238 results for 2010, Undergraduate

  • Vestibular Function in Vestibular Schwannoma

    Tranter-Entwistle, Isaac Brian (2016)

    Undergraduate thesis
    University of Otago

    Abstract Introduction: Traditionally vestibular function has been assessed using caloric irrigations; new methods have failed to reach the same level of accuracy. Vestibular nerve dysfunction occurs with ‘acoustic neuroma’ or ‘vestibular schwannoma. Quantitative testing of hearing by audiometry is much more widely available than quantitative vestibular testing, although consideration of vestibular dysfunction is part of clinical management. Validation of a new method of quantitative vestibular function testing could lead to more widespread integration into clinical practice and affect decision making (i.e. timing of surgery) Methods: A non-blind observational cohort study was undertaken in 31 participants. Study endpoints were either one or two separate participant measures in March/April 2013 the September/October 13. All participants underwent caloric and head impulse testing with video-oculography, while 10 underwent audiometric assessment. Repeat testing was performed for 10 subjects, including additional cognitive. The primary outcome was vestibular function test measures. Results: Video head impulse was strongly correlated with calorics (p=0.01) and showed good sensitivity (80%) and specificity (70%). Dizziness Handicap Inventory showed no correlation with other vestibular function measures. Participants showed reduced cognitive function tested using the CANTAB battery (p=0.01) Conclusion: Video head impulse testing is comparable to caloric testing to assess vestibular function. Vestibular lesions may lead to cognitive deficits. Further research is needed to better understand the role of video head impulse testing in vestibular schwannoma.

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

    Chen, Shumou (2014)

    Undergraduate thesis
    University of Otago

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

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  • 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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  • Quality of Diabetic Foot Care in Oman

    Al-Busaidi, Ibrahim Saleh (2014)

    Undergraduate thesis
    University of Otago

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

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

    Foster, Timothy John (2014)

    Undergraduate thesis
    University of Otago

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

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

    Hamilton, Katelin (2014)

    Undergraduate thesis
    University of Otago

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

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  • Bone Tissue Engineering: Generation of Autologous Bone from Mesenchymal Stem Cells

    Stace, Edward Thomas (2011)

    Undergraduate thesis
    University of Otago

    Bone tissue engineering is a developing technology with the promise of generating autologous bone grafts from small bone marrow samples. The ability to culture bone tissue from small marrow samples removes many of the problems associated with current autologous bone grafting techniques specifically donor site morbidity, supply and quality bone tissue. Whilst bone tissue engineering is being researched elsewhere, the exciting prospect of bone banking is novel. We see the cryopreservation of cultured bone for use in later life as an intriguing opportunity for people employed in hazardous jobs such as the armed forces and those engaging in potentially traumatic interests like skiing. To begin bone banking research, a successful bone tissue engineering protocol was required. There were three aspects to this work; defining a protocol for isolation of an appropriate cell population, generation of a suitable three dimensional scaffold and design of a perfusion culture system. This thesis examines these three initial aspects. Mesenchymal Stem Cells (MSCs), isolated from rat femoral bone marrow, were expanded and differentiated down the osteoblastic lineage by 28 days culture in a dexamethasone based osteogenic media. Over this osteogenic culture period, cells developed a cuboidal osteoblast-like morphology. Immunohistochemical staining showed these cells increased the expression of the known bone markers; collagen I, osteocalcin, osteopontin, osteonectin and bone sialoprotein. Additionally, osteogenic cultures showed a 200 fold increase in alkaline phosphatase (ALP) concentration. Scanning Electron Microscopy (SEM) showed the deposition of a highly fibrillar matrix surrounding the osteoblast-like cells in osteogenic cultures. Immunohistochemically, this matrix stained positively for collagen I and alizarin red staining showed mineralization of this matrix. Contrastingly, no change in morphology, no extracellular matrix and no increase in ALP concentration were noted in control conditions. For bone tissue culture, a chitosan-hydroxyapatite scaffold was generated through a freeze drying process. Micro Computer Tomography (µCT) and computer analysis showed the mean pore diameter was 228 µm. SEM surface analysis showed the hydroxyapatite distributed evenly within the scaffold. After the scaffold was subjected to degradation and cytotoxicity testing, MSCs were seeded onto cover slips coated in the chitosan-hydroxyapatite scaffold. MSCs were seen to adhere to and proliferate on this scaffold. MSCs were then seeded on to chitosan-hydroxyapatite scaffolds and cultured under perfusion conditions in the designed perfusion culture system. After a 10 day culture period no cells were detected on the scaffold. This is believed to be due to the low initial cell seeding density. This research has shown the successful differentiation of MSCs down the osteoblastic lineage, fabrication of a suitable chitosan-hydroxyapatite material, cell adherence to this scaffold material and development of a perfusion cell culture system. However, further optimisation of the perfusion culture protocol is needed. Successful perfusion culture would then allow experimentation with cryopreserved cultured bone and further investigation of the feasibility of bone banking.

