28 results for Undergraduate, 2011

  • 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 Effects of Comvita Manuka Honey on Oral Mucositis in Patients treated with Radiation Therapy to the Head and Neck

    Begley, Aubrey (2011)

    Undergraduate thesis
    University of Otago

    Mucositis is a common side effect of radiation therapy to the head and neck region and one that causes considerable discomfort for patients. Mucositis compromises patients? ability to eat, drink and talk thus affecting patient health and quality of life. Currently there is no worldwide standard for the prevention or treatment of oral mucositis; care is limited to symptom control. Honey has anti-bacterial and potential anti-inflammatory properties and three trials overseas investigated its effect on radiation-induced oral mucositis. The three trials conducted in Malaysia, Iran and Egypt found that honey did reduce the incidence of severe mucositis in head and neck patients. This New Zealand mucositis trial aimed to verify the results from the three overseas trials by comparing the effects of manuka honey with current best practice on oral mucositis in head and neck patients. This report analyses a sub-set of the patients recruited to the trial; those from the Wellington and Dunedin departments. A total of 14 patients were recruited to the trial from these departments, nine recruited to the honey arm and five to the control arm. Four honey patients withdrew from the trial due to issues with the honey application and one patient withdrew from the control arm. Honey arm patients were given manuka honey and instructed to swirl 20mL (amended to 10mL) around their oral cavity and then swallow three times a day, these patients also had access to the standard of care. The control arm patients were treated with the standard of care alone. Patient assessment involved three times weekly mucositis scoring using the TROG multi-site mucositis scoring system, weekly weight and fortnightly quality of life assessment using a 65 question form adapted from the European organisation for Research and Treatment of Cancer questionnaires (EORTC QLQ-C30 and EORTC QLQ HN35). Patients were also asked to fill in food and drug diaries to assess changes in food intake and pain medication. Results showed that manuka honey was not well tolerated by our patient cohort. Patients complained of extreme nausea and stinging sensations in the oral cavity. The honey had to be diluted to be better tolerated (1:3 with another liquid). Contradictory to previous studies, preliminary analysis showed that manuka honey did not affect the extent of oral mucositis in the small cohort of New Zealand head and neck patients when taken in addition to current best practice.

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  • Effects of Orf Virus on the Inflammation Signalling Pathways of the Toll-­like, Interleukin-­1 and Tumor Necrosis Factor Receptor Families

    Turner, Ethan (2011)

    Undergraduate thesis
    University of Otago

    Poxviruses are complex, large double stranded DNA viruses that replicate in the cytoplasm of infected cells. The early phase of infection involves a two step uncoating process, firstly releasing viral core structures, then later viral DNA into the host cell before completion of their replicative lifecycle. Orf virus is the type species of the parapoxvirus genus, which infects primarily sheep and goats in addition to humans by zoonosis transmission. Resulting symptoms are characterised by rapidly developing acute pustular lesions. Orf virus is known to encode several immuno-modulatory factors to permit its replication in the presence of strong host innate and inflammatory responses. Genomic sequencing and bioinformatics analysis have identified an orf virus gene (057) encoding a structural protein containing a phosphatase motif, with close homology to vaccinia virus VH1. As structural proteins, both 057 and VH1 are predicted to become immediately available in the host cytoplasm soon after virion uncoating, where targeted dephosphorylation of intracellular signalling pathways can ensue. This study set out to investigate the potential effect of an orf viral phosphatase on cell signalling pathways through central transcription factor NFĸB, a key upregulator of pro-inflammatory cytokine and chemokine genes. Several classes of receptors, notably, toll-like receptor 4, interleukin-1 and tumor necrosis factor receptor families strongly induce NFĸB by virtue of adapter proteins which transduce signals mediated via phosphorylation and ubiquitylation events. These pathways converge on the IKK complex, which in turn phosphorylates IĸB-α, an inhibitory protein that sequesters NFĸB-p65, resulting in ubiquityn-proteasomal targeted degradation of IĸB-α effectively liberating NFĸB-p65 to undergo nuclear translocation. Assays were performed in HeLa cells to establish stimulatory dynamics and kinetics of NFĸB-p65 activation through induction with respective ligands, LPS, IL-1β, and TNF-α of the aforementioned receptors. Lysates were prepared, resolved by SDS-page and western blot analysis to determine endogenous levels of IĸB-α, and in addition to phosphorylated levels of NFĸB-p65. Initial results from preliminary assays showed rapid phosphorylation kinetics of NFĸB-p65 observable within 10 minutes following induction. The effects upon infection of cells with orf virus were then examined. The most notable result revealed an apparent temporal delay in the maximal levels of phosphorylated NFĸB-p65 induced by LPS and TNF-α when comparing mock and orf virus infected cells with a shift in the accumulation time of maximal levels of phosphorylated NFĸB-p65. Although definitive results of the involvement of orf structural protein 057 in this observation remain inconclusive at this stage, this effect could potentially be attributed to an orf virion-associated phosphatase due to the occurrence preceding viral de novo protein synthesis which is known to begin approximately 4 hours post infection.

