UQ Theses Collection (RHD) - UQ staff and students only - UQ eSpace

The role of ChpA in Pseudomonas aeruginosa motility

Andrew James Leech — 2007-08-24T00:00:00Z

The role of complement factor C5a and its receptor; C5aR in the development of canine mammary tumours

Mohd Hezmee Mohd Noor — 2011-02-23T00:00:00Z

It was hypothesised that complement may play a role in the pathogenesis of canine mammary cancer. Complement activation is an integral pathway in the inflammatory process and recent studies had shown that selective inhibition of the C5a peptide had inhibited the progression of cervical cancer in mice. Before an effect of a mediator or pharmacological agent can be investigated, it should be determined if receptors for the mediator of interest are, indeed, found within the target tissue. Cultured cell lines were established from normal canine mammary tissue to focus experiments on the mammary epithelial cell from where mammary tumours primarily originate via neoplastic transformation of these particular cells. The use of cell lines would also restrict interference from other cell types normally found within the mammary gland, including fibroblasts and inflammatory cells. Immunohistochemical techniques were then applied to the cultured canine mammary epithelial cells and evidence of the expression of a C5a receptor (C5aR or CD88) was demonstrated. However, these results were inconclusive since the antibodies used for immunostaining had been validated for mice and could potentially bind to receptors other than for C5a in canine tissue. It was therefore decided to investigate whether C5a had direct effects on these isolated mammary cells using cell proliferation assays. A C5aR antagonist (ACF – [opdChaWR]; PMX53) resulted in a significant and dose-dependent inhibition of proliferation, while a C5aR agonist (YSFKPMPLar; EP54) had no effect on cell growth. Following on from the cell proliferation studies in normal mammary epithelial cells, it was decided to investigate if there was any change in C5aR expression in mammary tumour cells. Several mammary tissue samples from normal dogs (n=8) and dogs with various tumours (n=7) were collected from veterinary clinics. Immunohistochemistry was performed but, again, was inconclusive (described in Chapter 4). A quantitative reverse transcriptase-Polymerase Chain Reaction (qRT-PCR) assay was therefore developed using primers specific for canine C5aR. There was low but significant up-regulation of messenger RNA (mRNA) in all tumour samples, compared to no expression in the normal mammary samples. This was the first time that C5a had been shown to be involved in the pathogenesis of canine mammary cancer. The next logical step would have been using the C5a agonist and antagonist peptides in dogs. However, an unproven therapy would be unethical in dogs with spontaneously occurring mammary tumours, while the costs of these molecules would be prohibitive. It was therefore decided to explore a mouse model. Two mouse mammary tumour cells lines, 4T1 and EMT6, were selected and expression of C5aR/CD88 was confirmed by using an immunofluorescence assay with a monoclonal anti-mouse C5aR antibody. The effects of C5a on cell growth and viability were then investigated by using the agonist, EP54, and antagonist, PMX53, peptides. Treatment of the 4T1 cell line with EP54 lead to the death of cells in culture as showed by the Trypan Blue exclusion test and the ratio of live:dead cells using the Alamar Blue assay. Neither agonist nor antagonist treatment affected the growth or viability of the EMT6 cells. This was an unexpected result and showed that an agonist of C5a had a direct and deleterious effect on selected mammary tumour cells. The in vitro results differed with what had been reported in the literature and it was therefore uncertain if the anti-tumour activity of a C5a agonist was a direct or toxic effect on the tumour cells and if these results would persist in vivo. A model was developed where mammary tumours could reliably and predictably be induced by injecting either of the two mouse cell lines in BalbC mice (which is where these cell lines were originally isolated from). Mice then had tumours induced with injection of either EMT6 or 4T1 cells and, concurrently, were administered one of the following treatments daily for 14 days: (1) PMX53; (2) EP54; (3) meloxicam (representative of the oxicam’ group being investigated for tumour control by other groups), or (4) saline (control). An important outcome was that the agonist peptide significantly inhibited mammary tumour development in the mice. However, meloxicam and PMX53 did not inhibit tumour growth, compared to control, while PMX53 appeared to enhance tumour development. The study was repeated, although this time blinded to the lead investigator (Noor) and the following treatments administered: (1) PMX53; EP54; (3) EP141, or (4) saline (control). The EP141 peptide was used since it was an agonist of C3aR only, while EP54 was an agonist for both C5aR and C3aR, so it was hoped that a specific response to C5a could be differentiated. Again, EP54 inhibited tumour growth, while EP141 was not significantly different with control tumours. Furthermore, mice treated with EP54 generally appeared healthier, as judged by a health index based on appearance and activity, compared to control animals. These findings have never been reported previously and represent a major and important advance in cancer therapy.

The role of copper in the innate immune response to Salmonella enterica serovar Typhimurium

Sian Stafford — 2012-03-07T00:00:00Z

Salmonella enterica sv. Typhimurium (S. Typhimurium) causes gastroenteritis in humans and is used in the mouse as a model for human typhoid fever. It is an intracellular pathogen, which subverts normal phagolysosome formation to reside in specialized vesicles called Salmonella-containing vacuoles (SCV). Salmonella must acquire nutrients and defend against the antimicrobial action of the macrophage to survive in this intracellular environment. The importance of iron in Salmonella pathogenicity has long been recognized; in this study the role of copper in regulating Salmonella survival within macrophages was investigated. Infection of bone marrow-derived macrophages (BMM) with S. Typhimurium or treatment with LPS robustly increased the mRNA expression of genes encoding the copper transporters Ctr1, Ctr2 and Atp7a, divalent cation transporter Nramp1, copper chaperones Atox1, CCS and Cox17, as well as the multicopper ferroxidase Cp (ceruloplasmin), in both its secreted and anchored forms. mRNA expression of genes encoding metallothioneins Mt1 and Mt2 were also induced, as were the antimicrobial effectors gp91 (subunit of phox) and iNOS. Genes encoding Fpn (ferroportin1), Atp7b, Hamp (hepcidin) and Heph (hephaestin) were not induced by these stimuli. Despite being induced at the mRNA level, protein induction in BMM for Cp, Atp7a, Ctr1 and Ctr2 was not found under the conditions of a 24 hr time course after treatment with LPS. The addition of copper to S. Typhimurium-infected macrophages resulted in decreased intracellular bacterial loads, an effect that was rescued by the copper chelator Bathocuproinedisulfonic acid disodium salt (BCS). Infection of Nramp1+ compared to Nramp1- macrophages demonstrated the overriding importance of Nramp1 in the killing of intracellular pathogens. In an attempt to assess the importance of the copper transporters during infection, the use of siRNA for knockdown of mRNA of Ctr1, Ctr2 and Atp7a was employed. No significant effect on intracellular bacterial loads was observed. Consistent and sufficient reduction of the proteins proved difficult, which could have impacted on the results of the infection assays. The results show that the induction of genes involved in copper metabolism upon Salmonella infection. The antimicrobial effect of copper could indicate that this ion has a role in innate immune response to Salmonella enterica serovar Typhimurium. This may reflect the copper requirement for iron-efflux from the macrophage but it may also indicate that copper ions have a more direct role in the antimicrobial activity of macrophages.

The role of co-receptor ligation in the differentiation of cytokine-producing T lymphocytes

Campbell, Scott Bryan. — 2011-09-08T00:00:00Z

The Role of c-Src in E-Cadherin Activity

Robert Mclachlan — 2010-02-02T00:00:00Z

Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signalling, being enriched in tyrosine-phosphorylated proteins, tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signalling, however, is complex and incompletely understood. In my thesis I have tested the hypothesis that c-Src contributes positively to cadherin biology by functioning as part of an adhesion activated cell-signalling pathway. I found that c-Src is active at both established and reforming cell-cell contacts, and c-Src can be activated by homophilic ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signalling pathway serves positively to support cadherin function, while quantitative changes in signal strength may result in qualitative differences in functional outcome. Finally, my data implicated PI3-kinase signalling and cortactin as potential targets for cadherin-activated c-Src signalling. By inhibiting protein tyrosine phosphatases with pervanadate, I found that tyrosine phosphatase activity and not just protein binding was required to stimulate Src activity in response to cadherin ligation. I identified the tyrosine phosphatase RPTPα as a possible regulator of cadherin-activated Src signalling. RPTPα localises to cell-cell adhesions and it is found in a complex with E-cadherin and c-Src. Furthermore, knockdown of RPTPα disrupted the integrity of cadherin-based contacts and the activity of Src at these cell-cell contacts. This suggests that in response to cadherin-homophilic ligation PTP activity is required to stimulate Src signalling. Finally, I identified a novel pathway by which aberrant growth factor signalling could be downregulating cadherin function and promoting the invasion of epithelial cells. Stimulating cells with high levels of EGF revealed that aberrant epidermal growth factor signalling could disrupt cadherin-activated cell signalling. The integrity of cadherin-based contacts and the activity of Src at the cell-cell contacts were both disrupted in the presence of high levels of EGF. Analysis of E-cadherin and RPTPα immunoprecipitates suggested that activation of cadherin-bound EGFR might disrupt Src activation by displacing E-cadherin-RPTPα binding. Finally, analysing the subcellular distribution of these proteins revealed that, in response to high levels of EGF, E-cadherin, β-catenin, EGFR and pEGFR are internalised together in phospho-cortactin-rich endosomal-like structures. Therefore I propose that E-cadherin adhesion activates a cell-signalling pathway involving c-Src that functions to dynamically regulate the actin cytoskeleton and to maintain the adhesive strength of cell-cell adhesions. Perturbation of cadherin-activated Src signalling downregulates cadherin function and promotes the disassembly of cell-cell adhesive contacts. The concept of a cadherin-activated Src signalling pathway provides a new way to think about cadherin biology. Instead of merely functioning as passive glue holding two cells together, E-cadherin functions as an adhesion-activated signalling receptor. Dysregulation of E-cadherin-activated Src signalling and downregulation of cell-cell adhesions could be a mechanism promoting the invasion and metastasis of epithelial tumours.