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  • MANUKA HONEY: An Investigation into the Effect of Manuka Honey on Oral Mucositis in Patients Receiving Radiation Therapy to the Head and Neck

    Parsons, Emma Kay (2011)

    Undergraduate thesis
    University of Otago

    Head and neck cancer is the sixth most common type of cancer, with an estimated 650,000 registrations and 350,000 deaths worldwide annually (Parkin et al., 2005). The treatment for these types of cancers is becoming increasingly aggressive with the majority of patients receiving a combination of surgery, radiation therapy and chemotherapy to cure their cancer. Severe oral mucositis is a common side effect of these cytotoxic treatments with 60% of patients receiving radiation therapy and 92% of patients receiving chemoradiation developing it during the course of their treatment (Parulekar et al., 1998; Sonis, 1998; Dodd et al., 2000; Elting et al., 2003). Oral mucositis leads to many secondary complications including severe oral pain, difficulty in eating and swallowing, taste changes, infection, malnutrition and weight loss. Currently, there is no standard form of treatment for oral mucositis with the majority of treatments aimed at palliation of symptoms rather than preventing or treatment oral mucositis itself. The research presented in this thesis investigates the effect of manuka honey on the prevention and treatment of radiation induced oral mucositis in patients receiving radiation therapy and chemoradiation for head and neck cancers at the Palmerston North Oncology Department. The original study was designed as a stage II randomised single blinded trial where patients were randomised into one of two arms. Patients in the control arm were given the standard treatments for oral mucositis in New Zealand including Benzydamine Hydrochloride (HCL), bicarbonate rinses, pain killers and anti-fungals. Patients in the experimental arm were given all standard treatments and were asked to gargle 20mls of undiluted manuka honey three times per day. Patients oral mucositis was scored three times per week, they were weighed once per week and asked to fill out a food and drug diary everyday and a quality of life questionnaire once every fortnight during treatment. Due to poor patient compliance with the undiluted honey this trial was downgraded to a phase I pilot trial investigating the best way to administer manuka honey to treat oral mucositis. This thesis specifically reports the results for twelve patients recruited to this trial between March 2009 and December 2009. Due to the early downgrading of this trial from a randomised phase II trial to a pilot trial the effects of pure undiluted manuka honey on radiation induced oral mucositis could not be assessed. There was no statistically significant difference in the severity of oral mucositis reported between those taking diluted manuka honey and those using standard forms of treatment only. Patients taking diluted manuka honey appeared to have slightly less weight loss than those receiving standard treatments alone however this did not reach statistical significance. All patients, irrespective of whether they were taking honey or not, reported a severe decrease in quality of life throughout the course of their radiation therapy. There were large issues with patient compliance in this trial. Even when the honey had been diluted significantly patients complained the honey tasted too sweet, made them feel nauseous and stung their oral mucosa. Due to these issues with compliance, it was not deemed ethical to continue with the current trial unless the honey is given to patients is a way which is tolerated better.

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  • Effect of Delayed Treatment with Mesenchymal Stem Cells on Neonatal Hypoxic/Ischemic Brain Injury: A Behavioral and Stereological Study

    Alwakeel, Amr J. (2011)

