407 results for Undergraduate

  • A role for alpha-7 nAChRs at cerebellar synapses

    Kim, Yeri (2011)

    Undergraduate thesis
    University of Otago

    The cerebellum performs a well-known motor control function but accumulating evidence supports its role in cognitive functions. The nicotinic cholinergic system alters cognition through its actions elsewhere in the brain but its role in cerebellar processing is not understood. However, expression of a subtype of nicotinic acetylcholine receptors is increased in the cerebellum of human autism patients. Here, we explore the expression, localisation and functional contribution of these receptors at cerebellar synapses. Longitudinal cerebellar sections (30 μm) were prepared from C57BL/6 and Swiss Webster male mice (28-48 days old). Positive immunoreactivity for α7 nAChR (Abcam) and established excitatory synaptic proteins, Vesicular Glutamate Transporter 1 (VGLUT1; Synaptic Systems) and Post Synaptic Density-95 (PSD-95; Abcam) was visualised using secondary fluorescent antibodies Alexa488 and Alexa594 (Invitrogen) and confocal microscopy. Sagittal cerebellar slices (250 μm thick) (C57BL/6 male mice, 21-31 days old) were prepared in artificial cerebrospinal fluid (aCSF) for whole-cell patch clamp recordings from Purkinje neurons (PNs). We recorded excitatory post-synaptic currents (EPSCs) following parallel-fibre (PF) stimulation in aCSF (containing 50 µM picrotoxin to block GABA-A receptors) before, during and after 15 minutes application of 10 nM methyllycaconitine (MLA; Tocris) a potent α7 nAChR antagonist. Series and input resistances varied by < 10% throughout the recordings. The α7 nAChRs were abundantly expressed throughout the cerebellar cortex where they overlapped with the PF excitatory pre-synaptic marker protein VGLUT1 (10 ± 1%) and more strongly with the excitatory post-synaptic marker protein PSD-95 (54 ± 3%) (p < 0.0001, n = 3 animals, Mann-Whitney U-test). Overlapping expression of α7 nAChRs was not evident at inhibitory synapses. Electrophysiological recordings revealed that 10 nM MLA reduced EPSC amplitude, compared with controls, by 30 ± 10% (p < 0.05, n = 5, ANOVA). This study shows for the first time that α7 nAChR expression in the cerebellum contributes to excitatory synaptic transmission at the important PF-PN synapse. Our findings have wider implications for how nicotinic cholinergic inputs influence cerebellar processing.

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  • Hydrogen Sulfide: Investigation into the Effects of Different H2S Donors on the Upregulation of Inflammatory Cytokines

    Campbell, Elizabeth Julia (2011)

    Undergraduate thesis
    University of Otago

    Endogenous hydrogen sulfide (H2S) has recently been described as the third gaseous mediator of interest. It has been proposed in the literature [1] , that H2S has a role in inflammation, through the induction of upstream signals, which activate inflammatory mediators such as TNF-α, IL-1β and IL-6, and through this method H2S can give rise to a pro-inflammatory response. This study, therefore, investigated the mechanisms by which H2S contributes to inflammation in vitro, through the use of two sulfide donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na2S), to look at the inflammatory mediator release from differentiated U937 monocytic cells. Through the use of RT-PCR and ELISA experimentation, analysis was carried out using the two sulfide donors at different concentrations and time points. The results found, that there was no difference between the two sulfide donors in the upregulation of inflammatory cytokines. It was also found that the effects of concentration and time were significant in ELISA experiments, but not so with the PCR experiments. However, when either of these compounds were added into an in vitro system using differentiated U937 cells, there was an upregulation of both protein and mRNA expression relative to the untreated control group.

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  • Understanding Postpartum Mood Disorders: Characterization of Brain Nuclei Activated by Prolactin Induced Signalling

    Wojciechowski, Isabella Teresa (2011)

    Undergraduate thesis
    University of Otago

    "November 2011". University of Otago department: Anatomy

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  • Characterisation of Arkadia's RING domain

    Wright, Joshua Denis (2011)

    Undergraduate thesis
    University of Otago

    The attachment of ubiquitin to proteins (ubiquitylation) is a post-translational modification that regulates a diverse range of biological processes, including signal transduction, transcriptional regulation and receptor down-regulation. Many ubiquitylated proteins are subsequently degraded by the 26S proteasome. A cascade of enzymes comprising an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme and an E3 ubiquitin ligase facilitate ubiquitylation. E3s, which mediate the transfer of ubiquitin from the E2 to the substrate, are largely responsible for specifying substrates. Abnormalities in the regulation of E3s are associated with many diseases, including neurodegenerative diseases and cancers. RING (really interesting new gene) domains are one of three domains that possess E3 activity. There are over 500 RING-type E3s making them the largest group of E3s in humans. However, the mechanism by which RING-type E3s facilitate the transfer of ubiquitin from the E2 to the substrate is poorly understood. Arkadia is an ubiquitin E3 ligase with a C-terminal RING domain. This project aimed to characterise Arkadia’s RING domain to gain a better understanding of how RING-type E3s facilitate ubiquitin transfer. Of particular interest was mechanisms that regulate activity and distinguish monomeric RING domains from dimeric RING domains.

