402 results for Undergraduate

  • The effect of fetal exposure to androgen treatment on steroid receptors in the developing sheep ovary

    Vasilic, Mina (2013)

    Undergraduate thesis
    University of Otago

    Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility affecting more than 100 million women worldwide, yet its etiology remains unclear. Fetal exposure to elevated androgen levels has, however, become increasingly implicated in the development of the syndrome. It is hypothesized that increased exposure to androgens during fetal development affects the steroid receptors in the ovary. The aim of this study was to use a sheep model to determine the effect testosterone treatment to pregnant ewes has on the steroid receptors, ERα, ERβ and AR in the fetal ovary. Control and testosterone treated ovarian samples were collected at 90 days of gestation. Ovarian sections were examined histologically to determine the cell types and tissue organization at this stage of development and to observe if this was affected by testosterone treatment. Immunohistochemical staining was used for the protein localization of each steroid receptor. A radioactive in situ hybridisation (ISH) was undertaken to determine the localization of the mRNA transcripts. A quantitative analysis of the mRNA expression levels of each receptor was done using real-time quantitative PCT (qRT-PCR) (utilizing the fluorogenic dye, SYBR green). The histological organization of ovaries in this study was similar to previous descriptions that have investigated fetal ovarian histology in sheep. The day 90 ovary contained ovigerous cords with well-pronounced cell types both within and outside the cords. No differences in histology were observed between the control and testosterone treated ovaries. Immunohistochemistry (IHC) provided visualization of clear nuclear staining for each receptor. The staining was predominately observed in the ovarian cortex, specifically within the cells of the ovigerous cords. No observable were seen differences in the patterns of protein expression between control and testosterone treated ovaries, nor were differences in the intensities of staining apparent. This was consistent with all three steroid receptors (ERα, ERβ and AR). In situ hybridization provided evidence for the presence of mRNA transcripts of all three steroid receptors within the ovarian tissue but this method did not have the resolution to define specific cell expression. These findings were similar in both control and testosterone treated ovaries. High quality RNA was isolated from the ovaries and statistical analysis of the results obtained from the qRT-PCR provided evidence that there were no differences in mRNA expression levels of the receptors in control and testosterone treated ovaries. The results in the current study provided evidence that testosterone treatment does not effect the distribution or expression of the steroid receptors ER?, ER? and AR in fetal ovaries and thus does not support the hypothesis. This study provided further insight into the potential function of these steroid receptors in ovarian development. Specifically this highlighted their importance in the development of pre-granulosa cells from the ovarian surface epithelial cells, oogonia and their subsequent association to form primordial follicles.

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  • Immune suppression and cutaneous squamous cell carcinoma tumour biology

    Seddon, Annika (2013)

    Undergraduate thesis
    University of Otago

    Cutaneous squamous cell carcinoma is a non-melanoma skin cancer that is among the most common cancers capable of metastasis. The majority of cutaneous squamous cell carcinomas (cSCCs) are easily treated by simple surgical excision, but there exists a subset of cases (approximately five percent) that will become invasive and metastatic. Currently, there are no clinically relevant biomarkers to identify potentially aggressive cSCC, and the biological mechanisms remain unclear. Previous work by our laboratory found high levels of myeloid-derived suppressor cells (MDSCs), particularly the granulocytic subpopulation, in the circulating blood of renal transplant patients and patients with cSCC. MDSCs are a mixed group of immature immune cells (including a high proportion of immature granulocytes) that have been shown to inhibit anti-cancer immunosurveillance and hence facilitate tumour progression. This study analysed circulating and tumour infiltrating immunoregulatory cell populations (MDSCs, neutrophils and lymphocytes) in the blood and tumour samples from patients with cSCC who were not on immunosuppressive medications (non-immunosuppressed, n = 29) and immunosuppressed patients with cSCC (n = 18). The frequencies of MDSCs in the immunosuppressed group were significantly higher compared to the non-immunosuppressed group when analysed as a whole. When we split the non-immunosuppressed patient group by tumour stage (high-stage tumours (n =8) and low-stage tumours (n = 21)), we found that frequencies of total MDSCs and granulocytic MDSCs were significantly higher in the blood of both the high-stage tumour group and the immunosuppressed group compared to the low-stage tumour group. When the tumours of these patients were analysed, increased peritumoural and intratumoural levels of CD66b positive neutrophils as well as higher ratios of CD66b positive neutrophils to CD8 positive lymphocytes were observed as tumour stage increased. Moderate to strong correlations between levels of tumour-associated neutrophils and lymphocytes and levels of circulating MDSCs, were also observed. A clinical audit of cSCC patients treated in the Department of Plastic Surgery, Christchurch Hospital (2009 - 2011) was performed to investigate associations among immunoregulatory cell populations, high-risk tumour characteristics and survival in a larger cohort (n=168). Immunosuppressed patients (n = 39) had higher levels of circulating neutrophils and lower levels of lymphocytes in their blood compared to the non-immunosuppressed patients (n = 129). When the non-immunosuppressed patients were analysed independently, patients that had neutrophil counts of over 4.5 x 109/L, compared to those with lower neutrophil counts, had a significantly higher proportion of tumours that were greater than five millimetres in thickness (p = 0.03), a Clark level of five (p = 0.02) and had higher overall tumour stage (p = 0.04). Furthermore, a thickness of greater than five millimetres was the most significant predictor of overall survival in non-immunosuppressed patients, a characteristic that is given relatively little importance under the current staging system. None of the circulating immune cell populations investigated were associated with overall survival, however neutrophil levels in circulation showed some association with advanced tumour stage (p = 0.04). This is the first study to investigate MDSCs, neutrophils and lymphocytes with clinical information in patients with cSCC. Although patient numbers were small in the current study, and survival and recurrence data for this cohort is beyond the scope of this thesis, this work will be ongoing. As part of this ongoing research, the mechanisms by which neutrophils and granulocytic MDSCs might contribute to tumour progression should be explored. This research will lead to a greater understanding of the underlying pathology of aggressive cSCC, and may provide more informative biomarkers to help identify patients with high-risk cSCC.

