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.

An historical analysis of the development of thinking in the principal writings of Malcolm Knowles

Malcolm Shepherd Knowles was a key writer and theorist in the field of adult education in the United States. He died in 1997 and left a large legacy of books and journal articles. This thesis traced the development of his thinking over the 46-year period from 1950 to 1995. It examined the 25 works authored, co-authored, edited, reissued and revised by him during that period. The writings were scrutinised using a literature research methodology to expose the theoretical content, and a history of thought lens to identify and account for the development of major ideas. The methodology enabled a gradual unfolding of the history. A broadly-consistent and sequential pattern of thought focusing on the notion of andragogy emerged. The study revealed that after the initial phases of exploratory thinking, Knowles developed a practical-theoretical framework he believed could function as a comprehensive theory of adult learning. As his thinking progressed, his theory developed into a unified framework for human resource development and, later, into a model for the development of self-directed lifelong learners. The study traced the development of Knowles’ thinking through the phases of thought, identified the writings that belonged within each phase and produced a series of diagrammatic representations showing the evolution of his conceptual framework. The production of a history of the development of Knowles’ thought is the major outcome of the study. In addition to plotting the narrative sequence of thought-events, the history helps to explicate the factors and conditions that influenced Knowles’ thinking and to show the interrelationships between ideas. The study should help practitioners in their use and appreciation of Knowles’ works.

Statistical analysis and reduction of multiple access interference in MC-CDMA systems

Multicarrier code division multiple access (MC-CDMA) is a very promising candidate for the multiple access scheme in fourth generation wireless communi- cation systems. During asynchronous transmission, multiple access interference (MAI) is a major challenge for MC-CDMA systems and significantly affects their performance. The main objectives of this thesis are to analyze the MAI in asyn- chronous MC-CDMA, and to develop robust techniques to reduce the MAI effect. Focus is first on the statistical analysis of MAI in asynchronous MC-CDMA. A new statistical model of MAI is developed. In the new model, the derivation of MAI can be applied to different distributions of timing offset, and the MAI power is modelled as a Gamma distributed random variable. By applying the new statistical model of MAI, a new computer simulation model is proposed. This model is based on the modelling of a multiuser system as a single user system followed by an additive noise component representing the MAI, which enables the new simulation model to significantly reduce the computation load during computer simulations. MAI reduction using slow frequency hopping (SFH) technique is the topic of the second part of the thesis. Two subsystems are considered. The first sub- system involves subcarrier frequency hopping as a group, which is referred to as GSFH/MC-CDMA. In the second subsystem, the condition of group hopping is dropped, resulting in a more general system, namely individual subcarrier frequency hopping MC-CDMA (ISFH/MC-CDMA). This research found that with the introduction of SFH, both of GSFH/MC-CDMA and ISFH/MC-CDMA sys- tems generate less MAI power than the basic MC-CDMA system during asyn- chronous transmission. Because of this, both SFH systems are shown to outper- form MC-CDMA in terms of BER. This improvement, however, is at the expense of spectral widening. In the third part of this thesis, base station polarization diversity, as another MAI reduction technique, is introduced to asynchronous MC-CDMA. The com- bined system is referred to as Pol/MC-CDMA. In this part a new optimum com- bining technique namely maximal signal-to-MAI ratio combining (MSMAIRC) is proposed to combine the signals in two base station antennas. With the applica- tion of MSMAIRC and in the absents of additive white Gaussian noise (AWGN), the resulting signal-to-MAI ratio (SMAIR) is not only maximized but also in- dependent of cross polarization discrimination (XPD) and antenna angle. In the case when AWGN is present, the performance of MSMAIRC is still affected by the XPD and antenna angle, but to a much lesser degree than the traditional maximal ratio combining (MRC). Furthermore, this research found that the BER performance for Pol/MC-CDMA can be further improved by changing the angle between the two receiving antennas. Hence the optimum antenna angles for both MSMAIRC and MRC are derived and their effects on the BER performance are compared. With the derived optimum antenna angle, the Pol/MC-CDMA system is able to obtain the lowest BER for a given XPD.

