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.

A cost-based optimization of a fiberboard pressing plant using Monte-Carlo simulation (a reliability program)

In this research the reliability and availability of fiberboard pressing plant is assessed and a cost-based optimization of the system using the Monte- Carlo simulation method is performed. The woodchip and pulp or engineered wood industry in Australia and around the world is a lucrative industry. One such industry is hardboard. The pressing system is the main system, as it converts the wet pulp to fiberboard. The assessment identified the pressing system has the highest downtime throughout the plant plus it represents the bottleneck in the process. A survey in the late nineties revealed there are over one thousand plants around the world, with the pressing system being a common system among these plants. No work has been done to assess or estimate the reliability of such a pressing system; therefore this assessment can be used for assessing any plant of this type.

Intuitive interaction, prior experience and aging : an empirical study

This paper describes an experiment undertaken to investigate intuitive interaction, particularly in older adults. Previous work has shown that intuitive interaction relies on past experience, and has also suggested that older people demonstrate less intuitive uses and slower times when completing set tasks with various devices. Similarly, this experiment showed that past experience with relevant products allowed people to use the interfaces of two different microwaves more quickly, although there were no significant differences between the different microwaves. It also revealed that certain aspects of cognitive decline related to aging, such as central executive function, have more impact on time, correct uses and intuitive uses than chronological age. Implications of these results and further work in this area are discussed.

Concepts of intuitive interaction

Intuition is a type of cognitive processing that is often non-conscious and utilises stored experiential knowledge. Intuitive interaction involves the use of knowledge gained from other products and/or experiences. We have developed novel approaches and techniques for studying intuitive use of interfaces, and shown that intuitive interaction is based on past experience with similar artefacts. Based on our empirical work we have developed principles and tools for designers to assist them in making interfaces more intuitive. These principles are discussed in this paper.

Theoretical investigation of mechanisms of formation and interaction of nanoparticles

In this thesis an investigation into theoretical models for formation and interaction of nanoparticles is presented. The work presented includes a literature review of current models followed by a series of five chapters of original research. This thesis has been submitted in partial fulfilment of the requirements for the degree of doctor of philosophy by publication and therefore each of the five chapters consist of a peer-reviewed journal article. The thesis is then concluded with a discussion of what has been achieved during the PhD candidature, the potential applications for this research and ways in which the research could be extended in the future. In this thesis we explore stochastic models pertaining to the interaction and evolution mechanisms of nanoparticles. In particular, we explore in depth the stochastic evaporation of molecules due to thermal activation and its ultimate effect on nanoparticles sizes and concentrations. Secondly, we analyse the thermal vibrations of nanoparticles suspended in a fluid and subject to standing oscillating drag forces (as would occur in a standing sound wave) and finally on lattice surfaces in the presence of high heat gradients. We have described in this thesis a number of new models for the description of multicompartment networks joined by a multiple, stochastically evaporating, links. The primary motivation for this work is in the description of thermal fragmentation in which multiple molecules holding parts of a carbonaceous nanoparticle may evaporate. Ultimately, these models predict the rate at which the network or aggregate fragments into smaller networks/aggregates and with what aggregate size distribution. The models are highly analytic and describe the fragmentation of a link holding multiple bonds using Markov processes that best describe different physical situations and these processes have been analysed using a number of mathematical methods. The fragmentation of the network/aggregate is then predicted using combinatorial arguments. Whilst there is some scepticism in the scientific community pertaining to the proposed mechanism of thermal fragmentation,we have presented compelling evidence in this thesis supporting the currently proposed mechanism and shown that our models can accurately match experimental results. This was achieved using a realistic simulation of the fragmentation of the fractal carbonaceous aggregate structure using our models. Furthermore, in this thesis a method of manipulation using acoustic standing waves is investigated. In our investigation we analysed the effect of frequency and particle size on the ability for the particle to be manipulated by means of a standing acoustic wave. In our results, we report the existence of a critical frequency for a particular particle size. This frequency is inversely proportional to the Stokes time of the particle in the fluid. We also find that for large frequencies the subtle Brownian motion of even larger particles plays a significant role in the efficacy of the manipulation. This is due to the decreasing size of the boundary layer between acoustic nodes. Our model utilises a multiple time scale approach to calculating the long term effects of the standing acoustic field on the particles that are interacting with the sound. These effects are then combined with the effects of Brownian motion in order to obtain a complete mathematical description of the particle dynamics in such acoustic fields. Finally, in this thesis, we develop a numerical routine for the description of "thermal tweezers". Currently, the technique of thermal tweezers is predominantly theoretical however there has been a handful of successful experiments which demonstrate the effect it practise. Thermal tweezers is the name given to the way in which particles can be easily manipulated on a lattice surface by careful selection of a heat distribution over the surface. Typically, the theoretical simulations of the effect can be rather time consuming with supercomputer facilities processing data over days or even weeks. Our alternative numerical method for the simulation of particle distributions pertaining to the thermal tweezers effect use the Fokker-Planck equation to derive a quick numerical method for the calculation of the effective diffusion constant as a result of the lattice and the temperature. We then use this diffusion constant and solve the diffusion equation numerically using the finite volume method. This saves the algorithm from calculating many individual particle trajectories since it is describes the flow of the probability distribution of particles in a continuous manner. The alternative method that is outlined in this thesis can produce a larger quantity of accurate results on a household PC in a matter of hours which is much better than was previously achieveable.