The Role of Cultural Values and Race on Empathy towards Same and Other Race Individuals

Elsa Wong — 2011-04-05T00:00:00Z

Empathy is an affective reasoning ability that is crucial for the development of social competence. The present study examined whether differences in cultural perspectives (individualistic vs. collectivistic) and racial group membership impacts empathic responding. Caucasian and Asian participants (41 females, 30 males) read 4 different vignettes each describing a target character (Caucasian or Asian) who experienced distress related to a violation of either individualistic or collectivistic values. Racial group membership of the target was manipulated using either Caucasian or Asian names along with a photograph of the target with the relevant scenario. After reading each vignette, participants rated their emotional responses to the target's distress and completed questions designed to assess the extent to which the participant took the perspective of the target and perceived similarity with the target. Participants were divided post hoc into either "collectivist values" or "individualist values" groups based on a median split of their overall score on an established questionnaire measure of cultural values. It was predicted that when target race was the same as the participant's race and when target values matched the participant's values, then ratings of similarity, perspective taking and empathic responses would be higher compared to when target race and target values were different from the participant's own. These predictions were not supported, rather all participants tended to rate targets higher on the different dependent measures only when target's distress was due to a violation of individualistic values. The possible social impact of cultural values on empathy-related responses will be discussed.

The role of dietary factors in the aetiology of Nasopharyngeal carcinoma: a case-control study in Yogyakarta, Indonesia.

Syafiq, Ahmad — 2008-11-21T00:00:00Z

Nasopharyngeal carcinoma (NPC) is the most common cancer among males in Yogyakarta, Indonesia and rank second among males in Indonesia after skin cancer. NPC is associated with Epstein-Barr Virus, genetic susceptibility, and chemical carcinogens from occupational exposures and diet. Dietary factors might play role both as promoter and protector to NPC development. This study aims at investigating the role of dietary factors in NPC development in Yogyakarta, Indonesia. Design of the study was case-control with two types of controls, that is hospital controls and community controls. Cases were 73 histopathologically confirmed NPC patients, while hospital controls were 73 other cancer patients matched by diagnostic year and community controls were 73 healthy closest neighbours of cases matched for age and sex. Dietary exposures were collected retrospectively using specifically developed and validated food-frequency questionnaires. Design of the study also allows the collection of early information on survival analysis of NPC, which availability is very limited in Indonesia. The study was successfully developed food-frequencyquestionnaires modified from the available existing questionnaires through combination of different qualitative methods. In general, the questionnaires had a relatively moderate to high reliability and validity coefficients, although for some food items the reliability and validity was considered as poor. Results of the study showed that the risk of NPC was higher for those who consumed fermented salted fish during childhood period (OR: 9.3 CI 1.1-76.6). The risk of NPC was higher for those who consumed shrimp paste during the adult period (OR: 2.1 CI 1.1-4.0). Results on the fruit and vegetables hypotheses are a little mixed and uncertain because even though consumption of carrot during weaning period showed significant protective effect (OR: 0.4 CI 0.1-0.9), several other fruit and vegetable items showed significant risks as promotive factor of NPC. One possible explanation was related to low validity of measurement of distant past intake of fruit and vegetables. Hypotheses that we could not test due to small numbers of subjects were hypotheses related to duration of exposure and hypotheses related to dose response relationship.

The role of E2F in skin differentiation and carcinogenesis

Dicker, Anthony John. — 2010-12-02T00:00:00Z

The role of endothelin-1 in vascular remodelling of the internal mammary artery and its relationship to ischaemic heart disease

Sutherland, Allison J — 2013-02-15T12:13:03Z

THE ROLE OF EPHS AND EPHRINS IN MELANOMA METASTASIS AND AS POTENTIAL TARGETS FOR IMMUNOTHERAPY

Stylianou, Con — 2008-11-21T00:00:00Z

During development, Eph receptors and their ephrin ligands are expressed in a temporally and spatially restricted manner, governing the migration and positioning of cells, performing critical developmental roles including the formation of the vascular system. Ephs are expressed at far lower levels in adult tissues, and are ectopically overexpressed in a variety of malignancies where they consistently correlate with increased tumourigenesis. In this context, they are proposed to mediate metastasis, the most insidious aspect of cancer. Cytokines within the tumour microenvironment may facilitate tumourigenesis. The ability of cytokines to regulate the expression of Ephs and ephrins suggests there may be an underlying pathway linking these three factors. Further, the aberrant expression of Eph and ephrins in neoplastic tissues, coupled to their absence in adult tissues, makes them ideal candidates for targeted immunotherapy. Real Time PCR was used to determine if cytokines could regulate the expression of Eph and ephrin genes in three melanoma cell lines. Tumour necrosis factor (TNF) decreased EphA3 expression and Interferon-g (IFN-g) increased the expression of ephrin A4, and decreased the expression of ephrin A1 and EphB3. Insulin-like growth factor-1 (IGF) increased the expression of EphA3, EphA4, EphB1, ephrin A3 and ephrin B1. All-trans retinoic acid (RA) increased the expression of EphB2 whereas transforming growth factor-b (TGF-b), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) did not affect the expression of any Eph or ephrins. The potential role of EphA3, ephrins A1, A5, B2 and B3 in melanoma metastasis was examined in vivo following ectopic over-expression of these proteins in a nonmetastatic melanoma cell line (A02-JLO). The ectopic over-expression of these proteins did not confer to A02-JLO cells the ability to form metastases following subcutaneous or intravenous inoculation into nude mice. However, significant differences were noted in subcutaneous growth. EphA3, ephrin A1, ephrin B2 or ephrin B3 expression significantly attenuated, whereas ephrin A5 expression significantly increased tumour growth relative to parental or vector control cells. In vitro proliferation assays indicated all lines proliferated equally and CD31 immunostaining identified increased vessel density in EphA3, ephrin A1, ephrin B2 or ephrin B3 expressing tumours, suggesting the presence of functionally impaired vessels. The more rapidly growing ephrin A5 expressing tumours displayed similar blood vessel density to parental and vector control cells, however examination of H&E sections identified the presence of patterned networks indicative of a more aggressive tumour phenotype. Further, the lack of correlation between CD31 immunoreactivity and the number of vessel-like structures observed in H&E sections suggests the occurrence of vasculogenic mimicry. Finally, the therapeutic potential of an anti-EphA3 monoclonal antibody (monomeric or dimeric IIIA4) was determined both in vitro and in vivo. Real time-PCR (Q-PCR) of nude mouse tissues and an in vivo tissue distribution study of radiolabeled IIIA4 in an EphA3 positive melanoma xenograft model, confirmed the absence of EphA3 in adult nude mouse tissues, and the ability of IIIA4 to selectively target EphA3 positive melanoma xenografts. Prevention studies identified that IIIA4 dimer, but not IIIA4 monomer, was able to significantly attenuate EphA3 positive melanoma xenograft growth, which coincided with a significant increase in vessel number. In a model of disseminated leukaemia, monomeric IIIA4 and dimeric IIIA4 significantly attenuated leukaemic cell engraftment in the peripheral blood and infiltration into splenic tissue, whereas Mabthera (Rituxan®) could not attenuate either. In vitro assays determined that monomeric or dimeric IIIA4 failed to stimulate significant immune mediated lysis. Taken together, the above data suggests that Eph or ephrin expression alone is not sufficient to mediate melanoma metastasis. The present study identifies for the first time that ephrin A5 expression increases tumour aggressiveness and significantly alters tumour vascularisation. Further, manipulation of Eph or ephrin expression may lead to the formation of function-impaired blood vessels. Importantly, IIIA4 can effectively target EphA3 positive tumours and may be a powerful immunotherapeutic tool following humanisation to boost immunogenicity, or used as a vehicle to deliver cytotoxic drugs with unsurpassed avidity.

The role of epigenetic mediated gene repression in squamous differentiation and squamous cell carcinoma

Gannon, Orla Margaret — 2012-09-13T00:00:00Z

The role of epithiospecifier protein (ESP) in plant metabolism and its impact on the regulation of anti-cancer isothiocyanates in Brassica crops

David Williams — 2010-09-29T00:00:00Z

Abstract Glucosinolates are sulphur containing natural plant products found in Brassica vegetables as well as brassicaceous forages and oilseeds. Almost without exception they occur in the plant in conjunction with the hydrolytic enzyme, myrosinase. In intact tissues, the enzyme is stored separately from the glucosinolates. When tissue damage occurs glucosinolates are hydrolysed by the myrosinases to a range of breakdown products such as isothiocyanates, simple nitriles, epithionitriles and thiocyanates. The type of product that is formed depends on pH, structure of glucosinolate side chain and the presence or absence of supplementary specifier proteins such as epithiospecifier proteins (ESPs), thiocyanate-forming proteins (TFPs) and the recently identified nitrile-specifier proteins (AtNSPs). Knowledge of the factors controlling glucosinolate hydrolysis is critical for understanding the ecological roles and human health benefits of these compounds, because it is the hydrolysis products and not the glucosinolates themselves that have been found to be the most active. From a human health perspective, isothiocyanates are the most important because they are major inducers of carcinogen detoxifying enzymes. By contrast, most nitriles are ineffective as inducers of these detoxification enzymes and less effective as anti-proliferative agents. ESP operates in conjunction with myrosinase such that it directs the products of glucosinolate hydrolysis towards epithionitriles. ESP is not present in all brassicaceous plants and several studies have suggested that it is absent from those species that lack glucosinolates with terminally unsaturated side chains. This thesis describes investigations into the role of ESP in the formation of both simple nitriles and epithionitriles at the expense of isothiocyanates, the importance of the terminal alkenyl glucosinolate substrates and factors that influence ESP activity. This study provides strong evidence that simple nitrile formation is under ESP control in most Brassicas although the recent identification of nitrile specifier proteins in Arabidopsis thaliana demands a closer inspection of their possible role in simple nitrile formation. Fe2+ is an essential factor in ESP activity although several recent studies have highlighted discrepancies in our understanding of the ESP and Fe2+ interaction. To investigate further the role iron species play in regulating ESP activity, seedpowders were analysed for ESP and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of iron species, specific chelators and reducing agents. Aged seeds and 3 d seedlings were also tested to investigate the effects of seed storage and early plant development on iron levels and ESP activity. The ESP-containing plants gave two distinctive responses, thus providing strong evidence that ESPs viii vary markedly in their Fe2+ requirement for activity. The results also suggested that the Fe2+ concentration as indicated by the ratio of ferric to ferrous iron influences ESP activity. Consistent with this, reduction in the Fe2+ concentration was correlated with the loss of ESP activity during seed storage. Apparently the ESP-homologous AtNSPs were not dependent on iron for activity. When the likelihood of AtNSP activity contributing to simple nitrile formation was evaluated in this study the results obtained indicated only a minor, if any, contribution. Contrasting results have also been found regarding nitrile formation at different pH values. Various studies found nitrile production was favoured at low pH, while others reported predominant nitrile formation at neutral or even basic pH. As the measured hydrolysis pHs gave inconsistent results that varied markedly between species, a novel approach was used to describe these fluctuations. It is well known that proteins close to or at their isoelectric point have less biological activity. Using calculated pIs, based on amino acid sequences of several identified ESPs, differences between pH and pI were determined. The pH-pI values closely followed the variations in ESP activity in all the plant species analysed in this study. Epithionitrile formation has been shown to be heat sensitive in several species of Brassicas. Recent studies have shown that mild heating decreased ESP activity, with a subsequent increase in isothiocyanate levels, indicating that the myrosinase was unaffected. However, these studies were restricted to rapeseed and broccoli and the question was whether these temperatures apply to other Brassica vegetables during different stages of development. Experiments confirmed the results of these earlier studies but noted that both ESP and myrosinase activities of the seeds were more temperature resistant than those in the seedlings and mature plants.

