Mechanical influences on endothelial cell network formation in vitro

While both the restoration of the blood supply and an appropriate local mechanical environment are critical for uneventful bone healing, their influence on each other remains unclear. Human bone fracture haematomas (<72h post-trauma) were cultivated for 3 days in fibrin matrices, with or without cyclic compression. Conditioned medium from these cultures enhanced the formation of vessel-like networks by HMEC-1 cells, and mechanical loading further elevated it, without affecting the cells’ metabolic activity. While haematomas released the angiogenesis-regulators, VEGF and TGF-β1, their concentrations were not affected by mechanical loading. However, direct cyclic stretching of the HMEC-1 cells decreased network formation. The appearance of the networks and a trend towards elevated VEGF under strain suggested physical disruption rather than biochemical modulation as the responsible mechanism. Thus, early fracture haematomas and their mechanical loading increase the paracrine stimulation of endothelial organisation in vitro, but direct periodic strains may disrupt or impair vessel assembly in otherwise favourable conditions.

Active awareness : supporting the intentional disclosure of awareness information in collaborative systems

This thesis opens up the design space for awareness research in CSCW and HCI. By challenging the prevalent understanding of roles in awareness processes and exploring different mechanisms for actively engaging users in the awareness process, this thesis provides a better understanding of the complexity of these processes and suggests practical solutions for designing and implementing systems that support active awareness. Mutual awareness, a prominent research topic in the fields of Computer-Supported Cooperative Work (CSCW) and Human-Computer Interaction (HCI) refers to a fundamental aspect of a person’s work: their ability to gain a better understanding of a situation by perceiving and interpreting their co-workers actions. Technologically-mediated awareness, used to support co-workers across distributed settings, distinguishes between the roles of the actor, whose actions are often limited to being the target of an automated data gathering processes, and the receiver, who wants to be made aware of the actors’ actions. This receiver-centric view of awareness, focusing on helping receivers to deal with complex sets of awareness information, stands in stark contrast to our understanding of awareness as social process involving complex interactions between both actors and receivers. It fails to take into account an actors’ intimate understanding of their own activities and the contribution that this subjective understanding could make in providing richer awareness information. In this thesis I challenge the prevalent receiver-centric notion of awareness, and explore the conceptual foundations, design, implementation and evaluation of an alternative active awareness approach by making the following five contributions. Firstly, I identify the limitations of existing awareness research and solicit further evidence to support the notion of active awareness. I analyse ethnographic workplace studies that demonstrate how actors engage in an intricate interplay involving the monitoring of their co-workers progress and displaying aspects of their activities that may be of relevance to others. The examination of a large body of awareness research reveals that while disclosing information is a common practice in face-to-face collaborative settings it has been neglected in implementations of technically mediated awareness. Based on these considerations, I introduce the notion of intentional disclosure to describe the action of users actively and deliberately contributing awareness information. I consider challenges and potential solutions for the design of active awareness. I compare a range of systems, each allowing users to share information about their activities at various levels of detail. I discuss one of the main challenges to active awareness: that disclosing information about activities requires some degree of effort. I discuss various representations of effort in collaborative work. These considerations reveal that there is a trade-off between the richness of awareness information and the effort required to provide this information. I propose a framework for active awareness, aimed to help designers to understand the scope and limitations of different types of intentional disclosure. I draw on the identified richness/effort trade-off to develop two types of intentional disclosure, both of which aim to facilitate the disclosure of information while reducing the effort required to do so. For both of these approaches, direct and indirect disclosure, I delineate how they differ from related approaches and define a set of design criteria that is intended to guide their implementation. I demonstrate how the framework of active awareness can be practically applied by building two proof-of-concept prototypes that implement direct and indirect disclosure respectively. AnyBiff, implementing direct disclosure, allows users to create, share and use shared representations of activities in order to express their current actions and intentions. SphereX, implementing indirect disclosure, represents shared areas of interests or working context, and links sets of activities to these representations. Lastly, I present the results of the qualitative evaluation of the two prototypes and analyse the results with regard to the extent to which they implemented their respective disclosure mechanisms and supported active awareness. Both systems were deployed and tested in real world environments. The results for AnyBiff showed that users developed a wide range of activity representations, some unanticipated, and actively used the system to disclose information. The results further highlighted a number of design considerations relating to the relationship between awareness and communication, and the role of ambiguity. The evaluation of SphereX validated the feasibility of the indirect disclosure approach. However, the study highlighted the challenges of implementing cross-application awareness support and translating the concept to users. The study resulted in design recommendations aimed to improve the implementation of future systems.

