398 results for Undergraduate

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

    Chae, Matthew (2015)

    Undergraduate thesis
    University of Otago

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

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

    Deo, Manesh Shamal (2015)

    Undergraduate thesis
    University of Otago

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

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

    Meffan, Peter (2015)

    Undergraduate thesis
    University of Otago

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

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  • Investigating the pro-apoptotic properties of portimine

    Drake, Sarah Olivia Ann (2015)

    Undergraduate thesis
    University of Otago

    Portimine is a bioactive compound recently isolated from the marine dinoflagellate Vulcanodinium rugosum. It is a member of the fast acting class of toxins called cyclic imines, which are typically lethal to mammals at low doses. Portimine has been shown to have limited toxicity in mice, but it appears to be a very effective inducer of apoptosis in cultured cells. This study looked to understand more about the mechanism in which portimine mediates programmed cell death. Other cyclic imines are neurotoxic through binding of acetylcholine receptors. We hypothesized portimine also utilised a receptor to mediate cytotoxic effects, and therefore utilised the extracellular death receptor pathway. Jurkat T-lymphoma cells and mouse embryonic fibroblasts (MEFs) were exposed to portimine and the loss in cell viability was measured. Cell lines in which different members of apoptosis signalling pathways were knocked out or overexpressed were assessed to help determine the mechanism of apoptosis induction, and the effects of portimine was compared with other inducers of cell death in these cells. Portimine had an LC50 of 7.3 nM in wild-type Jurkat cells. It was unable to kill Jurkat cells overexpressing Bcl-2, even at very high concentrations. This contrasted dramatically with hydrogen peroxide, which could not kill Bcl-2 overexpressing Jurkat cells at low doses, but caused necrotic death at high concentrations. Therefore, portimine appears to kill via a selective apoptotic mechanism without causing significant cell damage. MEFs were also killed at low doses of portimine, with an LC50 of 5.2 nM. Interestingly, it took 48 h after portimine exposure to achieve optimal cell death. MEFs in which the apoptosis effectors Bax and Bak were knocked out were resistant to portimine, supporting an apoptosis-dependent mechanism. However, Bcl-2 was not protective as in Jurkat cells, and a Bid-/- cell line was also susceptible to portimine. Bid is important for the induction of death receptor-mediated apoptosis, suggesting our initial hypothesis is incorrect. There are potential applications for portimine or the novel pathway it uses, as a selective inducer of apoptosis in cells that display unregulated cell growth.

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  • Measuring Mitochondrial Dysfunction in Humans

    Brinsden, Mark (2015)

    Undergraduate thesis
    University of Otago

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

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  • Parents, siblings and pacifism : the Baxter family and others (World War One and World War Two)

    Cumming, Belinda C. (2007)

    Undergraduate thesis
    University of Otago

    Before she died, Millicent Baxter, wife of notorious New Zealand conscientious objector Archibald Baxter, wrote a letter confessing one of her final wishes: “I hope to live long enough to see the production of the documentary of my husband's book, We Will Not Cease ... I think it has a message for the young. The future of the world is in their hands …” Clearly pacifism was a shared commitment in her family, not just the passion of one individual member. This essay will seek to explore the importance of family in a pacifist stance. I will examine influences of, and effects on, various members of the family unit, and investigate the importance of familial support in aiding a conscientious objector to take the pacifist stance and cope with the consequent hardships confronted. My dissertation examines both the first and second World Wars. This focus on the family distinguishes my research from scholarship which precedes it.(…)While available scholarship provides a significant wealth of detail on conscientious objection in New Zealand, this narrow focus on administration has led to neglect of the families of the pacifists, the people who actually felt the affects of the policies formulated by politicians in Parliament and who witnessed the persecution and punishment of their loved ones. Attention has not been paid to the role of the family and I endeavour to remedy this neglect. I wish to explore how the family members in both wars were affected by the pacifist stance adopted by men in their families, and how they responded. (…) The stand of a conscientious objector is 'a protest against war and leads inevitably to conflict with the State.' 3 When a man elected to object to war and go against prevailing opinion at the time, he was putting himself forward to be ostracised, harassed and abused by the community, and be punished officially by the Government. This radical and life-changing decision to be a conscientious objector would likely have been a gradual process of thought and debate during the objector's life, not a spontaneous snap decision. I wish to explore the role that upbringing played in this decision-making process, and to investigate the influence that parents consciously or sub-consciously had over their children's views regarding peace and war. (…) By focusing on the family in research regarding conscientious objection in New Zealand over both Wars, this dissertation sheds light on hitherto neglected areas of investigation, and gives a voice to mothers, fathers, siblings and children who have until now often remained unheard. [extract from Introduction]

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

    Jones, Morgan Grace (2015)

    Undergraduate thesis
    University of Otago

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

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  • A search for synthetic lethality between Polo-like Kinase 3 and E- Cadherin

    Gaastra, Joel Bertus Ross (2015)

