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.

Soundness of workflow nets: classification, decidability, and analysis

Workflow nets, a particular class of Petri nets, have become one of the standard ways to model and analyze workflows. Typically, they are used as an abstraction of the workflow that is used to check the so-called soundness property. This property guarantees the absence of livelocks, deadlocks, and other anomalies that can be detected without domain knowledge. Several authors have proposed alternative notions of soundness and have suggested to use more expressive languages, e.g., models with cancellations or priorities. This paper provides an overview of the different notions of soundness and investigates these in the presence of different extensions of workflow nets.We will show that the eight soundness notions described in the literature are decidable for workflow nets. However, most extensions will make all of these notions undecidable. These new results show the theoretical limits of workflow verification. Moreover, we discuss some of the analysis approaches described in the literature.

The acquisition and exercise of nephrology nursing expertise : A grounded theory study

Aims and objectives. This purpose of this study was to describe the process of expertise acquisition in nephrology nursing practice. Background. It has been recognized for a number of decades that experts, compared with other practitioners in a number of professions and occupations, are the most knowledgeable and effective, in terms of both the quantity and quality of output. Studies relating to expertise have been undertaken in a range of nursing contexts and specialties; to date, however, none have been undertaken which focus on nephrology nursing. Design. This study, using grounded theory methodology, took place in one renal unit in New South Wales, Australia and involved six non-expert and 11 expert nurses. Methods. Simultaneous data collection and analysis took place using participant observation, semi-structured interviews and review of nursing documentation. Findings. The study revealed a three-stage skills-acquisitive process that was identified as non-expert, experienced non-expert and expert stages. Each stage was typified by four characteristics, which altered during the acquisitive process; these were knowledge, experience, skill and focus. Conclusion. This was the first study to explore nephrology nursing expertise and uncovered new aspects of expertise not documented in the literature and it also made explicit other areas, which had only been previously implied. Relevance to clinical practice. Of significance to nursing, the exercise of expertise is a function of the recognition of expertise by others and it includes the blurring of the normal boundaries of professional practice. © 2006 Blackwell Publishing Ltd.

The CDKN2A (p16) gene and human cancer

CDKN2A, the gene encoding the cell-cycle inhibitor p16CDKN2A, was first identified in 1994. Since then, somatic mutations have been observed in many cancers and germline alterations have been found in kindreds with familial atypical multiple mole/melanoma (FAMMM), also known as atypical mole syndrome. In this review we tabulate the known mutations in this gene and discuss specific aspects, particularly with respect to germline mutations and cancer predisposition.

The risks and returns of new public management : political business

Purpose – This article aims to consider success in terms of the financial returns and risks of new public management (NPM) in state-owned enterprises (SOEs). Design/methodology/approach – Financial returns of New Zealand SOEs were examined through a review of their annual reports over a five-year period. Dimensions of risk were examined through interviews conducted in two phases over a two-year period with senior executives from 12 of the (then) 17 SOEs operating in New Zealand. Findings – Findings indicate the potential for SOEs to operate as profitable government investments, with clear support for positive financial returns under NPM. However, variations noted within individual SOEs also indicate that profitable and commercial operations may not be possible in all cases. An examination of the risks associated with SOEs’ operations reveals a number of dimensions of risk, encompassing financial, political (including regulatory), reputational, and public accountability aspects. Practical implications – There is a need for an enhanced awareness on the part of internal and external stakeholders (such as the government and general public) of the risks SOEs face in pursuing higher levels of profitability. Also required, is a more acute understanding on the part of internal and external stakeholders (e.g. government and the public) of the need for SOEs to manage the range of risks identified, given the potentially delicate balance between risk and return. Originality/value – While previous studies have considered the financial returns of SOEs, or the risks faced by the public sector in terms of accountability, few have addressed the two issues collectively in a single context.

Human information behavior: Integrating diverse approaches and information use

For millennia humans have sought, organized, and used information as they learned and evolved patterns of human information behaviors to resolve their human problems and survive. However, despite the current focus on living in an "information age," we have a limited evolutionary understanding of human information behavior. In this article the authors examine the current three interdisciplinary approaches to conceptualizing how humans have sought information including (a) the everyday life information seeking-sense-making approach, (b) the information foraging approach, and (c) the problem-solution perspective on information seeking approach. In addition, due to the lack of clarity regarding the role of information use in information behavior, a fourth information approach is provided based on a theory of information use. The use theory proposed starts from an evolutionary psychology notion that humans are able to adapt to their environment and survive because of our modular cognitive architecture. Finally, the authors begin the process of conceptualizing these diverse approaches, and the various aspects or elements of these approaches, within an integrated model with consideration of information use. An initial integrated model of these different approaches with information use is proposed.

