421 results for Undergraduate

  • PKCε Phosphorylation of RyR2: A Novel Link between Diabetes and Arrhythmia.

    Wong, Alexander (2017)

    Undergraduate thesis
    University of Otago

    Cardiovascular disease (CVD) is the leading cause of death in the world. Arrhythmia is a type of CVD which can be caused by store overload induced calcium (Ca2+) release (SOICR) in cardiomyocytes. SOICR occurs through the cardiac ryanodine receptor (RyR2). RyR2 phosphorylation is known to be a cause of SOICR. Patients with diabetes (DM) have an increased risk of arrhythmia as well as an increase in RyR2 phosphorylation by certain kinases. One kinase activated in DM is Protein Kinase C (PKC). PKC isoforms; α,ε, β2 and δ have an increased activity in the DM heart. Our study aimed to determine the effect of PKC on RyR2 in regard to SOICR. We hypothesised that activation or overexpression of PKC would result in an increase in SOICR consistent with RyR2 phosphorylation by other kinases. SOICR was examined in HEK293 cells expressing RyR2 with or without PKC overexpression in the presence and absence of a PKC activator (Dic8) and inhibitor (Go6983). Dic8, as well as Go6983, resulted in an increase in the occurrence of SOICR. Recent studies in the lab show that ATP analogues directly affect RyR2 resulting in SOICR, making the results of Go6983 hard to interpret. Overexpression of PKCα, with or without Dic8, resulted in small increase in the occurrence of SOICR. However, overexpression of PKCε, with or without Dic8, resulted in a large increase in the occurrence of SOICR. The propensity for SOICR is determined by the sensitivity of RyR2 to sarcoplasmic reticulum (SR) Ca2+. To study if PKC altered the sensitivity of RyR2 to SR Ca2+a SR targeted Ca2+ sensing protein, D1ER, was used. Overexpression of PKCα resulted in no change in the sensitivity of RyR2 to SR Ca2+, however, consistent with the increase in the propensity for SOICR, PKCε resulted in an increase in the sensitivity of RyR2 to SR Ca2+. Our data indicate that akin to other kinases, PKCε can increase SOICR through the RyR2 due to an increase in RyR2’s sensitivity to SR Ca2+. These findings may represent a novel link through which DM mediated changes in cell signalling increase the risk of arrhythmias.

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  • The individual child : study of the development of social services in education in relation to the first Labour government's educational policy

    Goodyear, Rosemary Katherine (1987)

    Undergraduate thesis
    University of Otago

    The subject of this study is the effect which the policy of the individual child, as expressed by the Labour Government in 1939, had on the development of social services in education. This development was examined chiefly from 1935-1948, but the requirements of the study meant that the inclusion of material from outside this time period was necessary. Social services have been interpreted to mean those services which developed to cater for the emotional and physical well being of a child. The emphasis of this study is on the services which developed in the context of the primary and secondary school systems : health services in schools, Vocational guidance and careers advisory service, the Visiting Teachers service, and lastly the Psychological service. Since the Child Welfare Division of the Department of Education comes outside this definition, it is not specifically included in this study. A variety of primary sources form the basis of this work. The Appendices to the Journal of the House of Representatives proved a valuable source, and gave the basic facts of the development of social services in education. The substance of my essay was largely derived from the Education and Health Department files at National Archives in Wellington. Examination of these files was time consuming due to the large volume of material which had to be sifted through. This effort was amply rewarded by the insights gained into the inner workings of the services, the problems, personalities, and developments. Letters from the public included in these files also gave an account of how the community viewed these changes. Some of the material in Chapter IV was based on an oral history exercise on the development of the Visiting Teachers Service in Otago, which I researched in 1986. I placed great importance on my interview with Dr C.E. Beeby, and on his article in the Listener because he was Director of Education at the time. His contribution to the development of social services in education was decisive. Allowance had to be made for a natural bias, but he gave an insight into the changes in education, and contributed a sense of the personalities of the time. Some secondary sources were very useful in checking information. Education Today and Tomorrow provided a clear statement of the Labour Government's policy on education. Ralph Winterbourn's Guidance Services in New Zealand Education was a good reference book, since he was another important personality in education during this period. The development of the policy of the 'individual child' was extremely important since it set the theoretical basis in education until the present day. In 1986 Dr C.E. Beeby wrote "For me, the most important discovery in education over this century has been the discovery by the school system of the individual child".

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  • Predisposition to Non Alcoholic Fatty Liver Disease in Preterm Guinea Pigs

    Barnes, Heather (2017)

    Undergraduate thesis
    University of Otago

    Background: Preterm birth accounts for ~10% of all births and is now an established risk factor for the development of metabolic syndrome, a cluster of conditions responsible for significant morbidity and mortality in New Zealand. The mechanisms underlying the development of Non alcoholic fatty liver disease (NAFLD, the hepatic manifestation of metabolic syndrome) in the ex-preterm are unknown at present. This project investigated potential differences between term and preterm born guinea pigs in the biological pathways believed to be associated with non-alcoholic fatty liver disease, including metabolism, and molecular and physiologic changes within the liver. Methods: Guinea pigs were delivered spontaneously at term (~GA69days), or prematurely by pharmacological induction of labour (GA62; equivalent to 32 weeks in humans). Fasting blood sugar levels (BSL) were taken at birth, and weekly thereafter. At corrected postnatal age (CPNA) 28 days (equivalent to early childhood in humans), glucose tolerance tests were performed, followed by euthanasia and collection of tissues. The prevalence of NALFD at CPNA 28 days was investigated using a NAFLD activity score based on histological methods and stains to visualise steatosis (oil red o), fibrosis (Masson’s trichome) and inflammation (H&E) in the liver. To observe hepatic alterations at a molecular level, amino acid profiles were assessed by liquid chromotography-tandem mass spectroscopy (LC-MS/MS). Results: Lower BSL at birth was observed in preterms compared to terms (p=0.001), however, BSL was higher in preterms than term counterparts at TEA (p<0.001) an amino acid associated with hepatic steatosis. Conclusions: Prematurity results in significant changes to hepatic metabolic function in the neonate. This deficit is reduced upon reaching term equivalent age, however molecular and physiological changes within the liver persisted into early childhood. In identifying metabolic, molecular and physiological alterations in the preterm guinea pig this study has begun to elucidate the pathways involved in preterm susceptibility to NAFLD and provided a solid platform for treatment.

