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.

Safety culture research, lead indicators, and the development of safety effectiveness indicators in the construction sector

Construction sector application of Lead Indicators generally and Positive Performance Indicators (PPIs) particularly, are largely seen by the sector as not providing generalizable indicators of safety effectiveness. Similarly, safety culture is often cited as an essential factor in improving safety performance, yet there is no known reliable way of measuring safety culture. This paper proposes that the accurate measurement of safety effectiveness and safety culture is a requirement for assessing safe behaviours, safety knowledge, effective communication and safety performance. Currently there are no standard national or international safety effectiveness indicators (SEIs) that are accepted by the construction industry. The challenge is that quantitative survey instruments developed for measuring safety culture and/ or safety climate are inherently flawed methodologically and do not produce reliable and representative data concerning attitudes to safety. Measures that combine quantitative and qualitative components are needed to provide a clear utility for safety effectiveness indicators.

Dynamic visual features for audio-visual speaker verification

The cascading appearance-based (CAB) feature extraction technique has established itself as the state-of-the-art in extracting dynamic visual speech features for speech recognition. In this paper, we will focus on investigating the effectiveness of this technique for the related speaker verification application. By investigating the speaker verification ability of each stage of the cascade we will demonstrate that the same steps taken to reduce static speaker and environmental information for the visual speech recognition application also provide similar improvements for visual speaker recognition. A further study is conducted comparing synchronous HMM (SHMM) based fusion of CAB visual features and traditional perceptual linear predictive (PLP) acoustic features to show that higher complexity inherit in the SHMM approach does not appear to provide any improvement in the final audio-visual speaker verification system over simpler utterance level score fusion.

The QUT-NOISE-TIMIT corpus for the evaluation of voice activity detection algorithms

The QUT-NOISE-TIMIT corpus consists of 600 hours of noisy speech sequences designed to enable a thorough evaluation of voice activity detection (VAD) algorithms across a wide variety of common background noise scenarios. In order to construct the final mixed-speech database, a collection of over 10 hours of background noise was conducted across 10 unique locations covering 5 common noise scenarios, to create the QUT-NOISE corpus. This background noise corpus was then mixed with speech events chosen from the TIMIT clean speech corpus over a wide variety of noise lengths, signal-to-noise ratios (SNRs) and active speech proportions to form the mixed-speech QUT-NOISE-TIMIT corpus. The evaluation of five baseline VAD systems on the QUT-NOISE-TIMIT corpus is conducted to validate the data and show that the variety of noise available will allow for better evaluation of VAD systems than existing approaches in the literature.

Experiences of the FIRST LEGO League in China and Australia

FIRST (For Inspiration and Recognition of Science and Technology) was initiated in the U.S. by accomplished inventor Dean Kamen in 1989. FIRST LEGO League (FLL) is one of the five competitions conducted by this organization. Dean’s vision was “to create a world where science and technology are celebrated……where young people dream of becoming science and technology heroes”. Each year FLL creates opportunities for young people aged 9-16 to engage in problem solving, teamwork and collaborative learning around a real-world theme. In the 2009/2010 season, more than 145,000 young people in over 50 countries participated in this competition. As they tackle the challenges; they construct and de-construct their own knowledge through hands-on engagement in a constructivist learning environment. The challenges are presented at least eight weeks before the competition. In most events the participants are judged in four categories - robot game, robot design, team project and team challenge. “Gracious professionalism” is an essential element of the competition. This paper compares and contrasts the FLL in China and Australia and presents some of the achievements of the event. It also highlights some of the models which have been adopted in the two countries to facilitate participation. The educational benefits of embedding the FLL will also be discussed.

Recognising audio-visual speech in vehicles using the AVICAR database

Interacting with technology within a vehicle environment using a voice interface can greatly reduce the effects of driver distraction. Most current approaches to this problem only utilise the audio signal, making them susceptible to acoustic noise. An obvious approach to circumvent this is to use the visual modality in addition. However, capturing, storing and distributing audio-visual data in a vehicle environment is very costly and difficult. One current dataset available for such research is the AVICAR [1] database. Unfortunately this database is largely unusable due to timing mismatch between the two streams and in addition, no protocol is available. We have overcome this problem by re-synchronising the streams on the phone-number portion of the dataset and established a protocol for further research. This paper presents the first audio-visual results on this dataset for speaker-independent speech recognition. We hope this will serve as a catalyst for future research in this area.

The delta-phase spectrum with application to voice activity detection and speaker recognition

For several reasons, the Fourier phase domain is less favored than the magnitude domain in signal processing and modeling of speech. To correctly analyze the phase, several factors must be considered and compensated, including the effect of the step size, windowing function and other processing parameters. Building on a review of these factors, this paper investigates a spectral representation based on the Instantaneous Frequency Deviation, but in which the step size between processing frames is used in calculating phase changes, rather than the traditional single sample interval. Reflecting these longer intervals, the term delta-phase spectrum is used to distinguish this from instantaneous derivatives. Experiments show that mel-frequency cepstral coefficients features derived from the delta-phase spectrum (termed Mel-Frequency delta-phase features) can produce broadly similar performance to equivalent magnitude domain features for both voice activity detection and speaker recognition tasks. Further, it is shown that the fusion of the magnitude and phase representations yields performance benefits over either in isolation.

