426 results for Undergraduate

  • Milton, the rural depression experience

    Panjabi, Jayashree (1979)

    Undergraduate thesis
    University of Otago

    Description: iv, 65 leaves : photo (fold.) ; 30 cm. Notes: Tapes of the interviews accompany this thesis. Not to be quoted without the author's permission. Bibliography: leaves 61-65.

    View record details
  • Domestic disquiet? : New Zealand responses to conflict in Malaya/Malaysia 1954-1966

    Sargison, Georgina (2006)

    Undergraduate thesis
    University of Otago

    viii, 89 leaves, [9] leaves of plates :ill., facisms., ports. ; 30 cm. Bibliography: leaves 84-89. Typescript (photocopy).

    View record details
  • The Queenstown-Lakes District rural residential development debate : an analysis on the current debate about protecting outstanding natural landscapes and controlling rural residential development in the Wakatipu Basin

    Baudet, François Eugene Nicholas (2001)

    Undergraduate thesis
    University of Otago

    80 leaves ; 30 cm. Includes bibliographical references. University of Otago department: Surveying.

    View record details
  • 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.

    View record details
  • The Public Safety Conservation Act.

    Logan, Alastair John (1976)

    Undergraduate thesis
    University of Otago

    70 leaves :ill. ; 30 cm. Includes bibliographical references. Long essay.

    View record details
  • Rugby, racism and fear : the reaction of Invercargill people to the 1981 Springbok tour of New Zealand

    Arthur, Lucy (1998)

    Undergraduate thesis
    University of Otago

    iii, 93 leaves :ill., ; 30 cm. Includes bibliographical references (leaves 89-93). Typescript (photocopy).

    View record details
  • 'Our bounden duty' : an analysis of the arguments justifying the introduction of peacetime Compulsory Military Training in New Zealand, 1949

    Muir, John Robert (1995)

    Undergraduate thesis
    University of Otago

    168 leaves ; 30 cm. Bibliography: leaves 161-168.

    View record details
  • A Survey of Māori Medical Graduates from Te Whare Wānanga o Otāgo

    Barnett, Lauren Helena (2015)

    Undergraduate thesis
    University of Otago

    INTRODUCTION Māori health workforce development is an important part of the strategy for improving Māori health and reducing inequalities and Māori doctors are an important part of this workforce. This research aimed to identify the nature and level of contribution made to Māori health by Māori medical graduates from Otago. It further aimed to investigate Māori medical graduate perspectives in relation to their medical education, current work and professional development, expectations and needs as Māori health practitioners. METHODS Māori medical graduates from Otago were surveyed using either an internet-based or postal survey to determine their roles in Māori health and their perspectives. The University of Otago Alumni Database was used to identify this population and contact them. There were 77 respondents in total with 70 completing the questionnaire fully for analysis. A descriptive analysis of the data was undertaken using Survey Monkey, Excel and SPSS. Although mainly quantitative, free-text comments made by participants were also analysed for themes. RESULTS There was a 53.4% response rate for the internet survey and a 19.4% response rate for the postal Survey. Respondents reflected a diversity of Māori doctors across gender, age, stage in career, locality of work and roles in Māori health with the overall population being relatively youthful (67% under 40 years). Time spent in Māori health varied with 22% spending 50% or more of their time on Māori health and 44% working in areas of high Māori population. Respondents reported many additional roles in Māori health spanning teaching, public health and leadership roles. The number of roles was greater among senior doctors (mean number 4.75) when compared with junior doctors (1.55). Māori graduates reported high levels of expectations, a need to be culturally and clinically competent and a mixed-experience of colleges, training programmes, work places and medical school. CONCLUSION This research strongly supports the hypothesis that Māori doctors are contributing greatly to Māori health. This contribution includes working in areas of high Māori population, in working with Māori health providers and across a broad range of roles and responsibilities spanning community, professional, leadership, academic, public health and training support roles. The involvement in Māori health is spread across all areas of the Māori medical workforce and appears to begin during registrar training, growing as doctors’ progress in their careers. Findings from the perspectives of Māori doctors indicate high levels of passion for Māori health, a need for both clinical and cultural competency and high levels of expectation on Māori doctors to be competent in Māori health. Understanding and support of Māori doctors during training, in the Medical Colleges and workplaces varies considerably indicating a positive direction by some Colleges and workplaces however inconsistent progress in meeting the support needs of Māori doctors. These findings provide a valuable platform for discussion with a range of stakeholders about the needs of Māori doctors and a valuable platform for Māori health practitioner professional development, starting at University.

