373 results for Thesis, Undergraduate

  • Comparison of ground invertebrate assemblages across two types of natve forest fragment edge.

    Seldon, David (2002-06)

    Undergraduate thesis
    The University of Auckland Library

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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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  • 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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  • 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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  • The prevalence and level of education of Hepatitis C Virus among an asymptomatic population

    Vermunt, Jane (2014)

    Undergraduate thesis
    University of Otago

    Background The burden of Hepatitis C virus (HCV) is projected to increase substantially over the next 2 decades as a result of complications arising from chronically infected individuals who remain undiagnosed and untreated. Accurate epidemiological data on the prevalence and demographics of Hepatitis C is therefore needed to allow efficient planning of services and resource allocation for prevention and treatment management in the region. In order to minimise transmission and to recognise risk factors and symptoms of HCV infection, population-wide education is also essential. Aim This study aimed to identify the prevalence of Hepatitis C among the 40 to 59 year old population living in urban Dunedin. It also set about to identify gaps in knowledge about HCV in the target – assessment of HCV knowledge among this cohort was thought to be important to gaining better understanding any barriers to identification, diagnosis and treatment while concurrently raising awareness of the issue. Method A total of 1400 individuals aged between 40 and 59 living in urban Dunedin were randomly identified from the electorate role. A questionnaire was developed and posted to participants that explored risk profile, infection transmission, complications, symptoms and treatment. Participants were also asked to provide a blood sample for anti-HCV and HbsAg testing. Hepatitis B antigen testing (HbsAg) was also tested to allow comparison on prevalence and decrease perceived stigma of testing. Results Of the 1400 questionnaires sent, a total of 432 were returned completed and some 306 blood samples were analysed. The prevalence of HCV and Hepatitis B virus (HBV) was estimated to be less than 0.98%, based on a zero numerator. Significant knowledge gaps were identified. The average correct score from the questionnaire was 59.4%. Both adjusted and unadjusted logistic regression modelling showed that three demographics were statistically significant predictors of an individuals’ score. On average females scored 5.4% higher. Every increase in qualification level showed a 5.0% increase and a 4.8% increase was found between each occupation sector. No statistically significant relationships were found between socioeconomic status (SES) or age. The population sample recognised all the potential modes of HCV transmission. Only 23% correctly estimating the assumed prevalence of HCV. 93% of the sample population did not recognize that an acute or chronic infection may be asymptomatic and 97% were unaware that there could be no long term sequale to a chronic infection. 23.6% knew that it takes years rather than months weeks or days for symptoms of complications of a chronic infection to become apparent. Twenty-two per cent were aware that there is no available vaccine, 34.0% do not know that HCV can be treated and of those who do know, only 39.7% are aware that this is funded by the government. Conclusions The prevalence rate, although inferred, is lower than expected. Our group has thus committed to undertaken further work in this area to obtain a more representative sample of bloods from which to draw better prevalaence data – though completed, data was not ready for publication in this thesis. The lack of general knowledge about HCV is of concern as this population is at high risk of transmission and of developing complications related to unassumed chronic infection. It is clear that the majority of this population is unaware of the asymptomatic nature and when the nonspecific symptoms of an HBC or HCV infection are likely to manifest. Further, one-third of the population are unaware that HBV and HCV can be treated and two-thirds are unaware that treatment is fully funded. Well educated women working in the health or white collar sector have the best knowledge about risk of transmission, possible symptoms and treatment. Educational efforts to increase awareness empower people to be aware of symptoms, get diagnosed and undergo treatment needs to target all other population groups.

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  • The origin and development of Gore and its surrounding districts

    Kerse, Elizabeth (1943)

    Undergraduate thesis
    University of Otago

    vi, 117 leaves :ill. ; 30 cm. Includes bibliographical references (leaves 85-93) Typescript.

