Surgery (St Vincent's) - Theses
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ItemCharacterisation of endogenous repair mechanisms following endothelin-1 induced stroke in rats and the effects of human adult stem cell transplant to support brain reconstruction( 2014)Brain injury from stroke often results in permanent damage and disability due to neurons failing to re-establish lost connections. Potential for brain regeneration relies heavily on the surrounding microenvironment. Contributions from inflammatory cells, angiogenesis and stem cells all require collective consideration when investigating treatment options. Understanding this paradigm is critical to developing therapies that promote recovery. Cell-based therapies offer hope in rescuing the stroke affected brain and restoring function. However, differentiation of transplanted cells into significant neuronal populations is yet to be realised. The work presented in this thesis investigates cellular responses to brain injury and repair mechanisms activated following stroke using the endothelin-1 model of focal cerebral ischemia in conscious rats. Additionally these studies explore the potential of human adult neural progenitor cells to support brain repair. First, we investigated aspects of brain remodelling initiated following stroke, including the impact of lesion size on angiogenesis, cell responses within the subventricular zone (SVZ), inflammation, and scar formation. Immunohistochemical analysis revealed a positive correlation between stroke severity and the degree of pathological responses to recovery after stroke. Stroke severity was found to increase cell proliferation and migration from the SVZ, with many of these cells positive for GFAP and incorporated into the glial scar. Therefore, we highlight this as an important factor to consider when developing treatment strategies that stimulate cell responses within the neurogenic niche. Long term survival and success of non-autologous stem cell transplants requires use of immunosuppressive agents such as cyclosporine A (CsA). The influence of CsA on stroke outcome required investigation prior to commencing the intended stem cell transplant studies. We explored the effects of CsA administration on neurological and histological outcomes 7 days after stroke. Findings indicated CsA treatment significantly reduced the development of neurological deficits after stroke but did not affect infarct volume, activation of microglia/macrophages, or events within the neurogenic niche. CsA treatment did however attenuate reactive gliosis after stroke and retained pro-survival astrocytic phenotypes important for supporting neuronal rescue. Finally, we investigated the use of cell-based therapies to promote brain repair. SVZ-derived human neural progenitor cells (hNPCs) were isolated, characterized, and differentiated into GABAergic neurons. Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNPCs or media alone were transplanted into the rat brain 7 days after stroke during angiogenesis as it was hoped exogenous transplants would benefit from a redeveloped microvascular bed. GABAergic cell transplants were observed to accelerate functional recovery, showed evidence of maturation and promoted endogenous neurogenesis 28 days post-transplant. Undifferentiated hNPC transplants were predominantly GFAP positive and incorporated into the glial scar. These results suggest techniques aimed at differentiating cells into a neural lineage prior to transplant may be a favourable alternative for stroke treatment. Furthermore, targeting angiogenesis for cell-based treatments may offer greater survival of cells and support graft maturation for functional recovery. In conclusion, this work contributes significantly towards characterising endogenous cellular responses to stroke injury and recovery, and provides preclinical evidence for the benefits of directing exogenous stem cell differentiation towards neural lineage cells prior to transplant, resulting in accelerated recovery.
