51 results for Undergraduate, 2015

  • 11 week Beta-Hydroxy beta-Methylbutyric acid (HMB) supplementation: Effects on body composition and exercise performance in trained athletes.

    McIntosh, Nicholas Dean (2015)

    Undergraduate thesis
    University of Otago

    Background: Originally used in the farming industry to ‘bulk up’ cattle, interest in the leucine metabolite, beta-hydroxy beta-methylbutyrate (HMB), has been growing following a clinical trial which demonstrated significant improvements in strength and body composition in humans. Subsequent trials reaffirmed that previously untrained individuals benefitted from supplementation. However, trials involving athletes have demonstrated mixed results with short (0.05), nor was there a statistically significant difference with respect to skin fold measurements (p>0.05). Conclusion: The increase in body mass found in this study is consistent with other long term (>6 week) HMB supplementation studies. These gains in body mass may have influenced running performance as a larger mass is required to be moved. As no significant differences in body composition or strength were seen, the findings of this study suggest caution needs to be taken when supplementing with HMB as negative performance effects may occur. Therefore close attention to the type of activities required by the athlete needs to be considered prior to supplementation.

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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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  • 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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  • 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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  • 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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  • 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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  • Viral proteins as novel therapeutics in chronic horse wounds.

    Wakelin, Kirsty Anne (2015)

    Undergraduate thesis
    University of Otago

    This study examined the effects of two Orf virus-derived proteins, vIL-10 and VEGF-E, on chronic wounds in horses, to determine if they can improve vascularisation and re-epithelialisation while reducing scarring.

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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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  • Association of Interleukin-23 Receptor (IL-23R) gene variants with Gout and Rheumatic Heart Fever (RHF)

    Leaupepe, Keresoma (2015)

    Undergraduate thesis
    University of Otago

    Gout is an increasingly common form of inflammatory arthritis caused by the deposition of urate, leading to the formation of monosodium urate (MSU) crystals in joints and other body tissue. This results in subsequent recurrent acute inflammation attacks (1). Gout prevalence is increasing worldwide and has a particularly high prevalence in the Māori and Pacific populations of New Zealand (9.3 to 13.9% of Māori men and 14.9% of Pacific Island men) (2). Risk factors for gout development can be either genetic or environmental. The risk of gout is different between ancestral groups, suggesting that they have genetic differences (3). Rheumatic heart fever (RHF) is a systemic auto-inflammatory disease that is caused by infection of the upper respiratory tract (mainly the throat) by group A β-haemolytic streptococci (GABHS). RHF happens via antigen molecular mimicry and cross reactivity mechanisms between the host and bacteria. Cross reactivity of antibodies and/or T cells stimulates recognition between the S.pyrogenes peptides and the host protein and leads to inflammation and autoimmunity (4). RHF incidence and prevalence has steadily declined in developed countries since the early 1900s. However, it remains a leading cause of morbidity and mortality among young individuals (6 – 15 years) in developing countries. The risk of RHF can be familial or environmental e.g. as poor housing conditions, crowding, and poor health knowledge (5, 6). The IL23R gene codes for the interleukin 23 receptor. The receptor is located on the cell membrane of cells that are involved in the immune system, which provide defence mechanisms against infection and disease from foreign microbes. During the TH17 immune response, activation of IL23R from interaction with its subunit (IL23) initiates inflammation (7). Previous studies have shown that genetic variants derived from IL23R are associated with auto-inflammatory related diseases, such as rheumatoid arthritis, ankylosing spondylitis and inflammatory bowel disease. A study by Liu et al (2015) showed that the IL23R SNP rs7517847 minor allele G confers an association with gout in Chinese Han male population. 4 Our aim was to test IL23R gene variants (rs11209026, rs7517847 and rs11465804) for association with gout and RHF in European and Polynesian populations using case-control sample sets recruited within New Zealand and (for gout) Europe. To test this hypothesis, SNPs were genotyped using Taqman assay and statistical analysis was carried out using R studio logistic regression to test for association of SNPs with gout and RHF. Common confounders including ancestry, sex and age were adjusted for in the regression analysis. Gout results revealed that rs11465804 and rs11209026 in both European and Polynesian were not significantly associated with gout. However, the rs7517847 minor allele (G) showed a significant association with gout in Polynesian (Polynesian OR = 0.85, P = 0.04) (European OR = 0.94, P = 0.53), which is consistent with the Lui et al (2015) findings. These data replicate the Liu et al (2015) findings and support the claim that IL23R has a causal role in gout in people with Polynesian ancestry. Hence, the IL23R pathway is a target for gout treatment in the Polynesian population. RHF results revealed that only SNP rs11209026 shows evidence of association with a protective effect for the minor allele (A) (OR = 0.07, P-value = 0.002) after adjustment. Therefore the rs11209026 major allele (G) is in a susceptible direction. This provides evidence that IL23R has a casual role in RHF development risk in Polynesian people.

