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.

Gesturing elsewhere: the identity politics of the Balinese death/thrash metal scene

This essay explores the political significance of Balinese death/thrash fandom. In the early 1990s, the emergence of a death/thrash scene in Bali paralleled growing criticism of accelerated tourism development on the island. Specifically, locals protested the increasing ubiquity of Jakarta, 'the centre', cast as threatening to an authentically 'low', peripheral Balinese culture. Similarly, death/thrash enthusiasts also gravitated toward certain fringes, although they rejected dominant notions of Balinese-ness by gesturing elsewhere, toward a global scene. The essay explores the ways in which death/thrash enthusiasts engaged with local discourses by coveting their marginality, and aims to demonstrate how their articulations of 'alien-ness' contributed in important ways to a broader regionalism.

Regulating roles of the use of the balanced scorecard in shaping corporate identities

The literature on corporate identity management suggests that managing corporate identity is a strategically complex task embracing the shaping of a range of dimensions of organisational life. The performance measurement literature and its applications likewise now also emphasise organisational ability to incorporate various dimensions considering both financial and non-financial performance measures when assessing success. The inclusion of these soft non-financial measures challenges organisations to quantify intangible aspects of performance such as corporate identity, transforming unmeasurables into measurables. This paper explores the regulatory roles of the use of the balanced scorecard in shaping key dimensions of corporate identities in a public sector shared service provider in Australia. This case study employs qualitative interviews of senior managers and employees, secondary data and participant observation. The findings suggest that the use of the balanced scorecard has potential to support identity construction, as an organisational symbol, a communication tool of vision, and as strategy, through creating conversations that self-regulate behaviour. The development of an integrated performance measurement system, the balanced scorecard, becomes an expression of a desired corporate identity, and the performance measures and continuous process provide the resource for interpreting actual corporate identities. Through this process of understanding and mobilising the interaction, it may be possible to create a less obtrusive and more subtle way to control “what an organisation is”. This case study also suggests that the theoretical and practical fusion of the disciplinary knowledge around corporate identities and performance measurement systems could make a contribution to understanding and shaping corporate identities.

Aging in China and its impact on vehicle design

This study contributes to the growth of design knowledge in China, where vehicle design for the local, older user is in its initial developmental stages. Therefore, this research has explored the travel needs of older Chinese vehicle users in order to assist designers to better understand users’ current and future needs. A triangulation method consisting of interviews, logbook and co-discovery was used to collect multiple forms of data and so explore the research question. Grounded theory has been employed to analyze the research data. This study found that users’ needs are reflected through various ‘meanings’ that they attach to vehicles – meanings that give a tangible expression to their experiences. This study identified six older-user need categories: (i) safety, (ii) utility, (iii) comfort, (iv) identity, (v) emotion and (vi) spirituality. The interrelationships among these six categories are seen as an interactive structure, rather than as a linear or hierarchical arrangement. Chinese cultural values, which are generated from particular local context and users’ social practice, will play a dynamic role in linking and shaping the travel needs of older vehicle users in the future. Moreover, this study structures the older-user needs model into three levels of meaning, to give guidance to vehicle design direction: (i) the practical meaning level, (ii) the social meaning level and (ii) the cultural meaning level. This study suggests that a more comprehensive explanation exists if designers can identify the vehicle’s meaning and property associated with the fulfilled older users’ needs. However, these needs will vary, and must be related to particular technological, social, and cultural contexts. The significance of this study lies in its contributions to the body of knowledge in three areas: research methodology, theory and design. These theoretical contributions provide a series of methodological tools, models and approaches from a vehicle design perspective. These include a conditional/consequential matrix, a travel needs identification model, an older users’ travel-related needs framework, a user information structure model, and an Older-User-Need-Based vehicle design approach. These models suggest a basic framework for the new design process which might assist in the design of new vehicles to fulfil the needs of future, aging Chinese generations. The models have the potential to be transferred to other design domains and different cultural contexts.

Sexuality in a criminal justice curriculum : a study of student conceptualisations of gay identity

Heteronormative discourses provide the most common lens through which sexuality is understood within university curricula. This means that sexuality is discussed in terms of categories of identity, with heterosexuality accorded primacy while all 'others' are indeed 'othered'. This article draws on research carried out by the authors in a core first year university ethics class, in which a fictional text was introduced with the intention of unpacking these discourses. An ethnographic study was undertaken where both students and teachers engaged in discussions over, and personal written reflections on, the textual content. In reporting the results of that study this article uses a post-structural framework to identify how classroom and textual discourses might be used to break down socially constructed categories of sexuality and students' conceptualisations of non-heterosexual behaviour. It was found that engaging in discussion in the context of the fictional text allowed some students to begin to recognise their own heteronormative views and engage in an informed critique of them.

