The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

Publishing Engineering Education Resources: A Collaborative Web Project.

Web is a powerful hypermedia-based information retrieval mechanism that provides a user-friendly access across all major computer platforms connected over Internet. This paper demonstrates the application of Web technology when used as an educational delivery tool. It also reports on the development of a prototype electronic publishing project where Web technology was used to deliver power engineering educational resources. The resulting hyperbook will contain diverse teaching resources such as hypermedia-based modular educational units and computer simulation programs that are linked in a meaningful and structured way. The use of Web for disseminating information of this nature has many advantages that cannot possibly be achieved otherwise. PREAMBLE The continual increase of low-cost functionality available in desktop computing has opened up a new possibility in learning within a wider educational framework. This technology also is supported by enhanced features offered by new and ...

Application of K-tree to document clustering

Digital collections are growing exponentially in size as the information age takes a firm grip on all aspects of society. As a result Information Retrieval (IR) has become an increasingly important area of research. It promises to provide new and more effective ways for users to find information relevant to their search intentions. Document clustering is one of the many tools in the IR toolbox and is far from being perfected. It groups documents that share common features. This grouping allows a user to quickly identify relevant information. If these groups are misleading then valuable information can accidentally be ignored. There- fore, the study and analysis of the quality of document clustering is important. With more and more digital information available, the performance of these algorithms is also of interest. An algorithm with a time complexity of O(n2) can quickly become impractical when clustering a corpus containing millions of documents. Therefore, the investigation of algorithms and data structures to perform clustering in an efficient manner is vital to its success as an IR tool. Document classification is another tool frequently used in the IR field. It predicts categories of new documents based on an existing database of (doc- ument, category) pairs. Support Vector Machines (SVM) have been found to be effective when classifying text documents. As the algorithms for classifica- tion are both efficient and of high quality, the largest gains can be made from improvements to representation. Document representations are vital for both clustering and classification. Representations exploit the content and structure of documents. Dimensionality reduction can improve the effectiveness of existing representations in terms of quality and run-time performance. Research into these areas is another way to improve the efficiency and quality of clustering and classification results. Evaluating document clustering is a difficult task. Intrinsic measures of quality such as distortion only indicate how well an algorithm minimised a sim- ilarity function in a particular vector space. Intrinsic comparisons are inherently limited by the given representation and are not comparable between different representations. Extrinsic measures of quality compare a clustering solution to a “ground truth” solution. This allows comparison between different approaches. As the “ground truth” is created by humans it can suffer from the fact that not every human interprets a topic in the same manner. Whether a document belongs to a particular topic or not can be subjective.

Adapting a generic BEAMnrc model of the BrainLAB m3 micro-multileaf collimator to simulate a local collimation device

This work is focussed on developing a commissioning procedure so that a Monte Carlo model, which uses BEAMnrc’s standard VARMLC component module, can be adapted to match a specific BrainLAB m3 micro-multileaf collimator (μMLC). A set of measurements are recommended, for use as a reference against which the model can be tested and optimised. These include radiochromic film measurements of dose from small and offset fields, as well as measurements of μMLC transmission and interleaf leakage. Simulations and measurements to obtain μMLC scatter factors are shown to be insensitive to relevant model parameters and are therefore not recommended, unless the output of the linear accelerator model is in doubt. Ultimately, this note provides detailed instructions for those intending to optimise a VARMLC model to match the dose delivered by their local BrainLAB m3 μMLC device.

Reflections on the Hong Kong CIRC report

The Government of the Hong Kong SAR sponsored a report investigating the Hong Kong construction inudstry and published the investigating committee's findings in 2001 (HK CIRC 2001). Since then the Provisional Construction Industry Coordination Board (PCICB), and its successor, the Construction Industry Council (CIC), also set up by the Government, has made progress with the necessary reforms. Now that seven years have passed, it is time for an independent evaluation of the impact of the CIRC initiative in order to assist the CIC and the Government decision-makers in refining the efforts to improve the industry's performance. This paper reports on the interim results of a study that seeks to provide such an evaluation.

Architecture for a free subjectivity : Deleuze and Guattari at the Horizon of the Real

Architecture for a Free Subjectivity reformulates the French philosopher Gilles Deleuze's model of subjectivity for architecture, by surveying the prolific effects of architectural encounter, and the spaces that figure in them. For Deleuze and his Lacanian collaborator Félix Guattari, subjectivity does not refer to a person, but to the potential for and event of matter becoming subject, and the myriad ways for this to take place. By extension, this book theorizes architecture as a self-actuating or creative agency for the liberation of purely "impersonal effects." Imagine a chemical reaction, a riot in the banlieues, indeed a walk through a city. Simone Brott declares that the architectural object does not merely take part in the production of subjectivity, but that it constitutes its own.

