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.

Obstacle-free range determination for rail track maintenance vehicles

Maintenance trains travel in convoy. In Australia, only the first train of the convoy pays attention to the track sig- nalization (the other convoy vehicles simply follow the preceding vehicle). Because of human errors, collisions can happen between the maintenance vehicles. Although an anti-collision system based on a laser distance meter is already in operation, the existing system has a limited range due to the curvature of the tracks. In this paper, we introduce an anti-collision system based on vision. The two main ideas are, (1) to warp the camera image into an image where the rails are parallel through a projective transform, and (2) to track the two rail curves simultaneously by evaluating small parallel segments. The performance of the system is demonstrated on an image dataset.

RatSLAM : a hippocampal model for simultaneous localization and mapping

The work presents a new approach to the problem of simultaneous localization and mapping - SLAM - inspired by computational models of the hippocampus of rodents. The rodent hippocampus has been extensively studied with respect to navigation tasks, and displays many of the properties of a desirable SLAM solution. RatSLAM is an implementation of a hippocampal model that can perform SLAM in real time on a real robot. It uses a competitive attractor network to integrate odometric information with landmark sensing to form a consistent representation of the environment. Experimental results show that RatSLAM can operate with ambiguous landmark information and recover from both minor and major path integration errors.

Probabilistic visual recognition of artificial landmarks for simultaneous localization and mapping

Probabilistic robotics, most often applied to the problem of simultaneous localisation and mapping (SLAM), requires measures of uncertainly to accompany observations of the environment. This paper describes how uncertainly can be characterised for a vision system that locates coloured landmark in a typical laboratory environment. The paper describes a model of the uncertainly in segmentation, the internal camera model and the mounting of the camera on the robot. It =plains the implementation of the system on a laboratory robot, and provides experimental results that show the coherence of the uncertainly model,

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.

Preliminary modelling outcomes : calculation of holding costs in greenfield residential development

Many of the costs associated with greenfield residential development are apparent and tangible. For example, regulatory fees, government taxes, acquisition costs, selling fees, commissions and others are all relatively easily identified since they represent actual costs incurred at a given point in time. However, identification of holding costs are not always immediately evident since by contrast they characteristically lack visibility. One reason for this is that, for the most part, they are typically assessed over time in an ever-changing environment. In addition, wide variations exist in development pipeline components: they are typically represented from anywhere between a two and over sixteen years time period - even if located within the same geographical region. Determination of the starting and end points, with regards holding cost computation, can also prove problematic. Furthermore, the choice between application of prevailing inflation, or interest rates, or a combination of both over time, adds further complexity. Although research is emerging in these areas, a review of the literature reveals attempts to identify holding cost components are limited. Their quantification (in terms of relative weight or proportionate cost to a development project) is even less apparent; in fact, the computation and methodology behind the calculation of holding costs varies widely and in some instances completely ignored. In addition, it may be demonstrated that ambiguities exists in terms of the inclusion of various elements of holding costs and assessment of their relative contribution. Yet their impact on housing affordability is widely acknowledged to be profound, with their quantification potentially maximising the opportunities for delivering affordable housing. This paper seeks to build on earlier investigations into those elements related to holding costs, providing theoretical modelling of the size of their impact - specifically on the end user. At this point the research is reliant upon quantitative data sets, however additional qualitative analysis (not included here) will be relevant to account for certain variations between expectations and actual outcomes achieved by developers. Although this research stops short of cross-referencing with a regional or international comparison study, an improved understanding of the relationship between holding costs, regulatory charges, and housing affordability results.

Elements of public asset management framework for local governments in developing countries

There is a widespread recognition to the need of better manage municipal property in most cities in the world. Structural problems across regional, state, and territorial governments that have legal powers to own and maintain real property are similar, regardless of the level of development of each country. Start from a very basic level of property inventory records. The need for better manage to the local government owned property is the result of widespread decentralisation initiatives that often have devolved huge property portfolios from central to local governments almost “overnight”. At the same time municipal or regional governments were and continue to be unprepared to deal with multiple issues related to the role of property owners and managers. The lack of discussion of public asset management especially the elements that should be incorporated in the framework creates an important challenge to study the discipline of public asset management further. The aim of this paper is to study the practices of public asset management in developed countries, especially the elements of public asset management framework, and its transferability to developing countries. A case study was selected and conducted to achieve this aim. They involved interviews and a focus group. The study found that in public asset management framework, proper asset identification, public asset needs analysis, asset life cycle and performance measurements are an important element that should be incorporated in the framework. Those elements are transferable and applicable to developing countries’ local governments. Finally, findings from this study provide useful input for the local government policy makers, scholars and asset management practitioners to establish a public asset management framework toward more efficient and effective local governments in managing their assets as well as increasing public services quality.