    Undergraduate thesis
    University of Otago

    Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant condition caused by a mutation in the tumour suppressor gene E-cadherin, (CDH1). It predisposes to a 70% likelihood of developing highly penetrant diffuse gastric cancer. Because HDGC is driven by the absence of a tumour suppressor, conventional therapeutic approaches targeting tumour-promoting oncogenes cannot be used. Synthetic lethality is an approach that circumvents this issue by targeting vulnerabilities in cells that lack the functional tumour suppressor. This project aimed to investigate a potential synthetic lethal relationship between CDH1 and polo-like kinase 3 (PLK3), a cytoskeletal and cell cycle regulator. Lentiviral delivery of two shRNA were successful in knocking down PLK3 expression in isogenic MCF10A cell lines, with and without CDH1 expression (MCF10A and CDH1-/-). Viability was measured and confirmed as synthetic lethal (mutations in combination cause cells to be less viable) if the CDH1-/- cells were less viable with a ratio of ≤0.85. Results from one shRNA knockdown trended towards synthetic lethality (p > 0.05). Another shRNA resulted with a considerable reverse synthetic lethal effect, but was not statistically significant. PLK antagonists poloxipan and wortmannin were used to inhibit PLK3. Poloxipan induced reverse synthetic lethality with reduced viability in MCF10A cells at low concentrations. High concentrations produced a marginal synthetic lethal phenotype. Wortmannin’s effect on MCF10A and CDH1-/- cells also varied from synthetic lethal and reverse synthetic lethal. As the viability of CDH1 deficient cells could not be significantly reduced via PLK3 knockdown or inhibition, this candidate is no longer considered to be a potential therapeutic target for the treatment of HDGC.

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  • The effect of maternal obesity on neural stem cell proliferation in pre natal mice

    Gibbs, Jordan (2015)

    Undergraduate thesis
    University of Otago

    Autism Spectrum Disorder (ASD) is a complex neurological condition. It affects approximately 1 in 110 people world wide, with a 4:1 ratio of males to females affected. ASD presents with a wide range of psychological symptoms including deficits in speech, motor function and cognition. Interestingly, ASD first presents symptomatically in the first three years of life, sometimes even presenting as a regression in acquired skills, such as language. Research has shown a multitude of anatomical changes to the brain, creating a specific pathology seen in subjects with ASD. This includes overall changes to brain volume, with a lower volume at birth followed by an excessive overgrowth, and, consequently, excessive brain volume in the first few years of life. In addition to these findings, a set of regions in the brain have been repeatedly found to be associated with ASD, such as the amygdala, hippocampus and cerebellum. These findings suggest neural stem cells could be altered, leading to the changes seen in ASD, which are present right from birth. Due to the condition being psychiatric there is no way to identify ASD until behavioural symptoms appear. Consequently, there is no way to know what pre-natal ASD looks like, due to the lack of a biological marker. Though the origins of ASD are, as of yet, unknown, a wide variety of research has identified a number of associations that are potentially linked to ASD. One such association is maternal obesity, or obesity during pregnancy. This is associated with an increase in the risk for offspring to develop ASD. The aim of the present study was to test this hypothesis using immunohistochemistry. We analysed the effects of maternal obesity on neural stem proliferation in regions of the brain commonly associated with ASD. To examine this association, we used a mouse model of maternal obesity to generate prenatal offspring and compared them to control offspring undergoing gestation in a mother of normal weight. We used pups from late mouse gestation, GD15.5 and GD17.5, examined neural stem cell proliferation using immunohistochemistry to detect the cell proliferation marker Ki67. This was completed in 3 core areas associated with ASD; the cerebellum, amygdala and hippocampus. We identified a significant decrease in the number of cells proliferating in the amygdala at both ages in pups from obese mothers. Furthermore, we found sex differences in these results, with decreases in cell proliferation greater in males. We did not see any significant differences in the hippocampus or cerebellum. These results show that maternal obesity alters neural stem cell proliferation in the amygdala. The postnatal consequences of this are currently unclear, however, as the early neonatal ASD brain is smaller than neurologically normal controls, it is tempting to suggest that these findings represent pre-natal ASD. Given that the size of the brain is altered in ASD, which suggests a defect in the proliferation of neural stem cells, combined with the fact that maternal obesity is a predisposing risk factor, we hypothesised that when the fetal brain undergoes development in an obese mother there will be alterations in neural stem cell proliferation in regions of the brain commonly associated with ASD

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  • A tale of two fates: the role of histone modifications in establishing bipotentiality

    Sorensen, Summer Rose (2015)

