La réplication de l'ADN est un évènement majeur pour la cellule car elle permet la duplication fidèle du matériel génétique. Il s'agit d'une étape critique du cycle cellulaire, car les fourches de réplication rencontrent fréquemment des barrières naturelles ou des lésions d'origine endogènes (lésions oxydatives) ou exogènes (agents physiques ou chimiques), sources de cassures chromosomiques et donc d'instabilité génétique. Une des réponses à ces fourches bloquées est l'activation du point de contrôle (checkpoint) de la phase S du cycle cellulaire. Nous avons montré que l'ADN polymérase Kappa (pol Kappa), polymérase dite translesionnelle en raison de ses capacité à franchir des lésions sur l'ADN, s'avère être aussi un acteur du point de contrôle de phase S. En effet, la déplétion de pol Kappa par ARN interférence dans différentes lignées cellulaires ou par immunodépletion d'un extrait de Xénope, entraîne un défaut de phosphorylation de Chk1. Pol Kappa est impliquée dans la synthèse de brins naissant d'ADN au niveau des fourches bloquées, ce qui facilite le recrutement du complexe 9-1-1 composé des protéines Rad9, Rad1 et Hus1et permet alors, une activation correcte du checkpoint de phase S. Afin de décrypter le rôle de pol kappa, nous avons construits différents mutants et nous avons analysé leur capacité à former des foyers, à être recrutés à la chromatine et à interagir avec différents partenaires dans des conditions d'activation du point de contrôle de phase S. Nous avons pu constater que le mutant du domaine d'interaction à PCNA était incapable de former des foci foyers ?. Nous avons ensuite observé, qu'en condition de stress réplicatif, pol Kappa était recruté à la chromatine grâce à son domaine d'interaction à PCNA et par différentes approches biochimiques, nous avons pu voir que pol kappa interagissait avec Rad9 et Chk1. Nous avons également mis en évidence que le défaut d'activation de Chk1 en l'absence de pol kappa reflétait d'une diminution de son taux dans le noyau, suggérant une régulation commune entre Chk1 et pol Kappa. En effet, nous avons observé que pol Kappa, comme Chk1, était régulés par l'ubiquitine hydrolase USP7. En effet, l'interaction entre pol Kappa et USP7 est augmentée en condition de stress. Nous avons pu voir, qu'à l'instar de Chk1, l'absence de USP7 entrainait une baisse du niveau de pol kappa dans le noyau. Ainsi nous proposons qu'en réponse à un stress réplicatif, pol Kappa et Chk1 soient stabilisés via leur dé-ubiquitination par USP7, permettant leur recrutement à la chromatine et une activation correcte du checkpoint de phase S. Parallèlement à ces travaux, des publications récentes montrent une implication possible de pol Kappa au niveau des séquences répétées. Nous avons pu mettre en évidence une interaction entre pol Kappa et Cenpb, protéine centromérique reconnaissant une séquence de 17 paires de bases dans l'ADN a-satellite. Ces résultats préliminaires suggèrent que le rôle de pol Kappa dans le checkpoint de phase S s'adresse notamment aux régions d'hétérochromatine. L'ensemble des résultats obtenus montre l'importance de pol Kappa dans le maintien de la stabilité génomique, par son rôle dans le checkpoint de phase S, et par son implication dans la régulation de Chk1 en condition de stress réplicatif. / DNA replication is a major event for cells which allow the faithful duplication of genetic material. It is a critical step of cell cycle, because replication forks encounters frequently naturals barriers (non B-DNA structures), exogenous barriers (chemicals agents), or endogenous barriers (oxydatives lesions). These different barriers can be at the origin of chromosomes breaks and lead to genetic instability. To overcome the stalled forks, cells have evolved two major mechanisms: the induction of ATR replication checkpoint pathway and the recruitment of specialized DNA polymerase to perform the translesion synthesis. This two pathways are essential to maintain genomic stability. Human DNA polymerase Kappa (pol Kappa), the most conserved specialized DNA polymerase, is best known to participate to translesion synthesis. Recently, we have shown that pol kappa has a crucial role in the S-phase checkpoint activation. Indeed, pol Kappa is implicated in the synthesis of short DNA intermediates at the stalled forks, facilitating the recruitment of 9-1-1 clamp, and is required for an optimal phosphorylation of Chk1, the main effector of the S-phase checkpoint. Durant my PhD thesis, I explored the molecular mechanisms underlying this newly identified role. We have constructed several pol kappa mutants, and we have observed that for the mutation in the PCNA binding domain impeded pol kappa to form foci in response to replication stress. We also showed the requirement of this domain for pol Kappa recruitment on chromatin. By different experimental approaches, we have described a complex in which pol Kappa interacts with Rad9 and Chk1, two proteins required for the S-phase checkpoint activation. The Chk1 phosphorylation defect observed in absence of Kappa could also be the consequence of the Chk1 protein level decreased in the nucleus, meaning a potential common regulation between pol Kappa and Chk1. Based on this observation, we have studied how pol Kappa is regulated upon a replication stress and like Chk1, pol Kappa seems to be regulated by ubiquitination. We focused our attention on USP7 an ubiquitin hydrolase known to regulate Chk1. We have demonstrated an interaction between pol Kappa and USP7, which is stimulated after replication stress. Moreover, USP7 depletion leads to a decrease of pol Kappa level in the nucleus, suggesting that de-ubiquination of pol Kappa could to be a prerequisite for its checkpoint function and its stability.

