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.

How Australia’s Indigenous people are positioned within health service sites by our presence and by our absence

De Certeau (1984) constructs the notion of belonging as a sentiment which develops over time through the everyday activities. He explains that simple everyday activities are part of the process of appropriation and territorialisation and suggests that over time belonging and attachment are established and built on memory, knowledge and the experiences of everyday activities. Based on the work of de Certeau, non-Indigenous Australians have developed attachment and belonging to places based on the dispossession of Aboriginal people and on their everyday practices over the past two hundred years. During this time non-Indigenous people have marked their appropriation and territorialisation with signs, symbols, representations and images. In marking their attachment, they also define how they position Australia’s Indigenous people by both our presence and our absence. This paper will explore signs and symbols within spaces and places in health services and showcase how they reflect the historical, political, cultural, social and economic values, and power relations of broader society. It will draw on the voices of Aboriginal women to demonstrate their everyday experiences of such sites. It will conclude by highlighting how Aboriginal people assert their identities and un-ceded sovereignty within such health sites and actively resist on-going white epistemological notions of us and the logic of patriarchal white sovereignty.

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.

The effects of L1 orthography on processing an artificial logographic script

To date, studies have focused on the acquisition of alphabetic second languages (L2s) in alphabetic first language (L1) users, demonstrating significant transfer effects. The present study examined the process from a reverse perspective, comparing logographic (Mandarin-Chinese) and alphabetic (English) L1 users in the acquisition of an artificial logographic script, in order to determine whether similar language-specific advantageous transfer effects occurred. English monolinguals, English-French bilinguals and Chinese-English bilinguals learned a small set of symbols in an artificial logographic script and were subsequently tested on their ability to process this script in regard to three main perspectives: L2 reading, L2 working memory (WM), and inner processing strategies. In terms of L2 reading, a lexical decision task on the artificial symbols revealed markedly faster response times in the Chinese-English bilinguals, indicating a logographic transfer effect suggestive of a visual processing advantage. A syntactic decision task evaluated the degree to which the new language was mastered beyond the single word level. No L1-specific transfer effects were found for artificial language strings. In order to investigate visual processing of the artificial logographs further, a series of WM experiments were conducted. Artificial logographs were recalled under concurrent auditory and visuo-spatial suppression conditions to disrupt phonological and visual processing, respectively. No L1-specific transfer effects were found, indicating no visual processing advantage of the Chinese-English bilinguals. However, a bilingual processing advantage was found indicative of a superior ability to control executive functions. In terms of L1 WM, the Chinese-English bilinguals outperformed the alphabetic L1 users when processing L1 words, indicating a language experience-specific advantage. Questionnaire data on the cognitive strategies that were deployed during the acquisition and processing of the artificial logographic script revealed that the Chinese-English bilinguals rated their inner speech as lower than the alphabetic L1 users, suggesting that they were transferring their phonological processing skill set to the acquisition and use of an artificial script. Overall, evidence was found to indicate that language learners transfer specific L1 orthographic processing skills to L2 logographic processing. Additionally, evidence was also found indicating that a bilingual history enhances cognitive performance in L2.

Spectral coding methods for speech compression and speaker identification

This thesis investigates aspects of encoding the speech spectrum at low bit rates, with extensions to the effect of such coding on automatic speaker identification. Vector quantization (VQ) is a technique for jointly quantizing a block of samples at once, in order to reduce the bit rate of a coding system. The major drawback in using VQ is the complexity of the encoder. Recent research has indicated the potential applicability of the VQ method to speech when product code vector quantization (PCVQ) techniques are utilized. The focus of this research is the efficient representation, calculation and utilization of the speech model as stored in the PCVQ codebook. In this thesis, several VQ approaches are evaluated, and the efficacy of two training algorithms is compared experimentally. It is then shown that these productcode vector quantization algorithms may be augmented with lossless compression algorithms, thus yielding an improved overall compression rate. An approach using a statistical model for the vector codebook indices for subsequent lossless compression is introduced. This coupling of lossy compression and lossless compression enables further compression gain. It is demonstrated that this approach is able to reduce the bit rate requirement from the current 24 bits per 20 millisecond frame to below 20, using a standard spectral distortion metric for comparison. Several fast-search VQ methods for use in speech spectrum coding have been evaluated. The usefulness of fast-search algorithms is highly dependent upon the source characteristics and, although previous research has been undertaken for coding of images using VQ codebooks trained with the source samples directly, the product-code structured codebooks for speech spectrum quantization place new constraints on the search methodology. The second major focus of the research is an investigation of the effect of lowrate spectral compression methods on the task of automatic speaker identification. The motivation for this aspect of the research arose from a need to simultaneously preserve the speech quality and intelligibility and to provide for machine-based automatic speaker recognition using the compressed speech. This is important because there are several emerging applications of speaker identification where compressed speech is involved. Examples include mobile communications where the speech has been highly compressed, or where a database of speech material has been assembled and stored in compressed form. Although these two application areas have the same objective - that of maximizing the identification rate - the starting points are quite different. On the one hand, the speech material used for training the identification algorithm may or may not be available in compressed form. On the other hand, the new test material on which identification is to be based may only be available in compressed form. Using the spectral parameters which have been stored in compressed form, two main classes of speaker identification algorithm are examined. Some studies have been conducted in the past on bandwidth-limited speaker identification, but the use of short-term spectral compression deserves separate investigation. Combining the major aspects of the research, some important design guidelines for the construction of an identification model when based on the use of compressed speech are put forward.