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  • A novel genetically encoded voltage indicator for studying motor cortical circuitry

    Scholtz, David Johannes (2014)

    Undergraduate thesis
    University of Otago

    The primary motor cortex (M1) consists of layers that are occupied by distinctive excitatory pyramidal neuron and inhibitory interneuron populations. Neurons within each layer receive inputs from numerous cortical and subcortical structures that relay proprioceptive and sensory feedback to modulate motor outputs and facilitate motor learning. The neurons within the upper layers (layer 2/3) are linked with processing and integrating these inputs and activating the circuitry that generates motor output commands that drive voluntary movement. To date our understanding of how these circuits achieve this remains elusive. Our poor understanding arises from technical challenges associated with studying the simultaneous behaviour of the electrical activity of the vast diversity and complex connections of the neurons within these circuits. To overcome this limitation we aim to use a Genetically Encoded Voltage Indicator (GEVI) called VSFP-Butterfly 1.2 that is endogenously expressed in layer 2/3 pyramidal neurons M1 in a transgenic mouse. We aim to determine the fidelity of VSFP-Butterfly 1.2 expression in the transgenic mouse and its ability to report subthreshold synaptic fluctuations in electrical membrane potential as changes in fluorescence. VSFP-Butterfly 1.2 is engineered to be expressed in layer 2/3 pyramidal neurons downstream of the Ca2+ Calmodulin-dependent protein kinase 2 (CAMKII). Immunohistochemistry for CAMKII in layer 2/3 of M1 slices found that the majority of neurons that express VSFP-Butterfly 1.2 also clearly express CAMKII (99.24 ± 0.567 %, n = 9 slices from 6 mice). Simultaneous recording of local field potential (LFP) and VSFP-Butterfly 1.2 fluorescent optical signals from layer 2/3 of slices from the M1 in response to extracellular electrical stimulation revealed a clear voltage-response relationship for VSFP-Butterfly 1.2 (n = 8 slices from 4 mice). Pharmacological excitatory synaptic antagonists dampened both the optical VSFP-Butterfly 1.2 (P < 0.0001, One-way ANOVA multiple comparisons, n = 4 slices from 2 mice) signals and LFP responses (P < 0.0001, One-way ANOVA multiple comparisons, n = 4 slices from 2 mice); and all responses were eliminated by tetrodotoxin which is known to block all voltage dependent electrical activity in neurons (P < 0.05, One-way ANOVA multiple comparisons, n = 2 slices from 1 mouse). In addition, we provide evidence that VSFP-Butterfly 1.2 can report membrane potential fluctuations at distances as far as 793.6 μm from the recording column (P < 0.0001). Our results show that VSFP-Butterfly 1.2 is reliably expressed exclusively in layer 2/3 neurons of the M1 in the transgenic mouse where it accurately reports physiologically relevant electrical synaptic responses. Our validation supports the future use and exciting benefit of this mouse to begin to understand the basis of network and circuit connectivity during motor output and motor learning.

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  • Wnt signalling influences T cell phenotype in a novel intestinal immune system model

    Worters, Thomas David (2014)

    Undergraduate thesis
    University of Otago

    Inflammatory bowel disease (IBD) is a collective term for inflammatory conditions that affect the gastro-intestinal tract. These conditions feature a multifactorial etiology: an interplay of genetics, environmental exposures, the immune system and commensal microbiota, all of which converge at the intestinal epithelia. The ability to understand IBD is dependent on and limited by the model systems used to study it. We have developed an ex vivo model of the human intestine by co-culturing intestinal organoids and PBMCs from the same patient. As intestinal organoids accurately mimic the intestinal epithelia, this allows us to study the interaction of the epithelium and immune system on a controlled genetic background in both IBD patients and healthy individuals. I hypothesise that intestinal organoids cultured with immune cells will create an environment similar to the human intestinal immune environment. The aim of my research was to develop a platform to study the role of immune cells in this organoid culture. I analysed the effect of organoid culture conditions on the survival and phenotype of immune cells, measured by cytokine and cell surface receptor expression. PBMCs remained viable for four days when grown in DMEM, and this viability was not affected by suspending the cells in the Matrigel used for organoid culture. Freezing and thawing PBMCs, which is required to allow establishment of the organoids, only caused a slight reduction in viability, and did not affect the frequency of Th1, Th17 and regulatory T cells. Introduction of growth factors required for organoid culture did not affect viability of the PBMCs, however the frequencies of Th1 and Th17 but not regulatory T cells were reduced. Recombinant Wnt, a key component used to culture organoids, affected the ability of regulatory T cells to maintain but not differentiate their phenotype. Finally, viable T cells could be removed from a complete PBMC-organoid co-culture. These data indicate that PBMCs can be successfully cultured in conditions used to generate intestinal organoids without loss of viability or major changes in phenotype. Furthermore, this co-culture model will likely serve as an accurate model of the intestinal immune system and may aid in the search of an effective treatment for IBD.

