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.

A hydrogen tethered inhibitor of matrix metalloproteinases

During wound repair, the balance between matrix metalloproteinases (MMPs) and their natural inhibitors (the TIMPs) is crucial for the normal extra cellular matrix turnover. However, the over expression of several MMPs including MMP-1, 2, 3, 8, 9 and MMP-10, combined with abnormally high levels of activation or low expression of TIMPs, may contribute to excessive degradation of connective tissue and formation of chronic ulcers. There are many groups exploring strategies for promoting wound healing involving delivery of growth factors, cells, ECM components and small molecules. Our approach for improving the balance of MMPs is not to add anything more to the wound, but instead to neutralise the over-expressed MMPs using inhibitors tethered to a bandage-like hydrogel. Our in vitro experiments using designed synthetic pseudo peptide inhibitors have been demonstrated to inhibit MMP activity in standard solutions. These inhibitors have also been tethered to polyethylene glycol hydrogels using a facile reaction between the linker unit on the inhibitor and the gel. After tethering the inhibition of MMPs diminishes to some extent and we postulate that this arises due to poor diffusion of the MMPs into the gels. When the tethered inhibitors were tested against chronic wound fluid obtained against patients we observed over 40% inhibition in proteolytic activity suggesting our approach may prove useful in rebalancing MMPs within chronic wounds.

Outrage management in cases of sexual harassment as revealed in judicial decisions

Sexual harassment can be conceptualized as a series of interactions between harassers and targets that either inhibit or increase outrage by third parties. The outrage management model predicts the kinds of actions likely to be used by perpetrators to minimize outrage, predicts the consequences of failing to use these tactics—namely backfire, and recommends countertactics to increase outrage. Using this framework, our archival study examined outrage-management tactics reported as evidence in 23 judicial decisions of sexual harassment cases in Australia. The decisions contained precise, detailed information about the circumstances leading to the claim; the events which transpired in the courtroom, including direct quotations; and the judges' interpretations and findings. We found evidence that harassers minimize outrage by covering up the actions, devaluing the target, reinterpreting the events, using official channels to give an appearance of justice, and intimidating or bribing people involved. Targets can respond using countertactics of exposure, validation, reframing, mobilization of support, and resistance. Although there are limitations to using judicial decisions as a source of information, our study points to the value of studying tactics and the importance to harassers of minimizing outrage from their actions. The findings also highlight that, given the limitations of statutory and organizational protections in reducing the incidence and severity of sexual harassment in the community, individual responses may be effective as part of a multilevel response in reducing the incidence and impact of workplace sexual harassment as a gendered harm.

Examining psychosocial influences on speeding in Australian and Chinese contexts : a social learning approach

