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.

Men's intimate partner abuse and control : reconciling paradoxical masculinities and social contradictions

Intimate partner abuse and control is one of the most common forms of violence against women, and is considered an international problem of social, political, legal and human rights significance. Yet few studies have attempted to understand this problem from the perspective of male perpetrators. This gap is addressed by conducting in-depth interviews with 16 able-bodied men of white European ancestry born and educated in New Zealand or Australia, who have been physically violent and/or emotionally, intellectually, sexually or financially controlling of a live-in female partner. This thesis extends and deepens the dominant ways of thinking about men’s intimate partner abuse by utilising a new theoretical framework compatible with contemporary feminist scholarship. A synthesis of Connell’s theory of masculinities and Bourdieu’s field theory is utilised for the purpose of exploring more nuanced, complex understandings of manliness and men’s relationships with men, women and social structures. Through such an analysis, this thesis finds that men’s perpetration of power and control over women is driven by a need to avoid the stigma of appearing weak. As a consequence, their desire and ability to show love, care and empathy is suppressed in favour of a presumed honourable manliness, and their female partners are used as weapons in the pursuit of symbolic capital in the form of recognition, prestige and acceptance from real and/or imagined men. This research also uncovers the complex interplay between masculine practices and particular social contexts. For example, the norms of practice encountered from those in authority, such as teachers, sports coaches, police, court judges and workplace management, influences the decision making of the men in this study, to use, or not to use, physical violence, psychological abuse and structural control. The principal conclusion is that there is a repertoire of paradoxical masculinities and contradictory social messages available to the men in this study. But gender policing by other men, complicit women and those in authority provides little room for legitimate complexity in masculine practices. Perpetrators in this study reconcile these conflicts of interest by generally avoiding subordinated masculinity and possible ostracism, and instead practicing more heroic hegemonic masculinities by abusing and controlling women and particular other men. This thesis concludes that for intimate partner abuse and control to cease, changes in power structures have to occur at all levels of society.

Integrated Bayesian network frameworks for modelling complex ecological issues

Ecological problems are typically multi faceted and need to be addressed from a scientific and a management perspective. There is a wealth of modelling and simulation software available, each designed to address a particular aspect of the issue of concern. Choosing the appropriate tool, making sense of the disparate outputs, and taking decisions when little or no empirical data is available, are everyday challenges facing the ecologist and environmental manager. Bayesian Networks provide a statistical modelling framework that enables analysis and integration of information in its own right as well as integration of a variety of models addressing different aspects of a common overall problem. There has been increased interest in the use of BNs to model environmental systems and issues of concern. However, the development of more sophisticated BNs, utilising dynamic and object oriented (OO) features, is still at the frontier of ecological research. Such features are particularly appealing in an ecological context, since the underlying facts are often spatial and temporal in nature. This thesis focuses on an integrated BN approach which facilitates OO modelling. Our research devises a new heuristic method, the Iterative Bayesian Network Development Cycle (IBNDC), for the development of BN models within a multi-field and multi-expert context. Expert elicitation is a popular method used to quantify BNs when data is sparse, but expert knowledge is abundant. The resulting BNs need to be substantiated and validated taking this uncertainty into account. Our research demonstrates the application of the IBNDC approach to support these aspects of BN modelling. The complex nature of environmental issues makes them ideal case studies for the proposed integrated approach to modelling. Moreover, they lend themselves to a series of integrated sub-networks describing different scientific components, combining scientific and management perspectives, or pooling similar contributions developed in different locations by different research groups. In southern Africa the two largest free-ranging cheetah (Acinonyx jubatus) populations are in Namibia and Botswana, where the majority of cheetahs are located outside protected areas. Consequently, cheetah conservation in these two countries is focussed primarily on the free-ranging populations as well as the mitigation of conflict between humans and cheetahs. In contrast, in neighbouring South Africa, the majority of cheetahs are found in fenced reserves. Nonetheless, conflict between humans and cheetahs remains an issue here. Conservation effort in South Africa is also focussed on managing the geographically isolated cheetah populations as one large meta-population. Relocation is one option among a suite of tools used to resolve human-cheetah conflict in southern Africa. Successfully relocating captured problem cheetahs, and maintaining a viable free-ranging cheetah population, are two environmental issues in cheetah conservation forming the first case study in this thesis. The second case study involves the initiation of blooms of Lyngbya majuscula, a blue-green algae, in Deception Bay, Australia. L. majuscula is a toxic algal bloom which has severe health, ecological and economic impacts on the community located in the vicinity of this algal bloom. Deception Bay is an important tourist destination with its proximity to Brisbane, Australia’s third largest city. Lyngbya is one of several algae considered to be a Harmful Algal Bloom (HAB). This group of algae includes other widespread blooms such as red tides. The occurrence of Lyngbya blooms is not a local phenomenon, but blooms of this toxic weed occur in coastal waters worldwide. With the increase in frequency and extent of these HAB blooms, it is important to gain a better understanding of the underlying factors contributing to the initiation and sustenance of these blooms. This knowledge will contribute to better management practices and the identification of those management actions which could prevent or diminish the severity of these blooms.

