Centralized systems are easier to build and maintain as compared to completely distributed systems. However, distributed systems have the potential to be responsive and robust relative to centralized systems. This thesis proposes an architecture and concurrency algorithm for collaborative editing that lies between these extremes and preserves the advantages of both approaches while minimizing their shortcomings The Jupiter collaboration system at Xerox PARC uses a 2-party synchronization protocol for maintaining consistency between two users performing unconstrained edits to the document simultaneously. The primary goal of our work has been to extend this 2-party synchronization protocol to an n-way synchronization algorithm. NetEdit is a prototype collaborative editor built to demonstrate this n-way protocol. It uses a replicated architecture with the processing and data distributed across all the clients and the server. Due to replication, the response time of the local edits performed by the users is quite close to a single user editor. The clients do not need to be aware of other clients in the system since each of them synchronizes with their counterpart at the server. All communication regarding editing operations takes place through this server. As a result this system is quite scalable (linear growth) relative to distributed systems (quadratic growth) in terms of number of communication paths required as the number of clients grow. I discuss the details of this extension and illustrate it through an editing scenario. NetEdit uses groupware widgets (telepointers, and radarview) to distribute awareness information between participants. It supports completely unconstrained editing and allows late joining into a session. It does not assume any structure in terms of roles of participants or protocol for collaboration and thus allow users to form whatever protocol suits them. The results and conclusions derived from a preliminary usability study of NetEdit, discuss its efficacy. They also investigate the role of communication and its use in a groupware setting. / Master of Science

No / Shape memory polymers with surface micropatterns have seen rising demand for high value applications such as adjustable adherence surfaces, dynamic micro-geometries for cell culture studies and switchable information carriers. Recently, microinjection molding has emerged as an efficient way to manufacture devices which contain surface micro-features using a wide range of polymers with high accuracy. In this study, shape memory polyurethane-carbon nanotube composites were prepared by twin-screw melt extrusion and subsequently processed using microinjection molding to obtain components with surface micropatterns. Then an electro-activated surface micropattern tuning system was developed which could recover the original micropatterned surface of the components after a thermal deformation by applying a current which heats the component using resistive heating. In order to optimize the technique, three key areas were investigated in this work: conductivity of the microinjection molded microparts, the retention of shape memory micropatterns on the surface of microparts during annealing treatment, and the macroscopic area shrinkage of microparts after thermal treatment. It has been found that the electrical conductivity of microinjection molded parts is relatively low due to the high shear rates prevalent in the process. An annealing treatment improves the electrical conductivity by several orders of magnitude, but can be detrimental to the dimensional stability of the micropatterns, which depends significantly on the micro-injection molding parameters, especially the mold temperature. Increasing the mold temperature, melt temperature, injection speed and injection pressure result in better retention of the micropattern and improved dimension stability during annealing treatment. This work demonstrates the potential of electro-activated surface micropattern control for microinjection molded electrically conductive shape memory polymer composites, which could be a promising technology for a range of application areas including electro-adjustable adherence, information storage, and anti-counterfeiting technology.

Successful replication of the genome during each cell cycle requires that every replication fork merge with its opposing fork. However, lesions in the template DNA or protein-DNA barriers often impede replication forks and threaten the timely completion of genome duplication. If a fork encounters a replication fork barrier (RFB), it can be subject to a variety of fates. In some cases the replisome is maintained in a manner such that it can resume DNA synthesis when the barrier is removed. Alternatively the stalled fork is simply held in a competent state to merge with the opposing fork when it arrives. However, fork stalling can also precipitate dissociation of the replisome (fork collapse) or even fork breakage. If this happens the recombination machinery can intervene to restore DNA integrity and restart replication, albeit with a risk of causing deleterious genetic change if ectopic homologous sequences are recombined. I have exploited a site-specific RFB in fission yeast termed RTS1 to investigate the consequences of perturbing a single replication fork. RTS1 is a polar RFB (i.e. it blocks fork progression in a unidirectional fashion), enabling replication to be completed by the opposing fork. Despite this, fork blockage at RTS1 triggers a strong recombinational response that is able to restart DNA synthesis, which at least initially is highly error prone. Here, I present my work in establishing a live cell imaging approach to visualizing the recombinational response at the RTS1 RFB, demonstrating that the majority of cells initiate recombination-dependent replication (RDR). RDR begins within a few minutes of fork blockage and is only curtailed by the arrival of the opposing fork. It depends on the Rad52 protein, which remains associated with the restarted fork and whose presence correlates with its infidelity. I also illustrate the significance of various genetic factors, including Rad51, the Rad51 mediators, Fml1 helicase, Rad54 translocase, Pfh1 sweepase, and Cds1 checkpoint kinase, in modulating Rad52 localization and block-induced recombination at the RTS1 RFB.

