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.

Layered titanate nanofibers as efficient adsorbents for removal of toxic radioactive and heavy metal ions from water

Titanate nanofibers with two formulas, Na2Ti3O7 and Na1.5H0.5Ti3O7, respectively, exhibit ideal properties for removal of radioactive and heavy metal ions in wastewater, such as Sr2+ , Ba2+ (as substitute of 226Ra2+), and Pb2+ ions. These nanofibers can be fabricated readily by a reaction between titania and caustic soda and have structures in which TiO6 octahedra join each other to form layers with negative charges; the sodium cations exist within the interlayer regions and are exchangeable. They can selectively adsorb the bivalent radioactive ions and heavy metal ions from water through ion exchange process. More importantly, such sorption finally induces considerable deformation of the layer structure, resulting in permanent entrapment of the toxic bivalent cations in the fibers so that the toxic ions can be safely deposited. This study highlights that nanoparticles of inorganic ion exchangers with layered structure are potential materials for efficient removal of the toxic ions from contaminated water.

Dynamic load sharing for heavy vehicles : a new metric

Dynamic load sharing can be defined as a measure of the ability of a heavy vehicle multi-axle group to equalise load across its wheels under typical travel conditions; i.e. in the dynamic sense at typical travel speeds and operating conditions of that vehicle. Various attempts have been made to quantify the ability of heavy vehicles to equalise the load across their wheels during travel. One of these was the concept of the load sharing coefficient (LSC). Other metrics such as the dynamic load coefficient (DLC) have been used to compare one heavy vehicle suspension with another for potential road damage. This paper compares these metrics and determines a relationship between DLC and LSC with sensitivity analysis of this relationship. The shortcomings of these presently-available metrics are discussed with a new metric proposed - the dynamic load equalisation (DLE) measure.

Executive and organisational leadership development in universities : exploring key elements and perceptions

Organisational and leadership development is said to be one of the most challenging and important activities facing universities, particularly in the current environment of fast-paced change and accelerated age-related attrition. Succession leadership development being timely, the purpose of this study was to explore the nature of leadership development most suited to meeting the leadership and organisational development challenges for contemporary universities. A blend of literature-based and empirical research was undertaken. This resulted in seven papers submitted to internationally refereed journals; five papers published, one in press, and one under review. Six of these are sole authored papers and one is a co-authored paper. The papers identify some of the issues and challenges facing the tertiary sector. They shed light on factors influencing executive and organisational leadership development deriving from the literature review and from empirical research reporting the views of current university leaders. The papers and submission document herein include recommendations and suggested models informing executive and organisational leadership development in universities. The "Lantern" model - an Illuminated Model for Organisational Leadership Development - is a key original conceptual model framing the study.

The conceptualisation, sensitivity and measurement of holding costs and other selected elements impacting housing affordability (Literature review and research methodology for confirmation of PhD candidature)

It is widely held that strong relationships exist between housing, economic status, and well being. This is exemplified by widespread housing stock surpluses in many countries which threaten to destabilise numerous aspects related to individuals and community. However, the position of housing demand and supply is not consistent. The Australian position provides a distinct contrast whereby seemingly inexorable housing demand generally remains a critical issue affecting the socio-economic landscape. Underpinned by high levels of immigration, and further buoyed by sustained historically low interest rates, increasing income levels, and increased government assistance for first home buyers, this strong housing demand ensures elements related to housing affordability continue to gain prominence. A significant, but less visible factor impacting housing affordability – particularly new housing development – relates to holding costs. These costs are in many ways “hidden” and cannot always be easily identified. Although it is only one contributor, the nature and extent of its impact requires elucidation. In its simplest form, it commences with a calculation of the interest or opportunity cost of land holding. However, there is significantly more complexity for major new developments - particularly greenfield property development. Preliminary analysis conducted by the author suggests that even small shifts in primary factors impacting holding costs can appreciably affect housing affordability – and notably, to a greater extent than commonly held. Even so, their importance and perceived high level impact can be gauged from the unprecedented level of attention policy makers have given them over recent years. This may be evidenced by the embedding of specific strategies to address burgeoning holding costs (and particularly those cost savings associated with streamlining regulatory assessment) within statutory instruments such as the Queensland Housing Affordability Strategy, and the South East Queensland Regional Plan. However, several key issues require investigation. Firstly, the computation and methodology behind the calculation of holding costs varies widely. In fact, it is not only variable, but in some instances completely ignored. Secondly, some ambiguity exists in terms of the inclusion of various elements of holding costs, thereby affecting the assessment of their relative contribution. Perhaps this may in part be explained by their nature: such costs are not always immediately apparent. Some forms of holding costs are not as visible as the more tangible cost items associated with greenfield development such as regulatory fees, government taxes, acquisition costs, selling fees, commissions and others. Holding costs are also more difficult to evaluate since for the most part they must be ultimately assessed over time in an ever-changing environment, based on their strong relationship with opportunity cost which is in turn dependant, inter alia, upon prevailing inflation and / or interest rates. By extending research in the general area of housing affordability, this thesis seeks to provide a more detailed investigation of those elements related to holding costs, and in so doing determine the size of their impact specifically on the end user. This will involve the development of soundly based economic and econometric models which seek to clarify the componentry impacts of holding costs. Ultimately, there are significant policy implications in relation to the framework used in Australian jurisdictions that promote, retain, or otherwise maximise, the opportunities for affordable housing.

