In vitro cultivation of adult human chondrocytes : importance of culture system, oxygen and zonal differences

Articular cartilage exhibits limited intrinsic regenerative capacity and focal tissue defects can lead to the development of osteoarthritis (OA), a painful and debilitating loss of cartilage tissue. In Australia, 1.4 million people are affected by OA and its prevalence is increasing in line with current demographics. As treatment options are limited, new therapeutic approaches are being investigated including biological resurfacing of joints with tissue-engineered cartilage. Despite some progress in the field, major challenges remain to be addressed for large scale clinical success. For example, large numbers of chondrogenic cells are required for cartilage formation, but chondrocytes lose their chondrogenic phenotype (dedifferentiate) during in vitro propagation. Additionally, the zonal organization of articular cartilage is critical for normal cartilage function, but development of zonal structure has been largely neglected in cartilage repair strategies. Therefore, we hypothesised that culture conditions for freshly isolated human articular chondrocytes from non-OA and OA sources can be improved by employing microcarrier cultures and a reduced oxygen environment and that oxygen is a critical factor in the maintenance of the zonal chondrocyte phenotype. Microcarriers have successfully been used to cultivate bovine chondrocytes, and offer a potential alternative for clinical expansion of human chondrocytes. We hypothesised that improved yields can be achieved by propagating human chondrocytes on microcarriers. We found that cells on microcarriers acquired a flattened, polygonal morphology and initially proliferated faster than monolayercultivated cells. However, microcarrier cultivation over four weeks did not improve growth rates or the chondrogenic potential of non-OA and OA human articular chondrocytes over conventional monolayer cultivation. Based on these observations, we aimed to optimise culture conditions by modifying oxygen tension, to more closely reflect the in vivo environment. We found that propagation at 5% oxygen tension (moderate hypoxia) did not improve proliferation or redifferentiation capacity of human osteoarthritic chondrocytes. Moderate hypoxia increased the expression of chondrogenic markers during redifferentiation. However, osteoarthritic chondrocytes cultivated on microcarriers exhibited lower expression levels of chondrogenic surface marker proteins and had at best equivalent redifferentiation capacities compared to monolayer-cultured cells. This suggests that monolayer culture with multiple passaging potentially selects for a subpopulation of cells with higher differentiation capacity, which are otherwise rare in osteoarthritic, aged cartilage. However, fibroblastic proteins were found to be highly expressed in all cultures of human osteoarthritic chondrocytes indicating the presence of a high proportion of dedifferentiated, senescent cells with a chondrocytic phenotype that was not rescued by moderate hypoxia. The different zones of cartilage support chondrocyte subpopulations, which exhibit characteristic protein expression and experience varying oxygen tensions. We, therefore, hypothesised that oxygen tension affects the zonal marker expression of human articular chondrocytes isolated from the different cartilage layers. We found that zonal chondrocytes maintained these phenotypic differences during in vitro cultivation. Low oxygen environments favoured the expression of the zonal marker proteoglycan 4 in superficial cells, most likely through the promotion of chondrogenesis. The putative zonal markers clusterin and cartilage intermediate layer protein were found to be expressed by all subpopulations of human osteoarthritic chondrocytes ex vivo and, thus, may not be reliable predictors of in vitro stratification using these clinically relevant cells. The findings in this thesis underline the importance of considering low oxygen conditions and zonal stratification when creating native-like cartilaginous constructs. We have not yet found the right cues to successfully cultivate clinically-relevant human osteoarthritic chondrocytes in vitro. A more thorough understanding of chondrocyte biology and the processes of chondrogenesis are required to ensure the clinical success of cartilage tissue engineering.