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  • Voluntary Tremor Suppression in Parkinson's Disease

    Ha, William Anthony (2013)

    Undergraduate thesis
    University of Otago

    Parkinson’s disease (PD) is a common degenerative neurological disorder, and resting tremor is one of the main symptoms of this disease. It has been observed that some patients with Parkinsonian rest tremor are able to suppress their tremor voluntarily with mental concentration or by focusing attention on the affected limb. This process is not well understood and this study aims to describe and assess voluntary tremor suppression in patients with PD, as well as to identify the critical cortical or subcortical regions activated during this process. Methods: Nine participants with tremor-dominant PD were recruited for this study. These patients had unilateral rest tremors of the upper limb and were able to consciously stop their tremor for a period of time. Each patient was assessed using the Unified Parkinson’s Disease Rating Scale (UPDRS), movement tracking and functional imaging. Physical characteristics of the tremor such as amplitude and frequency were measured using a 3-D Polhemus Liberty electromagnetic movement tracking in the MoVELab. Functional imaging was undertaken using functional magnetic resonance imaging (fMRI) in a 3.0 Tesla scanner, with functional data collected with a standard T2 weighted MRI sequence along with T1 weighted 3-D anatomical data. Results: The extent of voluntary tremor suppression differed between the participants with some being able to suppress reliably for long periods of time, and others unable to do so consistently. Participants had slight to moderate tremors according to the UPDRS. The majority of participants described their method of suppression as concentrating on the affected limb and/or focusing on relaxing the limb. Movement tracking confirmed what was observed, with variation in tremor amplitude, and the extent of suppression. FMRI showed differing areas of activation involved in tremor suppression amongst the participants. Activated areas were generally contralateral to the tremor, and were widespread, including parts of the primary motor cortex, superior parietal lobule, supramarginal gyrus and middle frontal gyrus. Conclusion: This study was the first attempt at describing the process of voluntary tremor suppression in PD. The differing methods the participants used to suppress their tremor were recorded and described, and objective measures of the suppression taken. Functional imaging revealed a number of areas involved in tremor suppression.

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  • Predicting Steroid Responsiveness using Exhaled Nitric Oxide

    Rawcliffe, Laura Kay (2013)

    Undergraduate thesis
    University of Otago

    Background: In susceptible individuals, exercise can be a potent trigger of bronchoconstriction resulting in symptoms which are commonly diagnosed as exercise-induced asthma (EIA). Empiric trials of inhaled corticosteroids (ICS) are often employed as treatment in preventing EIA symptoms, yet there is marked variability in treatment response. This heterogeneity may be explained by the differing pathological mechanisms which predispose to exercise-induced bronchoconstriction (EIB). These include airway inflammation, which itself is heterogeneous. Patients with eosinophilic airway inflammation, compared to noneosinophilic, demonstrate greater protection against EIB with regular ICS therapy. Additionally, the degree of sputum eosinophilia correlates with the severity of EIB. Exhaled nitric oxide (FENO) is a non-invasive surrogate biomarker for eosinophilic airway inflammation. Increased levels of FENO are associated with the presence and severity of EIB, and in patients with non-specific respiratory symptoms can predict steroid responsiveness. Investigating the potential ability of FENO measurements to identify patients with EIA symptoms likely to have a favourable response to ICS would reduce empiric prescribing, and is therefore clinically important. Hypothesis: Patients with EIA symptoms and high FENO are more likely to respond to ICS treatment, compared to those with low FENO Aims: 1. Calculate the predictive utility of FENO measurements in patients with EIA symptoms and airway hyper-responsiveness (AHR) for response to ICS 2. Compare the effectiveness of ICS in the management of patients with EIA symptoms with low versus high FENO 3. Confirm that pre-treatment measurement of FENO is an important way to approach the management of patients with EIA symptoms. Methods: Patients with EIA symptoms and AHR to mannitol and/or exercise challenge were enrolled. A randomised, crossover, placebo-controlled trial of budesonide 800μg b.d was undertaken. Each treatment period was one month in duration, with an intervening two week washout. Patients were allocated to a low or high FENO group based on their pre-treatment off steroid measurement, using 45ppb as the cut-point. The following endpoints were measured at baseline and after each treatment arm: FENO, spirometry, AHR to mannitol and exercise challenges, Asthma Control Questionnaire score, and Borg Dyspnoea Score. Results: Forty five symptomatic patients were screened and seventeen fulfilled the eligibility criteria. FENO had a high predictive utility for steroid responsiveness (ROC AUC=0.833). The optimum cut-point for FENO to predict steroid responsiveness in this population was 41.0ppb with corresponding sensitivity, specificity, positive and negative predictive values of 78.6%, 66.7%, 91.7% and 40.0% respectively. Analyses by FENO stratification revealed there were no significant improvements in any of the measured endpoints following budesonide in the low FENO group, except for FENO itself (33.4ppb vs. 17.6ppb; p=0.006). In contrast, patients with a high baseline FENO, demonstrated a reduced FENO (76.5ppb vs. 36.1ppb; p=0.007) and reduced AHR to mannitol (PD15 to mannitol increased; 193mg vs. 443mg; p=0.010). Improvements in asthma control score and AHR to exercise challenge approached, but did not reach, significance (p=0.071 and 0.063 respectively). Conclusions: Patients with a high pre-treatment FENO demonstrated clinical improvements following treatment with budesonide, whereas those with a low FENO showed no improvement. Pre-treatment FENO measurements may be used to predict whether patients with EIA respiratory symptoms will respond to ICS treatment. These data support the use of this simple test to aid clinical decisions in the management EIA.