Are in vitro estimates of cell diffusivity and cell proliferation rate sensitive to assay geometry?

Cells respond to various biochemical and physical cues during wound–healing and tumour progression. In vitro assays used to study these processes are typically conducted in one particular geometry and it is unclear how the assay geometry affects the capacity of cell populations to spread, or whether the relevant mechanisms, such as cell motility and cell proliferation, are somehow sensitive to the geometry of the assay. In this work we use a circular barrier assay to characterise the spreading of cell populations in two different geometries. Assay 1 describes a tumour–like geometry where a cell population spreads outwards into an open space. Assay 2 describes a wound–like geometry where a cell population spreads inwards to close a void. We use a combination of discrete and continuum mathematical models and automated image processing methods to obtain independent estimates of the effective cell diffusivity, D, and the effective cell proliferation rate, λ. Using our parameterised mathematical model we confirm that our estimates of D and λ accurately predict the time–evolution of the location of the leading edge and the cell density profiles for both assay 1 and assay 2. Our work suggests that the effective cell diffusivity is up to 50% lower for assay 2 compared to assay 1, whereas the effective cell proliferation rate is up to 30% lower for assay 2 compared to assay 1.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Characterization of ENU-induced mutations in red blood cell structural proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

Supporting future curriculum leaders in embedding Indigenous knowledge on teaching practicum

This report documents the outcomes of the OLT funded project on Supporting Future Curriculum Leaders in Embedding Indigenous Knowledges on Teaching Practicum. This project investigated the learning and teaching relationships between pre-service teachers and their supervisors on practicum, with pre-service teachers who were specifically engaged (Aboriginal and Torres Strait Islander and non-Indigenous pre-service teachers studying the Indigenous Studies minor) with embedding Indigenous knowledge and perspectives in their teaching practice. It explored the negotiations of expectations, role modelling and the interactions that occur between pre-service teachers, their supervising teachers and QUT staff involved in supporting teaching practicum. The intent was to design a model to develop long term, future-oriented opportunities for teachers to develop expertise in embedding Indigenous knowledge and perspectives in curriculum, pedagogy and assessment.

Simulation of plant cell shrinkage during drying : A SPH–DEM approach

Plant based dried food products are popular commodities in global market where much research is focused to improve the products and processing techniques. In this regard, numerical modelling is highly applicable and in this work, a coupled meshfree particle-based two-dimensional (2-D) model was developed to simulate micro-scale deformations of plant cells during drying. Smoothed Particle Hydrodynamics (SPH) was used to model the viscous cell protoplasm (cell fluid) by approximating it to an incompressible Newtonian fluid. The visco-elastic characteristic of the cell wall was approximated to a Neo-Hookean solid material augmented with a viscous term and modelled with a Discrete Element Method (DEM). Compared to a previous work [H. C. P. Karunasena, W. Senadeera, Y. T. Gu and R. J. Brown, Appl. Math. Model., 2014], this study proposes three model improvements: linearly decreasing positive cell turgor pressure during drying, cell wall contraction forces and cell wall drying. The improvements made the model more comparable with experimental findings on dried cell morphology and geometric properties such as cell area, diameter, perimeter, roundness, elongation and compactness. This single cell model could be used as a building block for advanced tissue models which are highly applicable for product and process optimizations in Food Engineering.