Il a été montré récemment que l'instabilité génétique joue un rôle central dans les étapes précoces de la tumorigenèse. Celle-ci provoquerait une activation chronique des voies ATR/CHK1 et ATM/CHK2 dans les cellules précancéreuses, entrainant l'apoptose ou la sénescence des cellules concernées. Les mécanismes de checkpoint constituant une barrière contre la progression tumorale, toute mutation affectant ce checkpoint serait ainsi sélectionnée très tôt dans le processu s de tumorigenèse et faciliterait ensuite la progression tumorale. Ce modèle met en évidence le rôle central de l'instabilité génomique et du checkpoint dans la progression tumorale.Au cours de ma thèse, je me suis intéressé au complexe Claspine / Timeless / Tipin, initialement identifié comme médiateur de la voie ATR/CHK1 et qui est donc considéré comme ayant une fonction suppresseur de tumeur. Cependant, Claspine présente aussi des propriétés oncogéniques, puisque qu'elle est surexprimée dans de nombreuses lignées tumorales et cette surexpression est importante pour la prolifération cellulaire. Nous nous sommes donc demandé comment cette protéine pouvait être à la fois un oncogène et un suppresseur de tumeur. Chez la levure, l'homologue de Claspine est impliqué dans le maintien de la stabilité des fourches de réplication, indépendamment de sa fonction dans le checkpoint. Nous proposons que dans les cellules cancéreuses cette surexpression permette une meilleure stabilité des fourches, ce qui serait très important pour répliquer efficacement un génome soumis à un stress réplicatif constant. Au cours de ma thèse, nous avons construit et caractérisé un modèle de cellulescancéreuses HCT116 dans lesquelles nous avons diminué le niveau de Claspine ou deTimeless grâce à des shRNA, sans que cela n'affecte l'efficacité du checkpoint de réplication. Nous avons pu observer dans ces cellules un ralentissement de la progression des fourches de réplication et l'apparition d'une instabilité génétique. Il semblerait que spécifiquement dans le cas de Timeless, le ralentissement de la fourche de réplication et l'instabilité génomique se manifeste surtout dans les régions du génome répliquées tardivement. / The correct execution of the replication program is essential for the maintenance of genome integrity during S phase. Indeed, replication fork progression is frequently challenged by DNA lesions and by a variety of natural pause sites. Arrested forks are unstable structures, which represent a major threat for the genome integrity if they are not promptly stabilized and restarted. In response to replicative stress, cells activated the replication checkpoint to prevent collapse of stalled forks and promote fork recovery. Recent evidence indicates that spontaneous replication stress occurs in precancerous lesions and promotes the development of cancer. Identifying the origin of this replication stress would represent a better understanding of the early stages of tumorigenesis. We study the function of Claspin, a mediator of the replication checkpoint, which plays a key role in the response to replication stress. It is therefore acting as a tumor suppressor, preventing genomic instability during DNA replication. Intriguingly, recent evidence indicates that Claspin is overexpressed in different cancers and can also acts as an oncoprotein. Studies on Mrc1p, the yeast homologue of Claspin, have shown that Mrc1 is permanently associated to forks, where it forms a complex with two partners called Tof1p and Csm3p (homologues of human Tim and Tipin respectively). We have shown by DNA combing that the replicative function of Mrc1p, is critical for viability in the presence of a replication stress. In l ight of our results in yeast, we propose that the Claspin/Tim/Tipin complex may also play a direct role for the replication of human cells. If confirmed, this replication function may account for the role of this complex in the cancer process and tumor resistance to genotoxic agents commonly used in anticancer therapy.