Re-conceptualising IS function's support performance : a preliminary model

While IS function has gained widespread attention for over two decades, there is little consensus among information systems (IS) researchers and practitioners on how best to evaluate IS function's support performance. This paper reports on preliminary findings of a larger research effort proceeds from a central interest in the importance of evaluating IS function's support in organisations. This study is the first that attempts to re-conceptualise and conceive evaluate IS function's support as a multi- dimensional formative construct. We argue that a holistic measure for evaluating evaluate IS function's support should consist of dimensions that together assess the variety of the support functions and the quality of the support services provided to end-users. Thus, the proposed model consists of two halves, "Variety" and "Quality" within which resides seven dimensions. The Variety half includes five dimensions: Training; Documentation; Data- related Support, Software-related Support; and Hardware-related Support. The Quality half includes two dimensions: IS Support Staff and Support Services Performance. The proposed model is derived using a directed content analysis of 83 studies; from top IS outlets, employing the characteristics of the analytic theory and consistent with formative construct development procedures.

Validating the IS-Impact model in Australasian universities

This paper presents a novel study that aims to contribute to understanding the phenomenon of Enterprise Systems (ES) evaluation in Australasian universities. The proposed study addresses known limitations of arguably the most significant dependent variable in the Information System (IS) field - IS Success or IS-Impact. This study adopts the IS-Impact measurement model, reported by Gable et al. (2008), as the primary commencing theory-base and applies research extension strategy described by Berthon et al. (2002); extending both theory and the context. This study employs a longitudinal, multi-method research design, with two interrelated phases – exploratory and confirmatory. The exploratory phase aims to investigate the applicability and sufficiency of the IS-Impact dimensions and measures in the new context. The confirmatory phase will gather quantitative data to statistically validate IS-Impact model as a formative index.

Predicting adolescents’ use of social networking sites from an extended theory of planned behaviour perspective

The popularity of social networking sites (SNSs) among adolescents has grown exponentially, with little accompanying research to understand the influences on adolescent engagement with this technology. The current study tested the validity of an extended theory of planned behaviour model (TPB), incorporating the additions of group norm and self-esteem influences, to predict frequent SNS use. Adolescents (N = 160) completed measures assessing the standard TPB constructs of attitude, subjective norm, perceived behavioural control (PBC), and intention, as well as group norm and self-esteem. One week later, participants reported their SNS use during the previous week. Support was found for the standard TPB variables of attitude and PBC, as well as group norm, in predicting intentions to use SNS frequently, with intention, in turn, predicting behaviour. These findings provide an understanding of the factors influencing frequent engagement in what is emerging as a primary tool for adolescent socialisation.

An examination of the factors that influence drivers’ willingness to use hand-held mobile phones

Hand-held mobile phone use while driving is illegal throughout Australia yet many drivers persist with this behaviour. This study aims to understand the internal, driver-related and external, situational-related factors influencing drivers’ willingness to use a hand-held mobile phone while driving. Sampling 160 university students, this study utilised the Theory of Planned Behaviour (TPB) to examine a range of belief-based constructs. Additionally, drivers’ personality traits of neuroticism and extroversion were measured with the Neuroticism Extroversion Openness-Five Factor Inventory (NEO-FFI). In relation to the external, situational-related factors, four different driving-related scenarios, which were intended to evoke differing levels of drivers’ reported stress, were devised for the study and manipulated drivers’ time urgency (low versus high) and passenger presence (alone versus with friends). In these scenarios, drivers’ willingness to use a mobile phone in general was measured. Hierarchical regression analyses across the four different driving scenarios found that, overall, the TPB components significantly accounted for drivers’ willingness to use a mobile phone above and beyond the demographic variables. Subjective norms, however, was only a significant predictor of drivers’ willingness in situations where the drivers were driving alone. Generally, neuroticism and extroversion did not significantly predict drivers’ willingness above and beyond the TPB and demographic variables. Overall, the findings broaden our understanding of the internal and external factors influencing drivers’ willingness to use a hand-held mobile phone while driving despite the illegality of this behaviour. The findings may have important practical implications in terms of better informing road safety campaigns targeting drivers’ mobile phone use which, in turn, may contribute to a reduction in the extent that mobile phone use contributes to road crashes.