Literacy and identity : examining the metaphors in history and contemporary research

In this review, the authors interrogate the recent identity turn in literacy studies by asking the following: How do particular views of identity shape how researchers think about literacy and, conversely, how does the view of literacy taken by a researcher shape meanings made about identity? To address this question, the authors review various ways of conceptualizing identity by using five metaphors for identity documented in the identity literature: identity as (1) difference, (2) sense of self/subjectivity, (3) mind or consciousness, (4) narrative, and (5) position. Few literacy studies have acknowledged this range of perspectives on and views for conceptualizing identity and yet, subtle differences in identity theories have widely different implications for how one thinks about both how literacy matters to identity and how identity matters to literacy. The authors offer this review to encourage more theorizing of both literacy and identity as social practices and, most important, of how the two breathe life into each other.

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.

Is the Marlboro man the only alternative? The role of gender identity and self-construal salience in evaluations of male models

This research examines how men react to male models in print advertisements. In two experiments, we show that the gender identity of men influences their responses to advertisements featuring a masculine, feminine, or androgynous male model. In addition, we explore the extent to which men feel they will be classified by others as similar to the model as a mechanism for these effects. Specifically, masculine men respond most favorably to masculine models and are negative toward feminine models. In contrast, feminine men prefer feminine models when their private self is salient. Yet in a collective context, they prefer masculine models.These experiments shed light on how gender identity and self-construal influence male evaluations and illustrate the social pressure on men to endorse traditional masculine portrayals. We also present implications for advertising practice.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

“Queer” … does not have a uniform : articulations of queer identity in Australian queer student activist media

This article explores articulations of queer identity in recent Australian queer student media. Print media is of particular importance to queer communities because, as Cover argues, it provides a crucial grounding for community development and a model of queer to guide the positioning of identity and activism. This article uses discourse analysis of queer student activists’ media representations of diversity and inclusiveness to investigate the articulations of queer identity in one specific context: metropolitan Australian universities. This reveals real-life appropriations of this contentious term and contributes to a genealogy of sexuality, documenting one visible moment in history.

Identity and disciplinarity : Multi-disciplinary student cohorts in Higher Education [Abstract]

This paper will describe a research project that examines the implications of multidisciplinary student cohorts on teaching and learning within undergraduate and postgraduate units in higher education. Whist students generally specialise in one discipline, it is also common that, at some point during their degree, they will choose to undertake subjects that are outside their specialist area. Students may choose a multidisciplinary learning experience either out of interest or because the subject is seen as complementary to their core discipline. When the lens of identity is applied to the multi-disciplinary cohorts in undergraduate and postgraduate units, it assists in identifying learning needs. The nature of disciplinarity, and the impact it has on students’ academic identity, presents challenges to both students and teachers when they engage in teaching and learning, impacting on curriculum design, assessment practices and teaching delivery strategies (Winberg, 2008). This project aims to identify the barriers that exist to effective teaching and learning in units that have multidisciplinary student cohorts. It will identify the particular needs of students in multidisciplinary student cohorts and determine a teaching and learning model that meets the needs of such cohorts. References Becher, T. & Trowler, P.R. (2001). Academic tribes and territories: Intellectual enquiry and the culture of the discipline. Buckingham, UK: Open University Press. Light, G. & Cox, R. (2001). Learning and teaching in higher education: A reflective professional. Thousand Oaks, CA: Sage. Neumann, R. (2001). Disciplinary differences and university teaching. Studies in Higher Education, 26 (2), 135-46. Neumann, R., Parry, S. & Becher, T. (2002). Teaching and Learning in their disciplinary contexts: A conceptual analysis. Studies in Higher Education, 27(4), 405-417. Taylor, P.G. (1999) Making Sense of Academic Life: Academics, Universities and Change. Buckingham, UK: Open University Press. Winberg, C. (2008). Teaching engineering/engineering teaching: interdisciplinary collaboration and the construction of academic identities. Teaching in Higher Education, 13(3), 353 - 367.