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  • A novel technique to investigate coronary microvascular perfusion in diabetes

    Nissen, Hazel Merete (2014)

    Undergraduate thesis
    University of Otago

    Diabetes Mellitus (DM)-induced disease of the coronary microvessels contributes to the worldwide increase in cardiovascular morbidity and mortality in diabetic patients. These microvessels are vital to the regulation of regional blood flow, and facilitating oxygen and nutrient exchange within heart tissue. Whilst total coronary blood flow is readily measured, our limited ability to directly measure coronary microvascular perfusion has restricted our progress in understanding DM pathology. Therefore, I aimed to test the feasibility of adapting two techniques previously used in skeletal muscle, 1-methylxanthine metabolism and vascular casting, to measure coronary microvascular perfusion and assess how this is impaired in type 2 DM. Isolated rat hearts were perfused under physiological conditions, with assessment of cardiac contractility and total coronary flow. This was combined with investigation of both a chemical and physical approach to assess microvascular perfusion. Firstly, metabolism of exogenous 1-methylxanthine (1-MX) by xanthine oxidase on the endothelium was investigated as a measure of capillary surface area. In addition, casts of the physiological vascular structure were produced using rapidly setting dental acrylic injected into the coronary vasculature, and visualised with micro-computerised tomography. To examine the feasibility of these microvascular measures, protocols were developed to induce known perfusion changes in male Sprague Dawley rat hearts; isoproterenol (1x10-8M, vasodilation) and angiotensin II (1x10-7M, vasoconstriction) were applied. Secondly, a pilot study was conducted applying the 1-MX and casting techniques to compare 20-week-old male type 2 DM Zucker Diabetic Fatty (ZDF) rats to their non-diabetic littermates. In Sprague Dawley rats, isoproterenol significantly increased whilst, to a lesser extent, angiotensin II significantly decreased myocardial function and coronary flow (p≤0.05, n=6 and 7). Vascular casting produced promising results; a representative cast from the isoproterenol intervention displayed increased branching, and the angiotensin II intervention showed somewhat reduced branching of the microvessels relative to no intervention (n=1). However, 1-MX values did not reveal any changes between these interventions. Consequently, the 1-MX protocol was optimised to improve stability, before being applied in the type 2 DM pilot study. Under basal conditions, no significant difference was discerned between diabetic and non-diabetic rats in 1-MX disappearance (22.6±6.7nmol/min (n=5) vs. 23.4±3.9nmol/min (n=3); mean±SEM: n.s.), nor in measures of cardiac function. Likewise, no difference was discernible between a representative cast from the non-diabetic and diabetic group. However, a positive Spearman’s rank correlation was observed between coronary flow and 1-MX disappearance in the diabetic rats (rs=1, p≤0.05). With this study I have set up the foundations of using 1-MX metabolism and vascular casting, as techniques to examine coronary microvascular perfusion in the isolated heart. Conclusions regarding DM-induced changes cannot be drawn at this stage. However, pilot data provide valuable information on how to further develop these techniques. Novel measures of coronary microvascular perfusion have the potential to enhance our understanding of coronary microvascular pathology, and eventually reduce DM-induced cardiovascular complications.