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  • The effect of riboceine on lipoprotein(a)

    Kader, Tanjina (2013)

    Undergraduate thesis
    University of Otago

    Cardiovascular disease (CVD) is one of the leading causes of death worldwide. Atherosclerosis is characterized by the progressive formation of lipid plaques within blood vessel walls which eventually occlude the vessel lumen and block blood flow. Elevated concentrations of lipoprotein(a) [Lp(a)] are an independent risk factor for developing atherosclerosis and CVD. Lp(a) consists of a low-density lipoprotein like particle that is covalently linked to a unique glycoprotein apolipoprotein(a). Lp(a) possesses both atherogenic and thrombogenic properties. Lp(a) accumulates oxidised phospholipids (OxPL) from cell membranes of other lipoproteins and promotes lipid deposition and inflammation in the artery. Unfortunately, to date, there is no effective therapy available to lower Lp(a) or reduce these atherogenic properties of Lp(a). Riboceine is a cysteine analogue designed to increase synthesis of the antioxidant glutathione (GSH) by releasing L-cysteine, the precursor of GSH synthesis. This project aims to evaluate the potential of riboceine as a novel therapy to lower Lp(a) and increase GSH synthesis. As GSH is an essential cofactor for the reduction of OxPL, it may also reduce the OxPL content of Lp(a); thereby reduce atherogenicity of Lp(a). As OxPL are involved in the regulation of cholesterol levels, riboceine may also alter plasma cholesterol levels. This study showed that riboceine is not an ideal intervention for lowering Lp(a) levels as it reduces Lp(a) formation only by 15-20% and only at very high concentrations. Riboceine was shown to have no effect on already formed Lp(a). Riboceine showed a trend of increase in both plasma and liver GSH levels in vivo, indicating that riboceine may act as a promising therapeutic to increase GSH levels. Moreover, riboceine showed a trend of decrease in total plasma cholesterol levels in vivo. These results suggest that riboceine may increase GSH levels which could alter Lp(a) atherogenicity as well as reduce cholesterol levels, thus lowering the risk of atherosclerosis and heart disease. Future studies will require an increased sample size for both GSH and cholesterol studies in vivo. In addition, it will be required to measure cell membrane and Lp(a) OxPL content to confirm our hypothesis.

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  • Inhibiting the pro-inflammatory cytokine MIF

    Spencer, Emma Sue (2011)

    Undergraduate thesis
    University of Otago

    Macrophage migration inhibitory factor (MIF) is a highly conserved regulatory cytokine, known to exert pro-inflammatory effects. MIF biological activity is primarily regulated via interaction with the CD74 receptor. MIF also exhibits catalytic tautomerase activity via a conserved N-terminal proline residue. Interestingly, this proline is located within the region of MIF that binds to CD74, and small molecules designed to dock and inhibit tautomerase activity have been recognized to interfere with receptor binding. MIF knockout mice are protected in mouse models of rheumatoid arthritis, cardiovascular disease, sepsis, inflammatory bowel disease and cancer. Anti-MIF antibodies also display efficacy in these models. The first small molecule MIF inhibitor, ISO-1, also shows biological activity in disease models. Isothiocyanates are a new class of MIF inhibitors that have recently been discovered. Isothiocyanates are a class of phytochemicals, which have been shown to exhibit anti-cancer and anti-inflammatory activity. PEITC is able to covalently modify the N-terminal proline of MIF, resulting in the complete loss of catalytic tautomerase activity. This study investigated a structure-activity relationship of isothiocyanate inhibition of MIF tautomerase activity. PEITC had an IC50 value of 1.55μM for MIF tautomerase activity in Jurkat T-lymphoma cells, and an LD50 value of 8.4μM. This was 10-fold more effective than ISO-1 at inhibiting MIF tautomerase activity. Increasing the alkyl chain length of isothiocyanates did not greatly influence inhibitory capacity, although PHITC had an IC50 value of 4.2μM compared to that of BITC, with an IC50 value of just 0.54μM. Chlorine, amino and hydroxyl constituents showed minimal effect on the inhibitory capacity, however their cytotoxicity was significantly reduced, with LD50 values of 59μM for OH-PEITC and 60μM for NH2-PEITC. Aliphatic isothiocyanates varied widely in their inhibitory capacity. AITC was the most potent inhibitor with an IC50 value of 0.25μM, while SFN was less effective with an IC50 value of 2.2μM. Both showed very low cytotoxicity, with LD50 values above 100μM. Both AITC and PEITC were shown to inhibit the immunoreactivity of cellular MIF with a monoclonal antibody in a concentration dependent manner, while ISO-1 was shown to have no effect. Since cytokine inhibition therapy is widely considered to have great medicinal prospects, selective targeting of MIF with specific chemical inhibitors, such as isothiocyanates, might offer new therapeutic avenues for these disorders. This study lays the foundation for further drug design efforts.

