Determining the psychosocial predictors of living, living-related, and posthumous organ donation

The worldwide organ shortage occurs despite people’s positive organ donation attitudes. The discrepancy between attitudes and behaviour is evident in Australia particularly, with widespread public support for organ donation but low donation and communication rates. This problem is compounded further by the paucity of theoretically based research to improve our understanding of people’s organ donation decisions. This program of research contributes to our knowledge of individual decision making processes for three aspects of organ donation: (1) posthumous (upon death) donation, (2) living donation (to a known and unknown recipient), and (3) providing consent for donation by communicating donation wishes on an organ donor consent register (registering) and discussing the donation decision with significant others (discussing). The research program used extended versions of the Theory of Planned Behaviour (TPB) and the Prototype/Willingness Model (PWM), incorporating additional influences (moral norm, self-identity, organ recipient prototypes), to explicate the relationship between people’s positive attitudes and low rates of organ donation behaviours. Adopting the TPB and PWM (and their extensions) as a theoretical basis overcomes several key limitations of the extant organ donation literature including the often atheoretical nature of organ donation research, thefocus on individual difference factors to construct organ donor profiles and the omission of important psychosocial influences (e.g., control perceptions, moral values) that may impact on people’s decision-making in this context. In addition, the use of the TPB and PWM adds further to our understanding of the decision making process for communicating organ donation wishes. Specifically, the extent to which people’s registering and discussing decisions may be explained by a reasoned and/or a reactive decision making pathway is examined (Stage 3) with the novel application of the TPB augmented with the social reaction pathway in the PWM. This program of research was conducted in three discrete stages: a qualitative stage (Stage 1), a quantitative stage with extended models (Stage 2), and a quantitative stage with augmented models (Stage 3). The findings of the research program are reported in nine papers which are presented according to the three aspects of organ donation examined (posthumous donation, living donation, and providing consent for donation by registering or discussing the donation preference). Stage One of the research program comprised qualitative focus groups/interviews with university students and community members (N = 54) (Papers 1 and 2). Drawing broadly on the TPB framework (Paper 1), content analysed responses revealed people’s commonly held beliefs about the advantages and disadvantages (e.g., prolonging/saving life), important people or groups (e.g., family), and barriers and motivators (e.g., a family’s objection to donation), related to living and posthumous organ donation. Guided by a PWM perspective, Paper Two identified people’s commonly held perceptions of organ donors (e.g., altruistic and giving), non-donors (e.g., self-absorbed and unaware), and transplant recipients (e.g., unfortunate, and in some cases responsible/blameworthy for their predicament). Stage Two encompassed quantitative examinations of people’s decision makingfor living (Papers 3 and 4) and posthumous (Paper 5) organ donation, and for registering and discussing donation wishes (Papers 6 to 8) to test extensions to both the TPB and PWM. Comparisons of health students’ (N = 487) motivations and willingness for living related and anonymous donation (Paper 3) revealed that a person’s donor identity, attitude, past blood donation, and knowing a posthumous donor were four common determinants of willingness, with the results highlighting students’ identification as a living donor as an important motive. An extended PWM is presented in Papers Four and Five. University students’ (N = 284) willingness for living related and anonymous donation was tested in Paper Four with attitude, subjective norm, donor prototype similarity, and moral norm (but not donor prototype favourability) predicting students’ willingness to donate organs in both living situations. Students’ and community members’ (N = 471) posthumous organ donation willingness was assessed in Paper Five with attitude, subjective norm, past behaviour, moral norm, self-identity, and prior blood donation all significantly directly predicting posthumous donation willingness, with only an indirect role for organ donor prototype evaluations. The results of two studies examining people’s decisions to register and/or discuss their organ donation wishes are reported in Paper Six. People’s (N = 24) commonly held beliefs about communicating their organ donation wishes were explored initially in a TPB based qualitative elicitation study. The TPB belief determinants of intentions to register and discuss the donation preference were then assessed for people who had not previously communicated their donation wishes (N = 123). Behavioural and normative beliefs were important determinants of registering and discussing intentions; however, control beliefs influenced people’s registering intentions only. Paper Seven represented the first empirical test of the role of organ transplant recipient prototypes (i.e., perceptions of organ transplant recipients) in people’s (N = 465) decisions to register consent for organ donation. Two factors, Substance Use and Responsibility, were identified and Responsibility predicted people’s organ donor registration status. Results demonstrated that unregistered respondents were the most likely to evaluate transplant recipients negatively. Paper Eight established the role of organ donor prototype evaluations, within an extended TPB model, in predicting students’ and community members’ registering (n = 359) and discussing (n = 282) decisions. Results supported the utility of an extended TPB and suggested a role for donor prototype evaluations in predicting people’s discussing intentions only. Strong intentions to discuss donation wishes increased the likelihood that respondents reported discussing their decision 1-month later. Stage Three of the research program comprised an examination of augmented models (Paper 9). A test of the TPB augmented with elements from the social reaction pathway in the PWM, and extensions to these models was conducted to explore whether people’s registering (N = 339) and discussing (N = 315) decisions are explained via a reasoned (intention) and/or social reaction (willingness) pathway. Results suggested that people’s decisions to communicate their organ donation wishes may be better explained via the reasoned pathway, particularly for registering consent; however, discussing also involves reactive elements. Overall, the current research program represents an important step toward clarifying the relationship between people’s positive organ donation attitudes but low rates of organ donation and communication behaviours. Support has been demonstrated for the use of extensions to two complementary theories, the TPB and PWM, which can inform future research aiming to explicate further the organ donation attitude-behaviour relationship. The focus on a range of organ donation behaviours enables the identification of key targets for future interventions encouraging people’s posthumous and living donation decisions, and communication of their organ donation preference.

