862 results for Doctoral, 2011

  • The development and applications of a micro-gap perforated electrode flow through cell

    Nath, Hilary (2011)

    Doctoral thesis
    University of Waikato

    Electrochemical techniques provide convenient and environmentally compatible ways of bringing about chemical transformations. However they generally lose their economic viability when used with low conducting electrolyte systems. This has limited their usefulness in the treatment of water and wastewater. Increasing the electrolyte concentration of these systems is not an option as it is with industrial processes such as the chlor-alkali process. Cell resistance is the major limiting factor. Cell resistance can be reduced by reducing the inter-electrode gap. A novel micro gap perforated electrode flow through (PEFT) cell has been developed for efficient and cost effective treatment of aqueous systems of low ionic strength. The PEFT cell is an undivided flow through design which encompasses both parallel plate and porous electrode features. It consists of plate electrodes and flow is both through the electrodes and parallel to the surfaces of the two electrodes. The perforations in the electrodes and the short flow distance between the electrodes allow the inter-electrode gap to be reduced to 50 microns and less without causing excessive resistance to hydraulic flow. With reduced electrical resistance, effective electrochemical treatment of natural water and other low electrolyte systems is possible. The PEFT cell was first applied to overcome a local water supply problem, the Waikato region’s iron and manganese contaminated bore waters. These waters form stable colloidal suspensions during slow air oxidation. The problem can be overcome by rapid electrochemical oxidation using the PEFT cell. Electrochemical oxidation was found to be more effective and efficient than chemical oxidation allowing removal of iron and manganese to meet drinking water standards with minimal formation of disinfection by-products (DBP). Electrochemical oxidation of water and wastewater systems is brought about principally by chlorine mediated indirect oxidation processes. A 240 µm gap PEFT cell, with a graphite anode was used for chlorine generation. It produced chlorine at current efficiencies above 60% with an energy consumption of 4.83 kWh/kg of chlorine from a 0.5 mol/L NaCl solution. This result compares well with industrial hypochlorite production using an undivided cell. Chlorine mediated electro-oxidation of effluents was successfully demonstrated by the degradation of textile dyes in water. Complete single pass electrochemical decolourisation of indigo carmine (IC) dye effluent containing 0.35 mol/L NaCl was achieved using a graphite anode PEFT cell. Energy consumption was 0.8 kWh/m3 or 8.3 kWh/kg of dye. This is an order of magnitude less than the energy consumption reported for colour removal using graphite anodes. It is comparable or lower than most colour removal work carried out using metal oxide coated dimensionally stable anodes (DSAs) and boron doped diamond (BDD) anodes. Reduction of pH from 7 to 3 reduced the energy consumption for decolourisation of IC dye by 50% and also increased the TOC removal by 20%. When NaSO4 was used as the electrolyte rather than sodium chloride, colour removal was much less effective. A single pass through a 50 µm gap PEFT cell with a stainless steel cathode and a graphite anode operated at 5.5 V achieved a 6 log inactivation of Escherichia coli bacteria in a water sample containing only 1.7 mmol/L of chloride ions. The power consumption was 0.5 kWh/m3 of water. The narrow inter-electrode gap allows high electric fields to be produced from low applied voltages. When the cell was operated at above 5.0 volts, a synergistic electric field effect was observed. Specific lethality of the chlorine was increased to at least 50 L/(mgmin), approximately two orders of magnitude higher than in the absence of the field. Increased specific lethality means that disinfection can be achieved at much lower free available chlorine levels than previously possible. This reduces the risk of DBP formation. Improved current efficiencies and reduced energy consumption for electrolysis at low electrolyte concentrations were achieved by partial insulation of the active anode surface of a 50 µm gap PEFT cell. This electro-catalytic effect was consistent with enhanced transport of the electroactive species to the active part of the electrode, reducing concentration and resistance overpotentials. In the electrochemical production of chlorine from 0.85 mmol/L NaCl at a current density of 2 mA/cm2, current efficiency was tripled and power consumption was reduced by a factor of two, relative to the cell without the anode modification. The reduction in the inter-electrode gap to 50 µm and less has allowed the production of electric field strengths greater than 10 kV/cm from applied voltages of less than a 100V. Field strengths between 1and 10 kV/cm are known to cause reversible electroporation whereas irreversible electroporation occurs above 10 kV/cm. Evidence for irreversible electroporation was provided by the 6 log inactivation of Escherichia coli (in the absence of chlorine) at an applied electric field of 22.5 kV/cm generated in a 40 µm gap PEFT cell by a 90 V DC supply. The energy consumption was 430 J/mL and without cooling, the temperature remained below 42oC. Inactivation was achieved by 20 hydrodynamically generated DC pulses. The low applied voltage, the elimination of the need for pulsed electric fields, avoidance of external cooling and the simplicity of the experiment bring commercial non thermal electro-pasteurisation one step closer.

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  • Wavelets, ICA and statistical parametric mapping : with application to agitation-sedation modelling, detecting change points & to neuroinformatics

    Kang, In (2011)

    Doctoral thesis
    University of Canterbury Library

    The wavelet methods developed, advocated and used in this thesis are primarily based on the discrete wavelet transform (DWT), wavelet thresholding and density estimation via wavelet smoothing. First a suite of wavelet techniques are advocated, based on the DWT, and applied successfully to assess whether an ICU patient's simulated" agitation-sedation (A-S) status reflects their true dynamic A-S profile. The use of quantitative modelling to enhance understanding of the A-S system and the provision of an A-S simulation platform are key tools in this area of patient critical care. Secondly novel wavelet density metrics are developed, a wavelet time coverage index (WTCI) and a wavelet probability band (WPB), based on Bayesian density estimation. This led to the development of two numeric metrics, the average normalized wavelet density and the relative average normalized wavelet density; both shown to be in close agreement to our DWT and earlier metrics."The DWT and WPB approaches also yield excellent visual assessment tools and are generalisable to any study involving bivariate time series of a large number of units (patients, households etc) and of significant length. P Wavelet thresholding and independent component analysis (ICA) are tested as denoising methods, and applied to brain image data, as part of the neuro-informatics study of Turner et al. (2003). ICA methods are then implemented for denoising all the cerebral function data, at a voxel by voxel basis. This is performed as a preprocessing step to the creation of statistical parametric maps (SPMs), used to model brain function with respect to personality as non-linear models. The results derived from our novel SPM-ICA approach support the theory of a biological basis for personality and report more de/activation clusters in the brain, as related to specific personality traits than Turner et al. (2003). Our work gives credence to a growing body of thought for the need of non-linear modelling in psychometric research (Cloninger, 2008). Our work also has the potential to increase momentum for patient specific drugs for depressives. Lastly we develop a DWT based methodology for change point analysis that uses modified, maximal overlap DWT (MODWT) coefficients to link to a novel shifting DWT (SDWT) methodology - a combination of the DWT and MODWT for the change point detection problem, which is shown to provide an accurate and computationally efficient change point location method. SWDT can be generalized to find multiple change points, by way of a binary segmentation procedure and iteration.

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  • Thermomechanics, material flow and microstructure evolution during Friction Stir Processing of light cast alloys

    Cui, Song (2011-11-28)

    Doctoral thesis
    Auckland University of Technology

    Friction Stir Processing (FSP) is a solid-state processing technique which can be used to refine and modify as-cast microstructures for superior properties. The aim of the present research is to investigate the following fundamental aspects associated with FSP cast Al and Mg alloys: the quantitative relationships between processing speeds (rotation and linear speeds: ω and v) and the thermomechanical responses (tool torque–M, power–P, specific energy–Es and material flow volumes–Vflow); the details of material flow/deformation and microstructural evolution during FSP of a cast Al-Si alloy; the mechanism governing the removal of Beta-Mg17Al12 particles during FSP of cast Mg-Al alloys. Experimentally, FSP of A356 cast alloy were performed with wide ranges of ω and v. M was measured during each FSP experiment, and temperature (T) at various locations were monitored for selected experiments. Based on M, P and Es were calculated. Stir zone areas (Aflow) were measured using the metallographic samples to estimate Vflow values. FSP experiments, using tool-pin-breaking technique, were conducted on A356 plates under two representative conditions. Macro-scale flow, micro-scale deformation, and microstructural evolution were studied by means of Electron Backscatter Diffraction technique. Pin-breaking FSP experiments were also conducted on AZ91/AM60 cast alloys during which T was monitored, based on which the thermomechanical and metallurgical explanations for the removal of Beta-Mg17Al12 particles were investigated. The relationship between M and ω is found to be well described by an exponential decay function: M = Mo + Mfexp(–nω); while the influence of v on M can be described reasonably well by linearly relating Mo, Mf, and n to v. Together with the consideration of temperature data obtained, M is shown to intimately relate to material flow resistance to tool motion. Thus n and Mf can be adjusted for alloying effect in the low ω range, while such effect diminishes as ω increases. It is shown that tool shoulder flow volume generated per revolution (VS–rev = Ashouderv/ω) relates to M, which can be interpreted as energy input per revolution, in a form of M = Mo + Mf[1 – exp(–γVS–rev)]. The tool-pin flow volume does not require a proportional amount of energy input. The larger diameter of the shoulder compared to the pin, coupled with higher material flow stress near shoulder region are the fundamental causes of this. A new flow mechanism that explains the formation of the non-ring nugget during FSP A356 was identified. It is shown that regardless of the processing condition, the highly refined portion of the nugget zone clearly segregates from the less refined portion. How this macro-segregation relates to the difference in flow regime inside and outside thread spaces is demonstrated in detail. The deforming dendrites located ahead of the pin were traced and based on this the strain and strain rate during FSP were directly estimated. The mechanism governing the recrystallization of α-Al dendrites was identified primarily as Geometrical Dynamic Recrystallization. Recrystallized α-Al grains around the pin displayed a dominating “A” shear texture, although the local “A” texture must undergo a degree of rotation to obtain the ideal “A” texture due to the local texture frame misaligned with the ideal texture frame. It was found that this misalignment is closely related to the direction of material flow at the location under consideration. Finally, detailed evidences suggest that the major mechanism governing the removal of the eutectic beta-Mg17Al12 particles is through a sequence of incipient melting of beta-phase, Al rich liquid wetting recrystallized α-Mg grain boundaries thus leading to a significant increase in liquid/solid interface, transfer of Al solute from liquid to interiors of α-Mg and the growth of α-Mg into the liquid (resolidification).