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  • The Effect of Cell Metabolism on Epigenetic Processes in Cancer Cells

    Moustafa Albathish, Boushra (2014)

    Undergraduate thesis
    University of Otago

    Epigenetic marks are written on and erased from DNA through the activity of methylation and demethylation enzymes, thereby altering the level of gene expression. The methylase and demethylase enzymes respond to intrinsic and extrinsic factors, and their activity has been shown to be compromised under various pathological conditions, including cancer. The cycle of cytosine methylation and demethylation is a major epigenetic pathway for regulation of gene expression. DNA methyltransferase modifies cytosine, generating methylcytosine, which can be oxidised by the recently identified Ten Eleven Translocation enzymes (TET 1-3) to generate hydroxymethylcytosine, formylcytosine, and carboxycytosine. These oxidised methylcytosine products are intermediates in the cycle to regenerate unmodified cytosine. When present in gene regulatory regions, they also serve as stable epigenetic marks that alter gene expression. Aberrant methylation patterns, such as hyper- and hypo-methylation are a hallmark of cancer. This research project has focussed on the presence and activity of the TET enzymes in selected cancer cell lines. TET enzymes belong to the superfamily of proteins known as Fe(II) 2-oxoglutarate dependent dioxygenases. Proteins in this family act as metabolic sensors, because they require cofactors; iron and ascorbate, and co-substrates; oxygen and 2-oxoglutarate, for their activity. Changes in metabolic conditions and the availability of these metabolites modulate the activity of these proteins. In this project, I investigated the expression and activity of TET 1-3 isoforms in a range of human and mouse cancer cell lines. The cells were then subjected to different metabolic conditions, such as hypoxia, and ascorbate and iron deficiency. The effect of these conditions on TET activity and the epigenetic profile was evaluated using Western blotting and ELISA methods. Our preliminary results show varied expression of the TET isoforms across the selected cancer cell lines, with TET 3 expression being most prominent. Altering oxygen supply, iron and ascorbate appeared to affect the levels of 5mC and 5hmC. As these conditions affect cancer cells in vivo, we suggest that epigenetic changes in response to metabolic stress will affect genetic patterning in cancer.

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  • Novel Methods for the Rapid Identification and Susceptibility Testing of Blood Culture Isolates

    Robinson, Andrew Mark (2014)

    Undergraduate thesis
    University of Otago

    The increasing emergence of antimicrobial resistance, such as that mediated by extended-spectrum β-lactamase (ESBL)-producing gram-negative bacteria, make it more likely that patients with sepsis and bloodstream infections (BSI) will receive ineffective empirical treatment. Rapid identification of disease causing agents, coupled with early detection of antimicrobial resistance facilitates the optimisation of essential treatment decisions. Matrix-assisted laser desorption-ionization time of flight (MALDI-ToF) mass spectrometry has recently been applied to the identification of microorganisms directly from blood cultures, reducing the identification process by up to 24-hours. This study sought (i) to determine the optimal method for the rapid identification of isolates directly from blood cultures and (ii) to develop a rapid method to detect β-lactamase-mediated resistance to extended spectrum cephalosporins directly from blood cultures. Two in-house methods for sample preparation were optimized and compared to a commercially available method. Using the conventional scoring criteria, the differential centrifugation protocol correctly identified 86.8% and 67.9% of clinical isolates at the genus- and species- level. This was compared to a quicker method using Sodium dodecyl sulphate (SDS) to mediate blood cell lysis, which correctly identified 83.0% and 62.3% of clinical isolates to the genus- and species- level. Both methods performed similarly to the more expensive commercial method. Results also suggested that the scoring criteria could be altered to increase the number of species-level identification while maintaining accuracy, achieving up to 90.3% species level identifications. To rapidly detect β-lactamase-mediated resistance to extended spectrum cephalosporins, a high-performance liquid chromatography (HPLC) assay was developed and optimized to detect resistance directly from growth-positive blood cultures. With a 1-hour incubation of bacteria with cefotaxime, resistance could be detected with 95.5% sensitivity and 88.9% specificity. This method was better at detecting resistance mediated by group 1 and 9 CTX-M ESBLs, with reduced sensitivity for the detection of resistance mediated by AmpC β-lactamases. Further research is required to investigate additional markers that could improve the detection of other β-lactamases. Both of these methods could be rapidly integrated into the diagnostic microbiology laboratory, thus reducing the time to effective narrow spectrum antimicrobial therapy, and potentially improving patient outcomes and reducing the spread of antimicrobial resistance.