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No description available.

Exploateringsområdet Barkarbystaden är Nordens nuvarande största pågående exploateringsprojekt. Här väntas 14 000 nya bostäder vara klara till 2032. En beräknad mängd på ca 488 000 leveranser förväntas under byggtiden. Under denna byggtid verkar flera kvarter och olika byggherrar samtidigt. För att lyckas hantera allt detta ställs ett stort fokus på en fungerande logistiklösning med bra planering och framförhållning.Syftet med arbetet är att undersöka hur logistiklösningar påverkar produktionens effektivitet genom att undersöka och studera hur leveransplaneringen i Barkarbystaden fungerar. Målsättningen med arbetet är att komma fram till data och studier som bidrar till en effektivare och mer lönsam produktion i Barkarbystaden och för byggbranschen i helhet.Frågeställningarna examensarbetet utgår ifrån är följande: Hur skiljer sig leveransplaneringen gentemot det verkliga utfallet, hur har entreprenören anpassat leveransplaneringen och hur kan logistikplaneringen förbättras?För att uppnå ett resultat som besvarar examensarbetets frågeställningar har det genomförts intervjuer, en litteraturstudie samt en fallstudie. Examensarbetet är genomfört i samarbete med företaget Prolog som är involverade i logistiklösningen på projektet Barkarbystaden. Prolog har givit arbetet möjligheten att komma i kontakt med relevanta nyckelpersoner för intervjuer samt delat med sig av kunskap om projektet Barkarbystaden som varit till nytta för att genomföra fallstudien.Fallstudien resulterade i att planerad ledtid är nästan fyra gånger längre än den faktiska ledtiden. Vid mätningarna noterades klockslaget när transporterna anlände och när de lämnade. 39% av leveranserna lämnar efter planerad avfärdstid.Logistiklösningen i Barkarbystaden är endast dryga ett år gammal och är inte för alla entreprenörer ett tidigare känt arbetssätt. Entreprenörerna har haft olika stora utmaningar när det gäller att anpassa sig till logistiklösningen. Vissa entreprenörer ser utmaningar med ett annat arbetssätt men det blir med tiden. Entreprenörer som jobbat med liknande logistiklösningar på andra projekt har en fördel när de ska anpassa sig till Barkarbystadens logistiklösning.Logistikansvariga bör bli mer noggranna när dom bokar in respektive leverans med avseende på vad det är för transport och lossningsmetod som ska användas. Samt att korta ned den planerade ledtiden i bokingssystemet. Då skillnaden var markant mellan planerad respektive faktisk ledtid.Vid kontraktsskrivning mellan entreprenör och leverantör bör entreprenören kravställa att de ankommer inom angivet lossningsfönster och reglera konsekvenserna av för sen ankomst parterna sinsemellan. / The Barkarbystaden development area is the Nordic countries currently largest ongoing development project. Here, 14,000 new homes are expected to be ready by 2032. An estimated amount of approximately 488,000 deliveries is expected during the construction period. During this construction period, several blocks and different contractors operate simultaneously. In order to succeed in handling all this, great focus is placed on a working logistics solution with good planning and foresight.The purpose of the work is to investigate how logistics solutions affect production efficiency by examining and studying how delivery planning in Barkarbystaden works. The aim of the bachelor’s thesis is to obtain data and studies that contribute to a more efficient and profitable production in Barkarbystaden and for the construction industry as a whole.The questions the degree project is based on are the following: How does the delivery planning differ from the actual outcome; how has the contractor adapted the delivery planning and how can the logistics planning be improved?In order to achieve a result that answers the bachelor’s thesis questions; interviews, a literature study and a case study were conducted. The bachelor’s thesis has been carried out in collaboration with the company Prolog, which is involved in the logistics solution for the Barkarbystaden project. Prologue has given the work the opportunity to get in touch with relevant key people for interviews and shared knowledge about the Barkarbystaden project that has been useful for conducting the case study.The case study resulted in the planned lead time being almost four times longer than the actual lead time. During the measurements, the