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  • Neutrophil Activation in Inflammatory Bowel Disease

    Hoskin, Teagan Susan (2011)

    Undergraduate thesis
    University of Otago

    Abstract Inflammatory Bowel Disease (IBD) is an inflammatory condition in which neutrophils play an important role. Colonoscopy with biopsy is thought to be the best method for evaluating inflammation location, extent, and severity. However, the invasiveness of endoscopic examinations represents a strong limitation to their frequent use. Various studies have described faecal markers as powerful biomarkers of inflammation of the intestinal mucosa in patients with IBD. This thesis details the use of calprotectin and myeloperoxidase (MPO) as biomarkers of disease severity in patients with IBD, and aims to assess whether MPO is a superior biomarker to calprotectin. Abundant levels of both calprotectin and MPO are found in neutrophils. A total of 500 patients were recruited into the evaluation of Novel Biomarkers in Inflammatory Bowel Disease project cohort. However, only 100 of the initial stool samples from patients with or without IBD were used for this research. The samples had been stored at -80°C for up to one year. A short extraction procedure using 100 mg of faeces and approximately 4.9 mL of the appropriate extraction buffer was carried out. Levels of calprotectin and MPO protein were then measured by sandwich enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of MPO was also measured using 3,3’,5,5’-tetramethylbenzidine (TMB) as a reducing substrate. TMB forms a blue product when it reacts with peroxidase enzymes such as MPO. The resulting colour change was read on a microplate reader at a wavelength of 630 nm. Levels of calprotectin, MPO protein, and MPO TMB activity were significantly higher in IBD patients compared to controls. There were significant correlations between calprotectin, MPO protein and MPO TMB activity (p < 0.001). Levels of calprotectin and MPO protein correlated significantly with endoscopic disease severity in patients with CD (r = 0.487, p = 0.001, n = 41, r = 0.483, p = 0.001, n = 41, respectively) and UC (r = 0.677, p << 0.001, n = 35, r = 0.552, p < 0.001, n = 35, respectively). Consequently, both calprotectin and MPO protein were able to discriminate between IBD patients with inactive and high disease severity. However, MPO TMB activity failed to correlate with disease severity in CD and UC patients (r = 0.303, p = 0.054, n = 41; r = 0.258, p = 0.134, n = 35, respectively). The results obtained from this research show that calprotectin and MPO protein correlate strongly with each other. There was also a strong correlation between MPO protein and disease severity, and MPO could successfully distinguish between inactive and high disease severity in CD and UC patients. This suggests that MPO may be useful in the diagnosis and follow-up of patients with IBD. Although, the relationships between MPO TMB activity and disease severity in patients with CD and UC were not significant results were still very promising. This assay could prove to be a faster and more cost effective approach to aid in the diagnosis and follow up of patients with IBD in the future. However, further development and optimisation of the MPO ELISA and MPO TMB assay is required to validate the results from this research.

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  • Anti-Müllerian hormone: A missing link in prostate cancer?

