33 results for Undergraduate, 2014

  • 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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  • 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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  • The Epidemiology of Breast Cancer in Oman

    AL Farsi, Shamsa (2014)

    Undergraduate thesis
    University of Otago

    Background Breast cancer is the most common cancer in females around the world. Annually, more than one million women are diagnosed with breast cancer globally. In Oman, breast cancer is also the most common cancer in females and its incidence has been rising over the years. Published studies have shown that many Omani women have late stage breast cancer at presentation. Possible reasons for this have not been explored. Although Oman has in place a mortality registration database, no population-based mortality data from breast cancer are available. Likewise, the only data available about survival rate of people with breast cancer come from a hospital-based study. Aims The main aims of the research described in this thesis are: 1) To identify the extent of delay in breast cancer diagnosis in Oman. 2) To assess the relationship between delay and socio-demographic characteristics, medical and obstetric history, nature of presenting symptoms and women’s knowledge about breast cancer. 3) To identify reasons for delay in seeking medical help for self-detected breast cancer symptoms in Omani women. 4) To calculate the population-based survival rate from breast cancer in Oman. Methods Aims 1, 2 and 3 were addressed by a study of 150 patients attending oncology clinics in both the Royal Hospital and Sultan Qaboos University Hospital, who were interviewed using structured questionnaires. Patient delay was defined as a period of three months or more between an individual's first awareness of a sign or a symptom of illness and the initial medical consultation. In order to calculate survival rate, the National Cancer Registry records were to be linked to mortality databases in Oman (Directorate General of Civil Status and to the Parallel Mortality Database). Results The final analysis of delay included 144 patients with breast cancer. The median time taken by women in this sample between discovering the breast symptoms and seeing a doctor was 14 days. 56.9% of the patients had a medical consultation in less than a month after detecting symptoms, whereas 20.1 % had a consultation within 1 to 2 months. 22.9 % of the patients delayed consultation by ≥ 3 months. Of the socio-demographic characteristics examined in this study, it was observed that older age, low educational level and employment status were associated with patient delay. Practice of breast self-examination and having a history of chronic disease were also predictors of delay. 44% of patients had early stage disease (stage I/stage II) compared to 56% of patients with late stage disease (stage III/stage IV). However, patient delay was not associated with advanced stage cancer in this study. The main reasons given for delay were: failure to recognise the symptoms to be breast cancer, not seeing oneself at risk for breast cancer, fear and embarrassment, use of alternative therapy and family and work commitments. Due to ethical consideration, I was not able obtain data from Omani NCR and therefore the linking to mortality databases was not possible. Conclusion This study is the first in Oman to investigate the extent of patient delay for women with self-discovered breast symptoms and the factors that influence this delay. The findings of this study indicate the need for public education aimed at raising breast cancer awareness. Further, initiating a screening program in Oman should be considered to help women achieve diagnosis of the disease in its early stages. Population-based cancer research should be encouraged in Oman, and efforts should be taken to improve the quality and completeness of cancer data, which are important in providing on-going monitoring of cancer.

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  • Rates of fructose malabsorption in gout cases and non-gout controls in Christchurch, New Zealand

    Batt, Caitlin (2014)

    Undergraduate thesis
    University of Otago

    Background: Gout is a common form of inflammatory arthritis caused by the precipitation of monosodium urate crystals in the joints. The prevalence of gout in New Zealand is 9.62% in Māori men and 5.12% in European men. Hyperuricaemia is a risk factor for gout and has been associated with fructose, a sugar found in fruit and sweeteners. Fructose malabsorption has a prevalence of 34% in healthy individuals. The aim of this case-control study is to observe whether there is an association between fructose malabsorption and gout. Method: Cases (n=65) were those with clinically diagnosed gout. Controls (n=65) were age and gender matched from the general population. Fructose malabsorption was measured in cases and controls using a breath test that measures gas products of the metabolism of fructose by bacteria in the gastrointestinal tract. Results: The rate of fructose malabsorption in cases was 49.2% and in controls was 53.8%. The odds of malabsorbing fructose in those with gout compared to those without was 0.82 (95%CI 0.41-1.67). Conclusion: There is no significant difference in rates of fructose malabsorption between gout cases and controls. Future studies are required to determine whether the effect of fructose load on serum urate is different between malabsorbers and absorbers in gout cases and those without gout. In future practice dietary advice for gout could be individualised based on absorption status, improving compliance.

