SGs can assemble in response to oxidative stress (OS) during all the steps leading to senescence activation, their number significantly increases at late stage of senescence. This increase correlates with a rapid decrease in the expression of the cyclin kinase inhibitor p21, one of the main players in the activation of the senescence phenotype. Although the OS-induced recruitment of p21 mRNA to SGs correlates with a significant increase in its half-life, this translocation interferes with p21 translation only at late senescence. This translation inhibition could be explained by the co-recruitment of CUGBP1, a known translation activator during senescence of p21, and p21 mRNA to SGs. Therefore, our data suggest that SGs formation and the reduction in p21 protein levels represent two main events through which senescent cells respond to stress conditions.

HHV-6 is a ubiquitous human herpesvirus. Most individuals become infected at the age of 2 years. Primary infection by the virus causes a self-limiting febrile illness called exanthem subitum or roseola. In adults, primary infection may cause mononucleosis-like illnesses. The infection usually remains latent in healthy individuals, but often reactivates in immunocompromised individuals, for example, transplant patients and AIDS patients. The virus has also been associated with cancers and lymphoproliferative disorders. The virus encodes two proteins that interact with p53. However, little is known concerning the impact of the virus on cell cycle progression in human cells. The investigations reported in the thesis were focused on this issue. We show here that that HHV-6 infection delays the cell cycle progression in human T cell line HSB-2, as well as in primary human T cells and causes their accumulation in S and G2/M phase. By degrading the viral DNA in the virus-infected cells, we show that the infected cells accumulate in the G2/M and not in the S phase. We observed an increase in the kinase activity of cdc2 in virus-infected cells despite lower levels of its catalytic partners, cyclin A and cyclin B. We show here that the viral early antigen p41 associates with, and increases the kinase activity of, CDK1. Our studies have shown that there is a drastic reduction of p21 protein, despite the virus-induced stabilization and activation of p53 suggesting that p53 may be transcriptionally inactivated in the virus-infected cells. This decrease of p21 in infected cells was partially restored by proteasome inhibitors. These results suggest that HHV-6 causes perturbations in the normal progression of cell cycle in human T cells. Autophagy is a physiological cell process during which old cellular constituents and long-lived proteins in cells are degraded. This process is regulated in a cell cycle-dependent manner. We show here that infection with HHV-6 induces autophagy in HSB-2 cells. This was shown by the induction of LC-3 II as well as by the appearance of autophagic vacuoles in the virus-infected cells. However, we found that the virus inhibits fusion between autophagic vacuoles and lysosomes formed in infected cells, thus evading the autophagic response of infected host cells. Finally we tried to investigate replication of the virus in human cells in the absence of P53; a tumor suppressor gene which is also known as "the guardian of the genome ". During these investigations, we found that that inhibition of p53 gene expression mediated by siRNA as well as its inhibition by pharmacological inhibitors leads to massive cell death in human T cell line HSB-2 that carries a wild-type p53. We show that this death also occurs in another cell line CEM, which carries a transcriptionally mutated p53. Interestingly, the cell death could be prevented by pharmacological inhibitors of autophagy and necroptosis. Taken together, our results provide important novel insights concerning the impact of HHV-6 on cell cycle regulation and autophagy as well as of basal level p53 in cell survival. / HHV-6 est un virus herpès humain ubiquitaire. La plupart des individus