400 results for Undergraduate

  • Comparing subpopulations of gonadotropin-releasing hormone (GnRH) neurons with viral mediated cell-filling

    Marshall, Christopher Joseph (2014)

    Undergraduate thesis
    University of Otago

    Gonadotropin-releasing hormone (GnRH) neurons are the central regulators of reproductive function in all mammals. The cell bodies of GnRH neurons are unique in that they are not confined to a discrete nucleus, but rather exist as a scattered continuum of cells throughout the basal forebrain. In rodent species, most of these GnRH neurons reside within three anatomical divisions of the brain: the medial septum (MS), the rostral preoptic area (rPOA) and the anterior hypothalamic area (AHA). Typically, these neurons are thought of as one large homogenous group, serving similar functions, however, recent anatomical and functional evidence suggests that this is not the case. One way to distinguish subsets of neurons is by their patterns of projection throughout the brain. Until now, the projection patterns of GnRH neurons have only ever been assessed for the population as a whole, due to the lack of ability to distinguish subdivisions from one another. The recent development of novel transgenic tools has enabled us to visualize GnRH neurons and their projections in their anatomical subdivisions of the MS, rPOA or AHA for the first time. An adenovirus containing a transgene for enhanced, farnesylated green fluorescent protein (Ad-iZ/EGFPf) was injected intracranially at stereotaxic coordinates for the MS (n=4), rPOA (n=6) or AHA (n=4) into female transgenic GnRH-Cre mice. Using this approach, Ad-iZ/EGFPf was specifically targeted to GnRH neurons in each region of interest, in order to “fill” these cells to the most distal ends of their projections. The first aim of this project was to assess how accurately GnRH neurons in MS, rPOA and AHA could be specifically targeted. Injections filled between 5 and 20 cells in most animals with accurate injection sites. In animals that received MS injections, more GnRH neurons in the MS were filled than in the rPOA (P < 0.05) and AHA (P < 0.05). Similarly, animals that received rPOA injections had more filled cells in the rPOA compared with the MS (P < 0.0001) and AHA (P < 0.001). In animals injected in the AHA there was no significant difference in the number of filled cells in the AHA compared with the MS and rPOA. In wild-type controls (n=3) and animals that received off-target injections (n=3), no filled GnRH neurons or projections were present. In the second aim of this project the distribution of projections from Ad-iZ/EGFPf filled GnRH neurons residing in the MS, rPOA and AHA were mapped. Across all animals, GnRH neuron fibre projections that were positive for GFP were found in 120 different regions of the brain, including nuclei, subnuclei and white matter tracts. These regions were found across several major brain divisions, in the hypothalamus, septum, thalamus, cerebral cortex, pallidum, striatum, amygdala, hippocampus and midbrain. The broad distribution of GnRH neuron projections highlights the diverse functions that these neurons are potentially influencing within the brain, as well as the power of the viral cell-filling approach used to visualize the full extent of these neurons. In the third aim, the projection patterns from GnRH neurons in the MS, rPOA and AHA were compared. Remarkably, 60% of the brain regions that contained fibre projections only did so from one or a combination of any two subpopulations of GnRH neurons, indicating that the projection patterns of these subdivisions is not homogenous. Notably, fibre projections in the vomeronasal amygdala originated exclusively from GnRH neurons in the AHA. Areas involved with olfactory processing likewise only received projections from MS and AHA GnRH neurons. Surprisingly, the largest division of GnRH neurons, the rPOA, had the most confined pattern of projection, but projected robustly to the median eminence suggesting a primarily hypophysiotropic role. For the first time, it has been possible to interrogate the projection patterns of anatomical subdivisions of GnRH neurons, which has revealed that they are far from homogenous. Overall, these results provide strong support for the existence of GnRH neuron subpopulations, highlighting that these neurons should be treated as similar but separate entities. Identifying GnRH neural subpopulations and delineating their respective roles could have wide applications, from increasing reproductive success in livestock, to teasing apart the ongoing mysteries surrounding infertility in humans.

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  • A novel technique to investigate coronary microvascular perfusion in diabetes

    Nissen, Hazel Merete (2014)

    Undergraduate thesis
    University of Otago

    Diabetes Mellitus (DM)-induced disease of the coronary microvessels contributes to the worldwide increase in cardiovascular morbidity and mortality in diabetic patients. These microvessels are vital to the regulation of regional blood flow, and facilitating oxygen and nutrient exchange within heart tissue. Whilst total coronary blood flow is readily measured, our limited ability to directly measure coronary microvascular perfusion has restricted our progress in understanding DM pathology. Therefore, I aimed to test the feasibility of adapting two techniques previously used in skeletal muscle, 1-methylxanthine metabolism and vascular casting, to measure coronary microvascular perfusion and assess how this is impaired in type 2 DM. Isolated rat hearts were perfused under physiological conditions, with assessment of cardiac contractility and total coronary flow. This was combined with investigation of both a chemical and physical approach to assess microvascular perfusion. Firstly, metabolism of exogenous 1-methylxanthine (1-MX) by xanthine oxidase on the endothelium was investigated as a measure of capillary surface area. In addition, casts of the physiological vascular structure were produced using rapidly setting dental acrylic injected into the coronary vasculature, and visualised with micro-computerised tomography. To examine the feasibility of these microvascular measures, protocols were developed to induce known perfusion changes in male Sprague Dawley rat hearts; isoproterenol (1x10-8M, vasodilation) and angiotensin II (1x10-7M, vasoconstriction) were applied. Secondly, a pilot study was conducted applying the 1-MX and casting techniques to compare 20-week-old male type 2 DM Zucker Diabetic Fatty (ZDF) rats to their non-diabetic littermates. In Sprague Dawley rats, isoproterenol significantly increased whilst, to a lesser extent, angiotensin II significantly decreased myocardial function and coronary flow (p≤0.05, n=6 and 7). Vascular casting produced promising results; a representative cast from the isoproterenol intervention displayed increased branching, and the angiotensin II intervention showed somewhat reduced branching of the microvessels relative to no intervention (n=1). However, 1-MX values did not reveal any changes between these interventions. Consequently, the 1-MX protocol was optimised to improve stability, before being applied in the type 2 DM pilot study. Under basal conditions, no significant difference was discerned between diabetic and non-diabetic rats in 1-MX disappearance (22.6±6.7nmol/min (n=5) vs. 23.4±3.9nmol/min (n=3); mean±SEM: n.s.), nor in measures of cardiac function. Likewise, no difference was discernible between a representative cast from the non-diabetic and diabetic group. However, a positive Spearman’s rank correlation was observed between coronary flow and 1-MX disappearance in the diabetic rats (rs=1, p≤0.05). With this study I have set up the foundations of using 1-MX metabolism and vascular casting, as techniques to examine coronary microvascular perfusion in the isolated heart. Conclusions regarding DM-induced changes cannot be drawn at this stage. However, pilot data provide valuable information on how to further develop these techniques. Novel measures of coronary microvascular perfusion have the potential to enhance our understanding of coronary microvascular pathology, and eventually reduce DM-induced cardiovascular complications.

