<p>Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c.</p><p>The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are investigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and thereafter lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regulated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro-tein has been shown to repress the Pc promoter and thereby making tran-scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes.</p><p>In this work the importance of the native size for biologically active integrase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein-protein interfaces. In addition it is shown that P2 Cox and the P2 relative Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed.</p><p>The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.</p>

Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c. The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are investigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and thereafter lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regulated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro-tein has been shown to repress the Pc promoter and thereby making tran-scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes. In this work the importance of the native size for biologically active integrase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein-protein interfaces. In addition it is shown that P2 Cox and the P2 relative Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed. The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.

P2-like bacteriophages integrate their genome into the E. coli host cell by a site-specific recombination event upon lysogenization. The integrative recombination occurs between a specific sequence in the phage genome, attP, and a specific sequence in the host genome, attB, generating the host-phage junctions attL and attR. The integration is mediated by the phage enzyme integrase (Int) and the host factor IHF. The excisive recombination takes place between attL and attR, and is mediated by Int, IHF and phage encoded protein Cox. For safe integration of foreign genes into eukaryotic chromosome a recombinases is necessary which can perform the integration site-specifically. P2-like phage integrases have the potential to become tools for safe gene therapy. Their target is simple but specific, and once integration has occurred it is very stable in the absence of the Cox protein. The site-specific recombination mechanism has to be understood at the molecular level. Therefore, I have initiated the characterization of the site-specific recombination system of the P2-like phage ΦD145. In this work, Int and IHF are shown to bind to the different attachment sites cooperatively. One of two possible inverted repeats in attP is shown to be the Int core recognition site. The attP core of this phage has high identity with a site on human chromosome, denoted as ΨattB. In this study we have shown that in in vivo recombination ΦD145 Int can accept ΨattB in both bacteria and in eukaryotic cells. Also shown that Int consists of an intrinsic nuclear localization signal. A study also reveled that ΦD145 Int activity was affected by the Tyr-phosphorylation. Attempts have been made to change the specificity of the other P2-like phage P2 and WΦ integrases and also structural and functional analysis was done. A study on comparative analysis of Cox proteins and Cox binding sites gave us the basic information about the recombination mechanism. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

The use of site-specific data can enhance management decisions in the field. Three different uses of site-specific data were evaluated and their outcomes are promising. Historical yield data from yield monitors and height data from the HMAP (plant height mapping) system were used to select representative areas within the field, and areas of average conditions were used as sampling sites for COTMAN, a cotton management expert system. This proved to be effective, with predicted cutout dates and date of peak nodal development similar to the standard COTMAN approach. The HMAP system was combined with historical height data for variable rate application of mepiquat chloride, based on the plant growth rate. The system performance was evaluated, but weather conditions in 2004 did not allow a true evaluation of varying mepiquat chloride. A series of multi-spectral images were normalized utilizing the soil line transformation (SLT) technique and normalized difference vegetation index (NDVI) was calculated from the transformed images, from the raw image and for the true reflectance images. The SLT technique was effective in tracking the change in true reflectance NDVI in some images, but not all. Changes to the soil line extraction program are suggested so that it more effectively determines soil lines.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 82-99). Also available in electronic version. Access restricted to campus users.

Earthquake-induced landslides are a significant seismic hazard that can generate large economic losses. Predicting earthquake-induced landslides often involves an assessment of the expected sliding displacement induced by the ground shaking. A deterministic approach is commonly used for this purpose. This approach predicts sliding displacements using the expected ground shaking and the best-estimate slope properties (i.e., soil shear strengths, ground water conditions and thicknesses of sliding blocks), and does not consider the aleatory variability in predictions of ground shaking or sliding displacements or the epistemic uncertainties in the slope properties. In this dissertation, a probabilistic framework for predicting the sliding displacement of flexible sliding masses during earthquakes is developed. This framework computes a displacement hazard curve using: (1) a ground motion hazard curve from a probabilistic seismic hazard analysis, (2) a model for predicting the dynamic response of the sliding mass, (3) a model for predicting the sliding response of the sliding mass, and (4) a logic tree that incorporates the uncertainties in the various input parameters. The developed probabilistic framework for flexible sliding masses is applied to a slope at a site in California. The results of this analysis show that the displacements predicted by the probabilistic approach are larger than the deterministic approach due to the influence of the uncertainties in the slope properties. Reducing these uncertainties can reduce the predicted displacements. Regional maps of seismic landslide potential are used in land-use planning and to identify zones that require detailed, site-specific studies. Current seismic landslide hazard mapping efforts typically utilize deterministic approaches to estimate rigid sliding block displacements and identify potential slope failures. A probabilistic framework that uses displacement hazard curves and logic-tree analysis is developed for regional seismic landslide mapping efforts. A computationally efficient approach is developed that allows the logic-tree approach to be applied for regional analysis. Anchorage, Alaska is used as a study area to apply the developed approach. With aleatory variability and epistemic uncertainties considered, the probabilistic map shows that the area of high/very high hazard of seismic landslides increases by a factor of 3 compared with a deterministic map. / text

Many different approaches exist for engineering bacterial genomes. The most common current methods include transposons for random mutagenesis, recombineering for specific modifications in Escherichia coli, and targetrons for targeted knock-outs. Site-specific recombinases, which can catalyze a variety of large modifications at high efficiency, have been relatively underutilized in bacteria. Employing these technologies in combination could significantly expand and empower the toolkit available for modifying bacteria. Targetrons can be adapted to carry functional genetic elements to defined genomic loci. For instance, we re-engineered targetrons to deliver lox sites, the recognition target of the site-specific recombinase, Cre. We used this system on the E. coli genome to delete over 100 kilobases, invert over 1 megabase, insert a 12-kilobase polyketide-synthase operon, and translocate a 100 kilobase section to another site over 1 megabase away. We further used it to delete a 15-kilobase pathogenicity island from Staphylococcus aureus, catalyze an inversion of over 1 megabase in Bacillus subtilis, and simultaneously deliver nine lox sites to the genome of Shewanella oneidensis. This represents a powerful, versatile, and broad-host-range solution for bacterial genome engineering. We also placed lox sites on mariner transposons, which we leveraged to create libraries of millions of strains harboring rearranged genomes. The resulting data represents the most thorough search of the space of potential genomic rearrangements to date. While simple insertions were often most adaptive, the most successful modification found was an inversion that significantly improved fitness in minimal media. This approach could be pushed further to examine swapping or cutting and pasting regions of the genome, as well. As potential applications, we present work towards implementing and optimizing extracellular electron transfer in E. coli, as well as mathematical models of bacteria engineered to adhere to the principles of the economic concept of comparative advantage, which indicate that the approach is feasible, and furthermore indicate that economic cooperation is favored under more adverse conditions. Extracellular electron transfer has applications in bioenergy and biomechanical interfaces, while synthetic microbial economics has applications in designing consortia-based industrial bioprocesses. The genomic engineering methods presented above could be used to implement and optimize these systems. / text

No description available.

Thesis (M.S.)--Duquesne University, 2007. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 76-80) and index.

Honors Project--Smith College, Northampton, Mass., 2009. / Includes bibliographical references (p. 61).