No description available.

Free group outer automorphisms were shown by Bestvina and Randell to have fixed subgroups whose rank is bounded in terms of the rank of the underlying group. We consider the case where this upper bound is achieved and obtain combinatorial results about such outer automorphisms thus extending the work of Collins and Turner. We go on to show that such automorphisms can be represented by certain graph of group isomorphisms called Dehn Twists and also solve the conjuagacy problem in a restricted case, thus reproducing the work of Cohen and Lustig, but with different methods. We rely heavily on the relative train tracks of Bestvina and Randell and in fact go on to use an analogue of these for free product automorphisms developed by Collins and Turner. We prove an index theorem for such automorphisms which counts not only the group elements which are fixed but also the points which are fixed at infinity - the infinite reduced words. Two applications of this theorem are considered, first to irreducible free group automorphisms and then to the action of an automorphism on the boundary of a hyperbolic group. We reduce the problem of counting the number of points fixed on the. boundary to the case where the hyperbolic group is indecomposable and provide an easy application to virtually free groups.

Bacteroides fragilis is an important, anaerobic commensal of the human gastro-intestinal tract. As a Gram-negative bacterium, B. fragilis produces a large number of outer membrane vesicles (OMV), spherical globules consisting of outer membrane and periplasmic material, which have a range of potential functions and which are known to be able to deliver their cargo to host dendritic cells (DCs). One of the proteins believed to be packaged into the OMV of B. fragilis is BfUbb (encoded by the ubb gene) which shares 63% homology with human ubiquitin. Ubiquitin is a small, common, eukaryotic protein modifier, which is conjugated to target proteins via a series of activating, conjugating and ligating enzymes, and which has known roles in a wide range of eukaryotic cell processes. Due to key differences between the two proteins, BfUbb has the potential to act as a suicide substrate mimic of ubiquitin. BfUbb was therefore assayed for its ability to interact with ubiquitin E2 conjugating enzymes of the ubiquitylation cascade in vitro, and was found to covalently bind the majority of available enzymes in a DTT-sensitive manner. BfUbb showed a preference for three specific E2 enzymes, all of which are involved in the degradation of mitotic check point proteins, suggesting a role for BfUbb in the inhibition of cell cycle progression and, consequently, tumorigenesis. No binding partners of BfUbb were identified outside of the ubiquitylation cascade, however BfUbb was found to form spontaneous multimers in vitro, the biological function of which is unknown. This study also describes the construction of two sets of plasmids. The first set will allow the expression of untagged and fluorescently tagged forms of BfUbb for purification and use in biochemical assays. The second set will allow the expression of his-tagged and fluorescently tagged forms of BfUbb in mammalian cells, so that the effects of BfUbb on the host epithelial cells may be studied. The proteome of the OMV of B. fragilis was solved using LTQ-Orbitrap mass spectrometry. The identified proteins indicated several putative roles for B. fragilis OMV, including nutrient acquisition and protease inhibition. The suitability of techniques used during the isolation and proteomic analysis of OMV in different studies is discussed. BfUbb-carrying B. fragilis OMV were able to inhibit growth of Salmonella enterica Typhimurium, thus indicating a role for BfUbb in the inhibition of competing, pathogenic bacteria in the gastro-intestinal tract. The conclusions of this study are that the putative roles of both BfUbb and the OMV of B. fragilis may promote both survival of the bacterium and the gastro-intestinal health of the host.

