Crosstalk between developmental and tumour-specific signalling pathways : kallikrein-related serine peptidases and nodal in prostrate cancer

Prostate cancer is an important male health issue. The strategies used to diagnose and treat prostate cancer underscore the cell and molecular interactions that promote disease progression. Prostate cancer is histologically defined by increasingly undifferentiated tumour cells and therapeutically targeted by androgen ablation. Even as the normal glandular architecture of the adult prostate is lost, prostate cancer cells remain dependent on the androgen receptor (AR) for growth and survival. This project focused on androgen-regulated gene expression, altered cellular differentiation, and the nexus between these two concepts. The AR controls prostate development, homeostasis and cancer progression by regulating the expression of downstream genes. Kallikrein-related serine peptidases are prominent transcriptional targets of AR in the adult prostate. Kallikrein 3 (KLK3), which is commonly referred to as prostate-specific antigen, is the current serum biomarker for prostate cancer. Other kallikreins are potential adjunct biomarkers. As secreted proteases, kallikreins act through enzyme cascades that may modulate the prostate cancer microenvironment. Both as a panel of biomarkers and cascade of proteases, the roles of kallikreins are interconnected. Yet the expression and regulation of different kallikreins in prostate cancer has not been compared. In this study, a spectrum of prostate cell lines was used to evaluate the expression profile of all 15 members of the kallikrein family. A cluster of genes was co-ordinately expressed in androgenresponsive cell lines. This group of kallikreins included KLK2, 3, 4 and 15, which are located adjacent to one another at the centromeric end of the kallikrein locus. KLK14 was also of interest, because it was ubiquitously expressed among the prostate cell lines. Immunohistochemistry showed that these 5 kallikreins are co-expressed in benign and malignant prostate tissue. The androgen-regulated expression of KLK2 and KLK3 is well-characterised, but has not been compared with other kallikreins. Therefore, KLK2, 3, 4, 14 and 15 expression were all measured in time course and dose response experiments with androgens, AR-antagonist treatments, hormone deprivation experiments and cells transfected with AR siRNA. Collectively, these experiments demonstrated that prostatic kallikreins are specifically and directly regulated by the AR. The data also revealed that kallikrein genes are differentially regulated by androgens; KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 were modestly up-regulated, and KLK14 was repressed. Notably, KLK14 is located at the telomeric end of the kallikrein locus, far away from the centromeric cluster of kallikreins that are stimulated by androgens. These results show that the expression of KLK2, 3, 4, 14 and 15 is maintained in prostate cancer, but that these genes exhibit different responses to androgens. This makes the kallikrein locus an ideal model to investigate AR signalling. The increasingly dedifferentiated phenotype of aggressive prostate cancer cells is accompanied by the re-expression of signalling molecules that are usually expressed during embryogenesis and foetal tissue development. The Wnt pathway is one developmental cascade that is reactivated in prostate cancer. The canonical Wnt cascade regulates the intracellular levels of β-catenin, a potent transcriptional co-activator of T-cell factor (TCF) transcription factors. Notably, β-catenin can also bind to the AR and synergistically stimulate androgen-mediated gene expression. This is at the expense of typical Wnt/TCF target genes, because the AR:β-catenin and TCF:β-catenin interactions are mutually exclusive. The effect of β-catenin on kallikrein expression was examined to further investigate the role of β-catenin in prostate cancer. Stable knockdown of β-catenin in LNCaP prostate cancer cells attenuated the androgen-regulated expression of KLK2, 3, 4 and 15, but not KLK14. To test whether KLK14 is instead a TCF:β-catenin target gene, the endogenous levels of β-catenin were increased by inhibiting its degradation. Although KLK14 expression was up-regulated by these treatments, siRNA knockdown of β-catenin demonstrated that this effect was independent of β-catenin. These results show that β-catenin is required for maximal expression of KLK2, 3, 4 and 15, but not KLK14. Developmental cells and tumour cells express a similar repertoire of signalling molecules, which means that these different cell types are responsive to one another. Previous reports have shown that stem cells and foetal tissues can reprogram aggressive cancer cells to less aggressive phenotypes by restoring the balance to developmental signalling pathways that are highly dysregulated in cancer. To investigate this phenomenon in prostate cancer, DU145 and PC-3 prostate cancer cells were cultured on matrices pre-conditioned with human embryonic stem cells (hESCs). Soft agar assays showed that prostate cancer cells exposed to hESC conditioned matrices had reduced clonogenicity compared with cells harvested from control matrices. A recent study demonstrated that this effect was partially due to hESC-derived Lefty, an antagonist of Nodal. A member of the transforming growth factor β (TGFβ) superfamily, Nodal regulates embryogenesis and is re-expressed in cancer. The role of Nodal in prostate cancer has not previously been reported. Therefore, the expression and function of the Nodal signalling pathway in prostate cancer was investigated. Western blots confirmed that Nodal is expressed in DU145 and PC-3 cells. Immunohistochemistry revealed greater expression of Nodal in malignant versus benign glands. Notably, the Nodal inhibitor, Lefty, was not expressed at the mRNA level in any prostate cell lines tested. The Nodal signalling pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments triggered downstream phosphorylation of Smad2 in DU145 and LNCaP cells, and stably-transfected Nodal increased the clonogencity of LNCaP cells. Nodal was also found to modulate AR signalling. Nodal reduced the activity of an androgen-regulated KLK3 promoter construct in luciferase assays and attenuated the endogenous expression of AR target genes including prostatic kallikreins. These results demonstrate that Nodal is a novel example of a developmental signalling molecule that is reexpressed in prostate cancer and may have a functional role in prostate cancer progression. In summary, this project clarifies the role of androgens and changing cellular differentiation in prostate cancer by characterising the expression and function of the downstream genes encoding kallikrein-related serine proteases and Nodal. Furthermore, this study emphasises the similarities between prostate cancer and early development, and the crosstalk between developmental signalling pathways and the AR axis. The outcomes of this project also affirm the utility of the kallikrein locus as a model system to monitor tumour progression and the phenotype of prostate cancer cells.