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ItemInvestigations of the patello-femoral joint in the osteoarthritic knee( 2013)Background Patello-femoral arthritis and anterior knee pain is common in patients with tibio- femoral arthritis. It is presumed that anterior knee pain is caused by patello-femoral arthritis and or malalignment. The predictors of anterior knee pain post total knee arthroplasty are unknown. There are no studies describing the nature and location of anterior knee pain. Methods A retrospective analysis of a prospectively followed cohort of 334 patients undergoing knee arthroplasty was performed. Radiological analysis of measures of patello- femoral malalignment and joint space narrowing was performed. These measures were analysed for association with anterior knee pain and function. A prospective cohort study of 105 patients was performed with minimum 12 months follow-up to analyse post-operative patello-femoral alignment in knees with and without patellar resurfacing. Statistical analysis was performed for possible clinical and radiological predictors of post-operative anterior knee pain. The location of anterior knee pain was reported in this cohort via drawings performed by patients and analysis of pre and post arthroplasty anterior knee pain location was performed. Results Patello-femoral malalignment as measured by lateral patellar tilt and lateral displacement were strongly associated with severe patello-femoral arthritis. Anterior knee pain was not associated with arthritis or malalignment of the patello-femoral joint. Knees that underwent patellar resurfacing during total knee arthroplasty had better post-operative patellar alignment compared to patellae left non-resurfaced. Better patello-femoral alignment did not correlate with improvement in function or decrease in anterior knee pain. Female gender was an independent factor associated with anterior knee pain both before and after arthroplasty. Anterior knee pain is felt more often peripherally around the patella than centrally. Anterior knee pain post-arthroplasty is most commonly similar in nature and location to pain felt pre arthroplasty. Conclusion Patello-femoral arthritis is more severe in knees with patello-femoral malalignment. The cause of anterior knee pain is multi-factorial and predicting anterior knee pain after total knee arthroplasty is difficult. Female gender was the only strong statistical predictor of anterior knee pain pre and post arthroplasty. Better patellar alignment post arthroplasty does not guarantee better function or less anterior knee pain at 12 months follow-up.
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ItemRole of Epithelial Mesenchymal Plasticity associated cancer subpopulations in mammary tumourigenisis and chemoresistance( 2014)Tumour heterogeneity is a key characteristic of cancer and has significant implications relating to tumour response to chemotherapy as well as patient prognosis and potential relapse. It is increasingly accepted that tumours are clonal in origin, suggestive of a tumour arising from a deregulated or mutated cell. Cancer stem cells (CSC) possess/propagate these capabilities, and with appropriate intracellular triggers and/or signalling from extracellular environments, can ‘differentiate’ to initiate tumour formation. Additionally through epithelial mesenchymal plasticity (EMP), where cells gain and maintain characteristics of both epithelial and mesenchymal cell types, epithelial-derived tumour cells have been shown to ‘de-differentiate’ to acquire cancer stem attributes, which also imparts chemotherapy resistance. This new paradigm places EMP centrally in the process of tumour formation, propagation, progression and metastasis, as well as modulating drug response to current forms of chemotherapy. Furthermore, EMP and CSCs have been identified in cancers arising from different tissue types making them a possible generic therapeutic target in cancer biology. In this study, we expand on the relationship between tumour heterogeneity, EMP and CSC in BrCa through the identification and characterisation of epithelial and mesenchymal subpopulations within two BrCa cell lines. In addition, we demonstrate the plasticity that allows these cell populations to effectively regenerate the other cell populations with a particular emphasis on the CSC phenotype. Through a functional genomics screen, the importance of the mesenchymal phenotype in tumour initiation is demonstrated. Taken together, this study demonstrates that heterogeneity exists at a cell line level and this heterogeneity differs in different cellular systems. We also find evidence to suggest that BrCa cell lines can use multiple mechanisms to achieve an outcome such as tumour initiation or mammosphere formation, and subsequently emphasize the importance of phenotype specific drugs. This ideology of drug repurposing to identify phenotype specific drugs is explored through the use of the connectivity map database to identify new uses for previously established drugs to target these subpopulations find preliminary evidence for the role of HDACi to affect these EMP associated subpopulations in BrCa cell lines.