    View record details
  • Fatigue in Inflammatory Bowel Disease

    Al-Mandhari, Rashid Abdullah (2015)

    Undergraduate thesis
    University of Otago

    Introduction: Fatigue is a common yet undertreated symptom in patients with Inflammatory Bowel Disease (IBD). Treating underlying inflammation lead to improved health outcomes and fatigue. However, fatigue can persist in remission. Aim: The aim of this study is to investigate fatigue in IBD and relate to possible contributing factors. Method: Patients were identified through the Episoft database. Following consent, all patients were provided with a questionnaire regarding demographics, International Physical Activity Questionnaire, CDAI and SCCAI, Brief Fatigue Inventory (BFI), multi-dimensional Fatigue Inventory (MFI). Patients were asked to complete a full blood count and faecal calprotectin (FCP). Fatigued remission patients without evidence of anaemia and with depleted iron stores (serum ferritin <0.001). Disease indices and FCP but not CRP were significantly associated with fatigue. Younger patients, females, those with a shorter time since diagnosis and patients following a diet complained of more fatigue. There was no significant difference between diseases. Patients in remission but with depleted iron stores (n=13) complained of significantly more fatigue than those with sufficient iron stores (p=0.012). Of those, 7 patients received i.v. iron leading to significant improvement in fatigue levels after four weeks. Conclusion: Fatigue is multifactorial and highly prevalent in IBD even in remission. Reduced serum ferritin is associated with fatigue also in remission.

    View record details
  • The effect of maternal obesity on neural stem cell proliferation in pre natal mice

    Gibbs, Jordan (2015)

    Undergraduate thesis
    University of Otago

    Autism Spectrum Disorder (ASD) is a complex neurological condition. It affects approximately 1 in 110 people world wide, with a 4:1 ratio of males to females affected. ASD presents with a wide range of psychological symptoms including deficits in speech, motor function and cognition. Interestingly, ASD first presents symptomatically in the first three years of life, sometimes even presenting as a regression in acquired skills, such as language. Research has shown a multitude of anatomical changes to the brain, creating a specific pathology seen in subjects with ASD. This includes overall changes to brain volume, with a lower volume at birth followed by an excessive overgrowth, and, consequently, excessive brain volume in the first few years of life. In addition to these findings, a set of regions in the brain have been repeatedly found to be associated with ASD, such as the amygdala, hippocampus and cerebellum. These findings suggest neural stem cells could be altered, leading to the changes seen in ASD, which are present right from birth. Due to the condition being psychiatric there is no way to identify ASD until behavioural symptoms appear. Consequently, there is no way to know what pre-natal ASD looks like, due to the lack of a biological marker. Though the origins of ASD are, as of yet, unknown, a wide variety of research has identified a number of associations that are potentially linked to ASD. One such association is maternal obesity, or obesity during pregnancy. This is associated with an increase in the risk for offspring to develop ASD. The aim of the present study was to test this hypothesis using immunohistochemistry. We analysed the effects of maternal obesity on neural stem proliferation in regions of the brain commonly associated with ASD. To examine this association, we used a mouse model of maternal obesity to generate prenatal offspring and compared them to control offspring undergoing gestation in a mother of normal weight. We used pups from late mouse gestation, GD15.5 and GD17.5, examined neural stem cell proliferation using immunohistochemistry to detect the cell proliferation marker Ki67. This was completed in 3 core areas associated with ASD; the cerebellum, amygdala and hippocampus. We identified a significant decrease in the number of cells proliferating in the amygdala at both ages in pups from obese mothers. Furthermore, we found sex differences in these results, with decreases in cell proliferation greater in males. We did not see any significant differences in the hippocampus or cerebellum. These results show that maternal obesity alters neural stem cell proliferation in the amygdala. The postnatal consequences of this are currently unclear, however, as the early neonatal ASD brain is smaller than neurologically normal controls, it is tempting to suggest that these findings represent pre-natal ASD. Given that the size of the brain is altered in ASD, which suggests a defect in the proliferation of neural stem cells, combined with the fact that maternal obesity is a predisposing risk factor, we hypothesised that when the fetal brain undergoes development in an obese mother there will be alterations in neural stem cell proliferation in regions of the brain commonly associated with ASD

    View record details
  • Mutations in the WNT mediator, DVL1, cause an osteosclerotic form of Robinow Syndrome

    Bunn, Kieran James (2015)

