28 results for Undergraduate, 2011

  • Novel Organic Carbon Monoxide Releasing Molecules as a Potential Treatment for Triple Negative Breast Cancer

    Gunatunga, Kishan (2011)

    Undergraduate thesis
    University of Otago

    Carbon monoxide (CO) plays a role in many physiological and pathophysiological processes as a second messenger. Emerging evidence reveals the potential CO has as a therapeutic agent as it has been implicated in the modulation in a range of intracellular functions including apoptosis and proliferation. In the case of cancer, specifically triple negative breast cancer (TNBC), there is very little information regarding the effects of this molecule. Here we hypothesize that the targeted delivery of CO to a tumour will result in an anti-cancer effect in TNBC. The current study examines a novel class of compounds termed organic CO releasing molecules (CORMs) (CO-1 – CO-8) and previously published metal containing CORMs (CORM-2), as potential treatments for TNBC. Firstly a wide range of synthesised novel organic CORMs were screened for toxicity in MDA-MB-231 cells, a model for TNBC, and the lead compound CO-1 was identified from a range of 8 potential candidates (CO-1 – CO-8). Analysis of cell viability data revealed that CO-1 (1 – 200 μM) resulted in significant reductions in cell viability with an IC75 value of around 5 μM in the MDA-MB-231 TNBC cell line, while the by-product of CO-1, BP-1, demonstrated no residual cytotoxic effects. Time course and gas chromatograph-mass spectrometry (GC-MS) studies revealed that the compound released CO at a slow rate with a half-life in vitro between 9 and 24 hours. The ability of CO-1 and CORM-2 to modulate cell death via the induction of apoptosis was demonstrated using Annexin V conjugated to fluorescein (FITC) and propidium iodide (PI) staining followed by FACS analysis. CO-1 was able to induce apoptosis in MDA-MB-231 cells at both low (10 μM) and high (200 μM) concentrations (6% and 6% respectively) with no apoptotic or necrotic effects being observed when cells were treated with the by-product of CO-1, BP-1. The transition metal containing CORM-2 (200 μM) did not increase apoptotic markers compared to control, however treatment of cells with its “inactive” counterpart iCORM-2 (200 μM) resulted in a significant increase (7%) in apoptosis. In addition high (200 μM) but not low (5 and 10 μM) concentrations of CO-1 and CORM-2 produced a significant increase in the percentage of cells with a damaged mitochondrial membrane (3% and 5% for CO-1 and CORM-2 respectively), indicating that CO may have some concentration specific effects in vitro. High (200 μM) concentrations of both CO-1 and CORM-2 were also shown to induce mitochondrial damage in the MDA-MB-231 cell line and further to the potential anti-cancer effects of the novel compound CO-1, we have shown that low (10 μM) concentrations of the molecule causes a 1.2-fold and 1.4-fold increase in caspase 3 and p53 expression and a 1.2-fold increase in caspase 3 activation. The safety of both organic and transition metal CORMs were also assessed in the renal epithelial MDCK cell line. In MDCK cells treated with CO-1 (10 and 200 μM), COM-2 and iCORM-2 (20 and 100 μM) showed histopathological changes indicative of cell death were observed. These changes were not present in cells treated with the by-product of CO-1, BP-1. Interestingly the changes in histological architecture in MDCK cells treated with iCORM-2 appeared more extensive and severe that in cells treated with the active form of the compound CORM-2. Furthermore treatment of MDCK cells with low (10 μM) concentrations of CO-1, 20 and 200 μM CORM-2 and 200 μM iCORM-2 resulted in G2/M cell cycle arrest in the MDCK cell line. The current study proved CO-1, to be a safe and efficacious pharmacological agent with the ability to induce a cytotoxic and cytostatic effect in the MDA-MB-231 and MDCK cell line with no residual toxic effects resulting from treatment of cell with the by-product of CO-1 (BP-1). Our findings cast doubt over the notion that existing transition metal CORMs in their “inactive” form are not without biological effects. Therefore the current study has shown that novel organic CORMs have a combination of properties that translate into a desirable and potential treatment for TNBC.

