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.

Artful inquiry : an arts-based facilitation approach for individual and organisational learning and development

The research undertaken in these two major doctoral studies investigates the field of artsbased learning, a pedagogical approach to individual and organisational learning and development, my professional creative facilitation practice and development as a researcher. While the studies are stand-alone projects they are intended to build on each other in order to tell the evolving story of my research and professional practice. The first study combines The Role of Arts-based Learning in a Creative Economy; The Need for Artistry in Professional Education the art of knowing what to do when you don’t know what to do and Lines of Inquiry: Making Sense of Research and Professional Practice. The Role of Arts-based Learning in a Creative Economy provides an overview of the field of arts-based learning in business. The study focuses on the relevant literature and interviews with people working in the field. The paper argues that arts-based learning is a valuable addition to organisations for building a culture of creativity and innovation. The Need for Artistry in Professional Education continues that investigation. It explores the way artists approach their work and considers what skills and capabilities from artistic practice can be applied to other professions’ practices. From this research the Sphere of Professional Artistry model is developed and depicts the process of moving toward professional artistry. Lines of Inquiry: making sense of research and professional practice through artful inquiry is a self-reflective study. It explores my method of inquiry as a researcher and as a creative facilitation practitioner using arts-based learning processes to facilitate groups of people for learning, development and change. It discusses how my research and professional practice influence and inspire the other and draws on cased studies. The second major research study Artful Inquiry: Arts-based Learning for Inquiry, Reflection and Action in Professional Practice is a one year practice-led inquiry. It continues the research into arts-based and aesthetic learning experiences and my arts-based facilitation practice. The research is conducted with members of a Women’s Network in a large government service agency. It develops the concept of ‘Artful Inquiry’’ a creative, holistic, and embodied approach for facilitation, inquiry, learning, reflection, and action. Storytelling as Inquiry is used as a methodology for understanding participants’ experiences of being involved in arts-based learning experiences. The study reveals the complex and emergent nature of practice and research. It demonstrates what it can mean to do practice-led research with others, within an organisational context, and to what effect.

Work integrated learning for life : encouraging agentic engagement

In a rapidly changing world where new work patterns impact on our health, relationships and social fabric, it is critical that we reconsider the role universities could or should play in helping students prepare for the complexities of the 21st century. Efforts to respond to economic imperatives such as the skills shortage have seen a rush to embed work integrated and career development learning in the curriculum as well as a strengthening of the discourse that the university’s role is primarily to produce industry ready or ‘oven ready and self basting’ graduates (Atkins, 1999). This narrow focus on ‘giving industry what industry wants’ (Patrick, Peach & Pocknee, 2009) ignores the importance of helping students develop the types of skills and dispositions they will need. To enable students to thrive not just survive socially and economically in a radically unknowable world, where knowledge becomes obsolete, we need to be ready to develop new futures (Barnett, 2004). This paper considers the concept of ‘work’, the role it plays in our lives, and our aspirations to build sustainable, socially connected communities. We revisit the assumptions underlying the employability argument (Atkins, 1999) in the light of changing notions of work (Hagel, Seely Brown & Davison, 2010), and the need for higher education to contribute to a better and more sustainable society (Pocock, 2003). Specifically we present initiatives developed from work integrated learning (WIL) programs in the United Kingdom and Australia, where WIL programs are framed within the broader context of real world and life-wide curriculum (Jackson, 2010), and where transferable skills and elements of work-related learning programs prepare students for less certain job futures. Such approaches encourage students to take an agentic role (Billett & Pavlova, 2005) in selecting their work possibilities to develop resilience and capabilities to deal with new and challenging situations, assisting students to become who they want to be not just what they want to be. The theoretical and operational implications and challenges of shaping real world and life-wide curriculum will be investigated in more depth in the next phase of this research.

