Background: Escherichia coli (E.coli) O86a:K61 belongs to the Enteropathogenic E. coli (EPEC) group of pathogens. Acute gastroenteritis affects 2-4 billion people annually and EPEC is associated with 10-40% of hospitalized diarrhea cases globally. Coagulation Factor (F) V circulates as an inactive procofactor (Mr 330kDa) which upon thrombin activation to the active cofactor, FVa, functions in prothombinase to accelerate prothrombin to thrombin conversion by 300,000-fold. The ability of E.coli O86a:K61 to cause intestinal hemorrhage is of interest because previous research demonstrated that during E.coli O86a:K61 sepsis in baboons, a dose-dependent inactivation of FV was observed as the bacterial dose increased. These results suggested a secreted E.coli protease may have mediated this effect on FV. This research has focused on the purification, identification, and characterization of a secreted E. coli O86a:K61 protease that inactivates FV. The final partially-purified protease inactivated FV to a 250kDa product by immunoblotting, and possessed a 900-fold increase in specific activity versus FV in human plasma compared to the culture supernatant. At least 3 proteins were observed upon SDS-PAGE. Proteolytic inactivation of FV was activated by up to 500-fold with β-mercaptoethanol and 2-fold with 1M urea. The protease was heat stable retaining all of its activity versus FV after 1h at 70°C or 80°C, and partial activity (50%) at 95°C. Proteolysis of FV was blocked by 90% with alpha-1-protease inhibitor; however, the protease was resistant to 1.5 mM PMSF, and unaffected by E64, or iodoacetamide. FV is a major regulator of the coagulation process and its inactivation by the secreted E.coli protease would be expected to result in a net bleeding tendency which may contribute to the mucosal hemorrhage observed in humans with associated hemorrhagic colitis. Proteolytic inactivation of FV is predicted to result in decreased bacterial containment by host fibrin thereby increasing pathogen survival and growth. FV inactivation by the secreted E.coli protease may be part of a novel pathogenic virulence mechanism that deregulates the blood coagulation process to enhance bacterial infectivity and transmission. / UOIT

<p>My doctoral work stems from an original motivation to understand more closely why some areas of sandy coastlines erode and others accrete<—>an intriguing fundamental question and one of societal relevance wherever human coastal infrastructure exists. What are the physical processes driving shoreline change, and over what spatial and temporal scales are they manifest? If forces driving the littoral system change, how does the shoreline respond? Can we attribute observed patterns of shoreline change to a particular process?</p><p>Recent novel numerical shoreline-evolution modeling demonstrated that wave-driven gradients in alongshore sediment transport could produce self-organized, emergent features on spatial scales from sand waves to large-scale capes [<italic>Ashton et al.</italic>, 2001], introducing a new theoretical perspective to the cross-shore-oriented considerations of the coastal scientific community. The unexpected model results inspired fresh hypotheses about shoreline pattern formation and the forcing mechanisms behind them.</p><p>One overarching hypothesis was that under regimes of high- and low-angle deep-water incident waves, alongshore shoreline perturbations grow or diffuse away, respectively. To test the hypothesis we looked for a correlation between shoreline curvature (showing perturbations to a nearly straight coastline) and shoreline change in observed measurements. High-resolution topographic lidar surveys of the North Carolina Outer Banks from 1996<–>2006 allowed robust, quantitative comparisons between shoreline surveys spanning tens of kms. In Chapter 1 [<italic>Lazarus and Murray</italic>, 2007] we report that over the last decade, at multi-km scales along the barrier islands, convex-seaward promontories tended to erode and concave-seaward embayments accrete<—>a pattern of diffusion consistent with the smoothing effects of alongshore-transport gradients driven by a low-angle wave climate. Why then, after a decade or more of smoothing, do plan-view bumps in the shoreline still persist? In Chapter 2 [<italic>Lazarus et al.</italic>, in review] we compile evidence suggesting that (a) a framework of paleochannels may control the areas of persistent multi-km-scale shoreline convexity that (b) in turn drive decadal-term transient changes in shoreline morphology by (c) affecting gradients in wave-driven alongshore sediment transport.</p><p>In Chapter 3, a third investigation of large-scale coastal behavior, we explore an existing premise that shoreline change on a sandy coast is a self-affine signal wherein patterns of changes are scale-invariant, perhaps suggesting that a single process operates across the scales. Applying wavelet analysis<—>a mathematical technique involving scaled filter transforms<—>we confirm that a power law fits the average variance of shoreline change at alongshore scales spanning approximately three orders of magnitude (5<–>5000 m). The power law itself does not necessarily indicate a single dominant driver; beach changes across those scales likely result from a variety of cross-shore and alongshore hydrodynamic processes. A paired modeling experiment supports the conclusion that the power relationship is not an obvious function of wave-driven alongshore sediment transport alone.</p><p>Our tests of theory against field observations are middle steps in pattern-to-process attribution; they fit into a larger body of coastal morphodynamic research that in time may enable shoreline-change prediction. Present hydrodynamic models are still too limited in spatial and temporal scope to accommodate the extended scales at which large morphological changes occur, but more integrated quantitative models linking bathymetry, wave fields, and geologic substrate are underway and will set the next course of questions for the discipline.</p> / Dissertation

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 350-369). Available also in a digital version from Dissertation Abstracts.

