33 results for Undergraduate, 2014

  • The Epidemiology of Breast Cancer in Oman

    AL Farsi, Shamsa (2014)

    Undergraduate thesis
    University of Otago

    Background Breast cancer is the most common cancer in females around the world. Annually, more than one million women are diagnosed with breast cancer globally. In Oman, breast cancer is also the most common cancer in females and its incidence has been rising over the years. Published studies have shown that many Omani women have late stage breast cancer at presentation. Possible reasons for this have not been explored. Although Oman has in place a mortality registration database, no population-based mortality data from breast cancer are available. Likewise, the only data available about survival rate of people with breast cancer come from a hospital-based study. Aims The main aims of the research described in this thesis are: 1) To identify the extent of delay in breast cancer diagnosis in Oman. 2) To assess the relationship between delay and socio-demographic characteristics, medical and obstetric history, nature of presenting symptoms and women’s knowledge about breast cancer. 3) To identify reasons for delay in seeking medical help for self-detected breast cancer symptoms in Omani women. 4) To calculate the population-based survival rate from breast cancer in Oman. Methods Aims 1, 2 and 3 were addressed by a study of 150 patients attending oncology clinics in both the Royal Hospital and Sultan Qaboos University Hospital, who were interviewed using structured questionnaires. Patient delay was defined as a period of three months or more between an individual's first awareness of a sign or a symptom of illness and the initial medical consultation. In order to calculate survival rate, the National Cancer Registry records were to be linked to mortality databases in Oman (Directorate General of Civil Status and to the Parallel Mortality Database). Results The final analysis of delay included 144 patients with breast cancer. The median time taken by women in this sample between discovering the breast symptoms and seeing a doctor was 14 days. 56.9% of the patients had a medical consultation in less than a month after detecting symptoms, whereas 20.1 % had a consultation within 1 to 2 months. 22.9 % of the patients delayed consultation by ≥ 3 months. Of the socio-demographic characteristics examined in this study, it was observed that older age, low educational level and employment status were associated with patient delay. Practice of breast self-examination and having a history of chronic disease were also predictors of delay. 44% of patients had early stage disease (stage I/stage II) compared to 56% of patients with late stage disease (stage III/stage IV). However, patient delay was not associated with advanced stage cancer in this study. The main reasons given for delay were: failure to recognise the symptoms to be breast cancer, not seeing oneself at risk for breast cancer, fear and embarrassment, use of alternative therapy and family and work commitments. Due to ethical consideration, I was not able obtain data from Omani NCR and therefore the linking to mortality databases was not possible. Conclusion This study is the first in Oman to investigate the extent of patient delay for women with self-discovered breast symptoms and the factors that influence this delay. The findings of this study indicate the need for public education aimed at raising breast cancer awareness. Further, initiating a screening program in Oman should be considered to help women achieve diagnosis of the disease in its early stages. Population-based cancer research should be encouraged in Oman, and efforts should be taken to improve the quality and completeness of cancer data, which are important in providing on-going monitoring of cancer.

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  • Epigenetics and Expression of SFRP1 and PPARG and Epigenetic Effects of Glucocorticoids in B-cell Acute Lymphoblastic Leukaemia

    Foster, Timothy John (2014)

