Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Resistance : re-imagining innovation in higher education teaching and learning

The call to innovate is ubiquitous across the Australian educational policy context. The claims of innovative practices and environments that occur frequently in university mission statements, strategic plans and marketing literature suggest that this exhortation to innovate appears to have been taken up enthusiastically by the university sector. Throughout the history of universities, a range of reported deficiencies of higher education have worked to produce a notion of crisis. At present, it would seem that innovation is positioned as the solution to the notion of crisis. This thesis is an inquiry into how the insistence on innovation works to both enable and constrain teaching and learning practices in Australian universities. Alongside the interplay between innovation and crisis is the link between resistance and innovation, a link which remains largely unproblematized in the scholarly literature. This thesis works to locate and unsettle understandings of a relationship between innovation and Australian higher education. The aim of this inquiry is to generate new understandings of what counts as innovation within this context and how innovation is enacted. The thesis draws on a number of postmodernist theorists, whose works have informed firstly the research method, and then the analysis and findings. Firstly, there is an assumption that power is capillary and works through discourse to enact power relations which shape certain truths (Foucault, 1990). Secondly, this research scrutinised language practices which frame the capacity for individuals to act, alongside the language practices which encourage an individual to adopt certain attitudes and actions as one’s own (Foucault, 1988). Thirdly, innovation talk is read in this thesis as an example of needs talk, that is, as a medium through which what is considered domestic, political or economic is made and contested (Fraser, 1989). Fourthly, relationships between and within discourses were identified and analysed beyond cause and effect descriptions, and more productively considered to be in a constant state of becoming (Deleuze, 1987). Finally, the use of ironic research methods assisted in producing alternate configurations of innovation talk which are useful and new (Rorty, 1989). The theoretical assumptions which underpin this thesis inform a document analysis methodology, used to examine how certain texts work to shape the ways in which innovation is constructed. The data consisted of three Federal higher education funding policies selected on the rationale that these documents, as opposed to state or locally based policy and legislation, represent the only shared policy context for all Australian universities. The analysis first provided a modernist reading of the three documents, and this was followed by postmodernist readings of these same policy documents. The modernist reading worked to locate and describe the current truths about innovation. The historical context in which the policy was produced as well as the textual features of the document itself were important to this reading. In the first modernist reading, the binaries involved in producing proper and improper notions of innovation were described and analysed. In the process of the modernist analysis and the subsequent location of binary organisation, a number of conceptual collisions were identified, and these sites of struggle were revisited, through the application of a postmodernist reading. By applying the theories of Rorty (1989) and Fraser (1989) it became possible to not treat these sites as contradictory and requiring resolution, but rather as spaces in which binary tensions are necessary and productive. This postmodernist reading constructed new spaces for refusing and resisting dominant discourses of innovation which value only certain kinds of teaching and learning practices. By exploring a number of ironic language practices found within the policies, this thesis proposes an alternative way of thinking about what counts as innovation and how it happens. The new readings of innovation made possible through the work of this thesis were in response to a suite of enduring, inter-related questions – what counts as innovation?, who or what supports innovation?, how does innovation occur?, and who are the innovators?. The truths presented in response to these questions were treated as the language practices which constitute a dominant discourse of innovation talk. The collisions that occur within these truths were the contested sites which were of most interest for the analysis. The thesis concludes by presenting a theoretical blueprint which works to shift the boundaries of what counts as innovation and how it happens in a manner which is productive, inclusive and powerful. This blueprint forms the foundation upon which a number of recommendations are made for both my own professional practice and broader contexts. In keeping with the conceptual tone of this study, these recommendations are a suite of new questions which focus attention on the boundaries of innovation talk as an attempt to re-configure what is valued about teaching and learning at university.

