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.

An investigation into how the imagining of the nation is affected by understandings of the Internet

My research is located in an abiding concern with the nation and takes a special interest in the predictions and expectations surrounding the impact of the Internet on how this institution is lived. It is my hypothesis that the effects of the Internet are not limited only to those who are users but extends even to those who may never have witnessed its workings. The research question I began with: how is the imagining of the nation affected by our understandings and expectations of the Internet, developed through the writing of the first three chapters to: how is the living of the nation affected by the Internet’s inflection on lived time and lived space?

Mutual protection in a cloud computing environment

The term “cloud computing” has emerged as a major ICT trend and has been acknowledged by respected industry survey organizations as a key technology and market development theme for the industry and ICT users in 2010. However, one of the major challenges that faces the cloud computing concept and its global acceptance is how to secure and protect the data and processes that are the property of the user. The security of the cloud computing environment is a new research area requiring further development by both the academic and industrial research communities. Today, there are many diverse and uncoordinated efforts underway to address security issues in cloud computing and, especially, the identity management issues. This paper introduces an architecture for a new approach to necessary “mutual protection” in the cloud computing environment, based upon a concept of mutual trust and the specification of definable profiles in vector matrix form. The architecture aims to achieve better, more generic and flexible authentication, authorization and control, based on a concept of mutuality, within that cloud computing environment.

Identity fraud and torrens - minimising the risks and allocating the loss to achieve a sustainable system

Immediate indefeasibility is the cornerstone of the Torrens system of land registration. However, when combined with the apparent ease in which forged mortgages become registered, the operation of this doctrine can come into question. This article seeks to argue that, rather than question indefeasibility, the focus should be on the verification of identity of parties to land transactions. Whilst no system can ever be infallible, it is suggested that by correctly imposing the responsibility for identity verification on the appropriate individual, the Torrens system can retain immediate indefeasibility as its paramount principle, yet achieve the optimum level of fairness in terms of allocation of responsibility and loss. With the dawn of a new era of electronic conveyancing about to begin, the framework suggested here provides a model for minimising the risks of forged mortgages and appropriately allocating the loss.

The dynamic processes of visual, corporate, and organisational identity : an exploratory study

The purpose of this study is to contribute to the cross-disciplinary body of literature of identity and organisational culture. This study empirically investigated the Hatch and Schultz (2002) Organisational Identity Dynamics (OID) model to look at linkages between identity, image, and organisational culture. This study used processes defined in the OID model as a theoretical frame by which to understand the relationships between actual and espoused identity manifestations across visual identity, corporate identity, and organisational identity. The linking processes of impressing, mirroring, reflecting, and expressing were discussed at three unique levels in the organisation. The overarching research question of How does the organisational identity dynamics process manifest itself in practice at different levels within an organisation? was used as a means of providing empirical understanding to the previously theoretical OID model. Case study analysis was utilised to provide exploratory data across the organisational groups of: Level A - Senior Marketing and Corporate Communications Management, Level B - Marketing and Corporate Communications Staff, and Level C - Non-Marketing Managers and Employees. Data was collected via 15 in-depth interviews with documentary analysis used as a supporting mechanism to provide triangulation in analysis. Data was analysed against the impressing, mirroring, reflecting, and expressing constructs with specific criteria developed from literature to provide a detailed analysis of each process. Conclusions revealed marked differences in the ways in which OID processes occurred across different levels with implications for the ways in which VI, CI, and OI interact to develop holistic identity across organisational levels. Implications for theory detail the need to understand and utilise cultural understanding in identity programs as well as the value in developing identity communications which represent an actual rather than an espoused position.