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  • The effects of joint flight attendant and flight crew CRM training programmes on intergroup teamwork and communication

    Ford, Jane Rosemary (2011)

    Doctoral thesis
    University of Otago

    The aim of this research is to assess and evaluate the effectiveness of Crew Resource Management (CRM) training programmes for enhancing teamwork and cooperation between flight attendants and pilots. CRM programmes have been defined as the use of all available resources to achieve a safe flight (Helmreich, Merritt & Wilhelm, 1999). CRM programmes were developed for pilots following a series of accidents in the United States in the 1970s which were attributed to ineffective (or non-existent) communication within the flight decks. CRM programmes were extended to flight attendants in the 1990s after accident investigations had determined that some crashes could have been averted if flight attendants had passed on safety critical information to the pilots (e.g., the 1989 British Midlands crash at Kegworth). Human error is attributed to 60-80% of air accidents (Shappell and Wiegmann, 2004; von Thaden, 2008). Studies 1 and 2 involved a 36-item questionnaire for flight attendants which was administered before and after the introduction of the new CRM training programme for flight attendants at a South Pacific airline. The participating airline is a major air carrier so it was possible to obtain large samples (500+) for each of these quantitative studies. The results showed that there had been a significant attitude change in the positive direction. Multivariate analyses also revealed that there were significant differences between fleet type flown, crew position flown and length of service (seniority). As predicted crews with a greater length of service displayed safer attitudes as measured by the FSAQ (Flight Attendants) Crews on the narrow-bodied A320 and B737 showed safer attitudes than their colleagues on the wide-bodied long-haul aircraft. Flight attendants in senior positions (ISD, ISC, and Purser) also displayed safer attitudes. Study 3 followed up on the significant positive attitude changes through a series of seventeen focus groups which involved 100 flight attendants. The purpose was to obtain high quality qualitative data on perceived barriers (and solutions) to communication between pilots and flight attendants. The major barrier identified was the locked flight deck door which meant that flight attendants could not see periods of high workload on the flight deck and there was difficulty in communicating safety critical information over the interphone due to noise and the lack of face-to face contact. Flight attendants suggested that one possible solution would be to install CCVT cameras so that the pilots could see that it was safe to unlock the door or see the flight attendants face. Another barrier was seen to be the lack of a whole pre-flight briefing on long-haul aircraft as flight attendants rarely had the opportunity to even see the pilots on the large B747 aircraft. A solution would be to have the whole team assemble in the area nearest to the flight deck for a quick two-minute briefing. The full briefing would still be between the Captain and the lead flight attendant who would then brief the flight attendant team. These data were then used to develop Study 4 which consisted of a 14-item questionnaire (FSAQ-Pilots) which provided data on the ways pilots viewed the barriers (and solutions) to intergroup communication. The results from this study showed that pilots safety attitudes varied according to fleet type flown, length of service, and crew position flown. Captains, pilots on narrow-bodied aircraft and pilots with a greater length of service all displayed safer attitudes than their colleagues. The qualitative data displayed the same solutions to barriers as the flight attendants had shown. The major barrier was once more the locked flight deck door and the installation of CCTV cameras was recommended. A whole team pre-flight briefing was also recommended. Study 5 followed up on these data by developing a CD Rom which contained five scenarios presented in video clip format. These short video clips involved a landing gear malfunction, drunken passengers, a medical emergency and an explosive decompression followed by an emergency landing. All these provided opportunities for both pilots and flight attendants to identify how they would show intergroup communication and cooperative teamwork. Pilots and flight attendants identified very similar patterns of communication which showed effective intergroup teamwork. Pilots and flight attendants with seven or more years experience; those in leadership roles (Captains, lead flight attendants ); and crews on B737 , B767, and A320 fleets showed significantly lower perceived ratings of danger, volatility, complexity, the role of the captain,, flight attendants and communication in the majority of the five video clips (as described in Chapter 7). Study 6 was an experimental intervention based on the social identity and social categorization theories which formed the theoretical framework for this thesis. According to these theories flight attendants would be more willing to engage in cooperative teamwork behaviours when their social (as opposed to personal) identities had been primed. Three subscales were identified through factor analysis. The subscale labeled intergroup cooperation showed significant differences between the groups when social (as opposed to personal) had been primed. Flight attendants in the social priming condition indicated that they would be more willing to engage in intergroup teamwork. The results supported the main hypothesis. Social identity theory has not been applied to flight crew teamwork previously. These data showed that joint CRM training is valued by both flight attendants and pilots, especially when joint training sessions enabled both groups to meet and hence break down barriers to communication; a major aim of CRM programmes.

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  • The influence of exotic salmonids on native host-parasite dynamics

    Paterson, Rachel Anne (2011)

    Doctoral thesis
    University of Otago

    Native parasite acquisition provides introduced species with the potential to modify native host-parasite dynamics by acting as parasite reservoirs (with the ‘spillback’ of infection increasing the parasite burdens of native hosts) or sinks (with the ‘dilution’ of infection decreasing the parasite burdens of native hosts) of infection. Exotic salmonids are frequently shown to acquire native parasites; however, as research into the threats posed by exotic salmonids has largely focused on predation and competition, threats posed by shared native parasites are poorly understood. I used a multiple-pronged approach combining field observations, experimental infections and dynamic population modelling to investigate whether native parasite acquisition by exotic salmonids alters host-parasite dynamics in native fish populations from streams and lakes in New Zealand and Argentina. I also used a meta-analysis approach to investigate which trait(s) influence native parasite acquisition by exotic freshwater fish. My research demonstrated that two key factors strongly influence whether the dynamics of native parasites will be affected by exotic fish. On one hand, the competency of exotic fish for native parasites is an important determinant of whether native parasite populations are likely to increase or decrease. On the other hand, the relative abundance of the exotic species determines whether its competency for a native parasite will actually translate into altered native host-parasite dynamics, with highly abundant exotic species more likely to induce changes in native parasite dynamics. I also demonstrated how exotic species may be able to override the influence of low host abundance, or competency by altering native host behaviour. The meta-analysis suggested that traits known to influence parasite richness in native fish or invasion success of exotic species are not reliable predictors of native parasite acquisition by exotic fish. Instead, it is more likely that complex interactions between a variety of biological, geographical and historical factors govern parasite acquisition by exotic species, making it difficult to predict whether native parasites will be acquired.

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  • Neuropsychological Function of Children and Adolescents With ADHD: Group and Individual Change Four Years After Diagnosis

    Robinson, Thomas (2011)

    Doctoral thesis
    University of Otago

    The present study compared the intellectual, academic, and neuropsychological performance of 55 children diagnosed with attention deficit hyperactivity disorder (ADHD) with that of an age and gender matched control 4 years after initial diagnosis. The performance of the ADHD group at initial-assessment and at four years follow-up was also compared at both the group and individual levels of analysis. Cross-sectional comparisons indicated the ADHD sample performed less well than controls on measures of intellectual function, academic achievement, and on some neuropsychological measures. Subgroup analyses suggested participants whose symptoms had remitted were less impaired relative to controls. Longitudinal group comparisons found little evidence of change over the course of the study. However, higher than expected proportions of reliable change at an individual level were observed for intellectual function and especially for academic achievement.

