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.

A strategic marketing approach to national development initiatives involving large scale collaborative ventures (LSCV)

This paper relates to government initiatives which aim at advancing their country’s economic development and investor attractiveness. It identifies large scale projects that involve collaboration between government and business (termed: large scale collaborative venture – LSCV) as one aspect of competing in the new economy. The study pursued the research proposition that a LSCV can be effectively facilitated by following a theory based process similar to what is used in corporate practice. An approach to managing such ventures is outlined, based on strategic marketing theory applied to a major project, the Multifunction Polis. It is proposed that such an approach may enhance the success of a collaborative venture and thereby help countries participate 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.

Review article : ecological approach in studying the relation of perception-action : review of the theoretical and empirical data

In daily activities people are using a number of available means for the achievement of balance, such as the use of hands and the co-ordination of balance. One of the approaches that explains this relationship between perception and action is the ecological theory that is based on the work of a) Bernstein (1967), who imposed the problem of ‘the degrees of freedom’, b) Gibson (1979), who referred to the theory of perception and the way which the information is received from the environment in order for a certain movement to be achieved, c) Newell (1986), who proposed that movement can derive from the interaction of the constraints that imposed from the environment and the organism and d) Kugler, Kelso and Turvey (1982), who showed the way which “the degrees of freedom” are connected and interact. According to the above mentioned theories, the development of movement co-ordination can result from the different constraints that imposed into the organism-environment system. The close relation between the environmental and organismic constraints, as well as their interaction is responsible for the movement system that will be activated. These constraints apart from shaping the co-ordination of specific movements can be a rate limiting factor, to a certain degree, in the acquisition and mastering of a new skill. This frame of work can be an essential tool for the study of catching an object (e.g., a ball). The importance of this study becomes obvious due to the fact that movements that involved in catching an object are representative of every day actions and characteristic of the interaction between perception and action.

Interactive lounge : an interdisciplinary approach to the design of a gestural interaction device

Among the many new opportunities that digital technologies are enabling are an increased capacity for viewers to interact not only with the program content, but with an increasingly wide array of other digital applications. Within this context this project has developed a new interaction device (incorporating gestural platform technology) and user interfaces to facilitate interactive access to digital media in a lounge room setting. This paper provides an overview of an interdisciplinary design process applied by Australasian CRC for Interaction Design (ACID) researchers—in order to develop the device and present in detail its unique features.

A quasi stationary approach to drying kinetics of cylindrical food particulates in a heat pump asisted fluid bed dryer

Experiments were undertaken to study drying kinetics of moist cylindrical shaped food particulates during fluidised bed drying. Cylindrical particles were prepared from Green beans with three different length:diameter ratios, 3:1, 2:1 and 1:1. A batch fluidised bed dryer connected to a heat pump system was used for the experimentation. A Heat pump and fluid bed combination was used to increase overall energy efficiency and achieve higher drying rates. Drying kinetics, were evaluated with non-dimensional moisture at three different drying temperatures of 30, 40 and 50o C. Numerous mathematical models can be used to calculate drying kinetics ranging from analytical models with simplified assumptions to empirical models built by regression using experimental data. Empirical models are commonly used for various food materials due to their simpler approach. However problems in accuracy, limits the applications of empirical models. Some limitations of empirical models could be reduced by using semi-empirical models based on heat and mass transfer of the drying operation. One such method is the quasi-stationary approach. In this study, a modified quasi-stationary approach was used to model drying kinetics of the cylindrical food particles at three drying temperatures.

Prediction of fluidization behaviour and a quasi-stationary approach to drying kinetics of irregular particulate food materials

Changes in fluidization behaviour behaviour was characterised for parallelepiped particles with three aspect ratios, 1:1, 2:1 and 3:1 and spherical particles. All drying experiments were conducted at 500C and 15 % RH using a heat pump dehumidifier system. Fluidization experiments were undertaken for the bed heights of 100, 80, 60 and 40 mm and at 10 moisture content levels. Due to irregularities in shape minimum fluidisation velocity of parallelepiped particulates (potato) could not fitted to any empirical model. Also a generalized equation was used to predict minimum fluidization velocity. The modified quasi-stationary method (MQSM) has been proposed to describe drying kinetics of parallelepiped particulates at 30o C, 40o C and 50o C that dry mostly in the falling rate period in a batch type fluid bed dryer.

Towards model round-trip engineering : an abductive approach

Providing support for reversible transformations as a basis for round-trip engineering is a significant challenge in model transformation research. While there are a number of current approaches, they require the underlying transformation to exhibit an injective behaviour when reversing changes. This however, does not serve all practical transformations well. In this paper, we present a novel approach to round-trip engineering that does not place restrictions on the nature of the underlying transformation. Based on abductive logic programming, it allows us to compute a set of legitimate source changes that equate to a given change to the target model. Encouraging results are derived from an initial prototype that supports most concepts of the Tefkat transformation language

A multi-hypothesis topological SLAM approach for loop closing on edge-ordered graphs

We present a method for topological SLAM that specifically targets loop closing for edge-ordered graphs. Instead of using a heuristic approach to accept or reject loop closing, we propose a probabilistically grounded multi-hypothesis technique that relies on the incremental construction of a map/state hypothesis tree. Loop closing is introduced automatically within the tree expansion, and likely hypotheses are chosen based on their posterior probability after a sequence of sensor measurements. Careful pruning of the hypothesis tree keeps the growing number of hypotheses under control and a recursive formulation reduces storage and computational costs. Experiments are used to validate the approach.

Case studies on vibration based damage identification : multi-criteria approach

Vibration based damage identification methods examine the changes in primary modal parameters or quantities derived from modal parameters. As one method may have advantages over the other under some circumstances, a multi-criteria approach is proposed. Case studies are conducted separately on beam, plate and plate-on-beam structures. Using the numerically simulated modal data obtained through finite element analysis software, algorithms based on flexibility and strain energy changes before and after damage are obtained and used as the indices for the assessment of the state of structural health. Results show that the proposed multi-criteria method is effective in damage identification in these structures.

Improving the management of building life cycle : a data mining approach

The construction industry has adapted information technology in its processes in terms of computer aided design and drafting, construction documentation and maintenance. The data generated within the construction industry has become increasingly overwhelming. Data mining is a sophisticated data search capability that uses classification algorithms to discover patterns and correlations within a large volume of data. This paper presents the selection and application of data mining techniques on maintenance data of buildings. The results of applying such techniques and potential benefits of utilising their results to identify useful patterns of knowledge and correlations to support decision making of improving the management of building life cycle are presented and discussed.