The role of Fgf signalling in telencephalic midline development and astrogliogenesis

Gobius, Ilan — 2012-10-10T00:00:00Z

THE ROLE OF FIBROBLAST GROWTH FACTOR-1 IN HUMAN ADIPOGENESIS

Newell, Felicity Sara — 2008-11-21T00:00:00Z

Obesity has developed into a world-wide epidemic highlighting the necessity for basic research into the mechanisms that govern adipose tissue growth. The accumulation of adipose tissue mass, termed adipogenesis, is the result of both an increase in the triglyceride content of mature adipocytes and the proliferation and differentiation of the adipocyte precursors, preadipocytes. The features of murine adipogenesis have been extensively studied using in vitro models of differentiation, however human adipogenesis is poorly understood due to a lack of available cell lines and the often poor differentiation capacity of primary preadipocytes. In our laboratory, we have recently demonstrated that fibroblast growth factor- 1 (FGF-1) reproducibly increases the differentiation capacity of primary human preadipocytes (phPA). FGF-1 treatment increases phPA proliferation and during this period also primes cells for subsequent differentiation. The highest levels of phPA differentiation are obtained following FGF-1 treatment during both proliferation and differentiation. However it is not known how FGF-1 mediates these actions, or whether FGF-1 has a similar effect on other human preadipocyte models. Furthermore the features of preadipocyte differentiation in response to FGF-1 treatment have not been examined. Therefore, the aims of this thesis were to further characterise the features of human adipogenesis in FGF-1 treated human preadipocytes and to investigate the mechanisms of FGF-1 action in these cells. The features of differentiation were examined in FGF-1 treated phPA and FGF-1 treated SGBS PA (a human preadipocyte strain derived from an individual with Simpson-Golabi-Behmel syndrome). These models were compared with the well-characterised murine 3T3-L1 preadipocyte cell line (3T3-L1 PA). FGF-1 upregulated the adipogenic program in phPA, with increased expression of adipocyte markers during differentiation. Moreover, phPA differentiated in the presence of FGF-1 were more insulin responsive and secreted increased levels of adiponectin. FGF-1 treatment of SGBS PA elicited similar effects on cellular proliferation and differentiation, resulting in increased expression of adipocyte genes and an increased number of functional adipocytes. For the most part the adipogenic program in phPA and SGBS PA paralleled that observed in 3T3-L1 PA. However there was no evidence of mitotic clonal expansion in phPA, a process that is postulated to be necessary for differentiation in murine models and to involve the activation of ERK1/2 (extracellular signal-regulated kinase). In this thesis it was demonstrated that FGF-1 induced robust phosphorylation of ERK1/2 in early differentiation in phPA. Furthermore, inhibition of ERK1/2 activity significantly reduced phPA differentiation demonstrating that ERK1/2 activation is necessary for human adipogenesis in the absence of mitotic clonal expansion. The work in this thesis therefore highlights the existence of species specific differences with respect to adipogenesis and demonstrates that FGF-1 treated phPA and SGBS PA represent useful in vitro models for the study of human adipogenesis. The mechanisms of FGF-1 action in phPA and SGBS PA proliferation and differentiation were subsequently examined. FGF-1 is a member of the fibroblast growth factor family and elicits many of its effects by binding to cell surface tyrosine kinase FGF receptors (FGFR) and subsequently activating downstream signal transduction cascades. In this thesis, it was demonstrated that phPA expressed four FGF receptors (FGFR1-4), and that FGFR activation was required for phPA proliferation. FGFR activation in proliferation resulted in the stimulation of a number of downstream signalling pathways including the Ras-MAPK (mitogen activated protein kinase) pathway and the PI-3 kinase (phosphatidylinositol 3-kinase) pathway. Long term FGF-1 treatment of phPA during proliferation primes the cells for differentiation. In this thesis it was demonstrated that this priming resulted in changes in gene expression in confluent phPA, with alterations in the expression of other FGFs as well as genes required for differentiation. The role of FGF-1 in phPA differentiation was also examined. It was observed that, following induction of differentiation, FGF-1 increased signalling through several signal transduction pathways (the PI-3 kinase and Ras-MAP kinase pathways) which have postulated roles in human adipogenesis. Therefore, the work presented in this thesis serves to build on our earlier novel findings of the adipogenic role of FGF-1 in phPA and provides further evidence of the requirement for FGF-1 throughout phPA development. In addition, FGF-1 treated human preadipocytes offer an attractive in vitro model to study many features of adipogenesis in a human model and provide further insights into the processes that ultimately lead to obesity.

THE ROLE OF FLUENCY IN MATHEMATICAL DEVELOPMENT: FACTORS ASSOCIATED WITH EARLY LEARNING DIFFICULTIES IN MATHEMATICS

Finnane, Maureen Kathryn — 2008-11-21T00:00:00Z

This thesis explores factors associated with the development of early learning difficulties in mathematics from two perspectives: issues relating to mathematics development and early mathematics education, and models of memory and information processing. A principal aim of the research was to better understand the puzzling question of why a significant number of students with otherwise good reasoning capacity are unable to develop efficient, effective strategies for solving basic number combinations. The very poor mathematical understanding and skills of students who have been described as mathematically disabled (MD) or as having an arithmetic learning difficulty (ALD) or mathematical learning difficulty (MD) have been described in the research literature over a period of thirty years. The profile of the characteristics of MD students is well established, having been described with a consistent prevalence of 6-8% students in several countries. In spite of having average conceptual understanding in some areas of mathematics, these students face an outstanding difficulty in mastering basic arithmetic facts (Ginsburg, 1997), and continue to use very slow and ineffective strategies for solving even basic arithmetic problems, usually reliant on counting-by-ones finger strategies. More recent accounts of MD students also emphasize their poorly developed number sense and estimation skills. The development of effective interventions for mathematical learning difficulties is complicated by the fact that there are no distinctive diagnostic measures available for identifying MD (Geary, 2004). Furthermore, there are now strong indications that there are at least two distinctive forms of MD, with different instructional needs (Jordan, Hanich & Kaplan, 2003; Fuchs, Fuchs & Prentice, 2004). Accordingly, one goal of this thesis was to identify significant predictors of early mathematical learning difficulties, as a guide to identification and intervention. Based on previous research exploring factors associated with MD, a comprehensive range of assessment measures was administered to 68 children in three Year 2 classes in metropolitan Brisbane, Queensland. The measures included detailed assessments of mathematical skills and understanding, and a range of memory and processing tests, to capture underlying factors constraining early mathematical learning. Children at risk of early mathematical difficulties were identified by a statewide process of diagnosis in Queensland administered in the second year of formal schooling, known as the Year 2 Diagnostic Net (Numeracy). In view of the high rate of comorbidity of mathematical and literacy learning difficulties, data were also collected about which students failed the Year 2 Diagnostic Net (Literacy). For the same reason, the childrens speed of naming symbols, letters and words was measured at the same time as their fluency in identifying numbers. To understand better the nature of their learning difficulties, diagnostic teaching sessions were undertaken with ten of the students caught in the Year 2 Net (Numeracy), whom their teachers perceived as most in need of intensive learning support. In addition, an assessment and intervention were conducted with a Year 4 student at one of the study schools, who was facing significant mathematical and literacy learning difficulties. The study showed that forwards counting fluency and stage of strategy development (SEAL) on the Learning Framework in Number (Wright, Martland & Stanger, 2000) in the beginning of Year 2 were significant predictors of being caught in the Year 2 Net (Numeracy) at mid-year. Net status was also predicted by a students performance on the Make 10 test, a novel task designed by the researcher to assess fluency in retrieval of Ten Fact combinations. As well as the level of strategy use (SEAL), a students level of identification of written numerals was a further significant predictor of performance at the end of Year 2 on I Can Do Maths (Doig & de Lemos, 2000), a standardized test of early knowledge of Number, Measurement and Space concepts. The importance of identification and intervention for students at risk of MD was indicated by the increasing gap in performance between Net and normally achieving students on I Can Do Maths by the end of Year 2. The speed of processing of particular numerical skills were shown to be important indicators of early mathematical learning. Net students were significantly slower than their normally achieving peers in identifying 2-digit numbers on the CAAS 2-Digit task, and were slower on all the PAL rapid automatised naming RAN tasks - RAN Digits, Letters, Words, Words & Digits (Berninger, 2001). Fluency in rapidly naming and switching between 2-digit numbers and familiar words, as measured on the RAN Words & Digits task was also shown to be a significant predictor of performance on the One Minute Basic Number Facts Test of Addition. The level of Highest Forward Digit Span and poor performance on a working memory Counting Span task significantly discriminated between Net and normally achieving students. Information processing models and models of working memory provide useful explanatory frameworks to account for particular error patterns, and for the characteristic failure of MD students to learn basic arithmetic facts. Insights from the mathematics development and mathematics education literature add to our ability to understand the early impasse in learning of students at risk of MD. Work samples from diagnostic teaching sessions with five of the Intensive Net students further illustrate the influence of a low stage of counting fluency and strategy use on a students self-perception and ability to access the Year 2 mathematics curriculum. Processing strengths and weaknesses are discussed in relation to possible effects on the students mathematical learning. The detailed case study of the Year 4 student demonstrates how a researchbased intervention, which took into account cognitive factors, was successful in enabling learning of arithmetic facts and markedly changing the motivation and confidence of a student who was formerly very resistant to mathematics instruction. In particular, increased counting fluency and automatisation of addition facts appeared to free working memory resources for monitoring and self-correction by the student, and to facilitate a positive interest in exploring mathematical relationships. Based on the significant predictors of poor achievement in early mathematics, assessment measures are proposed which are critical for the identification of early mathematical learning difficulties. The thesis concludes with a number of recommendations for intervention, with accompanying requirements for pre-service and in-service training to assist effective implementation. Future directions for research in the identification and intervention of MD are proposed.