Strengthening the role of the adult education sector in training workers for the digital content industries in Australia and Singapore

In 2009, QUT’s Office of Research and the Institute for Adult Learning Singapore funded a six-month pilot project that represented the first stage of a larger international comparative study. The study is the first of its kind to investigate to what extent and how digital content workers’ learning needs are being met by adult education and training in Australia and Singapore. The pilot project involved consolidating key theoretical literature, studies, policies, programs and statistical data relevant to the digital content industries in Australia and Singapore. This had not been done before, and represented new knowledge generation. Digital content workers include professionals within and beyond the creative industries as follows: Visual effects and animation (including virtual reality and 3D products); Interactive multimedia (e.g. websites, CD-ROMs) and software development; Computer and online games; and Digital film & TV production and film & TV post-production. In the last decade, the digital content industries have been recognised as an industry sector of strong and increasing significance. The project compared Australia and Singapore on aspects of the digital content industries’ labour market, skill requirements, human capital challenges, the role of adult education in building a workforce for the digital content industries, and innovation policies. The consolidated report generated from the project formed the basis of the proposal for an ARC Linkage Project application submitted in the May 2010 round.

Costco and the Aussie shopper : a case study of the market entry of an international retailer

Retailing is a globalized industry yet retailers must respond to local shopping habits if they are to be perceived as legitimate by the host country customers. However, some retailers may be unable or unwilling to respond to all customer requirements. Costco, the membership warehouse club retailer, has been successful in its international expansion efforts, establishing its first Australian store in Melbourne in 2009. In the first twelve months of operation, the store became one of Costco’s top five stores in the world. We investigate this success focussing on the customer, and use institutional theory to analyze what concessions were made by the customer and the company. Data were collected from consumer interviews, site visits and secondary media and industry sources. Analysis revealed negotiations based on the rejection, acceptance or adaptation of the regulative, normative and cultural cognitive aspects of the Australian shopper and the Costco business model. Customers made concessions to accommodate the new business model, and Costco responded to entrenched Australian shopping habits. This case is the first to explore the outcome of retail internationalization from the customers’ perspective, revealing the concept of mutual concessions. The interaction and subsequent adaptation by both customer and retailer have resulted in the institutionalization of new shopping norms in the host country and success for the international retailer.

The role of cultural beliefs in contributing to risky road use behaviour in Pakistan

Background Pakistan has the highest population rate of road fatalities in South Asia (25.3 fatalities per 100,000 people: Global Status Report on Road Safety, WHO 2009). Along with road environment and vehicle factors, human factors make a substantial contribution to traffic safety in Pakistan. Beliefs about road crash causation and prevention have been demonstrated to contribute to risky road use behaviour and resistance to preventive measures in a handful of other developing countries, but has not been explored in Pakistan. In particular, fatalism (whether based on religion, other cultural beliefs or experience) has been highlighted as a barrier to achieving changes in attitudes and behaviour. Aims The research reported here aimed (i) to explore perceptions of road crash causation among policy makers, police officers, professional drivers and car drivers in Pakistan; (ii) to identify how cultural and religious beliefs influence road use behaviour in Pakistan; and (iii) to understand how fatalistic beliefs may work as obstacles to road safety interventions. Methods In-depth interviews were conducted by the primary author (mostly in Urdu) in Lahore, Rawalpindi and Islamabad with 12 professional drivers (taxi, bus and truck), 4 car drivers, 6 police officers, 4 policy makers and 2 religious orators. All but two were Muslim, two were female, and they were drawn from a wide range of ages (24 to 60) and educational backgrounds. The interviews were taped and transcribed, then translated into English and analysed for themes related to the aims. Results Fatalism emerged as a pervasive belief utilised to justify risky road use behaviour and to resist messages about preventive measures. There was a strong religious underpinning to the statement of fatalistic beliefs (this reflects popular conceptions of Islam rather than scholarly interpretations), but also an overlap with superstitious beliefs which have longer-standing roots in Pakistani culture. These beliefs were not limited to people of poor educational background or position. A particular issue which was explored in more detail was the way in which these beliefs and their interpretation within Pakistani society contributed to poor police reporting of crashes. Discussion and conclusions The pervasive nature of fatalistic beliefs in Pakistan affects road user behaviour by supporting continued risk taking behaviour on the road, and by interfering with public health messages about behaviours which would reduce the risk of traffic crashes. The widespread influence of these beliefs on the ways that people respond to traffic crashes and the death of family members contribute to low crash reporting rates and to a system which is difficult to change. The promotion of an evidence-based approach to road user behaviour is recommended, along with improved professional education for police and policy makers.

