Medicine (St Vincent's) - Theses
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ItemInvestigations into Mrpl44’s role in RNA processing and the regulation of the mitochondrial OXPHOS system( 2014)RNase III proteins are divalent metal ion‐dependent phosphodiesterases that specifically bind to and cleave double stranded (ds) RNA. In a search for proteins containing RNase III domains, we identified Mrpl44 as a potential RNase III protein. Mrpl44 has previously been found in association with the mitochondrial ribosome from bovine liver extracts; however, the precise Mrpl44 localization had been unclear. While Mrpl44 has been found to be crucial for the function of the mitochondrial ribosome and the translation of mitochondrial DNA (mtDNA)-encoded polypeptides, the function of RNase III of Mrpl44 is poorly understood. The focus of this thesis was to study the roles of Mrpl44 within the cell, particularly its RNA processing capability and its role in the mitochondrial ribosome. While Mrpl44 was first found in association with the mitochondrial ribosome, it has been captured in various studies with nuclear, cytoplasmic and mitochondrial baits. In this thesis, we showed by immunofluorescence microscopy and subcellular fractionation that Mrpl44 is localised to the matrix of the mitochondria. Using overexpression systems, we found that Mrpl44 forms multimers as part of the large subunit of the mitochondrial ribosome. While we also investigated the effect of Mrpl44 on the biogenesis of microRNAs (miRNAs), our studies using Mrpl44 knockdown models did not show Mrpl44 having any effect. To investigate the role of Mrpl44 within the mitochondria, more specifically, its role in the expression of the mitochondrial genome, we manipulated the expression of Mrpl44 within NIH3T3 cells. We showed that Mrpl44 has important functions in controlling the expression of mtDNA-encoded genes, at the level of RNA expression and protein translation. To determine whether Mrpl44 regulates mitochondrial gene expression by processing RNA, we tested whether Mrpl44 binds to or cleaves RNA within the mitochondria. We showed that Mrpl44 associates with mtDNA-encoded RNA but did not show any cleavage activity for Mrpl44. We subsequently investigated whether altered Mrpl44 expression affects mitochondrial function and found that it impacted the ATP synthesis capability and respiratory capacity of cells. These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial genome.
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ItemDefining the molecular profile of oral tongue squamous cell carcinomas and its impact on patient outcome( 2014)Amongst all head and neck squamous cell carcinomas (HNSCC), oral tongue carcinomas (OTSCC) have the worst prognosis for early stage disease. The current staging system is unable to consistently identify patients with high risk disease. In one of the largest comprehensively annotated OTSCC cohorts to date, this thesis examines a combination of literature identified candidate biomarkers, and also sought to determine if a novel prognostic molecular signature could be identified. Given the high frequency of reported CDKN2A alterations in HNSCC, a comprehensive analysis of the differential mechanisms of CDKN2A alteration was performed. Promoter methylation status, mutation status, copy number variation and protein expression were assessed. The majority of samples (95%) did not demonstrate p16 over-expression assessed by immunohistochemistry. Although disruption of CDKN2A was found to be a frequent event arising from a variety of mechanisms, no correlation between CDKN2A alteration and clinicopathological features was found. A panel of loci frequently reported to be hypermethylated in HNSCC was investigated within the OTSCC cohort, with three quantitative methodologies that assessed DNA methylation. In contrast to the literature, these loci were not commonly methylated. Findings were confirmed in an external cohort of HNSCC samples from The Cancer Genome Atlas (TCGA) that had methylation levels quantified with a fourth, orthogonal methodology. The use of non-quantitative methodology in the literature was the likely cause for the overestimation of significant methylation events, with this study highlighting the need for the cautious interpretation of this literature. Utilising a genome-scale wide methylation platform, a prognostic methylation signature was sought for the OTSCC cohort. Given the absence of consensus on data analysis, comprehensive bioinformatics analyses were performed utilising multiple contemporary R software library packages, to enable a thorough examination of the data with published algorithms used for pre-processing and downstream analysis. However, methylation assessed over greater than 450,000 CpG dinucleotides did not reveal a differentially methylated group of samples, and was not informative for clinicopathological variables. Furthermore, despite increasing the total number of samples to include the TCGA OTSCC dataset, a prognostic methylation signature was not identified in any cohort. Targeted mutational profiling of the cohort was also performed. A disproportionately large number of variant calls were identified on the initial processing of samples. Validation of a subset of variant calls with orthogonal methodology was able to confirm the presence of only 12/50 (24%) selected mutations. Despite pre-analytical quality assurance assessments, the replicated analysis of samples and the use of stringent filtering criteria, the presence of artefactual variant calls masked the identification of true mutations. The likely source of the large number of artefactual variant calls was from the PCR based amplification of artefact in DNA extracted from formalin-fixed, paraffin-embedded tissue. This thesis emphasises the importance and the impact of the choice of methodology on the successful identification of clinically relevant biomarkers. Within the limitations of current understanding and the size of the cohort examined, it also suggests that both CDKN2A alteration and DNA hypermethylation in isolation are not prognostically informative biomarkers for OTSCC. Further research is required into the prognostic value of other molecular alterations and the combined impact of simultaneous aberrations.