Coding speech signals at very low rates (below 1 kb/s) with high intelligibility

This thesis presents an original approach to parametric speech coding at rates below 1 kbitsjsec, primarily for speech storage applications. Essential processes considered in this research encompass efficient characterization of evolutionary configuration of vocal tract to follow phonemic features with high fidelity, representation of speech excitation using minimal parameters with minor degradation in naturalness of synthesized speech, and finally, quantization of resulting parameters at the nominated rates. For encoding speech spectral features, a new method relying on Temporal Decomposition (TD) is developed which efficiently compresses spectral information through interpolation between most steady points over time trajectories of spectral parameters using a new basis function. The compression ratio provided by the method is independent of the updating rate of the feature vectors, hence allows high resolution in tracking significant temporal variations of speech formants with no effect on the spectral data rate. Accordingly, regardless of the quantization technique employed, the method yields a high compression ratio without sacrificing speech intelligibility. Several new techniques for improving performance of the interpolation of spectral parameters through phonetically-based analysis are proposed and implemented in this research, comprising event approximated TD, near-optimal shaping event approximating functions, efficient speech parametrization for TD on the basis of an extensive investigation originally reported in this thesis, and a hierarchical error minimization algorithm for decomposition of feature parameters which significantly reduces the complexity of the interpolation process. Speech excitation in this work is characterized based on a novel Multi-Band Excitation paradigm which accurately determines the harmonic structure in the LPC (linear predictive coding) residual spectra, within individual bands, using the concept 11 of Instantaneous Frequency (IF) estimation in frequency domain. The model yields aneffective two-band approximation to excitation and computes pitch and voicing with high accuracy as well. New methods for interpolative coding of pitch and gain contours are also developed in this thesis. For pitch, relying on the correlation between phonetic evolution and pitch variations during voiced speech segments, TD is employed to interpolate the pitch contour between critical points introduced by event centroids. This compresses pitch contour in the ratio of about 1/10 with negligible error. To approximate gain contour, a set of uniformly-distributed Gaussian event-like functions is used which reduces the amount of gain information to about 1/6 with acceptable accuracy. The thesis also addresses a new quantization method applied to spectral features on the basis of statistical properties and spectral sensitivity of spectral parameters extracted from TD-based analysis. The experimental results show that good quality speech, comparable to that of conventional coders at rates over 2 kbits/sec, can be achieved at rates 650-990 bits/sec.

An examination of communication across cultures in news media and at informal/personal levels : with concentration on relations among two South East Asian countries and Australia and those two countries and Germany

In the age of globalisation dominated by mass communication, the flow of information contributes to a big extent to the worldviews of its "global citizens". From this point of view the mass media can be seen as one of the most salient sources of cross-cultural communication. This study investigates mass communication across cultures, focusing on South East Asia (Malaysia and Singapore), Australia and Germany. The centre of attention is the Western media coverage of South East Asia and vice versa. In this context a content analysis of newspapers of the three regions has been conducted. In addition, working practices and conditions of Western foreign correspondents in South East Asia have been examined. Apart from the investigation of inter-cultural media coverage, another focus of attention will be the examination of two levels of communication: The business level, concentrating on issues like e.g. the Asian business etiquette; and the private level, looking into the transition to a different culture from the perspective of Australian and German expatriates. Apart from investigating mass communication across cultures and to provide a written analysis of the findings, a series of radio documentaries in English and in German has been produced. They cover the following issues: Foreign correspondents in South East Asia, the expatriate-lifestyle of Australians and Germans in South East Asia, business etiquette in Asia, student exchange Germany-Asia, image and prejudices East-West and Tourism.