    Undergraduate thesis
    University of Otago

    Sex determination and differentiation is an essential process all mammals must undergo to reproduce. This process in mammals is initiated through the development of a bipotential tissue, the urogenital ridge (UGR), which differentiates into two morphologically different tissues; testes in male and ovaries in females. This is a genetically controlled process with the presence or absence of the Sry gene determining the sexual fate of an embryo. Many genetic pathways have been implicated in the development of the bipotential UGR but the epigenetic regulatory mechanisms involved are relatively unknown. Previously, to obtain a global view of genes active or actively repressed in the mouse UGR at E11.5, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) had been performed for active (H3K4me3) and repressive (H3K27me3) histone modifications. Analysis showed overrepresentation of genes linked to the Wnt signaling pathway targeted for epigenetic regulation from both histone modification data sets (Yang and Wilson, unpublished). Thus, we aimed to investigate the role of epigenetic mechanisms to histone proteins in the UGR prior to differentiation into a testis or ovary, on a set of targets involved in the Wnt signaling pathway. Four enrichment areas were chosen for ChIP-seq validation, Wnt10, Fzd6, Wnt4 and Fzd8. Significant enrichment for Wnt10a in association with H3K4me3 modifications and Wnt10a and Fzd6 in association with H3K27me3 modifications was observed through ChIP-qPCR (p<0.05). Expression patterns of Fzd6 and Wnt4 show stark differences in the UGR and surrounding mesonephros. Fzd6 expression was detected in the Wolffian duct and mesonephric tubules but not in the UGR itself whereas Wnt4 was restricted to the UGR. Further analysis of H3K4me3 and H3K27me3 modifications showed overlapping patterns of the distribution of these modifications in the UGR and mesonephric tubules Confirmation of Wnt signaling factors and their association with histone modifications when combined with the spatial patterns of the histone modifications, implements a mechanism by which the Wnt signaling pathway is regulated in the bipotential gonad. Motif analysis of regulatory gene regions enriched with the H3K27me3 histone modification (Yang and Wilson, unpublished) suggested that many of these genes are regulated by known pioneer factors, EBF1 and HNF6. RT-PCR confirmed the presence of EBF1 in the bipotential gonad at E11.5, however expression of HNF6 was not detected. Bioinformatic analysis using DAVID uncovered enrichment of genes involved in the MAPK signaling pathway along with processes relating to development and morphogenesis. The proposed model for the role of histone modifications in the regulation of gonad development is that they play an important role in maintaining the bipotential state of the gonad. Through the binding of EBF1, the gonad is kept in a poised transcriptional state, awaiting sex-specific signals to activate or repress pathways including the Wnt and MAPK signaling pathways

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  • Modulation of colonic stem cell proliferation by bacterial components

    van Hout, Isabelle (2015)

    Undergraduate thesis
    University of Otago

    The intestine is home to a large number of commensal microbes, which are restricted to the intestinal lumen by the epithelial barrier. In the colon, which has the greatest number of commensal microbes, the epithelial barrier is maintained by turnover of the epithelium every 3-5 days by a small population of stem cells found at the base of the crypts. If bacteria are present in the lamina propria, it indicates a loss of barrier integrity so it is important for the epithelium to respond to this, to increase proliferation and repair the barrier to prevent inflammation. Previous research indicates that stimulating pattern recognition receptors, found on colonic stem cells, may be a mechanism by which epithelial proliferation can be altered. Here we have used human colonic organoids to determine if the colonic bacteria modulate the proliferation of the colonic stem cells and hence the properties of the colonic epithelium. Colonic organoids were grown from crypts isolated from healthy patients and suspended in Matrigel® overlaid with a specific stem cell growth media. Two distinct organoid types were seen in culture enteroids, which have a well-developed columnar epithelium and so appear to be representative of the native colonic epithelium, and spheroids, which have a poorly differentiated epithelium. The effect of the removal of the growth factor Wnt3A, removal of the inhibitors of the TGF-β pathway LY2157299 and SB202190, induction of differentiation by the addition of the γ-secretase inhibitor DAPT (10µm, [N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), or inclusion of bacterial components including lipopolysaccharide (LPS), lipoteichoic acid (LTA) flagellin and muramyl dipeptide (MDP) (all 20ng ml-1), in the growth media, on cell proliferation in enteroids was assessed by measurement of the expression of proliferation-associated genes (Cylcin D, p15, LTBP1, Ki67 LGR5, FOXM1 and MYC) by qPCR. In addition, the incorporation of EdU (5-ethynul-2’-deoxyuridine) into the nuclei of dividing cells was used to quantify proliferation in whole mounts of both spheroids and enteroids. Statistical significance was determined by a Kruskal Wallis test and a Dunns post test or a Mann-Whitney test. Few changes in proliferation-associated genes at the transcript level in enteroids were seen, indicating environmental changes do not alter enteroid proliferation. Consistent with the qPCR findings, direct quantification of cell proliferation in whole mounts of enteroids, also found no significant change in proliferation between control and experimentally treated enteroids. In contrast, spheroids had a significantly higher rate of proliferation than enteroids (p<0.01) for 10 days, compared to control spheroids. These results show that environmental changes can affect spheroid proliferation; hence spheroids may be a better model of the crypt base, which is the site of proliferation in the native epithelium. They also show there is a basal level of proliferation maintained in the cultures, despite environmental manipulations. Finally, these results show that the bacterial components LPS and flagellin, which signal through TLR4 and TLR5 respectively, decrease colonic proliferation, by directly interacting with the epithelium. A decrease in proliferation under the conditions tested here was unexpected, and may highlight that other components such as immune or mesenchymal cells, may be important in controlling colonic proliferation.

