Towards automated and in-situ, near-real time 3-D reconstruction of coral reef environments

Coral reefs are biologically complex ecosystems that support a wide variety of marine organisms. These are fragile communities under enormous threat from natural and human-based influences. Properly assessing and measuring the growth and health of reefs is essential to understanding impacts of ocean acidification, coastal urbanisation and global warming. In this paper, we present an innovative 3-D reconstruction technique based on visual imagery as a non-intrusive, repeatable, in situ method for estimating physical parameters, such as surface area and volume for efficient assessment of long-term variability. The reconstruction algorithms are presented, and benchmarked using an existing data set. We validate the technique underwater, utilising a commercial-off-the-shelf camera and a piece of staghorn coral, Acropora cervicornis. The resulting reconstruction is compared with a laser scan of the coral piece for assessment and validation. The comparison shows that 77% of the pixels in the reconstruction are within 0.3 mm of the ground truth laser scan. Reconstruction results from an unknown video camera are also presented as a segue to future applications of this research.

Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

By the day nurses bank up in a casual pool!

We suspect that the array of silly names used to refer to temporary staff worldwide may be indicative of the extent to which these nurses have been relegated to, and we would argue, remain in, a type of underclass – relatively unsupported by employers in terms of professional practice and ipso facto excluded from contributing professionally to team work, practice development, clinical governance and evidence based practice. This may be acceptable to some but in a climate of risk averseness and in the interests of strategic planning we would suggest it is an accident waiting to happen. The recent UK Royal College of Nursing (RCN) (Ball & Pike, 2006) survey of bank and agency nurses brings a welcome focus on a group of nurses that make a significant contribution to the smooth running of health services in many countries.

The application of the statutory derivative action in Pt 2F.1A of the Corporations Act to companies in liquidation under Ch 5 : Well oiled machine or is Pt 2F.1A in need of a tune up?

The statutory derivative action was introduced in Australia in 2000. This right of action has been debated in the literature and introduced in a number of other jurisdictions as well. However, it is by no means clear that all issues have been resolved despite its operation in Australia for over 10 years. This article considers the application of Pt 2F.1A of the Corporations Act to companies in liquidation under Ch 5. It demonstrates that the application involves consideration of not only proper statutory interpretation but also policy matters around the role and the supervision by the court of a liquidator once a company has entered liquidation.

A transport protocol for real-time applications in wireless networked control systems

A Networked Control System (NCS) is a feedback-driven control system wherein the control loops are closed through a real-time network. Control and feedback signals in an NCS are exchanged among the system’s components in the form of information packets via the network. Nowadays, wireless technologies such as IEEE802.11 are being introduced to modern NCSs as they offer better scalability, larger bandwidth and lower costs. However, this type of network is not designed for NCSs because it introduces a large amount of dropped data, and unpredictable and long transmission latencies due to the characteristics of wireless channels, which are not acceptable for real-time control systems. Real-time control is a class of time-critical application which requires lossless data transmission, small and deterministic delays and jitter. For a real-time control system, network-introduced problems may degrade the system’s performance significantly or even cause system instability. It is therefore important to develop solutions to satisfy real-time requirements in terms of delays, jitter and data losses, and guarantee high levels of performance for time-critical communications in Wireless Networked Control Systems (WNCSs). To improve or even guarantee real-time performance in wireless control systems, this thesis presents several network layout strategies and a new transport layer protocol. Firstly, real-time performances in regard to data transmission delays and reliability of IEEE 802.11b-based UDP/IP NCSs are evaluated through simulations. After analysis of the simulation results, some network layout strategies are presented to achieve relatively small and deterministic network-introduced latencies and reduce data loss rates. These are effective in providing better network performance without performance degradation of other services. After the investigation into the layout strategies, the thesis presents a new transport protocol which is more effcient than UDP and TCP for guaranteeing reliable and time-critical communications in WNCSs. From the networking perspective, introducing appropriate communication schemes, modifying existing network protocols and devising new protocols, have been the most effective and popular ways to improve or even guarantee real-time performance to a certain extent. Most previously proposed schemes and protocols were designed for real-time multimedia communication and they are not suitable for real-time control systems. Therefore, devising a new network protocol that is able to satisfy real-time requirements in WNCSs is the main objective of this research project. The Conditional Retransmission Enabled Transport Protocol (CRETP) is a new network protocol presented in this thesis. Retransmitting unacknowledged data packets is effective in compensating for data losses. However, every data packet in realtime control systems has a deadline and data is assumed invalid or even harmful when its deadline expires. CRETP performs data retransmission only in the case that data is still valid, which guarantees data timeliness and saves memory and network resources. A trade-off between delivery reliability, transmission latency and network resources can be achieved by the conditional retransmission mechanism. Evaluation of protocol performance was conducted through extensive simulations. Comparative studies between CRETP, UDP and TCP were also performed. These results showed that CRETP significantly: 1). improved reliability of communication, 2). guaranteed validity of received data, 3). reduced transmission latency to an acceptable value, and 4). made delays relatively deterministic and predictable. Furthermore, CRETP achieved the best overall performance in comparative studies which makes it the most suitable transport protocol among the three for real-time communications in a WNCS.