Review article : ecological approach in studying the relation of perception-action : review of the theoretical and empirical data

In daily activities people are using a number of available means for the achievement of balance, such as the use of hands and the co-ordination of balance. One of the approaches that explains this relationship between perception and action is the ecological theory that is based on the work of a) Bernstein (1967), who imposed the problem of ‘the degrees of freedom’, b) Gibson (1979), who referred to the theory of perception and the way which the information is received from the environment in order for a certain movement to be achieved, c) Newell (1986), who proposed that movement can derive from the interaction of the constraints that imposed from the environment and the organism and d) Kugler, Kelso and Turvey (1982), who showed the way which “the degrees of freedom” are connected and interact. According to the above mentioned theories, the development of movement co-ordination can result from the different constraints that imposed into the organism-environment system. The close relation between the environmental and organismic constraints, as well as their interaction is responsible for the movement system that will be activated. These constraints apart from shaping the co-ordination of specific movements can be a rate limiting factor, to a certain degree, in the acquisition and mastering of a new skill. This frame of work can be an essential tool for the study of catching an object (e.g., a ball). The importance of this study becomes obvious due to the fact that movements that involved in catching an object are representative of every day actions and characteristic of the interaction between perception and action.

Translating tissue engineering technology platforms into cancer research

Technology platforms originally developed for tissue engineering applications produce valuable models that mimic three-dimensional (3D) tissue organization and function to enhance the understanding of cell/tissue function under normal and pathological situations. These models show that when replicating physiological and pathological conditions as closely as possible investigators are allowed to probe the basic mechanisms of morphogenesis, differentiation and cancer. Significant efforts investigating angiogenetic processes and factors in tumorigenesis are currently undertaken to establish ways of targeting angiogenesis in tumours. Anti-angiogenic agents have been accepted for clinical application as attractive targeted therapeutics for the treatment of cancer. Combining the areas of tumour angiogenesis, combination therapies and drug delivery systems is therefore closely related to the understanding of the basic principles that are applied in tissue engineering models. Studies with 3D model systems have repeatedly identified complex interacting roles of matrix stiffness and composition, integrins, growth factor receptors and signalling in development and cancer. These insights suggest that plasticity, regulation and suppression of these processes can provide strategies and therapeutic targets for future cancer therapies. The historical perspective of the fields of tissue engineering and controlled release of therapeutics, including inhibitors of angiogenesis in tumours is becoming clearly evident as a major future advance in merging these fields. New delivery systems are expected to greatly enhance the ability to deliver drugs locally and in therapeutic concentrations to relevant sites in living organisms. Investigating the phenomena of angiogenesis and anti-angiogenesis in 3D in vivo models such as the Arterio-Venous (AV) loop mode in a separated and isolated chamber within a living organism adds another significant horizon to this perspective and opens new modalities for translational research in this field.

Challenging endoscopy reprocessing guidelines: a prospective study investigating the safe shelf life of flexible endoscopes in a tertiary gastroenterology unit.

Professional prac− tice guidelines for endoscope reprocessing re− commend reprocessing endoscopes between each case and proper storage following repro− cessing after the last case of the list. There is lim− ited empirical evidence to support the efficacy of endoscope reprocessing prior to use in the first case of the day; however, internationally, many guidelines continue to recommend this practice. The aim of this study is to estimate a safe shelf life for flexible endoscopes in a high−turnover gastroenterology unit. Materials and methods: In a prospective obser− vational study, all flexible endoscopes in active service during the 3−week study period were mi− crobiologically sampled prior to reprocessing be− fore the first case of the day (n = 200). The main outcome variables were culture status, organism cultured, and shelf life. Results: Among the total number of useable samples (n = 194), the overall contamination rate was 15.5 %, with a pathogenic contamination rate of 0.5 %. Mean time between last case one day and reprocessing before the first case on the next day (that is, shelf life) was 37.62 h (SD 36.47). Median shelf life was 18.8 h (range 5.27± 165.35 h). The most frequently identified organ− ism was coagulase−negative Staphylococcus, an environmental nonpathogenic organism. Conclusions: When processed according to es− tablished guidelines, flexible endoscopes remain free from pathogenic organisms between last case and next day first case use. Significant re− ductions in the expenditure of time and resources on reprocessing endoscopes have the potential to reduce the restraints experienced by high−turnover endoscopy units and improve ser− vice delivery.