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  • The role of retromer in the epithelial sodium channel trafficking pathway

    Geda, Anna Caterina (2015)

    Undergraduate thesis
    University of Otago

    The epithelial sodium channel (ENaC) is a protein located at the apical membrane of polarised epithelial cells, primarily expressed in the epithelia of the gastrointestinal tract, lungs and kidney. ENaC's main function is that of absorbing sodium and it is strongly involved in regulating and maintaining total-body salt and water homeostasis, acting as the rate-limiting step for sodium reabsorption into the body. Its activity, therefore, is crucial for determining blood volume and, as a consequence, blood pressure. The sorting and trafficking of ENaC to the apical membrane is a tightly controlled process, requiring the interaction of multiple proteins and organelles. Although ENaC has been well-characterised, there are certain aspects about its trafficking which need to be clarified, such as defining the many proteins involved in the recycling of the channel to and from the apical membrane. A potential, novel candidate involved in ENaC recycling is the retromer complex. This endosome-associated protein complex has been shown to have a role in protein recycling, as well as maintaining cell polarity by assisting in the transport of proteins to and from their appropriate membrane. The aim of this study was to investigate whether retromer is involved in the recycling of ENaC in polarised epithelia, focusing on three specific proteins, namely ccdc22, Snx4 and KIBRA. Whilst ccdc22 is an established component of the retromer complex, Snx4 and KIBRA were hypothesised to be part of retromer, a plausible concept given their cellular localisation and proposed function. To test whether ccdc22, Snx4 and KIBRA were involved in ENaC recycling, their function was altered (via protein knockdown or overexpression) and the effects on ENaC trafficking were measured. Using transiently transfected HEK293 (human embryonic kidney) and FRT (Fischer rat thyroid) cells, semi-quantitative analysis was carried out with Western blots to visualise whether the knockdowns/overexpression of the proteins of interest were occurring. Then, Ussing chamber experiments were conducted to detect any changes in the ENaC channel’s activity at the apical membrane when a retromer protein was knocked down or over-expressed. Finally, GST-pulldown assays were performed to visualise whether the ENaC channel interacted with retromer through the protein KIBRA. Significant knockdowns were obtained of both Snx4 (ps blood pressure.

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  • 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.

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  • Cancer Stem Cells in Squamous Cell Carcinoma of the Oral Tongue

    Baillie, Ranui Francesca (2015)

