The nuclear lamina is a filamentous network that underlies the nuclear envelope. Lamina components include the family of LEM domain (LEM-D) proteins, named for LAP2, emerin and MAN1. Mutations in genes encoding LEM-D proteins cause tissue-restricted human disease, even though these genes are globally expressed. To understand the contributions of the LEM-D proteins to nuclear lamina function, investigations of the Drosophila LEM-D proteins was undertaken. The Drosophila genome encodes four LEM-D proteins and this thesis describes work done on the Drosophila homologues of MAN1 and emerin, Drosophila MAN1 (dMAN1) and Otefin (Ote). Chapter 2 describes the generation and phenotypic analyses of dMAN1 mutants. These mutants display a range of tissue-specific defects associated with an increase in BMP/Dpp signaling. This suggests that dMAN1 downregulates BMP/Dpp signaling at the nuclear periphery. Chapter 3 describes the identification and phenotypic analyses of ote mutants. Loss of Ote is associated with a tissue-specific defect of the female germline where ote mutant females display defects in germline stem cell (GSC) maintenance. Loss of Ote causes defects in the germline cells, the cap cells of GSC niche and an increased sensitivity to Dpp signaling in both germline and somatic cells. These findings support models suggesting that laminopathies arise from dysfunction of the homeostasis in stem cell populations. Taken together, these studies suggest that the nuclear lamina may play tissue-specific roles through regulation of signal transduction pathways. Our data also support the use of Drosophila as a system to elucidate the mechanistic basis of diseases associated with defects in the nuclear lamina.

Les cellules souches embryonnaires de souris (ESCs) et les cellules souches de l'épiblaste (EpiSCs) représentent, respectivement, les états naïf et amorcé de la pluripotence et sont maintenues in vitro par des voies de signalisation spécifiques. De plus les ESCs cultivées dans un milieu sans sérum avec deux inhibiteurs (2i) sont décrites comme étant les plus naïves. Plusieurs études ont suggéré que chaque type de cellules pluripotentes est caractérisé par une organisation différente de l'épigénome. Nous présentons ici une étude comparative de l'état épigénétique et transcriptionnel des séquences satellites répétées péricentromériques (PCH) entre les ESCs (2i et sérum) et EpiSCs. Nous montrons que H3K27me3 au PCH est très dynamique et peut discriminer les ESCs en 2i des autres cellules souches pluripotentes. Alors que la transcription des séquences satellites est élevée dans les ESCs en sérum, elle est plus faible dans ESCs en 2i et encore plus réprimée dans les EpiSCs. La suppression de la méthylation de l'ADN ou d'H3K9me3 dans les ESCs conduit à un dépôt important de H3K27me3 au PCH, mais peu de changements transcriptionnels de ces séquences. En revanche, l'absence d'H3K9me3 dans les EpiSCs n'empêche pas la méthylation de l'ADN au PCH, mais induit la transcription de ces séquences. La conversion in vitro des ESCs en EpiSCs est plus longue que le passage des cellules de l'ICM en épiblaste in vivo. Cette inefficacité ne peut pas être expliquée par une mise en place retardée du nouveau réseau transcriptionnel. Pour conclure notre étude a révélé que les EpiSCs ont perdu de la plasticité par rapport au ESCs sur l'hétérochromatine ainsi que l’euchromatine, comme le montre la réduction des niveaux d'H3K9ac et des domaines bivalents, étant ainsi plus proche épigénétiquement de cellules somatiques que de la pluripotence naïve. / Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent naïve and primed pluripotency states, respectively and are maintained in vitro using specific signaling pathways. Furthermore, ESCs cultured in serum-free medium with two inhibitors (2i) are described as being the most naïve. Several studies have suggested that each pluripotent cell type is characterized by a different epigenome organization. Here we present a comparative study of the epigenetic and transcriptional state of centromeric and pericentromeric (CH/PCH) satellite repeats in ESCs (2i and serum ones) and EpiSCs. We show that the pattern of H3K27me3 at PCH is highly dynamic and discriminate 2i-ESCs from the other pluripotent stem cells. Whereas satellites transcription is high in serum-ESCs, it is lower in 2i-ESCs and even more repressed in EpiSCs. Removal of either DNA methylation or H3K9me3 in ESCs leads to enhanced deposition of H3K27me3 but few changes in satellite transcription. By contrast, in EpiSCs removal of H3K9me3 does not prevent DNA methylation at PCH but de-represses the satellite transcription. In vitro conversion from naive to primed pluripotency showed an important delay compared to the in vivo development of ICM cells into post-implantation epiblast. Such inefficiency cannot be explained by a delayed switch to the new transcriptional network. Altogether our study reveals that EpiSCs have lost the chromatin plasticity of ESCs on heterochromatin as well as euchromatin, as shown by the reduction of H3K9ac levels and bivalent domains, thus being closer to somatic cells in terms of epigenetics than naive pluripotency.

