Whole Proteome Analysis of the Protozoan Parasite Trypanosoma brucei using Stable Isotope Labeling by Amino Acids in Cell Culture and Mass Spectrometry

The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time allowed for the characterization of the proteome from several different life stages of the parasite (1-3). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC; (4)) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knock-out approaches.

Culture conditions, mass spectrometric measurements and acclimation carbonate chemistry

- A combined increase in seawater [CO2] and [H+] was recently shown to induce a shift from photosynthetic HCO3- to CO2 uptake in Emiliania huxleyi. This shift occurred within minutes, whereas acclimation to ocean acidification (OA) did not affect the carbon source. - To identify the driver of this shift, we exposed low- and high-light acclimated E. huxleyi to a matrix of two levels of dissolved inorganic carbon (1400, 2800 lmol kg-1) and pH (8.15, 7.85) and directly measured cellular O2, CO2 and HCO3 fluxes under these conditions. - Exposure to increased [CO2] had little effect on the photosynthetic fluxes, whereas increased [H+] led to a significant decline in HCO3- uptake. Low-light acclimated cells overcompensated for the inhibition of HCO3- uptake by increasing CO2 uptake. High-light acclimated cells, relying on higher proportions of HCO3- uptake, could not increase CO2 uptake and photosynthetic O2 evolution consequently became carbon-limited. - These regulations indicate that OA responses in photosynthesis are caused by [H+] rather than by [CO2]. The impaired HCO3- uptake also provides a mechanistic explanation for lowered calcification under OA. Moreover, it explains the OA-dependent decrease in photosynthesis observed in high-light grown phytoplankton.

Exploring complex chemical systems: insights from mass spectrometry, electrochemistry and continuous cell culture

The work presented herein covers a broad range of research topics and so, in the interest of clarity, has been presented in a portfolio format. Accordingly, each chapter consists of its own introductory material prior to presentation of the key results garnered, this is then proceeded by a short discussion on their significance. In the first chapter, a methodology to facilitate the resolution and qualitative assessment of very large inorganic polyoxometalates was designed and implemented employing ion-mobility mass spectrometry. Furthermore, the potential of this technique for ‘mapping’ the conformational space occupied by this class of materials was demonstrated. These claims are then substantiated by the development of a tuneable, polyoxometalate-based calibration protocol that provided the necessary platform for quantitative assessments of similarly large, but unknown, polyoxometalate species. In addition, whilst addressing a major limitation of travelling wave ion mobility, this result also highlighted the potential of this technique for solution-phase cluster discovery. The second chapter reports on the application of a biophotovoltaic electrochemical cell for characterising the electrogenic activity inherent to a number of mutant Synechocystis strains. The intention was to determine the key components in the photosynthetic electron transport chain responsible for extracellular electron transfer. This would help to address the significant lack of mechanistic understanding in this field. Finally, in the third chapter, the design and fabrication of a low-cost, highly modular, continuous cell culture system is presented. To demonstrate the advantages and suitability of this platform for experimental evolution investigations, an exploration into the photophysiological response to gradual iron limitation, in both the ancestral wild type and a randomly generated mutant library population, was undertaken. Furthermore, coupling random mutagenesis to continuous culture in this way is shown to constitute a novel source of genetic variation that is open to further investigation.

Journalism, History and the Politics of Popular Culture

I argue that a divergence between popular culture as “object” and “subject” of journalism emerged during the nineteenth century in Britain. It accounts not only for different practices of journalism, but also for differences in the study of journalism, as manifested in journalism studies and cultural studies respectively. The chapter offers an historical account to show that popular culture was the source of the first mass circulation journalism, via the pauper press, but that it was later incorporated into the mechanisms of modern government for a very different purpose, the theorist of which was Walter Bagehot. Journalism’s polarity was reversed – it turned from “subjective” to “objective.” The paper concludes with a discussion of YouTube and the resurgence of self-made representation, using the resources of popular culture, in current election campaigns. Are we witnessing a further reversal of polarity, where popular culture and self-representation once again becomes the “subject” of journalism?

