Pre-replicative complex (pre-RC) components the origin recognition complex (ORC), Cdc6, and Cdt1, play key roles in the recruitment, and loading of the replicative helicase, the minichromosome maintenance complex (Mcm2-7), onto DNA to license origins for replication. Until recently, the prevailing model for pre-RC assembly predicted that once MCMs are loaded at origins, ORC, Cdc6, and Cdt1 are dispensible for replication. Contrary to this model, previous work has shown that Orc6 is required following origin licensing, for the continued association of the MCM complex in late G1 phase. In this study, a similar role in pre-RC maintenance has been demonstrated for Cdc6, and Cdt1. Chromatin immunoprecipitation (ChIP) analysis has shown that late G1 phase depletion of either Cdc6, or Cdt1 leads to the destabilization of MCMs from origins, although this destabilization is more pronounced for Cdc6 depletion than for Cdt1. Furthermore, the resynthesis of Cdc6 following its depletion, allows for the reassembly of pre-RCs in late G1 phase, and restores competence for DNA replication. In this study, a potential role for Orc6 in mitosis/cytokinesis in budding yeast has also been characterized, as research with both Drosophila and human cell lines has pointed to a role for Orc6 in these processes. Deleting HOF1 and CYK3 (two proteins involved in cytokinesis in budding yeast) leads to a synthetic lethal phenotype, suggesting that the resulting gene products function in redundant cytokinetic pathways. Indeed, Hof1 has been shown to be primarily involved in actin ring contraction, while Cyk3 functions in septum formation, both pathways of which are important for budding yeast cytokinesis. Interestingly, previous work has identified an Orc6-Hof1 interaction in budding yeast. In this study, it has been demonstrated that following Orc6 depletion in a GAL1-ORC6/Δcyk3 strain, fluorescence activated cell sorting (FACS) analysis is consistent with a stronger cytokinetic defect phenotype than observed for Δcyk3 cells. Preliminary cell counts indicate that following Orc6 depletion, a higher percentage of GAL1-ORC6/Δcyk3 cells display misshapen mother bud necks than in an isogenic Δcyk3 strain. Cell synchronization experiments have demonstrated that Orc6 depletion during a G2/M phase arrest, leads to a block in cell cycle progression following release.

Pre-replicative complex (pre-RC) components the origin recognition complex (ORC), Cdc6, and Cdt1, play key roles in the recruitment, and loading of the replicative helicase, the minichromosome maintenance complex (Mcm2-7), onto DNA to license origins for replication. Until recently, the prevailing model for pre-RC assembly predicted that once MCMs are loaded at origins, ORC, Cdc6, and Cdt1 are dispensible for replication. Contrary to this model, previous work has shown that Orc6 is required following origin licensing, for the continued association of the MCM complex in late G1 phase. In this study, a similar role in pre-RC maintenance has been demonstrated for Cdc6, and Cdt1. Chromatin immunoprecipitation (ChIP) analysis has shown that late G1 phase depletion of either Cdc6, or Cdt1 leads to the destabilization of MCMs from origins, although this destabilization is more pronounced for Cdc6 depletion than for Cdt1. Furthermore, the resynthesis of Cdc6 following its depletion, allows for the reassembly of pre-RCs in late G1 phase, and restores competence for DNA replication. In this study, a potential role for Orc6 in mitosis/cytokinesis in budding yeast has also been characterized, as research with both Drosophila and human cell lines has pointed to a role for Orc6 in these processes. Deleting HOF1 and CYK3 (two proteins involved in cytokinesis in budding yeast) leads to a synthetic lethal phenotype, suggesting that the resulting gene products function in redundant cytokinetic pathways. Indeed, Hof1 has been shown to be primarily involved in actin ring contraction, while Cyk3 functions in septum formation, both pathways of which are important for budding yeast cytokinesis. Interestingly, previous work has identified an Orc6-Hof1 interaction in budding yeast. In this study, it has been demonstrated that following Orc6 depletion in a GAL1-ORC6/Δcyk3 strain, fluorescence activated cell sorting (FACS) analysis is consistent with a stronger cytokinetic defect phenotype than observed for Δcyk3 cells. Preliminary cell counts indicate that following Orc6 depletion, a higher percentage of GAL1-ORC6/Δcyk3 cells display misshapen mother bud necks than in an isogenic Δcyk3 strain. Cell synchronization experiments have demonstrated that Orc6 depletion during a G2/M phase arrest, leads to a block in cell cycle progression following release.

