Development of a Rep-inducible, BBTV-based expression system in banana

Banana bunchy top is regarded as the most important viral disease of banana, causing significant yield losses worldwide. The disease is caused by Banana bunchy top virus (BBTV), which is a circular ssDNA virus belonging to the genus Babuvirus in the family Nanoviridae. There are currently few effective control strategies for this and other ssDNA viruses. “In Plant Activation” (InPAct) is a novel technology being developed at QUT for ssDNA virus-activated suicide gene expression. The technology exploits the rolling circle replication mechanism of ssDNA viruses and is based on a unique “split” gene design such that suicide gene expression is only activated in the presence of the viral Rep. This PhD project aimed to develop a BBTV-based InPAct system as a suicide gene strategy to control BBTV. The BBTV-based InPAct vector design requires a BBTV intergenic region (IR) to be embedded within an intron in the gene expression cassette. To ensure that the BBTV IR would not interfere with intron splicing, a TEST vector was initially generated that contained the entire BBTV IR embedded within an intron in a β-glucuronidase (GUS) expression vector. Transient GUS assays in banana embryogenic cell suspensions indicated that cryptic intron splice sites were present within the IR. Transcript analysis revealed two cryptic intron splice sites in the Domain III sequence of the CR-M within the IR. Removal of the CR-M from the TEST vector resulted in an enhancement of GUS expression suggesting that the cryptic intron splice sites had been removed. An InPAct GUS vector was subsequently generated that contained the modified BBTV IR, with the CR-M (minus Domain III) repositioned within the InPAct cassette. Using transient histochemical and fluorometric GUS assays in banana embryogenic cells, the InPAct GUS vector was shown to be activated in the presence of the BBTV Rep. However, the presence of both BBTV Rep and Clink was shown to have a deleterious effect on GUS expression suggesting that these proteins were cytotoxic at the levels expressed. Analysis of replication of the InPAct vectors by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector through the nicking/ligation activity of BBTV Rep. However, Rep-mediated episomal replicons, indicative of rolling circle replication of the released circularised cassettes, were not observed. The inability of the InPAct cassette to be replicated was further investigated. To examine whether the absence of Domain III of the CR-M was responsible, a suite of modified BBTV-based InPAct GUS vectors was constructed that contained the CR-M with the inclusion of Domain III, the CR-M with the inclusion of Domain III and additional upstream IR sequence, or no CR-M. Analysis of replication by Southern hybridisation revealed that neither the presence of Domain III, nor the entire CR-M, had an effect on replication levels. Since the InPAct cassette was significantly larger than the native BBTV genomic components (approximately 1 kb), the effect of InPAct cassette size on replication was also investigated. A suite of size variant BBTV-based vectors was constructed that increased the size of a replication competent cassette to 1.1 kbp through to 2.1 kbp.. Analysis of replication by Southern hybridisation revealed that an increase in vector size above approximately 1.5 - 1.7 kbp resulted in a decrease in replication. Following the demonstration of Rep-mediated release, circularisation and expression from the InPAct GUS vector, an InPAct vector was generated in which the uidA reporter gene was replaced with the ribonuclease-encoding suicide gene, barnase. Initially, a TEST vector was generated to assess the cytotoxicity of Barnase on banana cells. Although transient assays revealed a Barnase-induced cytotoxic effect in banana cells, the expression levels were sub-optimal. An InPAct BARNASE vector was generated and tested for BBTV Rep-activated Barnase expression using transient assays in banana embryogenic cells. High levels of background expression from the InPAct BARNASE vector made it difficult to accurately assess Rep-activated Barnase expression. Analysis of replication by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector but no Rep-mediated episomal replicons indicative of rolling circle replication of the released circularised cassettes were again observed. Despite the inability of the InPAct vectors to replicate to enable high level gene expression, the InPAct BARNASE vector was assessed in planta for BBTV Rep-mediated activation of Barnase expression. Eleven lines of transgenic InPAct BARNASE banana plants were generated by Agrobacterium-mediated transformation and were challenged with viruliferous Pentalonia nigronervosa. At least one clonal plant in each line developed bunchy top symptoms and infection was confirmed by PCR. No localised lesions were observed on any plants, nor was there any localised GUS expression in the one InPAct GUS line challenged with viruliferous aphids. The results presented in this thesis are the first study towards the development of a BBTV-based InPAct system as a Rep-activatable suicide gene expression system to control BBTV. Although further optimisation of the vectors is necessary, the preliminary results suggest that this approach has the potential to be an effective control strategy for BBTV. The use of iterons within the InPAct vectors that are recognised by Reps from different ssDNA plant viruses may provide a broad-spectrum resistance strategy against multiple ssDNA plant viruses. Further, this technology holds great promise as a platform technology for the molecular farming of high-value proteins in vitro or in vivo through expression of the ssDNA virus Rep protein.