Дизайнът на взаимодействията, масовата комуникация и предизвикателствата на споделеното експертно знание (Interaction Design, Mass Communication And the Challenge of Distributed Expertise)

This paper considers some of the implications of the rise of design as a master-metaphor of the information age. It compares the terms 'interaction design' and 'mass communication', suggesting that both can be seen as a contradiction in terms, inappropriately preserving an industrial-age division between producers and consumers. With the shift from mass media to interactive media, semiotic and political power seems to be shifting too - from media producers to designers. This paper argues that it is important for the new discipline of 'interactive design' not to fall into habits of thought inherited from the 'mass' industrial era. Instead it argues for the significance, for designers and producers alike, of what I call 'distributed expertise' -including social network markets, a DIY-culture, user-led innovation, consumer co-created content, and the use of Web 2.0 affordances for social, scientific and creative purposes as well as for entertainment. It considers the importance of the growth of 'distributed expertise' as part of a new paradigm in the growth of knowledge, which has 'evolved' through a number of phases, from 'abstraction' to 'representation', to 'productivity'. In the context of technologically mediated popular participation in the growth of knowledge and social relationships, the paper argues that design and media-production professions need to cross rather than to maintain the gap between experts and everyone else, enabling all the agents in the system to navigate the shift into the paradigm of mass productivity.

Europe and the Media: Building a new kind of Europe; is mass media the key?

The book is a joint effort of eight academics and journalists, Europe specialists from six countries (Australia, Germany, Poland, Slovenia, the United Kingdom and the United States). They give sometimes divergent views on the future of the so-called “European Project”, for building a common European economy and society, but agree that cultural changes, especially changes experienced through mass media, are rapidly taking place. One of the central interests of the book is the operation of the large media centre located at the European Commission in Brussels – the world’s largest gallery of permanently accredited correspondents. Jacket notes: The Lisbon Treaty of December 2009 is the latest success of the European Union’s drive to restructure and expand; yet questions persist about how democratic this new Europe might be. Will Brussels’ promotion of the “European idea” produce a common European culture and society? The authors consider it might, as a culture of everyday shared experience, though old ways are cherished, citizens forever thinking twice about committing to an uncertain future. The book focuses on mass media , as a prime agent of change, sometimes used deliberately to promote a “European project”; sometimes acting more naturally as a medium for new agendas. It looks at proposed media models for Europe, ranging from not very successful pan-European television, to the potentials of media systems based on national markets, and new media based on digital formats. It also studies the Brussels media service, the centre operated by the European Commission, which is the world’s largest concentration of journalists; and ways that dominant national media may come to serve the interests of communities now extending across frontiers. Europe and the Media notes change especially as encountered by new EU member countries of central and eastern Europe.

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.

The knock-on from the knock-off : recent shifts within Australian mass market fashion design practice

Australia’s mass market fashion labels have traditionally benefitted from their peripheral location to the world’s fashion centres. Operating a season behind, Australian mass market designers and buyers were well-placed to watch trends play out overseas before testing them in the Australian marketplace. For this reason, often a designer’s role was to source and oversee the manufacture of ‘knock-offs’, or close copies of Northern hemisphere mass market garments. Both Weller (2007) and Walsh (2009) have commented on this practice. The knock-on effect from this continues to be a cautious, derivative fashion sensibility within Australian mass market fashion design, where any new trend or product is first tested and proved overseas months earlier. However, there is evidence that this is changing. The rapid online dissemination of global fashion trends, coupled with the Australian consumer’s willingness to shop online, has meant that the ‘knock-off’ is less viable. For this reason, a number of mass market companies are moving away from the practice of direct sourcing and are developing product in-house under a Northern hemisphere model. This shift is also witnessed in the trend for mass market companies to develop collections in partnership with independent Australian designers. This paper explores the current and potential effects of these shifts within Australian mass market design practice, and discusses how they may impact on designers, consumers and on the wider culture of Australian fashion.