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ItemExamination of proteases and protease-activated receptors (PARs) in Barrett's oesophagus and oesophageal adenocarcinoma( 2014)Barrett’s oesophagus (BO), a metaplastic premalignant lesion, and gastroesophageal reflux disease (GORD) are two major risk factors for the development of oesophageal adenocarcinoma (OAC), an aggressive cancer the incidence of which is increasing faster than any other malignancy in the Western world. Proteases are known to be present in the refluxed contents of GORD patients and in inflamed BO and OAC tissue microenvironment. Yet, their role in these oesophageal pathologies is still poorly understood. Many (patho)-physiological responses induced by proteases can occur through their cell surface targets, the protease-activated receptors (PARs). Aberrant PAR expression and signalling have been reported in several malignancies. However, the tissue distribution patterns of PARs and their activating proteases as well as the effects of protease/PAR activation on oesophageal pathophysiology are largely unknown. Therefore, the general aim of this thesis was to investigate the role of PARs and proteases in BO and OAC pathogenesis. More specifically, this thesis aims to: 1) characterise the expression of PAR family members (PAR1-PAR4) in a panel of normal oesophageal mucosa, BO, and OAC tissues, epithelial cell lines, and tissue-derived fibroblasts; 2) characterise the expression and localisation of candidate PAR activating proteases - MMP1, trypsin-1, KLK5, and KLK6 in normal oesophageal mucosa, BO, and OAC tissues; and 3) investigate the signalling and functional effects of PAR1 and PAR2 activation in oesophageal epithelial cells. In Chapter 3, reverse transcription quantitative PCR (RT-qPCR) and immunohistochemical analyses revealed that PAR1 transcript and protein expression were significantly upregulated in BO and OAC tissues compared to normal oesophageal mucosa. PAR1 expression levels correlated significantly with disease progression. PAR3 and PAR4 mRNA overexpression were likewise detected; the former in BO and OAC tissues, and the later in tumour samples. PAR2 transcript and protein were found in all oesophageal tissue types with no statistically significant differences in expression levels. PAR1 and PAR2 expression within BO and OAC tissues were localised in various cell types but primarily in the epithelial cells. While BO and OAC tissues showed significant increase in MMP1 and trypsin-1 expression, comparable levels of KLK5 protein was noted in all tissue types (Chapter 4). KLK6 transcript was markedly downregulated in BO tissues. Strikingly, MMP1, trypsin-1, and KLK5 were frequently coexpressed with PAR1 and PAR2 in OAC and BO tissues. Analysis of the effects of PAR activation by the aforementioned proteases in a panel of representative epithelial cell lines revealed that PAR1- and PAR2-mediated signalling were dysregulated in OAC cells (Chapters 5 and 6). For instance, MMP1 but not thrombin induced Ca2+ signalling via PAR1 in OAC cells, whereas thrombin but not MMP1 evoked Ca2+ responses in normal and BO cells. In addition, while PAR2 activation triggered transient ERK1/2 signalling in normal and BO cells, ERK1/2 activation was sustained in OAC cells. Moreover, PAR1 and PAR2 stimulation activated PI3K/AKT signalling exclusively in OAC cells. Functionally, PAR1 and PAR2 activation promoted OAC cell invasion and chemotaxis, respectively. In all, these results highlight the PARs as potential players in OAC and BO pathogenesis and thus, potential therapeutic targets for these oesophageal pathologies.