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  • Stratigraphy, tectonics, and provenance of rocks in the Wether Hill area, Western Southland

    Willsman, Andrew (1991)

    Undergraduate thesis
    University of Otago

    Digital copy stored under Section 55 of the NZ Copyright Act.

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  • The geology of the Eastern Saddle Road area, Manawatu, N.I., N.Z.

    Grammer, Terrence Ronald (1971)

    Undergraduate thesis
    University of Otago

    Digital copy stored under Section 55 of the NZ Copyright Act.

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  • The effect of the trafficking protein p11 on the epithelial sodium channel

    Arora, Nikhil (2017)

    Undergraduate thesis
    University of Otago

    Regulation of renal sodium (Na+) excretion is crucial for the maintenance of extracellular salt and volume homeostasis and thus for blood pressure control. The epithelial sodium channel (ENaC) is composed of three subunits; α, β and γ; each subunit contains two transmembrane domains where both C- and N-terminal domains are cytoplasmic and allow interaction with regulatory proteins. For its sodium regulating properties, ENaC is principally present in the kidney collecting duct, reabsorbing ions from the urine to prevent unnecessary loss of salt and hence, water. This makes the channel vital for maintaining ECF homeostasis and consequently blood pressure. Channel activity is highly dependent on the density at the apical membrane, where sodium current is proportional to channel number. Dysregulation of ENaC or its associated trafficking proteins can lead to an array of problems which disrupt sodium homeostasis, leading to hypo/hypertension. Although extensive research has gone into unravelling the downregulation of ENaC by endocytosis, there has been significantly less research into its exocytosis. The p11 protein is known to promote exocytosis of a number of other membrane channels. We hypothesized that addition of p11 would cause an increase ENaC trafficking, and subsequently increase the amiloride sensitive current in Xenopus laevis oocytes. Previous research at the University of Otago confirmed the presence of an interaction between p11 and ENaC, and also identified that p11 is expressed endogenously within epithelial cells. To confirm a functional consequence of this interaction electrophysiological experiments were conducted. First, Xenopus laevis oocytes were injected with mRNA coding for α, β and γ-ENaC alone or together with mRNA coding for p11. Two electrode voltage clamp was carried out to measure ENaC current. Results from my experiments showed an increase in the amiloride sensitive current in the presence of p11 at both 0.75ng (12%) and 1.50ng (17%) p11 concentrations, however the results were insignificant for both 0.75ng (p=0.46) and 1.5ng (p=0.24). Preliminary results from the Condliffe lab show increased amiloride sensitive current for oocytes co-expressing ENaC + p11 as compared to oocytes expressing ENaC alone, indicating, that the presence of p11 promotes ENaC membrane insertion. Proteins from the oocytes were also used for western blotting to identify p11 within the oocytes, however, inconclusive results were obtained. Second, we wanted to determine if the amiloride sensitive current would reverse upon silencing of p11. Fisher rat thyroid epithelia were transfected with plasmids encoding ENaC subunits + si-p11 RNA, and their resistance and amiloride sensitive currents recorded using an Ussing chamber apparatus. Results show a significant decrease (average of 75%) (p=0.04) in the amiloride sensitive current for ENaC + si-p11, when compared to control (ENaC + si-control). Overall, it is confirmed that p11 interacts with ENaC. Furthermore, it is highly likely that p11 plays a role in aiding the exocytosis of ENaC, as concluded from both the overexpression and knockdown experiments. A significant lack of any previous research on the interaction between ENaC and p11 contributed to the difficulty of this project, however, the resultant information significantly aids our understanding of the exocytic process of ENaC and the individual proteins involved, such as p11. The real-world applications of this information span across a wide spectrum including therapeutic approaches for both hyper and hypotension which are large contributors to mortality around the world.

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  • Identification of Transporters Involved in Drug-drug Interactions During Gout Treatment in Primary Rat Hepatocytes

    Nguyen, Khanh Ho Kim (2017)