Individuals in a mass market environment: Australian bequest donors seek better communication from charities

Swelling social need and competing calls on government funds have heightened the philanthropic dollar’s value. Yet, Australia is not regarded as having a robust giving culture: while 86% of adults give, a mere 16% plan their giving with those who do donating four times as much as spontaneous givers (Giving Australia, 2005). Traditionally, the prime planned giving example is a charitable bequest, a revenue stream not prevalent here (Baker, 2007). In fact, Baker’s Victorian probate data shows under 5% of estates provide a charitable bequest and just over 1% of estate assets is bequeathed. The UK, in contrast, sources 30% and the US 10% of charitable income through bequests (NCVO, 2004; Sargeant, Wymer and Hilton,2006). Australian charities could boost bequest giving. Understanding the donor market, which has or may remember them in their will is critical. This paper reports donor perceptions of Australian charities’ bequest communication/ marketing. The data forms part of a wider study of Australian donors’ bequest attitudes and behaviour. Charities spend heavily on bequest promotion, from advertising to personal selling to public relations and promotion. Infrastructure funds are scarce so guidance on what works for donors is important. Guy and Patton (1988) made their classic call for a nonprofit marketing perspective and identify the need for charities to better understand the motivations and behaviour of their supporters. In similar vein, this study aims to improve the way nonprofits and givers interact; and ultimately, enhance the giving experience and thus multiply planned giving participation. Academically, it offers insights to Australian bequest motivations and attitudes not studied empirically before.

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

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

Making sense of mass education

Making Sense of Mass Education provides a comprehensive analysis of the field of mass education. The book presents new assessment of traditional issues associated with education – class, race, gender, discrimination and equity –to dispel myths and assumptions about the classroom. It examines the complex relationship between the media, popular culture and schooling, and places the expectations surrounding the modern teacher within ethical, legal and historical contexts. The book blurs some of the disciplinary boundaries within the field of education, drawing upon sociology, cultural studies, history, philosophy, ethics and jurisprudence to provide stronger analyses. The book reframes the sociology of education as a complex mosaic of cultural practices, forces and innovations. Engaging and contemporary, it is an invaluable resource for teacher education students, and anyone interested in a better understanding of mass education.

The knock-on from the knock-off : recent shifts within Australian mass market fashion design practice

Australia's mass market fashion labels have traditionally benefitted from their peripheral location to the world's fashion centres. Operating a season behind, Australian mass market designers and buyers were well-placed to watch trends play out overseas before testing them in the Australian marketplace. For this reason, often a designer's role was to source and oversee the manufacture of 'knock-offs', or close copies of northern hemisphere mass market garments. Both Weller and Walsh have commented on this practice.12 The knock-on effect from this continues to be a cautious, derivative fashion sensibility within Australian mass market fashion design, where any new trend or product is first tested and proved overseas months earlier. However, there is evidence that this is changing. The rapid online dissemination of global fashion trends, coupled with the Australian consumer’s willingness to shop online, has meant that the ‘knock-off’ is less viable. For this reason, a number of mass market companies are moving away from the practice of direct sourcing and are developing product in-house under a northern hemisphere model. This shift is also witnessed in the trend for mass market companies to develop collections in partnership with independent Australian designers. This paper explores the current and potential effects of these shifts within Australian mass market design practice, and discusses how they may impact on both consumers and on the wider culture of Australian fashion.

Promoting a more positive traffic safety culture in Australia : lessons learnt and future directions

Adopting a traffic safety culture approach, this paper identifies and discusses the ongoing challenge of promoting the road safety message in Australia. It is widely acknowledged that mass media and public education initiatives have played a critical role in the significant positive changes witnessed in community attitudes to road safety in the last three to four decades. It could be argued that mass media and education have had a direct influence on behaviours and attitudes, as well as an indirect influence through signposting and awareness raising functions in conjunction with enforcement. Great achievements have been made in reducing fatalities on Australia’s roads; a concept which is well understood among the international road safety fraternity. How well these achievements are appreciated by the general Australian community however, is not clear. This paper explores the lessons that can be learnt from successes in attitudinal and behaviour change in regard to seatbelt use and drink driving in Australia. It also identifies and discusses key challenges associated with achieving further positive changes in community attitudes and behaviours, particularly in relation to behaviours that may not be perceived by the community as dangerous, such as speeding and mobile phone use while driving. Potential strategies for future mass media and public education campaigns to target these challenges are suggested, including ways of harnessing the power of contemporary traffic law enforcement techniques, such as point-to-point speed enforcement and in-vehicle technologies, to help spread the road safety message.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.