Highly discriminatory single-nucleotide polymorphism interrogation of Escherichia coli by use of allele-specific real-time PCR and eBURST analysis

In total, 782 Escherichia coli strains originating from various host sources have been analyzed in this study by using a highly discriminatory single-nucleotide polymorphism (SNP) approach. A set of eight SNPs, with a discrimination value (Simpson's index of diversity [D]) of 0.96, was determined using the Minimum SNPs software, based on sequences of housekeeping genes from the E. coli multilocus sequence typing (MLST) database. Allele-specific real-time PCR was used to screen 114 E. coli isolates from various fecal sources in Southeast Queensland (SEQ). The combined analysis of both the MLST database and SEQ E. coli isolates using eight high-D SNPs resolved the isolates into 74 SNP profiles. The data obtained suggest that SNP typing is a promising approach for the discrimination of host-specific groups and allows for the identification of human-specific E. coli in environmental samples. However, a more diverse E. coli collection is required to determine animal- and environment-specific E. coli SNP profiles due to the abundance of human E. coli strains (56%) in the MLST database.

The uses of satire : unorthodox news, cultural chaos and the interrogation of power

This article focuses on the satirical Australian show The Chaser’s War on Everything, and uses it to critically assess the potential political and social ramifications of what McNair (2006) has called ‘cultural chaos’. Drawing upon and analysing several examples from this particular program, alongside interviews with its production team and qualitative audience research, this article argues that this TV show’s engagement with politicians and political issues, in a way that departs from the conventions of traditional journalism, offers a significant opportunity for the interrogation of power. The program’s use of often bizarre and unexpected comedic confrontation allows it to present a perhaps more authentic image of political agents than is often cultivated in mainstream journalism. This suggests therefore that the shift from homogeneity to heterogeneity in the news media – which McNair (2006) sees as a key feature of cultural chaos – presents a significant challenge to those who wish to retain control over what the public sees and understands about the political world, and is a development which should be viewed in positive terms.

Groundwater visualisation system (GVS) : a software framework for integrated display and interrogation of conceptual hydrogeological models, data and time-series animation

Management of groundwater systems requires realistic conceptual hydrogeological models as a framework for numerical simulation modelling, but also for system understanding and communicating this to stakeholders and the broader community. To help overcome these challenges we developed GVS (Groundwater Visualisation System), a stand-alone desktop software package that uses interactive 3D visualisation and animation techniques. The goal was a user-friendly groundwater management tool that could support a range of existing real-world and pre-processed data, both surface and subsurface, including geology and various types of temporal hydrological information. GVS allows these data to be integrated into a single conceptual hydrogeological model. In addition, 3D geological models produced externally using other software packages, can readily be imported into GVS models, as can outputs of simulations (e.g. piezometric surfaces) produced by software such as MODFLOW or FEFLOW. Boreholes can be integrated, showing any down-hole data and properties, including screen information, intersected geology, water level data and water chemistry. Animation is used to display spatial and temporal changes, with time-series data such as rainfall, standing water levels and electrical conductivity, displaying dynamic processes. Time and space variations can be presented using a range of contouring and colour mapping techniques, in addition to interactive plots of time-series parameters. Other types of data, for example, demographics and cultural information, can also be readily incorporated. The GVS software can execute on a standard Windows or Linux-based PC with a minimum of 2 GB RAM, and the model output is easy and inexpensive to distribute, by download or via USB/DVD/CD. Example models are described here for three groundwater systems in Queensland, northeastern Australia: two unconfined alluvial groundwater systems with intensive irrigation, the Lockyer Valley and the upper Condamine Valley, and the Surat Basin, a large sedimentary basin of confined artesian aquifers. This latter example required more detail in the hydrostratigraphy, correlation of formations with drillholes and visualisation of simulation piezometric surfaces. Both alluvial system GVS models were developed during drought conditions to support government strategies to implement groundwater management. The Surat Basin model was industry sponsored research, for coal seam gas groundwater management and community information and consultation. The “virtual” groundwater systems in these 3D GVS models can be interactively interrogated by standard functions, plus production of 2D cross-sections, data selection from the 3D scene, rear end database and plot displays. A unique feature is that GVS allows investigation of time-series data across different display modes, both 2D and 3D. GVS has been used successfully as a tool to enhance community/stakeholder understanding and knowledge of groundwater systems and is of value for training and educational purposes. Projects completed confirm that GVS provides a powerful support to management and decision making, and as a tool for interpretation of groundwater system hydrological processes. A highly effective visualisation output is the production of short videos (e.g. 2–5 min) based on sequences of camera ‘fly-throughs’ and screen images. Further work involves developing support for multi-screen displays and touch-screen technologies, distributed rendering, gestural interaction systems. To highlight the visualisation and animation capability of the GVS software, links to related multimedia hosted online sites are included in the references.