    Undergraduate thesis
    University of Otago

    Gout is one of the most common form of inflammatory arthritis, with hyperuricemia as the major risk factor. Chronic hyperuricemia, or high level of serum uric acid (SUA), can lead to the formation of monosodium urate crystals (MSU) in the joints, which can result in acute flares in gout. Allopurinol is the gold standard therapy for gout, with its active metabolite, oxypurinol, acts to inhibit the enzyme responsible for uric acid synthesis, xanthine oxidase (XO), thus lowering SUA level. Moreover, ~ 70% of US adults with gout also has hypertension, which is often treated with diuretics such as furosemide. However, concomitant treatment with furosemide compromises the therapeutic effects of allopurinol. The molecular mechanisms underlying this adverse drug-drug interaction are unknown. Based on current knowledge of the transport of furosemide and allopurinol/oxypurinol by transporters in the kidney, and the fact that similar transporter setups exists in the liver, as well as evidence from clinical studies, we hypothesise that transporters known to translocate these drugs in the kidney are responsible for the drug-drug interactions in the liver, where allopurinol/oxypurinol act to lower SUA. Hence, the aim of this project was to mimic the in vivo situation in gout patients with our cell model by treating cultured primary rat hepatocytes with gout-associated drugs. The functional outputs were assessed by measuring extra- (EUA) and intracellular uric acid (IUA) level. First, we were able to successfully establish a protocol to extract primary hepatocytes via in situ perfusion method. These extracted cells had polygonal morphology, characteristic of primary hepatocytes described in the literature. We characterised the gene expression profile of urate transporters and its converting enzymes in these hepatocytes and in the rat liver tissue. In both the extracted hepatocytes (n=3) and the tissue (n=6), qPCR analysis confirmed expression of the following genes: AOX1, XO, Oat2, Oat3, Glut9, Mrp4, Npt1, Npt4, and Abcg2. Furthermore, immunoblotting was carried out to confirm protein expression of our main proteins of interest: XO, Oat2, Glut9, Mrp4, and Abcg2. However, a temporal profile of the gene expression of the hepatocytes found that, after 48h there was a downregulation of most of the genes, except for Npt4 and Mrp4, which seemed to have not changed, and Abcg2 seemed to be upregulated (n = 1). From functional studies, we treated the cells with combinations of 250 μM allopurinol, 250 μM oxypurinol, 1 mM furosemide, and 1 mM probenecid. 24h after treatment, the cells and media were harvested for analysis. Our results showed that there was a significant decrease in EUA in cells treated with probenecid, which was in agreement with our model. However, due to low sample numbers, we were not able to draw any conclusion from these functional results. Additionally, a knockdown study was performed to evaluate the contribution of Oat2 and Glut9 to the transport of allopurinol and oxypurinol. Results from one set of experiment suggest that Oat2 might play a bigger role in transport of these drugs than Glut9. Further experiments are required to confirm transport of allopurinol/oxypurinol by these two transporters.

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  • Exploring the Different CaMKII Isoforms in the Vasculature

    Jagau, Kevin (2017)

    Undergraduate thesis
    University of Otago

    Coronary artery disease continues to be the leading cause of mortality in the world and a major source of disability, particularly for the aged population. The presence of vascular diseases such as atherosclerosis is a predisposition to life threatening events such as acute myocardial infarctions and strokes. Whilst the current best treatment is the use of statins, they still hold a significant residual cardiovascular risk. 1 in 4 people on statins still die as a result of cardiovascular disease, therefore there is much potential for supplementary treatments. Previous work in the Heather Lab has shown that the inhibition of the enzyme calcium/calmodulin dependent protein kinase 2 (CaMKII) through the administration of KN-93 reduces the atherosclerotic lesion size in ApoE-/- mice. The results of this study show the involvement of CaMKII in atherosclerosis, and thereby a potential target for treatment. Pathologies occurring in the vasculature such as atherosclerosis are characterised by endothelial dysfunction and increased migration and proliferation of vascular smooth muscle cells (VSMC). Different isoforms of CaMKII has emerged to play a role in the regulation of vascular homeostasis, namely the delta and gamma isoforms. Studies in rats using balloon angioplasty have shown that the CaMKII delta isoform is associated with adverse migration and proliferation of VSMC, whilst CaMKII gamma isoform is associated with decreased VSMC migration and proliferation. Determining which CaMKII isoforms are present in ApoE-/- mice, and their expression pattern during the development of atherosclerosis remains an active field of research, and will lead to a better understanding of the mechanism of atherosclerosis. It was hypothesised that as atherosclerosis progresses, the CaMKII delta isoform would both increase at the mRNA and protein level, whilst the CaMKII gamma isoform would decrease in the mouse aorta. To test this hypothesis, 13, 16 and 20 week old ApoE-/- mice had their whole aorta extracted and analysed for CaMKII protein and mRNA expression. The expression of protein and mRNA of both CaMKII delta and CaMKII gamma was also explored in human umbilical vein endothelial cells (HUVEC), human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC). It was hypothesised that there are differences in CaMKII isoform expression among the different human cell types of HUVEC, HCAEC and HCASMC.

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  • Determining the best combination of TLR Agonist and Tumour Peptide for Cancer Vaccination

    Gaskarth, Douglas (2017)

    Undergraduate thesis
    University of Otago

    Immunotherapy has revolutionised the treatment of cancer in recent years; significantly improving patient response and long-term survival. Though many immunotherapies focus on increasing the effector function of immune cells, the ability to generate and stimulate new tumour-specific immune cells has become an important topic for patients who do not respond to first line therapy. Previous work in our laboratory identified that intracellularly reversible conjugation of mode antigen ‘Ovalbumin’(OVA) to CpG B adjuvant improves the tumour specific response both in terms of immune cell activation, proliferation, and cytokine release, leading to complete tumour clearance in mice. This year I aimed to repeat this using the clinically significant melanoma antigen ‘gp100’ instead of OVA and to compare the use of CpG B adjuvant to CpG C adjuvant, which stimulates additional cytokine release, in the conjugate vaccine model. Using a combination of reversed phase high performance liquid chromatography (RP-HPLC) and cell culture, conjugates were produced and tested on their ability to activate Dendritic cells, induce their production of pro-inflammatory cytokines and induce T cell response in co-culture. Purification of gp100 conjugates was unsuccessful via RP-HPLC and testing reverted to the OVA model when comparing CpG conjugates. In antigen presenting cells, both conjugates induced similar levels of activation and antigen presentation but had unique cytokine profiles, with both conjugates trending towards higher levels of IL-1β and IL-12p70. Both CpG B-OVA and CpG C-OVA conjugates also induced a strong tumour-specific response with increased CD4+ and CD8+ T cell proliferation and significantly increased CD8+ T cell IFN-γ secretion. With these results in mind, both conjugates appear as strong candidates for therapeutic vaccination trials as either a monotherapy or a combined therapy with Checkpoint Blockade or Adoptive T Cell Therapy. In vivo testing using the CpG C construct is needed to assess its efficacy over CpG B.