    Undergraduate thesis
    University of Otago

    Hypoxia/ischemia is a major cause of acute neonatal brain injury and may lead to the development of neurological disabilities, mainly cerebral palsy. Hypoxic/ischemic (H/I) injury occurs as a result of decreased oxygen level in the brain and/or blood and reduced perfusion of the brain tissue. One of the main sites involved in neonatal H/I brain injury is the striatum. In children, injury to the striatum results in the muscular abnormalities of cerebral palsy. Medium-spiny neurons constitute the major neuronal population of the striatum in both primates and rodents. Hence, the rescue or restoration of the medium-spiny neuron population is a viable aim in treating neonatal H/I injury. Current evidence has shown hypothermia, a neuroprotective strategy, to be effective in treating H/I injury. However, hypothermia and other neuroprotective strategies can only be administered within 2 – 6 hours post-injury. The aim of this study was to investigate the therapeutic potential of a seven-day delay in treatment with mesenchymal stem cells (MSCs), a neurorestorative strategy, following hypoxia/ischemia in the neonatal rat. Furthermore, the effect of a subcutaneous injection of a high-dose (HD, 7.5 x 10^5 – 1 x 10^6) and of a low-dose (LD, 8.5 x 10^4 – 1.2 x 10^5) of MSCs was investigated. This was the first study to assess the efficacy of the subcutaneous route of delivery in mesenchymal stem cell (MSC) therapy following neonatal H/I injury. On postnatal day (PN) 7, male pups were exposed to H/I injury. After a seven-day delay (i.e. PN 14), pups were weight-matched in pairs or triplets and randomly assigned to either a diluent injection of Dulbecco's phosphate-buffered saline (DPBS) or a MSC injection. In the LD MSC experiment, five pups were administered the diluent while six pups received a LD MSC injection. In the HD MSC experiment, seven pups were administered the diluent while nine pups received a HD MSC injection. The therapeutic effect was assessed using behavioral testing, and stereological analysis of the absolute total number of striatal medium-spiny neurons. On PN 20, the functional outcome was assessed using the negative geotaxis, cylinder, elevated body swing and foot-fault tests. Each pup was sacrificed on PN 21 and their brain was dissected from the cranium. Injured hemispheres were subsequently embedded in Technovit, serially sectioned and stained. Sections were stereologically analyzed using the Cavalieri method and optical disector method to estimate the absolute number of striatal medium-spiny neurons between diluent- and MSC-receiving pups. To our knowledge, this was the first study that used unbiased modern stereological methods to quantify the absolute number of medium-spiny neurons in the striatum following MSC therapy in neonatal hypoxia/ischemia. A sub-aim of this study was to determine the efficacy of the negative geotaxis test in the study of neonatal H/I injury before the administration of any treatments. As such, pups were tested on the negative geotaxis apparatus on PN 12 and PN 14, prior to MSC and diluent injections on the afternoon of PN 14. The findings of this study showed that a seven-day delay in MSC treatment did not have a statistically significant improvement on the functional outcome following H/I injury. However, a positive trend was observed in the cylinder test in pups receiving MSCs. MSC administration resulted in a higher preference of using the contralateral injured limb over the ipsilateral uninjured limb when compared to the diluent-administered pups. This positive trend was more profound in the HD MSC group compared to the LD MSC pups. The stereological findings showed that delayed MSC therapy was effective in attenuating the loss in striatal medium-spiny neurons compared to diluent-receiving pups. This difference was found to be statistically significant. The HD MSCs were more effective than the LD MSCs and restored the number of striatal medium-spiny neurons to normal levels. The subcutaneous route was also shown to be an effective route in delivering MSCs. Finally, results from the negative geotaxis test showed that this test may not be an effective assessment in evaluating the functional outcome following neonatal H/I brain injury. In conclusion, the findings of this study suggest that delayed MSC therapy can be an effective tool in treating neonatal H/I brain injury. These findings may offer hope to children who have missed the critical period of 2 – 6 hours post-injury, which is limited to neuroprotective interventions.

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  • Measuring the Physical Activity of Children aged 3 to 7 years

    Ku, Hsi-Yu (2011)

    Undergraduate thesis
    University of Otago

    Introduction : Obesity has become epidemic throughout the world and is affecting both adults and children. New Zealand children have a high prevalence of being overweight, with estimates varying between 20% and 30%. Sedentary behaviour (SB) is an important mediator of successful prevention of developing overweight in children. However a reliable objective method for measuring SB is still lacking. Effective prevention of excessive weight gain could flow from having an objective device with a clear definition of SB. Accelerometers are motion sensing devices which have been used to study physical activity (PA) with promising validity in children. As one of the steps in establishing the utility of accelerometers in measuring SB, we aimed to assess the reliability and validity of the Actical accelerometer for its use in 3-7 year old children and to propose an appropriate cut-off that defines SB. Methods : Children (N=50) aged 3-7 year old were recruited in Dunedin, New Zealand, to participate in the study. The study was carried out at the participants’ preschool centre or school. The children were asked to wear the Actical accelerometer around their waist and to perform numerous selected activities of varying levels of intensity. At the same time, participants were video recorded for observational analysis to provide the criterion measure of PA. Activities performed during free play sessions at participants’ preschool centre or school were also measured. Reliability of the Actical accelerometers was assessed daily throughout the data collection phase using a custom-made motion generator. Validity of the accelerometer was assessed by comparing with activity levels measured by direct observation using the Children’s Activity Rating Score (CARS). The appropriate cut-off to define SB was determined by plotting the receiver operating characteristic curve, and the cut-off derived was then cross-validated by comparing with levels of SB measured by using the CARS. Results : Height, weight and BMI distributions of the children assessed (N=49) were comparable with published data on New Zealand children. Reliability tests during the data collection phase revealed high intra-instrument and inter-instrument reliabilities (r p-intra & r p-inter =1.0). Repeated measurements by the same accelerometer gave small differences (<0.05) and were categorised into four groups: inactivity (Sleep), sedentary level movement (Draw, Play Doh, Puzzle, Read, TV), light level activities (Toy Car), activities of higher intensities (Nintendo Wii and Free Play). Using the receiver operating characteristic curve (area under the curve: 0.843), a cut-off of 40 counts/15s was identified (sensitivity: 88.44% and specificity: 64.63%). For the children assessed by the CARS (N=9), correlation between Actical counts and CARS score was moderate (r p =0.56). The mean difference of percentage of time in sedentary activity judged by accelerometry compared to direct observation using CARS was 8.4%. There were no significant differences in the percentages of sedentary activity between accelerometer data versus CARS (p=0.055). Conclusions : Overall, the study has proposed a cut-off for SB of 40 counts/15s. Despite having obtained moderate correlation with the criterion measure, it appears that this cut-off tends to slightly under predict levels of SB and accurate prediction of SB is limited by sub-optimal inter-instrument agreement. Performance of the Actical could be improved if accurate calibration were possible outside the manufacturer. Utility of the cut-off could be further assessed by conducting a cross-validation of the cut-off with a larger sized sample. Outcome : The results of this study could be used in ongoing studies that use the Actical accelerometers to measure activity in children aged 3 to 7 years.