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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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  • Iatrogenic Upper Limb Nerve Injuries

    Zhang, John (2011)

    Undergraduate thesis
    University of Otago

    Background: Complications due to errors made by health practitioners are a major cause of concern and a source of distress, disability, and death in patients. In addition, they are associated with litigation and impose a major financial burden on healthcare budgets. Peripheral nerve injuries are among the most frequent iatrogenic complications. Recently, numerous studies have explored specific iatrogenic nerve injuries and possible ways of improving patient safety and preventing error. However, there are few data on the spectrum and relative frequency of iatrogenic nerve injuries and no national studies have been undertaken. Aim: To describe the current spectrum of iatrogenic upper limb nerve injuries in New Zealand, focusing particularly on injuries that have an anatomical and possibly preventable component. Methods: Three studies were undertaken. (1) A systematic review of English biomedical literature in the last ten years relating to major iatrogenic upper limb nerve injuries. The context, mechanism and frequency of nerve injuries were recorded. (2) A retrospective analysis of the Accident Compensation Corporation‟s (ACC) accepted claims database from the first six months of 2009, focusing on iatrogenic nerve injuries. (3) An educational poster targeted at operating staff using international recommendations was produced in consultation with local practising anaesthesiologists. Results: The systematic literature review revealed iatrogenic upper limb nerve injuries are relatively common and can affect patients in any surgical specialty. The spectrum of injuries has changed in parallel with technological advances in surgery and medicine. Analysis of the ACC‟s database revealed 151 successful treatment injury claims that could be classified as iatrogenic nerve injury during the study period. The majority of claimants were female (54.9%) and the elderly was over-represented with the median age being 51.5yrs, (range 0-83yrs). The five most frequent iatrogenic injuries were to the median nerve, sciatic nerve, common fibular nerve, radial nerve and ulnar nerve. An educational poster demonstrating the dos and don‟ts of upper limb positioning under general anaesthesia was successfully produced. Conclusion: This study has described for the first time the contemporary spectrum of iatrogenic nerve injuries in New Zealand as reported to the ACC. Appreciation and raising awareness of the risks associated with medical procedures is an essential first step in developing and implementing strategies to reduce iatrogenic injuries and improve patient safety. This study provides invaluable data by highlighting the procedures that need most attention. Future application of these results will hopefully benefit everyone involved in the New Zealand healthcare system.

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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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  • 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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  • 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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  • 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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  • Central venous surface anatomy: a critical reappraisal

    Hale, Samuel James Mitchell (2011)

