407 results for Undergraduate

  • Alpine fault pseudotachylytes

    Ritchie, Samuel David (2009)

    Undergraduate thesis
    University of Otago

    xvii, 171 leaves :col. ill., maps30 cm Includes bibliographical references. "October 2009". University of Otago department: Geology

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  • Evaluation of clinical ethics support needs and service provision at a tertiary hospital in New Zealand

    Dai, Libby (2013)

    Undergraduate thesis
    University of Otago

    Doctors often face ethical challenges in the course of clinical practice. Clinical ethics advisory services (CESS) provide a mechanism for supporting doctors facing these ethical dilemmas. In New Zealand, CESSs are relatively new and have emerged as a clinician-led initiative. This is an exciting time for CESSs in New Zealand, as their availability increases and their systems become increasingly formalised and integrated into the health care system. This thesis is the first in New Zealand to explore the clinical ethics needs of doctors and to evaluate how their clinical ethics needs could be most effectively met. The Capital and Coast District Health Board (CCDHB), comprising a major tertiary hospital and its satellite hospital, is used as a case study. Doctors at CCDHB have had access to a clinical ethics support service since 2010, when the CCDHB Clinical Ethics Advisory Group (CCDHB CEAG) was established. Many of the findings of the research would be applicable to CESSs throughout New Zealand. I developed a methodology to understand the clinical ethics needs of senior doctors at CCDHB and to evaluate how their needs could be better met. In-depth, semi-structured interviews were conducted with 14 senior doctors. The data were analysed using an iterative inductive strategy combining conceptual and normative analysis. My analysis draws on the current international literature of clinical ethics support, as well as my experience as a clinical medical student and my period of observation of the CCDHB Clinical Ethics Advisory Group. This study found that in the absence of formal services, doctors use ad hoc strategies of peer consultation to manage ethical issues. Not all doctors were equally able to access informal support, particularly junior doctors. Many participants were unaware that formal clinical ethics support was available to them and most did not know how formal ethics support worked. Some participants felt that to seek case consultation was to abrogate clinical responsibility and thought that doctors should be able to manage ethical issues themselves. Participants identified a need for improved strategies for clinically relevant ethics education. This study identifies five key recommendations to enhance clinical ethics support at Capital and Coast District Health Board: 1. CCDHB CEAG should formalise its activities, particularly case consultation, using a procedural justice model. 2. CCDHB CEAG should involve clinicians in the process of case consultation to increase user trust and to take advantage of case consultation’s educative value. 3. CCDHB CEAG should allow patients and their families and advocates the option of being involved in the process of case consultation, to ensure that patients feel that their perspectives have been adequately taken into account. 4. CCDHB CEAG should conduct monitoring and evaluation of its service to ensure that it achieves and maintains clinical relevance and normative robustness. 5. CCDHB CEAG should actively disseminate accurate and appropriate information about its aims and processes to all users and potential users to enhance trust in their service and to clarify misunderstandings about their role.

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  • Just prenatal testing? The science, ethics, and policy of testing for Down syndrome

    Cole, Robert (2013)