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  • Prolactin Regulation of Kisspeptin Neurons

    Crampton, Jessica Rose (2011)

    Undergraduate thesis
    University of Otago

    It is well established in both humans and rodents that the state of hyperprolactinaemia leads to reduced reproductive capacity, as a consequence of suppressed luteinising hormone secretion. Return of reproductive function can be achieved by physiological gonadotropin-releasing hormone (GnRH) administration in humans. In rodents, there is no decrease in pituitary GnRH responsiveness in the presence of elevated prolactin. These findings indicate that prolactin, an anterior pituitary hormone capable of direct action in the central nervous system, is affecting hypothalamic GnRH secretion, rather than pituitary gonadotropin secretion. However, as prolactin receptors are only expressed on a small minority of GnRH neurons, a direct suppressive action by prolactin on these neurons is unlikely. Hence, an indirect mechanism utilising neurons afferent to GnRH neurons may be in place. The neuropeptide kisspeptin has recently been discovered to be a key afferent regulator of GnRH secretion. Prolactin receptors are present on the majority of kisspeptin neurons, leading to the hypothesis of this thesis; that prolactin inhibits kisspeptin neurons, providing an indirect pathway through which prolactin alters GnRH output. To investigate this hypothesis, several experiments were carried out. Firstly, double-label immunohistochemistry, staining for pSTAT5 (an intracellular signal transducer of prolactin signalling) and kisspeptin, was performed throughout the AVPV/PeN and arcuate nuclei of the rat hypothalamus. Colocalisation of pSTAT5 nuclear-staining within kisspeptin neurons was evident in ovine prolactin (oPRL)-treated animals, indicating that the prolactin receptors expressed by kisspeptin neurons are functional in vivo. Secondly, Kiss1 mRNA expression in a lactational model of hyperprolactinaemia was analysed by qPCR. There was a significant suppression of Kiss1 mRNA expression in each nucleus during lactation compared to diestrous levels. This was not reversed by prolactin removal (by bromocriptine-treatment), suggesting a suckling-induced suppression not mediated solely by prolactin. A third treatment group, where pups were removed and oPRL was administered, however, suggested the presence of an additional suppressive effect of prolactin in the arcuate nucleus. Finally, in order to investigate the effects of hyperprolactinaemia without the confounding factors of a lactation, a nonlactational model of chronic hyperprolactinaemia was developed. This trial involved ovariectomy with low level oestradiol replacement, and oPRL administration every 8 hours for 48 hours. Serum oPRL concentration, profiled by serial blood sampling through indwelling jugular cannulae, was found to peak 1 -3 h post-injection (approximately 80 ng/ml) and drop to 0 ng/ml by 6 h post-injection. This oPRL-treatment did not suppress LH concentrations compared to vehicle-treated controls, and thus in this regard, the model was unsuccessful. Nevertheless, the hypothalamic tissue obtained was analysed by qPCR to investigate whether Kiss1 mRNA expression was altered by oPRL-treatment. No significant changes were detected in the AVPV/PeN, whilst in the arcuate, there was a significant four-fold increase in Kiss1 mRNA expression in vehicle-treated, ovariectomised rats. This increase was significantly dampened by approximately half, in oPRL-treated ovariectomised rats. Each of these experiments provide evidence in support of the hypothesis; indicating that prolactin does regulate kisspeptin neurons. This finding could hold important implications for further investigations into the use of kisspeptin as treatment of hyperprolactinaemic infertility, a condition that hinders many patients.

