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.

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.

Value-oriented process modeling : integrating financial perspectives into business process re-design

Purpose – Financial information about costs and return on investments are of key importance to strategic decision-making but also in the context of process improvement or business engineering. In this paper we propose a value-oriented approach to business process modeling based on key concepts and metrics from operations and financial management, to aid decision making in process re-design projects on the basis of process models. Design/methodology/approach – We suggest a theoretically founded extension to current process modeling approaches, and delineate a framework as well as methodical support to incorporate financial information into process re-design. We use two case studies to evaluate the suggested approach. Findings – Based on two case studies, we show that the value-oriented process modeling approach facilitates and improves managerial decision-making in the context of process re-design. Research limitations / implications – We present design work and two case studies. More research is needed to more thoroughly evaluate the presented approach in a variety of real-life process modeling settings. Practical implications – We show how our approach enables decision makers to make investment decisions in process re-design projects, and also how other decisions, for instance in the context of enterprise architecture design, can be facilitated. Originality/value – This study reports on an attempt to integrate financial considerations into the act of process modeling, in order to provide more comprehensive decision making support in process re-design projects.

Hypercapitalism: language, new media and social perceptions of value

Using examples from contemporary policy and business discourses, and exemplary historical texts dealing with the notion of value, I put forward an argument as to why a critical scholarship that draws on media history, language analysis, philosophy and political economy is necessary to understand the dynamics of what is being called 'the global knowledge economy'. I argue that the social changes associated with new modes of value determination are closely associated with new media forms.