Tag based collaborative filtering for recommender systems

Collaborative tagging can help users organize, share and retrieve information in an easy and quick way. For the collaborative tagging information implies user’s important personal preference information, it can be used to recommend personalized items to users. This paper proposes a novel tag-based collaborative filtering approach for recommending personalized items to users of online communities that are equipped with tagging facilities. Based on the distinctive three dimensional relationships among users, tags and items, a new similarity measure method is proposed to generate the neighborhood of users with similar tagging behavior instead of similar implicit ratings. The promising experiment result shows that by using the tagging information the proposed approach outperforms the standard user and item based collaborative filtering approaches.

Collaborative Filtering Recommender Systems based on Popular Tags

The social tags in web 2.0 are becoming another important information source to profile users' interests and preferences for making personalized recommendations. However, the uncontrolled vocabulary causes a lot of problems to profile users accurately, such as ambiguity, synonyms, misspelling, low information sharing etc. To solve these problems, this paper proposes to use popular tags to represent the actual topics of tags, the content of items, and also the topic interests of users. A novel user profiling approach is proposed in this paper that first identifies popular tags, then represents users’ original tags using the popular tags, finally generates users’ topic interests based on the popular tags. A collaborative filtering based recommender system has been developed that builds the user profile using the proposed approach. The user profile generated using the proposed approach can represent user interests more accurately and the information sharing among users in the profile is also increased. Consequently the neighborhood of a user, which plays a crucial role in collaborative filtering based recommenders, can be much more accurately determined. The experimental results based on real world data obtained from Amazon.com show that the proposed approach outperforms other approaches.

Printmaking as an expanding field in contemporary art practice : a case study of Japan, Australia and Thailand

This thesis proposes that contemporary printmaking, at its most significant, marks the present through reconstructing pasts and anticipating futures. It argues this through examples in the field, occurring in contexts beyond the Euramerican (Europe and North America). The arguments revolve around how the practice of a number of significant artists in Japan, Australia and Thailand has generated conceptual and formal innovations in printmaking that transcend local histories and conventions, whilst paradoxically, also building upon them and creating new meanings. The arguments do not portray the relations between contemporary and traditional art as necessarily antagonistic but rather, as productively dialectical. Furthermore, the case studies demonstrate that, in the 1980s and 1990s particularly, the studio practice of these printmakers was informed by other visual arts disciplines and reflected postmodern concerns. Departures from convention witnessed in these countries within the Asia-Pacific region shifted the field of the print into a heterogeneous and hybrid realm. The practitioners concerned (especially in Thailand) produced work that was more readily equated with performance and installation art than with printmaking per se. In Japan, the incursion of photography interrupted the decorative cast of printmaking and delivered it from a straightforward, craft-based aesthetic. In Australia, fixed notions of national identity were challenged by print practitioners through deliberate cultural rapprochements and technical contradictions (speaking across old and new languages).However time-honoured print methods were not jettisoned by any case study artists. Their re-alignment of the fundamental attributes of printmaking, in line with materialist formalism, is a core consideration of my arguments. The artists selected for in-depth analysis from these three countries are all innovators whose geographical circumstances and creative praxis drew on local traditions whilst absorbing international trends. In their radical revisionism, they acknowledged the specificity of history and place, conditions of contingency and forces of globalisation. The transformational nature of their work during the late twentieth century connects it to the postmodern ethos and to a broader artistic and cultural nexus than has hitherto been recognised in literature on the print. Emerging from former guild-based practices, they ambitiously conceived their work to be part of a continually evolving visual arts vocabulary. I argue in this thesis that artists from the Asia-Pacific region have historically broken with the hermetic and Euramerican focus that has generally characterised the field. Inadequate documentation and access to print activity outside the dominant centres of critical discourse imply that readings of postmodernism have been too limited in their scope of inquiry. Other locations offer complexities of artistic practice where re-alignments of customary boundaries are often the norm. By addressing innovative activity in Japan, Australia and Thailand, this thesis exposes the need for a more inclusive theoretical framework and wider global reach than currently exists for ‘printmaking’.