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  • Bowel Bacteria and Dendritic Cells in Ankylosing Spondylitis

    Peyroux, Estelle (2011)

    Undergraduate thesis
    University of Otago

    Ankylosing Spondylitis (AS) is chronic inflammatory arthritis of spine and sacroiliac joints with further peripheral manifestations. AS is a relatively common rheumatic disease affecting 0.1- 1.4% of the population. Early symptoms commence generally in the second decade of life, these include fatigue and pain which are managed by adherence to medication. Inflammation can result in fusion of the vertebrae in the spine. Susceptibility genes identified encode HLA-B27 and ERAP1 proteins, both essential components in antigen processing for immune response. An animal model of AS induced by the hla-B27 gene (present in 95% AS patients) indicated enteric bacteria triggers AS-like symptoms. Dendritic cells (DCs) are professional antigen presenting cells that direct immune responses. We generated monocyte-derived DCs (MoDCs) from venous blood of AS (n= 6) and control group (n= 8) and stimulated the cells with a range of gut bacteria- Bacteroides fragilis, Yersinia enterocolitica, Campylobacter jejuni and Helicobacter pylori. We measured expression of antigen presentation molecules MHC-I and MHC-II and CD83, a marker of activated DCs, by flow cytometry. Analysis of cytokine secretion in supernatants was assessed by Bioplex immunoassay. In addition to this preliminary experiments with inhibitors to MyD88 and TRIF adaptor molecules were used to identify the possible involvement of toll-like receptors in activation of DCs to bacterial treatments. The AS donors had a trend of lower basal expression of MHC-I and MHC-II. Our results indicated the level of MHC-I and MHC-II expression is significantly different (p< 0.0001) between AS and normal groups. The normal group’s up-regulation of MHC-I in response to C. jejuni was significantly greater (p= 0.03) compared to the AS group (p=0.1). Differences in MHC-II expression between AS and normal groups were just outside significance levels (p= 0.08) for both B. fragilis CAS10 and H. pylori treatments. CD83, a DC maturation marker, was expressed at similar levels for all AS-associated bacteria. H. pylori induced CD83 expression significantly (p= 0.03) in the normal group however the AS group did not up-regulate CD83 expression significantly (p= 0.2). Cytokine analysis showed a trend of decreased IL-12 and IL-10 production in AS-associated bacteria particularly in B. fragilis CAS10 and Y. enterocolitica. Our control bacteria, H. pylori, inversely induced higher IL-1β, IL-10 and IL-12. From these results we hypothesize that abnormal signaling through innate pattern recognition receptor signaling pathways induces aberrant activation of DCs in AS patients. These DCs in turn may direct cells of the immune system toward an inappropriate or misdirected immune response. We speculate that a low IL-10 production by DCs may result in a dysregulation of the immune response.

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  • The actions of Activin C and its mechanism towards the progression of Prostate disease

    Lee, Kai Lun (2011)

    Undergraduate thesis
    University of Otago

    The Transforming Growth Factor β (TGF β) superfamily is involved with regulating many biological and physiological processes. Activins are members of the TGF β superfamily. Four activin subunits (βA, βB, βC and βE) have been characterized in mammalian cells. Activin A is a negative growth regulator in the prostate and dysregulation is associated with prostate disease. Activin βC was discovered only in the last decade. Recently it was shown that activin βC regulated the expression and bioactivity of activin A, and over-expression led to the development of prostate disease in mice aged 3 months. The current study hypothesized that activin βC functions locally to antagonize the activity of activin A, and over expression of activin βC can therefore lead to the formation of overt prostate disease in aged mice. We used overexpressing activin βC transgenic mice (TG) aged 12 months, and studied the dorsal and ventral prostate. There were clear signs of hyperplasia in the TG prostate epithelial cells, and some abnormally stained nuclei indicative of mucinous metaplasia. The prostate weights had also increased due to a significant increase in epithelial cells. Immunohistochemistry was then performed for PCNA and the results indicated no increase in cellular proliferation. However, a decrease in apoptosis was also evident as was a decrease in p-SMAD 2 signaling in the dorsal prostate. Thus supporting our hypothesis that increased activin βC antagonized the growth inhibitory effects of activin A and this was the mechanism underlying the alterations in proliferation and apoptosis. Pathway focused gene expression was then undertaken in the dorsal prostate to assess the mechanism by which over-expression of activin βC decreased proliferation, apoptosis, and SMAD 2 signaling. An increase in gene expression of activin βA and βB, the activin receptors, as well as the SMAD signaling molecules were evident. In addition, an increase expression of gene in the negative regulation of cell cycle was observed in TG samples, indicating that these cells were signaled for growth arrest. This validates the decreased PCNA and apoptosis data in the TG prostate sections. Finally, correlation with human prostate pathology was determined by assessing the staining of activin βA and activin βC in human prostate disease arrays. Activin βA was increased in all prostate disease whereas activin βC was only increased in benign prostatic hyperplasia. Therefore we conclude that activin C is associated with benign disease and not prostate cancer in mice and men.