<p>The centromere is essential for chromosome segregation and genome stability. It is the site of kinetochore assembly and chromosome attachment to the spindle microtubules, and it is important for chromosome movement during mitosis and meiosis. Normal human chromosomes have one centromere, but genome rearrangements that occur with instability, aging, and disease often result in chromosomes with two centromeres, called dicentrics. Nearly seventy-five years ago, Barbara McClintock demonstrated that dicentric chromosomes in plants are associated with instability through mitotic "breakage-fusion-bridge" cycles. However, human dicentrics are unusually stable due to the poorly understood phenomenon of centromere inactivation. Centromere inactivation has been primarily studied in patient-derived dicentrics, limiting the derivation of a molecular pathway. Key centromere and kinetochore proteins are not present at inactive centromeres, but beyond these observations, the process of centromere inactivation is unclear. Epigenetic and sequence-dependent factors are known to contribute to centromere specification, but requirements for centromere assembly, maintenance, and suppression remain obscure. The aims of this research were to (1) determine the mechanism(s) by which de novo dicentric chromosomes are stabilized, (2) ascertain the factors influencing the involvement of specific chromosomes in de novo fusions, and (3) establish the epigenomic, temporal, and mechanistic basis of centromere inactivation. To uncover the mechanistic foundations of these processes, we developed in vitro cell culture systems to study the formation and stabilization of de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. This finding is notable since the most prevalent rearrangement in humans involves the acrocentrics and is called Robertsonian translocation (ROB). In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the centromeric DNA array is reduced compared to the same array before dicentric formation. Many functional dicentrics persist for months after formation. Our results indicate that dicentric human chromosomes undergo alternative fates after formation across a broad temporal window. During transient telomere disruption, we observed a dramatic change in nucleolar appearance. Nucleolar proteins did not coalesce into condensed structures, but appeared dispersed throughout the nucleus. This surprising alteration in nucleolar organization and nuclear architecture suggests remodeling of the nucleolus and subsequent effects on nucleolar-associated chromosomes, such as the acrocentrics, could contribute to the high incidence of ROB formation. Further studies and development of additional cell culture systems will allow us to evaluate current models of centromere assembly and disassembly and the importance of chromatin organization to centromere function and genome architecture.</p> / Dissertation

Le noyau des cellules est une structure très organisée, dont l'organisation joue un rôle important dans la régulation de l'expression des gènes. La compréhension des mécanismes à l'origine de cette organisation est donc essentielle à la compréhension du fonctionnement des génomes. De nombreuses expériences conduites chez la souris ont montré que les régions centromériques (RC) des chromosomes jouent un rôle dans l'organisation du noyau. L'organisation spatiale des RCs humaines est beaucoup moins étudiée, principalement à cause de la complexité des séquences qui les composent, qui rend plus difficile leur détection. Nous avons développé des outils de traitement et d'analyse quantitative d'image, qui, combinés à des nouveaux marqueurs des RCs humaines, nous ont permis de mieux décrire deux aspects de leur organisation spatiale. D'une part nous avons montré qu'elles se positionnent préférentiellement en périphérie du noyau ou aux bords des nucléoles, avec des fréquences qui dépendent des chromosomes. D'autre part nous avons montré qu'elles s'agrègent dans le noyau pour former un compartiment d'hétérochromatine, qui présente des caractéristiques similaires à celui observé dans d'autres espèces telles que la souris. Ces deux aspects sont tous deux inter-dépendants et varient au cours du cycle cellulaire. Cette description nouvelle met sur la piste de mécanismes responsables de l'organisation particulière des RCs, qui pourront être étudiés grâce à la méthode d'analyse et aux observables que nous avons développées. L'étude de ces mécanismes permettra de mieux comprendre la fonction des RCs humaines dans l'organisation du noyau. / The cell nucleus is a highly organized structure, playing an important role in gene