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  • Enhancing Adoptive Cell Transfer for the Treatment of Cancer

    Shields, Nicholas James (2014)

    Undergraduate thesis
    University of Otago

    Cancer is currently the leading cause of death in New Zealand, accounting for 30% of all deaths in 2010. Due to factors such as an ageing population and poor lifestyle choices, the incidence and mortality of this disease is set to increase dramatically in the coming decades. This will exert substantial stress on the healthcare system, highlighting the urgent need for improved cancer therapies. The adoptive transfer of tumour-specific T lymphocytes, known as adoptive cell transfer (ACT), is a promising new approach to the treatment of cancer that has proven effective for the treatment of haematological malignancies and metastatic melanoma, achieving long-lasting clinical responses in approximately 50% of patients. The challenge now remains to enhance the therapeutic efficacy of ACT to enable a broad application of this therapeutic approach to the treatment of multiple types of cancer and achieve clinical responses in all patients. Using ovalbumin as a model tumour-antigen, the aim of this study was to (a), compare the ability of dendritic cells (DCs) and macrophages to generate tumour-specific CD4+ and CD8+ T cells for use in ACT, and (b), assess the phenotype and function of these generated cells. Splenocytes isolated from OT-I (CD8+) and OT-II (CD4+) transgenic mice were cultured with dendritic cells pulsed with respective ovalbumin peptide epitopes. After 10 days, these cells were restimulated with either dendritic cells or macrophages and cultured for a further 10 days. Macrophages could effectively generate tumour-specific CD8+ T cell responses but were poor inducers of CD4+ T cell responses compared to dendritic cells. In the case of CD8+ T cells, antigen-experienced T cells that were restimulated with macrophages exhibited superior phenotypic characteristics for use in ACT compared to DC-restimulated cells. Both DCs and macrophages predominately generated effector memory CD4+ T cells (CD44+CD62L-) and effector memory CD8+ T cells (CD44+CD62L-). Further phenotypic analysis of in vitro-generated T cells revealed distinct expression patterns of markers associated with T cell dysfunction, survival and differentiation. In response to their cognate antigen, generated T cells produced high levels of TNF-α and IFN-ɣ, suggesting that these cells can mediate effective tumour destruction in vivo. Future experiments will determine the length of time that adoptively transferred T cells persist in vivo and assess the efficacy of ACT using generated CD4+ and CD8+ T cells in an in vivo murine B16-OVA melanoma model.

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  • The Effect of Cell Metabolism on Epigenetic Processes in Cancer Cells

    Moustafa Albathish, Boushra (2014)

    Undergraduate thesis
    University of Otago

    Epigenetic marks are written on and erased from DNA through the activity of methylation and demethylation enzymes, thereby altering the level of gene expression. The methylase and demethylase enzymes respond to intrinsic and extrinsic factors, and their activity has been shown to be compromised under various pathological conditions, including cancer. The cycle of cytosine methylation and demethylation is a major epigenetic pathway for regulation of gene expression. DNA methyltransferase modifies cytosine, generating methylcytosine, which can be oxidised by the recently identified Ten Eleven Translocation enzymes (TET 1-3) to generate hydroxymethylcytosine, formylcytosine, and carboxycytosine. These oxidised methylcytosine products are intermediates in the cycle to regenerate unmodified cytosine. When present in gene regulatory regions, they also serve as stable epigenetic marks that alter gene expression. Aberrant methylation patterns, such as hyper- and hypo-methylation are a hallmark of cancer. This research project has focussed on the presence and activity of the TET enzymes in selected cancer cell lines. TET enzymes belong to the superfamily of proteins known as Fe(II) 2-oxoglutarate dependent dioxygenases. Proteins in this family act as metabolic sensors, because they require cofactors; iron and ascorbate, and co-substrates; oxygen and 2-oxoglutarate, for their activity. Changes in metabolic conditions and the availability of these metabolites modulate the activity of these proteins. In this project, I investigated the expression and activity of TET 1-3 isoforms in a range of human and mouse cancer cell lines. The cells were then subjected to different metabolic conditions, such as hypoxia, and ascorbate and iron deficiency. The effect of these conditions on TET activity and the epigenetic profile was evaluated using Western blotting and ELISA methods. Our preliminary results show varied expression of the TET isoforms across the selected cancer cell lines, with TET 3 expression being most prominent. Altering oxygen supply, iron and ascorbate appeared to affect the levels of 5mC and 5hmC. As these conditions affect cancer cells in vivo, we suggest that epigenetic changes in response to metabolic stress will affect genetic patterning in cancer.

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  • Understanding Batten disease: CLN5 expression in CLN6 deficient ovine neural cultures.

    McIntyre, Kristina (2014)

    Undergraduate thesis
    University of Otago

    Neuronal Ceroid Lipofuscinoses (NCL) are a group of debilitating and fatal neurodegenerative diseases of childhood resulting from progressive brain atrophy. The human and ovine variant late infantile CLN6 (ceroid lipofuscinosis protein) forms of NCL result from mutations encoding the endoplasmic reticulum transmembrane protein CLN6. Mutations encoding the soluble lysosomal protein CLN5 also cause an NCL in humans and sheep. The functions of these proteins are not understood, however previous research suggests a relationship between them (Lyly et al., 2009). In CLN6-/- ovine neural tissue, CLN5 protein expression was reduced (McIntyre, K.M., summer studentship unpublished observations, 2013/2014). The aim of this study was to investigate this potential interaction in the ovine model of CLN6 NCL. In CLN6-/- and CLN6+/- control secondary ovine cultures derived from primary ovine neural cultures, concentrations of CLN5 mRNA were assessed by relative quantitative polymerase chain reaction (qPCR). Protein expression was assessed by 3, 3 diaminobenzidine (DAB) immunocytochemistry, immunofluorescence and western blotting techniques. To detect CLN5 protein, cultures were transduced with CLN5 lentivirus (pCDH.MND.CLN5 (VSVG)). Proteasome inhibitor MG132 was applied for 2 and 4 hours to determine whether the reduction in CLN5 was due to ERAD (Endoplasmic Reticulum Associated-Protein Degradation). Absence of GFAP (Glial Fibrillary Acidic Protein) and MAP2 (Microtubule-associated protein 2) immunofluorescence in cultures indicated a cell population devoid of astrocytes and neurons respectively. Relative to ATPase and RPLPO (Large Ribosomal Protein) (M-value 0.936), no statistical difference in CLN5 mRNA concentration was found between CLN6-/- and control cultures (p = 0.68, n = 3). DAB immunocytochemistry showed low CLN5 protein expression in non-transduced cultures; reduced CLN5 protein expression was not evident. In immunofluorescence studies, no significant difference in relative fluorescent intensity was seen in transduced cultures (p = 0.75, n = 3). Western blot analysis of overexpressed CLN5 protein between CLN6-/- and control cultures was inconclusive. In non-transduced cultures treated with MG132, CLN5 expression was not detectable by western blotting. Data from transduced cultures are currently inconclusive. These results suggest that in this cell population, CLN5 mRNA is unaltered in CLN6 NCL. These methods may subsequently be translated to primary cultures as a foundation for on-going investigations into NCL protein interactions in ovine cultures.