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  • Lhx9 is required for urogenital ridge development and ovarian function

    Workman, Stephanie (2017)

    Undergraduate thesis
    University of Otago

    While there has been extensive research into the differentiation of sexually dimorphic gonads, there is much to learn about the bi-potential structure they arise from, the urogenital ridge (UGR). This gap in knowledge is imperative in the contexts of disorders of sex development and infertility, of which many cases have unknown aetiology. The transcription factor LIM Homeobox 9 (Lhx9) has been shown to have a functional role in the development of the UGR. Lhx9 -/- mice display gonadal agenesis and complete male to female sex reversal. Little is known about the regulation of Lhx9 gene expression in the UGR or its role in the greater genetic networks of reproductive development and beyond. We hypothesised that Lhx9 expression is regulated by Notch signalling in the UGR. To investigate the regulation of Lhx9 in the UGR in situ hybridisation was used to analyse the expression patterns of Lhx9, Notch (receptor), and Hes1 (Notch downstream effector) in the embryonic mouse gonad. Overlapping expression patterns in the UGR suggested a coregulatory interaction between the genes. This was further demonstrated by explant culture of embryonic gonads in the presence of the Notch pathway inhibitor DAPT. RT-qPCR revealed reduced Lhx9 expression in RNA extracted from the treated gonads, providing a strong case for a regulatory relationship between Notch and Lhx9. Due to declines in Lhx9 heterozygote (Lhx9+/-) fertility and embryo viability we hypothesised that a reduction in Lhx9 expression would result in impaired fertility in the mouse model. RNA extracted from the gonads of Lhx9+/- embryos was used for RT-qPCR to determine the relative expression of markers of key cell types in the developing gonad. Significant changes in the expression of markers of both male and female somatic and germ cells were found. This raised the question of whether Lhx9 was expressed in the adult ovary, and if so were the observed fertility declines due to reduced Lhx9 expression. In situ hybridisation revealed the novel discovery of Lhx9 expression localised to the follicles of the ovary, this was confirmed by immunohistochemistry. RT-qPCR of heterozygote ovaries revealed trends of reduced expression of critical ovarian fertility genes, a finding reflected in abnormalities seen in histological analysis. These results provide significant evidence for the role of Lhx9 in UGR development and the adult ovary, and offer direction for further investigation into its potential role in the underlying genetic networks DSD and infertility.

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  • Ageing Astrocytes - Implications for Motoneuron Dysfunction and Sarcopenia

    Arpel, Caitlin (2017)

    Undergraduate thesis
    University of Otago

    Sarcopenia: The most conspicuous intractable feature of advancing age is the gradual loss of skeletal muscle mass and the resultant loss of strength and mobility, and this is a major driver of morbidity and mortality in our ageing population. Loss of muscle is accompanied by loss of motoneurons, however what causes the death of motoneurons with advancing age remains unknown. Astrocytes are the most numerous and diverse cell type in the central nervous system, and play a critical role in neuronal support. Recent research has revealed that in-vitro, ageing astrocytes become senescent and express a senescence-associated secretory phenotype (SASP) that confers a reduced neuroprotective capacity. However, culturing senescent astrocytes in Glial-Derived Neurotrophic Factor (GDNF), a trophic factor critical for survival and proliferation of neurons, reversed these effects. Whether any similar changes occur in vivo is unknown, which provides a need for further investigation. This study aimed to investigate whether astrocytes in ageing mouse spinal cord become senescent, and whether the senescence phenotype can be reversed or attenuated by exercise - a known stimulus of GDNF production. The second aim was to investigate whether GDNF levels in the lumbar spinal cord reduce with age, and whether any decline is reversed by exercise. To inform the aims of this experiment, Semi-Quantitative Immunohistochemistry (SQI) was performed on sections of spinal cord from young, elderly, and elderly exercised mice. The levels of three proteins of interest were measured: Glial-Fibrillary Acidic Protein (GFAP) an intermediate filament protein and marker of astrogliosis; p16, a marker of senescence; and the trophic factor GDNF. Here we report that levels of GFAP within astrocytes of the lumbar lateral motor column showed a trend of increasing with age, although this was not statistically significant (p=0.052). Exercise had no effect on GFAP levels. P16-positive cell nuclei were observed in sections of both elderly sedentary and elderly exercised, but these did not co-localise with GFAP immunostaining of astrocytes. Instead, p16-positive nuclei appeared to be that of motoneurons, a novel finding. GDNF levels showed no change with age, but were increased significantly in exercised animals compared to sedentary (p<0.0001), indicating that exercise exerts neuroprotective effects by skeletal muscle-derived GDNF production. These results indicate that astrocytes become reactive with age and as a result may show reduced neuroprotection of motoneurons, contributing to their demise associated with ageing and sarcopenia. Although exercise increased GDNF levels within spinal motoneurons, this did not correlate with a reduction in astrocyte reactivity or a reduction in the presence of p16-positive nuclei as hypothesized. Instead, GDNF may exert protective effects for motoneurons directly, attenuating their age-associated decline, and slowing the progression of sarcopenia.