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  • The Hamstring Muscles: Proximal Musculotendinous Junctions

    Storey, Richard Norman (2012)

    Undergraduate thesis
    University of Otago

    Background: Acute hamstring strains are common injuries in sports, responsible for excluding athletes for a considerable amount of time from participation and competition. This places a substantial burden on professional sporting organisations and healthcare systems. Aims: To describe patterns in the site and mechanism of hamstring strains and examine the morphology of the proximal tendon and musculotendinous junctions (MTJs) of these muscles using dissection and magnetic resonance imaging (MRI). Methods: Two systematic literature reviews were undertaken, firstly to ascertain the site and mechanism of hamstring strains in the medical imaging literature and secondly to review the anatomy of the hamstring muscles relevant to acute strains. The morphology of the proximal MTJs of biceps femoris long head (BFlh), semitendinosus (ST), and semimembranosus (SM) was investigated using two methods: (i) individual muscles in ten thighs from five male cadavers (aged 64-85 years) were dissected and measured in situ and then resected, embedded in jelly and serially sectioned for scaled photography; (ii) the hamstring muscles of 11 physically active men (aged 18-30 years) were imaged bilaterally using MRI. In each study group, the length, volume and cross-sectional area of the proximal tendon and muscle belly were measured. In addition, novel measurements of the muscle-tendon interface area at the proximal MTJs were taken in cadavers and the MTJs in both groups were reconstructed three-dimensionally (3-D) using Amira 4 software. The relative force of muscle contraction at the proximal MTJ was estimated by calculating a muscle belly volume to muscle-tendon interface area ratio. Comparisons were made between the three hamstring muscles and between study groups. Results: The literature review of hamstring strains indicated that the most frequently injured hamstring muscle is BFlh (65.8% of all acute strains). The MTJ was the site of injury in 73% of strains, usually the proximal MTJ. The site and mechanism of hamstring strains appear to be related: powerful eccentric contraction as occurs in sprinting is commonly associated with BFlh strains with or without additional pathology in ST, and, slow-speed stretching injuries predominantly affect SM but can also involve adjacent muscles such as quadratus femoris. Review of the anatomical literature showed that the morphology of these proximal structures is not well understood. Three-dimensional reconstruction showed a similar proximal tendon and MTJ morphology in elderly cadavers and young active men. In both groups SM consistently had the longest proximal tendon in all specimens and, on average, MTJ, the latter extending over 20 cm2 in both groups. In cadavers the MTJ of SM also had the greatest muscle-tendon interface area (84.6 ±31.5 cm2). Muscle belly volume was more than three times greater in young active men than in elderly male cadavers. Muscle belly volume/MTJ interface area ratios suggested that BFlh experiences the greatest forces at its proximal MTJ, with a ratio of 1.68, compared to 1.65 for the proximal region of ST, and 1.24 for SM. ST muscle fibres inserted proximally into both the common proximal tendon of BFlh and directly into the ischial tuberosity. The hierarchy of hamstring muscle peak cross-sectional area varied among the young active men but was constant in elderly cadavers. Conclusions: These results suggest that the morphology of the proximal MTJ is relatively consistent. However, key differences were evident between hamstring muscles. Estimation of force transfer at the proximal MTJ suggests that the architecture of the proximal MTJ in BFlh and ST may render them vulnerable to acute strains during powerful muscle contraction. In contrast, the larger muscle-tendon interface found in SM suggests less concentration of force at the MTJ and its longer proximal free tendon may explain its vulnerability to slow speed stretching injuries.