regulation. Understanding the underlying mechanisms is therefore essential for understanding genome function. Numerous studies conducted in mouse cells have shown that centromeric regions (RC) of chromosomes play a role in nuclear organization. The spatial organization of human RCs is less studied, mainly because of the complexity of the underlying DNA sequences that make them hard to detect. We have developed image processing and analysis tools, that, combined with new markers for human RCs, have allowed us to draw a better description of two features of their spatial organization. On the one hand, we have shown that they are preferentially located close to the nuclear periphery or nucleoli borders, with chromosome-dependent frequencies. On the other hand, we have shown that they cluster to form a heterochromatic compartment that displays similar properties as the one observed in other species such as mouse. Both features are inter-dependent, and vary throughout the cell-cycle. This new description puts on the track of mechanisms responsible for the peculiar organization of RCs. Those mechanisms could be studied using the methodology and the observables we have developed. The study of those mechanisms will provide a better understanding of human RC function in nuclear organization.

A cultura de segurança na área nuclear é definida como o conjunto de características e atitudes da organização e dos indivíduos que fazem que, com uma prioridade insuperável, as questões relacionadas à proteção e segurança nuclear recebam a atenção assegurada pelo seu significado. Até o momento, não existem instrumentos validados que permitam avaliar a cultura de segurança na área nuclear. Em vista disso, os resultados da definição de estratégias para o seu fortalecimento e o acompanhamento do desempenho das ações de melhorias tornam-se difíceis de serem avaliados. Este trabalho teve como objetivo principal desenvolver e validar um instrumento para a avaliação da cultura de segurança de organizações nucleares, utilizando o Instituto de Pesquisas Energéticas e Nucleares como unidade de pesquisa e coleta de dados. Os indicadores e variáveis latentes do instrumento foram definidos utilizando como referência modelos de avaliação de cultura de segurança da área da saúde e área nuclear. O instrumento de coleta de dados proposto inicialmente foi submetido à avaliação por especialistas da área nuclear e, posteriormente, ao pré-teste com indivíduos que pertenciam à população pesquisada. A validação do modelo foi feita por meio da modelagem por equações estruturais utilizando o método de mínimos quadrados parciais (Partial Least Square - Structural Equation Modeling PLS-SEM), no software SmartPLS. A versão final do instrumento foi composta por quarenta indicadores distribuídos em nove variáveis latentes. O modelo de mensuração apresentou validade convergente, validade discriminante e confiabilidade e, o modelo estrutural apresentou significância estatística, demonstrando que o instrumento cumpriu adequadamente todas as etapas de validação. / The safety culture in the nuclear area is defined as that assembly of characteristics and attitudes in organizations and individuals which establishes that, as an overriding priority, nuclear plant safety and protection issues receive the attention warranted by their significance. Until now, there are no validated instruments to evaluate the safety culture in the nuclear area. This fact makes it difficult to assess the results of strategies for its strengthening and of the improvement actions. The main objective of this work was to develop and validate an instrument for the evaluation of the safety culture of nuclear organizations, using the Instituto de Pesquisas Energéticas e Nucleares as a research unit and data collection. The indicators and latent variables of the instrument were defined using health and nuclear area models of safety culture evaluation as reference. The data collection instrument initially proposed was submitted to the evaluation by nuclear area experts and, subsequently, to the pretest with individuals who belonged to the researched population. The validation of the model was performed through structural equation modeling using the Partial Least Square - Structural Equation Modeling - PLS-SEM method in the SmartPLS software. The final version of the instrument was composed by forty indicators distributed in nine latent variables. The measurement model showed convergent validity, discriminant validity and composite reliability, and the structural model showed statistical significance. Therefore the overall model has successfully accomplished all the validation steps.