Learning approach and perception of learning context in economics education : a causality test

In the study of student learning literature, the traditional view holds that when students are faced with heavy workload, poor teaching, and content that they cannot relate to – important aspects of the learning context, they will more likely utilise the surface approach to learning due to stresses, lack of understanding and lack of perceived relevance of the content (Kreber, 2003; Lizzio, Wilson, & Simons, 2002; Ramdsen, 1989; Ramsden, 1992; Trigwell & Prosser, 1991; Vermunt, 2005). For example, in studies involving health and medical sciences students, courses that utilised student-centred, problem-based approaches to teaching and learning were found to elicit a deeper approach to learning than the teacher-centred, transmissive approach (Patel, Groen, & Norman, 1991; Sadlo & Richardson, 2003). It is generally accepted that the line of causation runs from the learning context (or rather students’ self reported data on the learning context) to students’ learning approaches. That is, it is the learning context as revealed by students’ self-reported data that elicit the associated learning behaviour. However, other research studies also found that the same teaching and learning environment can be perceived differently by different students. In a study of students’ perceptions of assessment requirements, Sambell and McDowell (1998) found that students “are active in the reconstruction of the messages and meanings of assessment” (p. 391), and their interpretations are greatly influenced by their past experiences and motivations. In a qualitative study of Hong Kong tertiary students, Kember (2004) found that students using the surface learning approach reported heavier workload than students using the deep learning approach. According to Kember if students learn by extracting meanings from the content and making connections, they will more likely see the higher order intentions embodied in the content and the high cognitive abilities being assessed. On the other hand, if they rote-learn for the graded task, they fail to see the hierarchical relationship in the content and to connect the information. These rote-learners will tend to see the assessment as requiring memorising and regurgitation of a large amount of unconnected knowledge, which explains why they experience a high workload. Kember (2004) thus postulate that it is the learning approach that influences how students perceive workload. Campbell and her colleagues made a similar observation in their interview study of secondary students’ perceptions of teaching in the same classroom (Campbell et al., 2001). The above discussions suggest that students’ learning approaches can influence their perceptions of assessment demands and other aspects of the learning context such as relevance of content and teaching effectiveness. In other words, perceptions of elements in the teaching and learning context are endogenously determined. This study attempted to investigate the causal relationships at the individual level between learning approaches and perceptions of the learning context in economics education. In this study, students’ learning approaches and their perceptions of the learning context were measured. The elements of the learning context investigated include: teaching effectiveness, workload and content. The authors are aware of existence of other elements of the learning context, such as generic skills, goal clarity and career preparation. These aspects, however, were not within the scope of this present study and were therefore not investigated.

Raman and infrared study of phyllosilicates containing heavy metals (Sb, Bi) : bismutoferrite and chapmanite.

The kaolinite-like phyllosilicate minerals bismutoferrite BiFe3+2Si2O8(OH) and chapmanite SbFe3+2Si2O8(OH) have been studied by Raman spectroscopy and complemented with infrared spectra. Tentatively interpreted spectra were related to their molecular structure. The antisymmetric and symmetric stretching vibrations of the Si-O-Si bridges,  SiOSi and  OSiO bending vibrations,  (Si-Oterminal)- stretching vibrations,  OH stretching vibrations of hydroxyl ions, and  OH bending vibrations were attributed to observed bands. Infrared bands 3289-3470 cm-1 and Raman bands 1590-1667 cm-1 were assigned to adsorbed water. O-H...O hydrogen bond lengths were calculated from the Raman and infrared spectra.