Speeding remains a significant contributing factor to road trauma internationally, despite increasingly sophisticated speed management strategies being adopted around the world. Increases in travel speed are associated with increases in crash risk and crash severity. As speed choice is a voluntary behaviour, driver perceptions are important to our understanding of speeding and, importantly, to designing effective behavioural countermeasures. The four studies conducted in this program of research represent a comprehensive approach to examining psychosocial influences on driving speeds in two countries that are at very different levels of road safety development: Australia and China. Akers’ social learning theory (SLT) was selected as the theoretical framework underpinning this research and guided the development of key research hypotheses. This theory was chosen because of its ability to encompass psychological, sociological, and criminological perspectives in understanding behaviour, each of which has relevance to speeding. A mixed-method design was used to explore the personal, social, and legal influences on speeding among car drivers in Queensland (Australia) and Beijing (China). Study 1 was a qualitative exploration, via focus group interviews, of speeding among 67 car drivers recruited from south east Queensland. Participants were assigned to groups based on their age and gender, and additionally, according to whether they self-identified as speeding excessively or rarely. This study aimed to elicit information about how drivers conceptualise speeding as well as the social and legal influences on driving speeds. The findings revealed a wide variety of reasons and circumstances that appear to be used as personal justifications for exceeding speed limits. Driver perceptions of speeding as personally and socially acceptable, as well as safe and necessary were common. Perceptions of an absence of danger associated with faster driving speeds were evident, particularly with respect to driving alone. An important distinction between the speed-based groups related to the attention given to the driving task. Rare speeders expressed strong beliefs about the need to be mindful of safety (self and others) while excessive speeders referred to the driving task as automatic, an absent-minded endeavour, and to speeding as a necessity in order to remain alert and reduce boredom. For many drivers in this study, compliance with speed limits was expressed as discretionary rather than mandatory. Social factors, such as peer and parental influence were widely discussed in Study 1 and perceptions of widespread community acceptance of speeding were noted. In some instances, the perception that ‘everybody speeds’ appeared to act as one rationale for the need to raise speed limits. Self-presentation, or wanting to project a positive image of self was noted, particularly with respect to concealing speeding infringements from others to protect one’s image as a trustworthy and safe driver. The influence of legal factors was also evident. Legal sanctions do not appear to influence all drivers to the same extent. For instance, fear of apprehension appeared to play a role in reducing speeding for many, although previous experiences of detection and legal sanctions seemed to have had limited influence on reducing speeding among some drivers. Disregard for sanctions (e.g., driving while suspended), fraudulent demerit point use, and other strategies to avoid detection and punishment were widely and openly discussed. In Study 2, 833 drivers were recruited from roadside service stations in metropolitan and regional locations in Queensland. A quantitative research strategy assessed the relative contribution of personal, social, and legal factors to recent and future self-reported speeding (i.e., frequency of speeding and intentions to speed in the future). Multivariate analyses examining a range of factors drawn from SLT revealed that factors including self-identity (i.e., identifying as someone who speeds), favourable definitions (attitudes) towards speeding, personal experiences of avoiding detection and punishment for speeding, and perceptions of family and friends as accepting of speeding were all significantly associated with greater self-reported speeding. Study 3 was an exploratory, qualitative investigation of psychosocial factors associated with speeding among 35 Chinese drivers who were recruited from the membership of a motoring organisation and a university in Beijing. Six focus groups were conducted to explore similar issues to those examined in Study 1. The findings of Study 3 revealed many similarities with respect to the themes that arose in Australia. For example, there were similarities regarding personal justifications for speeding, such as the perception that posted limits are unreasonably low, the belief that individual drivers are able to determine safe travel speeds according to personal comfort with driving fast, and the belief that drivers possess adequate skills to control a vehicle at high speed. Strategies to avoid detection and punishment were also noted, though they appeared more widespread in China and also appeared, in some cases, to involve the use of a third party, a topic that was not reported by Australian drivers. Additionally, higher perceived enforcement tolerance thresholds were discussed by Chinese participants. Overall, the findings indicated perceptions of a high degree of community acceptance of speeding and a perceived lack of risk associated with speeds that were well above posted speed limits. Study 4 extended the exploratory research phase in China with a quantitative investigation involving 299 car drivers recruited from car washes in Beijing. Results revealed a relatively inexperienced sample with less than 5 years driving experience, on average. One third of participants perceived that the certainty of penalties when apprehended was low and a similar proportion of Chinese participants reported having previously avoided legal penalties when apprehended for speeding. Approximately half of the sample reported that legal penalties for speeding were ‘minimally to not at all’ severe. Multivariate analyses revealed that past experiences of avoiding detection and punishment for speeding, as well as favourable attitudes towards speeding, and perceptions of strong community acceptance of speeding were most strongly associated with greater self-reported speeding in the Chinese sample. Overall, the results of this research make several important theoretical contributions to the road safety literature. Akers’ social learning theory was found to be robust across cultural contexts with respect to speeding; similar amounts of variance were explained in self-reported speeding in the quantitative studies conducted in Australia and China. Historically, SLT was devised as a theory of deviance and posits that deviance and conformity are learned in the same way, with the balance of influence stemming from the ways in which behaviour is rewarded and punished (Akers, 1998). This perspective suggests that those who speed and those who do not are influenced by the same mechanisms. The inclusion of drivers from both ends of the ‘speeding spectrum’ in Study 1 provided an opportunity to examine the wider utility of SLT across the full range of the behaviour. One may question the use of a theory of deviance to investigate speeding, a behaviour that could, arguably, be described as socially acceptable and prevalent. However, SLT seemed particularly relevant to investigating speeding because of its inclusion of association, imitation, and reinforcement variables which reflect the breadth of factors already found to be potentially influential on driving speeds. In addition, driving is a learned behaviour requiring observation, guidance, and practice. Thus, the reinforcement and imitation concepts are particularly relevant to this behaviour. Finally, current speed management practices are largely enforcement-based and rely on the principles of behavioural reinforcement captured within the reinforcement component of SLT. Thus, the application