Investigating epidermogenesis in a human skin equivalent model

Skin is the largest, and arguably, the most important organ of the body. It is a complex and multi-dimensional tissue, thus making it essentially impossible to fully model in vitro in conventional 2-dimensional culture systems. In view of this, rodents or pigs are utilised to study wound healing therapeutics or to investigate the biological effects of treatments on skin. However, there are many differences between the wound healing processes in rodents compared to humans (contraction vs. re-epithelialisation) and there are also ethical issues associated with animal testing for scientific research. Therefore, the development of skin equivalent (HSE) models from surgical discard human skin has become an important area of research. The studies in this thesis compare, for the first time, native human skin and the epidermogenesis process in a HSE model. The HSE was reported to be a comparable model for human skin in terms of expression and localisation of key epidermal cell markers. This validated HSE model was utilised to study the potential wound healing therapeutic, hyperbaric oxygen (HBO) therapy. There is a significant body of evidence suggesting that lack of cutaneous oxygen results in and potentiates the chronic, non-healing wound environment. Although the evidence is anecdotal, HBO therapy has displayed positive effects on re-oxygenation of chronic wounds and the clinical outcomes suggest that HBO treatment may be beneficial. Therefore, the HSE was subjected to a daily clinical HBO regime and assessed in terms of keratinocyte migration, proliferation, differentiation and epidermal thickening. HBO treatment was observed to increase epidermal thickness, in particular stratum corneum thickening, but it did not alter the expression or localisation of standard epidermal cell markers. In order to elucidate the mechanistic changes occurring in response to HBO treatment in the HSE model, gene microarrays were performed, followed by qRT-PCR of select genes which were differentially regulated in response to HBO treatment. The biological diversity of the HSEs created from individual skin donors, however, overrode the differences in gene expression between treatment groups. Network analysis of functional changes in the HSE model revealed general trends consistent with normal skin growth and maturation. As a more robust and longer term study of these molecular changes, protein localisation and expression was investigated in sections from the HSEs undergoing epidermogenesis in response to HBO treatment. These proteins were CDCP1, Metallothionein, Kallikrein (KLK) 1 and KLK7 and early growth response 1. While the protein expression within the HSE models exposed to HBO treatment were not consistent in all HSEs derived from all skin donors, this is the first study to detect and compare both KLK1 and CDCP1 protein expression in both a HSE model and native human skin. Furthermore, this is the first study to provide such an in depth analysis of the effect of HBO treatment on a HSE model. The data presented in this thesis, demonstrates high levels of variation between individuals and their response to HBO treatment, consistent with the clinical variation that is currently observed.

In Plant Activation: An inducible, hyperexpression platform for recombinant protein production in plants

In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the b-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.

MMP-9 and the epidermal growth factor signal pathway in non-small cell lung cancer

Background Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type-IV collagen. Enhanced expression has been related to tumour progression in a number of systems. The control of MMP expression is complex, but recently epidermal growth actor receptor (EGFR) activity has been implicated in up-regulation of MMP-9 in tumour cells in vitro. Aims To evaluate interrelations between MMP-9 and EGFR expression in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of 152 patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Minimum follow-up was 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-9 and EGFR. Results: MMP-9 was expressed in the tumour cells of 79/152 (52%) cases. EGFR expression was found in 86/152 (57%) cases [membranous 51/152 (34%), cytoplasmic 35/152 (23%)]. MMP-9 expression was associated with poor outcome (p=0.04). Membranous, cytoplasmic and overall EGFR expression were not associated with outcome (p=0.29, p=0.85 and p=0.41 respectively). There was a strong correlation between MMP-9 expression and EGFR expression (p=0.001) and EGFR membranous expression (p=0.01) but not with cytoplasmic EGFR expression (p=0.28). Co-expression of MMP-9 and EGFR (36%) conferred a worse prognosis (p=0.003). Subset analysis revealed only MMP-9 and membranous EGFR co-expression (22%) was associated with poor outcome (p=0.008). Conclusions Our results show that MMP-9 and EGFR are co-expressed in NSCLC. This finding suggests the EGFR signalling pathway may play an important role in the invasive behaviour of NSCLC via specific upregulation of MMP-9. The co-expression of these markers also confers a poor prognosis.

