Medicine (Austin & Northern Health) - Theses
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ItemThe role of Hepatitis B surface antigen in the development of severe liver disease and hepatocellular carcinoma( 2016)Hepatitis B virus (HBV) infection continues to be a major public health issue worldwide, affecting an estimated 2 billion people globally. In spite of safe vaccination and effective therapy, there is still a large burden of disease due to chronic hepatitis B (CHB), affecting an estimated 240 million people, which may lead to cirrhosis and or the development of hepatocellular carcinoma (HCC). Liver cancer is rapidly emerging as the single greatest challenge in hepatology and liver transplantation and the burden of disease is set to explode in the next 20-30 years. Viral hepatitis is ranked as the seventh leading cause of death worldwide, resulting in at least 1.2 million deaths annually from liver cirrhosis and HCC. HCC is currently the sixth most common cancer and the second leading cause of cancer-related deaths globally. Case-control studies have shown that chronic HBV carriers have more than 100-fold increased risk of HCC compared with non-infected individuals. Approximately 80% of all HCC cases can be attributed to viral hepatitis, more than half of that due to HBV, which is the second most potent carcinogen after tobacco (World Health Organization), and this is despite remarkable improvement in therapy with the advent of nucleos(t)ide analogues (NA). The global reservoir of HBV infection serves as the basis for the generation of HBV variants via recombination and a high frequency of mutation in the HBV genome. Due to the inherent molecular biology of this virus which replicates its DNA genome via a low fidelity viral reverse transcriptase (rt)/polymerase, a population of closely related genetic variants known as a quasispecies is produced. The last two decades have seen a significant increase in the emergence of mutants as the virus responds to selective pressures, such as vaccination and antiviral therapy. Surveillance for clinically significant HBV mutations and an improved understanding of the impact of these emerging variants on the natural history of the disease and its diagnosis, control and management will pose a challenge to global health care in the foreseeable future. This is because these mutants have the potential to alter current diagnostic and treatment algorithms. NA therapy was approved in 1998, which efficiently lowers the HBV DNA viral load in HBV-infected patients. Given that the HCC risk is particularly high in the presence of cirrhosis and/or persistent high HBV DNA replication, NA should be a rational treatment to prevent liver disease progression including liver cancer in such patients. However since the introduction of antiviral therapy, the HCC incidence has continued to rise. Registration for the liver transplant waiting list due to HBV-related HCC in the U.S. has increased in the NA era, which may in part be due to patients no longer dying of liver disease. There is clearly a need for a greater understanding of the role that NA therapy might play in the development of HCC. Numerous risk factors for HBV-related HCC development have been identified. However the relationships are complex. Older patient age, liver cirrhosis, DNA viral load, hepatitis e antigen (HBeAg) status, HBV genotype, gender and family history have all been found to be important risk factors for HCC in most studies. The exact mechanism of HBV-related carcinogenesis is not fully elucidated, however it is likely a multi-factorial process reliant on a combination of mechanisms which include: ongoing inflammation, cycles of damage and regeneration of hepatocytes, increased chromosomal instability through multiple HBV DNA integrations, oxidative stress as a consequence of NA driven alterations to the HBV life cycle, and finally any direct effects of the virus or viral proteins themselves. The aim of this study was to determine the role that the hepatitis B surface antigen (HBsAg) might play in patients with CHB in the development of severe liver disease and HCC in the era of hepatitis B virus (HBV)-specific NA therapy. The hypothesis tested was that the development of HCC may be associated with altered expression of phenotype and abnormal cellular distribution of HBsAg, and that NA-induced drug resistance can influence this process. This is an important question given the rising incidences of HCC. This study will investigate the effects of NA treatment on HBV replication and any subsequent variation in oncogenic potential from common NA resistant variants. This study will also use various novel techniques such as HBV splicing and next generation sequencing to explore new approaches for the molecular pathogenesis of CHB. The findings are important as virological factors may have prognostic importance in patients developing HCC, which may impact existing CHB treatment strategies and professional practice guidelines.