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  • The Cytotoxicity and Action of Curcumin Derivatives against Fn14+ Triple Negative Breast Cancer through repression of NF-κB p65

    Azam, Mayur (2017)

    Undergraduate thesis
    University of Otago

    Triple negative breast cancer (TNBC) is an aggressive subtype of the disease which lacks options for targeted systemic therapies due to a lack of actionable clinical targets. Molecular analysis has revealed that Fn14, a cytokine receptor, is over-expressed in 75% of invasive breast cancers but not in normal mammary epithelia. Ectopic Fn14 overexpression has been shown to induce canonical NF-κB signalling, which in response enhances Fn14 expression configuring an auto-regulatory loop that drives breast cancer cell malignant behavior. We hypothesised that suppression of the Fn14/NF-κB positive feedback loop may reduce Fn14 expression and the associated pro-migratory and pro- invasive characteristics of TNBC. Previously synthesised curcumin derivatives RL66 and RL71 have been shown to inhibit Fn14 and NF-κB p65 expression in triple negative BT-549 and MDA-MB-231 cell lines, while RL121 and RL118 have been shown to inhibit NF-κB activity in in vitro models of prostate cancer. Thus, we postulated that RL121 and RL118 would modulate Fn14 expression in TNBC. Investigations were conducted using two highly invasive Fn14+ TNBC cell lines, MDA-MB-231 and BT-549. RL121 and RL118 had a similar potency to previous derivatives in MDA-MB-231, IC50 0.57 μM and 0.45 μM respectively, and BT-549 cells, 0.16 μM and 0.30 μM respectively. Following 24 hr treatment with RL121, there was a 65% decrease in Fn14 and a 57% decrease in NF-κB p65 in MDA-MB-231 cells. In parallel, RL121 reduced both the invasive and migration capacity in vitro by 50% and 56%, respectively. RL118 was not found to effectively reduce NF-κB p65 or Fn14 expression and associated invasion and migration in either cell line. RL121-mediated reduction in Fn14 expression and associated reduction in migration and invasion is likely due to our observed suppression of NF-κB p65 expression, which consequently prevents expression of pro-migratory and pro-invasive genes including Fn14. RL121 and RL118 exhibited contrasting mechanisms of action, while both drugs were cytotoxic, only RL121 inhibited expression of NF-κB p65 and Fn14, and reduced in vitro invasion and migration. The cytotoxic pharmacodynamics of RL118 in TNBC requires further investigation. Our findings provide evidence that RL121 has potent anti-invasive activity in Fn14+ TNBC cells. Further investigation regarding the temporal downregulation of Fn14 and NF-κB p65, and identification of which invasive genes are being modulated are required.

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  • Investigating the role of peroxiredoxin in hydrogen peroxide signalling

    Carrad, Rose Jane (2017)

    Undergraduate thesis
    University of Otago

    Hydrogen peroxide (H2O2) was viewed as an unwanted by-product of aerobic respiration for many years but it is now well-established as being involved in many different signalling pathways. H2O2 is reduced to water by peroxiredoxins (Prdx), which make up a family of antioxidant enzymes. Due to high rate constants and the abundancy of Prdx in cells, H2O2 is quickly reduced to water. Therefore, signalling proteins have to compete with Prdx to be oxidised by H2O2. This has resulted in two conflicting theories for how Prdx are involved in H2O2 signalling. The first is the floodgate model, suggesting that Prdx are inactivated by high levels of H2O2, allowing for direct H2O2 oxidation of signalling proteins. The second model, signal peroxidase model, suggests that Prdx are more directly involved in the reversible oxidative activation of specific non-peroxidase thiol-containing proteins. Prdx1, a ubiquitously expressed cytoplasmic enzyme, is one of six isoforms of the Prdx family. Prdx1 has been characterised as acting in a redox relay mechanism in the H2O2 activation of the apoptosis signal-regulating kinase 1 (ASK1) apoptotic pathway. ASK1 activates apoptosis via the phosphorylation and activation of p38. This set of experiments aims to characterise the role of Prdx1 in H2O2 signal transduction, looking specifically at H2O2 induced apoptosis via the phosphorylation and activation of p38. The model systems used for this set of experiments were Hap1 wild type and Prdx1 knockout cells. Experiments carried out to measure the effect that Prdx1 knockout has on cell growth were done through the observation of Prdx1 knockout and WT growth rates. There was no observed difference in growth rates providing evidence that Prdx1 knockout does not affect cell growth. Secondly the effect that Prdx1 has on H2O2 induced phosphorylation and activation of p38 was determined through the comparison of p-p38 in WT and Prdx1 knockout cell lines that had been treated with H2O2. Due to nonspecific binding of the anti-phoshpo-p38 antibody these experiments remain inconclusive. Therefore, H2O2 induced activation of apoptosis was measured in Prdx1 knockout and WT cell lines by caspase-3 activity. Again, there was no observed difference of H2O2 induced apoptosis between the Prdx1 knockout and WT cell lines. This result gives evidence that Prdx1 does not have a critical role in the H2O2 signalling cascade that activates apoptosis. Further work to supplement these experiments is to observe H2O2 induced phosphorylation of a broad range of human kinases between Prdx1 knockout and WT cell lines. There were various kinases that were seen to be differentially phosphorylated in Prdx1 knockout cells. C-Jun, Hck, HSP60, PYK2, STAT2, STAT5a and STAT5a/b were seen to be differentially phosphorylated in Prdx1 knockout cells when treated with H2O2. In conclusion, this set of experiments gives evidence that Prdx1 does not have a crucial role in cell growth or H2O2 induced apoptosis in Hap1 cells. Therefore, these results do not support either theory for how Prdx are involved in H2O2 signalling in the cell. Preliminary phospho-kinase array results show a possible role of Prdx1 in signal transduction as per the signal peroxidase model.