Eucharistic rituals in Catholic primary schools in the Archdiocese of Brisbane : a study of administrators' conceptions

The focus of this study is the celebration of Eucharist in Catholic primary schools within the Archdiocese of Brisbane. The context of the contemporary Australian Catholic primary school embodies certain 'problematical realities' in relation to the time-honoured way in which school Eucharistic rituals have been celebrated. These contemporary realities raise a number of issues that impact on school celebrations of Eucharist. The purpose of this study is to explore administrators' differing conceptions of school Eucharistic rituals in an attempt to investigate some of these issues and assist members of individual school communities as they strive to make celebrations of Eucharist appropriate and meaningful for the group gathered. The phenomenographic research approach was adopted, as it is well suited to the purpose of this study and the nature of the research question. Phenomenography is essentially a study of variation. It attempts to map the 'whole' phenomenon under investigation by describing on equal terms all conceptions of the phenomenon and establishing an ordered relationship among them. The purpose of this study and the nature of the research question necessitate an approach that allows the identification and description of the different ways in which administrators' experience school Eucharistic rituals. Accordingly, phenomenography was selected. Members of the Administration Team, namely the principal, the APRE (Assistant to the Principal Religious Education) and, in larger primary schools, the AP A (Assistant to the Principal Administration) share responsibility for leading change in Catholic primary schools in the Archdiocese of Brisbane. In practice, however, principals delegate the role of leading the development of the school's religion program and providing leadership in the religious life of the school community to the APRE (Brisbane Catholic Education, 1997). Informants in this study are nineteen APREs from a variety of Catholic primary schools in the Archdiocese of Brisbane. These APREs come from schools across the archdiocese, rather than from within one particular region. Several significant findings resulted from this study. Firstly, the data show that there are significant differences in how APREs' experience school Eucharistic rituals, although the number of these qualitatively different conceptions is quite limited. The study identifies and describes six distinct yet related conceptions of school Eucharistic rituals. The logical relationship among these conceptions (the outcome space) is presented in the form of a diagram with accompanying explication. The variation among the conceptions is best understood and described in terms of three dimensions of the role of Eucharist in the Catholic primary school and is represented on the model of the outcome space. Individual transcripts suggest that individual APREs tend to emphasise some conceptions more than others. It is the contention of the present study that change in the practice of school Eucharistic rituals is unlikely to occur until all of a school community's conceptions are brought out into the open and articulated. As leaders of change, APREs need to be alerted to their own biases and become aware of alternative ways of conceiving school Eucharistic ritual. It is proposed that the different categories of description and dimensions, represented by the model of the outcome space, can be used to help in the process of articulating a school community's conceptions of Eucharist, with the APRE as facilitator of this process. As a result, the school community develops a better understanding of why their particular school does what it does in relation to school Eucharistic rituals.

The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

Crosstalk between developmental and tumour-specific signalling pathways : kallikrein-related serine peptidases and nodal in prostrate cancer

