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.

TV stories : from representation to productivity

This chapter revisits the concept of the ‘bardic function’ (Fiske & Hartley 1978), using historical analysis of the oral bardic institutions to re-theorise it for the era of interactive media and digital storytelling. It shows how ‘representative’ storytelling has transformed into self-representation, and proposes that the ‘bardic function’ can be divided into three types: representative (the ‘Taliesin function’); pedagogic (the ‘Gandalf function’); and self-organised (the ‘eisteddfod function’).

Computational power meets human contact

This chapter traces the development of the global digital storytelling movement from its origins in California to its adoption by the BBC in the UK and its subsequent dispersal around the world. It identifies the foundational practices, uneven development and diffusion, and emergent practices internationally.

Digital storytelling as participatory public history in Australia

This chapter discusses digital storytelling as a methodology for participatory public history through a detailed reflection on an applied research project that integrated both public history and digital storytelling in the context of a new master-planned urban development: the Kelvin Grove Urban Village Sharing Stories project.

Regulating Multi-National Corporations Through State-Based Laws : Problems with Enforcing Human Rights Under The Alien Tort Statute

Under the Alien Tort Statute United States of America (“America”) Federal Courts have the jurisdiction to hear claims for civil wrongs, committed against non-American citizens, which were perpetrated outside America’s national borders. The operation of this law has confronted American Federal Courts with difficulties on how to manage conflicts between American executive foreign policy and judicial interpretations of international law. Courts began to pass judgment over conduct which was approved by foreign governments. Then in 2005 the American Supreme Court wound back the scope of the Alien Tort Statute. This article will review the problems with the expansion of the Alien Tort Statute and the reasons for its subsequent narrowing.

Non-adversarial advocates and gatekeepers : lawyers, FDR practitioners and co-operative post-separation parenting

The compulsory dispute resolution requirements in family law parenting cases create new roles and obligations for both lawyers and family dispute resolution (FDR) practitioners. This article will discuss how the legislative provisions impact on both sets of professionals in practice. It will also highlight the increased non-adversarial role of lawyers and a new role for FDR practitioners as “gatekeepers” to family courts in cases requiring FDR certificates.

Documentary practice in a participatory culture

Debates concerning the veracity, ethics and politics of the documentary form circle endlessly around the function of those who participate in it, and the meaning attributed to their participation. Great significance is attached to the way that documentary filmmakers do or do not participate in the world they seek to represent, just as great significance is attached to those subjects whose participation extends beyond playing the part of eyewitness or expert, such that they become part of the very filmmaking process itself. This Ph.D. explores the interface between documentary practice and participatory culture by looking at how their practices, discursive fields and histories intersect, but also by looking at how participating in one might mean participating in the other. In short, the research is an examination of participatory culture through the lens of documentary practice and documentary criticism. In the process, however, this examination of participatory culture will in turn shed light on documentary thinking, especially the meaning and function of ‘the participant’ in contemporary documentary practice. A number of ways of conceiving of participation in documentary practice are discussed in this research, but one of the ideas that gives purpose to that investigation is the notion that the participant in contemporary documentary practice is someone who belongs to a participatory culture in particular. Not only does this mean that those subjects who play a part in a documentary are already informed by their engagement with a range of everyday media practices before the documentary apparatus arrives, the audience for such films are similarly informed and engaged. This audience have their own expectations about how they should be addressed by media producers in general, a fact that feeds back into their expectations about participatory approaches to documentary practice too. It is the ambition of this research to get closer to understanding the relationship between participants in the audience, in documentary and ancillary media texts, as well as behind the camera, and to think about how these relationships constitute a context for the production and reception of documentary films, but also how this context might provide a model for thinking about participatory culture itself. One way that documentary practice and participatory culture converge in this research is in the kind of participatory documentary that I call the ‘Camera Movie’, a narrow mode of documentary filmmaking that appeals directly to contemporary audiences’ desires for innovation and participation, something that is achieved in this case by giving documentary subjects control of the camera. If there is a certain inevitability about this research having to contend with the notion of the ‘participatory documentary’, the ‘participatory camera’ also emerges strongly in this context, especially as a conduit between producer and consumer. Making up the creative component of this research are two documentaries about the reality television event Band In A Bubble, and participatory media practices more broadly. The single-screen film, Hubbub , gives form to the collective intelligence and polyphonous voice of contemporary audiences who must be addressed and solicited in increasingly innovative ways. One More Like That is a split-screen, DVD-Video with alternate audio channels selected by a user who thereby chooses who listens and who speaks in the ongoing conversation between media producers and media consumers. It should be clear from the description above that my own practice does not extend to highly interactive, multi-authored or web-enabled practices, nor the distributed practices one might associate with social media and online collaboration. Mine is fundamentally a single authored, documentary video practice that seeks to analyse and represent participatory culture on screen, and for this reason the Ph.D. refrains from a sustained discussion of the kinds of collaborative practices listed above. This is not to say that such practices don’t also represent an important intersection of documentary practice and participatory culture, they simply represent a different point of intersection. Being practice-led, this research takes its procedural cues from the nature of the practice itself, and sketches parameters that are most enabling of the idea that the practice sets the terms of its own investigation.

