51 results for Undergraduate, 2015

  • Rib Fractures in Infants: Retrospective Survey of Fractures and Biomechanical Study.

    Blackburne, William Bligh (2015)

    Undergraduate thesis
    University of Otago

    Literature suggests that rib fractures are highly associated with abuse and the present understanding is that antero-posterior compression associated with the ‘shaken baby syndrome’ is their cause. However, this mechanism rests on a number of assumptions with little experimental data to support them. Recent work using a porcine model of fractures suggests that, in the case of lateral fractures this may be highly unlikely. This work shows a feasible alternate mechanism, that of blunt force trauma (BFT), for the cause of these lateral fractures. A piglet model is used and shows the ease with which ribs fracture as a result of BFT, compared to the difficulty of fracture seen previously in compressive injury. The initial development of a computational simulation of these ribs for use in injury scenarios is also outlined here. Secondly, skeletal surveys from New Zealand’s largest children’s care facility, Starship Hospital, were examined to give a picture of non-accidental injury (NAI) and how its patterns compare with accidental injury in New Zealand. It has been found that, as in foreign studies, there are a number of lesions highly associated with abuse and these include rib fractures, which are highly specific (97%) for NAI. Unusuallyhigh frequencies of lateral-type rib fractures (46.4%) were found and half the cases were found to be unilateral. This is not wholly in line with the currently accepted idea that rib fracture is due to antero-posterior compression, in which bilateral, posterior fractures are said to be most common. Overall, this work brings into question the traditional mechanism of rib fractures, provides a highly useful snapshot of abusive injury in NZ and also sets a strong foundation for future work.

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  • The role of cytoskeletal elements in the trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells

    Farquhar, Rachel (2015)

    Undergraduate thesis
    University of Otago

    The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane in polarized epithelia where it plays an essential role in promoting trans-epithelial ion transport. KCa3.1 is found in many tissues in the body and plays an important role in many physiological and pathological processes (e.g., regulation of salt and fluid transport in the gastrointestinal tract, atherosclerosis, sickle cell disease and asthma). Functional KCa3.1 must be targeted to the basolateral membrane, a process that is dependent upon proper cytoskeletal function. The cytoskeleton is comprised of actin and microtubule filaments. Actin filaments are comprised of polymerised G-actin monomers bound to form filamentous F-actin strands. Microtubules are long filamentous structures comprised of tubulin subunits, made from α-tubulin and β-tubulin monomers. This study examines the role of microfilaments and microtubules in the trafficking of KCa3.1 to the basolateral membrane of polarised epithelial cells. To address this, Fischer Rat Thyroid cells grown on filter inserts to form a confluent epithelium were stably transfected with the Biotin Ligase Acceptor Peptide (BLAP)-KCa3.1 construct. This construct allowed for the selective labeling of basolaterally expressed KCa3.1 using streptavidin. Selective labeling of membrane bound KCa3.1 allowed for the measurement of changes in KCa3.1 expression, in response to drugs that disrupt cytoskeletal elements, to reflect changes in KCa3.1 located on the basolateral membrane. This measure allowed for a direct correlation to be drawn between targeted disruption of specific cytoskeletal elements, e.g. microtubules and microfilaments, and expression of basolaterally-located KCa3.1. PCR was used to determine the mRNA expression levels of KCa3.1 in stably transfected cell lines and SDS-PAGE techniques were employed to investigate protein expression levels of KCa3.1. Western blotting was used to explore the effects of Cytochalasin D (Cyto D), Latrunculin A (Lat A), and Myosin Light Chain Inhibitor-7 (ML-7) which inhibit the function of actin (Cyto D, Lat A) and myosin light chain kinase (ML-7) respectively. Toxicity tests were performed to determine cell survival under a range concentrations of 0-20 μM (0, 3, 5 hr) for all three drugs with cell survival reduced with 20 μM at t = 5 hr for Cyto D and Lat A. Cyto D was administered over intervals of 0, 3 and 5 hr at 10 μM resulting in a decreased relative expression of KCa3.1 (compared to control) of 0.6±0.14 at t = 3 and further decrease in the expression of the channel at t = 5 hr with a relative expression of 0.12±0.035 (n = 5, p < 0.05). Lat A was also administered over intervals of 0, 3 and 5 hr at 10 μM causing a relative reduction in the expression of KCa3.1 at the basolateral membrane compared to the control. At t = 3 hr the expression of KCa3.1 was reduced to 0.7±0.065 and decreased to 0.3±0.049 at t = 5 hr (n = 4, p < 0.001). Finally, cells treated with microtubule inhibitor ML-7 showed a relative reduction in KCa3.1 expression of 0.55±0.12 at t = 3 hr, the expression was further decreased to 0.33±0.11 at t = 5 hr compared to the control. These data confirm that microtubules and microfilaments of the cytoskeleton are crucial in trafficking KCa3.1 to the basolateral membrane of polarised epithelial cells.

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  • 11 week Beta-Hydroxy beta-Methylbutyric acid (HMB) supplementation: Effects on body composition and exercise performance in trained athletes.

    McIntosh, Nicholas Dean (2015)

    Undergraduate thesis
    University of Otago

    Background: Originally used in the farming industry to ‘bulk up’ cattle, interest in the leucine metabolite, beta-hydroxy beta-methylbutyrate (HMB), has been growing following a clinical trial which demonstrated significant improvements in strength and body composition in humans. Subsequent trials reaffirmed that previously untrained individuals benefitted from supplementation. However, trials involving athletes have demonstrated mixed results with short (0.05), nor was there a statistically significant difference with respect to skin fold measurements (p>0.05). Conclusion: The increase in body mass found in this study is consistent with other long term (>6 week) HMB supplementation studies. These gains in body mass may have influenced running performance as a larger mass is required to be moved. As no significant differences in body composition or strength were seen, the findings of this study suggest caution needs to be taken when supplementing with HMB as negative performance effects may occur. Therefore close attention to the type of activities required by the athlete needs to be considered prior to supplementation.