of SLT to a behaviour such as speeding offers promise in advancing our understanding of the factors that influence speeding, as well as extending our knowledge of the application of SLT. Moreover, SLT could act as a valuable theoretical framework with which to examine other illegal driving behaviours that may not necessarily be seen as deviant by the community (e.g., mobile phone use while driving). This research also made unique contributions to advancing our understanding of the key components and the overall structure of Akers’ social learning theory. The broader SLT literature is lacking in terms of a thorough structural understanding of the component parts of the theory. For instance, debate exists regarding the relevance of, and necessity for including broader social influences in the model as captured by differential association. In the current research, two alternative SLT models were specified and tested in order to better understand the nature and extent of the influence of differential association on behaviour. Importantly, the results indicated that differential association was able to make a unique contribution to explaining self-reported speeding, thereby negating the call to exclude it from the model. The results also demonstrated that imitation was a discrete theoretical concept that should also be retained in the model. The results suggest a need to further explore and specify mechanisms of social influence in the SLT model. In addition, a novel approach was used to operationalise SLT variables by including concepts drawn from contemporary social psychological and deterrence-based research to enhance and extend the way that SLT variables have traditionally been examined. Differential reinforcement was conceptualised according to behavioural reinforcement principles (i.e., positive and negative reinforcement and punishment) and incorporated concepts of affective beliefs, anticipated regret, and deterrence-related concepts. Although implicit in descriptions of SLT, little research has, to date, made use of the broad range of reinforcement principles to understand the factors that encourage or inhibit behaviour. This approach has particular significance to road user behaviours in general because of the deterrence-based nature of many road safety countermeasures. The concept of self-identity was also included in the model and was found to be consistent with the definitions component of SLT. A final theoretical contribution was the specification and testing of a full measurement model prior to model testing using structural equation modelling. This process is recommended in order to reduce measurement error by providing an examination of the psychometric properties of the data prior to full model testing. Despite calls for such work for a number of decades, the current work appears to be the only example of a full measurement model of SLT. There were also a number of important practical implications that emerged from this program of research. Firstly, perceptions regarding speed enforcement tolerance thresholds were highlighted as a salient influence on driving speeds in both countries. The issue of enforcement tolerance levels generated considerable discussion among drivers in both countries, with Australian drivers reporting lower perceived tolerance levels than Chinese drivers. It was clear that many drivers used the concept of an enforcement tolerance in determining their driving speed, primarily with the desire to drive faster than the posted speed limit, yet remaining within a speed range that would preclude apprehension by police. The quantitative results from Studies 2 and 4 added support to these qualitative findings. Together, the findings supported previous research and suggested that a travel speed may not be seen as illegal until that speed reaches a level over the prescribed enforcement tolerance threshold. In other words, the enforcement tolerance appears to act as a ‘de facto’ speed limit, replacing the posted limit in the minds of some drivers. The findings from the two studies conducted in China (Studies 2 and 4) further highlighted the link between perceived enforcement tolerances and a ‘de facto’ speed limit. Drivers openly discussed driving at speeds that were well above posted speed limits and some participants noted their preference for driving at speeds close to ‘50% above’ the posted limit. This preference appeared to be shaped by the perception that the same penalty would be imposed if apprehended, irrespective of what speed they travelling (at least up to 50% above the limit). Further research is required to determine whether the perceptions of Chinese drivers are mainly influenced by the Law of the People’s Republic of China or by operational practices. Together, the findings from both studies in China indicate that there may be scope to refine enforcement tolerance levels, as has happened in other jurisdictions internationally over time, in order to reduce speeding. Any attempts to do so would likely be assisted by the provision of information about the legitimacy and purpose of speed limits as well as risk factors associated with speeding because these issues were raised by Chinese participants in the qualitative research phase. Another important practical implication of this research for speed management in China is the way in which penalties are determined. Chinese drivers described perceptions of unfairness and a lack of transparency in the enforcement system because they were unsure of the penalty that they would receive if apprehended. Steps to enhance the perceived certainty and consistency of the system to promote a more equitable approach to detection and punishment would appear to be welcomed by the general driving public and would be more consistent with the intended theoretical (deterrence) basis that underpins the current speed enforcement approach. The use of mandatory, fixed penalties may assist in this regard. In many countries, speeding attracts penalties that are dependent on the severity of the offence. In China, there may be safety benefits gained from the introduction of a similar graduated scale of speeding penalties and fixed penalties might also help to address the issue of uncertainty about penalties and related perceptions of unfairness. Such advancements would be in keeping with the principles of best practice for speed management as identified by the World Health Organisation. Another practical implication relating to legal penalties, and applicable to both cultural contexts, relates to the issues of detection and punishment avoidance. These two concepts appeared to strongly influence speeding in the current samples. In Australia, detection avoidance strategies reported by participants generally involved activities that are not illegal (e.g., site learning and remaining watchful for police vehicles). The results from China were similar, although a greater range of strategies were reported. The most common strategy reported in both countries for avoiding detection when speeding was site learning, or familiarisation with speed camera locations. However, a range of illegal practices were also described by Chinese drivers (e.g., tampering with or removing vehicle registration plates so as to render the vehicle unidentifiable on camera and use of in-vehicle radar detectors). With regard to avoiding punishment when apprehended, a range of strategies were reported by drivers from both countries, although a greater range of strategies were reported by Chinese drivers. As the results of the current research indicated that detection avoidance was strongly associated with greater self-reported speeding in both samples, efforts to reduce avoidance opportunities are strongly recommended. The practice of randomly scheduling speed camera locations, as is current practice in Queensland, offers one way to minimise site learning. The findings of this research indicated that this practice should continue. However, they also indicated that additional strategies are needed to reduce opportunities to evade detection. The use of point-to-point speed detection (also known as sectio

