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.

Face authentication test on the BANCA database

This work details the results of a face authentication test (FAT2004) (http://www.ee.surrey.ac.uk/banca/icpr2004) held in conjunction with the 17th International Conference on Pattern Recognition. The contest was held on the publicly available BANCA database (http://www.ee.surrey.ac.uk/banca) according to a defined protocol (E. Bailly-Bailliere et al., June 2003). The competition also had a sequestered part in which institutions had to submit their algorithms for independent testing. 13 different verification algorithms from 10 institutions submitted results. Also, a standard set of face recognition software packages from the Internet (http://www.cs.colostate.edu/evalfacerec) were used to provide a baseline performance measure.

From margins to mainstream : how screen and creative industries developed in the Northern Rivers region of NSW : 2000–2010

This thesis explores the proposition that growth and development in the screen and creative industries is not confined to the major capital cities. Lifestyle considerations, combined with advances in digital technology, convergence and greater access to broadband are altering requirements for geographic location, and creative workers are being drawn away from the big metropolises to certain regional areas. Regional screen industry enclaves are emerging outside of London, in the Highlands and Islands of Scotland, in Nova Scotia in Canada and in New Zealand. In the Australian context, the proposition is tested in an area regarded as a ‘special case’ in creative industry expansion: the Northern Rivers region of NSW. A key feature of the ‘specialness’ of this region is the large number of experienced, credited producers who live and operate their businesses within the region. The development of screen and creative industries in the Northern Rivers over the decade 2000 – 2010 has implications for regional regeneration and offers new insights into the rapidly changing screen industry landscape. This development also has implications for creative industry discourse, especially the dominance of the urban in creative industries thought. The research is pioneering in a number of ways. Building on the work conducted for my Masters thesis in 2000, a second study was conducted during the research phase, adapting creative industries theory and mapping methods, which have been largely city and nation-centric, and applying them to a regional context. The study adopted an action research approach as an industry development strategy for screen industries, while at the same time developing fine-grained ground up methods for collecting primary quantitative data on the size and scope of the creative industries. In accordance with the action research framework, the researcher also acted in the dual roles of industry activist and screen industry producer in the region. The central focus of the research has been both to document and contribute to the growth and development of screen and creative industries over the past decade in the Northern Rivers region. These interventions, along with policy developments at both a local and national level, and broader global shifts, have had the effect of repositioning the sector from a marginal one to a priority area considered integral to the future economic and cultural life of the region. The research includes a detailed mapping study undertaken in 2005 with comparisons to an earlier 2000 study and to ABS data for 2001 and 2006 to reveal growth trends. It also includes two case studies of projects that developed from idea to production and completion in the region during the decade in question. The studies reveal the drivers, impediments and policy implications for sustaining the development of screen industries in a regional area. A major finding of the research was the large and increasing number of experienced producers who operate within the region and the leadership role they play in driving the development of the emerging local industry. The two case studies demonstrate the impact of policy decisions on local screen industry producers and their enterprises. A brief overview of research in other regional areas is presented, including two international examples, and what they reveal about regional regeneration. Implications are drawn for creative industries discourse and regional development policy challenges for the future.

Functional fixation : experimental evidence on the presentation of financial information through different digital formats

This study examines whether, in the presentation of financial information, digital formats address the concern over users’ functional fixation. The accounting literature indicates that the presentation of financial information either within the financial statements or in the notes to the financial statements often creates functional fixation where users of financial statements fail to adjust for differences in accounting policy. This leads users to judge what would otherwise be identical financial situations as being different due to the different accounting policies and methods adopted. It has been suggested that the use of digital formats in presenting financial reports may overcome functional fixation. Using an experimental design involving accountants in public practice, the results indicate that the use of digital formats to present financial reports does not fully overcome the issue of functional fixation in the processing of financial information. Although the participants were able to identify and extract relevant information, irrespective of whether or not the information was presented within the financial statements or in the notes to the accounts, the evidence indicates that functional fixation remained when the participants made final decisions based on available information. This suggests that functional fixation may not be caused by access to or extraction of information but by the level of perceived significance based on where the information is reported in the financial statements. In general, the results indicate that current technology may not be able to fully reduce functional fixation in the evaluation of financial information prepared in accordance with different accounting policies and methods.

The role of life friendly policies on employees’ work-life balance

Employees' inability to balance work and non-work related responsibilities have resulted in an increase in stress related illnesses. Historically, research into the relationship between work and non-work has primarily focused on work/family conflict, predominately investigating the impact of this conflict on parents, usually mothers. To date research has not sufficiently examined the management practices that enable all 'individuals' to achieve a 'balance' between work and life. This study explores the relationship between contemporary life friendly HR management policies and work/life balance for individuals as well as the effect of managerial support to the policies. Self-report questionnaire data from 1,241 men and women is analysed and discussed to enable organizations to consider the use of life friendly policies and thus create a convergence between the well-being of employees and the effectiveness of the organization.