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  • Effects of Orf Virus on the Inflammation Signalling Pathways of the Toll-­like, Interleukin-­1 and Tumor Necrosis Factor Receptor Families

    Turner, Ethan (2011)

    Undergraduate thesis
    University of Otago

    Poxviruses are complex, large double stranded DNA viruses that replicate in the cytoplasm of infected cells. The early phase of infection involves a two step uncoating process, firstly releasing viral core structures, then later viral DNA into the host cell before completion of their replicative lifecycle. Orf virus is the type species of the parapoxvirus genus, which infects primarily sheep and goats in addition to humans by zoonosis transmission. Resulting symptoms are characterised by rapidly developing acute pustular lesions. Orf virus is known to encode several immuno-modulatory factors to permit its replication in the presence of strong host innate and inflammatory responses. Genomic sequencing and bioinformatics analysis have identified an orf virus gene (057) encoding a structural protein containing a phosphatase motif, with close homology to vaccinia virus VH1. As structural proteins, both 057 and VH1 are predicted to become immediately available in the host cytoplasm soon after virion uncoating, where targeted dephosphorylation of intracellular signalling pathways can ensue. This study set out to investigate the potential effect of an orf viral phosphatase on cell signalling pathways through central transcription factor NFĸB, a key upregulator of pro-inflammatory cytokine and chemokine genes. Several classes of receptors, notably, toll-like receptor 4, interleukin-1 and tumor necrosis factor receptor families strongly induce NFĸB by virtue of adapter proteins which transduce signals mediated via phosphorylation and ubiquitylation events. These pathways converge on the IKK complex, which in turn phosphorylates IĸB-α, an inhibitory protein that sequesters NFĸB-p65, resulting in ubiquityn-proteasomal targeted degradation of IĸB-α effectively liberating NFĸB-p65 to undergo nuclear translocation. Assays were performed in HeLa cells to establish stimulatory dynamics and kinetics of NFĸB-p65 activation through induction with respective ligands, LPS, IL-1β, and TNF-α of the aforementioned receptors. Lysates were prepared, resolved by SDS-page and western blot analysis to determine endogenous levels of IĸB-α, and in addition to phosphorylated levels of NFĸB-p65. Initial results from preliminary assays showed rapid phosphorylation kinetics of NFĸB-p65 observable within 10 minutes following induction. The effects upon infection of cells with orf virus were then examined. The most notable result revealed an apparent temporal delay in the maximal levels of phosphorylated NFĸB-p65 induced by LPS and TNF-α when comparing mock and orf virus infected cells with a shift in the accumulation time of maximal levels of phosphorylated NFĸB-p65. Although definitive results of the involvement of orf structural protein 057 in this observation remain inconclusive at this stage, this effect could potentially be attributed to an orf virion-associated phosphatase due to the occurrence preceding viral de novo protein synthesis which is known to begin approximately 4 hours post infection.

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  • The Effects of Comvita Manuka Honey on Oral Mucositis in Patients treated with Radiation Therapy to the Head and Neck

    Begley, Aubrey (2011)

    Undergraduate thesis
    University of Otago

    Mucositis is a common side effect of radiation therapy to the head and neck region and one that causes considerable discomfort for patients. Mucositis compromises patients? ability to eat, drink and talk thus affecting patient health and quality of life. Currently there is no worldwide standard for the prevention or treatment of oral mucositis; care is limited to symptom control. Honey has anti-bacterial and potential anti-inflammatory properties and three trials overseas investigated its effect on radiation-induced oral mucositis. The three trials conducted in Malaysia, Iran and Egypt found that honey did reduce the incidence of severe mucositis in head and neck patients. This New Zealand mucositis trial aimed to verify the results from the three overseas trials by comparing the effects of manuka honey with current best practice on oral mucositis in head and neck patients. This report analyses a sub-set of the patients recruited to the trial; those from the Wellington and Dunedin departments. A total of 14 patients were recruited to the trial from these departments, nine recruited to the honey arm and five to the control arm. Four honey patients withdrew from the trial due to issues with the honey application and one patient withdrew from the control arm. Honey arm patients were given manuka honey and instructed to swirl 20mL (amended to 10mL) around their oral cavity and then swallow three times a day, these patients also had access to the standard of care. The control arm patients were treated with the standard of care alone. Patient assessment involved three times weekly mucositis scoring using the TROG multi-site mucositis scoring system, weekly weight and fortnightly quality of life assessment using a 65 question form adapted from the European organisation for Research and Treatment of Cancer questionnaires (EORTC QLQ-C30 and EORTC QLQ HN35). Patients were also asked to fill in food and drug diaries to assess changes in food intake and pain medication. Results showed that manuka honey was not well tolerated by our patient cohort. Patients complained of extreme nausea and stinging sensations in the oral cavity. The honey had to be diluted to be better tolerated (1:3 with another liquid). Contradictory to previous studies, preliminary analysis showed that manuka honey did not affect the extent of oral mucositis in the small cohort of New Zealand head and neck patients when taken in addition to current best practice.

