416 results for Undergraduate

  • A new target for treating arrhythmia? Interaction of triadin with the cardiac ryanodine receptor reduces Store Overload Induced Calcium Release

    Deo, Manesh Shamal (2015)

    Undergraduate thesis
    University of Otago

    Arrhythmia occurs in a number of heart diseases including heart failure (HF) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). It has been shown that Store Overload Induced Calcium Release (SOICR) is a common mechanism underlying many types of arrhythmia. SOICR occurs due to the inappropriate opening of the cardiac ryanodine receptor (RyR2) once calcium in the sarcoplasmic reticulum (SR) reaches a certain threshold. RyR2 forms a large macromolecular complex with other proteins. One such protein is triadin; which is known to be lost from the RyR2 complex in HF and CPVT. The loss of triadin is also linked to an increase in arrhythmias, suggesting its loss may lead to SOICR. However, triadin has also been shown to have a role in maintaining the ultrastructure of cardiomyocytes, therefore it is unclear whether arrhythmia occurs as a direct result of the loss of interaction between the RyR2 and triadin or simply due to structural changes in the cell. In the present study, we investigated whether the direct interaction between RyR2 and triadin alters the propensity for SOICR. Single cell cytosolic imaging in HEK 293 cells stably expressing RyR2 with or without triadin, using the high affinity calcium indicator Fluo4-AM, showed that the presence of triadin reduced the propensity of SOICR events, suggesting an inhibitory role for triadin. This was confirmed by single cell luminal calcium imaging, using the endoplasmic reticulum (ER) targeted Ca2+ indicator protein D1ER which showed that the expression of triadin increased ER calcium threshold at which SOICR occurred. These results provide evidence that the direct interaction between RyR2 and triadin reduces the propensity for SOICR by increasing the threshold that SR calcium must reach to trigger SOICR. Conversely, this suggests that loss of the direct interaction between RyR2 and triadin in HF and CPVT will be arrhythmogenic. Therefore, stablising this interaction in patients susceptible to arrhythmias is likely to be therapeutic.

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  • Time to Safe Driving after Total Hip and Knee Replacement Surgery

    Meffan, Peter (2015)

    Undergraduate thesis
    University of Otago

    Introduction: Osteoarthritis is the most common form of arthritis in the western world. With its increasing prevalence, total joint replacement is in high demand. After total hip replacement (THR) and total knee replacement (TKR) a period of driving cessation is necessary. For many patients, a period of driving cessation creates financial stress and threatens independence. The current recommendation for driving cessation following THR or TKR surgery is 6 weeks, however the literature surrounding this is varied. Aims: To measure the average time it takes for a patient following either a THR or TKR to return to operating a motor vehicle safely; based on recovery of their pre-operative baseline transfer time to within 10%. Methods: The transfer time from accelerator to a brake force of 100N (transfer time) was measured on a custom built rig. Patients were tested pre-operatively, and 1, 2, 4, and 6 weeks post-operatively The time in weeks was measured to return to within 10% of their pre-operative transfer time. A quantitative questionnaire was used at each test to establish patient perception to the impediments to safe and confident driving. Results: The median time to return to baseline in THR was 2.0 weeks (95%CI 1.3-2.7) and in TKR 5.5 weeks (Log rank score 0.034). 14.3% of THR and 62.5% of TKR patients failed to reach baseline in the test period. The median time to return to baseline for all operation groups was 3.3 weeks in males (2.6 – 4.0) and 2.0 weeks in females (1.4 – 2.6) (Log rank 0.67). 18.2% of males and 45.5% of females failed to return to baseline in the test period. Males recorded faster transfer times at baseline than females (414ms and 573 respectively). Joint pain decreased markedly over the test period when scored by visual analogue score. TKR felt more joint pain at 1 week postoperative than THR (VAS 5.26 and 2.0 respectively). Perceived driving confidence had improved to baseline by 2 weeks postoperative. When asked, patients reported joint stiffness as the most troublesome symptoms in the postoperative period. Discussion: THR recovered to their baseline transfer time significantly quicker than TKR. This may be due to TKR experiencing more pain in the immediate post-operative period. Females recovered to baseline quicker than males, however this observation was not statistically significant. There was significant loss to follow up in this study, which particularly affected the TKR group. There were a significant number of TKR patients who did not reach baseline in the study period. Without data past 6 weeks follow up it is difficult to make recommendations for the TKR group. We suggest that THR are safe to return driving 3-4 weeks after their operation date. Due to the time limitations of the BMedSci (Hons) programme the study size was smaller than desired. Following completion of this thesis data collection will continue to strengthen the findings of this study.

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  • Psychosocial determinants of glycaemic control in children and adolescents with Type One Diabetes Mellitus

    Chae, Matthew (2015)