time was noted when the transports arrived and when they left. 39% of deliveries leave after the planned departure time.The logistics solution in Barkarbystaden is only just over a year old and is not a previously known way of working for all contractors. This means that the contractors have had various challenges when it comes to adapting to the logistics solution. Where some have said that from the beginning it was difficult with a different way of working but that it got better and better with time. While some of the respondents said that they had worked with a similar logistics solution on previous projects, which obviously means an advantage when they have to adapt to Barkarbystaden's logistics solution.Logistics managers should be more careful when booking lead times for each delivery. By careful it is meant that logistics managers should take more account of what kind of transport and unloading method is to be used and to generally shorten the planned lead time in the booking system. This is because the difference was significant between the planned and actual lead time. When writing a contract between contractor and supplier, the contractor should require them to arrive within the specified unloading window and regulate the consequences of late arrival between the parties.

La granulatimide et l'isogranulatimide sont deux alcaloïdes marins de structure imidazo[4,5a]pyrrolo[3,4- c]carbazole décrits comme inhibiteurs du checkpoint G2 agissent spécifiquement sur la checkpoint kinase 1. De tels inhibiteurs utilisés en association avec un génotoxique pourraient potentialiser l'action de celui-ci permettant ainsi d'en réduire les doses et de diminuer ses effets secondaires. Deux types d'analogues inhibiteurs potentiels du checkpoint G2 ont été étudiés. Le premier correspond à une structure pentacyclique diversement substituées au niveau du cycle indole dans laquelle le cycle imidazole a été remplacé par un cycle pyrrole ou pyrazole et. Le second type est une structure tetracyclique ou le noyau indole a été substitué par un cyclopentène. Les différents composés, préparés en adaptant des méthodes de la littérature, ont été évalués autant d'un point de vue de leur cytotoxicité que d'un point de vue de leur inhibition du checkpoint G2.

Large-scale changes are common in genomes, and are often associated with pathological disorders. In the work presented in this dissertation, I provide insights into how inverted repeat sequences in budding yeast fuse during replication. Fusion leads to the formation of dicentric chromosomes, a translocation, and other chromosomal rearrangements.Using extensive genetics and some molecular analyses, I demonstrate that dicentric chromosomes are key intermediates in genome instability of a specific chromosome in budding yeast. I provide three pieces of evidence that is consistent with this conclusion. First, I detect a recombination fusion junction that is diagnostic of a dicentric chromosome (using a PCR technique). Second, I show a strong correlation between the amount of the dicentric fragment and the frequency of instability of the entire chromosome. Third, I demonstrate that a mutant known to stabilize dicentric chromosomes suppress instability. Based on these observations, I conclude that dicentric chromosomes are intermediates in causing genome instability in this system.Next, we demonstrate that fusion of inverted repeats is general. Both endogenous and synthetic nearby inverted repeats can fuse. Using genetics, I also show that many DNA repair and checkpoint pathways suppress fusion of nearby inverted repeats and genome instability. Based on our analysis, we propose a novel mechanism for fusion of inverted repeats that we term `faulty template switching.'Lastly, I discuss two genes that are necessary for fusion of nearby inverted repeats. I identified a mutant of the Exonuclease 1 (Exo1) and a mutant of anaphase inhibitor securin (Pds1) that suppress nearby inverted repeat fusion and genome instability. Studies of Exo1 and Pds1 provide us with insights into the molecular mechanisms of fusion.Our finding that nearby inverted repeats can fuse to form dicentric chromosomes that lead to genome instability may have great implications. The generality of this fusion reaction raises the possibility that dicentric chromosomes formed by inverted repeats can lead to genome instability in mammalian cells, and thereby contribute to a cancer phenotype.