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  • Self gelling microemulsion systems for vaccine delivery

    Singh, Rinku (2011)

    Doctoral thesis
    University of Otago

    Purpose: The increasing interest in new generation vaccines is based upon utilising highly purified proteins and peptides as antigens. However, a disadvantage of these subunit vaccines is that they are often poorly immunogenic. Therefore there is a need to develop new formulation strategies which can generate the desired immune responses and are both safe and efficacious. One important formulation strategy involves incorporating antigens in polymeric nanoparticles, while another formulation strategy involves the dispersion of antigens in a sustained release carrier with the aim of increasing the size of the immune response generated and perhaps avoiding the need for multiple immunisations. In this thesis the aim was to combine these two approaches by dispersion of polymeric nanoparticles loaded with the antigen into a sustained release delivery systems (a self gelling microemulsion). Two polymeric nanoparticles (poly(ethylcyanoacrylate), PECA and chitosan nanoparticles, CNP) were dispersed in self gelling microemulsion (ME) templates. Ovalbumin (Ova) and Quil A were used as model antigen and adjuvant respectively. Biocompatible microemulsions were developed by establishing pseudoternary phase diagrams and these were then characterised at 37 ˚C. Lamellar liquid crystalline gel regions were found adjacent to the microemulsion regions in the phase diagrams. Upon addition of water or body fluids a phase transition from microemulsions to liquid crystalline systems may occur, which may make these systems suitable for sustained antigen delivery, as the rate of diffusion within the liquid crystalline phase is slower as compared to liquid vehicles. The study investigated the influence of the oil and water composition of the microemulsions on viscosity and release of antigen dispersed in the microemulsion in molecular form and incorporated into nanoparticles from the microemulsions and liquid crystalline gels. The ability of these formulations to generate immune responses towards Ova was also investigated in vivo. Methods: The ME components consisted of isopropyl myristate, lecithin, ethanol, water and either decyl glucoside (DG) or capryl-caprylyl glucoside (CCG). The selected microemulsions had a surfactant: water (S:W) ratio of 9:1 and a surfactant: oil (S:O) ratio of 5.2: 4.8. Formulations were loaded with fluorescently labelled Ova (FITC-Ova) and used as polymerisation templates for the preparation of PECA nanoparticles by interfacial polymerisation. CNP were prepared separately by a precipitation/coacervation method facilitated by sodium sulphate. The viscosity of one phase and two phase liquid crystalline gels (with and without nanoparticles incorporated) was determined using a cone and plate rheometer. A fluorometric assay was used to determine entrapment and in vitro release of FITC-Ova. An HPLC method was used to determine entrapment of Quil A in PECA nanoparticles and CNP. Self-gelling microemulsion templates containing Ova and Quil A either free or incorporated in nanoparticles were subsequently investigated in an in vivo mouse model with respect to their ability to induce a sustained immune response in comparison to nanoparticles in aqueous dispersions. Result and discussions: Biocompatible pseudoternary phase diagrams were established and characterised at 37 ˚C. Lamellar liquid crystalline gel regions and two phase turbid gel regions were found adjacent to a microemulsion region. This raised the possibility to formulate microemulsion templates converting into one phase and two phase liquid crystalline gels upon aqueous dilution. The appearance of a birefringent texture, characteristic for lamellar liquid crystals, after injection of the microemulsions into HPMC gels supported the hypothesis of an in situ phase transition. The average size of the PECA nanoparticles containing FITC-Ova was found to be in the range of 244-339 nm. The size of the nanoparticles was found to be larger in DG based microemulsions than CCG based microemulsions. The viscosity of microemulsions and liquid crystalline gels was found to show Newtonian and pseudoplastic flow behaviour, respectively. The viscosity of the microemulsion templates did not show any significant differences with increasing oil or water content, either in the presence or absence of nanoparticles. Further, the viscosity of one phase liquid crystalline gels was found to increase with increases in water content and to decrease with increases in oil content, both in the presence and absence of nanoparticles. In contrast, the viscosities of two phase liquid crystalline gels decreased with increases in water content and increased with increases in oil content. When the viscosity of one phase and two phase liquid crystalline gels were compared for systems formulated with the two different types of surfactants, the viscosity was found to be higher for DG based systems as compared to CCG based systems. The release of FITC-Ova from the selected microemulsion templates did not show any statistical differences due to the non-significant differences in their viscosity. The release of FITC-Ova from both DG and CCG based microemulsion templates was slow for the first 18 h with a rapid increase to 24 h, after which they attained a constant profile. In contrast, FITC-Ova release from one phase and two phase liquid crystalline gels was sustained for both antigen encapsulated in the nanoparticles and for free FITC-Ova. When the release from microemulsions, one phase and two phase liquid crystalline gels was compared for systems formulated with the two different types of surfactants used, release was found to be similar from microemulsion templates but was higher from CCG based one phase and two phase gels as compared to DG based gels. This was due to the lower viscosity of CCG based gels as compared to DG based gels. The entrapment of FITC-Ova in PECA nanoparticles was found to be in the range of 37.3% to 54.6% determined by a direct assay and 41.3% to 58.9% determined by an indirect assay, respectively. FITC-Ova entrapment determined by both methods was thus found to be in fairly good agreement. FITC-Ova entrapment in PECA nanoparticles however, did not follow any detectable trend with increasing oil or water content in the various formulations. The DG containing microemulsions and liquid crystalline gel formulations from the in vitro characterisation studies were selected and used to examine their ability to stimulate an effective immune response in an in vivo study. In vitro characterisation of the selected formulation was again carried out in terms of entrapment and in vitro FITC-Ova release. Additionally, entrapment of Quil A in PECA nanoparticles dispersed in the formulation was determined and found to be 46%. For comparison, a CNP dispersion in the same microemulsion formulation was prepared and entrapment of Quil A and FITC-Ova was found to be 27% and 56% repectively. The release of free FITC-Ova from the formulations was found to be faster than for encapsulated FITC-Ova for both PECA and CNP containing microemulsions. Surprisingly, a reduced T cell expansion and T cell proliferation were seen in lymph nodes and spleen after subcutaneous administration of microemulsions containing particulate formulations compared to aqueous nanoparticle dispersions. Cytokine levels, especially IFN-γ, were found either lower or comparable in mice immunised with sustained release microemulsion vaccine formulations as compared to nanoparticle dispersions. Ova specific IgG antibody titres however, showed lower antibody responses in mice immunised with particulate vaccines as compared to microemulsion based formulations, but this result cannot be further interpreted as higher response were also obtained with antigen dissolved in PBS and in the presence of alum (i.e. the control groups). Of major importance however was that the microemulsion formulations induced significant injection site reactions curtailing further animal experiments. Conclusions: Microemulsion formulations were successfully prepared and modified to incorporate FITC-Ova into PECA nanoparticles. The microemulsions demonstrated phase transition capability to liquid crystalline gels upon absorption of small amounts of water. The nanoparticles containing microemulsions can thus be used as in situ gelling slow release formulation for antigen, encapsulated in nanoparticles as a sustained release formulation to deliver antigen for a prolonged period of time. However, an advantage of these systems to stimulate higher immune responses, compared to aqueous antigen containing nanoparticle dispersions, was not found, as the latter showed better immune responses as compared to particle containing microemulsions. The reasons for this may be that the release of encapsulated antigen from the in situ forming liquid crystal is too slow due to the slow diffusion of the nanoparticles and that the release of free antigen is also slow due to extensive protein-gel binding.

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  • An Epigenetic Analysis of the Human Placenta

    Macaulay, Erin Cuffe (2011)