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  • Effects of Male Age on Reproductive Success and Offspring Fitness in Zebrafish (Danio rerio)

    Gardiner, Thomas Clarence (2014)

    Undergraduate thesis
    University of Otago

    Many studies have observed that sperm quality decreases both in humans and other animals as individual’s age; however, the direct effects of age on reproduction and offspring fitness are not yet fully understood. A preliminary study in zebrafish has shown some survival advantages in offspring from old males despite reduced sperm quality. However, what effects, if any, this has on subsequent generations is not clear. Sperm samples from the offspring of old and young males (F1 generation) were collected and in-vitro fertilisations (IVFs) used to produce an F2 generation. Fitness parameters (e.g., fertilisation success, hatching success, and survival) and sperm traits (e.g., motility and swimming velocity; measured using computer assisted sperm analysis) of F1 from young and old males were compared. Results demonstrate a significantly higher proportion of fertilised embryos produced from IVFs using the sperm of the offspring of old males compared to the young males. But, we see no significant differences between the two groups in the proportion of fertilised embryos that hatch or survive. There was a significant difference in the semen volume expelled during sampling where the F1 of old males tended to produce more sperm than the F1 from young males. But, there was no difference in any other sperm traits and no sperm traits were significant predictors of fertilisation success. These data suggest that there is a reproductive advantage acquired from old males in the form of greater sperm production and increased rates of fertilisation. Overall, the data are consistent with previous findings suggesting that old males deliver fitness benefits.

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  • Gene expression profiling of mesenteric lymph nodes in red deer (Cervus elaphus) resilient or susceptible to Johne's disease

    Ranson, Courtenay Suzanne (2014)

    Undergraduate thesis
    University of Otago

    Johne’s disease is a chronic wasting granulomatous enteritis of ruminants caused by infection with Mycobacterium avium subsp. paratuberculosis (MAP). Previous research has largely focused on the immune-associated gene expression profiles of the peripheral blood as a measure of the complex immunological response to MAP, with less information on the immune response occurring at the site of infection; the mesenteric lymph nodes. The DRL has in previous work established breed lines of deer that naturally express paternally heritable resilient (R) or susceptible (S) phenotypes to Johne’s disease. Differential expression levels of specific immune genes have been linked to R/S phenotypes which could ultimately be used in the diagnosis of S animals, and the selective breeding of R animals. In this present study, samples from the posterior jejunal lymph nodes of 20 red deer (Cervus elaphus) experimentally challenged with MAP were taken at 13 weeks post- infection, and at 40 weeks following slaughter. Quantitative-PCR was utilised to determine expression profiles of candidate innate and adaptive immunity-associated genes. Aside from increased IFNG in intermediate (I) and S animals at 40 weeks p.i., no significant differential expression was observed between R, I and S animals in the expression of pro-inflammatory Th1, Th17 and anti-inflammatory Th2 and Treg candidate genes at 13 and 40 weeks p.i.. While this study failed to identify biomarkers associated with R/S phenotypes, a dramatic differential expression of the candidate genes between 13 and 40 weeks was observed, which may provide insights into the role and progression of immune parameters as the diseases progresses over time.