Gender

The OED informs us that “gender” has at its root the Latin genus, meaning “race, kind,” and emerges as early as the fifth century as a term for differentiating between types of (especially) people and words. In the following 1500 years, gender appears in linguistic and biological contexts to distinguish types of words and bodies from one another, as when words in Indo-European languages were identified as masculine, feminine, or neuter, and humans were identified as male or female. It is telling that gender has historically (whether overtly or covertly) been a tool of negotiation between our understandings of bodies, and meanings derived from and attributed to them. Within the field of children’s literature studies, as in other disciplines, gender in and of itself is rarely the object of critique. Rather, specific constructions of gender structure understandings of subjectivity; allow or disallow certain behaviors or experiences on the basis of biological sex; and dictate a specific vision of social relations and organization. Critical approaches to gender in children’s literature have included linguistic analysis (Turner-Bowker; Sunderland); analysis of visual representations (Bradford; Moebius); cultural images of females (Grauerholz and Pescosolido); consideration of gender and genre (Christian-Smith; Stephens); ideological (Nodelman and Reimer); psychoanalytic (Coats); discourse analysis (Stephens); and masculinity studies (Nodelman) among others. In the adjacent fields of education and literacy studies, gender has been a sustained point of investigation, often deriving from perceived gendering of pedagogical practices (Lehr) or of reading preferences and competencies, and in recent years, perceptions of boys as “reluctant readers” (Moss). The ideology of patriarchy has primarily come under critical scrutiny 2 because it has been used to locate characters and readers within the specific binary logic of gender relations that historically subordinated the feminine to the masculine. Just as feminism might be broadly defined as resistance to existing power structures, a gendered reading might be broadly defined as a “resistant reading” in that it most often reveals or contests that which a text assumes to be the norm.

Toward robust image detection of crown-of-thorns starfish for autonomous population monitoring

Robust texture recognition in underwater image sequences for marine pest population control such as Crown-Of-Thorns Starfish (COTS) is a relatively unexplored area of research. Typically, humans count COTS by laboriously processing individual images taken during surveys. Being able to autonomously collect and process images of reef habitat and segment out the various marine biota holds the promise of allowing researchers to gain a greater understanding of the marine ecosystem and evaluate the impact of different environmental variables. This research applies and extends the use of Local Binary Patterns (LBP) as a method for texture-based identification of COTS from survey images. The performance and accuracy of the algorithms are evaluated on a image data set taken on the Great Barrier Reef.