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  • Is Prolactin Action on Forebrain Neurons During Pregnancy Important for Maternal Neurogenesis and Behaviours?

    Cashen, Sarah Christina (2014)

    Undergraduate thesis
    University of Otago

    Pregnancy is associated with a dramatic increase in serum prolactin levels that is essential for the normal increase in neurogenesis in the maternal brain, and for the associated expression of maternal behaviours after birth. As yet, it is not known which neurons prolactin acts on in the maternal brain to induce these changes, but it has been shown that prolactin receptors are present on GABAergic neurons in forebrain nuclei implicated in the regulation of maternal behaviour. This study aimed to examine the effect that prolactin action on forebrain neurons has on expression of maternal behaviour and on levels of neurogenesis, employing conditional deletion of the prolactin receptor using cre-lox technology. This technology allowed the creation of mice with prolactin receptors removed from CaM kinase II containing neurons (most forebrain neurons; CKC mice), and more specifically from neurons containing the vesicular GABA transporter (vGAT mice). When cre positive mice and controls (wildtype C57BL/6J and cre negative mice), were reproductively mature (6-8 weeks old), some mice were placed in individual cages and mated with a stud male. Neurogenesis was assessed on day seven of pregnancy, using injections of BrdU, which labels newly dividing cells. Other groups of mice were allowed to continue the pregnancy until term, and the day of parturition was designated as postpartum day one (PPD1). Maternal behaviour was tested on PPD2 by placing the mouse and three pups in a clean novel cage and observing all behaviours. On PPD3 maternal behaviour was tested in a similar manner in the home cage. Mice were also tested for anxiety on PPD4 in the light-dark box. Both CKC and vGAT cre positive mice showed significantly impaired maternal behaviour in the home cage compared with control mice, but the impairment was greater in the CKC positive mice than the vGAT cre positive mice. While all mice showed impairment in the novel cage, maternal behaviour was once again significantly more impaired in the CKC positive mice. CKC positive mice mostly ignored the pups, but at times were aggressive towards them, either eating pups before, or after death. Consequently, pup survival was dramatically reduced in CKC cre positive mice, compared with controls. There was no difference in levels of anxiety between the groups postpartum, suggesting that prolactin acts elsewhere to exert the postpartum anxiolytic effect. Surprisingly, given the maternal behaviour results, the levels of neurogenesis on day seven of pregnancy in the transgenic mice were not reduced as expected. There was no significant decrease in neurogenesis in either cre positive group compared with the controls. These findings suggest that prolactin acts through receptors on GABAergic neurons to stimulate some aspects of maternal behaviour, but other neurons must also contribute, and that prolactin acts somewhere other than forebrain neurons to induce neurogenesis.

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  • Multiple Dosing of RHDV VLP to Enhance the Anti-tumour Response

    Sadrolodabai, Yasmin (2014)

    Undergraduate thesis
    University of Otago

    Metastatic melanoma has a poor prognosis, with a median survival of 13 months after diagnosis. New Zealand has among the highest melanoma rates in the world with more than 2000 cases registered every year. Metastatic melanoma continues to be a challenging disease to treat, but recent immunotherapeutic approaches have demonstrated promising results. Our laboratory has developed a cancer vaccine using a virus-like particle (VLP) derived from Rabbit hemorrhagic disease virus (RHDV), which acts as a highly immunogenic scaffold to deliver tumour-associated antigens (TAAs) to the immune system. In vivo studies to date have shown that one or two doses of the VLP carrying gp100 (a melanoma-associated antigen) can specifically activate an anti-tumour response and trigger the formation of immunological memory against gp100 to prevent tumour recurrence. Administering multiple doses of a vaccine often achieves better responses in vivo, so the key aim of this study was to determine what effect multiple dosing of RHDV VLP coupled to gp100 would have on the anti-tumour response. RHDV VLP was successfully synthesized in a baculovirus expression system using Sf9 insect cells and subsequently used in a series of in vivo experiments. C57BL/6 mice were used in all experiments, receiving either 1, 3 or 6 doses of gp100-carrying VLP (n = 5 per group). An in vivo cytotoxicity assay showed that 3 doses of the VLP vaccine given 5 days apart elicited the highest levels of antigen-specific lysis in a target cell population, compared to a single dose or controls. Therapeutic tumour trials also showed that multiple dosing elicited a stronger anti-tumour response – mice that were first inoculated with B16 melanoma cells and then received 3 or 6 doses of the vaccine 5 days apart had the best overall survival, compared to controls and those that received a single dose. Mice that were tumour-free for 50 days were then rechallenged with B16 cells to assess the immunological memory response, and were found to have increased overall survival, with one mouse from the 3 dose cohort remaining tumour-free. The antibody response against the VLP in these mice was also examined via indirect ELISA. It was found that high levels of antibody against the capsid protein of the VLP were produced in all treated mice, which increased with each additional dose of the vaccine administered. A VLP uptake assay identified that the presence of antibody against the VLP can enhance the early uptake of VLP by DCs, but whether this has an effect on the anti-tumour response remains unclear. Overall, these preliminary results show that treatment involving multiple dosing of RHDV VLP coupled to the melanoma-associated antigen gp100 does somewhat enhance the anti-tumour response in vivo compared to treatment with a single dose, but the reasons for this need to be investigated further. Future work will focus on determining the role that the antibodies against the VLP play in the anti-tumour response, especially in relation to antigen-presenting cells, and further optimizing the vaccine for clinical trials.

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  • Phenotyping Langerhans cell like cell treated with microparticles from keratinocytes expressing human papilloma viruse16 E7 oncoprotein

    Zhang , Junda (2014)

    Undergraduate thesis
    University of Otago

    Cervical cancer in females is a worldwide health issue. High risk subtype of human papilloma viruses (HPV) are involved as a major risk factor. HPV oncogenes, E7 and E6 which are over-expressed in the host cells and promote malignant transformation. There is also evidence of HPVE7 involvement in immune modulation. Microparticles (MP), a type of small membrane fragments (0.1um ~1um) released by activated cells, have been implicated in suppressing immunity following interaction with antigen presenting cells. Our laboratory has previously reported up-regulation of microparticle secretion by HPV16E7 expressing keratinocytes. A component of this study is to advancing the understanding of the effect of keratinocyte microparticle on the phenotype of langerhans cells. Increasingly, roles for p53 in regulation of the immune response is being recognized. In this project, the effect of p53 status in langerhans cells on maintianing the immune phenotype will also be tested. The HPV16E7 oncoprotein expressing mouse keratinocyte (E7-PDV) cell line was established following lentiviral transduction, for microparticle (MP) production. Wild-type (p53+) Langerhans cell-like cells (LCLC) and p53 deficient (p53-) LCLC, which were generated from murine bone marrow cells resembling the phenotype of epidermal LC, were used in this study. The effect of MP on LCLC phenotype (CD40, CD86, E-cadherin and cytokine production) was investigated following 48 h co-culture. Compared to the control PDV cell lines, HPV16E7 expressing PDV were found to produce more MP, suggesting a poteintial role for oncoprotein HPV16E7 in inducing MP production. In response to lipopolysacharride stimulation, the up-regulation of inflammatory surface marker CD40 on p53- LCLC was abrogated compared to wild-type LCLC and E-cadherin expression was also found to be low compare to that of wild-type LCLC. This suggests, to some extend, a potential role for p53 protein in maintaining the proper immune phenotype of normal LCLCs. Moreover, comparing to control groups, the same amount of MP from E7-PDV found to have an inhibitory effect on CD40 expression and it also reduced inflammatory cytokine interleukin 12 productions in LPS-stimulated LCLC. The altered LCLC phenotypes subject to MP treatment had confirmed the hypothesis that HPV16E7 induced MP had a modulating effect on the phenotype of LCLC. Finally, the combined down-regulating effect on CD40 expression was observed when MP treatment was applied to p53- LCLC suggested that the MP effect on CD40 expression was p53 independent. The findings of phenotypic alteration of LCLC subject to MP treatment has unveiled the potential role of HPV-induced MP in immune supression that might provide a mechanism to contribute HPV persistence in the skin.  

