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.

Promoting sustainable urban development in fast growing city-regions : practices from Kuala Lumpur and Hong Kong

The Asia‐Pacific region is characterised by rapid population growth and urbanisation. These trends often result in an increasing consumption of land, which in turn lead to spatially expansive and discontinuous urban development. As a consequence, local communities and the environment face strong pressures. Many cities in the region have developed policies to tackle the issue of rapid growth and its associated consequences, for example climate change. The broad aim of this paper is to identify the nature, trends and strategies of growth management in major Asia‐Pacific city‐regions, and their implications for natural resource management and infrastructure provision. More specifically, this research seeks to provide insights on sustainable urban development practice, particularly on the promotion of compact urbanisation within the Asia‐Pacific’s fastest growing regions. The methodology of the paper includes a detailed literature review and a comparative analysis of existing strategies and policies. The literature review focuses on the key concepts related to sustainable urban growth management. It also includes existing applications of urban growth management approaches and planning information system in managing growth. Following the literature review, the paper undertakes a comparative analysis of the strategies of major Asia‐Pacific city‐regions of Kuala Lumpur and Hong Kong in terms of their approaches to sustainable urban development. The findings of the paper provide a clear understanding of the necessity of sustainable urban development practices. It contributes to the development of a substantial base for further research. Ultimately, this research aims to shed light on sustainable urban development by providing insights on the management of growth, natural resources and urban infrastructures.

Emerging strategies for sponsorship on the Internet

Internet sponsorship is a rapidly evolving communications platform at a fledgling stage for sponsorship-linked marketing. The complex and involving nature of the phenomena offers new research opportunities and the potential for innovative marketing strategies. The Internet presents the opportunity to combine aspects of traditional event/activity sponsorship and media sponsorship innovatively and to extend marketing strategy in new directions. This paper presents an overview of emerging Internet sponsorships strategies, identifies virtual and reality sponsorship opportunities, and explores emerging sponsorship extension strategies that relate to media, brand, channel development, consumer experience, and networking. Finally, future directions for researchers and practitioners are suggested.

User-designer collaboration during the early stage of the product development process

As an understanding of users' tacit knowledge and latent needs embedded in user experience has played a critical role in product development, users’ direct involvement in design has become a necessary part of the design process. Various ways of accessing users' tacit knowledge and latent needs have been explored in the field of user-centred design, participatory design, and design for experiencing. User-designer collaboration has been used unconsciously by traditional designers to facilitate the transfer of users' tacit knowledge and to elicit new knowledge. However, what makes user-designer collaboration an effective strategy has rarely been reported on or explored. Therefore, interaction patterns between the users and the designers in three industry-supported user involvement cases were studied. In order to develop a coding system, collaboration was defined as a set of coordinated and joint problem solving activities, measured by the elicitation of new knowledge from collaboration. The analysis of interaction patterns in the user involvement cases revealed that allowing users to challenge or modify their contextual experiences facilitates the transfer of knowledge and new knowledge generation. It was concluded that users can be more effectively integrated into the product development process by employing collaboration strategies to intensify the depth of user involvement.

Enhancing research and development: designing collaborative environments for innovation

Work environments have previously been studied to identify the strategies, structures and processes which increase the likelihood of creativity, innovation and collaboration for productive workplaces. A number of perspectives have emerged which identify social and cognitive factors known to contribute to or to restrict innovation and collaboration. Recently more attention has been given to designing physical environments to encourage processes relevant to innovation such as creativity (McCoy & Evans, 2002) knowledge sharing (Hemlin, Allwood & Martin, 2008) and collaboration (Bozeman & Corley, 2004). Some attention has been given specifically to research and development environments (Boutellier et al, 2008) but little integration of this research has occurred. In the context of the construction of new purpose-built premises which will bring together under one roof separate public sector agencies engaged in research and development in agriculture, natural resource systems and the environment, this paper examines the extant literature and develops initial propositions for research relevant to the transition, collaboration and performance of research and development in new organizational environments where traditional boundaries have been redrawn.