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  • Child Mortality after Discharge from a Health Facility Following Suspected Pneumonia, Meningitis and Septicaemia in Rural Gambia

    Chhibber, Aakash Varun (2015)

    Undergraduate thesis
    University of Otago

    Background Two years away from 2015, the decline in child mortality is not fast enough to reach Millennium Development Goal 4. The Integrated Management of Childhood Illness (IMCI) is a strategy that simplifies management of child health. Beyond effective disease management, IMCI recommendations for care following illnesses are based on limited evidence from the field. The aim of this project was to find (1) the magnitude of and (2) risk factors for child mortality following discharge from a health facility in a low-income setting. Methods This study used an established population-based surveillance system for suspected invasive pneumococcal disease in Upper River Region, The Gambia, West Africa. Children that survived admission for suspected pneumonia, meningitis or septicaemia at the Region’s only referral centre (Basse Major Health Centre, Upper River Region) were followed for 180 days after discharge. Vitality status monitored by the DSS informed time-to-death information in a survival analysis that identified predictors of post-discharge mortality. Two multivariable Cox proportional hazards models were constructed. Model A described the clinical syndrome on admission (provisional diagnosis) and risk of post-discharge mortality. Model B used a reverse step-wise approach to find pre-discharge risk factors for mortality following discharge. Results The cohort that survived admission had higher mortality rates than the background rate in the community. Overall, 105 (2.8%) of 3735 patients died during the 6 months of follow-up. Half of the deaths occurred within 45 days of discharge. Approximately half as many patients died in the six months following discharge as died during hospital admission. Age stratified post-discharge mortality rates were three to six times higher than community mortality rates. In addition to demonstrating the protective effect of increasing age at discharge (HR 0.98 [95%CI: 0.96, 0.99] for every month increase in age), Model A showed that, compared to pneumonia alone, a provisional diagnosis of: pneumonia with visible signs of severe malnutrition had a HR 8.74 (95%CI: 4.93, 15.49); meningitis with visible signs of severe malnutrition had a HR of 13.90 (95%CI: 5.43, 35.58); sepsis with visible signs of severe malnutrition had a HR 18.79 (95%CI: 11.65, 30.32). Model B showed independent risk factors associated with post-discharge mortality were: the presence of neck stiffness on assessment (HR 17.60 [95%CI: 7.36, 42.10]); low mid-upper arm circumference (MUAC) (<10.5cm, HR 11.52 [4.59, 28.90]); visible signs of severe malnutrition (HR 3.94 [95%CI: 2.11, 7.36]); non- medical discharge (HR 6.22 [95%CI: 2.98, 13.01]); discharge during dry season (HR 2.33 [95%CI: 1.44, 3.77]); decreasing peripheral arterial haemoglobin oxygen saturation (HR 0.95 [95%CI: 0.93, 0.98] per percent increase); decreasing haemoglobin concentration (HR 0.82 [95%CI: 0.74, 0.90]) per unit g/dL increase); and decreasing axillary temperature (HR 0.70 [0.58, 0.84] per unit oC increase). Conclusion Gambian children in Upper River Region with suspected invasive pneumococcal disease are at increased risk of death following discharge from a health facility, and most of these deaths occur early. There are identifiable risk factors for death, including neck stiffness, low MUAC, visible signs of severe malnutrition, non-medical discharge, discharge during dry season, decreasing peripheral arterial haemoglobin oxygen saturation, decreasing haemoglobin concentration and decreasing axillary temperature. These data add to the evidence base needed to inform the development key guidelines and may be helpful towards development of a tool with clinical utility to identify children for intervention after discharge from hospital.

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  • Falcon Forestry Carriage Series 2 : a case study of productivity and operation.

    Bolitho, Callum (2015)

    Undergraduate thesis
    University of Canterbury Library

    The multiple drivers of workplace safety and increasing productivity are resulting in increased mechanisation within the forestry industry. The use of motorised grapples in cable harvesting is an applicable mechanisation method to the large proportion of steep terrain harvesting in New Zealand. In this dissertation a time study of the Falcon Forestry Carriage Series 2 has been undertaken in order to access its productivity and operation. Mean values of productivity were found to be 54.9m³/PMH for wood extracted from the ground, 64.6m³/PMH for bunched wood and 75.6m³/PMH for excavator fed wood after adjustment for the cycle distance and accumulation type. Longer cycles were found to decrease productivity by 0.15m³/PMH for each meter of cycle distance. Utilisation in the study was found to be 56% of total time which was similar to previous studies. 15% of total study time was accounted for by operational delays, 7% by personal delays and 23% by mechanical delays. Mechanical problems with the carriage occurred 6 times and accounted for 171 minutes or 13.4% of total delay time. Mechanical delay breakdown was similar to that found by McFadzean (2012) who recorded that 15% of total delay time was attributable to carriage mechanical delays. During a study of Operator effect it was found that the inexperienced Operator 3 and Operator 4 had a productivity of 52.2% (not statistically significantly different) and 18.5% (p value <0.05). The effects of accumulation method and cycle distance upon productivity were found to be similar to the results of previous studies, as was the utilisation of time within the study.

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  • Understanding demand for wood products in New Zealand’s major log markets.