Preventing sexual violence?

This article critically assesses the main social policy responses to preventing rape following much feminist struggle to make sexual violence a public matter of legitimate concern. It considers the preventative potential of legal measures, anti-violence campaigns waged by feminist and men's groups in the US and Australia, public education campaigns in Schools and Universities, and public awareness campaigns sponsored by the state.We argue that sexual violence is not amenable to quick fix strategies that place responsibility for prevention entirely on individual men or women. While we recognise that responsibilising victims and individualising offenders is consistent with wider global shifts in social policy calling upon individuals to manage their own risk, we argue that the increasing reliance on such neo-liberal social policy is especially problematic in preventing rape. The paper suggests ways to resist this which place greater emphasis on the promotion of sexual ethics; the eroticisation of consent; the reinvention of the norms of romance to include both these, and the complete separation of the psycho-social-symbolic connections between sex and violence, and ultimately the re-evaluation of the cultural expectations of masculinity and femininity.

Sex trafficking : the global market in women and children

This book is one of a series on contemporary social issues. It provides a painstakingly researched analysis of the contemporary phenomenon of sex trafficking. As the author Kathryn Farr points out, the phenomenon is not all that contemporary, as women and children have historically been trafficked and enslaved for the purposes of prostitution, particularly during war: in World War II on the southern islands of Okinawa, the Philippines, Hawaii, Liberia, Japan, the Korean war, the Vietnam war, and more recently in Bosnia and Rwanda. Farr links the phenomenon to military socialization, especially to its patriarchal culture which celebrates hyper-masculinity, eroticizes violence, desensitizes soldiers to suffering and brutality and treats women as sex objects.

Gender

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

Effects of lead exposure on hippocampal metabotropic glutamate receptor subtype 3 and 7 in developmental rats

Background A complete explanation of the mechanisms by which Pb2+ exerts toxic effects on developmental central nervous system remains unknown. Glutamate is critical to the developing brain through various subtypes of ionotropic or metabotropic glutamate receptors (mGluRs). Ionotropic N-methyl-D-aspartate receptors have been considered as a principal target in lead-induced neurotoxicity. The relationship between mGluR3/mGluR7 and synaptic plasticity had been verified by many recent studies. The present study aimed to examine the role of mGluR3/mGluR7 in lead-induced neurotoxicity. Methods Twenty-four adult and female rats were randomly selected and placed on control or 0.2% lead acetate during gestation and lactation. Blood lead and hippocampal lead levels of pups were analyzed at weaning to evaluate the actual lead content at the end of the exposure. Impairments of short -term memory and long-term memory of pups were assessed by tests using Morris water maze and by detection of hippocampal ultrastructural alterations on electron microscopy. The impact of lead exposure on mGluR3 and mGluR7 mRNA expression in hippocampal tissue of pups were investigated by quantitative real-time polymerase chain reaction and its potential role in lead neurotoxicity were discussed. Results Lead levels of blood and hippocampi in the lead-exposed rats were significantly higher than those in the controls (P < 0.001). In tests using Morris Water Maze, the overall decrease in goal latency and swimming distance was taken to indicate that controls had shorter latencies and distance than lead-exposed rats (P = 0.001 and P < 0.001 by repeated-measures analysis of variance). On transmission electron microscopy neuronal ultrastructural alterations were observed and the results of real-time polymerase chain reaction showed that exposure to 0.2% lead acetate did not substantially change gene expression of mGluR3 and mGluR7 mRNA compared with controls. Conclusion Exposure to lead before and after birth can damage short-term and long-term memory ability of young rats and hippocampal ultrastructure. However, the current study does not provide evidence that the expression of rat hippocampal mGluR3 and mGluR7 can be altered by systemic administration of lead during gestation and lactation, which are informative for the field of lead-induced developmental neurotoxicity noting that it seems not to be worthwhile to include mGluR3 and mGluR7 in future studies. Background

