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.

Cosmopolitanism, the nation-state and imaginative realism

Contemporary writing on cosmopolitanism has asserted the need for a new sociological toolkit to deal with an emergent post-national social order. At the heart of this agenda is a misunderstanding about the role of the nation-state, which has led to some rather unhelpful theorizations. The state is assumed to be a dead hand in the development of post-national sentiments or an increasingly irrelevant social structure. We argue that the superseding of the nation-state is not necessary for the development of cosmopolitan sentiments of solidarity. In addition to classical sociology, it is work surrounding the concepts of cosmopolitan democracy and constitutional patriotism and the public sphere that can assist us in theorizing cosmopolitanism. What distinguishes this tradition is the utilization of social science concepts such as democracy, state, public sphere and law in an attempt to ground the idea of cosmopolitanism within the context of existing social structures.

The efficacy of an augmented virtual reality system to alleviate pain in children undergoing burns dressing changes : a randomised controlled trial

In children, the pain and anxiety associated with acute burn dressing changes can be severe, with drug treatment alone frequently proving to be inadequate. Virtual reality (VR) systems have been successfully trialled in limited numbers of adult and paediatric burn patients. Augmented reality (AR) differs from VR in that it overlays virtual images onto the physical world, instead of creating a complete virtual world. This prospective randomised controlled trial investigated the use of AR as an adjunct to analgesia and sedation in children with acute burns. Forty-two children (30 male and 12 female), with an age range of 3–14 years (median age 9 years) and a total burn surface area ranging from 1 to 16% were randomised into a treatment (AR) arm and a control (basic cognitive therapy) arm after administration of analgesia and/or sedation. Pain scores, pulse rates (PR), respiratory rates (RR) and oxygen saturations (SaO2) were recorded pre-procedurally, at 10 min intervals and post-procedurally. Parents were also asked to grade their child's overall pain score for the dressing change. Mean pain scores were significantly lower (p = 0.0060) in the AR group compared to the control group, as were parental pain assessment scores (p = 0.015). Respiratory and pulse rates showed significant changes over time within groups, however, these were not significantly different between the two study groups. Oxygen saturation did not differ significantly over time or between the two study groups. This trial shows that augmented reality is a useful adjunct to pharmacological analgesia.

Near real-time prediction of Zenith Tropospheric Delays for position estimation over long-baseline CORS network

This paper presents the preliminary results in establishing a strategy for predicting Zenith Tropospheric Delay (ZTD) and relative ZTD (rZTD) between Continuous Operating Reference Stations (CORS) in near real-time. It is anticipated that the predicted ZTD or rZTD can assist the network-based Real-Time Kinematic (RTK) performance over long inter-station distances, ultimately, enabling a cost effective method of delivering precise positioning services to sparsely populated regional areas, such as Queensland. This research firstly investigates two ZTD solutions: 1) the post-processed IGS ZTD solution and 2) the near Real-Time ZTD solution. The near Real-Time solution is obtained through the GNSS processing software package (Bernese) that has been deployed for this project. The predictability of the near Real-Time Bernese solution is analyzed and compared to the post-processed IGS solution where it acts as the benchmark solution. The predictability analyses were conducted with various prediction time of 15, 30, 45, and 60 minutes to determine the error with respect to timeliness. The predictability of ZTD and relative ZTD is determined (or characterized) by using the previously estimated ZTD as the predicted ZTD of current epoch. This research has shown that both the ZTD and relative ZTD predicted errors are random in nature; the STD grows from a few millimeters to sub-centimeters while the predicted delay interval ranges from 15 to 60 minutes. Additionally, the RZTD predictability shows very little dependency on the length of tested baselines of up to 1000 kilometers. Finally, the comparison of near Real-Time Bernese solution with IGS solution has shown a slight degradation in the prediction accuracy. The less accurate NRT solution has an STD error of 1cm within the delay of 50 minutes. However, some larger errors of up to 10cm are observed.