A cultura de segurança na área nuclear é definida como o conjunto de características e atitudes da organização e dos indivíduos que fazem que, com uma prioridade insuperável, as questões relacionadas à proteção e segurança nuclear recebam a atenção assegurada pelo seu significado. Até o momento, não existem instrumentos validados que permitam avaliar a cultura de segurança na área nuclear. Em vista disso, os resultados da definição de estratégias para o seu fortalecimento e o acompanhamento do desempenho das ações de melhorias tornam-se difíceis de serem avaliados. Este trabalho teve como objetivo principal desenvolver e validar um instrumento para a avaliação da cultura de segurança de organizações nucleares, utilizando o Instituto de Pesquisas Energéticas e Nucleares como unidade de pesquisa e coleta de dados. Os indicadores e variáveis latentes do instrumento foram definidos utilizando como referência modelos de avaliação de cultura de segurança da área da saúde e área nuclear. O instrumento de coleta de dados proposto inicialmente foi submetido à avaliação por especialistas da área nuclear e, posteriormente, ao pré-teste com indivíduos que pertenciam à população pesquisada. A validação do modelo foi feita por meio da modelagem por equações estruturais utilizando o método de mínimos quadrados parciais (Partial Least Square - Structural Equation Modeling PLS-SEM), no software SmartPLS. A versão final do instrumento foi composta por quarenta indicadores distribuídos em nove variáveis latentes. O modelo de mensuração apresentou validade convergente, validade discriminante e confiabilidade e, o modelo estrutural apresentou significância estatística, demonstrando que o instrumento cumpriu adequadamente todas as etapas de validação. / The safety culture in the nuclear area is defined as that assembly of characteristics and attitudes in organizations and individuals which establishes that, as an overriding priority, nuclear plant safety and protection issues receive the attention warranted by their significance. Until now, there are no validated instruments to evaluate the safety culture in the nuclear area. This fact makes it difficult to assess the results of strategies for its strengthening and of the improvement actions. The main objective of this work was to develop and validate an instrument for the evaluation of the safety culture of nuclear organizations, using the Instituto de Pesquisas Energéticas e Nucleares as a research unit and data collection. The indicators and latent variables of the instrument were defined using health and nuclear area models of safety culture evaluation as reference. The data collection instrument initially proposed was submitted to the evaluation by nuclear area experts and, subsequently, to the pretest with individuals who belonged to the researched population. The validation of the model was performed through structural equation modeling using the Partial Least Square - Structural Equation Modeling - PLS-SEM method in the SmartPLS software. The final version of the instrument was composed by forty indicators distributed in nine latent variables. The measurement model showed convergent validity, discriminant validity and composite reliability, and the structural model showed statistical significance. Therefore the overall model has successfully accomplished all the validation steps.