An in-depth study of a teacher engaged in an innovative primary science trial professional development project

The implementation of effective science programmes in primary schools is of continuing interest and concern for professional developers. As part of the Australian Academy of Science's approach to creating an awareness of Primary Investigations, a project team trialled a series of satellite television broadcasts of lessons related to two units of the curriculum for Year 3 and 4 children in 48 participating schools. The professional development project entitled Simply Science, included a focused component for the respective classroom teachers, which was also conducted by satellite. This paper reports the involvement of a Year 4 teacher in the project and describes her professional growth. Already an experienced and confident teacher, no quantitative changes in science teaching self efficacy were detected. However, her pedagogical content knowledge and confidence to teach science in the concept areas of matter and energy were enhanced. Changes in the teacher's views about the co-operative learning strategies espoused by Primary Investigations were also evident. Implications for the design of professional development programmes for primary science teachers are discussed.

Targeting injury prevention strategies to adolescents of Pacific Islander descent : what will work?

Background: Ethnicity is rarely considered in injury prevention program development, even though this is known to impact on participation in injury risk behaviour. An understanding of injury, risk behaviour and risk and protective factors specific to adolescents of Pacific Islander descent will inform the development of prevention strategies appropriate to this group.----- Aims: To determine patterns of injury and associated risk behaviour among adolescents of Pacific Islander descent, and to understand the risk and protective factors that influence injury rates among this group.----- Methods: A total of 875 Year 9 students from five Queensland high schools completed a survey during health classes. Seventy-one students (n = 38 male) identified as Pacific Islander. The survey consisted of scales examining injury, risk taking behaviour, and relationships with family, school and police.----- Results: The leading causes of injury among adolescents of Pacific Islander descent were sports (48%) and transport (e.g. 45% reported bicycle injuries). Interpersonal violence related injuries were also relatively frequent, with 28% having been injured in a fight. Reports of alcohol use were relatively low (20% c.f. 40% of the remaining sample), however reports of other risk behaviours were relatively high (e.g. 43% c.f. 25% of remaining sample reported a group fight).----- Discussion and conclusions: Conclusions will be drawn regarding risk-related injuries reported by adolescents of Pacific Islander descent and those of other ethnic backgrounds. Additionally, risk and protective factors relating to family, school and police will be explored, in order to inform prevention strategies appropriate to this group.

Transitional employment aspirations for bridging retirement : implications for training and development

Purpose: The aim of this study was to identify the transitional employment aspirations and training and development needs of older and younger workers at risk of early retirement due to limited education and/or employment in blue collar occupations. Design/ Methodology/ Approach: A computer based methodology was used to evaluate the demographic effects of gender, education level and occupation group on aspirations pertaining to transitional employment and training and development in a sample of over 1000 Local Government employees. Findings: Older blue collar, secondary school educated and younger workers were less interested in transitional employment than older workers with higher levels of education or from white collar backgrounds. The early retirement risk factors of blue collar work and secondary school education had a more limited effect on perceived training and development needs for older workers. However for younger workers, these risk factors provided the impetus to undertake training to move into less physically demanding or more challenging roles as their careers progressed. Practical Implications: Via the identification of education level and occupation types groups’ transitional employment aspirations and perceptions of preparatory training and development within younger and older cohorts, long term strategies to develop and retain staff may be formulated. Originality/ Value: Past studies of transitional employment have rarely included younger workers or older workers at risk of early retirement. Preparatory training and development for transitional employment roles has not been considered in the literature.

The relationship between construction firm strategies and innovation outcomes

Survey results provide a preliminary assessment of the relative contribution of a range of tactical business strategies to innovation performance by firms in the Australian construction industry. Over 1,300 firms were surveyed in 2004, resulting in a response rate of 29%. Respondents were classified as high, medium or low innovators according to an innovation index based on the novelty and impact of their innovations and their adoption of listed technological and organizational advances. The relative significance of 23 business strategies concerning (1) employees, (2) marketing, (3) technology, (4) knowledge and (5) relationships was examined by determining the extent to which they distinguished high innovators from low innovators. The individual business strategies that most strongly distinguished high innovators were (1) ‘investing in R&D’, (2) ‘participating in partnering and alliances on projects’, (3) ‘ensuring project learnings are transferred into continuous business processes’, (4) ‘monitoring international best practice’, and (5) ‘recruiting new graduates’. Of the five types of strategies assessed, marketing strategies were the least significant in supporting innovation. The results provide practical guidance to managers in project-based industries wishing to improve their innovation performance.