Kallikrein-related peptidase 4 activation of protease-activated receptor family members and association with prostate cancer

Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.

Robot path planning in dynamic environments using a simulated annealing based approach

Mobile robots are widely used in many industrial fields. Research on path planning for mobile robots is one of the most important aspects in mobile robots research. Path planning for a mobile robot is to find a collision-free route, through the robot’s environment with obstacles, from a specified start location to a desired goal destination while satisfying certain optimization criteria. Most of the existing path planning methods, such as the visibility graph, the cell decomposition, and the potential field are designed with the focus on static environments, in which there are only stationary obstacles. However, in practical systems such as Marine Science Research, Robots in Mining Industry, and RoboCup games, robots usually face dynamic environments, in which both moving and stationary obstacles exist. Because of the complexity of the dynamic environments, research on path planning in the environments with dynamic obstacles is limited. Limited numbers of papers have been published in this area in comparison with hundreds of reports on path planning in stationary environments in the open literature. Recently, a genetic algorithm based approach has been introduced to plan the optimal path for a mobile robot in a dynamic environment with moving obstacles. However, with the increase of the number of the obstacles in the environment, and the changes of the moving speed and direction of the robot and obstacles, the size of the problem to be solved increases sharply. Consequently, the performance of the genetic algorithm based approach deteriorates significantly. This motivates the research of this work. This research develops and implements a simulated annealing algorithm based approach to find the optimal path for a mobile robot in a dynamic environment with moving obstacles. The simulated annealing algorithm is an optimization algorithm similar to the genetic algorithm in principle. However, our investigation and simulations have indicated that the simulated annealing algorithm based approach is simpler and easier to implement. Its performance is also shown to be superior to that of the genetic algorithm based approach in both online and offline processing times as well as in obtaining the optimal solution for path planning of the robot in the dynamic environment. The first step of many path planning methods is to search an initial feasible path for the robot. A commonly used method for searching the initial path is to randomly pick up some vertices of the obstacles in the search space. This is time consuming in both static and dynamic path planning, and has an important impact on the efficiency of the dynamic path planning. This research proposes a heuristic method to search the feasible initial path efficiently. Then, the heuristic method is incorporated into the proposed simulated annealing algorithm based approach for dynamic robot path planning. Simulation experiments have shown that with the incorporation of the heuristic method, the developed simulated annealing algorithm based approach requires much shorter processing time to get the optimal solutions in the dynamic path planning problem. Furthermore, the quality of the solution, as characterized by the length of the planned path, is also improved with the incorporated heuristic method in the simulated annealing based approach for both online and offline path planning.

Planning for knowledge-based urban development : global perspectives

Purpose: The purpose of this article is to investigate the engineering of creative urban regions through knowledge-based urban development. In recent years city administrators realised the importance of engineering and orchestrating knowledge city formation through visioning and planning for economic, socio-cultural and physical development. For that purpose a new development paradigm of ‘‘knowledge-based urban development’’ is formed, and quickly finds implementation ground in many parts of the globe.----- Design/methodology/approach: The paper reviews the literature and examines global best practice experiences in order to determine how cities are engineering their creative urban regions so as to establish a base for knowledge city formation.----- Findings: The paper sheds light on the different development approaches for creative urban regions, and concludes with recommendations for urban administrations planning for knowledge-based development of creative urban regions.----- Originality/value: The paper provides invaluable insights and discussion on the vital role of planning for knowledge-based urban development of creative urban regions.