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  • Characterization of Alkyltriphenylphosphonium Cations and Their Interaction with Bacterial Cells

    Dunn, Elyse (2011)

    Undergraduate thesis
    University of Otago

    Tuberculosis (TB) is a difficult to treat disease caused by the bacterium Mycobacterium tuberculosis. M. tuberculosis is able to shut down its metabolism in response to diverse environmental cues and enter a stage of non-replicating persistence that makes it resistant to many frontline TB drugs. This is further compounded by the “walling off” of M. tuberculosis in granulomatous lesions during infection. New drugs and strategies are in desperate need to combat TB, which currently kills two million people a year. The goal of this thesis was to explore the chemotherapeutic potential of alkyltriphenylphosphonium (alkylTPP) cations; lipophilic positively charged molecules known to accumulate at biological membranes in response to the membrane potential. To address this goal, a structure-function analysis of alkylTPP cations was carried out against several clinically important microorganisms: Mycobacterium tuberculosis, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli; and the non-pathogenic Mycobacterium smegmatis. In addition, we determined if these compounds were toxic to murine RAW macrophages. A series of alkylTPP cations ranging in lipophilicity were characterized, where their toxicity against each cell type was used as a measure of effective accumulation. AlkylTPP cations were shown to be highly toxic to bacteria and mammalian macrophages at concentrations of as low as 1 – 2 μg/mL, where this toxicity increased with respect to lipophilicity. This was deemed an important structure- function relationship for their efficacy. The alkylTPP cation Aa10 was shown to be an effective inhibitor of all bacterial strains used in this study, where it elicited bactericidal killing in M. smegmatis and collapsed the membrane potential. On the basis of these data it is proposed that Aa10 inhibits bacterial growth in a bactericidal manner by dissipating the membrane potential. At toxic concentrations this is due to the accumulation of positively charged alkylTPP cations in the cytoplasmic membrane, where the specifics of this mechanism are yet to be defined. This is validated by the ability of Aa10 to effectively inhibit the anaerobic growth of E. faecalis JH2-2, implying that the action of Aa10 is not dependent on an electron transport chain. Future work will focus on investigating other structure- function relationships that attribute to effective alkylTPP cation toxicity. This includes the addition of different substituents around the central phosphonium ion and variations of the central cation (such as ammonium). Defining these relationships is key in developing alkylTPP cations for a therapeutic application.

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  • 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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  • 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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  • 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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  • Prevalence and characteristics of acute headaches and dizziness in mild head injury

    Cowley, Tessa Katarina (2012)

    Undergraduate thesis
    University of Otago

    Little research has been undertaken into the prevalence and characteristics of the acute symptoms following mild head injury, especially within the first month. This study recruited 75 mild head injury patients identified by the Christchurch Hospital Emergency Department and followed them up at 1 week, 1 month and 3 months via questionnaire over the phone. Participants described their symptoms in their own words before being asked more specific questions by the researcher, who then determined which type of headache and dizziness were present, along with the prevalence of other symptoms. The major causes of these mild head injuries were physical altercation (22, 29%) and sports injury (22, 29%), with the remainder due to household accidents (7, 9%), traffic accidents (6, 8%) and “other” (18, 24%). Forty-seven participants were male and 28 were female, and the response rate was 56.4%. The number of participants with headaches decreased with each follow up, 55 (73%) on presentation, 37 (49%) at one week, 22 (32%) at one month, and 11 (27%) at three months. The most common type of headache was consistently tension-type (one week, 35%, one month, 26%, three months, 36%), with the amount of other types varying at each follow up. The number of participants with dizziness was 14 (19%) on presentation, 18 (24%) at one week, 9 (13%) at one month and 3 (6%) at three months. The number of participants whose dizziness occurred with position change at one week was 12 (16%), at one month was 7 (10%), and at three months was 3 (6%). The data provided in this study contributed to the literature surrounding mild head injury and the acute symptoms, especially headaches and dizziness, following it. The fact that tension-type headaches are the most common type of acute post-traumatic headache is of great interest, as is the information that the majority of all headaches have resolved by the three month follow up. Novel information was also reported for dizziness. The prevalence of dizziness increased from presentation to the one week follow up and the resolution differed between dizziness occurring with position change and dizziness occurring without position change, with dizziness occurring with position change being the only type to persist past the 3 month follow up. An extension of this study with greater numbers is indicated.

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  • Body Weight Regulation During Pregnancy in the mouse

    Fieldwick, Diana Maria (2013)

    Undergraduate thesis
    University of Otago

    Leptin is an adipose-derived hormone that acts in the hypothalamus to regulate energy homeostasis by decreasing appetite and increasing metabolic rate. During pregnancy, food intake and fat deposition increases, despite elevated leptin levels, suggesting a state of leptin resistance. The aim of this thesis is to determine whether mice become resistant to leptin during pregnancy. This would facilitate use of transgenic animals to elucidate the mechanism of pregnancy-induced leptin resistance. To examine this we looked at food intake in response to leptin administration, and the effect of leptin on the expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in the hypothalamus of pregnant mice. To establish normal eating patterns throughout the mouse reproductive cycle, food, water intake and body weight were monitored during the estrous cycle, pregnancy and early lactation. To investigate whether pregnant mice remained responsive to the satiety effects of leptin, food intake was measured after an intraperitoneal (i.p.) leptin or vehicle injection in fasted mid-pregnant (day 13) mice and compared to non-pregnant (diestrous) mice. Leptin treatment significantly reduced food intake in the non-pregnant mice while no effect was seen in the pregnant mice. This indicates that mice become resistant to the appetite suppressant effects of leptin during pregnancy. To quantify hypothalamic leptin resistance in the pregnant mouse, pSTAT3, a marker of leptin signal transduction, was measured in mid-pregnant and non-pregnant mice following i.p. administration of leptin. In all regions examined in both the pregnant and non-pregnant mice, leptin treatment induced significant pSTAT3 expression compared to vehicle treatment. However, in the ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH), leptin treatment induced significantly less pSTAT3 expression in pregnant compared to nonpregnant mice. In contrast, in the arcuate nucleus there was no significant difference in expression of pSTAT3 following leptin treatment between pregnant and non-pregnant mice. These data support the hypothesis that pregnancy in the mouse is a leptin resistant state associated with impaired leptin-induced signal transduction, involving the JAK/STAT pathway, specifically in the VMH and DMH. This is an adaptive maternal response to provide sufficient energy stores for the metabolically demanding tasks of pregnancy and lactation. Maternal obesity is a significant health concern that is exacerbated by this physiological adaptation with considerable side effects for both mother and baby. Gaining a better understanding of pregnancy-induced leptin resistance provides an opportunity to help in the management of this topical issue.