The student as customer model and its impact on the academic leadership role in higher education

This paper posits that the 'student as customer' model has a negative impact upon the academic leadership which in turn is responsible for the erosion of objectivity in the assessment process in the higher education sector. The paper draws on the existing literature to explore the relationship between the student as customer model, academic leadership, and student assessment. The existing research emanating from the literature provides the basis from which the short comings of the student as customer model are exposed. From a practical perspective the arguments made in this paper provide the groundwork for possible future research into the adverse affects of the student as customer model on academic leadership and job satisfaction in the academic work force. The concern for quality may benefit from empirical investigation of the relationship between the student as customer model and quality learning and assessment outcomes in the higher education sector. The paper raises awareness of the faults with the present reliance on the student as customer model and the negative impact on both students and academic staff. The issues explored have the potential to influence the future directions of the higher education sector with regard to the social implications of their quest for quality educational outcomes. The paper addresses a gap in the literature in regard to use of the student as customer model and the subsequent adverse affect on academic leadership and assessment in higher education.

Investigation of the effect of organics on the water uptake of marine aerosols

Water uptake refers to the ability of atmospheric particles to take up water vapour from the surrounding atmosphere. This is an important property that affects particle size and phase and therefore influences many characteristics of aerosols relevant to air quality and climate. However, the water uptake properties of many important atmospheric aerosol systems, including those related to the oceans, are still not fully understood. Therefore, the primary aim of this PhD research program was to investigate the water uptake properties of marine aerosols. In particular, the effect of organics on marine aerosol water uptake was investigated. Field campaigns were conducted at remote coastal sites on the east coast of Australia (Agnes Water; March-April 2007) and west coast of Ireland (Mace Head; June 2007), and laboratory measurements were performed on bubble-generated sea spray aerosols. A combined Volatility-Hygroscopicity-Tandem Differential Mobility Analyser (VH-TDMA) was employed in all experiments. This system probes the changes in the hygroscopic properties of nanoparticles as volatile organic components are progressively evaporated. It also allows particle composition to be inferred from combined volatility-hygroscopicity measurements. Frequent new particle formation and growth events were observed during the Agnes Water campaign. The VH-TDMA was used to investigate freshly nucleated particles (17-22.5 nm) and it was found that the condensation of sulphate and/or organic vapours was responsible for driving particle growth during the events. Aitken mode particles (~40 nm) were also measured with the VH-TDMA. In 3 out of 18 VH-TDMA scans evaporation of a volatile, organic component caused a very large increase in hygroscopicity that could only be explained by an increase in the absolute water uptake of the particle residuals, and not merely an increase in their relative hygroscopicity. This indicated the presence of organic components that were suppressing the hygroscopic growth of mixed particles on the timescale of humidification in the VH-TDMA (6.5 secs). It was suggested that the suppression of water uptake was caused by either a reduced rate of hygroscopic growth due to the presence of organic films, or organic-inorganic interactions in solution droplets that had a negative effect on hygroscopicity. Mixed organic-inorganic particles were rarely observed by the VH-TDMA during the summer campaign conducted at Mace Head. The majority of particles below 100 nm in clean, marine air appeared to be sulphates neutralised to varying degrees by ammonia. On one unique day, 26 June 2007, particularly large concentrations of sulphate aerosol were observed and identified as volcanic emissions from Iceland. The degree of neutralisation of the sulphate aerosol by ammonia was calculated by the VH-TDMA and found to compare well with the same quantity measured by an aerosol mass spectrometer. This was an important verification of the VH-TMDA‘s ability to identify ammoniated sulphate aerosols based on the simultaneous measurement of aerosol volatility and hygroscopicity. A series of measurements were also conducted on sea spray aerosols generated from Moreton Bay seawater samples in a laboratory-based bubble chamber. Accumulation mode sea spray particles (38-173 nm) were found to contain only a minor organic fraction (< 10%) that had little effect on particle hygroscopicity. These results are important because previous studies have observed that accumulation mode sea spray particles are predominantly organic (~80% organic mass fraction). The work presented here suggests that this is not always the case, and that there may be currently unknown factors that are controlling the transfer of organics to the aerosol phase during the bubble bursting process. Taken together, the results of this research program have significantly improved our understanding of organic-containing marine aerosols and the way they interact with water vapour in the atmosphere.