    Undergraduate thesis
    University of Otago

    B-cell Acute Lymphoblastic Leukaemia (B-ALL), a cancer of immature B-lymphocytes, is the most common cancer in childhood. This cancer is characterised by widespread abnormalities of DNA methylation, when compared with non-cancerous blood cells. DNA methylation is a chemical modification of the cytosine residues of DNA, and only cytosine residues immediately followed by guanine residues (so called CpG sequences or sites) undergo methylation. Methylation of CpG sites in gene promoter regions leads to non-expression of the methylated gene. DNA methylation abnormalities in cancers (such as B-ALL) have received significant attention over recent years, and have been shown to have significant biological effects in tumour cells, due to abnormal expression of the aberrantly methylated genes. This project aimed to show that the putative tumour suppressor genes, SFRP1 and PPARG, showed increased DNA methylation in B-ALL cells, when compared with normal blood cells and that this was associated with reduced expression of these genes in B-ALL. The methylation of the gene promoters was determined by bisulphite sequencing and gene expression by qRT-PCR. The results showed that PPARG and SFRP1 both show increased methylation in the gene promoter regions of B-ALL cells, when compared with normal blood cells. SFRP1 has previously been shown to show reduced expression in B-ALL and the qRT-PCR results showed that the PPARγ-1 transcript from the PPARG gene showed reduced expression in B-ALL cells, when compared with B-cells from normal blood as well as normal whole blood. Overall, it was concluded, on the basis of these results and others’, that SFRP1 and PPARG show reduced expression compared with normal blood cells, due to promoter methylation in B-ALL. It has also been suggested in the literature that glucocorticoid drugs (analogues to the steroid hormone cortisol) can alter the methylation of CpG sites in individual genes (in non B-ALL cells). This is of interest in the context of B-ALL, as glucocorticoids are well known to be strong anti-leukaemia agents and are used in B-ALL treatment. Glucocorticoids are also known to affect the expression of many genes, an effect that is compatible with changing the DNA methylation of cells. Therefore, this project also aimed to show that the glucocorticoid dexamethasone could induce changes in DNA methylation in many genes within the genomes of B-ALL cells. Multi-gene methylation was measured using the, relatively new, RRBS technique with the NALM-6 human B-ALL cell-line with or without exposure to dexamethasone acting as my model of B-ALL. The results showed a number of methylation changes throughout the genome, with some particularly strong methylation changes observed in the promoter regions of the genes SPINT2, GATA3, IRX5, SOX13, GATM, PDGFA and DOCK10; genes implicated in cancer or in steroid-sensitive metabolism (such as energy metabolism). These results suggest that steroids do indeed alter the DNA methylation of B-ALL cells, which, if these results are replicated, is a novel mode of action of glucocorticoids in B-ALL treatment.

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  • Abduction Strength Deficiency: How Common, How Early and How Amendable?

    Chen, Shumou (2014)

    Undergraduate thesis
    University of Otago

    Gluteus medius strength deficiency has been linked to various injuries of the lower limb (Fairclough et al., 2007, Bullock-Saxton et al., 1993, Powers et al., 2003, Williams and Cohen, 2009). However there is limited information in the literature about the prevalence of this condition among healthy individuals. When observing peoples’ walking patterns, it is common to see excess side to side movement indicative of abduction strength deficiencies. However the conventional dynamometry strength testing generally show normal results despite the person having an abnormal gait pattern and the conventional exercise used to treat this condition is not yet proven to be effective. A recently published study on Australian Rules footballers suggested that hip abduction weakness does occur in healthy people when a previously unpublished test was used. It uncovered the weakness and using the same position as an exercise was capable of correcting it (Osborne et al., 2012). The current study investigated the testing position against conventional testing positions and the exercise against conventional exercises. This study also investigated the possibility of growth related hip abduction strength deficiency in high school aged males. Three studies were used to investigate the new testing position and exercise. An observational study among 101 healthy adults was completed to investigate the prevalence of hip abduction strength deficiency and compare the new hip abduction testing position to conventional hip abduction testing positions. An interventional study was completed to investigate the effects of the new abduction exercise against a conventional abduction exercise and an adduction exercise as controls. This study involved three 1st XV rugby teams with a intervention period of two months. The third study was also an observational study involving 105 high school students. This study investigated the prevalence of abduction strength deficiency in relation to growth among high school aged males. In the study involving healthy adults, it was found that people tested the weakest in the new testing position. When the new hip abduction exercise was compared to conventional hip abduction exercises and an addcution exercise as a control, there were no significant strength improvements. The third study also found no hip abduction strength deifciency realted to growth among high school aged males. The recently published testing position may be a useful tool in uncovering hip abduction strength deficiency but as an exercise it did not produce any significant strength gains. Although a recently published study on Australian Rules Footballers suggested that hip abduction strength deficiency may occur due to growth (Osborne et al., 2012), this study suggested there were no growth related hip abduction strength deficiency.