    Undergraduate thesis
    University of Otago

    Introduction Type 1 Diabetes Mellitus (T1DM) is a chronic life-long condition that commonly presents in children and adolescents. It is a condition that requires close monitoring, as poor management may lead to severe complications later in life. Psychosocial factors are an aspect that must be considered when making management plans, as it can impact health outcomes for these individuals. This study aims to explore three psychosocial measures: Chaos Hubbub and Order Scale (CHAOS); Intuitive Eating; and temperament/self-control, in children and adolescents with T1DM, and investigate any relationship between these measures and their glycaemic control. In doing so, we hope to contribute to the growing body of literature aiming to improve glycaemic control in children and adolescents with T1DM. Methods This was a cross sectional study involving 74 eligible families of children/adolescents with T1DM. The Child/adolescent were required to be between ages 3-18 and had to be out of their honeymoon phase (> 0.5 units of insulin per kg per day). Each parent (both maternal and paternal) were asked to fill out a demographic questionnaire, a CHAOS questionnaire, self-reported Adult Temperament Questionnaire and an age appropriate Temperament Questionnaire on their child. The Child/adolescent were asked to also fill-out a demographic questionnaire, participate in the Heads knees shoulders and Toes (HKST) test, and fill out an Intuitive Eating Scale if they were above the age of 12. HbA1c was used as a measure of glycaemic control. An age and sex matched control group was recruited through participating families and the University of Otago’s Psychology Database. These Control families were not required to participate in the HKST test nor the Temperament questionnaire. Results Maternal CHAOS (MCHAOS) and Paternal CHAOS (PCHAOS) were both negatively associated with HbA1c after controlling for age, self-monitoring of Blood Glucose (SMBG), insulin therapy and Body Mass Index (for paternal only). The Odds Ratio generated by the multivariate model for MCHAOS and PCHAOS with HbA1c, was 1.30 (95% CI: 1.02 – 1.65) and 1.28 (95% CI: 1.01 – 1.62) respectively. MCHAOS showed limited utility as a tool to detect poor or acceptable glycaemic control with a sensitivity and specificity of 52.9% (95% CI: 35.10 – 70.20%) and 85.7% (95% CI: 67.30 – 96.00%), respectively. However PCHAOS showed potential to be used as a screening tool with a sensitivity and specificity of 92% (95% CI: 74 – 99%) and 37.5% (95% CI:18.80 – 59.4%), respectively. No statistically significant difference was observed between MCHAOS of families with T1DM (mean: 18.02) and their controls (17.40) with a 95% confidence interval for the t-test of -0.341 – 1.581. additionally, PCHAOS was reported to be significantly higher in T1DM (mean: 18.51) than controls (mean: 17.25) with a 95% confidence interval for the t-test of 0.052 – 2.468. Out of the four scales of intuitive eating, Eating for physical rather than emotional reasons, was found to be negatively associated with HbA1c with an odds ratio of 0.27 and a 95% CI ranging from 0.08 – 0.85. No significant difference was found in measures of intuitive eating between adolescents with T1DM and their matched controls. Maternal and Paternal report of Child effortful control were both negatively associated with HbA1c after controlling for age, SMBG, insulin therapy and BMI (for paternal and paternally reported child measures only). Maternally and paternally reported child effortful control had an odds ratio of 0.42 (95% CI: 0.20 – 0.89) and 0.21 (95% CI: 0.06 – 0.71)respectively. Maternally reported child attention was also negatively associated with HbA1c (Odds Ratio:0.44, 95% CI: 0.22 – 0.91), with paternally reported child attention approaching significance (p-value of 0.08). In addition, self-reported maternal activity and attention were both found to be negatively associated with HbA1c, even after controlling for age, SMBG and Insulin Therapy. The odds ratio for Maternal activity and attention were 0.36 (95% CI: 0.17 – 0.78) and 0.43 (95% CI: 0.21 – 0.87) respectively. The HKST test failed to show any significant relationship with HbA1c. With a median score of 57 out of 60, it was found to be inappropriate for this age group. Conclusion: This is the first study to investigate the concept of intuitive eating and CHAOS in Type One diabetes mellitus. We have provided evidence indicating that these measures are predictors of HbA1c, and paternal CHAOS in particular, has clinical potential to aid the process of identifying populations at risk of poor glycaemic control. The present study is also the first study to use the Mary Rothbart’s Temperament scales in this population, and has found that child effortful control (Cognitive regulation) is associated with good glycaemic control. In addition to this, maternal measures of temperament, in particular activity and attention levels were also found to be predictors of glycaemic control. This study has explored three novel psychosocial tools in diabetes, and provides preliminary evidence warranting further research in these areas.

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  • Mutant p53 and pro-inflammatory gene expression in colorectal cancer cells

    Saha, Elora Konok (2015)

    Undergraduate thesis
    University of Otago

    p53 gene is the most commonly mutated gene in cancer, and in many malignancies a loss of p53 promotes cancer progression. However recent evidence has shown that missense mutations in p53, particularly those that lead to the protein acquiring abilities not seen in the wild-type form, may actually aid oncogenesis. These so-called ‘gain of function’ mutations are thought to upregulate many pro-inflammatory cytokines, which can enhance tumour growth, and inhibit other proteins, such as TP63 – which has been shown to have anti-oncogenic properties. The central question of this project is to establish whether gain-of-function mutations in p53 influence the expression of pro-inflammatory genes in colorectal cancer (CRC) cells. To address this question, shRNA constructs against MDM2 were generated to stablise mutant p53, which were then transfected into various CRC cell lines that either harboured a gain-of-function mutation, had wildtype p53, or were void of all p53. In addition, a puromycin-resistance gene was included allowing for the selection of positively transfected cells using puromycin. qPCR primer sets were also developed for measuring the expression of several selected chemokines once the CRC lines had been transfected. To assess the ability of mutant p53 to functionally interact with p63, I attempted to generate a puromycin-selectable p63 expression construct. Resulting data suggest that gain-of-function p53 mutations may indeed heighten expression of various chemokines.

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  • Measuring Mitochondrial Dysfunction in Humans

    Brinsden, Mark (2015)