    Doctoral thesis
    University of Otago

    The human placenta is a highly specialized organ that is responsible for the survival of pregnancy. During its development, placental trophoblast cells invade into the uterine wall to establish a blood supply for the growing fetus. Previous studies have suggested similarities between the invasive phenotypes of trophoblasts and tumour cells; however, a key difference is that trophoblast invasion is under strict control. Given that epigenetic mechanisms have been linked with the silencing of key regulatory genes in cancer, we hypothesized that the epigenetic regulation of first-trimester placental trophoblasts may provide a mechanistic relationship between placental and cancer growth. Further, although the hypomethylated environment within the pseudo-malignant placenta is unique, its role in facilitating placental function is poorly understood. We sought to document placental-specific epigenetic modifications, taking into account that the origin of the placenta is determined during the earliest stages of embryonic development, when the inner-cell mass is first distinguished from the trophectoderm, and when the inner-cell mass further differentiates into the primitive endoderm and the epiblast. A genome-wide methylation analysis was performed using methylated DNA immunoprecipitation (MeDIP) combined with hybridisation to promoter microarrays to identify differentially methylated gene promoters between first-trimester human placenta and peripheral blood DNA. The promoter methylation of 29 candidate genes was then quantified using Sequenom MassARRAY®. Differential methylation patterns were detected in placental tissues compared to both fetal and adult somatic tissues. The relationship between promoter methylation and gene expression was then assessed using real-time PCR and immunohistochemistry. The promoter methylation of one gene, KCNH5, was found to be lineage-specific: low in all tissues derived from the extra-embryonic lineages (trophectoderm and primitive endoderm) and very high in tissues derived from the embryonic (epiblast) lineage. The dichotomous promoter methylation of KCNH5 was found to regulate the lineage-specific expression of alternative gene transcripts. Interestingly, the KCNH5 promoter that is used in tissues derived from the extra-embryonic lineages, and which shows dichotomous methylation, has recently evolved from a SINE retrotransposon that is present in only humans, old world monkeys and apes. To our knowledge, this the first example of a human transcript derived from the insertion of a SINE element. Finally, the lineage origin of the extra-embryonic mesenchyme has been a topic of longstanding debate. The combined epigenetic and expression profiles of KCNH5 in placental villous stroma provide compelling evidence that the extra-embryonic mesenchyme is derived from the primitive endoderm. Retrotransposons are normally silenced by methylation to prevent genome dysfunction. However, the placenta is becoming increasingly known as a tissue in which retrotransposons are actively transcribed. We observed that the absence of retrotransposon-silencing by methylation permitted the emergence of a placental-specific transcript by allowing the retrotransposon to serve as an alternative promoter for KCNH5. Examination of additional retrotransposon-derived genes in the placenta (INSL4 and ERVWE1) confirmed that dichotomous methylation between embryonic and extra-embryonic lineages is a feature of early development. The finding that the retro-elements in these genes have escaped the normal silencing mechanism suggests that they may have functional roles that are unique to the invasive placentas of humans and recent primates.

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  • Role of NK cells in DC-based immunotherapy of melanoma

    Bouwer, Anthea Lynne (2011)

    Doctoral thesis
    University of Otago

    Natural killer (NK) cells were first identified by their ability to kill tumour or virally infected cells without prior sensitization. In spite of this, the actual role of NK cells in tumour immunotherapy remains controversial. This study therefore set out to investigate the potential of Streptococcus salivarius K12, a gram-positive bacterium that has a history of commercial application as a probiotic in New Zealand, for use as a NK cell adjuvant, applying the therapy using B16.OVA melanoma as a model. To confirm that S. salivarius K12 was able to induce efficient activation of NK cells, I first screened a number of gram-positive and gram-negative bacteria for their ability to induce IFNγ release from NK cells. Using ELISA and fluorescence activated cell sorting (FACS) I found that gram-positive bacteria stimulated a rapid release ( through interaction with self MHC during development. Therefore having a setting where the addition of S. salivarius K12 activates NK cells, I investigated whether these NK cells were recruited to the draining lymph nodes where they could potentially influence the adaptive immune response. A range of adjuvant-activated and S. salivarius K12-activated DC were injected subcutaneously into the flanks of mice and tested their ability to recruit NK cells to the draining lymph node. The adjuvants differed markedly in their ability to recruit NK cells with S. salivarius K12 being the most effective. To determine if activated NK cells would be of benefit in tumour immunotherapy, I investigated the ability of bacterially activated DCs to elicit anti-tumour responses in a B16.OVA melanoma model. Utilizing a therapeutic tumour model where treatment was started three days following tumour inoculation, I found a significant delay of tumour growth in mice that were immunized with ovalbumin-pulsed DC that had been treated for 4 hours with S. salivarius K12 as opposed to other adjuvants tested. I also determined that in vivo depletion of NK cells completely abolished the benefit of DC immunotherapy. A therapeutic tumour experiment where DC were primed in the presence or absence of tumour antigen showed that while NK cells were critical for the antigen-dependent anti-tumour response they did not appear to exert an effector function. To investigate the role of NK cells in priming the anti-tumour response I next utilized a prophylactic setting, where mice were challenged with tumours sixty days after DC immunization. By depleting CD4+/CD8+ T cells and NK cells before time of priming or challenge, I tentatively showed that all three subsets of cells play a role in the anti-tumour response, although NK cells may play a greater role at time of challenge.

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  • Raising the comprehension skills of Thai tertiary readers of English through strategies-based instruction

    Akkakoson, Songyut (2011)

    Doctoral thesis
    University of Otago

    This quasi-experimental study researches the efficacy of strategies-based instruction on the L2 and L1 reading proficiency and reading strategy use of Thai students. The subjects were 164 tertiary students of scientific and technological domains at King Mongkut’s University of Technology North Bangkok (KMUTNB), Thailand. A programme of strategy training was introduced to an experimental cohort of 82 students while another 82 students were taught in a control condition using traditional, teacher-fronted methods. A mixed research approach using both quantitative and qualitative procedures was adopted. A standardised test of English reading comprehension, a test of Thai reading comprehension, and a strategy use questionnaire were administered to all subjects as pre-post measures. A post-lesson interview was conducted with three students from each cohort during the course. A concept interview was conducted with all students from both cohorts to form a post-course survey. As out-of-class assignments, all students were required to produce portfolio entries giving their retrospective accounts of strategy use for 11 weeks. The quantitative findings revealed a significantly higher gain in English and Thai reading abilities in favour of the experimental cohort. Both cohorts reported more frequent strategy use after the course, but there were no statistically-significant differences on the post-survey between the two cohorts. The findings indicated a significant correlation between strategy use and English reading proficiency as well as between English and Thai reading proficiency. The qualitative results indicated that the experimental cohort developed more strategic awareness, and appeared to use a wider range of strategies when reading in other situations. The results also show that, in the process of learning to use reading strategies, EFL learners with higher reading proficiency are more efficient at manipulating strategies than those with lower reading proficiency. Overall, the results of this study support the effectiveness of activating metacognitive awareness and of explicit instruction in reading strategy use.

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  • RAGE Biology and Inflammation

    Park, Sun-Jin (2011)

    Doctoral thesis
    University of Otago

    The receptor for advanced glycation end products (RAGE) is a cell surface signal transduction receptor that amplifies inflammation, principally through the activation of nuclear factor (NF)-κB. A circulating soluble form of RAGE (sRAGE) acts as a “decoy” receptor to alleviate the pro-inflammatory activity mediated by membrane-bound RAGE (mRAGE)-ligand interaction. N-linked glycosylation of RAGE plays an important role in the regulation of ligand binding. Two sites for N-linked glycosylation, at Asn25 and Asn81, are implicated, one of which is potentially influenced by a naturally occurring polymorphism that substitutes Gly82 with Ser (G82S). The G82S RAGE polymorphic variant is associated with reduced plasma sRAGE levels and enhanced ligand-binding. As a consequence, there is increased pro-inflammatory activity associated with the G82S variant. The overall aims of this study were to establish the effects of the G82S polymorphism on RAGE glycosylation and the consequences for sRAGE production and mRAGE-ligand binding. To compare the glycosylation patterns of the G82S variant of RAGE with that for wild-type (WT) receptor, WT-mRAGE or G82S-mRAGE were produced by transfecting human embryonic kidney 293 (HEK293) cells and the glycosylation patterns of expressed proteins were compared. Enzymatic deglycosylation showed that WT-mRAGE and the G82S-mRAGE are glycosylated to the same extent. Furthermore, various mutant forms of mRAGE (N25Q, N81Q, N25Q+G82S, and N25Q+N81Q) were produced to further investigate the complexity of the RAGE glycosylation patterns. Cell surface biotinylation and flow cytometry analysis was used to establish the sub-cellular distribution of WT and all of the mutant forms of mRAGE. All forms reached the cell surface when expressed by HEK293 cells and African Green Monkey SV40-transfected kidney fibroblast cells (COS-7). Mutagenesis and mass spectrometry analysis showed that Asn25 is always “fully” glycosylated in both WT-mRAGE and G82S-mRAGE. However, Asn81 may or may not be glycosylated in WT-mRAGE, whereas in G82S-mRAGE, Asn81 is always “fully” glycosylated. Combined these data indicate that the G82S polymorphism promotes N-linked glycosylation of Asn81. Subsequently, the effects of the enhanced Asn81 glycosylation were investigated. The production of sRAGE via mRAGE ectodomain shedding and the binding of ligand to WT and the variously glycosylated, mutant forms of mRAGE were investigated. Human embryonic kidney 293 cells were transiently transfected with plasmids containing WT or various mutant forms of mRAGE cDNA. To assess matrix metalloproteinase (MMP)-induced mRAGE shedding, surface-expressed mRAGE was biotinylated and transfected cells were treated with potential “shedding inducers” - 4-aminophenylmercuric acetate (APMA) or phorbol 12-myristate 13-acetate (PMA). Levels and component species of mRAGE remaining on the cell surface and of sRAGE released into the cell culture medium were compared by western blot analysis. The data show that reduced amounts of sRAGE were released from transfected cells expressing G82S-mRAGE, when compared to WT receptor. It appears that enhanced N-linked glycosylation associated with G82S-mRAGE reduces the efficiency with which the mRAGE ectodomain is shed from the cell surface. Increased G82S-mRAGE remained on the cell surface. The combination of reduced sRAGE production and retention of cell surface mRAGE reveals one mechanism whereby the G82S polymorphic variant is associated with increased pro-inflammatory activity. Finally, binding of WT and mutant forms of mRAGE by S100B and high mobility group B (HMGB)-1 ligands were compared. Rather than consider the ligand binding directly, the consequences for NF-κB activation were assessed by NF-κB p65 nuclear translocation and western blot analysis. The data show that with WT-mRAGE, S100B requires Asn25 glycosylation for binding while glycosylation of both Asn25 and Asn81 is required for the mRAGE-HMGB-1 interaction. In contrast, N-linked glycosylation of Asn81 was sufficient to mediate S100B and HMGB-1 binding to G82S-mRAGE. The results suggest that the enhanced Asn81 glycosylation seen in G82S-mRAGE introduces an additional point for mRAGE-ligand interaction. This may contribute to the increased ligand binding displayed by the G82S variant of RAGE. In support of this an adapted surface molecule structure model for RAGE shows Asn81 is located in close proximity to the ligand-binding region of RAGE. Therefore the glycosylation of Asn81 may have an impact on RAGE structure. Given that the glycosylation does appear to influence RAGE-ligand interaction, Asn81 is ideally positioned to perform a sentinel role, controlling RAGE receptor function. The results in this thesis highlight different N-linked glycosylation patterns for WT-mRAGE and G82S-mRAGE, and provide detail of how N-linked glycosylation regulates aspects of RAGE biology. Enhanced N-linked glycosylation, promoted by the G82S polymorphism contributes to reduced sRAGE production and mRAGE-ligand interaction. The data go some way to explaining why there is increased inflammation associated with the G82S RAGE polymorphic variant.