    Drummond, Ryan C.M. (2015)

    Undergraduate thesis
    University of Canterbury Library

    New Zealand’s forestry sector is largely reliant on the presence of a strong export market with 57% of the volume harvested being exported of which 99% goes to Japan, the Republic of Korea, China and India. This identifies the need to analyse demand in these countries to better understand their needs in the future. Consumption of wood products per capita is a commonly used metric for estimating demand and was used in this research. Volumes of imports, exports and production were collected from the Food and Agricultural Organisation of the United Nations (FAO) and data for a range of explanatory variables was collected from a variety of official sources. Historical trends in consumption identified that as countries develop socially and economically their consumption shifts from largely solid wood products such as sawn timber to more processed products such as wood-based panels and paper and paperboard. Consumption was modelled using linear regression techniques to develop models which could be used to forecast consumption in the future. A wide variety of potential explanatory variables were considered and the models presented represent the most effective of these. GDP per capita was found to be the single most effective explanatory variable being highly significant (p<0.01) in all models. Price was also found to be a strong determinant of consumption, understandable as price is a major component of supply and demand dynamics. Measures of construction activity were found to be related to consumption of sawn timber in all studied countries and for wood-based panels in Japan. Forecasts produced for consumption in Japan should be used as only an example of the capability of the models presented herein. More work is required to develop these equations into a form where they can be used to more accurately estimate future consumption.

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  • The performance of blocks of clones in a radiata pine production forest.

    Farmery, Acacia (2015)

    Undergraduate thesis
    University of Canterbury Library

    Problem: Genetically identical clones of Pinus radiata are being planted in New Zealand plantation forests. There have been many clonal trials carried out; however there is a weakness in published literature surrounding the performance of clones in production blocks. Method: Five comparisons in four of Pan Pac Forests Products production forests were measured. Three comparisons were measured at age 4.5 years old and two were measured at 7.5 years old. There were six Forest Genetics clones and three different control-pollinated seedlots measured in these comparisons. Each comparison had a different number and selection of seedlots. There were six different traits measured for the trees; diameter at breast height over bark, height, acoustic velocity, straightness, branching habit, and malformation. The different traits were compared between the seedlots within each comparison. The differences in variation for diameter at breast height and modulus of elasticity were compared between clones and control-pollinated seedlots. Finally, the results by clone for the traits, excluding height, were compared to the expected performance supplied by Forest Genetics. Results: There were differences in performance between seedlots. Four clones performed well across a range of traits. One clone performed well in the 7.5 year old blocks but not in the 4.5 year old blocks. One clone did not perform well in size and stiffness. Clones were significantly less variable than control-pollinated seedlots for diameter at breast height but not for modulus of elasticity. The performance of each clone in Pan Pac Forest Products forests was very similar to the expected performance provided by Forest Genetics. Implications: There are clones that can produce desired yield, stiffness and form. Clones will provide a more uniform crop in diameter than control-pollinated seedlots. Pan Pac Forest Products can rely on Forest Genetics prediction of clonal performance as a guide to performance in their forests.

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  • A new target for treating arrhythmia? Interaction of triadin with the cardiac ryanodine receptor reduces Store Overload Induced Calcium Release

    Deo, Manesh Shamal (2015)

    Undergraduate thesis
    University of Otago

    Arrhythmia occurs in a number of heart diseases including heart failure (HF) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). It has been shown that Store Overload Induced Calcium Release (SOICR) is a common mechanism underlying many types of arrhythmia. SOICR occurs due to the inappropriate opening of the cardiac ryanodine receptor (RyR2) once calcium in the sarcoplasmic reticulum (SR) reaches a certain threshold. RyR2 forms a large macromolecular complex with other proteins. One such protein is triadin; which is known to be lost from the RyR2 complex in HF and CPVT. The loss of triadin is also linked to an increase in arrhythmias, suggesting its loss may lead to SOICR. However, triadin has also been shown to have a role in maintaining the ultrastructure of cardiomyocytes, therefore it is unclear whether arrhythmia occurs as a direct result of the loss of interaction between the RyR2 and triadin or simply due to structural changes in the cell. In the present study, we investigated whether the direct interaction between RyR2 and triadin alters the propensity for SOICR. Single cell cytosolic imaging in HEK 293 cells stably expressing RyR2 with or without triadin, using the high affinity calcium indicator Fluo4-AM, showed that the presence of triadin reduced the propensity of SOICR events, suggesting an inhibitory role for triadin. This was confirmed by single cell luminal calcium imaging, using the endoplasmic reticulum (ER) targeted Ca2+ indicator protein D1ER which showed that the expression of triadin increased ER calcium threshold at which SOICR occurred. These results provide evidence that the direct interaction between RyR2 and triadin reduces the propensity for SOICR by increasing the threshold that SR calcium must reach to trigger SOICR. Conversely, this suggests that loss of the direct interaction between RyR2 and triadin in HF and CPVT will be arrhythmogenic. Therefore, stablising this interaction in patients susceptible to arrhythmias is likely to be therapeutic.

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  • Time to Safe Driving after Total Hip and Knee Replacement Surgery

    Meffan, Peter (2015)