    Undergraduate thesis
    University of Otago

    Mitochondria are essential organelles found in almost every cell in the human body. They host a number of important metabolic pathways and carry out essential biological functions such as ATP synthesis and regulating cell death. There is a slow decline in mitochondrial function associated with ageing and mitochondrial dysfunction is proposed to act causally in a number of diseases. Previously it has been difficult to measure the health of human mitochondria as tests have required tissue from invasive muscle biopsies. The Seahorse XF Analyser is a recent technological advance that enables researchers to test mitochondrial function in small numbers of live cells. Recently, using the Seahorse analyser, peripheral blood cells such as platelets, monocytes and lymphocytes have been shown to display individually distinct bioenergetic profiles. During circulation, these cells are exposed to metabolic or environmental stressors throughout the body, potentially allowing them to act as biomarkers of bioenergetic health and ageing. Different cell preparations were trialled to purify and isolate platelets, monocytes and lymphocytes from freshly drawn whole blood. These protocols succeeded in preparing platelet and T-lymphocyte samples for XF analysis, however inconsistent results indicated that the protocols need further development. The Seahorse XF analyser was used to measure bioenergetic function in human platelets and T-lymphocytes from healthy donors ranging from 21 to 56 years of age. Each cell type required optimisation experiments to determine the optimal inhibitor and substrate concentrations to generate a meaningful bioenergetic profile. Similarly, seeding densities were determined to ensure oxygen consumption values that were suitable to the instruments sensitivity. These cell types have elastic metabolic phenotypes, and appeared sensitive to metabolic switching during early stages of the XF assay. Platelets were particularly difficult to work with because of their inclination to cease using oxidative phosphorylation and switch metabolism to using purely glycolytic pathways. Platelet susceptibility for metabolic switching is undetermined at this point. Lymphocyte optimization experiments also indicated possible premature activation during the assay. Further work is needed to fine tune this protocol to ensure consistent and uniform measurements before accurate BHI values are calculated for donors of different ages.

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  • Exploring Molecular Links between Obesity and Breast Cancer

    Crake, Rebekah Lee Isla (2015)

    Undergraduate thesis
    University of Otago

    Obesity is associated with a high risk of incidence of, and mortality for, postmenopausal breast cancer. Despite this well-established link, the molecular and mechanistic basis of the obesity and breast cancer association still remains unclear. In obesity research, genetic variation due to copy number differences has become increasingly popular. The salivary amylase gene, AMY1, is well-known for its extensive copy number variation (CNV) in the human genome and has previously been correlated with a genetic predisposition toward obesity; however, research surrounding this association is controversial. Despite an established relationship between obesity and breast cancer risk, the recently reported genetic association between AMY1 CNV and obesity has not yet been examined in normal and obese breast cancer patients. Furthermore, gene expression changes in breast tumours from obese women remain poorly characterised. We hypothesise that obese breast cancer patients are associated with (1) low AMY1 copy number and (2) differential expression of candidate genes in the breast tumour. This study included 55 post-menopausal breast cancer patients from The Cancer Society Tissue Bank, with a BMI (body mass index)> 30 (obese; n=28) or BMI < 25 (healthy; n=27). Quantitative PCR (qPCR) assessment of germline AMY1 copy number status from blood showed that obese breast cancer patients have a lower average copy number of AMY1 compared to normal weight patients. Examining breast tumour expression profiles of obese and non-obese patients from two published studies, identified four candidate genes (GRIA2, DUSP4, NR2F1, and ADH1B) shared between both studies. Analysis of gene expression data from The Cancer Genome Atlas (TCGA) indicated that these four genes are differentially expressed within clinically relevant breast tumour subtypes characterised by oestrogen receptor, progesterone receptor and HER2 status. qPCR analysis of each candidate gene within our study cohort showed that the average expression of GRIA2, DUSP4, NR2F1 and ADH1B was lower in obese compared to healthy breast tumours, but these results were not statistically significant. My study indicated that BMI may be associated with lower germline copy number of AMY1 in post-menopausal breast cancer patients; however, further work with a larger cohort is needed to establish if GRIA2, DUSP4, NR2F1 and ADH1B are associated with obesity related breast cancer.

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  • Does fat provide energy for breast tumour cell invasion and metastasis?

    Jones, Morgan Grace (2015)

    Undergraduate thesis
    University of Otago

    Breast cancer is the most commonly diagnosed cancer in New Zealand women. Obese breast cancer patients are more likely to have tumours with advanced clinical stage and high vascular and lymph node involvement. The tumour microenvironment provides vital support for tumours during development and progression of cancer, yet the local effects of stromal adipocytes on breast cancer cells have been largely overlooked. Recent studies by Dirat et al. (2011) and Bochet et al. (2011) have shown that breast cancer cells co-cultured with adipocytes become more resistant to radio- and chemotherapy, and more invasive. However, little is known about the metabolic changes that occur in breast cancer cells when they are cultured with adipocytes. Nieman et al. (2011) determined that lipids were transferred from omental adipocytes to ovarian cancer cells and were consequently used in β-oxidation. It was hypothesised that β-oxidation is increased in breast cancer to exploit the glycerol and fatty acids released by lipolysis from adipocytes in order to support the migration and invasion of breast cancer cells. In this study, breast cancer cell lines MCF7 (ER+; oestrogen receptor positive) and MDA-MB-231 (ER-/PR-/HER2-; oestrogen, progesterone and human epidermal growth factor receptor negative) were co-cultured with adipocytes isolated from breast adipose tissue. Adipose tissue samples were collected via the Cancer Society Tissue Bank from patients at Christchurch Hospital undergoing surgery for therapeutic mastectomy, prophylactic mastectomy and breast reductions. A Seahorse XF24 Analyser was used to measure oxygen consumption and extracellular acidification, as indicators of oxidative phosphorylation and glycolysis, respectively, in breast cancer cells grown alone or in co-culture with human breast adipocytes. MCF7 cells were found to have upregulated glycolysis after co-culture with adipocytes. Western blotting was used to assess differences in the expression of proteins involved in β-oxidation between breast cancer cells grown alone or in co-culture with adipocytes. Levels of carnitine palmitoyltransferase 1 (CPT1A), a protein involved in translocation of fatty acids into the mitochondrial matrix for β-oxidation, showed no change. However, phosphorylated acetyl-CoA carboxylase (ACC), a key metabolic enzyme that when inhibited relieves inhibition of CPT1A to allow fatty acid translocation into mitochondria, showed increased levels in both MCF7 and MDA-MB-231 cells after co-culture with adipocytes. These results support the concept that breast cancer cell metabolism, specifically glycolysis and β-oxidation, is being altered in the presence of adipocytes to utilise fatty acids and glycerol released by adipocytes during lipolysis.