Prostate cancer is an important male health issue. The strategies used to diagnose and treat prostate cancer underscore the cell and molecular interactions that promote disease progression. Prostate cancer is histologically defined by increasingly undifferentiated tumour cells and therapeutically targeted by androgen ablation. Even as the normal glandular architecture of the adult prostate is lost, prostate cancer cells remain dependent on the androgen receptor (AR) for growth and survival. This project focused on androgen-regulated gene expression, altered cellular differentiation, and the nexus between these two concepts. The AR controls prostate development, homeostasis and cancer progression by regulating the expression of downstream genes. Kallikrein-related serine peptidases are prominent transcriptional targets of AR in the adult prostate. Kallikrein 3 (KLK3), which is commonly referred to as prostate-specific antigen, is the current serum biomarker for prostate cancer. Other kallikreins are potential adjunct biomarkers. As secreted proteases, kallikreins act through enzyme cascades that may modulate the prostate cancer microenvironment. Both as a panel of biomarkers and cascade of proteases, the roles of kallikreins are interconnected. Yet the expression and regulation of different kallikreins in prostate cancer has not been compared. In this study, a spectrum of prostate cell lines was used to evaluate the expression profile of all 15 members of the kallikrein family. A cluster of genes was co-ordinately expressed in androgenresponsive cell lines. This group of kallikreins included KLK2, 3, 4 and 15, which are located adjacent to one another at the centromeric end of the kallikrein locus. KLK14 was also of interest, because it was ubiquitously expressed among the prostate cell lines. Immunohistochemistry showed that these 5 kallikreins are co-expressed in benign and malignant prostate tissue. The androgen-regulated expression of KLK2 and KLK3 is well-characterised, but has not been compared with other kallikreins. Therefore, KLK2, 3, 4, 14 and 15 expression were all measured in time course and dose response experiments with androgens, AR-antagonist treatments, hormone deprivation experiments and cells transfected with AR siRNA. Collectively, these experiments demonstrated that prostatic kallikreins are specifically and directly regulated by the AR. The data also revealed that kallikrein genes are differentially regulated by androgens; KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 were modestly up-regulated, and KLK14 was repressed. Notably, KLK14 is located at the telomeric end of the kallikrein locus, far away from the centromeric cluster of kallikreins that are stimulated by androgens. These results show that the expression of KLK2, 3, 4, 14 and 15 is maintained in prostate cancer, but that these genes exhibit different responses to androgens. This makes the kallikrein locus an ideal model to investigate AR signalling. The increasingly dedifferentiated phenotype of aggressive prostate cancer cells is accompanied by the re-expression of signalling molecules that are usually expressed during embryogenesis and foetal tissue development. The Wnt pathway is one developmental cascade that is reactivated in prostate cancer. The canonical Wnt cascade regulates the intracellular levels of β-catenin, a potent transcriptional co-activator of T-cell factor (TCF) transcription factors. Notably, β-catenin can also bind to the AR and synergistically stimulate androgen-mediated gene expression. This is at the expense of typical Wnt/TCF target genes, because the AR:β-catenin and TCF:β-catenin interactions are mutually exclusive. The effect of β-catenin on kallikrein expression was examined to further investigate the role of β-catenin in prostate cancer. Stable knockdown of β-catenin in LNCaP prostate cancer cells attenuated the androgen-regulated expression of KLK2, 3, 4 and 15, but not KLK14. To test whether KLK14 is instead a TCF:β-catenin target gene, the endogenous levels of β-catenin were increased by inhibiting its degradation. Although KLK14 expression was up-regulated by these treatments, siRNA knockdown of β-catenin demonstrated that this effect was independent of β-catenin. These results show that β-catenin is required for maximal expression of KLK2, 3, 4 and 15, but not KLK14. Developmental cells and tumour cells express a similar repertoire of signalling molecules, which means that these different cell types are responsive to one another. Previous reports have shown that stem cells and foetal tissues can reprogram aggressive cancer cells to less aggressive phenotypes by restoring the balance to developmental signalling pathways that are highly dysregulated in cancer. To investigate this phenomenon in prostate cancer, DU145 and PC-3 prostate cancer cells were cultured on matrices pre-conditioned with human embryonic stem cells (hESCs). Soft agar assays showed that prostate cancer cells exposed to hESC conditioned matrices had reduced clonogenicity compared with cells harvested from control matrices. A recent study demonstrated that this effect was partially due to hESC-derived Lefty, an antagonist of Nodal. A member of the transforming growth factor β (TGFβ) superfamily, Nodal regulates embryogenesis and is re-expressed in cancer. The role of Nodal in prostate cancer has not previously been reported. Therefore, the expression and function of the Nodal signalling pathway in prostate cancer was investigated. Western blots confirmed that Nodal is expressed in DU145 and PC-3 cells. Immunohistochemistry revealed greater expression of Nodal in malignant versus benign glands. Notably, the Nodal inhibitor, Lefty, was not expressed at the mRNA level in any prostate cell lines tested. The Nodal signalling pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments triggered downstream phosphorylation of Smad2 in DU145 and LNCaP cells, and stably-transfected Nodal increased the clonogencity of LNCaP cells. Nodal was also found to modulate AR signalling. Nodal reduced the activity of an androgen-regulated KLK3 promoter construct in luciferase assays and attenuated the endogenous expression of AR target genes including prostatic kallikreins. These results demonstrate that Nodal is a novel example of a developmental signalling molecule that is reexpressed in prostate cancer and may have a functional role in prostate cancer progression. In summary, this project clarifies the role of androgens and changing cellular differentiation in prostate cancer by characterising the expression and function of the downstream genes encoding kallikrein-related serine proteases and Nodal. Furthermore, this study emphasises the similarities between prostate cancer and early development, and the crosstalk between developmental signalling pathways and the AR axis. The outcomes of this project also affirm the utility of the kallikrein locus as a model system to monitor tumour progression and the phenotype of prostate cancer cells.

