In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

Relational contracting and its efficacy of project performance in the Australian construction industry

The Australian construction industry is characterized as being a competitive and risky business environment due to lack of cooperation, insufficient trust, ineffective communication and adversarial relationships which are likely lead to poor project performance. Relational contracting (RC) is advocated by literature as an innovative approach to improve the procurement process in the construction industry. Various studies have collectively added to the current knowledge of known RC norms, but there seem to be little effort on investigating the determinants of RC and its efficacy on project outcomes. In such circumstances, there is lack of evidence and explanation on the manner on how these issues lead to different performance. Simultaneously, the New Engineering Contract (NEC) that embraced the concept of RC is seen as a modern way of contracting and also considered as one of the best approaches to the perennial problem of improving adversarial relationships within the industry. The reality of practice of RC in Australia is investigated through the lens of the NEC. A synthesis of literature views on the concept, processes and tools of RC is first conducted to develop the framework of RC. A case study approach is proposed for an in-depth analysis to explore the critical issues addressed by RC in relation to project performance. Understanding the realities of RC will assist stakeholders in the construction industry with their investment in RC.

Exporting educational services : an Australian university’s experience in delivering offshore project management programs

Increasingly, Australian universities are facing the challenges of global education. While overseas students studying in Australia provide the primary source of export earnings for educational institutions, a number of institutions, including QUT, are also involved in international trade in educational services by providing services offshore. This paper discusses driving forces behind moves by Australian universities to enter the international education market. It then briefly describes Queensland University of Technology’s internationalisation strategy. The paper concludes with a case study describing how the School of Construction Management has pioneered the development of offshore courses at QUT. The introduction of the Master of Project Management and Graduate Diploma of Project Management programs in Singapore in November 1993 represented QUT’s first experience in this area. With the experience of 18 months of operation, the School now has the opportunity to reflect on the outcomes of this venture and consider future options.

Construction project delivery and industry skills : crafting a better fit

The construction industry should be a priority to all governments because it impacts economically and socially on all citizens. Sector turnover in industrialised economies typically averages 8-12% of GDP. Further, construction is critical to economic growth. Recent Australian studies estimate that a 10% gain in efficiency in construction translates to a 2.5% increase in GDP ----- ----- Inefficiencies in the Australian construction industry have been identified by a number of recent studies modelling the building process. They have identified potential savings in time of between 25% and 40% by reducing non-value added steps in the process. A culture of reform is now emerging in the industry – one in which alternate forms of project delivery are being trial. ----- ----- Government and industry have identified Alliance Contracting as a means to increase efficiency in the construction industry as part of a new innovative procurement environment. Alliance contracting requires parties to form relationships and work cooperatively to provide a more complete service. This is a significant cultural change for the construction industry, with its well-known adversarial record in traditional contracting. Alliance contracts offer enormous potential benefits, but the Australian construction industry needs to develop new skills to effectively participate in the new relationship environment. ----- ----- This paper describes a collaborative project identifying skill needs for clients and construction professionals to more effectively participate in an increasingly sophisticated international procurement environment. The aim of identifying these skill needs is to assist industry, government, and skill developers to prepare the Australian construction workforce for the future. The collaborating Australian team has been fortunate to secure the Australian National Museum in Canberra as its live case study. The Acton Peninsula Development is the first major building development in the world awarded on the basis of a joint alliance contract.

Project alliancing at National Museum of Australia : the collaborative process

Project alliancing is a new alternative to traditional project delivery systems, especially in the commercial building sector. The Collaborative Process is a theoretical model of people and systems characteristics that are required to reduce the adversarial nature of most construction projects. Although developed separately, both are responses to the same pressures. Project alliancing was just used successfully to complete the National Museum of Australia. This project was analyzed as a case study to determine the extent to which it could be classified as a “collaborative project”. Five key elements of The Collaborative Process were reviewed and numerous examples from the management of this project were cited that support the theoretical recommendations of this model. In the case of this project, significant added value was delivered to the client and many innovations resulted from the collective work of the parties to the contract. It was concluded that project alliances for commercial buildings offer many advantages over traditional project delivery systems, which are related to increasing the levels of collaboration among a project management team.

Project managers and technical change : curriculum development in strategic technology management

Traditional business approaches do not take account of the rapid technological developments underpinning today's world. Further understanding the role of technology and its efficient management to build and maintain a competitive edge in business can allow project managers to more successfully manage organisations, and to adapt to and capitalise on, today’s rapidly changing environment. Strategic Technology Management links engineering, science and management principles to identify, choose, and implement the most effective means of attaining compatibility between internal skills and resources of an organisation and its competitive, economic and social environment. This paper reviews the rationale and the development of a new Strategic Technology Management subject in QUT’s Master of Project Management program. It discusses recent developments in the area of technology management from an international perspective, provides details of the curriculum developed and discusses the experience of completing two years of teaching the new program.

Profiling outdoor leadership

This paper examines the role outdoor recreation and education plays in the development of generic leaders who have a positive relationship to the natural world. Three questionnaires (Multifactor Leadership Questionnaire - MLQ; the New Ecological Paradigm Scale - NEP; and the Connectedness to Nature Scale - CNS) were administered online to 104 international outdoor leaders through five online networks. The three instruments assessed the nexus of transformational leadership theory and outdoor leadership. A descriptive analysis of early findings from the project are outlined in this paper. The results can be viewed as an appropriate platform for understanding outdoor recreation and education leaders’ ecological perspectives and the generic, transformational leadership skills.