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  • Searching for a Functional Variant in a Non-Coding Genomic Region Associated with Serum Urate

    Dowdle, Amy Evaline (2015)

    Undergraduate thesis
    University of Otago

    Gout was historically known as the king of diseases and disease of kings, reserved for those of wealthy, extravagant lifestyles. Far from being resigned to history books, gout is on the rise worldwide and in New Zealand – where we have the highest rate of gout in the world – Maori and Polynesian populations have a greatly elevated risk of experiencing the debilitating disease. Gout arises as a direct result of increased urate in the blood, and as such many studies worldwide have investigated gout, using serum urate levels as a proxy measure. Recently, two important genome-wide association studies were published – these compare the genomes of those with elevated serum urate to the genomes of control subjects and identify differences. Single nucleotide polymorphisms (SNPs) were identified upstream of MAF, a gene that had not before been associated with serum urate levels. These SNPs were in what is known as a non-protein-coding region, which often play an important role in gene regulation. MAF is a transcription factor that is expressed in the lens of the eye and immune cells, and many studies into MAF have focused on these aspects. However, MAF has also been shown to be expressed in the developing kidney (in zebrafish and mice), which is of great relevance in a study of gout. Therefore, it was hypothesised that one or more of the SNPs upstream of MAF alter regulation of the MAF gene in the kidney, resulting in a change in serum urate levels. An in silico analysis was carried out, using publicly available datasets to produce candidate causal variants upstream of the MAF gene. When multiple datasets were combined and meta-analysed, no specific candidates were produced – however, some SNPs approached significance. This is noteworthy in such a small sample set (n = 18,503, versus 71,149 and >140,000 in the GWAS analyses), and the analysis needs to be repeated on a larger scale. Based on ENCODE annotations, candidate SNPs were selected in areas that looked to be involved in regulation of gene expression. To investigate the putative cis-regulatory role of SNPs, reporter constructs were used in human cell lines. This enhancer assay indicated that one element acts to enhance gene expression, and that the SNPs within this element increased this effect. Finally, a chromosome conformation capture (3C) experiment aimed to determine the interactions occurring between the MAF promoter and these upstream regions, but unfortunately the results were inconclusive. This study contributes to a growing field of research investigating the effects of non-coding DNA on gene expression, in the context of an important disease affecting the lives of New Zealanders everyday. Future findings may provide a novel mechanism by which non-coding variants affect serum urate levels in a system that has not previously been characterised.

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  • Development of a Metabolic Syndrome Mouse Model of Breast Cancer

    Mandani, Anishah Nanji Devji (2015)

    Undergraduate thesis
    University of Otago

    Metabolic syndrome is a cluster of disorders, including obesity, atherosclerosis, inflammation and insulin resistance. It is associated with increased risk of various types of cancers including breast cancer. Obesity in particular is a risk factor for an aggressive tumour phenotype and reduced survival of patients with breast cancer. To understand the underlying mechanisms I aimed to develop and characterise a metabolic syndrome mouse with an orthotopic model of breast cancer. Apolipoprotein E (ApoE) is involved in the catabolism of triglycerides and cholesterol, and the ApoE knockout mouse model is prone to obesity and development of atherosclerosis. The double knockout ApoE/ArKO mouse displays all features of metabolic syndrome. At 6 months of age, wild type, ApoE and ApoE/ArKO C57BL/6 mice were inoculated with the murine breast cancer cell line E0771. Growth of tumours in the mammary fat pad and mouse weight were measured until tumours reached ethical endpoint. The hypoxia marker, pimonidazole, was injected 90min prior to euthanasia, and plasma, organs and tumours were harvested and weighed. Half of each tumour was formalin fixed and paraffin embedded for Immunohistochemical (IHC) analysis of cancer associated adipocytes (perilipin), proliferation (phosphohistone-H3), estrogen receptor status (ERα) and hypoxia (pimonidazole adducts). The other half was frozen and processed for tumour lysates, which were used to measure hypoxia inducible factor 1 (HIF-1α) by Western blotting, and adipokines, using an antibody array. Vascular endothelial growth factor (VEGF) and Insulin-like growth factor binding protein 5 (IGFBP5) concentrations were further analysed by an ELISA assay. HIF-1α levels in EO771 cells were analysed by subjecting the cells to hypoxic conditions. ApoE mice weighed more than wild type and ApoE/ArKO mice, and showed increased cellular proliferation. ApoE/ArKO mice had the least omental fat and the smallest tumours. IHC analysis showed that EO771 tumours in ApoE mice had the highest number of intratumoral, perilipin positive adipocytes (p<0.01). My findings show that breast tumours grown in ApoE/ArKO mice have an aggressive tumour phenotype, with increased proliferation, tumour hypoxia and VEGF concentration. These models represent valuable tools for research that will bridge the gap between cell culture models and breast cancer patients.

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  • Serum biomarkers and paracetamol during post-chemotherapy infections

    Bowden, Emily Ellen (2015)