    Undergraduate thesis
    University of Otago

    Background: Given the discovery of cancer stem cells (CSCs) within haematological and solid tumours and the expression of primitive markers by infantile haemangioma, it was hypothesised that a CSC population would also be present in oral tongue squamous cell carcinoma (OTSCC). A deeper understanding of the cells that drive tumourigenesis is required for the development of mechanism-based therapies for OTSCC. Aims: This study aimed to identify and characterise the CSC population within OTSCC based on their protein and gene expression profiles. Methods: Immunohistochemical staining, Western Blotting, mass spectrometry and Nanostring analysis were employed to investigate the expression of a panel of proteins and genes in formalin-fixed paraffin-embedded tissues, and snap frozen tissues of OTSCC from 21 patients. Markers used include epithelial cancer markers (p63 and EMA), CSC markers (CD44, CD133 and SOX2) and embryonic stem cell (ESC) markers (Oct-4, Nanog and pSTAT3). Results: Widespread and overlapping expression of p63, EMA, CD44, SOX2, pSTAT3, Nanog and Oct-4 was identified within the OTSCC. Co-expression of CD44, SOX2, Oct-4, Nanog and pSTAT3, was found within the OTSCC cells, while cells scattered within the peri-tumoural stroma expressed CD44, CD133, pSTAT3, Nanog and Oct-4. A CD44+/SOX2+/Nanog+/Oct-4+/pSTAT3+ but CD133-/EMA-/p63- CSC population was identified in OTSCC. Expression of CSC and ESC markers by differentiated phenotype structures, and the presence of polyploid giant cancer cells that are CD44+/Nanog+ were novel findings. Conclusions: The unique widespread distribution of the CSC population with co-expression of epithelial cancer cell, CSC and ESC markers within OTSCC suggests that these cells are comprised of several overlapping sub-populations that are organised hierarchically within the tumour. The lack of CD133 expression in OTSCC tumour cells brings into question the utility of this protein as a CSC marker. The findings demonstrate a unique expression signature for the CSC population within the OTSCC samples investigated and support a hierarchical CSC model of carcinogenesis, while the novel findings provide interesting avenues for further research.

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  • 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.

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  • 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.

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  • Child Mortality after Discharge from a Health Facility Following Suspected Pneumonia, Meningitis and Septicaemia in Rural Gambia

    Chhibber, Aakash Varun (2015)

    Undergraduate thesis
    University of Otago

    Background Two years away from 2015, the decline in child mortality is not fast enough to reach Millennium Development Goal 4. The Integrated Management of Childhood Illness (IMCI) is a strategy that simplifies management of child health. Beyond effective disease management, IMCI recommendations for care following illnesses are based on limited evidence from the field. The aim of this project was to find (1) the magnitude of and (2) risk factors for child mortality following discharge from a health facility in a low-income setting. Methods This study used an established population-based surveillance system for suspected invasive pneumococcal disease in Upper River Region, The Gambia, West Africa. Children that survived admission for suspected pneumonia, meningitis or septicaemia at the Region’s only referral centre (Basse Major Health Centre, Upper River Region) were followed for 180 days after discharge. Vitality status monitored by the DSS informed time-to-death information in a survival analysis that identified predictors of post-discharge mortality. Two multivariable Cox proportional hazards models were constructed. Model A described the clinical syndrome on admission (provisional diagnosis) and risk of post-discharge mortality. Model B used a reverse step-wise approach to find pre-discharge risk factors for mortality following discharge. Results The cohort that survived admission had higher mortality rates than the background rate in the community. Overall, 105 (2.8%) of 3735 patients died during the 6 months of follow-up. Half of the deaths occurred within 45 days of discharge. Approximately half as many patients died in the six months following discharge as died during hospital admission. Age stratified post-discharge mortality rates were three to six times higher than community mortality rates. In addition to demonstrating the protective effect of increasing age at discharge (HR 0.98 [95%CI: 0.96, 0.99] for every month increase in age), Model A showed that, compared to pneumonia alone, a provisional diagnosis of: pneumonia with visible signs of severe malnutrition had a HR 8.74 (95%CI: 4.93, 15.49); meningitis with visible signs of severe malnutrition had a HR of 13.90 (95%CI: 5.43, 35.58); sepsis with visible signs of severe malnutrition had a HR 18.79 (95%CI: 11.65, 30.32). Model B showed independent risk factors associated with post-discharge mortality were: the presence of neck stiffness on assessment (HR 17.60 [95%CI: 7.36, 42.10]); low mid-upper arm circumference (MUAC) (<10.5cm, HR 11.52 [4.59, 28.90]); visible signs of severe malnutrition (HR 3.94 [95%CI: 2.11, 7.36]); non- medical discharge (HR 6.22 [95%CI: 2.98, 13.01]); discharge during dry season (HR 2.33 [95%CI: 1.44, 3.77]); decreasing peripheral arterial haemoglobin oxygen saturation (HR 0.95 [95%CI: 0.93, 0.98] per percent increase); decreasing haemoglobin concentration (HR 0.82 [95%CI: 0.74, 0.90]) per unit g/dL increase); and decreasing axillary temperature (HR 0.70 [0.58, 0.84] per unit oC increase). Conclusion Gambian children in Upper River Region with suspected invasive pneumococcal disease are at increased risk of death following discharge from a health facility, and most of these deaths occur early. There are identifiable risk factors for death, including neck stiffness, low MUAC, visible signs of severe malnutrition, non-medical discharge, discharge during dry season, decreasing peripheral arterial haemoglobin oxygen saturation, decreasing haemoglobin concentration and decreasing axillary temperature. These data add to the evidence base needed to inform the development key guidelines and may be helpful towards development of a tool with clinical utility to identify children for intervention after discharge from hospital.