    Undergraduate thesis
    University of Otago

    Introduction: Osteoarthritis is the most common form of arthritis in the western world. With its increasing prevalence, total joint replacement is in high demand. After total hip replacement (THR) and total knee replacement (TKR) a period of driving cessation is necessary. For many patients, a period of driving cessation creates financial stress and threatens independence. The current recommendation for driving cessation following THR or TKR surgery is 6 weeks, however the literature surrounding this is varied. Aims: To measure the average time it takes for a patient following either a THR or TKR to return to operating a motor vehicle safely; based on recovery of their pre-operative baseline transfer time to within 10%. Methods: The transfer time from accelerator to a brake force of 100N (transfer time) was measured on a custom built rig. Patients were tested pre-operatively, and 1, 2, 4, and 6 weeks post-operatively The time in weeks was measured to return to within 10% of their pre-operative transfer time. A quantitative questionnaire was used at each test to establish patient perception to the impediments to safe and confident driving. Results: The median time to return to baseline in THR was 2.0 weeks (95%CI 1.3-2.7) and in TKR 5.5 weeks (Log rank score 0.034). 14.3% of THR and 62.5% of TKR patients failed to reach baseline in the test period. The median time to return to baseline for all operation groups was 3.3 weeks in males (2.6 – 4.0) and 2.0 weeks in females (1.4 – 2.6) (Log rank 0.67). 18.2% of males and 45.5% of females failed to return to baseline in the test period. Males recorded faster transfer times at baseline than females (414ms and 573 respectively). Joint pain decreased markedly over the test period when scored by visual analogue score. TKR felt more joint pain at 1 week postoperative than THR (VAS 5.26 and 2.0 respectively). Perceived driving confidence had improved to baseline by 2 weeks postoperative. When asked, patients reported joint stiffness as the most troublesome symptoms in the postoperative period. Discussion: THR recovered to their baseline transfer time significantly quicker than TKR. This may be due to TKR experiencing more pain in the immediate post-operative period. Females recovered to baseline quicker than males, however this observation was not statistically significant. There was significant loss to follow up in this study, which particularly affected the TKR group. There were a significant number of TKR patients who did not reach baseline in the study period. Without data past 6 weeks follow up it is difficult to make recommendations for the TKR group. We suggest that THR are safe to return driving 3-4 weeks after their operation date. Due to the time limitations of the BMedSci (Hons) programme the study size was smaller than desired. Following completion of this thesis data collection will continue to strengthen the findings of this study.

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  • Psychosocial determinants of glycaemic control in children and adolescents with Type One Diabetes Mellitus

    Chae, Matthew (2015)

    Undergraduate thesis
    University of Otago

    Introduction Type 1 Diabetes Mellitus (T1DM) is a chronic life-long condition that commonly presents in children and adolescents. It is a condition that requires close monitoring, as poor management may lead to severe complications later in life. Psychosocial factors are an aspect that must be considered when making management plans, as it can impact health outcomes for these individuals. This study aims to explore three psychosocial measures: Chaos Hubbub and Order Scale (CHAOS); Intuitive Eating; and temperament/self-control, in children and adolescents with T1DM, and investigate any relationship between these measures and their glycaemic control. In doing so, we hope to contribute to the growing body of literature aiming to improve glycaemic control in children and adolescents with T1DM. Methods This was a cross sectional study involving 74 eligible families of children/adolescents with T1DM. The Child/adolescent were required to be between ages 3-18 and had to be out of their honeymoon phase (> 0.5 units of insulin per kg per day). Each parent (both maternal and paternal) were asked to fill out a demographic questionnaire, a CHAOS questionnaire, self-reported Adult Temperament Questionnaire and an age appropriate Temperament Questionnaire on their child. The Child/adolescent were asked to also fill-out a demographic questionnaire, participate in the Heads knees shoulders and Toes (HKST) test, and fill out an Intuitive Eating Scale if they were above the age of 12. HbA1c was used as a measure of glycaemic control. An age and sex matched control group was recruited through participating families and the University of Otago’s Psychology Database. These Control families were not required to participate in the HKST test nor the Temperament questionnaire. Results Maternal CHAOS (MCHAOS) and Paternal CHAOS (PCHAOS) were both negatively associated with HbA1c after controlling for age, self-monitoring of Blood Glucose (SMBG), insulin therapy and Body Mass Index (for paternal only). The Odds Ratio generated by the multivariate model for MCHAOS and PCHAOS with HbA1c, was 1.30 (95% CI: 1.02 – 1.65) and 1.28 (95% CI: 1.01 – 1.62) respectively. MCHAOS showed limited utility as a tool to detect poor or acceptable glycaemic control with a sensitivity and specificity of 52.9% (95% CI: 35.10 – 70.20%) and 85.7% (95% CI: 67.30 – 96.00%), respectively. However PCHAOS showed potential to be used as a screening tool with a sensitivity and specificity of 92% (95% CI: 74 – 99%) and 37.5% (95% CI:18.80 – 59.4%), respectively. No statistically significant difference was observed between MCHAOS of families with T1DM (mean: 18.02) and their controls (17.40) with a 95% confidence interval for the t-test of -0.341 – 1.581. additionally, PCHAOS was reported to be significantly higher in T1DM (mean: 18.51) than controls (mean: 17.25) with a 95% confidence interval for the t-test of 0.052 – 2.468. Out of the four scales of intuitive eating, Eating for physical rather than emotional reasons, was found to be negatively associated with HbA1c with an odds ratio of 0.27 and a 95% CI ranging from 0.08 – 0.85. No significant difference was found in measures of intuitive eating between adolescents with T1DM and their matched controls. Maternal and Paternal report of Child effortful control were both negatively associated with HbA1c after controlling for age, SMBG, insulin therapy and BMI (for paternal and paternally reported child measures only). Maternally and paternally reported child effortful control had an odds ratio of 0.42 (95% CI: 0.20 – 0.89) and 0.21 (95% CI: 0.06 – 0.71)respectively. Maternally reported child attention was also negatively associated with HbA1c (Odds Ratio:0.44, 95% CI: 0.22 – 0.91), with paternally reported child attention approaching significance (p-value of 0.08). In addition, self-reported maternal activity and attention were both found to be negatively associated with HbA1c, even after controlling for age, SMBG and Insulin Therapy. The odds ratio for Maternal activity and attention were 0.36 (95% CI: 0.17 – 0.78) and 0.43 (95% CI: 0.21 – 0.87) respectively. The HKST test failed to show any significant relationship with HbA1c. With a median score of 57 out of 60, it was found to be inappropriate for this age group. Conclusion: This is the first study to investigate the concept of intuitive eating and CHAOS in Type One diabetes mellitus. We have provided evidence indicating that these measures are predictors of HbA1c, and paternal CHAOS in particular, has clinical potential to aid the process of identifying populations at risk of poor glycaemic control. The present study is also the first study to use the Mary Rothbart’s Temperament scales in this population, and has found that child effortful control (Cognitive regulation) is associated with good glycaemic control. In addition to this, maternal measures of temperament, in particular activity and attention levels were also found to be predictors of glycaemic control. This study has explored three novel psychosocial tools in diabetes, and provides preliminary evidence warranting further research in these areas.