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  • Functional Analysis of Disease-Associated 3’ UTR Variants: the CXCL12β 3’ UTR G801A Polymorphism

    Jin, Chan (2014)

    Undergraduate thesis
    University of Otago

    In recent years, the previously neglected 3’ untranslated region (UTR) has gained recognition as a critical region in the regulation of gene expression. Numerous studies have been conducted on disease-associated 3’ UTR variants to elucidate unidentified regulatory elements within the 3’ UTR. An example of an unresolved 3’ UTR variant is the G801A polymorphism (rs1801157) occurring within the 3’ UTR of the CXCL12β transcript isoform. The CXCL12 gene encodes a C-X-C motif chemokine 12 (CXCL12) that functions as a ligand for the chemokine (C-X-C motif) receptor 4 (CXCR4), which is involved in the entry of the human immunodeficiency virus (HIV) into host cells. Correspondingly, previous association studies have indicated a potential correlation between the A allele of the G801A polymorphism and increased resistance to the progression of acquired immunodeficiency syndrome (AIDS). This suggests that the G801A polymorphism may be linked to a regulatory element in the 3’ UTR, which modulates the expression of CXCL12β, and therefore plays an intermediary role in its reported association with HIV/AIDS. In order to identify the regulatory elements that may be associated with the G801A polymorphism, a bioinformatic approach was adopted by utilising a webserver developed in the Brown Lab called ‘Scan For Motifs’. However, only a 26 base pair (bp) region of high conservation was identified at the site of the G801A polymorphism. Consequently, three variable lengths of the CXCL12β 3’ UTR, containing the highly conserved region with either alleles at the G801A polymorphism, were cloned downstream from the luciferase gene (luc+) in pGL3MS2site/basic cloning vectors to yield three sets of reporter gene constructs. The desired CXCL12β 3’ UTR inserts were prepared by either the polymerase chain reaction (PCR) amplification of said sequences from HepG2 genomic DNA (gDNA) or appropriate oligonucleotide design. Site-directed mutagenesis was implemented when applicable using PCR-based methods such as the ‘megaprimer’ method and the overlap extension method, to create a polymorphic counterpart for each reporter gene construct. Prepared reporter gene constructs and previously characterised control plasmids were transiently co-transfected with a renilla luciferase reporter gene construct (phRL-SV40) using Lipofectamine® 2000 into HeLa cells and HEK293T. The transfected cells were harvested after a 48-hour incubation period for subsequent luciferase assays in order to measure differences in gene expression with respect to the G801A polymorphism. Changes in gene expression in response to different experimental conditions were also analysed through the application of two independent stimuli: the replenishment of foetal bovine/calf serum (FBS/FCS) (10%) and the premature harvest of cells after 24 hours. The amount of DNA transfected was also standardised through the co-transfection of pUC18, a non-coding plasmid. In contrast to initial expectations, different levels of gene expression were not observed between each pair of reporter gene constructs with allelic differences at the G801A polymorphism. In support of the experimental results, levels of gene expression in response to two different conditions behaved in agreement with initial expectations. Likewise, the standardisation of the amount of DNA transfected using pUC18 co-transfection only increased the gene expression associated with the larger plasmid relative to the other smaller DNA constructs. Despite the negative results obtained from a series of luciferase assays, the functional implications associated with the G801A polymorphism was not conclusively determined as absent given the conflicting indications reported in the wider literature. Further experimental analyses are required to confirm or reject the functional consequences of the G801A polymorphism. If there is a functional impact associated with allelic differences at the G801A polymorphism, the next step would be to elucidate the responsible regulatory element in the context of its associated diseases.

