Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

How to tell the difference between production and consumption : a case study in Doctor Who fandom

A television series is tagged with the label "cult" by the media, advertisers, and network executives when it is considered edgy or offbeat, when it appeals to nostalgia, or when it is considered emblematic of a particular subculture. By these criteria, almost any series could be described as cult. Yet certain programs exert an uncanny power over their fans, encouraging them to immerse themselves within a fictional world.In Cult Television leading scholars examine such shows as The X-Files; The Avengers; Doctor Who, Babylon Five; Star Trek; Xena, Warrior Princess; and Buffy the Vampire Slayer to determine the defining characteristics of cult television and map the contours of this phenomenon within the larger scope of popular culture.Contributors: Karen Backstein; David A. Black, Seton Hall U; Mary Hammond, Open U; Nathan Hunt, U of Nottingham; Mark Jancovich; Petra Kuppers, Bryant College; Philippe Le Guern, U of Angers, France; Alan McKee; Toby Miller, New York U; Jeffrey Sconce, Northwestern U; Eva ViethSara Gwenllian-Jones is a lecturer in television and digital media at Cardiff University and co-editor of Intensities: The Journal of Cult Media.Roberta E. Pearson is a reader in media and cultural studies at Cardiff University. She is the author of the forthcoming book Small Screen, Big Universe: Star Trek and Television.

Entering the era of post production the role visual effects will play in documentary

Creating an acceptance of Visual Effects (VFX) as an effective non-fiction communication tool has the potential to significantly boost return on investment for filmmakers producing documentary. Obtaining this acceptance does not necessarily mean rethinking the way documentary is defined, however, the need to address negative perceptions presently dominant within the production industry does exist; specifically, the misguided judgement that use of sequences which include visual effects discredits a filmmaker's attempt to represent reality. After completing a documentary utilising a traditional model of production for methodology, the question of how to increase this film's marketability is then examined by testing the specific assertion that Visual Effects is capable of increasing the level of appeal inherent within the documentary genre. Whilst this area of research is speculative, qualifying Visual Effects as an acceptable communication tool in non-fiction narratives will allow the documentary sector to benefit from increased production capabilities.

Alternative film production

Typically a film producer expects the director and actors to 'do their job' within a scheduled timeframe. Rather than expecting the creative principals to just deliver, a production model can be tailored to help this creative team produce successful outcomes. This research paper contrasts alternative production models with a traditional (or standard) production and presents possibilities for producers to emphasise the collaborative potential for their production.

Relational factors that explain supply chain relationships

Purpose: The purpose of this paper is to gain a better understanding of the types of relationships that exist along the supply chain and the capabilities that are needed to manage them effectively. ---------- Design/methodology/approach: This is exploratory research as there has been little empirical research into this area. Quantitative data were gathered by using a self-administered questionnaire, using the Australian road freight industry as the context. There were 132 usable responses. Inferential and descriptive analysis, including factor analysis, confirmatory factor and regression analysis was used to examine the predictive power of relational factors in inter-firm relationships. ---------- Findings: Three factors were identified as having significant influence on relationships: sharing, power and interdependency. “Sharing” is the willingness of the organisation to share resources with other members of the supply chain. “Power” relates to exercising control based on experience, knowledge and position in the supply chain. “Interdependency” is the relative levels of dependency along the supply chain. ---------- Research limitations/implications: The research only looks at the Australian road freight industry; a wider sample including other industries would help to strengthen the generalisability of the findings. ---------- Practical implications: When these factors are correlated to the types of relationship, arm's length, cooperation, collaboration and alliances, managerial implications can be identified. The more road freight businesses place importance on power, the less they will cooperate. The greater the importance of sharing and interdependency, the greater is the likelihood of arm's length relationships. ---------- Originality/value: This paper makes a contribution by describing empirical work conducted in an under-researched but important area – supply chain relationships in the Australian road freight industry.

