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.

Amblyopia treatment : an evidence-based approach to maximising treatment outcome

The basis of treatment for amblyopia (poor vision due to abnormal visual experience early in life) for 250 years has been patching of the unaffected eye for extended times to ensure a period of use of the affected eye. Over the last decade randomised controlled treatment trials have provided some evidence on how to tailor amblyopia therapy more precisely to achieve the best visual outcome with the least negative impact on the patient and the family. This review highlights the expansion of knowledge regarding treatment for amblyopia and aims to provide optometrists with a summary of research evidence to enable them to better treat amblyopia. Treatment for amblyopia is effective, as it reduces overall prevalence and severity of visual loss in this population. Correction of refractive error alone significantly improves visual acuity, sometimes to the point where further amblyopia treatment is not required. Atropine penalisation and patch occlusion are effective in treating amblyopia. Lesser amounts of occlusion or penalisation have been found to be just as effective as greater amounts. Recent evidence has highlighted that occlusion or penalisation in amblyopia treatment can create negative changes in behaviour in children and impact on family life. These complications should be considere when prescribing treatment because they can negatively affect compliance. Studies investigating the maximum age at which treatment of amblyopia can still be effective and the importance of near activities during occlusion are ongoing.

Competing in the new economy through large scale collaborative ventures (LSCV): a strategic marketing approach based on a study of the multifunction polis.

This paper draws on a study of government initiat ives aimed at facilitating economic development, specifically the Multifunction Polis Feasibility Study involving the governments and business enterprises of Australia and Japan (1987-1991). Large scale projects that involve collaboration between gove rnment and business (termed: large scale collaborative venture LSCV)are identified as one aspect of competing in the new economy . The study pursued the research propos ition that a LSCV can be effectively facilitated by following a theory based process similar to those in corporate practice. An approach to managing such ventures is outlined, based on strategic marketing theory that may enhance their success and thereby help countries part icipate more successfully in global competition through such ventures.

Gendered perceptions of experiential value in using web-based retail channels

Purpose – This paper compares the experiential consumption values that motivate consumer choice to purchase online for both male and female purchasers and non-purchasers. Design/methodology/approach – Using the theory of consumption value the study examines gendered perceptions of the functional, social and conditional value of using a virtual consumption setting for purchasing. Data was collected through an online survey and analysed using multiple discriminant analysis to determine meaningful differences between male and female purchasers and non-purchasers. Findings – The findings show that male online purchasers are discriminated from female purchasers by social value and from male non-purchasers by conditional value. Female purchasers are discriminated from male purchasers by functional value and from female non-purchasers by social value. Female non-purchasers are discriminated from female purchasers by conditional value. Male non-purchasers are discriminated from male purchasers by functional and social value. Research limitations/implications – Limitations include using an Internet survey and an Australian sample which may impact the generalisability of the findings to a wider population of Internet users. Future research should involve replication of the study in a country more or less developed in terms of gender composition of internet users to extend the generalisability of the findings. Additionally, researchers should examine whether other dimensions of consumption value,such as social influence through on- and off-line communication networks, may influence consumer choice to purchase online. Practical implications – The study provides practical implications for marketers to leverage consumption values that influence male and female consumers’ choice to purchase online and then drive their behaviour online through integrated marketing campaigns that involve both on- and offline strategies. Originality/value – The research makes an original contribution to the consumer behaviour literature as to date, no research has been found that undertakes such a comprehensive gender-based comparison of the perceived value of using a virtual consumption setting for purchasing.

Knowledge-Based Urban Development : Planning and Applications in the Information Era

In the globalizing world, knowledge and information (and the social and technological settings for their production and communication) are now seen as keys to economic prosperity. The economy of a knowledge city creates value-added products using research, technology, and brainpower. The social benefit of knowledge-based urban development (KBUD); however, extends beyond aggregate economic growth.

Now everything looks like a game : Mobile phones and location-based games.

This paper explores how we may transform peoples’ perceived access to cultural participation by exploiting the possible relationships between place, play and mobile devices. It presents SCOOT; a location-based game in order to investigate how aspects of game-play can be employed to evoke at once playful and culturally meaningful experiences of place. In particular this paper is concerned with how the portable, communicative and social affordances of mobile phones are integral to making a “now everything looks like a game” experience.

Cipher cities : effective user interface design for building a digital social network around location-based gaming

In this paper we explore what is required of a User Interface (UI) design in order to encourage participation around playing and creating Location-Based Games (LBGs). To base our research in practice, we present Cipher Cities, a web based system. Through the design of this system, we investigate how UI design can provide tools for complex content creation to compliment and encourage the use of mobile phones for designing, distributing, and playing LBGs. Furthermore we discuss how UI design can promote and support socialisation around LBGs through the design of functional interface components and services such as groups, user profiles, and player status listings.