Investigation of molecular mechanisms regulating biomineralization of pearl oyster Pinctada maxima

Biomineralization is a process encompassing all mineral containing tissues produced within an organism. The most dynamic example of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this remarkable architecture. Subsequently, for the past decade considerable research have been undertaken to identify and characterize the protein components involved in biomineralization. Despite these efforts the general understanding of the process remains ambiguous. This study employs a novel molecular approach to further the elucidation of the shell biomineralization. A microarray platform has been custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from the mantle, an organ involved in shell formation. This microarray has been used as the primary tool for three separate investigations in an effort to associate transcriptional gene expression from P. maxima to the process of shell biomineralization. The first investigation analyzes the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and each analyzed for gene expression with PmaxArray 1.0. Over 2000 ESTs were differentially expressed among the tissue sections, identifying five major expression regions. Three of these regions have been proposed to have shell formation functions belonging to nacre, prismatic calcite and periostracum. The spatial gene expression map was confirmed by in situ hybridization, localizing a subset of ESTs from each expression region to the same mantle area. Comparative sequence analysis of ESTs expressed in the proposed shell formation regions with the BLAST tool, revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell formation genes. The second investigation correlates temporal EST expression during P. maxima larval ontogeny with transitions in shell mineralization during the same period. A timeline documenting the morphologicat microstructural and mineralogical shell characteristics of P. maxima throughout larval ontogeny has been established. Three different shell types were noted based on the physical characters and termed, prodissoconch I, prodissoconch 11 and dissoconch. PmaxArray 1.0 analyzed ESTs expression of animals throughout the larval development of P. maxima, noting up-regulation of 359 ESTs in association with the shell transitions from prodissoconch 1 to prodissoconch 11 to dissoconch. Comparative sequence analysis of these ESTs indicates a number of the transcripts are novel as well as showing significant sequence similarities between ESTs and known shell matrix associated genes and proteins. These ESTs are discussed in relation to the shell characters associated with their temporal expression. The third investigation uses PmaxArray 1.0 to analyze gene expression in the mantle tissue of P. maxima specimens exposed to sub-lethal concentrations of a shell-deforming toxin, tributyltin (TBT). The shell specific effects of TBT are used in this investigation to interpret differential expression of ESTs with respect to shell formation functions. A lethal and sublethal TBT concentration range was established for P. maxima, noting a concentration of 50 ng L- 1 TBT as sub-lethal over a 21 day period. Mantle tissue from P. maxima animals treated with 50 ng L- 1 TBT was assessed for differential EST expression with untreated control animals. A total of 102 ESTs were identified as differentially expressed in association with TBT exposure, comparative sequence identities included an up-regulation of immunity and detoxification related genes and down-regulation of several shell matrix genes. A number of transcripts encoding novel peptides were additionally identified. The potential actions of these genes are discussed with reference to TBT toxicity and shell biomineralization. This thesis has used a microarray platform to analyze gene expression in spatial, temporal and toxicity investigations, revealing the involvement of numerous gene transcripts in specific shell formation functions. Investigation of thousands of transcripts simultaneously has provided a holistic interpretation of the organic components regulating shell biomineralization.

The new epidemics

In developed countries we once thought that the scourge of infectious diseases was tamed. Antibiotics were controlling infection in individual patients, vaccines were preventing illness and great faith was placed in the capacity of science to confound the most cunning organism. However, things have changed and in the new millennium we are confronting a host of challenges to public health from infectious diseases. Epidemics mean an excess of cases in the community from that normally expected or the appearance of a new infection (Webber ####, 22) Chapter 11 outlined the background to infectious diseases and the individual strategies directed towards the control and management of infectious diseases. The aim of this chapter is to outline the impact that infectious diseases have on population health, to identify the risks of major outbreaks and to identify the strategies required to reduce the risk and to manage any possible outbreak.