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  • Mutant p53 and pro-inflammatory gene expression in colorectal cancer cells

    Saha, Elora Konok (2015)

    Undergraduate thesis
    University of Otago

    p53 gene is the most commonly mutated gene in cancer, and in many malignancies a loss of p53 promotes cancer progression. However recent evidence has shown that missense mutations in p53, particularly those that lead to the protein acquiring abilities not seen in the wild-type form, may actually aid oncogenesis. These so-called ‘gain of function’ mutations are thought to upregulate many pro-inflammatory cytokines, which can enhance tumour growth, and inhibit other proteins, such as TP63 – which has been shown to have anti-oncogenic properties. The central question of this project is to establish whether gain-of-function mutations in p53 influence the expression of pro-inflammatory genes in colorectal cancer (CRC) cells. To address this question, shRNA constructs against MDM2 were generated to stablise mutant p53, which were then transfected into various CRC cell lines that either harboured a gain-of-function mutation, had wildtype p53, or were void of all p53. In addition, a puromycin-resistance gene was included allowing for the selection of positively transfected cells using puromycin. qPCR primer sets were also developed for measuring the expression of several selected chemokines once the CRC lines had been transfected. To assess the ability of mutant p53 to functionally interact with p63, I attempted to generate a puromycin-selectable p63 expression construct. Resulting data suggest that gain-of-function p53 mutations may indeed heighten expression of various chemokines.

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  • Measuring Mitochondrial Dysfunction in Humans

    Brinsden, Mark (2015)

    Undergraduate thesis
    University of Otago

    Mitochondria are essential organelles found in almost every cell in the human body. They host a number of important metabolic pathways and carry out essential biological functions such as ATP synthesis and regulating cell death. There is a slow decline in mitochondrial function associated with ageing and mitochondrial dysfunction is proposed to act causally in a number of diseases. Previously it has been difficult to measure the health of human mitochondria as tests have required tissue from invasive muscle biopsies. The Seahorse XF Analyser is a recent technological advance that enables researchers to test mitochondrial function in small numbers of live cells. Recently, using the Seahorse analyser, peripheral blood cells such as platelets, monocytes and lymphocytes have been shown to display individually distinct bioenergetic profiles. During circulation, these cells are exposed to metabolic or environmental stressors throughout the body, potentially allowing them to act as biomarkers of bioenergetic health and ageing. Different cell preparations were trialled to purify and isolate platelets, monocytes and lymphocytes from freshly drawn whole blood. These protocols succeeded in preparing platelet and T-lymphocyte samples for XF analysis, however inconsistent results indicated that the protocols need further development. The Seahorse XF analyser was used to measure bioenergetic function in human platelets and T-lymphocytes from healthy donors ranging from 21 to 56 years of age. Each cell type required optimisation experiments to determine the optimal inhibitor and substrate concentrations to generate a meaningful bioenergetic profile. Similarly, seeding densities were determined to ensure oxygen consumption values that were suitable to the instruments sensitivity. These cell types have elastic metabolic phenotypes, and appeared sensitive to metabolic switching during early stages of the XF assay. Platelets were particularly difficult to work with because of their inclination to cease using oxidative phosphorylation and switch metabolism to using purely glycolytic pathways. Platelet susceptibility for metabolic switching is undetermined at this point. Lymphocyte optimization experiments also indicated possible premature activation during the assay. Further work is needed to fine tune this protocol to ensure consistent and uniform measurements before accurate BHI values are calculated for donors of different ages.

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  • Exploring Molecular Links between Obesity and Breast Cancer

    Crake, Rebekah Lee Isla (2015)

    Undergraduate thesis
    University of Otago

    Obesity is associated with a high risk of incidence of, and mortality for, postmenopausal breast cancer. Despite this well-established link, the molecular and mechanistic basis of the obesity and breast cancer association still remains unclear. In obesity research, genetic variation due to copy number differences has become increasingly popular. The salivary amylase gene, AMY1, is well-known for its extensive copy number variation (CNV) in the human genome and has previously been correlated with a genetic predisposition toward obesity; however, research surrounding this association is controversial. Despite an established relationship between obesity and breast cancer risk, the recently reported genetic association between AMY1 CNV and obesity has not yet been examined in normal and obese breast cancer patients. Furthermore, gene expression changes in breast tumours from obese women remain poorly characterised. We hypothesise that obese breast cancer patients are associated with (1) low AMY1 copy number and (2) differential expression of candidate genes in the breast tumour. This study included 55 post-menopausal breast cancer patients from The Cancer Society Tissue Bank, with a BMI (body mass index)> 30 (obese; n=28) or BMI < 25 (healthy; n=27). Quantitative PCR (qPCR) assessment of germline AMY1 copy number status from blood showed that obese breast cancer patients have a lower average copy number of AMY1 compared to normal weight patients. Examining breast tumour expression profiles of obese and non-obese patients from two published studies, identified four candidate genes (GRIA2, DUSP4, NR2F1, and ADH1B) shared between both studies. Analysis of gene expression data from The Cancer Genome Atlas (TCGA) indicated that these four genes are differentially expressed within clinically relevant breast tumour subtypes characterised by oestrogen receptor, progesterone receptor and HER2 status. qPCR analysis of each candidate gene within our study cohort showed that the average expression of GRIA2, DUSP4, NR2F1 and ADH1B was lower in obese compared to healthy breast tumours, but these results were not statistically significant. My study indicated that BMI may be associated with lower germline copy number of AMY1 in post-menopausal breast cancer patients; however, further work with a larger cohort is needed to establish if GRIA2, DUSP4, NR2F1 and ADH1B are associated with obesity related breast cancer.