Development of rapid and highly resolving combinatorial genotyping schemes for Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni followed by Campylobacter coli contribute substantially to the economic and public health burden attributed to food-borne infections in Australia. Genotypic characterisation of isolates has provided new insights into the epidemiology and pathogenesis of C. jejuni and C. coli. However, currently available methods are not conducive to large scale epidemiological investigations that are necessary to elucidate the global epidemiology of these common food-borne pathogens. This research aims to develop high resolution C. jejuni and C. coli genotyping schemes that are convenient for high throughput applications. Real-time PCR and High Resolution Melt (HRM) analysis are fundamental to the genotyping schemes developed in this study and enable rapid, cost effective, interrogation of a range of different polymorphic sites within the Campylobacter genome. While the sources and routes of transmission of campylobacters are unclear, handling and consumption of poultry meat is frequently associated with human campylobacteriosis in Australia. Therefore, chicken derived C. jejuni and C. coli isolates were used to develop and verify the methods described in this study. The first aim of this study describes the application of MLST-SNP (Multi Locus Sequence Typing Single Nucleotide Polymorphisms) + binary typing to 87 chicken C. jejuni isolates using real-time PCR analysis. These typing schemes were developed previously by our research group using isolates from campylobacteriosis patients. This present study showed that SNP + binary typing alone or in combination are effective at detecting epidemiological linkage between chicken derived Campylobacter isolates and enable data comparisons with other MLST based investigations. SNP + binary types obtained from chicken isolates in this study were compared with a previously SNP + binary and MLST typed set of human isolates. Common genotypes between the two collections of isolates were identified and ST-524 represented a clone that could be worth monitoring in the chicken meat industry. In contrast, ST-48, mainly associated with bovine hosts, was abundant in the human isolates. This genotype was, however, absent in the chicken isolates, indicating the role of non-poultry sources in causing human Campylobacter infections. This demonstrates the potential application of SNP + binary typing for epidemiological investigations and source tracing. While MLST SNPs and binary genes comprise the more stable backbone of the Campylobacter genome and are indicative of long term epidemiological linkage of the isolates, the development of a High Resolution Melt (HRM) based curve analysis method to interrogate the hypervariable Campylobacter flagellin encoding gene (flaA) is described in Aim 2 of this study. The flaA gene product appears to be an important pathogenicity determinant of campylobacters and is therefore a popular target for genotyping, especially for short term epidemiological studies such as outbreak investigations. HRM curve analysis based flaA interrogation is a single-step closed-tube method that provides portable data that can be easily shared and accessed. Critical to the development of flaA HRM was the use of flaA specific primers that did not amplify the flaB gene. HRM curve analysis flaA interrogation was successful at discriminating the 47 sequence variants identified within the 87 C. jejuni and 15 C. coli isolates and correlated to the epidemiological background of the isolates. In the combinatorial format, the resolving power of flaA was additive to that of SNP + binary typing and CRISPR (Clustered regularly spaced short Palindromic repeats) HRM and fits the PHRANA (Progressive hierarchical resolving assays using nucleic acids) approach for genotyping. The use of statistical methods to analyse the HRM data enhanced sophistication of the method. Therefore, flaA HRM is a rapid and cost effective alternative to gel- or sequence-based flaA typing schemes. Aim 3 of this study describes the development of a novel bioinformatics driven method to interrogate Campylobacter MLST gene fragments using HRM, and is called ‘SNP Nucleated Minim MLST’ or ‘Minim typing’. The method involves HRM interrogation of MLST fragments that encompass highly informative “Nucleating SNPS” to ensure high resolution. Selection of fragments potentially suited to HRM analysis was conducted in silico using i) “Minimum SNPs” and ii) the new ’HRMtype’ software packages. Species specific sets of six “Nucleating SNPs” and six HRM fragments were identified for both C. jejuni and C. coli to ensure high typeability and resolution relevant to the MLST database. ‘Minim typing’ was tested empirically by typing 15 C. jejuni and five C. coli isolates. The association of clonal complexes (CC) to each isolate by ‘Minim typing’ and SNP + binary typing were used to compare the two MLST interrogation schemes. The CCs linked with each C. jejuni isolate were consistent for both methods. Thus, ‘Minim typing’ is an efficient and cost effective method to interrogate MLST genes. However, it is not expected to be independent, or meet the resolution of, sequence based MLST gene interrogation. ‘Minim typing’ in combination with flaA HRM is envisaged to comprise a highly resolving combinatorial typing scheme developed around the HRM platform and is amenable to automation and multiplexing. The genotyping techniques described in this thesis involve the combinatorial interrogation of differentially evolving genetic markers on the unified real-time PCR and HRM platform. They provide high resolution and are simple, cost effective and ideally suited to rapid and high throughput genotyping for these common food-borne pathogens.

Implications of faecal indicator bacteria for the microbiological assessment of roof harvested rainwater quality in Southeast Queensland, Australia

The study aimed to evaluate the suitability of Escherichia coli, enterococci and C. perfringens to assess the microbiological quality of roof harvested rainwater, and to assess whether the concentrations of these faecal indicators can be used to predict the presence or absence of specific zoonotic bacterial or protozoan pathogens. From a total of 100 samples tested, respectively 58%, 83% and 46% of samples were found to be positive for E. coli, enterococci and C. perfringens spores, as determined by traditional culture based methods. Additionally, in the samples tested, 7%, 19%, 1%, 8%, 17%, and 15% were PCR positive for A. hydrophila lip, C. coli ceuE, C. jejuni mapA, L. pneumophila mip, Salmonella invA, and G. lamblia β-giardin genes. However, none of the samples was positive for E. coli O157 LPS, VT1, VT2 and C. parvum COWP genes. The presence or absence of these potential pathogens did not correlate with any of the faecal indicator bacterial concentrations as determined by a binary logistic regression model. The roof-harvested rainwater samples tested in this study appear to be of poor microbiological quality and no significant correlation was found between the concentration of faecal indicators and pathogenic microorganisms. The use of faecal indicator bacteria raises questions regarding their reliability in assessing the microbiological quality of water and particularly their poor correlation with pathogenic microorganisms. The presence of one or more zoonotic pathogens suggests that the microbiological analysis of water should be performed, and appropriate treatment measures should be undertaken especially in tanks where the water is used for drinking.