    Masterton, Sarah Lorraine (2011)

    Undergraduate thesis
    University of Otago

    Prostate cancer is the most frequently diagnosed cancer in men in many countries including the USA, France, Germany and New Zealand (Health, 2002, Jemal et al., 2008, Ferlay et al., 2010). The growth of human prostate tissue and the development of prostate cancer is an intricate process maintained by the fine balance of a variety of factors including androgens, estrogens, stromal-epithelial interactions, nervous system input and growth factors (Massague, 1987, Moses et al., 1990, Ittman and Mansukhani, 1997, McVary et al., 1998, Giri et al., 1999, Khandwala et al., 2000, Renehan et al., 2004). Anti-Müllerian hormone (AMH) is one growth factor that may be involved in prostate cancer progression. Previously Segev et al. (2002) showed that AMH inhibits the growth of prostate cancer cells, and Loo et al. (2008) and Segev et al. (2002) showed that its type II receptor (AMHRII) was expressed by the prostate in mice and humans, respectively. Consequently, this project sought to begin to investigate the role of AMH in prostate cancer by examining the localisation and quantifying the expression of AMH propeptide, AMH and AMHRII in benign tissue, low-grade and high-grade cancer of the human prostate using human prostate tissue samples and cells. Immunohistochemistry was used to determine the localisation of AMH propeptide, AMH and AMHRII in human prostate tissue samples, which included various disease states. Double immunofluorescence was then carried out to confirm the location of AMH and AMHRII in human prostate tissue samples. Assessment of immunoreactivity and Western blot analysis were used to quantify the expression of AMH propeptide, AMH and AMHRII expression in human prostate tissue samples. The localisation and expression of AMH and AMHRII in normal and malignant stromal and epithelial human prostate cells was also determined using immunocytochemistry, Western blot analysis and assessment of immunoreactivity, respectively. The results of this project show that AMH propeptide, AMH and AMHRII are located in human prostate stroma and epithelium at all stages of disease. Immunohistochemistry and double immunofluorescence of human prostate tissue samples (which included benign tissue, low-grade cancer and high-grade cancer) exhibited AMH propeptide, AMH and AMHRII immunoreactivity of stroma and epithelium. Immunocytochemistry of normal and malignant stromal and epithelial human prostate cells also demonstrated AMH and AMHRII immunoreactivity. Furthermore, Western blot analysis of benign and malignant human prostate tissue samples and cells revealed immunoreactive bands that may correspond to cleaved and uncleaved forms of AMH and AMHRII. This project also found that there was no significant difference in expression of AMH propeptide or AMHRII in the stroma and epithelium of benign and malignant human prostate tissue samples, as indicated by assessment of immunoreactivity and Western blot analysis. However, there was evidence to suggest that AMH expression is increased in malignancy because AMH immunoreactivity was significantly increased in the epithelium of low-grade and high-grade human prostate tissue samples. Therefore, it seems that AMH may be involved in an autocrine/paracrine growth regulatory mechanism in the human prostate, as has been demonstrated in the human testes (Racine et al., 1998), brain motor neurons (Wang et al., 2005a), and female endometrium (Wang et al., 2009a). This implies AMH may function as a growth-inhibitor (as was indicated by Segev et al.'s (2002) findings on the effect of AMH on prostate cancer cells) or as a growth-stimulator depending on the cellular context. However, further research is needed to confirm the expression pattern of AMH and AMHRII in human prostate cancer progression, and to investigate the effect of AMH peptide on normal and malignant human prostate cell growth both in vivo and in vitro.

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  • Inhibiting the pro-inflammatory cytokine MIF

    Spencer, Emma Sue (2011)