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  • Does fat provide energy for breast tumour cell invasion and metastasis?

    Jones, Morgan Grace (2015)

    Undergraduate thesis
    University of Otago

    Breast cancer is the most commonly diagnosed cancer in New Zealand women. Obese breast cancer patients are more likely to have tumours with advanced clinical stage and high vascular and lymph node involvement. The tumour microenvironment provides vital support for tumours during development and progression of cancer, yet the local effects of stromal adipocytes on breast cancer cells have been largely overlooked. Recent studies by Dirat et al. (2011) and Bochet et al. (2011) have shown that breast cancer cells co-cultured with adipocytes become more resistant to radio- and chemotherapy, and more invasive. However, little is known about the metabolic changes that occur in breast cancer cells when they are cultured with adipocytes. Nieman et al. (2011) determined that lipids were transferred from omental adipocytes to ovarian cancer cells and were consequently used in β-oxidation. It was hypothesised that β-oxidation is increased in breast cancer to exploit the glycerol and fatty acids released by lipolysis from adipocytes in order to support the migration and invasion of breast cancer cells. In this study, breast cancer cell lines MCF7 (ER+; oestrogen receptor positive) and MDA-MB-231 (ER-/PR-/HER2-; oestrogen, progesterone and human epidermal growth factor receptor negative) were co-cultured with adipocytes isolated from breast adipose tissue. Adipose tissue samples were collected via the Cancer Society Tissue Bank from patients at Christchurch Hospital undergoing surgery for therapeutic mastectomy, prophylactic mastectomy and breast reductions. A Seahorse XF24 Analyser was used to measure oxygen consumption and extracellular acidification, as indicators of oxidative phosphorylation and glycolysis, respectively, in breast cancer cells grown alone or in co-culture with human breast adipocytes. MCF7 cells were found to have upregulated glycolysis after co-culture with adipocytes. Western blotting was used to assess differences in the expression of proteins involved in β-oxidation between breast cancer cells grown alone or in co-culture with adipocytes. Levels of carnitine palmitoyltransferase 1 (CPT1A), a protein involved in translocation of fatty acids into the mitochondrial matrix for β-oxidation, showed no change. However, phosphorylated acetyl-CoA carboxylase (ACC), a key metabolic enzyme that when inhibited relieves inhibition of CPT1A to allow fatty acid translocation into mitochondria, showed increased levels in both MCF7 and MDA-MB-231 cells after co-culture with adipocytes. These results support the concept that breast cancer cell metabolism, specifically glycolysis and β-oxidation, is being altered in the presence of adipocytes to utilise fatty acids and glycerol released by adipocytes during lipolysis.

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  • Searching for a Functional Variant in a Non-Coding Genomic Region Associated with Serum Urate

    Dowdle, Amy Evaline (2015)

    Undergraduate thesis
    University of Otago

    Gout was historically known as the king of diseases and disease of kings, reserved for those of wealthy, extravagant lifestyles. Far from being resigned to history books, gout is on the rise worldwide and in New Zealand – where we have the highest rate of gout in the world – Maori and Polynesian populations have a greatly elevated risk of experiencing the debilitating disease. Gout arises as a direct result of increased urate in the blood, and as such many studies worldwide have investigated gout, using serum urate levels as a proxy measure. Recently, two important genome-wide association studies were published – these compare the genomes of those with elevated serum urate to the genomes of control subjects and identify differences. Single nucleotide polymorphisms (SNPs) were identified upstream of MAF, a gene that had not before been associated with serum urate levels. These SNPs were in what is known as a non-protein-coding region, which often play an important role in gene regulation. MAF is a transcription factor that is expressed in the lens of the eye and immune cells, and many studies into MAF have focused on these aspects. However, MAF has also been shown to be expressed in the developing kidney (in zebrafish and mice), which is of great relevance in a study of gout. Therefore, it was hypothesised that one or more of the SNPs upstream of MAF alter regulation of the MAF gene in the kidney, resulting in a change in serum urate levels. An in silico analysis was carried out, using publicly available datasets to produce candidate causal variants upstream of the MAF gene. When multiple datasets were combined and meta-analysed, no specific candidates were produced – however, some SNPs approached significance. This is noteworthy in such a small sample set (n = 18,503, versus 71,149 and >140,000 in the GWAS analyses), and the analysis needs to be repeated on a larger scale. Based on ENCODE annotations, candidate SNPs were selected in areas that looked to be involved in regulation of gene expression. To investigate the putative cis-regulatory role of SNPs, reporter constructs were used in human cell lines. This enhancer assay indicated that one element acts to enhance gene expression, and that the SNPs within this element increased this effect. Finally, a chromosome conformation capture (3C) experiment aimed to determine the interactions occurring between the MAF promoter and these upstream regions, but unfortunately the results were inconclusive. This study contributes to a growing field of research investigating the effects of non-coding DNA on gene expression, in the context of an important disease affecting the lives of New Zealanders everyday. Future findings may provide a novel mechanism by which non-coding variants affect serum urate levels in a system that has not previously been characterised.