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  • Childhood Amnesia and Episodic Memory: A Developmental Perspective

    Tustin, Karen (2011)

    Doctoral thesis
    University of Otago

    The phenomenon of childhood amnesia has been well-documented over the last century; in general, adults are unable to recall events that occurred during their infancy and early childhood. Although researchers have proposed a wide variety of theoretical accounts, the specific mechanism responsible for childhood amnesia is unknown. One theory of childhood amnesia involves the development of episodic memory. The term episodic memory is used to refer to the recollection of personal, past experiences. Tulving (1972, 1983) originally coined the term; he argued that episodic memory is memory for information about the what, when, and where components of an event. More recently, Tulving (1985, 2002a, 2005) has also argued that episodic memory is accompanied by autonoetic consciousness. The critical distinction of autonoetic consciousness is that the individual must remember something that happened to him or her in the past and not simply know that it happened. Tulving has argued that episodic memory skill is a uniquely human ability. Within the context of childhood amnesia, Tulving has also argued that infants and children under the age of 4 years lack the ability to form episodic memories, which renders them amnesic for events that took place during their infancy and early childhood. Although this theory has been widely accepted in the psychological literature, there is no empirical evidence to support the notion that young children are incapable of autonoetic consciousness and episodic memory. The experiments presented in this thesis were designed to examine episodic memory in young children. In Experiment 1, a new Timeline procedure was developed to directly compare the age and density of the early memories reported by children, adolescents, and adults. Overall, the proportion of memories reported before the age of 3 years was greater for the children and adolescents relative to the adults. In addition, the single earliest memory reported by children and adolescents was younger than that reported by adults. Importantly, regardless of the age of the rememberer, participants’ early memories had the same episodic and autonoetic characteristics. Experiments 2 and 3 involved a prospective design that provided the opportunity to eliminate alternative interpretations of the results of Experiment 1. Young children were tested using an operant train procedure originally developed by Rovee-Collier. Children’s verbal recall of the event was assessed after a 24-hour delay (Experiment 2) and after a 1-year delay (Experiment 3). Irrespective of the delay, children reported a large amount of information about the event. In addition, after both delays, children provided a significant amount of autonoetic, episodic information about their memories of the train event. In contrast to Tulving's account of childhood amnesia, the results of the present experiments indicate that children as young as 3 are able to encode episodic information and to report that information when they are tested after a significant delay. Although children and adolescents retain access to these very early episodic memories (at least during childhood), they are eventually forgotten. Clearly, childhood amnesia cannot be explained by an inability to form episodic memories early in development. Instead, children’s growing ability to use language to encode, store, and retain fuller representations of their episodic memories may make their memories less susceptible to forgetting over the long-term.

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  • Influence of the gut microbiota and probiotics on selenium metabolism in the rat: In vitro and in vivo studies

    Krittaphol, Woravimol (2011)

    Doctoral thesis
    University of Otago

    Selenium plays a major role in the immune system and in decreasing the risk of cancer. Plasma selenium levels are low in patients with certain gastrointestinal disorders suggesting a role for the gut microbiota in selenium metabolism and disposition. Probiotic treatment can modulate the gut microbiota but the effect of such treatment on the metabolism of selenium supplements is unknown. The present study investigated the metabolism of L-selenomethionine (L-SeMet) and selenite, commonly used as selenium supplements, by probiotic bacteria in vitro and by rat gut contents ex vivo. The effect of probiotic treatment on the disposition of selenium after oral dosing with L-SeMet and selenite in rats was also investigated. After anaerobic incubation of L-SeMet (0.51 mM) with 10% w/w suspensions of the contents of jejunum, ileum, caecum and colon from male Wistar rats at 37°C for 3 h, L-SeMet metabolism (30%) was greatest in caecum contents followed by colon, ileum and jejunum. Dimethyldiselenide (DMDSe) was produced to the extent of 8.7% of the L-SeMet added and 28.9% of the L-SeMet lost. A similar result was obtained after incubation of selenite (0.58 mM) with metabolism being complete in caecum contents and almost complete in colon. Dimethylselenide (DMSe) (5.7% of the selenite added) was produced accompanied by a red precipitate of elemental selenium. When L-SeMet (0.51 mM) was incubated anaerobically with individual antibiotic-resistant probiotic strains (Streptococcus salivarius K12, Lactobacillus rhamnosus 67B, Lactobacillus acidophilus L10 and Bifidobacterium lactis LAFTI® B94) (1 - 5x1010 cfu/mL) and with a mixture of the four probiotic strains (ca. 3x1010 cfu/mL) at 37°C for 24 h, 10 - 18% was metabolised with 36-80% of L-SeMet being converted to DMDSe and DMSe. In similar incubations with selenite (0.58 mM), metabolism was more extensive (26 - 100%) particularly by the lactobacilli with 0-4.8% of selenite being converted to DMSe and DMDSe accompanied by the formation of elemental selenium. Metabolism of L-SeMet or selenite in incubations with a combination of gut contents and the four probiotic strains indicated some suppression of L-SeMet metabolism and enhancement of selenite metabolism. These results suggest probiotics and gut microorganisms interact in relation to selenium metabolism in the gut. In the in vivo study, three groups of rats (n = 3/group) were given saline or a single oral dose of 2 mg selenium/kg as L-SeMet or selenite by gavage (untreated rats). Another four groups of rats (n = 6/group) were given the same dose of either L-SeMet or selenite (2 mg selenium/kg) at the time of the last dose of treatment with 3 mL of a mixture containing equal numbers of the four antibiotic-resistant probiotic strains (total cell count ca. 1x1010 cfu/mL) or vehicle (a mixture of the lyoprotectants trehalose, maltodextrin and lactitol) every 12 h for three days (treated rats). Blood was collected from five rats in each treatment group over 24 h and serum analysed for selenium along with samples of liver and kidney obtained at 24 h. The sixth rat in each treatment group was used to determine the counts of total bacteria and of each of the antibiotic-resistant probiotic strains in the four segments of the gut at 24 h. Serum selenium concentrations over 24 h were not significantly different between probiotic and vehicle treated rats but were more sustained after L-SeMet or significantly higher after selenite than in untreated rats. In the liver and kidney of probiotic treated rats at 24 h, L-SeMet produced a significantly higher selenium level in the liver and lower selenium level in the kidney than in untreated rats. A similar trend was observed in rats given selenite but the differences were not significant. Total bacterial counts in corresponding segments of probiotic and vehicle treated rats were similar to each other and to corresponding counts in normal rats except in jejunum which were 103 - 104 cfu/g higher. In rats treated with probiotics, only L. rhamnosus 67B and L. acidophilus L10 were detected in all gut segments in approximately equal numbers which were 102 - 104 cfu/g less than the corresponding total bacterial counts. The present study indicates that: L-SeMet and selenite are metabolised in rat gut contents ex vivo to form volatile methylated selenium compounds and, in the case of selenite, elemental selenium; probiotic bacteria can metabolise L-SeMet and selenite in a manner similar to the gut microbiota; oral treatment with lyoprotectants stimulates bacterial growth in the gut leading to changes in the metabolism of L-SeMet and selenite; and treatment with probiotics and lyoprotectants exerts an effect on selenium disposition over and above that produced by lyoprotectants alone. Overall these results suggest that the gut microbiota play an important role in metabolising selenium supplements and that treatment with probiotics and lyoprotectants can influence selenium disposition after oral doses of L-SeMet and selenite. Whether these changes indicate a beneficial role for combination treatment with probiotics and selenium supplements requires further research.

