Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Wandering and the physical environment

Background/Rationale Guided by the need-driven dementia-compromised behavior (NDB) model, this study examined influences of the physical environment on wandering behavior. Methods Using a descriptive, cross-sectional design, 122 wanderers from 28 long-term care (LTC) facilities were videotaped 10 to 12 times; data on wandering, light, sound, temperature and humidity levels, location, ambiance, and crowding were obtained. Associations between environmental variables and wandering were evaluated with chi-square and t tests; the model was evaluated using logistic regression. Results In all, 80% of wandering occurred in the resident’s own room, dayrooms, hallways, or dining rooms. When observed in other residents’ rooms, hallways, shower/baths, or off-unit locations, wanderers were likely (60%-92% of observations) to wander. The data were a good fit to the model overall (LR [logistic regression] χ2 (5) = 50.38, P < .0001) and by wandering type. Conclusions Location, light, sound, proximity of others, and ambiance are associated with wandering and may serve to inform environmental designs and care practices.

Robot building for preschoolers

This paper describes Electronic Blocks, a new robot construction element designed to allow children as young as age three to build and program robotic structures. The Electronic Blocks encapsulate input, output and logic concepts in tangible elements that young children can use to create a wide variety of physical agents. The children are able to determine the behavior of these agents by the choice of blocks and the manner in which they are connected. The Electronic Blocks allow children without any knowledge of mechanical design or computer programming to create and control physically embodied robots. They facilitate the development of technological capability by enabling children to design, construct, explore and evaluate dynamic robotics systems. A study of four and five year-old children using the Electronic Blocks has demonstrated that the interface is well suited to young children. The complexity of the implementation is hidden from the children, leaving the children free to autonomously explore the functionality of the blocks. As a consequence, children are free to move their focus beyond the technology. Instead they are free to focus on the construction process, and to work on goals related to the creation of robotic behaviors and interactions. As a resource for robot building, the blocks have proved to be effective in encouraging children to create robot structures, allowing children to design and program robot behaviors.

Receptor-DNA interactions: EMSA and footprinting

Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these proteins modulate the expression of their specific target genes. The purpose of this chapter is to provide the reader with a detailed guide with respect to the materials and the key methods required to perform this type of DNA-binding analysis. Irrespective of whether starting with purified DNA-binding proteins or somewhat crude cellular extracts, the tried-and-true procedures described here will enable one to accurately access the capacity of specific proteins to bind to DNA as well as to determine the exact sequences and DNA contact nucleotides involved. For illustrative purposes, we primarily have used the interaction of the androgen receptor with the rat probasin proximal promoter as our model system.

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.

Tactical driver lane change model using forward search

This paper describes the development and evaluation of a tactical lane change model using the forward search algorithm, for use in a traffic simulator. The tactical lane change model constructs a set of possible choices of near-term maneuver sequences available to the driver and selects the lane change action at the present time to realize the best maneuver plan. Including near term maneuver planning in the driver behavior model can allow a better representation of the complex interactions in situations such as a weaving section and high-occupancy vehicle (HOV) lane systems where drivers must weave across several lanes in order to access the HOV lanes. To support the investigation, a longitudinal control model and a basic lane change model were also analyzed. The basic lane change model is similar to those used by today's commonly-used traffic simulators. Parameters in all models were best-fit estimated for selected vehicles from a real-world freeway vehicle trajectory data set. The best-fit estimation procedure minimizes the discrepancy between the model vehicle and real vehicle's trajectories. With the best fit parameters, the proposed tactical lane change model gave a better overall performance for a greater number of cases than the basic lane change model.