Visual control of robots : high-performance visual servoing

The application of high-speed machine vision for close-loop position control, or visual servoing, of a robot manipulator. It provides a comprehensive coverage of all aspects of the visual servoing problem: robotics, vision, control, technology and implementation issues. While much of the discussion is quite general the experimental work described is based on the use of a high-speed binary vision system with a monocular "eye-in-hand" camera.

Art in early childhood : the discourse of 'proper' teaching

This thesis is a problematisation of the teaching of art to young children. To problematise a domain of social endeavour, is, in Michel Foucault's terms, to ask how we come to believe that "something ... can and must be thought" (Foucault, 1985:7). The aim is to document what counts (i.e., what is sayable, thinkable, feelable) as proper art teaching in Queensland at this point ofhistorical time. In this sense, the thesis is a departure from more recognisable research on 'more effective' teaching, including critical studies of art teaching and early childhood teaching. It treats 'good teaching' as an effect of moral training made possible through disciplinary discourses organised around certain epistemic rules at a particular place and time. There are four key tasks accomplished within the thesis. The first is to describe an event which is not easily resolved by means of orthodox theories or explanations, either liberal-humanist or critical ones. The second is to indicate how poststructuralist understandings of the self and social practice enable fresh engagements with uneasy pedagogical moments. What follows this discussion is the documentation of an empirical investigation that was made into texts generated by early childhood teachers, artists and parents about what constitutes 'good practice' in art teaching. Twenty-two participants produced text to tell and re-tell the meaning of 'proper' art education, from different subject positions. Rather than attempting to capture 'typical' representations of art education in the early years, a pool of 'exemplary' teachers, artists and parents were chosen, using "purposeful sampling", and from this pool, three videos were filmed and later discussed by the audience of participants. The fourth aspect of the thesis involves developing a means of analysing these texts in such a way as to allow a 're-description' of the field of art teaching by attempting to foreground the epistemic rules through which such teacher-generated texts come to count as true ie, as propriety in art pedagogy. This analysis drew on Donna Haraway's (1995) understanding of 'ironic' categorisation to hold the tensions within the propositions inside the categories of analysis rather than setting these up as discursive oppositions. The analysis is therefore ironic in the sense that Richard Rorty (1989) understands the term to apply to social scientific research. Three 'ironic' categories were argued to inform the discursive construction of 'proper' art teaching. It is argued that a teacher should (a) Teach without teaching; (b) Manufacture the natural; and (c) Train for creativity. These ironic categories work to undo modernist assumptions about theory/practice gaps and finding a 'balance' between oppositional binary terms. They were produced through a discourse theoretical reading of the texts generated by the participants in the study, texts that these same individuals use as a means of discipline and self-training as they work to teach properly. In arguing the usefulness of such approaches to empirical data analysis, the thesis challenges early childhood research in arts education, in relation to its capacity to deal with ambiguity and to acknowledge contradiction in the work of teachers and in their explanations for what they do. It works as a challenge at a range of levels - at the level of theorising, of method and of analysis. In opening up thinking about normalised categories, and questioning traditional Western philosophy and the grand narratives of early childhood art pedagogy, it makes a space for re-thinking art pedagogy as "a game oftruth and error" (Foucault, 1985). In doing so, it opens up a space for thinking how art education might be otherwise.