    Undergraduate thesis
    University of Otago

    Background: Febrile neutropenia (FN) is a common complication of cancer chemotherapy defined as fever with neutropenia below 1.0 x109 /L. Prompt antibiotic treatment is life-saving. Antipyretics (e.g. paracetamol) are commonly used during antibiotic treatment to reduce temperature and discomfort. A phase II randomised, placebo-controlled double-blinded trial of paracetamol during FN was completed at Wellington Hospital. This study aimed to determine whether paracetamol affects temperature or quality of life (QoL) during FN, and to assess biomarkers as potential secondary endpoints. Methods: Participants received 1g oral paracetamol or placebo six hourly for 42 h. Tympanic temperature was monitored four hourly. Blood was taken 0, 4, 24 and 72 h after FN presentation. In the current study cytokine bead array was used to determine levels of TNF-α, IL-6, IL-8 and IL-10, procalcitonin (PCT) was assessed by ELISA, and C-reactive protein (CRP) using an immunoturbidimetric method. Participants completed the EQ-5D-5L QoL questionnaire daily and the FACT-N questionnaire on day 3. Results: Of 37 enrolled patients, 22 participants developed FN and received at least one dose of paracetamol (n = 13) or placebo (n = 9). Treatment groups had comparable demographics and vital signs at baseline. Per pre-determined criteria, 23% and 33% of patients had successful treatment in the paracetamol and placebo groups respectively (not significant). Peak temperature was significantly lower in paracetamol- than placebo-treated patients on days 1 and 2 (difference 0.7°C and 0.6°C, respectively, p < 0.01 and p = 0.03), but not on day 3. Average daily temperature was also significantly lower in the paracetamol than placebo group. IL-6, IL-8, IL-10 and TNF-α were raised at baseline and/or 4 h and declined thereafter. PCT peaked at 24 h. Presentation and 4 h levels of IL-6, IL-8, IL-10, PCT and TNF-α, as well as 24 h PCT and 72 h IL-8 levels, were associated with adverse outcome. IL-6 was higher in the placebo than paracetamol group at 24 h (p <0.02). QoL scores were worse in the paracetamol group during the first two days of treatment (difference not significant). Conclusions: Paracetamol was an effective antipyretic during FN. Serum biomarkers change during FN, and IL-6 and IL-8 are promising secondary outcome measures for future trials. The adverse impact of paracetamol on QoL scores was unexpected and requires confirmation in a larger study.

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  • Determining biomarkers for a diagnostic test of Chronic Fatigue Syndrome / Myalgic Encephalomyelitis

    Denny, Lisa (2015)

    Undergraduate thesis
    University of Otago

    Chronic fatigue syndrome / Myalgic encephalomyelitis (CFS/ME) is an unexplained chronic multi-system illness, which leads to a lifetime of impairment. Symptoms indicative of the disease include immunological dysregulation, incapacitating fatigue, cognitive impairments, pain in the lymph nodes, and post-exertional sickness. The pathophysiology of CFS/ME is unknown, however several potential causes of development of the disease have been speculated. Consequently, due to a lack of understanding both medically and scientifically, there are no validated laboratory tests for diagnosis or management. Filling this gap of knowledge by finding an appropriate biomarker would aid the medical community in determining appropriate treatment. This pilot study aims to evaluate whether certain molecules in the blood of CFS/ME patients may be biomarkers to aid in diagnosis. This involves analysis of cytokine levels in CFS/ME patients with matched controls, and changes in cytokine levels following a pre-determined exercise regimen, in which patients are known to perform poorly. Additionally, the ratio of translational initiation factor eIF2α, to its stress activated phosphorylated peIF2α derivative, will be examined in white blood cells of patients and controls. We hypothesise that these molecules have the potential to be informative in relation to immune deterioration, a hallmark of CFS/ME. To test this hypothesis we obtained blood samples from 10 CFS/ME patients and 10 matched controls according to the International Consensus Criteria. In addition, following a separate exercise study with 11 CFS/ME patients and 3 MS patients as controls, all samples were fractionated to separate plasma, lymphocyte and neutrophils. The prepared plasma samples from all participants were simultaneously examined for expression of 27 cytokines. Statistical analysis revealed Interleukin-9 (IL9) and vascular endothelial growth factor (VEGF) expressed at significantly higher levels in CFS/ME patients compared to healthy controls (P<0.05). IL-9 and IL-13 were represented in both analyses, which indicates their potential as biomarkers for CFS/ME. Western analysis of proteins isolated from white blood cells detects both phosphorylated and unphosphorylated states of eIF2α with respective purified antibodies. Western analysis showed protein peIF2α to be slightly higher in patient lymphocytes compared to controls, though further experiments will need to be undertaken to determine the value of this result. This work suggests potential biomarkers that can be seen in blood, and Western blot analyses of additional patient and control samples will define whether a change in molecular state of eIF2α could be developed into a diagnostic test. This study gives a means to form a larger cohort for analyses on CFS/ME patients to enhance on current results in terms of identifying a possible biomarker for the disease.

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  • Impact of G-quadruplex structures and DNA methylation on allelic drop-out during in vitro amplification of imprinted genes

    Taylor, Millie Grace (2015)

    Undergraduate thesis
    University of Otago

    Diagnostic testing for genetic disorders or techniques such as preimplantation genetic diagnosis (PGD) both require accurate PCR genotyping (1). The failure of amplification of one allele, referred to as allelic drop-out (ADO) can confound genotyping results by falsely identifying heterozygotes as homozygous (2). A unique ADO mechanism has previously been demonstrated to occur consistently in the imprinted MEST gene, where both DNA methylation and G-quadruplex (G4) DNA structure contributed to allele loss (3). G4s are alternative DNA structures that form in G-rich regions due to the self-associating ability of guanine. Under certain ionic conditions, four guanine residues bind together either within or between strands to form a G-quartet, which can then stack upon one another to form the higher order structure. Such structures have the ability to act as a steric block to Taq polymerase. This effect is exacerbated when the G4 is methylated due to an increased thermal stability (4). This thesis explored the hypothesis that ADO via this mechanism occurs more widely throughout the imprinted genome. To test this, 22 target loci containing G4-DNA motifs were selected from 16 imprinted genes and an assay designed to detect ADO during PCR was developed. This required the creation of two variant alleles via the introduction of a single nucleotide polymorphism (SNP) with differential primer design. Both variants were then subjected to in vitro methylation and template mixing PCR experiments followed by Sanger sequencing to reveal mono-allelic or bi-allelic amplification. Of the 22 amplicons initially selected, only 14 were able to be consistently amplified and were thus used for this analysis. This method revealed that MEST is not alone in being susceptible to ADO events, with nine other amplicons showing either complete or partial mono-allelic amplification when methylated G4s were present. To confirm that the predicted G4 motifs did adopt the structure, CD spectroscopy was used. This revealed that these motifs were capable of forming the secondary structure and therefore contributing to ADO events. This work confirms that the effect of cytosine methylation and G4 regions on ADO that was previously observed (3) occurs more widely throughout the imprinted genome, and further highlights the need for diligence in both a diagnostic and research setting when analysing imprinted genes or other methylated regions.