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  • Cranberry Capsules: The efficacy of cranberry capsules in the management of acute radiation cystitis in men with prostate cancer

    Hamilton, Katelin (2014)

    Undergraduate thesis
    University of Otago

    Background: Acute radiation-induced cystitis is a common side effect of radiation therapy (RT) to the pelvis, with up to 40-50% of prostate cancer patients suffering from cystitis to some extent. Acute symptoms can occur within weeks of radiation treatment and include urinary urgency, frequency, dysuria, and hematuria. Currently there is no effective treatment for radiation cystitis. Here, in a double-blinded pilot study, we investigated the effect of standardised cranberry capsules on the extent of radiation-induced cystitis, and how this impacts on quality of life in prostate cancer patients. Methodology: A total of 41 men receiving RT for prostate cancer at the Southern Blood and Cancer Center (SBCC) in Dunedin participated in this trial, which opened in May 2012. The men took one capsule a day during breakfast from their first day of treatment until two weeks after completion of treatment. This took place regardless of which arm they were randomised to. Cranberry capsules contained 72mg of proanthocyanidins (PACs) each and were indistinguishable from placebo capsules. Patients, clinicians and research assistants were blinded to the content of the capsules. Severity of cystitis was assessed using a modified urinary domain of the Expanded Prostate Cancer Index Composite (EPIC) scale. Items included severity of symptoms (pain, blood in urine, leakage, urinary frequency in day and night) use of pads and symptomatic relief (URAL), as well as the effect of these symptoms on daily life. Results: This thesis analysed the results of the first 10 cranberry and 10 control patients who presented with low baseline EPIC scores. The results showed that cranberry capsules seem to decrease certain aspects of radiation cystitis both with regard to physical symptoms and the effect on quality of life. However results in this small cohort did not generally reach statistical significance and limitations of the trial methodology have been recognised. Conclusion: In light of the limitations of this trial and the positive trends in the results, further investigation is warranted. Future research should focus particularly on establishing consistent hydration levels, regulating the use of symptomatic relief and developing improved methods for assessing the level of acute radiation cystitis.

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  • Quality of Diabetic Foot Care in Oman

    Al-Busaidi, Ibrahim Saleh (2014)

    Undergraduate thesis
    University of Otago

    Background Diabetes mellitus is a common and increasingly important chronic disease worldwide. In Oman, the setting of this thesis, the prevalence of diabetes was 12.3% in 2008. Diabetes causes substantial morbidity and mortality, with diabetic foot disease (DFD) being one of the most serious and costly complications of diabetes. Good preventive foot care measures, patient and provider education and adherence to proper foot self-care practices can reduce the risk of developing DFD by up to 85.0%. No published study has investigated diabetic foot care in Oman. Objectives The aim of this study was to explore the quality of diabetic foot care provided by primary and secondary health care professionals in an area of Muscat, Oman. The specific objectives were: 1) To ascertain the level of foot self-care amongst people with diabetes; 2) To determine the level of foot care education for people with diabetes provided by primary and secondary health care professionals; 3) To determine the level of professional foot care services provided to people with diabetes; and 4) To examine the association between foot self-care practices and known risk factors for diabetes-related foot disease (DRFD). Methods The study setting was eight primary health care clinics and one polyclinic in Alseeb, Muscat, Oman. A convenience sample of 350 Omani patients with diabetes (310 from primary health care and 40 from the polyclinic) were invited to participate in the study. A questionnaire developed from two pre-existing questionnaires and pre-tested and translated into Arabic, was administered by author of this thesis and research assistants. The questionnaire included six domains including demographic details, patient-reported DRFD, foot self-care, foot care education, and professional foot care. Data were checked, entered into Excel spreadsheet, and analysed using STATA Statistical Software version 12.0 (2012). Proportions and means were calculated as appropriate for variables of interest. To examine the association between dependent and independent variables, a one-way analysis of variance was used for categorical variables and product-moment correlation test for continuous variables. Ethical approval was obtained from the Medical Research and Ethics Review Committee, Ministry of Health, Oman. Results Of the 350 participants, 62.3% were female and more than half of the patients were illiterate (52.9%). DRFD was found to be common in this population with more than 55.0% of the study population reported having at least one or more sensory peripheral neuropathy symptoms, and almost half (49.1%) complained of one or more peripheral vascular disease symptoms in the last month. In spite of this, patients often did not adopt all recommended behavioural foot care practices. For example, 54.7% did not look at the bottoms of their feet daily, 58.4% reported using moisturising creams or lotions between their toes daily, and 46.0% reported wearing traditional Omani sandals which do not offer protection from injuries. Fewer than half of the participants reported receiving advice or information on recommended foot care practices from their diabetes health care professionals. Professional diabetes foot care services were suboptimal. For example, 20.4% of participants reported never being asked about numbness in their feet and 21.7% reported having been seen by a podiatrist during the previous year. In the final model, a statistically significant association was found between foot self-care scores and level of formal education, diabetes treatment and professional foot care. Conclusions and recommendations Despite the presence of DRFD in this Omani population with diabetes, the overall quality of diabetic foot care was suboptimal. From the patient perspective there is a need for high quality diabetic foot care education to improve patients’ foot care awareness and self-management. Patient education requires good communication skills and an understanding of patients’ education levels, and the influence of cultural, social and religious practices. A multidisciplinary team approach and ongoing foot care education for health care professionals is needed in order to improve their diabetic foot care knowledge and skills. To better understand the context, barriers to regular recommended foot self-care practices needs to be explored further, and the reasons for non-adherence to the Omani diabetes foot care guidelines by health care professionals requires further clarification. Nevertheless, findings from this study will be useful for health care planners and policy makers in Oman and neighbouring countries with similar health systems for improving the overall quality of diabetes foot care.