Effect of the mediator in feedback mode-based SECM interrogation of indium tin-oxide and boron-doped diamond electrodes

Indium tin-oxide (ITO) and polycrystalline boron-doped diamond (BDD) have been examined in detail using the scanning electrochemical microscopy technique in feedback mode. For the interrogation of electrodes made from these materials, the choice of mediator has been varied. Using Ru(CN) 4− 6 (aq), ferrocene methanol (FcMeOH), Fe(CN) 3− 6 (aq) and Ru(NH 3) 3+ 6 (aq), approach curve experiments have been performed, and for purposes of comparison, calculations of the apparent heterogeneous electron transfer rates (k app) have been made using these data. In general, it would appear that values of k app are affected mainly by the position of the mediator reversible potential relative to the relevant semiconductor band edge (associated with majority carriers). For both the ITO (n type) and BDD (p type) electrodes, charge transfer is impeded and values are very low when using FcMeOH and Fe(CN) 3− 6 (aq) as mediators, and the use of Ru(NH 3) 3+ 6(aq) results in the largest value of k app. With ITO, the surface is chemically homogeneous and no variation is observed for any given mediator. Data is also presented where the potential of the ITO electrode is fixed using a ratio of the mediators Fe(CN) 3− 6(aq) and Fe(CN) 4− 6(aq). In stark contrast, the BDD electrode is quite the opposite and a range of k app values are observed for all mediators depending on the position on the surface. Both electrode surfaces are very flat and very smooth, and hence, for BDD, variations in feedback current imply a variation in the electrochemical activity. A comparison of the feedback current where the substrate is biased and unbiased shows a surprising degree of proportionality.

Negotiating multiple identities between school and the outside world : A critical discourse analysis

This article examines interview talk of three students in an Australian high school to show how they negotiate their young adult identities between school and the outside world. It draws on Bakhtin’s concepts of dialogism and heteroglossia to argue that identities are linguistically and corporeally constituted. A critical discourse analysis of segments of transcribed interviews and student-related public documents finds a mismatch between a social justice curriculum at school and its transfer into students’ accounts of outside school lived realities. The article concludes that a productive social justice pedagogy must use its key principles of (con)textual interrogation to engage students in reflexive practice about their positioning within and against discourses of social justice in their student and civic lives. An impending national curriculum must decide whether or not it negotiates the discursive divide any better.

The use of narrative text for injury surveillance research : a systematic review

Objective: To summarise the extent to which narrative text fields in administrative health data are used to gather information about the event resulting in presentation to a health care provider for treatment of an injury, and to highlight best practise approaches to conducting narrative text interrogation for injury surveillance purposes.----- Design: Systematic review----- Data sources: Electronic databases searched included CINAHL, Google Scholar, Medline, Proquest, PubMed and PubMed Central.. Snowballing strategies were employed by searching the bibliographies of retrieved references to identify relevant associated articles.----- Selection criteria: Papers were selected if the study used a health-related database and if the study objectives were to a) use text field to identify injury cases or use text fields to extract additional information on injury circumstances not available from coded data or b) use text fields to assess accuracy of coded data fields for injury-related cases or c) describe methods/approaches for extracting injury information from text fields.----- Methods: The papers identified through the search were independently screened by two authors for inclusion, resulting in 41 papers selected for review. Due to heterogeneity between studies metaanalysis was not performed.----- Results: The majority of papers reviewed focused on describing injury epidemiology trends using coded data and text fields to supplement coded data (28 papers), with these studies demonstrating the value of text data for providing more specific information beyond what had been coded to enable case selection or provide circumstantial information. Caveats were expressed in terms of the consistency and completeness of recording of text information resulting in underestimates when using these data. Four coding validation papers were reviewed with these studies showing the utility of text data for validating and checking the accuracy of coded data. Seven studies (9 papers) described methods for interrogating injury text fields for systematic extraction of information, with a combination of manual and semi-automated methods used to refine and develop algorithms for extraction and classification of coded data from text. Quality assurance approaches to assessing the robustness of the methods for extracting text data was only discussed in 8 of the epidemiology papers, and 1 of the coding validation papers. All of the text interrogation methodology papers described systematic approaches to ensuring the quality of the approach.----- Conclusions: Manual review and coding approaches, text search methods, and statistical tools have been utilised to extract data from narrative text and translate it into useable, detailed injury event information. These techniques can and have been applied to administrative datasets to identify specific injury types and add value to previously coded injury datasets. Only a few studies thoroughly described the methods which were used for text mining and less than half of the studies which were reviewed used/described quality assurance methods for ensuring the robustness of the approach. New techniques utilising semi-automated computerised approaches and Bayesian/clustering statistical methods offer the potential to further develop and standardise the analysis of narrative text for injury surveillance.