Monte Carlo modelling of X-ray absorptiometry and its application to body composition studies

This thesis applies Monte Carlo techniques to the study of X-ray absorptiometric methods of bone mineral measurement. These studies seek to obtain information that can be used in efforts to improve the accuracy of the bone mineral measurements. A Monte Carlo computer code for X-ray photon transport at diagnostic energies has been developed from first principles. This development was undertaken as there was no readily available code which included electron binding energy corrections for incoherent scattering and one of the objectives of the project was to study the effects of inclusion of these corrections in Monte Carlo models. The code includes the main Monte Carlo program plus utilities for dealing with input data. A number of geometrical subroutines which can be used to construct complex geometries have also been written. The accuracy of the Monte Carlo code has been evaluated against the predictions of theory and the results of experiments. The results show a high correlation with theoretical predictions. In comparisons of model results with those of direct experimental measurements, agreement to within the model and experimental variances is obtained. The code is an accurate and valid modelling tool. A study of the significance of inclusion of electron binding energy corrections for incoherent scatter in the Monte Carlo code has been made. The results show this significance to be very dependent upon the type of application. The most significant effect is a reduction of low angle scatter flux for high atomic number scatterers. To effectively apply the Monte Carlo code to the study of bone mineral density measurement by photon absorptiometry the results must be considered in the context of a theoretical framework for the extraction of energy dependent information from planar X-ray beams. Such a theoretical framework is developed and the two-dimensional nature of tissue decomposition based on attenuation measurements alone is explained. This theoretical framework forms the basis for analytical models of bone mineral measurement by dual energy X-ray photon absorptiometry techniques. Monte Carlo models of dual energy X-ray absorptiometry (DEXA) have been established. These models have been used to study the contribution of scattered radiation to the measurements. It has been demonstrated that the measurement geometry has a significant effect upon the scatter contribution to the detected signal. For the geometry of the models studied in this work the scatter has no significant effect upon the results of the measurements. The model has also been used to study a proposed technique which involves dual energy X-ray transmission measurements plus a linear measurement of the distance along the ray path. This is designated as the DPA( +) technique. The addition of the linear measurement enables the tissue decomposition to be extended to three components. Bone mineral, fat and lean soft tissue are the components considered here. The results of the model demonstrate that the measurement of bone mineral using this technique is stable over a wide range of soft tissue compositions and hence would indicate the potential to overcome a major problem of the two component DEXA technique. However, the results also show that the accuracy of the DPA( +) technique is highly dependent upon the composition of the non-mineral components of bone and has poorer precision (approximately twice the coefficient of variation) than the standard DEXA measurements. These factors may limit the usefulness of the technique. These studies illustrate the value of Monte Carlo computer modelling of quantitative X-ray measurement techniques. The Monte Carlo models of bone densitometry measurement have:- 1. demonstrated the significant effects of the measurement geometry upon the contribution of scattered radiation to the measurements, 2. demonstrated that the statistical precision of the proposed DPA( +) three tissue component technique is poorer than that of the standard DEXA two tissue component technique, 3. demonstrated that the proposed DPA(+) technique has difficulty providing accurate simultaneous measurement of body composition in terms of a three component model of fat, lean soft tissue and bone mineral,4. and provided a knowledge base for input to decisions about development (or otherwise) of a physical prototype DPA( +) imaging system. The Monte Carlo computer code, data, utilities and associated models represent a set of significant, accurate and valid modelling tools for quantitative studies of physical problems in the fields of diagnostic radiology and radiography.