Finding order in chaos : a problem based collaborative assignment for 1300 students across 19 disciplines

The use of collaborative assignments for assessment is a risky undertaking for students and course designers. Yet the benefits, in terms of core learning outcomes, competencies, collaborative sense making and student involvement, suggest that the effort is worthwhile. Formal descriptions and rules do little to ameliorate the perception of risk and increased anxiety by students. (Ryan, 2007). BEB100 Introducing Professional Learning is a faculty-wide foundation unit with over 1300 students from 19 disciplines across the Faculty of the Built Environment and Engineering (“BEE”) at the Queensland University of Technology (“QUT”), Brisbane, Australia. Finding order in chaos outlines the approach and justification, assessment criteria, learning resources, teamwork tools, tutorial management, communication strategies, 2007-09 Student Learning Experience Survey results, annual improvements, findings and outcomes.

Queensland's Smart State Initiative : a successful knowledge based urban development strategy

The chapter will set out to explain the KBUD and urban policy making processes in Queensland, Australia. This chapter will draw on providing a clear understanding on policy frameworks and relevant ICT applications of the Queensland ‘Smart State’ experience. The chapter is consisted of six sections. The first section following the introduction provides background information. The second section focuses on the KBUD processes in Queensland. The third section offers a comprehensive analysis of the ‘Queensland Smart State’ initiative, and it also identifies actors and goals of the agenda of Smart State experience. The fourth section reviews knowledge based development and ICT applications and policies of the Queensland Smart State and Brisbane Smart City experiences, and their impacts on Brisbane’s successful KBUD. The fifth section discusses knowledge hubs and ICT developments within the Brisbane metropolitan area. Then the chapter concludes with future trends and conclusion sections.

Knowledge-based strategic planning: harnessing (in)tangible assets of city-regions

Purpose – The aim of this paper is to investigate the ways of best managing city-regions’ valuable tangible and intangible assets while pursuing a knowledge-based urban development that is sustainable and competitive. Design/methodology/approach – The paper provides a theoretical framework to conceptualise a new strategic planning mechanism, knowledge-based strategic planning, which has been emerged as a planning mechanism for the knowledge-based urban development of post-industrial city-regions. Originality/value – The paper develops a planning framework entitled 6K1C for knowledge-based strategic planning to be used in the analysis of city-regions’ tangible and intangible assets. Practical implications – The paper discusses the importance of asset mapping of cityregions, and explores the ways of successfully managing city-regions’ tangible/intangible assets to achieve an urban development that is sustainable and knowledge-based. Keywords – Knowledge-based urban development, Knowledge-based strategic planning, Tangible assets, Intangible assets, City-regions. Paper type – Academic Research Paper

Research Monograph: Building Online Participatory Systems: Towards the Community Based Interactive Environmental Decision Support Systems

The importance of broadening community participation in environmental decision-making is widely recognized and lack of participation in this process appears to be a perennial problem. In this context, there have been calls from some academics for the more extensive use of geographic information systems (GIS) and distance learning technologies, accessible via the Internet, as a possible means to inform and empower communities. However, a number of problems exist. For instance, at present the scope for online interaction between policy-makers and citizens is currently limited. Contemporary web-based environmental information systems suffer from this lack of interactivity on the one hand and on the other hand from the apparent complexity for the lay user. This paper explores the issue of online community participation at the local level and attempts to construct a framework for a new (and potentially more effective) model of online participatory decision-making. The key components, system architecture and stages of such a model are introduced. This model, referred to as a ‘Community Based Interactive Environmental Decision Support System’, incorporates advanced information technologies, distance learning and community involvement tools which will be applied and evaluated in the field through a pilot project in Tokyo in the summer of 2002.

Education for all : providing curriculum for students with disabilities in a standards-based environment : implications for Queensland schools

This report investigates lessons learned by educators in the United States when providing a standards-based curriculum for all students including Students with Disabilities (SWD). Assumptions about implementation of these lessons are then made to the Queensland school system. Queensland mainstream schools currently provide a standards-based curriculum for over sixteen thousand-four hundred students with mild-moderate disabilities and appear to be challenged by this new educational reform and its implications to school and teacher practices, beliefs and attitudes. The analysis of US research, literature and educational policy for this report, has provided some implications for Queensland schools in the areas of student participation, achievement and curriculum planning to provide an “education for all”. The analysis and comparison of legislation and policy, which demonstrates some significant similarities, provides greater validity for the application of lessons learned in the United States to the Queensland context. The key findings about lessons learned provides Queensland schools with some assumptions as to why and how they need to refocus school leader and teachers’ practices, beliefs and attitudes to provide an “education for all”. These lessons infer that school leaders and teachers to explicitly focus on equity, expectation, accountability, performance, alignment and collaboration so that effective curriculum is provided for SWD, indeed all students, in the Queensland standards-based curriculum environment.

Linking resource acquisition and development processes to resource-based advantage : bricolage and the resource-based view

We investigate the relationship between bricolage – an approach to a firm’s resource development – and the firm’s strategic resource position as depicted by the resource-based view (RBV). The RBV is concerned with the resource characteristics of firms that lead to sustainable competitive advantage. Alternatively, bricolage is a process of resource use and development characterised by using resources at hand, recombining resources and making do. Based on a sample of approximately 700 nascent and 700 young firms we find that higher levels of bricolage behaviour tend to lead to more advantageous strategic resource positions.