Whatever it takes : an exploration of writing tools and strategies for completing a novel

This thesis consists of a novel written with the express purpose of exploring what practices and strategies are most useful in writing novel-length fiction as well as an exegesis which discusses the process. By its very nature, an undergraduate degree in Creative Writing is broad and general in approach. The Creative Writing undergraduate is being trained to manage many and varying writing tasks but none of them larger than can be readily marked and assessed in class quantities. This does not prepare the writing graduate for the gargantuan task of managing a project as large as a single title novel which can be up to 100,000 words and often is more. This study explores the question of what writing tools and practices best equip an emerging writer to begin, write and manage a long narrative within a deadline.

Development of rapid and highly resolving combinatorial genotyping schemes for Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni followed by Campylobacter coli contribute substantially to the economic and public health burden attributed to food-borne infections in Australia. Genotypic characterisation of isolates has provided new insights into the epidemiology and pathogenesis of C. jejuni and C. coli. However, currently available methods are not conducive to large scale epidemiological investigations that are necessary to elucidate the global epidemiology of these common food-borne pathogens. This research aims to develop high resolution C. jejuni and C. coli genotyping schemes that are convenient for high throughput applications. Real-time PCR and High Resolution Melt (HRM) analysis are fundamental to the genotyping schemes developed in this study and enable rapid, cost effective, interrogation of a range of different polymorphic sites within the Campylobacter genome. While the sources and routes of transmission of campylobacters are unclear, handling and consumption of poultry meat is frequently associated with human campylobacteriosis in Australia. Therefore, chicken derived C. jejuni and C. coli isolates were used to develop and verify the methods described in this study. The first aim of this study describes the application of MLST-SNP (Multi Locus Sequence Typing Single Nucleotide Polymorphisms) + binary typing to 87 chicken C. jejuni isolates using real-time PCR analysis. These typing schemes were developed previously by our research group using isolates from campylobacteriosis patients. This present study showed that SNP + binary typing alone or in combination are effective at detecting epidemiological linkage between chicken derived Campylobacter isolates and enable data comparisons with other MLST based investigations. SNP + binary types obtained from chicken isolates in this study were compared with a previously SNP + binary and MLST typed set of human isolates. Common genotypes between the two collections of isolates were identified and ST-524 represented a clone that could be worth monitoring in the chicken meat industry. In contrast, ST-48, mainly associated with bovine hosts, was abundant in the human isolates. This genotype was, however, absent in the chicken isolates, indicating the role of non-poultry sources in causing human Campylobacter infections. This demonstrates the potential application of SNP + binary typing for epidemiological investigations and source tracing. While MLST SNPs and binary genes comprise the more stable backbone of the Campylobacter genome and are indicative of long term epidemiological linkage of the isolates, the development of a High Resolution Melt (HRM) based curve analysis method to interrogate the hypervariable Campylobacter flagellin encoding gene (flaA) is described in Aim 2 of this study. The flaA gene product appears to be an important pathogenicity determinant of campylobacters and is therefore a popular target for genotyping, especially for short term epidemiological studies such as outbreak investigations. HRM curve analysis based flaA interrogation is a single-step closed-tube method that provides portable data that can be easily shared and accessed. Critical to the development of flaA HRM was the use of flaA specific primers that did not amplify the flaB gene. HRM curve analysis flaA interrogation was successful at discriminating the 47 sequence variants identified within the 87 C. jejuni and 15 C. coli isolates and correlated to the epidemiological background of the isolates. In the combinatorial format, the resolving power of flaA was additive to that of SNP + binary typing and CRISPR (Clustered regularly spaced short Palindromic repeats) HRM and fits the PHRANA (Progressive hierarchical resolving assays using nucleic acids) approach for genotyping. The use of statistical methods to analyse the HRM data enhanced sophistication of the method. Therefore, flaA HRM is a rapid and cost effective alternative to gel- or sequence-based flaA typing schemes. Aim 3 of this study describes the development of a novel bioinformatics driven method to interrogate Campylobacter MLST gene fragments using HRM, and is called ‘SNP Nucleated Minim MLST’ or ‘Minim typing’. The method involves HRM interrogation of MLST fragments that encompass highly informative “Nucleating SNPS” to ensure high resolution. Selection of fragments potentially suited to HRM analysis was conducted in silico using i) “Minimum SNPs” and ii) the new ’HRMtype’ software packages. Species specific sets of six “Nucleating SNPs” and six HRM fragments were identified for both C. jejuni and C. coli to ensure high typeability and resolution relevant to the MLST database. ‘Minim typing’ was tested empirically by typing 15 C. jejuni and five C. coli isolates. The association of clonal complexes (CC) to each isolate by ‘Minim typing’ and SNP + binary typing were used to compare the two MLST interrogation schemes. The CCs linked with each C. jejuni isolate were consistent for both methods. Thus, ‘Minim typing’ is an efficient and cost effective method to interrogate MLST genes. However, it is not expected to be independent, or meet the resolution of, sequence based MLST gene interrogation. ‘Minim typing’ in combination with flaA HRM is envisaged to comprise a highly resolving combinatorial typing scheme developed around the HRM platform and is amenable to automation and multiplexing. The genotyping techniques described in this thesis involve the combinatorial interrogation of differentially evolving genetic markers on the unified real-time PCR and HRM platform. They provide high resolution and are simple, cost effective and ideally suited to rapid and high throughput genotyping for these common food-borne pathogens.