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ItemThe development and regulation of islet-specific T cells in an experimental model of autoimmune diabetes( 2014)Type 1 diabetes (T1D) is an autoimmune disease. T cells specific for β-cell antigens such as proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) are important in mediating the disease. The aims of this thesis were to study the development and regulation of T cells in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Chapter 2 describes the development of IGRP-specific CD8+ T cells in autoimmune diabetes. IGRP-specific T cells in the mouse were tracked using a sensitive MHC-tetramer based magnetic enrichment. There was an increase in the number of IGRP-specific T cells in the peripheral blood and lymphoid tissue as mice age, and the increase correlated with insulitis progression. These cells had an effector-memory phenotype, which was only acquired in the inflammatory environment of the islets, and not the draining lymph nodes. Islet-specific T cells could also migrate from islets into the periphery. In the development of autoimmune diabetes, important changes to IGRP-specific T cells during the pathogenesis of diabetes occur not in the draining lymph nodes but in the islets, where they expand and differentiate into effector-memory T cells, and emigrate to the periphery, where they can report progression of islet pathology. Tumour Necrosis Factor (TNF) is an inflammatory cytokine that has been implicated in the pathogenesis of autoimmune diabetes. In chapter 3, we investigate the effects of TNF-TNFR1 signalling deficiency on the development of autoimmune diabetes, by using a NOD mouse deficient in TNF receptor 1 (TNFR1). TNFR1-/- islets grafted onto kidney capsule of diabetic mice were destroyed, showing that TNFR1 deficiency on β-cell did not confer protection against immune destruction. The specific effects of TNFR1 deficiency on the immune system of NOD mice were also examined. Adoptively transferred β-cell specific T cells proliferated normally in the pancreatic lymph nodes, but failed to migrate into the pancreas of TNFR1-/- recipient mice. Notably, analysis of immune cell subsets by flow cytometry showed an increased percentage of CD4+CD25+Foxp3+ T regulatory cells in TNFR1 deficient mice. Depletion of CD4+CD25+ regulatory T cells using GK1.5 CD4 depleting mAb restored diabetes in NOD8.3/TNFR1-/- mice. These results suggest that blockade of TNF signalling suppresses diabetes by increasing regulatory functions of the immune system. T cell responses to insulin (INS) are crucial in development of T1D. Chapter 4 of the thesis examines insulin-specific T cells in NOD mice, and in NOD mice tolerant to proinsulin II (NODPI), which do not develop diabetes or insulitis. There was no significant difference in the absolute number of insulin-specific CD8+ T-cells in NOD and NODPI mice. INS-specific CD8+ T-cells in NOD mice expanded significantly more in response to stimulation by peptide compared to NODPI. In vivo cytotoxic activity in NODPI was reduced compared to NOD. The absolute number of INS-specific CD4+ T-cells in NOD and NODPI mice was similar. The proportion of regulatory INS-specific CD4+ T cells that were Foxp3+ was also similar. INS-specific CD4+ T cells in the NOD and NODPI were tested on whether they could help CD8+ T-cells mediate diabetes. NODRAG1-/-/8.3 developed diabetes rapidly after NOD CD4+ T-cells were transferred (median=32days). 7/8 of recipients did not develop diabetes when NODPI CD4+ T-cells were transferred. In a NOD mouse tolerant to proinsulin, insulin-specific CD4+ and CD8+ T-cells were detected, suggesting that the main mechanism of tolerance is not deletion. It is more likely that these cells could have impaired function.
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ItemA genetic study of familial Barrett’s oesophagus and oesophageal adenocarcinoma using whole exome sequencing( 2014)The goal of this thesis was to explore the genetic variants underlying familial Barrett’s oesophagus (BO) and oesophageal adenocarcinoma (OAC). From cases with a strong family history, genetic variants were identified from germline DNA by Whole Exome Sequencing. For the genetic variants common between the sequenced family members, bioinformatic analysis and filtering based on population data and segregation analysis identified candidate genes associated with familial BO/OAC, which may imply their role in the development of this disease.