Chez la levure à bourgeon, l’établissement de domaines silencieux pour la transcription nécessite la formation d’une structure, de type hétérochromatine, formée par le complexe SIR (Silencing Information Regulator). Les gènes soumis à la répression transcriptionnelle par ce complexe se trouvent aux sites cryptiques de détermination du type sexuel (HM) et dans les régions subtélomériques localisées à la périphérie nucléaire. Le recrutement des protéines Sir à ces sites nécessite la présence de séquences en cis comme les silencers ou les répétitions télomériques. Mon travail de thèse s’est attaché à l’étude d’une nouvelle voie d’établissement de la répression transcriptionnelle des gènes. En effet, nous avons démontré que la répétition en tandem de protéines fortement liées à l’ADN constitue un stress pour la fibre de chromatine. Ce stress induit le recrutement du complexe SIR favorisant ainsi la formation d’hétérochromatine et la mise en silence des gènes dans des régions normalement actives du génome. De plus, nous avons observé qu’en absence de l’ADN hélicase Rrm3, dont la fonction est de faciliter la progression de la fourche de réplication le long de la fibre de chromatine, la répression induite par ces complexes est exacerbée. Ce lien entre stress réplicatif et établissement de la répression transcriptionnelle a été observé, dans un premier temps, grâce à l’utilisation de systèmes artificiels (systèmes d’étiquetage des gènes : lacO/LacI et tetO/TetR). En outre, nous avons montré qu’un site naturel de pause de la réplication, tel qu’un gène codant un ARN de transfert, peut également favoriser la répression par les protéines Sir. De manière intéressante, à l’échelle du génome, nous avons pu observer le recrutement des protéines Sir dans des régions où la progression de la fourche de réplication est ralentie. Ainsi, nos données révèlent une nouvelle voie de mise en silence des gènes liant stress réplicatif et répression transcriptionnelle. / In budding yeast, the heterochromatin-like structure formed by the SIR complex (Silencing Information Complex) represses transcription. SIR mediated repression occurs at the cryptic mating type loci (HM) and subtelomeric regions localized at the nuclear periphery. The recruitment of the Sir proteins is induced by the presence of cis-acting elements as silencers or telomeric repeats.My doctorate work was focused on the characterization of a novel pathway of silencing establishment. Indeed, we have shown that arrays of tight DNA-proteins complexes lead to a chromatin stress. This stress induces the recruitment of the SIR complex and the establishment of stable heterochromatin-like domain at ectopic sites in the budding yeast genome. Moreover, this heterochromatinization is enhanced in cells mutated for Rrm3, a specialized DNA helicase acting ahead the fork to remove replication-impeding structures. Thus, we first observed a link between replication stress and silencing establishment by using artificial systems (gene tagging systems: lacO/LacI and tetO/TetR). Further, we have shown that tRNA genes, which are known to act as replication pause sites, can favor SIR-mediated repression. Interestingly, we found that Sir proteins are recruited where the replication fork progression is impeded at the genome wide scale. All together, these data reveal a novel mechanism for heterochromatin formation linking replication stress with gene repression.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

La réplication de l’ADN est un processus essentiel qui doit avoir lieu une seule fois par cycle cellulaire. Ce processus hautement régulé et très conservé chez les eucaryotes, assure une complète duplication et donc une totale transmission de l’information génétique. Des changements dans le programme de réplication, qui est définit par le moment d’activation et la fréquence d’utilisation de l’ensemble des origines, ont été observés lors du développement, après induction de la différenciation chez des cellules souches embryonnaires de souris, ainsi que dans un grand nombre de cancers. La régulation de la réplication de l’ADN est donc un processus essentiel pour le maintien de l’intégrité du génome et le programme de réplication pourrait y contribuer de manière importante. Cependant, en dépit d’un grand nombre de travaux sur les différentes protéines et modifications impliquées dans la sélection des origines, les principaux déterminants ainsi que leur interdépendance restent étonnement méconnus. Mon projet de thèse se focalise sur l’identification des paramètres clés qui régulent le programme de réplication, en utilisant comme modèle la levure de fission, Schizosaccharomyces pombe. Premièrement, je me suis intéressé au rôle de la dynamique de l’activité des CDKs lors de la phase G1 ainsi que de leur niveau d’activité à la frontière G1/S dans la sélection des origines. J’ai démontré que changer la longueur de la phase G1 à travers la modulation de l’activité des CDKs se traduit par une modification du profil de réplication tout au long du génome. Plus précisément, les origines inefficaces sont utilisées plus fréquemment alors que les origines efficaces ont une activité réduite. D’un