The hallmark of Gram-negative bacteria is the presence of an outer membrane (OM) surrounding the cytoplasmic membrane (here called the inner membrane [IM]) and the cell wall. The OM is a unique asymmetric bilayer with an inner leaflet consisting of phospholipid and an outer leaflet consisting of lipopolysaccharide (LPS). LPS is a large anionic molecule that typically contains six fatty acyl chains and up to several hundred sugar residues. This chemical structure explains why the OM is relatively impermeable to large hydrophobic molecules, such as detergents, bile salts, and high molecular weight antibiotics, which readily cross a normal phospholipid bilayer. LPS and the OM are essential to the viability of most Gram-negative organisms, including major human pathogens. LPS molecules are biosynthesized at the IM and subsequently exported out of the IM, across the intermembrane space (the periplasm) and through the OM to their final position at the cell surface. In Escherichia coli, the essential LPS transport proteins, LptA-G, are required for this process. This Lpt pathway includes an IM adenosine triphosphate binding cassette (ABC) transporter, LptBFG, which is associated with an additional IM protein, LptC; a periplasmic protein, LptA; and an OM complex consisting of the lipoprotein LptE and the transmembrane \(\beta\)-barrel protein LptD. All seven Lpt proteins associate as a single complex that spans the cell envelope. However, little is known about how these proteins work together to transport LPS. Here, we use in vivo and in vitro biochemical studies to probe the organization, function, and assembly of the Lpt machine. In Chapter 2, we show that LptE forms a plug within the LptD \(\beta\)-barrel and present a model for how this unusual structure can move LPS from the periplasm directly into the outer leaflet of the OM. In Chapter 3, we demonstrate that the Lpt transenvelope bridge consists of a series of structurally homologous domains – LptC, LptA, and the N-terminal domain of LptD – stacked in a head-to-tail orientation, providing a route for LPS from the IM to the OM. Finally, in Chapter 4, we connect these two sets of results by showing how the assembly of the Lpt transenvelope bridge is regulated by that of the LptD/E complex in the OM. Together, these findings explain how the functions of the Lpt proteins are coordinated to ensure delivery of LPS to the correct cellular compartment. A fundamental understanding of LPS biogenesis will contribute to the development of new therapies against Gram-negative infections.

<p>Outer membrane vesicles are an important constitutive product of all Gram-negative bacteria. Bacteria have evolved many responses to alleviate all different types of stress. The primary objective of this dissertation is to investigate the role of outer membrane vesicles (OMVs) as a method by which Gram-negative bacteria can quickly act to protect themselves against particular threats. Generally, we find that stressors whose primary effect is on the outer membrane can be protected against by OMVs. Throughout this study, a variety of different microbiological and biochemical methods are used to answer key questions in the innate ability of OMVs to protect against particular antimicrobials. Using Escherichia coli as well as Pseudomonas aeruginosa as model organisms we tested the ability of purified vesicles from each species to protect themselves and other hosts. Using bacteriophage T4, we investigated the ability of OMVs purified from E. coli to adsorb phage as well as how this interaction affected the efficiency of infection. We found that OMVs are protective against antimicrobial peptides, as well as bacteriophage. In the course of understanding this protection we also observed and characterized the cross species effects of both OMV protection as well as phage infection. Where typically a phage infects a specific species, we found that T4 associated OMVs treating a non-native host P. aeruginosa resulted in the production of a novel prophage. Upon further examination, we determined that this induction was occurring via a novel pathway that we attempted to further characterize by performing a genetic screen to identify genes important to this induction. The work within this dissertation fully supports the hypothesis of a regulated response to outer membrane acting stimuli, resulting in the induction of vesiculation and the adsorption of stressor in the extra-cellular milieu. This model of protection agrees with the idea of a bacterial innate defense system, which acts in the short term before the adaptive response can fully occur, resulting in a bridge between the untreated to the treated and resistant culture.</p> / Dissertation