deviennent séropositifs à l’âge de 2 ans. L’infection primaire par HHV-6 donne lieu à une maladie fébrile chez les enfants, appelée exanthème subitum ou la roséole. Par contre, chez l'adulte, cette infection cause des maladies de type mononucléose. L'infection reste généralement latente chez les individus sains, mais elle se réactive souvent chez les personnes immunodéprimées, par exemple, chez les personnes greffées et les patients atteints du sida. HHV-6 a été associé à plusieurs types de cancers et de désordres lymphoprolifératifs. Ce virus induit l’immunosuppression et inhibe la prolifération des lymphocytes par les mitogènes. C’est pour toutes ces raisons que nous voulions savoir si ce virus dérègle le cycle cellulaire des cellules qu’il infecte. Les travaux réalisés durant cette thèse ont porté sur les changements induits dans les cellules humaines par ce virus au cours de la progression du cycle cellulaire. Nous avons montré que l'infection par HHV-6 retarde la progression du cycle cellulaire dans la lignée cellulaire T humaine HSB-2, ainsi que dans les lymphocytes T primaires humains pour les accumuler dans les phases S et G2/M. Cependant, après avoir traité les cellules avec la nucléase du Micrococcus, nous avons constaté que le cycle cellulaire des cellules infectées s’accumulait plutôt dans la phase G2/M. La nucléase dégrade préférentiellement l’ADN virale. Nous avons observé une augmentation de l'activité kinase de cdc2 dans les cellules infectées malgré une baisse des niveaux de ses partenaires catalytiques, la cycline A et la cycline B. Nos études ont montré qu’il y a une diminution drastique de la protéine p21 dans les cellules infectées, en dépit de la stabilisation et de l'activation de p53 induite dans ces cellules. Ce qui laisse penser que la protéine p53 pourrait être inactive sur le plan transcriptionnel dans les cellules infectées. Cette diminution de p21 dans les cellules infectées est partiellement restaurée après incubation des cellules dans un milieu de culture contenant des inhibiteurs du protéasome. En plus, nous démontrons ici qu’une protéine virale précoce, p41, s’associe et se fixe avec cdc2 et augmente son activité kinase. Tous ces résultats suggèrent que HHV-6 provoque des perturbations énormes dans la progression normale du cycle cellulaire dans les cellules T humaines. Dans ces études, nous avons démontré aussi que l’infection par HHV-6 induit l'autophagie dans les cellules HSB-2, comme il a été démontré par l’induction de LC-3 II et par la formation de vacuoles autophagiques dans les cellules qui sont infectées. Nos résultats indiquent que HHV-6 inhibe la fusion entre les vacuoles autophagiques formées et les lysosomes dans les cellules infectées modulant ainsi la réponse autophagique des cellules hôtes infectées. Nous avons trouvé aussi que l’inhibition de ce processus par un inhibiteur pharmacologique diminue la réplication virale. L'autophagie est un processus physiologique cellulaire pendant lequel les vieux constituants cellulaires (mitochondries, protéines cellulaires, etc) se dégradent. Le fait que ce processus soit modulé dans les cellules dépendantes des différentes phases du cycle cellulaire, nous a poussé à l’étudier. Enfin, nous essayons d’investiguer la réplication virale dans les cellules dépourvues de p53, le gène suppresseur de tumeur, qui contrôle la progression de cycle cellulaire. Nous avons émis l’hypothèse suivante, que ces virus peuvent mieux se répliquer dans les cellules n’exprimant pas le gène p53. En vérifiant cette hypothèse, nous avons trouvé que l'inhibition de l’expression de p53 provoquée par siRNA ou par un agent pharmacologique conduit à une mort cellulaire massive dans une lignée de cellules T humaines ayant un gène p53 de type sauvage. Nous démontrons que cette mort se produit aussi dans une autre lignée cellulaire dont le p53 est muté et qu’elle pourrait être évitée par des inhibiteurs d'autophagie ou de nécroptose. Nos observations mettent en évidence qu’un niveau d’expression basale de p53 est nécessaire à la survie cellulaire.