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  • The Activity of the JAK-STAT Pathway in Infantile Haemangioma and the Haemogenic Potential of Infantile Haemangioma Explant Derived Cells

    Sulzberger, Lucy Isabelle (2017)

    Undergraduate thesis
    University of Otago

    Background: Stem cells have been identified within proliferating infantile haemangioma (IH), the most common tumour of infancy, and have been demonstrated to play a critical role in the rapid proliferation and gradual involution of this tumour. There is accumulating evidence showing that IH is caused by aberrant proliferation and differentiation of a haemogenic endothelium (HE). This HE possesses a functional capacity to undergo primitive erythropoiesis in vitro. Short chain fatty acid (SCFA) derivatives have been shown to stimulate cell proliferation and induce STAT-5 activation in various haematopoietic cell lines. Aims: The aims of this study were to investigate (1) the activity of the components of JAK-STAT pathway within the three phases of IH development; (2) the haematopoietic capacity of IH in vitro; and (3) the effects of SCFAs, butyric acid and propionic acid, to induce erythroid differentiation of explant-derived cells (IHEDCs) in culture. Methods: The presence of pSTAT proteins in proliferating, involuted and involuting IH were investigated using 3,3'-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining, 1-DE Western Blotting, and NanoString analysis. Proliferating IH explants were cultured using an in vitro model and the IHEDCs emanating from the explants were harvested. Cell suspension of volume equivalent to 5x105 live cells was plated on Matrigel and incubated in 0.05-1mM butyric acid, RPMI and 0.05-1mM propionic acid, and 0.05M DMSO (positive control) in each of RPMI media only, RPMI enriched with iron and MCDB media. Media was changed daily and cells were extracted and quantified following 24-72 hours in culture. Differentiated IHEDCs were characterised by IF immunocytochemical (ICC) staining with glycophorin-A. Results: Protein and genomic data reveal the expression of STATs 1, 3 and 5 which are activated in IH, particularly in the proliferative phase, with expression tapering as the lesion involutes. pSTAT3 is expressed most abundantly with pSTAT5 the least abundant. Low concentrations of both butyric acid and propionic acid significantly increased proliferation and differentiation of IHEDCs into blast colonies and the production of bi-concave cells within 72 hours in culture. These enucleated bi-concave cells expressed the erythrocyte-specific marker, glycophorin-A. Conclusion: The findings of SCFAs promoting proliferation and differentiation of IHEDCs into blast colonies and differentiated erythrocytes reveal a novel role for SCFAs in human haematopoietic differentiation, possibly via pSTAT-5 signalling. IH offers a simple and novel in vitro model for generating haematopoietic precursors and production of human erythrocytes.

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  • Caesarean Delivery on Maternal Request: A New Zealand Perspective

    Dwight, Emily May (2017)

    Undergraduate thesis
    University of Otago

    In many countries, including New Zealand, the caesarean delivery rate far exceeds the current WHO recommendation of 10-15% of live births. This is causing concern amongst a number of parties. One of the explanations for the rate increasing so quickly and to such an extent is Caesarean Delivery on Maternal Request (CDMR). There have been no studies conducted on CDMR in a New Zealand context to date. This qualitative study explored the perceptions of a group of New Zealand obstetricians’ and midwives’ on CDMR. The information was obtained via 12 face-to-face semi-structured interviews. The maternity providers were asked if they had ever encountered requests, whether they believed that it was ever reasonable to accede to a request for a caesarean section in a low-risk pregnancy, and whether there was a place for these procedures in the public healthcare system. An ethical analysis followed thematic analysis of the data. The ethical justification for the interviewee’s responses was analysed in the light of the four principles of biomedical ethics as articulated by Beauchamp and Childress; autonomy, beneficence, non-maleficence and justice. The results of this study show that there is no standardized approach to CDMR in New Zealand, raising concerns about equity of access. For this reason the development and implementation of a national care pathway for CDMR is commendable.

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  • Modulation of DNA methylation by L-ascorbate and 5-aza-2’-deoxycytidine in murine embryonic stem cells

    Bridgman, Luke David (2017)

    Undergraduate thesis
    University of Otago

    Cytosine methylation, normally found on cytosine residues adjacent to guanine (i.e., a CpG dinucleotide), is one means by which long-term gene repression occurs. Immediately after semi-conservative DNA replication, CpG dinucleotides on the replicated daughter strand are unmethylated, giving the “hemimethylated” state, where one DNA strand is methylated and one unmethylated. Hemimethylation is usually corrected through complementary methylation by Dnmt1 maintenance methyltransferase, but oxidative stress can inhibit Dnmt1, and so replicated DNA will remain hemimethylated. The Morison laboratory had new evidence that this aberrant hemimethylated DNA is “corrected” by the Tet family of enzymes, which actively catalyse the conversion of parent strand 5-mC to 5-carboxylcytosine (5-cC), that is subsequently replaced with cytosine by base excision repair. This study developed a murine embryonic stem cell model through which the molecular basis of active demethylation could be investigated. It was hypothesised that Tet family enzymes actively demethylate DNA, following oxidative stress-mediated inhibition of Dnmt1; i.e., after the generation of hemimethylated DNA. To model oxidative stress-induced hemimethylation the Dnmt1 inhibitor decitabine was used. The effect of ascorbate on Tet activity, both alone and in conjunction with decitabine was also assessed. Ascorbate increases Tet activity by increasing regeneration of Fe2+, whilst decitabine (5-aza-2’-deoxycytidine) inhibits Dnmt1 by binding and sequestering it. We hypothesise that when Tet is activated using ascorbate and Dnmt1 is inhibited by decitabine the proportion of unmethylated DNA should increase, due to the creation of hemimethylated DNA by decitabine and the upregulation of Tet activity by ascorbate. To perform this research, murine embryonic stem (ES) cells were maintained and manipulated in cell culture. Cell lines were synchronised to G1 by thymidine block. ES cells received differential treatments: control culture, + decitabine, + ascorbate, and + decitabine / + ascorbate. Samples were extracted at 2, 4 and 6 hours post-release from thymidine synchronisation. Hairpin linkers were used to maintain the connection between complementary DNA strands throughout PCR amplification, allowing comparison of parent-daughter strand methylation to identify hemimethylated sequences. Hairpin linkers were synthesised for three highly methylated genes: Asz1, Ckt2 and Kcnv2. Two next-generation sequencing libraries were prepared, containing 144 and 288 samples respectively, and sequenced using the high-throughput Illumina MiSeq platform. Bisulfite-converted sequences were aligned using BiQ Analyzer HT software, and methylation symmetry in complementary DNA strands determined using RStudio. Methylation proportions were averaged and/or correlated with results for each replicate, and plotted. This project identified that ascorbate induced marked demethylation in ES cells, whilst decitabine caused large increases in hemimethylation, but no increase in demethylation. When decitabine was added in conjunction with ascorbate, increasing demethylation was observed. These findings demonstrate that decitabine has the potential to induce marked hemimethylation, even in wild-type cells, in ascorbate-deficient culture. There was also evidence to suggest that in the presence of hemimethylated target sequence, ascorbate is necessary to allow Tet activity. This was seen in Tet-Triple knockout ES cells, where ascorbate failed to induce demethylation. Overall, the results support the hypothesis that hemimethylated DNA has the potential to induce Tet enzyme activity.

