Development of a 3D culture system to study the skeletal metastasis of prostate cancer

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex

This study describes the design of a biphasic scaffold composed of a Fused Deposition Modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) for periodontal regeneration. In order to achieve simultaneous alveolar bone and periodontal ligament regeneration a cell-based strategy was carried out by combining osteoblast culture in the bone compartment and placement of multiple periodontal ligament (PDL) cell sheets on the electrospun membrane. In vitro data showed that the osteoblasts formed mineralized matrix in the bone compartment after 21 days in culture and that the PDL cell sheet harvesting did not induce significant cell death. The cell-seeded biphasic scaffolds were placed onto a dentin block and implanted for 8 weeks in an athymic rat subcutaneous model. The scaffolds were analyzed by μCT, immunohistochemistry and histology. In the bone compartment, a more intense ALP staining was obtained following seeding with osteoblasts, confirming the μCT results which showed higher mineralization density for these scaffolds. A thin mineralized cementum-like tissue was deposited on the dentin surface for the scaffolds incorporating the multiple PDL cell sheets, as observed by H&E and Azan staining. These scaffolds also demonstrated better attachment onto the dentin surface compared to no attachment when no cell sheets were used. In addition, immunohistochemistry revealed the presence of CEMP1 protein at the interface with the dentine. These results demonstrated that the combination of multiple PDL cell sheets and a biphasic scaffold allows the simultaneous delivery of the cells necessary for in vivo regeneration of alveolar bone, periodontal ligament and cementum. © 2012 Elsevier Ltd.

A case of community safety : displacing complex ‘social’ problems in Fortitude Valley

Public dialogue regarding the high concentration of drug use and crime in inner city locations is frequently legitimised through visibility of drug-using populations and a perception of high crime rates. The public space known as the Brunswick Street Mall (Valley mall), located in the inner city Brisbane suburb of Fortitude Valley, has long provided the focal point for discussions regarding the problem of illicit drug use and antisocial behaviour in Brisbane. During the late 1990s a range of stakeholders in Fortitude Valley became mobilised to tackle crime and illicit drugs. In particular they wanted to dismantle popular perceptions of the area as representing the dark and unsafe side of Brisbane. The aim of this campaign was to instil a sense of safety in the area and dislodge Fortitude Valley from its reputation as a =symbolic location of danger‘. This thesis is a case study about an urban site that became contested by the diverse aims of a range of stakeholders who were invested in an urban renewal program and community safety project. This case study makes visible a number of actors that were lured from their existing roles in an indeterminable number of heterogeneous networks in order to create a community safety network. The following analysis of the community safety network emphasises some specific actors: history, ideas, technologies, materialities and displacements. The case study relies on the work of Foucault, Latour, Callon and Law to draw out the rationalities, background contingencies and the attempts to impose order and translate a number of entities into the community safety project in Fortitude Valley. The results of this research show that the community safety project is a case of ontological politics. Specifically the data indicates that both the (reality) problem of safety and the (knowledge) solution to safety were created simultaneously. This thesis explores the idea that while violence continues to occur in the Valley, evidence that community safety got done is located through mapping its displacement and eventual disappearance. As such, this thesis argues that community safety is a =collateral reality‘.

Wing shape as a potential discriminator of morphologically similar pest taxa within the Bactrocera dorsalis species complex (Diptera: Tephritidae)

Four morphologically cryptic species of the Bactrocera dorsalis fruit fly complex (B. dorsalis s.s., B. papayae, B. carambolae and B. philippinensis) are serious agricultural pests. As they are difficult to diagnose using traditional taxonomic techniques, we examined the potential for geometric morphometric analysis of wing size and shape to discriminate between them. Fifteen wing landmarks generated size and shape data for 245 specimens for subsequent comparisons among three geographically distinct samples of each species. Intraspecific wing size was significantly different within samples of B. carambolae and B. dorsalis s.s. but not within samples of B. papayae or B. philippinensis. Although B. papayae had the smallest wings (average centroid size=6.002 mm±0.061 SE) and B. dorsalis s.s. the largest (6.349 mm±0.066 SE), interspecific wing size comparisons were generally non-informative and incapable of discriminating species. Contrary to the wing size data, canonical variate analysis based on wing shape data discriminated all species with a relatively high degree of accuracy; individuals were correctly reassigned to their respective species on average 93.27% of the time. A single sample group of B. carambolae from locality 'TN Malaysia' was the only sample to be considerably different from its conspecific groups with regards to both wing size and wing shape. This sample was subsequently deemed to have been originally misidentified and likely represents an undescribed species. We demonstrate that geometric morphometric techniques analysing wing shape represent a promising approach for discriminating between morphologically cryptic taxa of the B. dorsalis species complex.