Architectural designers often need to strike an uneasy balance between idealism and reality. Under most circumstances, architects are restricted by clients, budgets, and available technologies. However, divorced from traditional constraints, visionary concepts of new dwellings, new cities, and new “worlds” will spark greater forms of innovation and drive creativity for future generations. The exploration of new spatial boundaries and conceptual environments for design will irrevocably alter the human experience while adapting new challenging roles for future architects. Architecture can be understood in part as the art of organizing spaces through the manipulation of materials and forms. Designed spaces are arranged to provide unique sensory reactions for their occupants while emotionally and physically orientating them on Earth. As a catalyst towards the awareness of one’s surroundings, architecture has always had to contend with the many limiting factors imposed by the forces on Earth. These include, but are not limited to, gravity and climate. On Earth, structurally sound construction is limited by the forces of gravity as it influences design capabilities by standardizing forms, functions, and structural elements of architectural spaces. New design challenges and opportunities arrive when we look to create structures outside of Earth’s boundaries. This thesis proposes a futuristic model of an efficient and unique passenger transport system that connects Earth-based hybrid air/space ports with an outer space orbital infrastructural hub. This modern intervention will allow for new outer space industries, such as transit, tourism, and hospitality, which will provide unique opportunities for the future of humanity. Additionally, the thesis studies the positive architectural and experiential potentials for the future living occupancy of outer space. In recognizing the financial and logistical limitations of current space constructions, such as the International Space Station, the thesis looks beyond the limitations of current technologies and towards designs that are driven by the fulfillment of human experiences in space. Life in space, the thesis envisions, will spark new human experiences and rituals while necessitating new forms and designs in architecture. Weightlessness and its related spatial disorientations, in addition to the many other unique conditions in this unfamiliar territory, will inspire a new conceptual language for architecture and human cultures. The thesis will demonstrate that spaces designed for extraterrestrial experiences can be innovatively dynamic as they respond to new cultures and activities that evolve as a reaction to extreme conditions. Introducing humans to the environs of orbital space will be the initial stage in a long-term phasing tactic to colonize and commercialize beyond the expanse of Earth, eventually extending humanity to the remote neighbouring planets of the universe.

Lipopolysaccharide (LPS) is the glycolipid that comprises the outer leaflet of the Gram-negative outer membrane (OM). Because it is essential in nearly all Gram-negative species, and because it is responsible for making these bacteria impervious to many types of antibiotics, LPS biogenesis has become an important area of research. While its biosynthesis at the cytoplasmic face of the inner membrane (IM) is well studied, the process by which it is removed from the IM, transported across the aqueous periplasmic compartment, and specifically inserted into the outer leaflet of the OM is only beginning to be understood. This transport process is mediated by the essential seven-protein LPS transport (Lpt) complex, LptA/B/C/D/E/F/G. The OM portion of the exporter, LptD/E, is a unique plug-and-barrel protein complex in which LptE, a lipoprotein, sits inside of LptD, a β-barrel integral membrane protein. LptD is of particular interest, as it is the target of an antibiotic in Pseudomonas aeruginosa. Part I of this thesis investigates how the cell forms the two non-consecutive disulfide bonds that connect LptD's C-terminal β-barrel to its N-terminal soluble domain. These disulfides, one of which is almost universally conserved among Gram-negatives, are essential for cell viability. Here, we show that an intermediate oxidation state with non-native disulfide bonds accumulates in the absence of LptE and in strains defective in either LptE or LptD. We then demonstrate that this observed intermediate is on-pathway and part of the native LptD oxidative folding pathway. Using a defective mutant of DsbA, the protein that introduces disulfide bonds into LptD, we are able to identify additional intermediates in the LptD oxidative folding pathway. We ultimately demonstrate that the disulfide rearrangement that activates the LptD/E complex occurs following an exceptionally slow β-barrel assembly step and is dependent on the presence of LptE. Part II describes work towards obtaining X-ray crystal structures of the LptD N-terminal domain and LptD/E complex. Expression construct and purification optimization enabled the production of stable LptD/E in quantities that make crystallography feasible. Numerous precipitants, detergents, and additives were screened, ultimately resulting in protein crystals that diffract to a resolution of 3.85 Å. / Chemistry and Chemical Biology