The politics of literacy in David Almond and Dave McKean’s "The Savage" (2008)

Competency in language and literacy are central to contemporary debates about education in Anglophone nations around the world. This paper suggests that such debates are informing not just educational policy but children’s literature itself as can be seen in Almond and McKean’s The Savage. This hybrid text combines prose and graphic narrative and narration in order to tell the story of Blue, a young British boy negotiating his identity in the aftermath of his father's death. While foregrounding a narrative of ideal masculinity, The Savage enacts and privileges a formal and thematic ideal of literacy as index of individual agency and development. Almond and McKean produce a politicised understanding of language and literacy that simultaneously positions The Savage in a textual tradition of socio-culturally disenfranchised youth, and intervenes in that tradition to (perhaps ironically) affirm the very conditions previously critiqued by that very tradition. Where earlier authors such as Barry Hines sought to challenge normative accounts of language and literacy in order to indict educational policy and praxes, Almond and McKean work to naturalise the very logics of education and agency by which their protagonist has been disenfranchised. In doing so, The Savage exemplifies current approaches to education which claim to value social and cultural diversity while imposing national standardised testing predicated on assumptions about the legitimacy of uniform standards and definitions of literacy.

How to be masculine in the block area

This article focuses on the social interactions of several boys aged 3-5 years in the block area of a preschool classroom in a childcare setting. Using transcripts of video segments showing these boys engaged in daily play and interactions, the article analyses two episodes that occurred in the first weeks of the school year. At first glance, both episodes appear chaotic, with little appearance of order among the players. A closer analysis reveals a finely organized play taking place, with older boys teaching important lessons to the newcomers about how to be masculine in the block area. These episodes illustrate that masculinity is not a fixed character trait, but is determined through practice and participation in the activities of masculinity. Play and conflict are the avenues through which this occurs.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

Re-thinking home economics : from modern to postmodern accounts of pedagogical bodies

Two perceptions of the marginality of home economics are widespread across educational and other contexts. One is that home economics and those who engage in its pedagogy are inevitably marginalised within patriarchal relations in education and culture. This is because home economics is characterised as women's knowledge, for the private domain of the home. The other perception is that only orthodox epistemological frameworks of inquiry should be used to interrogate this state of affairs. These perceptions have prompted leading theorists in the field to call for non-essentialist approaches to research in order to re-think the thinking that has produced this cul-de-sac positioning of home economics as a body of knowledge and a site of teacher practice. This thesis takes up the challenge of working to locate a space outside the frame of modernist research theory and methods, recognising that this shift in epistemology is necessary to unsettle the idea that home economics is inevitably marginalised. The purpose of the study is to reconfigure how we have come to think about home economics teachers and the profession of home economics as a site of cultural practice, in order to think it otherwise (Lather, 1991). This is done by exploring how the culture of home economics is being contested from within. To do so, the thesis uses a 'posthumanist' approach, which rejects the conception of the individual as a unitary and fixed entity, but instead as a subject in process, shaped by desires and language which are not necessarily consciously determined. This posthumanist project focuses attention on pedagogical body subjects as the 'unsaid' of home economics research. It works to transcend the modernist dualism of mind/body, and other binaries central to modernist work, including private/public, male/female,paid/unpaid, and valued/unvalued. In so doing, it refuses the simple margin/centre geometry so characteristic of current perceptions of home economics itself. Three studies make up this work. Studies one and two serve to document the disciplined body of home economics knowledge, the governance of which works towards normalisation of the 'proper' home economics teacher. The analysis of these accounts of home economics teachers by home economics teachers, reveals that home economics teachers are 'skilled' yet they 'suffer' for their profession. Further,home economics knowledge is seen to be complicit in reinforcing the traditional roles of masculinity and femininity, thereby reinforcing heterosexual normativity which is central to patriarchal society. The third study looks to four 'atypical'subjects who defy the category of 'proper' and 'normal' home economics teacher. These 'atypical' bodies are 'skilled' but fiercely reject the label of 'suffering'. The discussion of the studies is a feminist poststructural account, using Russo's (1994) notion of the grotesque body, which is emergent from Bakhtin's (1968) theory of the carnivalesque. It draws on the 'shreds' of home economics pedagogy,scrutinising them for their subversive, transformative potential. In this analysis, the giving and taking of pleasure and fun in the home economics classroom presents moments of surprise and of carnival. Foucault's notion of the construction of the ethical individual shows these 'atypical' bodies to be 'immoderate' yet striving hard to be 'continent' body subjects. This research captures moments of transgression which suggest that transformative moments are already embodied in the pedagogical practices of home economics teachers, and these can be 'seen' when re-looking through postmodemist lenses. Hence, the cultural practices ofhome economics as inevitably marginalised are being contested from within. Until now, home economics as a lived culture has failed to recognise possibilities for reconstructing its own field beyond the confines of modernity. This research is an example of how to think about home economics teachers and the profession as a reconfigured cultural practice. Future research about home economics as a body of knowledge and a site of teacher practice need not retell a simple story of oppression. Using postmodemist epistemologies is one way to provide opportunities for new ways of looking.