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  • Development of a Metabolic Syndrome Mouse Model of Breast Cancer

    Mandani, Anishah Nanji Devji (2015)

    Undergraduate thesis
    University of Otago

    Metabolic syndrome is a cluster of disorders, including obesity, atherosclerosis, inflammation and insulin resistance. It is associated with increased risk of various types of cancers including breast cancer. Obesity in particular is a risk factor for an aggressive tumour phenotype and reduced survival of patients with breast cancer. To understand the underlying mechanisms I aimed to develop and characterise a metabolic syndrome mouse with an orthotopic model of breast cancer. Apolipoprotein E (ApoE) is involved in the catabolism of triglycerides and cholesterol, and the ApoE knockout mouse model is prone to obesity and development of atherosclerosis. The double knockout ApoE/ArKO mouse displays all features of metabolic syndrome. At 6 months of age, wild type, ApoE and ApoE/ArKO C57BL/6 mice were inoculated with the murine breast cancer cell line E0771. Growth of tumours in the mammary fat pad and mouse weight were measured until tumours reached ethical endpoint. The hypoxia marker, pimonidazole, was injected 90min prior to euthanasia, and plasma, organs and tumours were harvested and weighed. Half of each tumour was formalin fixed and paraffin embedded for Immunohistochemical (IHC) analysis of cancer associated adipocytes (perilipin), proliferation (phosphohistone-H3), estrogen receptor status (ERα) and hypoxia (pimonidazole adducts). The other half was frozen and processed for tumour lysates, which were used to measure hypoxia inducible factor 1 (HIF-1α) by Western blotting, and adipokines, using an antibody array. Vascular endothelial growth factor (VEGF) and Insulin-like growth factor binding protein 5 (IGFBP5) concentrations were further analysed by an ELISA assay. HIF-1α levels in EO771 cells were analysed by subjecting the cells to hypoxic conditions. ApoE mice weighed more than wild type and ApoE/ArKO mice, and showed increased cellular proliferation. ApoE/ArKO mice had the least omental fat and the smallest tumours. IHC analysis showed that EO771 tumours in ApoE mice had the highest number of intratumoral, perilipin positive adipocytes (p<0.01). My findings show that breast tumours grown in ApoE/ArKO mice have an aggressive tumour phenotype, with increased proliferation, tumour hypoxia and VEGF concentration. These models represent valuable tools for research that will bridge the gap between cell culture models and breast cancer patients.

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  • Serum biomarkers and paracetamol during post-chemotherapy infections

    Bowden, Emily Ellen (2015)

    Undergraduate thesis
    University of Otago

    Background: Febrile neutropenia (FN) is a common complication of cancer chemotherapy defined as fever with neutropenia below 1.0 x109 /L. Prompt antibiotic treatment is life-saving. Antipyretics (e.g. paracetamol) are commonly used during antibiotic treatment to reduce temperature and discomfort. A phase II randomised, placebo-controlled double-blinded trial of paracetamol during FN was completed at Wellington Hospital. This study aimed to determine whether paracetamol affects temperature or quality of life (QoL) during FN, and to assess biomarkers as potential secondary endpoints. Methods: Participants received 1g oral paracetamol or placebo six hourly for 42 h. Tympanic temperature was monitored four hourly. Blood was taken 0, 4, 24 and 72 h after FN presentation. In the current study cytokine bead array was used to determine levels of TNF-α, IL-6, IL-8 and IL-10, procalcitonin (PCT) was assessed by ELISA, and C-reactive protein (CRP) using an immunoturbidimetric method. Participants completed the EQ-5D-5L QoL questionnaire daily and the FACT-N questionnaire on day 3. Results: Of 37 enrolled patients, 22 participants developed FN and received at least one dose of paracetamol (n = 13) or placebo (n = 9). Treatment groups had comparable demographics and vital signs at baseline. Per pre-determined criteria, 23% and 33% of patients had successful treatment in the paracetamol and placebo groups respectively (not significant). Peak temperature was significantly lower in paracetamol- than placebo-treated patients on days 1 and 2 (difference 0.7°C and 0.6°C, respectively, p < 0.01 and p = 0.03), but not on day 3. Average daily temperature was also significantly lower in the paracetamol than placebo group. IL-6, IL-8, IL-10 and TNF-α were raised at baseline and/or 4 h and declined thereafter. PCT peaked at 24 h. Presentation and 4 h levels of IL-6, IL-8, IL-10, PCT and TNF-α, as well as 24 h PCT and 72 h IL-8 levels, were associated with adverse outcome. IL-6 was higher in the placebo than paracetamol group at 24 h (p <0.02). QoL scores were worse in the paracetamol group during the first two days of treatment (difference not significant). Conclusions: Paracetamol was an effective antipyretic during FN. Serum biomarkers change during FN, and IL-6 and IL-8 are promising secondary outcome measures for future trials. The adverse impact of paracetamol on QoL scores was unexpected and requires confirmation in a larger study.

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  • Determining biomarkers for a diagnostic test of Chronic Fatigue Syndrome / Myalgic Encephalomyelitis

    Denny, Lisa (2015)