Market access of Papua New Guinea bananas (Musa spp.) with particular respect to banana fly (Bactrocera musae (Tryon)) (Diptera: Tephritidae)

International market access for fresh commodities is regulated by international accepted phytosanitary guidelines, the objectives of which are to reduce the biosecurity risk of plant pest and disease movement. Papua New Guinea (PNG) has identified banana as a potential export crop and to help meet international market access requirements, this thesis provides information for the development of a pest risk analysis (PRA) for PNG banana fruit. The PRA is a three step process which first identifies the pests associated with a particular commodity or pathway, then assesses the risk associated with those pests, and finally identifies risk management options for those pests if required. As the first step of the PRA process, I collated a definitive list on the organisms associated with the banana plant in PNG using formal literature, structured interviews with local experts, grey literature and unpublished file material held in PNG field research stations. I identified 112 organisms (invertebrates, vertebrate, pathogens and weeds) associated with banana in PNG, but only 14 of these were reported as commonly requiring management. For these 14 I present detailed information summaries on their known biology and pest impact. A major finding of the review was that of the 14 identified key pests, some research information occurs for 13. The single exception for which information was found to be lacking was Bactrocera musae (Tryon), the banana fly. The lack of information for this widely reported ‘major pest on PNG bananas’ would hinder the development of a PNG banana fruit PRA. For this reason the remainder of the thesis focused on this organism, particularly with respect to generation of information required by the PRA process. Utilising an existing, but previously unanalysed fruit fly trapping database for PNG, I carried out a Geographic Information System analysis of the distribution and abundance of banana in four major regions of PNG. This information is required for a PRA to determine if banana fruit grown in different parts of the country are at different risks from the fly. Results showed that the fly was widespread in all cropping regions and that temperature and rainfall were not significantly correlated with banana fly abundance. Abundance of the fly was significantly correlated (albeit weakly) with host availability. The same analysis was done with four other PNG pest fruit flies and their responses to the environmental factors differed to banana fly and each other. This implies that subsequent PRA analyses for other PNG fresh commodities will need to investigate the risk of each of these flies independently. To quantify the damage to banana fruit caused by banana fly in PNG, local surveys and one national survey of banana fruit infestation were carried out. Contrary to expectations, infestation was found to be very low, particularly in the widely grown commercial cultivar, Cavendish. Infestation of Cavendish fingers was only 0.41% in a structured, national survey of over 2 700 banana fingers. Follow up laboratory studies showed that fingers of Cavendish, and another commercial variety Lady-finger, are very poor hosts for B. musae, with very low host selection rates by female flies and very poor immature survival. An analysis of a recent (within last decade) incursion of B. musae into the Gazelle Peninsula of East New Britain Province, PNG, provided the final set of B. musae data. Surveys of the fly on the peninsular showed that establishment and spread of the fly in the novel environment was very rapid and thus the fly should be regarded as being of high biosecurity concern, at least in tropical areas. Supporting the earlier impact studies, however, banana fly has not become a significant banana fruit problem on the Gazelle, despite bananas being the primary starch staple of the region. The results of the research chapters are combined in the final Discussion in the form of a B. musae focused PRA for PNG banana fruit. Putting the thesis in a broader context, the Discussion also deals with the apparent discrepancy between high local abundance of banana fly and very low infestation rates. This discussion focuses on host utilisation patterns of specialist herbivores and suggests that local pest abundance, as determined by trapping or monitoring, need not be good surrogate for crop damage, despite this linkage being implicit in a number of international phytosanitary protocols.

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.

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Correcting mean-field approximations for birth-death-movement processes

On the microscale, migration, proliferation and death are crucial in the development, homeostasis and repair of an organism; on the macroscale, such effects are important in the sustainability of a population in its environment. Dependent on the relative rates of migration, proliferation and death, spatial heterogeneity may arise within an initially uniform field; this leads to the formation of spatial correlations and can have a negative impact upon population growth. Usually, such effects are neglected in modeling studies and simple phenomenological descriptions, such as the logistic model, are used to model population growth. In this work we outline some methods for analyzing exclusion processes which include agent proliferation, death and motility in two and three spatial dimensions with spatially homogeneous initial conditions. The mean-field description for these types of processes is of logistic form; we show that, under certain parameter conditions, such systems may display large deviations from the mean field, and suggest computationally tractable methods to correct the logistic-type description.