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ItemCardiac surgery using cardiopulmonary bypass in adult patients, effects of pre-operative insulin resistance and the metabolic syndrome, dimethylamine dimethylaminohydrolase activity, and vasoreactivity of regional adipose tissue( 2013)Introduction: Atherosclerosis and coronary artery disease (CAD) are accelerated in Diabetes mellitus (DM) and the Metabolic Syndrome (MS). Insulin Resistance (IR) is thought to be central in its pathogenesis. Recently, Asymmetric Dimethylarginine (ADMA) has linked IR and CAD. ADMA inhibits the vasodilating and vasoprotective agent Nitric Oxide (NO), while the enzyme Dimethylarginine Dimethylaminohydrolase (DDAH)reduces circulating levels of ADMA. As an increase in visceral adiposity is associated with MS and IR, we sought to determine if there were clinically relevant differences in ADMA and DDAH levels in adipose tissue from patients undergoing cardiac surgery, with and without DM and MS. Adipose tissue may alter vascular function via production of vasoreactive paracrine factors. This may explain the acceleration of atherosclerosis in patients with DM and IR. We sought to determine if vasoreactive factors were altered in the adipose tissue of patients with IR or the MS. Methods: Between January 2002 and December 2011, clinical data was prospectively collected on all patients undergoing open cardiac surgery at St Vincent’s Hospital. During 2012, 40 patients undergoing non-emergency cardiac surgery were recruited and adipose tissue was harvested intra-operatively, as well as peri-operative blood and IR parameters. Adipose samples were analysed for DDAH activity, and serum was processed for measurement of ADMA levels. Adipose tissue was incubated with culture medium, and the resulting conditioned medium was used for vasoreactivity experiments. Rat aortic, as well as autologous bypass conduit vessel vasoreactivity was measured using organ bath methodology in the presence of conditioned medium. Results: Of 4542 patients who underwent cardiac surgery in the last 10 years, 1640 (36.1%) had 3 or more surrogate markers of the MS, and 1360 (29.9%) had DM. There was a greater incidence of atrial fibrillation (43.3% vs. 38.6%, p=0.026), renal failure (6.5% vs. 3.8%, p=0.004) and low systemic vascular resistance (16.5% vs. 11.7%, p=0.04) in elective patients undergoing CABG who had the MS. Pre-operative fasting glucose (r=0.63, p<0.001) and HbA1c (r=0.57, p=0.001) correlated with intra-operative hyperglycaemia. HbA1c also predicted post-operative hyperglycaemia (r=0.40, p=0.02). IR measures involving serum insulin (HOMA-IR, QUICKI, LogHOMA) did not predict peri-operative dysglycaemia. Erythrocyte DDAH activity did not appear to change over the course of Cardio-Pulmonary Bypass (CPB). There was an increase in subcutaneous adipose DDAH activity over the course of CPB in patients with the MS (0.02419-0.04318 μg Cit/μg protein/min, p=0.047, n=6), but no change in mediastinal or epicardial adipose DDAH activity was detected. There was also an increase in subcutaneous adipose DDAH activity in diabetic patients (0.02024-0.03127μg Cit/μg protein/min, p=0.013, n=10).There were positive correlations between pre- and post-operative DDAH activity in all depots in patients undergoing CABG with CPB, but not in visceral depots in patients who did not have CPB (OPCAB). Organ bath experiments revealed a significant factor in culture medium that delayed vaso-relaxation to acetylcholine. Vaso-constrictive responses to phenylephrine were not significantly different when conditioned medium was added to the organ bath. Conclusions: There is an increased incidence of post-operative renal failure, atrial fibrillation and low vascular resistance in patients with the MS undergoing CABG. These factors are common with the literature, and may reflect an increased inflammatory response to CPB and surgery. Simple measures of pre-operative dysglycaemia (fasting glucose and HbA1c) correlate with peri-operative dysglycaemia. Levels of DDAH activity in human subjects sustaining an inflammatory stimulus are reported, and appear to increase in subcutaneous adipose tissue. Culture medium utilised for adipose culture contains a significant vaso-active substance that needs to be considered in future experiments.