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  • Recombinant Virus-Like Particles Presenting Epitopes for Antibody Generation

    McCulloch, Tim (2012)

    Undergraduate thesis
    University of Otago

    Virus-like particles (VLP) have been shown to be effective vessels for drug or gene delivery, and vaccination. Much research has focused on the generation of VLP carrying heterologous tumour antigens to initiate cell-mediated immunity against tumours, where the VLP enhances the immunogenicity of the attached antigen. However, VLP can also be modified to incorporate an antigen on the surface to facilitate antibody generation. Purification tags or specific coupling sites could also be incorporated onto the surface of the particles, improving their functionality. This study aimed to utilise the immune stimulatory properties of VLP to generate antibodies against a heterologous model antigen, producing a scaffold for delivery and enhancement of antibody mediated subunit vaccines. More specifically, Human Norovirus (HuNV) and Rabbit hemorrhagic disease virus (RHDV) capsid proteins were engineered to express the YG1 epitope from human tumour necrosis factor-alpha (hTNF-α) at positions predicted to be displayed on the surface of the VLP. Amino acids positions 368 of HuNV and 306 of RHDV capsid proteins were identified in the literature as confirmed sites of peptide insertion, with each locating to the exposed loops of the capsid protein P domain. Recombinant gene constructs expressing the YG1 peptide at these sites were produced by PCR. VLP were expressed in a baculovirus expression system and purified using differential centrifugation and cesium chloride gradient separation. Following expression, recombinant VLP assembly was confirmed by electron microscopy, and while the RHDV VLP was less stable than HuNV, they were both able to form particles. Rats immunised with these recombinant VLP generated IgA and IgG antibodies specific for both the native VLP carrier and the YG1 epitope. Thus initial data indicates that HuNV and RHDV VLP can be genetically engineered to act as vaccine delivery systems, leading to enhanced peptide based subunit vaccines. The confirmed insertion sites could also be used to incorporate purification tags, specific coupling sites, or allow multi-epitope capability, where peptides can be included at both the N-terminus and on the surface of the same VLP.

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  • Allocation and Ageism in Aotearoa: An exploration of the ethical justification for age-based healthcare rationing

    Lambie, Deborah (2013)

    Undergraduate thesis
    University of Otago

    New Zealand’s population, like many first world countries, is ‘ageing’. This will place our healthcare system under increasing and changing pressures, as there will be a greater proportion of older people in our population who have particular health needs and generally require greater levels of care over longer periods. These projected changes have resulted in calls to ration healthcare resources to the elderly. This is driven in part by concerns that the elderly will consume resources at the expense of other generations. Such proposals have been met with staunch criticism, most of which focuses on the idea that age-based allocation devalues those discriminated against, viz. the elderly. In this thesis I argue that this criticism can theoretically be overcome if the principles of age prioritisation are applied consistently over the lifetime of members of society, so that everyone is equally affected. I then introduce Norman Daniels’ ‘Prudential Lifespan Account’, which provides a moral basis from which such principles can be derived. This shows us that as a society we should protect the ‘normal range of opportunity’ available to citizens. This creates a need for resources to be distributed fairly between generations in order to ensure that each is given the best chance possible of having the normal range of opportunity. Under this theory, certain forms of age-based allocation can be justified. I go on to outline Daniel Callahan’s view of the nature of mortality and end-of-life care, which incorporates Daniels’ Prudential Lifespan Account. This provides a helpful framework for reflecting on our collective response to ageing and dying, which will place certain limits on the amount spent on care for the elderly. However, these limits will not ultimately address the challenges our healthcare sector is currently facing due to the ageing population. With this in mind, I consider whether a straightforward cut-off for healthcare allocation is ethically justifiable. I argue that it cannot because it contradicts the basic principles of justice underpinning Norman Daniels’ theory, and so lacks an adequate moral foundation. Some other responses must be taken in order to ensure that members of our society receive their share of opportunity. In the final chapter I outline some possible strategies that are consistent with the ideals put forward by both Daniels and Callahan.

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  • CT Investigation of Thoracic Intervertebral Disc Morphometry

    Fletcher, Justin Geoffrey Revell (2013)