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  • The role of anti-Müllerian hormone in the brain’s response to steroid hormones

    Sirisomboonwong, Korawan Erika (2017)

    Undergraduate thesis
    University of Otago

    Many sexual dimorphisms that exist in the brain develop in the postnatal period. For males, this is when testosterone levels are minimal and anti-Müllerian hormone (AMH) is elevated. Testosterone requires the enzyme aromatase to undergo conversion to oestradiol to exert masculinising effects on the brain. Hence, the effect of AMH on aromatase was investigated. Transgenic mice for the Type 2 AMH receptor (AMHR2) were used, with male AMHR2-/- mice compared to male and female AMHR2+/+ mice. Experimental groups included perinatal, pre-pubescent and adult mice to assess different hormonal environments. In situ hybridisation was utilised to assess the presence of aromatase. Radioactive in situ hybridisation showed that at 2 days old, there was a wide variation of aromatase expression within each genotype group. There were also similar levels of aromatase between the male AMHR2+/+ and AMHR2-/- mice. This indicates that the role of AMH is independent from aromatase. To confirm this, larger sample sizes would be used so statistical analysis could be performed. Location of AMH intracellular signalling molecules could be identified and genomic RNA studies could be used to assess the effect AMH has on neurons.

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  • Nanopore Sequencing of RNA from Breast Cancer Genes

    de Jong, Lucy Clair (2016)

    Undergraduate thesis
    University of Otago

    Abnormal mRNA splicing can disrupt gene function and influence the course of disease. Analysis of abnormal splicing is an important part of determining whether a particular genetic variant found in the population is pathogenic or not. However, to correctly identify abnormal splicing, we must first understand what is normal. This project assessed the isoforms of the genes BRCA1 and BARD1, which are particularly relevant to the onset of breast cancer. BRCA1 is a tumour suppressor gene implicated in breast cancer onset. BARD1 codes for a protein that interacts with BRCA1 and produces a smaller mRNA transcript. Normal exon skipping events have been identified for both BRCA1 and BARD1, however, current methods are unable to reliably identify full transcripts. This has resulted in knowledge of individual exon skipping events but often does not tell us whether multiple events occur in the same transcript. The MinION nanopore sequencer (Oxford Nanopore Technologies), uses a nanopore to produce long-read, single molecule sequences. This has great potential for identifying multiple long isoforms, which is not practical using current technologies. The aim of this project was to examine the ability of the MinION to identify mRNA splicing patterns of transcripts derived from BRCA1 and BARD1. All mRNA from a normal lymphoblastoid cell line was converted to cDNA and targeted genes of interest were amplified by polymerase chain reaction (PCR). All potential isoforms generated from BRCA1 and BARD1 were then pooled and analysed using the MinION sequencer. After trialling many different analysis methods, the read data was analysed using the BLAST-like Alignment Tool (BLAT) with two outputs, a tabular and a graphical format. The tabular format grouped reads into potential isoforms, while the graphical format allowed visualisation of these isoforms and identified the exon/intron boundaries. Using both these formats 34 BRCA1 isoforms and 39 BARD1 isoforms were identified, 24 and 17 of which were potential novel isoforms respectively. Two of these novel isoforms from the BRCA1 dataset (Δ10-17 and Δ11q21) were further verified using Sanger sequencing. This was a proof of principle research project that demonstrated the potential use of the MinION nanopore sequencer for successful characterisation of multiple mRNA isoforms. This research has successfully identified a number of novel isoforms from the BRCA1 and BARD1 genes using the MinION sequencing device.

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  • The effect of Mepitel Film on skin reaction severity in patients undergoing radiation therapy for head and neck cancer: a feasibility study

    Wooding, Hayley (2017)