    Undergraduate thesis
    University of Otago

    Prenatal testing for Down syndrome (DS) has been available for over forty years. With the development of screening technologies, testing is now offered to all pregnant women. But is the increasing use of these tests really a good thing? Is a test for DS just another test; or is it another form of disability prevention, one with unsettling and discriminatory overtones? In this thesis I argue that whether testing is permissible or not is dependent in part on the morality of abortion. After an examination of arguments regarding fetal viability, sentience, and potential, I conclude that a fetus with DS may have less right to life than a fetus without DS, but this is dependent upon one’s perception of what constitutes ‘the good life’. That raising a child with DS may result in significant disruption to parents’ lives is an additional factor to take into account. If we hold that an abortion for social reasons is an acceptable expression of reproductive autonomy, then abortion for DS should be viewed as a morally permissible act. While prenatal testing for congenital conditions is distinct from many routine pregnancy tests, it does not follow that testing for DS should be restricted. It is conceded that prenatal testing may offend some people with DS (and their families), on the ground that DS may have considerable bearing on the development of one’s identity. But while some people take offence at the prospect of testing and abortion, it is not clear that they should; for the decision to terminate a pregnancy is a complex one, based upon many different factors. As new technologies become available, it is likely that more women will choose prenatal tests. More testing may mean that the numbers of those with DS will fall. However, this possibility should not preclude women access to testing. Pregnant women do not have a responsibility to ensure the continued prevalence of any trait or disorder, and DS is no exception. The treatment of people with disabilities has improved over recent decades, and this is something one would expect to continue. Though the number of people with DS may fall, there is increasing likelihood that social support will be more comprehensive than in the past. Ensuring that this occurs is of utmost importance. By using the controversial case of testing for DS, important issues surrounding all forms of testing in pregnancy come to light. This thesis concludes that two criteria must be met for a condition to be suitable for prenatal testing: that the condition significantly disrupts parents’ lives, and that the condition significantly limits a child’s open future. Although the existing framework may need improvement, the current practice of prenatal testing enhances reproductive autonomy. It provides information on one’s pregnancy which many women find important, and enables decision-making which is otherwise unavailable. Though not without its challenges, prenatal testing for DS is morally defensible, and should continue.

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

    Vasilic, Mina (2013)

    Undergraduate thesis
    University of Otago

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

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  • Evaluating molecular markers of breast tumour progression

    Gerring, Zachary (2013)

    Undergraduate thesis
    University of Otago

    Background: Tumour cell proliferation has emerged as a major prognostic factor in breast cancer. The immunohistochemical (IHC) proliferation marker Ki67 has been extensively investigated, but has not gained widespread clinical acceptance. Phosphohistone H3 (PHH3) is a new immunohistochemical marker for quantifying mitoses, however there is limited information on its prognostic value in breast cancer. This study performed a head-to-head comparison of Ki67 and phosphohistone H3. The aims were to establish the marker of greatest prognostic value, and to compare Ki67 protein expression and Ki67 mRNA levels in breast tumours. Methods: Tumour samples from 108 women with breast cancer were constructed as tissue microarrays (TMAs) and evaluated using immunohistochemistry. Cox proportional hazards regression was used to evaluate the prognostic significance of Ki67 and phosphohistone H3. C-statistics, integrated discrimination improvement (IDI), and net reclassification improvement (NRI) were used to evaluate the discriminatory ability of various prediction models. Ki67 mRNA from 30 matched fresh/frozen tumour specimens was analysed using quantitative polymerase chain reaction (qPCR), and was compared with Ki67 protein expression. Results: Phosphohistone H3 had greater prognostic value than Ki67 in a multivariable model that adjusted for traditional prognostic variables in breast cancer (hazard ratio: 3.32; 95\% confidence interval: 1.51--7.30). A risk prediction model that incorporated phosphohistone H3 efficiently separated patients at low risk and high risk of death at 5-years after diagnosis. The correlation between Ki67 protein expression and Ki67 mRNA levels was weak. Conclusions: Phosphohistone H3 outperforms Ki67 as an independent predictor of survival in breast cancer. Phosphohistone H3 may provide important information for counselling patients about the likely outcome of their disease, and accurately classifying groups of patients for clinical trials.

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  • Immune suppression and cutaneous squamous cell carcinoma tumour biology

    Seddon, Annika (2013)

    Undergraduate thesis
    University of Otago

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

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  • The Paediatric Outpatient Coding Pilot Study

    Kerr, Neal Alexander Moealia (2013)

    Undergraduate thesis
    University of Otago

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

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  • Gene transfer to restore vitamin C synthesis in human cell line

    Flett, Teresa Joy (2013)