Reduced Thrombospondin-1 at presentation predicts disease progression in superficial bladder cancer

Objectives: Superficial bladder cancer (SBC) presents a difficult clinical dilemma at diagnosis as only a small subgroup of patients will subsequently develop invasive disease. Study of cancer biology has found that angiogenesis is central to growth and spread. This study examines the relationship between the angiogenic inhibitory factor Thrombospondin-1 (TSP-1) at initial presentation and subsequent progression of SBC. Methods: Using immunohistochemistry, 220 cases of SBC were examined for pattern and extent of expression of TSP-1 at initial presentation. Results: TSP-1 was detected in perivascular tissue, at the epithelial-stromal junction, in the stroma and in tumour cells and reduced perivascular TSP-1 staining at presentation was an independent predictive factor for the subsequent development of muscle invasive or metastatic disease. Conclusion: This adds further weight to the theory that TSP-1 plays a major part in the biology of bladder cancer possibly through the control of angiogenesis. © 2002 Elsevier Science B.V. All rights reserved.

Media and information literacy : policy and strategy guidelines

In the evolving knowledge societies of today, some people are overloaded with information, others are starved for information. Everywhere, people are yearning to freely express themselves,to actively participate in governance processes and cultural exchanges. Universally, there is a deep thirst to understand the complex world around us. Media and Information Literacy (MIL) is a basis for enhancing access to information and knowledge, freedom of expression, and quality education. It describes skills, and attitudes that are needed to value the functions of media and other information providers, including those on the Internet, in societies and to find, evaluate and produce information and media content; in other words, it covers the competencies that are vital for people to be effectively engaged in all aspects of development.

Improved molecular tools for sugar cane biotechnology

Sugar cane is a major source of food and fuel worldwide. Biotechnology has the potential to improve economically-important traits in sugar cane as well as diversify sugar cane beyond traditional applications such as sucrose production. High levels of transgene expression are key to the success of improving crops through biotechnology. Here we describe new molecular tools that both expand and improve gene expression capabilities in sugar cane. We have identified promoters that can be used to drive high levels of gene expression in the leaf and stem of transgenic sugar cane. One of these promoters, derived from the Cestrum yellow leaf curling virus, drives levels of constitutive transgene expression that are significantly higher than those achieved by the historical benchmark maize polyubiquitin-1 (Zm-Ubi1) promoter. A second promoter, the maize phosphonenolpyruvate carboxylate promoter, was found to be a strong, leaf-preferred promoter that enables levels of expression comparable to Zm-Ubi1 in this organ. Transgene expression was increased approximately 50-fold by gene modification, which included optimising the codon usage of the coding sequence to better suit sugar cane. We also describe a novel dual transcriptional enhancer that increased gene expression from different promoters, boosting expression from Zm-Ubi1 over eightfold. These molecular tools will be extremely valuable for the improvement of sugar cane through biotechnology.

Reflective Practice in the Arts

In creative disciplines, reflective practice is an integral and cumulative form of learning. Reflective learning generates knowledge that is specific to oneself and is a form of evidence upon which to analyse and change one’s practice. Critical reflection requires a deep knowledge of the discipline and an awareness of one’s positioning within that discipline and in relation to one’s creative performance. Meaning making through performative expression allows for personal transformation through acute awareness of and reflection on one’s own beliefs, knowledges and values through the process of creating artistic work. Self-awareness and identity are significant both in the study of the arts and in becoming an artist, as aesthetic inquiry and performance are constituted by subjective self-expression in relation to objective conditions. Reflection can be expressed using symbols or semiotic systems other than language. Depending on the disciplinary context, particular modes or forms of expression will be privileged, including material forms of practice, still and moving images, music and sound, live action and digital code. This chapter explores the problematics of what counts as reflection in the arts and how reflection is represented, expressed and performed in discursive and non-discursive ways in becoming arts literate.

The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit

The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-β) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-β), and epsilon carotene hydroxylase (CRH-ε) showed some variation in gene expression. The LCY-β gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-β gene.