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  • Cellular Mechanisms of Prolactin Regulation of Oxytocin Neurons in Reproduction

    Alyousif, Yousif (2011)

    Undergraduate thesis
    University of Otago

    The hormone oxytocin is secreted from nerve terminals of oxytocin magnocellular cells (MNCs) in the posterior pituitary gland and is important in the timing of birth and is essential for milk secretion. Another reproductive hormone, prolactin, is secreted from the anterior pituitary gland and is critical for breast development during pregnancy, as well as for milk synthesis during lactation. Oxytocin MNCs of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus undergo significant plasticity during pregnancy and lactation. Prolactin receptors are expressed by oxytocin neurons in both of these nuclei and prolactin has been shown to inhibit oxytocin MNCs in virgin rats. This project aimed to test two hypotheses. The first hypothesis was that the inhibitory effects of endogenous prolactin on the electrical activity of oxytocin MNCs will be reduced over the course of pregnancy or early lactation. To test this hypothesis, virgin (dioestrous) and lactating (day 6-12 post-partum) female rats were anaesthetised with urethane and extracellular singleunit recordings were made from identified oxytocin (and vasopressin) MNCs. Prolactin (1 μg in 1 μl intracerebroventricular) reduced the firing rate of oxytocin MNCs in virgin, but not lactating, rats. The second hypothesis was that reproduction-induced adaptations in oxytocin MNC responses to prolactin might be mediated by changes in second messenger systems downstream of the prolactin receptor. Double labelled (for oxytocin and phosphorylated signal transducer and activator of transcription 5 (pSTAT5)) immunohistochemistry was used to examine prolactin-induced activation of the Jak/STAT5 pathway in oxytocin MNCs. pSTAT5 expression was significantly increased in oxytocin MNCs of virgin rats treated with prolactin, while both the vehicle and prolactin treated lactating females had high levels of pSTAT5 in their oxytocin MNCs. Together, these data provide evidence that prolactin may directly and specifically regulates activity of oxytocin MNCs. However, the significance of this regulation remains to be elucidated.

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  • Early Vascular Disease in Children with Epilepsy

    Keenan, Ngaire Fiona (2011)

    Undergraduate thesis
    University of Otago

    Background: Mortality from cardiovascular and cerebrovascular disease in patients with epilepsy is up to five times that seen in the general population. It is postulated that this elevation in cardiovascular disease is partly due to elevation of the cardiovascular risk factor Total Plasma Homocyst(e)ine (tHcy). Elevated tHcy is frequently elevated in adolescents and adults with epilepsy as a result of Antiepileptic Drug (AED) induced B-vitamin deficiencies, particularly folate, vitamin B12 and vitamin B6 that are important cofactors for homocysteine metabolism. It has been recommended by previous investigators that all children with epilepsy should receive vitamin supplementation to reduce their cardiovascular risk. Other cardiovascular risk factors, such as oxidative stress, hyperinsulinaemia and hyperlipidaemia are also frequently reported in patients with epilepsy treated with AEDs. As early cardiovascular disease, especially in children, is potentially reversible, we plan to investigate endothelial function and structure as well as biochemical cardiovascular risk factors, such as tHcy and lipid levels, in children with epilepsy treated with AED therapy. Methods: Thirty children with idiopathic or symptomatic epilepsy who had been on AED treatment for at least twelve months were recruited from paediatric outpatient clinics in Wellington, New Zealand. Thirty age, sex and BMI matched healthy controls were also recruited. Fasting tHcy, serum folate, red blood cell folate, Pyridoxal-5-Phosphate (PLP), vitamin B12, plasma glucose and lipid levels were measured in each participant. Endothelial function and structure through Flow-Mediated Dilation (FMD) and Intima-Media Thickness (IMT) of the carotid and aortic arteries were measured in subjects and controls. Results: No statistical differences in tHcy, serum folate, red blood cell folate and PLP concentrations were observed between the epilepsy and control groups. Sub group analysis of individual AED therapies also showed no differences. Vitamin B12 levels were elevated in children with epilepsy compared to controls, particularly in the Sodium Valproate (VPA) monotherapy group. Marginally significantly lower fasting glucose was apparent in children with epilepsy compared to controls. This was seen to be primarily due to VPA monotherapy. Lipid and lipoprotein concentrations in children with epilepsy were statistically comparable to controls. After analysis of individual AED treatments however elevated total cholesterol, total cholesterol/ HDL cholesterol ratio, free triglycerides and lipoprotein B levels were evident in children treated with Carbamazepine monotherapy. No statistical differences were apparent in FMD, carotid IMT and aortic IMT between children with epilepsy and controls. Conclusions: We were unable to demonstrate elevated tHcy in our children with epilepsy and so not surprisingly their endothelial function and structure was also unremarkable. Given our findings vitamin supplementation in all children with epilepsy would appear unnecessary. It is likely that our population has diets with vitamin intakes adequate to compensate for any loss of vitamins induced by AED therapy and subsequently the threshold needed to produce elevated tHcy was not reached. Therefore, vitamin supplementation may only be indicated in populations with lower nutritional intakes and adults who naturally have lower B-vitamin levels compared to children. We conclude that recommendations of diets high in B-vitamins by paediatricians and neurologists would be of benefit.