Practices of alternative schools in Queensland in supporting Aboriginal and Torres Strait Islander young people to remain engaged in education

Alternative schools are an emerging model of education offered to young people who have been disenfranchised from conventional schooling opportunities. The body of literature on alternative schools in Australia has not identified how many Indigenous young people are engaged with alternative schools and how alternative schools are supporting Indigenous young people to remain engaged in education. It is well documented that Aboriginal and Torres Strait Islander people experience significant disadvantage including poorer educational outcomes than their non-Indigenous peers. This study seeks to contribute to improving educational outcomes for Aboriginal and Torres Strait Islander young people through exploring Aboriginal and Torres Strait Islander interactions with alternative schools in Queensland and investigating the practices of alternative school leaders in terms of how they are supporting Indigenous young people to remain engaged in education. Critical race theory informed the development of this study. An Aboriginal researcher sought to shift the focus of this study away from Indigenous young people to Principals; to explore their perspective of their own knowledge and practices in supporting Aboriginal and Torres Strait Islander young people at their school. Using survey methodology, a web-based questionnaire was developed to survey Principals’ providing data on alternative schools in Queensland including the demographics of the alternative school; self-reported knowledge of Indigenous cultures and communities and practices that support Aboriginal and Torres Strait Islander young people at their alternative school. There are nine key findings that emerged through the analysis of this study: key finding one is the high percentage of Aboriginal and Torres Strait Islander young people enrolled in schools surveyed; key finding two is there is a high percentage of Aboriginal and Torres Strait Islander staff employed in the schools; key finding three is the majority of the schools are located in low socio-economic areas; key finding four is the strong willingness of Principals’ in this study to engage in self-directed learning in relation to Aboriginal and Torres Strait Islander cultures; key finding five is the limited demonstration of understandings of Aboriginal and Torres Strait Islander cultures and communities; key finding six is the most prevalent practice of Principals’ in this study is the celebration of cultural events and cultural activities; key finding seven is the limited Principal engagement with Aboriginal and Torres Strait Islander young people, their families and the local community; key finding eight is the practice of alternative schools provides limited support and nurturing of Aboriginal and Torres Strait Islander young person’s cultural identity and key finding nine is that Principals’ are relying heavily on informal discussions with staff to know what their staff’s knowledge and skills are in relation to supporting Aboriginal and Torres Strait Islander young people. There are multiple implications that have arisen from this study. The data demonstrated high numbers of Aboriginal and Torre Strait Islander students and staff. The data also revealed that Principal’s demonstration of knowledge in relation to Indigenous cultures and communities was limited, as well as limited Principal engagement with Indigenous young peoples, families and communities. Therefore a major practical implication of this study is the urgent need for quality cultural learning opportunities for leaders of alternative schools to improve practices. Additionally, the implications of this study support an urgent need for further research on the role alternative schools are playing in supporting Indigenous young people to remain engaged in education.

Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis.

Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden

Over 80% of women diagnosed with advanced-stage ovarian cancer die as a result of disease recurrence due to failure of chemotherapy treatment. In this study, using two distinct ovarian cancer cell lines (epithelial OVCA 433 and mesenchymal HEY) we demonstrate enrichment in a population of cells with high expression of CSC markers at the protein and mRNA levels in response to cisplatin, paclitaxel and the combination of both. We also demonstrate a significant enhancement in the sphere forming abilities of ovarian cancer cells in response to chemotherapy drugs. The results of these in vitro findings are supported by in vivo mouse xenograft models in which intraperitoneal transplantation of cisplatin or paclitaxel-treated residual HEY cells generated significantly higher tumor burden compared to control untreated cells. Both the treated and untreated cells infiltrated the organs of the abdominal cavity. In addition, immunohistochemical studies on mouse tumors injected with cisplatin or paclitaxel treated residual cells displayed higher staining for the proliferative antigen Ki67, oncogeneic CA125, epithelial E-cadherin as well as cancer stem cell markers such as Oct4 and CD117, compared to mice injected with control untreated cells. These results suggest that a short-term single treatment of chemotherapy leaves residual cells that are enriched in CSC-like traits, resulting in an increased metastatic potential. The novel findings in this study are important in understanding the early molecular mechanisms by which chemoresistance and subsequent relapse may be triggered after the first line of chemotherapy treatment.