<p>Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCF<super>Cdc4</super> ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that in the budding yeast,<italic> Saccharomyces cerevisiae</italic>, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APC<super>Cdh1</super>). I have determined, however, that the Kip1 and Cin8 proteins are actually present at wild-type levels in yeast in the absence of Clb/Cdc28 kinase activity. Here, I show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28, and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's <italic>in vivo</italic> localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, I have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCF<super>Cdc4</super> target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, I propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors. </p><p>In addition to the positive regulation of kinesin-5 function in spindle assembly, I have also found evidence that suggests CDK phosphorylation of kinesin-5 motors at different sites negatively regulates kinesin-5 activity to prevent premature spindle pole separation. I have also begun to characterize a novel putative role for the kinesins-5 in mitochondrial genome inheritance in <italic>S. cerevisiae</italic> that may also be regulated by CDK phosphorylation. </p><p>In the course of my dissertation research, I encountered problems with several established molecular biology tools used by yeast researchers that I have tried to address. I have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in <italic>S. cerevisiae</italic> that can be propagated in the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes <italic>HIS2</italic> and <italic>ADE1</italic>, respectively. My new pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed in order to increase the availability of unique restriction sites within their polylinker regions. Hence, my pRSII plasmids are a complete set of integrating, centromere and 2&#61549; episomal plasmids with the biosynthetic marker genes <italic>ADE2</italic>, <italic>HIS3</italic>, <italic>TRP1</italic>, <italic>LEU2</italic>, <italic>URA3</italic>, <italic>HIS2</italic> and <italic>ADE1</italic> and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, I have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin and bialaphos. Finally, in the process of generating the new plasmids, I have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using updated plasmid sequences, I constructed pRS plasmid backbones with a unique restriction site for inserting new markers in order to facilitate future expansion of the pRS/pRSII series.</p> / Dissertation

In vertebrate oocytes, meiotic progression is driven by the sequential translational activation of maternal messenger RNAs stored in the cytoplasm. This activation is mainly induced by the cytoplasmic elongation of their poly(A) tails, which is mediated by the cytoplasmic polyadenylation element (CPE) present in their 3’ untranslated regions (3´ UTRs). Sequential, phase-specific translation of these maternal mRNAs is required to complete the two meiotic divisions. Although the earlier polyadenylation events in prophase I and metaphase I are driven by the CPE-binding protein 1 (CPEB1), 90% of this protein is degraded by the anaphase promoting complex in the first meiotic division. The low levels of CPEB1 during interkinesis and in metaphase II raise the question of how the cytoplasmic polyadenylation required for the second meiotic division is achieved. In this work, we demonstrate that CPEB1 activates the translation of the maternal mRNA encoding CPEB4, which, in turn, recruits the cytoplasmic poly(A) polymerase GLD2 to “late” CPE-regulated mRNAs driving the transition from metaphase I to metaphase II, and, therefore, replacing CPEB1 for “late” meiosis polyadenylation.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 196-214.

In eukaryotes, cell proliferation and cell cycle transitions are strictly controlled by the anaphase-promoting complex/cyclosome (APC/C). The APC/C is an E3 ubiquitin ligase that regulates chromatid segregation at the metaphase to anaphase transition, exit from mitosis and the establishment and maintenance of G1. The APC/C’s catalytic activity and substrate specificity are controlled by its interactions with two coactivators, Cdc20 and Cdh1. In contrast to Cdh1, APC/C activation by Cdc20 during mitosis requires hyper-phosphorylation of APC/C subunits by cyclin-dependent kinase (Cdk) and polo kinase. The aim of the first part of this thesis was to understand how mitotic phosphorylation regulates APC/C activity. Using cryo-electron microscopy and biochemical analysis, we found that an auto-inhibitory segment of the Apc1 subunit acts as a molecular switch that in apo unphosphorylated APC/C interacts with a coactivator-binding site (C-box binding site), thereby obstructing engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box binding site to relieve APC/C auto-inhibition. Efficient phosphorylation of the auto-inhibitory segment requires the recruitment of the kinase Cdk-cyclin-Cks to a hyper-phosphorylated loop of Apc3. In addition to regulation of APC/C activity by phosphorylation, ordered cell progression is ensured by the ability of the APC/C to target substrate degradation in a defined order. At mitosis onset, degradation of securin and cyclin B1 is inhibited by the spindle assembly checkpoint, exerted by the mitotic checkpoint complex (MCC), whereas both cyclin A2 and Nek2A are not subject to MCC inhibition. The aim of the second part of the thesis was to elucidate the mechanism of how the APC/C achieves its substrate specificity. Our biochemical analysis showed that the resistance of cyclin A2 to MCC inhibition is due to its ABBA motif and the Cdk-associated Cks2 subunit. Furthermore, we found that it is the Cdc20 molecule of the MCC that binds to the ABBA motif to allow for cyclin A2 ubiquitination. Strikingly, mutating all three known degrons (KEN box, D box and ABBA motif) of cyclin A did not affect its ubiquitination by APC/CCdc20. Deletion of a potential novel degron found within residues 60-80 of cyclin A2 impaired cyclin A2 ubiquitination.