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  • The Paediatric Outpatient Coding Pilot Study

    Kerr, Neal Alexander Moealia (2013)

    Undergraduate thesis
    University of Otago

    In New Zealand, recent literature would suggest that children with chronic health conditions and disabilities, and their families are in need of greater support. The primary aims of this thesis were to; determine whether the information currently available on these children was sufficient for service planning; explore the options for information collection; assess whether paediatric outpatient diagnostic coding could be used to inform planning and funding decisions for this group. These aims were addressed by examination of the national information sources on children with chronic health conditions and disabilities and assessing their ability to inform population Health Needs Assessment for service planning within District Health Boards. The data within the National Non-Admitted Patient Collection was then analysed in detail to determine whether it could yield findings on children with chronic health conditions and disabilities useful for service planning. Finally, a pilot study was undertaken to determine whether clinician based diagnostic coding of paediatric outpatient services comparing the use of ICD-10-AM and SNOMED-CT to yield data on these children is suitable for service planning and funding decisions. Review of the available information on children with chronic health conditions and disabilities in New Zealand confirmed that these children and their families are in need of greater support but that there are no sources currently in use which are suitable to inform service planning and funding decisions at the local District Health Board level (e.g. Timely, Comprehensive, Consistent, Regionally Specific, Diagnostically Defined and with associated Demographic Elements). The National Non-Admitted Patient Collection was shown to have many of the elements required to inform planning and funding decisions but lacked diagnostic information. Preliminary analysis of the Paediatric Outpatient Coding Pilot Study findings from the Southern District Health Board indicated that on average clinician based coding was faster with SNOMED-CT (1.1minutes) per visit than ICD-10-AM (1.6 minutes) or than Trained Clinical Coders using ICD-10-AM (3.1 minutes). Trained Clinical Coders using ICD-10-A M were able to assign diagnostic codes to the most common conditions more consistently than clinicians using ICD-10-AM or SNOMED-CT. Overall, 85 percent of participating clinicians either supported or strongly supported the national routine collection of paediatric outpatient diagnostic codes. A number of conclusions were evident from the research. The information currently available on children with chronic health conditions and disabilities is adequate to demonstrate that they need greater support. However, the information is not sufficient to inform local service planning to address this need. The preliminary findings of the Paediatric Outpatient Coding Pilot Study would suggest that, with further development in IT systems particularly, paediatric outpatient diagnostic coding combined with the National Non-Admitted Patient Collection, would constitute an ideal approach to capturing information on children with chronic health conditions and disabilities suitable to inform planning of local and national level health support services.

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  • An in vivo investigation into the morphology of whole GnRH neurons

    Neill, Angus (2013)

    Undergraduate thesis
    University of Otago

    The ability to pass on genetic information through reproduction represents nature’s basic element of existence. Central to the control of reproduction is gonadotropin-releasing hormone (GnRH), a neuropeptide synthesized and released from GnRH neurons in the mammalian hypothalamus. Our understanding of these neurons has traditionally been limited to examination of the cell body and very proximal dendrite. However, recent technological advances have revealed that GnRH neurons possess extensive dendrites with numerous morphological features that aid in our understanding of their physiology. Despite this, current research has been limited to in vitro brain slice preparations that do not always encompass the entire extent of GnRH neurons and limit our ability to investigate specific in vivo physiological time points. In light of this, the first aim of the present study was to image the morphology of whole GnRH neurons in optically cleared, adenoviral injected mouse brains. We optimized a technical approach for studying GnRH neurons that combines in vivo cell-filling of GnRH neurons with optical tissue clearing and long working distance confocal microscopy. This novel approach utilized adenoviral vector-mediated expression of farnesylated enhanced green fluorescent protein (Ad-iZ/EGFPf) to “fill” whole GnRH neurons in vivo via a cranial injection. Following Ad-iZ/EGFPf injection, perfusion fixed mouse brains were made transparent by immersion in Scaleview-A2, a chemical solution that reduces the light absorbance of tissue, enabling imaging of deeper structures in thick tissue samples. Tissue absorbance was reduced from an average of 2.060 ± 0.09au in uncleared samples to 0.4914 ± 0.08au in Scaleview-A2 treated samples, at a wavelength of 490nm. In addition, we found injections of Ad-iZ/EGFPf to the rostral pre-optic area resulted in the expression of EGFPf along the entire cell membrane of a small population of GnRH neurons. After brains had been cleared for 14 days, long working distance confocal microscopy enabled visualisation of somal, dendritic and axonal compartments of GnRH neurons that could be traced on a millimetre scale, a feat that has challenged scientists in recent years. We were also able to identify microscopic features of GnRH neurons at a depth of >400μm through the brain tissue. The second objective of this study was to investigate the changes in GnRH neuron morphology associated with neuronal activation at the pre-ovulatory surge, a physiologically critical time point in females. The compatibility of chemical clearing with immunohistochemistry was confirmed with staining for c-Fos, a marker of neuronal activation. The LH plasma concentration from estradiol benzoate treated (n=5) and vehicle treated animals (n=5) was analysed to confirm the induction of the pre-ovulatory surge, before 1.5mm thick sagittal sections were immunostained for c-Fos. Tissue samples displayed c-Fos staining to an average depth of 130μm, however, some filled GnRH neurons were found at 400μm. Consequently no c-Fos positive GnRH neurons were identified meaning they could not be explicitly categorized as either activated or non-activated. Despite our inability to investigate changes in morphological features of GnRH neurons at this time point, unique features of the GnRH neurons identified with this novel technique were explored. Overall, the establishment of in vivo cell-filling in large volume tissue samples, and the subsequent ability to visualise and image entire GnRH neurons represents a great leap in GnRH research, taking us beyond the soma and proximal dendrite, and beyond in vitro brain slice preparations.