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.

In vivo knockdown of the androgen receptor results in growth inhibition and regression of well-established, castration-resistant prostate tumours

Purpose: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors. Experimental Design: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors. Results:In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm3. In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA. Conclusions: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.

The hedgehog pathway inhibitor GDC-0449 shows potential in skin and other cancers

Background: The hedgehog signaling pathway is vital in early development, but then becomes dormant, except in some cancer tumours. Hedgehog inhibitors are being developed for potential use in cancer. Objectives/Methods: The objective of this evaluation is to review the initial clinical studies of the hedgehog inhibitor, GDC-0449, in subjects with cancer. Results: Phase I trials have shown that GDC-0449 has benefits in subjects with metastatic or locally advanced basal-cell carcinoma and in one subjects with medulloblastoma. GDC-0449 was well tolerated. Conclusions: Long term efficacy and safety studies of GDC-0449 in these conditions and other solid cancers are now underway. These clinical trials with GDC-0449, and trials with other hedgehog inhibitors, will reveal whether it is beneficial and safe to inhibit the hedgehog pathway, in a wide range of solid tumours or not.

Multi-criteria assessment of innovative technology in the beef industry

In Australia rural research and development corporations and companies expended over $AUS500 million on agricultural research and development. A substantial proportion of this is invested in R&D in the beef industry. The Australian beef industry exports almost $AUS5billionof product annually and invest heavily in new product development to improve the beef quality and improve production efficiency. Review points are critical for effective new product development, yet many research and development bodies, particularly publicly funded ones, appear to ignore the importance of assessing products prior to their release. Significant sums of money are invested in developing technological innovations that have low levels and rates of adoption. The adoption rates could be improved if the developers were more focused on technology uptake and less focused on proving their technologies can be applied in practice. Several approaches have been put forward in an effort to improve rates of adoption into operational settings. This paper presents a study of key technological innovations in the Australian beef industry to assess the use of multiple criteria in evaluating the potential uptake of new technologies. Findings indicate that using multiple criteria to evaluate innovations before commercializing a technology enables researchers to better understand the issues that may inhibit adoption.

