A humanized tissue-engineered in vivo model to dissect interactions between human prostate cancer cells and human bone

Currently used xenograft models for prostate cancer bone metastasis lack the adequate tissue composition necessary to study the interactions between human prostate cancer cells and the human bone microenvironment. We introduce a tissue engineering approach to explore the interactions between human tumor cells and a humanized bone microenvironment. Scaffolds, seeded with human primary osteoblasts in conjunction with BMP7, were implanted into immunodeficient mice to form humanized tissue engineered bone constructs (hTEBCs) which consequently resulted in the generation of highly vascularized and viable humanized bone. At 12 weeks, PC3 and LNCaP cells were injected into the hTEBCs. Seven weeks later the mice were euthanized. Micro-CT, histology, TRAP, PTHrP and osteocalcin staining results reflected the different characteristics of the two cell lines regarding their phenotypic growth pattern within bone. Microvessel density, as assessed by vWF staining, showed that tumor vessel density was significantly higher in LNCaP injected hTEBC implants than in those injected with PC3 cells (p\0.001). Interestingly, PC3 cells showed morphological features of epithelial and mesenchymal phenotypes suggesting a cellular plasticity within this microenvironment. Taken together, a highly reproducible humanized model was established which is successful in generating LNCaP and PC3 tumors within a complex humanized bone microenvironment. This model simulates the conditions seen clinically more closely than any other model described in the literature to date and hence represents a powerful experimental platform that can be used in future work to investigate specific biological questions relevant to bone metastasis.

Antidepressants, but not antipsychotics, modulate GR function in human whole blood: An insight into molecular mechanisms

Clinical studies have demonstrated an impairment of glucocorticoid receptor (GR)-mediated negative feedback on the hypothalamic-pituitary-adrenal (HPA) axis in patients with major depression (GR resistance), and its resolution by antidepressant treatment. Recently, we showed that this impairment is indeed due to a dysfunction of GR in depressed patients (Carvalho et al., 2009), and that the ability of the antidepressant clomipramine to decrease GR function in peripheral blood cells is impaired in patients with major depression who are clinically resistant to treatment (Carvalho et al. 2008). To further investigate the effect of antidepressants on GR function in humans, we have compared the effect of the antidepressants clomipramine, amytriptiline, sertraline, paroxetine and venlafaxine, and of the antipsychotics, haloperidol and risperidone, on GR function in peripheral blood cells from healthy volunteers (n=33). GR function was measured by glucocorticoid inhibition of lypopolysaccharide (LPS)-stimulated interleukin-6 (IL-6) levels. Compared to vehicle-treated cells, all antidepressants inhibited dexamethasone (DEX, 10-100nM) inhibition of LPS-stimulated IL-6 levels (p values ranging from 0.007 to 0.1). This effect was specific to antidepressants, as antipsychotics had no effect on DEX-inhibition of LPS-stimulated IL-6 levels. The phosphodiesterase (PDE) type 4 inhibitor, rolipram, potentiated the effect of antidepressants on GR function, while the GR antagonist, RU-486, inhibited the effect of antidepressants on GR function. These findings indicate that the effect of antidepressants on GR function are specific for this class of psychotropic drugs, and involve second messenger pathways relevant to GR function and inflammation. Furthermore, it also points towards a possible mechanism by which one maybe able to overcome treatment-resistant depression. Research in this field will lead to new insights into the pathophysiology and treatment of affective disorders.

B-adrenoceptor determinants of contractility in the human heart: The role of phosphodiesterase enzymes

This thesis investigated how enzymes called phosphodiesterases control changes in contractility mediated by noradrenaline and adrenaline through activation of β1- and β2-adrenoceptors in live human heart tissue from patients with advanced heart failure undergoing transplantation. The study compared patients who had been administered β-blocker medicines metoprolol or carvedilol or no β-blocker treatment. This work helped to further elucidate the complex roles of target receptors and enzymes that are integral to the progression of heart failure, to compare the mechanisms of action of β-blockers currently used to manage heart failure and to identify new drug targets for heart failure treatment.

Design of a tablet sized human-computer interface and its effect on situation awareness

Emergency Response Teams increasingly use interactive technology to help manage information and communications. The challenge is to maintain a high situation awareness for different interactive devices sizes. This research specifically compared a handheld interactive device in the form of an iPad with a large interactive multi-touch tabletop. A search and rescue inspired simulator was designed to test operator situation awareness for the two sized devices. The results show that operators had better situation awareness on the tabletop device when the operation related to detecting of moving targets, searching target locations, distinguishing target types, and comprehending displayed information.

Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: Implications for xenograft models of human prostate cancer

Background Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. Methods An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. Results At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. Conclusion These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.

Environmental crime, human rights and green criminology

This chapter is concerned with the prospects for a safe and sustainable environment in a fair and just world. At present, these prospects look bleak. However there are a number of legal developments and ethical principles on which to build, including the European Convention on the Protection of the Environment through Criminal Law, notions of environmental, ecological and species justice, and conceptions of human rights. The chapter considers these in five sections: first providing an overview and exploring the links between human rights and environmental issues; then examining examples of environmental crimes / harms and attempts to regulate or criminalise these; before outlining the development of a Green Criminology and proposals for an international law against ecocide as a framework for addressing this range of challenges. Finally, concluding comments draw attention to debates and directions for discussion and research.

Blue future: Maude Barlow, water rights, investor clauses, and trade deals

Maude Barlow is the chairperson of the Council of Canadians, and the founder of the Blue Planet Project. She is a recipient of Sweden’s Right Livelihood Award, and a Lannan Cultural Freedom Fellowship. As well as being a noted human rights and trade activist, Barlow is the author of a number of books on water rights — including Blue Gold, Blue Covenant, and Blue Future. She has been particularly vocal on the impact of trade and investment agreements upon water rights. Barlow has been critical of the push to include investor-state dispute settlement clauses in trade agreements — such as the Comprehensive Economic and Trade Agreement (CETA) between Canada and the European Union, the Trans-Pacific Partnership (TPP), and the Trans-Atlantic Trade and Investment Partnership Agreement (TTIP). She has also been concerned by the Trade in Services Agreement (TISA) leaked by WikiLeaks.

Michael Kirby's challenge: Intellectual property, HIV/AIDS, and human rights

In July 2014, Melbourne hosted the 20th International AIDS Conference. The event opened, paying tribute to the late Dutch HIV/AIDS researcher Professor Joep Lange, with his image projected onto a screen, with the accompanying quotation: ‘If we can bring a bottle of Coke to every corner of Africa, we should be able to also deliver antiretroviral drugs.’

Lock the gate: Fracking, investor-state dispute settlement, and the Trans-Pacific Partnership

There has been much debate about the relationship between international trade, the environment, biodiversity protection, and climate change.The Obama Administration has pushed such issues into sharp relief, with its advocacy for sweeping international trade agreements, such as the Trans-Pacific Partnership and the Trans-Atlantic Trade and Investment Partnership. There has been much public concern about the impact of the mega-trade deals upon the protection of the environment. In particular, there has been a debate about whether the Trans-Pacific Partnership will promote dirty fracking. Will the Trans-Pacific Partnership transform the Pacific Rim into a Gasland?There has been a particular focus upon investor-state dispute settlement being used by unconventional mining companies. Investor-state dispute settlement is a mechanism which enables foreign investors to seek compensation from national governments at international arbitration tribunals. In her prescient 2009 book, The Expropriation of Environmental Governance, Kyla Tienhaara foresaw the rise of investor-state dispute resolution of environmental matters. She observed:'Over the last decade there has been an explosive increase of cases investment arbitration. This is significant in terms of not only the number of disputes that have arisen and the number of states that have been involved, but also the novel types of dispute that have emerged. Rather than solely involving straightforward incidences of nationalization or breach of contract, modern disputes often revolve around public policy measures and implicate sensitive issues such as access to drinking water, development on sacred indigenous sites and the protection of biodiversity.'In her study, Kyla Tienhaara observed that investment agreements, foreign investment contracts and investment arbitration had significant implications for the protection for the protection of the environment. She concluded that arbitrators have made it clear that they can, and will, award compensation to investors that claim to have been harmed by environmental regulation. She also found that some of the cases suggest that the mere threat of arbitration is sufficient to chill environmental policy development. Tienhaara was equally concerned by the possibility that a government may use the threat of arbitration as an excuse or cover for its failure to improve environmental regulation. In her view, it is evident that arbitrators have expropriated certain fundamental aspects of environmental governance from states. Tienhaara held: As a result, environmental regulation has become riskier, more expensive, and less democratic, especially in developing countries. This article provides a comparative analysis of the battles over fracking, investment, trade, and the environment in a number of key jurisdictions including the United States, Canada, Australia, and New Zealand. Part 1 focuses upon the United States. Part 2 examines the dispute between the Lone Pine Resources Inc. and the Government of Canada over a fracking moratorium in Quebec. Part 3 charts the rise of the Lock the Gate Alliance in Australia, and its demands for a moratorium in respect of coal seam gas and unconventional mining. Part 4 focuses upon parallel developments in New Zealand. This article concludes that Pacific Rim countries should withdraw from investor-state dispute settlement procedures, because of the threat posed to environmental regulation in respect of air, land, and water.