A new multilevel converter configuration for high power and high quality applications

The Queensland University of Technology (QUT) allows the presentation of theses for the Degree of Doctor of Philosophy in the format of published or submitted papers, where such papers have been published, accepted or submitted during the period of candidature. This thesis is composed of ten published /submitted papers and book chapters of which nine have been published and one is under review. This project is financially supported by an Australian Research Council (ARC) Discovery Grant with the aim of investigating multilevel topologies for high quality and high power applications, with specific emphasis on renewable energy systems. The rapid evolution of renewable energy within the last several years has resulted in the design of efficient power converters suitable for medium and high-power applications such as wind turbine and photovoltaic (PV) systems. Today, the industrial trend is moving away from heavy and bulky passive components to power converter systems that use more and more semiconductor elements controlled by powerful processor systems. However, it is hard to connect the traditional converters to the high and medium voltage grids, as a single power switch cannot stand at high voltage. For these reasons, a new family of multilevel inverters has appeared as a solution for working with higher voltage levels. Besides this important feature, multilevel converters have the capability to generate stepped waveforms. Consequently, in comparison with conventional two-level inverters, they present lower switching losses, lower voltage stress across loads, lower electromagnetic interference (EMI) and higher quality output waveforms. These properties enable the connection of renewable energy sources directly to the grid without using expensive, bulky, heavy line transformers. Additionally, they minimize the size of the passive filter and increase the durability of electrical devices. However, multilevel converters have only been utilised in very particular applications, mainly due to the structural limitations, high cost and complexity of the multilevel converter system and control. New developments in the fields of power semiconductor switches and processors will favor the multilevel converters for many other fields of application. The main application for the multilevel converter presented in this work is the front-end power converter in renewable energy systems. Diode-clamped and cascade converters are the most common type of multilevel converters widely used in different renewable energy system applications. However, some drawbacks – such as capacitor voltage imbalance, number of components, and complexity of the control system – still exist, and these are investigated in the framework of this thesis. Various simulations using software simulation tools are undertaken and are used to study different cases. The feasibility of the developments is underlined with a series of experimental results. This thesis is divided into two main sections. The first section focuses on solving the capacitor voltage imbalance for a wide range of applications, and on decreasing the complexity of the control strategy on the inverter side. The idea of using sharing switches at the output structure of the DC-DC front-end converters is proposed to balance the series DC link capacitors. A new family of multioutput DC-DC converters is proposed for renewable energy systems connected to the DC link voltage of diode-clamped converters. The main objective of this type of converter is the sharing of the total output voltage into several series voltage levels using sharing switches. This solves the problems associated with capacitor voltage imbalance in diode-clamped multilevel converters. These converters adjust the variable and unregulated DC voltage generated by renewable energy systems (such as PV) to the desirable series multiple voltage levels at the inverter DC side. A multi-output boost (MOB) converter, with one inductor and series output voltage, is presented. This converter is suitable for renewable energy systems based on diode-clamped converters because it boosts the low output voltage and provides the series capacitor at the output side. A simple control strategy using cross voltage control with internal current loop is presented to obtain the desired voltage levels at the output voltage. The proposed topology and control strategy are validated by simulation and hardware results. Using the idea of voltage sharing switches, the circuit structure of different topologies of multi-output DC-DC converters – or multi-output voltage sharing (MOVS) converters – have been proposed. In order to verify the feasibility of this topology and its application, steady state and dynamic analyses have been carried out. Simulation and experiments using the proposed control strategy have verified the mathematical analysis. The second part of this thesis addresses the second problem of multilevel converters: the need to improve their quality with minimum cost and complexity. This is related to utilising asymmetrical multilevel topologies instead of conventional multilevel converters; this can increase the quality of output waveforms with a minimum number of components. It also allows for a reduction in the cost and complexity of systems while maintaining the same output quality, or for an increase in the quality while maintaining the same cost and complexity. Therefore, the asymmetrical configuration for two common types of multilevel converters – diode-clamped and cascade converters – is investigated. Also, as well as addressing the maximisation of the output voltage resolution, some technical issues – such as adjacent switching vectors – should be taken into account in asymmetrical multilevel configurations to keep the total harmonic distortion (THD) and switching losses to a minimum. Thus, the asymmetrical diode-clamped converter is proposed. An appropriate asymmetrical DC link arrangement is presented for four-level diode-clamped converters by keeping adjacent switching vectors. In this way, five-level inverter performance is achieved for the same level of complexity of the four-level inverter. Dealing with the capacitor voltage imbalance problem in asymmetrical diodeclamped converters has inspired the proposal for two different DC-DC topologies with a suitable control strategy. A Triple-Output Boost (TOB) converter and a Boost 3-Output Voltage Sharing (Boost-3OVS) converter connected to the four-level diode-clamped converter are proposed to arrange the proposed asymmetrical DC link for the high modulation indices and unity power factor. Cascade converters have shown their abilities and strengths in medium and high power applications. Using asymmetrical H-bridge inverters, more voltage levels can be generated in output voltage with the same number of components as the symmetrical converters. The concept of cascading multilevel H-bridge cells is used to propose a fifteen-level cascade inverter using a four-level H-bridge symmetrical diode-clamped converter, cascaded with classical two-level Hbridge inverters. A DC voltage ratio of cells is presented to obtain maximum voltage levels on output voltage, with adjacent switching vectors between all possible voltage levels; this can minimize the switching losses. This structure can save five isolated DC sources and twelve switches in comparison to conventional cascade converters with series two-level H bridge inverters. To increase the quality in presented hybrid topology with minimum number of components, a new cascade inverter is verified by cascading an asymmetrical four-level H-bridge diode-clamped inverter. An inverter with nineteen-level performance was achieved. This synthesizes more voltage levels with lower voltage and current THD, rather than using a symmetrical diode-clamped inverter with the same configuration and equivalent number of power components. Two different predictive current control methods for the switching states selection are proposed to minimise either losses or THD of voltage in hybrid converters. High voltage spikes at switching time in experimental results and investigation of a diode-clamped inverter structure raised another problem associated with high-level high voltage multilevel converters. Power switching components with fast switching, combined with hard switched-converters, produce high di/dt during turn off time. Thus, stray inductance of interconnections becomes an important issue and raises overvoltage and EMI issues correlated to the number of components. Planar busbar is a good candidate to reduce interconnection inductance in high power inverters compared with cables. The effect of different transient current loops on busbar physical structure of the high-voltage highlevel diode-clamped converters is highlighted. Design considerations of proper planar busbar are also presented to optimise the overall design of diode-clamped converters.