    Undergraduate thesis
    University of Otago

    Background: Despite being prone to degenerative disease and other pathology, relatively little is known of the morphology and morphometry of thoracic intervertebral discs in comparison to discs in the cervical and lumbar spines. The few studies of thoracic intervertebral disc morphometry that have been performed have included relatively small numbers of cadavers. No previous studies have attempted to investigate how thoracic intervertebral disc morphometry varies with sex and age. Aims: This study aimed to describe the normal morphometry of adult thoracic intervertebral discs in subjects with no known spinal disease and investigate potential associations with sex, disc level and age. Methods: Computed tomography (CT) scans on 128 recently deceased unembalmed cadavers (70 males, 58 females, aged 20-79 years, divided into 10-year age cohorts) with no known spinal pathology were obtained from the archives of the Victorian Institute of Forensic Medicine, Australia. Intervertebral disc height, anteroposterior and transverse disc dimensions, wedge-index and other morphometric parameters were measured at alternate intervertebral discs throughout the thoracic spine, beginning at T2-3. Data were analysed to determine associations between intervertebral disc parameters and sex, disc level and age (10-year age groups). Linear mixed models were fitted to anterior and posterior disc height and anteroposterior and transverse disc dimensions. Intra- and inter-rater variation were assessed by intraclass correlation coefficients (ICCs) performed on 25% and 18% of the whole sample, respectively. Measurement reliability was further assessed by investigating agreement between radiographic and equivalent measures performed directly on two cadaver thoracic spine specimens. Results: Intervertebral disc heights and axial dimensions were all significantly larger in males than females except middle disc height (anterior disc height 4.0 ± 1.4mm vs 3.6 ± 1.3mm; posterior disc height 3.6 ± 0.90 vs 3.4 ± 0.93, both p<0.05). Discs in the upper and lower thoracic spine were significantly more wedge-shaped and displayed greater convexity than those in the mid-thoracic spine (T6-7). Except at T2-3, anterior disc height decreased with advancing age while anteroposterior and transverse disc dimensions increased; posterior and middle disc heights and indices of disc shape showed no consistent statistically significant change. Most measured parameters showed substantial to almost perfect agreement for both intra-rater and inter-rater reliability while cadaver validation showed fair to almost perfect agreement between radiographic and anatomic measures. Conclusions: Thoracic intervertebral disc morphometry varies significantly and consistently with sex, disc level and age. This study provides unique data on normal thoracic intervertebral disc morphometry of adults, which should be useful in the detection of pathological changes and for informing biomechanical and functional studies.

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  • Brain Regions Responding to Endogenous Prolactin during Pregnancy and Lactation

    McFadden, Sarah Louise (2013)

    Undergraduate thesis
    University of Otago

    There is an increased incidence of dysfunctional mood disorders postpartum, affecting approximately 10-20% of all women. Postpartum mood disorders can have devastating effects on the mother, and lead to altered relationships with offspring, an increased incidence of child abuse, and increased conflict in relationships leading to a higher rate of separation. It has been discovered that low levels of prolactin during early pregnancy prevent the normal increase in neurogenesis in the subventricular zone (SVZ) from occurring, resulting in markedly increased anxiety postpartum in mice. However, it has been observed that the SVZ does not express prolactin receptor mRNA, nor prolactin-induced phosphorylation of the transcription factor STAT5 (pSTAT5), a marker of prolactin mediated signal transduction. Therefore, we hypothesized that the normal elevation of prolactin that occurs in early pregnancy initiates changes in other areas of the maternal brain that indirectly lead to increased neurogenesis in the SVZ. The aim of this study was to characterize which areas of the brain are activated in response to the normal surges of prolactin that occur during early pregnancy, and also if there is a difference in the regions of the brain that are activated during pregnancy and in the postpartum period. A group of mice were killed on day 3 of pregnancy at a time when prolactin levels were low, and another group of mice were killed during a surge when prolactin levels were high. Two groups of mice were also killed on day 7, one at a time when prolactin was low, and the other when prolactin was high. The final two groups were killed on postpartum day 4; one group had been exposed to pups, and the other had not. Using immunohistochemistry we analyzed levels of pSTAT5 throughout the mouse brain. We found that expression of pSTAT5 was significantly increased when prolactin levels were high compared to when they were low during pregnancy. Expression of pSTAT5 was seen in regions of the brain known to be crucial for regulation of mood and behaviour, such as the medial preoptic area, and also in the Arcuate nucleus and the VMH (ventromedial hypothalamic nucleus). In the lactating mice, a higher expression of pSTAT5 was seen in these areas, as well as in additional areas not seen during pregnancy, such as the Bed Nucleus of the Stria Terminalis, the Amygdala and the Dorsomedial Hypothalamic Nucleus. These areas may be areas necessary for maternal behaviours at the time of lactation. We also stained for c-fos, a marker of early gene activation in neurons to establish if there were nuclei that are indirectly responding to prolactin. The c-fos data showed that it was not a good marker for prolactin activation, but it was able to show areas of the brain important for maternal behaviour. In conclusion, this study confirmed that prolactin does not activate pSTAT5 in the sub ventricular zone during pregnancy, consistent with the hypothesis that prolactin induced increase in neurogenesis in this area is mediated indirectly. We identified the MPA, the arcuate and the VMH as the major areas responsive to endogenous prolactin in early pregnancy. Moreover, we found that there were more prolactin-responsive areas in the maternal brain during lactation.

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