autre coté, nous avons également montré que le nombre d’origines actives pour une phase S donnée, dépend du niveau d’activité des CDKs lors de l’entrée en phase S, suggérant ainsi que cette activité est un facteur limitant dans la régulation de l’initiation de la réplication. Dans un second temps, j’ai utilisé une approche dans laquelle les cellules établissent un programme de réplication de novo après la sortie de quiescence, afin d’étudier les premières étapes de la sélection des origines de réplication, en se focalisant sur l’importance du recrutement de ORC (Origin Recognition Complex) aux origines. L’analyse du profil de liaison de ORC révèle une forte corrélation entre le niveau de liaison de ORC aux origines et l’efficacité de ces dernières, démontrant pour la première fois que ORC n’est pas simplement un marqueur des sites d’initiation potentiels mais plutôt un déterminant crucial dans l’établissement du programme de réplication. Finalement, j’ai observé que les origines efficaces ont tendance à être organisées en groupes tout au long du génome, suggérant que l’organisation chromosomique pourrait être importante dans la sélection des origines de réplication. Afin d’étudier cela, j’ai généré des souches contenant différents réarrangements chromosomiques. Nos résultats indiquent que la position relative d’une origines par rapport à son contexte chromosomique, joue un rôle important dans la régulation de son efficacité et que des régions distinctes peuvent avoir des effets opposés sur la sélection des origines en étant soit activatrices ou inhibitrices. / DNA replication is an essential process that occurs only once in a cell cycle before cell division. Replication is highly regulated through conserved mechanisms to ensure the faithful duplication and transmission of genetic information. Interestingly, changes in the replication program, defined by the temporal and spatial pattern of replication origin activation, have been observed during development in distinct cell types, after induction of differentiation in mouse embryonic stem cells, and in various cancers. The regulation of DNA replication is therefore essential for ensuring the integrity of the genome, and the program of origin activation may be an important contributor to this process. However, despite a large body of work on the many enzymes and modifications involved in origin selection, the critical determinants as well as their interdependence remain surprisingly unknown. My thesis project focuses on identifying the key parameters that regulate the replication program, taking advantage of unique approaches using the fission yeast Schizosaccharomyces pombe as a model system. First, we investigated the qualitative and quantitative aspects of the role of CDK activity in determining the program of DNA replication. We demonstrated that changing the length of G1 phase through modulation of CDK activity has an impact on the profile of replication initiation along the chromosome. More specifically, inefficient origins show increases in their usage, while efficient origins have reduced activities. Moreover, we have shown that cells are highly sensitive to differences in CDK activity levels at the G1/S transition, which result in genome-wide changes in replication initiation across the entire spectrum of efficiencies. This suggests that CDK activity is a dose-dependent, limiting factor in the regulation of origin usage. Thus, our study establishes the integration of both temporal and quantitative regulation of CDK activity as a key determinant in defining the program of genome duplication. Second, using an approach in which cells establish a replication program de novo after exit from quiescence, we investigated the critical first steps of origin selection. We focused on the importance of the essential Origin Recognition Complex, whose recruitment to origins is required for the subsequent assembly of replication complexes. Our analysis reveals a strong correspondence between the level of ORC binding at origins and the efficiency of these origins in both cells exiting quiescence as well as those in vegetative growth conditions. Therefore, we demonstrate for the first time that ORC is not simply a marker of potential initiation sites but rather a crucial determinant in the program of origin usage.Finally, our observation that efficient origins are organized in distinct clusters in the de novo replication program suggested that chromosomal organization may be important for origin selection. To address this question, we have generated strains containing a series of distinct chromosomal rearrangements and assessed their origin efficiency profiles. Our findings indicate that the localization of an origin with respect to its chromosomal context plays an important role in regulating its efficiency. Moreover, distinct regions may have different effects on origin selection by being permissive or inhibitory for origin activity. Those observations could indicate a role for the spatial organization of the genome in origin selection and thus led us to study chromosome and nuclear organization in conditions where the replication program is different.