From the cathedral to the classroom : the emergence of new discourses on religious education

In 2009, Religious Education is a designated key learning area in Catholic schools in the Archdiocese of Brisbane and, indeed, across Australia. Over the years, though, different conceptualisations of the nature and purpose of religious education have led to the construction of different approaches to the classroom teaching of religion. By investigating the development of religious education policy in the Archdiocese of Brisbane from 1984 to 2003, the study seeks to trace the emergence of new discourses on religious education. The study understands religious education to refer to a lifelong process that occurs through a variety of forms (Moran, 1989). In Catholic schools, it refers both to co-curricula activities, such as retreats and school liturgies, and the classroom teaching of religion. It is the policy framework for the classroom teaching of religion that this study explores. The research was undertaken using a policy case study approach to gain a detailed understanding of how new conceptualisations of religious education emerged at a particular site of policy production, in this case, the Archdiocese of Brisbane. The study draws upon Yeatman’s (1998) description of policy as occurring “when social actors think about what they are doing and why in relation to different and alternative possible futures” (p. 19) and views policy as consisting of more than texts themselves. Policy texts result from struggles over meaning (Taylor, 2004) in which specific discourses are mobilised to support particular views. The study has a particular interest in the analysis of Brisbane religious education policy texts, the discursive practices that surrounded them, and the contexts in which they arose. Policy texts are conceptualised in the study as representing “temporary settlements” (Gale, 1999). Such settlements are asymmetrical, temporary and dependent on context: asymmetrical in that dominant actors are favoured; temporary because dominant actors are always under challenge by other actors in the policy arena; and context - dependent because new situations require new settlements. To investigate the official policy documents, the study used Critical Discourse Analysis (hereafter referred to as CDA) as a research tool that affords the opportunity for researchers to map and chart the emergence of new discourses within the policy arena. As developed by Fairclough (2001), CDA is a three-dimensional application of critical analysis to language. In the Brisbane religious education arena, policy texts formed a genre chain (Fairclough, 2004; Taylor, 2004) which was a focus of the study. There are two features of texts that form genre chains: texts are systematically linked to one another; and, systematic relations of recontextualisation exist between the texts. Fairclough’s (2005) concepts of “imaginary space” and “frameworks for action” (p. 65) within the policy arena were applied to the Brisbane policy arena to investigate the relationship between policy statements and subsequent guidelines documents. Five key findings emerged from the study. First, application of CDA to policy documents revealed that a fundamental reconceptualisation of the nature and purpose of classroom religious education in Catholic schools occurred in the Brisbane policy arena over the last twenty-five years. Second, a disjuncture existed between catechetical discourses that continued to shape religious education policy statements, and educational discourses that increasingly shaped guidelines documents. Third, recontextualisation between policy documents was evident and dependent on the particular context in which religious education occurred. Fourth, at subsequent links in the chain, actors created their own “imaginary space”, thereby altering orders of discourse within the policy arena, with different actors being either foregrounded or marginalised. Fifth, intertextuality was more evident in the later links in the genre chain (i.e. 1994 policy statement and 1997 guidelines document) than in earlier documents. On the basis of the findings of the study, six recommendations are made. First, the institutional Church should carefully consider the contribution that the Catholic school can make to the overall pastoral mission of the diocese in twenty-first century Australia. Second, policymakers should articulate a nuanced understanding of the relationship between catechesis and education with regard to the religion classroom. Third, there should be greater awareness of the connections among policies relating to Catholic schools – especially the connection between enrolment policy and religious education policy. Fourth, there should be greater consistency between policy documents. Fifth, policy documents should be helpful for those to whom they are directed (i.e. Catholic schools, teachers). Sixth, “imaginary space” (Fairclough, 2005) in policy documents needs to be constructed in a way that allows for multiple “frameworks for action” (Fairclough, 2005) through recontextualisation. The findings of this study are significant in a number of ways. For religious educators, the study highlights the need to develop a shared understanding of the nature and purpose of classroom religious education. It argues that this understanding must take into account the multifaith nature of Australian society and the changing social composition of Catholic schools themselves. Greater recognition should be given to the contribution that religious studies courses such as Study of Religion make to the overall religious development of a person. In view of the social composition of Catholic schools, there is also an issue of ecclesiological significance concerning the conceptualisation of the relationship between the institutional Catholic Church and Catholic schools. Finally, the study is of significance because of its application of CDA to religious education policy documents. Use of CDA reveals the foregrounding, marginalising, or excluding of various actors in the policy arena.