The role of frataxin in mitochondrial iron and haem metabolism and the development of iron chelators as potential therapeutic agents for the treatment of Friedreich's ataxia

Becker, Erika Michelle. — 2011-09-28T00:00:00Z

THE ROLE OF GENERALISATION AND CONTEXTUAL CUES IN MOTOR ADAPTATION TO NOVEL VISUAL ENVIRONMENTS

Woolley, Daniel Graham — 2008-11-21T00:00:00Z

The series of experiments which form this thesis investigate the generalisation of visuomotor learning. The thesis consists of seven chapters, with the first chapter providing a general introduction to the research program, and the final chapter a summary of the main research findings. Chapters 2 to 6 each represent stand-alone scientific works. Chapter 2 is a review of previous work examining the constraints imposed by the central nervous system on the generalisation of visuomotor learning in behavioural tasks. Particular consideration is given to tasks which have investigated how adaptation to a visuomotor rotation transfers to new visual target locations and movement directions. The role of contextual information in facilitating dual adaptation to opposing novel environments which are initially prone to interference is also discussed. The study described in Chapter 3 first assessed if movements made with a custom designed two degree of freedom isometric torque manipulandum would manifest a typical pattern of motor adaptation when subjects were exposed to a single visuomotor rotation. Two groups of subjects were trained to a single visuomotor rotation, which were of a different magnitude and in opposite directions for each group. Following a rapid improvement in performance to the rotated environment over the course of the training block, directional after-effects were observed on return to the non-rotated environment. Once it was confirmed that our task produced a typical pattern of adaptation to a single visuomotor rotation, we then examined if colour cues provided a suitable source of contextual information to facilitate dual adaptation to two directionally opposed visuomotor rotations of unequal magnitude, which were concurrently presented to the same set of visual targets. A performance improvement over the course of training occurred to only one of the visuomotor rotations, with the possibility of a performance decrement to the other. The absence of after-effects following training indicated that adaptation did not occur in a similar manner to that observed in the single visuomotor rotation groups. The study presented in chapter 4 further investigated constraints on the ability of subjects to dual adapt to opposing visuomotor rotations. We tested if colour cues, a separation in workspace, and presentation schedule, could be used to distinguish between two opposing visuomotor rotations and enable dual adaptation. The opposing visuomotor rotations were either presented in the same region of workspace and each associated with colour cues, presented in different regions of workspace in addition to colour cues, or presented in different regions of workspace only. We also assessed the effectiveness of the workspace separation with both randomised and alternating presentation schedules. The results indicated that colour cues were not effective at enabling dual adaptation when each of the visuomotor rotations was associated with the same region of workspace. When associated with different regions of workspace, however, dual adaptation to the opposing rotations was successful regardless of whether colour cues were present or the type of presentation schedule. The purpose of the study described in chapter 5 was to determine if target amplitude would serve as an appropriate source of contextual information to enable dual adaptation. This was tested by associating two opposing visuomotor rotations with a set of visual targets that were matched in angular direction but of different amplitude. The two sets of visual target amplitudes also resulted in movements of different amplitude. The results indicated that visual target and movement amplitude is not a suitable source of contextual information to enable dual adaptation. Interference was observed in groups who were exposed to opposing visuomotor rotations, or a visuomotor rotation and no rotation, both when the onset of the visuomotor rotations was sudden, or occurred gradually over the course of training. Furthermore, the pattern of interference indicated that the inability to dual adapt was the result of the generalisation of learning between the two visual target and movement amplitudes. Chapter 6 describes a series of experiments in which we used a dual adaptation paradigm to assess if distinct patterns of generalisation for visuomotor learning were manifested in the visual and motor workspaces. By manipulating the position of the visual targets and the magnitude and direction of the visuomotor rotations, we were able to independently determine the extent of generalisation in the visual and motor workspaces during the adaptation phase of the task. When the set of targets associated with each of the opposing visuomotor rotations were near in the visual workspace and separate in the motor workspace, extensive interference was observed. In the reverse scenario when the targets were separate in the visual workspace and near in the motor workspace, only a limited degree of interference was present. The dominance of generalisation in the visual workspace was supported by the result that dual adaptation to opposing visuomotor rotations was found to occur when separate targets in the visual workspace required movements to overlapping targets in the motor workspace.

The role of genetic and environmental variation on thermal tolerance of a reef-building coral, Acropora millepora

Smith, Carolyn — 2013-02-15T13:05:44Z

The role of glutamine in exercise-induced changes in immune function

Hiscock, Natalie. — 2007-08-24T00:00:00Z

The role of group I metabotropic glutamate receptors in neuronal excitotoxicity in Alzheimer's disease

Tsai, Wang Wei Vicky. — 2007-08-24T18:46:58Z

THE ROLE OF HABITAT STRUCTURE AND COMPETITION IN THE ECOLOGY OF LISTERIA SPECIES IN FOOD-RELATED AND OTHER ENVIRONMENTS

Sally Chiu — 2010-06-25T00:00:00Z

Listeria monocytogenes is a foodborne pathogen with a high mortality rate in susceptible populations and is of great public health concern with regard to food safety. The ability to grow at refrigeration temperatures during storage and at low pH levels during food processing has enabled the species to establish and sustain growth on processed food. Some food products particularly at risk of contamination by L. monocytogenes are deli or processed meat products, seafood, processed vegetables, dairy products and other food that do not require heating or reheating before consumption. The aims of this study are therefore to investigate firstly the prevalence rates of the species in high risk food products and a food processing plant in Brisbane. Secondly, to determine whether food isolates are better than environmental isolates at surviving the stress factors in food processing environments, or if their lineage groupings are a better indicator of their survival. Thirdly, to compare the survival of food and environmental isolates under temperature stress in co-cultures. A survey of more than 100 high-risk food products at supermarkets was carried out to investigate the prevalence of L. monocytogenes and other Listeria species in food. Isolates were also obtained from a food processing plant during routine tests. This study has found a low prevalence rate (under 10%) of L. monocytogenes in the processed vegetables and meat products tested. Other products tested included processed and raw seafood and processed fresh fruit. More L. monocytogenes isolates were isolated from the food processing plant (101) than from the food survey (25). Listeria grayi (73 isolates), a non-pathogenic species, was more frequently isolated from the food survey. The characterisation of those isolates has revealed their lineage groupings and REP-PCR profiles, which did not appear to be related to their sources. A selected group of 25 isolates were also serotyped for further identification. A larger number of lineage II isolates (70) were found compared to lineage I isolates (25), and were more common in food than the environments; while some (7) produced inconclusive results in the lineage PCR. The REP-PCR did not separate isolates of different sources, lineages or serotypes. In order to investigate the survival fitness of L. monocytogenes isolates whilst under environmental stress relevant to food safety, ten isolates from the food survey and food processing plant were chosen. Five isolates each from lineages I and II were subjected to temperatures ranging from 4ºC to 30ºC and pH levels from 4.0 to 6.0 for two weeks continuously, with their growth monitored by either optical density or plate counts. It was found that the isolates were most susceptible at the combination of pH 4.0 and 4ºC, where the growth of the isolates was completely inhibited. Again no relationship was observed between the lineage or the sources and the survival fitness of the chosen isolates. Due to the frequency of L. monocytogenes being co-isolated with other Listeria species as well as other food-borne pathogens, the relative competitive fitness of four of the isolates from the survival fitness experiment were compared in co-cultures at 4ºC and 30ºC at pH 7.4 in a small-scale preliminary study. The four isolates from food and environments were grown in broth cultures in pairs with the plate counts performed on antibiotic-supplemented selective TSA agar. The isolates were distinguished on agar supplemented with tetracycline which the isolates had acquired resistance to for this purpose. No significant difference (P>0.05) was observed between the lineages or the sources and the competitive fitness of the isolates in this study. The isolates always produced slightly more colonies in the antibioticresistant form compared to the wildtype form but did not seem to relate to the competitive fitness of the isolates. It would seemed that within the scope of this study, neither the lineage, serotype nor source of the isolates indicated any isolate with a better ability of survival while at low temperatures and low pH levels in pure and mixed cultures. However, other classifying groups such as serotypes, RAPD profiles may reveal possible co-relations, as well as a wider isolate pool. Furthermore, different stress factors could be included as part of an investigation on the survival of L. monocytogenes, as this study focused on food safety during processing.

The role of HCV core protein in the regulation of HCV replication

Li, Dongsheng — 2007-08-24T00:00:00Z

The role of higher education for knowledge on and for Africa: A historical critique

Adelino Chissale — 2009-12-10T00:00:00Z

This thesis investigates the ways in which higher education in Africa has been construing its mission for Africa’s development and how such constructions are shaped by particular global regimes of knowledge on development. The thesis unpacks the ways in which such regimes are deployed using specific technologies: neo-liberal precepts on economic development. To that end, I pose a set of questions which can be summarized in these two: How has higher education in Africa discursively construed Africa’s experiences? Second, in which terms such constructions have helped responding to Africa’s problems of development? Taking Mozambican higher education as a unit of analysis, I used postcolonial theory to unsettle neo-liberal regimes of development and to show how contingent they are. Methodologically, a historical critique was carried out to historize neo-liberal globalization as a contingent process and to understand multiple possibilities of construing Africa’s experiences. My data consisted of texts discussing ways in which Africa is discursively understood by both, African and Western scholarship, higher education policy in Mozambique, interviews with senior administrators of some Mozambican higher education institutions and text materials from higher education institutions’ websites in Mozambique. The findings suggest that, on the one hand, constructions of Africa as being in crisis are not new. In fact, for centuries Africa has always been a subject of knowledge from which the West constructs its differences. It is from such differences that the West assumed a civilizing mission in order to integrate African peoples in the world order. On the other hand, African scholars’ responses to Western constructions of Africa’s experiences end up building another crisis at the theoretical level: the difficulties of thinking effectively on Africa so as to solve its problems. The second finding is that Mozambican higher education’s responses to the crisis have been marked by a development agenda within the broader context of Mozambique’s history from late the 1970s onwards: first, within the socialist model of central planning economy and, second, within the international agenda of global neo-liberal market economy. My analysis suggests that both development practices reflect, to some extent, continuities of colonial regimes of development which did not take into account the contextualities of the colonized. Finally, my investigation found that higher education institutions in Mozambique are responding to development challenges based on very technological conceptions of development following global trends. The thesis contends that an engagement with the ethics of knowledge and development would lead to a development model more preoccupied with the social contexts beyond market rationalities.