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  • Inactivation of a Thiol-Dependent Enzyme by Urate Hydroperoxide

    Hamzah, Melanie Rosina (2015)

    Undergraduate thesis
    University of Otago

    There are links between high serum urate (hyperuricemia) and many inflammatory diseases, yet the mechanism is obscure. Urate, the product of purine and ATP break down, builds up in plasma because humans lack the enzyme uricase to convert it to allantoin, which is freely excreted. Urate may benefit health by acting as an antioxidant that scavenges reactive oxygen species. However, hyperuricemia is associated with gout, metabolic syndrome and cardiovascular disease. Oxidative stress is also associated with all these inflammatory diseases. During oxidative stress urate is converted to several reactive electrophiles, including urate hydroperoxide. This novel oxidant could contribute to the adverse effects of urate. Urate hydroperoxide is formed when urate is oxidized to a radical that subsequently combines with superoxide. Activated white blood cells called neutrophils, and xanthine oxidase along with myeloperoxidase/lactoperoxidase, can produce urate hydroperoxide. Previous studies characterized the formation of urate hydroperoxide and its oxidation of small biomolecules. In this investigation, I explored oxidation of thiols and the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by urate hydroperoxide. The effectiveness of urate hydroperoxide as a thiol oxidant was compared with taurine chloramine. Ellman’s assay for reduced thiols was used to measure depletion of cysteine residues on GAPDH by urate hydroperoxide and taurine chloramine. GAPDH was exposed to oxidants in a dose-dependent manner, then assayed by measuring its ability to catalyse the production of NADH. Mass spectrometry was used to identify specific modifications of GAPDH. Urate hydroperoxide oxidized exposed thiols on GAPDH and fully inactivated the enzyme at a ratio of about 5:1. Half of its activity was recovered by reduction with DTT. In comparison, taurine chloramine inactivated GAPDH at approximately 10:1 and DTT reduction recovered all activity. Hence, urate hydroperoxide inactivates GAPDH by reversible and irreversible routes. GAPDH increased in molecular mass by 132 Da with exposure to urate hydroperoxide, indicating the formation of a GAPDH-urate adduct. However, I could not identify which residue was modified with a tryptic digest. Formation of urate hydroperoxide during inflammation and its subsequent oxidative reactions may explain some of the adverse effects of hyperuricemia.

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  • Identifying the functional role of miRNA-34a in diabetic cardiac stem cells

    Gandhi, Sophie (2015)

    Undergraduate thesis
    University of Otago

    Transplantation of resident cardiac stem cells (CSCs) to the injured myocardium presents the potential to improve functioning of the diabetic heart in patients post-myocardial infarction. However, this therapy is not effective in people with diabetes due to reduced number and impaired functionality of CSCs and the molecular mechanisms underlying this impairment remain obscure. Recent studies have shown marked modulation of microRNAs (miRs) before the development of structural and functional changes in the diabetic heart. Among several miRs, microRNA-34a (miR-34a) is highly expressed in the diseased heart and differentially expressed in various stem cells. The aim of this study is to determine the role of miR-34a in diabetic CSCs in both acute and chronic diabetic states. We hypothesised an increase in miR-34a expression under both conditions and detrimental effect of miR-34a activation on CSC function. CSCs isolated from Type 2 diabetic (BKS.Cg-m+Leprdb/J) db/db mice demonstrated a 3 fold increase in miR-34a expression (p<0.05). Surprisingly, after miR-34a inhibition, CSCs in the acute and chronic diabetic models demonstrated a trend towards a 3.5% and 13% increase in apoptosis respectively. Furthermore, the acute and diabetic models demonstrated a trend towards a decrease in proliferation following miR-34a inhibition. Interestingly, expression of senescent gene marker TP53, a gene encoding for p53 protein, showed a trend towards a 15% decrease in TP53 expression in the non-diabetic model following miR-34a inhibition. Unexpectedly, the acute and chronic diabetic models demonstrated trend towards a 30% and 90% increase in TP53 respectively. However, we observed a decrease in p53 protein expression in the non-diabetic and diabetic models following miR-34a inhibition. Interestingly, these findings suggest miR-34a plays a dual role in the regulation of TP53 in non-diabetic and diabetic conditions. Findings from this study suggest a protective role of miR-34a in CSCs of the diabetic heart and modulation of miR-34a expression may improve diabetic CSC function.

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  • The Use of the Ankle Brachial Pressure Index (ABPI) in General Practice: A Mixed Methods Study

    Ding, Thomas Gregory (2016)

    Undergraduate thesis
    University of Otago

    Background Peripheral Arterial Disease (PAD) is an increasingly prevalent long-term illness globally. The Ankle Brachial Pressure Index (ABPI) is a well-established, simple, relatively quick, inexpensive and non-invasive assessment useful in diagnosing and quantifying PAD with varying symptomology. Though literature has documented many theoretical benefits associated with its use, ABPIs are still underutilised in general practice. Aims The study aimed to investigate the usefulness of ABPIs in general practice. Methods This study used quantitative analysis of routine electronic practice data from Mosgiel Health Centre between 2006-2015. The study also analysed thirteen one-to-one interviews with healthcare professionals regarding views on the usefulness of the ABPI in general practice. Results The quantitative arm showed that of 379 patients having ABPI tests over ten years, half were completed to investigate venous compression therapy eligibility and half investigated arterial-related symptoms. Of all patients who had ABPIs, 26% were referred to the vascular department. ABPIs helped to prevent inappropriate referrals in over 70% of cases. Patients living in more socially deprived areas were over-represented in the ABPI group. The qualitative arm showed that GPs are aware that ABPIs are beneficial to patients: through ruling in or out PAD to aid management, to aid triage for hospital-based systems and to implement immediate therapy in the community. Practical barriers were discussed, including cost, time and low patient need. Conclusion There are marked benefits associated with ABPI use in general practice. However, practical barriers need to be overcome before ABPIs are considered as a viable investigation for some GPs.