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ItemThe effect of inhibiting pro-apoptotic BH3-only proteins on beta cell loss and glucose homeostasis in type 2 diabetes( 2014)Type 2 diabetes is caused by a combination of insulin resistance, as well as dysfunction and loss of beta cell mass. Around 30-70% of beta cell mass is lost in type 2 diabetes and this is due to apoptosis, induced mainly by chronic hyperglycaemia. High concentrations of glucose activate the intrinsic apoptosis pathway in beta cells and this requires the pro-apoptotic Bcl-2 family proteins Bim and Puma. Glucose-induced production of reactive oxygen species may also activate the NLRP3-inflammasome pathway, resulting in caspase-1 cleavage, and production of IL-1β. This thesis aimed to identify the stress pathways that mediate glucose toxicity of beta cells. In addition, possible benefits of apoptosis inhibition on glucose homeostasis and beta cell function were examined using mouse models. Chapter 2 describes the role of endoplasmic reticulum (ER) stress and oxidative stress in mediating glucose toxicity of islets. Exposure of islets to high concentrations of glucose induced oxidative and ER stress and increased expression of the pro-apoptotic ER factor CHOP. This caused downstream activation of Bim and Puma-mediated apoptosis and was inhibited by chemical chaperones or antioxidant treatment. Further, increased expression of Bim and Puma mRNA was observed in human islets in type 2 diabetes. Inhibition of ER and oxidative stress could be an effective strategy to reduce beta cell apoptosis in type 2 diabetes. In Chapter 3, the controversial subject of activation of the NLRP3-inflammasome in islets by glucose toxicity-induced ER and oxidative stress was studied. Apoptosis induced by ER or oxidative stress inducing agents was similar in islets isolated from wild-type, NLRP3 or caspase-1 deficient mice. Loss of NLRP3 did not protect islets from glucose, ribose or gluco-lipotoxicity. The presence of a hyperactive mutant of NLRP3 in beta cells had no effect on ribose toxicity or ribose-induced IL-1β secretion. These results suggest that activation of the inflammsome does not mediate glucose toxicity in islets. In Chapter 4, the metabolic effects of apoptosis inhibition were studied in Bimdeficient mice. Bim deficiency resulted in reduced adiposity, improved insulin sensitivity and glucose tolerance in mice. Islet size and function was normal suggesting that phenotype was not beta cell-dependent. Study of mice in metabolic cages revealed that loss of Bim resulted in increased lipid oxidation and this may explain the metabolic changes. The effects of apoptosis inhibiton in beta cells were studied in Chapter 5 by analysing Bim-deficient Leprdb/db mice. Loss of Bim resulted in increased body weight but normalized fasting blood glucose and improved glucose tolerance in Leprdb/db mice. These changes were likely due to a two-fold increase in islet area and volume, and a similar increase in serum insulin concentration. Therefore, loss of Bim alters the balance between apoptosis and replication, thereby making it a possible therapeutic target to prevent beta cell loss in type 2 diabetes. Overall, work presented in this thesis shows that inhibition of apoptosis or its upstream activators ER and oxidative stress may prevent beta cell loss in type 2 diabetes. Inhibiting pro-apoptotic proteins could also have additional benefits such as increased lipid oxidation and improved insulin sensitivity.
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ItemThe 78kDa glucose regulated protein (GRP78) interacts with thrombomodulin on endothelial surface and demonstrates antithrombotic activity( 2014)Thrombomodulin (TM) is a multi-domain glycoprotein expressed primarily on vascular endothelial cells. Recent research has demonstrated that the N-terminal module, the lectin-like domain (LLD), exhibits anti-inflammatory properties. In this project, a specific interaction between the LLD and the 78kDa glucose regulated protein (GRP78) was identified. This interaction was confirmed by reciprocal immunoprecipitation of both proteins and by colocalization on cell surface. GRP78 is mostly an intracellular protein, best known for its function as a chaperone, guiding the unfolded protein response. Extracellular GRP78 has been demonstrated to inhibit tissue factor (TF)-mediated coagulation. In this project, recombinant GRP78 (full length, various mutants and individual domains) was generated, and its effect on haemostasis was studied in vitro and in vivo. GRP78 prolonged the TF-dependent coagulation in a coagulation assay as well as the biochemical analysis of spectrozyme Xa. Contact-initiated clotting was not altered and there was no effect on thrombin time. Recombinant GRP78 (8 to 10μg/ml) was shown to inhibit platelet aggregation significantly in response to collagen (1U/mL), TRAP (1μM) and ADP (10μM) but not with Ristocetin. Pre-administration of GRP78 (1-5μg/g mouse body weight) demonstrated dose-dependent prolongation of mouse tail bleeding time. Antithrombotic activity of GRP78 was tested in vivo in a model of pulmonary thrombosis, induced by collagen (1.2μg/g) infusion into the jugular vein of anaesthetised mice with monitoring for morbidity and survival at 30 min. GRP78 demonstrated significant dose-dependent (1-8μg/g mouse body weight) improvement in survival from acute pulmonary thrombosis as compared to buffer control.TM is already known to participate in a clinically relevant anticoagulant pathway: the generation of activated protein C (APC) by thrombin binding to the epidermal growth factor-like modules of TM and consequent inhibition of procoagulant proteins Va and VIIIa. Here, another novel mechanism has been identified by which TM can maintain the endogenous antithrombotic phenotype of endothelial cells: the interaction between the LLD and GRP78 recruits the capacity to inhibit TF-mediated coagulation and inhibit platelet aggregation to the site of a developing thrombus. The clinical importance and detailed dissection of mechanisms is ongoing in the laboratory.
