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.

Screening for drugs in oral fluid : illicit drug use and drug driving in a sample of urban and regional Queensland motorists

Police services in a number of Australian states and overseas jurisdictions have begun to implement or consider random road-side drug testing of drivers. This paper outlines research conducted to provide an estimate of the extent of drug driving in a sample of Queensland drivers in regional, rural and metropolitan areas. Oral fluid samples were collected from 2657 Queensland motorists and screened for illicit substances including cannabis (delta 9 tetrahydrocannibinol [THC]), amphetamines, ecstasy, and cocaine. Overall, 3.8% of the sample (n = 101) screened positive for at least one illicit substance, although multiple drugs were identified in a sample of 23 respondents. The most common drugs detected in oral fluid were ecstasy (n = 53), and cannabis (n = 46) followed by amphetamines (n = 23). A key finding was that cannabis was confirmed as the most common self-reported drug combined with driving and that individuals who tested positive to any drug through oral fluid analysis were also more likely to report the highest frequency of drug driving. Furthermore, a comparison between drug vs. drink driving detection rates for one region of the study, revealed a higher detection rate for drug driving (3.8%) vs. drink driving (0.8%). This research provides evidence that drug driving is relatively prevalent on Queensland roads, and may in fact be more common than drink driving. This paper will further outline the study findings’ and present possible directions for future drug driving research.

Orals ain't orals : how instruction and assessment practices affect delivery choices with prepared student oral presentations.

Despite an ostensibly technology-driven society, the ability to communicate orally is still seen as an essential ability for students at school and university, as it is for graduates in the workplace. The need to develop effective oral communication skills is often tied to future work-related tasks. One tangible way that educators have assessed proficiency in this area is through prepared oral presentations. While some use the terms oral communication and oral presentation interchangeably, other writers question the role more formal presentations play in the overall development of oral communication skills. Adding to the discussion, this paper is part of a larger study examining the knowledge and skills students bring into the academy from previous educational experiences. The study examines some of the teaching and assessment methods used in secondary schools to develop oral communication skills through the use of formal oral presentations. Specifically, it will look at assessment models and how these are used as a form of instruction as well as how they contribute to an accurate evaluation of student abilities. The purpose of this paper is to explore key terms and identify tensions between expectations and practice. Placing the emphasis on the ‘oral’ aspect of this form of communication this paper will particularly look at the ‘delivery’ element of the process.

Quality-based benefit design in health insurance : the impact of a product benefit design change on the utilisation of oral health services by members of a private health insurance fund in regional and rural New South Wales, Australia

Objective: To examine the impact on dental utilisation following the introduction of a participating provider scheme (Regional and Rural Oral Health Program {RROHP)). In this model dentists receive higher third party payments from a private health insurance fund for delivering an agreed range of preventive and diagnostic benefits at no out-ofpocket cost to insured patients. Data source/Study setting: Hospitals Contribution Fund of Australia (HCF) dental claims for all members resident in New South Wales over the six financial years from l99811999 to 200312004. Study design: This cohort study involves before and after analyses of dental claims experience over a six year period for approximately 81,000 individuals in the intervention group (HCF members resident in regional and rural New South Wales, Australia) and 267,000 in the control group (HCF members resident in the Sydney area). Only claims for individuals who were members of HCF at 31 December 1997 were included. The analysis groups claims into the three years prior to the establishment of the RROHP and the three years subsequent to implementation. Data collection/Extraction methods: The analysis is based on all claims submitted by users of services for visits between 1 July 1988 and 30 June 2004. In these data approximately 1,000,000 services were provided to the intervention group and approximately 4,900,000 in the control group. Principal findings: Using Statistical Process Control (SPC) charts, special cause variation was identified in total utilisation rate of private dental services in the intervention group post implementation. No such variation was present in the control group. On average in the three years after implementation of the program the utilisation rate of dental services by regional and rural residents of New South Wales who where members of HCF grew by 12.6%, over eight times the growth rate of 1.5% observed in the control group (HCF members who were Sydney residents). The differences were even more pronounced in the areas of service that were the focus of the program: diagnostic and preventive services. Conclusion: The implementation of a benefit design change, a participating provider scheme, that involved the removal of CO-payments on a defined range of preventive and diagnostic dental services combined with the establishment and promotion of a network of dentists, appears to have had a marked impact on HCF members' utilisation of dental services in regional and rural New South Wales, Australia.

Integrating concrete and virtual materials in an elementary mathematics classroom : a case study of success with fractions

This paper describes an approach to introducing fraction concepts using generic software tools such as Microsoft Office's PowerPoint to create "virtual" materials for mathematics teaching and learning. This approach replicates existing concrete materials and integrates virtual materials with current non-computer methods of teaching primary students about fractions. The paper reports a case study of a 12-year-old student, Frank, who had an extremely limited understanding of fractions. Frank also lacked motivation for learning mathematics in general and interacted with his peers in a negative way during mathematics lessons. In just one classroom session involving the seamless integration of off-computer and on-computer activities, Frank acquired a basic understanding of simple common equivalent fractions. Further, he was observed as the session progressed to be an enthusiastic learner who offered to share his learning with his peers. The study's "virtual replication" approach for fractions involves the manipulation of concrete materials (folding paper regions) alongside the manipulation of their virtual equivalent (shading screen regions). As researchers have pointed out, the emergence of new technologies does not mean old technologies become redundant. Learning technologies have not replaced print and oral language or basic mathematical understanding. Instead, they are modifying, reshaping, and blending the ways in which humankind speaks, reads, writes, and works mathematically. Constructivist theories of learning and teaching argue that mathematics understanding is developed from concrete to pictorial to abstract and that, ultimately, mathematics learning and teaching is about refinement and expression of ideas and concepts. Therefore, by seamlessly integrating the use of concrete materials and virtual materials generated by computer software applications, an opportunity arises to enhance the teaching and learning value of both materials.

Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

A qualitative study of the experience of oral cancer among Taiwanese men

The incidence and mortality of oral cancer in Taiwanese men have increased over the past decade, primarily associated with a surge in the popularity of betel quid chewing. The aim of this study was to examine the experience of six Taiwanese men with oral cancer, who were aged between 40 and 60 years, using a qualitative approach. The three major themes emerging from the data include: (i) understanding the cancer diagnosis; (ii) the challenges of cancer treatment; and (iii) adapting to difference. Increasing nurses' understanding of the experiential aspects of oral cancer in this population is required if nurses are to develop successful health promotion programmes and nursing interventions to meet these patients' needs.