1,500 results for The University of Auckland Library, 2007

  • The Special Court for Sierra Leone: Justice for whom?

    Mahony, Christopher (2007)

    Masters thesis
    The University of Auckland Library

    The thesis examined the divergence of conceptions of justice between civil society actors in Sierra Leone and personnel working at the Special Court for Sierra Leone.

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  • Structural studies of the sugar-binding protein from the pneumococcal raffinose transport system

    Paterson, Neil (2007)

    Doctoral thesis
    The University of Auckland Library

    Streptococcus pneumoniae (the pneumococcus) is a Gram-positive bacterium responsible for a large number of deaths annually due to pneumonia, septicaemia and meningitis, mainly among young, elderly and immunocompromised populations. The primary virulence factor is the polysaccharide capsule that surrounds the cell and confers protection from phagocytosis; in addition the organism surface is decorated with a variety of proteins attached by both covalent and non-covalent means, with many of these also being involved in virulence. The pneumococcus is highly dependent on a wide range of carbohydrates for energy and growth with both phosphotransferase systems (PTS) and adenosine triphosphate binding cassette (ABC) transport systems utilised in sugar importation. Included among these is an ABC transport system, the Raf system, responsible for the importation and initial metabolism of the trisaccharide raffinose (α-D-Galp-(1→6)-α- D-Glcp-(1→2)-β- D-Fruf) that is highly sequentially homologous to the multiple sugar metabolism (Msm) system from S. mutans. The transporter itself comprises an extracellular raffinose binding protein (RafE), two membrane permease domains (RafF and RafG) and a protein responsible for adenosine triphosphate (ATP) binding and hydrolysis that has yet to be definitively identified. The 46.6 kDa substrate binding protein from the Raf system, RafE, is attached to the surface of the cell by means of a posttranslational lipoprotein modification and is responsible for the initial detection and capture of raffinose and conveying it to the transmembrane domains. RafE has been successfully overexpressed and purified to homogeneity using a novel interaction with a gel filtration matrix. Biophysical characterisation of RafE revealed the protein to be monomeric with one raffinose binding site per molecule and an affinity of 337μM for raffinose. Affinity for melibiose (α-D-Galp-(1→6)-α- D- Glcp), a substrate of the Msm system, was determined to be 6.84mM although the Raf system is incapable melibiose transport. Purified RafE was crystallised in both native and selenomethionine-labelled forms allowing solution of the phase problem by single wavelength anomalous dispersion (SAD) to a resolution of 2.90Å. Subsequent rational truncation and a change of construct to facilitate polyhistidine tag removal has produced two different crystal forms diffracting to a resolution of 1.04Å with the purification tag in place and 1.40Å iii following tag cleavage. An original solution to ice-ring diffraction was utilised for collection of this latter dataset which obviated the need for potentially problematic cryoprotectant solution. The crystal structures of RafE reveal that the protein adopts the periplasmic binding protein-like II fold, in common with a number of other substrate binding components of ABC transport systems, comprising two α/β/α sandwich domains joined by a hinge region consisting of three cross-linking peptide chains with the active site located in the cleft formed between the domains. These structures allowed identification of key active site residues and also revealed a range of conformational motion between the two domains. The active site is formed from a large aromatic patch, formed from three tryptophan residues aligned with their aromatic faces exposed to the solvent, surrounded by polar and charged residues. To assess the interaction with raffinose, polyhistidine-tag cleaved RafE was crystallised in the presence of raffinose and diffraction data collected to a resolution of 2.80Å. Structure solution using molecular replacement of the separate domains revealed a substantial conformational shift compared to the apo structures, with the two domains rotated approximately 29o towards each other, closing the active site region and trapping raffinose. RafE forms direct hydrogen bonding contacts between nine residues and the hydroxyl groups of the carbohydrate coupled with stacking of the sugar rings to the aromatic surface of the tryptophan residues. Molecular modelling of MsmE based on the raffinose bound RafE was used to try to assess the differences contributing to different substrate specificity and revealed changes in the residues forming contacts to the galactose moiety of raffinose but these did not appear to fully explain the dissimilar specificities. As an additional project, work was carried out in an attempt to obtain a crystal structure, with a view to functional elucidation of the choline-binding protein CbpI. This protein is a member of a highly important family for pneumococcal virulence with proteins of diverse function sharing a common surface attachment domain that binds to the choline moieties of teichoic and lipoteichoic acids that intersperse the cell wall. CbpI has been successfully overexpressed, purified and crystallised with diffraction data measured to a resolution of 3.50Å. The diffraction data however display very high mosaic spread and structure solution by molecular replacement has not been successful.