Applied batch cryptography

The material presented in this thesis may be viewed as comprising two key parts, the first part concerns batch cryptography specifically, whilst the second deals with how this form of cryptography may be applied to security related applications such as electronic cash for improving efficiency of the protocols. The objective of batch cryptography is to devise more efficient primitive cryptographic protocols. In general, these primitives make use of some property such as homomorphism to perform a computationally expensive operation on a collective input set. The idea is to amortise an expensive operation, such as modular exponentiation, over the input. Most of the research work in this field has concentrated on its employment as a batch verifier of digital signatures. It is shown that several new attacks may be launched against these published schemes as some weaknesses are exposed. Another common use of batch cryptography is the simultaneous generation of digital signatures. There is significantly less previous work on this area, and the present schemes have some limited use in practical applications. Several new batch signatures schemes are introduced that improve upon the existing techniques and some practical uses are illustrated. Electronic cash is a technology that demands complex protocols in order to furnish several security properties. These typically include anonymity, traceability of a double spender, and off-line payment features. Presently, the most efficient schemes make use of coin divisibility to withdraw one large financial amount that may be progressively spent with one or more merchants. Several new cash schemes are introduced here that make use of batch cryptography for improving the withdrawal, payment, and deposit of electronic coins. The devised schemes apply both to the batch signature and verification techniques introduced, demonstrating improved performance over the contemporary divisible based structures. The solutions also provide an alternative paradigm for the construction of electronic cash systems. Whilst electronic cash is used as the vehicle for demonstrating the relevance of batch cryptography to security related applications, the applicability of the techniques introduced extends well beyond this.

Hierarchical models for 2D presence/absence data having ambiguous zeroes: With a biogeographical case study on dingo behaviour

This dissertation is primarily an applied statistical modelling investigation, motivated by a case study comprising real data and real questions. Theoretical questions on modelling and computation of normalization constants arose from pursuit of these data analytic questions. The essence of the thesis can be described as follows. Consider binary data observed on a two-dimensional lattice. A common problem with such data is the ambiguity of zeroes recorded. These may represent zero response given some threshold (presence) or that the threshold has not been triggered (absence). Suppose that the researcher wishes to estimate the effects of covariates on the binary responses, whilst taking into account underlying spatial variation, which is itself of some interest. This situation arises in many contexts and the dingo, cypress and toad case studies described in the motivation chapter are examples of this. Two main approaches to modelling and inference are investigated in this thesis. The first is frequentist and based on generalized linear models, with spatial variation modelled by using a block structure or by smoothing the residuals spatially. The EM algorithm can be used to obtain point estimates, coupled with bootstrapping or asymptotic MLE estimates for standard errors. The second approach is Bayesian and based on a three- or four-tier hierarchical model, comprising a logistic regression with covariates for the data layer, a binary Markov Random field (MRF) for the underlying spatial process, and suitable priors for parameters in these main models. The three-parameter autologistic model is a particular MRF of interest. Markov chain Monte Carlo (MCMC) methods comprising hybrid Metropolis/Gibbs samplers is suitable for computation in this situation. Model performance can be gauged by MCMC diagnostics. Model choice can be assessed by incorporating another tier in the modelling hierarchy. This requires evaluation of a normalization constant, a notoriously difficult problem. Difficulty with estimating the normalization constant for the MRF can be overcome by using a path integral approach, although this is a highly computationally intensive method. Different methods of estimating ratios of normalization constants (N Cs) are investigated, including importance sampling Monte Carlo (ISMC), dependent Monte Carlo based on MCMC simulations (MCMC), and reverse logistic regression (RLR). I develop an idea present though not fully developed in the literature, and propose the Integrated mean canonical statistic (IMCS) method for estimating log NC ratios for binary MRFs. The IMCS method falls within the framework of the newly identified path sampling methods of Gelman & Meng (1998) and outperforms ISMC, MCMC and RLR. It also does not rely on simplifying assumptions, such as ignoring spatio-temporal dependence in the process. A thorough investigation is made of the application of IMCS to the three-parameter Autologistic model. This work introduces background computations required for the full implementation of the four-tier model in Chapter 7. Two different extensions of the three-tier model to a four-tier version are investigated. The first extension incorporates temporal dependence in the underlying spatio-temporal process. The second extensions allows the successes and failures in the data layer to depend on time. The MCMC computational method is extended to incorporate the extra layer. A major contribution of the thesis is the development of a fully Bayesian approach to inference for these hierarchical models for the first time. Note: The author of this thesis has agreed to make it open access but invites people downloading the thesis to send her an email via the 'Contact Author' function.

