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 epistemology that maintains white race privilege, power and control of Indigenous Studies and Indigenous peoples' participation in universities

This paper represents my attempt to turn the gaze and demonstrate how Indigenous Studies is controlled in some Australian universities in ways that witness Indigenous peoples being further marginalised, denigrated and exploited. I have endeavoured to do this through sharing an experience as a case study. I have opted to write about it as a way of exposing the problematic nature of racism, systemic marginalisation, white race privilege and radicalised subjectivity played out within an Australian higher education institution and because I am dissatisfied with the on-going status quo. In bringing forth analysis to this case study, I reveal the relationships between oppression, white race privilege and institutional privilege and the epistemology that maintains them. In moving from the position of being silent on this experience to speaking about it, I am able to move from the position of object to subject and to gain a form of liberated voice (hooks 1989:9). Furthermore, I am hopeful that it will encourage others to examine their own practices within universities and to challenge the domination that continues to subjugate Indigenous peoples.

Comparing international television news : the case of violence presentation in Germany, Great Britain and Russia -- [in German : Gewalt in internationalen Fernsehnachrichten - eine komparative Analyse medialer Gewaltpräsentation in Deutschland, Großbritannien und Russland]

This is an important book that ought to launch a debate about how we research our understanding of the world, it is an innovative intervention in a vital public issue, and it is an elegant and scholarly hard look at what is actually happening. Jean Seaton, Prof of Media History, U of Westminster, UK & Official Historian of the BBC -- Summary: This book investigates the question of how comparative studies of international TV news (here: on violence presentation) can best be conceptualized in a way that allows for crossnational, comparative conclusions on an empirically validated basis. This book shows that such a conceptualization is necessary in order to overcome existing restrictions in the comparability of international analysis on violence presentation. Investigated examples include the most watched news bulletins in Great Britain (10o'clock news on the BBC), Germany (Tagesschau on ARD) and Russia (Vremja on Channel 1). This book highlights a substantial cross-national violence news flow as well as a cross-national visual violence flow (key visuals) as distinct transnational components. In addition, event-related textual analysis reveals how the historical rootedness of nations and its symbols of power are still manifested in televisual mediations of violence. In conclusion, this study lobbies for a conscientious use of comparative data/analysis both in journalism research and practice in order to understand what it may convey in the different arenas of today’s newsmaking.

Catalogue essay : a thin white line

Objects have consequences, seemingly. They move, atomic, formlessly – when static they are seen. That they vibrate constantly, that they are NOW present, is something we will have to trust the physicists on. They only seem here. Now is their moment of form, but later, who knows? Things SEEM when we recognise our own transience and temporary-ness. We call upon a bevy of senses that forever frustrate us with their limitation, despite our little understanding of what we actually have – is this here? So some forms seem to be telling us to trust our senses – that this world IS as it seems. Their form constantly refines and is refined and refined until in its essentialness it cannot be doubted – it absolutely IS. Is this our eyes? Can we only see it? But light is also a particle, if I remember correctly, so there is some weight to seeing. So to SEEM is also to FEEL,as this light imposes its visual weight upon our skins – we see with every pore of our body.

White possession : the legacy of Cook's choice

Book Synopsis: From Terra Nullius to Land of Opportunities and Last Frontier, the European dream has constructed and deconstructed Australia to feed its imagination of new societies. At the same time Australia has over the last two centuries forged and re-invented its own liaisons with Europe arguably to carve out its identity. From the arts to social sciences, to society itself, a complex dynamic has grown between the two continents in ways that invite study and discussion. A transnational research group has begun its collective investigation project of which this first volume is the outcome. The book is a substantial multidisciplinary collection of current research and offers critical perspectives on culture, literature and history around themes at the heart of the Imagined Australia project. The essays instigate reflection, discovery and discussion of how reciprocal imagining between Australia and Europe has articulated itself and ways and dimensions in which a relationship between communities, imagined and not, has unfolded.

Too great expectations?

Teachers have always been charged with the task of turning out the next generation of citizens—educated, healthy in mind, and healthy in body. The central question here is: how far should this responsibility extend? Just how much can we reasonably expect teachers to be responsible for? And just what should schools be responsible for? Obviously, teachers ought to be responsible for the fundamentals of learning, but should they be held accountable for what the children eat, or how they choose to behave, or for every single risk, direct or indirect, that could conceivably occur within the school grounds? These are precisely the kinds of expectation that have become part of educational life. Bit by bit, new forms of responsibility are being added to the site of the school and, more specifically, to the professional life of the teacher. The intention here is not necessarily to challenge any of these diverse changes, but rather to express concern about their cumulative effect on the ability of the school to do its primary job effectively.

Southern theory and its dynamics for postcolonial education

This paper discusses how the exploration of social texts and historical contexts from the global 'South', as put forward in Raewyn Connell's study 'Southern Theory' (2007), can improve the theoretical tools used in postcolonial education analysis. Connell analyses a selection of excellent and compelling social theory texts written by scholars in Africa, India, Iran, Latin America and Australia to show how they challenge and counter the silences, distortions and plain lies of dominant Western social theory. These texts of the global South do not mince words in laying bare the role of the institutions and elites of the West in the destruction, dispossession, and bloodshed involved in creating the world in which we live, and in perpetuating its catastrophes. The texts also reveal intense debates between scholars over their conceptualisations of local, national and global society. My paper argues that this kind of work is of vital importance to postcolonial studies in education. It helps education scholars to uncover the problematic assumptions and distortions of dominant education thought, and understand different ways of seeing. Postcolonial educators could use this to help both students and teacher unlearn many of our taught perceptions of the world, whether in the global North or the global South. Developing a countervailing social theory in education would sharpen our questioning of the structures of schooling as they relate to society, and tease out new dimensions of postcolonial leadership for education.

Under great pressure, a diamond is being formed: The SBS over time

This article observes a paradox in the recent history of the Special Broadcasting Service. It is argued that, in contrast to the Australian Broadcasting Corporation, the role and general direction of SBS were not extensively debated as part of the ‘culture wars’ that occurred during the years of the Howard government. While that made SBS a less fraught space during that period, it may now be a factor in the comparative lack of support being given by the Rudd Labor government to SBS in comparison with the ABC, as some of the ‘special’ status of SBS has been blunted by its drift towards more mainstream programming and a mixed economy of commercial advertising, as well as government funding.

Great adaptations : China's creative clusters and the new social contract

The transformation of China's urban landscape has witnessed a boom in cultural adaptation, namely the adaptation of a Western idea, the creative cluster. This chapter examines the formatting of hundreds of creative clusters-art centres, animation bases, cultural zones, and incubators. The cluster has important implications for how we understand China going forward into the second decade of the 21st century. The cluster phenomenon has resulted in to a substantive remaking of the social contract, between officials, entrepreneurs, local residents, academics-and most significantly cultural producers. However, these processes of adaption are mostly driven by real estate developers working in partnership with local government officials. Cut and paste design is the fast road to completion. In this sense, the description 'creative' may well be redundant.