Experiments in visual localisation around underwater structures

Localisation of an AUV is challenging and a range of inspection applications require relatively accurate positioning information with respect to submerged structures. We have developed a vision based localisation method that uses a 3D model of the structure to be inspected. The system comprises a monocular vision system, a spotlight and a low-cost IMU. Previous methods that attempt to solve the problem in a similar way try and factor out the effects of lighting. Effects, such as shading on curved surfaces or specular reflections, are heavily dependent on the light direction and are difficult to deal with when using existing techniques. The novelty of our method is that we explicitly model the light source. Results are shown of an implementation on a small AUV in clear water at night.

Effect of rain on travel demand and traffic accidents

This paper studies the effect of rain on travel demand measured on the Tokyo Metropolitan Expressway (MEX). Rainfall data monitored by the Japan Meteorological Agency's meso-scale network of weather stations are used. This study found that travel demand decreases during rainy days and, in particular, larger reductions occur over the weekend. The effect of rainfall on the number of accidents recorded on 10 routes on the MEX is also analysed. Statistical testing shows that the average frequency of accidents, during periods of rainfall, is significantly different from the average frequency at other times.

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

Evaluating the effect of a patient education brochure on patients’ expectations and satisfaction with emergency department service

Introduction: The purpose of this study was to assess the capacity of a written intervention, in this case a patient information brochure, to improve patient satisfaction during an Emergency Department (ED) visit. For the purpose of measuring the effect of the intervention the ED journey was conceptualised as a series of distinct areas of service comprising waiting time, service by the triage nurse, care from doctors and nurses and information giving Background of study: Research into patient satisfaction has become a widespread activity endorsed by both governments and hospital administrations. The literature on ED patient satisfaction has consistently indicated three primary areas of patient dissatisfaction: waiting time, nursing care and communication. Recent developments in the literature on patient satisfaction studies however have highlighted the relationship between patients. expectations of a service encounter and their consequent assessment of the experience as dissatisfying or satisfying. Disconfirmation theory posits that the degree to which expectations are confirmed will affect subsequent levels of satisfaction. The conceptual framework utilised in this study is Coye.s (2004) model of disconfirmation. Coye while reiterating satisfaction is a consequence of the degree expectations are either confirmed or disconfirmed also posits that expectations can be modified by interventions. Coye.s work conceptualises these interventions as intra encounter experiences (cues) which function to adjust expectations. Coye suggests some cues are unintended and may have a negative impact which also reinforces the value of planned cues intended to meet or exceed consumer expectations. Consequently the brochure can be characterized as a potentially positive cue, encouraging the patient to understand processes and to orient them in what can be a confronting environment. Only a limited number of studies have examined the effect of written interventions within an ED. No studies could be located which have tested the effect of ED interventions using a conceptual framework which relates the effect of the degree to which expectations are confirmed or disconfirmed in terms of satisfaction with services. Method: Two studies were conducted. Study One used qualitative methods to explore patients. expectations of the ED from the perspective of both patients and health care professionals. Study One was used in part to direct the development of the intervention (brochure) in Study Two. The brochure was an intervention designed to modify patients. expectations thus increasing their satisfaction with the provision of ED service. As there was no existing tools to measure ED patients. expectations and satisfaction a new tool was also developed based on the findings and the literature of Study One. Study Two used a non-randomised, quasi-experimental