33 results for Undergraduate, 2014

  • 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on Australian Rules footballers suggested that hip abduction weakness does occur in healthy people when a previously unpublished test was used.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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  • 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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  • 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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  • 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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  • 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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  • 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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  • 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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  • 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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  • 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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  • Understanding Batten disease: CLN5 expression in CLN6 deficient ovine neural cultures.

    McIntyre, Kristina (2014)

    Undergraduate thesis
    University of Otago

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 In CLN6-/- ovine neural tissue, CLN5 protein expression was reduced (McIntyre, K.M., summer studentship unpublished observations, 2013/2014).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

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  • Is Prolactin Action on Forebrain Neurons During Pregnancy Important for Maternal Neurogenesis and Behaviours?

    Cashen, Sarah Christina (2014)

    Undergraduate thesis
    University of Otago

    Pregnancy is associated with a dramatic increase in serum prolactin levels that is essential for the normal increase in neurogenesis in the maternal brain, and for the associated expression of maternal behaviours after birth. As yet, it is not known which neurons prolactin acts on in the maternal brain to induce these changes, but it has been shown that prolactin receptors are present on GABAergic neurons in forebrain nuclei implicated in the regulation of maternal behaviour. This study aimed to examine the effect that prolactin action on forebrain neurons has on expression of maternal behaviour and on levels of neurogenesis, employing conditional deletion of the prolactin receptor using cre-lox technology. This technology allowed the creation of mice with prolactin receptors removed from CaM kinase II containing neurons (most forebrain neurons; CKC mice), and more specifically from neurons containing the vesicular GABA transporter (vGAT mice). When cre positive mice and controls (wildtype C57BL/6J and cre negative mice), were reproductively mature (6-8 weeks old), some mice were placed in individual cages and mated with a stud male. Neurogenesis was assessed on day seven of pregnancy, using injections of BrdU, which labels newly dividing cells. Other groups of mice were allowed to continue the pregnancy until term, and the day of parturition was designated as postpartum day one (PPD1). Maternal behaviour was tested on PPD2 by placing the mouse and three pups in a clean novel cage and observing all behaviours. On PPD3 maternal behaviour was tested in a similar manner in the home cage. Mice were also tested for anxiety on PPD4 in the light-dark box. Both CKC and vGAT cre positive mice showed significantly impaired maternal behaviour in the home cage compared with control mice, but the impairment was greater in the CKC positive mice than the vGAT cre positive mice. While all mice showed impairment in the novel cage, maternal behaviour was once again significantly more impaired in the CKC positive mice. CKC positive mice mostly ignored the pups, but at times were aggressive towards them, either eating pups before, or after death. Consequently, pup survival was dramatically reduced in CKC cre positive mice, compared with controls. There was no difference in levels of anxiety between the groups postpartum, suggesting that prolactin acts elsewhere to exert the postpartum anxiolytic effect. Surprisingly, given the maternal behaviour results, the levels of neurogenesis on day seven of pregnancy in the transgenic mice were not reduced as expected. There was no significant decrease in neurogenesis in either cre positive group compared with the controls. These findings suggest that prolactin acts through receptors on GABAergic neurons to stimulate some aspects of maternal behaviour, but other neurons must also contribute, and that prolactin acts somewhere other than forebrain neurons to induce neurogenesis.

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

    Sadrolodabai, Yasmin (2014)

    Undergraduate thesis
    University of Otago

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

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

    Zhang , Junda (2014)

    Undergraduate thesis
    University of Otago

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

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  • Non-Alcoholic Fatty Liver Disease and Elevated Alanine Aminotransferase in New Zealand: An Examination of the New Zealand Adult Nutrition Survey (2008/09).

    Heron, Robert Campbell Roydon (2014)