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  • Non-Alcoholic Fatty Liver Disease and Elevated Alanine Aminotransferase in New Zealand: An Examination of the New Zealand Adult Nutrition Survey (2008/09).

    Heron, Robert Campbell Roydon (2014)

    Undergraduate thesis
    University of Otago

    BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is common world-wide and associated with obesity, diabetes mellitus, cardiovascular disease, hyperuricaemia and dyslipidaemia. These conditions are common in New Zealand, but the prevalence of NAFLD in New Zealand is unknown. New Zealand has a high prevalence of overweight and obesity, so it is likely that NAFLD is also common. The nationally representative 2008/09 New Zealand Adult Nutrition Survey (NZANS) provided an opportunity to estimate the prevalence of NAFLD as defined by elevated alanine aminotransferase (ALT), a measure of liver damage. AIMS: The aims of this study were to estimate the prevalence of elevated ALT and examine associations between elevated ALT levels and demographic, clinical and nutritional factors in New Zealand adults using data from the 2008/09 NZANS. METHODS: 4,721 New Zealand civilians aged 15 years or older participated in the 2008/09 NZANS, of which 3,035 agreed to have a blood test. Serum ALT was not measured as part of the main survey, and was measured using remaining blood samples. Sociodemographic, medical history, anthropometric and blood pressure measures, blood test results and dietary intake data were extracted and analysed. Elevated ALT was defined as an ALT activity > 29 units/L for men, and ALT > 22 units/L for women. The prevalence of elevated ALT was calculated for different groups, including age, ethnicity, and body mass index categories. Odds ratios were also calculated. Mean energy intake and intakes of both macro- and micronutrients (as measured by 24-hour dietary recall) were calculated and compared between participants with and without elevated ALT. RESULTS: The prevalence of elevated ALT was 13.1% (16.9% for men and 9.7% for women). The prevalence of elevated ALT was high among Maori (18.0%) and Pacific (18.6%) compared with New Zealand European and Other ethnicities (12.2%). NAFLD was more common in overweight (13.5%) and obese persons (20.1%) than in those with a normal weight (6.2%). Male sex, age 25 – 34 years, increased BMI and increased waist circumference were significantly associated with having an elevated ALT after adjustment. Ethnicity was not associated with the odds of having elevated ALT after adjustment. There were few differences in dietary intakes between those with elevated ALT and those with normal ALT. Mean cholesterol intake was higher (338 mg vs. 273 mg, P = 0.003) and thiamine intake was lower (1.3 mg vs. 1.5 mg, P = 0.014) in those with elevated ALT compared with the normal ALT group, respectively. CONCLUSIONS: Liver damage as defined by an elevated ALT, is common in New Zealand and is comparable to that of the United States. The prevalence of elevated ALT among those classified as obese was particularly high. Given the high prevalence of obesity and associated conditions such as diabetes in New Zealand, a large proportion of those with an elevated ALT are likely to have NAFLD. NAFLD is an increasing cause for concern worldwide, and further research using more sensitive and specific diagnostic method(s) is needed to more clearly understand the epidemiology of NAFLD in New Zealand.

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  • Functional Analysis of Disease-Associated 3’ UTR Variants: the CXCL12β 3’ UTR G801A Polymorphism

    Jin, Chan (2014)

    Undergraduate thesis
    University of Otago

    In recent years, the previously neglected 3’ untranslated region (UTR) has gained recognition as a critical region in the regulation of gene expression. Numerous studies have been conducted on disease-associated 3’ UTR variants to elucidate unidentified regulatory elements within the 3’ UTR. An example of an unresolved 3’ UTR variant is the G801A polymorphism (rs1801157) occurring within the 3’ UTR of the CXCL12β transcript isoform. The CXCL12 gene encodes a C-X-C motif chemokine 12 (CXCL12) that functions as a ligand for the chemokine (C-X-C motif) receptor 4 (CXCR4), which is involved in the entry of the human immunodeficiency virus (HIV) into host cells. Correspondingly, previous association studies have indicated a potential correlation between the A allele of the G801A polymorphism and increased resistance to the progression of acquired immunodeficiency syndrome (AIDS). This suggests that the G801A polymorphism may be linked to a regulatory element in the 3’ UTR, which modulates the expression of CXCL12β, and therefore plays an intermediary role in its reported association with HIV/AIDS. In order to identify the regulatory elements that may be associated with the G801A polymorphism, a bioinformatic approach was adopted by utilising a webserver developed in the Brown Lab called ‘Scan For Motifs’. However, only a 26 base pair (bp) region of high conservation was identified at the site of the G801A polymorphism. Consequently, three variable lengths of the CXCL12β 3’ UTR, containing the highly conserved region with either alleles at the G801A polymorphism, were cloned downstream from the luciferase gene (luc+) in pGL3MS2site/basic cloning vectors to yield three sets of reporter gene constructs. The desired CXCL12β 3’ UTR inserts were prepared by either the polymerase chain reaction (PCR) amplification of said sequences from HepG2 genomic DNA (gDNA) or appropriate oligonucleotide design. Site-directed mutagenesis was implemented when applicable using PCR-based methods such as the ‘megaprimer’ method and the overlap extension method, to create a polymorphic counterpart for each reporter gene construct. Prepared reporter gene constructs and previously characterised control plasmids were transiently co-transfected with a renilla luciferase reporter gene construct (phRL-SV40) using Lipofectamine® 2000 into HeLa cells and HEK293T. The transfected cells were harvested after a 48-hour incubation period for subsequent luciferase assays in order to measure differences in gene expression with respect to the G801A polymorphism. Changes in gene expression in response to different experimental conditions were also analysed through the application of two independent stimuli: the replenishment of foetal bovine/calf serum (FBS/FCS) (10%) and the premature harvest of cells after 24 hours. The amount of DNA transfected was also standardised through the co-transfection of pUC18, a non-coding plasmid. In contrast to initial expectations, different levels of gene expression were not observed between each pair of reporter gene constructs with allelic differences at the G801A polymorphism. In support of the experimental results, levels of gene expression in response to two different conditions behaved in agreement with initial expectations. Likewise, the standardisation of the amount of DNA transfected using pUC18 co-transfection only increased the gene expression associated with the larger plasmid relative to the other smaller DNA constructs. Despite the negative results obtained from a series of luciferase assays, the functional implications associated with the G801A polymorphism was not conclusively determined as absent given the conflicting indications reported in the wider literature. Further experimental analyses are required to confirm or reject the functional consequences of the G801A polymorphism. If there is a functional impact associated with allelic differences at the G801A polymorphism, the next step would be to elucidate the responsible regulatory element in the context of its associated diseases.