Testing a belief-based intervention encouraging sun safety among adolescents in a high risk area

Objective. To provide a preliminary test of a Theory of Planned Behavior (TPB) belief-based intervention to increase adolescents’ sun protective behaviors in a high risk area, Queensland, Australia. Methods. In the period of October-November, 2007 and May-June, 2008, 80 adolescents (14.53 ± 0.69 years) were recruited from two secondary schools (one government and one private) in Queensland after obtaining student, parental, and school informed consent. Adolescents were allocated to either a control or intervention condition based on the class they attended. The intervention comprised three, one hour in-school sessions facilitated by Cancer Council Queensland employees with sessions covering the belief basis of the TPB (i.e., behavioral, normative, and control [barrier and motivator] sun-safe beliefs). Participants completed questionnaires assessing sun-safety beliefs, intentions, and behavior pre- and post-intervention. Repeated Measures Multivariate Analysis of Variance was used to test the effect of the intervention across time on these constructs. Results. Students completing the intervention reported stronger sun-safe normative and motivator beliefs and intentions and the performance of more sun-safe behaviors across time than those in the control condition. Conclusion. Strengthening beliefs about the approval of others and motivators for sun protection may encourage sun-safe cognitions and actions among adolescents.

Looking through the corridor : Malaysia and the MSC

As part of a development plan-in-progress spanning a total of 25 years(1996 to 2020), Malaysia’s Multimedia Super Corridor (MSC) provides a unique opportunity to witness a brief and microcosmic unfolding of the reciprocally formative process between society and technology that Lewis Mumford lays out in exhaustive detail in Technics and Civilization (Mumford, 1963). The interlocking of national imagining, destiny and progress with a specific group of technologies, information and communication technologies (ICT) is, in itself, worthy of interest. However, what renders the MSC doubly remarkable is its introduction in Malaysia, one of the most well established of contemporary ethnocracies. This chapter reads the development and implementation of the MSC as the text through which the association between nation and ethnicity is examined. Broadly speaking I argue here that the MSC inflects the imagining(s) of Malaysia at two levels. At the first level where the MSC is understood to be the insertion of a new policy into Malaysia’s pre-existent ethnocratic climate, I contend the MSC inflects the nation through its incongruence with prevalent conditions. At the second level, where the MSC is viewed through the position of its Chinese populace, I suggest that the MSC inflects Malaysia (perhaps to a lesser degree) through the re-emphasis it lends to issues of transnationalism and belonging for the Malaysian Chinese.