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ItemInvestigating the relative contribution of obesity and glucose in the development of β-cell dysfunction( 2016)Insulin resistance and impaired insulin secretion are hallmark features that contribute to the development of hyperglycaemia in type 2 diabetes (T2D) and other complications such as kidney failure, stroke and cardiovascular disease. Obesity is considered to be one of the main drivers in expediting hyperglycaemia by inducing insulin resistance in the liver, muscle and fat. These defects place additional stress on the β-cell to increase insulin output to compensate for the prevailing glucose and over time, can result in declined β-cell function and T2D. Pre-clinical and clinical studies investigating interventions that reduce obesity in pre-diabetes have shown that the incidence of T2D can be attenuated by preserving β-cell function through enhanced insulin sensitivity. However, as obesity clearly induces hyperglycaemia, it has become inherently difficult to dissociate the relative contribution of each in the progression of impaired glucose tolerance (IGT) to T2D. Accordingly, the scope of this thesis was to use dietary and pharmacological interventions to determine the contribution of obesity and glucose in the development of metabolic defects associated with T2D, namely glucose intolerance, insulin resistance and β-cell dysfunction. The overall hypothesis was that both obesity and excess glucose contribute to these defects. In order to address the overall aim of this thesis, it was necessary to firstly characterise a pre-clinical model that does not rely on the presence of obesity to drive hyperglycaemia and subsequently, insulin resistance and β-cell dysfunction. We therefore utilised the phosphoenolpyruvate carboxykinase (PEPCK) transgenic rat, which is characterised by a 2-3 fold induction of PEPCK in the liver and kidney that leads to the impaired suppression of endogenous glucose production. Through the use of in vivo and in vitro experimental techniques, we show that the PEPCK transgenic rat develops defective glucose-stimulated insulin secretion in parallel with the worsening of glucose tolerance at 14 weeks of age, and that this is primarily due to the significant reduction in β-cell Glut2 gene expression and the inability of the constituents that make up the K+ATP channel, Sur1 and Kir6.2, to function properly. This defect in insulin secretion progressively worsens by 20 weeks due to the combination of β-cell dysregulation and reduced β-cell mass. We next investigated the potential of the selective glucose-lowering SGLT2 inhibitor, dapagliflozin, in preventing the progression of insulin resistance and β-cell dysfunction in the PEPCK transgenic rat. We show that in older animals with established insulin resistance and β-cell dysfunction, dapagliflozin treatment for 6 weeks resulted in lower body weight gain despite the compensatory increase in food intake due to energy loss from the urine, reduced plasma glucose and insulin levels, and improvements in glucose tolerance which was associated with enhanced insulin sensitivity and glucose uptake in muscle and fat. In addition, dapagliflozin treatment in PEPCK transgenic rats significantly improved GLUT4 protein content in fat while adipocyte number was increased and the size reduced. A subset of PEPCK transgenic rats were also calorie-restricted in order to prevent further weight gain so that they could be used to account for any potential weight-induced insulin-sensitising benefits seen with dapagliflozin treatment. The prevention of weight gain in these PEPCK transgenic rats greatly enhanced peripheral insulin sensitivity to levels comparable with dapagliflozin treatment. Interestingly, dapagliflozin treatment did not preserve β-cell mass or improve the insulin secretory response to glucose. These data suggest that dapagliflozin elicits its effects on the β-cell in an indirect manner by increasing insulin sensitivity and providing an islet β-cell sparring effect. To dissociate the relative contribution of obesity and glucose per se in IGT and β-cell dysfunction, obesity and glucose excess were prevented by commencing calorie-restriction and dapagliflozin treatment prior to any metabolic defects in 5 week-old PEPCK transgenic rats. Our findings show that preventing either obesity or glucose improves glucose tolerance but does not directly increase the insulin secretory capacity when assessed by a hyperglycaemic clamp or static incubation of isolated islets. These results support the hypothesis that both obesity and glucose per se contribute to the development of glucose intolerance, insulin resistance and β-cell dysfunction in the PEPCK transgenic rat and that enhancing insulin sensitivity with either intervention could effectively prevent the decline in β-cell function with age.
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ItemNo Preview AvailableNovel approaches to circulating tumour markers for non-invasive monitoring of melanoma progression and therapy response( 2016)Although the number of treatment options for advanced melanoma has been increasing, highly reliable methods for assessing treatment response and disease progression are still lacking. There is an urgent need for tumour markers that can rapidly and sensitively inform clinicians if the treatment is effective and whether there is evidence of resistance to treatment. There is also a demand for effective tools that can monitor the tumour specific cellular changes in response to treatment, which would allow us to gain a deeper understanding of the mechanisms utilized by tumour cells to resist treatment. We have developed three blood-based methods namely circulating tumour DNA (ctDNA) quantification by droplet digital PCR, circulating tumour cell (CTC) quantification and characterisation by Nanoshearing microfluidic platform, and CTC in vivo tracking with Surface-enhanced Raman Spectroscopy (SERS) immunogold nanotechnology. In this thesis, I will evaluate their effectiveness using a combination of cell lines and clinical patient samples. With these methods, I was able to easily assess tumour responses to treatment and detect early disease progression. Furthermore, the SERS technology allowed me to interrogate CTCs for the expression of multiple (surface) markers at once, giving insight into phenotypic changes over treatment and during the emergence of resistance. This should allow future drug development and personalised treatment planning.
