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.

How can entrepreneurial musicians use electronic social networks to diffuse their music

Principal Topic: ''In less than ten years music labels will not exist anymore.'' Michael Smelli, former Global COO Sony/BMG MCA/QUT IMP Business Lab Digital Music Think Thanks 9 May 2009, Brisbane Big music labels such as EMI, Sony BMG and UMG have been responsible for promoting and producing a myriad of stars in the music industry over the last decades. However, the industry structure is under enormous threat with the emergence of a new innovative era of digital music. Recent years have seen a dramatic shift in industry power with the emergence of Napster and other file sharing sites, iTunes and other online stores, iPod and the MP3 revolution. Myspace.com and other social networking sites are connecting entrepreneurial artists with fans and creating online music communities independent of music labels. In 2008 the digital music business internationally grew by around 25% to 3.7 Billion US-Dollar. Digital platforms now account for around 20% of recorded music sales, up from 15 % in 2007 (IFPI Digital music report 2009). CD sales have fallen by 40% since their peak levels. Global digital music sales totalled an estimated US$ 3 Billion in 2007, an increase of 40% on 2006 figures. Digital sales account for an estimated 15% of global market, up from 11% in 2006 and zero in 2003. The music industry is more advanced in terms of digital revenues than any other creative or entertainment industry (except games). Its digital share is more than twice that of newspapers (7%), films (35) or books (2%). All these shifts present new possibilities for music entrepreneurs to act entrepreneurially and promote their music independently of the major music labels. Diffusion of innovations has a long tradition in both sociology (e.g. Rogers 1962, 2003) and marketing (Bass 1969, Mahajan et al., 1990). The context of the current project is theoretically interesting in two respects. First, the role of online social networks replaces traditional face-to-face word of mouth communications. Second, as music is a hedonistic product, this strongly influences the nature of interpersonal communications and their diffusion patterns. Both of these have received very little attention in the diffusion literature to date, and no studies have investigated the influence of both simultaneously. This research project is concerned with the role of social networks in this new music industry landscape, and how this may be leveraged by musicians willing to act entrepreneurially. Our key research question we intend to address is: How do online social network communities impact the nature, pattern and speed that music diffuses? Methodology/Key Propositions : We expect the nature/ character of diffusion of popular, generic music genres to be different from specialized, niche music. To date, only Moe & Fader (2002) and Lee et al. (2003) investigated diffusion patterns of music and these focus on forecast weekly sales of music CDs based on the advance purchase orders before the launch, rather than taking a detailed look at diffusion patterns. Consequently, our first research questions are concerned with understanding the nature of online communications within the context of diffusion of music and artists. Hence, we have the following research questions: RQ1: What is the nature of fan-to-fan ''word of mouth'' online communications for music? Do these vary by type of artist and genre of music? RQ2: What is the nature of artist-to-fan online communications for music? Do these vary by type of artist and genre of music? What types of communication are effective? Two outcomes from research social network theory are particularly relevant to understanding how music might diffuse through social networks. Weak tie theory (Granovetter, 1973), argues that casual or infrequent contacts within a social network (or weak ties) act as a link to unique information which is not normally contained within an entrepreneurs inner circle (or strong tie) social network. A related argument, structural hole theory (Burt, 1992), posits that it is the absence of direct links (or structural holes) between members of a social network which offers similar informational benefits. Although these two theories argue for the information benefits of casual linkages, and diversity within a social network, others acknowledge that a balanced network which consists of a mix of strong ties, weak ties is perhaps more important overall (Uzzi, 1996). It is anticipated that the network structure of the fan base for different types of artists and genres of music will vary considerably. This leads to our third research question: RQ3: How does the network structure of online social network communities impact the pattern and speed that music diffuses? The current paper is best described as theory elaboration. It will report the first exploratory phase designed to develop and elaborate relevant theory (the second phase will be a quantitative study of network structure and diffusion). We intend to develop specific research propositions or hypotheses from the above research questions. To do so we will conduct three focus group discussions of independent musicians and three focus group discussions of fans active in online music communication on social network sites. We will also conduct five case studies of bands that have successfully built fan bases through social networking sites (e.g. myspace.com, facebook.com). The idea is to identify which communication channels they employ and the characteristics of the fan interactions for different genres of music. We intend to conduct interviews with each of the artists and analyse their online interaction with their fans. Results and Implications : At the current stage, we have just begun to conduct focus group discussions. An analysis of the themes from these focus groups will enable us to further refine our research questions into testable hypotheses. Ultimately, our research will provide a better understanding of how social networks promote the diffusion of music, and how this varies for different genres of music. Hence, some music entrepreneurs will be able to promote their music more effectively. The results may be further generalised to other industries where online peer-to-peer communication is common, such as other forms of entertainment and consumer technologies.