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  • Bowel Bacteria and Dendritic Cells in Ankylosing Spondylitis

    Peyroux, Estelle (2011)

    Undergraduate thesis
    University of Otago

    Ankylosing Spondylitis (AS) is chronic inflammatory arthritis of spine and sacroiliac joints with further peripheral manifestations. AS is a relatively common rheumatic disease affecting 0.1- 1.4% of the population. Early symptoms commence generally in the second decade of life, these include fatigue and pain which are managed by adherence to medication. Inflammation can result in fusion of the vertebrae in the spine. Susceptibility genes identified encode HLA-B27 and ERAP1 proteins, both essential components in antigen processing for immune response. An animal model of AS induced by the hla-B27 gene (present in 95% AS patients) indicated enteric bacteria triggers AS-like symptoms. Dendritic cells (DCs) are professional antigen presenting cells that direct immune responses. We generated monocyte-derived DCs (MoDCs) from venous blood of AS (n= 6) and control group (n= 8) and stimulated the cells with a range of gut bacteria- Bacteroides fragilis, Yersinia enterocolitica, Campylobacter jejuni and Helicobacter pylori. We measured expression of antigen presentation molecules MHC-I and MHC-II and CD83, a marker of activated DCs, by flow cytometry. Analysis of cytokine secretion in supernatants was assessed by Bioplex immunoassay. In addition to this preliminary experiments with inhibitors to MyD88 and TRIF adaptor molecules were used to identify the possible involvement of toll-like receptors in activation of DCs to bacterial treatments. The AS donors had a trend of lower basal expression of MHC-I and MHC-II. Our results indicated the level of MHC-I and MHC-II expression is significantly different (p< 0.0001) between AS and normal groups. The normal group’s up-regulation of MHC-I in response to C. jejuni was significantly greater (p= 0.03) compared to the AS group (p=0.1). Differences in MHC-II expression between AS and normal groups were just outside significance levels (p= 0.08) for both B. fragilis CAS10 and H. pylori treatments. CD83, a DC maturation marker, was expressed at similar levels for all AS-associated bacteria. H. pylori induced CD83 expression significantly (p= 0.03) in the normal group however the AS group did not up-regulate CD83 expression significantly (p= 0.2). Cytokine analysis showed a trend of decreased IL-12 and IL-10 production in AS-associated bacteria particularly in B. fragilis CAS10 and Y. enterocolitica. Our control bacteria, H. pylori, inversely induced higher IL-1β, IL-10 and IL-12. From these results we hypothesize that abnormal signaling through innate pattern recognition receptor signaling pathways induces aberrant activation of DCs in AS patients. These DCs in turn may direct cells of the immune system toward an inappropriate or misdirected immune response. We speculate that a low IL-10 production by DCs may result in a dysregulation of the immune response.

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  • The actions of Activin C and its mechanism towards the progression of Prostate disease

    Lee, Kai Lun (2011)

    Undergraduate thesis
    University of Otago

    The Transforming Growth Factor β (TGF β) superfamily is involved with regulating many biological and physiological processes. Activins are members of the TGF β superfamily. Four activin subunits (βA, βB, βC and βE) have been characterized in mammalian cells. Activin A is a negative growth regulator in the prostate and dysregulation is associated with prostate disease. Activin βC was discovered only in the last decade. Recently it was shown that activin βC regulated the expression and bioactivity of activin A, and over-expression led to the development of prostate disease in mice aged 3 months. The current study hypothesized that activin βC functions locally to antagonize the activity of activin A, and over expression of activin βC can therefore lead to the formation of overt prostate disease in aged mice. We used overexpressing activin βC transgenic mice (TG) aged 12 months, and studied the dorsal and ventral prostate. There were clear signs of hyperplasia in the TG prostate epithelial cells, and some abnormally stained nuclei indicative of mucinous metaplasia. The prostate weights had also increased due to a significant increase in epithelial cells. Immunohistochemistry was then performed for PCNA and the results indicated no increase in cellular proliferation. However, a decrease in apoptosis was also evident as was a decrease in p-SMAD 2 signaling in the dorsal prostate. Thus supporting our hypothesis that increased activin βC antagonized the growth inhibitory effects of activin A and this was the mechanism underlying the alterations in proliferation and apoptosis. Pathway focused gene expression was then undertaken in the dorsal prostate to assess the mechanism by which over-expression of activin βC decreased proliferation, apoptosis, and SMAD 2 signaling. An increase in gene expression of activin βA and βB, the activin receptors, as well as the SMAD signaling molecules were evident. In addition, an increase expression of gene in the negative regulation of cell cycle was observed in TG samples, indicating that these cells were signaled for growth arrest. This validates the decreased PCNA and apoptosis data in the TG prostate sections. Finally, correlation with human prostate pathology was determined by assessing the staining of activin βA and activin βC in human prostate disease arrays. Activin βA was increased in all prostate disease whereas activin βC was only increased in benign prostatic hyperplasia. Therefore we conclude that activin C is associated with benign disease and not prostate cancer in mice and men.

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  • Establishment and validation of a telomere length assay

    Jodczyk, Sarah (2011)