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  • Exploring the physiological and functional differences between stem cells isolated from the human right atrium and left ventricle

    Donnelly, Hayden (2016)

    Undergraduate thesis
    University of Otago

    Recent discovery of resident cardiac stem cells (CSCs), capable of cardiac lineage differentiation and paracrine mediated effects, suggest promise for cardiac tissue repair. The best site in the heart for obtaining CSCs remains to be determined. We therefore aim to investigate the differences between CSCs isolated from the right atrial appendage (RAA) and the left ventricle (LV) in patients undergoing on-pump cardiac surgery. Isolated CSCs were characterized using flow cytometry for the CSC specific surface markers CD90 and CD105. Hematopoietic lineage cells were identified using the CD34 surface marker. To date, approximately 80% of isolated cells (n=10) have been confirmed to be CD34 negative and CD105 positive. To exclude fibroblast contamination, immunofluorescence was performed to detect the expression of β-MHC, confirming the ability of the CSCs to differentiate into a cardiomyocyte lineage. Typically, administered CSCs undergo massive cell death in the first six weeks. Therefore, survival and paracrine factor production under conditions mimicking the microenvironment of the myocardial infarct territory were investigated. This involved measuring levels of apoptosis and production of important paracrine factors, SDF-1α and VEGF-A under conditions of hypoxia (1% O2) and serum starvation for 3 days. This was done via a luminescent caspase 3/7 assay and sandwich ELISA respectively. Results show (n = 3) lower relative caspase activity for RAA compared to LV at a ratio of 0.88 (95% CI 0.71 to 1.07, p = 0.107) and in the setting of serum starvation, hypoxia had a protective effect at a ratio of 0.79 and 0.87 for RAA and LV respectively (95% CI 0.56 to 1.11, p = 0.149 and 95% CI 0.79 to 0.95, p = 0.027). The relative expansion potential was also observed over the first 8 weeks (n=3). However, no statistically significant difference between RAA and LV expansion potential was shown. This is a preliminary study, establishing a foundation for a fair head-to-head comparison of RAA and LV. It is my hope that data collection will continue and with more statistical power, stronger statements will be made about either the equivalency, or the superiority of, one or the other cell types.¬

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  • Vestibular Function in Vestibular Schwannoma

    Tranter-Entwistle, Isaac Brian (2016)

    Undergraduate thesis
    University of Otago

    Abstract Introduction: Traditionally vestibular function has been assessed using caloric irrigations; new methods have failed to reach the same level of accuracy. Vestibular nerve dysfunction occurs with ‘acoustic neuroma’ or ‘vestibular schwannoma. Quantitative testing of hearing by audiometry is much more widely available than quantitative vestibular testing, although consideration of vestibular dysfunction is part of clinical management. Validation of a new method of quantitative vestibular function testing could lead to more widespread integration into clinical practice and affect decision making (i.e. timing of surgery) Methods: A non-blind observational cohort study was undertaken in 31 participants. Study endpoints were either one or two separate participant measures in March/April 2013 the September/October 13. All participants underwent caloric and head impulse testing with video-oculography, while 10 underwent audiometric assessment. Repeat testing was performed for 10 subjects, including additional cognitive. The primary outcome was vestibular function test measures. Results: Video head impulse was strongly correlated with calorics (p=0.01) and showed good sensitivity (80%) and specificity (70%). Dizziness Handicap Inventory showed no correlation with other vestibular function measures. Participants showed reduced cognitive function tested using the CANTAB battery (p=0.01) Conclusion: Video head impulse testing is comparable to caloric testing to assess vestibular function. Vestibular lesions may lead to cognitive deficits. Further research is needed to better understand the role of video head impulse testing in vestibular schwannoma.

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  • Engineering electrospun nanofibres with orf virus proteins for improved wound healing

    Wilkins, Samuel John Dunbar (2015)

    Undergraduate thesis
    University of Otago

    Wounded skin, in patients with underlying disease, can become inflamed and unable to heal (1). The economic burden of chronic wounds is significant and expected to rise substantially in the future. Current treatments are often ineffective, and even when successful there is a high recurrence rate (2). Growth factors and cytokines are critical for generating an efficient wound healing response, and may potentially be exploited to improve wound healing (3). However to-date, delivery of growth factors and cytokines to wounds has been largely unsuccessful, likely due to increased levels of proteolysis in the wound environment (4). Orf virus is a zoonotic parapoxvirus which encodes homologues of two proteins that are important in wound healing; vascular endothelial growth factor A (VEGF-A) and interleukin 10 (IL-10). Orf viral VEGF (VEGF-E) increases angiogenesis and endothelial cell migration and proliferation thus accelerating wound closure, while the viral IL-10 (ovIL-10) limits inflammation and reduces scarring (5, 6). These viral proteins are therefore promising wound therapeutics; however a delivery system is required which will protect these proteins from degradation while also enabling their controlled release at physiological concentrations. We hypothesised that emulsion electrospun nanofibres would be a suitable platform for VEGF-E and ovIL-10 delivery to wounds. This study aimed to develop methods to incorporate the viral proteins into nanofibres and to evaluate their release and bioactivity. Various FDA-approved synthetic polymers were tested for their ability to form electrospun nanofibres, the morphologies of which were confirmed by scanning electron microscopy (SEM). By loading the fibres with a fluorescently labelled test protein (ovalbumin) we were able to determine the release rate for each nanofibre and found that a fibre comprised of a poly(ethylene)oxide and poly(ɛ-caprolactone) (PEO:PCL) blend produced nanofibres with suitable protein release kinetics. To enhance detection, VEGF-E and ovIL-10 were conjugated with near infrared (NIR) markers. Labelling VEGF-E did not significantly affect its bioactivity. Labelling of ovIL-10 however, did significantly but not completely reduce its bioactivity. The labelled proteins were then incorporated into the PEO:PCL nanofibres. Confocal scanning laser microscopy was used to demonstrate protein localisation within the core of the fibres, however a continuous core-shell structure was not observed. In solution, VEGF-E (8%) and ovIL-10 (19%) were released within 2 hr, and released proteins were intact when visualised using SDS-PAGE. Following release, VEGF-E bioactivity was confirmed using a receptor binding ELISA. SEM confirmed that Keratinocytes adhered to fibres, while MTT assays indicated that cells grown on the fibres were viable. This work identified and characterised a method by which VEGF-E and ovIL-10 can be encapsulated into biocompatible nanofibres to both protect the proteins and control their release. Future studies will optimise the release kinetics and therapeutic utility of the nanofibres, in the hope that this will translate into enhanced healing of various types of wounds, particularly chronic wounds.