    Undergraduate thesis
    University of Otago

    This project aimed to clinically and biochemically characterise a novel phenotype which we have named Robinow Syndrome – Osteosclerotic type (RS-OS). Robinow Syndrome (RS) is a rare form of mesomelic dwarfism defined by a distinctive facial gestalt known as “fetal facies” – a combination of features including hypertelorism and midface hypoplasia. RS can be caused by loss-of-function mutations in genes encoding components of planar cell polarity WNT signalling. Osteosclerosis is associated with gain-of-function mutations in mediators of a different aspect of WNT signalling, the canonical WNT pathway. This thesis details three sporadic cases of RS-OS. Before this project began a combination of next generation and Sanger sequencing identified that two of these individuals had similar heterozygous de novo mutations in gene for the pan-WNT component DVL1, which has not previously been associated with RS. A third case was identified during this project and Sanger sequencing revealed another, similar, de novo mutation in DVL1. All three DVL1 mutations fall within the 14th exon and cause a -1 frameshift which predicts a DVL1 product with the wild type C-terminal sequence replaced by a novel amino acid sequence, which is shared across all three affected individuals. We hypothesised that these DVL1 mutations cause osteosclerosis through a gain-of-function leading to an increase in canonical WNT signalling. Transcript from cell lysates of cultured dermal fibroblasts taken from an affected individual were analysed by Sanger sequencing and restriction enzyme digest revealed that the mutation-bearing mRNA was endogenously expressed. Transient transfection of mouse C2C12 cells with EGFP-tagged DVL1 constructs showed similar protein levels between mutant and wild type DVL1 with fluorescent Western blotting. Taken together these experiments showed that the mutant allele is endogenously transcribed as a persistent mRNA, and that the majority of the protein product of that transcript is stable. Canonical WNT signalling was studied with a transient transfection-based TOPFlash assay. These revealed that, paradoxical to the osteosclerotic phenotype, the mutant DVL1 was significantly less active in the canonical WNT pathway than wild type DVL1. However the co-expression of the mutant DVL1 alongside the wild type DVL1 lead to a significant (~3-fold, P < 0.01) increase in canonical WNT activity over the same amount of wild type DVL1 expressed alone. This co-expression may explain the clinical osteosclerosis: the affected individuals are heterozygous for the DVL1 mutations thus are likely to co-express mutant and wild type DVL1. This work establishes that novel mutations in DVL1, a gene previously not associated with RS, causes an osteosclerotic form of RS. These mutations are likely to lead to osteosclerosis through a gain-of-function mechanism, with an increase in canonical WNT signalling. However this gain-of-function, in vitro, depends upon the presence of wild type DVL1 alongside the mutant DVL1. This wild type-dependent gain-of-function is, to our knowledge, unique amongst Mendelian disorders.

    View record details
  • The transition from acute to chronic post-surgical pain – A prospective cohort study

    MacLachlan, Campbell (2016)