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  • An investigation of the correlations between subjective and objective measures of bowel inflammation in Spondyloarthritis

    Bloomer, Chris (2012)

    Undergraduate thesis
    University of Otago

    Introduction: Spondyloarthritis (SpA) and Inflammatory Bowel Disease (IBD) are related conditions of unknown aetiology demonstrating both common clinical features and common genetic and immunological pathomechanisms. Patients with SpA frequently exhibit intestinal inflammation and many develop significant gastrointestinal symptoms. Conversely, many patients with IBD develop an inflammatory arthritis. It has been proposed that an increase in intestinal permeability is an important mechanism in the aetiology of both conditions. However, to date the association between symptoms, intestinal pathology and altered gut permeability has been poorly elucidated. Methods: Patients who fulfilled the Assessments in Spondyloarthritis Internation Society (ASAS) criteria for axial spondyloarthritis were recruited to the study. Gastrointestinal symptoms were assessed using the Dudley Inflammatory bowel symptom Disease Questionnaire (DISQ). Intestinal permeability was measured using the three sugars test, which measures the differential urine recovery of ingested sucralose, L-rhamnose, and lactulose. Small-intestinal lesions were assessed with wireless capsule endoscopy (WCE). An indirect measure of intestinal inflammation (faecal calprotectin) was also used. Drug therapy – including the use and dose of NSAIDs was recorded. Patients with SpA were assessed clinically including a measure of disease activity - the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and CRP. Results: In total, 35 patients and 15 healthy controls completed the DISQ and underwent a sucralose, lactulose and L-rhamnose absorption test to assess intestinal permeability. Ten patients underwent WCE. The majority of patients were taking NSAIDs (25/35). DISQ scores were significantly increased in patients compared to controls (p<0.001). Intestinal permeability was not significantly different between patients and controls, and was not associated with DISQ or BASDAI scores. Faecal calprotectin results were high in some patients, but the correlation with DISQ scores was not significant (p=0.169) although a trend was apparent. WCE showed mild to severe ulcerations/erosions to be remarkably common (8 out of 9 complete studies). Macroscopic lesions of the duodenum appeared to be associated with bowel symptoms, while some severe lesions in the jejunum and ileum 3 were often asymptomatic. Conclusions: We conclude that both gastrointestinal symptoms and intestinal lesions are common in SpA patients. The use of non-steroidal anti-inflammatory drugs appears to be a associated with intestinal symptoms in SpA patients, but ileocolonic ulceration is commonly asymptomatic. The DISQ appears to be a good screening tool for identifying patients with bowel symptoms which are more common with upper GI involvement and especially with lesions in the duodenum. Faecal calprotectin and WCE identified a high proportion of SpA patients as having asymptomatic lesions of the jejunum and ileum, common sites of inflammation in IBD.

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  • What’s the harm in waiting? Patient harms in the referral waiting gap

    Patel, Vimal (2016)

    Undergraduate thesis
    University of Otago

    Background Patient safety research seeks to improve the delivery of care, and ensure that patients’ risk of injury from healthcare itself is minimised. Referral between primary healthcare, specialist diagnostic agencies (such as community medical laboratories and radiological centres), and hospital based healthcare is common and important in primary care, yet patients have highly variable waiting times before receiving their care. However, there is almost no research exploring what happens to patients while they wait. Aims This study aims to investigate patient’s waiting periods between referral from their General Practitioner (GP) and receiving specialist healthcare. Specifically, this study aims to determine if patients come to any harm in this waiting gap, and if so, which patients are harmed and what types of harm happen. Methods I reviewed 5 years (2003-2007) of healthcare records of 201 general practice patient’s notes. Each consultation record was examined to identify the types of referral that were made and to find evidence of harms while the patient was waiting for referred healthcare. A subset of 101 of these patients also had the records reviewed for investigation types and evidence of harm while waiting for investigation. A broad definition of harm was used to capture a greater number of harms. Harms were categorised as related to referral for investigation, referral to medical specialty or referral to other non-medical specialty. Harms were also graded in severity (mild, moderate and severe) and were described under the following: ‘continued symptoms’, ‘delay in subsequent management’, ‘deterioration of condition’, ‘financial cost to patient’, ‘anxiety/mental harm’ or ‘other’. Comparisons were made between patients whose referrals had evidence of harm in the waiting gap with patients who did not. Comparisons included length of waiting gap, age, gender and specialty referred to and used t-tests or non-parametric tests, as appropriate. Results 5003 Consultation records were reviewed. A referral rate of 0.21 per person per year for medical and non-medical specialties was found, and a referral rate of 1.00 per person per year for investigations was found. 45 of 183 (25.5%) of referrals to medical or non-medical specialties had evidence of harm in the waiting gap, whereas 9 of 105 (1.8%) of referrals for investigation had harm in the waiting gap. Of the 58 total harms, 43 (74.1%) of harms were minor, 12 (20.5%) were moderate and 3 (5.2%) were severe. The largest broad classification of harm was “continued symptoms” with 38 harms (65.5%), followed by “delay in subsequent management” with 14 harms (24.1%) and “deterioration in condition” with 14 harms (24.1%). There were no statistically significant relationships between the age of patient nor sex of patient nor length of waiting time and the incidence of harm in the waiting gap. Conclusion This is the first study of harm in the referral waiting gap. The findings indicate that harm does happen while patients wait for referred care, and more research is needed to explore these harms. While the relatively small number of patients in this study limits the ability to draw robust implications for changed clinical practice, it is a strong starting point for larger, future research.