Governance and effectiveness in regional planning: an analysis of North American, European, and Australasian practice

Over the past several years, there has been resurgent interest in regional planning in North America, Europe and Australasia. Spurred by issues such as metropolitan growth, transportation infrastructure, environmental management and economic development, many states and metropolitan regions are undertaking new planning initiatives. These regional efforts have also raised significant question about governance structures, accountability and measures of effectiveness.n this paper, the authors conducted an international review of ten case studies from the United States, Canada, England, Belgium, New Zealand and Australia to explore several critical questions. Using qualitative data template, the research team reviewed plans, documents, web sites and published literature to address three questions. First, what are the governance arrangements for delivering regional planning? Second, what are the mechanisms linking regional plans with state plans (when relevant) and local plans? Third, what means and mechanisms do these regional plans use to evaluate and measure effectiveness? The case study analysis revealed several common themes. First, there is an increasing focus on goverance at the regional level, which is being driven by a range of trends, including regional spatial development initiatives in Europe, regional transportation issues in the US, and the growth of metropolitan regions generally. However, there is considerable variation in how regional governance arrangements are being played out. Similarly, there is a range of processes being used at the regional level to guide planning that range from broad ranging (thick) processes to narrow and limited (thin) approaches. Finally, evaluation and monitoring of regional planning efforts are compiling data on inputs, processes, outputs and outcomes. Although there is increased attention being paid to indicators and monitoring, most of it falls into outcome evaluations such as Agenda 21 or sustainability reporting. Based on our review we suggest there is a need for increased attention on input, process and output indicators and clearer linkages of these indicators in monitoring and evaluation frameworks. The focus on outcome indicators, such as sustainability indicators, creates feedback systems that are too long-term and remote for effective monitoring and feedback. Although we found some examples of where these kinds of monitoring frameworks are linked into a system of governance, there is a need for clearer conceptual development for both theory and practice.

Critically evaluating rural women’s participation and empowerment : An interdisciplinary feminist framework for research and analysis

Community development is increasingly using participatory processes that aim to be inclusive and empowering. However, researchers have found that such processes can have contradictory effects. Australian research has highlighted the significant leadership of rural women in sustainable community and economic development and in the adoption of new communication technologies such as the Internet. A focus on gender in participatory development may therefore lead to more effective programs and policies. This chapter outlines an interdisciplinary feminist framework for critically evaluating the participation and empowerment of rural women. This framework was found effective in evaluating an Australian project that aimed to enhance rural women’s access to communication technologies and to empower its participants. Its multiple theoretical and methodological approaches are outlined. The framework advocates an analysis of diversity and difference and the macro and micro contexts. Some principles and strategies for rural women’s inclusion, participation, empowerment, and for participatory feminist evaluation are outlined.

Exploratory spatial analysis of social and environmental factors associated with the incidence of Ross River Virus in Brisbane, Australia

We used geographic information systems and a spatial analysis approach to explore the pattern of Ross River virus (RRV) incidence in Brisbane, Australia. Climate, vegetation and socioeconomic data in 2001 were obtained from the Australian Bureau of Meteorology, the Brisbane City Council and the Australian Bureau of Statistics, respectively. Information on the RRV cases was obtained from the Queensland Department of Health. Spatial and multiple negative binomial regression models were used to identify the socioeconomic and environmental determinants of RRV transmission. The results show that RRV activity was primarily concentrated in the northeastern, northwestern, and southeastern regions in Brisbane. Multiple negative binomial regression models showed that the spatial pattern of RRV disease in Brisbane seemed to be determined by a combination of local ecologic, socioeconomic, and environmental factors.

