416 results for Undergraduate

  • 'Je pense, donc je suis les traces' : a literary and historical analysis of the enlightenment, modernity and detective fiction in French

    Caswell, Erin Hubbard (2006)

    Undergraduate thesis
    University of Otago

    ii, 25 leaves ; 29 cm. Bibliography: leaves 19-20. University of Otago department: French. "October 2006."

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  • Another Look at the Faunal Remains of CA-SCR-9

    Nims, Reno (2011-06)

    Undergraduate thesis
    The University of Auckland Library

    CA-SCR-9 is an important early Middle Period (3100-2800 cal BP) site from the California central coast region that has been used to characterize residential base camps from that time. Previous studies have attempted to analyze the fauna using incomplete and non-representative samples, creating multiple, contradictory conclusions about the foodways of Middle Period peoples. The goal of this study was to synthesize and analyze all identified material to answer questions about the seasonal use of SCR-9, differences between two possible phases of occupation, and the adaptive strategies of Middle Period peoples on the California central coast. Using a representative sample of the fauna, this paper finds that SCR-9???s inhabitants primarily preyed upon mule deer. However, diverse species of marine mammals, leporids, terrestrial carnivores, birds, and marine fishes were also deposited at SCR-9, and inland site. The faunal remains from SCR-9 alone are not enough to identify relationships between sites, but these marine materials suggest that SCR-9 may have functioned as a seasonal or year round habitation site from which Middle Period peoples traveled to coastal sites such as SMA-218, which is nearly contemporaneous with SCR-9. Other writers have argued that two separate phases are represented ad SCR-9, including the Sand Hill Bluff Phase and the later A??o Nuevo Phase. The fauna from these two phases is extraordinarily homogenous, suggesting there were no changes in adaptive strategy, or that rodent activity has mixed the materials, making it impossible to compare fauna from the Sand Hill Bluff and A??o Nuevo phases. Fortunately, the assemblage does shed light on differential handling of taxa, and raises questions about the nature of bone grease extraction practices.

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  • Reforming New Zealand's Legislative Council: a study of constitutional change, 1891 and 1912-1920

    Roberts, Marcus (2008)

    Undergraduate thesis
    The University of Auckland Library

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  • Budget deficits and interest rates a bounds testing approach evidence from New Zealand

    McKenzie, Paxton L R (2000-10)

    Undergraduate thesis
    University of Otago

    This empirical study investigates the impact of budget deficits on long-term interest rates in a New Zealand context. Two measures of inflationary expectations are generated, one using the low frequency component of the CPI, provided by the Hodrick-Prescott filter, and the other using a survey based measure. To overcome unit root pre-testing uncertainty, a 'bounds testing approach' is employed to test for cointegration. A single-equation error correction model is then used to test the relationship. Evidence is found in favour of a positive long run relationship between budget deficits and long-term interest rates, suggesting a 'crowding out' effect.

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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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  • Molecular phylogenetics of Antarctic Sea spiders (Pycnogonida)

    Nielsen, Johanna Fønss (2005)