The development of Lactococcus lactis as an antimicrobial agent

Non-pathogenic lactic acid bacteria are economically important Gram-positive bacteria used extensively in the food industry. Due to their “generally regarded as safe” status, certain species from the genera Lactobacillus and Lactococcus are also considered desirable as candidates for the production and secretion of recombinant proteins, particular those with therapeutic applications. The hypothesis examined by this thesis is that Lactococcus lactis can be modified to be an effective antimicrobial agent. Therefore, the aims of this thesis were to investigate the optimisation of the expression, secretion and/or activities of potential heterologous antimicrobial proteins by the model lactic acid bacterium, Lactococcus lactis subsp. cremoris MG1363. L. lactis strains were engineered to express and secrete the recombinant CyuC surface protein from Lactobacillus reuteri BR11, and a fusion protein consisting of CyuC and lysostaphin using the Sep promoter and secretion signal. CyuC has been characterised as a cystine-binding protein, but has also been demonstrated to have fibronectin binding activity. Lysostaphin is a bacteriolytic enzyme with specific activity against the Gram-positive pathogen, Staphylococcus aureus. These modified L. lactis strains were then investigated to see if they had the ability to inhibit the adhesion of S. aureus to host extracellular matrix (ECM) proteins. It was observed that the cell extracts of the L. lactis strain with the vector only (pGhost9:ISS1) was able to inhibit the adhesion of S. aureus to fibronectin, whilst the cell extracts of the L. lactis strain expressing lysostaphin was able to inhibit adhesion to keratin. Finally, this thesis has identified specific lactococcal genes that affect the secretion of lysostaphin through the use of random transposon mutagenesis. Ten mutants with higher lysostaphin activity contained insertions in four different genes encoding: (i) an uncharacterised putative transmembrane protein (llmg_0609), (ii) an enzyme catalysing the first step in peptidoglycan biosynthesis (murA2), (iii) a homolog of the oxidative defence regulator (trmA), and (iv) an uncharacterised putative enzyme involved in ubiquinone biosynthesis (llmg_2148). The higher lysostaphin activity observed in these mutants was found to be due to higher amounts of lysostaphin being secreted. The findings of this thesis contribute to the development of this organism as an antimicrobial agent and also to our understanding of L. lactis genetics.

Engineering an Ecosystem : Taking Cues from Nature’s Paradigm to Build Tissue in the Lab and the Body

This manuscript took a 'top down' approach to understanding survival of inhabitant cells in the ecosystem bone, working from higher to lower length and time scales through the hierarchical ecosystem of bone. Our working hypothesis is that nature “engineered” the skeleton using a 'bottom up' approach,where mechanical properties of cells emerge from their adaptation to their local me-chanical milieu. Cell aggregation and formation of higher order anisotropic struc- ture results in emergent architectures through cell differentiation and extracellular matrix secretion. These emergent properties, including mechanical properties and architecture, result in mechanical adaptation at length scales and longer time scales which are most relevant for the survival of the vertebrate organism [Knothe Tate and von Recum 2009]. We are currently using insights from this approach to har-ness nature’s regeneration potential and to engineer novel mechanoactive materials [Knothe Tate et al. 2007, Knothe Tate et al. 2009]. In addition to potential applications of these exciting insights, these studies may provide important clues to evolution and development of vertebrate animals. For instance, one might ask why mesenchymal stem cells condense at all? There is a putative advantage to self-assembly and cooperation, but this advantage is somewhat outweighed by the need for infrastructural complexity (e.g., circulatory systems comprised of specific differentiated cell types which in turn form conduits and pumps to overcome limitations of mass transport via diffusion, for example; dif-fusion is untenable for multicellular organisms larger than 250 microns in diameter. A better question might be: Why do cells build skeletal tissue? Once cooperatingcells in tissues begin to deplete local sources of food in their aquatic environment, those that have evolved a means to locomote likely have an evolutionary advantage. Once the environment becomes less aquarian and more terrestrial, self-assembled organisms with the ability to move on land might have conferred evolutionary ad-vantages as well. So did the cytoskeleton evolve several length scales, enabling the emergence of skeletal architecture for vertebrate animals? Did the evolutionary advantage of motility over noncompliant terrestrial substrates (walking on land) favor adaptations including emergence of intracellular architecture (changes in the cytoskeleton and upregulation of structural protein manufacture), inter-cellular con- densation, mineralization of tissues, and emergence of higher order architectures?How far does evolutionary Darwinism extend and how can we exploit this knowl- edge to engineer smart materials and architectures on Earth and new, exploratory environments?[Knothe Tate et al. 2008]. We are limited only by our ability to imagine. Ultimately, we aim to understand nature, mimic nature, guide nature and/or exploit nature’s engineering paradigms without engineer-ing ourselves out of existence.