The development of a clinical learning environment scale

Within nursing, there is a strong demand for high-quality, cost-effective clinical education experiences that facilitate student learning in the clinical setting The clinical learning environment (CLE) is the interactive network of forces within the clinical setting that influence the students'clinical learning outcomes The identification of factors that characterize CLE could lead to strategies that foster the factors most predictive of desirable student learning outcomes and ameliorate those which may have a negative impact on student outcomes The CLE scale is a 23-item instrument with five subscales staff–student relationships, nurse manager commitment, patient relationships, interpersonal relationships, and student satisfaction These factors have strong substantive face validity and construct validity, as determined by confirmatory factor analysis Reliability coefficients range from high (0 85) to marginal (0 63) The CLE scale provides the educator with a valid and reliable instrument to evaluate affectively relevant factors in the CLE, direct resources to areas where improvement may be required, and nurture those areas functioning well It will assist in the application of resources in a cost-effective, efficient, productive manner, and will ensure that the clinical learning experience offers the nursing student the best possible learning outcomes

How to progress forward in improving safety outcomes in the work-related driving field : a review of work-related driving research and development of an intervention framework [Extended abstract]

Road crashes are now the most common cause of work-related injury, death and absence in a number of countries. Given the impact of workrelated driving crashes on social and economic aspects of business and the community, workrelated road safety and risk management has received increasing attention in recent years. However, limited academic research has progressed on improving safety within the work-related driving sector. The aim of this paper is to present a review of work-related driving safety research to date, and provide an intervention framework for the future development and implementation of workrelated driving safety intervention strategies.

Establishment of a preclinical ovine model for tibial segmental bone defect repair by applying bone tissue engineering strategies

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence commercialization, is referred to as the ‘Valley of Death’ and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes (GMP) and scalable designs and to apply these in pre-clinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopaedic bone engineering from bench to bedside byestablishing a pre-clinical ovine critical-sized tibial segmental bone defect model and discuss our preliminary data relating to this decisive step.

Communicating ecologically positive development

It is difficult to present a paradigm shift from resource efficient to ecologically sustainable design, when many students have not yet thought about what sustainability is, let alone what it implies for the design of the built environment ‘Positive Development’ requires students to think beyond green building to something that does not yet exist. The concept of ecologically positive development suggests a product, building, system or urban area that leaves the ecological base and public estate better off than if no development had occurred. For some years now, I have experimented with communicating this paradigm shift in design to students and professionals ‐ with mixed results. This paper discusses some of the challenges, failures and successes in shifting design studio work from environmentally‐sensitive to eco-positive. The framework underlying this exploration is action research. Conclusions about the success of the strategies used for overcoming perceptual barriers to new typologies of architecture are drawn from recent student feedback. The talk will show examples of student projects that attempt eco-positive development projects.

Cooperative learning in Thailand : professional development to enhance primary education

The overall purpose of this study was to develop a model to inform the design of professional development programs and the implementation of cooperative learning within Thai primary school mathematics classrooms. Action research design, with interviews, surveys and observations, was used for this study. Survey questionnaires and classroom observations investigated the factors that influence the implementation of cooperative learning strategies and academic achievement in Thai primary school mathematics classrooms. The teachers’ interviews and classroom observation also examined the factors that need to be addressed in teacher professional development programs in order to facilitate cooperative learning in Thai mathematics classrooms. The outcome of this study was a model consisting of two sets of criteria to inform the successful implementation of cooperative learning in Thai primary schools. The first set of criteria was for proposers and developers of professional development programs. This set consists of macro- and micro-level criteria. The macro-level criteria focus on the overall structure of professional development programs and how and when the professional development programs should be implemented. The micro-level criteria focused on the specific topics that need to be included in professional development programs. The second set of criteria was for Thai principals and teachers to facilitate the introduction of cooperative learning in their classrooms. The research outcome also indicated that the attainment of these cooperative learning strategies and skills had a positive impact on the students’ learning of mathematics.