The Dragon stream cipher: Design, analysis and implementation issues

Dragon is a word-based stream cipher. It was submitted to the eSTREAM project in 2005 and has advanced to Phase 3 of the software profile. This paper discusses the Dragon cipher from three perspectives: design, security analysis and implementation. The design of the cipher incorporates a single word-based non-linear feedback shift register and a non-linear filter function with memory. This state is initialized with 128- or 256-bit key-IV pairs. Each clock of the stream cipher produces 64 bits of keystream, using simple operations on 32-bit words. This provides the cipher with a high degree of efficiency in a wide variety of environments, making it highly competitive relative to other symmetric ciphers. The components of Dragon were designed to resist all known attacks. Although the design has been open to public scrutiny for several years, the only published attacks to date are distinguishing attacks which require keystream lengths greatly exceeding the stated 264 bit maximum permitted keystream length for a single key-IV pair.

Phylogenetic analysis of polyomaviruses based on their complete genomes

Perez-Losada et al. [1] analyzed 72 complete genomes corresponding to nine mammalian (67 strains) and 2 avian (5 strains) polyomavirus species using maximum likelihood and Bayesian methods of phylogenetic inference. Because some data of 2 genomes in their work are now not available in GenBank, in this work, we analyze the phylogenetic relationship of the remaining 70 complete genomes corresponding to nine mammalian (65 strains) and two avian (5 strains) polyomavirus species using a dynamical language model approach developed by our group (Yu et al., [26]). This distance method does not require sequence alignment for deriving species phylogeny based on overall similarities of the complete genomes. Our best tree separates the bird polyomaviruses (avian polyomaviruses and goose hemorrhagic polymaviruses) from the mammalian polyomaviruses, which supports the idea of splitting the genus into two subgenera. Such a split is consistent with the different viral life strategies of each group. In the mammalian polyomavirus subgenera, mouse polyomaviruses (MPV), simian viruses 40 (SV40), BK viruses (BKV) and JC viruses (JCV) are grouped as different branches as expected. The topology of our best tree is quite similar to that of the tree constructed by Perez-Losada et al.

Polybrominated diphenyl ether (PBDE) concentrations decrease with age : analysis of pooled human blood serum in the Australian population

Introduction Polybrominated diphenyl ethers (PBDEs) are considered to be a cost effective and efficient way to reduce the possibility of product ignition and inhibit the spread of fire, thereby limiting harm caused by fires. PBDEs are incorporated into a wide variety of manufactured products and are now considered an ubiquitous contaminant found worldwide in biological and environmental samples . In comparison to “traditional” persistent organic pollutants (POPs), the exposure modes of PBDEs in humans are less well defined, although dietary sources, inhalation (air/particulate matter) and dust ingestion have been reported 2-4. Limited investigations of population specific factors such as age or gender and PBDE concentrations report: no conclusive correlation by age in adults ; higher concentrations in children ; similar concentrations in maternal and cord blood ; and no gender differences . After preliminary findings of higher PBDE concentrations in children than in adults in Australia11 we sought to investigate at what age the PBDE concentrations peaked in an effort to focus exposure studies. This investigation involved the collection of blood samples from young age groups and the development of a simple model to predict PBDE concentrations by age in Australia.

Identification of factors predicting clickthrough in Web searching using neural network analysis

In this research, we aim to identify factors that significantly affect the clickthrough of Web searchers. Our underlying goal is determine more efficient methods to optimize the clickthrough rate. We devise a clickthrough metric for measuring customer satisfaction of search engine results using the number of links visited, number of queries a user submits, and rank of clicked links. We use a neural network to detect the significant influence of searching characteristics on future user clickthrough. Our results show that high occurrences of query reformulation, lengthy searching duration, longer query length, and the higher ranking of prior clicked links correlate positively with future clickthrough. We provide recommendations for leveraging these findings for improving the performance of search engine retrieval and result ranking, along with implications for search engine marketing