Noise robust voice activity detection using normal probability testing and time-domain histogram analysis

This paper presents a method of voice activity detection (VAD) suitable for high noise scenarios, based on the fusion of two complementary systems. The first system uses a proposed non-Gaussianity score (NGS) feature based on normal probability testing. The second system employs a histogram distance score (HDS) feature that detects changes in the signal through conducting a template-based similarity measure between adjacent frames. The decision outputs by the two systems are then merged using an open-by-reconstruction fusion stage. Accuracy of the proposed method was compared to several baseline VAD methods on a database created using real recordings of a variety of high-noise environments.

Slum free India program. Report on the findings and recommendations of QUT BEE workshop to investigate strategies to deliver better outcomes

As a result of a broad invitation extended by Professor Martin Betts, Executive Dean of the Faculty of Built Environment and Engineering, to the community of interest at QUT, a cross-disciplinary collaborative workshop was conducted to contribute ideas about responding to the Government of India’s urgent requirement to implement a program to re-house slum dwellers. This is a complex problem facing the Indian Ministry of Housing. Not only does the government aspire to eradicate existing slum conditions and to achieve tangible results within five years, but it must also ensure that slums do not form in the future. The workshop focused on technological innovation in construction to deliver transformation from the current unsanitary and overcrowded informal urban settlements to places that provide the economically weaker sections of Indian society with healthy, environmentally sustainable, economically viable mass housing that supports successful urban living. The workshop was conducted in two part process as follows: Initially, QUT academics from diverse fields shared current research and provided technical background to contextualise the challenge at a pre-workshop briefing session. This was followed by a one-day workshop during which participants worked intensively in multi-disciplinary groups through a series of exercises to develop innovative approaches to the complex problem of slum redevelopment. Dynamic, compressed work sessions, interspersed with cross-functional review and feedback by the whole group took place throughout the day. Reviews emphasised testing the concepts for their level of complexity, and likelihood of success. The two-stage workshop process achieved several objectives:  Inspired a sense of shared purpose amongst a diverse group of academics  Built participants’ knowledge of each other’s capacity  Engaged multi disciplinary team in an innovative design research process  Built participants’ confidence in the collaborative process  Demonstrated that collaborative problem solving can create solutions that represent transformative change.  Developed a framework of how workable solutions might be developed for the program through follow up workshops and charrettes of a similar nature involving stakeholders drawn from the context of the slum housing program management.

Implementing low intensity CBT in case management of clients with severe mental illness

In a previous chapter (Dean and Kavanagh, Chapter 37), the authors made a case for applying low intensity (LI) cognitive behaviour therapy (CBT) to people with serious mental illness (SMI). As in other populations, LI CBT interventions typically deal with circumscribed problems or behaviours. LI CBT retains an emphasis on self-management, has restricted content and segment length, and does not necessarily require extensive CBT training. In applying these interventions to SMI, adjustments may be needed to address cognitive and symptomatic difficulties often faced by these groups. What may take a single session in a less affected population may require several sessions or a thematic application of the strategy within case management. In some cases, the LI CBT may begin to appear more like a high-intensity (HI) intervention, albeit simple and with many LI CBT characteristics still retained. So, if goal setting were introduced in one or two sessions, it could clearly be seen as an LI intervention. When applied to several different situations and across many sessions, it may be indistinguishable from a simple HI treatment, even if it retains the same format and is effectively applied by a practitioner with limited CBT training. ----- ----- In some ways, LI CBT should be well suited to case management of patients with SMI. treating staff typically have heavy workloads, and find it difficult to apply time-consuming treatments (Singh et al. 2003). LI CBT may allow provision of support to greater numbers of service users, and allow staff to spend more time on those who need intensive and sustained support. However, the introduction of any change in practice has to address significant challenges, and LI CBT is no exception. ----- ----- Many of the issues that we face in applying LI CBT to routine case management in a mnetal health service and their potential solutions are essentially the same as in a range of other problem domains (Turner and Sanders 2006)- and, indeed, are similar to those in any adoption of innovation (Rogers 2003). Over the last 20 years, several commentators have described barriers to implementing evidence-based innovations in mental health services (Corrigan et al. 1992; Deane et al. 2006; Kavanagh et al. 1993). The aim of the current chapter is to present a cognitive behavioural conceptualisation of problems and potential solutions for dissemination of LI CBT.

Stages of knowledge management systems in policing financial crime

A stage model for knowledge management systems in policing financial crime is developed in this paper. Stages of growth models enable identification of organizational maturity and direction. Information technology to support knowledge work of police officers is improving. For example, new information systems supporting police investigations are evolving. Police investigation is an information-rich and knowledge-intensive practice. Its success depends on turning information into evidence. This paper presents an organizing framework for knowledge management systems in policing financial crime. Future case studies will empirically have to illustrate and validate the stage hypothesis developed in this paper.

Extending the task of diarization to speaker attribution

In this paper we extend the concept of speaker annotation within a single-recording, or speaker diarization, to a collection wide approach we call speaker attribution. Accordingly, speaker attribution is the task of clustering expectantly homogenous intersession clusters obtained using diarization according to common cross-recording identities. The result of attribution is a collection of spoken audio across multiple recordings attributed to speaker identities. In this paper, an attribution system is proposed using mean-only MAP adaptation of a combined-gender UBM to model clusters from a perfect diarization system, as well as a JFA-based system with session variability compensation. The normalized cross-likelihood ratio is calculated for each pair of clusters to construct an attribution matrix and the complete linkage algorithm is employed to conduct clustering of the inter-session clusters. A matched cluster purity and coverage of 87.1% was obtained on the NIST 2008 SRE corpus.