No description available.

No description available.

The gene encoding the Dictyostelium replication protein A large subunit (DdRPA1) has been cloned by screening of an EcoR I partial genomic library and a Hind III genomic sub-library. The complete nucleotide sequence, including the promoter region of the gene has been obtained by sequencing. Though the DdRPA1 protein has a size shift during development, 62 kDa in undifferentiated cells and 81 kDa in differentiated cells; they are the products of the same gene. Northern blot analysis revealed that the expression level of the DdRPA1 was constant throughout differentiation and the size of mRNA is the same at all stages, corresponding to a 81 kDa protein. Thus, it seems that the size change between the 62 kDa and 81 kDa is probably due to posttranslational modification, most likely, proteolytic cleavage. The transcription start site for both sizes of DdRPA1 has been identified at 306 bp upstream of the coding sequence by primer extension reaction. A PCR fragment representing 27% of the gene encoding the DdRPA middle size subunit (DdRPA2) has been generated by using the degenerate primers. This PCR fragment has been cloned and sequenced. The mRNA for this subunit corresponds to a protein of about 35 kDa. A decrease of the DdRPA2 mRNA expression level during differentiation was found by comparison between undifferentiated and differentiated cells. In Dictyostelium, replication protein A is a heterotrimeric protein that can bind with specific DNA sequences in a stage-dependent pattern. These DNA sequences were identified as the cis-acting regulatory sites in differentiation-related genes, including the glycogen phosphorylase 2 gene (gp2). Therefore, it is possible that DdRPA is not only a single-stranded DNA binding protein that is used in multiple essential DNA metabolic processes, such as DNA replication, repair and recombination in undifferentiated cells, but also involved in the transcriptional regulation process during differentiation. / Master of Science

The RNA world hypothesis posits that at some stage in the development of life, RNA functioned as both an informational polymer and a catalyst for important reactions. However, many questions remain as to how RNA molecules might have evolved into living organisms. This thesis uses computer simulations to model processes thought to be important to the development of an RNA world. First, a model is discussed which describes non-enzymatic polymerization of single-stranded RNA from different kinds of activated nucleotides, a necessary first step towards an RNA world. It was found that a system undergoing polymerization of RNA from 5′-activated triphosphates or imidazolides behaves differently from an equilibrium system undergoing reversible polymerization reactions from 2′,3′-cyclic monophosphates, for example. In the 5′-triphosphate case, the system is not in equilibrium but rather in a state of circular reaction flux that must be maintained by an external source of phosphates. This model is then adapted to investigate non-enzymatic template-directed replication of RNA strands. It is found that this process fulfills all the necessary requirements to function as a metabolism which maintains a difference between the outside non-living environment and the internal environment of the cell. Finally, byproducts arising from the template copying mechanism in this model are discussed, including the development of highly regular sequence patterns in the strand population due to selection for the ability to form duplexes with neighbouring strands. Altogether, this thesis illustrates new implications, potential pitfalls, and possibilities of the RNA world hypothesis for the origin of life. In particular, it emphasizes the fundamental link between the processes of replication and metabolism, both of which must have been crucial to the functioning of the earliest protocells. This link has been largely overlooked in scientific literature on the topic to date. / Thesis / Master of Science (MSc) / For millennia, humanity has told stories about the origin of life. Since the 1960s, scientists have hypothesized that RNA is a key player in this origin story. RNA can both hold information and catalyze chemical reactions, meaning only one molecule is needed for both these crucial functions. However, many questions remain about how this would work in practice. This project used computer simulations to model steps along the path from RNA to living organisms. First, a model was developed for the formation of single-stranded RNA from building block molecules. The model was then expanded to include copying of existing RNA strands, and it was found that this process constitutes a metabolism. Finally, it was discovered that over time the copying process produces simple patterns in the sequence of building blocks that make up the RNA strands. Altogether, these findings emphasize the link between replication and metabolism in early cells.