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ItemAntioxidants as potential anti-osteolytic therapies for breast cancer bone metastasis( 2013)Reactive oxygen species (ROS) are known to play a crucial role in different stages of breast cancer progression, in particular in the maturation and activation of bone-resorbing osteoclast cells. In support of this, several recent reports have shown that antioxidants suppress bone resorption in mouse models of osteoporosis. On this basis, antioxidants may have therapeutic potential in inhibiting breast cancer-induced osteolysis. While measurement of antioxidant activity in cell culture is relatively easy, the assessment of antioxidant activity and ROS levels in human and animal tissue is far more problematic. The constant challenge of antioxidant studies is a) to demonstrate treatment effect and b) providing evidence that the observed effect was indeed caused by antioxidant activity. To investigate the impact of antioxidant treatment on breast cancer primary tumour, metastasis to secondary organs and cancer-induced osteolysis, we employed the orally available antioxidant N-acetylcysteine (NAC) in appropriate pre-clinical mouse models, using grafted mouse (4T1.2) and human (MDA-MB-231, MDA-MB-468) cell lines. Fluorescent-tagged (mCherry) breast cancer cells were inoculated into the mammary fat pad of Balb/C wild type (syngeneic) or Balb/C nu/nu (xenograft) mice. Tumour growth rates for the three cell lines grafted were similar for Placebo (water)-treated mice as well as the doses of NAC examined (0.1 to 2 g/kg/day). Tumour architecture and ratio of necrotic to viable cell mass in the MDA-468-mCherry tumours was unaffected by NAC treatment. Metastatic foci in the lungs of 4T1.2-mCherry and MDA-468-mCherry inoculated mice formed within 30 and 100 days respectively. However, overall tumour burden was not reduced by NAC treatment in the 4T1.2-mCherry model and sub-optimal duration of tumour growth limited the detection of metastatic lung lesions in the MDA-468-mCherry and MDA-231-mCherry models. Contrary to recent reports in the literature, antioxidant treatment did not promote metastasis in our pre-clinical breast cancer models. NAC, when combined with the chemotherapeutic agent doxorubicin (DOX) had no impact on the efficacy of the anthracycline. Supporting in vitro studies demonstrated that NAC at sub-lethal concentration (10 mM) has no effect on DOX-induced inhibition of clonogenicity in MDA-231-mCherry cells, but the antioxidant has an additive, positive impact on the DOX-induced inhibition of clonogenicity when the same assessment is conducted with murine 4T1.2-mCherry cancer cells. Breast cancer-induced osteolysis was initiated by inoculation of 4T1.2-mCherry and MDA-231-mCherry into the tibial marrow of syngeneic and xenograft mice respectively. Treatment with NAC inhibited the onset of osteolysis in the xenograft model and showed mild suppression of osteolysis in the syngeneic mice. To assess impact of NAC treatment on anti-osteolytic therapy with the bisphosphonate Zometa, mice were inoculated with the 4T1.2-mCherry and MDA-231-mCherry breast cancer cell lines following combination treatment with the two agents. Zometa inhibited osteolysis in the MDA-231-mCherry model and supplementation with NAC appeared to increase the inhibition, although the added bone protection did not reach statistical significance. Zometa therapy reduced bone resorption in the 4T1.2-mCherry model, but this effect was only statistically significant when Zometa and 30 mM NAC treatment were combined. This finding suggests that supplementing bisphosphonate therapy with antioxidants like NAC may increase efficacy in anti-osteolysis therapies, including therapies against breast cancer-induced bone resorption. An important part of our studies was the evaluation of pharmacodynamic assays that directly or indirectly measure antioxidant activity in vivo. A modified prooxidant/antioxidant balance (PAB) assay proved useful to measure antioxidant capacity in the NAC drug formulation and in plasma from NAC-treated, non-tumour challenged mice. The dihydroethidium (DHE) assay was employed to detect superoxide formation in kidneys from NAC-treated and control mice. We were able to demonstrate that the fluorescent oxidation product 2-hydroxyethidium can be detected by other means other than high performance liquid chromatography (HPLC) and mass spectrometry, which supports the aim to establish reliable pharmacodynamic assays that can be employed by research and clinical laboratories without expensive instrumentation or specialist skills. The thiobarbituric acid reactive substances (TBARS) assay identified higher malondialdehyde (MDA) levels in tumour challenged mice compared to non-tumour-challenged mice, indicating breast cancer-induced oxidative stress in this model. Furthermore, the assay identified significantly lower MDA levels after treatment with the antioxidant Trolox, in comparison with other antioxidants, including NAC. An assessment of 8-OH-dG urine levels in the same mice revealed significantly higher antioxidant activity in the Olive leaf extract- and Curcumin-treated mice when compared with the control group and NAC-treated mice. This offers the possibility to assess antioxidant impact on breast cancer progression and in particular breast cancer-induced osteolysis with potentially stronger antioxidants.