Algebraic attacks on clock-controlled stream ciphers

Stream ciphers are encryption algorithms used for ensuring the privacy of digital telecommunications. They have been widely used for encrypting military communications, satellite communications, pay TV encryption and for voice encryption of both fixed lined and wireless networks. The current multi year European project eSTREAM, which aims to select stream ciphers suitable for widespread adoptation, reflects the importance of this area of research. Stream ciphers consist of a keystream generator and an output function. Keystream generators produce a sequence that appears to be random, which is combined with the plaintext message using the output function. Most commonly, the output function is binary addition modulo two. Cryptanalysis of these ciphers focuses largely on analysis of the keystream generators and of relationships between the generator and the keystream it produces. Linear feedback shift registers are widely used components in building keystream generators, as the sequences they produce are well understood. Many types of attack have been proposed for breaking various LFSR based stream ciphers. A recent attack type is known as an algebraic attack. Algebraic attacks transform the problem of recovering the key into a problem of solving multivariate system of equations, which eventually recover the internal state bits or the key bits. This type of attack has been shown to be effective on a number of regularly clocked LFSR based stream ciphers. In this thesis, algebraic attacks are extended to a number of well known stream ciphers where at least one LFSR in the system is irregularly clocked. Applying algebriac attacks to these ciphers has only been discussed previously in the open literature for LILI-128. In this thesis, algebraic attacks are first applied to keystream generators using stop-and go clocking. Four ciphers belonging to this group are investigated: the Beth-Piper stop-and-go generator, the alternating step generator, the Gollmann cascade generator and the eSTREAM candidate: the Pomaranch cipher. It is shown that algebraic attacks are very effective on the first three of these ciphers. Although no effective algebraic attack was found for Pomaranch, the algebraic analysis lead to some interesting findings including weaknesses that may be exploited in future attacks. Algebraic attacks are then applied to keystream generators using (p; q) clocking. Two well known examples of such ciphers, the step1/step2 generator and the self decimated generator are investigated. Algebraic attacks are shown to be very powerful attack in recovering the internal state of these generators. A more complex clocking mechanism than either stop-and-go or the (p; q) clocking keystream generators is known as mutual clock control. In mutual clock control generators, the LFSRs control the clocking of each other. Four well known stream ciphers belonging to this group are investigated with respect to algebraic attacks: the Bilateral-stop-and-go generator, A5/1 stream cipher, Alpha 1 stream cipher, and the more recent eSTREAM proposal, the MICKEY stream ciphers. Some theoretical results with regards to the complexity of algebraic attacks on these ciphers are presented. The algebraic analysis of these ciphers showed that generally, it is hard to generate the system of equations required for an algebraic attack on these ciphers. As the algebraic attack could not be applied directly on these ciphers, a different approach was used, namely guessing some bits of the internal state, in order to reduce the degree of the equations. Finally, an algebraic attack on Alpha 1 that requires only 128 bits of keystream to recover the 128 internal state bits is presented. An essential process associated with stream cipher proposals is key initialization. Many recently proposed stream ciphers use an algorithm to initialize the large internal state with a smaller key and possibly publicly known initialization vectors. The effect of key initialization on the performance of algebraic attacks is also investigated in this thesis. The relationships between the two have not been investigated before in the open literature. The investigation is conducted on Trivium and Grain-128, two eSTREAM ciphers. It is shown that the key initialization process has an effect on the success of algebraic attacks, unlike other conventional attacks. In particular, the key initialization process allows an attacker to firstly generate a small number of equations of low degree and then perform an algebraic attack using multiple keystreams. The effect of the number of iterations performed during key initialization is investigated. It is shown that both the number of iterations and the maximum number of initialization vectors to be used with one key should be carefully chosen. Some experimental results on Trivium and Grain-128 are then presented. Finally, the security with respect to algebraic attacks of the well known LILI family of stream ciphers, including the unbroken LILI-II, is investigated. These are irregularly clock- controlled nonlinear filtered generators. While the structure is defined for the LILI family, a particular paramater choice defines a specific instance. Two well known such instances are LILI-128 and LILI-II. The security of these and other instances is investigated to identify which instances are vulnerable to algebraic attacks. The feasibility of recovering the key bits using algebraic attacks is then investigated for both LILI- 128 and LILI-II. Algebraic attacks which recover the internal state with less effort than exhaustive key search are possible for LILI-128 but not for LILI-II. Given the internal state at some point in time, the feasibility of recovering the key bits is also investigated, showing that the parameters used in the key initialization process, if poorly chosen, can lead to a key recovery using algebraic attacks.