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  • Characterization Of In Vitro Generated Autologous Osteoid Tissue

    Reilly, Terrence (2016)

    Undergraduate thesis
    University of Otago

    Bone disease and injury is expected to increase in the near future due to an ageing population, causing social and economic burden due to disability and lowered quality of life. Critical sized defects, the smallest bony injury that will not heal completely over the lifetime of an individual, are significant orthopedic and oral-maxillofacial issues, as they require bone grafts to facilitate repair. As the gold standard autograft is in short supply with additional disadvantages, bone tissue engineering seeks to alleviate this demand. It combines cells isolated from patients with biomaterials to produce bone tissue. This research looks into the feasibility of bone banking: the cryopreservation and storage of a patient’s cells that can be reanimated at the time of injury or disease and the engineering of a bone graft in vitro. Chitosan and nano-hydroxyapatite composite scaffolds were fabricated to produce appropriate pore diameters and porosities. A final concentration of 8% (w/v) chitosan and 5% (w/v) nano-hydroxyapatite were used in the final scaffold, where imaging studies (scanning electron microscopy, micro-computed tomography) showed pore diameters with a 106 µm average with 79% porosity. These scaffolds were subjected to testing, such as degradation, pH, sterilization, swelling studies, Fourier transform infrared spectroscopy, and energy dispersive X-ray spectroscopy in order to characterize physical and chemical properties. Using an osteogenic sarcoma cell line (SAOS-2), scaffolds were then subjected to in vitro cell studies including the LIVE/DEAD® viability assay and the MTS cell proliferation assay to characterize biocompatibility. Results indicate scaffolds are biocompatible and non-cytotoxic. Further three-dimensional in vitro testing was performed using SAOS-2 in a custom-made perfusion bioreactor for 14 days. After this period isolated cells were detected on the scaffold using histological and fluorescent microscopy techniques. Further in vitro testing was investigated using mesenchymal stem cells isolated from rats. However, these cells reached senescence rapidly, therefore, the supply of cells were limited and differentiation down the osteoblastic lineage and three-dimensional culture was unable to be accomplished. This research demonstrated the development of a biocompatible scaffold with appropriate structural parameters that promote viable cells when cultured in a bioreactor. Future study should investigate the scaffolds mechanical and structural properties to determine suitability for bearing load and the production of a homogenous scaffold matrix that results in homogenous osteoid tissue. Cell culture optimization in terms of mesenchymal stem cell proliferation and extracellular matrix production also needs to be investigated in terms of modifying tissue culture factors such as shear stress and seeding density.

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  • Comparison of Spironolactone versus Amlodipine in Hypertensive Cardiorenal Pathology

    Strickland, James Todd Langdale (2016)

    Undergraduate thesis
    University of Otago

    Hypertension is endemic worldwide, afflicting up to 40% of the global population over the age of 25. Haemodynamic stretch stress from raised blood pressure is a driver for organ damage in the heart and kidney, contributing to potentially fatal conditions. Aldosterone activated mineralocorticoid receptors have also been shown to contribute to cardiorenal damage in a hypertensive state. This study compared the effects of spironolactone, a mineralocorticoid receptor antagonist (MRA), and amlodipine, a calcium channel blocker, when administered in a chronic hypertensive Cyp1a1-Ren2 transgenic rat model. Cyp1a1-Ren2 rats characteristically develop hypertension in a dose-dependent manner in response to dietary indole-3-carbinol (I3C). Male rats (19 week old) were assigned to either standard or I3C (0.167% w/w) diet, 2 weeks prior to experimental day 0. At experimental day 40, rats in both dietary groups were assigned into three drug treatment groups (n=4/group): spironolactone (8.82 mg/kg/day), amlodipine (0.44 mg/kg/day) or vehicle control oral dosing until termination at experimental day 85. Systolic blood pressures recorded at day 0 and day 85 were significantly higher in the I3C diet group compared to standard diet. Spironolactone and amlodipine did not reduce systolic blood pressure at day 85 compared to control groups. Urine volume, proteinuria, left ventricular wall thickness, glomerulosclerosis, renal fibrosis and renal populations of both macrophages and myofibroblasts were increased in the I3C dietary group compared to standard diet; however none of these variables were significantly reduced with either spironolactone or amlodipine treatment compared to the control group in the I3C diet group. A reduction in renal fibrosis and renal cortex macrophage infiltration was observed with spironolactone treatment compared to control in the I3C diet group; however this was not statistically significant. A blood pressure independent reduction in proteinuria was seen in the spironolactone treated rats compared to control, but was also not statistically significant. The trends identified with spironolactone treatment in this study were consistent with many other published works; however these studies suggest spironolactone has been shown to have many other protective characteristics unidentified in this study. Internal validity issues arose from initial stress-related difficulties, compromising intra-group comparisons. This and the underpowered nature of this study limit the interpretation of the study’s results. Therefore this study came to no conclusion in regards to blood pressure independent benefits of addition of spironolactone to a systemic hypertensive situation