What it means to become a public relations professional: student perceptions of professional identity through real-world learning

Public relations educators need new solutions to prepare students to become tomorrow's practitioner today. Managers and employers in the new creative workforce (McWilliam, 2008) expect graduates to be problem solvers, critical and creative thinkers, reflective, and self reliant (Barrie, 2008; David, 2004). Enabling students to develop these attributes requires a collaborative and creative approach to pedagogy (Jeffrey & Craft, 2001, 2004). A model for the next generation of public relations education was developed to integrate industry partnerships as a way to bridge pedagogy and professional practice. The model suggests (a) that industry partnerships be embedded in learning activities, (b) that assessment items be considered on a continuum and delivered incrementally across a course of study, and (c) that connections between classroom and workplace activities are clearly signposted for students.

Surrogacy : is it harder to relinquish genes?

Surrogacy has produced some positive outcomes creating an opportunity for otherwise childless couples to live their dream of parenthood. However it has also been problematic, particularly where the surrogate mother fails to relinquish a child born as a result of the surrogacy arrangement. This article examines whether a surrogate mother, who is genetically related to the child she delivers, is less likely to relinquish the child than one who has no genetic ties. An examination of empirical evidence provides support for this argument. Legislation and case law in three jurisdictions, Australia, the United States and the United Kingdom, is examined to determine which, if any, of these jurisdictions take into account the existence, or otherwise, of a genetic link between the surrogate mother and the child she bears. The article concludes that surrogacy legislation should, subject to exceptional circumstances, encourage surrogacy arrangements where the child and the surrogate are not genetically related.

What is food literacy and does it influence what we eat : a study of Australian food experts

This report details the findings of the Food Literacy Delphi Study. This study was the first of three studies commissioned by Health Promotion Queensland, Queensland Health. It examines the views of experts from around Australia about food literacy; its meaning, potential components and relationship to nutrition.

Quantifying in-plane deformation of plate elements using vibration characteristics

Plate elements are used in many engineering applications. In-plane loads and deformations have significant influence on the vibration characteristics of plate elements. Numerous methods have been developed to quantify the effects of in-plane loads and deformations of individual plate elements with different boundary conditions based on their natural frequencies. However, these developments cannot be applied to the plate elements in a structural system as the natural frequency is a global parameter for the entire structure. This highlights the need for a method to quantify in-plane deformations of plate elements in structural framing systems. Motivated by this gap in knowledge, this research has developed a comprehensive vibration based procedure to quantify in-plane deformation of plate elements in a structural framing system. This procedure with its unique capabilities to capture the influence of load migration, boundary conditions and different tributary areas is presented herein and illustrated through examples.

Mapping the language of science and science teaching practices : a case study of early childhood school science