The study of outer radiation belt dynamics has been ongoing for over 5 decades. Outer radiation belt dropouts involve the rapid lost of electron fluxes at the main phase of a storm and subsequent recovery. The characteristics of the dropouts are many and varied. This study examines the Geosynchronous Earth Orbit (GEO) and the ionospheric responses during Stream Interface (SI) andMagnetic Cloud (MC) events, using a combination of ground based and satellite instruments. SI- and MC-driven dropout events were inspected from summary plots of the Synchronous Orbit Particle Analyser (SOPA) instruments from the year 1996 to 2007. Comprehensive studies were done on six selected events. Analysis of the data from the instruments indicate that SIs and MCs are important lost drivers with significant differences in GEO and ionospheric environment. To validate the data and test for consistent response of the events, the Superposed Epoch Analysis (SEA) technique was used. The ground based measurements also revealed how the absorption peaks in the ionosphere correlated with dropouts and geophysical activity. Ionospheric absorption during SI associated dropouts was enhanced for 5 < L < 6, while significant peaks in ionospheric absorption extended to lower L during MC driven dropouts. Wave-particle interactions and southward Interplanetary Magnetic Field (IMF-Bz) are apparent causes for the precipitation. This analysis showed that, within the confines of the selected events, SI driven dropouts were more dominant at the declining phase of the solar cycle while the MC driven dropouts were more dominant during solar maximum. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Despite being amongst the most common zoonotic diseases in the world, brucellosis is a neglected disease for which an approved vaccine for human use does not exist. Thus far, the traditional approaches to Brucella antigen selection for subunit vaccine development have yielded unacceptable results. In this work, we evaluated the predictive ability of a multistep Brucella antigen selection process with in vitro immunological and invasion assays and in vivo protection experiments. Initial in silico screening for antigens was performed via genomic sequence analysis where 27 Brucella melitensis open reading frames (ORF) coding for outer membrane proteins bearing MHC epitopes, adhesin and conserved properties were identified. Evidence for a role in any aspect of Brucella virulence (i.e., invasion, co-regulation/expression with known Brucella virulence factors, intracellular adaptation) was then used to narrow the list of candidate antigens. To further increase confidence in the candidate ORF putative role in Brucella pathogenesis, differential expression of candidate ORF was evaluated using previously generated global transcriptomics data in in vitro HeLa and in vivo bovine models of acute Brucella infection. Protein expression in the E. coli heterologous system resulted in the successful expression of OmpW, BtuB, Omp22, Hia, and FlgK. With regards to virulence, the two proteins with the highest predicted adhesin scores conferred an invasive phenotype to the non-invasive BL-21 E. coli strain in alveolar epithelial cells. From an immunogenicity standpoint, all proteins elicited IgG production in Brucella-exposed goats, mouse and humans. Antigen-specific recall responses in splenocytes from C57BL/6 mice immunized with a cocktail of the three proteins with highest MHC scores revealed a mixed Th1/Th2 response with a comparatively greater Th1 response. In protection studies, subcutaneous (SQ) immunization with BtuB, Hia and FlgK, individually, promoted bacterial clearance following a robust intraperitoneal challenge dose of Brucella melitensis 16M. In addition, single SQ inoculation of FlgK enhanced protective efficacy of the vaccine strain B. abortus S19. In contrast, immunization of mice with the three protective antigens in a cocktail formulation elicited immune responses but no protection against intraperitoneal challenge with Brucella melitensis 16M in the spleen and liver. In conclusion, our results indicate that our combinatorial in silico, in vitro and in vivo antigen selection and identification modeling approach provides strong evidence for prediction of Brucella protective antigens, and represent a novel strategy with broad application to other major pathogens.

This thesis addresses the legal and policy issues relating to what may be the most exciting prospect in the history of the human civilisation: the commercial exploitation of natural resources in outer space. The thesis is based on the hypothesis that such ventures are inhibited not by physical, technological and economic factors, but by the inadequacies and uncertainties present in the current body of space law and policy. Consequently, a new international legal framework and a policy consensus are required to provide a legal environment favourable for such a valuable and necessary development. To substantiate this hypothesis, the thesis begins by establishing the economic necessity and technical feasibility of space mining today, an estimate of the financial commitments required. This is followed by a risk analysis of a typical commercial mining venture in space, identifying the economic and legal risks. This leads to the recognition that the legal risks must be minimised to enable such enormous financial commitments to be made. What then follows is a detailed analysis of the legal framework for such activities as well as identifying the inadequacies of space law for the commercial exploitation of celestial resources. This is achieved through a discussion of the general principles of international space law, particularly dealing with state responsibility and international liability, as well as some of the issues arising from space mining activities. Much detail is devoted to the analysis of the content of the “common heritage of mankind” doctrine in international law and the effect of international disagreement over its application to celestial bodies. Having established the relevant legal issues, the thesis then turns to consider the past failures in reach similar agreements and the competing policy interests that have prevented the success of such agreements. It attempts to balance such interests in creating a legal and policy compromise that may be acceptable to a majority of the international community and provide some practical proposals on the structural, procedural, administrative and judicial aspects of creating and implementing a new legal framework.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1971. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 203-207).