    Undergraduate thesis
    University of Otago

    Background: Surface anatomy is an essential part of safe clinical practice, and a key component of physical examination. Our knowledge of surface anatomy has been primarily derived from cadaver studies, with all their associated limitations. Surface anatomy needs to be reappraised in the light of modern imaging techniques in healthy living subjects to ensure that it is fit for purpose in the modern evidence based era. Aims: (i) To determine the consistency with which surface anatomy relevant to cardiothoracic medicine and surgery is reported in contemporary anatomical reference texts, and (ii) to establish evidence based surface markings for the central veins using computerised tomography and ultrasound. Methods: (i) Major surface anatomy landmarks reported in ten contemporary anatomical reference texts and three popular clinical examination texts were analysed to assess consistency. These were compared to evidence based landmarks derived from scientific studies. (ii) 103 computerised tomographic (CT) scans of the chest (52 female; mean age 64 years [range 19–89 years]) were analysed to establish evidence based surface markings for the central veins and the cardiac apex. In addition, the surface anatomy of the subclavian veins was examined bilaterally using ultrasound in 50 healthy volunteers (25 female; mean age 35 years [range 19–68 years]; mean BMI 24.0 [16.5–37]). The relationship of the subclavian vein to the clavicle was examined both with and without passive shoulder retraction with 10º of head down tilt. Results: (i) There are numerous inconsistencies in the reporting of many surface anatomy landmarks both between and within reference texts. Few texts address variation with age, gender, ethnicity, body habitus, posture and phase of respiration. Clinical examination texts contain comparatively little surface anatomy. (ii) In most living adults, the brachiocephalic veins are formed posterior to the ipsilateral sternoclavicular joint, the superior vena cava is formed posterior to the right second costal cartilage and enters the right atrium behind the right fourth costal cartilage. The azygos vein typically joins the superior vena cava at the level of the lower half of the fifth thoracic vertebra, 2 cm below the sternal angle. The cardiac apex lies on average in the left fifth intercostal space close to the midclavicular line, about 9 cm from the midline. The subclavian vein lies closest to the clavicle approximately 7 cm from the midline; it has an average diameter of 10 mm, decreasing by approximately 10% after passive shoulder retraction. Age, gender and body mass index affect these variables. Conclusions: Whilst some commonly accepted thoracic surface markings appear to be reliable when examined in living subjects using modern imaging techniques, others are inaccurate. This is not only relevant to ensuring that modern anatomy teaching is fit for purpose but also important for practical procedures such as central venous catheterisation. Rather than improving successful subclavian vein puncture, passive shoulder retraction may reduce the chances of successful catheterisation by reducing venous diameter. Surface anatomy must be reappraised in living subjects using modern imaging techniques if it is to be accurate and remain useful in clinical practice.

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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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  • 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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  • Investigation of a mutation in CYP26B1

    Eerens, Yoska (2011)

    Undergraduate thesis
    University of Otago

    Retinoic acid is known to have several important roles in embryonic development, including limb and hindbrain development and has been shown to induce neural tube defects when it is present at teratogenic levels. The CYP26 enzyme family are known to metabolise retinoic acid and also play an important role in embryonic development as has been illustrated through several animal knockout studies. A distinct phenotype was observed in two fetuses of a consanguineous Somalian family. The phenotype displayed severe congenital abnormalities of the skeleton and the brain. A sequence variant was identified in CYP26B1 and was investigated as the cause of the observed phenotype. The 1088G>T sequence variant predicts an amino acid substitution, R363L, within the highly conserved EXXR motif that is present in almost all of the enzymes belonging to the cytochrome super family. The aim of this study is to determine whether the CYP26B1 R363L variant identified lacks normal enzymatic activity. Stable expression constructs of the wildtype, mutant and empty control vector were transfected into HEK293 cells. The stable cell lines were treated with retinoic acid, RNA extracted and used to create cDNA. qRT-PCR was used to measure the up regulation of the RA responsive genes, ICER and CYP26A1. This showed that the mutant does indeed lack activity when using CYP26A1 as a test gene and that ICER was not a good gene for measuring the catabolism of RA by CYP26B1. This evidence is consistent with the hypothesis that CYP26B1 R363L is the cause of the phenotype observed.

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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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  • Effect of chronic renal denervation on cardiac response to β-adrenergic stimulation in a diabetic nephropathy rat model

    Thaung, Hnin Pyu Aye (2011)