Strengthening and formally verifying privacy in identity management systems

In a digital world, users’ Personally Identifiable Information (PII) is normally managed with a system called an Identity Management System (IMS). There are many types of IMSs. There are situations when two or more IMSs need to communicate with each other (such as when a service provider needs to obtain some identity information about a user from a trusted identity provider). There could be interoperability issues when communicating parties use different types of IMS. To facilitate interoperability between different IMSs, an Identity Meta System (IMetS) is normally used. An IMetS can, at least theoretically, join various types of IMSs to make them interoperable and give users the illusion that they are interacting with just one IMS. However, due to the complexity of an IMS, attempting to join various types of IMSs is a technically challenging task, let alone assessing how well an IMetS manages to integrate these IMSs. The first contribution of this thesis is the development of a generic IMS model called the Layered Identity Infrastructure Model (LIIM). Using this model, we develop a set of properties that an ideal IMetS should provide. This idealized form is then used as a benchmark to evaluate existing IMetSs. Different types of IMS provide varying levels of privacy protection support. Unfortunately, as observed by Jøsang et al (2007), there is insufficient privacy protection in many of the existing IMSs. In this thesis, we study and extend a type of privacy enhancing technology known as an Anonymous Credential System (ACS). In particular, we extend the ACS which is built on the cryptographic primitives proposed by Camenisch, Lysyanskaya, and Shoup. We call this system the Camenisch, Lysyanskaya, Shoup - Anonymous Credential System (CLS-ACS). The goal of CLS-ACS is to let users be as anonymous as possible. Unfortunately, CLS-ACS has problems, including (1) the concentration of power to a single entity - known as the Anonymity Revocation Manager (ARM) - who, if malicious, can trivially reveal a user’s PII (resulting in an illegal revocation of the user’s anonymity), and (2) poor performance due to the resource-intensive cryptographic operations required. The second and third contributions of this thesis are the proposal of two protocols that reduce the trust dependencies on the ARM during users’ anonymity revocation. Both protocols distribute trust from the ARM to a set of n referees (n > 1), resulting in a significant reduction of the probability of an anonymity revocation being performed illegally. The first protocol, called the User Centric Anonymity Revocation Protocol (UCARP), allows a user’s anonymity to be revoked in a user-centric manner (that is, the user is aware that his/her anonymity is about to be revoked). The second protocol, called the Anonymity Revocation Protocol with Re-encryption (ARPR), allows a user’s anonymity to be revoked by a service provider in an accountable manner (that is, there is a clear mechanism to determine which entity who can eventually learn - and possibly misuse - the identity of the user). The fourth contribution of this thesis is the proposal of a protocol called the Private Information Escrow bound to Multiple Conditions Protocol (PIEMCP). This protocol is designed to address the performance issue of CLS-ACS by applying the CLS-ACS in a federated single sign-on (FSSO) environment. Our analysis shows that PIEMCP can both reduce the amount of expensive modular exponentiation operations required and lower the risk of illegal revocation of users’ anonymity. Finally, the protocols proposed in this thesis are complex and need to be formally evaluated to ensure that their required security properties are satisfied. In this thesis, we use Coloured Petri nets (CPNs) and its corresponding state space analysis techniques. All of the protocols proposed in this thesis have been formally modeled and verified using these formal techniques. Therefore, the fifth contribution of this thesis is a demonstration of the applicability of CPN and its corresponding analysis techniques in modeling and verifying privacy enhancing protocols. To our knowledge, this is the first time that CPN has been comprehensively applied to model and verify privacy enhancing protocols. From our experience, we also propose several CPN modeling approaches, including complex cryptographic primitives (such as zero-knowledge proof protocol) modeling, attack parameterization, and others. The proposed approaches can be applied to other security protocols, not just privacy enhancing protocols.

QUT Speaker Identity Verification system for EVALITA 2009

This document outlines the system submitted by the Speech and Audio Research Laboratory at the Queensland University of Technology (QUT) for the Speaker Identity Verification: Application task of EVALITA 2009. This competitive submission consisted of a score-level fusion of three component systems; a joint-factor analysis GMM system and two SVM systems using GLDS and GMM supervector kernels. Development evaluation and post-submission results are presented in this study, demonstrating the effectiveness of this fused system approach. This study highlights the challenges associated with system calibration from limited development data and that mismatch between training and testing conditions continues to be a major source of error in speaker verification technology.

Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje

Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns—they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.

Children re-read and re-write their neighborhoods : critical literacies and identity work

This chapter considers the complex literate repertoires of 21st century children in multicultural primary classrooms in Adelaide South Australia. It draws on the curricular and pedagogical work of two experienced primary school teachers who explore culture, race and class, by positioning children as textual producers across a variety of media. In particular we discuss two child-authored texts – A is for Arndale – a local alphabet book co-authored by children aged between eight and ten, and – Cooking Afghani Style - a magazine style film produced by a multi-aged class of children (aged eight to thirteen) recently arrived in Australia. In the process of making these texts, primary children engaged in reading as a cultural practice – re-reading and re-writing their neighbourhoods and identities (both individual and collective). This involved frequent excursions to local key sites, both familiar and unfamiliar to the children. They investigated how diverse children experienced and lived their lives in particular places within changing communities.

Endochondral ossification in vitro is influenced by mechanical bending

Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000-1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration.

Trans digital storytelling : everyday activism, mutable identity and the problem of visibility

Mainstream representations of trans people typically run the gamut from victim to mentally ill and are almost always articulated by non-trans voices. The era of user-generated digital content and participatory culture has heralded unprecedented opportunities for trans people who wish to speak their own stories in public spaces. Digital Storytelling, as an easy accessible autobiographic audio-visual form, offers scope to play with multi-dimensional and ambiguous representations of identity that contest mainstream assumptions of what it is to be ‘male’ or ‘female’. Also, unlike mainstream media forms, online and viral distribution of Digital Stories offer potential to reach a wide range of audiences, which is appealing to activist oriented storytellers who wish to confront social prejudices. However, with these newfound possibilities come concerns regarding visibility and privacy, especially for storytellers who are all too aware of the risks of being ‘out’ as trans. This paper explores these issues from the perspective of three trans storytellers, with reference to the Digital Stories they have created and shared online and on DVD. These examplars are contextualised with some popular and scholarly perspectives on trans representation, in particular embodied and performed identity. It is contended that trans Digital Stories, while appearing in some ways to be quite conventional, actually challenge common notions of gender identity in ways that are both radical and transformative.