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  • Patient autonomy in end of life decisions

    Chamberlain , Joshua (2014)

    Undergraduate thesis
    University of Otago

    End of life scenarios must be patient driven and focused. In this thesis it is argued that patient intention is often misrepresented by being assimilated to extreme and clear-cut cases where patients are committed to a certain path of action. The role of the patient in decision-making, a role that has developed through the advent of autonomy and subsequently informed consent, has created an environment that is so dependent upon the patient that our duty of care and the role of experience in difficult clinical decisions can be obscured or forgotten. Given the experience of clinicians and the conflicting duties to which they often find themselves subject, questions have been raised over the intent of physicians in end of life situations but the extent to which these should lead to the very same questions being asked of patients is under-explored in the literature. The varying end of life decisions and the moral acceptability attached to them is a concept that is poorly understood by a mainstream audience. This, among other things is part of the misconceptions that are commonly held about euthanasia. Finally I explore what the future holds for euthanasia and end of life decisions. The part the patient plays in these decisions is coming under more scrutiny and as it does, the role of the physician is being examined as well. Happenings in the Netherlands over the last decade have prompted a new surge of calls for reform in this most ancient of debates, this surge I propose could lead to a new era of dialogue and relationship focused care in end of life, where a one size fits all approach is not welcome.

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  • Endogenous Immune Responses to Colorectal Cancer

    Laws, Gemma (2014)

    Undergraduate thesis
    University of Otago

    An ideal immunotherapy for colorectal cancer would target an endogenous tumour antigen. Endogenous antigens produce a more physiologically relevant immune response than model antigens traditionally used to study anti-tumour immune responses. CD8+ T cells play an important role in cancer control as they are cytotoxic and recognise antigens on target tumour cells. This study aims to develop a model of endogenous immune responses to an endogenous colorectal cancer antigen. Lymphocytes responding to the endogenous antigen AH1 on the murine colon carcinoma CT26 were investigated in vitro using proliferation assays to measure expansion, flow cytometry to measure activation, and a pentamer to measure AH1-specificity. These results were incorporated into a mouse model of colorectal cancer, in vivo via a subcutaneous injection, and the response to endogenous AH1 antigen measured. CD25+ CD44+ and CD62Llow CD8 T cells increased in frequency and total number after two stimulations with AH1 peptide, indicating T cells were activated and undergoing clonal expansion. Pentamer staining revealed CD8+ T cells specific for this endogenous AH1 antigen. An even higher activation was seen when αCD3/28 was added to the primary stimulation. In vivo experiments also demonstrated CD8+ T cell responses to the AH1 peptide indicating this antigen can trigger an immune response, and therefore it is a potential target for immunotherapy applications. This research develops a physiologically relevant model of endogenous immune responses to an endogenous colorectal cancer antigen. AH1 is a possible target for immunotherapy. Data gained from this study brings us closer to immunotherapy development.

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  • Role of the Host Microtubule Cytoskeleton in Infection of Human Cells by Yersinia enterocolitica

    Kean, Bernard (2014)

    Undergraduate thesis
    University of Otago

    The bacterial pathogen Yersinia enterocolitica causes food-borne illnesses resulting in gastroenteritis, metastatic abscesses and sepsis. Yersinia promotes its internalisation into human cells through the binding of the bacterial surface protein invasin to the host receptor beta 1 integrin. The interaction of invasin with the beta 1 integrin receptor stimulates host signalling pathways that promote cell surface changes driving bacterial uptake. It has previously been shown that the signalling protein type IA phosphoinositide 3-kinase (PI3K) plays a critical role in invasin-mediated entry. Findings in the Ireton lab show that of the downstream effectors of PI3K, the proteins PHLDB1 and CLASP1 were required for invasin-mediated entry (Dr. G. Dowd and Prof. K. Ireton, unpublished data). These two proteins are known components of the ELKS complex – a complex involved in microtubule stabilisation. Subsequent experiments which showed that destabilisation of microtubules caused inhibition of bacterial entry led to the hypothesis that microtubules may promote invasin-mediated internalisation through the ELKS complex. To test this hypothesis I performed an RNA interference based screen to identify components of the ELKS complex that play a role in invasin-mediated entry into the human cell line HeLa. Of the 10 genes targeted, the results showed that at least 2 of the proteins encoded have a potential role in invasin-mediated entry. These human proteins are CLIP-170, a protein that bind CLASPs and regulates localisation of dynein, and KANK1, a regulator of actin polymerisation. Also investigated in this research was the possible recruitment of ELKS components during invasin-mediated internalisation. Using confocal microscopy, it was found that PHLDB1 and the truncated PH domain would localise near bacteria during infection. These findings suggest evidence that the ELKS complex is involved in invasin-mediated internalisation into host cells that is induced by Y. enterocolitica. This work provides a further insight into the complex signalling pathway induced by Yersinia.