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  • Efficacy of B16OVA Tumour Cell Lysate Conjugated to Rabbit Haemorrhagic Disease Virus Virus-Like Particles as an Anti-Tumour Vaccine

    Grant, Melanie (2011)

    Undergraduate thesis
    University of Otago

    By presenting antigen to T cells dendritic cells (DC) carry out a central role in the activation of T cell mediated immunity to cancer. Tumour cell lysate (TL) as a source of tumour antigens offers the advantage over single, defined tumour-associated antigens (TAA) of being able to stimulate polyclonal T cell responses to heterogeneous tumours containing both known and unknown antigens. However TL alone does not generate a robust, long-lasting anti-tumour response. Virus-like particles (VLP) coupled to defined TAA have been shown to stimulate strong anti-tumour responses but the majority of cancer antigens remain undefined. This project aimed to develop VLP-antigen conjugates by coupling unidentified TAAs in TL to Rabbit Haemorrhagic Disease Virus (RHDV) VLP. TL was generated from the melanoma cell line B16OVA that secretes the model antigen, ovalbumin (OVA). Bone-marrow derived DCs (BMDC) were pulsed with VLP-TL and the BMDC maturation response evaluated by assessing upregulation of the key DC surface markers, CD40, CD80, CD86 and MHC-II by flow cytometry. Subsequently antigen-pulsed DC were co-cultured with OVA MHC-I and MHC-II peptide-specific OT-I and OT-II splenocytes and the T cell proliferative and cytotoxic response measured. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled OT-I splenocytes proliferated in response to VLP-TL indicating T cell activation; OT-II splenocytes on the other hand showed no such response. IFN-γ was detected in the supernatant of both OT-I and OT-II co-cultures, indicating a cytotoxic response. Inhibition of T cell proliferation and cytotoxicity was seen in the presence of VLP or TL alone and VLP-TL was sometimes able to overcome this inhibition. In vivo CTL-mediated cytotoxicity was also examined with VLP-TL vaccinated mice showing a significant TL-specific cytotoxic response, demonstrating proof of principle for future in vivo assays of VLP-TL. These results indicate that VLP-TL may have a beneficial effect on the ability of DC to stimulate T cell proliferation and anti-tumour cytotoxicity. Further investigations with increased dosages are warranted to ascertain whether or not the effect seen is dose-dependent.

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  • Optimal Management of ST-Segment Elevation Myocardial Infarction

    McKay, Emma (2012)

    Undergraduate thesis
    University of Otago

    Background: ST-segment elevation myocardial infarction (STEMI) is a high-risk clinical scenario within the acute coronary syndrome spectrum. It is associated with poor clinical outcomes, particularly if acute reperfusion and appropriate care protocols are not initiated in a timely fashion. Consequently, optimal STEMI management represents a significant challenge to modern health-care provision. Aims: This thesis intends to provide insight into contemporary, optimal STEMI management, with a specific focus on documented quality indicators. Methods: Chapter 1 is a discussion of the modern approach to STEMI management. We explored particularly how primary percutaneous coronary intervention (PCI) and thrombolysis are integrated as acute reperfusion options into systems of care. Chapter 2 comprises a systematic review concerning the evolving use of pre-hospital electrocardiograph (ECG) technology in improving reperfusion times and clinical outcomes, with specific consideration as to how this may be used in pathways of care. In Chapter 3, a four-year study of reperfusion-eligible patients presenting to Dunedin Hospital between 2004 and 2008 was performed. Morbidity and mortality outcomes were assessed with one-year follow-up. Cases were analysed by age, gender, and hour of presentation to observe any effect these factors had on quality of care and clinical outcomes. Results: Eighteen studies describing the use of pre-hospital ECG in improving door to balloon times or mortality outcomes were identified after a systematic literature search. While these studies were generally small and methodologically represented lower level evidence, they appeared to support the utility of this evolving technology in newer models of care to significantly reduce reperfusion times; however, there were limited reported data on mortality or changes in other outcomes. Variable approaches to pre-hospital ECG use, reporting of outcomes, and study quality precluded formal quantitative meta-analysis. A four-year review of Dunedin Hospital STEMI care identified a cohort ranging between 60 to 79 cases each year. There was a significant increase in the use of primary PCI across the years 2004–2008 (12.9% to 81.0%, p<0.001), with an accompanying reduction in the use of thrombolytic (lytic) drugs and non-use of acute reperfusion. Apart from an increase in the use of evidence-based therapies, no other significant changes were identified over the study period. In-hospital mortality ranged from 3.3% to 9.7% (p=0.511); at one year this ranged from 5.0% to 19.6% (p=0.125), and unadjusted for differing baseline characteristics did not differ between lytic or primary PCI therapy. Rates of bleeding were notably high, particularly in patients receiving thrombolysis. Older patients fared worse in many clinical outcomes assessed, as did women. Out-of-hours presentation was not observed to be associated with differences in institutional performance or clinical outcomes. Conclusions: The systematic review of pre-hospital ECGs supports use in improving reperfusion times, and potentially as a means to facilitate regional cardiac networks. There remain some uncertainties about the specifics of technology, implementation, and cost effectiveness. It is technology that is potentially rapidly evolving and advancing, but already appears to be practicable with well-described examples of implementation reducing reperfusion times. Larger studies will be required to demonstrate ability to improve hard clinical outcomes. Review of real-world outcomes at this institution indicates results latterly consistent with benchmarks of international trial and registry data. That there were no differences in mortality between thrombolysis and primary PCI supports the mixed-strategy approach, assuming continued allowance for the individual patient context. Identification of potential unequal provision of care due to age or gender provides potential targets for future quality improvement initiatives.