This dissertation centers on the study of epithelial to mesenchymal cell transition (EMT) in the heart model of valve development. EMT is a process used by specific cells to invade adjacent matrix in order to differentiate into a three-dimensional structure. The first section of the project includes a study on the effects of inorganic arsenic on EMT and therefore the environmental concerns produced by deleterious effects on EMT. The second section focuses on the discovery of an intrinsic regulator of EMT, olfactomedin-1 (OLFM1). The discovery of a novel regulator of EMT in the atrioventricular canal is interesting, by itself, as it allows us to better understand the intrinsic molecular regulation of EMT in valve formation of the heart. The activity of this protein, as a regulator of cell invasion, identifies an important checkpoint in EMT. Because OFLM1 is conserved across many species, including humans, it may be a common or shared regulator of all types of EMT including cancer. Therefore, OLFM1 represents a promising new target for an anti-cancer agent as well as a potential clinical inducer of EMT to repair congenital heart disease that include valve defects.

<p>Bud formation by Saccharomyces cerevisiae is a fundamental process for yeast proliferation. Bud emergence is initiated by the polarization of the cytoskeleton, leading to local secretory vesicle delivery and gulcan synthase activity. The master regulator of polarity establishment is a small Rho-family GTPase – Cdc42. Cdc42 forms a clustered patch at the incipient budding site in late G1 and mediates downstream events which lead to bud emergence. Cdc42 promotes morphogenesis via its various effectors. PAKs (p21-activated kinases) are important Cdc42 effectors which mediate actin cytoskeleton polarization and septin filament assembly. The PAKs Cla4 and Ste20 share common binding domains for GTP-Cdc42 and they are partially redundant in function. However, we found that Cla4 and Ste20 behaved differently during the polarization and this depended on their different membrane interaction domains. Also, Cla4 and Ste20 compete for a limited number of binding sites at the polarity patch during bud emergence. These results suggest that PAKs may be differentially regulated during polarity establishment.</p><p>Morphogenesis of yeast must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site prior to bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor”. Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed, and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence.</p> / Dissertation

Les relations entre les histones qui composent les nucléosomes et le processus de transcription des gènes codants, sont à la fois multiples et extrêmement complexes. Au cours de ma thèse, je me suis intéressé à deux de ces relations. Tout d’abord, une première étude a été réalisée en collaboration avec les laboratoires de Franck Holstege et de Catherine Dargemont. Ce travail permet de préciser clairement l’effet des différentes formes de méthylation de la lysine 4 de l’histone H3 sur l’activité transcriptionnelle. Dans cette étude nous démontrons que la méthylation de H3K4 n’influence la transcription que d’un nombre très limité de gènes. Concernant ces gènes, un profil non conventionnel de distribution des formes de méthylation de H3K4 a été identifié par la présence inhabituelle d’un enrichissement en 3’ de ces gène des formes di- et triméthylées de H3K4.L’effet majoritaire de cette marque est d’induire une répression transcriptionnelle selon au moins deux mécanismes distincts. L’enrichissement atypique de la triméthylation de H3K4 influence négativement l’expression des gènes via la production d’ARN non codant anti-sens. Concernant l’effet répressif associé à la diméthylation de H3K4, la quantité d’ARN anti-sens ainsi que sa production ne sont pas impliquées.Dans une seconde étude réalisée en collaboration avec les laboratoires de Sebastian Chavez etd’Akash Gunjan, nous nous sommes intéressés au complexe FACT qui est impliqué dans l’assemblage et le désassemblage des nucléosomes lors du passage de l’ARN polymérase II. Jusqu’alors, un défaut de croissance chez les mutants thermosensibles du complexe FACT avait pu être observé. Dans notre étude, nous montrons que l’altération de FACT conduit, lors de la transcription, à l’éviction d’histones normalement incorporées à la chromatine. L’accumulation de ces histones libres à fort potentiel toxique, induit une répression spécifique de CLN3 qui code pour la première cycline dephase G1. Pour la première fois, nous mettons en évidence dans cette étude l’existence d’un mécanisme de surveillance moléculaire du cycle cellulaire induit par l’excès d’histones non incorporées à la chromatine / Relationships between histones, components of nucleosomes, and the transcription process of coding genes are both multiple and extremely complex. During my Thesis, I looked at twoof these relationships. First, we performed a study in collaboration with the Franck Holstedge and Catherine Dargemont labs. This work has allowed us to clearly define the effect of various methylation forms of the lysine 4 of the histone 3 on gene transcription. In this study we have shownthat H3K4 methylation influences the transcription of only a very limited number of genes. For these genes, a non conventional