New strength of competition and innovation : China’s independent television production

The state-owned media system in China has evolved considerably since 1994 when the first independent TV production company was officially registered. Today, there are thousands of independent TV production companies looking for market opportunities in China. Independent production companies have facilitated the circulation of program trade and investment, and in the process have encouraged innovation and professionalization. This paper focuses on the evolution of independents and the changing face of the television market. It discusses the ecology of independent television companies in China and how government regulations are impacting on the TV production market. It argues that independent TV is providing a new strength for China‟s TV market, one often suspected of being imitative, propagandistic and lacking colour.

Service identification through value chain analysis and prioritization

In a resource constrained business world, strategic choices must be made on process improvement and service delivery. There are calls for more agile forms of enterprises and much effort is being directed at moving organizations from a complex landscape of disparate application systems to that of an integrated and flexible enterprise accessing complex systems landscapes through service oriented architecture (SOA). This paper describes the deconstruction of an enterprise into business services using value chain analysis as each element in the value chain can be rendered as a business service in the SOA. These business services are explicitly linked to the attainment of specific organizational strategies and their contribution to the attainment of strategy is assessed and recorded. This contribution is then used to provide a rank order of business service to strategy. This information facilitates executive decision making on which business service to develop into the SOA. The paper describes an application of this Critical Service Identification Methodology (CSIM) to a case study.

The effects of pore architecture in silk fibroin scaffolds on the growth and differentiation of mesenchymal stem cells expressing BMP7

The pore architecture of scaffolds is known to play a critical role in tissue engineering as it provides the vital framework for seeded cells to organize into a functioning tissue. In this report we have investigated the effects of different concentrations of silk fibroin protein on three-dimensional (3D) scaffold pore microstructure. Four pore size ranges of silk fibroin scaffolds were made by the freeze drying technique, with the pore sizes ranging from 50 to 300 lm. The pore sizes of the scaffolds decreased as the concentration of fibroin protein increased. Human bone marrow mesenchymal stromal cells (BMSC) transfected with the BMP7 gene were cultured in these scaffolds. A cell viability colorimetric assay, alkaline phosphatase assay and reverse transcription-polymerase chain reaction were performed to analyze the effect of pore size on cell growth, the secretion of extracellular matrix (ECM) and osteogenic differentiation. Cell migration in 3D scaffolds was confirmed by confocal microscopy. Calvarial defects in SCID mice were used to determine the bone forming ability of the silk fibroin scaffolds incorporating BMSC expressing BMP7. The results showed that BMSC expressing BMP7 preferred a pore size between 100 and 300 lm in silk fibroin protein fabricated scaffolds, with better cell proliferation and ECM production. Furthermore, in vivo transplantation of the silk fibroin scaffolds combined with BMSC expressing BMP7 induced new bone formation. This study has shown that an optimized pore architecture of silk fibroin scaffolds can modulate the bioactivity of BMP7-transfected BMSC in bone formation.

A two-step mutual authentication protocol based on randomized hash-lock for small RFID networks

RFID has been widely used in today's commercial and supply chain industry, due to the significant advantages it offers and the relatively low production cost. However, this ubiquitous technology has inherent problems in security and privacy. This calls for the development of simple, efficient and cost effective mechanisms against a variety of security threats. This paper proposes a two-step authentication protocol based on the randomized hash-lock scheme proposed by S. Weis in 2003. By introducing additional measures during the authentication process, this new protocol proves to enhance the security of RFID significantly, and protects the passive tags from almost all major attacks, including tag cloning, replay, full-disclosure, tracking, and eavesdropping. Furthermore, no significant changes to the tags is required to implement this protocol, and the low complexity level of the randomized hash-lock algorithm is retained.