Expert elicitation and its interface with technology : a review with a view to designing Elicitator

Expert knowledge is valuable in many modelling endeavours, particularly where data is not extensive or sufficiently robust. In Bayesian statistics, expert opinion may be formulated as informative priors, to provide an honest reflection of the current state of knowledge, before updating this with new information. Technology is increasingly being exploited to help support the process of eliciting such information. This paper reviews the benefits that have been gained from utilizing technology in this way. These benefits can be structured within a six-step elicitation design framework proposed recently (Low Choy et al., 2009). We assume that the purpose of elicitation is to formulate a Bayesian statistical prior, either to provide a standalone expert-defined model, or for updating new data within a Bayesian analysis. We also assume that the model has been pre-specified before selecting the software. In this case, technology has the most to offer to: targeting what experts know (E2), eliciting and encoding expert opinions (E4), whilst enhancing accuracy (E5), and providing an effective and efficient protocol (E6). Benefits include: -providing an environment with familiar nuances (to make the expert comfortable) where experts can explore their knowledge from various perspectives (E2); -automating tedious or repetitive tasks, thereby minimizing calculation errors, as well as encouraging interaction between elicitors and experts (E5); -cognitive gains by educating users, enabling instant feedback (E2, E4-E5), and providing alternative methods of communicating assessments and feedback information, since experts think and learn differently; and -ensuring a repeatable and transparent protocol is used (E6).

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Androgen-regulated genes differentially modulated by the androgen receptor coactivator L-dopa decarboxylase in human prostate cancer cells

Background The androgen receptor is a ligand-induced transcriptional factor, which plays an important role in normal development of the prostate as well as in the progression of prostate cancer to a hormone refractory state. We previously reported the identification of a novel AR coactivator protein, L-dopa decarboxylase (DDC), which can act at the cytoplasmic level to enhance AR activity. We have also shown that DDC is a neuroendocrine (NE) marker of prostate cancer and that its expression is increased after hormone-ablation therapy and progression to androgen independence. In the present study, we generated tetracycline-inducible LNCaP-DDC prostate cancer stable cells to identify DDC downstream target genes by oligonucleotide microarray analysis. Results Comparison of induced DDC overexpressing cells versus non-induced control cell lines revealed a number of changes in the expression of androgen-regulated transcripts encoding proteins with a variety of molecular functions, including signal transduction, binding and catalytic activities. There were a total of 35 differentially expressed genes, 25 up-regulated and 10 down-regulated, in the DDC overexpressing cell line. In particular, we found a well-known androgen induced gene, TMEPAI, which wasup-regulated in DDC overexpressing cells, supporting its known co-activation function. In addition, DDC also further augmented the transcriptional repression function of AR for a subset of androgen-repressed genes. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time RT-PCR. Conclusion Taken together, our results provide evidence for linking DDC action with AR signaling, which may be important for orchestrating molecular changes responsible for prostate cancer progression.

The inadequacy of embedded markup for cultural heritage texts

Embedded generalized markup, as applied by digital humanists to the recording and studying of our textual cultural heritage, suffers from a number of serious technical drawbacks. As a result of its evolution from early printer control languages, generalized markup can only express a document’s ‘logical’ structure via a repertoire of permissible printed format structures. In addition to the well-researched overlap problem, the embedding of markup codes into texts that never had them when written leads to a number of further difficulties: the inclusion of potentially obsolescent technical and subjective information into texts that are supposed to be archivable for the long term, the manual encoding of information that could be better computed automatically, and the obscuring of the text by highly complex technical data. Many of these problems can be alleviated by asserting a separation between the versions of which many cultural heritage texts are composed, and their content. In this way the complex inter-connections between versions can be handled automatically, leaving only simple markup for individual versions to be handled by the user.

A myosin from a higher plant has structural similarities to class V myosins

In plant cells, myosin is believed to be the molecular motor responsible for actin-based motility processes such as cytoplasmic streaming and directed vesicle transport. In an effort to characterize plant myosin, a cDNA encoding a myosin heavy chain was isolated from Arabidopsis thaliana. The predicted product of the MYA1 gene is 173 kDa and is structurally similar to the class V myosins. It is composed of the highly-conserved NH2-terminal "head" domain, a putative calmodulin-binding "neck" domain an alpha-helical coiled-coil domain, and a COOH-terminal domain. Northern blot analysis shows that the Arabidopsis MYA1 gene is expressed in all the major plant tissues (flower, leaf, root, and stem). We suggest that the MYA1 myosin may be involved in a general intracellular transport process in plant cells.