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  • Comparing subpopulations of gonadotropin-releasing hormone (GnRH) neurons with viral mediated cell-filling

    Marshall, Christopher Joseph (2014)

    Undergraduate thesis
    University of Otago

    Gonadotropin-releasing hormone (GnRH) neurons are the central regulators of reproductive function in all mammals. The cell bodies of GnRH neurons are unique in that they are not confined to a discrete nucleus, but rather exist as a scattered continuum of cells throughout the basal forebrain. In rodent species, most of these GnRH neurons reside within three anatomical divisions of the brain: the medial septum (MS), the rostral preoptic area (rPOA) and the anterior hypothalamic area (AHA). Typically, these neurons are thought of as one large homogenous group, serving similar functions, however, recent anatomical and functional evidence suggests that this is not the case. One way to distinguish subsets of neurons is by their patterns of projection throughout the brain. Until now, the projection patterns of GnRH neurons have only ever been assessed for the population as a whole, due to the lack of ability to distinguish subdivisions from one another. The recent development of novel transgenic tools has enabled us to visualize GnRH neurons and their projections in their anatomical subdivisions of the MS, rPOA or AHA for the first time. An adenovirus containing a transgene for enhanced, farnesylated green fluorescent protein (Ad-iZ/EGFPf) was injected intracranially at stereotaxic coordinates for the MS (n=4), rPOA (n=6) or AHA (n=4) into female transgenic GnRH-Cre mice. Using this approach, Ad-iZ/EGFPf was specifically targeted to GnRH neurons in each region of interest, in order to “fill” these cells to the most distal ends of their projections. The first aim of this project was to assess how accurately GnRH neurons in MS, rPOA and AHA could be specifically targeted. Injections filled between 5 and 20 cells in most animals with accurate injection sites. In animals that received MS injections, more GnRH neurons in the MS were filled than in the rPOA (P < 0.05) and AHA (P < 0.05). Similarly, animals that received rPOA injections had more filled cells in the rPOA compared with the MS (P < 0.0001) and AHA (P < 0.001). In animals injected in the AHA there was no significant difference in the number of filled cells in the AHA compared with the MS and rPOA. In wild-type controls (n=3) and animals that received off-target injections (n=3), no filled GnRH neurons or projections were present. In the second aim of this project the distribution of projections from Ad-iZ/EGFPf filled GnRH neurons residing in the MS, rPOA and AHA were mapped. Across all animals, GnRH neuron fibre projections that were positive for GFP were found in 120 different regions of the brain, including nuclei, subnuclei and white matter tracts. These regions were found across several major brain divisions, in the hypothalamus, septum, thalamus, cerebral cortex, pallidum, striatum, amygdala, hippocampus and midbrain. The broad distribution of GnRH neuron projections highlights the diverse functions that these neurons are potentially influencing within the brain, as well as the power of the viral cell-filling approach used to visualize the full extent of these neurons. In the third aim, the projection patterns from GnRH neurons in the MS, rPOA and AHA were compared. Remarkably, 60% of the brain regions that contained fibre projections only did so from one or a combination of any two subpopulations of GnRH neurons, indicating that the projection patterns of these subdivisions is not homogenous. Notably, fibre projections in the vomeronasal amygdala originated exclusively from GnRH neurons in the AHA. Areas involved with olfactory processing likewise only received projections from MS and AHA GnRH neurons. Surprisingly, the largest division of GnRH neurons, the rPOA, had the most confined pattern of projection, but projected robustly to the median eminence suggesting a primarily hypophysiotropic role. For the first time, it has been possible to interrogate the projection patterns of anatomical subdivisions of GnRH neurons, which has revealed that they are far from homogenous. Overall, these results provide strong support for the existence of GnRH neuron subpopulations, highlighting that these neurons should be treated as similar but separate entities. Identifying GnRH neural subpopulations and delineating their respective roles could have wide applications, from increasing reproductive success in livestock, to teasing apart the ongoing mysteries surrounding infertility in humans.