Architectural designers often need to strike an uneasy balance between idealism and reality. Under most circumstances, architects are restricted by clients, budgets, and available technologies. However, divorced from traditional constraints, visionary concepts of new dwellings, new cities, and new “worlds” will spark greater forms of innovation and drive creativity for future generations. The exploration of new spatial boundaries and conceptual environments for design will irrevocably alter the human experience while adapting new challenging roles for future architects. Architecture can be understood in part as the art of organizing spaces through the manipulation of materials and forms. Designed spaces are arranged to provide unique sensory reactions for their occupants while emotionally and physically orientating them on Earth. As a catalyst towards the awareness of one’s surroundings, architecture has always had to contend with the many limiting factors imposed by the forces on Earth. These include, but are not limited to, gravity and climate. On Earth, structurally sound construction is limited by the forces of gravity as it influences design capabilities by standardizing forms, functions, and structural elements of architectural spaces. New design challenges and opportunities arrive when we look to create structures outside of Earth’s boundaries. This thesis proposes a futuristic model of an efficient and unique passenger transport system that connects Earth-based hybrid air/space ports with an outer space orbital infrastructural hub. This modern intervention will allow for new outer space industries, such as transit, tourism, and hospitality, which will provide unique opportunities for the future of humanity. Additionally, the thesis studies the positive architectural and experiential potentials for the future living occupancy of outer space. In recognizing the financial and logistical limitations of current space constructions, such as the International Space Station, the thesis looks beyond the limitations of current technologies and towards designs that are driven by the fulfillment of human experiences in space. Life in space, the thesis envisions, will spark new human experiences and rituals while necessitating new forms and designs in architecture. Weightlessness and its related spatial disorientations, in addition to the many other unique conditions in this unfamiliar territory, will inspire a new conceptual language for architecture and human cultures. The thesis will demonstrate that spaces designed for extraterrestrial experiences can be innovatively dynamic as they respond to new cultures and activities that evolve as a reaction to extreme conditions. Introducing humans to the environs of orbital space will be the initial stage in a long-term phasing tactic to colonize and commercialize beyond the expanse of Earth, eventually extending humanity to the remote neighbouring planets of the universe.

As the debate over demarcation between airspace and outer space remains unresolved, advancements in technology are bringing these two realms of flight closer than ever before. Rather than relying on traditional functional or spatial approaches to define the legal framework of flight, this paper proposes a completely new legal system based on orbital status known as "Orbit Law." / The first chapter examines the functional versus spatial debate, and highlights those aspects of existing International Air Law and Space Law which may be useful to an Orbit Law regime. Chapter II studies the science bridging air flight with space flight, and proposes the standardization of safety requirements for all suborbital and orbital flights. Finally Chapter III outlines the new legal principles of Orbit Law, highlighting innovative submissions for suborbital and orbital flights, solutions to issues of liability, and "Open Skies" for all flights.

Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism.

Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study, the gene encoding a 29.7 kDa outer membrane lipoprotein of the causative intestinal spirochaete Brachyspira hyodysenteriae, was identified and sequenced. An 816 bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site, and putative –10 and –35 promoter regions upstream from the start of the ORF. The 29.7 kDa outer membrane lipoprotein was designated Bhlp29.7 and the encoding gene named bhlp29.7. The amino acid sequence of Bhlp29.7 included a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. In silico analysis of this protein together with lipidation studies further supported its probable outer membrane localisation. Comparison of the Bhlp29.7 sequence with public sequence databases showed that it had up to 40% similarity with the D-methionine substrate-binding outer membrane lipoprotein (MetQ) of a number of bacterial pathogens. The Bhlp29.7 gene was detected in all 48 strains of B. hyodysenteriae examined, and in Brachyspira innocens strain B256T, but not in 10 other strains of B. innocens or in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence identity and had 97.5-99.5% identity with the gene of B. innocens strain B256T. The Bhlp29.7 gene was subsequently cloned and expressed as a histidine fusion protein in an Escherichia coli expression system. An ELISA test using recombinant his-tagged Bhlp29.7 (His6-Bhlp29.7) as the detecting antigen was developed and evaluated. The threshold value of the test was chosen to provide a highly stringent assessment of the disease status of a herd. The sensitivity and specificity of the test was 100%. When the test was applied to sera from eight herds with suspected SD, four gave ELISA values indicating that the herds were diseased. The remaining four herds gave ELISA values below the threshold value. These results indicated that the Bhlp29.7-ELISA was useful as an indirect test for exposure of a herd to B. hyodysenteriae and may be a helpful complement to current methods of SD diagnosis. Recombinant His6-Bhlp29.7 was evaluated as a vaccine subunit for prevention of SD. The His6-Bhlp29.7 was shown to be immunogenic in mice following two intramuscular injections. Vaccination of mice with His6-Bhlp29.7 provided full protection after oral challenge with B. hyodysenteriae. In two experiments, intramuscular and oral vaccination of pigs with the His6-Bhlp29.7 resulted in a 50% reduction in incidence of SD compared to unvaccinated control pigs (P=0.047). This is the first subunit vaccine shown to provide pigs with protection from SD. Further work is needed to optimise delivery routes and adjuvants for commercial development of the vaccine.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/01/15). Includes bibliographical references.