    Undergraduate thesis
    University of Otago

    Vitamin C (ascorbate) is deficient in humans due to the lack in the final step in the biosynthesis pathway, Gulonolactone oxidase (Gulo). Ascorbate has been studied as a possible cancer therapy since the 1970’s, however results have been inconclusive. One of the reasons for this is the variability in ascorbate transport to the tumour. This study aims to design a biological tool that allows the study of ascorbate in cancer without transport being a variable. HepG2 cells were transfected with pCMV6-­‐Gulo and tested for genome incorporation by PCR, Gulo enzyme expression by western blot and functionality by HPLC-­‐ECD. One successful clone was established and a level of ascorbate comparable to previous studies was observed when the Gulo substrate (gulonolactone) was added. Transfected cells were assayed for HIF-­1α levels as a sign of tumour aggression. When ascorbate is added to the cells in 1% oxygen HIF-­1α levels are reduced. Interestingly a similar level of reduction was also observed when gulonolactone is added to the cells

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  • Functional consequences of lowering mitochondrial peroxiredoxin 3 expression

    Vick, Kate Angela (2013)

    Undergraduate thesis
    University of Otago

    Peroxiredoxin 3 (Prx 3) is an abundant mitochondrial protein that is very effective at scavenging hydroperoxides. The overexpression of Prx 3 has been shown to protect cells from oxidative stress and apoptosis, and knockout studies show an increase in intracellular ROS levels and oxidative stress. However, the knockout phenotype is mild and there has been no in-depth characterization of the effects of Prx 3 knockdown on mitochondrial function in response to oxidative stress. In this study, Prx 3 expression was knocked down to approximately 10-20% in a HeLa cell line, over a 5-6 day period. An inducible small hairpin RNA (shRNA) system was used to achieve this and no significant compensatory increase by other members of the Prx family was observed. These cells showed no difference in cell viability in response to exogenous H2O2, or during increased mitochondrial H2O2 production by the respiratory chain inhibitors; rotenone and antimycin A. Mitochondrial respiration was measured in intact cells with a Seahorse Extracellular Flux Analyzer and while the Prx 3 knockdown cells appeared to have normal rates of O2 consumption under basal conditions, mitochondrial stress was more detrimental in these cells. Overall, Prx 3 knockdown had a relatively limited phenotype, suggesting that low levels of Prx 3 are sufficient to protect cells from oxidative stress, or that Prx 3 may have a more important role as a sensor and signalling molecule than as a direct antioxidant protein.

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  • Voluntary Tremor Suppression in Parkinson's Disease

    Ha, William Anthony (2013)

    Undergraduate thesis
    University of Otago

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

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  • Investigating the Early Immune Response to Mycobacterial Infection in Mice

    Hall, Stephen (2013)

    Undergraduate thesis
    University of Otago

    Mycobacterium tuberculosis is an air-borne pathogen that causes more death each year than any other bacterial infection. The current vaccine, Bacillus Calmette-Guerin (BCG), is considered one of the most widely distributed in the world, yet the incidence of tuberculosis (TB) remains a huge problem. In order to improve TB vaccines, a greater understanding of the immune response to mycobacteria is required. Often, the innate immune response is able to reduce the mycobacterial burden in the lungs but not completely clear the invading organism. Dendritic cell (DC) subsets in the murine lung have been investigated during late mycobacterial infection; however, the early immune response to mycobacteria is an under-investigated field. This thesis describes a flow cytometry gating strategy to identify the main DC subsets in the lungs of mice. These subsets were examined for signs of activation following intra-nasal infection of mice with BCG. It was found that only CD11b+ myeloid DC exhibited upregulation of any co-stimulatory molecules. A fluorescent strain of BCG was used to identify the primary cell targets of mycobacteria during the first week of infection. These targets were found to be lung macrophages and CD11b+ myeloid DC. A sustantial decrease in bacterial load in the lungs was also observed over this time period which could be explained by early innate killing of mycobacteria. Finally, preliminary tests with fluorescent mycobacteria suggests that a positive signal may be detected through a thin layer of murine tissue in vivo. Overall, this thesis highlights the need for further study into the early immune response to mycobacteria and provides preliminary examination of the dynamics of lung DC subsets in this context. These findings contribute to a greater understanding of the immune system and its initial response to mycobacteria.