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  • Understanding Batten disease: CLN5 expression in CLN6 deficient ovine neural cultures.

    McIntyre, Kristina (2014)

    Undergraduate thesis
    University of Otago

    Neuronal Ceroid Lipofuscinoses (NCL) are a group of debilitating and fatal neurodegenerative diseases of childhood resulting from progressive brain atrophy. The human and ovine variant late infantile CLN6 (ceroid lipofuscinosis protein) forms of NCL result from mutations encoding the endoplasmic reticulum transmembrane protein CLN6. Mutations encoding the soluble lysosomal protein CLN5 also cause an NCL in humans and sheep. The functions of these proteins are not understood, however previous research suggests a relationship between them (Lyly et al., 2009). In CLN6-/- ovine neural tissue, CLN5 protein expression was reduced (McIntyre, K.M., summer studentship unpublished observations, 2013/2014). The aim of this study was to investigate this potential interaction in the ovine model of CLN6 NCL. In CLN6-/- and CLN6+/- control secondary ovine cultures derived from primary ovine neural cultures, concentrations of CLN5 mRNA were assessed by relative quantitative polymerase chain reaction (qPCR). Protein expression was assessed by 3, 3 diaminobenzidine (DAB) immunocytochemistry, immunofluorescence and western blotting techniques. To detect CLN5 protein, cultures were transduced with CLN5 lentivirus (pCDH.MND.CLN5 (VSVG)). Proteasome inhibitor MG132 was applied for 2 and 4 hours to determine whether the reduction in CLN5 was due to ERAD (Endoplasmic Reticulum Associated-Protein Degradation). Absence of GFAP (Glial Fibrillary Acidic Protein) and MAP2 (Microtubule-associated protein 2) immunofluorescence in cultures indicated a cell population devoid of astrocytes and neurons respectively. Relative to ATPase and RPLPO (Large Ribosomal Protein) (M-value 0.936), no statistical difference in CLN5 mRNA concentration was found between CLN6-/- and control cultures (p = 0.68, n = 3). DAB immunocytochemistry showed low CLN5 protein expression in non-transduced cultures; reduced CLN5 protein expression was not evident. In immunofluorescence studies, no significant difference in relative fluorescent intensity was seen in transduced cultures (p = 0.75, n = 3). Western blot analysis of overexpressed CLN5 protein between CLN6-/- and control cultures was inconclusive. In non-transduced cultures treated with MG132, CLN5 expression was not detectable by western blotting. Data from transduced cultures are currently inconclusive. These results suggest that in this cell population, CLN5 mRNA is unaltered in CLN6 NCL. These methods may subsequently be translated to primary cultures as a foundation for on-going investigations into NCL protein interactions in ovine cultures.

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  • Multiple Dosing of RHDV VLP to Enhance the Anti-tumour Response

    Sadrolodabai, Yasmin (2014)