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

    Vermunt, Jane (2014)

    Undergraduate thesis
    University of Otago

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

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  • Rates of fructose malabsorption in gout cases and non-gout controls in Christchurch, New Zealand

    Batt, Caitlin (2014)

    Undergraduate thesis
    University of Otago

    Background: Gout is a common form of inflammatory arthritis caused by the precipitation of monosodium urate crystals in the joints. The prevalence of gout in New Zealand is 9.62% in Māori men and 5.12% in European men. Hyperuricaemia is a risk factor for gout and has been associated with fructose, a sugar found in fruit and sweeteners. Fructose malabsorption has a prevalence of 34% in healthy individuals. The aim of this case-control study is to observe whether there is an association between fructose malabsorption and gout. Method: Cases (n=65) were those with clinically diagnosed gout. Controls (n=65) were age and gender matched from the general population. Fructose malabsorption was measured in cases and controls using a breath test that measures gas products of the metabolism of fructose by bacteria in the gastrointestinal tract. Results: The rate of fructose malabsorption in cases was 49.2% and in controls was 53.8%. The odds of malabsorbing fructose in those with gout compared to those without was 0.82 (95%CI 0.41-1.67). Conclusion: There is no significant difference in rates of fructose malabsorption between gout cases and controls. Future studies are required to determine whether the effect of fructose load on serum urate is different between malabsorbers and absorbers in gout cases and those without gout. In future practice dietary advice for gout could be individualised based on absorption status, improving compliance.

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  • Investigating and enhancing rural communities’ existing mental health service networks.

    Erskine, Nicholas Robert (2014)

    Undergraduate thesis
    University of Otago

    Mental illness is a growing concern throughout the world. The burden of mental illness is increasing globally, currently representing 7.4% of the total years of life either lost due to premature mortality or lived with disability. In New Zealand, the estimated life-time risk of meeting the criteria for one or more mental illnesses is 46.6%. It is therefore essential to have a secure and effective mental healthcare system throughout the country. This can be particularly challenging to deliver in rural communities due to the unique circumstances that they face. Previous studies have found that rural residents have decreased access to mental health specialist services, and increased exposure to mental health risk factors. However, there is ongoing dispute as to rurality’s overall effects on mental health status. Further research is required. Despite the urgent need to better understand the interactions between rurality and mental health, there is a severe paucity of research in the area on a national and global scale. This action research project helped investigate the impact of rurality on mental health within New Zealand by interviewing mental health and wellbeing service providers operating in Wanaka and Balclutha. The information gathered from these rural providers included their difficulties with interagency collaboration, the effects caused by rural providers working beyond their role descriptions, rural community characteristics that impact on service access, and service deficits within rural communities. This data was used in conjunction with established literature in order to help understand several of the issues that are important to mental healthcare in rural New Zealand communities. From this background, attempts were made to help improve Balclutha’s existing mental health service network. These attempts included facilitating networking between services, creating a community-specific service information package, and encouraging the use of minimal-contact guided self-help therapies in a community-appropriate manner. By investigating issues that are pertinent to rural mental health in New Zealand, this project helps address a major gap in current knowledge. The feasibility testing and process evaluation of enhancing an existing mental health service network will also serve to greatly benefit ongoing research and action in rural New Zealand.