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

    Neill, Angus (2013)

    Undergraduate thesis
    University of Otago

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

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  • Predicting Steroid Responsiveness using Exhaled Nitric Oxide

    Rawcliffe, Laura Kay (2013)

    Undergraduate thesis
    University of Otago

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

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  • Regulation of voltage-gated calcium channels in PC12 cells by Leucine Rich Repeat Kinase 2

    Bedford, Cade Henry (2013)

    Undergraduate thesis
    University of Otago

    Leucine rich repeat kinase two (LRRK2) is a widely expressed protein belonging to the Roco family of proteins, mutations in which have recently been discovered as a cause of familial Parkinson’s disease (PD). Despite an array of interacting proteins having been identified across multiple cellular systems, LRRK2’s functional role remains to be determined. Manipulation of LRRK2 expression disrupts many Ca2+ dependent cellular processes. It, therefore, may act as an upstream regulator of initial Ca2+ signalling events, which could explain LRRK2’s widespread effects. The central aim of this study was to determine whether LRRK2 alters endogenous voltage-gated Ca2+ (CaV) channel function in PC12 cells using whole cell patch clamp electrophysiology. Additionally, transiently transfected PC12 cells underwent epifluorescence imaging to identify morphological changes and identify any effects of L-type Ca2+ blockers on morphology. Peak CaV channel currents in LRRK2 transfected cells showed a significantly (p=0.0025, n≥7, one way ANOVA with Tukeys post-hoc test) higher current density across a number of holding voltages relative to untransfected and EGFP transfected controls. These results indicate that LRRK2 up regulates endogenous CaV channel function. Morphological assessment, however showed no significant effect of LRRK2 transfection on morphological parameters relative to EGFP transfected and non-transfected controls (N≥63, Kruskal-Wallis test). Furthermore, addition of the L-type Ca2+ channel blocker nifedipine had no significant effect relative to untransfected and ethanol vehicle controls (N≥43,Kruskal-Wallis test). These results suggest that LRRK2 dependent modulation of CaV channel function does not affect neurite differentiation. Overall, this study has identified a novel effect of LRRK2 on CaV channels, which may explain how LRRK2 has such widespread cellular effects and advances our understanding of LRRK2s functional role. If the effect of LRRK2 on CaV channels is responsible for pathology, CaV channel blockers currently being investigated for Parkinson’s therapy may be particularly effective in Parkinson’s patients harbouring LRRK2 mutations.

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  • The effect of riboceine on lipoprotein(a)

    Kader, Tanjina (2013)

    Undergraduate thesis
    University of Otago

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

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  • Phenotypic and functional characterisation of human macrophages – A potential role for gut resident macrophages in colorectal cancer.

    Norton, Samuel (2013)

    Undergraduate thesis
    University of Otago

    High infiltration of macrophages in colorectal cancer is associated with improved patient prognosis. This association is the opposite of what is observed in other cancers, however, we are yet to understand why. Previous approaches to study this have been too superficial and overlooked the complexity of macrophages. As a result current data is conflicting. This study aims to characterise human macrophages in greater detail, and to therefore resolve these ambiguities. Multicolour flow cytometry, ELISAs, Greiss assays and qPCR, have been used to define a more detailed phenotype of human macrophage populations in vitro and also in colorectal cancer patient tissue samples. Using these methods, a further level of phenotypic complexity and plasticity, which was previously overlooked, has been unveiled. Macrophage phenotypes were defined, and the plasticity of these phenotypes under different stimuli was assessed. M1 macrophages were found to be CD11b+, CD64+, CD14-, CD206+/lo and CD163-. M2 populations were found to be CD11b+, CD64+, CD14hi, CD206+ and CD163. These were subsequently detected, using the same approach, in both tumour tissue and non-transformed bowel tissue from colorectal cancer patients. Analysis of this tissue also revealed a large population of gut resident macrophages, with a unique phenotype, present in both tumour and non-transformed bowel tissue. This study highlights the complexity of macrophages and also provides methods to examine them ex vivo from human tissue. Data gained from this work could be used for future work looking at macrophages as both prognostic and therapeutic targets.