Simple rules to contain an invasive species with a complex life cycle and high dispersal capacity

Designing practical rules for controlling invasive species is a challenging task for managers, particularly when species are long-lived, have complex life cycles and high dispersal capacities. Previous findings derived from plant matrix population analyses suggest that effective control of long-lived invaders may be achieved by focusing on killing adult plants. However, the cost-effectiveness of managing different life stages has not been evaluated. We illustrate the benefits of integrating matrix population models with decision theory to undertake this evaluation, using empirical data from the largest infestation of mesquite (Leguminosae: Prosopis spp) within Australia. We include in our model the mesquite life cycle, different dispersal rates and control actions that target individuals at different life stages with varying costs, depending on the intensity of control effort. We then use stochastic dynamic programming to derive cost-effective control strategies that minimize the cost of controlling the core infestation locally below a density threshold and the future cost of control arising from infestation of adjacent areas via seed dispersal. Through sensitivity analysis, we show that four robust management rules guide the allocation of resources between mesquite life stages for this infestation: (i) When there is no seed dispersal, no action is required until density of adults exceeds the control threshold and then only control of adults is needed; (ii) when there is seed dispersal, control strategy is dependent on knowledge of the density of adults and large juveniles (LJ) and broad categories of dispersal rates only; (iii) if density of adults is higher than density of LJ, controlling adults is most cost-effective; (iv) alternatively, if density of LJ is equal or higher than density of adults, management efforts should be spread between adults, large and to a lesser extent small juveniles, but never saplings. Synthesis and applications.In this study, we show that simple rules can be found for managing invasive plants with complex life cycles and high dispersal rates when population models are combined with decision theory. In the case of our mesquite population, focussing effort on controlling adults is not always the most cost-effective way to meet our management objective.

Vapour phase assembly of a halogen bonded complex of an isoindoline nitroxide and 1,2-diiodotetrafluorobenzene

Vapour phase assembly has been used for the first time to prepare co-crystals in which the primary intermolecular interaction is halogen bonding. Co-crystals of the nitroxide 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) and 1,2-diiodotetrafluorobenzene (1,2-DITFB) are readily formed under standard sublimation conditions. Single crystal X-ray diffraction confirmed the structure of a 2:2 cyclic tetramer, (TMIO)2·(1,2-DITFB)2, which exhibits a new halogen bonding motif, with each nitroxide oxygen atom accepting two halogen bonds. Powder X-ray diffraction confirmed the homogeneity of the bulk sample. The crystalline complex was further characterized in the solid state using thermal analysis and vibrational spectroscopy (infrared and Raman). Density functional theory calculations were also used to evaluate the enthalpy of formation, electrostatic potential and unpaired electron density of the complex. These findings illustrate the preparation of co-crystals where solution state methodology is problematic and the potential of this approach for the formation of novel organic spin systems.

Reconstructing the event stratigraphy from the complex structural - stratigraphic architecture of an Archaean volcanic – intrusive – sedimentary succession : the Boorara Domain, Eastern Goldfields Superterrane, Western Australia

Two main deformational phases are recognised in the Archaean Boorara Domain of the Kalgoorlie Terrane, Eastern Goldfields Superterrane, Yilgarn Craton, Western Australia, primarily involving southover- north thrust faulting that repeated and thickened the stratigraphy, followed by east northeast – west-southwest shortening that resulted in macroscale folding of the greenstone lithologies. The domain preserves mid-greenschist facies metamorphic grade, with an increase to lower amphibolite metamorphic grade towards the north of the region. As a result of the deformation and metamorphism, individual stratigraphic horizons are difficult to trace continuously throughout the entire domain. Volcanological and sedimentological textures and structures, primary lithological contacts, petrography and geochemistry have been used to correlate lithofacies between faultbounded structural blocks. The correlated stratigraphic sequence for the Boorara Domain comprises quartzo-feldspathic turbidite packages, overlain by high-Mg tholeiitic basalt (lower basalt), coherent and clastic dacite facies, intrusive and extrusive komatiite units, an overlying komatiitic basalt unit (upper basalt), and at the stratigraphic top of the sequence, volcaniclastic quartz-rich turbidites. Reconstruction of the stratigraphy and consideration of emplacement dynamics has allowed reconstruction of the emplacement history and setting of the preserved sequence. This involves a felsic, mafic and ultramafic magmatic system emplaced as high-level intrusions, with localised emergent volcanic centres, into a submarine basin in which active sedimentation was occurring.