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  • Characterization of Siderophores in the Southern Ocean

    Velasquez, Imelda (2011)

    Doctoral thesis
    University of Otago

    Iron is an essential but limiting nutrient for phytoplankton growth in the marine environment. In some oceanic bodies like the Southern Ocean, the chlorophyll levels are low even if the nutrients are replete in these waters. These areas, otherwise known as high nutrient low chlorophyll (HNLC) regions, have very low Fe levels (Butler 2005). Previous works have shown evidence that dissolved Fe is generally present complexed with organic ligands (Rue and Bruland, 1997; Boye et al., 2001; Vraspir and Butler, 2009). These ligands prevent the Fe from precipitating under aerobic conditions and at near-neutral pH and thereby make them more bioavailable to organisms. The similarities of the stability constants of these ligands with the stability constants of known siderophores, or those that were isolated from cultured organisms, led to the assumption that these ligands may be or include siderophores (Rue and Bruland, 1995; Macrellis et al., 2001; Gledhill et al., 2004). Siderophores are low molecular-weight, organic compounds with a high affinity to Fe3+. Due to contamination problems and the difficulty in sampling, only a small number of marine siderophores have been chemically characterized. However, taking into consideration that these organic compounds could possibly be one of the driving factors that control productivity in the ocean and have influence on Fe biogeochemical cycling, a study focusing on the determination of their chemical nature is very important. In the present study, both fieldwork and experiments were conducted to determine the chemical nature of the marine siderophores in different water masses around New Zealand. Analytical methods were initially tested by characterizing siderophores biosynthesized by cultured bacteria isolated from thermoalkaphilic terrestrial system and marine bacteria grown under low Fe media. Catechol and hydroxamate moieties were detected from the thermoalkaphilc Caldalkalibacillus thermarum strain TA2.A1 by chemical assays. However, only a hydroxamate-type siderophore was identified by HPLC-MS analysis. A catechol siderophore was produced and detected from the heterotrophic marine organism Vibrio alginolyticus PWH3a. The enterobactin hydrolysis dimer, dihydroxy benzoylserine ((DHBS)2) was recognized from the MSMS fragmentation of the compound. Furthermore, a carboxylate-type similar to the known siderophore rhizofferin was detected from a culture of the aerobic marine proteobacteria roseobacter Silicibacter pomeroyi DSS-3. In neritic and Sub-Antarctic waters off the south eastern coast of New Zealand, a total of six hydroxamate type siderophores were detected and identified in surface waters. The Sub-Antarctic water represents a Fe-deplete body of water while neritic waters were considered Fe-replete. A large proportion of strong L1 class Fe-binding ligands were found in surface waters corroborating the detection of siderophores at the same depths and the assumptions that most of the strong ligands will be dominant at the surface. Furthermore, seasonal and water-mass-specific differences were observed for the ratio of L1 to the sum of ligands present in the surface water. In the mesotrophic and Fe replete subtropical waters off the eastern coast of New Zealand, hydroxamate-type siderophores, which all exhibit ferrioxamine fragmentation patterns, were identified together with their Na adducts in surface waters. Two among the five hydroxamate siderophores were suspected to also contain carboxylate groups, however, due to sample limitation this was not confirmed. The results were strongly supported by chemical assays, estimates of siderophore- producing bacterial abundance using CAS agar plate experiments and electrochemical measurements of the complexing capacity. The strong L1 class was found to dominate the Fe-binding ligand distribution in surface waters. Furthermore, a catechol-type siderophore was likewise qualitatively detected using chemical assay but as with neritic and Sub-Antarctic samples, this was not confirmed or detected by HPLC separation and mass spectrometry measurements. On board incubation experiments focusing on the origin of the ligands were also conducted during the Fe Cycle II cruise in the sub tropical waters off the eastern coast of New Zealand. The goal was to determine whether siderophores are produced during the bioremineralization of marine particles. Results showed the presence of a known open-chained ferrioxamine B and G siderophore among two more hydroxamate siderophore types found. The chemical assays and CAS agar test experiments strongly agreed with the HPLC-MS results. The electrochemistry analysis, however, failed to detect strong L1 class Fe-binding ligands. In general, this study has successfully identified the major functional groups of siderophores found in both Fe-deplete and Fe-replete water bodies around New Zealand. It can be concluded that hydroxamate (mainly ferrioxamine types) siderophores are present and potentially dominant in this area. The exact chemical structures of most siderophore were not confirmed. However, two known open- chained ferrioxamines (i.e. B and G) were detected from the particle remineralization experiment conducted during the Fe Cycle II cruise in sub tropical waters.

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  • The Marine Biogeochemisty of Cadmium: Studies of Cadmium Isotopic Variations in the Southern Ocean

    Gault-Ringold, Melanie (2011)

    Doctoral thesis
    University of Otago

    Cadmium (Cd) in the oceans closely mimics the behavior of the macronutrient phosphate (PO43-) and can be used in the enzyme carbonic anhydrase (CA), suggesting a biological uptake of Cd. This relationship between Cd and PO43- has been used extensively as a paleoproxy for historic nutrient cycling. However, the validity of this proxy is questionable due to the complexity of the Cd /PO43- relationship. To this end, Cd isotopic studies can provide critical insight into the mechanism controlling Cd uptake and may, in itself, be a useful paleoproxy for historic primary production. In this study, multiple collector inductively coupled plasma mass spectrometry (MC-ICPMS), combined with double spiking techniques, was used to determine Cd isotopic compositions in surface waters from the subtropical convergence and Southern Ocean phytoplankton cultures. These analytical techniques were improved to increase the precision on low (<0.2 nmol kg-1) year round, diffusion limitation of both Cd and Zn may be governing biological uptake. To provide further supporting evidence for this hypothesis, the Cd isotopic composition was also determined, for the first time, in a cultured marine phytoplankton (Proboscia inermis). The effects of Zn and/or Fe limitation on Cd uptake were investigated, and Cd isotopic compositions were used to calculate isotopic fractionation factors. Results showed that marine phytoplankton preferentially remove light isotopes from the medium, and the most fractionated Cd isotopic compositions were observed in Zn-replete samples despite higher cellular Cd concentrations in Zn-limited cultures. This suggests that phytoplankton have at least two different uptake mechanisms with one more highly selective for light isotopes than the other and that these mechanisms are dependent on Zn concentrations.

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  • Mild traumatic brain injury: A prospective repeated measures study investigating the influence of illness perceptions and coping on clinical outcome.

    Snell, Deborah Lee (2011)

    Doctoral thesis
    University of Otago

    Background: Mild traumatic brain injury (MTBI) results in persisting disability for a significant minority and factors influencing non-recovery remain poorly understood. Descriptive recovery models suggest psychological factors become increasingly important with time but these models lack empirical support. Research in other health conditions suggests patient perceptions of symptoms and recovery can have powerful explanatory utility but such constructs have not been systematically explored in MTBI. Accordingly, a theoretically derived model of health behaviour such as Leventhal’s common sense model (CSM) could refine understandings about MTBI recovery. The objective of this research is to examine associations between components of the CSM (injury perceptions, distress, coping) and outcome after MTBI. The key clinical applications of this research are the earlier identification of both those at risk for atypical recovery and potential treatment targets, thus improving the effectiveness of MTBI interventions. Study Design: In a prospective observational cohort study with repeated measures, participants age 16 or older (n = 147) were recruited within three months following a MTBI and seen again six months later. Clinical and demographic information was collected and participants completed questionnaires at both visits (Revised Illness Perceptions Questionnaire (IPQ-R), Brief COPE, Hospital Anxiety and Depression Scale (HADS), Rivermead Post-Concussion Symptoms Questionnaire, Rivermead Head Injury Follow-Up Questionnaire). Associations between components of the CSM, and conservative MTBI outcome criteria were examined using univariate (Chi square, t-test) and multivariate (multiple regressions) statistical approaches. Results: Demographic and injury related variables were not associated with MTBI outcomes over time. However components of the CSM were significantly associated with outcomes. Participants endorsing unhelpful injury perceptions at time one, that is stronger beliefs about symptoms attributed to the injury (Odds Ratio (OR) 2.9, 95% Confidence Interval (CI) 1.2 to 6.7; p < 0.05), severity of expected consequences (OR 2.0, 95% CI 0.9 to 4.7; p = 0.09), chronicity of disability (OR 2.0, 95% CI 0.9 to 4.7, p = 0.09) and emotional impact (OR 3.4, 95% CI 1.4 to 8.0; p < 0.01), had significantly greater odds of poor outcome six months later. There were also significant associations between high distress at time one (HADS anxiety and depression scores) and poor outcomes over time. Associations between coping and outcome were inconsistent and problems with the way coping was conceptualised and measured may have contributed to variability in the results. Conclusions: Consistent with Leventhal’s CSM, those at baseline visit who attributed many symptoms to their MTBI, expected this to have severe and lasting consequences, and greater emotional impact, were more likely to have poor clinical outcomes six months later. Coping appeared to have important associations with outcome but the results were inconsistent. More research is required to examine whether coping mediates or influences outcomes more directly. A theoretically derived, coherent model of health behaviour such as Leventhal’s CSM, offers a reasoned approach to examining psychological factors with potential for both predicting those at risk for atypical MTBI recoveries, and development of clinical and research approaches to intervention.