The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.

MicroRNAs (miRNAs) are non-coding small RNAs that regulate eukaryotic gene activity at the post-transcriptional level by a process termed miRNA gene suppression. MicroRNA-122 (miR-122) is predominantly expressed in human liver cells and recent studies indicated that miR-122 promotes Hepatitis C Virus (HCV) replication and translation through physical interaction with two tandem binding sites located in the 5 untranslated region (5UTR) of the HCV genome (Jopling, et al., 2006; Jopling, et al., 2008). It has been reported that host genes that are also implicated in the miRNA gene suppression pathway are key regulators of HCV replication (Randall, et al., 2007). Two proteins, Dicer, a key RNaseIII enzyme, and its binding partner TRBP are essential proteins for miRNA activity. They are part of a protein complex called the RNA induced silencing complex (RISC) which also includes Argonaute proteins, and function in miRNA biogenesis loading the miRNA into RISC. As such, they are intriguing targets to study host-viral interplay during HCV replication.<p> In our study, we designed siRNAs to knock down Dicer and TRBP and then observed the effects of gene knockdown on full length J6/JFH-1-RLuc HCV (genotype 2a chimeric genome) replication and translation. The results showed that knocking down Dicer and TRBP reduced wild type (wt) J6/JFH-1-RLuc replication but had almost no effects on HCV translation in human liver cells. However, since knocking down Dicer and TRBP did not significantly alter miR-122 levels in the cell, it appears that the role of Dicer and TRBP was not solely the biogenesis of miR-122. This was confirmed by an experiment in which we observed that knocking down Dicer and TRBP also attenuated replication of a mutant virus in which replication is dependent on a exogenously supplied miRNA instead of endogenous miR-122.<p> Taken together, the results supported the hypotheses that Dicer and TRBP facilitate HCV infection mainly through HCV replication but not translation. The effects of Dicer and TRBP on HCV replication are not solely due to miR-122 biogenesis, and may be due to RISC loading functions in steps of miRNA gene suppression.<p> This study has set some essential groundwork for investigating potential roles of host factors in the RNAi machinery modulating HCV replication/translation and exploring novel antiviral targets.

<p>RecQ4, a member of the conserved RecQ family of helicases, is involved in replication and associated with several clinical syndromes. Although biologically important, the biochemistry of RecQ4 has remained elusive. We have expressed and purified Drosophila melanogaster RecQ4 from a baculovirus expression system. Biochemical characterization of the helicase, ATP hydrolysis, annealing, and binding activities of the enzyme has been performed, using native and non-native gel electrophoresis and thin layer chromatography, among other techniques. These reveal that RecQ4 is a 3' to 5' helicase that is stimulated by the presence of single-stranded DNA 3' of the duplex DNA region to be unwound. The enzyme is also capable of annealing complementary DNA strands, though this is inhibited by AMPPNP, a non-hydrolyzable analog of ATP. RecQ4 also forms a stable complex with single-stranded DNA in the presence of AMPPNP. We argue that the helicase activity of RecQ4 is important to the process of DNA replication. This leads to the conclusion that two helicases, RecQ4 and the Mcm2-7 complex, are involved in replication. The manner of their simultaneous involvement is not intuitive, and so models by which the two enzymes may cooperate are discussed.</p> / Dissertation