Achieving a responsive industrial relations environment for construction industry workers – a project alliancing case study

This paper outlines how the project agreement operating on the Australian National Museum project in Canberra, Australia facilitated a responsible and responsive workplace environment for construction workers. A project alliancing approach was adopted and designed to encourage industrial relations innovation in the workplace. The trigger for this approach was the perceived success of the alliancing working arrangements between key project delivery teams and a desire to extend this arrangement to subcontractors, suppliers and the workforce. Changes in the Australian workplace relations environment and introduction of a national code of practice for the Australian construction industry provided impetus for reaching a new type of workplace agreement. The workplace culture and characteristics of relationships formed between workers and management on that site shaped the agreed terms and conditions of work. It also spurred the pursuit of innovative approaches to project delivery from a technology, management and workplace culture perspective.

Project alliancing Vs project partnering : a case study of the Australian National Museum project

Significant differences between project partnering and project alliancing occur in the selection process, management structure of the organisations undertaking the project and nature of risk and reward incentives. This paper helps clarify the nature of project alliancing and how alliance member organisations were selected for this case study. A core issue that differentiates between the two approaches is that in partnering, partners may reap rewards at the expense of other partners. In alliancing each alliance member places their profit margin and reward structure ÁÁat riskÂÂ. Thus in alliancing, the entire alliance entity either benefits together or not all. This fundamentally changes the motivation and dynamics of the relationship between alliance members.

Innovative project procurement in the Queensland government : the Woodford Correctional Centre

Much of the effort of the construction industry is directed towards the provision of services and products, many with substantial long term implications. Systems and procedures have evolved over centuries to provide these services and products, but inefficiencies have developed. One strategy for improving the efficiency of the construction industry is to restructure the systems and procedures which deliver projects so that improved benefits to the end users are provided. In this paper, contemporary systems and procedures for the delivery of projects are reviewed and the roles of the major stakeholders are examined. The recent construction of Woodford Correctional Centre in Queensland is reviewed as a case study in restructuring the delivery process and the lessons learned from this successful project are summarised.

The Blair Witch Project : a marketing case study

The Blair Witch Project was a low budget movie made by student filmmakers that become an international box office hit in 1999. Blair Witch was a landmark in movie marketing and distribution because it was the first time that any movie had successfully leveraged the Internet as a marketing platform to reach a wide audience. The marketing team employed a range of innovative strategies and tactics to stimulate audience demand. This case study describes and analyses the success of the marketing launch of The Blair Witch Project.It also provides an Instructors booklet comprising seven questions and answers related to the marketing success of the movie.

The Blair Witch Project : Forming strong attitudes, beliefs and consumer intentions from a myth

This brief consumer marketing case study was published in a consumer marketing text book.

Australian ePortfolio Project–Stage 2 ePortfolio use by university students in Australia : Developing a sustainable community of practice Case studies extracted from the final project report December 2009

Fourteen sase studies extracted from the final project report - December 2009 Australian Flexible Learning Framework: E-portfolios Community of Practice (Aus) Personal learning plans and ePortfolio (Aus) RMIT University: Introducing ePortfolios (Aus) ePortfolio Practice: ALTC Exchange (Aus) Australian PebblePad User Group (APpUG) (Aus) ePortfolios in the library and information services sector (Aus) PDP and ePortfolios UK (UK) SURF NL Portfolio (Netherlands) University of Canterbury ePortfolio (NZ) AAEEBL: Association for Authentic, Experiential and Evidence-Based Learning (USA) Midlands Eportfolio Group, West Midlands(UK) EPAC: Electronic Portfolio Action and Communication (USA) Scottish Higher Education PDP Forum (UK) Centre for Recording Achievement (CRA)(UK)

Ecological leadership : a new perspective on leadership

Given today's focus on the state of the environment and the developing role of corporate social leadership in could be argued that there is a need for the development of successful business leaders who have a positive relationship to the natural world. Gifford (2007) argued that any real change in sustainable practice will most likely happen at an individual level, through changes in attitudes and everyday behaviour. For this change to happen, an individual will need to feel connected to the natural world (Dunbar, 2004; Schroll, 2007). Roszak (1992) developed the notion of ecopsychology specifically to explore this relationship and suggest new ways to generate greater environmental awareness as well as ameliorate psychological problems caused or exacerbated by widespread alienation from nature. From this perspective it seems imperative that we develop people centred leader’s who feel connected to the natural world whilst demonstrating solid performance, as measured by organisational and social indicators. This paper presents information from an International research project that might add further insights into the role outdoor education plays in the development of generic leaders who have a positive relationship to the natural world. Three questionnaires, an established measurement of generic transformational leadership (MLQ) and two established measurement of attitudes to and feelings about the natural world (the New Ecological Paradigm Scale and the Connectedness to Nature Scale), were administered to 214 (males, n=138 and females, n=76) International outdoor leaders with the implicit aim of assessing the nexus of transformational leadership theory and adventure based leadership development. The large and diverse cohort of participants has provided ground-breaking insights into transformational and ecological leadership styles. This paper outlines a descriptive analysis of findings and offers valuable information for those involved in training leaders. Throughout this presentation participants will be encouraged to contextualise the information for their specific circumstance.