Looking through the haze of discontent : smokers as a data source

In the case of industrial relations research, particularly that which sets out to examine practices within workplaces, the best way to study this real-life context is to work for the organisation. Studies conducted by researchers working within the organisation comprise some of the (broad) field’s classic research (cf. Roy, 1954; Burawoy, 1979). Participant and non-participant ethnographic research provides an opportunity to investigate workplace behaviour beyond the scope of questionnaires and interviews. However, we suggest that the data collected outside a workplace can be just as important as the data collected inside the organisation’s walls. In recent years the introduction of anti-smoking legislation in Australia has meant that people who smoke cigarettes are no longer allowed to do so inside buildings. Not only are smokers forced outside to engage in their habit, but they have to smoke prescribed distances from doorways, or in some workplaces outside the property line. This chapter considers the importance of cigarette-smoking employees in ethnographic research. Through data collected across three separate research projects, the chapter argues that smokers, as social outcasts in the workplace, can provide a wealth of important research data. We suggest that smokers also appear more likely to provide stories that contradict the ‘management’ or ‘organisational’ position. Thus, within the haze of smoke, researchers can uncover a level of discontent with the ‘corporate line’ presented inside the workplace. There are several aspects to the increased propensity of smokers to provide a contradictory or discontented story. It may be that the researcher is better able to establish a rapport with smokers, as there is a removal of the artificial wall a researcher presents as an outsider. It may also be that a research location physically outside the boundaries of the organisation provides workers with the freedom to express their discontent. The authors offer no definitive answers; rather, this chapter is intended to extend our knowledge of workplace research through highlighting the methodological value in using smokers as research subjects. We present the experience of three separate case studies where interactions with cigarette smokers have provided either important organisational data or alternatively a means of entering what Cunnison (1966) referred to as the ‘gossip circle’. The final section of the chapter draws on the evidence to demonstrate how the community of smokers, as social outcasts, are valuable in investigating workplace issues. For researchers and practitioners, these social outcasts may very well prove to be an important barometer of employee attitudes; attitudes that perhaps cannot be measured through traditional staff surveys.

Curriculum mapping of the Spatial Science Program, QUT : what was the cartography journey?

There are currently a number of issues of great importance affecting universities and the way in which their programs are now offered. Many issues are largely being driven top-down and impact both at a university-wide and at an individual discipline level. This paper provides a brief history of cartography and digital mapping education at the Queensland University of Technology (QUT). It also provides an overview of what is curriculum mapping and presents some interesting findings from the program review process. Further, this review process has triggered discussion and action for the review, mapping and embedding of graduate attributes within the spatial science major program. Some form of practical based learning is expected in vocationally oriented degrees that lead to professional accreditation and are generally regarded as a good learning exposure. With the restructure of academic programs across the Faculty of Built Environment and Engineering in 2006, spatial science and surveying students now undertake a formal work integrated learning unit. There is little doubt that students acquire the skills of their discipline (mapping science, spatial) by being immersed in the industry culture- learning how to process information and solve real-world problems within context. The broad theme of where geo-spatial mapping skills are embedded in this broad-based tertiary education course are examined with some focused discussion on the learning objectives, outcomes and examples of some student learning experiences