    Undergraduate thesis
    University of Otago

    Telomeres are specialised structures, consisting of tracts of simple DNA sequence repeats that cap the ends of chromosomes. These structures play a vital role in maintaining the integrity of the genome and prevent the loss of genetic material that resides near the ends of the chromosome. As cells age and divide, the length of telomeric DNA sequences reduces and the progressive shortening of telomeres eventually leads to cell senescence and death. At the whole organism level telomere length appears to reflect cumulative lifetime stresses, and in humans, telomere shortening is associated with reduced longevity, many disease states including cancers, cardiovascular disease, mental illness, cognitive function and other phenotypes. The primary goals of this thesis were to establish and validate a method for measuring average telomere length, then apply it to examine a large longitudinal birth cohort for whom a great deal of data has been gathered over a period of 30 years. The telomere length assay is known as the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay (1). This method provides relative quantification of average telomere length in a genomic DNA sample, by measuring the number of telomere repeats (T) normalised to the single copy reference gene albumin (S), and expressed as a T/S ratio. Signficant technical issues were encountered during the establishment of the assay, and it was found that the choice of hot-start polymerase was essential to the success of the assay. This was because the design of the telomere primers (1) necessitated an overlap of 3bp on their 3’ terminus, which led to primer dimer formation with six of fifteen hot-start polymerases tested. Data acquisition and calculation of T/S ratios was also challenging, and required export of data from the real-time PCR platform used (Roche LightCycler LC480), because the machine software was unable to handle the required analyses. Once established, the reproducibility of the assay was tested and it was found that the assay variation was sufficiently low to enable the detection of differences in telomere length between individuals, using duplicate measures for each sample. Further validation was attempted using Southern blotting of genomic DNA for comparison, but the number of samples obtained was insufficient to validate the assay. As telomere length is known to decrease with age, the performance of the assay was tested by comparing telomere length for subjects of different ages (seven, 25-40 and 50 years). A difference in telomere length was found in the 50 year old age group compared to younger age groups (P=0.001, 0.017). In addition, mouse embryonic stem cell DNA, reported to have ultra-long telomeres, was also examined and these samples did show very long telomere length in both the MMqPCR assay and Southern Blot. The MMqPCR assay was then applied to the Christchurch Health and Development Study (CHDS) which is a prospective longitudinal health study of over 1000 New Zealanders that have been closely followed in intimate detail from birth until the present time. This cohort provides a novel opportunity to examine associations between telomere length and life stress in a longitudinal setting, which should provide superior outcomes to the more typical cross-sectional or retrospective studies that form the bulk of the telomere biomarker literature. Preliminary MMqPCR data from a subset of the CHDS samples (n=255) was generated. Attempts to test associations between general measures of stress and telomere length were underpowered due to the small initial sample set used and the complexity and heterogeneity of available stress measures. However, a significant association was observed between individuals with the shortest telomeres and the highest rates of dependence using the single stress measures of nicotine and alcohol dependence, and the collective measure of substance dependence (P=0.0124, 0.0362 and 0.0008 respectively). No significant difference in telomere length was found with the stress measure of illicit drug dependence, but this group is of a small size. With the observed variability seen in the subset of samples assayed from the CHDS cohort (T/S ratios of 0.04-10.48) and the associations made between individual stressors and telomere length, there appears to be great potential in the use of telomere length as a biomarker of stress.

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  • Neutrophil Activation in Inflammatory Bowel Disease

    Hoskin, Teagan Susan (2011)

    Undergraduate thesis
    University of Otago

    Abstract Inflammatory Bowel Disease (IBD) is an inflammatory condition in which neutrophils play an important role. Colonoscopy with biopsy is thought to be the best method for evaluating inflammation location, extent, and severity. However, the invasiveness of endoscopic examinations represents a strong limitation to their frequent use. Various studies have described faecal markers as powerful biomarkers of inflammation of the intestinal mucosa in patients with IBD. This thesis details the use of calprotectin and myeloperoxidase (MPO) as biomarkers of disease severity in patients with IBD, and aims to assess whether MPO is a superior biomarker to calprotectin. Abundant levels of both calprotectin and MPO are found in neutrophils. A total of 500 patients were recruited into the evaluation of Novel Biomarkers in Inflammatory Bowel Disease project cohort. However, only 100 of the initial stool samples from patients with or without IBD were used for this research. The samples had been stored at -80°C for up to one year. A short extraction procedure using 100 mg of faeces and approximately 4.9 mL of the appropriate extraction buffer was carried out. Levels of calprotectin and MPO protein were then measured by sandwich enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of MPO was also measured using 3,3’,5,5’-tetramethylbenzidine (TMB) as a reducing substrate. TMB forms a blue product when it reacts with peroxidase enzymes such as MPO. The resulting colour change was read on a microplate reader at a wavelength of 630 nm. Levels of calprotectin, MPO protein, and MPO TMB activity were significantly higher in IBD patients compared to controls. There were significant correlations between calprotectin, MPO protein and MPO TMB activity (p < 0.001). Levels of calprotectin and MPO protein correlated significantly with endoscopic disease severity in patients with CD (r = 0.487, p = 0.001, n = 41, r = 0.483, p = 0.001, n = 41, respectively) and UC (r = 0.677, p << 0.001, n = 35, r = 0.552, p < 0.001, n = 35, respectively). Consequently, both calprotectin and MPO protein were able to discriminate between IBD patients with inactive and high disease severity. However, MPO TMB activity failed to correlate with disease severity in CD and UC patients (r = 0.303, p = 0.054, n = 41; r = 0.258, p = 0.134, n = 35, respectively). The results obtained from this research show that calprotectin and MPO protein correlate strongly with each other. There was also a strong correlation between MPO protein and disease severity, and MPO could successfully distinguish between inactive and high disease severity in CD and UC patients. This suggests that MPO may be useful in the diagnosis and follow-up of patients with IBD. Although, the relationships between MPO TMB activity and disease severity in patients with CD and UC were not significant results were still very promising. This assay could prove to be a faster and more cost effective approach to aid in the diagnosis and follow up of patients with IBD in the future. However, further development and optimisation of the MPO ELISA and MPO TMB assay is required to validate the results from this research.