The impact of brand and product placements in electronic games

In an environment where it has become increasingly difficult to attract consumer attention, marketers have begun to explore alternative forms of marketing communication. One such form that has emerged is product placement, which has more recently appeared in electronic games. Given changes in media consumption and the growth of the games industry, it is not surprising that games are being exploited as a medium for promotional content. Other market developments are also facilitating and encouraging their use, in terms of both the insertion of brand messages into video games and the creation of brand-centred environments, labelled ‘advergames’. However, while there is much speculation concerning the beneficial outcomes for marketers, there remains a lack of academic work in this area and little empirical evidence of the actual effects of this form of promotion on game players. Only a handful of studies are evident in the literature, which have explored the influence of game placements on consumers. The majority have studied their effect on brand awareness, largely demonstrating that players can recall placed brands. Further, most research conducted to date has focused on computer and online games, but consoles represent the dominant platform for play (Taub, 2004). Finally, advergames have largely been neglected, particularly those in a console format. Widening the gap in the literature is the fact that insufficient academic attention has been given to product placement as a marketing communication strategy overall, and to games in general. The unique nature of the strategy also makes it difficult to apply existing literature to this context. To address a significant need for information in both the academic and business domains, the current research investigates the effects of brand and product placements in video games and advergames on consumer attitude to the brand and corporate image. It was conducted in two stages. Stage one represents a pilot study. It explored the effects of use simulated and peripheral placements in video games on players’ and observers’ attitudinal responses, and whether these are influenced by involvement with a product category or skill level in the game. The ability of gamers to recall placed brands was also examined. A laboratory experiment was employed with a small sample of sixty adult subjects drawn from an Australian east-coast university, some of who were exposed to a console video game on a television set. The major finding of study one is that placements in a video game have no effect on gamers’ attitudes, but they are recalled. For stage two of the research, a field experiment was conducted with a large, random sample of 350 student respondents to investigate the effects on players of brand and product placements in handheld video games and advergames. The constructs of brand attitude and corporate image were again tested, along with several potential confounds. Consistent with the pilot, the results demonstrate that product placement in electronic games has no effect on players’ brand attitudes or corporate image, even when allowing for their involvement with the product category, skill level in the game, or skill level in relation to the medium. Age and gender also have no impact. However, the more interactive a player perceives the game to be, the higher their attitude to the placed brand and corporate image of the brand manufacturer. In other words, when controlling for perceived interactivity, players experienced more favourable attitudes, but the effect was so weak it probably lacks practical significance. It is suggested that this result can be explained by the existence of excitation transfer, rather than any processing of placed brands. The current research provides strong, empirical evidence that brand and product placements in games do not produce strong attitudinal responses. It appears that the nature of the game medium, game playing experience and product placement impose constraints on gamer motivation, opportunity and ability to process these messages, thereby precluding their impact on attitude to the brand and corporate image. Since this is the first study to investigate the ability of video game and advergame placements to facilitate these deeper consumer responses, further research across different contexts is warranted. Nevertheless, the findings have important theoretical and managerial implications. This investigation makes a number of valuable contributions. First, it is relevant to current marketing practice and presents findings that can help guide promotional strategy decisions. It also presents a comprehensive review of the games industry and associated activities in the marketplace, relevant for marketing practitioners. Theoretically, it contributes new knowledge concerning product placement, including how it should be defined, its classification within the existing communications framework, its dimensions and effects. This is extended to include brand-centred entertainment. The thesis also presents the most comprehensive analysis available in the literature of how placements appear in games. In the consumer behaviour discipline, the research builds on theory concerning attitude formation, through application of MacInnis and Jaworski’s (1989) Integrative Attitude Formation Model. With regards to the games literature, the thesis provides a structured framework for the comparison of games with different media types; it advances understanding of the game medium, its characteristics and the game playing experience; and provides insight into console and handheld games specifically, as well as interactive environments generally. This study is the first to test the effects of interactivity in a game environment, and presents a modified scale that can be used as part of future research. Methodologically, it addresses the limitations of prior research through execution of a field experiment and observation with a large sample, making this the largest study of product placement in games available in the literature. Finally, the current thesis offers comprehensive recommendations that will provide structure and direction for future study in this important field.