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  • Neuroendocrine Control of Fertility by Leptin and Insulin via Glutamate Neurons

    Roberts, Amy (2012)

    Undergraduate thesis
    University of Otago

    Obesity is a growing problem in the western world, and this is accompanied by an increase in infertility. In fact, the link between nutrition and fertility occurs at both ends of the body weight spectrum: severely obese or undernourished individuals have altered puberty onset and impaired fertility. The pathways used by hormones known to signal metabolic status to modulate fertility are currently unknown. There is evidence that leptin and insulin act in the brain to affect the GnRH (gonadotropin releasing hormone) neurons that control the HPG (hypothalamic-pituitary-gonadal) axis. However, it is now known that they do not target GnRH neurons directly, but through intermediate neurons. These intermediate neurons have not yet been determined, although a number of neuronal subtypes have been tested. A key area thought to be involved in the nutritional control of fertility is the PMv (ventral premammillary nucleus). This nucleus responds to leptin, and has been shown to contain neurons that innervate sites of GnRH neurons. The PMv is also known to contain an abundance of glutamate neurons. This has led to the hypothesis that leptin (and possibly insulin) acts through glutamatergic neurons in the PMv to control fertility. The hypothesis that leptin and insulin modulate fertility via glutamatergic neurons was tested using a conditional knock-out mouse model, where insulin or leptin receptors were knocked out of glutamate neurons using the Cre-Lox system. This was confirmed using a GFP-reporter mouse, to verify that the Cre recombinase used to induce the knock-out had occurred in brain regions known to express glutamate. The fertility of these mice was then examined. The onset of puberty was measured in females by determining the age at vaginal opening and first estrus, and in males by the date of the first successful mating. There was no effect on puberty onset when leptin or insulin receptors were knocked out. Estrous cycles were then examined in the female mice, but there was no difference when compared to control animals. A major part of this study was to examine the fertility and fecundity of the knock-out mice. The mice were paired with a wild-type mate, and the dates of birth of the litters used to determine litter-to-litter interval, and therefore the fertility, and the litter size was used to determine fecundity. However, there were no fertility defects in either the leptin or insulin receptor knock-out mice when compared to the controls. The conclusions from this study are therefore that leptin and insulin do not target glutamate neurons (such as those in the PMv) in the control of fertility. This result has made it easier to narrow down the targets used, since a large group of neurons can now be excluded. Concurrent research in our laboratory has highlighted GABAergic neurons as a likely alternate candidate for mediating leptin’s effects on fertility. If the targets of metabolic hormones in the control of fertility can be determined this may have important implications for designing novel fertility treatments.

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  • The role of prolactin in pregnancy-induced changes in food intake

    Stenhouse, Natasha Danielle (2012)