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  • The role of Δ122p53 in B cell development

    Roth, Imogen Margaret (2011)

    Undergraduate thesis
    University of Otago

    p53 is a crucial tumour suppressor, as evidenced by three important observations: p53 is lost or mutated in half of all human cancers, an inheritable p53 mutation predisposes to an early onset of multiple cancer types, and mice which lack p53 entirely all develop cancer. p53 is thus the ‘guardian of the genome’ (Lane, 1992), and prevents tumourigenesis by inducing the transcription of genes that carry out cell cycle arrest, apoptosis, DNA repair, and cellular senescence. p53 is normally present as a number of isoforms in healthy tissue, though some, including Δ133p53, are elevated in some cancer types. A mouse model of the Δ133p53 isoform, Δ122p53, has been shown to act as an oncogene and have a pro-inflammatory profile. Δ122p53 mice also have a distinctive B cell tumour spectrum largely composed of diffuse large B cell lymphomas, which was the starting point of this research project. The aims of this project were to elucidate the fundamental phenotypic differences between wild type and Δ122p53 mice, largely focusing on differences in cell surface marker expression. We also sought to determine the differences between wild type and Δ122p53 mice in their basal cell cycle profiles and p53-mediated response to DNA damage. In addition, we attempted to follow these differences as the mice aged to determine the potential onset of tumourigenesis in Δ122p53 mice. The results of this project show that Δ122p53 mice have a greater spleen mass than wild type mice, and the bone marrow and spleen have an elevated G2/M population and undergo less apoptosis than wild type mice. While none of the cell surface markers showed consistent differences in expression between wild type and Δ122p53 mice, there were several Δ122p53 mice that had alterations in the expression of the lineage cocktail and Ig κ light chain markers compared to their age matched wild types. The results of an earlier study were replicated, showing that Δ122p53 mice have elevated levels of serum IL-6, a cytokine implicated in many cancer types including those commonly found in Δ122p53 mice. This project also showed a reduction in serum IgG and IgM levels in Δ122p53 mice, suggesting that the Δ122p53 isoform is preventing B cell maturation. This project concludes that the Δ122p53 isoform affects p53 mediated responses and B cell development. We propose that the lineage cocktail and Ig κ light chain markers are potential markers of the tumour precursor population in Δ122p53 mice. Further studies are needed to confirm this, which we anticipate will demonstrate the role of the Δ122p53 isoform in B cell development. This will provide insight into an oncogenic p53 isoform in mice, closely related to the Δ133p53 isoform found in humans, and may provide clues to potential cancer therapies.

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  • Anti-Müllerian hormone: A missing link in prostate cancer?

    Masterton, Sarah Lorraine (2011)

    Undergraduate thesis
    University of Otago

    Prostate cancer is the most frequently diagnosed cancer in men in many countries including the USA, France, Germany and New Zealand (Health, 2002, Jemal et al., 2008, Ferlay et al., 2010). The growth of human prostate tissue and the development of prostate cancer is an intricate process maintained by the fine balance of a variety of factors including androgens, estrogens, stromal-epithelial interactions, nervous system input and growth factors (Massague, 1987, Moses et al., 1990, Ittman and Mansukhani, 1997, McVary et al., 1998, Giri et al., 1999, Khandwala et al., 2000, Renehan et al., 2004). Anti-Müllerian hormone (AMH) is one growth factor that may be involved in prostate cancer progression. Previously Segev et al. (2002) showed that AMH inhibits the growth of prostate cancer cells, and Loo et al. (2008) and Segev et al. (2002) showed that its type II receptor (AMHRII) was expressed by the prostate in mice and humans, respectively. Consequently, this project sought to begin to investigate the role of AMH in prostate cancer by examining the localisation and quantifying the expression of AMH propeptide, AMH and AMHRII in benign tissue, low-grade and high-grade cancer of the human prostate using human prostate tissue samples and cells. Immunohistochemistry was used to determine the localisation of AMH propeptide, AMH and AMHRII in human prostate tissue samples, which included various disease states. Double immunofluorescence was then carried out to confirm the location of AMH and AMHRII in human prostate tissue samples. Assessment of immunoreactivity and Western blot analysis were used to quantify the expression of AMH propeptide, AMH and AMHRII expression in human prostate tissue samples. The localisation and expression of AMH and AMHRII in normal and malignant stromal and epithelial human prostate cells was also determined using immunocytochemistry, Western blot analysis and assessment of immunoreactivity, respectively. The results of this project show that AMH propeptide, AMH and AMHRII are located in human prostate stroma and epithelium at all stages of disease. Immunohistochemistry and double immunofluorescence of human prostate tissue samples (which included benign tissue, low-grade cancer and high-grade cancer) exhibited AMH propeptide, AMH and AMHRII immunoreactivity of stroma and epithelium. Immunocytochemistry of normal and malignant stromal and epithelial human prostate cells also demonstrated AMH and AMHRII immunoreactivity. Furthermore, Western blot analysis of benign and malignant human prostate tissue samples and cells revealed immunoreactive bands that may correspond to cleaved and uncleaved forms of AMH and AMHRII. This project also found that there was no significant difference in expression of AMH propeptide or AMHRII in the stroma and epithelium of benign and malignant human prostate tissue samples, as indicated by assessment of immunoreactivity and Western blot analysis. However, there was evidence to suggest that AMH expression is increased in malignancy because AMH immunoreactivity was significantly increased in the epithelium of low-grade and high-grade human prostate tissue samples. Therefore, it seems that AMH may be involved in an autocrine/paracrine growth regulatory mechanism in the human prostate, as has been demonstrated in the human testes (Racine et al., 1998), brain motor neurons (Wang et al., 2005a), and female endometrium (Wang et al., 2009a). This implies AMH may function as a growth-inhibitor (as was indicated by Segev et al.'s (2002) findings on the effect of AMH on prostate cancer cells) or as a growth-stimulator depending on the cellular context. However, further research is needed to confirm the expression pattern of AMH and AMHRII in human prostate cancer progression, and to investigate the effect of AMH peptide on normal and malignant human prostate cell growth both in vivo and in vitro.