Dynamic analysis of on-board mass data to determine tampering in heavy vehicle on-board mass systems

Transport Certification Australia Limited, jointly with the National Transport Commission, has undertaken a project to investigate the feasibility of on-board mass monitoring (OBM) devices for regulatory purposes. OBM increases jurisdictional confidence in operational heavy vehicle compliance. This paper covers technical issues regarding potential use of dynamic data from OBM systems to indicate that tampering has occurred. Tamper-evidence and accuracy of current OBM systems needed to be determined before any regulatory schemes were put in place for its use. Tests performed to determine potential for, and ease of, tampering. An algorithm was developed to detect tamper events. Its results are detailed.

Static accuracy and precision of heavy vehicle on-board mass systems

Objective • Feasibility programme for on-board mass (OBM) monitoring of heavy vehicles (HVs) • Australian road authorities through Transport Certification Australia (TCA) • Accuracy of contemporary, commercially-available OBM units in Australia • Results need to be addressed/incorporated into specifications for Stage 2 of Intelligent Access Program (IAP) by Transport Certification Australia

A vibration based method to update axial shortening of load bearing elements

Axial shortening in vertical load bearing elements of reinforced concrete high-rise buildings is caused by the time dependent effects of shrinkage, creep and elastic shortening of concrete under loads. Such phenomenon has to be predicted at design stage and then updated during and after construction of the buildings in order to provide mitigation against the adverse effects of differential axial shortening among the elements. Existing measuring methods for updating previous predictions of axial shortening pose problems. With this in mind, a innovative procedure with a vibration based parameter called axial shortening index is proposed to update axial shortening of vertical elements based on variations in vibration characteristics of the buildings. This paper presents the development of the procedure and illustrates it through a numerical example of an unsymmetrical high-rise building with two outrigger and belt systems. Results indicate that the method has the capability to capture influence of different tributary areas, shear walls of outrigger and belt systems as well as the geometric complexity of the building.