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.

Use of electronic travel diaries and vehicle instrumentation packages in the year 2000 : Atlanta Regional Household Travel Survey

The Georgia Institute of Technology is currently performing research that will result in the development and deployment of three instrumentation packages that allow for automated capture of personal travel-related data for a given time period (up to 10 days). These three packages include: A handheld electronic travel diary (ETD) with Global Positioning System (GPS) capabilities to capture trip information for all modes of travel; A comprehensive electronic travel monitoring system (CETMS), which includes an ETD, a rugged laptop computer, a GPS receiver and antenna, and an onboard engine monitoring system, to capture all trip and vehicle information; and a passive GPS receiver, antenna, and data logger to capture vehicle trips only.

Stretch : episodes of a recreation in progress

An episodic recreation of Hibberd's Stretch of the Imagination presented as a performance extract as part of the Enter the New Wave Symposium at Melbourne University September 2007.

Examination of relationships between urban form, household activities, and time allocation in the Atlanta Metropolitan Region

A substantial body of research is focused on understanding the relationships between socio-demographics, land-use characteristics, and mode specific attributes on travel mode choice and time-use patterns. Residential and commercial densities, inter-mixing of land uses, and route directness in conjunction with transportation performance characteristics interact to influence accessibility to destinations as well as time spent traveling and engaging in activities. This study uniquely examines the activity durations undertaken for out-of-home subsistence; maintenance, and discretionary activities. Also examined are total tour durations (summing all activity categories within a tour). Cross-sectional activities are obtained from household activity travel survey data from the Atlanta Metropolitan Region. Time durations allocated to weekdays and weekends are compared. The censoring and endogeneity between activity categories and within individuals are captured using multiple equations Tobit models. The analysis and modeling reveal that land-use characteristics such as net residential density and the number of commercial parcels within a kilometer of a residence are associated with differences in weekday and weekend time-use allocations. Household type and structure are significant predictors across the three activity categories, but not for overall travel times. Tour characteristics such as time-of-day and primary travel mode of the tours also affect traveler's out-of-home activity-tour time-use patterns.

BLAST Atlas : a function based multiple genome browser

BLAST Atlas is a visual analysis system for comparative genomics that supports genome-wide gene characterisation, functional assignment and function-based browsing of one or more chromosomes. Inspired by applications such as the WorldWide Telescope, Bing Maps 3D and Google Earth, BLAST Atlas uses novel three-dimensional gene and function views that provide a highly interactive and intuitive way for scientists to navigate, query and compare gene annotations. The system can be used for gene identification and functional assignment or as a function-based multiple genome comparison tool which complements existing position based comparison and alignment viewers.

From Jindabyne to 'Jindabyne' : immigrant and indigenous specters

This paper reads Ray Lawrence's film Jindabyne (2006) in order to consider how "Australian" connotes as "immigrant" or "Indigenous" in contemporary Australia. Although the film is an adaptation of an American short story, it exploits an existing grammar of place in Australian cultural production in order to interrogate this very culture. If questions of race, gender, and class have haunted post-settlement Australia, Lawrence's film simultaneously stages these spectres and gestures towards the necessary failure of any attempt to exorcise them in a place where indigeneity is invisible. Thus, the location of Jindabyne, the town drowned in the name of progress, offers an exemplary visual metaphor for the failed project of identity formation in a place where forgetting is a survival tool.

The play's the thing? : Hamlet, Slings & Arrows, and the authority of theatrical consumption

This paper reads season 1 of the critically-acclaimed Canadian television series “Slings & Arrows” (2003). This six-episode series is set in a fictionalised version of the Stratford Festival, and tells the story of a plagued production of Shakespeare’s Hamlet. It follows the play’s rehearsal after the death of the festival’s artistic director; Geoffrey Tennant (himself a plagued Hamlet) takes over the role of director, and must face his past in order to produce a Hamlet that will save the festival, redeem his reputation, and repair his interpersonal relationships. Drawing on popular and theatrical understandings of Shakespeare’s play, the series negotiates tropes of metatheatre, filiality, cultural production and consumption, in order to demonstrate the ongoing relevance and legitimacy of “Shakespeare” in the twenty-first century. The “Slings & Arrows” narrative revolves around the doubled-plot of Hamlet and the experiences of the company mounting Hamlet. In quite obvious ways, the show thus thematises ways in which Shakespeare can be used to read one’s own life and world. In the broader sense, however, the show also offers theatre/performance as a catalyst for affect. In doing so, the show functions as a relatively straight adaptation of Hamlet, and a metatheatrical/metafictional commentary on the functions of Hamlet within contemporary culture. In Shakespeare’s play, the production of “The Mouse-Trap” proves, both to Hamlet and the audience, the legitimacy of the ghost’s claims. Similarly, in “Slings & Arrows”, the successful performance of Hamlet legitimises Geoffrey’s position as artistic director of the festival, and affirms for the viewer the value of Shakespearean production in contemporary culture. In each text, theatre/performance enables and legitimises a son carrying out a dead father’s wishes in order to restore or reproduce socio-cultural order. The metatheatrics of these gestures engage the reader/viewer in a self-reflexive process whereby the ‘value’ of theatre is thematised and performed, and the consumer is positioned as the arbiter and agent of that value: complicit in its production even as they are the site of its consumption.

Blast atlas: a virtual observatory for genomes

Following the completion of the draft Human Genome in 2001, genomic sequence data is becoming available at an accelerating rate, fueled by advances in sequencing and computational technology. Meanwhile, large collections of astronomical and geospatial data have allowed the creation of virtual observatories, accessible throughout the world and requiring only commodity hardware. Through a combination of advances in data management, data mining and visualization, this infrastructure enables the development of new scientific and educational applications as diverse as galaxy classification and real-time tracking of earthquakes and volcanic plumes. In the present paper, we describe steps taken along a similar path towards a virtual observatory for genomes – an immersive three-dimensional visual navigation and query system for comparative genomic data.

Longitudinal investigation of wandering behavior in department of veterans affairs nursing home care units

Objectives To explore the extent of and factors associated with male residents who change wandering status post nursing home admission. Design Longitudinal design with secondary data analyses. Admissions over a 4-year period were examined using repeat assessments with the Minimum Data Set (MDS) to formulate a model understanding the development of wandering behavior. Setting One hundred thirty-four Veterans Administration (VA) nursing homes throughout the United States. Participants: Included 6673 residents admitted to VA nursing homes between October 2000 and October 2004. Measurements MDS variables (cognitive impairment, mood, behavior problems, activities of daily living and wandering) included ratings recorded at residents’ admission to the nursing home and a minimum of two other time points at quarterly intervals. Results The majority (86%) of the sample were classified as non wanderers at admission and most of these (94%) remained non wanderers until discharge or the end of the study. Fifty one per cent of the wanderers changed status to non wanderers with 6% of these residents fluctuating in status more than two times. Admission variables associated with an increased risk of changing status from non-wandering to wandering included older age, greater cognitive impairment, more socially inappropriate behavior, resisting care, easier distractibility, and needing less help with personal hygiene. Requiring assistance with locomotion and having three or more medical comorbidities were associated with a decreased chance of changing from non-wandering to wandering status. Conclusion A resident’s change from non-wandering to wandering status may reflect an undetected medical event that affects cognition, but spares mobility.