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  • Access to elective and acute orthopaedic surgery in Dunedin: an analysis of surgical delay

    Ramage, Dean James (2016)

    Undergraduate thesis
    University of Otago

    Background: Delay in surgical intervention may lead to significant personal, medical and economical consequences. For individuals undergoing treatment in the public health system, there is inevitably a delay in access to surgery. Access to elective joint replacement surgery can take anywhere from three months, to more than a year. Acute orthopaedic procedures may also be delayed due to clinical and logistical reasons. Thus the aim of this study was to compare outcomes of patients undergoing total hip and knee replacement surgery in the public and private health sectors. In addition to this, the contributing factors to acute surgical delay, and the cost of any surgical delay in Dunedin Hospital has also been investigated along with patients’ perspective of the time spent waiting for surgery. Methods: For the elective arm of the study, 232 public and 231 private patients were retrospectively recruited using New Zealand Joint Registry data. This included 253 that had total hip joint arthroplasty and 210 that had total knee joint arthroplasty. Six-month Oxford Hip and Knee Scores were the primary measure used to compare outcomes between the two groups where the 0-48 scoring system is used. NHI numbers were also used to gain access to other surgical data. For the acute arm of the study, 472 patients who underwent a total of 507 acute orthopaedic procedures were recruited retrospectively. Patients were recruited using the Dunedin Hospital surgical theatre management database and NHI numbers were used to access additional clinical information. In addition to this 47 patients were recruited prospectively to undergo a questionnaire of their perspective of waiting for surgery in Dunedin Hospital. Results: The elective arm of this study showed that private patients achieve better six month postoperative outcomes than public patients. For those undergoing THJR, private patients had a mean score of 41.74 (SD=5.7) whereas the mean for public patients was 37.94 (SD=9.1). For TKJR patients, private patients had a mean six-month postoperative score of 40.82 (SD=6.0) and public patients mean was 36.55 (SD=8.1). These differences in score persisted after adjustment for age and gender. For the acute arm of this study, 66.7% of the 507 procedures reviewed were delayed. Of the cases that were delayed, 84.3% were delayed due to a logistical reason such as unavailability of theatre time. Other factors associated with surgical delay included the priority grade, day of the week surgery was booked, and the anatomical location of the trauma. The total cost of delay was found to be approximately $145,800 for all surgeries delayed due to a logistical reason. Conclusion: The difference seen between public and private groups for hip replacement surgery supports the limited amount of prior literature. This study is the first that shows a difference in postoperative outcome for knee replacement surgery between public and private patients. In addition to this, findings show that there is a large amount of surgical delay currently present in Dunedin Hospital that comes at a high cost to the healthcare system. Prior literature suggests that a dedicated orthopaedic trauma operating theatre will reduce surgical delay. Finally, patient satisfaction with their time waited for surgery is associated with the timeliness of their surgery, and the communication they receive from medical staff.

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  • Umbilical Cord Serum Chemokines and the Development of Atopic Dermatitis

    Townsley, Hermaleigh (2016)

    Undergraduate thesis
    University of Otago

    Background: Atopic dermatitis (AD) is a chronic skin condition characterised by the development of pruritic and inflamed lesions. One key component of AD pathogenesis is a cutaneous hyper-reactivity to allergens influenced by a Th2-polarised immune response. Macrophage-derived chemokine (MDC) and Thymus and activation-regulated chemokine (TARC) are two chemokines involved in this pathological immune response. Research has shown a strong association between MDC and TARC levels in blood and AD severity. Recently, some cohort studies have suggested that levels of MDC and TARC in umbilical cord blood (UCB) may be predictive of whether infants will develop AD during childhood. This cohort study aimed to further investigate the potential predictive value of UCB MDC and TARC for AD development in childhood. Methods: This project involved a retrospective analysis of data obtained from the NZA2CS population birth cohort study. Information about AD-related outcomes was gathered using questionnaires at various time points, along with physical examination of flexural dermatitis and measurement of total and specific IgE levels at age six. UCB MDC levels were measured using enzyme-linked immunosorbent assay (ELISA) techniques for a total of 647 participants. Haemolysis of UCB samples was found to affect TARC measurement; therefore fewer (n = 270) samples were analysed for TARC. Haemolysis of UCB samples did not affect measurement of MDC concentration. Logistic regression was used to calculate odds ratios to determine the association between UCB chemokine levels and development of AD- related outcomes in childhood. Results: UCB MDC and TARC levels were not predictive of development of AD at age six. Neither were they consistently significantly associated with the development of AD-related outcomes such as an itchy rash or atopy. Some statistically significant associations were found, although their value is difficult to interpret as these were isolated findings. UCB MDC levels were significantly associated with the development of an itchy rash at four years of age (p = 0.027) and with the level of specific IgE to cat allergen at age six (p = 0.05). When the data was segregated by sex, UCB MDC levels in males were consistently significantly associated with development of an itchy rash during childhood (p < 0.05). Conclusion: UCB MDC and TARC concentrations are unlikely to be clinically useful biomarkers for the development of AD in childhood. This was the largest cohort study so far to investigate cord MDC and TARC levels as predictors of future AD onset, and the findings are concordant one other large cohort study. Therefore, these results do not warrant further research into UCB MDC and TARC as predictive biomarkers of AD development.

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