A qualitative analysis of local community communications

This paper explores how people communicate in reference to local interests and suggests information and communication technology (ICT) design for enhancement of local community networks. Qualitative data was gathered from participant observations of local community collective action and open interviews with active community members. Data analysis revealed concepts, leading to categories in relation to local interactions and interests. Design suggestions consider introducing people to local community private-strategic activity via public displays that indicate simple entry points to active participation, and creating information collections according to local community perspectives for long-term reference.

Co-creating games: A co-evolutionary analysis

ABSTRACT. The phenomenon of consumer co-creation is often framed in terms of whether either economic market forces or socio-cultural non-market forces ultimately dominate. We propose an alternate model of consumer co-creation in terms of co-evolution between markets and non-markets. Our model is based on a recent ethnographic study of a massively multiplayer online game through its development, release and ultimate failure, and cast in terms of two explanatory models: multiple games and social network markets. We conclude that consumer co-creation is indeed complex, but in ways that relate to both emergent market expectations and the evolution of markets, not to the transcendence of markets.

Search engine content analysis

Search engines have forever changed the way people access and discover knowledge, allowing information about almost any subject to be quickly and easily retrieved within seconds. As increasingly more material becomes available electronically the influence of search engines on our lives will continue to grow. This presents the problem of how to find what information is contained in each search engine, what bias a search engine may have, and how to select the best search engine for a particular information need. This research introduces a new method, search engine content analysis, in order to solve the above problem. Search engine content analysis is a new development of traditional information retrieval field called collection selection, which deals with general information repositories. Current research in collection selection relies on full access to the collection or estimations of the size of the collections. Also collection descriptions are often represented as term occurrence statistics. An automatic ontology learning method is developed for the search engine content analysis, which trains an ontology with world knowledge of hundreds of different subjects in a multilevel taxonomy. This ontology is then mined to find important classification rules, and these rules are used to perform an extensive analysis of the content of the largest general purpose Internet search engines in use today. Instead of representing collections as a set of terms, which commonly occurs in collection selection, they are represented as a set of subjects, leading to a more robust representation of information and a decrease of synonymy. The ontology based method was compared with ReDDE (Relevant Document Distribution Estimation method for resource selection) using the standard R-value metric, with encouraging results. ReDDE is the current state of the art collection selection method which relies on collection size estimation. The method was also used to analyse the content of the most popular search engines in use today, including Google and Yahoo. In addition several specialist search engines such as Pubmed and the U.S. Department of Agriculture were analysed. In conclusion, this research shows that the ontology based method mitigates the need for collection size estimation.

An interpretative phenomenological analysis of adult clients’ experience of My Career Chapter

This paper presents an investigation into the properties of a new narrative technique for career assessment and counselling, My Career Chapter: A Dialogical Autobiography. This technique is used to facilitate clients’ construction of a meaningful career-related autobiography. Previous research indicates the usefulness of My Career Chapter for adult clients and its alignment with recommendations for the development and application of qualitative assessment and counselling techniques. This study specifically commences research into the technique’s applicability for adolescents. A focus group, comprised of guidance counselling professionals whose work primarily pertained to the needs of adolescents, found that there is potential to develop a version of My Career Chapter that is suitable for adolescents.

Context and models for the analysis of individual and group needs

This paper summarizes the papers presented in the thematic stream Models for the Analysis of Individual and Group Needs, at the 2007 IAEVG-SVP-NCDA Symposium: Vocational Psychology and Career Guidance Practice: An International Partnership. The predominant theme which emerged from the papers was that theory and practice need to be positioned within their contexts. For this paper, context has been formulated as a dimension ranging from the individual’s experience of himself or herself in conversations, including interpersonal transactions and body culture, through to broad higher levels of education, work, nation, and economy.

Using ontology for the representational analysis of process modelling techniques

Selecting an appropriate business process modelling technique forms an important task within the methodological challenges of a business process management project. While a plethora of available techniques has been developed over the last decades, there is an obvious shortage of well-accepted reference frameworks that can be used to evaluate and compare the capabilities of the different techniques. Academic progress has been made at least in the area of representational analyses that use ontology as a benchmark for such evaluations. This paper reflects on the comprehensive experiences with the application of a model based on the Bunge ontology in this context. A brief overview of the underlying research model characterizes the different steps in such a research project. A comparative summary of previous representational analyses of process modelling techniques over time gives insights into the relative maturity of selected process modelling techniques. Based on these experiences suggestions are made as to where ontology-based representational analyses could be further developed and what limitations are inherent to such analyses.