Investigation into the introduction of clonal strains of Pseudomonas aeruginosa to the cystic fibrosis population by consumption of raw salad vegetables

Most salad vegetables are eaten fresh by consumers. However, raw vegetables may pose a risk of transmitting opportunistic bacteria to immunocompromised people, including cystic fibrosis (CF) patients. In particular, CF patients are vulnerable to chronic Pseudomonas aeruginosa lung infections and this organism is the primary cause of morbidity and mortality in this group. Clonal variants of P. aeruginosa have been identified as emerging threats to people afflicted with CF; however it has not yet been proven from where these clones originate or how they are transmitted. Due to the organisms‟ aquatic environmental niche, it was hypothesised that vegetables may be a source of these clones. To test this hypothesis, lettuce, tomatoes, mushrooms and bean sprout packages (n = 150) were analysed from a green grocer, supermarket and farmers‟ market within the Brisbane region, availability permitting. The internal and external surfaces of the vegetables were separately analysed for the presence of clonal strains of P. aeruginosa using washings and homogenisation techniques, respectively. This separation was in an attempt to establish which surface was contaminated, so that recommendations could be made to decrease or eliminate P. aeruginosa from these foods prior to consumption. Soil and water samples (n = 17) from local farms were also analysed for the presence of P. aeruginosa. Presumptive identification of isolates recovered from these environmental samples was made based on growth on Cetrimide agar at 42°C, presence of the cytochrome-oxidase enzyme and inability to ferment lactose. P. aeruginosa duplex real-time polymerase chain reaction assay (PAduplex) was performed on all bacterial isolates presumptively identified as P. aeruginosa. Enterobacterial repetitive intergenic consensus strain typing PCR (ERIC-PCR) was subsequently performed on confirmed bacterial isolates. Although 72 P. aeruginosa were isolated, none of these proved to be clonal strains. The significance of these findings is that vegetables may pose a risk of transmitting sporadic strains of P. aeruginosa to people afflicted with CF and possibly, other immunocompromised people.

Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima

Background: Biomineralization is a process encompassing all mineral containing tissues produced within an organism. One of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this architecture. However general understanding of how this process is achieved remains ambiguous. The mantle is a conserved organ involved in shell formation throughout molluscs. Specifically the mantle is thought to be responsible for secreting the protein component of the shell. This study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization. Results: A microarray platform was custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from mantle tissue. This microarray was used to analyze the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and analyzed for differential gene expression with PmaxArray 1.0. Over 2000 ESTs were determined to be differentially expressed among the tissue sections, identifying five major expression regions. In situ hybridization validated and further localized the expression for a subset of these ESTs. Comparative sequence similarity analysis of these ESTs revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes.

Under the carpet : the politics and trauma of patient harm

Few studies have investigated iatrogenic outcomes from the viewpoint of patient experience. To address this anomaly, the broad aim of this research is to explore the lived experience of patient harm. Patient harm is defined as major harm to the patient, either psychosocial or physical in nature, resulting from any aspect of health care. Utilising the method of Consensual Qualitative Research (CQR), in-depth interviews are conducted with twenty-four volunteer research participants who self-report having been severely harmed by an invasive medical procedure. A standardised measure of emotional distress, the Impact of Event Scale (IES), is additionally employed for purposes of triangulation. Thematic analysis of transcript data indicate numerous findings including: (i) difficulties regarding patients‘ prior understanding of risks involved with their medical procedure; (ii) the problematic response of the health system post-procedure; (iii) multiple adverse effects upon life functioning; (iv) limited recourse options for patients; and (v) the approach desired in terms of how patient harm should be systemically handled. In addition, IES results indicate a clinically significant level of distress in the sample as a whole. To discuss findings, a cross-disciplinary approach is adopted that draws upon sociology, medicine, medical anthropology, psychology, philosophy, history, ethics, law, and political theory. Furthermore, an overall explanatory framework is proposed in terms of the master themes of power and trauma. In terms of the theme of power, a postmodernist analysis explores the politics of patient harm, particularly the dynamics surrounding the politics of knowledge (e.g., notions of subjective versus objective knowledge, informed consent, and open disclosure). This analysis suggests that patient care is not the prime function of the health system, which appears more focussed upon serving the interests of those in the upper levels of its hierarchy. In terms of the master theme of trauma, current understandings of posttraumatic stress disorder (PTSD) are critiqued, and based on data from this research as well as the international literature, a new model of trauma is proposed. This model is based upon the principle of homeostasis observed in biology, whereby within every cell or organism a state of equilibrium is sought and maintained. The proposed model identifies several bio-psychosocial markers of trauma across its three main phases. These trauma markers include: (i) a profound sense of loss; (ii) a lack of perceived control; (iii) passive trauma processing responses; (iv) an identity crisis; (v) a quest to fully understand the trauma event; (vi) a need for social validation of the traumatic experience; and (vii) posttraumatic adaption with the possibility of positive change. To further explore the master themes of power and trauma, a natural group interview is carried out at a meeting of a patient support group for arachnoiditis. Observations at this meeting and members‘ stories in general support the homeostatic model of trauma, particularly the quest to find answers in the face of distressing experience, as well as the need for social recognition of that experience. In addition, the sociopolitical response to arachnoiditis highlights how public domains of knowledge are largely constructed and controlled by vested interests. Implications of the data overall are discussed in terms of a cultural revolution being needed in health care to position core values around a prime focus upon patients as human beings.