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  • Rib Fractures in Infants: Retrospective Survey of Fractures and Biomechanical Study.

    Blackburne, William Bligh (2015)

    Undergraduate thesis
    University of Otago

    Literature suggests that rib fractures are highly associated with abuse and the present understanding is that antero-posterior compression associated with the ‘shaken baby syndrome’ is their cause. However, this mechanism rests on a number of assumptions with little experimental data to support them. Recent work using a porcine model of fractures suggests that, in the case of lateral fractures this may be highly unlikely. This work shows a feasible alternate mechanism, that of blunt force trauma (BFT), for the cause of these lateral fractures. A piglet model is used and shows the ease with which ribs fracture as a result of BFT, compared to the difficulty of fracture seen previously in compressive injury. The initial development of a computational simulation of these ribs for use in injury scenarios is also outlined here. Secondly, skeletal surveys from New Zealand’s largest children’s care facility, Starship Hospital, were examined to give a picture of non-accidental injury (NAI) and how its patterns compare with accidental injury in New Zealand. It has been found that, as in foreign studies, there are a number of lesions highly associated with abuse and these include rib fractures, which are highly specific (97%) for NAI. Unusuallyhigh frequencies of lateral-type rib fractures (46.4%) were found and half the cases were found to be unilateral. This is not wholly in line with the currently accepted idea that rib fracture is due to antero-posterior compression, in which bilateral, posterior fractures are said to be most common. Overall, this work brings into question the traditional mechanism of rib fractures, provides a highly useful snapshot of abusive injury in NZ and also sets a strong foundation for future work.

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  • Review of a multidisciplinary team approach to patient positioning in head-and-neck cancer: a quantitative analysis

    Moore, Sarah (2015)

    Undergraduate thesis
    University of Otago

    Accurate patient positioning is extremely important in radiation therapy for head-and-neck cancer. With the introduction of three-dimensional cone beam computed tomography (CBCT) at the Wellington Blood and Cancer Centre (WBCC), it was agreed that positioning accuracy required improvement. This led to the establishment of a multidisciplinary team (MDT) focused on patient positioning. Following a number of process changes made by the MDT, improvements in setup accuracy were observed but not formally quantified. The aim of this thesis was to retrospectively quantify setup accuracy at WBCC, using CBCT images of 96 patients treated for head-and-neck cancer. On average 7 CBCT scans per patient were sequentially registered using each of the following match structures: C1-C3, C3-C5, C5-C7, C7-caudal, mandible occipital bone and the larynx. This enabled quantification of patient deformation as the measure of setup accuracy, by calculating the position of each structure relative to C1-C3. Statistical Process Control (SPC) was then used to assess trends in setup accuracy over time, allowing identification of specific time points where improvements occurred and correlation with process changes to be made. The multiple rigid registration protocol and deformation values calculated for this patient cohort clearly demonstrated the relative movement of anatomical sub regions in the head-and-neck. SPC charts showed that a significant and consistent reduction in deformation was achieved since the instigation of the MDT. A reduction in the magnitude and variation of the patient systematic 3D-deformation vector was observed, from 2.8 mm ± 0.1 mm (1 S.D.) in 2011 to 0.9 ± 0.0 mm (1 S.D.) in late 2013/early 2014. Statistical correlation analysis revealed that the introduction of new head supports (p = 0.003), as well as retraining of staff in making immobilisation equipment (p = 0.003) had a significant impact on patient systematic deformation. Both of these changes were actions initiated by the multidisciplinary team, which shows that a multidisciplinary approach to patient positioning had a positive impact on setup accuracy in our department.

