416 results for Undergraduate

  • The Therapeutic Role of NK Cells in DC Immunotherapy

    Caldwell, Felicity Jane (2013)

    Undergraduate thesis
    University of Otago

    Dendritic cell (DC) immunotherapy is a promising treatment for cancer. Despite now being used in the clinic, the role and mechanisms of lymphocyte subsets stimulated by DC immunotherapy are not completely understood. Potential mechanisms for anti-tumour responses include cytokine (e.g. interferon-gamma; IFN-γ) or direct cell-killing mechanisms (e.g. perforin) performed by natural killer (NK) cells, NKT cells or CD4+ and CD8+ T cells. This current study used a B16/OVA-melanoma tumour model with bacteria-stimulated dendritic cells, coupled with antibody-mediated depletions (NK cells, CD4+ and CD8+ T cells and IFN-γ) and mice genetically deficient in IFN-γ and perforin to determine the mechanism of successful DC immunotherapy. In agreement with previous work, CD4+ T cell, CD8+ T cell and NK cell depletion resulted in a loss in efficacy of DC immunotherapy, although in this study, this effect failed to reach statistical significance for all lymphocytes. We found that DC immunotherapy induced an IFN-γ, not perforin, mediated anti-tumour response against B16/OVA melanoma tumour. Determining the role and mechanism of killing tumour cells in DC immunotherapy is of great importance and could lead to improvements in DC immunotherapy strategies. Showing particular promise is optimisation of IFN-γ release to enhance a potent anti-tumour response.

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  • Characterising the role of human mTOR in Listeria monocytogenes infection

    Law, Daria Rose (2013)

    Undergraduate thesis
    University of Otago

    The pathogenic food bourne bacterium Listeria monocytogenes has the potential to cause gastroenteritis, meningitis and abortion in an infected individual. L. monocytogenes is a facultative intracellular bacterium that facilitates its internalisation into some host cells by binding of the bacterial surface protein InlB with its cognate human receptor, the receptor tyrosine kinase Met. This binding stimulates a number of cellular signaling events, which ultimately lead to host cell membrane rearrangement and engulfment of the bacterium. One human signaling protein the Mammalian target of Rapamycin (mTOR) was recently shown to be important in the InlB mediated internalisation of L. monocytogenes. However the molecular mechanism underlying mTOR’s role in internalisation is not understood. Using an RNA interference based method, this project set out to elucidate which components of mTOR are important in L. monocytogenes internalisation into the human epithelial cell line HeLa. Here we find that the mammalian target of rapamycin complex 2 (mTORC2) and its downstream substrate PKC-α are involved in the InlB mediated internalisation of L. monocytogenes into the epithelial cell line HeLa.

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

    Foster, Timothy John (2014)

    Undergraduate thesis
    University of Otago

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

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

    Chen, Shumou (2014)

    Undergraduate thesis
    University of Otago

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

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

    Hamilton, Katelin (2014)

    Undergraduate thesis
    University of Otago

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

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  • Quality of Diabetic Foot Care in Oman

    Al-Busaidi, Ibrahim Saleh (2014)

    Undergraduate thesis
    University of Otago

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

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  • 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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