Application of the analytic hierarchy process (AHP) in multi-criteria analysis of the selection of intelligent building systems

The availability of innumerable intelligent building (IB) products, and the current dearth of inclusive building component selection methods suggest that decision makers might be confronted with the quandary of forming a particular combination of components to suit the needs of a specific IB project. Despite this problem, few empirical studies have so far been undertaken to analyse the selection of the IB systems, and to identify key selection criteria for major IB systems. This study is designed to fill these research gaps. Two surveys: a general survey and the analytic hierarchy process (AHP) survey are proposed to achieve these objectives. The first general survey aims to collect general views from IB experts and practitioners to identify the perceived critical selection criteria, while the AHP survey was conducted to prioritize and assign the important weightings for the perceived criteria in the general survey. Results generally suggest that each IB system was determined by a disparate set of selection criteria with different weightings. ‘Work efficiency’ is perceived to be most important core selection criterion for various IB systems, while ‘user comfort’, ‘safety’ and ‘cost effectiveness’ are also considered to be significant. Two sub-criteria, ‘reliability’ and ‘operating and maintenance costs’, are regarded as prime factors to be considered in selecting IB systems. The current study contributes to the industry and IB research in at least two aspects. First, it widens the understanding of the selection criteria, as well as their degree of importance, of the IB systems. It also adopts a multi-criteria AHP approach which is a new method to analyse and select the building systems in IB. Further research would investigate the inter-relationship amongst the selection criteria.

Electronic workplace surveillance and employee privacy : a comparative analysis of privacy protection in Australia and the United States

More than a century ago in their definitive work “The Right to Privacy” Samuel D. Warren and Louis D. Brandeis highlighted the challenges posed to individual privacy by advancing technology. Today’s workplace is characterised by its reliance on computer technology, particularly the use of email and the Internet to perform critical business functions. Increasingly these and other workplace activities are the focus of monitoring by employers. There is little formal regulation of electronic monitoring in Australian or United States workplaces. Without reasonable limits or controls, this has the potential to adversely affect employees’ privacy rights. Australia has a history of legislating to protect privacy rights, whereas the United States has relied on a combination of constitutional guarantees, federal and state statutes, and the common law. This thesis examines a number of existing and proposed statutory and other workplace privacy laws in Australia and the United States. The analysis demonstrates that existing measures fail to adequately regulate monitoring or provide employees with suitable remedies where unjustifiable intrusions occur. The thesis ultimately supports the view that enacting uniform legislation at the national level provides a more effective and comprehensive solution for both employers and employees. Chapter One provides a general introduction and briefly discusses issues relevant to electronic monitoring in the workplace. Chapter Two contains an overview of privacy law as it relates to electronic monitoring in Australian and United States workplaces. In Chapter Three there is an examination of the complaint process and remedies available to a hypothetical employee (Mary) who is concerned about protecting her privacy rights at work. Chapter Four provides an analysis of the major themes emerging from the research, and also discusses the draft national uniform legislation. Chapter Five details the proposed legislation in the form of the Workplace Surveillance and Monitoring Act, and Chapter Six contains the conclusion.

Review of vibration-based damage detection and condition assessment of bridge structures using structural health monitoring

As a part of vital infrastructure and transportation networks, bridge structures must function safely at all times. However, due to heavier and faster moving vehicular loads and function adjustment, such as Busway accommodation, many bridges are now operating at an overload beyond their design capacity. Additionally, the huge renovation and replacement costs always make the infrastructure owners difficult to undertake. Structural health monitoring (SHM) is set to assess condition and foresee probable failures of designated bridge(s), so as to monitor the structural health of the bridges. The SHM systems proposed recently are incorporated with Vibration-Based Damage Detection (VBDD) techniques, Statistical Methods and Signal processing techniques and have been regarded as efficient and economical ways to solve the problem. The recent development in damage detection and condition assessment techniques based on VBDD and statistical methods are reviewed. The VBDD methods based on changes in natural frequencies, curvature/strain modes, modal strain energy (MSE) dynamic flexibility, artificial neural networks (ANN) before and after damage and other signal processing methods like Wavelet techniques and empirical mode decomposition (EMD) / Hilbert spectrum methods are discussed here.