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  • Patient autonomy in end of life decisions

    Chamberlain , Joshua (2014)

    Undergraduate thesis
    University of Otago

    End of life scenarios must be patient driven and focused. In this thesis it is argued that patient intention is often misrepresented by being assimilated to extreme and clear-cut cases where patients are committed to a certain path of action. The role of the patient in decision-making, a role that has developed through the advent of autonomy and subsequently informed consent, has created an environment that is so dependent upon the patient that our duty of care and the role of experience in difficult clinical decisions can be obscured or forgotten. Given the experience of clinicians and the conflicting duties to which they often find themselves subject, questions have been raised over the intent of physicians in end of life situations but the extent to which these should lead to the very same questions being asked of patients is under-explored in the literature. The varying end of life decisions and the moral acceptability attached to them is a concept that is poorly understood by a mainstream audience. This, among other things is part of the misconceptions that are commonly held about euthanasia. Finally I explore what the future holds for euthanasia and end of life decisions. The part the patient plays in these decisions is coming under more scrutiny and as it does, the role of the physician is being examined as well. Happenings in the Netherlands over the last decade have prompted a new surge of calls for reform in this most ancient of debates, this surge I propose could lead to a new era of dialogue and relationship focused care in end of life, where a one size fits all approach is not welcome.

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  • Characteristics of road traffic injuries and potential risk factors in Oman

    Al-Risi, Ahmed (2014)

    Undergraduate thesis
    University of Otago

    Background: Globally, road traffic injuries have increased by almost 46% in the twenty years prior to 2010. This makes road traffic crashes (RTC) the tenth leading cause of death in the world and the leading cause of death of young people. Oman, a wealthy country where motorising is increasing rapidly, has a very high road traffic mortality. In Oman, road traffic deaths and injuries are the main external cause of morbidity in young adults and have a direct effect on the economic and health resources of the country. Road traffic crash research has only recently begun in Oman. The modifiable causes of this problem and most effective response have not yet been well-explored. Aims: The main aims of the research described in this thesis were: - To describe the distribution of road traffic crashes in Oman by time, person and place using the data collected by the Royal Oman Police and examine their assessment of reasons for crashes, from 1985-2010. - To collect data in order to estimate the prevalence and distribution of known risk factors in road traffic crashes in Oman, focusing specifically on Sohar. Methods: Two studies were conducted to achieve the aims of this thesis. Firstly, a retrospective case series used the Royal Oman Police data from 1985-2010. This examined trends in the police data on road traffic crashes across the whole time period and then summarised the most recent available year of data (2010) in more detail. Secondly, a prospective case series of injured drivers admitted to Sohar Hospital Emergency Department was carried out. Between 20 February 2012 and 20 March 2012, consecutive injured drivers admitted to Sohar Emergency Department were recruited to the study. Questionnaire-based face-to-face interviews were held to collect data on socio-demographics, circumstances of the crash, and known risk factors for road traffic injuries, including risk behaviours at the time of the crash and usual behaviours. All admitted injured drivers were recruited apart from the most seriously injured drivers who were taken by ambulance to the capital city Muscat for further investigations, and those who died from their injures. Results: According to Royal Oman Police data (1985-2010), total deaths and injuries from RTCs have increased by almost 300% in Oman since 1985. An element of speeding was reported for all the crashes since 1992. The victims of road traffic crashes were mostly the young age group (21-30 years). More drivers have been killed than any other road user group, constituting 43.8% of total road traffic deaths, and passengers have been the most injured, constituting 48.2% of total road traffic injuries. In the Sohar study, 250 injured drivers, 75% males and 25% females, were interviewed with a 100% response rate. Interviewed injured drivers were found to have spent long hours on the roads and had driven for long distances. Overall, less than 5% of injured drivers were over the age of 35 years. There was a marked difference in the age distribution of male and female injured drivers. Among men almost half (49.5%) of the injured drivers were 18-25 years old and 45.7% were 25-35. Among women, 95% of injured drivers were 25-35 years old. Generally, male drivers had more traffic violations than female drivers with 83% of males reporting at least three traffic violations over the past five years, whereas almost half of the females reported one or no, traffic violation over the same period. The highest frequency of crashes occurred on Saturdays and Thursdays (18.8% and 17.6% respectively) and the majority of the injured drivers were either familiar or very familiar with the roads on which they crashed. It was clear that the injured drivers in Sohar routinely ignored the traffic laws and reported risky driving behaviours. For instance, less than 10% of injured male drivers and only 56% of female drivers were wearing their seatbelts at the time of the crash. Also, a high proportion of both male and female drivers were travelling at a speed of 100-140 km/hour at the time of their crash (65.4% male and 58% female drivers). Moreover, 31.4% of injured male drivers and 24% of injured female drivers were using their cell phones at the time of their crash. When describing their usual driving behaviour, 52.8% of all interviewed drivers reported that they never or almost never wear seatbelts while driving. Conclusions: Overall, the incidence of road traffic crash injuries and deaths is high and increasing in Oman with a high prevalence of known risk factors for road traffic injuries even where protective legislation exists. Even though this case series cannot establish that these risk factors cause road traffic injuries in this population, experience from other countries suggests that appropriate legislation and increased enforcement could reduce road traffic injuries in Oman.