    Undergraduate thesis
    The University of Auckland Library

    Whole document restricted, but available by request, use the feedback form to request access. Sea spiders, or pycnogonids, are a unique group of exclusively marine invertebrates that are found worldwide. A scarcity of pycnogonid research is reflected in the unclear position of this group with regards to the phylum Arthropoda and lack of certainty in their family-level phylogeny. Traditionally, the pycnogonid phylogeny has relied on the external morphological characters of temperate, shallow water species. The Antarctic sea spider fauna displays a high degree of endemism and a number of species have the potential to address several long-standing questions regarding the pycnogonid evolution. This research uses new sequence data from Antarctic species to provide the most complete molecular phylogenetic reconstructions of the Pycnogonida, and is the first study to formally test a number of alternative hypotheses on the interfamilial relationships of this group of organisms. The BioRoss 2004 pycnogonid collection was classified into 18 different OTUs (5 families & 10 genera) and used, in combination with publicly accessible sequences, to provide samples for this study. Partial regions of the nuclear 18S and 28S rDNA, mitochondrial 12S and 16S rDNA and protein coding COI loci were sequenced for each dataset, and the concatenated data tested for incongruence using the Partition of Homogeneity test. The distance based Neighbour Joining and character based Maximum Likelihood tree-building algorithms were used to reconstruct the pycnogonid phylogeny for each locus independently and as a concatenated dataset. A series of alternative evolutionary hypotheses based on previous studies were examined via the Shimodaira-Hasegawa test. The primary hypothesis examined was the cephalic appendage reductive trend, which implies that ancestral sea spider taxa possess the greatest complexity of anterior appendages. On all the individual locus trees the family Nymphonidae were the earliest diverged lineage of pycnogonids, although low resolution at the roots of the trees implies that the data are not strong enough to reject an alternative hypothesis of a basal Ammotheidae group. Pycnogonidae is not the most recently derived sea spider family and the cephalic appendage loss hypothesis is thus rejected. None of the phylogenies supported a close relationship between the Colossendeidae and Nymphonidae families and doubt is raised over the true identification of several GenBank sequences. Polymerous species do not form a combined, ancestral group but are instead more likely to represent recent divergences from three separate families. Strong evidence supports the placement of the transient Austropallene genus (Callipallenidae) at the base of the Nymphonidae family. This study, and ongoing work, has generated large amounts of new sequence data. This can be used in future pycnogonid phylogenetic research and/or in investigations on the highly contentious position of the Pycnogonida with regards to the phylum Arthropoda. A DNA Surveillance website has been created to assist in the molecular identification of pycnogonids from future benthic bio-discovery expeditions (http://www.dna-surveillance.auckland.ac.nz).

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  • The geology and eruptive history of the Table Mountain region, Coromandel Peninsula

    Hayward, Bruce W. (Bruce William) (1971)

    Undergraduate thesis
    The University of Auckland Library

    The Table Mountain region covers an area of 2,200 hectares, 17 kilometres north-east of Thames, and straddles the main Coromandel Peninsula Divide between the headwaters of the Kauaeranga and Waiwawa Rivers. It is a region of steeply dissected, bush clad slopes and rugged bluffs composed of andesite, rhyolite and sediments. These rocks belong to three Groups. The oldest group of rocks consists of andesite lavas, breccias and sediments that form the upper part of the Beesons Island Volcanics sequence and were erupted during the upper Miocene and lowermost Pliocene. Unconformably overlying these is the mid Pliocene Whitianga Group containing rhyolitic lavas and sediments. In the Table Mt. Region this Group has been divided into the Minden Rhyolites and two informal sedimentary formations. The Wainora Formation contains basal volcanic breccias and freshwater, carbonaceous, epiclastic sediments that were deposited in two lakes on the dissected surface of the older andesites. This formation contains impressions of fresh-water mussels and numerous leaves, as well as considerable amounts of silicified wood. Conformably overlying the Wainora Formation are the thicker and more extensive water and aerially deposited pyroclastic sediments and rarer ignimbrites of the Waiwawa Formation. Many of the water laid deposits are inferred to have been formed by hot pyroclastic flows entering a lake. Minden Rhyolite domes were produced, by endogenous and exogenous growth, towards the end of this phreatic eruptive period. Hydrothermal alteration is inferred to be closely associated with the four Minden Rhyolite domes of this region. During the upper Pliocene to lower Pleistocene, the Omahia Andesite Group was intruded. The narrow Waiwawa Intrusive came up along an old intrusive contact between a Minden Rhyolite dome and the Waiwawa Formation sediments. The large Table Mt. andesite mass is believed to have formed by a combination of upwelling of lava along a fissure and actual intrusion. Both the Waiwawa and Table Mt. Intrusives spilled small amounts of lava out over the surface as lava flows. In the two million years since the cessation of volcanic activity in this region, erosion has greatly altered the landscape and emphasized the harder rock masses.

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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 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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  • 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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  • Evaluating the Seeding Genetic Algorithm

    Meadows, Benjamin (2012)

    Undergraduate thesis
    The University of Auckland Library

    This work is largely motivated by the PhD thesis of Cameron Skinner [Skinner, 2009], which features a rigorous mathematical and empirical approach to understanding the underlying mechanism behind the functioning of the genetic algorithm. The results are a new understanding of the algorithm in terms of the notions of discovery, selection and combination. Skinner uses these notions to create a modification to the genetic algorithm: the ???seeding??? genetic algorithm. We recognise this innovation as an important contribution to the field of evolutionary algorithms, and our focus in this dissertation will be to test its successes, failures, and the scope of its applicability.

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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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  • 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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  • 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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