approach using a non-equivalent post-test only comparison group design used to investigate the effect of the patient education brochure (Stommel and Wills, 2004). The brochure was disseminated to one of two study groups (the intervention group). The effect of the brochure was assessed by comparing the data obtained from both the intervention and control group. These two groups consisted of 150 participants each. It was expected that any differences in the relevant domains selected for examination would indicate the effect of the brochure both on expectation and potentially satisfaction. Results: Study One revealed several areas of common ground between patients and nurses in terms of relevant content for the written intervention, including the need for information on the triage system and waiting times. Areas of difference were also found with patients emphasizing communication issues, whereas focus group members expressed concern that patients were often unable to assimilate verbal information. The findings suggested the potential utility of written material to reinforce verbal communication particularly in terms of the triage process and other ED protocols. This material was synthesized within the final version of the written intervention. Overall the results of Study Two indicated no significant differences between the two groups. The intervention group did indicate a significant number of participants who viewed the brochure of having changed their expectations. The effect of the brochure may have been obscured by a lack of parity between the two groups as the control group presented with statistically significantly higher levels of acuity and experienced significantly shorter waiting times. In terms of disconfirmation theory this would suggest expectations that had been met or exceeded. The results confirmed the correlation of expectations with satisfaction. Several domains also indicated age as a significant predictor with older patients tending to score higher satisfaction results. Other significant predictors of satisfaction established were waiting time and care from nurses, reinforcing the combination of efficient service and positive interpersonal experiences as being valued by patients. Conclusions: Information presented in written form appears to benefit a significant number of ED users in terms of orientation and explaining systems and procedures. The degree to which these effects may interact with other dimensions of satisfaction however is likely to be limited. Waiting time and interpersonal behaviours from staff also provide influential cues in determining satisfaction. Written material is likely to be one element in a series of coordinated strategies to improve patient satisfaction during periods of peak demand.

Randomized trial of effect of bifocal and prismatic bifocal spectacles on myopic progression

Objective: To determine whether bifocal and prismatic bifocal spectacles could control myopia in children with high rates of myopic progression. ---------- Methods: This was a randomized controlled clinical trial. One hundred thirty-five (73 girls and 62 boys) myopic Chinese Canadian children (myopia of 1.00 diopters [D]) with myopic progression of at least 0.50 D in the preceding year were randomly assigned to 1 of 3 treatments: (1) single-vision lenses (n = 41), (2) +1.50-D executive bifocals (n = 48), or (3) +1.50-D executive bifocals with a 3–prism diopters base-in prism in the near segment of each lens (n = 46). ---------- Main Outcome Measures: Myopic progression measured by an automated refractor under cycloplegia and increase in axial length (secondary) measured by ultrasonography at 6-month intervals for 24 months. Only the data of the right eye were used. ---------- Results: Of the 135 children (mean age, 10.29 years [SE, 0.15 years]; mean visual acuity, –3.08 D [SE, 0.10 D]), 131 (97%) completed the trial after 24 months. Myopic progression averaged –1.55 D (SE, 0.12 D) for those who wore single-vision lenses, –0.96 D (SE, 0.09 D) for those who wore bifocals, and –0.70 D (SE, 0.10 D) for those who wore prismatic bifocals. Axial length increased an average of 0.62 mm (SE, 0.04 mm), 0.41 mm (SE, 0.04 mm), and 0.41 mm (SE, 0.05 mm), respectively. The treatment effect of bifocals (0.59 D) and prismatic bifocals (0.85 D) was significant (P < .001) and both bifocal groups had less axial elongation (0.21 mm) than the single-vision lens group (P < .001). ---------- Conclusions: Bifocal lenses can moderately slow myopic progression in children with high rates of progression after 24 months.