La duplication de l'ADN au cours de la phase S est initiée à partir de l'activation de plusieurs dizaines de milliers d'origines de réplication. La mise en place des origines a lieu au cours de la phase G1 sous la forme de complexe de pré-réplication (pré-RC) et leur activation est orchestrée par un programme spatio-temporel. La régulation spatiale détermine les origines qui seront activées et la régulation temporelle, ou timing de réplication, détermine le moment de leur activation. En effet, toutes ces origines ne sont pas activées en même temps durant la phase S : certaines origines seront activées en début de phase S, d'autre en milieu, ou d'autre à la fin. Ce programme est établi en tout début de phase G1, au " point de décision du timing ". C'est un programme très robuste qui signe l'identité d'une cellule, son état de différenciation et le type cellulaire à laquelle elle appartient. Il a aussi été montré qu'il est altéré dans des situations pathologiques, en particulier le cancer, sans qu'on ne comprenne très bien les raisons mécanistiques. De manière générale, les mécanismes moléculaires qui régulent le timing de réplication sont méconnus. Le premier volet de ma thèse a permis l'identification d'un nouveau régulateur du timing de réplication : il s'agit de l'ADN polymérase spécialisée Thêta. Recrutée à la chromatine très tôt en phase G1, elle interagit avec des composants du pré-RC, et régule le recrutement des hélicases réplicatives à la chromatine. Enfin, sa déplétion ou sa surexpression entraîne une modification du timing de réplication à l'échelle du génome. Dans la deuxième partie de ma thèse, j'ai exploré les mécanismes qui régulent ce programme temporel d'activation des origines suite à un stress réplicatif. J'ai identifié un mécanisme de régulation transgénérationnel inédit : la modification du timing de réplication de domaines chromosomiques ayant subi un stress réplicatif au cycle cellulaire précédent. Des cellules-filles issues d'une cellule ayant subi des problèmes de réplication dans des domaines fragiles (riches en AT, et donc potentiellement structurés, et pauvres en origines) présentent un timing plus précoce de l'activation des origines au niveau de ces domaines. Ce nouveau processus biologique d'adaptation est particulièrement intéressant dans un contexte tumoral de haut stress réplicatif chronique car ce pourrait être un moyen pour la cellule tumorale de survivre à son propre stress réplicatif mais aussi aux thérapies antitumorales qui sont nombreuses à cibler la réplication de l'ADN. / DNA duplication in S phase starts from thousands of initiation sites called DNA replication origins. These replication origins are set in G1 as pre-replication complexes (pre-RC) and fired in S phase following a spatio-temporal program of activation. This program determines which origins will be fired and when. Indeed, all the origins are not fired in the same time and we can distinguish early, middle and late replication origins. This temporal regulation is called "replication timing" and is determined at the "timing decision point" (TDP) in early G1. It's a robust program, which participates to the definition of cell identity, in term of differentiation state or cell type. However, the precise molecular mechanisms involved are poorly understood. Defective timing program has been evidenced in pathological contexts, in particular in cancers, but the mechanisms of this deregulation remain unclear. In the first part of my PhD, I contributed to the discovery of a new regulator of the origin timing program: the specialized DNA polymerase Theta (Pol Theta). Pol Theta is loaded onto chromatin in early G1, coimmunoprecipitates with pre-RC components and modulates the recruitment of Mcm helicases at TDP. Moreover, depletion or overexpression of Pol Theta modifies the timing of replication at a fraction of chromosomal domains. The second part of my work aimed at exploring the mechanisms that regulates replication timing after a replicative stress. I identified a totally new transgenerational adaptive mechanism of DNA replication timing regulation: the modification of the timing of origin activation at chromosomal domains that have suffered from a replicative stress during the previous cell cycle. Daughter cells from a cell that has experienced replication stress at particular domains (late replicating domains, AT rich so they can form structured DNA, and poor in origin density) shows advanced origin activation within these regions. This new biological process in response to replicative stress could be of particular interest in the context of cancer since, tumor cells are characterized by high level of intrinsic chronic replicative stress. This new mechanism may favor cancer cell survival despite replication stress, particularly upon treatments with anti-tumor agents that target DNA.

No description available.