The role of hnRNPs in a2re-mediated rna trafficking in oligodendrocytes and neurons

Shan, Jianguo — 2007-08-24T00:00:00Z

The role of immune disturbances in the pathology of heat stroke : the dual pathway model of heat stroke

Lim, Chin Leong — 2008-11-21T00:00:00Z

The Role of Institutions in Economic Development: An Empirical Analysis of State Capacity and Economic Development in a Small Island State in the Pacific

Scott Hook — 2012-03-10T00:00:00Z

This thesis reviews the experience of Fiji in the context of the evolution of development thinking in the last 20 years. The rise and decline of the Washington Consensus, the rise of institutional analysis and the new direction in development economics has meant that, in the twenty-first century, there is a different understanding about development. In particular, there is greater need to understand the capacity of states to develop and implement any reform agenda and an acceptance that building on local institutions is more likely to succeed. This thesis argues that the failure of the Pacific reform agendas in the past has been because of a poor understanding of the capabilities of organisations in states and the capacity of the people to implement reform. In particular, there has been little assessment of alternative approaches to policy implementation such as the capacity of local and indigenous institutions to guide the policy development and implementation process more effectively. The importance of state institutional capacity to reform program outcomes has long been underestimated by the designers of policy reform programs. North (1993) suggested that successful economic growth is the story of the evolution of more complex institutions, composed of rules, norms of behaviour and the way they are enforced. These institutions make the level of cooperative relations necessary for businesses and individuals to trade and participate in economic activity with confidence. Thus, without an understanding of the development of institutions and their role in better outcomes, there will be inadequate outcomes and an absence of support for the reform process. Fiji has had several coups that have precipitated three changes in the highest law: the constitution. This presents problems for state institutions and organisations to operate as the changes can lead to different roles and particular movements in power and influence. Changes in constitutions, instability in government and weakening of bureaucratic capacity have made reform more difficult. At the other end of the scale, village communities have changed little and retain many traditional governance structures. External groups can supply alternative institutions but the reality of development is that it is the demand side of governance that holds the potential for a different development path. The LMMA process reflects the reality that local level demand for change is effective for good policy. Thus, a governance and institutional reform strategy focused on promoting links between communities and government to make government more accountable, and supporting the emergence of civil society institutions such as non-government organisations, the media and the private sector to engage proactively with government in shaping the development process is likely to lead to better policy outcomes. The success of any reform, economic or otherwise, will be determined primarily by the political and cultural climate in which it operates: a ‘one-size-fits-all’ reform recipe simply does not exist. This needs to be backed by representative, democratic institutions and driven by political direction. Reform in Fiji may need to be somewhat slower to ensure that the transaction costs do not outweigh the benefits expected from reform. Charting such a course requires better skills and understanding by donor nations, non-government organisations and international financial institutions. The research reported in this thesis reinforces the view that one of the key reasons some groups block change or make it difficult is because it typically erodes the potential for power to be secured and utilised. These responses can also facilitate the entry of competitors, potentially displacing incumbents or requiring new power sharing arrangements. It is towards a better understanding of such factors that this thesis contributes.

The role of intracellular storage products in biological nutrient removal

Zeng, Raymond Jianxiong. — 2007-08-24T00:00:00Z

The role of MEK1 in progression through G2/M phase of the cell cycle

Tanya Pike — 2011-06-15T00:00:00Z

The primary endpoint for signalling through the canonical Ras-Raf-MEK-MAP kinase cascade is ERK activation. The work undertaken in this thesis describes an alternative outcome for signalling through this pathway which utilises a proteolytically truncated form of MEK1to inhibit activation of the pathways' recognised downstream effector, ERK. Activation of the mitogen activated protein kinase (MAPK) pathway by growth factors during G1 phase promotes cell cycle progression but activation of the MAPK pathway during G2 phase by growth factors (EGF) or phorbol esters (TPA) results in delayed G2/M phase progression. We have previously identified a novel proteolytically truncated form of MEK1 (tMEK - truncated MEK1) (Harding, Giles et al. 2003), which is cleaved to remove its N-terminal ERK binding domain. We show that induction of tMEK with activation of MAPK signalling during G2 phase, delays entry into mitosis. RNAi depletion in somatic cells or knockout in mouse embryonic fibroblasts of MEK1 but not MEK2, results in loss of the G2 delay. The G2 delay can be recapitulated with addition of recombinant tMEK protein. Investigation of G2 checkpoint proteins demonstrated a lack of involvement of classical G2 phase DNA damage/stress response molecules such as p38MAPK, Chk1/2 and ATM/ATR kinases. Examination of the proximal mediators of mitotic entry, the Cdc25 family of dual specificity phosphatases, revealed the rapid degradation of Cdc25B in response to exogenous growth factor or phorbol ester addition. This degradation could be inhibited by the addition of the proteasome subunit inhibitor MG132 or interestingly, the addition of the MEK1/2 inhibitor U0126. A potential ERK dependent phosphorylation site proximal to a β-TRCPSCF binding motif which targets Cdc25B to the proteasome for degradation was identified. Phosphorylation of Cdc25B at Ser249 occurs with TPA treatment and could be inhibited by the MEK1/2 inhibitor, U0126, strongly suggesting that this site is phosphorylated by ERK in response to TPA treatment. Addition of recombinant tMEK protein also resulted in reduced levels of the Cdc25B isoform indicating that tMEK mediates its effect of delaying G2/M progression through regulating the protein level of the key cell cycle regulator, Cdc25B. Cdc25B is, by nature, an unstable protein, which begs the question: why further destabilise an already unstable protein? What we observed was, after the initial activation of ERK following mitogenic stimulation; ERK activity was rapidly down-regulated. This correlated with the maintenance of decreased Cdc25B protein levels and concomitant G2 phase delay. The data presented herein led us to hypothesise that the role of tMEK is to inhibit de novo synthesis of critical cell cycle proteins required for progression from G2 phase to mitosis. Further investigation into the role of protein synthesis during G2 phase revealed a marked reduction in the phosphorylation and corresponding inhibition of the activity of key components of the translational initiation machinery and mRNA cap binding proteins in response to TPA treatment. The existence of a novel form of MEK1 which functions to inhibit its normal effector ERK is of great biological interest. In many cancers, signalling through the MAPK pathway involving Ras-Raf-MEK-ERK is enhanced, usually via oncogenic mutations in Ras or Raf, providing proliferative and pro-survival advantages to cells. The existence of another function for components of this pathway provides an entirely novel spectrum of possible outcomes for aberrant activation of this pathway.

The Role of Microphthalmia Transcription Factor (Mitf) in Osteoclast Gene Regulation

Meadows, Nicholas Axel — 2008-11-21T00:00:00Z

Microphthalmia transcription factor (Mitf) has been shown to be a key regulator of osteoclast function by activating genes like TRAP and cathepsin K. Mitf is a member of the helix-loop-helix leucine zipper family of transcription factors and regulates its target genes by binding as a homo- or heterodimer to an E-box consensus sequence termed the M-Box (T/CANNTG/A). In order to identify the suite of genes that are regulated by Mitf in macrophages and osteoclasts, the level of Mitf activity has been modulated in the pre-osteoclastic macrophage cell line, RAW/C4. Stable transfectant cell lines expressing the Mitf-A isoform (RAW/C4-Mitf-A/pEF6) and the dominant negative isoform, mi (RAW/C4-mi/pEF6), under the control of the EF1a promoter, were prepared. Both the expression and function of the exogenous genes were validated using western blotting, immunocytochemical staining, Q-PCR and promoter-reporter transfection analysis. The genes differentially regulated by the alteration of the level of Mitf in these cells were expression profiled by microarray analysis. The 22 000 element murine Compugen array was hybridized with RNA prepared from the RAW/C4-Mitf/pEF6 and RAW/C4-mi/pEF6 cell lines before and after induction of osteoclast differentiation with RANKL. Genes identified include those either up- or down-regulated during osteoclast differentiation, as well as genes with differential expression as a result of overexpression of Mitf protein. A bioinformatic analysis was undertaken in parallel to identify candidate M-box-containing genes that may be Mitf targets. A large-scale Blast technique was developed to download 2 kb of DNA sequence 5 of the translation start site for all the genes on the Compugen array. These promoter regions were searched for the presence of M-boxes and a subset of potential Mitf target genes was compiled and compared with targets generated from the microarray analysis. This approach identified eight genes that were indicated to be Mitf regulated based on their expression profile and the presence of a M-box within their proximal promoter region. The expression of these genes was validated using Q-PCR and following a literature review of their functional biology, five genes remained. Ccl9, Clcn7, Eos, Fra1 and Ostm1 all met the stringent criteria necessary for selection for further investigation. A bioinformatic analysis of the promoter regions for these genes was undertaken to determine mouse-human evolutionary conservation and establish a common mechanism for transcriptional regulation of each gene. Common conserved transcription factor binding sites were identified and candidate M-boxes within their promoter regions were assayed for Mitf binding and transcription activation using a series of in vitro techniques on the cell lines. Electrophoretic mobility shift and promoter-reporter luciferase assays were performed on RAW/C4 cells to demonstrate binding site specificity and promoter activation by Mitf. Consequently osteopetrosis associated protein 1, Ostm1 and chloride channel 7, Clcn7 were identified as specific targets for Mitf transcriptional regulation. Primary cells were also used for Q-PCR and chromatin immunoprecipitation assays, which confirmed Mitf regulation of Clcn7. Both Ostm1 and Clcn7 have known mutations that can cause a failure of osteoclast activity and severe osteopetrosis, a phenotype characteristic of particular Mitf mutations. These findings validate the combined bioinformatic and microarray approach used to identify novel Mitf targets during osteoclastogenesis. The identification of two genes, whose expression appears to be regulated by Mitf, that are directly involved with osteoclast resorption suggests the role of Mitf in controlling bone resorption might be more significant than previously perceived. These results indicate that Mitf may be a master regulator of osteoclast bone resorption and genes regulated by Mitf during osteoclast activation may be considered therapeutic targets for the treatment of bone resorption disorders.