Investigating users’ intuitive interaction with complex artefacts

This paper examines the role of intuition in the way that people operate unfamiliar devices. Intuition is a type of cognitive processing that is often non-conscious and utilises stored experiential knowledge. Intuitive interaction involves the use of knowledge gained from other products and/or experiences. Two initial experimental studies revealed that prior exposure to products employing similar features helped participants to complete set tasks more quickly and intuitively, and that familiar features were intuitively used more often than unfamiliar ones. A third experiment confirmed that performance is affected by a person's level of familiarity with similar technologies, and also revealed that appearance (shape, size and labelling of features) seems to be the variable that most affects time spent on a task and intuitive uses during that time. Age also seems to have an effect. These results and their implications are discussed.

In Interaction

The field was the design of cross-cultural media art exhibition outcomes for the Japanese marketplace. The context was improved understandings of spatial, temporal and contextual exhibition design procedures as they ultimately impact upon the augmentation of cross-cultural understanding. The research investigated cross-cultural new media exhibition practices suited to the specific sensitivies of Japanese exhibition practices. The methodology was principally practice-led. The research drew upon seven years of prior exhibition design practices in order to generate new Japanese exhibition design methodologies. It also empowered Zaim Artpsace’s Japanese curators to later present a range of substantial new media shows. The project also succeeded in developing new cross-cultural alliances that led to significant IDA projects in Beijing, Australia and Europe in the years 2008-10. Through invitations from external curators the new versions of the exhibition work subsequently travelled to 4 other major venues including the prestigious Songzhang Art Museum, Beijing in 07/08, the Block, QUT, Brisbane and the Tokyo International Film festival. Inspiration Art Press printed a major catalogue for the event extensively featuring this exhibition. This project also led to the Sudamalis (2007) paper, ‘Building Capacity: Literacy And Creative Workforce Development Through International Digital Arts Projects’ (IDAprojects) Exhibition Programs And Partnerships’.

Interaction and mediation in preadmission clinics : implications for the design of a telehealth stethoscope

A telehealth stethoscope would make it possible for doctors to perform physical examinations on patients at great distances. In order to develop a useful and usable telehealth stethoscope we have conducted fieldwork observations of existing anaesthetic preadmission clinics to understand how stethoscopes are currently used. Both face-to-face consultations and videoconference consultations have been studied. Our results indicate that the stethoscope plays a minor role in the consultation and that consultations are mediated by the administrative work that is the reason for the consultation. We suggest that a stethoscope plays an infrastructural role in the consultation. The implications of considering stethoscopes as infrastructure are explored and considered in the context of a future telehealth stethoscope.

PIPP #1 - Pedestrian Interaction Patch Project

The Pedestrian Interaction Patch Project (PIPP) seeks to exert influence over and encourage abnormal pedestrian behavior. By placing an unadvertised (and non recording) interactive video manipulation system and projection source in a high traffic public area, the PIPP allows pedestrians to privately (and publically) re-engage with a previously inactive physical environment, like a commonly used walkway or corridor. This system, the results of which are projected in real time on the architectural surface, inadvertently provides pedestrians with questions around preconceived notions of self and space. In an attempt to re-activate our relationship with the physical surrounds we occupy each day the PIPP creates a new set of memories to be recalled as we re-enter known environments once PIPP has moved on and as such re-enlivens our relationship with the everyday architecture we stroll past everyday. The PIPP environment is controlled using the software program Isadora, devised by Mark Coniglio at Troika Ranch, and contains a series of video manipulation patches that are designed to not only grab the pedestrians attention but to also encourage a sense of play and interaction between the architecture, the digital environment, the initially unsuspecting participant(s) and the pedestrian audience. The PIPP was included as part of the planned walking tour for the “Playing in Urban Spaces” seminar day, and was an installation that ran for the length of the symposium in a reclaimed pedestrian space that was encountered by both the participants and general public during the course of the day long event. Ideally once discovered PIPP encouraged pedestrians to return through the course of the seminar day to see if the environmental patches had changed or altered, and changed their standard route to include the PIPP installation or to avoid it, either way, encouraging an active response to the pathways normally traveled or newly discovered each day.

Assessment of oral proficiency in EAP programs : a case for pair interaction

Reliability and validity in the testing of spoken language are essential in order to assess learners' English language proficiency as evidence of their readiness to begin courses in tertiary institutions. Research has indicated that the task chosen to elicit language samples can have a marked effect on both the nature of the interaction, including the power differential, and assessment, raising the issue of ethics. This exploratory studey, with a group of 32 students from the Peoples's Republic of China preparing for tertiary study in Singapore, compares test-takers' reactions to the use of an oral proficiency interview and a pair interaction.

Interaction between disinhibition and restraint: Implications for body weight and eating disturbance

An increase in obesity is usually accompanied by an increase in eating disturbances. Susceptibility to these states may arise from different combinations of underlying traits: Three Factor Eating Questionnaire (TFEQ) Restraint and Disinhibition. Two studies were conducted to examine the interaction between these traits; one on-line study (n=351) and one laboratory-based study (n=120). Participants completed a battery of questionnaires and provided self-report measures of body weight and physical activity. A combination of high Disinhibition and high Restraint was associated with a problematic eating behaviour profile (EAT-26), and a higher rate of smoking and alcohol consumption. A combination of high Disinhibition and low Restraint was associated with a higher susceptibility to weight gain and a higher sedentary behaviour. These data show that different combinations of Disinhibition and Restraint are associated with distinct weight and behaviour outcomes.