An evaluation of IS-impact utility and intuitiveness

This study is conducted within the IS-Impact Research Track at Queensland University of Technology (QUT). The goal of the IS-Impact Track is, "to develop the most widely employed model for benchmarking information systems in organizations for the joint benefit of both research and practice" (Gable et al, 2006). IS-Impact is defined as "a measure at a point in time, of the stream of net benefits from the IS [Information System], to date and anticipated, as perceived by all key-user-groups" (Gable Sedera and Chan, 2008). Track efforts have yielded the bicameral IS-Impact measurement model; the "impact" half includes Organizational-Impact and Individual-Impact dimensions; the "quality" half includes System-Quality and Information-Quality dimensions. The IS-Impact model, by design, is intended to be robust, simple and generalisable, to yield results that are comparable across time, stakeholders, different systems and system contexts. The model and measurement approach employs perceptual measures and an instrument that is relevant to key stakeholder groups, thereby enabling the combination or comparison of stakeholder perspectives. Such a validated and widely accepted IS-Impact measurement model has both academic and practical value. It facilitates systematic operationalisation of a main dependent variable in research (IS-Impact), which can also serve as an important independent variable. For IS management practice it provides a means to benchmark and track the performance of information systems in use. From examination of the literature, the study proposes that IS-Impact is an Analytic Theory. Gregor (2006) defines Analytic Theory simply as theory that ‘says what is’, base theory that is foundational to all other types of theory. The overarching research question thus is "Does IS-Impact positively manifest the attributes of Analytic Theory?" In order to address this question, we must first answer the question "What are the attributes of Analytic Theory?" The study identifies the main attributes of analytic theory as: (1) Completeness, (2) Mutual Exclusivity, (3) Parsimony, (4) Appropriate Hierarchy, (5) Utility, and (6) Intuitiveness. The value of empirical research in Information Systems is often assessed along the two main dimensions - rigor and relevance. Those Analytic Theory attributes associated with the ‘rigor’ of the IS-Impact model; namely, completeness, mutual exclusivity, parsimony and appropriate hierarchy, have been addressed in prior research (e.g. Gable et al, 2008). Though common tests of rigor are widely accepted and relatively uniformly applied (particularly in relation to positivist, quantitative research), attention to relevance has seldom been given the same systematic attention. This study assumes a mainly practice perspective, and emphasises the methodical evaluation of the Analytic Theory ‘relevance’ attributes represented by the Utility and Intuitiveness of the IS-Impact model. Thus, related research questions are: "Is the IS-Impact model intuitive to practitioners?" and "Is the IS-Impact model useful to practitioners?" March and Smith (1995), identify four outputs of Design Science: constructs, models, methods and instantiations (Design Science research may involve one or more of these). IS-Impact can be viewed as a design science model, composed of Design Science constructs (the four IS-Impact dimensions and the two model halves), and instantiations in the form of management information (IS-Impact data organised and presented for management decision making). In addition to methodically evaluating the Utility and Intuitiveness of the IS-Impact model and its constituent constructs, the study aims to also evaluate the derived management information. Thus, further research questions are: "Is the IS-Impact derived management information intuitive to practitioners?" and "Is the IS-Impact derived management information useful to practitioners? The study employs a longitudinal design entailing three surveys over 4 years (the 1st involving secondary data) of the Oracle-Financials application at QUT, interspersed with focus groups involving senior financial managers. The study too entails a survey of Financials at four other Australian Universities. The three focus groups respectively emphasise: (1) the IS-Impact model, (2) the 2nd survey at QUT (descriptive), and (3) comparison across surveys within QUT, and between QUT and the group of Universities. Aligned with the track goal of producing IS-Impact scores that are highly comparable, the study also addresses the more specific utility-related questions, "Is IS-Impact derived management information a useful comparator across time?" and "Is IS-Impact derived management information a useful comparator across universities?" The main contribution of the study is evidence of the utility and intuitiveness of IS-Impact to practice, thereby further substantiating the practical value of the IS-Impact approach; and also thereby motivating continuing and further research on the validity of IS-Impact, and research employing the ISImpact constructs in descriptive, predictive and explanatory studies. The study also has value methodologically as an example of relatively rigorous attention to relevance. A further key contribution is the clarification and instantiation of the full set of analytic theory attributes.