Remembering beliefs

Optimal decision-making requires us to accurately pinpoint the basis of our thoughts, e.g. whether they originate from our memory or our imagination. This paper argues that the phenomenal qualities of our subjective experience provide permissible evidence to revise beliefs, particularly as it pertains to memory. I look to the source monitoring literature to reconcile circumstances where mnemic beliefs and mnemic qualia conflict. By separating the experience of remembering from biological facts of memory, unusual cases make sense, such as memory qualia without memory (e.g. déjà vu, false memories) or a failure to have memory qualia with memory (e.g. functional amnesia, unintentional plagiarism). I argue that a pragmatic, probabilistic approach to belief revision is a way to rationally incorporate information from conscious experience, whilst acknowledging its inherent difficulties as an epistemic source. I conclude with a Bayesian defense of source monitoring based on C.I. Lewis’ coherence argument for memorial knowledge.

Requirements for enhancing trust, security and integrity of GNSS location services

The paper describes a number of requirements for enhancing the trust of location acquisition from Satellite Navigation Systems, particularly for those applications where the location is monitored through a remote GNSS receiver. We discuss how the trust of a location acquisition could be propagated to an application through the use of a proposed tamper-­resistant GNSS receiver which quantifies the trust of a location solution from the signaling used (ie. P(Y) code, Galileo SOL, PRS, CS) and provides a cryptographic proof of this to a remote application. The tamper­-resistance state of the receiver is also included in this cryptographic proof.

Petri net modeling of spatial and temporal constraints in track allocation problem at railway stations

The track allocation problem (TAP) at a multi-track, multi-platform mainline railway station is defined by the station track layout and service timetable, which implies combinations of spatial and temporal conflicts. Feasible solutions are available from either traditional planning or advanced intelligent searching methods and their evaluations with respect to operational requirements are essential for the operators. To facilitate thorough analysis, a timed Coloured Petri Nets (CPN) model is presented here to encapsulate the inter-relationships of the spatial and temporal constraints in the TAP.

Fostering cultural awareness in pre-service teacher education : an experiment in Asia literacy capacity building in an Australian university

This paper presents the findings of a small pilot study conducted with a group of final year pre-service teachers studying a secondary social science curriculum method unit in an Australian university. One of the study’s research objectives aimed at identifying how students responded to efforts to embed intercultural understanding through Studies of Asia in their final curriculum method unit. The 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 regional and global concerns in their Australian secondary school classrooms. This pilot study’s research methodology was located within the qualitative framework of a participatory action research model whereby the lecturer who designed, coordinated and taught the unit was also the researcher. Its scope was limited to one semester with volunteer students. The pilot study sought to investigate responses to several issues, and this paper reports on pre-service teacher reflections on the content, pedagogy and learning they experienced in their weekly sessions with specific reference to cultural understanding, Studies of Asia and the development of Asia literacy. It also reports on pre-service teacher reflections about their own evolving capacity as beginning teachers. The findings indicate that pre-service teachers valued the opportunity to engage with learning experiences which enhanced their conceptual understandings about culture whilst also extending their pedagogical and content knowledge.

A prioritised fuzzy constraint satisfaction approach to model agent negotiation for railway scheduling

With the recent regulatory reforms in a number of countries, railways resources are no longer managed by a single party but are distributed among different stakeholders. To facilitate the operation of train services, a train service provider (SP) has to negotiate with the infrastructure provider (IP) for a train schedule and the associated track access charge. This paper models the SP and IP as software agents and the negotiation as a prioritized fuzzy constraint satisfaction (PFCS) problem. Computer simulations have been conducted to demonstrate the effects on the train schedule when the SP has different optimization criteria. The results show that by assigning different priorities on the fuzzy constraints, agents can represent SPs with different operational objectives.

Cultural determinants of stress in the construction industry

The need to better understand and deal with workplace stress has major implications for the construction industry, especially on a project level, because of its potential to directly impact on site productivity and safety, and ultimately, the achievement of project objectives. While there has been some understanding of the effect of workplace stress within the construction industry, the majority of these studies have explored individual determinants of workplace stress among construction professionals such as architects, engineers, quantity surveyors etc. To date, very little research has focused on workplace stress as encountered by construction site operatives. This is an important research deficiency as construction site operatives typically make up a significant percentage of on-site workforce and contribute most directly to project success. To address this imbalance in research, this paper proposes a theoretical framework to better understand site operatives’ experience of stress from a cultural perspective on three levels: individual, project and organizational which has been largely neglected in previous studies.

Simulation and measurement of an electrostatic discharge

Electrostatic discharge is the sudden and brief electric current that flashes between two objects at different voltages. This is a serious issue ranging in application from solid-state electronics to spectacular and dangerous lightning strikes (arc flashes). The research herein presents work on the experimental simulation and measurement of the energy in an electrostatic discharge. The energy released in these discharges has been linked to ignitions and burning in a number of documented disasters and can be enormously hazardous in many other industrial scenarios. Simulations of electrostatic discharges were designed to specifications by IEC standards. This is typically based on the residual voltage/charge on the discharge capacitor, whereas this research examines the voltage and current in the actual spark in order to obtain a more precise comparative measurement of the energy dissipated.