Values, wellness and the social sciences curriculum

Recent initiatives in values education in Australia emphasise the importance of the process of valuing and general methodologies that foster this in the classroom. Although a range of strategies are available, this chapter argues that inquiry-based approaches in the Social Sciences play a significant role in linking valuing processes with decision making skills. Collectively, these approaches prompt the development of reasoning and self awareness which also impact on student wellness. This chapter proposes some curriculum approaches to foreground values education in the Australian Social Sciences classroom. It argues that valuing is an important life skill that can be cultivated in the classroom through specific valuing strategies. It contends that the development of the capacity to make informed value choices is a critical factor in promoting wellness and resilience in students and in preparing them for the decision making skills required for effective participation in society.

Embedding values through global education in social sciences pre-service teacher education : linking the head and heart

This chapter aims to situate values education as a core component of social science pre-service teacher education. In particular, it reflects on an experiment in embedding a values laden Global Education perspective in a fourth year social science curriculum method unit. This unit was designed and taught by the researcher on the assumption that beginning social science teachers need to be empowered with pedagogical skills and new dispositions to deal with value laden emerging global and regional concerns in their secondary school classrooms. Moreover, it was assumed that when pre-service teachers engage in dynamic and interactive learning experiences in their curriculum unit, they commence the process of ‘capacity building’ those skills which prepare them for their own lifelong professional learning. This approach to values education also aimed at providing pre-service teachers with opportunities to ‘create deep understandings of teaching and learning’ (Barnes, 1989, p. 17) by reflecting on the ways in which ‘pedagogy can be transformative’ (Lovat and Toomey, 2011 add page no from Chapter One). It was assumed that this tertiary experience would foster the sine qua non of teaching – a commitment to students and their learning. Central to fostering new ‘dispositions’ through this approach, was the belief in the power of pedagogy to make the difference in enhancing student participation and learning. In this sense, this experiment in values education in secondary social science pre-service teacher education aligns with the Troika metaphor for a paradigm change, articulated by Lovat and Toomey (2009) in Chapter One.

Innovation in construction : a case study of the Australian context

The dominant economic paradigm currently guiding industry policy making in Australia and much of the rest of the world is the neoclassical approach. Although neoclassical theories acknowledge that growth is driven by innovation, such innovation is exogenous to their standard models and hence often not explored. Instead the focus is on the allocation of scarce resources, where innovation is perceived as an external shock to the system. Indeed, analysis of innovation is largely undertaken by other disciplines, such as evolutionary economics and institutional economics. As more has become known about innovation processes, linear models, based on research and development or market demand, have been replaced by more complex interactive models which emphasise the existence of feedback loops between the actors and activities involved in the commercialisation of ideas (Manley 2003). Currently dominant among these approaches is the national or sectoral innovation system model (Breschi and Malerba 2000; Nelson 1993), which is based on the notion of increasingly open innovation systems (Chesbrough, Vanhaverbeke, and West 2008). This chapter reports on the ‘BRITE Survey’ funded by the Cooperative Research Centre for Construction Innovation which investigated the open sectoral innovation system operating in the Australian construction industry. The BRITE Survey was undertaken in 2004 and it is the largest construction innovation survey ever conducted in Australia. The results reported here give an indication of how construction innovation processes operate, as an example that should be of interest to international audiences interested in construction economics. The questionnaire was based on a broad range of indicators recommended in the OECD’s Community Innovation Survey guidelines (OECD/Eurostat 2005). Although the ABS has recently begun to undertake regular innovation surveys that include the construction industry (2006), they employ a very narrow definition of the industry and only collect very basic data compared to that provided by the BRITE Survey, which is presented in this chapter. The term ‘innovation’ is defined here as a new or significantly improved technology or organisational practice, based broadly on OECD definitions (OECD/Eurostat 2005). Innovation may be technological or organisational in nature and it may be new to the world, or just new to the industry or the business concerned. The definition thus includes the simple adoption of existing technological and organisational advancements. The survey collected information about respondents’ perceptions of innovation determinants in the industry, comprising various aspects of business strategy and business environment. It builds on a pilot innovation survey undertaken by PricewaterhouseCoopers (PWC) for the Australian Construction Industry Forum on behalf of the Australian Commonwealth Department of Industry Tourism and Resources, in 2001 (PWC 2002). The survey responds to an identified need within the Australian construction industry to have accurate and timely innovation data upon which to base effective management strategies and public policies (Focus Group 2004).

Aboriginal public health and education : has it made a difference in practice?