Professional ethics : the IT experience

IT professionals work in an environment which is under continual innovation, exerts a global influence and yet is relatively young. In such a context, how can professionals be effectively supported in their ethical practice? IT professional ethics has predominantly focussed on external standards or internal reasoning. However, attention also has to be paid to professionals’ experience of ethics, which influences how they interpret standards and construct reasoning. A comprehensive experience of ethics will embrace the professional’s inner circle, their employer, their client and humanity. In order to promote such a comprehensive view, we need to re-conceptualise a) the IT discipline, focussing on information users; b) professional ethics, adopting other-centred attitudes; and c) professional development, pursuing a change in lived experience. This book is written for those interested in professional ethics (practitioners, educators, professional bodies and employers), especially in the computing field.

The failure of Australian legislation on indirect discrimination to detect the systemic racism which prevents Aboriginal people from fully participating in the workforce

Government figures put the current indigenous unemployment rate at around 23%, 3 times the unemployment rate for other Australians. This thesis aims to assess whether Australian indirect discrimination legislation can provide a remedy for one of the causes of indigenous unemployment - the systemic discrimination which can result from the mere operation of established procedures of recruitment and hiring. The impact of those practices on indigenous people is examined in the context of an analysis of anti-discrimination legislation and cases from all Australian jurisdictions from the time of the passing of the Racial Discrimination Act by the Commonwealth in 1975 to the present. The thesis finds a number of reasons why the legislation fails to provide equality of opportunity for indigenous people seeking to enter the workforce. In nearly all jurisdictions it is obscurely drafted, used mainly by educated middle class white women, and provides remedies which tend to be compensatory damages rather than change to recruitment policy. White dominance of the legal process has produced legislative and judicial definitions of "race" and "Aboriginality" which focus on biology rather than cultural difference. In the commissions and tribunals complaints of racial discrimination are often rejected on the grounds of being "vexatious" or "frivolous", not reaching the required standard of proof, or not showing a causal connection between race and the conduct complained of. In all jurisdictions the cornerstone of liability is whether a particular employment term, condition or practice is reasonable. The thesis evaluates the approaches taken by appellate courts, including the High Court, and concludes that there is a trend towards an interpretation of reasonableness which favours employer arguments such as economic rationalism, the maintenance of good industrial relations, managerial prerogative to hire and fire, and the protection of majority rights. The thesis recommends that separate, clearly drafted legislation should be passed to address indigenous disadvantage and that indigenous people should be involved in all stages of the process.

The impact of “empirical facts” on legal scholarship and legal research training

Lawyers have traditionally viewed law as a closed system, and doctrinal research has been the research methodology used most widely in the profession. This reflects traditional concepts of legal reasoning. There is a wealth of reliable and valid social science data available to lawyers and judges. Judges in fact often refer to general facts about the world, society, institutions and human behaviour (‘empirical facts’). Legal education needs to prepare our students for this broader legal context. This paper examines how ‘empirical facts’ are used in Australian and other common law courts. Specifically, the paper argues that there is a need for enhanced training in non-doctrinal research methodologies across the law school curriculum. This should encompass a broad introduction to social science methods, with more attention being paid to a cross-section of methodologies such as content analysis, comparative law and surveys that are best applied to law.

Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Submission to Travelsafe : vehicle impoundment for drink drivers

This submission has been prepared in response to the Parliamentary Travelsafe Committee's Inquiry into vehicle impoundment for drink drivers to address research relevant to the committee’s investigation into whether: • Drink drivers in Queensland continue to drive illegally after being apprehended by police or disqualified from driving by the courts; • The incidence of repeat drink driving undermines the effectiveness of existing penalties for drink driving offences; and • Vehicle impoundment and/or ignition key confiscation are cost-effective deterrents that will reduce drink driving recidivism, relating to other existing or potential methods of managing offenders.