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.

A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

An egg apparatus-specific enhancer of Arabidopsis, identified by enhancer detection

Despite a central role in angiosperm reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer-detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis (Arabidopsis thaliana). The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5' and 3' deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp. Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This EASE works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE. The potential application of the EASE element and similar control elements as part of an open-source biotechnology toolkit for apomixis is discussed.

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were colleted during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples colleted during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both, C. jejuni mapA and Salmonella invA genes were detected in 10% and 10% of samples, respectively collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 102 to 4.3 × 102 genomic copies per 500 ml of water G. lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.

Deriving grassland management factors for a carbon accounting method developed by the intergovernmental panel on climate change

Grassland management affects soil organic carbon (SOC) storage and can be used to mitigate greenhouse gas emissions. However, for a country to assess emission reductions due to grassland management, there must be an inventory method for estimating the change in SOC storage. The Intergovernmental Panel on Climate Change (IPCC) has developed a simple carbon accounting approach for this purpose, and here we derive new grassland management factors that represent the effect of changing management on carbon storage for this method. Our literature search identified 49 studies dealing with effects of management practices that either degraded or improved conditions relative to nominally managed grasslands. On average, degradation reduced SOC storage to 95% +/- 0.06 and 97% +/- 0.05 of carbon stored under nominal conditions in temperate and tropical regions, respectively. In contrast, improving grasslands with a single management activity enhanced SOC storage by 14% 0.06 and 17% +/- 0.05 in temperate and tropical regions, respectively, and with an additional improvement(s), storage increased by another 11% +/- 0.04. We applied the newly derived factor coefficients to analyze C sequestration potential for managed grasslands in the U.S., and found that over a 20-year period changing management could sequester from 5 to 142 Tg C yr(-1) or 0.1 to 0.9 Mg C ha(-1) yr(-1), depending on the level of change. This analysis provides revised factor coefficients for the IPCC method that can be used to estimate impacts of management; it also provides a methodological framework for countries to derive factor coefficients specific to conditions in their region.

Expected safety performance of rural signalized intersections in South Korea

Understanding the expected safety performance of rural signalized intersections is critical for (a) identifying high-risk sites where the observed safety performance is substantially worse than the expected safety performance, (b) understanding influential factors associated with crashes, and (c) predicting the future performance of sites and helping plan safety-enhancing activities. These three critical activities are routinely conducted for safety management and planning purposes in jurisdictions throughout the United States and around the world. This paper aims to develop baseline expected safety performance functions of rural signalized intersections in South Korea, which to date have not yet been established or reported in the literature. Data are examined from numerous locations within South Korea for both three-legged and four-legged configurations. The safety effects of a host of operational and geometric variables on the safety performance of these sites are also examined. In addition, supplementary tables and graphs are developed for comparing the baseline safety performance of sites with various geometric and operational features. These graphs identify how various factors are associated with safety. The expected safety prediction tables offer advantages over regression prediction equations by allowing the safety manager to isolate specific features of the intersections and examine their impact on expected safety. The examination of the expected safety performance tables through illustrated examples highlights the need to correct for regression-to-the-mean effects, emphasizes the negative impacts of multicollinearity, shows why multivariate models do not translate well to accident modification factors, and illuminates the need to examine road safety carefully and methodically. Caveats are provided on the use of the safety performance prediction graphs developed in this paper.

Across-country comparisons of selected infant and young child feeding indicators and associated factors in four South Asian countries

Background: Information on infant and young child feeding is widely available in Demographic and Health Surveys and National Family Health Surveys for countries in South Asia; however, infant and young child feeding indicators from these surveys have not been compared between countries in the region. Objective. To compare the key indicators of breastfeeding and complementary feeding and their determinants in children under 24 months of age between four South Asian countries. Methods: We selected data sets from the Bangladesh Demographic and Health Survey 2004, the India National Family Health Survey (NFHS-03) 2005–06, the Nepal Demographic and Health Survey 2006, and the Sri Lanka 2000 Demographic and Health Survey. Infant feeding indicators were estimated according to the key World Health Organization indicators. Results: Exclusive breastfeeding rates were 42.5% in Bangladesh, 46.4% in India, and 53.1% in Nepal. The rate of full breastfeeding ranged between 60.6% and 73.9%. There were no factors consistently associated with the rate of no exclusive breastfeeding across countries. Utilization of health services (more antenatal clinic visits) was associated with higher rates of exclusive breastfeeding in India but lower rates in Nepal. Delivery at a health facility was a negative determinant of exclusive breastfeeding in India. Postnatal contacts by Public Health Midwives were a positive factor in Sri Lanka. A considerable proportion of infants under 6 months of age had been given plain water, juices, or other nonmilk liquids. The rate of timely first suckling ranged from 23.5% in India to 56.3% in Sri Lanka. Delivery by cesarean section was found to be a consistent negative factor that delayed initiation of breastfeeding. Nepal reported the lowest bottle-feeding rate of 3.5%. Socioeconomically privileged mothers were found to have higher bottlefeeding rates in most countries. Conclusions: Infant and young child feeding practices in the South Asia region have not reached the expected levels that are required to achieve a substantial reduction in child mortality. The countries with lower rates of exclusive breastfeeding have a great potential to improve the rates by preventing infants from receiving water and water-based or other nonmilk liquids during the first 6 months of life.