Development and validation of novel methods for microbial source tracking based on Escherichia coli as an indicator of water quality

Microbial pollution in water periodically affects human health in Australia, particularly in times of drought and flood. There is an increasing need for the control of waterborn microbial pathogens. Methods, allowing the determination of the origin of faecal contamination in water, are generally referred to as Microbial Source Tracking (MST). Various approaches have been evaluated as indicatorsof microbial pathogens in water samples, including detection of different microorganisms and various host-specific markers. However, until today there have been no universal MST methods that could reliably determine the source (human or animal) of faecal contamination. Therefore, the use of multiple approaches is frequently advised. MST is currently recognised as a research tool, rather than something to be included in routine practices. The main focus of this research was to develop novel and universally applicable methods to meet the demands for MST methods in routine testing of water samples. Escherichia coli was chosen initially as the object organism for our studies as, historically and globally, it is the standard indicator of microbial contamination in water. In this thesis, three approaches are described: single nucleotide polymorphism (SNP) genotyping, clustered regularly interspaced short palindromic repeats (CRISPR) screening using high resolution melt analysis (HRMA) methods and phage detection development based on CRISPR types. The advantage of the combination SNP genotyping and CRISPR genes has been discussed in this study. For the first time, a highly discriminatory single nucleotide polymorphism interrogation of E. coli population was applied to identify the host-specific cluster. Six human and one animal-specific SNP profile were revealed. SNP genotyping was successfully applied in the field investigations of the Coomera watershed, South-East Queensland, Australia. Four human profiles [11], [29], [32] and [45] and animal specific SNP profile [7] were detected in water. Two human-specific profiles [29] and [11] were found to be prevalent in the samples over a time period of years. The rainfall (24 and 72 hours), tide height and time, general land use (rural, suburban), seasons, distance from the river mouth and salinity show a lack of relashionship with the diversity of SNP profiles present in the Coomera watershed (p values > 0.05). Nevertheless, SNP genotyping method is able to identify and distinquish between human- and non-human specific E. coli isolates in water sources within one day. In some samples, only mixed profiles were detected. To further investigate host-specificity in these mixed profiles CRISPR screening protocol was developed, to be used on the set of E. coli, previously analysed for SNP profiles. CRISPR loci, which are the pattern of previous DNA coliphages attacks, were considered to be a promising tool for detecting host-specific markers in E. coli. Spacers in CRISPR loci could also reveal the dynamics of virulence in E. coli as well in other pathogens in water. Despite the fact that host-specificity was not observed in the set of E. coli analysed, CRISPR alleles were shown to be useful in detection of the geographical site of sources. HRMA allows determination of ‘different’ and ‘same’ CRISPR alleles and can be introduced in water monitoring as a cost-effective and rapid method. Overall, we show that the identified human specific SNP profiles [11], [29], [32] and [45] can be useful as marker genotypes globally for identification of human faecal contamination in water. Developed in the current study, the SNP typing approach can be used in water monitoring laboratories as an inexpensive, high-throughput and easy adapted protocol. The unique approach based on E. coli spacers for the search for unknown phage was developed to examine the host-specifity in phage sequences. Preliminary experiments on the recombinant plasmids showed the possibility of using this method for recovering phage sequences. Future studies will determine the host-specificity of DNA phage genotyping as soon as first reliable sequences can be acquired. No doubt, only implication of multiple approaches in MST will allow identification of the character of microbial contamination with higher confidence and readability.