The role of MicroRNA in head and neck cancer

Anderson, Shane Robert — 2013-03-07T12:18:13Z

The role of microsatellite instability in Hepatocarcinogenesis

Macdonald, Graeme Alistair. — 2011-09-03T00:00:00Z

The Role of Middle Managers in Organisational Change

Herzig, Sharyn Elizabeth — 2008-11-21T00:00:00Z

Change is ever-present in todays fast-paced organisations (Lewis, 2000). Market demands, the introduction of new technologies and an internal push for growth are some of the major factors driving change in organisations. However the failure rate, reported as high as 50% (Majchrzak, 1988) to 70% (Burnes, 2002), needs to be addressed (Marks & Mirvis, 2000). In particular, adequate coverage has not been given to the role middle managers play in organisational change (Huy, 2002; McKinley & Scherer, 2000). Middle management can be broadly defined as managers holding positions between the first-level supervisors and the level of executives, below those who have company-wide responsibilities (Frohman & Johnson, 1992). In the 1990s, middle management was typically viewed as resistant to change (Wai-Kwong, Priem, & Cycyota, 2001). It is important to reduce negative views held of middle management and promote their positive input into organisational change processes. Middle managers have been identified as contributing to higher levels of organisational performance when involved in strategy-making (Wai-Kwong et al., 2001) and the positive emotional management of their employees (Huy, 2002). Thus, the aim of the current body of research was to make a contribution to the existing deficient literature on middle managers in organisational change by exploring the strategic role of middle managers. Factors which facilitate organisational change and several underlying psychological states were also investigated. In addition, a measure of perceived change success was developed. Three studies were undertaken to investigate the role of middle managers. Study 1 was an exploratory study investigating middle managers perceptions of their role in change. It was qualitative in nature and examined a number of different types of change. Study 2 also was from the point of view of middle managers, while Study 3 investigated the impact of middle managers roles on employees. Both Studies 2 and 3 were quantitative and focused on transitional change. Study 1 involved interviews with 40 middle managers from a range of organisations. Thematic analysis revealed that, at the pre-implementation stage, the psychological state of uncertainty experienced by middle managers focused on the strategic concept of the change. During implementation however, uncertainty related to the appropriate procedures to implement. Middle managers strategic role was classified into three categories labelled creators, designers, and implementers. Those with higher strategic input (i.e., the creators and designers), reported lower levels of uncertainty than those with low strategic input (i.e., implementers). The factors identified as being facilitators to uncertainty management (i.e., communication with senior management and their own staff, support from senior management, role conflict, and peer interaction) along with other psychological states (i.e., change efficacy and commitment) were expressed in themes in the qualitative study. From middle managers responses regarding their perceptions of change success, an outcome measure of change was developed and employed in Studies 2 and 3. Study 2 extends the findings of Study 1 by means of quantitative research. A questionnaire was utilised in a cross-sectional study of 123 middle managers to examine the role of middle management during organisational change at a large mining company. The change context was the implementation of Six Sigma projects. Change outcome variables included perceived change success and importance placed on Six Sigma values. In addition, an independent measure of success was provided by the company via an internal audit. Using hierarchical multiple regression, factors which facilitated the implementation of projects by middle managers were examined (including communication with leaders, support from leaders, communication with employees, peer interaction and training). The psychological states (uncertainty, change efficacy and commitment) were found to play a mediating role in some relationships between the facilitators and the change outcomes. Also of interest was the role middle managers adopted during change, determined by strategic design input and Six Sigma project member role. Analysis of variance (ANOVAs) revealed that higher levels of strategic input and higher level Six Sigma roles were associated with more positive perceptions of a number of factors (e.g., more positive perceptions of communication, support, perceived change success and reduced strategic uncertainty) compared to those middle managers with lower levels of strategic input and lower level Six Sigma roles. A third study was conducted to further examine the findings related to communication with employees and to further explore the effect middle managers have on employees during change. One hundred and six employees were surveyed at one point in time regarding their past experiences of technological change at a tourism organisation. A series of hierarchical multiple regression analyses revealed that, at the pre-implementation stage of change, communication from middle managers was positively related to employees perceptions of change success. This relationship was partially mediated by employee readiness for change. During implementation, transformational leadership and transactional leadership were introduced as factors of theoretical interest and both were found to be positively related to employees perceptions of change success and this relationship was mediated by communication from middle managers. Overall, these studies support the important positive role of middle managers in organisational change, including creating a readiness for change in employees and leading to more successful perceptions of change for both middle managers and employees. The findings suggest that a greater strategic involvement of middle managers was associated with more successful change outcomes. Also identified were numerous important facilitators to the role of middle managers and their relationship to psychological states. Another key contribution was the development of the organisational change outcome measure, titled perceived change success. The relevance of this measure for future research is discussed.

The role of mononuclear phagocytes in dengue immunopathogenesis

Bhatia, Kanika Devi. — 2007-08-24T00:00:00Z

The role of mosquitoes in the emergence of Japanese encephalitis virus in Australia

van den Hurk, Andrew Francis. — 2007-08-24T00:00:00Z

The role of multicultural festivals in a society in transition: From being ethnically homogeneous to becoming multicultural

Insun Sunny Lee — 2012-03-28T00:00:00Z

While there are numerous studies focusing on various aspects of festivals in general, research on particular types of festivals, including multicultural festivals, remains limited. However, with the emerging cultural diversity of many societies worldwide, the importance of building well-integrated multicultural societies, as well as understanding this process, is increasing. Multicultural festivals have emerged in many locations, in particular in societies still at the beginning of becoming multi-ethnic, as a useful instrument for promoting social harmony and integration. The evaluation on this role, however, remains poorly explored. This study aimed to explore the role of multicultural festivals in a society in transit from being ethnically homogeneous to multicultural, and to offer insights into the contribution of multicultural festivals as an instrument within a multicultural policy to the development of successful multiculturalism. To evaluate the role of multicultural festivals as an instrument for the development of multiculturalism, the experiences and benefits visitors gain from attending a multicultural festival, and how these experiences and benefits contribute to building better multiculturalism must be considered. By adopting a Benefits-Based Management (BBM) framework to the multicultural festival field, this study explores the festival experiences that visitors had at a multicultural festival, the benefits gained from those festival experiences, and what particular experience leads to which benefit. To achieve the aim and objectives of this study, a mixed method research approach was used in the research design. Quantitative data were collected and analysed in the first phase, followed by qualitative interviews that helped to elaborate the initial findings. In the first phase, a total of 420 valid questionnaires from 176 Koreans and 244 ethnic minorities were collected at three multicultural festivals: the ‘Open multicultural festival’, ‘Siheung multicultural festival’ and ‘Colourful multicultural festival’, in South Korea, from April to June 2010. In the second phase of the qualitative study, a total of 44 visitors, 24 Korean and 20 ethnic minority visitors, were interviewed after the festival from April to June 2010. Findings from the quantitative data revealed that visitors had emotional experience as the most common experience, followed by social and learning experiences at multicultural festivals, and transformational benefit was identified as the greatest benefit the majority of visitors gained, followed by cognitive, social and affective benefits. It was also found that different visitors across the ethnic and behavioural variables had different experiences and benefits. Visitors gained transformational benefit mostly from social experience at a multicultural festival, cognitive benefit from emotional and social experiences, affective benefit from social experience, and social benefit from having learning and emotional experiences. It also appeared that the impacts of experiences on benefits were different for different ethnic and behavioural groups. By demonstrating the use of multicultural festivals as a socio-political strategy for the development of successful multiculturalism, this study encourages governments and policy makers to consider multicultural festivals as an important instrument for the development of successful multiculturalism. Identifying visitor experiences and benefits from a visit to a multicultural festival can be used as a barometer to measure the development of multiculturalism, and may indeed demonstrate that such festivals assist in enhancing social integration. The understanding of what experience leads to which benefit is of use in the management of festivals, providing additional information to event organisers for the planning and implementation of strategies to strengthen specific visitor benefits at future festivals. This study also has significance examining the festival experience and benefit from visitor perspective by adopting a BBM approach to the festival context, the multicultural festival setting in particular. Testing a BBM approach to festivals in this study with quantitative data enables research on understanding festival visitor experience to be advanced, and qualitative study in this study contributes to expand the knowledge and understanding of visitor experiences and benefit at multicultural festivals in a society in transition from being ethnically homogeneous to becoming multicultural in particular. As the findings of this study are delimited to multicultural festivals in societies in transit from ethnically homogeneous to becoming multicultural, future research may compare the roles of multicultural festivals in different societies, one in a society in transit to becoming a multicultural society, and one in a society in which multiculturalism is well established. As the BBM approach has been initially applied to the festival context in this study, continued applications of the BBM approach in a broad range of festivals and events are also valid topic for future festival visitor studies.

The role of Neogenin during neurogenesis and migration in the embryonic forebrain

Stacey Cole — 2010-10-13T00:00:00Z

The proliferation and differentiation of neural progenitors during neurogenesis, and their subsequent migration and positioning within the embryonic forebrain, are key processes of cortical development. The neuronal progenitors of the cortex are the radial glia, which give rise to pyramidal neurons that then migrate towards the outer surface. In the ventral forebrain, progenitors give rise to interneurons that migrate tangentially into the developing cortex and integrate into the circuitry of the brain. Abnormal neuronal migration is the underlying cause of several human disorders, including lissencephaly and epilepsy. Neogenin is a member of the immunoglobulin (Ig) superfamily and is closely related to the axon guidance receptor Deleted in Colorectal Cancer (DCC). Neogenin has been identified as a receptor for members of the Netrin and Repulsive Guidance Molecule (RGM) ligand families, and has been shown to regulate a range of developmental processes including axon guidance, neural tube closure, and mammary gland development. In this thesis we investigate the role of Neogenin during development of the mouse forebrain. The precise mRNA expression and protein localisation patterns of Neogenin in the developing forebrain are described, with Neogenin detected in both progenitor and neuronal populations. We also characterise the Neogenin (Neo1Gt) loss-of-function mouse, where it was shown that the Neogenin mutation in these animals was hypomorphic, with full-length protein detected in embryonic mice homozygous for the mutation. As a result, we determined that these animals displayed variable phenotypic defects with low penetrance in the developing forebrain. It was also shown that Neogenin played a role in neuronal differentiation. In vitro, Neogenin regulated interneuron differentiation in the presence of RGMa, a ligand for Neogenin, with an increase in interneuron number when cells were cultured in the presence of high levels of the ligand. Furthermore, this thesis also demonstrated that RGMa was involved in the regulation of neurite outgrowth in neuronal cultures. Finally, using an explant assay this study demonstrated that RGMa induced a chemorepulsive response in interneurons migrating from the medial ganglionic eminences. This suggests that Neogenin plays a role in interneuron migration in the ventral forebrain. To determine the precise role of Neogenin during neurogenesis and migration, further work utilising siRNA Neogenin knockdown in an optimised lentivirus system would be required. This would confirm a role for Neogenin in neuronal differentiation, migration and neurite outgrowth in the developing mammalian forebrain.