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  • The involvement of mGluR1 hyper-activation in the progression of cerebellar ataxia in SCA1 mice

    Desai, Heena Nitin (2014)

    Undergraduate thesis
    University of Otago

    Spinocerebellar ataxia type 1 (SCA1) is an incurable, autosomal dominant progressive neurodegenerative disorder characterised by ataxia, progressive motor deterioration and selective neuronal loss in the cerebellum. It results from a CAG trinucleotide repeat expansion within the SCA1 gene product, ataxin-1. Metabotropic glutamate receptors type 1 (mGluR1) mobilise calcium from intracellular stores as part of their key role in cerebellar synaptic plasticity and motor learning and may be involved in the progression of ataxic symptoms. In this study, we use an 82Q transgenic mouse model of SCA1 where the CAG expansion is restricted to mouse cerebellar Purkinje neurons (PNs), the primary site of SCA1 pathology. This restricted expression in the PNs is achieved by tetracycline-controlled system. We use doxycycline to repress transgene expression at early (6 weeks) and mid (12 weeks) stages of the disease. Our aim is to use this model to identify potential mechanisms that contribute to the early stages of the progression of SCA1. We hypothesise that changes in mGluR1 expression underlie the progression from early pre-symptomatic to ataxia symptoms. Behavioural testing involved using an accelerating rotarod apparatus to assess motor performance and learning. 6 week old SCA1 transgenic mice exhibited mild ataxic motor symptoms (P < 0.01, two way ANOVA, n = 12) that progressed further at 12 weeks of age (P < 0.05, two way ANOVA, n = 7). Doxycycline treatment to repress the transgene expression prevented the mild ataxic symptoms at 6 weeks and reversed the progressively worse ataxic symptoms at 12 weeks of age. Immunohistochemistry experiments showed an increase in mGluR1 expression specifically in the molecular layer of 12 week old SCA1 mice (P < 0.05, two way ANOVA, n = 4). Doxycycline treatment did not prevent this enhanced expression of mGluR1, suggesting that enhanced mGluR1 expression may precede the onset of behavioural ataxia. Cell attached patch clamp recordings from PNs in SCA1 transgenic mice showed a decrease in instantaneous action potential (spike) firing frequency in comparison to PNs from FVB mice (P < 0.01, unpaired t-test with Welch’s correction, FVB: n = 3, SCA: n = 10). The application of Picrotoxin (PTX), a GABAA receptor antagonist resulted in: a non-significant trend towards an increase in instantaneous frequency and decrease in instantaneous firing irregularity of PNs from SCA1 mice. These data suggest a more powerful inhibitory influence in the cerebellar cortex of SCA1 mice compared with FVB mice. Overall, these results suggest that enhanced mGluR1 expression may disrupt PN calcium homeostasis leading to changes in PN firing and cerebellar output that drives the progression of SCA1. Our findings have important implications for the treatment of this rare but incurable human ataxia. The mGluR1 may be a potential therapeutic target for treating patients that are mildly symptomatic in the early stages of the disease.

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  • Characteristics of road traffic injuries and potential risk factors in Oman

    Al-Risi, Ahmed (2014)

    Undergraduate thesis
    University of Otago

    Background: Globally, road traffic injuries have increased by almost 46% in the twenty years prior to 2010. This makes road traffic crashes (RTC) the tenth leading cause of death in the world and the leading cause of death of young people. Oman, a wealthy country where motorising is increasing rapidly, has a very high road traffic mortality. In Oman, road traffic deaths and injuries are the main external cause of morbidity in young adults and have a direct effect on the economic and health resources of the country. Road traffic crash research has only recently begun in Oman. The modifiable causes of this problem and most effective response have not yet been well-explored. Aims: The main aims of the research described in this thesis were: - To describe the distribution of road traffic crashes in Oman by time, person and place using the data collected by the Royal Oman Police and examine their assessment of reasons for crashes, from 1985-2010. - To collect data in order to estimate the prevalence and distribution of known risk factors in road traffic crashes in Oman, focusing specifically on Sohar. Methods: Two studies were conducted to achieve the aims of this thesis. Firstly, a retrospective case series used the Royal Oman Police data from 1985-2010. This examined trends in the police data on road traffic crashes across the whole time period and then summarised the most recent available year of data (2010) in more detail. Secondly, a prospective case series of injured drivers admitted to Sohar Hospital Emergency Department was carried out. Between 20 February 2012 and 20 March 2012, consecutive injured drivers admitted to Sohar Emergency Department were recruited to the study. Questionnaire-based face-to-face interviews were held to collect data on socio-demographics, circumstances of the crash, and known risk factors for road traffic injuries, including risk behaviours at the time of the crash and usual behaviours. All admitted injured drivers were recruited apart from the most seriously injured drivers who were taken by ambulance to the capital city Muscat for further investigations, and those who died from their injures. Results: According to Royal Oman Police data (1985-2010), total deaths and injuries from RTCs have increased by almost 300% in Oman since 1985. An element of speeding was reported for all the crashes since 1992. The victims of road traffic crashes were mostly the young age group (21-30 years). More drivers have been killed than any other road user group, constituting 43.8% of total road traffic deaths, and passengers have been the most injured, constituting 48.2% of total road traffic injuries. In the Sohar study, 250 injured drivers, 75% males and 25% females, were interviewed with a 100% response rate. Interviewed injured drivers were found to have spent long hours on the roads and had driven for long distances. Overall, less than 5% of injured drivers were over the age of 35 years. There was a marked difference in the age distribution of male and female injured drivers. Among men almost half (49.5%) of the injured drivers were 18-25 years old and 45.7% were 25-35. Among women, 95% of injured drivers were 25-35 years old. Generally, male drivers had more traffic violations than female drivers with 83% of males reporting at least three traffic violations over the past five years, whereas almost half of the females reported one or no, traffic violation over the same period. The highest frequency of crashes occurred on Saturdays and Thursdays (18.8% and 17.6% respectively) and the majority of the injured drivers were either familiar or very familiar with the roads on which they crashed. It was clear that the injured drivers in Sohar routinely ignored the traffic laws and reported risky driving behaviours. For instance, less than 10% of injured male drivers and only 56% of female drivers were wearing their seatbelts at the time of the crash. Also, a high proportion of both male and female drivers were travelling at a speed of 100-140 km/hour at the time of their crash (65.4% male and 58% female drivers). Moreover, 31.4% of injured male drivers and 24% of injured female drivers were using their cell phones at the time of their crash. When describing their usual driving behaviour, 52.8% of all interviewed drivers reported that they never or almost never wear seatbelts while driving. Conclusions: Overall, the incidence of road traffic crash injuries and deaths is high and increasing in Oman with a high prevalence of known risk factors for road traffic injuries even where protective legislation exists. Even though this case series cannot establish that these risk factors cause road traffic injuries in this population, experience from other countries suggests that appropriate legislation and increased enforcement could reduce road traffic injuries in Oman.