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  • The Role of Dendritic Cell Subsets in the Early Immune Response to Mycobacterial Infection

    Manners, Kate Meredith (2014)

    Undergraduate thesis
    University of Otago

    The early immune response to tuberculosis (TB) is poorly understood. However, recent evidence has shown that dendritic cells (DC) are important for both control of bacterial growth and for activating adaptive immunity. DC are divided into functionally and phenotypically distinct subsets, but the role of each of these subsets in the early immune response to tuberculosis remains unknown. This study aimed to investigate the roles of DC subsets in a murine model of mycobacterial infection. Use of a fluorescent strain of the attenuated TB vaccine strain Mycobacterium bovis BCG, combined with multicolour flow cytometry, enabled detection of BCG associated DC subsets within the murine lung during the first 14 days of infection. The early immune response to mycobacterial infection was found to be highly dynamic, with significant variation in the proportion of BCG associated cells in each DC subset of the lung during infection. CD11b+ conventional DC (cDC) and plasmacytoid DC (pDC) were found to be associated with BCG during the first 14 days of infection, whereas CD103+ cDC were not. Differential expression of the cell surface markers GR-1, CD11b and CD11c detected within previously described DC subsets correlated with their propensity to associate with BCG. Conventional DC and pDC were found to respond differently to mycobacterial infection; cDC upregulated expression of CD86 during infection whereas pDC were found to do the opposite, suggesting that these subsets have distinct roles during early infection. Importantly, and contrary to current dogma, alveolar macrophages were not found to associate with BCG during early infection. This study highlights the distinct roles of different DC subsets during mycobacterial infection. This research may have implications for the development of a novel vaccine or therapy for tuberculosis, for which there is a significant need.

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  • Involvement of mitochondria and peroxiredoxin in TNF-mediated necroptosis

    Zawari, Masuma (2014)

    Undergraduate thesis
    University of Otago

    Necroptosis is a form of regulated necrosis considered to be involved in several pathophysiological conditions such as inflammation and ischaemia-reperfusion injury. The cellular events involved in the initiation of necroptosis are unclear, but recent studies indicate that the pseudokinase mixed lineage kinase domain like (MLKL) plays an important role. Understanding the molecular mechanisms regulating necroptosis is therefore an important priority that may lead to the development of new therapies. Mitochondria and reactive oxygen species generation has been reported in necroptosis, however it is not clear whether these play a direct role in the death process. These changes were investigated in a TNF-dependent model of necroptosis using mouse dermal fibroblasts (MDF) from wild-type and MLKL knock-out animals. Viability analysis by FACS and live cell imaging indicated early signs of cell death by 60 min and by three hours about half of the population were executed by necroptosis in an MLKL dependent manner. TMRE labelled cells showed a loss of mitochondrial membrane potential before cell death. Rapid increase in oxygen consumption 30 min after the injection of TNF indicated a direct link between the necrosome and downstream signalling to mitochondria for efficient necroptosis to occur. This is the first report of MLKL dependant effects on mitochondrial function. Western blot analysis indicated a rapid accumulation of oxidized mitochondrial peroxiredoxin in an MLKL dependent manner. Timing of mitochondrial changes and oxidation of peroxiredoxin suggest they may be early markers or even executors of cell death rather than a consequence of cellular damage. However further investigation is required to determine the importance of these changes in cell death.

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