The development of Lactococcus lactis as an antimicrobial agent

Non-pathogenic lactic acid bacteria are economically important Gram-positive bacteria used extensively in the food industry. Due to their “generally regarded as safe” status, certain species from the genera Lactobacillus and Lactococcus are also considered desirable as candidates for the production and secretion of recombinant proteins, particular those with therapeutic applications. The hypothesis examined by this thesis is that Lactococcus lactis can be modified to be an effective antimicrobial agent. Therefore, the aims of this thesis were to investigate the optimisation of the expression, secretion and/or activities of potential heterologous antimicrobial proteins by the model lactic acid bacterium, Lactococcus lactis subsp. cremoris MG1363. L. lactis strains were engineered to express and secrete the recombinant CyuC surface protein from Lactobacillus reuteri BR11, and a fusion protein consisting of CyuC and lysostaphin using the Sep promoter and secretion signal. CyuC has been characterised as a cystine-binding protein, but has also been demonstrated to have fibronectin binding activity. Lysostaphin is a bacteriolytic enzyme with specific activity against the Gram-positive pathogen, Staphylococcus aureus. These modified L. lactis strains were then investigated to see if they had the ability to inhibit the adhesion of S. aureus to host extracellular matrix (ECM) proteins. It was observed that the cell extracts of the L. lactis strain with the vector only (pGhost9:ISS1) was able to inhibit the adhesion of S. aureus to fibronectin, whilst the cell extracts of the L. lactis strain expressing lysostaphin was able to inhibit adhesion to keratin. Finally, this thesis has identified specific lactococcal genes that affect the secretion of lysostaphin through the use of random transposon mutagenesis. Ten mutants with higher lysostaphin activity contained insertions in four different genes encoding: (i) an uncharacterised putative transmembrane protein (llmg_0609), (ii) an enzyme catalysing the first step in peptidoglycan biosynthesis (murA2), (iii) a homolog of the oxidative defence regulator (trmA), and (iv) an uncharacterised putative enzyme involved in ubiquinone biosynthesis (llmg_2148). The higher lysostaphin activity observed in these mutants was found to be due to higher amounts of lysostaphin being secreted. The findings of this thesis contribute to the development of this organism as an antimicrobial agent and also to our understanding of L. lactis genetics.

Enhancing pathways for ACE learners : Building the capacity of Adult and Community Education (ACE) providers to offer non-accredited courses and VET modules with pathways into VET programs.

This report presents findings from a project that considered a) the current capacity of Adult and Community Education (ACE) providers to offer non-accredited courses and single modules of accredited learning that provide pathways into full scale accredited VET programs, and b) the factors that aid and inhibit this from occurring. Based on the findings, suggestions are made as to what needs to be done to extend this capacity and thereby to achieve the goals outlined in the 2008 Ministerial Declaration on Adult Community Education.

Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

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

Introduction Polybrominated diphenyl ethers (PBDEs) are considered to be a cost effective and efficient way to reduce the possibility of product ignition and inhibit the spread of fire, thereby limiting harm caused by fires. PBDEs are incorporated into a wide variety of manufactured products and are now considered an ubiquitous contaminant found worldwide in biological and environmental samples1 . In comparison to “traditional” persistent organic pollutants (POPs), the exposure modes of PBDEs in humans are less well defined, although dietary sources, inhalation (air/particulate matter) and dust ingestion have been reported 2-4. Limited investigations of population specific factors such as age or gender and PBDE concentrations report: no conclusive correlation by age in adults; higher concentrations in children ; similar concentrations in maternal and cord blood; and no gender differences. After preliminary findings of higher PBDE concentrations in children than in adults in Australia11 we sought to investigate at what age the PBDE concentrations peaked in an effort to focus exposure studies. This investigation involved the collection of blood samples from young age groups and the development of a simple model to predict PBDE concentrations by age in Australia.

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

Pressure, oxygen tension and temperature in the periosteal callus during bone healing - An in vivo study in sheep

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

Inhibition of integrative cartilage repair by proteoglycan 4 in synovial fluid

To determine the effects of the articular cartilage surface, as well as synovial fluid (SF) and its components, specifically proteoglycan 4 (PRG4) and hyaluronic acid (HA), on integrative cartilage repair in vitro. Methods. Blocks of calf articular cartilage were harvested, some with the articular surface intact and others without. Some of the latter types of blocks were pretreated with trypsin, and then with bovine serum albumin, SF, PRG4, or HA. Immunolocalization of PRG4 on cartilage surfaces was performed after treatment. Pairs of similarly treated cartilage blocks were incubated in partial apposition for 2 weeks in medium supplemented with serum and 3 H-proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength, and protein biosynthesis and deposition were determined by incorporated 3 H-proline. Results. Samples with articular surfaces in apposition exhibited little integrative repair compared with samples with cut surfaces in apposition. PRG4 was immunolocalized at the articular cartilage surface, but not in deeper, cut surfaces (without treatment). Cartilage samples treated with trypsin and then with SF or PRG4 exhibited an inhibition of integrative repair and positive immunostaining for PRG4 at treated surfaces compared with normal cut cartilage samples, while samples treated with HA exhibited neither inhibited integrative repair nor PRG4 at the tissue surfaces. Deposition of newly synthesized protein was relatively similar under conditions in which integration differed significantly. Conclusion. These results support the concept that PRG4 in SF, which normally contributes to cartilage lubrication, can inhibit integrative cartilage repair. This has the desirable effect of preventing fusion of apposing surfaces of articulating cartilage, but has the undesirable effect of inhibiting integrative repair.