Playpolis : transyouth and urban networking in Seoul

The overarching aim of this study is to create new knowledge about how playful interactions (re)create the city via ubiquitous technologies, with an outlook to apply the knowledge for pragmatic innovations in relevant fields such as urban planning and technology development in the future. The study looks at the case of transyouth, the in-between demographic bridging youth and adulthood in Seoul, one of the most connected, densely populated, and quickly transforming metropolises in the world. To unravel the elusiveness of ‘play’ as a subject and the complexity of urban networks, this study takes a three-tier transdisciplinary approach comprised of an extensive literature review, Shared Visual Ethnography (SVE), and interviews with leading industry representatives who design and develop the playscape for Seoul transyouth. Through these methodological tools, the study responds to the following four research aims: 1. Examine the sociocultural, technological, and architectural context of Seoul 2. Investigate Seoul transyouth’s perception of the self and their technosocial environment 3. Identify the pattern of their playful interaction through which meanings of the self and the city are recreated 4. Develop an analytical framework for enactment of play This thesis argues that the city is a contested space that continuously changes through multiple interactions among its constituents on the seam of control and freedom. At the core of this interactive (re)creation process is play. Play is a phenomenon that is enacted at the centre of three inter-related elements of pressure, possibility, and pleasure, the analytical framework this thesis puts forward as a conceptual apparatus for studying play across disciplines. The thesis concludes by illustrating possible trajectories for pragmatic application of the framework for envisioning and building the creative, sustainable, and seductive city.

Using digital rights management for securing data in a medical research environment

We propose a digital rights management approach for sharing electronic health records in a health research facility and argue advantages of the approach. We also give an outline of the system under development and our implementation of the security features and discuss challenges that we faced and future directions.

Music, Mime & Metamorphosis: Interdisciplinary intersections, interactions and influences between music, mime and corporate communication

This doctoral thesis comprises three distinct yet related projects which investigate interdisciplinary practice across: music collaboration; mime performance; and corporate communication. Both the processes and underpinning research of these projects explore, expose and exploit areas where disparate and apparently conflicting fields of professional practice successfully and effectively; intersect, interact, and inform each other - rather than conflict - thereby enhancing each, both individually and collectively. Informed by three decades of professional practice across: music; stage performance; television; corporate communication; design; and tertiary education, the three projects have produced innovative, creative, and commercial viable outcomes, manifest in a variety of media including: music; written text; digital, audio/visual; and internet. In exploring new practice and creating new knowledge, these project outcomes clearly demonstrate the value and effectiveness of reconciling disparate fields of practice through the application of inter-disciplinary creativity and innovation to professional practice.

Implications for the design of a digital stethoscope for anaesthetic preadmission consultations

Background This research addresses the development of a digital stethoscope for use with a telehealth communications network to allow doctors to examine patients remotely (a digital telehealth stethoscope). A telehealth stethoscope would allow remote auscultation of patients who do not live near a major hospital. Travelling from remote areas to major hospitals is expensive for patients and a telehealth stethoscope could result in significant cost savings. Using a stethoscope requires great skill. To design a telehealth stethoscope that meets doctors’ expectations, the use of existing stethoscopes in clinical contexts must be examined. Method Observations were conducted of 30 anaesthetic preadmission consultations. The observations were video- taped. Interaction between doctor, patient and non-human elements in the consultation were “coded” to transform the video into data. The data were analysed to reveal essential aspects of the interactions. Results The analysis has shown that the doctor controls the interaction during auscultation. The conduct of auscultation draws heavily on the doctor’s tacit knowledge, allowing the doctor to treat the acoustic stethoscope as infrastructure – that is, the stethoscope sinks into the background and becomes completely transparent in use. Conclusion Two important, and related, implications for the design of a telehealth stethoscope have arisen from this research. First, as a telehealth stethoscope will be a shared device, doctors will not be able to make use of their existing expertise in using their own stethoscopes. Very simply, a telehealth stethoscope will sound different to a doctor’s own stethoscope. Second, the collaborative interaction required to use a telehealth stethoscope will have to be invented and refined. A telehealth stethoscope will need to be carefully designed to address these issues and result in successful use. This research challenges the concept of a telehealth stethoscope by raising questions about the ease and confidence with which doctors could use such a device.

Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.