The role of neogenin in the adult brain: neuronal migration, differentiation and implications for adult neurogenesis

Dana Bradford — 2011-03-06T00:00:00Z

The ability of the adult brain to produce neurons is well established. The molecular mechanisms controlling differentiation to a neural fate and those guiding migration of neurons from a proliferative zone to their final destination are still poorly understood. The largest proliferative region in the adult brain is the subventricular zone (SVZ) where cells differentiate into neuronal precursors. These new neurons migrate along the rostral migratory stream (RMS) and integrate into the neuronal circuitry of the olfactory bulb. Therefore the RMS is an ideal system in which to study neuronal differentiation and cell migration. Neogenin is a multi-functional receptor with diverse developmental roles including axon guidance, differentiation and migration. Neogenin has been shown to interact with two ligand families — Netrins, which are classic guidance cues well described in embryogenesis, and the Repulsive Guidance Molecules (RGMs) which have recently reported roles in axon guidance. This Thesis addresses the functional role of neogenin in the adult mouse forebrain and cerebellum. Defects in differentiation in neogenin loss of function mice (neogt/gt) are described and their mechanisms discussed. Neogenin is expressed on neural stem cells and progenitor cells in the SVZ. In vitro analysis of these cells showed significantly fewer neurons generated from neogt/gt mice, demonstrating a role for neogenin in neuronal differentiation. In vitro and in vivo, the subpopulation of neurons affected was found to be the adult-born calretinin-positive granule cell population. A corresponding decrease in granule cell layer density in both the olfactory bulb and cerebellum of neogt/gt mice was also observed. Further analysis suggests that RGMa is a candidate ligand for regulating this effect. Neuroblasts in the RMS also express neogenin, suggesting neogenin may have a role in cell migration along the RMS. The expression pattern of netrin-1 in the adult forebrain coupled with disrupted migration in mice heterozygous for the netrin-1 mutation indicates a potential role for netrin-1 in guiding migration in this system. Finally, preliminary studies in human forebrain tissue show the expression of neogenin and RGMa are conserved, suggesting their function is the same in human brains.

The role of neutrophil primining and neutrophil antibodies in the pathogenesis of Transfusion-Related Acute Lung Injury (TRALI)

Yoke Lin Fung — 2008-10-28T00:00:00Z

THE ROLE OF NMYC AND CMYC IN SKIN DEVELOPMENT AND IN HEDGEHOG PATHWAY-INDUCED TUMORIGENESIS

RHONDA HUI PING KAN — 2012-02-08T00:00:00Z

Members of the Myc family of proteins namely Nmyc, Cmyc and Lmyc are a group of basic helix-loop-helix transcription factors which have been implicated in the regulation of a plethora of cellular functions and whose dysregulated expression has been associated with a wide variety of human diseases. Given that the endogenous role of Myc family members in the skin has been poorly defined, particularly that of Nmyc, the role of Nmyc and Cmyc in postnatal skin development and homeostasis was first investigated. This was genetically tested via conditional deletion of Nmyc and/or Cmyc in the skin using the epithelial-specific K14Cre recombinase. While Nmyc and Cmyc expression levels were significantly reduced, Nmyc and/or Cmyc deficient mice exhibited normal postnatal skin histology with normal hair follicle (HF) cycling and did not result in transcriptional upregulation of other Myc family members. Hedgehog (Hh) signalling is required for proper embryonic development and postnatal homeostasis and its dysregulation is associated with a growing list of congenital disorders and human cancers including basal cell carcinoma (BCC). The majority of sporadic/familial forms of human BCCs have been linked to mutations in the HH signal receptor, PATCHED 1(PTCH1), which results in constitutive HH pathway activation. Nmyc has previously been shown to be ectopically expressed throughout BCC-like lesions that developed upon loss of Ptch1 activity in the skin. Further detailed investigation into Nmyc expression in Ptch1 deleted skin confirmed that Nmyc is upregulated specifically in late, but not initial, stages of BCC development. However, the functional significance of elevated Nmyc expression in Hh pathway-induced BCC is still unclear. Therefore, to examine the requirement for Nmyc and its cellular homologue Cmyc at different stages of BCC development, Nmyc and/or Cmyc were simultaneously deleted together with Ptch1 in the mouse skin using K14Cre. Deletion of Nmyc alone did not rescue the phenotypes of hyperproliferation, expansion of progenitor cell compartment and BCC-like lesion formation, which occur following loss of Ptch1 activity. Similarly, removal of Cmyc alone did not modify the early effects induced by loss of Ptch1. However, its function in the progression of BCC development could not be determined given these mice do not survive into adulthood. Transcriptional analyses in the skin of K14Cre;Nmyc;Ptch1 and K14Cre;Cmyc;Ptch1 mice identified increased expression of other Myc family members to compensate for their individual loss. Interestingly, although inactivation of both Nmyc and Cmyc did not attenuate the early Ptch1 deficient phenotype, their combined loss strongly suppresses the progression of BCC lesion formation namely by limiting the expansion and hyperproliferation of the epidermal progenitor cell compartment. In addition to the well-established cell autonomous role of mutation-driven activation of Hh pathway in cancers, there is accumulating evidence to support the importance of the stromal environment in supporting Hh pathway-induced tumour growth and progression. However, at the time when this project commenced, very little was known about the participation of the Hh pathway in regulating epithelial-mesenchymal interaction in BCC development. The paucity of knowledge about this interaction is mainly attributed to the fact that the analysis of such complex reciprocal interactions is difficult in vivo. Therefore, the aim of this part of the project was to establish an in vitro organotypic skin co-culture model which serves as a powerful tool to elucidate the role of Hh signalling in epithelial-mesenchymal interaction in the skin. Using a combined approach of an organotypic co-culture model and Ptch1 deleted cells, it was found that Hh pathway activation in either the epidermal or dermal compartment alone was unable to trigger the development of characteristic BCC-like lesions. Based on this observation, it was predicted that BCC development likely requires a complex reciprocal tumour-stroma interaction. In summary, this thesis demonstrates for the first time that although Nmyc and Cmyc are not functionally required in postnatal skin under homeostatic (physiological) conditions, overall Myc expression levels are critical determinants in driving progression, but not initiation, of Hh pathway-induced BCC tumour development.

The Role of Non-Structural Protein NS2A in Flavivirus Assembly and Secretion

Jason Leung — 2010-08-14T00:00:00Z

Flaviviruses are a group of medically relevant pathogens, known to cause serious disease in animals and humans. The previously defined roles of the flavivirus non-structural protein 2A (NS2A) in RNA replication, and modulation of the host antiviral response, has recently been extended to include virus assembly and secretion. In West Nile virus subtype Kunjin (KUN), an Isoleucine (I)-to-Asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. In this study, prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59, but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59, or the compensatory T149P mutation into NS2A-I59N replicon RNA, restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. These results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for these induced membranes in virus assembly. To further our understanding of how mutations within NS2A are able to affect the induction of virus-induced membranes, leading to a block in virus assembly, the membrane topology of KUN NS2A was investigated. Using a plasmid encoding NS1 and NS2A proteins with C-terminal c-myc and FLAG epitopes, NS2A proteins containing N-linked acceptor sites and C-terminal truncations were generated. Assays were performed to identify the subcellular localization of specific sequences within NS2A by Western blot and immunofluorescence analyses. While the membrane topology could not be determined experimentally, the findings of this study support the assertion that cleavage at the NS1/NS2A junction requires the majority, if not all of the NS2A protein for proper processing to occur, and suggests that the interaction between hydrophilic loops and -helical transmembrane segments plays an important role in the formation and stability of the flavivirus NS2A protein topology. Based on the knowledge of polyprotein processing events, and utilizing a range of software packages, a topology model of NS2A was predicted. The likelihood of additional sequences within NS2A affecting the ability to induce virus-specific membranes, and facilitate virion assembly, has led to the development of an invasive bacterial screening system, as a delivery vehicle to screen libraries of mutated KUN replicon clones. Using these invasive bacteria to deliver mutated KUN replicons into BHK-derived packaging cells, mutations causing a deficiency in either RNA replication or encapsidation can be identified by performing -gal assays on cells maintained in the presence, or absence of Doxycycline (suppressing the expression of structural proteins), respectively. Furthermore, this system was adapted for use in a 96-well plate format, allowing for high-throughput screening. Thus, KUN replicon clones capable of RNA replication, but unable to assemble and secrete virus-like particles can be identified and further analyzed, in the hope of mapping amino acid residues and motifs involved in encapsidation of flavivirus RNA. Finally, a range of hypotheses are discussed, explaining the possible mechanisms by which NS2A is involved in flavivirus assembly. A number of future directions and applications are also presented.

The role of norms in intergroup discrimination : a social identity pesrspective

Wellen, Jackie Melissa. — 2007-08-24T00:00:00Z

The role of nuclear factor one (NFI) in cortical development

Mason, Sharon Anne — 2012-09-27T00:00:00Z

The role of obesity in the development and progression of chronic liver diseases

Hickman, Ingrid Joy — 2007-08-24T00:00:00Z

The role of Patched1 during development of the mouse cerebellum and regulation of neural stem cells in medulloblastoma