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  • XIAP, a potential regulator of follicular atresia in the sheep ovary

    Douglas, Hollian Raphaelle (2011)

    Doctoral thesis
    University of Otago

    X-linked inhibitor of apoptosis (XIAP) is a strong inhibitor of initiator (-9) and executer (-3 and -7) caspase activity (Eckelman and Salvesen, 2006). In female reproductive research, XIAP has received minimal attention considering the ability to arrest caspases provides a potential mechanism for regulation of follicular atresia. This study aimed to test the hypotheses that XIAP is indicative of follicular health and regulates follicular atresia and to investigate the novel idea that prolactin (PRL) stimulates XIAP expression in the sheep ovary. Estrous cycles of adult Romney ewes (N=31) were synchronized with Estrumate®. Tissue and blood samples were subsequently collected on either days 14, 15 and 16 or at twelve hour intervals during the follicular phase. Initially analysis involved developing a plasma PRL profile by radioimmunoassay. The presence and localization patterns of XIAP and prolactin receptor (PRLR) isoforms protein and/or mRNA were determined by RT-PCR, in situ hybridization histochemistry and immunohistochemistry. This was followed by a comparative study evaluating levels of active caspase-3 immunoreactivity and XIAP mRNA (N=22) or protein (N=23) expression in adjacent sections containing the same day 14, 15 and 16 antral follicles. Triple label immunofluorescence and confocal microscopy were subsequently used to further clarify XIAP and active caspase-3 protein localization and to establish the presence of an inverse expression relationship in granulosa and thecal cells. Differing doses of Ovine FSH and PRL, as potential stimuli of XIAP upregulation, were trialed in cultured granulosa cells. Finally, a provisional attempt to develop a granulosa cell death model for determination of PRL’s ability to upregulate XIAP was undertaken. This study showed widespread XIAP expression during both luteal and follicular phases of the estrous cycle. XIAP protein was detected from the primary follicle stage onwards, whereas the mRNA was only evident in antral follicles, likely due to limited sensitivity of the in situ hybridization histochemical technique. This also proved problematic for detection of PRLR isoform mRNA, which was localized in luteal cells and three antral follicles only. In the comparative analysis, all day 14 antral follicles showed positive XIAP mRNA expression irrespective of active caspase-3 levels. Over 50% of the day 15 and 16 antral follicles scored, however, showed an inverse relationship between XIAP mRNA/protein and active caspase-3 protein levels in, as did XIAP protein in day 14 antral follicles. Interestingly, in healthy follicles XIAP expression in thecal tissue was widespread, becoming increasingly localized to interna layers as active caspase-3 immunopositive granulosa cells became prevalent, despite a lack of caspase activity in the theca itself. Confocal microscopy showed active caspase- 3 and XIAP colocalization in granulosa cells. No significant negative correlation in expression patterns was observed, possibly due to brief active caspase-3 expression at apoptosis onset and/or individual follicle properties. Increasing plasma PRL levels including two peaks were apparent during the follicular phase, however, limited granulosa cell culture lifespan prevented conclusive results concerning PRL-induced changes in XIAP levels in vitro. Overall the results indicate XIAP directly interacts with active caspase-3 in granulosa cells, thereby promoting follicle heath and functioning as a regulator of follicular atresia in the sheep ovary.

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  • Non-Psychotic Traits of Schizophrenia as Abnormalities of Cerebral Asymmetry: Development of a Potential Early Screening Instrument

    Ball, Kate Louise (2011)

    Doctoral thesis
    University of Otago

    The main aim of this research is to provide the basis for future development of an early screening instrument to identify individuals who may be at risk of developing schizophrenia (or related disorders). It is based on a neurodynamic theory of schizophrenia (Miller, 2008) which accounts for the non-psychotic trait abnormalities that are characteristic of the disorder. These abnormalities are enduring traits present before, during and after a psychotic episode and may become manifest in teenage years. Hence there is a potential to use these traits to identify risk at this stage for the purpose of intervening early with supportive, and/or educative resources to guide at-risk individuals to utilise their potential strengths, and help with the inevitable challenges that such a disorder may bring. The theory is based on a premise that there is a relative absence of rapidly conducting cortico-cortical axons thought to be associated with functions normally preferred by the right hemisphere. Most functions (but not all), that signify a trait abnormality in schizophrenia, come into this category. This suggests that schizophrenia has homogenous origins that manifest in a number of different impairments. The traits correspond broadly to two cortical states, the ‘upstate’ and the ‘downstate’. They were resolved further into 16 ‘a priori’ subject areas of function. These a priori categories were assessed in an instrument (the Schizophrenia Traits Questionnaire, or STQ), consisting of 96 statements about everyday experiences, to be rated on a 5-point Likert scale. In order to test the theory as the basis for a potential early screening instrument, it was necessary to test the large-scale correlational structure of the 96 items deriving from it, using Factor analysis. A second aim was to assess whether the 16 a priori categories could reliably distinguish schizophrenia and normal. The third aim was to find the combination of the items in the STQ which can best predict whether an individual will fall into the schizophrenia group or normal group with a high level of accuracy. Two versions of the STQ (‘STQ1’ AND ‘STQ2’) were tested on 300 mental health users (MHU) and 300 non-mental health users (non-MHU). There was some overlap in respondents from STQ1 to STQ2 (40 identified in the MHU group). For both STQ1 AND STQ2, factor analysis involved a 2-step process (due to the requirements of factor analysis for 5-10 respondents per item). Step 1 consisted of 16 separate factor analyses from which 26 factors emerged overall. Summary scores of the 150 MHU and 150 non-MHU participants on each factor, were used for the second step in a Grand Factor Analysis to assess the overall conceptual structure. Both step 1 and step 2 of the factor analyses supported the underlying theory. Power analyses were performed on each item in STQ1 to determine how many respondents were needed for the comparison t-tests to have ‘power’ (to reject the null hypothesis). Paired t-tests were performed on all 96 items of STQ1 to compare MHU and non-MHU participants. 30 items were eliminated from STQ1 on the basis of 5 criteria. 1.Item loading above 0.400 on its factor 2. P-value (in relation to T value) ≤0.05; 3. Number of subjects needed for comparison from power calculations for each item, 4. Conceptual coherency, 5. Specificity and relevance to the underlying theory. On the basis of this process, items in STQ1were changed and refined in developing STQ2. Consent to seek a verified diagnosis (including some associated information: see appendix 1c) was given by 136/150 MHUs. 75/136 had a verified diagnosis of schizophrenia. Factor analysis was performed on STQ2 and gave the underlying theory further support. The results support the corresponding 2 factors (of ‘disorganisation’ and ‘psychomotor poverty’) of Liddle’s (1987) 3-factor classification of the symptoms of chronic schizophrenia. Paired t-tests were then performed on the 75 schizophrenia respondents matched to 75 normal respondents for age, sex and number of years at secondary school. These comparisons revealed statistically significant differences between groups in 56 of the STQ2 items, with no items giving significant challenge to the theory. Paired t-tests were also performed on a smaller group of 29 with a verified diagnosis of unipolar depression and 29 matched with schizophrenia and also 8 with diagnoses of bipolar disorder matched with 8 with schizophrenia. This was for the purpose of distinguishing statistically between schizophrenia and other mental health groups to eliminate the possibility that traits are associated with mental health users in general rather than schizophrenia in particular. Reliability statistics (kappa) were performed on 62 repeat (normal respondents) over a 6 to 12 month period. Using the results of the factor analyses, t-tests and kappa statistics, groups of items were chosen to perform a Discriminant Function Analysis on 75 schizophrenia respondents and 150 normal respondents. A combination of 13 items was able to predict whether an individual would fall into the schizophrenia or normal group to 85% accuracy. Strengths that stand out from other early intervention instruments are: (1) The STQ is based on a psychobiological theory backed by empirical evidence (2) The theory defines trait abnormalities that can be independently assessed by questionnaire (3) The traits are enduring and not dependent on prodromal symptoms but on everyday activities enabling earlier detection (4) STQ is easily administered, inexpensive, with innocuous items potentially suited to young people (5) Thirteen of the items in combination accurately predicted schizophrenia to 85% accuracy in this sample with no mention of psychotic symptoms. In studying the theoretical background to the STQ, it was possible to link it to a previous work of the author (Smith 1998) on the sense of self in schizophrenia. It is therefore suggested that there is an imbalance in a ‘now experiencing I’ and an ‘objectified me’ in schizophrenia. There is justification for this, given that in schizophrenia there is difficulty in the self interacting in the moment as subject, until experience can be conceptualised and the self becomes object (to itself). This ‘I/me’ split has been further realised in the current work on trait abnormality: The functions normally preferred by the right hemisphere coincide with the function of the ‘I’ as the ‘subject’ of experience, which is impaired in a manner similar to other trait abnormalities. The strength of the conceptualising left hemisphere to compensate for impairment in the right corresponds to the ‘me’ as ‘object’ (and to a trait that is potentially better than normal in schizophrenia, the tendency to ‘long trains of thought’). Points are raised in regard to the ‘gap’ between the analysis of psychological or brain mechanisms and ‘whole person psychology’, which corresponds to the psychiatrist’s world of hard science while dealing with human beings, and the philosophical dilemma of mechanism versus meaning.