Development and evaluation of hydrocarbon sorbent materials with the aid of chemometrics

Hydrocarbon spills on roads are a major safety concern for the driving public and can have severe cost impacts both on pavement maintenance and to the economy through disruption to services. The time taken to clean-up spills and re-open roads in a safe driving condition is an issue of increasing concern given traffic levels on major urban arterials. Thus, the primary aim of the research was to develop a sorbent material that facilitates rapid clean-up of road spills. The methodology involved extensive research into a range of materials (organic, inorganic and synthetic sorbents), comprehensive testing in the laboratory, scale-up and field, and product design (i.e. concept to prototype). The study also applied chemometrics to provide consistent, comparative methods of sorbent evaluation and performance. In addition, sorbent materials at every stage were compared against a commercial benchmark. For the first time, the impact of diesel on asphalt pavement has been quantified and assessed in a systematic way. Contrary to conventional thinking and anecdotal observations, the study determined that the action of diesel on asphalt was quite rapid (i.e. hours rather than weeks or months). This significant finding demonstrates the need to minimise the impact of hydrocarbon spills and the potential application of the sorbent option. To better understand the adsorption phenomenon, surface characterisation techniques were applied to selected sorbent materials (i.e. sand, organo-clay and cotton fibre). Brunauer Emmett Teller (BET) and thermal analysis indicated that the main adsorption mechanism for the sorbents occurred on the external surface of the material in the diffusion region (sand and organo-clay) and/or capillaries (cotton fibre). Using environmental scanning electron microscopy (ESEM), it was observed that adsorption by the interfibre capillaries contributed to the high uptake of hydrocarbons by the cotton fibre. Understanding the adsorption mechanism for these sorbents provided some guidance and scientific basis for the selection of materials. The study determined that non-woven cotton mats were ideal sorbent materials for clean-up of hydrocarbon spills. The prototype sorbent was found to perform significantly better than the commercial benchmark, displaying the following key properties: • superior hydrocarbon pick-up from the road pavement; • high hydrocarbon retention capacity under an applied load; • adequate field skid resistance post treatment; • functional and easy to use in the field (e.g. routine handling, transportation, application and recovery); • relatively inexpensive to produce due to the use of raw cotton fibre and simple production process; • environmentally friendly (e.g. renewable materials, non-toxic to environment and operators, and biodegradable); and • rapid response time (e.g. two minutes total clean-up time compared with thirty minutes for reference sorbents). The major outcomes of the research project include: a) development of a specifically designed sorbent material suitable for cleaning up hydrocarbon spills on roads; b) submission of patent application (serial number AU2005905850) for the prototype product; and c) preparation of Commercialisation Strategy to advance the sorbent product to the next phase (i.e. R&D to product commercialisation).

Single nucleotide polymorphism (SNP) - genotyping of Community Acquired Methicillin-Resistant Staphylococcus aureus, including the subtyping of PVL toxin producers using Real-Time PCR

Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.