    Undergraduate thesis
    University of Otago

    BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is common world-wide and associated with obesity, diabetes mellitus, cardiovascular disease, hyperuricaemia and dyslipidaemia. These conditions are common in New Zealand, but the prevalence of NAFLD in New Zealand is unknown. New Zealand has a high prevalence of overweight and obesity, so it is likely that NAFLD is also common. The nationally representative 2008/09 New Zealand Adult Nutrition Survey (NZANS) provided an opportunity to estimate the prevalence of NAFLD as defined by elevated alanine aminotransferase (ALT), a measure of liver damage. AIMS: The aims of this study were to estimate the prevalence of elevated ALT and examine associations between elevated ALT levels and demographic, clinical and nutritional factors in New Zealand adults using data from the 2008/09 NZANS. METHODS: 4,721 New Zealand civilians aged 15 years or older participated in the 2008/09 NZANS, of which 3,035 agreed to have a blood test. Serum ALT was not measured as part of the main survey, and was measured using remaining blood samples. Sociodemographic, medical history, anthropometric and blood pressure measures, blood test results and dietary intake data were extracted and analysed. Elevated ALT was defined as an ALT activity > 29 units/L for men, and ALT > 22 units/L for women. The prevalence of elevated ALT was calculated for different groups, including age, ethnicity, and body mass index categories. Odds ratios were also calculated. Mean energy intake and intakes of both macro- and micronutrients (as measured by 24-hour dietary recall) were calculated and compared between participants with and without elevated ALT. RESULTS: The prevalence of elevated ALT was 13.1% (16.9% for men and 9.7% for women). The prevalence of elevated ALT was high among Maori (18.0%) and Pacific (18.6%) compared with New Zealand European and Other ethnicities (12.2%). NAFLD was more common in overweight (13.5%) and obese persons (20.1%) than in those with a normal weight (6.2%). Male sex, age 25 – 34 years, increased BMI and increased waist circumference were significantly associated with having an elevated ALT after adjustment. Ethnicity was not associated with the odds of having elevated ALT after adjustment. There were few differences in dietary intakes between those with elevated ALT and those with normal ALT. Mean cholesterol intake was higher (338 mg vs. 273 mg, P = 0.003) and thiamine intake was lower (1.3 mg vs. 1.5 mg, P = 0.014) in those with elevated ALT compared with the normal ALT group, respectively. CONCLUSIONS: Liver damage as defined by an elevated ALT, is common in New Zealand and is comparable to that of the United States. The prevalence of elevated ALT among those classified as obese was particularly high. Given the high prevalence of obesity and associated conditions such as diabetes in New Zealand, a large proportion of those with an elevated ALT are likely to have NAFLD. NAFLD is an increasing cause for concern worldwide, and further research using more sensitive and specific diagnostic method(s) is needed to more clearly understand the epidemiology of NAFLD in New Zealand.

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  • Functional Analysis of Disease-Associated 3’ UTR Variants: the CXCL12β 3’ UTR G801A Polymorphism

    Jin, Chan (2014)

    Undergraduate thesis
    University of Otago

    In recent years, the previously neglected 3’ untranslated region (UTR) has gained recognition as a critical region in the regulation of gene expression. Numerous studies have been conducted on disease-associated 3’ UTR variants to elucidate unidentified regulatory elements within the 3’ UTR. An example of an unresolved 3’ UTR variant is the G801A polymorphism (rs1801157) occurring within the 3’ UTR of the CXCL12β transcript isoform. The CXCL12 gene encodes a C-X-C motif chemokine 12 (CXCL12) that functions as a ligand for the chemokine (C-X-C motif) receptor 4 (CXCR4), which is involved in the entry of the human immunodeficiency virus (HIV) into host cells. Correspondingly, previous association studies have indicated a potential correlation between the A allele of the G801A polymorphism and increased resistance to the progression of acquired immunodeficiency syndrome (AIDS). This suggests that the G801A polymorphism may be linked to a regulatory element in the 3’ UTR, which modulates the expression of CXCL12β, and therefore plays an intermediary role in its reported association with HIV/AIDS. In order to identify the regulatory elements that may be associated with the G801A polymorphism, a bioinformatic approach was adopted by utilising a webserver developed in the Brown Lab called ‘Scan For Motifs’. However, only a 26 base pair (bp) region of high conservation was identified at the site of the G801A polymorphism. Consequently, three variable lengths of the CXCL12β 3’ UTR, containing the highly conserved region with either alleles at the G801A polymorphism, were cloned downstream from the luciferase gene (luc+) in pGL3MS2site/basic cloning vectors to yield three sets of reporter gene constructs. The desired CXCL12β 3’ UTR inserts were prepared by either the polymerase chain reaction (PCR) amplification of said sequences from HepG2 genomic DNA (gDNA) or appropriate oligonucleotide design. Site-directed mutagenesis was implemented when applicable using PCR-based methods such as the ‘megaprimer’ method and the overlap extension method, to create a polymorphic counterpart for each reporter gene construct. Prepared reporter gene constructs and previously characterised control plasmids were transiently co-transfected with a renilla luciferase reporter gene construct (phRL-SV40) using Lipofectamine® 2000 into HeLa cells and HEK293T. The transfected cells were harvested after a 48-hour incubation period for subsequent luciferase assays in order to measure differences in gene expression with respect to the G801A polymorphism. Changes in gene expression in response to different experimental conditions were also analysed through the application of two independent stimuli: the replenishment of foetal bovine/calf serum (FBS/FCS) (10%) and the premature harvest of cells after 24 hours. The amount of DNA transfected was also standardised through the co-transfection of pUC18, a non-coding plasmid. In contrast to initial expectations, different levels of gene expression were not observed between each pair of reporter gene constructs with allelic differences at the G801A polymorphism. In support of the experimental results, levels of gene expression in response to two different conditions behaved in agreement with initial expectations. Likewise, the standardisation of the amount of DNA transfected using pUC18 co-transfection only increased the gene expression associated with the larger plasmid relative to the other smaller DNA constructs. Despite the negative results obtained from a series of luciferase assays, the functional implications associated with the G801A polymorphism was not conclusively determined as absent given the conflicting indications reported in the wider literature. Further experimental analyses are required to confirm or reject the functional consequences of the G801A polymorphism. If there is a functional impact associated with allelic differences at the G801A polymorphism, the next step would be to elucidate the responsible regulatory element in the context of its associated diseases.

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

    Desai, Heena Nitin (2014)

    Undergraduate thesis
    University of Otago

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

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  • 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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  • 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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  • 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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