    Undergraduate thesis
    University of Otago

    Metastatic melanoma has a poor prognosis, with a median survival of 13 months after diagnosis. New Zealand has among the highest melanoma rates in the world with more than 2000 cases registered every year. Metastatic melanoma continues to be a challenging disease to treat, but recent immunotherapeutic approaches have demonstrated promising results. Our laboratory has developed a cancer vaccine using a virus-like particle (VLP) derived from Rabbit hemorrhagic disease virus (RHDV), which acts as a highly immunogenic scaffold to deliver tumour-associated antigens (TAAs) to the immune system. In vivo studies to date have shown that one or two doses of the VLP carrying gp100 (a melanoma-associated antigen) can specifically activate an anti-tumour response and trigger the formation of immunological memory against gp100 to prevent tumour recurrence. Administering multiple doses of a vaccine often achieves better responses in vivo, so the key aim of this study was to determine what effect multiple dosing of RHDV VLP coupled to gp100 would have on the anti-tumour response. RHDV VLP was successfully synthesized in a baculovirus expression system using Sf9 insect cells and subsequently used in a series of in vivo experiments. C57BL/6 mice were used in all experiments, receiving either 1, 3 or 6 doses of gp100-carrying VLP (n = 5 per group). An in vivo cytotoxicity assay showed that 3 doses of the VLP vaccine given 5 days apart elicited the highest levels of antigen-specific lysis in a target cell population, compared to a single dose or controls. Therapeutic tumour trials also showed that multiple dosing elicited a stronger anti-tumour response – mice that were first inoculated with B16 melanoma cells and then received 3 or 6 doses of the vaccine 5 days apart had the best overall survival, compared to controls and those that received a single dose. Mice that were tumour-free for 50 days were then rechallenged with B16 cells to assess the immunological memory response, and were found to have increased overall survival, with one mouse from the 3 dose cohort remaining tumour-free. The antibody response against the VLP in these mice was also examined via indirect ELISA. It was found that high levels of antibody against the capsid protein of the VLP were produced in all treated mice, which increased with each additional dose of the vaccine administered. A VLP uptake assay identified that the presence of antibody against the VLP can enhance the early uptake of VLP by DCs, but whether this has an effect on the anti-tumour response remains unclear. Overall, these preliminary results show that treatment involving multiple dosing of RHDV VLP coupled to the melanoma-associated antigen gp100 does somewhat enhance the anti-tumour response in vivo compared to treatment with a single dose, but the reasons for this need to be investigated further. Future work will focus on determining the role that the antibodies against the VLP play in the anti-tumour response, especially in relation to antigen-presenting cells, and further optimizing the vaccine for clinical trials.

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  • The involvement of mGluR1 hyper-activation in the progression of cerebellar ataxia in SCA1 mice

    Desai, Heena Nitin (2014)

    Undergraduate thesis
    University of Otago

    Spinocerebellar ataxia type 1 (SCA1) is an incurable, autosomal dominant progressive neurodegenerative disorder characterised by ataxia, progressive motor deterioration and selective neuronal loss in the cerebellum. It results from a CAG trinucleotide repeat expansion within the SCA1 gene product, ataxin-1. Metabotropic glutamate receptors type 1 (mGluR1) mobilise calcium from intracellular stores as part of their key role in cerebellar synaptic plasticity and motor learning and may be involved in the progression of ataxic symptoms. In this study, we use an 82Q transgenic mouse model of SCA1 where the CAG expansion is restricted to mouse cerebellar Purkinje neurons (PNs), the primary site of SCA1 pathology. This restricted expression in the PNs is achieved by tetracycline-controlled system. We use doxycycline to repress transgene expression at early (6 weeks) and mid (12 weeks) stages of the disease. Our aim is to use this model to identify potential mechanisms that contribute to the early stages of the progression of SCA1. We hypothesise that changes in mGluR1 expression underlie the progression from early pre-symptomatic to ataxia symptoms. Behavioural testing involved using an accelerating rotarod apparatus to assess motor performance and learning. 6 week old SCA1 transgenic mice exhibited mild ataxic motor symptoms (P < 0.01, two way ANOVA, n = 12) that progressed further at 12 weeks of age (P < 0.05, two way ANOVA, n = 7). Doxycycline treatment to repress the transgene expression prevented the mild ataxic symptoms at 6 weeks and reversed the progressively worse ataxic symptoms at 12 weeks of age. Immunohistochemistry experiments showed an increase in mGluR1 expression specifically in the molecular layer of 12 week old SCA1 mice (P < 0.05, two way ANOVA, n = 4). Doxycycline treatment did not prevent this enhanced expression of mGluR1, suggesting that enhanced mGluR1 expression may precede the onset of behavioural ataxia. Cell attached patch clamp recordings from PNs in SCA1 transgenic mice showed a decrease in instantaneous action potential (spike) firing frequency in comparison to PNs from FVB mice (P < 0.01, unpaired t-test with Welch’s correction, FVB: n = 3, SCA: n = 10). The application of Picrotoxin (PTX), a GABAA receptor antagonist resulted in: a non-significant trend towards an increase in instantaneous frequency and decrease in instantaneous firing irregularity of PNs from SCA1 mice. These data suggest a more powerful inhibitory influence in the cerebellar cortex of SCA1 mice compared with FVB mice. Overall, these results suggest that enhanced mGluR1 expression may disrupt PN calcium homeostasis leading to changes in PN firing and cerebellar output that drives the progression of SCA1. Our findings have important implications for the treatment of this rare but incurable human ataxia. The mGluR1 may be a potential therapeutic target for treating patients that are mildly symptomatic in the early stages of the disease.