    Undergraduate thesis
    University of Otago

    Macrophage migration inhibitory factor (MIF) is a highly conserved regulatory cytokine, known to exert pro-inflammatory effects. MIF biological activity is primarily regulated via interaction with the CD74 receptor. MIF also exhibits catalytic tautomerase activity via a conserved N-terminal proline residue. Interestingly, this proline is located within the region of MIF that binds to CD74, and small molecules designed to dock and inhibit tautomerase activity have been recognized to interfere with receptor binding. MIF knockout mice are protected in mouse models of rheumatoid arthritis, cardiovascular disease, sepsis, inflammatory bowel disease and cancer. Anti-MIF antibodies also display efficacy in these models. The first small molecule MIF inhibitor, ISO-1, also shows biological activity in disease models. Isothiocyanates are a new class of MIF inhibitors that have recently been discovered. Isothiocyanates are a class of phytochemicals, which have been shown to exhibit anti-cancer and anti-inflammatory activity. PEITC is able to covalently modify the N-terminal proline of MIF, resulting in the complete loss of catalytic tautomerase activity. This study investigated a structure-activity relationship of isothiocyanate inhibition of MIF tautomerase activity. PEITC had an IC50 value of 1.55μM for MIF tautomerase activity in Jurkat T-lymphoma cells, and an LD50 value of 8.4μM. This was 10-fold more effective than ISO-1 at inhibiting MIF tautomerase activity. Increasing the alkyl chain length of isothiocyanates did not greatly influence inhibitory capacity, although PHITC had an IC50 value of 4.2μM compared to that of BITC, with an IC50 value of just 0.54μM. Chlorine, amino and hydroxyl constituents showed minimal effect on the inhibitory capacity, however their cytotoxicity was significantly reduced, with LD50 values of 59μM for OH-PEITC and 60μM for NH2-PEITC. Aliphatic isothiocyanates varied widely in their inhibitory capacity. AITC was the most potent inhibitor with an IC50 value of 0.25μM, while SFN was less effective with an IC50 value of 2.2μM. Both showed very low cytotoxicity, with LD50 values above 100μM. Both AITC and PEITC were shown to inhibit the immunoreactivity of cellular MIF with a monoclonal antibody in a concentration dependent manner, while ISO-1 was shown to have no effect. Since cytokine inhibition therapy is widely considered to have great medicinal prospects, selective targeting of MIF with specific chemical inhibitors, such as isothiocyanates, might offer new therapeutic avenues for these disorders. This study lays the foundation for further drug design efforts.

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  • Characterization of Alkyltriphenylphosphonium Cations and Their Interaction with Bacterial Cells

    Dunn, Elyse (2011)

    Undergraduate thesis
    University of Otago

    Tuberculosis (TB) is a difficult to treat disease caused by the bacterium Mycobacterium tuberculosis. M. tuberculosis is able to shut down its metabolism in response to diverse environmental cues and enter a stage of non-replicating persistence that makes it resistant to many frontline TB drugs. This is further compounded by the “walling off” of M. tuberculosis in granulomatous lesions during infection. New drugs and strategies are in desperate need to combat TB, which currently kills two million people a year. The goal of this thesis was to explore the chemotherapeutic potential of alkyltriphenylphosphonium (alkylTPP) cations; lipophilic positively charged molecules known to accumulate at biological membranes in response to the membrane potential. To address this goal, a structure-function analysis of alkylTPP cations was carried out against several clinically important microorganisms: Mycobacterium tuberculosis, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli; and the non-pathogenic Mycobacterium smegmatis. In addition, we determined if these compounds were toxic to murine RAW macrophages. A series of alkylTPP cations ranging in lipophilicity were characterized, where their toxicity against each cell type was used as a measure of effective accumulation. AlkylTPP cations were shown to be highly toxic to bacteria and mammalian macrophages at concentrations of as low as 1 – 2 μg/mL, where this toxicity increased with respect to lipophilicity. This was deemed an important structure- function relationship for their efficacy. The alkylTPP cation Aa10 was shown to be an effective inhibitor of all bacterial strains used in this study, where it elicited bactericidal killing in M. smegmatis and collapsed the membrane potential. On the basis of these data it is proposed that Aa10 inhibits bacterial growth in a bactericidal manner by dissipating the membrane potential. At toxic concentrations this is due to the accumulation of positively charged alkylTPP cations in the cytoplasmic membrane, where the specifics of this mechanism are yet to be defined. This is validated by the ability of Aa10 to effectively inhibit the anaerobic growth of E. faecalis JH2-2, implying that the action of Aa10 is not dependent on an electron transport chain. Future work will focus on investigating other structure- function relationships that attribute to effective alkylTPP cation toxicity. This includes the addition of different substituents around the central phosphonium ion and variations of the central cation (such as ammonium). Defining these relationships is key in developing alkylTPP cations for a therapeutic application.