Wide-baseline keypoint detection and matching with wide-angle images for vision based localisation

This thesis addresses the problem of detecting and describing the same scene points in different wide-angle images taken by the same camera at different viewpoints. This is a core competency of many vision-based localisation tasks including visual odometry and visual place recognition. Wide-angle cameras have a large field of view that can exceed a full hemisphere, and the images they produce contain severe radial distortion. When compared to traditional narrow field of view perspective cameras, more accurate estimates of camera egomotion can be found using the images obtained with wide-angle cameras. The ability to accurately estimate camera egomotion is a fundamental primitive of visual odometry, and this is one of the reasons for the increased popularity in the use of wide-angle cameras for this task. Their large field of view also enables them to capture images of the same regions in a scene taken at very different viewpoints, and this makes them suited for visual place recognition. However, the ability to estimate the camera egomotion and recognise the same scene in two different images is dependent on the ability to reliably detect and describe the same scene points, or ‘keypoints’, in the images. Most algorithms used for this purpose are designed almost exclusively for perspective images. Applying algorithms designed for perspective images directly to wide-angle images is problematic as no account is made for the image distortion. The primary contribution of this thesis is the development of two novel keypoint detectors, and a method of keypoint description, designed for wide-angle images. Both reformulate the Scale- Invariant Feature Transform (SIFT) as an image processing operation on the sphere. As the image captured by any central projection wide-angle camera can be mapped to the sphere, applying these variants to an image on the sphere enables keypoints to be detected in a manner that is invariant to image distortion. Each of the variants is required to find the scale-space representation of an image on the sphere, and they differ in the approaches they used to do this. Extensive experiments using real and synthetically generated wide-angle images are used to validate the two new keypoint detectors and the method of keypoint description. The best of these two new keypoint detectors is applied to vision based localisation tasks including visual odometry and visual place recognition using outdoor wide-angle image sequences. As part of this work, the effect of keypoint coordinate selection on the accuracy of egomotion estimates using the Direct Linear Transform (DLT) is investigated, and a simple weighting scheme is proposed which attempts to account for the uncertainty of keypoint positions during detection. A word reliability metric is also developed for use within a visual ‘bag of words’ approach to place recognition.

A comparative study of the effect of calibration conditions on the water equivalence of a range of gel dosimeters

Gel dosimeters are of increasing interest in the field of radiation oncology as the only truly three-dimensional integrating radiation dosimeter. There are a range of ferrous-sulphate and polymer gel dosimeters. To be of use, they must be water-equivalent. On their own, this relates to their radiological properties as determined by their composition. In the context of calibration of gel dosimeters, there is the added complexity of the calibration geometry; the presence of containment vessels may influence the dose absorbed. Five such methods of calibration are modelled here using the Monte Carlo method. It is found that the Fricke gel best matches water for most of the calibration methods, and that the best calibration method involves the use of a large tub into which multiple fields of different dose are directed. The least accurate calibration method involves the use of a long test tube along which a depth dose curve yields multiple calibration points.

A comparative study of the effect of calibration conditions on the water equivalence of a range of gel dosimeters

Gel dosimeters are of increasing interest in the field of radiation oncology as the only truly three-dimensional integrating radiation dosimeter. There are a range of ferrous-sulphate and polymer gel dosimeters. To be of use, they must be water-equivalent. On their own, this relates to their radiological properties as determined by their composition. In the context of calibration of gel dosimeters, there is the added complexity of the calibration geometry; the presence of containment vessels may influence the dose absorbed. Five such methods of calibration are modelled here using the Monte Carlo method. It is found that the Fricke gel best matches water for most of the calibration methods, and that the best calibration method involves the use of a large tub into which multiple fields of different dose are directed. The least accurate calibration method involves the use of a long test tube along which a depth dose curve yields multiple calibration points.

The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000 ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000 ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.