Wayfinding : a simple concept, a complex process

Wayfinding is the process of finding your way to a destination in a familiar or unfamiliar setting using any cues given by the environment. Due to its ubiquity in everyday life, wayfinding appears on the surface to be a simply characterised and understood process, however this very ubiquity and the resulting need to refine and optimise wayfinding has lead to a great number of studies that have revealed that it is in fact a deeply complex exercise. In this paper we examine the motivations for investigating wayfinding, with particular attention being paid to the unique challenges faced in transportation hubs, and discuss the associated principles and factors involved as they have been perceived from different research perspectives.We also review the approaches used to date in the modelling of wayfinding in various contexts. We attempt to draw together the different perspectives applied to wayfinding and postulate the importance of wayfinding and the need to understand this seemingly simple, but concurrently complex, process.

Teachers’ detecting and reporting child abuse and neglect : research into the determinants of complex professional behavior

Child abuse and neglect is prevalent and entails significant costs to children, families and society. Teachers are responsible for significant proportions of official notifications to statutory child protection agencies. Hence, their accurate and appropriate reporting is crucial for well-functioning child protection systems. Approximately one-quarter of Australian teachers indicate never detecting a case of child maltreatment across their careers, while a further 13-15% admit to not reporting suspected cases in some circumstances. The detection and reporting of child abuse and neglect are complex decision-making behaviors, influenced by: the nature of the maltreatment itself; the characteristics of the teacher; the school environment; and the broader legislative and policy environment. In this chapter, the authors provide a background to teachers’ involvement in detecting and reporting child abuse and neglect, and an overview of the role of teachers is provided. Results are presented from three Australian studies that examine the unique contributions of: case; teacher; and contextual characteristics to detection and reporting behaviors. The authors conclude by highlighting the key implications for enhancing teacher training in child abuse and neglect, and outline future research directions.

Human proximal tubule epithelial cells modulate autologous dendritic cell function

Background We have previously demonstrated that human kidney proximal tubule epithelial cells (PTEC) are able to modulate autologous T and B lymphocyte responses. It is well established that dendritic cells (DC) are responsible for the initiation and direction of adaptive immune responses and that these cells occur in the renal interstitium in close apposition to PTEC under inflammatory disease settings. However, there is no information regarding the interaction of PTEC with DC in an autologous human context. Methods Human monocytes were differentiated into monocyte-derived DC (MoDC) in the absence or presence of primary autologous activated PTEC and matured with polyinosinic:polycytidylic acid [poly(I:C)], while purified, pre-formed myeloid blood DC (CD1c+ BDC) were cultured with autologous activated PTEC in the absence or presence of poly(I:C) stimulation. DC responses were monitored by surface antigen expression, cytokine secretion, antigen uptake capacity and allogeneic T-cell-stimulatory ability. Results The presence of autologous activated PTEC inhibited the differentiation of monocytes to MoDC. Furthermore, MoDC differentiated in the presence of PTEC displayed an immature surface phenotype, efficient phagocytic capacity and, upon poly(I:C) stimulation, secreted low levels of pro-inflammatory cytokine interleukin (IL)-12p70, high levels of anti-inflammatory cytokine IL-10 and induced weak Th1 responses. Similarly, pre-formed CD1c+ BDC matured in the presence of PTEC exhibited an immature tolerogenic surface phenotype, strong endocytic and phagocytic ability and stimulated significantly attenuated T-cell proliferative responses. Conclusions Our data suggest that activated PTEC regulate human autologous immunity via complex interactions with DC. The ability of PTEC to modulate autologous DC function has important implications for the dampening of pro-inflammatory immune responses within the tubulointerstitium in renal injuries. Further dissection of the mechanisms of PTEC modulation of autologous immune responses may offer targets for therapeutic intervention in renal medicine.