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  • The Cost Effectiveness of Implantable Cardioverter Defibrillator Therapy in New Zealand

    De Silva, Praveen Handunnethi (2012)

    Undergraduate thesis
    University of Otago

    The Implanted Cardioverter Defibrillator (ICD) has emerged as one of the strongest therapeutic options for patients at high risk of SCD [65-67]. Despite this its high cost, in relation to other therapies, has made its use controversial in many patient groups. The repercussions are particularly visible in smaller countries like New Zealand where limited resources and doubts on cost effectiveness have resulted in lower implantation rates. The aims of this Thesis were to provide an estimation of ICD cost effectiveness in a New Zealand context and label the strongest and weakest influencers of cost effectiveness. ICD costs and efficacy data, for Secondary prevention patients and Primary prevention patient with LV dysfunction, were collected through a review of international literature and analysis of New Zealand data collected from 2000-2007. These were then entered into a computer model and run over a twenty year lifetime. Model inputs included the efficacy of an ICD in secondary and primary prevention, measured through Life Years Gained, implant mortality, the cost of an ICD, costs of adverse events, lead/battery replacement costs and cost of annual follow up. Results showed the average cost effectiveness of an ICD was NZ $107,191/ LYG, when used in secondary prevention and $178,291/ LYG in primary prevention. The overall range of cost effectiveness across all model inputs was $85,799/ LYG to $207,301/LYG. The strongest factors influencing cost effectiveness were efficacy of the device in both groups, the cost of battery replacements, longevity of ICD leads, mortality rate of primary prevention patients and the annual cost of follow up. The weakest influencers of cost effectiveness included the costs of initial ICD and implant procedure and the cost of adverse events occurring from ICDs. These findings correlated well with previous studies conducted overseas. New Zealand showed a similar range of cost effectiveness, when compared to international trials. Our estimate remained outside the range of cost effectiveness thresholds considered economically attractive however when assessed alongside other therapies was comparative to renal dialysis for end stage disease. The results of this thesis encourage an increase in implantation rates to match other Westernised countries and detail areas that need to be addressed to further improve cost effectiveness.

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  • Histamine mediated neuropeptide gene expression in bovine chromaffin cells

    Kaur, Joydeep (2012)

    Undergraduate thesis
    University of Otago

    The adrenal medulla regulates the acute stress response and is itself controlled through a variety of signals, including neuronal, endocrine and paracrine factors. Additionally, recent literature suggests that adrenal medullary function may also be influenced by signals from the immune system, such as interleukin-1, interleukin-6 and tumor necrosis factor-α. Secretions from the adrenal medulla may in return influence the immune response thereby suggesting a bi-directional relationship between the immune system and the stress response. Recent microarray data from our laboratory have indicated that isolated bovine adrenal medullary chromaffin cells are also responsive to histamine, another immune-derived signal. Exposure to histamine results in marked changes to their neuropeptide gene expression. The aims of this thesis were to investigate this histamine-mediated neuropeptide gene expression by using real time PCR to validate the microarray results, to examine the signalling mechanisms involved and to explore any interactions between histamine and other known regulators of adrenal medulla. Cultured bovine chromaffin cells were incubated in absence or presence of histamine for 6-48h. The mRNA was extracted and analysed using qRT-PCR for the neuropeptides which were vasoactive intestinal peptide (VIP) and galanin. Additional experiments were performed using protein kinase inhibitors to examine the signalling pathways involved. Immunocytochemistry was also carried out in an attempt to localise the histamine-induced changes VIP protein expression. The results confirm histamine induces an increase in both VIP and galanin mRNA, the latter being mediated by protein kinase C. The pathway responsible for stimulating VIP mRNA was not positively identified but does not involve protein kinase A or C. Synergistic interactions were seen between histamine and other adrenal medullary activators (PACAP and IL-6) for VIP but not galanin mRNA. Immunocytochemistry however did not show any localisation of VIP, which was probably due to the VIP antibody being very non-specific. Thus whether the changes in neuropeptide gene levels result in alterations in protein expression remains unresolved. Given that VIP and galanin are both implicated in anti-inflammatory roles these results are consistent with the concept of a bi-directional relationship between stress response and the immune system.

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  • 17α-hydroxylase and the androgen-treated sheep ovary – a model for Polycystic Ovary Syndrome (PCOS)

    Caldwell, Aimée Sarah Lee (2012)