    Undergraduate thesis
    University of Otago

    Pregnancy is associated with a significant increase in food intake. This occurs in order to supply the mother with sufficient energy to support both herself and the developing fetus throughout pregnancy and lactation when metabolic demand is high. This increase in food intake occurs despite an elevation in circulating levels of the appetite-suppressing hormone leptin. Hence, pregnancy is considered a state of leptin resistance. The hormone prolactin, the levels of which are significantly increased during pregnancy, is thought to be involved in increasing food intake during pregnancy. Exogenous administration of prolactin in rodents is associated with increased food intake and reduced response to leptin. As such, it is possible that the pregnancy-induced increases in prolactin may have the same effect. We hypothesised that prolactin acts centrally to mediate the changes in food intake that occur during pregnancy. Thus, the aim of this experiment was to measure food intake during pregnancy in mice that specifically lack prolactin receptors in the brain. To characterise food intake in these mice, food intake and bodyweight were measured daily in non-pregnant, pregnant and lactating neuron-specific prolactin receptor knockout mice and controls. This neuron-specific prolactin receptor knockout model has lost prolactin receptors throughout the brain and thus exhibits impaired central prolactin response. Immunohistochemistry for phosphorylated signal transducer and activator of transcription 5 (pSTAT5), a marker of prolactin receptor activation, was performed in order to assess the success of the knockout animal. Finally, because there appeared to be a difference in bodyweight in the non-pregnant knockout animals, non-pregnant mice were subjected to a fast and refeed protocol and then placed on a high fat diet in order to further characterise their food intake regulatory systems. Food intake increased significantly over pregnancy and was higher still during lactation in both animal groups. Bodyweight also increased over pregnancy, then remained stable during the lactation period. Food intake was significantly higher throughout pregnancy in the knockout animals compared with the controls. Unexpectedly, food intake and bodyweight were significantly higher in the non-pregnant knockout animals compared with the controls suggesting a basal difference in bodyweight regulation. Immunohistochemistry for pSTAT5 demonstrated significant, but not complete loss, of the prolactin receptor throughout the brain, specifically in the arcuate nucleus and medial preoptic area. There was no significant difference between the animal groups in the fast and refeed protocol. The high fat diet data demonstrated that the knockout animals on a high fat diet gained significantly more weight than the control animals on a control diet. The results obtained did not support our hypothesis that prolactin action in the brain critically mediates changes in food intake during pregnancy. However, interpretation of these results was complicated by the non-pregnant animal results and the incomplete nature of the knockout. It remains possible the remaining prolactin-responsive neurons in the brain may mediate prolactin action to stimulate food intake. This latter interpretation is supported by the changes in bodyweight in the non-pregnant knockout animals. Despite the failure to support or disprove our hypothesis, a significant step towards characterisation of this knockout model was made.

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  • 17α-hydroxylase and the androgen-treated sheep ovary – a model for Polycystic Ovary Syndrome (PCOS)

    Caldwell, Aimée Sarah Lee (2012)

    Undergraduate thesis
    University of Otago

    Polycystic Ovary Syndrome (PCOS) is the most common cause of infertility in women of reproductive age, with an estimated five to ten percent of women worldwide affected by the endocrine condition. The aetiology of PCOS remains, for the most part unclear, however, androgens have been implicated in the development of PCOS with hyperandrogenism being one of the most consistent PCOS traits. The products of a complex steroid pathway, androgens, in particular androstenedione and testosterone, are found to be significantly increased in many PCOS patients. The aim of this study was to investigate the activity of a key enzyme in the steroid pathway, 17α-hydroxylase, in the prenatally testosterone treated (T-treatment) sheep ovary and subsequently measure levels of androstenedione present in blood samples taken from the jugular and ovarian veins. In addition to this, a histological study of follicular mapping provided useful insights into the effect of T-treatment on aspects of ovine follicular development. Sheep ovaries and blood samples were collected from pre-pubertal (5 month old) and pubertal (10 month old) animals. Serial sections were examined to study follicle numbers and morphology. A radioactive in situ hybridisation (ISH) was undertaken to determine tissue location and levels of 17α-hydroxylase (17α-OH) mRNA. Androstenedione levels in blood were determined using ELISA. Results showed that while body weight was similar in both T-treated and control groups, T-treatment was associated with an increase in mean ovarian weight in both pre-pubertal and pubertal age groups. T-treated animals experienced some masculinisation of the external genitalia, which has also been reported by other researchers using this model. In pre-pubertal animals, T-treatment was associated with a significant increase in the total number of growing ovarian Type 3-5 follicles within the ovary. When analysed individually, all eight categories of follicles in this study were found to be increased with T-treatment however these proved to be not statistically significant, with the exception of degenerate follicles where a 14-fold increase was seen in T-treated ovaries. Pre-pubertal T-treated ovaries had a significant accumulation of antral follicles ≥4mm, while pubertal T-treated ovaries displayed an increase in follicles ≥1mm. During the isolation of these follicles, it was found that T-treated follicles were more likely to be unhealthy or haemorrhagic than those from control ovaries. Thecal tissue measurements showed that Type 4 and small Type 5 follicles from T-treated ovaries had a significantly larger thecal tissue layer – the site of androstenedione synthesis. In situ hybridisation results showed that expression of 17α-OH mRNA in the theca was significantly increased in Type 4 T-treated follicles and appeared stronger in Type 5 T-treated follicles. Despite 17α-OH being a key enzyme in the production of androstenedione, these results did not translate into a detectable difference in blood levels of androstenedione in both pre-pubertal and pubertal animals. The results of this study suggest that elements of a PCOS phenotype are evident in pre-pubertal, prenatally androgenised sheep. Further study of this model may provide more answers as to the development of PCOS and it’s symptoms and the role that androgens play in the development of the syndrome and the consequent infertility.