The transformation of banana with potential virus resistance genes

One approach to reducing the yield losses caused by banana viral diseases is the use of genetic engineering and pathogen-derived resistance strategies to generate resistant cultivars. The development of transgenic virus resistance requires an efficient banana transformation method, particularly for commercially important 'Cavendish' type cultivars such as 'Grand Nain'. Prior to this study, only two examples of the stable transformation of banana had been reported, both of which demonstrated the principle of transformation but did not characterise transgenic plants in terms of the efficiency at which individual transgenic lines were generated, relative activities of promoters in stably transformed plants, and the stability of transgene expression. The aim of this study was to develop more efficient transformation methods for banana, assess the activity of some commonly used and also novel promoters in stably transformed plants, and transform banana with genes that could potentially confer resistance to banana bunchy top nanovirus (BBTV) and banana bract mosaic potyvirus (BBrMV). A regeneration system using immature male flowers as the explant was established. The frequency of somatic embryogenesis in male flower explants was influenced by the season in which the inflorescences were harvested. Further, the media requirements of various banana cultivars in respect to the 2,4-D concentration in the initiation media also differed. Following the optimisation of these and other parameters, embryogenic cell suspensions of several banana (Musa spp.) cultivars including 'Grand Nain' (AAA), 'Williams' (AAA), 'SH-3362' (AA), 'Goldfinger' (AAAB) and 'Bluggoe' (ABB) were successfully generated. Highly efficient transformation methods were developed for both 'Bluggoe' and 'Grand Nain'; this is the first report of microprojectile bombardment transformation of the commercially important 'Grand Nain' cultivar. Following bombardment of embryogenic suspension cells, regeneration was monitored from single transfom1ed cells to whole plants using a reporter gene encoding the green fluorescent protein (gfp). Selection with kanamycin enabled the regeneration of a greater number of plants than with geneticin, while still preventing the regeneration of non-transformed plants. Southern hybridisation confirmed the neomycin phosphotransferase gene (npt II) was stably integrated into the banana genome and that multiple transgenic lines were derived from single bombardments. The activity, stability and tissue specificity of the cauliflower mosaic virus 358 (CaMV 35S) and maize polyubiquitin-1 (Ubi-1) promoters were examined. In stably transformed banana, the Ubi-1 promoter provided approximately six-fold higher p-glucuronidase (GUS) activity than the CaMV 35S promoter, and both promoters remained active in glasshouse grown plants for the six months they were observed. The intergenic regions ofBBTV DNA-I to -6 were isolated and fused to either the uidA (GUS) or gfjJ reporter genes to assess their promoter activities. BBTV promoter activity was detected in banana embryogenic cells using the gfp reporter gene. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that generated by the maize Ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter (BT6.1) was restricted to the phloem of leaves and roots, stomata and root meristems. The activity of the BT6.1 promoter was enhanced by the inclusion of intron-containing fragments derived from the maize Ubi-1, rice Act-1, and sugarcane rbcS 5' untranslated regions in GUS reporter gene constructs. In transient assays in banana, the rice Act-1 and maize Ubi-1 introns provided the most significant enhancement, increasing expression levels 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about 10-fold. In stably transformed banana plants, the maize Ubi-1 intron enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35S promoter, but did not appear to alter the tissue specificity of the promoter. Both 'Grand Nain' and 'Bluggoe' were transformed with constructs that could potentially confer resistance to BBTV and BBrMV, including constructs containing BBTV DNA-1 major and internal genes, BBTV DNA-5 gene, and the BBrMV coat protein-coding region all under the control of the Ubi-1 promoter, while the BT6 promoter was used to drive the npt II selectable marker gene. At least 30 transgenic lines containing each construct were identified and replicates of each line are currently being generated by micropropagation in preparation for virus challenge.

The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

Economic growth, inequality and technology adoption in transitional economies

The stylized facts that motivate this thesis include the diversity in growth patterns that are observed across countries during the process of economic development, and the divergence over time in income distributions both within and across countries. This thesis constructs a dynamic general equilibrium model in which technology adoption is costly and agents are heterogeneous in their initial holdings of resources. Given the households‟ resource level, this study examines how adoption costs influence the evolution of household income over time and the timing of transition to more productive technologies. The analytical results of the model constructed here characterize three growth outcomes associated with the technology adoption process depending on productivity differences between the technologies. These are appropriately labeled as „poverty trap‟, „dual economy‟ and „balanced growth‟. The model is then capable of explaining the observed diversity in growth patterns across countries, as well as divergence of incomes over time. Numerical simulations of the model furthermore illustrate features of this transition. They suggest that that differences in adoption costs account for the timing of households‟ decision to switch technology which leads to a disparity in incomes across households in the technology adoption process. Since this determines the timing of complete adoption of the technology within a country, the implications for cross-country income differences are obvious. Moreover, the timing of technology adoption appears to be impacts on patterns of growth of households, which are different across various income groups. The findings also show that, in the presence of costs associated with the adoption of more productive technologies, inequalities of income and wealth may increase over time tending to delay the convergence in income levels. Initial levels of inequalities in the resources also have an impact on the date of complete adoption of more productive technologies. The issue of increasing income inequality in the process of technology adoption opens up another direction for research. Specifically increasing inequality implies that distributive conflicts may emerge during the transitional process with political- economy consequences. The model is therefore extended to include such issues. Without any political considerations, taxes would leads to a reduction in inequality and convergence of incomes across agents. However this process is delayed if politico-economic influences are taken into account. Moreover, the political outcome is sub optimal. This is essentially due to the fact that there is a resistance associated with the complete adoption of the advanced technology.

Repair of calvarial defects with customized tissue-engineered bone grafts - I. Evaluation of osteogenesis in a three-dimensional culture system

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.

A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.