National Aboriginal and Torres Strait Islander women's health strategy

The Strategy presented in this report was developed through the Australian Women’s Health Network Talking Circle in 2009-2010. Over 400 Aboriginal and Torres Strait Islander women were involved in the consultations. The Action Areas and Recommendations presented in this Strategy were raised and discussed by the women who contributed to the Talking Circle. This Strategy is not intended to replace any other national or state/territory identified priorities or needs. Instead, this Strategy supplements other work. Aboriginal and Torres Strait Islander women experience extremely poor health outcomes. They have a right to determine for themselves what their health system will look like. This Strategy is part of that process. If Aboriginal and Torres Strait Islander women continue to have their sense of identity marginalised and eroded, they will continue to have the poorest health of any group of women in Australian society.

Re-generating the praxis of academic staff development through arts-based learning : beyond paradox

Effective staff development remains a challenge in higher education. This paper examines the non-traditional methodology of arts-based staff development, its potential to foster transformational learning and the practice of professional artistry, through perceptions of program impact. Over a three year period, eighty academics participated in one metropolitan Australian university’s arts-based academic development program. The methodology used one-on-one hermeneutic-based conversations with fifteen self-selected academics and a focus group with twenty other academics from all three years. The paper presents a learning model to engender academic professional artistry. The findings provide developers with support for using a non-traditional strategy of transformational learning.

A principal at work : a story of leadership for building sustainable capacity of a school

This study explores through a lifestream narrative how the life experiences of a female primary school principal are organised as practical knowledge, and are used to inform action that is directed towards creating a sustainable school culture. An alternative model of school leadership is presented which describes the thinking and activity of a leader as a process. The process demonstrates how a leader's practical knowledge is dynamic, broadly based in experiential life, and open to change. As such, it is described as a model of sustainable leadership-in-process. The research questions at the heart of this study are: How does a leader construct and organize knowledge in the enactment of the principal ship to deal with the dilemmas and opportunities that arise everyday in school life? And: What does this particular way of organising knowledge look like in the effort to build a sustainable school community? The sustainable leadership-in-process thesis encapsulates new ways of leading primary schools through the principalship. These new ways are described as developing and maintaining the following dimensions of leadership: quality relationships, a collective (shared vision), collaboration and partnerships, and high achieving learning environments. Such dimensions are enacted by the principal through the activities of conversations, performance development, research and data-driven action, promoting innovation, and anticipating and predicting the future. Sustainable leadership-in-process is shared, dynamic, visible and transparent and is conducted through the processes of positioning, defining, organising, experimenting and evaluating in a continuous and iterative way. A rich understanding of the specificity of the life of a female primary school principal was achieved using story telling, story listening and story creation in a collaborative relationship between the researcher and the researched participant. as a means of educational theorising. Analysis and interpretation were undertaken as a recursive process in which the immediate interpretations were shared with the researched participant. The view of theorising adopted in this research is that of theory as hermeneutic; that is, theory is generated out of the stories of experiential life, rather than discovered in the stories.

After the marketplace: evidence, social science and educational research

This paper is an essay on the state of Australian education that frames new directions for educational research. It outlines three challenges faced by Australian educators: highly spatialised poverty with particularly strong mediating effects on primary school education; the need for intellectual and critical depth in pedagogy, with a focus in the upper primary and middle years; and the need to reinvent senior schooling to address emergent pathways from school to work and civic life. It offers a narrative description of the dynamics of policy making in Australia and North America and argues for an evidence-based approach to social and educational policy – but one quite unlike current test and market-based approaches. Instead, it argues for a multidisciplinary approach to a broad range of empirical and case-based evidence that subjects these to critical, hermeneutic social sciences. Such an approach would join educational policy with educational research, and broader social, community and governmental action with the aim of reorganising and redistributing material, cultural and social resources.