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  • Histamine mediated neuropeptide gene expression in bovine chromaffin cells

    Kaur, Joydeep (2012)

    Undergraduate thesis
    University of Otago

    The adrenal medulla regulates the acute stress response and is itself controlled through a variety of signals, including neuronal, endocrine and paracrine factors. Additionally, recent literature suggests that adrenal medullary function may also be influenced by signals from the immune system, such as interleukin-1, interleukin-6 and tumor necrosis factor-α. Secretions from the adrenal medulla may in return influence the immune response thereby suggesting a bi-directional relationship between the immune system and the stress response. Recent microarray data from our laboratory have indicated that isolated bovine adrenal medullary chromaffin cells are also responsive to histamine, another immune-derived signal. Exposure to histamine results in marked changes to their neuropeptide gene expression. The aims of this thesis were to investigate this histamine-mediated neuropeptide gene expression by using real time PCR to validate the microarray results, to examine the signalling mechanisms involved and to explore any interactions between histamine and other known regulators of adrenal medulla. Cultured bovine chromaffin cells were incubated in absence or presence of histamine for 6-48h. The mRNA was extracted and analysed using qRT-PCR for the neuropeptides which were vasoactive intestinal peptide (VIP) and galanin. Additional experiments were performed using protein kinase inhibitors to examine the signalling pathways involved. Immunocytochemistry was also carried out in an attempt to localise the histamine-induced changes VIP protein expression. The results confirm histamine induces an increase in both VIP and galanin mRNA, the latter being mediated by protein kinase C. The pathway responsible for stimulating VIP mRNA was not positively identified but does not involve protein kinase A or C. Synergistic interactions were seen between histamine and other adrenal medullary activators (PACAP and IL-6) for VIP but not galanin mRNA. Immunocytochemistry however did not show any localisation of VIP, which was probably due to the VIP antibody being very non-specific. Thus whether the changes in neuropeptide gene levels result in alterations in protein expression remains unresolved. Given that VIP and galanin are both implicated in anti-inflammatory roles these results are consistent with the concept of a bi-directional relationship between stress response and the immune system.

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  • Repetitive Transcranial Magnetic Stimulation; A Comparative Trial of Low Frequency Treatments

    Heaton, James (2012)

    Undergraduate thesis
    University of Otago

    Background and Aims: Repetitive Transcranial Magnetic Stimulation (rTMS) is a non‐invasive technique capable of altering cortical excitability and treating disease through effects on long term potentiation and depression. In general, high frequency stimulation is thought to produce facilitation, whereas low frequencies (1 Hz or less) produce inhibition. Few studies have investigated inhibitory rTMS using frequencies less than 1 Hz. One potential indication for inhibitory rTMS is hemispheric stroke, where hyperexcitability in the unaffected cortex may be detrimental, due to upregulated transcallosal inhibitory influences. Inhibitory rTMS to the unaffected hemisphere may be of benefit. Simple motor reaction times SRT) are slower when performed bimanually versus unimanually, in an effect termed Bimanual Cost (BC). BC is probably mediated by similar transcallosal mechanisms. The aims of this study were firstly to compare the effects of different low frequency rTMS protocols, and secondly, to determine if BC as a functional model for transcallosal effects in stroke could be modulated by rTMS. Methods: Three stimulation frequencies (1, 0.2 and 0.05 Hz) delivered to the right motor cortex at two intensities (110% and 80% of resting motor threshold) were compared in 20 normal volunteers aged 45 to 75 years, using a counterbalanced cross over design. TMS was carried out using a 70 mm figure of eight coil and was preceded and followed by assessment of neurophysiology with single pulse TMS, SRT and strength dynamometry. Each subject received four out of six possible combinations of frequency and intensity in sessions spaced at least 4 days apart. Results: Contrary to expectations, the predominant effect of rTMS protocols was to increase resting MEP (RMEP) amplitudes, indicating cortical excitation rather than inhibition. Comparison of protocols using a mixed linear statistical model revealed significant effects of frequency and intensity. Frequency was a significant determinant of RMEP (p= 0.026), CSP (p< 0.001) and unimanual SRT (Right and left hand, p= 0.02 and p< 0.001). Low intensity was more effective than high intensity at raising RMEP (p< 0.001) and caused a slight reduction in pinch grip strength (p= 0.044). There was significant interaction between frequency and intensity (p< 0.001) and the 1 Hz low intensity combination was the most effective at raising RMEP amplitude (p< 0.001). Conclusions: In this study, low frequency rTMS produced excitatory rather than inhibitory effects. Possible reasons for this are the older age group of participants and biphasic rather than monophasic stimulation. This study highlights the unreliability of low frequency rTMS as a means of inducing cortical inhibition and the need for better protocols. It also demonstrates that rTMS can modulate BC in a way analogous to hemispheric stroke, providing further support for the hypothesis that hyperexcitability of the unaffected motor cortex may have detrimental effects on recovery.

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