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  • HCO3— secretion is not impaired in inflamed mouse anterior proximal colon

    Lai, David (2013)

    Undergraduate thesis
    University of Otago

    One of the characteristics of inflammatory bowel disease (IBD) is an impaired, diminished or absent mucus barrier. For mucus to be effective in restricting commensal bacteria to the intestinal lumen, it must be expanded or hydrated. Mucus is secreted in a compacted state, and bound by H+ and Ca2+ ions, which shield the protein core of mucus consisting of many negative charges. A proposed mechanism for mucus expansion, is that HCO3— is secreted along with mucus into the intestinal lumen and it binds Ca2+ and neutralizes H+ ions. However, a recent study revealed a reduction in expression of the NaHCO3 cotransporter, NBCe1 in inflamed proximal colon. NBCe1 is thought to be involved in intestinal HCO3— secretion, therefore reduced secretion of HCO3— may be the cause of the impaired mucus barrier in IBD. To test this, we investigated whether HCO3— secretion was compromised in the proximal colon, specifically in the anterior proximal colon (APC), in an animal model of IBD, IL10-/- mice infected with Helicobacter typhlonius. By using the Ussing short circuit technique and pH stat technique, the electrogenic and electroneutral components of HCO3— secretion were measured. The results reveal that there was no appreciable electrogenic HCO3— secretion, and the majority of the electrogenic anion secretion, in the APC was a result of electrogenic Cl— secretion. In addition, although, electroneutral HCO3— secretion was present in the APC, it was very small, it was not stimulated by forskolin, and it was unaffected by inflammation. The results indicate that, little if any HCO3— secretion occurs in the anterior proximal colon, and that which does occur does not involve NBCe1. This suggests that the hydration of mucus in the proximal colon does not involve HCO3— and NBCe1 has some other role, possibly regulation of cell pH.

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  • Genome Architecture and Phenotypic Plasticity: Is the Lethal (2) Essential for Life cluster epigenetically regulated during ovary activation in the honeybee, Apis mellifera?

    Lovegrove, Mackenzie R. (2013)

    Undergraduate thesis
    University of Otago

    Phenotypic plasticity is the ability of an organism to alter its phenotype, without altering its genome, in response to environmental cues. There is mounting evidence it is involved in human development, where it has been implicated in the risk of developing noncommunicable adult diseases. Studying the molecular basis of this in mammals can be difficult, particularly separating out single influences from complex environmental interactions. The honey bee, Apis mellifera, provides a useful model in which to study plasticity because of its well-controlled, easily triggered plastic responses. Queen bees are normally the only reproductively active females within a hive, but workers can activate their ovaries in response to the loss of the queen. During this process, over a third of the genome shows altered gene expression, implying that coordinated gene regulation within a chromatin domain may play a role. We have identified a candidate cluster for investigating this hypothesis, the Lethal (2) Essential for Life (L(2)efl) group. The genes of which are down-regulated as the workers undergo ovary activation. The findings of this study show that the original boundaries of the chromatin domain had been underestimated, and that the CTCF insulator element binding sites which flank the genes of the Lethal(2)efl cluster, LOC100576174 and Gmap, appear to be the boundaries of the coordinated regulation. All of the genes within these sites show co-ordinated regulation, with expression occurring in the terminal filament cells of the ovary in queens, workers and active workers. As ovary activation is a phenotypically plastic response to an environmental cue, it was hypothesised that the mechanisms which underlie it are epigenetic in nature, with previous work identifying the repressive histone mark H3K27me3 as likely playing a role in ovary activation. Potential binding sites for the ecdysteroid-regulated transcription factors BR-C Z1 and Z4 were found for all of the genes within the CTCF binding sites, and none directly outside it (LOC411452 and LOC412824). The proposed model for the coordinated regulation of the genes within the chromatin domain containing the L(2)efl group is through an interaction of both histone modifications and ecdysteroid-regulated transcription factors. This work provides evidence for large scale, coordinated changes in gene expression leading to phenotypic plasticity in response to an environmental influence.