A practical implementation of a continuous isotropic spherical omnidirectional drive

This paper presents a continuous isotropic spherical omnidirectional drive mechanism that is efficient in its mechanical simplicity and use of volume. Spherical omnidirectional mechanisms allow isotropic motion, although many are limited from achieving true isotropic motion by practical mechanical design considerations. The mechanism presented in this paper uses a single motor to drive a point on the great circle of the sphere parallel to the ground plane, and does not require a gearbox. Three mechanisms located 120 degrees apart provide a stable drive platform for a mobile robot. Results show the omnidirectional ability of the robot and demonstrate the performance of the spherical mechanism compared to a popular commercial omnidirectional wheel over edges of varying heights and gaps of varying widths.

Civic engagement and associationalism : the impact of group membership scope versus intensity of participation

The relationship between participation in civic and political activities and membership of voluntary associations is now well established. What is less clear is the relative impacts of how much time people spend on group activities (associational intensity), and the number and type of groups that individuals are involved with (associational scope). Does it matter in terms of civic engagement, for example, whether one is a member of a quilting-circle or trade union? Does it matter whether association ‘membership’ is simply an annual payment or a major commitment of time and energy? In this article, we use a large survey to explore these questions empirically by focusing on the membership patterns and civic engagement practices of 4,001 citizens drawn from eight suburbs across Greater Melbourne, Australia. Our findings indicate that, while associational intensity is positively related to civic engagement, associational scope (the number of group memberships per person), is a more influential determinant of the level of civic and political participation. The results also suggest that while all forms of associationalism are important in terms of fostering greater levels of civic activity, not all forms have the same impact.

Molecular characterisation of six badnavirus species associated with leaf streak disease of banana in East Africa

Banana leaf streak disease, caused by several species of Banana streak virus (BSV), is widespread in East Africa. We surveyed for this disease in Uganda and Kenya, and used rolling-circle amplification (RCA) to detect the presence of BSV in banana. Six distinct badnavirus sequences, three from Uganda and three from Kenya, were amplified for which only partial sequences were previously available. The complete genomes were sequenced and characterised. The size and organisation of all six sequences was characteristic of other badnaviruses, including conserved functional domains present in the putative polyprotein encoded by open reading frame (ORF) 3. Based on nucleotide sequence analysis within the reverse transcriptase/ribonuclease H-coding region of open reading frame 3, we propose that these sequences be recognised as six new species and be designated as Banana streak UA virus, Banana streak UI virus, Banana streak UL virus, Banana streak UM virus, Banana streak CA virus and Banana streak IM virus. Using PCR and species-specific primers to test for the presence of integrated sequences, we demonstrated that sequences with high similarity to BSIMV only were present in several banana cultivars which had tested negative for episomal BSV sequences.

Reflections from Senior Women Academics

The Tiddas Showin’ Up, Talkin’ Up and Puttin’ Up: Indigenous Women and Educational Leadership project was led by Flinders University in partnership with the Australian Catholic University through the Indigenous Higher Education Centres located in Adelaide (Yunggorendi First Nations Centre) and Brisbane (Weemala Indigenous Unit) (Bunda and White 2009). At the beginning of the project, two levels of governance were established: a Circle of Senior Indigenous Women from the higher education sector, and a Steering Committee of Senior Executive Women from partner universities. The Circle of Senior Indigenous Women included experts in the fields of learning and teaching, scholarship, administration and management, and community engagement. The Circle’s members were: • Professor Wendy Brady, Charles Darwin University • Dr Jackie Huggins, University of Queensland • Ms Angela Leitch, Education Queensland • Professor Aileen Moreton-Robinson, Queensland University of Technology • Dr Bronwyn Fredericks, Queensland University of Technology and Monash University The Steering Committee members were: • Professor Gabrielle McMullen, Australian Catholic University • Professor Marie Emmit, Australian Catholic University • Professor Faith Trent, Flinders University • Dr Jane Robbins, Flinders University The two levels of governance provided advice to the project leaders throughout the project. Three of these women share their reflections in this paper.