The quest for authenticity in the west – negotiating the self in late modern cultures

Across western culture in the late modern era a number of phenomena have emerged that seek to challenge mainstream consumer capitalism and its effects on everyday lifestyles. Two of these movements labelled as Seachange (Amenity Migration) and Downshifting have grown steadily in popularity within the public sphere and also academic discourse. In this thesis these phenomena are investigated further using a Durkheimian platform for theoretical interrogation. It is argued that while previous research accomplishes much in the investigation of Seachange and Downshifting, there is a significant gap in theoretical explanation and synthesis that requires filling. Thus in this research, it is argued that the concept of self-authenticity assists in the fulfilment of this aim. It is shown here that authenticity guides the construction, negotiation and experience of the phenomena which serves to authenticate the self. It is further argued however that Downshifting and Seachange reflect a wider theme of the self where the individual seeks protection from the profane impacts of advanced capitalism. Subsequently, the thesis aims not only to reveal the underlying principles which feed each phenomenon, but also relate them back to a wider cultural narrative of the sacred self.

On location : the imagined private interior in public life

This paper reveals the interior landscapes of selected contemporary Australian films, such as The Caterpillar Wish and Bad Boy Bubby, to develop a number of thematic influences on the manner in which domestic and private lives are constructed through filmic imagination. The research uncovers the conditions that contribute to particular scenographic representations of the humble interiors that act as both backdrop and performer to subtle and often troubled narratives. Such readings are informed by the theoretical works of writer Gertrude Stein, among others, who explore the relationships between the scenographic third dimension and the fourth dimensional performance in the representation of narrative space. A further theoretical thread lies in Giuliana Bruno’s work on the tension between private and public filmic space, which is explored through the public outing of intensely private spaces generated through narratives framed by the specificities of found interiors. Beyond the interrogation of qualities of imagined filmic space is the condition whereby locations, once transformed by the event of movie making are consequently forever revised. These altered conditions subsequently reinvest the lives of those who return to the location with layered narratives of occupation. Situationally, the now reconverted interior performs as contributor to subsequent private inhabitation, even if only as imagined space. The possibility here is that the qualities of the original may be superimposed and recontextualised to invest post-produced interiors with the qualities of the other space as imagined. This reading of film space explores new theoretical design scenarios for imagined and everyday interior landscapes.