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ItemThe effect of testosterone treatment on glucose metabolism and other health outcomes in men with type 2 diabetes: a randomized controlled trial( 2016)Context: The efficacy and safety of testosterone treatment in men with type 2 diabetes mellitus (T2D) and low to low-normal circulating testosterone levels is unknown. The number of testosterone prescriptions has increased significantly throughout the developed world whilst the prevalence of traditional indications for treatment is unchanged. This in part reflects increased prescription for novel indications such as T2D in the absence of sufficient efficacy and safety data. The effects of testosterone treatment on insulin resistance and other health outcomes in men with T2D were studied using rigorous methodology in this randomized controlled trial (RCT). Objective and Patients: In this 40-week RCT of 88 men with T2D, we investigated the effect of intramuscular testosterone undecanoate on insulin resistance as measured by homeostasis model assessment of insulin resistance (HOMA2-IR). Effects on glycemic control, body composition, sexual and constitutional symptoms and cardiac biomarkers were also assessed. Results: Testosterone treatment did not improve insulin resistance (Mean adjusted difference (MAD) for HOMA2-IR compared with placebo over 40 weeks -0.08 [95% CI -0.31 to 0.47] p=0.23) or glycemic control (MAD HbAlc 0.36% [0.0,0.7] p=0.05) despite a decrease in fat mass (MAD -2.38kg [-3.10,-1.66] p<0.001) and an increase in lean mass (MAD 2.08kg [1.52, 2.64] p<0.001). Testosterone treatment reduced subcutaneous abdominal adipose tissue volume (MAD SAT -320 cm3 [-477,-163] p<0.001) but not visceral abdominal adipose tissue volume (MAD VAT 140cm3 [-89,369] p=0.90) and did not increase adiponectin levels (MAD -3.3 ug/ml [-8.2,1.6] p=0.42). Testosterone treatment did not improve constitutional symptoms as measured by Aging Male Symptom (AMS) score (MAD AMS -0.9 [-4.1,2.2] p=0.67) but reduced erectile function as measured by International Index of Erectile Function abridged version 5 (IIEF-5) (MAD IIEF-5 -2.0 [-3.4,-0.6] p<0.02). No significant difference between baseline and 40 week IIEF-5 scores were noted when both groups were analyzed separately. At baseline symptoms were worse in men with depression (AMS 39 vs. 31 p=0.002, IIEF-5 17 vs. 18 p=0.53) and microvascular complications (Neuropathy AMS 38 vs. 32 p=0.04, IIEF-5 8 vs. 18 p=0.03, Retinopathy AMS 40 vs. 33 p=0.22, IIEF-5 7 vs. 18 p=0.02) but did not correlate with testosterone levels (Total testosterone AMS -0.02 p=0.78, IIEF-5 0.09, p=0.37). Testosterone treatment reduced N terminal pro brain natriuretic peptide (NT-proBNP) levels (MAD -17.9 ng/L [-32.4,-3.5] p=0.047) but did not change high sensitivity cardiac troponin T (hs cTnT) levels (MAD 0.41ng/L [-0.56,1.39] p=0.62). Six men, three in each group experienced an adverse cardiac event, displaying already higher baseline NT-proBNP (p<0.01) and hs cTnT levels (p=0.01). Conclusions: In our studies, testosterone treatment did not improve insulin resistance or glycemic control, visceral abdominal adipose tissue volume, constitutional or sexual symptoms in a cohort of obese men with T2D and modest reductions in circulating testosterone levels that are typical for men with T2D. While the long term risks and benefits of testosterone treatment in men without classical hypogonadism due to organic gonadal axis pathology remain uncertain, T2D in itself should not be an indication for testosterone treatment. Further study of the relationship between exogenous testosterone and long term health outcomes is required to better inform us of the risk benefit ratio of testosterone treatment in men in general and in the diabetic subpopulation.
