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.

Improvement to Chinese information retrieval by incorporating word segmentation and query expansion

The increasing diversity of the Internet has created a vast number of multilingual resources on the Web. A huge number of these documents are written in various languages other than English. Consequently, the demand for searching in non-English languages is growing exponentially. It is desirable that a search engine can search for information over collections of documents in other languages. This research investigates the techniques for developing high-quality Chinese information retrieval systems. A distinctive feature of Chinese text is that a Chinese document is a sequence of Chinese characters with no space or boundary between Chinese words. This feature makes Chinese information retrieval more difficult since a retrieved document which contains the query term as a sequence of Chinese characters may not be really relevant to the query since the query term (as a sequence Chinese characters) may not be a valid Chinese word in that documents. On the other hand, a document that is actually relevant may not be retrieved because it does not contain the query sequence but contains other relevant words. In this research, we propose two approaches to deal with the problems. In the first approach, we propose a hybrid Chinese information retrieval model by incorporating word-based techniques with the traditional character-based techniques. The aim of this approach is to investigate the influence of Chinese segmentation on the performance of Chinese information retrieval. Two ranking methods are proposed to rank retrieved documents based on the relevancy to the query calculated by combining character-based ranking and word-based ranking. Our experimental results show that Chinese segmentation can improve the performance of Chinese information retrieval, but the improvement is not significant if it incorporates only Chinese segmentation with the traditional character-based approach. In the second approach, we propose a novel query expansion method which applies text mining techniques in order to find the most relevant words to extend the query. Unlike most existing query expansion methods, which generally select the highly frequent indexing terms from the retrieved documents to expand the query. In our approach, we utilize text mining techniques to find patterns from the retrieved documents that highly correlate with the query term and then use the relevant words in the patterns to expand the original query. This research project develops and implements a Chinese information retrieval system for evaluating the proposed approaches. There are two stages in the experiments. The first stage is to investigate if high accuracy segmentation can make an improvement to Chinese information retrieval. In the second stage, a text mining based query expansion approach is implemented and a further experiment has been done to compare its performance with the standard Rocchio approach with the proposed text mining based query expansion method. The NTCIR5 Chinese collections are used in the experiments. The experiment results show that by incorporating the text mining based query expansion with the hybrid model, significant improvement has been achieved in both precision and recall assessments.

Computer profiling for forensic purposes

Computer forensics is the process of gathering and analysing evidence from computer systems to aid in the investigation of a crime. Typically, such investigations are undertaken by human forensic examiners using purpose-built software to discover evidence from a computer disk. This process is a manual one, and the time it takes for a forensic examiner to conduct such an investigation is proportional to the storage capacity of the computer's disk drives. The heterogeneity and complexity of various data formats stored on modern computer systems compounds the problems posed by the sheer volume of data. The decision to undertake a computer forensic examination of a computer system is a decision to commit significant quantities of a human examiner's time. Where there is no prior knowledge of the information contained on a computer system, this commitment of time and energy occurs with little idea of the potential benefit to the investigation. The key contribution of this research is the design and development of an automated process to describe a computer system and its activity for the purposes of a computer forensic investigation. The term proposed for this process is computer profiling. A model of a computer system and its activity has been developed over the course of this research. Using this model a computer system, which is the subj ect of investigation, can be automatically described in terms useful to a forensic investigator. The computer profiling process IS resilient to attempts to disguise malicious computer activity. This resilience is achieved by detecting inconsistencies in the information used to infer the apparent activity of the computer. The practicality of the computer profiling process has been demonstrated by a proof-of concept software implementation. The model and the prototype implementation utilising the model were tested with data from real computer systems. The resilience of the process to attempts to disguise malicious activity has also been demonstrated with practical experiments conducted with the same prototype software implementation.

A practical example of a siphon at work

In this paper, some classroom experiments are described for correcting the common misconception that the operation of a siphon depends on atmospheric pressure. One experiment makes use of a chain model of a siphon and another demonstrates that flow rate is dependent on the height difference between the inflow and outflow of a siphon and not atmospheric pressure. A real-life example of the use of a siphon to refill a lake in South Australia is described, demonstrating that the siphon is not only of academic interest but has practical applications.