The last taboo: Patenting human beings

This article considers the efforts of the Australian Law Reform Commission to clarify the meaning of section 18(2) of the Australian Patents Act 1990 (Cth): ’Human beings and the biological processes for their generation are not patentable inventions.' It provides a critique of the proposals of the Commission with respect to patent law and stem cell research. The Commission has recommended that IP Australia should develop examination guidelines to explain how the criteria for patentability apply to inventions involving stem cell technologies. It has advised the Australian Government that the practice code of the United Kingdom Patent Office (UKPO) would be a good model for such guidelines, with its distinction between totipotent and pluripotent stem cells. Arguably, though, there is a need to codify this proposal in a legislative directive, and not merely in examination guidelines. The Commission has been reluctant to take account of the ethical considerations with respect to patent law and stem cell research. There could be greater scope for such considerations, by the use of expert advisory boards, opposition proceedings and the requirement of informed consent. The Commission has put forward a number of general and specific recommendations to enhance access to patented stem cell technologies. It recommends the development of a research exemption, and the modernisation of compulsory licensing and crown use provisions. It also explores the establishment of a stem cell bank and the promulgation of guidelines by funding agencies. Such proposals to promote greater public access to stem cell research are to be welcomed.

Comparison of human and animal Chlamydia pneumoniae responses to interferon gamma and penicillin treatment

This thesis has made a significant contribution to future chlamydial research by uncovering the chlamydial pathogenic mechanisms which will potentially help in the development of targeted vaccine against the pathogen. This thesis has made important new contributions to our understanding of Chlamydia pneumoniae specific adaptations to stress responses and has provided new perspectives on the survival of this successful pathogen. This thesis has used two well established microbial stressors and has identified major differences in stress responses between human and animal Chlamydia pneumoniae isolates.

Development of a new ultrasound-based system for tracking motion of the human lumbar spine: Reliability, stability and repeatability during forward bending movement trials

The aim of this study was to develop a new method for quantifying intersegmental motion of the spine in an instrumented motion segment L4–L5 model using ultrasound image post-processing combined with an electromagnetic device. A prospective test–retest design was employed, combined with an evaluation of stability and within- and between-day intra-tester reliability during forward bending by 15 healthy male patients. The accuracy of the measurement system using the model was calculated to be ± 0.9° (standard deviation = 0.43) over a 40° range and ± 0.4 cm (standard deviation = 0.28) over 1.5 cm. The mean composite range of forward bending was 15.5 ± 2.04° during a single trial (standard error of the mean = 0.54, coefficient of variation = 4.18). Reliability (intra-class correlation coefficient = 2.1) was found to be excellent for both within-day measures (0.995–0.999) and between-day measures (0.996–0.999). Further work is necessary to explore the use of this approach in the evaluation of biomechanics, clinical assessments and interventions.

Hierarchical topological network analysis of anatomical human brain connectivity and differences related to sex and kinship

Modern non-invasive brain imaging technologies, such as diffusion weighted magnetic resonance imaging (DWI), enable the mapping of neural fiber tracts in the white matter, providing a basis to reconstruct a detailed map of brain structural connectivity networks. Brain connectivity networks differ from random networks in their topology, which can be measured using small worldness, modularity, and high-degree nodes (hubs). Still, little is known about how individual differences in structural brain network properties relate to age, sex, or genetic differences. Recently, some groups have reported brain network biomarkers that enable differentiation among individuals, pairs of individuals, and groups of individuals. In addition to studying new topological features, here we provide a unifying general method to investigate topological brain networks and connectivity differences between individuals, pairs of individuals, and groups of individuals at several levels of the data hierarchy, while appropriately controlling false discovery rate (FDR) errors. We apply our new method to a large dataset of high quality brain connectivity networks obtained from High Angular Resolution Diffusion Imaging (HARDI) tractography in 303 young adult twins, siblings, and unrelated people. Our proposed approach can accurately classify brain connectivity networks based on sex (93% accuracy) and kinship (88.5% accuracy). We find statistically significant differences associated with sex and kinship both in the brain connectivity networks and in derived topological metrics, such as the clustering coefficient and the communicability matrix.