Design after design to bridge between people living with cognitive or sensory impairments, their friends and proxies

A carer or teacher often plays the role of proxy or spokesperson for a person living with an intellectual disability or form of cognitive or sensory impairment. Our research undertook co-design with people living with cognitive and sensory impairments and their proxies in order to explore new ways of facilitating communication. We developed simple functioning interactive prototypes to support people with a diverse range of competencies to communicate and explore their use. Deployment of the prototypes enabled use, appropriation and design after design by our two participant groups; adults living with cognitive or sensory impairments and children identified with language delays and autism spectrum disorder. The prototypes supported concrete expression of likes, dislikes, capabilities, emotional wants and needs and forms of expression that hitherto had not been fostered, further informing design. Carers and designers were surprised at the ways in which the technology was used and how it fostered new forms of social interaction and expression. We elaborate on how design after design can be an effective approach for engaging people living with intellectual disabilities, giving them greater capacity for expression and power in design and offering the potential to expand and deepen their social relationships.

FaceXpress : an integrated software suite for facial emotion stimulus manipulation and facial measurement

The characterisation of facial expression through landmark-based analysis methods such as FACEM (Pilowsky & Katsikitis, 1994) has a variety of uses in psychiatric and psychological research. In these systems, important structural relationships are extracted from images of facial expressions by the analysis of a pre-defined set of feature points. These relationship measures may then be used, for instance, to assess the degree of variability and similarity between different facial expressions of emotion. FaceXpress is a multimedia software suite that provides a generalised workbench for landmark-based facial emotion analysis and stimulus manipulation. It is a flexible tool that is designed to be specialised at runtime by the user. While FaceXpress has been used to implement the FACEM process, it can also be configured to support any other similar, arbitrary system for quantifying human facial emotion. FaceXpress also implements an integrated set of image processing tools and specialised tools for facial expression stimulus production including facial morphing routines and the generation of expression-representative line drawings from photographs.

Trailing Touch

Trailing Touch (2014) is a twenty minute classically-based dance work set to music by composers Carter Burwell and Hildur Gudnadottir. It is an abstract work that explores images based on imperfect patterns and seeks to transmit the sensations felt when clusters form and disperse through the space or crisscross to create swirling wave-like reactions in the dancers’ tulle skirts. These simple references are inspired by the lyrical use of arms found in ballet, particular to the ballet aesthetic. Trailing Touch was created in collaboration with QUT’s third-year BFA (Dance Performance) students and performed as part of Dance14 at QUT’s Gardens Points Theatre from the 4th to 8th November 2014 and was performed in Singapore as part of Contact Contemporary Dance Festival on 30th November, 2014. Additionally, the creative process of Trailing Touch (2014) forms the initial project of Phase III of my PhD research, Writing the Dance Score in the Twenty-first Century: An approach for the Independent Choreographer. This PhD research will examine the potential of dance scores as a suite of choreographic strategies to map key aspects of the choreographic process. While a certain degree of ambiguity drives the creative process, the suite of choreographic strategies attempt to capture what is transmitted through the lived experience of dance. “[T]hese documents harbor a force of expression, a visual energy related to the body and the movement” (Louppe 1994, 7) that triggers movement responses, unforeseen intensities and enables personal interpretation. Consequently, Phase III will test and evaluate the relevance of Phase II research within the pressures of mainstream dance rehearsal and performance contexts. In Project One Trailing Touch this was demonstrated in the dance scores produced by the choreographer and interpreted by the dancers within the performance. By drawing from both the theoretical and practical, it is anticipated that this research will suggest a form of languaging movement that is not reliant on images or numbers, but generated in response to the intuitive and complex process underpinning choreographic practice. Rather than constructing a codified dance notation system, it will focus on strategies that reveal movement, its spatial patterns, qualities and intensities of expression and the procedures underlying key choreographic concepts. The outcome of this research project aims to support the independent choreographer in two major areas, by facilitating and enriching the choreographic process for both the performers and choreographer, and by strengthening artistic development and performance outcomes.