Over the years, public health in relation to Australian Aboriginal people has involved many individuals and groups including health professionals, governments, politicians, special interest groups and corporate organisations. Since colonisation commenced until the1980s, public health relating to Aboriginal and Torres Strait Islander people was not necessarily in the best interests of Aboriginal and Torres Strait Islander people, but rather in the interests of the non-Aboriginal population. The attention that was paid focussed more generally around the subject of reproduction and issues of prostitution, exploitation, abuse and venereal diseases (Kidd, 1997). Since the late 1980s there has been a shift in the broader public health agenda (see Baum, 1998) along with public health in relation to Aboriginal and Torres Strait Islander people (NHMRC, 2003). This has been coupled with increasing calls to develop appropriate tertiary curriculum and to educate, train, and employ more Aboriginal and Torres Strait Islander and non-Aboriginal people in public health (Anderson et al., 2004; Genat, 2007; PHERP, 2008a, 2008b). Aboriginal and Torres Strait Islander people have been engaged in public health in ways in which they are in a position to influence the public health agenda (Anderson 2004; 2008; Anderson et al., 2004; NATSIHC, 2003). There have been numerous projects, programs and strategies that have sought to develop the Aboriginal and Torres Strait Islander Public Health workforce (AHMAC, 2002; Oldenburg et al., 2005; SCATSIH, 2002). In recent times the Aboriginal community controlled health sector has joined forces with other peak bodies and governments to find solutions and strategies to improve the health outcomes of Aboriginal and Torres Strait Islander peoples (NACCHO & Oxfam, 2007). This case study chapter will not address these broader activities. Instead it will explore the activities and roles of staff within the Public Health and Research Unit (PHRU) at the Victorian Aboriginal Community Controlled Health Organisation (VACCHO). It will focus on their experiences with education institutions, their work in public health and their thoughts on gaps and where improvements can be made in public health, research and education. What will be demonstrated is the diversity of education qualifications and experience. What will also be reflected is how people work within public health on a daily basis to enact change for equity in health and contribute to the improvement of future health outcomes of the Victorian Aboriginal community.

Music is a Wordless Knowing of Others : Resilience in Virtual Ensembles

Music making affects relationships with self and others by generating a sense of belonging to a culture or ideology (Bamford, 2006; Barovick, 2001; Dillon & Stewart, 2006; Fiske, 2000; Hallam, 2001). Whilst studies from arts education research present compelling examples of these relationships, others argue that they do not present sufficiently validated evidence of a causal link between music making experiences and cognitive or social change (Winner & Cooper, 2000; Winner & Hetland, 2000a, 2000b, 2001). I have suggested elsewhere that this disconnection between compelling evidence and observations of the effects of music making are in part due to the lack of rigor in research and the incapacity of many methods to capture these experiences in meaningful ways (Dillon, 2006). Part of the answer to these questions about rigor and causality lay in the creative use of new media technologies that capture the results of relationships in music artefacts. Crucially, it is the effective management of these artefacts within computer systems that allows researchers and practitioners to collect, organize, analyse and then theorise such music making experiences.

Semantic search and inferencing in health informatics

Consider a person searching electronic health records, a search for the term ‘cracked skull’ should return documents that contain the term ‘cranium fracture’. A information retrieval systems is required that matches concepts, not just keywords. Further more, determining relevance of a query to a document requires inference – its not simply matching concepts. For example a document containing ‘dialysis machine’ should align with a query for ‘kidney disease’. Collectively we describe this problem as the ‘semantic gap’ – the difference between the raw medical data and the way a human interprets it. This paper presents an approach to semantic search of health records by combining two previous approaches: an ontological approach using the SNOMED CT medical ontology; and a distributional approach using semantic space vector space models. Our approach will be applied to a specific problem in health informatics: the matching of electronic patient records to clinical trials.

Voices of the Unheard

Adversity has the effect of eliciting talents, which, in prosperous circumstances, would have lain dormant. Horace - Roman lyric poet and satirist 65BC – 8 BC This quotation from Horace could well be the chorus to a medley of songs sung by people who face extraordinary adversity and have gained emotional resilience through music making. In this chapter we present three composition ventures that are stories or verses in a new song and whose chorus summarises the nature of the resilience factors present in the narratives. We are aware that words on a page like this can have the effect of filtering out the engaging nature of musical experience and reduce music to a critique or an evaluation of its aesthetic value. This disjuncture between language and the ephemeral, embodied experience is a problem for those who use these creative processes in therapeutic and salutogenic ways (Antonovsky, 1996) for public health. The notion of salutogenic health, put simply, delineates it from therapy in that the processes focus upon wellness rather than therapy. Whilst we include evidence from the fields of community music therapy (Pavlicevic, 2004; Leitschuh et al., 1991), neuroscience (Bittman et al., 2001) and community music (Bartleet et al., 2009) the framework for a salutogenic health outcome in community music is one which seeks to employ music practices and the qualities of music making that provide positive health benefit to communities –to enhance health and well being rather than the “treatment” of disorders. It is essentially a holistic and interdisciplinary study. Therapy and salutogenic health are not mutually exclusive as both depend upon the qualities of music experience to affect change. Collecting, analysing and presenting evidence of change in human behaviour that can be directly attributed to creative music making is a problem of evaluation.