L'ARN d'interférence (ARNi) est un mécanisme de régulation du gène qui permet un ciblage spécifique d'ARNmessager (ARNm) par reconnaissance de séquence. Les effecteurs de l'ARNi sont de petites molécules d'ARN non codants (siARN, microARN et piARN). Evoluant dans le contexte de l'ARNi, les virus de différentes familles ont adopté des stratégies afin utiliser l'ARNipour leur propre bénéfice. Le principal objectif de ma thèse a été d'étudier la fonction de l' ARNmiRTAR, un miARN viral dérivé de l'extrémité 3 'de l'ARN VIH-1 TAR dans la réplication du VIH-1. Nous avons constaté que miRTAR réguleà la fois l'activité basale et la transactivation induite par Tat du promoteur du VIH-1. L'effet de miRTAR ne nécessite pas de sa liaison à l'ARN TAR. miRTAR agit en augmentant l'activité de NF-kB, un facteur important pour la transcription du promoteur du VIH-1. En effet, la mutation des sites NF-kB, mais pas des sites Sp1 dans le LTR, abroge l'amélioration miRTARmédiée par la transcription. De plus, l'inhibition de l'expression de NF-kBpardes siARNspécifiquesdes les sous-unités p50 et p65, entraîne une perte d'activité dumiRTAR. Bien que nous n'avons pas pu identifier le(s) gène(s) cellulaire(s) ciblé par miRTAR, sa surexpression conduit à l'activation de la voie NF-kB. Mutation de l'extrémité 3 'du VIH-1 dans les résultats TAR réduction spectaculaire de la réplication virale Tat sans affecter médiée par la transcription, en raison de la perte de production de miRTARsauvage. Enfin, la surexpression de miRTARrestaure la réplication de ce virus contenant une mutation au niveau de la séquence TAR. En conclusion, nos résultats démontrent clairement que lemiRTARencodé par le VIH-1 joue un rôle clé dans la réplication du virus. Sur la base de ces résultats, nous proposons le modèle suivant pour la fonction de miRTAR. La transcription du LTR du VIH-1 conduit à la production de courts ARN dénommés TAR contenant une structure en épingle à cheveux. TAR est « processé » pour générer le miARN miRTAR. L'ARN miRTAR est ensuite chargé dans le complexe RISC et régule l'expression de plusieurs gènes cellulaires impliqués dans la régulation négative de NF-kB. miRTARmédiée par l'activation de NF-kB résultats dans la régulation de gènes viraux et par conséquent augmente la production de virus. L'ensemble de nos résultats montrent que le VIH-1 utilise la voie de l'ARNiinterférence pour optimiser l'environnement intracellulaire requis pour une réplication optimale. / RNA interference (RNAi) is a gene regulatory mechanism that offers a sequence specific targeting of mRNA. Evolving in the context of RNAi, viruses of different families adopted strategies to use RNAi for their benefit. The main objective of my thesis was to understand the function of miRTAR, a viral miRNA derived from 3' end of the HIV-1 TAR RNA, in HIV-1 replication. We found that miRTAR regulates both basal and Tat-mediated transactivation of HIV-1 promoter. The effect of miRTAR does not require its binding to TAR RNA. miRTAR acts by inducing NF-κB transcription factor important for the LTR activity. Indeed, mutation of NF-κB sites but not Sp1 sites within the LTR abrogate miRTAR-mediated enhancement of transcription from the LTR. Additionally, Inhibition of NF-κB using specific siRNA directed against p50 and p65 subunits results in loss of miRTAR activity. Although, we were unable to identify the cellular gene(s) targeted by miRTAR, its overexpression lead to the activation of NF-κB pathway. Mutation of the 3' end of HIV-1 TAR results in dramatic reduction of viral replication without affecting Tat-mediated transcription. Importantly, overexpression of miRTAR rescued the replication of miRTAR HIV-1 mutant virus.In conclusion, our results strongly demonstrate that the HIV-1 encoded miRTAR plays a key role in virus replication. On the basis of these findings, we propose the following model for the function of miRTAR. Transcription of the HIV-1 LTR leads to production of short, TAR containing, RNA hairpin sequences. TAR is processed to generate miRNA, miRTAR. miRTAR is then loaded into the RISC complex and regulates the expression of several cellular genes involved in the negative regulation of NF-κB. miRTAR-mediated activation of NF-κB results in up regulation of viral genes and consequently enhances virus production. Taken together, our results demonstrate that HIV-1 uses RNAi pathway to optimize the intracellular environment for optimal replication.