Learning power and language expressiveness

The topic of the present work is to study the relationship between the power of the learning algorithms on the one hand, and the expressive power of the logical language which is used to represent the problems to be learned on the other hand. The central question is whether enriching the language results in more learning power. In order to make the question relevant and nontrivial, it is required that both texts (sequences of data) and hypotheses (guesses) be translatable from the “rich” language into the “poor” one. The issue is considered for several logical languages suitable to describe structures whose domain is the set of natural numbers. It is shown that enriching the language does not give any advantage for those languages which define a monadic second-order language being decidable in the following sense: there is a fixed interpretation in the structure of natural numbers such that the set of sentences of this extended language true in that structure is decidable. But enriching the original language even by only one constant gives an advantage if this language contains a binary function symbol (which will be interpreted as addition). Furthermore, it is shown that behaviourally correct learning has exactly the same power as learning in the limit for those languages which define a monadic second-order language with the property given above, but has more power in case of languages containing a binary function symbol. Adding the natural requirement that the set of all structures to be learned is recursively enumerable, it is shown that it pays o6 to enrich the language of arithmetics for both finite learning and learning in the limit, but it does not pay off to enrich the language for behaviourally correct learning.

Gene transfer to plants by diverse species of bacteria

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology1. However, the complexity of the patent landscape2 has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile ‘open source’ platform for plant biotechnology, may lead to new uses of natural bacteria– plant interactions to achieve plant transformation.

Applications on fault tree analysis in railway power supply systems

Fault tree analysis (FTA) is presented to model the reliability of a railway traction power system in this paper. First, the construction of fault tree is introduced to integrate components in traction power systems into a fault tree; then the binary decision diagram (BDD) method is used to evaluate fault trees qualitatively and quantitatively. The components contributing to the reliability of overall system are identified with their relative importance through sensitivity analysis. Finally, an AC traction power system is evaluated by the proposed methods.

Comparing charge transport predictions for a ternary electrolyte using the Maxwell–Stefan and Nernst–Planck equations

In this work, we investigate and compare the Maxwell–Stefan and Nernst–Planck equations for modeling multicomponent charge transport in liquid electrolytes. Specifically, we consider charge transport in the Li+/I−/I3−/ACN ternary electrolyte originally found in dye-sensitized solar cells. We employ molecular dynamics simulations to obtain the Maxwell–Stefan diffusivities for this electrolyte. These simulated diffusion coefficients are used in a multicomponent charge transport model based on the Maxwell– Stefan equations, and this is compared to a Nernst–Planck based model which employs binary diffusion coefficients sourced from the literature. We show that significant differences between the electrolyte concentrations at electrode interfaces, as predicted by the Maxwell–Stefan and Nernst–Planck models, can occur. We find that these differences are driven by a pressure term that appears in the Maxwell–Stefan equations. We also investigate what effects the Maxwell–Stefan diffusivities have on the simulated charge transport. By incorporating binary diffusivities found in the literature into the Maxwell–Stefan framework, we show that the simulated transient concentration profiles depend on the diffusivities; however, the simulated equilibrium profiles remain unaffected.