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  • The Use of Type 1 Cytokines to Modulate Immune Responses Raised by the Gene Gun Method of DNA Delivery

    Williman, Jonathan (2007-05)

    Doctoral thesis
    The University of Auckland Library

    Since its discovery 15 years ago there has been an explosion of research in the field of DNA immunisation. Unfortunately despite early promises that DNA immunisation had the potential to cure almost any infectious disease, autoimmune disease or even cancer, progress towards clinical trials has been slow. This has been due in part to the huge range of permutations possible in delivering the DNA. One approach is to deliver the DNA by gene gun. Gene gun delivery is a very efficient way of transfecting cells however also has a number of possible disadvantages. These drawbacks include a weak immunogenicity in larger animals as well as the tendency to bias towards the development of a strong type 2 response. In an effort to enhance antigen-specific immune responses and counter the type 2 polarisation of gene gun delivery, a series of DNA vaccines were created where the extracellular portion of the hemagglutinin (HA) gene from influenza A/PR8/34 virus was genetically fused the type 1 cytokines IFNγ, IL-12 and IL-23. Interleukin-23 has been recently discovered and even though both IL-12 and IL-23 contain the p40 subunit they seem to have dissimilar functions. The vaccine constructs were first tested in cellular assays in vitro to ensure correct production and biological activity of the attached cytokines. They were then delivered in various combinations to groups of BALB/c mice to test development of immune responses and the effect of different delivery regimes. Finally mice were immunised then challenged with live influenza virus to determine the different DNA vaccines’ protective efficacy. DNA vaccines containing the HA gene alone (pHA) or fused to IFNγ (pIFNγHA), IL-12 (pIL-12HA) or IL-23 (pIL-23HA) were successfully constructed. The fusion of the HA gene to the genes for IFNγ, IL-12 or IL-23 did not significantly disturb the structure of the antigen or prevent the biological actions of the cytokines. Mice immunised three times with pHA had high titres of serum IgG1 antibody and their splenocytes produced approximately equal amounts of IFNγ and IL-5. Co-delivery of IFNγ was unable to alter immune responses regardless of whether it was delivered at the first, last or during all immunisations. Surprisingly co-delivery of IL-12 acted to suppress both antibody and cellular immune responses, possibly through an IFNγ/nitric oxide feedback loop. On the other hand co-delivery of IL-23 tended to enhanced immune responses and, while it did not significantly alter the type 1 to type 2 balance, it was able to increase the ability of mice to clear live influenza virus from their lungs when they were challenged 26 weeks after immunisation. This protection was associated with increased levels of neutralising antibody in the serum of pIL-23HA immunised mice. This research has illuminated several of the pitfalls in the development of DNA vaccines and the use of cytokine as adjuvants. However it has also broadened our understanding of IL-23 and implies that IL-23 could be effectively used to increase the development of longterm immunity after immunisation.