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  • A role for alpha-7 nAChRs at cerebellar synapses

    Kim, Yeri (2011)

    Undergraduate thesis
    University of Otago

    The cerebellum performs a well-known motor control function but accumulating evidence supports its role in cognitive functions. The nicotinic cholinergic system alters cognition through its actions elsewhere in the brain but its role in cerebellar processing is not understood. However, expression of a subtype of nicotinic acetylcholine receptors is increased in the cerebellum of human autism patients. Here, we explore the expression, localisation and functional contribution of these receptors at cerebellar synapses. Longitudinal cerebellar sections (30 μm) were prepared from C57BL/6 and Swiss Webster male mice (28-48 days old). Positive immunoreactivity for α7 nAChR (Abcam) and established excitatory synaptic proteins, Vesicular Glutamate Transporter 1 (VGLUT1; Synaptic Systems) and Post Synaptic Density-95 (PSD-95; Abcam) was visualised using secondary fluorescent antibodies Alexa488 and Alexa594 (Invitrogen) and confocal microscopy. Sagittal cerebellar slices (250 μm thick) (C57BL/6 male mice, 21-31 days old) were prepared in artificial cerebrospinal fluid (aCSF) for whole-cell patch clamp recordings from Purkinje neurons (PNs). We recorded excitatory post-synaptic currents (EPSCs) following parallel-fibre (PF) stimulation in aCSF (containing 50 µM picrotoxin to block GABA-A receptors) before, during and after 15 minutes application of 10 nM methyllycaconitine (MLA; Tocris) a potent α7 nAChR antagonist. Series and input resistances varied by < 10% throughout the recordings. The α7 nAChRs were abundantly expressed throughout the cerebellar cortex where they overlapped with the PF excitatory pre-synaptic marker protein VGLUT1 (10 ± 1%) and more strongly with the excitatory post-synaptic marker protein PSD-95 (54 ± 3%) (p < 0.0001, n = 3 animals, Mann-Whitney U-test). Overlapping expression of α7 nAChRs was not evident at inhibitory synapses. Electrophysiological recordings revealed that 10 nM MLA reduced EPSC amplitude, compared with controls, by 30 ± 10% (p < 0.05, n = 5, ANOVA). This study shows for the first time that α7 nAChR expression in the cerebellum contributes to excitatory synaptic transmission at the important PF-PN synapse. Our findings have wider implications for how nicotinic cholinergic inputs influence cerebellar processing.

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  • Establishment and validation of a telomere length assay

    Jodczyk, Sarah (2011)

    Undergraduate thesis
    University of Otago

    Telomeres are specialised structures, consisting of tracts of simple DNA sequence repeats that cap the ends of chromosomes. These structures play a vital role in maintaining the integrity of the genome and prevent the loss of genetic material that resides near the ends of the chromosome. As cells age and divide, the length of telomeric DNA sequences reduces and the progressive shortening of telomeres eventually leads to cell senescence and death. At the whole organism level telomere length appears to reflect cumulative lifetime stresses, and in humans, telomere shortening is associated with reduced longevity, many disease states including cancers, cardiovascular disease, mental illness, cognitive function and other phenotypes. The primary goals of this thesis were to establish and validate a method for measuring average telomere length, then apply it to examine a large longitudinal birth cohort for whom a great deal of data has been gathered over a period of 30 years. The telomere length assay is known as the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay (1). This method provides relative quantification of average telomere length in a genomic DNA sample, by measuring the number of telomere repeats (T) normalised to the single copy reference gene albumin (S), and expressed as a T/S ratio. Signficant technical issues were encountered during the establishment of the assay, and it was found that the choice of hot-start polymerase was essential to the success of the assay. This was because the design of the telomere primers (1) necessitated an overlap of 3bp on their 3’ terminus, which led to primer dimer formation with six of fifteen hot-start polymerases tested. Data acquisition and calculation of T/S ratios was also challenging, and required export of data from the real-time PCR platform used (Roche LightCycler LC480), because the machine software was unable to handle the required analyses. Once established, the reproducibility of the assay was tested and it was found that the assay variation was sufficiently low to enable the detection of differences in telomere length between individuals, using duplicate measures for each sample. Further validation was attempted using Southern blotting of genomic DNA for comparison, but the number of samples obtained was insufficient to validate the assay. As telomere length is known to decrease with age, the performance of the assay was tested by comparing telomere length for subjects of different ages (seven, 25-40 and 50 years). A difference in telomere length was found in the 50 year old age group compared to younger age groups (P=0.001, 0.017). In addition, mouse embryonic stem cell DNA, reported to have ultra-long telomeres, was also examined and these samples did show very long telomere length in both the MMqPCR assay and Southern Blot. The MMqPCR assay was then applied to the Christchurch Health and Development Study (CHDS) which is a prospective longitudinal health study of over 1000 New Zealanders that have been closely followed in intimate detail from birth until the present time. This cohort provides a novel opportunity to examine associations between telomere length and life stress in a longitudinal setting, which should provide superior outcomes to the more typical cross-sectional or retrospective studies that form the bulk of the telomere biomarker literature. Preliminary MMqPCR data from a subset of the CHDS samples (n=255) was generated. Attempts to test associations between general measures of stress and telomere length were underpowered due to the small initial sample set used and the complexity and heterogeneity of available stress measures. However, a significant association was observed between individuals with the shortest telomeres and the highest rates of dependence using the single stress measures of nicotine and alcohol dependence, and the collective measure of substance dependence (P=0.0124, 0.0362 and 0.0008 respectively). No significant difference in telomere length was found with the stress measure of illicit drug dependence, but this group is of a small size. With the observed variability seen in the subset of samples assayed from the CHDS cohort (T/S ratios of 0.04-10.48) and the associations made between individual stressors and telomere length, there appears to be great potential in the use of telomere length as a biomarker of stress.