Wavelet based approaches and high resolution methods for complex process models

Many industrial processes and systems can be modelled mathematically by a set of Partial Differential Equations (PDEs). Finding a solution to such a PDF model is essential for system design, simulation, and process control purpose. However, major difficulties appear when solving PDEs with singularity. Traditional numerical methods, such as finite difference, finite element, and polynomial based orthogonal collocation, not only have limitations to fully capture the process dynamics but also demand enormous computation power due to the large number of elements or mesh points for accommodation of sharp variations. To tackle this challenging problem, wavelet based approaches and high resolution methods have been recently developed with successful applications to a fixedbed adsorption column model. Our investigation has shown that recent advances in wavelet based approaches and high resolution methods have the potential to be adopted for solving more complicated dynamic system models. This chapter will highlight the successful applications of these new methods in solving complex models of simulated-moving-bed (SMB) chromatographic processes. A SMB process is a distributed parameter system and can be mathematically described by a set of partial/ordinary differential equations and algebraic equations. These equations are highly coupled; experience wave propagations with steep front, and require significant numerical effort to solve. To demonstrate the numerical computing power of the wavelet based approaches and high resolution methods, a single column chromatographic process modelled by a Transport-Dispersive-Equilibrium linear model is investigated first. Numerical solutions from the upwind-1 finite difference, wavelet-collocation, and high resolution methods are evaluated by quantitative comparisons with the analytical solution for a range of Peclet numbers. After that, the advantages of the wavelet based approaches and high resolution methods are further demonstrated through applications to a dynamic SMB model for an enantiomers separation process. This research has revealed that for a PDE system with a low Peclet number, all existing numerical methods work well, but the upwind finite difference method consumes the most time for the same degree of accuracy of the numerical solution. The high resolution method provides an accurate numerical solution for a PDE system with a medium Peclet number. The wavelet collocation method is capable of catching up steep changes in the solution, and thus can be used for solving PDE models with high singularity. For the complex SMB system models under consideration, both the wavelet based approaches and high resolution methods are good candidates in terms of computation demand and prediction accuracy on the steep front. The high resolution methods have shown better stability in achieving steady state in the specific case studied in this Chapter.

The utilisation of skilled immigrants and skilled Refugees in countries of destination

about 82 million immigrants in the OECD area; and worldwide, there are about 191 million immigrants and displaced persons, and some 30-40 million unauthorised immigrants. Also according to recent OECD report, little in-depth research has been carried out to-date to help decision makers in government, business, and society at large, to better understand the complexities and wider consequences of future migration flows. Literatures have also indicated that the lack of a skilled population in muchneeded occupations in countries of destination have contributed to the need to bring in skilled foreign workers. Furthermore, despite current global financial crisis, some areas of occupation are in need of skilled workers such that in a job-scarce environment jobs become fewer and employers are more likely to demand skilled workers from both natives and immigrants. Global competition for labour is expected to intensify, especially for top talent, highly qualified and semi-skilled individuals. This exacerbate the problems faced by current skilled immigrants and skilled refugees, particularly those from non-main English speaking countries who are not employed at optimal skill level in countries of destination. The research study investigates whether skilled immigrants are being effectively utilised in their countries of destination, in the context of employment. In addition to skilled immigrants, data sampling will also include skilled refugees who, although arriving under the humanitarian program, possess formal qualifications from their country of origin. Underlying variables will be explored such as the strength of social capital or interpersonal ties; and human capital in terms of educational attainment and proficiency in the English Language. The aim of the study is to explain the relationship between the variables; and whether the variables influence the employment outcomes. A broad-ranging preliminary literature review has been undertaken to explore the substantial bodies of knowledge on skilled immigrants worldwide, including skilled refugees; and to investigate whether the utilisation issues are universal or specific to a country. In addition, preliminary empirical research and analysis has been undertaken, to set the research focus and to identify the problems beyond literature. Preliminary findings have indicated that immigrants and refugees from non-main English speaking countries are particularly impacted by employment issues regardless of their skills and qualifications acquired in their country of origins; compared with immigrants from main-English speaking countries. Preliminary findings from the literature review also indicate that gaps in knowledge still exist. Although the past two decades have witnessed a virtual explosion of theory and research on international migration, no in-depth research has been located that specifically links immigrants and refugees social and human capitals in terms of employment outcomes. This research study aims to fill these gaps in research; and subsequently contribute to contemporary body of knowledge in literatures on the utilisation of skilled immigrants and skilled refugees, specifically those from non-main English speaking countries. A mixed methods design will be used, which incorporates techniques from both quantitative and qualitative research traditions that will be triangulated at the end of the data collection stage.