    Undergraduate thesis
    University of Otago

    Background: Chronic pain is pain which persists beyond the normal physiological healing timeframe. It affects up to 20% of the Australian and New Zealand populations. Chronic pain is also costly, not only in terms of healthcare costs, but also in lost work days and decreased productivity. Surgery is one of the leading causes of chronic pain, highlighting the need to understand and prevent the transition from acute to chronic post-surgical pain. This study has documented the onset and development of subacute pain and the development of chronic pain. The undertaking of this study may have added to the arsenal of tools used to predict which patients will go on to develop chronic pain. By predicting chronic pain development in advance, interventions may be developed to prevent the development of persistent post-surgical neuropathic pain. Considering the rising costs of healthcare, and the growing socioeconomic burden of chronic pain, strategies to prevent chronic post-surgical pain need to be carefully considered. Hypothesis: That one or more of the proposed factors measured perioperatively will predict continued pain at six weeks and three months after gynaecological surgery. Aims: 1) To determine the prevalence of acute persistent pain at 6 weeks following surgery 2) To determine the prevalence of chronic pain at 3 months following surgery 3) To determine the extent to which (if at all) the perioperative factors measured predict continued pain at six weeks and three months. Methods: Patients were assessed at the Christchurch Women’s Hospital Pre-Admission clinic and approached for written informed consent. Those agreeing to participate were given validated questionnaires to complete in order to document their physical, emotional and functional state pre-surgery. In addition a cold pressor test was carried out to determine pain threshold and tolerance. The cold pressor test involves placing a hand in cold water containing ice. The pain slowly builds until the participant can bear it no longer, at which point it is voluntarily removed from the stimulus. Intraoperative factors (anaesthetic techniques, surgical techniques, and analgesic use) were measured, as well as patient-controlled analgesia (PCA) use, and medications prescribed on discharge. At 6 weeks, participants were telephoned, and validated questionnaires completed. At 3 months, all participants were again telephoned, and validated questionnaires were completed. Results and conclusions: Of the 54 participants 15.7% were deemed to be experiencing significant pain at 6 weeks post-operatively; 8.2% of participants were deemed to be experiencing significant pain at 3 months postoperatively. The psychometric questionnaires used often found differences between those experiencing pain and those not experiencing pain at given observation points, but only the Brief Illness Perception Questionnaire (BIPQ) appears to be predictive of developing prolonged postoperative pain. The mean difference (7.4 on a 0-50) scale may even be enough to see it used clinically alongside other predictive measures. Many of the demographic factors correlated with the experience of pain at a given time point(s), but none were predictive of the development of prolonged pain to significant levels. The cold pressor test did not show any significant differences between those in pain at baseline, and those that are not in pain at baseline. However, pain threshold as measured by this test was shown to predict prolonged pain outcomes. Pain tolerance and pain endurance followed the same trend, but were not statistically significant. No surgical approach or group was significantly more likely to develop a prolonged pain state than the others. However, it did seem as though laparotomy may be associated with poorer pain outcomes, which is supported by the literature. Intraoperatively, those who would later develop prolonged pain states were less likely to receive opioids, but possibly likely to receive greater doses if they did. Perhaps worryingly, those who would later develop prolonged pain states were significantly more likely to receive tramadol intraoperatively. There were no statistically significant trends in postoperative inpatient or discharge prescribing, however non-significant trends were noted across both in terms of prescription of specific non-steroidal anti-inflammatory drugs (NSAIDs) and weak opioids. Introduction to this thesis: Chapters One, Two, and Four of this thesis review the relevant literature for the following: the pathophysiology and epidemiology of pain (Chapter One); the tools currently available for the management of pain (Chapter Two); the tools used in this study to measure pain; and the tools used in this study to measure pain related behaviour (Chapter Four). Chapter Three gives a brief overview of the relevant aspects of the gynaecological surgical types used in this study. Chapter Five of this thesis outlines how the protocol for this study was designed and tested, while Chapter Six outlines the hypothesis, specific aims, and methods used in data collection and analysis. Chapter Seven details the results discovered using the aforementioned methods. Chapter Eight discusses the results found in Chapter Seven, the limitations and strengths of the present study, and draws conclusions based on these. From this, Chapter Eight makes appropriate recommendations for future research and notes potential influences on clinical practice.

    View record details
  • Measuring Markers of Immune Response in Patients Treated with Nivolumab (Opdivo®) and Pembrolizumab (Keytruda®)

    Butt, Leah (2017)

    Undergraduate thesis
    University of Otago

    Immune checkpoint inhibitors (ICIs) have drastically improved the clinical outcome for many cancer patients. However, not all patients treated with ICIs show a clinical response and the development of a unique spectrum of adverse events, termed immune-related adverse events (irAEs), restricts their oncological applicability. Additionally, the production of anti-drug antibodies (ADAs) may negatively influence patient outcome, but their role during therapy is yet to be established due to limitations in standard detection techniques. The primary aim of this exploratory study was to identify biomarkers that predict patient outcome to prevent a proportion of individuals exposed to a potentially ineffective and/or harmful therapy. It was hypothesized that patients who develop anti-drug antibodies experience a decrease in treatment efficacy and/or an increase in toxicity. Furthermore, patients with differing outcomes in terms of response, survival and the development of toxicity may display distinct clinicopathological characteristics. A retrospective review was performed on 32 patients undergoing nivolumab or pembrolizumab monotherapy for metastatic melanoma. Blood serum trough samples from 8 pembrolizumab-treated patients were analysed using in-house developed ELISA’s to measure pembrolizumab and anti-pembrolizumab antibody levels. Of the patients reviewed, 23 (72%) were ineligible for inclusion in initial clinical trials of ICI drugs. 29 patients (91%) experienced irAEs and 13 (41%) progressed during treatment. No clinicopathological variables were found to significantly predict patient outcome. Anti-pembrolizumab antibodies were detected in one patient and correlated with decreased blood serum drug levels. In this patient case, the individual responded to treatment according to RECIST, but, developed irAEs (pneumonitis and infusion reactions). This study indicates that, patients who are ineligible for initial clinical trials may effectively be treated with immune checkpoint inhibitors. Further investigation in a larger cohort is required to determine the prevalence and role of anti-ICI antibodies during ICI-therapy.

    View record details
  • 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.

    View record details
  • 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.

    View record details
  • 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.

    View record details
  • 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.

    View record details
  • 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.

    View record details
  • 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.

    View record details
  • 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.

    View record details