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.

Elucidating the links between UV radiation and vitamin D synthesis : using an in vitro model

Ultraviolet radiation (UV) is the carcinogen that causes the most common malignancy in humans ��� skin cancer. However, moderate UV exposure is essential for producing vitaminDin our skin. VitaminDincreases the absorption of calcium from the diet, and adequate calcium is necessary for the building and maintenance of bones. Thus, low levels of vitamin D can cause osteomalacia and rickets and contribute to osteoporosis. Emerging evidence also suggests vitamin D may protect against falls, internal cancers, psychiatric conditions, autoimmune diseases and cardiovascular diseases. Since the dominant source of vitamin D is sunlight exposure, there is a need to understand what is a ���balanced��� level of sun exposure to maintain an adequate level of vitamin D but minimise the risks of eye damage, skin damage and skin cancer resulting from excessive UV exposure. There are many steps in the pathway from incoming solar UV to the eventual vitamin D status of humans (measured as 25-hydroxyvitamin D in the blood), and our knowledge about many of these steps is currently incomplete. This project begins by investigating the levels of UV available for synthesising vitamin D, and how these levels vary across seasons, latitudes and times of the day. The thesis then covers experiments conducted with an in vitro model, which was developed to study several aspects of vitamin D synthesis. Results from the model suggest the relationship between UV dose and vitamin D is not linear. This is an important input into public health messages regarding ���safe��� UV exposure: larger doses of UV, beyond a certain limit, may not continue to produce vitamin D; however, they will increase the risk of skin cancers and eye damage. The model also showed that, when given identical doses of UV, the amount of vitamin D produced was impacted by temperature. In humans, a temperature-dependent reaction must occur in the top layers of human skin, prior to vitamin D entering the bloodstream. The hypothesis will be raised that cooler temperatures (occurring in winter and at high latitudes) may reduce vitamin D production in humans. Finally, the model has also been used to study the wavelengths of UV thought to be responsible for producing vitamin D. It appears that vitamin D production is limited to a small range of UV wavelengths, which may be narrower than previously thought. Together, these results suggest that further research is needed into the ability of humans to synthesise vitamin D from sunlight. In particular, more information is needed about the dose-response relationship in humans and to investigate the proposed impact of temperature. Having an accurate action spectrum will also be essential for measuring the available levels of vitamin D-effective UV. As this research continues, it will contribute to the scientific evidence-base needed for devising a public health message that will balance the risks of excessive UV exposure with maintaining adequate vitamin D.

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.

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.

Merging fact with fiction : a study of year 9 students' developing scientific literacy through the writing of hybridised scientific narratives on a socioscientific issue

International assessments of student science achievement, and growing evidence of students' waning interest in school science, have ensured that the development of scientific literacy continues to remain an important educational priority. Furthermore, researchers have called for teaching and learning strategies to engage students in the learning of science, particularly in the middle years of schooling. This study extends previous national and international research that has established a link between writing and learning science. Specifically, it investigates the learning experiences of eight intact Year 9 science classes as they engage in the writing of short stories that merge scientific and narrative genres (i.e., hybridised scientific narratives) about the socioscientific issue of biosecurity. This study employed a triangulation mixed methods research design, generating both quantitative and qualitative data, in order to investigate three research questions that examined the extent to which the students' participation in the study enhanced their scientific literacy; the extent to which the students demonstrated conceptual understanding of related scientific concepts through their written artefacts and in interviews about the artefacts; and the extent to which the students' participation in the project influenced their attitudes toward science and science learning. Three aspects of scientific literacy were investigated in this study: conceptual science understandings (a derived sense of scientific literacy), the students' transformation of scientific information in written stories about biosecurity (simple and expanded fundamental senses of scientific literacy), and attitudes toward science and science learning. The stories written by students in a selected case study class (N=26) were analysed quantitatively using a series of specifically-designed matrices that produce numerical scores that reflect students' developing fundamental and derived senses of scientific literacy. All students (N=152) also completed a Likert-style instrument (i.e., BioQuiz), pretest and posttest, that examined their interest in learning science, science self-efficacy, their perceived personal and general value of science, their familiarity with biosecurity issues, and their attitudes toward biosecurity. Socioscientific issues (SSI) education served as a theoretical framework for this study. It sought to investigate an alternative discourse with which students can engage in the context of SSI education, and the role of positive attitudes in engaging students in the negotiation of socioscientific issues. Results of the study have revealed that writing BioStories enhanced selected aspects of the participants' attitudes toward science and science learning, and their awareness and conceptual understanding of issues relating to biosecurity. Furthermore, the students' written artefacts alone did not provide an accurate representation of the level of their conceptual science understandings. An examination of these artefacts in combination with interviews about the students' written work provided a more comprehensive assessment of their developing scientific literacy. These findings support extensive calls for the utilisation of diversified writing-to-learn strategies in the science classroom, and therefore make a significant contribution to the writing-to-learn science literature, particularly in relation to the use of hybridised scientific genres. At the same time, this study presents the argument that the writing of hybridised scientific narratives such as BioStories can be used to complement the types of written discourse with which students engage in the negotiation of socioscientific issues, namely, argumentation, as the development of positive attitudes toward science and science learning can encourage students' participation in the discourse of science. The implications of this study for curricular design and implementation, and for further research, are also discussed.

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.