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  • The Effects of Pharmacological Preconditioning with GYKI-52466 and Domoic Acid on LTP and LTD Induction in the Rat Hippocampus

    Macindoe, Jessica Ellen (2013)

    Undergraduate thesis
    University of Otago

    Many neurodegenerative diseases are associated with severe memory loss and cognitive impairment, highlighting a critical demand for the development of neuroprotectants and nootropics. It has been shown that certain compounds can trigger lasting neuroprotective mechanisms. This phenomenon is called ‘pharmacological preconditioning,’ and it has recently been suggested that preconditioning may also enhance cognitive function. Indeed, preconditioning with GYKI-52466 and domoic acid (DOM) has prophylactic neuroprotective efficacy in vivo and in vitro, and preliminary in vitro results demonstrate their ability to enhance long term synaptic potentiation (LTP) and long term depression (LTD), thus denoting nootropic potential. The aim of the present study was develop an effective in vitro preconditioning strategy using GYKI-52466 or DOM, and clarify their effects on LTP and LTD induction in the rat hippocampus. Hippocampal slices from male Sprague Dawley rats were subject to acute or chronic preconditioning with 6 μM GYKI-52466, or acute preconditioning with 50 nM DOM. Control slices were not preconditioned. Slices subsequently underwent LTP or LTD induction, and electrophysiological techniques were used to assess the response to this. Orthodromic Schaffer collateral-evoked CA1 population spikes and field excitatory postsynaptic potentials (fEPSP) were monitored before and after LTP or LTD induction. Data were expressed as mean percentage change from baseline (± SEM) and group differences compared to controls at a 30 minute time-point post LTP or LTD induction was determined by an unpaired student’s t-test at a confidence level of P<0.05. GYKI-52466 and DOM preconditioning failed to enhance LTP and LTD induction. Both control and preconditioned slices exhibited comparable magnitudes of LTP and LTD for population spike amplitude, area and fEPSP slope, with no significant differences between control and preconditioned slices evident at a 30 minute time-point. These findings suggest that preconditioning with GYKI-52466 and DOM would not confer nootropic potential.

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  • Isolation and characterization of human dental pulp derived stem cells

    Kang, Isaac (Jinho) (2013)

    Undergraduate thesis
    University of Otago

    Background: Dental caries remains a major public health concern. Dental endodontics (root canal) therapy involves extirpating the dental pulp and replacing with inert materials. For severe tooth decay, it is the only available treatment; however, it fails to restore the biological functions and vitality of the dental tissues and may ultimately leads to tooth loss. To overcome these shortcomings, dental pulp stem cells (DPSCs) are being investigated as a novel prospective approach to regenerate the dental tissue. In this study, we isolated and purified DPSCs and characterized the purified cells. Objectives: The aims of this study were as follows: (i) to rapidly extirpate dental pulp tissues from human third molar teeth under sterile conditions; (ii) to isolate, characterize, and purify a heterogeneous population of DPSCs using mesenchymal stem cell markers; (iii) to determine the ability of DPSCs to differentiate down an odontoblastic lineage. Design: DPSCs were mechanically and chemically isolated from human impacted third molar teeth. Cells were expanded, passaged, and a heterogeneous population of DPSCs isolated using a cloning cylinder. DPSCs were characterized and purified by flow cytometry using the mesenchymal stem cell markers, STRO-1, CD44, and CD146. DPSCs were induced under two different odontogenic conditions comprising different concentrations of beta-glycerophosphate, and dexamethasone. DPSCs were analysed for morphology, proliferation potential, collagen formation, mineralization characteristics, and expression of the dentin-specific markers dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1), using immunohistochemistry. Results: DPSCs were positive for the mesenchymal stem cell markers STRO-1, CD44, and CD146, although two populations of cells showed different levels of STRO-1 expression. Differentiated DPSCs (dDPSCs) demonstrated a significant increase in alkaline phosphatase concentration between days 14 and 21, while a similar increase in collagen deposition, mineralization, and calcification was also observed on day 28. The proliferation rate of dDPSCs decreased with time. Odontoblast characteristics of dDPSCs were observed, with increased expression of the dentin-specific markers DSPP and DMP-1. Conclusions: This investigation demonstrated successful isolation of DPSCs and differentiation of DPSCs down an odontoblastic lineage, indicating that DPSCs represent a promising approval for the regeneration of lost dental tissues.

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