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  • The role of lipopolysaccharide/Toll-like Receptor 4 signalling pathway during liver regeneration after partial hepatectomy in mice

    Muhamad, Marlini (2011)

    Doctoral thesis
    University of Otago

    The process of liver regeneration after partial hepatectomy is very complex and is associated with signalling cascades involving initiation signals, transcription factors, cytokines, growth factors, tissue remodelling and termination of growth related signals. To date the exact mechanism of liver regeneration remains poorly understood. Toll-like receptor 4 (TLR4) acts as a sensor for immune signals and plays a critical role in host defence. It is known that lipopolysaccharide (LPS) is one of the ligands for TLR4. Binding of LPS to TLR4 leads to activation of transcription factor, nuclear factor kappa B (NF-κB) via the intracellular adaptor molecule, myeloid differentiation factor 88 (MyD88), which in turn activates the production of proinflammatory cytokines, TNF-α and IL-6. Evidence suggests that LPS/TLR4 signalling may be involved in liver regeneration following partial hepatectomy, as delayed liver regeneration and impaired cytokine responses were observed in C3H/HeJ mice, a mouse that is hyporesponsive to LPS due to a point mutation in the tlr4 gene. In mice lacking TLR4 receptor (TLR4 knockout mice), the early phase of the regenerative response was found to be normal, indicating TLR4 might not be involved in the early phase of liver regeneration. However, the involvement of the TLR4 signalling in the late phase of liver regeneration has never been elucidated. Therefore, we investigated whether LPS/TLR4 signalling is critical for liver regeneration after partial hepatectomy. In mice that are LPS-insensitive due to a mutation in the tlr4 gene (C3H/HeJ mice) or due to TLR4 receptor deficiency (TLR4 knockout/TLR4-/- mice) and in normal, LPS-sensitive mice (C3H/HeN and C57BL/6), hepatocyte proliferation and liver mass restoration, TNF-α and IL-6 production and NF-κB activation in the liver were assessed following partial hepatectomy. In addition, microcirculation and structural changes of the regenerating liver were examined in these mice following partial hepatectomy using the intravital fluorescence microscopy (IVFM). In the TLR4-/- mice, secondary gene expression changes deriving from the original signalling blockade was never considered. In light of this, hepatocyte proliferation and liver mass restoration following partial hepatectomy were examined in the LPS-sensitive mice (C3H/HeN) pretreated with anti-TLR4 monoclonal antibody (MTS510). Furthermore, in order to investigate the role of LPS in liver regeneration, hepatocyte proliferation and liver mass restoration were assessed following partial hepatectomy in the LPS-sensitive mice (C3H/HeN) treated with antibiotics, to restrict the availability of gram-negative bacteria in the gut (i.e. the main source of LPS). In the antibiotic treated C3H/HeN mice, hepatocyte proliferation and liver mass restoration following partial hepatectomy were found to be impaired, indicating the importance of LPS in liver regeneration. In addition, hepatocyte proliferation after partial hepatectomy was significantly reduced in the anti-TLR4 antibody treated C3H/HeN mice compared to the non-treated group. In LPS-insensitive C3H/HeJ mice, hepatocyte proliferation and liver mass restoration were delayed, which were associated with delayed TNF-α and IL-6 responses, impaired early activation of NF-κB in the liver, altered red blood cell (RBC) velocity and delayed regenerative structural changes following partial hepatectomy. In LPS-insensitive TLR4-/- mice, normal hepatocyte proliferation and liver mass restoration were observed, which were associated with an early upregulation of the IL-6 response, rapid activation of NF-κB in the liver and increased RBC velocity and sinusoidal blood flow following partial hepatectomy. However, in the TLR4-/- mice, the TNF-α response was blunted, late phase activation of NF-κB in the liver was impaired and hepatocyte hypertrophy and hepatocyte clusters remained at the late phase following partial hepatectomy, which indicate a delay in hepatic tissue remodelling in the TLR4-/- mice. In conclusion, the thesis has demonstrated that LPS/TLR4 signalling may be essential for liver regeneration following partial hepatectomy. The finding that TLR4 is involved in the hepatic tissue remodelling at the later phase of the regenerative process, most likely via TNF-α and subsequent late phase activation of NF-κB, is novel. In addition, the thesis has established differences between LPS-insensitive, C3H/HeJ and TLR4-/- mice, particularly in liver regeneration following partial hepatectomy.

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  • The Meaning and Discourses of Ethical Consumption and Production in Fair Trade Tourism

    Boluk, Karla Aileen (2011)

    Doctoral thesis
    University of Otago

    This thesis investigates a new and sustainable approach to tourism Fair Trade Tourism (FTT). FTT emerged as a mechanism to not only foster the sustainable operation of tourism businesses, but to also significantly attend to Pro-Poor Tourism (PPT) strategies. Fair Trade Tourism South Africa (FTTSA) is a certification programme that emerged in 2002. It is equally focused on the social, environmental and economic conduct of businesses. In comparison to the many other tourism approaches FTTSA’s distinction is that it is a certification. It provides a clear framework for implementation, evaluation and review. Such a certification in the context of post-apartheid South Africa facilitates improved conditions contributing to positive on-going transformation and creates conditions for the practice of a fairer tourism. The aim of this study is to investigate the consumption and production of FTT from the perspective of two informant groups, consumers and producers. The study investigates the following research questions: How do tourism consumers perceive fair trade and what is their level of involvement in ethical consumption? What was the motivation for FTTSA members to apply for FTTSA certification? Three objectives support the aim of this study: to explore consumers’ and producers’ understanding of the concept of ethical consumption as it relates to fair trade and FTTSA; to explore the discourses that inform the production and consumption of FTT in South Africa; to explore FTT in the context of a consumer’s wider ethical consumptive behaviour. The study utilizes two methods; namely Heuristic Inquiry (HI) and Critical Discourse Analysis (CDA). HI was used to guide the collection of the data and create a binary focus on both the researcher and the informant groups. As such I tracked my personal experiences and utilized a reflective approach to illustrate how my thought processes changed throughout the investigation and time spent in the field. CDA was used to analyse the data collected and reveal the multi-layers inherent in informants’ discourse. Specifically the use of CDA elicited three forms of constructions that influence informants’ discourses such as subject positions, linguistic repertoires and alternative subject positions. Data collection took place in South Africa over two phases. This research is concerned with the value associated with FTTSA membership from the perspective of tour operators and traveller’s awareness of FTTSA and their involvement in ethical consumption. Fundamentally, the research found that consumers had a good understanding of fair trade and they participated in ethical consumption back home, although, consumers had no prior knowledge of FTTSA before arriving in South Africa. Therefore, consumer fair trade awareness did not influence their consumptive behaviour while in South Africa. FTTSA members were interested in membership based on the opportunities the certification process provided to improve their businesses and create tangible benchmarks to ensure their social progress. Although informant groups demonstrated virtuous and collective interests, at times they contradicted such social concerns. The presentation of the data in the analysis chapters takes the form of two interpretations. Informants’ subject positions or micro discourses reveal their merits regarding their concerns and actively make contributions to their communities. A re-interpretation of the data identified some of the socio-cultural influences which affected informants’ language and consequently contradicted their exemplary micro positions creating tensions. Producers’ macro discourses illustrated soft paternalistic tendencies which may have been a consequence of their colonial guilt. Sometimes consumers demonstrated a moral high ground in some of their behaviours based on their level of education and specific lifestyle choices. Furthermore, they may have been concerned with demonstrating their ethical selves. Both producers’ and consumers’ macro discourses revealed a neo-colonial perspective based on their capitalist conditioning. Such macro discourses also influenced my discourse and this is discussed throughout the analysis chapters. Inherent contradictions in informants’ discourses demonstrate the intricate meanings and various motivations for tour operators’ interests in taking up FTTSA certification and consumers’ participation in ethical consumption.

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