Accommodating serendipity in the active studio

This paper presents a series of ongoing experiments to facilitate serendipity in the design studio through a diversity of delivery modes. These experiments are conducted in a second year architectural design studio, and include physical, dramatic and musical performance. The act of designing is always exploratory, always seeking an unknown resolution, and the ability to see and capture the value in the unexpected is a critical aspect of such creative design practice. Engaging with the unexpected is however a difficult ability to develop in students. Just how can a student be schooled in such abilities when the challenge and the context are unforeseeable? How can students be offered meaningful feedback about an issue that cannot be predicted, when feedback comes in the form of extrinsic assessment from a tutor? This project establishes a number of student activities that seek to provide intrinsic feedback from the activity itself. Further to this, the project seeks to heighten student engagement with the project through physical expression and performance: utilising more of the students’ senses than just vision and hearing. Diana Laurillard’s theories of conversational frameworks (2002) are used to interrogate the act of dramatic performance as an act of learning, with particular reference to the serendipitous activities of design. Such interrogation highlights the feedback mechanisms that facilitate intrinsic feedback and fast, if not instantaneous, cycles of learning. The physical act of performance itself provides a learning experience that is not replicable in other modes of delivery. Student feedback data and independent assessment of project outcomes are used to assess the success of this studio model.

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.

Fiber Bragg Grating sensors for railway systems

Fiber Bragg grating (FBG) sensor technology has been attracting substantial industrial interests for the last decade. FBG sensors have seen increasing acceptance and widespread use for structural sensing and health monitoring applications in composites, civil engineering, aerospace, marine, oil & gas, and smart structures. One transportation system that has been benefitted tremendously from this technology is railways, where it is of the utmost importance to understand the structural and operating conditions of rails as well as that of freight and passenger service cars to ensure safe and reliable operation. Fiberoptic sensors, mostly in the form of FBGs, offer various important characteristics, such as EMI/RFI immunity, multiplexing capability, and very long-range interrogation (up to 230 km between FBGs and measurement unit), over the conventional electrical sensors for the distinctive operational conditions in railways. FBG sensors are unique from other types of fiber-optic sensors as the measured information is wavelength-encoded, which provides self-referencing and renders their signals less susceptible to intensity fluctuations. In addition, FBGs are reflective sensors that can be interrogated from either end, providing redundancy to FBG sensing networks. These two unique features are particularly important for the railway industry where safe and reliable operations are the major concerns. Furthermore, FBGs are very versatile and transducers based on FBGs can be designed to measure a wide range of parameters such as acceleration and inclination. Consequently, a single interrogator can deal with a large number of FBG sensors to measure a multitude of parameters at different locations that spans over a large area.

Red cap

The field of research is contemporary theatre practice with a community focus. In 2007, La Boite Theatre Company partnered with the Queensland Music Festival to produce an operatic representation of the 1964 Mt Isa industrial dispute, focussed on the charismatic figure of Pat Mackie. “Community theatre” is often criticised on grounds that the work aims only to satisfy community outcomes. This work explored whether a story from a specific location, which is very much an embedded story in the culture of the Mt Isa community, could be told in such a way as to appeal to, resonate with, and have relevance for, broader national and international audiences. To address this question required rigorous interrogation of both content and form. The play was researched through interviews with members of the Mt Isa community, political leadership at the time of the dispute, and participants of the dispute, including Pat Mackie himself. The production was then framed as an oratorio. Uniquely, the play had two back-to-back seasons; the first in Mount Isa (3 shows: 1500 people including a significant number of school children) and a 4-week season at the Roundhouse Theatre, Brisbane (over 5,000 attendances). In each location, a chorale was formed of community participants who, alongside the professional cast, performed the work. The production and its complementary exhibition had a significant local and national profile. The project was featured in The Australian newspaper’s Queensland Music Festival wrap-up as an exemplar of successful community engagement and creative adventure. Playlab Press has since published the script.