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  • Enhancing Students Conceptual Understanding of Chemistry through the SOLO Taxonomy

    Gan, Joo (2007)

    Book item
    The University of Auckland Library

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  • Engaging pedagogies for teacher education: Considering a modest critical pedagogy for preparing tomorrow's teachers

    Tinning, Richard (2007)

    Book item
    The University of Auckland Library

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  • Constitutive expression of SOCS-3 in skeletal muscle induces obesity in mice

    Cognard, Emmanuelle (2007-12-21)

    Doctoral thesis
    The University of Auckland Library

    Type 1 or type 2 diabetes is a metabolic syndrome characterized by chronic hyperglycemia. The World Health Organization estimates that there are more than 180 millions of diabetic people worldwide and this number should double by 2030. The prevalence of diabetes strongly increases in all countries and nowadays its development is considered epidemic. Type 2 diabetes or non-insulin-dependent diabetes represents 95% of diabetic patients. Most of the time, it is associated with obesity. Type 2 diabetes occurs when insulin-sensitive tissues become insulin-resistant and pancreatic β-cells secrete less insulin. SOCS (Suppressor Of Cytokine Signaling) proteins have been discovered because of their negative feedback loop on cytokine signaling but they are also important inhibitors of the insulin signaling pathway (SOCS-3 in particular). Insulin actually induces SOCS-3 expression, which then interacts with the insulin receptor. SOCS-3 prevents the interaction between the receptor and its substrates, which decreases the upstream signal. Therefore SOCS-3 could play an important role in the development of insulin resistance. Liver, skeletal muscle and adipose tissue are the main target-tissue of insulin. It was shown in vitro that SOCS-3 inhibits insulin signaling in these tissues. However, SOCS-3 role in vivo and the mechanisms involved in the process are not fully understood yet. Furthermore we do not have a clear picture of the exact role of each tissue in the development of insulin resistance. The aims of this study are 1) to find out if constitutive expression of SOCS-3 in skeletal muscle could induce insulin resistance and type 2 diabetes, 2) to analyze the underlying molecular mechanisms. To do this, we have generated transgenic mice, which constitutively express SOCS-3 specifically in skeletal muscle (MLC/SOCS-3 mice). We have analyzed the phenotype of the transgenic mice compared to their wild type littermates and we have performed a molecular analysis of the muscles. Interestingly, MLC/SOCS-3 mice have more adipose tissue, result of an increase in the size of the adipocytes. Food intake is the same in the transgenic mice compared to the wild type littermate. However, we found a decrease in energy expenditure for the transgenic mice, which could be due, at least in part, to a decrease of physical activity. Transgenic mice have a normal glucose tolerance and no hyperglycemia. So MLC/SOCS-3 mice do not develop type 2 diabetes. Insulin tolerance is nevertheless reduced and this correlates with the degree of obesity of the transgenic mice. Molecular analysis of skeletal muscle did not show any decrease in insulin signaling in the muscle of MLC/SOCS-3 mice. However, we observed a decrease in SOCS-2 expression, another isoform of the SOCS family. SOCS-2 is a potential insulin inhibitor and so this decrease in SOCS-2 expression could compensate the increase in SOCS-3 expression and in this manner insulin signaling would not be disturbed. In conclusion, this study shows new evidence for the role of SOCS-3 in insulin resistance, as well as its possible involvement in muscle physiology. We have shown that, if constitutive expression of SOCS-3 in the skeletal muscle does not lead to type 2 diabetes, this induces an increase in adipose tissue, which is due in part to a decrease in physical activity. Molecular mechanisms for the role of SOCS-3 in physical activity remain to be defined.

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  • Critical pedagogy: where are we now?

    McLaren, Peter (2007)

    Book
    The University of Auckland Library

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  • Synergistic Induction of Differentiation in Leukemia Cells by All-Trans Retinoic Acid (ATRA) and Plant polyphenols: Involvment of Retinoic Acid Receptor Beta (RARβ)

    Steiner, Michael; Danilenko, M; Levy, J; Sharoni, Y (2007)

