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.

Effect of spacing of reinforcement on the behaviour of partially grouted masonry shear walls

Partially Grouted Reinforced Masonry (PGRM) shear walls perform well in places where the cyclonic wind pressure dominates the design. Their out-of-plane flexural performance is better understood than their inplane shear behaviour; in particular, it is not clear whether the PGRM shear walls act as unreinforced masonry (URM) walls embedded with discrete reinforced grouted cores or as integral systems of reinforced masonry (RM) with wider spacing of reinforcement. With a view to understanding the inplane response of PGRM shear walls, ten full scale single leaf, clay block walls were constructed and tested under monotonic and cyclic inplane loading cases. It has been shown that where the spacing of the vertical reinforcement is less than 2000mm, the walls behave as an integral system of RM; for spacing greater than 2000mm, the walls behave similar to URM with no significant benefit from the reinforced cores based on the displacement ductility and stiffness degradation factors derived from the complete lateral load – lateral displacement curves.

Response to "a step in the right direction : commentary on expected values for pedometer-determined physical activity in youth"

The figure Beets took exception to displays sex‐ and age‐specific median values of aggregated published expected values for pedometer determined physical activity.

Attitudes to living and working in pandemic conditions among emergency prehospital medical care personnel

Introduction: Little is known about the risk perceptions and attitudes of healthcare personnel, especially of emergency prehospital medical care personnel, regarding the possibility of an outbreak or epidemic event. Problem: This study was designed to investigate pre-event knowledge and attitudes of a national sample of the emergency prehospital medical care providers in relation to a potential human influenza pandemic, and to determine predictors of these attitudes. Methods: Surveys were distributed to a random, cross-sectional sample of 20% of the Australian emergency prehospital medical care workforce (n = 2,929), stratified by the nine services operating in Australia, as well as by gender and location. The surveys included: (1) demographic information; (2) knowledge of influenza; and (3) attitudes and perceptions related to working during influenza pandemic conditions. Multiple logistic regression models were constructed to identify predictors of pandemic-related risk perceptions. Results: Among the 725 Australian emergency prehospital medical care personnel who responded, 89% were very anxious about working during pandemic conditions, and 85% perceived a high personal risk associated with working in such conditions. In general, respondents demonstrated poor knowledge in relation to avian influenza, influenza generally, and infection transmission methods. Less than 5% of respondents perceived that they had adequate education/training about avian influenza. Logistic regression analyses indicate that, in managing the attitudes and risk perceptions of emergency prehospital medical care staff, particular attention should be directed toward the paid, male workforce (as opposed to volunteers), and on personnel whose relationship partners do not work in the health industry. Conclusions: These results highlight the potentially crucial role of education and training in pandemic preparedness. Organizations that provide emergency prehospital medical care must address this apparent lack of knowledge regarding infection transmission, and procedures for protection and decontamination. Careful management of the perceptions of emergency prehospital medical care personnel during a pandemic is likely to be critical in achieving an effective response to a widespread outbreak of infectious disease.

Development of the response coefficient-root function method for the transient vibration serviceability design of flat concrete floors

The vibration serviceability limit state is an important design consideration for two-way, suspended concrete floors that is not always well understood by many practicing structural engineers. Although the field of floor vibration has been extensively developed, at present there are no convenient design tools that deal with this problem. Results from this research have enabled the development of a much-needed, new method for assessing the vibration serviceability of flat, suspended concrete floors in buildings. This new method has been named, the Response Coefficient-Root Function (RCRF) method. Full-scale, laboratory tests have been conducted on a post-tensioned floor specimen at Queensland University of Technology’s structural laboratory. Special support brackets were fabricated to perform as frictionless, pinned connections at the corners of the specimen. A series of static and dynamic tests were performed in the laboratory to obtain basic material and dynamic properties of the specimen. Finite-element-models have been calibrated against data collected from laboratory experiments. Computational finite-element-analysis has been extended to investigate a variety of floor configurations. Field measurements of floors in existing buildings are in good agreement with computational studies. Results from this parametric investigation have led to the development of new approach for predicting the design frequencies and accelerations of flat, concrete floor structures. The RCRF method is convenient tool to assist structural engineers in the design for the vibration serviceability limit-state of in-situ concrete floor systems.