    Undergraduate thesis
    University of Otago

    Polycystic Ovary Syndrome (PCOS) is the most common cause of infertility in women of reproductive age, with an estimated five to ten percent of women worldwide affected by the endocrine condition. The aetiology of PCOS remains, for the most part unclear, however, androgens have been implicated in the development of PCOS with hyperandrogenism being one of the most consistent PCOS traits. The products of a complex steroid pathway, androgens, in particular androstenedione and testosterone, are found to be significantly increased in many PCOS patients. The aim of this study was to investigate the activity of a key enzyme in the steroid pathway, 17α-hydroxylase, in the prenatally testosterone treated (T-treatment) sheep ovary and subsequently measure levels of androstenedione present in blood samples taken from the jugular and ovarian veins. In addition to this, a histological study of follicular mapping provided useful insights into the effect of T-treatment on aspects of ovine follicular development. Sheep ovaries and blood samples were collected from pre-pubertal (5 month old) and pubertal (10 month old) animals. Serial sections were examined to study follicle numbers and morphology. A radioactive in situ hybridisation (ISH) was undertaken to determine tissue location and levels of 17α-hydroxylase (17α-OH) mRNA. Androstenedione levels in blood were determined using ELISA. Results showed that while body weight was similar in both T-treated and control groups, T-treatment was associated with an increase in mean ovarian weight in both pre-pubertal and pubertal age groups. T-treated animals experienced some masculinisation of the external genitalia, which has also been reported by other researchers using this model. In pre-pubertal animals, T-treatment was associated with a significant increase in the total number of growing ovarian Type 3-5 follicles within the ovary. When analysed individually, all eight categories of follicles in this study were found to be increased with T-treatment however these proved to be not statistically significant, with the exception of degenerate follicles where a 14-fold increase was seen in T-treated ovaries. Pre-pubertal T-treated ovaries had a significant accumulation of antral follicles ≥4mm, while pubertal T-treated ovaries displayed an increase in follicles ≥1mm. During the isolation of these follicles, it was found that T-treated follicles were more likely to be unhealthy or haemorrhagic than those from control ovaries. Thecal tissue measurements showed that Type 4 and small Type 5 follicles from T-treated ovaries had a significantly larger thecal tissue layer – the site of androstenedione synthesis. In situ hybridisation results showed that expression of 17α-OH mRNA in the theca was significantly increased in Type 4 T-treated follicles and appeared stronger in Type 5 T-treated follicles. Despite 17α-OH being a key enzyme in the production of androstenedione, these results did not translate into a detectable difference in blood levels of androstenedione in both pre-pubertal and pubertal animals. The results of this study suggest that elements of a PCOS phenotype are evident in pre-pubertal, prenatally androgenised sheep. Further study of this model may provide more answers as to the development of PCOS and it’s symptoms and the role that androgens play in the development of the syndrome and the consequent infertility.

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  • Phenotyping Asthma using an Electronic Nose

    Liley, Albert James (2012)

    Undergraduate thesis
    University of Otago

    Rationale: Asthma is a chronic respiratory condition affecting many millions of people worldwide. The recommended long-term preventative treatment for asthma is corticosteroid medication. Improvement in asthma symptoms in response to corticosteroids is not universal, and prescription to non-responsive patients is costly and potentially dangerous. The assessment of whether patients will respond to corticosteroid treatment is an important clinical problem. This study investigated the utility of a gas sensor array, the ‘Electronic nose’, in predicting response to steroid among asthmatics and in differentiating asthmatics from healthy controls using exhaled breath. The anti-inflammatory actions of steroids and the biochemical basis of steroid response are complex and may be better quantified by the electronic nose than by single biomarkers. In parallel with the assessment of steroid response a study was conducted on the ability of the electronic nose to predict sputum eosinophil counts among asthmatics. Eosinophil count is a predictor of steroid response and can be used as a guide to asthma treatment. Statement of problems: Can steroid-responsive and non-steroid responsive asthmatics be distinguished using electronic nose analysis of exhaled breath? Is sputum eosinophil count able to be predicted by electronic nose analysis of exhaled breath? Methods: 47 patients (27 asthmatics, 20 healthy controls) participated in the study. Asthmatic patients completed a two-week trial of oral prednisone. Asthmatics were classified as steroid responsive if FEV1 improved by 15%, PC20AMP improved by >300%, or ACQ (asthma control questionnaire) improved by >0.5 points over the steroid course. 16 asthmatics were steroid responsive and 11 asthmatics were steroid unresponsive. Breath samples were taken before and after the steroid course. A sputum sample was taken prior to the steroid course. Asthmatics were defined as eosinophilic if their sputum cell count contained more than 3% eosinophils. Healthy controls provided a single breath sample and sputum sample. Main results: Steroid responsive and non-steroid responsive asthmatics were unable to be distinguished either before or after the steroid course (no significant principal component differences, Mdistances0.2). Healthy controls were significantly differentiated from pre-steroid asthmatics (PC2, PC4, PC6, p=0.0090, 0.000060, 0.0090 respectively, M-distance=4.66,p=0.031) and less differentiated from post-steroid asthmatics (PC6, p=0.0016, Mdistance=3.80,p=0.16). A multilayer perceptron based predictive model for the comparison was associated with a cross-validation value (CVV) of 83% between controls and pre-steroid samples and 70% between controls and post-steroid samples. Improvement in ACQ (PC6, p=0.0041) and improvement in FEV1 (PC6, p=0.045) could be detected based on differences in samples before and after the steroid course. A principal component from pre-steroid asthmatics was strongly correlated with sputum eosinophil counts (coefficient=0.615, p=0.0082). Breathprints from eosinophilic and noneosinophilic asthmatics were significantly differentiated (PC4, p=0.0033, M-distance=2.00, p=0.0020). A predictive model for the comparison was associated with a CVV of 77%. Conclusions: Steroid responsive and non-steroid responsive asthmatics cannot be differentiated on the basis of exhaled breath analysis by electronic nose. Healthy controls and asthmatics can be significantly differentiated on this basis. Electronic nose readings are correlated with sputum eosinophil counts and eosinophilic and non-eosinophilic asthma can be significantly differentiated.