Testing the effectiveness of a Practice Development intervention on changing the culture of evidence based practice in an acute care environment

In this age of evidence-based practice, nurses are increasingly expected to use research evidence in a systematic and judicious way when making decisions about patient care practices. Clinicians recognise the role of research when it provides valid, realistic answers in practical situations. Nonetheless, research is still perceived by some nurses as external to practice and implementing research findings into practice is often difficult. Since its conceptual platform in the 1960s, the emergence and growth of Nursing Development Units, and later, Practice Development Units has been described in the literature as strategic, organisational vehicles for changing the way nurses think about nursing by promoting and supporting a culture of inquiry and research-based practice. Thus, some scholars argue that practice development is situated in the gap between research and practice. Since the 1990s, the discourse has shifted from the structure and outcomes of developing practice to the process of developing practice, using a Practice Development methodology; underpinned by critical social science theory, as a vehicle for changing the culture and context of care. The nursing and practice development literature is dominated by descriptive reports of local practice development activity, typically focusing on reflection on processes or outcomes of processes, and describing perceived benefits. However, despite the volume of published literature, there is little published empirical research in the Australian or international context on the effectiveness of Practice Development as a methodology for changing the culture and context of care - leaving a gap in the literature. The aim of this study was to develop, implement and evaluate the effectiveness of a Practice Development model for clinical practice review and change on changing the culture and context of care for nurses working in an acute care setting. A longitudinal, pre-test/post-test, non-equivalent control group design was used to answer the following research questions: 1. Is there a relationship between nurses' perceptions of the culture and context of care and nurses' perceptions of research and evidence-based practice? 2. Is there a relationship between engagement in a facilitated process of Practice Development and change in nurses' perceptions of the culture and context of care? 3. Is there a relationship between engagement in a facilitated process of Practice Development and change in nurses' perceptions of research and evidence-based practice? Through a critical analysis of the literature and synthesis of the findings of past evaluations of Nursing and Practice Development structures and processes, this research has identified key attributes consistent throughout the chronological and theoretical development of Nursing and Practice Development that exemplify a culture and context of care that is conducive to creating a culture of inquiry and evidence-based practice. The study findings were then used in the development, validation and testing of an instrument to measure change in the culture and context of care. Furthermore, this research has also provided empirical evidence of the relationship of the key attributes to each other and to barriers to research and evidence-based practice. The research also provides empirical evidence regarding the effectiveness of a Practice Development methodology in changing the culture and context of care. This research is noteworthy in its contribution to advancing the discipline of nursing by providing evidence of the degree to which attributes of the culture and context of care, namely autonomy and control, workplace empowerment and constructive team dynamics, can be connected to engagement with research and evidence-based practice.

Dealing with Post-GFC stress disorder : Bwembya Chikolwa quoted by Christine Retschlag for Property Australia magazine 4th March 2010

Has the GFC really changed the thinking of the property industry? Or are investment managers suffering from post-GFC stress disorder fated to repeat the mistakes of the past? Christine Retschlag reports on the mindset of the market.

Thin film deposition and characterization of pure and iron-doped electron-beam evaporated tungsten oxide for gas sensors

Pure Tungsten Oxide (WO3) and Iron-doped (10 at%) Tungsten Oxide (WO3:Fe) nanostructured thin films were prepared using a dual crucible Electron Beam Evaporation techniques. The films were deposited at room temperature in high vacuum condition on glass substrate and post-heat treated at 300 oC for 1 hour. From the study of X-ray diffraction and Raman the characteristics of the as-deposited WO3 and WO3:Fe films indicated non-crystalline nature. The surface roughness of all the films showed in the order of 2.5 nm as observed using Atomic Force Microscopy (AFM). X-Ray Photoelectron Spectroscopy (XPS) analysis revealed tungsten oxide films with stoichiometry close to WO3. The addition of Fe to WO3 produced a smaller particle size and lower porosity as observed using Transmission Electron Microscopy (TEM). A slight difference in optical band gap energies of 3.22 eV and 3.12 eV were found between the as-deposited WO3 and WO3:Fe films, respectively. However, the difference in the band gap energies of the annealed films were significantly higher having values of 3.12 eV and 2.61 eV for the WO3 and WO3:Fe films, respectively. The heat treated samples were investigated for gas sensing applications using noise spectroscopy and doping of Fe to WO3 reduced the sensitivity to certain gasses. Detailed study of the WO3 and WO3:Fe films gas sensing properties is the subject of another paper.

Implementing change within a school science department: progressive and dissonant voices

The purpose of this study was to describe the teaching and leadership experiences of a science teacher who, as head of department, was preparing to introduce changes in the science department of an independent school in response to the requirements of the new junior science syllabus in Queensland, Australia. This teacher consented to classroom observations and interviews with the researchers where his beliefs about teaching practice and change were explored. Other science teachers at the school also were interviewed about their reactions to the planned changes. Interpretive analysis of the data provides an account of the complex interactions, negotiations, compromises, concessions, and trade-offs faced by the teacher during a period of education reform. Perceived barriers existing within the school that impeded proposed change are identified