    Undergraduate thesis
    University of Otago

    Abstract Increased cardiac sympathetic drive as a consequence of increased renal sympathetic activity is an important contributor to the development of cardiovascular complications in patients with diabetic nephropathy. Bilateral renal denervation (BRD), by reducing systemic sympathetic output, has been shown to normalize blood pressure and reduce left ventricular hypertrophy in clinical and experimental studies of hypertension. Therefore, this study aimed to determine the effect of BRD on cardiac response to β-adrenergic stimulation in a diabetic model with underlying hypertension using the transgenic (mRen-2)27 rats. We hypothesize that BRD will improve cardiac response to β-adrenergic stimulation in a diabetic nephropathy rat model. Following streptozotocin (STZ) induction of diabetes, BRD or sham surgeries were conducted repeatedly (at the 3rd, 6th, and 9th week following induction) in both non-diabetic and diabetic 6 week old female Ren-2 rats. Blood glucose was maintained at 25-30 mmol/L using s.c. insulin. Cardiac function was determined in isolated hearts perfused in the Langendorff mode at 12th week following STZ induction. Normalized left ventricular developed pressure (LVDP) was recorded in response to cumulative concentrations of isoproterenol (nonspecific β1- and β2-agonist) (10-10 to 5×10-8 M) and BRL 37344 (specific β3-agonist) (10-12 to 10-6 M). No significant differences in basal cardiac characteristics were observed in non-diabetic and diabetic rats. LVDP response to isoproterenol stimulation was reduced in the diabetic compared to non-diabetic group. However, stimulation with BRL 37344 had no significant effect on LVDP response in both non-diabetic and diabetic rats. In addition, BRD had no effect on LVDP response to isoproterenol and BRL 37344 stimulations compared to the innervated animals in both non-diabetic and diabetic rats. Interestingly, there was a significant increase in diastolic pressure-volume and hence filling pressure in diabetic rats, but a significant increase in diastolic pressure-volume in non-diabetic rats, after renal denervation. In conclusion, renal denervation did not restore the attenuated cardiac response to β-adrenergic stimulation in the diabetic hypertensive rats. This suggests that in the diabetic hypertensive rat hearts, reducing renal sympathetic activity does not decrease sympathetic drive to the heart, but may contribute to a reduction in underlying cardiac remodeling.

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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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  • Hydrogen Sulfide: Investigation into the Effects of Different H2S Donors on the Upregulation of Inflammatory Cytokines

    Campbell, Elizabeth Julia (2011)

    Undergraduate thesis
    University of Otago

    Endogenous hydrogen sulfide (H2S) has recently been described as the third gaseous mediator of interest. It has been proposed in the literature [1] , that H2S has a role in inflammation, through the induction of upstream signals, which activate inflammatory mediators such as TNF-α, IL-1β and IL-6, and through this method H2S can give rise to a pro-inflammatory response. This study, therefore, investigated the mechanisms by which H2S contributes to inflammation in vitro, through the use of two sulfide donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na2S), to look at the inflammatory mediator release from differentiated U937 monocytic cells. Through the use of RT-PCR and ELISA experimentation, analysis was carried out using the two sulfide donors at different concentrations and time points. The results found, that there was no difference between the two sulfide donors in the upregulation of inflammatory cytokines. It was also found that the effects of concentration and time were significant in ELISA experiments, but not so with the PCR experiments. However, when either of these compounds were added into an in vitro system using differentiated U937 cells, there was an upregulation of both protein and mRNA expression relative to the untreated control group.

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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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  • Characterisation of Arkadia's RING domain

    Wright, Joshua Denis (2011)

    Undergraduate thesis
    University of Otago

    The attachment of ubiquitin to proteins (ubiquitylation) is a post-translational modification that regulates a diverse range of biological processes, including signal transduction, transcriptional regulation and receptor down-regulation. Many ubiquitylated proteins are subsequently degraded by the 26S proteasome. A cascade of enzymes comprising an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme and an E3 ubiquitin ligase facilitate ubiquitylation. E3s, which mediate the transfer of ubiquitin from the E2 to the substrate, are largely responsible for specifying substrates. Abnormalities in the regulation of E3s are associated with many diseases, including neurodegenerative diseases and cancers. RING (really interesting new gene) domains are one of three domains that possess E3 activity. There are over 500 RING-type E3s making them the largest group of E3s in humans. However, the mechanism by which RING-type E3s facilitate the transfer of ubiquitin from the E2 to the substrate is poorly understood. Arkadia is an ubiquitin E3 ligase with a C-terminal RING domain. This project aimed to characterise Arkadia’s RING domain to gain a better understanding of how RING-type E3s facilitate ubiquitin transfer. Of particular interest was mechanisms that regulate activity and distinguish monomeric RING domains from dimeric RING domains.

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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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