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ItemCharacterisation, optimization and transplantation of a tissue engineered cardiac muscle flap( 2014)The field of tissue engineering presents a new means of generating tissues for reconstruction. Engineering functional myocardium de novo can potentially address the current challenges faced in the field of heart transplantation and congenital cardiac abnormalities. However, heart tissue is metabolically demanding, and therefore highly susceptible to ischaemia. As the main strategies to engineer myocardium rely on assembling cardiomyocytes in vitro using scaffolds (e.g. polymer based or hydrogel based) or in a form of scaffoldless cell-sheets, its survival through implantation in vivo relies on neovascularisation from the recipient bed. This presents a major hurdle for cardiac tissue engineers, as these ‘cardiac grafts’ are unlikely to survive, especially, in the harsh environment of ischaemic tissue post- myocardial infarction. Publications quantifying the survival of cardiac tissues after implantation in vivo are scarce but conservative estimates suggest that less than one-tenth of such grafted cardiac tissue will survive. Using an in vivo vascularisation approach at our laboratory, by placing a microsurgically fabricated arteriovenous loop into a polycarbonate chamber (AV loop tissue engineering chamber), cardiomyocytes suspended in a hydrogel-based scaffold assembled in vivo into an ‘engineered vascularised cardiac muscle flap’ that is potentially transplantable. While this concept seemed an attractive solution to the problem facing cardiac tissue engineering, several questions have yet to be investigated: 1. Will the tissue engineered cardiac muscle flap suffer minimal tissue loss, after transplantation? 2. Following transplantation to the epicardium, will the engineered cardiac muscle tissue integrate with the myocardium? In this thesis, a series of experiments were performed to answer these questions. The information obtained in the studies can be summarised as follows: A. Seeding syngeneic neonatal rat cardiomyocytes into the AV loop tissue engineering chamber with MatrigelTM as a scaffold, it is possible to generate a contractile cardiac muscle flap in Sprague Dawley rats. Immunohistological examination showed no signs of acute rejection. This implies the AV loop approach may be suitable when autologous cell sources are available for implantation. B. In a dose-response experiment, seeding 6 million cardiomyocytes per chamber was found to produce a small variance and a significant mean volume. This has important implications for the outcome of histomorphometric analysis of the flap. It was also observed that the cardiac tissue volume generated seemed to demonstrate a ‘dose-response trend’, that was not seen in other existing cardiac tissue engineered approaches. C. When investigating two isoforms of an enzyme system, NADPH oxidase, in hope of boosting angiogenesis to generate robust cardiac tissues, it was found that the angiogenic environment in the tissue engineering chamber was too complex to be altered by simply targeting a single factor. D. Subjecting the tissue engineered cardiac muscle flap to ischaemia time and conditions similar to that seen in the heterotopic rat heart model, the cardiac muscle flap did not show any quantitative loss or morphological changes when transplanted to an ectopic site. E. The small size of the Sprague Dawley rats and its anatomical features did not allow the cardiac muscle flap to be transplanted in an autologous fashion to the heart. A novel model using two syngeneic adult rats, allowed transplantation of the cardiac muscle flap based on a long pedicle from one rat to the other’s heart. The method was feasible and reproducible. Histological examination of the transplanted flap showed connective tissue integration of the flap with the host’s heart, however, the flap’s cardiac tissue remained separated from the myocardium by some collagenous tissue. In summary, a syngeneic cardiac muscle flap was generated in the AV loop tissue engineering chamber and the concept of a transplantable vascularised cardiac muscle flap was demonstrated. The novel allogeneic transplant model may have application as a platform for testing functionality of various cardiomyogenic stem cell sources. While much is there to overcome, this is a step forward in translating this approach from the bench to bedside.