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  • Cardiac function and β-adrenergic receptor responsiveness in the isolated human diabetic myocardium

    Wang, Heng-Yu Simon (2012)

    Undergraduate thesis
    University of Otago

    The prevalence of type 2 diabetic mellitus is closely associated with cardiovascular complications. Diabetic patients with preserved ejection fraction (EF) are at high risk of developing heart failure. Until now, little is known about the etiology of the impaired cardiac performance in diabetic patients with preserved EF. Hence this study aimed to address this by investigating the cardiac function of these diabetic patients. We hypothesised that isolated cardiac muscles from diabetic patients will have reduced cardiac performance both at basal conditions, and after β-adrenoceptor (β-AR) stimulation mimicking a physiological stress response. Using isolated cardiac muscles obtained from right atrial appendages of patients undergoing coronary artery bypass grafting, functional characteristics of non-diabetic (n = 8) and diabetic myocardium (n = 6) were compared. (Samples from patients who had acute coronary artery disease and reduced EF (< 40%) were excluded.) Contractile and relaxation parameters of both cohorts were first determined under basal conditions, then in response to a β-AR agonist (dobutamine, 1x10-7 to 1x10-5 M). In addition, Langendorff-perfused isolated hearts from non-diabetic and diabetic ZDF rats were also implemented to compare cardiac function. Compared to non-diabetics patients, the developed force (Fdev) of diabetic human cardiac muscles was not different. However, a slower rate of maximum contraction (+dF/dtmax) and relaxation (-dF/dtmax), as well as prolonged relaxation times during the force-length relationship were observed in diabetic muscles (p < 0.05). A significantly longer relaxation time was also observed during force-frequency relationship in the diabetic muscles (p < 0.05). Moreover, diabetic cardiac muscles were less responsive to β-AR stress response, as shown by the reduced increase in Fdev, +dF/dtmax and -dF/dtmax, as well as smaller reduction in relaxation times. These observations were consistent with the results obtained from the isolated rat hearts. Ultimately, this study showed that diabetic patients with adequate systolic function, as indicated by preserved ejection fraction, suffer from diastolic dysfunctional characteristics and exhibit a reduction in the β-AR.

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  • The role of prolactin in pregnancy-induced changes in food intake

    Stenhouse, Natasha Danielle (2012)

    Undergraduate thesis
    University of Otago

    Pregnancy is associated with a significant increase in food intake. This occurs in order to supply the mother with sufficient energy to support both herself and the developing fetus throughout pregnancy and lactation when metabolic demand is high. This increase in food intake occurs despite an elevation in circulating levels of the appetite-suppressing hormone leptin. Hence, pregnancy is considered a state of leptin resistance. The hormone prolactin, the levels of which are significantly increased during pregnancy, is thought to be involved in increasing food intake during pregnancy. Exogenous administration of prolactin in rodents is associated with increased food intake and reduced response to leptin. As such, it is possible that the pregnancy-induced increases in prolactin may have the same effect. We hypothesised that prolactin acts centrally to mediate the changes in food intake that occur during pregnancy. Thus, the aim of this experiment was to measure food intake during pregnancy in mice that specifically lack prolactin receptors in the brain. To characterise food intake in these mice, food intake and bodyweight were measured daily in non-pregnant, pregnant and lactating neuron-specific prolactin receptor knockout mice and controls. This neuron-specific prolactin receptor knockout model has lost prolactin receptors throughout the brain and thus exhibits impaired central prolactin response. Immunohistochemistry for phosphorylated signal transducer and activator of transcription 5 (pSTAT5), a marker of prolactin receptor activation, was performed in order to assess the success of the knockout animal. Finally, because there appeared to be a difference in bodyweight in the non-pregnant knockout animals, non-pregnant mice were subjected to a fast and refeed protocol and then placed on a high fat diet in order to further characterise their food intake regulatory systems. Food intake increased significantly over pregnancy and was higher still during lactation in both animal groups. Bodyweight also increased over pregnancy, then remained stable during the lactation period. Food intake was significantly higher throughout pregnancy in the knockout animals compared with the controls. Unexpectedly, food intake and bodyweight were significantly higher in the non-pregnant knockout animals compared with the controls suggesting a basal difference in bodyweight regulation. Immunohistochemistry for pSTAT5 demonstrated significant, but not complete loss, of the prolactin receptor throughout the brain, specifically in the arcuate nucleus and medial preoptic area. There was no significant difference between the animal groups in the fast and refeed protocol. The high fat diet data demonstrated that the knockout animals on a high fat diet gained significantly more weight than the control animals on a control diet. The results obtained did not support our hypothesis that prolactin action in the brain critically mediates changes in food intake during pregnancy. However, interpretation of these results was complicated by the non-pregnant animal results and the incomplete nature of the knockout. It remains possible the remaining prolactin-responsive neurons in the brain may mediate prolactin action to stimulate food intake. This latter interpretation is supported by the changes in bodyweight in the non-pregnant knockout animals. Despite the failure to support or disprove our hypothesis, a significant step towards characterisation of this knockout model was made.

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  • St. Margaret's College : a study of women students in relationship to their environment.

    McClymont, Maris Laline (1940)

    Undergraduate thesis
    University of Otago

    With: Simplex Group Intelligence scale. (13 p.)

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  • The geology of the Round Hill area, upper Hakataramea Valley

    Falconer, Mark Lloyd (2000)

    Undergraduate thesis
    University of Otago

    viii, 79 [7] leaves ; 26 cm. Bibliography: l. [84-86] University of Otago department: Geology.

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