Herpes B virus, otherwise known as Macacine herpesvirus 1, is a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Simplex, and is closely related to human herpes simplex viruses 1 and 2 (HSV1 and HSV2). B virus is endemic in macaque monkeys, but is capable of zoonotic transmission to humans resulting in fatality in greater than 80% of untreated cases. The goal of our lab is to understand the disparity in the outcome of infection between the natural host- macaques and the foreign host- humans. An important barrier to progress is the lack of understanding of host cell: B virus interactions in response to infection. An important pathway activated by stress, known as the mitogen activated protein kinase (MAPK) p38 pathway, is activated by B virus infection. Of particular interest is its role in regulating cellular translation via stimulation of activation of the eukaryotic initiation factor 4E (eIF4E). The activation of eIF4E is a vital rate-limiting step in translation, which can be manipulated by a variety of viruses. For example HSV1 can activate eIF4E through the p38 pathway but in the absence of this pathway eIF4E activity and viral titers are decreased. Because of the effect HSV1 has on the p38 pathway, and because B virus is a close relative of HSV1, we hypothesized that B virus also utilizes the p38 pathway to activate eIF4E in a host-dependent manner. In this dissertation, we show that the role of MAPK p38 with regard to translation is crucial to cellular processes that reduce virus replication in natural host cells, but within human cells this stress pathway appears not to play a role in reducing B virus replication. Data generated for this dissertation suggest that the p38 pathway is responsible in part for controlling the virus infection and spread within the natural host, but does not dampen virus replication in human host cells encountering the virus. Taken together, our results suggest that this pathway has at least one host-specific defense to combat B virus infection and that both cellular and viral proteins require the presence or absence of this pathway to function.

published_or_final_version / Pathology / Master / Master of Philosophy

Mitochondrial DNA encodes thirteen protein subunits in the oxidative phosphorylation system (OXPHOS) that is responsible for cellular energy production. Mitochondrial disorders have been identified to be associated with mtDNA mutations. However, the molecular mechanisms of specific mtDNA mutations are still being explored in order to establish causative links. This study tries to elucidate the mutational effects of mtDNA on OXPHOS complex activities and cell growths. Using mouse 3T3 fibroblasts as a cell model, single-cell clones with different growth rates were isolated. The entire mtDNA genome was sequenced for mutations. The enzymatic activities of OXPHOS complex I to V were analysed. Three growth patterns represented by five clones were identified. Three clones (clone #2, #3, and #6) had the shortest doubling times (11.5 - 14.9 hours). Clone #1 had a medium growth rate (19.2 hous); and clone #5 had a significantly slow growth rate (22 hours). MtDNA sequencing results revealed that clone #5 had several heteroplasmic mutations (one in 16S rRNA, two in tRNAser (UCN), three in tRNAasp, one in tRNAlys, one in COI, five in COII, and one in ATPase8) while the other four clones showed sequence homology. Enzymatic analyses showed that on average clone #5 had significantly low complex III, IV, and V activities (p < 0.05). Changes in biochemical properties and protein structure were analyzed to deduct possible mechanisms for reduced respiration. In conclusion, the slow growth rate is associated with reduced OXPHOS enzyme functions. It is most likely that the combination of COI and COII mutations resulted in the reduction of complex IV function. It is still unclear whether the ATPase8 mutation (T7869A) in the non-conserved region alone can have such a pronounced phenotypic effect. A reduction in complex III also cannot be explained since there were no mutations in the only mtDNA-encoded complex III gene, but it is possible that there are mutations in the nDNA-encoded complex III genes. Mutations in tRNA and rRNA genes may also be responsible for reduced protein syntheses and consequently reduced OXPHOS activities. It is unclear why complex I activity was not affected. Although the mutational effect of individual mtDNA mutation observed cannot be clearly identified, this study establishes a correlation between mtDNA mutation and cell energy production and growth.

The main purpose of this research is to incorporate additional higher-level semantics into the existing data replication strategies in such a way that their flexibility and performance can be improved in favour of both data providers and consumers. The resulting approach from this research is referred to as the selective data replication system. With this system, the data that has been updated by a data provider is captured and batched into messages known as update notifications. Once update notifications are received by data consumers, they are used to evaluate so-called update policies, which are specified by data consumers containing details on when data replications need to occur and what data needs to be updated during the replications.