Design on the future: developing and enacting sustainable living programs in primary schools

Sustainable living is high on the international agenda (Ginsberg & Frame, 2004; Sutton, 2004). If education is fundamental to global transformation towards sustainability, then schools are in strategic positions to facilitate this change. Over recent years, schools in Australia have become more active in encouraging sustainability with the implementation of programs such as Science Education for Sustainable Living (SESL) that focus on topics such as energy efficiency, recycling, enhancing biodiversity, protecting species, and managing resources. This paper reports on a government funded Australian School Innovation in Science, Technology and Mathematics (ASISTM) project titled “Integrating science, technology and mathematics for understanding sustainable living” in which teachers, preservice teachers and other science professionals worked collaboratively to plan and enact a range of SESL programs for primary school students. Participants in this study included: 6 teachers, 5 preservice teachers, 2 university partners, 2 scientists, 4 consultants, and over 250 primary students. The findings from this qualitative study revealed a need for: (1) professional development for understanding SESL, (2) procedures for establishing and implementing SESL, and (3) strategies to devise, implement and evaluate SESL units of work.

Young, inexperienced and on the road: Do novice drivers comply with road rules?

Young novice drivers are at considerable risk of injury and fatality, particularly when they first drive independently. Graduated Driver Licensing (GDL) has been introduced in numerous jurisdictions to allow more driving experience in conditions of reduced risk and increasing driving privileges over a longer duration. Queensland, Australia, enhanced GDL July 2007. Learners must record 100 hours in a logbook (10 hours at night) over 1 year, no mobile handsfree/loudspeaker by driver or any passenger. Provisional 1 (P1) drivers must not carry 2 or more peer passengers 11pm - 5am, no mobile handsfree/loudspeaker by any passenger. Self-reported compliance with new GDL and general road rules has not been examined.

Searching for salvation: An analysis of US religious searching on the World Wide Web

The goals of this research were to answer three questions. How predominant is religious searching online? How do people interact with Web search engines when searching for religious information? How effective are these interactions in locating relevant information? Specifically, referring to a US demographic, we analyzed five data sets from Web search engine, collected between 1997 and 2005, of over a million queries each in order to investigate religious searching on the Web. Results point to four key findings. First, there is no evidence of a decrease in religious Web-searching behaviors. Religious interest is a persistent topic of Web searching. Second, those seeking religious information on the Web are becoming slightly more interactive in their searching. Third, there is no evidence for a move away from mainstream religions toward non-mainstream religions since the majority of the search terms are associated with established religions. Fourth, our work does not support the hypothesis that traditional religious affiliation is associated with lower adoption of or sophistication with technology. These factors point to the Web as a potentially usefully communication medium for a variety of religious organizations. © 2009 Elsevier Ltd. All rights reserved.

Teaching English : some remarks on the emergence of the sympathetic teacher in the English classroom

The chapter explores the concept of the teacher-student relationship within the English classroom and proposes that this relationship has a specific purpose in building a civil society by inculcating practices of self-reflection, empathy and ethics. The topic is explored through the example of teaching Australian literature, specifically, Tim Winton’s Lockie Leonard, Human Torpedo to secondary school students. Ian Hunter’s work on literature education and the construction of socio-ethical practices provides a conceptual framework for the chapter.

Estimation on the mortality and disease burden attributed to selected risk factors in Shandong province [山东省主要危险因素的归因死亡和疾病负担分析]

Objective: To determine the major health related risk factors and provide evidence for policy-making,using health burden analysis on selected factors among general population from Shandong province. Methods: Based on data derived from the Third Death of Cause Sampling Survey in Shandong. Years of life lcrat(YLLs),yearS Iived with disability(YLDs)and disability-adjusted life years(DALYs) were calculated according to the GBD ethodology.Deaths and DALYs attributed to the selected risk factors were than estimated together with the PAF data from GBD 2001 study.The indirect method was employed to estimate the YLDs. Results: 51.09%of the total dearlls and 31.83%of the total DALYs from the Shandong population were resulted from the 19 selected risk factors.High blood pre.ure,smoking,low fruit and vegetable intake,aleohol consumption,indoor smoke from solid fuels,high cholesterol,urban air pollution, physical inactivity,overweight and obesity and unsafe injections in health care settings were identified as the top 10 risk faetors for mortality which together caused 50.21%of the total deaths.Alcohol use,smoking,high blood pressure,Low fruit and vegetable intake, indoor smoke from solid fuels, overweight and obesity,high cholesterol, physical inactivity,urban air pollution and iron-deficiency anemia were proved as the top 10 risk factors related to disease burden and were responsible for 29.04%of the total DALYs. Conclusion: Alcohol use.smoking and high blood pressure were determined as the major risk factors which influencing the health of residents in Shandong. The mortality and burden of disease could be reduced significantly if these major factors were effectively under control.

On the robustness and security of digital image watermarking

In most of the digital image watermarking schemes, it becomes a common practice to address security in terms of robustness, which is basically a norm in cryptography. Such consideration in developing and evaluation of a watermarking scheme may severely affect the performance and render the scheme ultimately unusable. This paper provides an explicit theoretical analysis towards watermarking security and robustness in figuring out the exact problem status from the literature. With the necessary hypotheses and analyses from technical perspective, we demonstrate the fundamental realization of the problem. Finally, some necessary recommendations are made for complete assessment of watermarking security and robustness.