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  • Novel Methods for the Rapid Identification and Susceptibility Testing of Blood Culture Isolates

    Robinson, Andrew Mark (2014)

    Undergraduate thesis
    University of Otago

    The increasing emergence of antimicrobial resistance, such as that mediated by extended-spectrum β-lactamase (ESBL)-producing gram-negative bacteria, make it more likely that patients with sepsis and bloodstream infections (BSI) will receive ineffective empirical treatment. Rapid identification of disease causing agents, coupled with early detection of antimicrobial resistance facilitates the optimisation of essential treatment decisions. Matrix-assisted laser desorption-ionization time of flight (MALDI-ToF) mass spectrometry has recently been applied to the identification of microorganisms directly from blood cultures, reducing the identification process by up to 24-hours. This study sought (i) to determine the optimal method for the rapid identification of isolates directly from blood cultures and (ii) to develop a rapid method to detect β-lactamase-mediated resistance to extended spectrum cephalosporins directly from blood cultures. Two in-house methods for sample preparation were optimized and compared to a commercially available method. Using the conventional scoring criteria, the differential centrifugation protocol correctly identified 86.8% and 67.9% of clinical isolates at the genus- and species- level. This was compared to a quicker method using Sodium dodecyl sulphate (SDS) to mediate blood cell lysis, which correctly identified 83.0% and 62.3% of clinical isolates to the genus- and species- level. Both methods performed similarly to the more expensive commercial method. Results also suggested that the scoring criteria could be altered to increase the number of species-level identification while maintaining accuracy, achieving up to 90.3% species level identifications. To rapidly detect β-lactamase-mediated resistance to extended spectrum cephalosporins, a high-performance liquid chromatography (HPLC) assay was developed and optimized to detect resistance directly from growth-positive blood cultures. With a 1-hour incubation of bacteria with cefotaxime, resistance could be detected with 95.5% sensitivity and 88.9% specificity. This method was better at detecting resistance mediated by group 1 and 9 CTX-M ESBLs, with reduced sensitivity for the detection of resistance mediated by AmpC β-lactamases. Further research is required to investigate additional markers that could improve the detection of other β-lactamases. Both of these methods could be rapidly integrated into the diagnostic microbiology laboratory, thus reducing the time to effective narrow spectrum antimicrobial therapy, and potentially improving patient outcomes and reducing the spread of antimicrobial resistance.

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  • The prevalence and level of education of Hepatitis C Virus among an asymptomatic population

    Vermunt, Jane (2014)

    Undergraduate thesis
    University of Otago

    Background The burden of Hepatitis C virus (HCV) is projected to increase substantially over the next 2 decades as a result of complications arising from chronically infected individuals who remain undiagnosed and untreated. Accurate epidemiological data on the prevalence and demographics of Hepatitis C is therefore needed to allow efficient planning of services and resource allocation for prevention and treatment management in the region. In order to minimise transmission and to recognise risk factors and symptoms of HCV infection, population-wide education is also essential. Aim This study aimed to identify the prevalence of Hepatitis C among the 40 to 59 year old population living in urban Dunedin. It also set about to identify gaps in knowledge about HCV in the target – assessment of HCV knowledge among this cohort was thought to be important to gaining better understanding any barriers to identification, diagnosis and treatment while concurrently raising awareness of the issue. Method A total of 1400 individuals aged between 40 and 59 living in urban Dunedin were randomly identified from the electorate role. A questionnaire was developed and posted to participants that explored risk profile, infection transmission, complications, symptoms and treatment. Participants were also asked to provide a blood sample for anti-HCV and HbsAg testing. Hepatitis B antigen testing (HbsAg) was also tested to allow comparison on prevalence and decrease perceived stigma of testing. Results Of the 1400 questionnaires sent, a total of 432 were returned completed and some 306 blood samples were analysed. The prevalence of HCV and Hepatitis B virus (HBV) was estimated to be less than 0.98%, based on a zero numerator. Significant knowledge gaps were identified. The average correct score from the questionnaire was 59.4%. Both adjusted and unadjusted logistic regression modelling showed that three demographics were statistically significant predictors of an individuals’ score. On average females scored 5.4% higher. Every increase in qualification level showed a 5.0% increase and a 4.8% increase was found between each occupation sector. No statistically significant relationships were found between socioeconomic status (SES) or age. The population sample recognised all the potential modes of HCV transmission. Only 23% correctly estimating the assumed prevalence of HCV. 93% of the sample population did not recognize that an acute or chronic infection may be asymptomatic and 97% were unaware that there could be no long term sequale to a chronic infection. 23.6% knew that it takes years rather than months weeks or days for symptoms of complications of a chronic infection to become apparent. Twenty-two per cent were aware that there is no available vaccine, 34.0% do not know that HCV can be treated and of those who do know, only 39.7% are aware that this is funded by the government. Conclusions The prevalence rate, although inferred, is lower than expected. Our group has thus committed to undertaken further work in this area to obtain a more representative sample of bloods from which to draw better prevalaence data – though completed, data was not ready for publication in this thesis. The lack of general knowledge about HCV is of concern as this population is at high risk of transmission and of developing complications related to unassumed chronic infection. It is clear that the majority of this population is unaware of the asymptomatic nature and when the nonspecific symptoms of an HBC or HCV infection are likely to manifest. Further, one-third of the population are unaware that HBV and HCV can be treated and two-thirds are unaware that treatment is fully funded. Well educated women working in the health or white collar sector have the best knowledge about risk of transmission, possible symptoms and treatment. Educational efforts to increase awareness empower people to be aware of symptoms, get diagnosed and undergo treatment needs to target all other population groups.