Image synthesis based on a model of human vision

Modern computer graphics systems are able to construct renderings of such high quality that viewers are deceived into regarding the images as coming from a photographic source. Large amounts of computing resources are expended in this rendering process, using complex mathematical models of lighting and shading. However, psychophysical experiments have revealed that viewers only regard certain informative regions within a presented image. Furthermore, it has been shown that these visually important regions contain low-level visual feature differences that attract the attention of the viewer. This thesis will present a new approach to image synthesis that exploits these experimental findings by modulating the spatial quality of image regions by their visual importance. Efficiency gains are therefore reaped, without sacrificing much of the perceived quality of the image. Two tasks must be undertaken to achieve this goal. Firstly, the design of an appropriate region-based model of visual importance, and secondly, the modification of progressive rendering techniques to effect an importance-based rendering approach. A rule-based fuzzy logic model is presented that computes, using spatial feature differences, the relative visual importance of regions in an image. This model improves upon previous work by incorporating threshold effects induced by global feature difference distributions and by using texture concentration measures. A modified approach to progressive ray-tracing is also presented. This new approach uses the visual importance model to guide the progressive refinement of an image. In addition, this concept of visual importance has been incorporated into supersampling, texture mapping and computer animation techniques. Experimental results are presented, illustrating the efficiency gains reaped from using this method of progressive rendering. This visual importance-based rendering approach is expected to have applications in the entertainment industry, where image fidelity may be sacrificed for efficiency purposes, as long as the overall visual impression of the scene is maintained. Different aspects of the approach should find many other applications in image compression, image retrieval, progressive data transmission and active robotic vision.

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

Automatic tracking, super-resolution and recognition of human faces from surveillance video

Identifying an individual from surveillance video is a difficult, time consuming and labour intensive process. The proposed system aims to streamline this process by filtering out unwanted scenes and enhancing an individual's face through super-resolution. An automatic face recognition system is then used to identify the subject or present the human operator with likely matches from a database. A person tracker is used to speed up the subject detection and super-resolution process by tracking moving subjects and cropping a region of interest around the subject's face to reduce the number and size of the image frames to be super-resolved respectively. In this paper, experiments have been conducted to demonstrate how the optical flow super-resolution method used improves surveillance imagery for visual inspection as well as automatic face recognition on an Eigenface and Elastic Bunch Graph Matching system. The optical flow based method has also been benchmarked against the ``hallucination'' algorithm, interpolation methods and the original low-resolution images. Results show that both super-resolution algorithms improved recognition rates significantly. Although the hallucination method resulted in slightly higher recognition rates, the optical flow method produced less artifacts and more visually correct images suitable for human consumption.

Curriculum design innovation in flexible science teaching

In this paper you will be introduced to a number of principles which can be used to inform good teaching practice and rigorous curriculum design. Principles relate to: * Application of a common sequence of events for how learners learn; * Accommodating different learning styles; * Adopting a purposeful approach to teaching and learning; * Using assessment as a central driving force in the curriculum and as an organising structure leading to coherence of teaching and learning approach; and * The increasing emphasis that is being placed on the development of generic graduate competencies over and above discipline content knowledge. The principles are particularly significant in relation to adult learning. The paper will use three specific applications as illustrations to help you to learn how these principles can be applied. The illustrations are taken from a second year subject in supercomputing that uses scientific case studies. The subject has been developed (with support from Silicon Graphics Inc. and Intel) to be taught entirely via the Internet.

If teams are so good.. : science teachers' conceptions of teams and teamwork

The focus of this study is the phenomenon of teams and teamwork. Currently the Professional Standards of Queensland’s teachers state that teams are critical to teachers’ work. This study uses a phenomenographic approach to investigate science teachers’ conceptions of teams and teamwork in the science departments of fifteen Queensland State secondary schools. The research identifies eight conceptions of teams and teamwork. The research findings suggest that the team represents a collective of science teachers bounded by the Science Department and their current timetabled subject. Collaboration was found in the study to be an activity that occurred between teachers in the same social space. The research recognises a new category of relationship between teachers, designated as ‘ask-and-receive’. The research identifies a lack of teamwork within the science department and the school. There appears to be no teaming with other subject departments. The research findings highlight the non-supportive team and teamwork policies, procedures and structures in the schools and identify the lack of recognition of the specialised skills of science teachers. The implications for the schools and science teachers are considerable, as the current Professional Standards of Education Queensland and the Queensland College of Teachers provide benchmarks of knowledge and practice of teams and teamwork for teachers. The research suggests that the professional standards relating to teams and teamwork cannot be achieved in the present school environment.

Using concept mapping to scaffold learning for students who experience learning difficulties in science classes

In order to develop scientific literacy students need the cognitive tools that enable them to read and evaluate science texts. One cognitive tool that has been widely used in science education to aid the development of conceptual understanding is concept mapping. However, it has been found some students experience difficulty with concept map construction. This study reports on the development and evaluation of an instructional sequence that was used to scaffold the concept-mapping process when middle school students who were experiencing difficulty with science learning used concept mapping to summarise a chapter of a science text. In this study individual differences in working memory functioning are suggested as one reason that students experience difficulty with concept map construction. The study was conducted using a design-based research methodology in the school’s learning support centre. The analysis of student work samples collected during the two-year study identified some of the difficulties and benefits associated with the use of scaffolded concept mapping with these students. The observations made during this study highlight the difficulty that some students experience with the use of concept mapping as a means of developing an understanding of science concepts and the amount of instructional support that is required for such understanding to develop. Specifically, the findings of the study support the use of multi-component, multi-modal instructional techniques to facilitate the development of conceptual understanding with students who experience difficulty with science learning. In addition, the important roles of interactive dialogue and metacognition in the development of conceptual understanding are identified.