Mental harm, rescuers and the Civil Liability Act 2002 (NSW)

At common law, a duty of care may be owed to a claimant who suffers nervous shock or pure mental harm due to witnessing, or hearing about, physical injury caused to another due to a defendant’s negligence. “Pure mental harm” is the ‘impairment of a person’s mental condition’ that is not suffered as a consequence of any other kind of personal injury to them. However, as many accidents have the potential to create a wide circle of mental suffering to bystanders, family members or others not physically injured themselves, it has traditionally been ‘thought impolitic that everybody so affected should be able to recover damages from the tortfeasor.’ ‘To allow such extended recovery would stretch liability too far.’ Nevertheless, whilst adopting a restrictive approach to liability, the common law courts have recognised that a defendant might owe a duty in relation to the pure mental harm suffered by one who foreseeably attends an accident scene to rescue another from a situation created by the defendant’s negligence.

Re-generating the praxis of academic staff development

Effective academic workforce staff development remains a challenge in higher education. This thesis-by-publication examined the importance of alternative paradigms for academic staff development, focusing specifically on arts-based learning as a non-traditional approach to transformative learning for management and self-development within the business of higher education. The research question asked was whether or not the facilitation of staff development through the practice of arts-based transformational learning supported academic aims in higher education, based on data obtained with the participants of the academic staff development program at one Australian university over a three year period. Over that three year period, eighty academics participated in one large metropolitan Australian university’s arts-based academic development program. The research approach required analysis of the transcribed one-on-one hermeneutic-based conversations with fifteen self-selected academics, five from each year, and with a focus group of twenty other self-selected academics from all three years. The study’s findings provided evidence that supported the need for academic staff development that prepared academics to be engaged and creative and therefore more likely to be responsive to emerging issues and to be innovative in the presence of constraints, including organisational constraints. The qualitative participative conversation transcription data found that arts-based lifelong learning processes provided participant perception of enhanced capabilities for self-creation and clarity of transformational action in academic career management. The study presented a new and innovative Artful Learning Wave Trajectory learning model to engender academic professional artistry. The findings provided developers with support for using a non-traditional strategy of transformational learning.

Teaching sustainability : vehicle or endpoint?

In 2006, the Faculty of Built Environment and Engineering introduced the first faculty wide unit dedicated to sustainability at any Australian University. BEB200 Introducing Sustainability has semester enrolments of up to 1500 students. Instruments such as lectures, readings, field visits, group projects and structured tutorial activities are used and have evolved over the last five years in response to student and staff feedback and attempts to better engage students. More than seventy staff have taught in the unit, which is in its final offering in this form in 2010. This paper reflects on the experiences of five academics who have played key roles in the development and teaching of this unit over the last five years. They argue that sustainability is a paradigm that allows students to explore other ways of knowing as they engage with issues in a complex world, not an end in itself. From the students’ perspective, grappling with such issues enables them to move towards a context in which they can understand their own discipline and its role in the contradictory and rapidly changing professional world. Insights are offered into how sustainability units may be developed in the future.

Alternative futures of globalisation a socio-ecological study of the world social forum process

Inspired by the initial World Social Forum in Porto Alegre Brazil, over the past decade over 200 local and regional social forums have been held, on five continents. This study has examined the nature of this broader social forum process, in particular as an aspect of the movement for 'another globalisation'. I discuss both the discourses for 'another world', as well as the development of an Alternative Globalisation Movement. As an action research study, the research took place within a variety of groups and networks. The thesis provides six accounts of groups and people striving and struggling for 'another world'. I provide a macro account of the invention and innovation of the World Social Forum. A grassroots film-makers collective provides a window into media. A local social forum opens up the radical diversity of actors. An activist exchange circle sheds light on strategic aspects of alternative globalisation. An educational initiative provides a window into transformations in pedagogy. And a situational account (of the G20 meeting in Melbourne in 2006) provides an overview of the variety of metanetworks that converge to voice demands for global justice and sustainability. In particular, this study has sought to shed light on how, within this process, groups and communities develop 'agency', a capacity to respond to the global challenges they / we face. And as part of this question, I have also explored how alternatives futures are developed and conceived, with a re-cognition of the importance of histories and geo-political (or 'eco-political') structures as contexts. I argue the World Social Forum Process is prefigurative, as an interactional process where many social alternatives are conceived, supported, developed and innovated into the world. And I argue this innovation process is meta-formative, where convergences of diverse actors comprise ‘social ecologies of alternatives’ which lead to opportunities for dynamic collaboration and partnership.