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  • The effect of polymeric formula on enterocyte differentiation

    Budd, Gabrielle (2016)

    Undergraduate thesis
    University of Otago

    Exclusive Enteral Nutrition (EEN) is commonly used in the management of Crohn's disease (CD). Polymeric formulae (PF), comprising whole proteins along with the daily requirements of vitamins and minerals, are commonly used for EEN. The mechanism by which several weeks of PF treatment induces remission is incompletely understood and appears to be a combination of modulation of the intestinal microbiota and anti-inflammatory effects on the intestinal mucosa. These include a reduction in inflammatory cytokine production and improved barrier function.The hypothesis for the current work was that PF caused an increased rate of differentiation of enterocytes at physiological concentrations. This was tested using Caco-2 cell culture and murine enteroids as in vitro models of the intestine.When Caco-2 cells were treated with PF, it caused an increase in intestinal alkaline phosphatase (IAP) expression and activity, a marker of enterocyte differentiation. This was associated with a decrease in cell proliferation and viability. Murine enteroids dissociated after several hours of exposure to PF, possibly due to interference with Wnt signalling. It was hypothesised that the observed effect of PF on Caco-2 cells might be mediated via the vitamin D receptor (VDR). However, when the activity of this receptor was reduced through use of an inhibitor, no change in PF-induced IAP activity and/or decrease in proliferation was observed. This suggests that the VDR pathway is not the primary driver of the accelerated differentiation seen in PF-treated cells.

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  • The Overlap of Hyperventilation Syndrome with Asthma and Anxiety

    Shin, Hayden Hyunjae (2016)

    Undergraduate thesis
    University of Otago

    Hyperventilation Syndrome (HVS) is a functional breathing disorder characterised by recurrent or chronic changes in breathing pattern causing respiratory and non-respiratory complaints that cannot be attributed to any specific medical diagnosis. HVS is thought to be common yet often unrecognised or misdiagnosed as ‘difficult-to-control’ asthma. HVS can co-exist with asthma and is also a differential diagnosis of asthma. There are also similarities and overlaps between HVS and anxiety disorders. There is no gold standard for the diagnosis of HVS, but the Nijmegen Questionnaire (NQ), a 16-item symptom checklist for HVS symptoms, is most commonly used in both clinical and research settings. A high NQ indicates HVS. However, this questionnaire includes several symptoms that are also associated with asthma and anxiety disorders. This casts doubts as to whether the NQ can distinguish HVS as a distinct clinical entity. Some view HVS as undiagnosed asthma, while others consider that it is a form of panic disorder. The aim of this thesis was to explore the overlap of HVS with asthma and anxiety in a general population sample. The prevalence of HVS and its association with asthma diagnosis, symptoms, objective markers of severity, and anxiety diagnosis at age 38 years were studied in the Dunedin Multidisciplinary Health and Development Study, a longitudinal investigation of 1,037 individuals born between 1972 and 1973 in Dunedin, New Zealand. Nearly one in 10 Study members had HVS according to the NQ and it was approximately twice as common in women as men (12.1% vs. 6.5%, respectively; P = 0.003). There was a substantial overlap between HVS and asthma with up to one-third of asthmatic women (35.4%) and one-eighth of asthmatic men (13.5%) potentially affected with co-morbid HVS. A different pattern of NQ symptom scores was found in people with only HVS compared to that of people with only asthma. Additionally, the objective markers of asthma including FEV1/FVC ratio and bronchodilator response differed significantly between those with only HVS and those with only asthma, suggesting that HVS can be distinguished from asthma using the NQ and is not merely undiagnosed asthma that is falsely detected by the NQ due to overlapping symptoms. Asthmatics with high NQ scores showed higher symptom scores for all 16 NQ items, rather than scoring high only for the asthma-like symptoms suggesting that these high NQ scores represented co-morbid HVS rather than more severe asthma. Further, there were no differences observed in the objective markers of asthma between those with both asthma and HVS and those with asthma alone. A large overlap was also found between HVS and anxiety disorders, especially with panic disorder (PD). The high NQ scores found in those with both HVS and panic disorder could not solely be explained by co-morbid panic disorder diagnosis: despite a large overlap in symptoms, only approximately half of the total NQ score could be attributed to panic disorder and HVS is thus likely to be distinct from panic. This is the first descriptive study of the prevalence and the overlap of HVS with asthma and anxiety in a large population-based sample. A substantial number of young adults in the general population have HVS and it is more common in those with asthma or anxiety disorders. It is important to assess for, and recognise HVS in these contexts.

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