To maintain genomic integrity, all cells must accurately duplicate their genetic material in order to provide intact and complete copies to each daughter cell following cell division. Successful inheritance of chromosomal information without changing even a single nucleotide requires accurate and robust DNA replication. This requires that cells tightly control replication initiation from the origin(s), processive elongation of the replisome, and the completion of DNA replication by resolving convergent replication forks ensuring that each sequence is duplicated without alteration. Unlike initiation and elongation, the process by which replication forks converge and are resolved into two discrete, inheritable DNA molecules is not well understood. This process must be remarkably efficient, occurring thousands of times per cell division in human cells, and is likely to be a fundamental step in regulating genome stability in all cells. In this dissertation I address how DNA replication completes in the model system Escherichia coli. To achieve this, I examined candidate mutants for impairments in the completion of DNA replication. By evaluating growth, viability, chromosomal copy number, and plasmid stability I identified a requirement for the proteins RecBCD, ExoI, and SbcCD in the completion reaction. SbcCD and ExoI act before RecBCD in the completion reaction and process the DNA intermediates arising as replication forks converge. These enzymes act in the completion reaction without recombination or RecA, but in the absence of the normal process recombination is required to complete DNA replication via an aberrant pathway that results in genomic instability.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 169-183).

No description available.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xiii, 196 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical reference.

La sénescence, considérée comme étant un arrêt irréversible du cycle cellulaire, se caractérise par des changements drastiques de l'expression génique et de l'organisation de la structure de la chromatine. En effet, il se forme des foyers denses d'hétérochromatine au sein du noyau (SAHF) et ces modifications s'accompagnent d'un déclin progressif de la capacité à dupliquer le génome. Au cours de ma thèse, j'ai voulu savoir si ces modifications de la chromatine induite par les SAHF pouvaient influer sur le programme de réplication et changer la distribution des origines de réplication sur le génome au cours du processus d'entrée en sénescence réplicative des cellules. Nous avons donc, dans un premier temps, comparé par peignage moléculaire de l'ADN réplicatif la distribution des origines de réplication de cellules primaires prolifératives et sénescentes. Nous avons également cartographié l'ensemble de leurs origines de réplication sur la totalité du génome en purifiant les brins naissants aux origines de réplication que nous avons couplé à une analyse de séquençage à haut débit.Les protéines HMGA1 et HMGA2 étant des éléments précurseurs essentiels à la mise en place des SAHF, nous avons créé des lignées cellulaires qui, en sur-exprimant de façon inductibles ces protéines, induit une sénescence prématurée. Nous avons réalisé le même type d'analyses sur ces cellules afin de mettre en évidence le rôle de ces protéines dans les modifications du programme de réplication que nous avons observé au cours de l'entrée en sénescence de ces différents types cellulaires. / Senescence, considered as an irreversible cell cycle arrest, is characterized by dramatic changes in genes expression and chromatin organisation forming dense heterochromatic foci (SAHF). These changes are concomitant to a progressive decline of the capactity to replicate the genome. My PhD topic was to investigate whether the chromatin changes induced by SAHF formation could influence the replication program and modify the origin distribution along the genome at replicative senescence. We first compared the origin distribution of proliferative and pre-senescent primary fibroblasts by DNA molecular combing. Then, we mapped the origins positions in whole human genome by using the nascent strand purification assay coupled to deep sequencing.As HMGA1 and HMGA2 proteins are essential to induce SAHF formation, we designed inducible cell lines wich overexpress these proteins, triggering premature senescence. We made the same type of experiments in these cell lines in order to investigate the implication of these proteins on the changes of the replication program we observed during senescence.