As broadband connections to the Internet become more common, new information sharing applications that provide rich services to distributed users will emerge. Furthermore, as computing devices become pervasive and better connected, the scalability requirements for Internet-based services are also increasing. Distributed object middleware has been widely used to develop such applications since it made it easier to rapidly develop distributed applications for heterogeneous computing and communication systems. As the application's scale increases, however, the client/server architecture limits the performance due to the bottleneck at the centralized servers. The recent development in peer-to-peer technologies creates a new opportunity for addressing scalability and performance problems for services that are used by many nodes. In a peer-to-peer system, peer nodes can contribute a fraction of their resources to the system, enabling more flexible and extended sharing between the entities in the system. When peer nodes are required to contribute their resources by replicating a service for self and others, however, several new challenges arise. Our thesis is that non-dedicated resources in a distributed system can be utilized to replicate shared objects dynamically so that the quality and scalability of a distributed service can be achieved with lower cost by replicating the objects at right places and updates to those shared objects can be disseminated efficiently and quickly. The following are the contributions of our work that has been done to validate the thesis. 1. A new fair and self-managing replication algorithm that allows distributed non-dedicated resources to be used to improve service performance with lower cost. 2. A multicast grouping algorithm that is used to disseminate updates to the shared objects among a large set of heterogeneous peer nodes to keep consistent view for all peer nodes. It groups nodes with similar interests into same group and multicasts all the required data to the group so that the unwanted data received by each node can be minimized. 3. An overlay construction algorithm that aims at reducing both network latency and total network traffic when delivering data through the built overlay network. 4. An implementation of a distributed object framework, GT-RMI, that allows peer nodes to invoke dynamically replicated objects transparently. The framework can be configured for a particular peer node through a policy file.

Because of the maturation of the VoIP technique, VoIP can not only satisfy the basic requirement of telecommunication but also provide multimedia communication services. As a result, it is very attractive in recent years. Through VoIP, the cost of communication can be saved. It can be very competitive. In addition, VoIP can be combined with PSTN (Public Switched Telephone Network). This helps traditional PSTN users to be able to use traditional telephones to make VoIP calls.Besides, VoIP can also extend other services. It can achieve diversification of services, comfortable using and reducing the cost requirement. Moreover, with the increasing of the VoIP population, the traditional method using single server is unable to afford so much loading. It is possible that the large load makes the service stop anytime. This makes the usability and the reliability decrease. To make the VoIP service work anytime, we implement a method in both client side and server side to achieve the goal of continuous providing of the service. From this implementation, the service of VoIP can be provided anytime. The users, however, have no need to be aware of the different operation style in VoIP.

Certain scientific application domains, such as High-Energy Physics or Earth Observation, are expected to produce several Petabytes (220 Gigabyes) of data that is analyzed and evaluated by the scientists all over the world. In the context of data grid technology, data replication is mostly used to reduce access latency and bandwidth consumption. In this thesis, we adopt the typical Data Grid architecture, three kinds of nodes: server, cache, and client nodes. A server node represents a main storage site. A client node represents a site where data access requests are generated, and a cache node represents an intermediate storage site. However, the access latency of the hierarchical storage system may be of the order of seconds up to hours. The static replication strategy can be used to improve such long delay; however, it cannot adapt to changes of users¡¦ behaviors. Therefore, the dynamic data replication strategy is used in Data Grids. Three fundamental design issues in a dynamic replication strategy are: (1) when to create the replicas, (2) which files to be replicated, and (3) where the replicas to be placed. Two of well known replication strategies are Fast-Spread and Cascading, which can work well for different kinds of access patterns individually. For example, the Fast-Spread strategy works well for random access patterns, and the Cascading strategy works well for the patterns with the properties of localities. However, for so many different access patterns, if we use a strategy for one kind of access patterns and another strategy for another kind of access patterns, the system may become too complex. Therefore, in this thesis, we propose one strategy which can work for any kind of access patterns. We propose a replication approach, a Different Threshold (DT) approach to data replication in Data Grids, which can be dynamically adapted to several kinds of access patterns and provide even better performance than Cascading and Fast-Spread strategies. In our approach, there are different thresholds for different layers. Based on this approach, first, we propose a static DT strategy in which the threshold at each layer is fixed. So, by carefully adjusting the difference between the thresholds Ti, where i is the i-th layer of the tree structure, we can even provide the better performance than the above two well-known strategies. Moreover, among large amount of different data files, there may exist some hot data files. Those files which have been mostly requested are hot data files. To reduce the number of requests for the hot files, next, we propose the dynamic DT strategy. In the dynamic DT strategy, each data file even has its own threshold. We let data replication of hot files occur earlier than others by decreasing the thresholds of hot files earlier than the normal ones. From our simulation results, we show that the response time in our static DT strategy is less than that in the Cascading and the Fast-Spread strategies. Moreover, we can show that the performance of the dynamic DT strategy is better than that of the static DT strategy.

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Initiation of DNA replication. Department of Biochemistry and Molecular Genetics. Includes bibliographical references (142-171). Also available online through Digital Dissertations.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 139-153). Also available in electronic version. Access restricted to campus users.