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  • Wnt signalling influences T cell phenotype in a novel intestinal immune system model

    Worters, Thomas David (2014)

    Undergraduate thesis
    University of Otago

    Inflammatory bowel disease (IBD) is a collective term for inflammatory conditions that affect the gastro-intestinal tract. These conditions feature a multifactorial etiology: an interplay of genetics, environmental exposures, the immune system and commensal microbiota, all of which converge at the intestinal epithelia. The ability to understand IBD is dependent on and limited by the model systems used to study it. We have developed an ex vivo model of the human intestine by co-culturing intestinal organoids and PBMCs from the same patient. As intestinal organoids accurately mimic the intestinal epithelia, this allows us to study the interaction of the epithelium and immune system on a controlled genetic background in both IBD patients and healthy individuals. I hypothesise that intestinal organoids cultured with immune cells will create an environment similar to the human intestinal immune environment. The aim of my research was to develop a platform to study the role of immune cells in this organoid culture. I analysed the effect of organoid culture conditions on the survival and phenotype of immune cells, measured by cytokine and cell surface receptor expression. PBMCs remained viable for four days when grown in DMEM, and this viability was not affected by suspending the cells in the Matrigel used for organoid culture. Freezing and thawing PBMCs, which is required to allow establishment of the organoids, only caused a slight reduction in viability, and did not affect the frequency of Th1, Th17 and regulatory T cells. Introduction of growth factors required for organoid culture did not affect viability of the PBMCs, however the frequencies of Th1 and Th17 but not regulatory T cells were reduced. Recombinant Wnt, a key component used to culture organoids, affected the ability of regulatory T cells to maintain but not differentiate their phenotype. Finally, viable T cells could be removed from a complete PBMC-organoid co-culture. These data indicate that PBMCs can be successfully cultured in conditions used to generate intestinal organoids without loss of viability or major changes in phenotype. Furthermore, this co-culture model will likely serve as an accurate model of the intestinal immune system and may aid in the search of an effective treatment for IBD.

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  • Determining Drivers of Community Structuring and Denitrification Potential Within a Shallow Coastal Lake/Lagoon (Lake Ellesmere) Exposed to Anthropogenic Nutrient Deposition

    Highton, Matthew Paul (2014)

    Undergraduate thesis
    University of Otago

    As a result of human agricultural activity, large amounts of the potent greenhouse gas nitrous oxide (N2O) are entering our atmosphere. In NZ, urine from dairy cows contributes large loads of nitrogen to the surrounding land and waterways via leaching or surface runoff. Lake Ellesmere, a coastal lake draining farmland, maintains lower nitrogen levels in its waters than inflowing rivers suggesting a nitrogen removal mechanism, present within the lake. It is likely that much of the incoming nitrogen could be emitted from the lake as N2 or N2O microbial denitrification. We investigated the microbial community structure and denitrification potential across 18 sites in Lake Ellesmere by using 16S ribosomal rRNA gene sequencing, denitrification enzyme assays and quantification of key nitrogen cycling genes, with a focus on denitrification genes, by quantitative PCR. Physical and chemical analyses (water salinity, temperature, clarity, total nitrogen, nitrate/nitrite, dissolved reactive phosphorous, ammonia, sediment organic matter, and sediment texture class classification) were also carried out simultaneously on the same sites. Analyses identified sediment texture class and organic matter content as the strongest drivers of bacterial community composition within the lake. Water column nutrients such as Total Nitrogen and Phosphorous were only weakly correlated to community structure suggesting they functioned as weaker modifiers of the system at the time of sampling. Analysis of discrete functional populations by qPCR (e.g. nitrogen fixers, denitrifiers) demonstrated significant variation of gene copy number within the lake sites, however no significant drivers of these populations were identified for the denitrifiers. Nitrogen fixers were found to be enriched in high silt sediments. These results demonstrate the difficulty in analyzing these diverse, variable systems. Nevertheless they restate the importance of sediment texture class as a strong determinant for microbial community structure. Further work will need to focus on temporal variability within the system and the production of supporting evidence through controlled testing of denitrification.

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  • Regulation of voltage-gated calcium channels in PC12 cells by Leucine Rich Repeat Kinase 2

    Bedford, Cade Henry (2013)

    Undergraduate thesis
    University of Otago

    Leucine rich repeat kinase two (LRRK2) is a widely expressed protein belonging to the Roco family of proteins, mutations in which have recently been discovered as a cause of familial Parkinson’s disease (PD). Despite an array of interacting proteins having been identified across multiple cellular systems, LRRK2’s functional role remains to be determined. Manipulation of LRRK2 expression disrupts many Ca2+ dependent cellular processes. It, therefore, may act as an upstream regulator of initial Ca2+ signalling events, which could explain LRRK2’s widespread effects. The central aim of this study was to determine whether LRRK2 alters endogenous voltage-gated Ca2+ (CaV) channel function in PC12 cells using whole cell patch clamp electrophysiology. Additionally, transiently transfected PC12 cells underwent epifluorescence imaging to identify morphological changes and identify any effects of L-type Ca2+ blockers on morphology. Peak CaV channel currents in LRRK2 transfected cells showed a significantly (p=0.0025, n≥7, one way ANOVA with Tukeys post-hoc test) higher current density across a number of holding voltages relative to untransfected and EGFP transfected controls. These results indicate that LRRK2 up regulates endogenous CaV channel function. Morphological assessment, however showed no significant effect of LRRK2 transfection on morphological parameters relative to EGFP transfected and non-transfected controls (N≥63, Kruskal-Wallis test). Furthermore, addition of the L-type Ca2+ channel blocker nifedipine had no significant effect relative to untransfected and ethanol vehicle controls (N≥43,Kruskal-Wallis test). These results suggest that LRRK2 dependent modulation of CaV channel function does not affect neurite differentiation. Overall, this study has identified a novel effect of LRRK2 on CaV channels, which may explain how LRRK2 has such widespread cellular effects and advances our understanding of LRRK2s functional role. If the effect of LRRK2 on CaV channels is responsible for pathology, CaV channel blockers currently being investigated for Parkinson’s therapy may be particularly effective in Parkinson’s patients harbouring LRRK2 mutations.

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  • The Effect of Mepilex Lite Dressings on Acute Radiation-Induced Skin Reactions in Women Receiving Post-Mastectomy Chest wall Irradiation

    Poonam, Prashika (2013)

    Undergraduate thesis
    University of Otago

    Acute radiation-induced skin reactions are the most common side-effect of external beam radiation therapy ranging from changes in skin colour to dry and flaky skin, leading to moist desquamation (ulceration). Severe skin reactions compromise quality of life; however, there is currently no standard treatment. Treatment is therefore based on historical and/or anecdotal data which has resulted in substantial variation in skin care practice. A multicentre, intra-individually controlled, randomised trial was conducted to investigate whether Mepilex Lite dressings are superior to the standard departmental care in reducing the extent of acute radiation-induced skin reactions in patients receiving treatment for breast cancer post-mastectomy. Mepilex Lite (Mölnlycke Health Care LTD, Göteborg, Sweden) is a thin, self-adhering, absorbent, soft silicone dressing which was hypothesised to reduce reactions by protecting the irradiated skin against mechanical damage caused by friction and abrasion from clothing or adjacent tissue. This thesis analyses the results of 13 patients recruited at the Regional Cancer Treatment Services who were a subset of the large 80 patient multicentre trial. From the first sign of erythema on the chest wall, the erythematous patch was divided into two equal halves; one half was randomly assigned to be covered in Mepilex Lite dressings, the other to be treated with aqueous cream. Once erythema advanced to moist desquamation, skin under the control patch was dressed with dressings that were standard to the department while the intervention patch continued to be treated with Mepilex Lite dressings. The Modified Radiation-Induced Skin Reaction Assessment Scale (RISRAS) was used to assess the visual signs (researcher component) of the skin reaction while the patient component assessed symptomatic changes for at least three times a week during radiation therapy and once a week post-treatment until all reactions were resolved. An exit questionnaire was filled out by each patient upon completion of the trial allowing them to comment on the different aspects of the trial including their experience with using the Mepilex Lite dressings. Mepilex Lite dressings decreased the severity of skin reactions by 38% (p=0.002) based on the mean combined (patients and researcher) RISRAS scores. Patient RISRAS scores heavily influenced this score, showing a decrease of 77% (p=0.004) compared to the researcher scores which showed a decrease of 19% (p=0.008). Analysis of the peak RISRAS scores to assess the difference in the maximum severity of the skin reactions under each arm showed a similar trend. Combined peak RISRAS showed a decrease of 43% (p=0.005), with a patient component of 74% (p=0.006) and a researcher component of 20% (p=0.026). Analysis of moist desquamation scores alone showed a decrease in both the mean and peak RISRAS scores (38% (p=0.04); 46% (p=0.02) respectively) in favour of the Mepilex Lite dressings. Exit questionnaires highlighted that the silicon dressing was easy to use and comfortable to wear and most patients preferred the dressings over the cream. The findings of this thesis demonstrates that Mepilex Lite dressings reduce the visible signs of radiation-induced acute skin reactions and cause a substantial decrease in patient discomfort and subjective symptoms.