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

    Scholtz, David Johannes (2014)

    Undergraduate thesis
    University of Otago

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

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

    Worters, Thomas David (2014)

    Undergraduate thesis
    University of Otago

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

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  • A novel technique to investigate coronary microvascular perfusion in diabetes

    Nissen, Hazel Merete (2014)

    Undergraduate thesis
    University of Otago

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

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  • The Role of Dendritic Cell Subsets in the Early Immune Response to Mycobacterial Infection

    Manners, Kate Meredith (2014)

    Undergraduate thesis
    University of Otago

    The early immune response to tuberculosis (TB) is poorly understood. However, recent evidence has shown that dendritic cells (DC) are important for both control of bacterial growth and for activating adaptive immunity. DC are divided into functionally and phenotypically distinct subsets, but the role of each of these subsets in the early immune response to tuberculosis remains unknown. This study aimed to investigate the roles of DC subsets in a murine model of mycobacterial infection. Use of a fluorescent strain of the attenuated TB vaccine strain Mycobacterium bovis BCG, combined with multicolour flow cytometry, enabled detection of BCG associated DC subsets within the murine lung during the first 14 days of infection. The early immune response to mycobacterial infection was found to be highly dynamic, with significant variation in the proportion of BCG associated cells in each DC subset of the lung during infection. CD11b+ conventional DC (cDC) and plasmacytoid DC (pDC) were found to be associated with BCG during the first 14 days of infection, whereas CD103+ cDC were not. Differential expression of the cell surface markers GR-1, CD11b and CD11c detected within previously described DC subsets correlated with their propensity to associate with BCG. Conventional DC and pDC were found to respond differently to mycobacterial infection; cDC upregulated expression of CD86 during infection whereas pDC were found to do the opposite, suggesting that these subsets have distinct roles during early infection. Importantly, and contrary to current dogma, alveolar macrophages were not found to associate with BCG during early infection. This study highlights the distinct roles of different DC subsets during mycobacterial infection. This research may have implications for the development of a novel vaccine or therapy for tuberculosis, for which there is a significant need.

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  • Enhancing Adoptive Cell Transfer for the Treatment of Cancer

    Shields, Nicholas James (2014)

    Undergraduate thesis
    University of Otago

    Cancer is currently the leading cause of death in New Zealand, accounting for 30% of all deaths in 2010. Due to factors such as an ageing population and poor lifestyle choices, the incidence and mortality of this disease is set to increase dramatically in the coming decades. This will exert substantial stress on the healthcare system, highlighting the urgent need for improved cancer therapies. The adoptive transfer of tumour-specific T lymphocytes, known as adoptive cell transfer (ACT), is a promising new approach to the treatment of cancer that has proven effective for the treatment of haematological malignancies and metastatic melanoma, achieving long-lasting clinical responses in approximately 50% of patients. The challenge now remains to enhance the therapeutic efficacy of ACT to enable a broad application of this therapeutic approach to the treatment of multiple types of cancer and achieve clinical responses in all patients. Using ovalbumin as a model tumour-antigen, the aim of this study was to (a), compare the ability of dendritic cells (DCs) and macrophages to generate tumour-specific CD4+ and CD8+ T cells for use in ACT, and (b), assess the phenotype and function of these generated cells. Splenocytes isolated from OT-I (CD8+) and OT-II (CD4+) transgenic mice were cultured with dendritic cells pulsed with respective ovalbumin peptide epitopes. After 10 days, these cells were restimulated with either dendritic cells or macrophages and cultured for a further 10 days. Macrophages could effectively generate tumour-specific CD8+ T cell responses but were poor inducers of CD4+ T cell responses compared to dendritic cells. In the case of CD8+ T cells, antigen-experienced T cells that were restimulated with macrophages exhibited superior phenotypic characteristics for use in ACT compared to DC-restimulated cells. Both DCs and macrophages predominately generated effector memory CD4+ T cells (CD44+CD62L-) and effector memory CD8+ T cells (CD44+CD62L-). Further phenotypic analysis of in vitro-generated T cells revealed distinct expression patterns of markers associated with T cell dysfunction, survival and differentiation. In response to their cognate antigen, generated T cells produced high levels of TNF-α and IFN-ɣ, suggesting that these cells can mediate effective tumour destruction in vivo. Future experiments will determine the length of time that adoptively transferred T cells persist in vivo and assess the efficacy of ACT using generated CD4+ and CD8+ T cells in an in vivo murine B16-OVA melanoma model.