    Doctoral thesis
    The University of Auckland Library

    Current cancer therapies are highly toxic and often nonspecific. A potentially less toxic approach to treating this prevalent disease employs agents that modify cancer cell differentiation, termed "differentiation therapy". Acute promyelocytic leukemia (APL), bearing a characteristic t(15;17) chromosomal translocation with breakpoints within the retinoic acid receptor alpha (RARα) and promyelocytic leukemia (PML) gene, is the best example of a malignant disease successfully treated by differentiation-inducing agents, e.g. all-trans retinoic acid (ATRA). The specific sensitivity of APL cells to ATRA has modified the therapeutic approach of APL resulting in 90% complete remission. However, prolonged treatment with high (therapeutic) ATRA doses (~ 1 μM) results in retinoid resistance syndrome and relapses usually occur within months in nearly all patients who are initially sensitive to ATRA. Another powerful differentiation agent, 1,25- dihydroxyvitamin D3 (1,25D3), can induce differentiation in various myeloid leukemia cells in vitro. However, it is profoundly toxic (hypercalcemic) at pharmacologically active doses. Recently, we have shown that several dietary antioxidant micronutrients, such as the tomato carotenoid lycopene and the polyphenol carnosic acid (CA) found in rosemary, substantially enhance the differentiating and anti-proliferative effects of low, non-toxic concentrations of ATRA and 1,25-dihydroxyvitamin D3 in HL-60 and U937 human myeloid leukemia cells (in vitro) and in the mouse-leukemia model (in vivo). This enhancement of antileukemic effect in vitro was associated with an increase in the levels of nuclear receptors for retinoids and vitamin D (VDR). The current study characterized strong anti-proliferative and differentiation effects of combined treatment of different human myeloid leukemia cell lines and cells obtained from leukemic patients with low concentrations of ATRA (0.3-1 nM) and CA (2.5-5 µM), and explored the molecular mechanism underlying these effects. A synergistic cell growth inhibitory effect of CA/ATRA combinations in different leukemic cell lines correlated with cell accumulation in G0/G1 phase of cell cycle and was not associated with cell death. Furthermore, a synergistic induction of differentiation was observed, as was evident by the changes in cell morphology, reorganization of nuclear bodies, induction of CD11b, CD11c and CD18 surface markers and oxygen burst activity. Importantly, combinations of ATRA and CA showed anti-proliferative and differentiation effects on ATRA-resistant cell lines (NB4-R1 and HL-60R). Importantly, the above antileukemic effects of inducer/polyphenol combinations in all leukemia cell lines (NB4, HL-60, U937, PLB-985) and ex vivo cells obtained from leukemia patients correlated with strong induction of RAR and, in NB4 cells (the APL cell line), with PML-RAR degradation and RAR stabilization. RAR gene expression is often lost or reduced in human carcinoma cells, suggesting a tumor suppressor role of RAR. Interestingly, the resistance of NB4 cells to 1,25D3, and of HT-29 colon cancer cells, and some types of ex vivo leukemic cells (M2 and M4) to polyphenols, ATRA, 1,25D3 and their combinations were associated with undetectable levels of RAR in these cells that were not changed by the indicated agents. Overexpression of RAR, after transfection of the RAR-expression vector into HT-29 cells, enhanced cell growth inhibition induced by CA, ATRA and their combination by 20-40%. Overexpression of the same vector in U937 cells enhanced the ATRA-induced growth inhibition and differentiation by 20-30%, whereas the effects of CA/ATRA combinations were not increased. These data indicate that substantial upregulation of RAR by the CA/ATRA combination results in a maximal cell differentiation response which is not further enhanced by the ectopic expression of RAR. The opposite effects on cell growth and differentiation were detected after RAR downregulation. Expression of the dominant negative RAR, mutated in its DNA-binding domain, in HL-60 cells decreased the cell growth inhibition, induced by the CA/ATRA combination (by 20%), and differentiation induced by both ATRA at high concentration (by 30%) and the combination (by 25%). Even stronger reduction of ATRA- and CA/ATRA-induced differentiation (by about 40%) was obtained by silencing of RAR induced by RAR-siRNA oligos transfected into HL-60 cells. The mechanism, by which plant polyphenols synergistically enhance the anticancer effects of ATRA and 1,25D3, is largely unknown. Our data indicate the involvement of retinoic acid receptor regulation at both transcriptional (mRNA) and protein levels in the CA-ATRA synergism. This is evidenced by the correlation between the enhanced antileukemic effect of the CA/ATRA combination and upregulation and stabilization of RAR, induction of RAR, and reduction in PML- RAR levels. In addition, strong upregulation of vitamin D receptor (VDR) and its heterodimeric partner, retinoid X receptor (RXR), correlated with the polyphenol potentiation of the antileukemic effect of 1,25D3 in patient-derived leukemic cells. Besides CA-induced upregulation of RAR at the transcriptional level, as seen by mRNA induction and transactivation of RAR response element (DR5) derived from RAR gene promoter, the possible mechanism of nuclear receptor modulation may involve the inhibition by polyphenols of proteasome-dependent or -independent degradation of these receptors. Indeed, treatment of NB4 cells with the proteasome inhibitor, MG132, dramatically elevated RAR levels. However, in contrast to CA, MG123 only slightly increased the ATRA-induced differentiation, indicating that the upregulation of RAR alone is not sufficient for the differentiation enhancement. An additional putative mechanism, which emerged from this study and may contribute to the polyphenol-inducer synergism, is CA-induced inhibition of the ATRA degradation pathway. Our preliminary results obtained by RT-PCR indicate that in ATRA treated NB4 cells, CA downregulates CYP26, the member of the cytochrome P450 enzyme family which is responsible for specific oxidation and degradation of ATRA. CYP26 downregulation is likely to increase cellular levels of ATRA which, in turn, would enhance the differentiation response. These data are supported by the evidence that a synergistic potentiation by CA of ATRA-induced CD11b induction in NB4 cells was mimicked by ketoconazole, a broad range inhibitor of P450 related enzymes. Taken together, the results of the current mechanistic study suggest that the enhanced cooperative anticancer effects of the combinations of polyphenols and differentiation inducers in both leukemic and non-leukemic cancer cells are mediated by multiple complementing mechanisms (e.g., nuclear receptor upregulation and stabilization of inducer concentrations). This study may provide the basis for the development of strategies for the treatment of myeloid leukemias and other malignant diseases, both sensitive and resistant to ATRA treatment, using combinations of ATRA and dietary polyphenols. This work supports the important role of RAR as the therapeutic target in cancer prevention and treatment.