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  • Effect of Toll-like Receptor agonists on platelet activation in Acute Coronary Syndromes

    Chen-Xu, Michael Xin Hua (2013)

    Undergraduate thesis
    University of Otago

    Introduction: Platelets express functional Toll-like receptors (TLRs), and numerous studies have found that certain TLR agonists and bacteria are capable of inducing thrombotic and inflammatory responses from isolated platelets. Few studies have examined the functionality of the platelet-expressed TLRs in the clinical context or in whole blood. Platelets and their thromboinflammatory functions are central to the pathogenesis of atherosclerosis and the acute coronary syndromes (ACS). As a first step to understanding the effects of TLR agonists on platelets in ACS, we aimed to develop a flow cytometry-based protocol to examine platelet activation responses to TLR agonist stimulation. Methods: A flow cytometry-based protocol to measure the expression of platelet activation markers PAC-1, P-selectin (CD62p) and CD40-ligand (CD40L) in response to stimulation by TLR agonists was developed. Fourteen patients presenting to Wellington Regional Hospital with ACS pretreated with aspirin and clopidogrel (dual antiplatelet therapy, DAPT) were recruited prospectively. Fourteen age- and sex- matched healthy volunteers not on antiplatelet medications were recruited prospectively as controls. Whole blood samples from ACS patients and controls were each separately stimulated with agonists for TLR4 (lipopolysaccharide, LPS), TLR2/1 (Pam3CysSerLys4, PAM3CSK4) and TLR2/6 (fibroblast stimulating ligand-1, FSL-1). Thrombin Receptor Activating Peptide (TRAP) was used a positive control. Following stimulation, samples were analysed by flow cytometry. Results: There were no differences in the platelet activation states between groups for the unstimulated samples. Stimulation of whole blood from the control group by the TLR agonists tested resulted in statistically significant increases in PAC-1 and CD62p. Stimulation of whole blood from the ACS patients on DAPT with TLR agonists did not result in statistically significant increases in the expression of the platelet-activation markers tested, although trends towards significance were noted at the higher concentrations of agonists tested. Platelet responses were lower in the ACS group relative to the controls. For the maximal LPS concentration used, the %-positive expressiondifferences between the control group and ACS group for PAC-1 and CD62p were 29.3 (p = 0.002) and 7.2 (p = 0.02), respectively. For the maximal concentration of PAM3CSK4, the differences for PAC-1 and CD62p were 16.4 (p < 0.001) and 8.4 (p = 0.04), respectively. For the maximal concentration of FSL-1, the differences for PAC-1 and P-selectin were 18.7 (p < 0.001) and 7.8 (p = 0.04), respectively. Conclusions: TLR agonist stimulation increased platelet activation in healthy volunteers, although it was unclear whether this was due to a direct effect on the platelet surface-expressed TLRs or an indirect effect on platelets via leucocytes, erythrocytes or plasma proteins. DAPT appeared to inhibit TLR agonist-induced platelet activation in the ACS patients. Although we found no statistically significant platelet activation responses to the TLR agonists tested in the ACS patients on DAPT, this will require confirmation in larger sample sets as our study may have been underpowered. If so, current DAPT regimens may be insufficient to prevent ischaemic events in settings of infection for patients at risk of arteriothromboses involving platelets.

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  • Evaluation of clinical ethics support needs and service provision at a tertiary hospital in New Zealand

    Dai, Libby (2013)

    Undergraduate thesis
    University of Otago

    Doctors often face ethical challenges in the course of clinical practice. Clinical ethics advisory services (CESS) provide a mechanism for supporting doctors facing these ethical dilemmas. In New Zealand, CESSs are relatively new and have emerged as a clinician-led initiative. This is an exciting time for CESSs in New Zealand, as their availability increases and their systems become increasingly formalised and integrated into the health care system. This thesis is the first in New Zealand to explore the clinical ethics needs of doctors and to evaluate how their clinical ethics needs could be most effectively met. The Capital and Coast District Health Board (CCDHB), comprising a major tertiary hospital and its satellite hospital, is used as a case study. Doctors at CCDHB have had access to a clinical ethics support service since 2010, when the CCDHB Clinical Ethics Advisory Group (CCDHB CEAG) was established. Many of the findings of the research would be applicable to CESSs throughout New Zealand. I developed a methodology to understand the clinical ethics needs of senior doctors at CCDHB and to evaluate how their needs could be better met. In-depth, semi-structured interviews were conducted with 14 senior doctors. The data were analysed using an iterative inductive strategy combining conceptual and normative analysis. My analysis draws on the current international literature of clinical ethics support, as well as my experience as a clinical medical student and my period of observation of the CCDHB Clinical Ethics Advisory Group. This study found that in the absence of formal services, doctors use ad hoc strategies of peer consultation to manage ethical issues. Not all doctors were equally able to access informal support, particularly junior doctors. Many participants were unaware that formal clinical ethics support was available to them and most did not know how formal ethics support worked. Some participants felt that to seek case consultation was to abrogate clinical responsibility and thought that doctors should be able to manage ethical issues themselves. Participants identified a need for improved strategies for clinically relevant ethics education. This study identifies five key recommendations to enhance clinical ethics support at Capital and Coast District Health Board: 1. CCDHB CEAG should formalise its activities, particularly case consultation, using a procedural justice model. 2. CCDHB CEAG should involve clinicians in the process of case consultation to increase user trust and to take advantage of case consultation’s educative value. 3. CCDHB CEAG should allow patients and their families and advocates the option of being involved in the process of case consultation, to ensure that patients feel that their perspectives have been adequately taken into account. 4. CCDHB CEAG should conduct monitoring and evaluation of its service to ensure that it achieves and maintains clinical relevance and normative robustness. 5. CCDHB CEAG should actively disseminate accurate and appropriate information about its aims and processes to all users and potential users to enhance trust in their service and to clarify misunderstandings about their role.

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