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  • The role of cytoskeletal elements in the trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells

    Farquhar, Rachel (2015)

    Undergraduate thesis
    University of Otago

    The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane in polarized epithelia where it plays an essential role in promoting trans-epithelial ion transport. KCa3.1 is found in many tissues in the body and plays an important role in many physiological and pathological processes (e.g., regulation of salt and fluid transport in the gastrointestinal tract, atherosclerosis, sickle cell disease and asthma). Functional KCa3.1 must be targeted to the basolateral membrane, a process that is dependent upon proper cytoskeletal function. The cytoskeleton is comprised of actin and microtubule filaments. Actin filaments are comprised of polymerised G-actin monomers bound to form filamentous F-actin strands. Microtubules are long filamentous structures comprised of tubulin subunits, made from α-tubulin and β-tubulin monomers. This study examines the role of microfilaments and microtubules in the trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells. To address this, Fischer Rat Thyroid cells grown on filter inserts to form a confluent epithelium were stably transfected with the Biotin Ligase Acceptor Peptide (BLAP)-KCa3.1 construct. This construct allowed for the selective labeling of basolaterally expressed KCa3.1 using streptavidin. Selective labeling of membrane bound KCa3.1 allowed for the measurement of changes in KCa3.1 expression, in response to drugs that disrupt cytoskeletal elements, to reflect changes in KCa3.1 located on the basolateral membrane. This measure allowed for a direct correlation to be drawn between targeted disruption of specific cytoskeletal elements, e.g. microtubules and microfilaments, and expression of basolaterally-located KCa3.1. PCR was used to determine the mRNA expression levels of KCa3.1 in stably transfected cell lines and SDS-PAGE techniques were employed to investigate protein expression levels of KCa3.1. Western blotting was used to explore the effects of Cytochalasin D (Cyto D), Latrunculin A (Lat A), and Myosin Light Chain Inhibitor-7 (ML-7) which inhibit the function of actin (Cyto D, Lat A) and myosin light chain kinase (ML-7) respectively. Toxicity tests were performed to determine cell survival under a range concentrations of 0-20 μM (0, 3, 5 hr) for all three drugs with cell survival reduced with 20 μM at t = 5 hr for Cyto D and Lat A. Cyto D was administered over intervals of 0, 3 and 5 hr at 10 μM resulting in a decreased relative expression of KCa3.1 (compared to control) of 0.6±0.14 at t = 3 and further decrease in the expression of the channel at t = 5 hr with a relative expression of 0.12±0.035 (n = 5, p < 0.05). Lat A was also administered over intervals of 0, 3 and 5 hr at 10 μM causing a relative reduction in the expression of KCa3.1 at the basolateral membrane compared to the control. At t = 3 hr the expression of KCa3.1 was reduced to 0.7±0.065 and decreased to 0.3±0.049 at t = 5 hr (n = 4, p < 0.001). Finally, cells treated with microtubule inhibitor ML-7 showed a relative reduction in KCa3.1 expression of 0.55±0.12 at t = 3 hr, the expression was further decreased to 0.33±0.11 at t = 5 hr compared to the control. These data confirm that microtubules and microfilaments of the cytoskeleton are crucial in trafficking KCa3.1 to the basolateral membrane of polarised epithelial cells.

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