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  • The role of Δ122p53 in B cell development

    Roth, Imogen Margaret (2011)

    Undergraduate thesis
    University of Otago

    p53 is a crucial tumour suppressor, as evidenced by three important observations: p53 is lost or mutated in half of all human cancers, an inheritable p53 mutation predisposes to an early onset of multiple cancer types, and mice which lack p53 entirely all develop cancer. p53 is thus the ‘guardian of the genome’ (Lane, 1992), and prevents tumourigenesis by inducing the transcription of genes that carry out cell cycle arrest, apoptosis, DNA repair, and cellular senescence. p53 is normally present as a number of isoforms in healthy tissue, though some, including Δ133p53, are elevated in some cancer types. A mouse model of the Δ133p53 isoform, Δ122p53, has been shown to act as an oncogene and have a pro-inflammatory profile. Δ122p53 mice also have a distinctive B cell tumour spectrum largely composed of diffuse large B cell lymphomas, which was the starting point of this research project. The aims of this project were to elucidate the fundamental phenotypic differences between wild type and Δ122p53 mice, largely focusing on differences in cell surface marker expression. We also sought to determine the differences between wild type and Δ122p53 mice in their basal cell cycle profiles and p53-mediated response to DNA damage. In addition, we attempted to follow these differences as the mice aged to determine the potential onset of tumourigenesis in Δ122p53 mice. The results of this project show that Δ122p53 mice have a greater spleen mass than wild type mice, and the bone marrow and spleen have an elevated G2/M population and undergo less apoptosis than wild type mice. While none of the cell surface markers showed consistent differences in expression between wild type and Δ122p53 mice, there were several Δ122p53 mice that had alterations in the expression of the lineage cocktail and Ig κ light chain markers compared to their age matched wild types. The results of an earlier study were replicated, showing that Δ122p53 mice have elevated levels of serum IL-6, a cytokine implicated in many cancer types including those commonly found in Δ122p53 mice. This project also showed a reduction in serum IgG and IgM levels in Δ122p53 mice, suggesting that the Δ122p53 isoform is preventing B cell maturation. This project concludes that the Δ122p53 isoform affects p53 mediated responses and B cell development. We propose that the lineage cocktail and Ig κ light chain markers are potential markers of the tumour precursor population in Δ122p53 mice. Further studies are needed to confirm this, which we anticipate will demonstrate the role of the Δ122p53 isoform in B cell development. This will provide insight into an oncogenic p53 isoform in mice, closely related to the Δ133p53 isoform found in humans, and may provide clues to potential cancer therapies.

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  • Understanding Postpartum Mood Disorders: Characterization of Brain Nuclei Activated by Prolactin Induced Signalling

    Wojciechowski, Isabella Teresa (2011)

    Undergraduate thesis
    University of Otago

    "November 2011". University of Otago department: Anatomy

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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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