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  • Phenotyping Langerhans cell like cell treated with microparticles from keratinocytes expressing human papilloma viruse16 E7 oncoprotein

    Zhang , Junda (2014)

    Undergraduate thesis
    University of Otago

    Cervical cancer in females is a worldwide health issue. High risk subtype of human papilloma viruses (HPV) are involved as a major risk factor. HPV oncogenes, E7 and E6 which are over-expressed in the host cells and promote malignant transformation. There is also evidence of HPVE7 involvement in immune modulation. Microparticles (MP), a type of small membrane fragments (0.1um ~1um) released by activated cells, have been implicated in suppressing immunity following interaction with antigen presenting cells. Our laboratory has previously reported up-regulation of microparticle secretion by HPV16E7 expressing keratinocytes. A component of this study is to advancing the understanding of the effect of keratinocyte microparticle on the phenotype of langerhans cells. Increasingly, roles for p53 in regulation of the immune response is being recognized. In this project, the effect of p53 status in langerhans cells on maintianing the immune phenotype will also be tested. The HPV16E7 oncoprotein expressing mouse keratinocyte (E7-PDV) cell line was established following lentiviral transduction, for microparticle (MP) production. Wild-type (p53+) Langerhans cell-like cells (LCLC) and p53 deficient (p53-) LCLC, which were generated from murine bone marrow cells resembling the phenotype of epidermal LC, were used in this study. The effect of MP on LCLC phenotype (CD40, CD86, E-cadherin and cytokine production) was investigated following 48 h co-culture. Compared to the control PDV cell lines, HPV16E7 expressing PDV were found to produce more MP, suggesting a poteintial role for oncoprotein HPV16E7 in inducing MP production. In response to lipopolysacharride stimulation, the up-regulation of inflammatory surface marker CD40 on p53- LCLC was abrogated compared to wild-type LCLC and E-cadherin expression was also found to be low compare to that of wild-type LCLC. This suggests, to some extend, a potential role for p53 protein in maintaining the proper immune phenotype of normal LCLCs. Moreover, comparing to control groups, the same amount of MP from E7-PDV found to have an inhibitory effect on CD40 expression and it also reduced inflammatory cytokine interleukin 12 productions in LPS-stimulated LCLC. The altered LCLC phenotypes subject to MP treatment had confirmed the hypothesis that HPV16E7 induced MP had a modulating effect on the phenotype of LCLC. Finally, the combined down-regulating effect on CD40 expression was observed when MP treatment was applied to p53- LCLC suggested that the MP effect on CD40 expression was p53 independent. The findings of phenotypic alteration of LCLC subject to MP treatment has unveiled the potential role of HPV-induced MP in immune supression that might provide a mechanism to contribute HPV persistence in the skin.  

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  • Structure, stratigraphy and metamorphism in the upper Hakataramea valley area, South Canterbury, New Zealand

    Fagan, Robert Keith (1971)

    Undergraduate thesis
    University of Otago

    vi, 73 leaves ; 30 cm. Includes bibliographical references. University of Otago department: Geology.

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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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  • 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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