Semantic and context-based retrieval of digital cultural objects [Published in Chinese]

Cultural objects are increasingly generated and stored in digital form, yet effective methods for their indexing and retrieval still remain an important area of research. The main problem arises from the disconnection between the content-based indexing approach used by computer scientists and the description-based approach used by information scientists. There is also a lack of representational schemes that allow the alignment of the semantics and context with keywords and low-level features that can be automatically extracted from the content of these cultural objects. This paper presents an integrated approach to address these problems, taking advantage of both computer science and information science approaches. We firstly discuss the requirements from a number of perspectives: users, content providers, content managers and technical systems. We then present an overview of our system architecture and describe various techniques which underlie the major components of the system. These include: automatic object category detection; user-driven tagging; metadata transform and augmentation, and an expression language for digital cultural objects. In addition, we discuss our experience on testing and evaluating some existing collections, analyse the difficulties encountered and propose ways to address these problems.

Link-the-Wiki : performance evaluation based on frequent phrases

In this paper, we discuss our participation to the INEX 2008 Link-the-Wiki track. We utilized a sliding window based algorithm to extract the frequent terms and phrases. Using the extracted phrases and term as descriptive vectors, the anchors and relevant links (both incoming and outgoing) are recognized efficiently.

Biosecurity threats : the design of surveillance systems, based on power and risk

We consider the problem of designing a surveillance system to detect a broad range of invasive species across a heterogeneous sampling frame. We present a model to detect a range of invertebrate invasives whilst addressing the challenges of multiple data sources, stratifying for differential risk, managing labour costs and providing sufficient power of detection.We determine the number of detection devices required and their allocation across the landscape within limiting resource constraints. The resulting plan will lead to reduced financial and ecological costs and an optimal surveillance system.

Towards an automatic road lane marks extraction based on ISODATA segmentation and shadow detection from large-scale aerial images

The automatic extraction of road features from remote sensed images has been a topic of great interest within the photogrammetric and remote sensing communities for over 3 decades. Although various techniques have been reported in the literature, it is still challenging to efficiently extract the road details with the increasing of image resolution as well as the requirement for accurate and up-to-date road data. In this paper, we will focus on the automatic detection of road lane markings, which are crucial for many applications, including lane level navigation and lane departure warning. The approach consists of four steps: i) data preprocessing, ii) image segmentation and road surface detection, iii) road lane marking extraction based on the generated road surface, and iv) testing and system evaluation. The proposed approach utilized the unsupervised ISODATA image segmentation algorithm, which segments the image into vegetation regions, and road surface based only on the Cb component of YCbCr color space. A shadow detection method based on YCbCr color space is also employed to detect and recover the shadows from the road surface casted by the vehicles and trees. Finally, the lane marking features are detected from the road surface using the histogram clustering. The experiments of applying the proposed method to the aerial imagery dataset of Gympie, Queensland demonstrate the efficiency of the approach.

Passenger experience in an airport : an activity-centred approach

An airport is one of the largest and most complex systems in modern society. Observational field studies have been conducted to investigate passenger experiences in, and interactions within, an international airport. Based on these studies, this paper discusses how activities mediate people’s experiences in the airport. For example, moving through the security screening process is discussed from both passenger and staff perspectives. The applied coding scheme ensured research rigor. The findings illustrate that passenger activities are complex and shared, and only partially supported by current terminal design. Thus, this research has the potential to impact on airport design to facilitate passenger flow through airport precincts.

Visualisation of groundwater flow using texture based visualisation techniques

Texture based techniques for visualisation of unsteady vector fields have been applied for the visualisation of a Finite volume model for variably saturated groundwater flow through porous media. This model has been developed by staff in the School of Mathematical Sciences QUT for the study of salt water intrusion into coastal aquifers. This presentation discusses the implementation and effectiveness of the IBFV algorithm in the context of visualisation of the groundwater simulation outputs.

Mixing colours : an ICT tool based on a semiotic framework for mathematical meaning-making about ratio and fractions

This paper reports on the research and development of an ICT tool to facilitate the learning of ratio and fractions by adult prisoners. The design of the ICT tool was informed by a semiotic framework for mathematical meaning-making. The ICT tool thus employed multiple semiotic resources including topological, typological, and social-actional resources. The results showed that individual semiotic resource could only represent part of the mathematical concept, while at the same time it might signify something else to create a misconception. When multiple semiotic resources were utilised the mathematical ideas could be better learnt.

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.