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

    Moustafa Albathish, Boushra (2014)

    Undergraduate thesis
    University of Otago

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

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  • Effects of Male Age on Reproductive Success and Offspring Fitness in Zebrafish (Danio rerio)

    Gardiner, Thomas Clarence (2014)

    Undergraduate thesis
    University of Otago

    Many studies have observed that sperm quality decreases both in humans and other animals as individual’s age; however, the direct effects of age on reproduction and offspring fitness are not yet fully understood. A preliminary study in zebrafish has shown some survival advantages in offspring from old males despite reduced sperm quality. However, what effects, if any, this has on subsequent generations is not clear. Sperm samples from the offspring of old and young males (F1 generation) were collected and in-vitro fertilisations (IVFs) used to produce an F2 generation. Fitness parameters (e.g., fertilisation success, hatching success, and survival) and sperm traits (e.g., motility and swimming velocity; measured using computer assisted sperm analysis) of F1 from young and old males were compared. Results demonstrate a significantly higher proportion of fertilised embryos produced from IVFs using the sperm of the offspring of old males compared to the young males. But, we see no significant differences between the two groups in the proportion of fertilised embryos that hatch or survive. There was a significant difference in the semen volume expelled during sampling where the F1 of old males tended to produce more sperm than the F1 from young males. But, there was no difference in any other sperm traits and no sperm traits were significant predictors of fertilisation success. These data suggest that there is a reproductive advantage acquired from old males in the form of greater sperm production and increased rates of fertilisation. Overall, the data are consistent with previous findings suggesting that old males deliver fitness benefits.

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  • Involvement of mitochondria and peroxiredoxin in TNF-mediated necroptosis

    Zawari, Masuma (2014)

    Undergraduate thesis
    University of Otago

    Necroptosis is a form of regulated necrosis considered to be involved in several pathophysiological conditions such as inflammation and ischaemia-reperfusion injury. The cellular events involved in the initiation of necroptosis are unclear, but recent studies indicate that the pseudokinase mixed lineage kinase domain like (MLKL) plays an important role. Understanding the molecular mechanisms regulating necroptosis is therefore an important priority that may lead to the development of new therapies. Mitochondria and reactive oxygen species generation has been reported in necroptosis, however it is not clear whether these play a direct role in the death process. These changes were investigated in a TNF-dependent model of necroptosis using mouse dermal fibroblasts (MDF) from wild-type and MLKL knock-out animals. Viability analysis by FACS and live cell imaging indicated early signs of cell death by 60 min and by three hours about half of the population were executed by necroptosis in an MLKL dependent manner. TMRE labelled cells showed a loss of mitochondrial membrane potential before cell death. Rapid increase in oxygen consumption 30 min after the injection of TNF indicated a direct link between the necrosome and downstream signalling to mitochondria for efficient necroptosis to occur. This is the first report of MLKL dependant effects on mitochondrial function. Western blot analysis indicated a rapid accumulation of oxidized mitochondrial peroxiredoxin in an MLKL dependent manner. Timing of mitochondrial changes and oxidation of peroxiredoxin suggest they may be early markers or even executors of cell death rather than a consequence of cellular damage. However further investigation is required to determine the importance of these changes in cell death.

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

    Robinson, Andrew Mark (2014)

    Undergraduate thesis
    University of Otago

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

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

    Marshall, Christopher Joseph (2014)

    Undergraduate thesis
    University of Otago

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

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