Computational micromodel for epigenetic mechanisms

Characterization of the epigenetic profile of humans since the initial breakthrough on the human genome project has strongly established the key role of histone modifications and DNA methylation. These dynamic elements interact to determine the normal level of expression or methylation status of the constituent genes in the genome. Recently, considerable evidence has been put forward to demonstrate that environmental stress implicitly alters epigenetic patterns causing imbalance that can lead to cancer initiation. This chain of consequences has motivated attempts to computationally model the influence of histone modification and DNA methylation in gene expression and investigate their intrinsic interdependency. In this paper, we explore the relation between DNA methylation and transcription and characterize in detail the histone modifications for specific DNA methylation levels using a stochastic approach.

The Black Label: trade mark dilution, culture jamming, and the no logo movement

This article considers the ongoing debate over the appropriation of well-known and famous trade marks by the No Logo Movement for the purposes of political and social critique. It focuses upon one sensational piece of litigation in South Africa, Laugh It Off Promotions v. South African Breweries International (Finance) B.V. t/a Sabmark International. In this case, a group called Laugh It Off Promotions subjected the trade marks of the manufacturers of Carling Beer were subjected to parody, social satire, and culture jamming. The beer slogan “Black Label” was turned into a T-Shirt entitled “Black Labour/ White Guilt”. In the ensuing litigation, the High Court of South Africa and the Supreme Court of Appeal were of the opinion that the appropriation of the mark was a case of hate speech. However, the Constitutional Court of South Africa disagreed, finding that the parodies of a well-known, famous trade mark did not constitute trade mark dilution. Moseneke J observed that there was a lack of evidence of economic or material harm; and Sachs J held that there is a need to provide latitude for parody, laughter, and freedom of expression. The decision of the Constitutional Court of South Africa provides some important insights into the nature of trade mark dilution, the role of parody and satire, and the relevance of constitutional protections of freedom of speech and freedom of expression. Arguably, the ruling will be of help in the reformation of trade mark dilution law in other jurisdictions – such as the United States. The decision in Laugh It Off Promotions v. South African Breweries International demonstrates that trade mark law should not be immune from careful constitutional scrutiny.

Pack

Inspiration for this work came from my recent experience of living in Beijing, China. Having a massive population of over 21 million people, one cannot feel but just another number within the crowd of this hectic megalopolis. Society is conditioned to obey, to conform, with some cultures being more hard lined with their citizens than others. However, there is a desire within each of us to have freedom of expression… to project an identity, to be an individual… nevertheless a desire not to be different… to follow trends and fads… a desire to fit in and connect. Within the pack there is also the outcast… the one who is different… the one who does not fit in. Pack has been created to be absorbed and interpreted openly by each individual. Driven by repetition and cycles, I have used this choreographic approach as a metaphor for the daily routine inherent in one’s life.

Epithelial-mesenchymal axis in head and neck cancer cell lines

Objectives: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous tumour type which necessitates multiple invitro models to attain an appreciation of its multiple subtypes. The phenomenon of epithelial-mesenchymal transition (EMT) isimportant to the development of a metastatic cancer cell phenotype being relevant to the ability of cancer cells to intravasate intovasculature and to invade tissues. The role of EMT in human papilloma virus (HPV) positive HNSCC is not well understood. Thispaper aims to characterize seven HNSCC cell lines (FaDu, SCC-25, SCC-15, CAL27, RPMI2650) including two new HPV-16positive HNSCC cell lines (UD-SCC2, 93-VU-147T) for their epithelial and mesenchymal properties. Materials and methods: A panel of HNSCC cell lines from multiple head and neck anatomical sites were profiled for basalexpression of epithelial and mesenchymal characteristics at mRNA, protein and functional levels (proliferative, migratory andinvasive properties). Furthermore, 3D spheroid forming capabilities were investigated. Results: We found that the HPV-16 positive cell line, in particular UD-SCC2 demonstrated a more invasive and mesenchymalphenotype at the molecular and functional levels suggesting HPV infection may mediate some of these cellular properties.Moreover, HPV-negative cell lines were not strictly epithelial presenting with a dynamic range of expression. Conclusions: This study presents the molecular and phenotypic diversity of HNSCC cell lines. It highlights the need formore studies in this field and a scoring system where HNSCC cell lines are ranked according to their respective epithelial andmesenchymal nature. This data will be useful to anyone modelling HNSCC behaviour, providing a molecular context which willenable them to decipher cell phenotypes and to develop therapies which block EMT progression.