Teachers’ work in curricular markets : conditions of design and relations between the International Baccalaureate Diploma and the local curriculum

School level strategy enabled by neoliberal choice policies can produce internal curricular markets whereby branded curricula such as the International Baccalaureate are offered alongside the local government curriculum in the same school. This project investigated how such curricular markets operating in Australian schools impacted on teachers’ work. This paper reports on teachers work in three case study schools that offered both the International Baccalaureate Diploma program and the local senior schooling curriculum, then draws on an online survey of 225 teachers in 26 such schools across Australia. The analysis reveals the impact of curricular markets along two dimensions: the curriculum’s internal design; and the relational aspects of how schools manage to deliver tandem offerings within institutional constraints. Teachers working in the IBD Diploma program were shown to relish its design, despite additional demands, while teachers working in just the local curriculum reported more relational issues. The paper argues that these trends suggest that there are winners and losers emerging in the work conditions produced by curricular markets.  

Building prosperous knowledge cities : policies, plans and metrics

This unique book reveals the procedural aspects of knowledge-based urban planning, development and assessment. Concentrating on major knowledge city building processes, and providing state-of-the-art experiences and perspectives, this important compendium explores innovative models, approaches and lessons learnt from a number of key case studies across the world. Many cities worldwide, in order to brand themselves as knowledge cities, have undergone major transformations in the 21st century. This book provides a thorough understanding of these transformations and the key issues in building prosperous knowledge cities by focusing particularly on the policy-making, planning process and performance assessment aspects. The contributors reveal theoretical and conceptual foundations of knowledge cities and their development approach of knowledge-based urban development. They present best-practice examples from a number of key case studies across the globe. This important book provides readers with a thorough understanding of the key issues in planning and developing prosperous knowledge cities of the knowledge economy era, which will prove invaluable to national, state/regional and city governments’ planning and development departments. Academics, postgraduate and undergraduate students of regional and urban studies will also find this path-breaking book an intriguing read.

Staying in touch and crafting mementoes

Designers of systems that attempt to support people in maintaining social connectedness should pay attention to the physical interaction characteristics of the systems they create. Systems today mostly accept the dominant computer interface configuration of screens, keyboards and mice, which are not always a good fit for settings outside the office, such as the home. We argue by reference to two design exemplars for a design philosophy that engages a broader range of people's abilities for skilled physical action when designing for social connectedness.

Indian students at QUT : expectations and experiences

India currently ranks among the top source countries for the Australian education industry and therefore, a better understanding of the concerns and challenges confronted by Indian students is essential. This study was undertaken to assess the needs and expectations of Indian students enrolled at Queensland University of Technology (QUT) that would inform the formulation of strategies to provide superior service to the current and future cohorts of students coming from India. Data collection was undertaken through surveys and focus group meetings. The findings reveal the acute need for more effective dissemination of information prior to the students commencing their programs on both academic and non-academic aspects of university life as well as the resources and support available at QUT. Usage of English in an academic setting, career related services, accommodation, and networking opportunities were identified as some of the key areas of concern by the participants.

Sustainable infrastructure operations : a review of assessment schemes and decision support

Infrastructure forms a vital component in supporting today’s way of life and has a significant role or impact on economic, environmental and social outcomes of the region around it. The design, construction and operation of such assets are a multi-billion dollar industry in Australia alone. Another issue that will play a major role in our way life is that of climate change and the greater concept of sustainability. With limited resources and a changing natural world it is necessary for infrastructure to be developed and maintained in a manner that is sustainable. In order to achieve infrastructure sustainability in operations it is necessary for there to be: a sustainability assessment scheme that provides a scientifically sound and realistic approach to measuring an assets level of sustainability; and, systems and tools to support the making of decisions that result in sustainable outcomes by providing feedback in a timely manner. Having these in place will then help drive the consideration of sustainability during the decision making process for infrastructure operations and maintenance. In this paper we provide two main contributions; a comparison and review of sustainability assessment schemes for infrastructure and their suitability for use in the operations phase; and, a review of decision support systems/tools in the area of infrastructure sustainability in operations. For this paper, sustainability covers not just the environment, but also finance/economic and societal/community aspects as well. This is often referred to as the Triple Bottom Line and forms one of the three dimensions of corporate sustainability [Stapledon, 2004].