Workplace design in the knowledge economy : a case of the NetWorkPlace™©

This thesis explores a way to inform the architectural design process for contemporary workplace environments. It reports on both theoretical and practical outcomes through an exclusively Australian case study of a network enterprise comprised of collaborative, yet independent business entities. The internet revolution, substantial economic and cultural shifts, and an increased emphasis on lifestyle considerations have prompted a radical re-ordering of organisational relationships and the associated structures, processes, and places of doing business. The social milieu of the information age and the knowledge economy is characterised by an almost instantaneous flow of information and capital. This has culminated in a phenomenon termed by Manuel Castells as the network society, where physical locations are joined together by continuous communication and virtual connectivity. A new spatial logic encompassing redefined concepts of space and distance, and requiring a comprehensive shift in the approach to designing workplace environments for today’s adaptive, collaborative organisations in a dynamic business world, provides the backdrop for this research. Within the duality of space and an augmentation of the traditional notions of place, organisational and institutional structures pose new challenges for the design professions. The literature revealed that there has always been a mono-organisational focus in relation to workplace design strategies. The phenomenon of inter-organisational collaboration has enabled the identification of a gap in the knowledge relative to workplace design. This new context generated the formulation of a unique research construct, the NetWorkPlace™©, which captures the complexity of contemporary employment structures embracing both physical and virtual work environments and practices, and provided the basis for investigating the factors that are shaping and defining interactions within and across networked organisational settings. The methodological orientation and the methods employed follow a qualitative approach and an abductively driven strategy comprising two distinct components, a cross-sectional study of the whole of the network and a longitudinal study, focusing on a single discrete workplace site. The complexity of the context encountered dictated that a multi-dimensional investigative framework was required to be devised. The adoption of a pluralist ontology and the reconfiguration of approaches from traditional paradigms into a collaborative, trans-disciplinary, multi-method epistemology provided an explicit and replicatable method of investigation. The identification and introduction of the NetWorkPlace™© phenomenon, by necessity, spans a number of traditional disciplinary boundaries. Results confirm that in this context, architectural research, and by extension architectural practice, must engage with what other disciplines have to offer. The research concludes that no single disciplinary approach to either research or practice in this area of design can suffice. Pierre Bourdieau’s philosophy of ‘practice’ provides a framework within which the governance and technology structures, together with the mechanisms enabling the production of social order in this context, can be understood. This is achieved by applying the concepts of position and positioning to the corporate power dynamics, and integrating the conflict found to exist between enterprise standard and ferally conceived technology systems. By extending existing theory and conceptions of ‘place’ and the ‘person-environment relationship’, relevant understandings of the tensions created between Castells’ notions of the space of place and the space of flows are established. The trans-disciplinary approach adopted, and underpinned by a robust academic and practical framework, illustrates the potential for expanding the range and richness of understanding applicable to design in this context. The outcome informs workplace design by extending theoretical horizons, and by the development of a comprehensive investigative process comprising a suite of models and techniques for both architectural and interior design research and practice, collectively entitled the NetWorkPlace™© Application Framework. This work contributes to the body of knowledge within the design disciplines in substantive, theoretical, and methodological terms, whilst potentially also influencing future organisational network theories, management practices, and information and communication technology applications. The NetWorkPlace™© as reported in this thesis, constitutes a multi-dimensional concept having the capacity to deal with the fluidity and ambiguity characteristic of the network context, as both a topic of research and the way of going about it.

Measuring the success of ABC training in South Africa: A case study in the production of Western Liberal broadcast news values

In 1993 the Australian Broadcasting Corporation was contracted by the Australian Government to assist in the reshaping of the South African Broadcasting Corporation from a state-run broadcaster to a respected and trusted national broadcaster for all people in the newly democratic South Africa. Broadcast journalism training was identified by ABC consultant Bob Wurth as possibly the greatest need for SABC Radio. This thesis examines the ABC's role in South Africa and the effectiveness of its radio journalism training project considering the organisational, structural, cultural and political constraints of the SABC. This thesis will show through interviews and participant observation the difficulties in achieving the production of Western Liberal journalism values at the SABC within the time constraints set by the project funded by the Australian Government and the particular South African morays.