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  • Life in schools: an introduction to critical pedagogy in the foundations of education

    McLaren, Peter (2007)

    Book
    The University of Auckland Library

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  • Nietzsche's Evolutionary Ethics

    Small, Robin (2007)

    Book item
    The University of Auckland Library

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  • Jade Taniwha: Māori-Chinese Identity and Schooling. Auckland: Rautaki.

    Lee, Jennifer (2007)

    Book
    The University of Auckland Library

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  • On the Beach: Or the 'Unbearable Scandal' of Shrinking Swimwear

    Daley, Caroline (2007)

    Book item
    The University of Auckland Library

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  • Tertiary teaching matters: political economy of a New Zealand centre for tertiary teaching excellence

    Jesson, Jocelyn; Smith, R (2007)

    Book item
    The University of Auckland Library

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  • Psychiatry and the Law

    Brookbanks, Warren; Simpson, AIF (2007-05)

    Book
    The University of Auckland Library

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  • Maori and the Criminal Justice System

    Quince, Khylee (2007)

    Book item
    The University of Auckland Library

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  • Maori Disputes and Their Resolution

    Quince, Khylee (2007)

    Book item
    The University of Auckland Library

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  • Xenophon on Government

    Gray, Vivienne (2007)

    Book
    The University of Auckland Library

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  • International Encyclopaedia of Laws: Conflict of Laws: South Africa

    Schoeman, Elsabe; Roodt, HC (2007)

    Book
    The University of Auckland Library

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  • Nietzsche and Rée: A Star Friendship

    Small, Robin (2007)

    Book
    The University of Auckland Library

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  • Lecture on Ethics: Introduction, Interpretation and Complete Text

    Wittgenstein, Ludwig (2007)

    Book
    The University of Auckland Library

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