distribution profile of H3K4 methylation forms has been identified by the presence of an unusual enrichment in di- and trimethylated H3K4 in the 3’ of these genes. The principal effect of this mark is to promote transcriptional repression by at least two distinct mechanisms. The atypical enrichment of H3K4 trimethylation negatively influences gene expressionvia the production of non coding antisense RNA. For the repressive effect associated with dimethylH3K4, the quantity of antisense RNA as well as its production are not involved. We propose severalh ypotheses that link our results to the data known on this subject. In a second study performed incollaboration with the Sebastian Chavez and Akash Gunjan labs, we concentrated on the FACT complex that is involved in the assembly and disassembly of nucleosomes as RNA polymerase IImoves past. Previously, a growth defect in thermosensitive mutants of the FACT complex had been observed. In our study, we show that FACT deterioration leads to the eviction of histones that arenormally incorporated into chromatin during transcription. The accumulation of these free histones,which have a high toxic potential, induces the specific repression of CLN3 which encodes for the firstcyclin of G1 phase. For the first time, we show in this study the existence of a cell cycle molecular surveillance mechanism that is induced by an excess of free histones

<p>The DNA of a cell operates as its blueprint, providing coded information for the production of the RNA and proteins that allow the cell to function. Cells can face a myriad of insults to their genomic integrity during their lifetimes, from simple errors during growth and division to reactive oxygen species to chemotherapeutic reagents. To deal with these mutagenic insults and avoid passing them on to progeny, cells are equipped with multiple defenses. Checkpoints can sense problems and halt a cell’s progression through the cell cycle in order to allow repairs. More drastically, cells can also prevent passing on mutations to progeny by triggering apoptosis, or programmed cell death. This work will present two separate discoveries regarding the regulation of DNA damage-induced apoptosis and the regulation of the spindle checkpoint.</p><p> The protease caspase-2 has previously been shown to be an important regulator of DNA damage-induced apoptosis. In unstressed cells caspase-2 is present as an inactive monomer, but upon sensing a stress caspase-2 dimerizes and becomes catalytically active. The mechanisms that regulate this dimerization are poorly understood. The first research chapter details our development of a novel method to study dimerized caspase-2, which in turn identified TRAF2 as a direct activator of caspase-2. Specifically, we utilized the Bimolecular Fluorescence Complementation technique, wherein complementary halves of the Venus fluorophore are fused to caspase-2: when caspase-2 dimerizes, the non-fluorescent halves fold into a functional Venus fluorophore. We combined this technique with a Venus-specific immunoprecipitation that allowed the purification of caspase-2 dimers. Characterization of the caspase-2 dimer interactome by MS/MS identified several members of the TNF Receptor Associated Factor (TRAF) family, specifically TRAF1, 2, and 3. Knockdown studies revealed that TRAF2 plays a primary role in promoting caspase-2 dimerization and downstream apoptosis in response to DNA damage. Identification of a TRAF Interacting Motif (TIM) on caspase-2 indicates that TRAF2 directly acts on caspase-2 to induce its activation. TRAF2 is known to act as an E3 ubiquitin ligase as well as a scaffold for other E3 ubiquitin ligases. Indeed, we identified three lysine residues in the caspase-2 prodomain (K15, K152, and K153) important for its ubiquitination and complex formation. Together these results revealed a novel role for TRAF2 as a direct activator of caspase-2 apoptosis triggered by DNA damage.</p><p> During mitosis, when the cell prepares to divide, great care is taken to ensure that the chromosomes are properly segregated between the two daughter cells by the mitotic spindle. This is primarily accomplished through the spindle checkpoint, which becomes activated when the mitotic spindle is not properly attached to each chromosome’s kinetochore. When activated, the primary effector of the spindle checkpoint, the mitotic checkpoint complex (MCC), inhibits the anaphase-promoting complex (APC/C) by binding to the APC/C co-activator, CDC20. This prevents the APC/C from targeting critical pro-mitotic proteins, like cyclin B and securin, to promote mitotic exit. Although the function of the MCC is well understood, its regulation is not, especially in regard to protein phosphatases To investigate this, we activated the spindle checkpoint with microtubule inhibitors and then treated with a variety of phosphatase inhibitors, examining the effect on the MCC and APC/C. We found that two separate inhibitors, calyculin A and okadaic acid (1uM), were able to promote the dissociation of the MCC. This led to the activation of the APC/C, but the cells remained in mitosis as evidenced by high levels of Cdk1 activity and chromosome condensation. This is the first time that phosphatases have been shown to be essential to maintaining the MCC and an active spindle checkpoint.</p> / Dissertation