    Undergraduate thesis
    University of Otago

    Radiation skin reactions are a common side effect of radiation therapy and can be distressing and painful for patients. Head and neck cancer patients receive a high dose of radiation to the skin and are therefore at high risk of acute skin toxicity. There have been many clinical trials investigating topical agents to reduce or prevent these reactions but the evidence to date is lacking and many centres still base their practice on anecdotal evidence. Recently clinical trials in breast cancer patients have shown that using Mepitel Film® (Mölnlycke Health Care AB, Gothenburg, Sweden) reduced skin reaction severity and stopped the development of moist desquamation when used prophylactically (from the first day of radiation therapy). Mepitel Film and other soft silicone dressings that adhere very closely to the folds of the skin, have been hypothesized to decrease skin reaction severity by stopping friction by clothing and allow the radiation damaged skin to repair itself. The aim of this randomised controlled feasibility study in this thesis was to investigate whether Mepitel Film dressings were superior to Sorbolene cream in reducing or managing radiation-induced skin reactions in patients with head and neck cancer Head and neck cancer patients are prescribed a higher dose than breast cancer patients, have an uneven surface for the Mepitel Film to adhere to and have complex non-homogenous dose distributions, This means that testing the effect of Mepitel Film in this cohort would be challenging. Despite this, it was hypothesised that Mepitel Film was superior to standard Sorbolene cream in decreasing the severity of acute radiation-induced skin reaction in patients receiving radiation therapy for head and neck cancer. In order to test this hypothesis a randomised, controlled, multi-centre, international, open label intra-patient feasibility study was conducted in New Zealand and China. This thesis analyses a subset of 12 patients recruited at the Canterbury Regional Cancer and Haematology Service (CRCHS) at Christchurch Public Hospital. For the first six patients, the study area was chosen as the area of first erythema which was divided into equal halves. Each half was randomised to either Mepitel Film or Sorbolene cream. Mepitel Film was applied as soon as erythema was visible (management protocol). For the next six patients, the study area was chosen at the planning stage to include an area of relatively uniform high dose (>40Gy). This area was divided into two equal halves; one half was randomised to Mepitel Film the other half to Sorbolene cream. Mepitel Film was applied from day one of radiation therapy treatment (prophylactic protocol). Sorbolene cream was applied twice a day by the patient. The Modified Radiation-induced Skin Reaction Assessment Scale (RISRAS) and the Modified Radiation Therapy Oncology Group (RTOG) skin toxicity score were used to assess skin reaction severity three times a week. Patients also filled out the New Zealand validated Distress screening tool once a week and completed exit questionnaires at the end of the follow-up period. Thermoluminescent dosimeters (TLDs) were used to measure the actual dose to the skin underneath Mepitel Film and the control cream for all patients. When results of all 12 patients were combined, there was a statistically significant decrease in skin reaction severity in favour of Mepitel Film of 29% for combined scores, of 15% for researcher scores and of 49% for patients’ scores (p= 0.001, 0.002 and 0.004 respectively). The difference in peak RISRAS score between skin covered with Mepitel Film and control skin covered in cream was also significantly lower (p=0.02). The results were disappointing compared to those reported by the breast cancer trial where skin reaction severity was reduced by more than 90% when Mepitel Film was used prophylactically. Several factors may explain the lack of effectiveness of the Mepitel Film in this patient cohort. Dose to the skin was significantly higher in head and neck cancer patients and Mepitel Film did not adhere well to skin with heavy beard stubble, which meant Mepitel Film needed to be replaced almost daily for the first few weeks of radiation therapy. The latter may also explain why there was no difference in the Mepitel Film effect between the skin of patients on the management protocol and those on the prophylactic protocol which should have had the strongest skin protective effect. In addition, compared with skin covering the breast area, skin in the neck area may be “tougher” and less likely to benefit from “friction protection”. The results suggest that Mepitel Film does reduce skin reaction severity in head and neck cancer patients but the increase in skin folds, beard growth and high skin dose mean that the protective effects of Mepitel Film are limited, particularly in men with heavy beard growth. Mepitel Film appeared to be more effective in women but there were too few women in this trial to perform a statistically meaningful analysis. Future research should include clinical studies in different cohorts of head and neck patients, such as in women and men with less beard growth.

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  • Predictors of Referral Delay and Treatment Response to Intravitreal Bevacizumab for Wet Age-Related Macular Degeneration

    Ang, Wee Choen (Sebastian) (2017)

    Undergraduate thesis
    University of Otago

    Background: Recent years have seen anti-vascular endothelial growth factor (VEGF) agents revolutionize the treatment of wet age-related macular degeneration (AMD). To ensure optimal outcomes for this time-sensitive disease, current guidelines recommend that patients should be seen in clinic within two weeks of referral and treated with a loading phase of three injections, followed by a follow-up visit for reassessment. However, meeting these guidelines is challenging due to the increasing global prevalence of wet AMD and the accumulating cohort of patients requiring treatment. In addition, services with inefficient referral pathways and limited treatment capacities have been shown to threaten patient access to prompt treatment and increase adverse patient outcomes. Aims and Methods: This project is a two-year retrospective audit conducted at the Eye Department in Dunedin Public Hospital with the aims to (1) assess the efficiency of the current wet AMD referral pathway against contemporary guidelines and (2) identify any risk factors that are predictive of referral delay and treatment outcome. Relevant patient, referrer, referral and clinical characteristics were retrieved from retrospective analysis of clinical records and OCT scans of 113 patients. The outcome measures were referral delay (i.e. duration from point of referral to first assessment clinic); and treatment response measured by changes in visual acuity, changes in central macular thickness (CMT) and clearance of macular fluid. All potential predictors of these outcome measures were analysed via multivariable binomial logistic regression. Results: Only 49% of patients at Dunedin Public Hospital met referral guidelines, but 85% met treatment guidelines. Overall median time from point of referral to first treatment was 10 days. A loading phase of three bevacizumab injections significantly improved mean visual acuity by 5 ± 24 letters (p=0.03) and reduced mean central macular thickness (CMT) by 55μm (p85 years) were associated with an increased likelihood of CMT reduction (OR 9.157; 95% CI 2.6, 31.747; p<0.001 and OR 4.79; 95% CI 1.11, 20.7; p=0.036 respectively), whereas patients with longer duration of symptoms (1 to 3 months) were significantly associated with a decreased likelihood of CMT reduction compared to patients with symptoms of less than one month (OR 1.65; 95% CI 0.044, 0.616, p value = 0.007). Thicker baseline CMT was also found to be significantly associated with a greater reduction of macular fluid (OR 1.475; 95% CI 0.75, 4.578; p=0.017) after treatment, and so were females compared to males (OR 3.9; 95% CI 1.2, 12.58; p=0.02). Conclusion: This study identified that the current wet AMD referral pathway at Dunedin Public Hospital can and should be more efficient, and quality improvement work is warranted to improve compliance to contemporary guidelines.

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