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.

The condition, development and suggestions of policy of the highway express freight transportation system in China

The highway express freight transportation (HEFT) is a new transportation organization form separated from the common freight transportation with economic development and incessant adjustment of highway transportation structure in China. At present, the phenomenon of inadaptability still exists in the HEFT system of China, from foundation structure like highways, parking lots and stations to transportation equipments and transportation organizing. In order to develop the HEFT system more rationally and effectively, we should start with the structure of the system, conform the resources existing, and consummate the freight transport system. In due course, relevant policies and measures to supervise, lead and support are necessary and important. This paper analyzes the existing problems of HEFT system in our country, based on its characteristics, development situation and adaptability, and presents the policy and measures of promoting and leading the development of the HEFT system.

Queensland Emergency Medical System : a structural and organizational model for the emergency medical system in Australia

Objective: The emergency medical system (EMS) can be defined as a comprehensive, coordinated and integrated system of care for patients suffering acute illness and injury. The aim of the present paper is to describe the evolution of the Queensland Emergency Medical System (QEMS) and to recommend a strategic national approach to EMS development. Methods: Following the formation of the Queensland Ambulance Service in 1991, a state EMS committee was formed. This committee led the development and approval of the cross portfolio QEMS policy framework that has resulted in dynamic policy development, system monitoring and evaluation. This framework is led by the Queensland Emergency Medical Services Advisory Committee. Results: There has been considerable progress in the development of all aspects of the EMS in Queensland. These developments have derived from the improved coordination and leadership that QEMS provides and has resulted in widespread satisfaction by both patients and stakeholders. Conclusions: The strategic approach outlined in the present paper offers a model for EMS arrangements throughout Australia. We propose that the Council of Australian Governments should require each state and Territory to maintain an EMS committee. These state EMS committees should have a broad portfolio of responsibilities. They should provide leadership and direction to the development of the EMS and ensure coordination and quality of outcomes. A national EMS committee with broad representation and broad scope should be established to coordinate the national development of Australia's EMS.

The role of the research system in 'Learning Economies'

This paper provides a framework for analysing the role of Australia’s research system in promoting national economic development. The paper is in two parts. Part One focuses on knowledge diffusion and technological development and emphasises the systemic nature of innovation processes within the emerging context of ‘learning economies’. The key understandings relevant to a nation’s research system are drawn out from contemporary developments in the international literature on ‘learning economies’. Some of the implications for Australia are discussed. The aim is to provide readers with some indications of what to look for in considering options for the future of Australia’s research system. More detailed information on relevant aspects of Australia’s industrial and trade structure, the extent of the R&D effort in industry and on issues such as management capability can be obtained from (Marceau et al 1997). In the second part, broad elements of the Australian research system are reviewed in the light of findings from the literature. The central role of universities in the innovation and research systems is described. Actions that can be taken by both universities and governments are suggested, particularly regarding the need to build and maintain efficient information flows at local, national and international levels. The paper briefly points to the nature of a research system capable of contributing effectively to the wealth of the nation and indicates some possible directions for enabling Australia to meet the demands of, and profit from, a knowledge-based economy.

Implementing an employee performance management system in a nonprofit organization

Nonprofit organizations are not exempt from the imperatives of employee attraction, retention, and motivation. As competition for staff, donors, and funding increases, the need to manage employee performance will continue to be a critical human resource management issue. This article outlines a study of the introduction of a performance management system in an Australian nonprofit organization and analyzes its design and implementation. It explores how performance management can be introduced and used effectively within a nonprofit environment to benefit staff and the organization. However, the use of performance management is not without its challenges, and the research also identified initial employee resistance and a resulting initial spike in labor turnover. However, findings indicate that if nonprofit organizations are willing to undertake consultation with staff and ensure that the organization's specific context, values, and mission are reflected in the performance management system, it can be a useful tool for managers and a direct benefit to employees.

Implementing design build project delivery system in Indonesian road infrastructure projects

Design-Build (DB) project delivery systems have increasingly been adopted by many private and public sector organizations worldwide due to its many advantages. However, many Indonesian road infrastructure projects are still delivered using the traditional design-bid-build (DBB) project delivery system. This paper reviews the existing literature to explore factors that can influence the successful implementation of DB project delivery system in Indonesian road infrastructure projects. It founds the lack of clarification in existing legislations as well as the lack of experiences, knowledge and skill as the main obstacles in implementing DB systems in Indonesia. To overcome these obstacles, this paper proposes (1) A relook at existing legislation in term of providing more guidance on determining projects appropriate for the DB, procedures for implementing DB, and the structure of builder entity; (2) To develop the skills and knowledge of DB to all stakeholders through communications, knowledge sharing and training. The outcome of this review can serve as a guide to development a framework for the implementation of the design-build project delivery system in Indonesian road infrastructure projects.

United we stand, divided we fall: the failure of an accounting information system in a major radiology provider

This article explores the way in which a major Australian radiology organization implemented a complex accounting information system and how workers in the 72 radiology practises that had to use it resisted the change. The study reports on the issues that led to the circumvention of the system by individuals and, after only three years, complete withdrawal of the accounting information system by the parent organization. This article has implications for firms in the health care and other sectors considering implementing new accounting information systems. Organizations need to incorporate change management techniques and provide open communication to all stakeholders to minimize disruption and potential problems.