Early osteogenic response of osteoprogenitor cells on modified titanium implant surfaces leads to improved osteogenesis

Background: Implant surface micro-roughness and hydrophilicity are known to improve the osteogenic differentiation potential of osteoprogenitor cells. This study was aimed to determine whether topographically and chemically modified titanium implant surfaces stimulate an initial osteogenic response in osteoprogenitor cells, which leads to their improved osteogenesis. ----- ----- Methods: Statistical analysis of microarray gene expression profiling data available from studies (at 72 hours) on sand-blasted, large grit acid etched (SLA) titanium surfaces was performed. Subsequently, human osteoprogenitor cells were cultured on SLActive (hydrophilic SLA), SLA and polished titanium surfaces for 24 hours, 3 days and 7 days. The expression of BMP2, BMP6, BMP2K, SP1, ACVR1, FZD6, WNT5A, PDLIM7, ITGB1, ITGA2, OCN, OPN, ALP and RUNX2 were studied using qPCR. ----- ----- Results: Several functional clusters related to osteogenesis were highlighted when genes showing statistically significant differences (from microarray data at 72 hours) in expression on SLA surface (compared with control surface) were analysed using DAVID (online tool). This indicates that differentiation begins very early in response to modified titanium surfaces. At 24 hours, ACVR1 (BMP pathway), FZD6 (Wnt pathway) and SP1 (TGF-β pathway) were significantly up-regulated in cultures on the SLActive surface compared to the other surfaces. WNT5A and ITGB1 also showed higher expression on the modified surfaces. Gene expression patterns on Day 3 and Day 7 did not reveal any significant differences.----- ----- Conclusion: These results suggest that the initial molecular response of osteoprogenitor cells to modified titanium surfaces may be responsible for an improved osteogenic response via the BMP and Wnt signalling pathways.

From the bottom-up : redesigning the international legal response to anthropogenic climate change

The design of the Kyoto Protocol renders it incapable of effectively responding to the problem of anthropogenic climate change. Therefore, this article explores the opportunity to construct a new, principled legal approach to respond to climate change that is premised on nationally derived legal responses. To do so, this article considers the theoretical foundation of the international legal response to climate change – Hardin's "The Tragedy of the Commons‟ – and the systemic design faults of the Kyoto Protocol. This article also suggests four principles – a judicious mix of legal instruments, flexibility, intrinsic legal coherence, and quantifiable and achievable targets for the reduction of greenhouse gas intensity – that are necessary to guide the creation of a nationally derived legal response to climate change. This approach is intended to provide the catalyst for new bilateral and multilateral arrangements that can, with the passing of time, generate sufficient momentum to drive the creation of a new and effective cooperative international legal framework to mitigate anthropogenic climate change.

Implications of the National Rental Affordability Scheme on the valuation of residential property

The provision of shelter is a basic need and in Australia there has been a history of home ownership. However recent economic growth and rising construction costs, particularly over the past decade, has placed home ownership out of reach for some. In response to increased affordability pressures, the Australian Federal Government established the National Rental Affordability Scheme (NRAS) in 2008. The aim of establishing the NRAS initiative is to stimulate the supply of new affordable rental dwellings, targeting 50,000 new properties by June 2012, through the provision of a National Rental Incentive for each “approved” dwelling. To be approved the dwelling must be newly constructed and subsequently rented to eligible low and moderate income households at rentals no greater than 80 percent of market rates. There is a further requirement that the accommodation be provided as part of the scheme for no less than 10 years. The requirement to provide new residential accommodation at below market rentals for no less than 10 years has an impact on value and as such the valuation methodologies employed. To give guidance to valuers this paper investigates the scheme, the impact on value and expectations for the future.