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ItemMolecular phenotypes of ascites cells in drug resistant ovarian carcinomas( 2013)Epithelial ovarian cancer (EOC) is an aggressive form of cancer diagnosed at an advanced-stage. A manifestation of this disease is the accumulation of ascites fluid in the abdominal cavity. Surgery and systemic administration of platinum/taxol based combination therapy eliminates most tumour cells, however, resistant residual tumour cells eventually emerge. It is hypothesised that cancer stem cells (CSC) together with epithelial-mesenchymal transition- (EMT) and mesenchymal-epithelial transition (MET)-associated mechanisms contribute to drug resistance. The molecular characteristics accompanying these phenotypes have yet to be fully determined. Hence, the aims of this study were to: • separate and characterise the epithelial and mesenchymal ascites cells prior to and following chemotherapy, • apply gene expression microarray technology to chemonaive (CN) and chemoresistant (CR) samples to determine the gene expression signatures of the epithelial tumour and stromal cells, • determine the role of EMT and CSCs marker profiles in facilitating cisplatin resistance, • establish the role of the JAK2/STAT3 pathway in cisplatin resistant EOC cells by utilising a novel inhibitor (CYT387) which inhibits JAK2/STAT3 pathway and sensitises EOC cells to cisplatin by suppressing CSC-like phenotype and • determine the tumourigenicity of cisplatin treated and untreated EOC cells by inoculating nude mice with cisplatin-treated surviving cells exhibiting features of CSC-like cells. Tumourigenic and non-tumourigenic ascites cells were separated by a novel method developed during this study and were characterised by in vitro assays including wound healing, cell growth and chemosensitivity assays. The expression of genetic markers was assessed using immunofluorescence, flow cytometry, Western blot, qPCR and gene expression microarray. Tumourigenicity of EOC cells was assessed by xenograft studies in mouse. Hematoxylin and Eosin staining performed on histology section of mice organs was used to determined invasive tumour behaviours. The significant findings were; • Separation of the epithelial and mesenchymal ascites cells isolated from high-grade EOC patients prior to and following chemotherapy. • Molecular and gene expression profiles of epithelial cells, which were tumourigenic and expressed markers such as CA125, EpCAM, STAT3, Oct4 and E-cadherin, while non-tumourigenic stromal cells exhibited a mesenchymal phenotype and were enriched for CD44, MMP2, MMP9, FSP, CD90, CD105, CD73, N-cadherin and vimentin. • Genetic signatures of the epithelial tumours and stromal cells obtained from gene expression microarray technology applied to CN and CR samples. • EMT and CSCs marker profiles were determined to play an important role in facilitating cisplatin resistance. • The JAK2/STAT3 pathway was implicated in cisplatin resistance EOC cells as the CYT387 inhibitor sensitised EOC cells to cisplatin by suppressing CSC-like phenotype. • Tumourigenicity of cisplatin treated and untreated EOC cells in nude mice - surviving cells exhibited features of EMT- and CSC. Unique populations of EOC cells exhibiting characteristics of CSC-like cells are capable of undergoing cellular changes in line with EMT-like (during cisplatin treatment) and enhanced epithelial phenotypes (in in vivo settings). Together these traits contribute to drug resistance and recurrence, and targeting pathways that facilitate these phenotypes may improve treatment.