Artefact ecologies : supporting embodied meeting practices with distance access

Frameworks such as activity theory, distributed cognition and structuration theory, amongst others, have shown that detailed study of contextual settings where users work (or live) can help the design of interactive systems. However, these frameworks do not adequately focus on accounting for the materiality (and embodiment) of the contextual settings. Within the IST-EU funded AMIDA project (Augmented Multiparty Interaction with Distance Access) we are looking into supporting meeting practices with distance access. Meetings are inherently embodied in everyday work life and that material artefacts associated with meeting practices play a critical role in their formation. Our eventual goal is to develop a deeper understanding of the dynamic and embodied nature of meeting practices and designing technologies to support these. In this paper we introduce the notion of "artefact ecologies" as a conceptual base for understanding embodied meeting practices with distance access. Artefact ecologies refer to a system consisting of different digital and physical artefacts, people, their work practices and values and lays emphasis on the role artefacts play in embodiment, work coordination and supporting remote awareness. In the end we layout our plans for designing technologies for supporting embodied meeting practices within the AMIDA project.

Automatic classification of human epithelial type 2 cell indirect immunofluorescence images using cell pyramid matching

This paper describes a novel system for automatic classification of images obtained from Anti-Nuclear Antibody (ANA) pathology tests on Human Epithelial type 2 (HEp-2) cells using the Indirect Immunofluorescence (IIF) protocol. The IIF protocol on HEp-2 cells has been the hallmark method to identify the presence of ANAs, due to its high sensitivity and the large range of antigens that can be detected. However, it suffers from numerous shortcomings, such as being subjective as well as time and labour intensive. Computer Aided Diagnostic (CAD) systems have been developed to address these problems, which automatically classify a HEp-2 cell image into one of its known patterns (eg. speckled, homogeneous). Most of the existing CAD systems use handpicked features to represent a HEp-2 cell image, which may only work in limited scenarios. We propose a novel automatic cell image classification method termed Cell Pyramid Matching (CPM), which is comprised of regional histograms of visual words coupled with the Multiple Kernel Learning framework. We present a study of several variations of generating histograms and show the efficacy of the system on two publicly available datasets: the ICPR HEp-2 cell classification contest dataset and the SNPHEp-2 dataset.

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.

Differentiation state and invasiveness of human breast cancer cell lines

Eighteen breast cancer cell lines were examined for expression of markers of epithelial and fibroblastic differentiation: E-cadherin, desmoplakins, ZO- 1, vimentin, keratin and β1 and β4 integrins. The cell lines were distributed along a spectrum of differentiation from epithelial to fibroblastic phenotypes. The most well-differentiated, epithelioid cell lines contained proteins characteristic of desmosomal, adherens and tight junctions, were adherent to one another on plastic and in the basement membrane matrix Matrigel and were keratin-positive and vimentin-negative. These cell lines were all weakly invasive in an in vitro chemoinvasion assay. The most poorly-differentiated, fibroblastic cell lines were E-cadherin-, desmoplakin- and ZO-1-negative and formed branching structures in Matrigel. They were vimentin-positive, contained only low levels of keratins and were highly invasive in the in vitro chemoinvasion assay. Of all of the markers analyzed, vimentin expression correlated best with in vitro invasive ability and fibroblastic differentiation. In a cell line with unstable expression of vimentin, T47D(CO), the cells that were invasive were of the fibroblastic type. The differentiation markers described here may be useful for analysis of clinical specimens and could potentially provide a more precise measure of differentiation grade yielding more power for predicting prognosis.

Sphingosine-1-phosphate, a novel second messenger involved in cell growth regulation and signal transduction, affects growth and invasiveness of human breast cancer cells

This review will focus on the role of sphingosine and its phosphorylated derivative sphingosine-1-phosphate (SPP) in cell growth regulation and signal transduction. We will show that many of the effects attributed to sphingosine in quiescent Swiss 3T3 fibroblasts are mediated via its conversion to SPP. We propose that SPP has appropriate properties to function as an intracellular second messenger based on the following: it elicits diverse cellular responses; it is rapidly produced from sphingosine by a specific kinase and rapidly degraded by a specific lyase; its concentration is low in quiescent cells but increases rapidly and transiently in response to the growth factors, fetal calf serum (FCS) and platelet derived growth factor (PDGF); it releases Ca2+ from internal sources in an InsP3-independent manner; and finally, it may link sphingolipid signaling pathways to cellular ras-mediated signaling pathways by elevating phosphatidic acid levels. The effects of this novel second messenger on growth, differentiation and invasion of human breast cancer cells will be discussed. © 1994 Kluwer Academic Publishers.