Jonathan Robson — 2010-12-24T00:00:00Z

The Sonic Hedgehog (Shh) signalling pathway plays a critical role in the development of many organs including the brain, lungs and skin. Of particular interest to this project, aberrant activity of the Shh pathway has been shown to be involved in the development of the paediatric cerebellar tumour medulloblastoma, typically through loss of function of the Shh receptor Patched 1 (Ptc1). Binding of Shh to Ptc1, or ablation of Ptc1 function, results in Smoothened (Smo) activation resulting in the activation of a number of downstream transcription factors responsible for cell proliferation and migration. In addition to its role in cerebellar development the Shh/Ptc1 pathway has also been associated with neural stem cell regulation although direct assessment of this relationship is difficult given the lack of in vivo neural stem cell markers. The aim of this project was to quantitatively assess the effect increased Shh/Ptc1 pathway activity has on neural stem cells via in vitro stem and progenitor cell assays, and determine if neural stem cells are the cell of origin of medulloblastoma. In order to determine how the Shh/Ptc1 pathway regulates neural stem cell fates Ptc1 function was ablated in the neocortex and subventricular zones of the developing mouse embryo through the use of an inducible Nestin Cre recombinase/Ptc1 conditional transgenic mouse line. Using in vitro neural stem cell assays it was identified that constitutive activation of the Shh/Ptc1 pathway in the neocortex and subventricular zone of the developing brain resulted in increased stem and progenitor cell numbers in vivo. Concomitant with an increase in Gli1 expression in the neocortex, an increase in the rate of symmetric neural stem cell division was observed. While sufficient to increase stem cell and progenitor cell numbers, increased activity of the Shh/Ptc1 pathway was not sufficient to rescue the loss of stem cells observed in neocortices devoid of Notch1 signalling. Although studies are beginning to elucidate the role of the Shh/Ptc1 signalling pathway in medulloblastoma formation little is known about the primary cell type responsible for tumour initiation. The observation that Shh/Ptc1 constitutive activation increased both stem and progenitor cell numbers and increased the rate of cell division prompted us to investigate the possibility that Ptc1 ablated neural stem cells may be a cellular origin of medulloblastoma, whereby the Shh/Ptc1 pathway maintains neural cells in a stem like fate. To identify the relationship between neural stem cells and medulloblastoma formation Ptc1 function was ablated in the developing cerebellum under the control of GFAP Cre recombinase which resulted in constitutive activation of the Shh/Ptc1 pathway in the cerebellar stem cell compartment, leading to medulloblastoma. It was shown that homozygous loss of Ptc1 in the cerebellar ventricular zone and developing external granule layer resulted in increased cell proliferation concomitant with increased Gli1 expression. Neural stem-like cells were isolated from the adult Ptc1 deleted medulloblastoma but not from equivalently aged wild type mice. Unlike primary Ptc1 deleted cells, isolated neurospheres from Ptc1 deleted medulloblastomas failed to recapitulate tumour formation in vivo. In contrast to primary Ptc1 deleted cells, Ptc1 deleted neurospheres showed significantly decreased Shh/Ptc1 pathway expression concomitant with a loss of primary cilia in vitro. To further investigate the possibility of a cancer stem cell as a cell of origin of medulloblastoma the primary Ptc1 deleted cell population was assayed for cancer stem cell markers through Fluorescent Activated Cell Sorting (FACS) of a panel of CD antibodies. This project identified 5 antibodies, CD38, CD44, CD90.2, CD117 and CD121b to have lower binding detection in Ptc1 deleted medulloblastoma compared to wild type cerebella and 3 antibodies, CD1d, CD24 and CD81 which have greater binding detection in Ptc1 deleted medulloblastomas compared to wild type cerebella. Of these, CD24 positive cells were identified to have increased tumour forming potential compared to CD24 negative cells identifying a possible medulloblastoma initiation marker in Ptc1 deleted stem/progenitor cells. Co-localised with CD15, a recently hypothesised neural stem cell marker, the CD24 positive population was found to be split into two distinct cell populations which led to the hypothesis that CD24/CD15 co-expression may identify the tumour forming cell population in medulloblastoma. Collectively these data suggest that medulloblastoma resulting from aberrant Shh/Ptc1 signalling may be due to a persistent stem-like cell whose tumorigenic potential depends on the presence of primary cilia and may be detected in vivo through specific expression profiles of cell surface markers.

The role of patched1 in mammalian facial dysmorphology

Metzis, Vicki — 2012-10-31T00:00:00Z

The role of p120-ctn in regulating E-cadherin-mediated adhesion

Marita Kathleen Goodwin — 2007-08-24T00:00:00Z

The role of Peroxisome proliferator-activated receptors in the rat brain

Smith, Steven Andrew — 2007-08-24T00:00:00Z

THE ROLE OF PHASE VARIABLE TYPE III RESTRICTION-MODIFICATION SYSTEMS IN GENE REGULATION

Srikhanta, Yogitha — 2008-11-21T00:00:00Z

Phase variation, the high frequency reversible switching of gene expression, is a common feature of host-adapted bacterial pathogens and is generally associated with genes encoding surface factors. Phase variation results in genetically and phenotypically diverse populations, providing a strategy for rapid adaptation to changes within the host environment and evading immune responses. However, in a growing number of host-adapted pathogens, phase variation has been found to occur in genes encoding methyltransferases (mod genes) associated with type III restrictionmodification (R-M) systems. R-M systems traditionally confer protection against foreign DNA, and several roles have been proposed for phase variable R-M systems based on DNA restriction function. The existence of phase variable methyltransferases raises the possibility of further functions for R-M systems such as gene regulation. In this thesis the role of a phase variable methyltransferase (mod) associated with a type III R-M system of Haemophilus influenzae strain Rd was investigated. Microarray expression analysis comparing a wild-type strain expressing mod to a mod knockout mutant strain, revealed altered expression of 15 genes under Mod control, some of which were virulence associated. This key finding confirmed that this phase variable methyltransferase coordinates the random switching of expression of multiple genes. Phylogenetic studies were used to analyse phase variable mod genes associated with type III R-M systems in the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae revealing that these organisms have two distinct mod genes - modA and modB. There are also distinct alleles of modA and modB that differ only in their DNA recognition domain. Phylogenetic analysis was also used to create an up-to-date list of potentially phase variable type III R-M systems present within other host-adapted organisms. To confirm whether phase variable methyltransferases controlled gene expression in other pathogens, the phase variable modA genes of Neisseria were studied. Mutant strains lacking the modA11, modA12 or modA13 genes were made and their phenotype analysed. Microarray analysis revealed that in all three modA alleles multiple genes were either up- or down-regulated, some of which were virulence associated. For example, in N. meningitidis (modA11), 80 genes were differentially expressed including the vaccine antigen candidates lactoferrin binding proteins A and B. Functional studies in N. gonorrhoeae confirmed that wild-type FA1090 modA13 ON and FA1090modA13::kan mutant strains have distinct phenotypes in antimicrobial resistance, a primary human cervical epithelial cell model of infection and biofilm formation. In summary, this thesis provides experimental confirmation that in three important human pathogens, H. influenzae, N. meningitidis and N. gonorrhoeae, alteration of expression of a type III DNA-methyltransferase causes switching of multiple genes. This novel genetic system has been termed the phasevarion (phase variable regulon). The wide distribution of phase variable mod genes indicates that this may be a common strategy used by host-adapted bacterial pathogens to randomly switch between distinct differentiated cell types.

THE ROLE OF PROTEASE IN UNLOCKING THE NUTRITIVE VALUE OF SORGHUM GRAIN FOR PIGS

Avril Finn — 2011-10-18T00:00:00Z

In Australia, and especially Queensland, sorghum is an important animal feed grain due to its drought resistance and heat tolerance. In many instances sorghum is thought to have a lower nutritive value compared to other cereal grains by pig producers due to poor pig performance when fed the grain with respect to rate of gain (ROG), average daily intake (ADI) and feed conversion ratio (FCR). An experiment was conducted on 640 Large White pigs (7.0 to 9.2 kgs) to evaluate the effects of different grain types and cultivars on performance of weaned pigs. The pigs were fed complete diets for 21 days containing 65% of one of 32 test grains (11 sorghum, 9 wheat, 7 barley and 5 triticale) with a calculated DE of 14 MJ/kg and lysine content of 0.90 g/MJ DE. Overall, sorghum was shown to have a significantly (P<0.05) negative effect on pig performance when compared to wheat, barley and triticale. However, it was also shown that some sorghum cultivars could perform just as well as the other grain types. To determine whether pig performance was affected by animal age, a second experiment was conducted on 120 Large White male pigs (16 to 25 kgs) to evaluate the effects of different sorghum cultivars. The pigs were fed complete diets for 21 days containing 82% of one of 10 sorghum grains (one yellow and nine red) with a calculated DE of 14 MJ/kg and lysine content of 1.13 g/MJ DE. Grower pigs fed different varieties of sorghum grains showed statistically significant (P<0.05) differences in ROG, but not ADI or FCR. The performance of the grower pigs compared to the weaner pigs on the same 10 test sorghum grains showed no correlation which leads to the conclusion that the age of a pig can affect its growth performance when fed the same grain. However, in contrast to the modest growth performance of pigs fed sorghum, the total digestible energy (DE) of sorghum is either similar or higher than some grains e.g. wheat. Therefore a third experiment was conducted on 42 Large White male pigs (32 to 37 kgs) to investigate whether DE values in grower pigs from both apparent ileal digested (AID) energy and apparent total tract digested (ATTD) energy measurements vary between different cereal grains. The pigs were fed complete diets containing 95% of each of the 32 grains used earlier with a calculated DE of 13 MJ/kg and lysine content of 0.70 g/MJ DE. There were highly significant (P<0.001) differences in the DE between grain types. The highest DE was from sorghum and wheat, followed by triticale, and the lowest for barley. Grain cultivar had a highly significant effect (P<0.001) on the ATTD energy for the diet on an as received (AR) basis, and the ATTD energy for the grain as an ingredient on an AR and dry matter (DM) basis. However, grain cultivar had no effect on the AID energy for the diet. There was also a significant (P<0.05) effect of the grain variety on the ratio of the diet AID to ATTD with wheat having the highest ratio followed by sorghum, triticale, and barley. One reason for poorer pig performance, when fed sorghum may be due to starch/protein interactions in the endosperm matrix restricting digestion of both starch and (kafirin) protein. Therefore an in vitro experiment was conducted using two sorghum cultivars, the yellow seeded Liberty and a red seed coat hybrid (Red1) to determine the efficacy of specific exogenous enzymes on the digestion of sorghum kafirins. Five exogenous enzymes, xylanase, beta-glucanase, protease, amylase and phytase were used in combination and individually. Enzymes were added to hammer milled whole grain then a protein extraction and sodium dodecyl sulfate polycrylamide gel electrophoresis (SDS-PAGE) analysis was undertaken, which showed that digestion of sorghum α-, β- and γ-kafirins was improved by the addition of protease either on its own or in combination with other enzymes. However, in combination with xylanase, beta-glucanase and amylase it appeared to lose its efficacy. Due to the positive effect of exogenous protease in vitro, an in vivo experiment with 144 Large White male pigs (7 to 9 kgs) was conducted to evaluate the addition of protease enzyme at four different levels (0ppm, 50ppm, 100ppm and 500ppm) to a complete diet containing 65% of either Liberty (yellow), Buster (red) sorghum and a wheat control, with a calculated digestible energy (DE) of 14.57 MJ/kg and an available lysine content of 1.55 g/MJ DE. At 0-21 days, for the highest protease level (500ppm) both sorghums performed better than in the absence of protease, and similarly to the wheat control in terms of FCR. In conclusion, although sorghum and wheat show higher AID and ATTD energy compared to triticale and barley, sorghum typically gives poorer pig performance than wheat, barley and triticale. However, selection of appropriate sorghum cultivars and, particularly, treatment with exogenous protease show promise as means of obtaining pig growth performance and feed conversion efficiency similar to or better than that of other cereals used in Australia.

The role of retinoic acid receptor related orphan nuclear receptor ROR[alpha]1 in muscle differentiation

Lau, Wai Fun Patrick — 2010-12-07T00:00:00Z