Concerns raised in educational reports about school science in terms of students. outcomes and attitudes, as well as science teaching practices prompted investigation into science learning and teaching practices at the foundational level of school science. Without science content and process knowledge, understanding issues of modern society and active participation in decision-making is difficult. This study contended that a focus on the development of the language of science could enable learners to engage more effectively in learning science and enhance their interest and attitudes towards science. Furthermore, it argued that explicit teaching practices where science language is modelled and scaffolded would facilitate the learning of science by young children at the beginning of their formal schooling. This study aimed to investigate science language development at the foundational level of school science learning in the preparatory-school with students aged five and six years. It focussed on the language of science and science teaching practices in early childhood. In particular, the study focussed on the capacity for young students to engage with and understand science language. Previous research suggests that students have difficulty with the language of science most likely because of the complexities and ambiguities of science language. Furthermore, literature indicates that tensions transpire between traditional science teaching practices and accepted early childhood teaching practices. This contention prompted investigation into means and models of pedagogy for learning foundational science language, knowledge and processes in early childhood. This study was positioned within qualitative assumptions of research and reported via descriptive case study. It was located in a preparatory-school classroom with the class teacher, teacher-aide, and nineteen students aged four and five years who participated with the researcher in the study. Basil Bernstein.s pedagogical theory coupled with Halliday.s Systemic Functional Linguistics (SFL) framed an examination of science pedagogical practices for early childhood science learning. Students. science learning outcomes were gauged by focussing a Hallydayan lens on their oral and reflective language during 12 science-focussed episodes of teaching. Data were collected throughout the 12 episodes. Data included video and audio-taped science activities, student artefacts, journal and anecdotal records, semi-structured interviews and photographs. Data were analysed according to Bernstein.s visible and invisible pedagogies and performance and competence models. Additionally, Halliday.s SFL provided the resource to examine teacher and student language to determine teacher/student interpersonal relationships as well as specialised science and everyday language used in teacher and student science talk. Their analysis established the socio-linguistic characteristics that promoted science competencies in young children. An analysis of the data identified those teaching practices that facilitate young children.s acquisition of science meanings. Positive indications for modelling science language and science text types to young children have emerged. Teaching within the studied setting diverged from perceived notions of common early childhood practices and the benefits of dynamic shifting pedagogies were validated. Significantly, young students demonstrated use of particular specialised components of school-science language in terms of science language features and vocabulary. As well, their use of language demonstrated the students. knowledge of science concepts, processes and text types. The young students made sense of science phenomena through their incorporation of a variety of science language and text-types in explanations during both teacher-directed and independent situations. The study informs early childhood science practices as well as practices for foundational school science teaching and learning. It has exposed implications for science education policy, curriculum and practices. It supports other findings in relation to the capabilities of young students. The study contributes to Systemic Functional Linguistic theory through the development of a specific resource to determine the technicality of teacher language used in teaching young students. Furthermore, the study contributes to methodology practices relating to Bernsteinian theoretical perspectives and has demonstrated new ways of depicting and reporting teaching practices. It provides an analytical tool which couples Bernsteinian and Hallidayan theoretical perspectives. Ultimately, it defines directions for further research in terms of foundation science language learning, ongoing learning of the language of science and learning science, science teaching and learning practices, specifically in foundational school science, and relationships between home and school science language experiences.

The challenge of assessing professional competence in work integrated learning

A fundamental aspect of work integrated learning (WIL) is the development of professional competence, the ability of students to perform in the work place. Alignment theory therefore suggests that the assessment of WIL should include an assessment of students’ demonstration of professional competence in the workplace. The assessment of professional competence in WIL is, however, problematic. It may be impractical for the academic supervisor to directly assess professional competence if there is a large number of students in external placements. If evidence of professional competence is provided by the student, the student’s ability to articulate his or her own capabilities will interfere with the validity of the assessment. If evidence of professional competency is provided by the supervisor then the assessment is heavily dependent on the individual supervisor and may be unreliable. This paper will examine the literature relating to the assessment of professional competence in WIL. The paper will be informed by the author’s experience in coordinating a WIL subject in an undergraduate law course. It will recommend that a mix of evidence provided by the student, the workplace supervisor and the academic supervisor should be used to assess professional competence in WIL.

Competences and capabilities for innovation in supply chain relationships

There is little empirical research on the relational capabilities and competences in supply chain relationships, and the important links that exist between relational capabilities, competences and supply chain innovation. This study addresses some of that knowledge gap gained through a case study of the Australian freight services sector. Findings suggest that different competences developed by supply chain participants support different ways to enable operational effectiveness. Road freight businesses should acquire knowledge of how to select and manage inter-firm relationships that provide the greatest benefit, acquiring competences that either improve their current capabilities or assist them to explore strategies that ensure incremental innovation and supply chain performance objectives are met.

Reconfiguration or innovation in supply chains?

This research explores supply chain competitiveness and dynamic capabilities. It examines a pilot group of Australian supply chain organisations to understand the importance of dynamic capability in building innovation capacity for competitive advantage, and the concept of adopting a strategic approach to supply chain relationship building. A supply chain is after all a group of intra and interorganisational relationships delivering demand to end-users. This exploratory study confirms a positive relationship between the variables indicating both a strategic intent to develop relational capability, and very strong predictive linkages between the importance placed on developing supply chain dynamic capability and achieving supply chain innovation capacity as a competitive advantage.