    Undergraduate thesis
    University of Otago

    Chronic fatigue syndrome / Myalgic encephalomyelitis (CFS/ME) is an unexplained chronic multi-system illness, which leads to a lifetime of impairment. Symptoms indicative of the disease include immunological dysregulation, incapacitating fatigue, cognitive impairments, pain in the lymph nodes, and post-exertional sickness. The pathophysiology of CFS/ME is unknown, however several potential causes of development of the disease have been speculated. Consequently, due to a lack of understanding both medically and scientifically, there are no validated laboratory tests for diagnosis or management. Filling this gap of knowledge by finding an appropriate biomarker would aid the medical community in determining appropriate treatment. This pilot study aims to evaluate whether certain molecules in the blood of CFS/ME patients may be biomarkers to aid in diagnosis. This involves analysis of cytokine levels in CFS/ME patients with matched controls, and changes in cytokine levels following a pre-determined exercise regimen, in which patients are known to perform poorly. Additionally, the ratio of translational initiation factor eIF2α, to its stress activated phosphorylated peIF2α derivative, will be examined in white blood cells of patients and controls. We hypothesise that these molecules have the potential to be informative in relation to immune deterioration, a hallmark of CFS/ME. To test this hypothesis we obtained blood samples from 10 CFS/ME patients and 10 matched controls according to the International Consensus Criteria. In addition, following a separate exercise study with 11 CFS/ME patients and 3 MS patients as controls, all samples were fractionated to separate plasma, lymphocyte and neutrophils. The prepared plasma samples from all participants were simultaneously examined for expression of 27 cytokines. Statistical analysis revealed Interleukin-9 (IL9) and vascular endothelial growth factor (VEGF) expressed at significantly higher levels in CFS/ME patients compared to healthy controls (P<0.05). IL-9 and IL-13 were represented in both analyses, which indicates their potential as biomarkers for CFS/ME. Western analysis of proteins isolated from white blood cells detects both phosphorylated and unphosphorylated states of eIF2α with respective purified antibodies. Western analysis showed protein peIF2α to be slightly higher in patient lymphocytes compared to controls, though further experiments will need to be undertaken to determine the value of this result. This work suggests potential biomarkers that can be seen in blood, and Western blot analyses of additional patient and control samples will define whether a change in molecular state of eIF2α could be developed into a diagnostic test. This study gives a means to form a larger cohort for analyses on CFS/ME patients to enhance on current results in terms of identifying a possible biomarker for the disease.

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  • Impact of G-quadruplex structures and DNA methylation on allelic drop-out during in vitro amplification of imprinted genes

    Taylor, Millie Grace (2015)

    Undergraduate thesis
    University of Otago

    Diagnostic testing for genetic disorders or techniques such as preimplantation genetic diagnosis (PGD) both require accurate PCR genotyping (1). The failure of amplification of one allele, referred to as allelic drop-out (ADO) can confound genotyping results by falsely identifying heterozygotes as homozygous (2). A unique ADO mechanism has previously been demonstrated to occur consistently in the imprinted MEST gene, where both DNA methylation and G-quadruplex (G4) DNA structure contributed to allele loss (3). G4s are alternative DNA structures that form in G-rich regions due to the self-associating ability of guanine. Under certain ionic conditions, four guanine residues bind together either within or between strands to form a G-quartet, which can then stack upon one another to form the higher order structure. Such structures have the ability to act as a steric block to Taq polymerase. This effect is exacerbated when the G4 is methylated due to an increased thermal stability (4). This thesis explored the hypothesis that ADO via this mechanism occurs more widely throughout the imprinted genome. To test this, 22 target loci containing G4-DNA motifs were selected from 16 imprinted genes and an assay designed to detect ADO during PCR was developed. This required the creation of two variant alleles via the introduction of a single nucleotide polymorphism (SNP) with differential primer design. Both variants were then subjected to in vitro methylation and template mixing PCR experiments followed by Sanger sequencing to reveal mono-allelic or bi-allelic amplification. Of the 22 amplicons initially selected, only 14 were able to be consistently amplified and were thus used for this analysis. This method revealed that MEST is not alone in being susceptible to ADO events, with nine other amplicons showing either complete or partial mono-allelic amplification when methylated G4s were present. To confirm that the predicted G4 motifs did adopt the structure, CD spectroscopy was used. This revealed that these motifs were capable of forming the secondary structure and therefore contributing to ADO events. This work confirms that the effect of cytosine methylation and G4 regions on ADO that was previously observed (3) occurs more widely throughout the imprinted genome, and further highlights the need for diligence in both a diagnostic and research setting when analysing imprinted genes or other methylated regions.

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  • Inactivation of a Thiol-Dependent Enzyme by Urate Hydroperoxide

    Hamzah, Melanie Rosina (2015)

    Undergraduate thesis
    University of Otago

    There are links between high serum urate (hyperuricemia) and many inflammatory diseases, yet the mechanism is obscure. Urate, the product of purine and ATP break down, builds up in plasma because humans lack the enzyme uricase to convert it to allantoin, which is freely excreted. Urate may benefit health by acting as an antioxidant that scavenges reactive oxygen species. However, hyperuricemia is associated with gout, metabolic syndrome and cardiovascular disease. Oxidative stress is also associated with all these inflammatory diseases. During oxidative stress urate is converted to several reactive electrophiles, including urate hydroperoxide. This novel oxidant could contribute to the adverse effects of urate. Urate hydroperoxide is formed when urate is oxidized to a radical that subsequently combines with superoxide. Activated white blood cells called neutrophils, and xanthine oxidase along with myeloperoxidase/lactoperoxidase, can produce urate hydroperoxide. Previous studies characterized the formation of urate hydroperoxide and its oxidation of small biomolecules. In this investigation, I explored oxidation of thiols and the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by urate hydroperoxide. The effectiveness of urate hydroperoxide as a thiol oxidant was compared with taurine chloramine. Ellman’s assay for reduced thiols was used to measure depletion of cysteine residues on GAPDH by urate hydroperoxide and taurine chloramine. GAPDH was exposed to oxidants in a dose-dependent manner, then assayed by measuring its ability to catalyse the production of NADH. Mass spectrometry was used to identify specific modifications of GAPDH. Urate hydroperoxide oxidized exposed thiols on GAPDH and fully inactivated the enzyme at a ratio of about 5:1. Half of its activity was recovered by reduction with DTT. In comparison, taurine chloramine inactivated GAPDH at approximately 10:1 and DTT reduction recovered all activity. Hence, urate hydroperoxide inactivates GAPDH by reversible and irreversible routes. GAPDH increased in molecular mass by 132 Da with exposure to urate hydroperoxide, indicating the formation of a GAPDH-urate adduct. However, I could not identify which residue was modified with a tryptic digest. Formation of urate hydroperoxide during inflammation and its subsequent oxidative reactions may explain some of the adverse effects of hyperuricemia.

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