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ItemMuscle effects of androgen deprivation therapy in men with prostate cancer( 2016)BACKGROUND: Androgen deprivation therapy (ADT) is an effective treatment for prostate cancer but has many adverse effects consequent to severe testosterone deficiency including decreases in muscle and bone and increases in fat mass. Despite the decline in muscle mass, previous studies of muscle function have not demonstrated consistent deficits, likely due to imprecise methodology, which was insensitive to detect functional changes associated with ADT. We hypothesised that firstly, ADT causes clinically relevant deficits in the lower-limb muscles and has differential effects on individual muscles; secondly, ADT decreases muscle mass, increases frailty, insulin resistance and impairs quality of life (QoL); thirdly, ADT leads to changes in testosterone-regulated genes in skeletal muscle. METHODS: My colleagues and I conducted a 12-month prospective, observational case-control study of 63 men with non-metastatic prostate cancer at a tertiary hospital. Men newly commencing androgen deprivation therapy (ADT) (n=34) were compared to age-and radiotherapy-matched prostate cancer controls (n=29) using a linear mixed model. Motion capture and ground reaction force data were combined with computational musculoskeletal modeling to assess lower-limb muscle function whilst walking on level ground at self-selected speed in a Biomotion Laboratory. The following primary outcomes were determined: 1) Peak joint torques developed about the hip, knee and ankle, and corresponding individual muscle forces. 2) Individual muscle contributions to the accelerations of the body’s centre of mass. 3) Walking speed, stride length and step width. Secondary outcomes included handgrip strength, insulin resistance (updated homeostatic model assessment for insulin resistance: HOMA2-IR), Fried’s frailty criteria and QoL. Next-generation RNA sequencing was performed on skeletal muscle biopsies (n=9) to identify differentially expressed genes with ADT. RESULTS: Compared to controls over 12 months, men receiving ADT had more marked decreases in peak hip flexor torque and knee extensor torque, with mean differences of -0.11newtons/kg [-0.19, -0.03], p=0.01 (-14% of the initial mean value) and -0.11newtons/kg [-0.20, -0.02], p=0.02 (-16% of the initial mean value), respectively. Correspondingly, iliopsoas force decreased by 14% (p=0.006) and quadriceps force decreased by 11%, although this narrowly missed statistical significance (p=0.07). Soleus decreased contribution to forward acceleration of the body’s centre of mass by 17% (p<0.01) in the ADT group compared with controls. Furthermore, step width increased by 18% (p=0.042) with no change in stride length or walking speed. Insulin resistance increased which was related to increased fat mass, p=0.003, but not decreased lean mass (p=0.09) and less so to testosterone levels (p=0.088). Visceral fat was unchanged. QoL decreased in the ADT group, with predominant effects on physical and sexual subdomains. Actin alpha cardiac muscle 1 (ACTC1) gene was upregulated in skeletal muscle of men undergoing ADT. CONCLUSION: Testosterone deprivation causes selective functional deficits on lower-limb muscles, predominantly affecting iliopsoas, quadriceps and soleus, muscles involved in supporting body weight and accelerating the body forward during walking, and may also affect balance. As gain in fat mass mediates insulin resistance, implementing lifestyle measures to prevent obesity in men commencing ADT is paramount. Future exercise studies or promyogenic interventions to mitigate ADT-associated sarcopaenia and obesity should target the deficits described to maximise muscle function and QoL in men commencing ADT.
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ItemThe role of muscle glycogen synthase in glucose metabolism and exercise capacity( 2016)Muscle glucose storage and muscle glycogen synthase (gys1) defects have been associated with insulin resistance and reduced exercise capacity. As there are multiple mechanisms for these defects, the specific role of impaired glycogen synthase is not clear. The focus of the current PhD dissertation is to conduct optimisation, molecular and physiological characterisation of the novel muscle-specific gys1-KO model regarding glucose and exercise metabolism, exercise and endurance capacity and exploration of any potential mechanism able to explain the defects. 8-10 week-old (adult) mice were fed a diet containing 1 mg/g tamoxifen for 8 weeks followed by a 4-week recovery period on tamoxifen-free chow diet. gys1-KO mice displayed 88% reduced gys1 mRNA levels translated into 85% reduced muscle gys1 protein concentration, preserved for up to 4 weeks following tamoxifen withdrawal, while there was no difference between the groups in all other tissues expressing gys1. The reduced muscle gys1 levels led to a 66-71% reduction in skeletal muscle glycogen concentration, with slightly modified levels according to the fasting state of the animals. In contrast, gys1-KO mice showed increased liver glycogen concentration under non-fasting conditions, but no significant difference after a 6-hour and overnight fasting period. All physiological experiments were conducted in 22-week-old gys1-KO and littermate control mice. Insulin Tolerance Tests (ITTs), Oral and Intraperitoneal Glucose Tolerance Tests (OGTTs and IPGTTs respectively), hyperinsulinaemic-euglycaemic clamps under two different insulin concentrations and 2-deoxyglucose kinetics were utilised to assess glucose tolerance, insulin sensitivity, basal and insulin-stimulated glucose turnover, plasma and tissue glucose uptake. Furthermore, three-day acclimatisation to a treadmill, followed by three different exercise tests (incremental, low intensity, high intensity), were performed to assess their exercise and endurance capacity. Post-exercise IPGTTs after the high-intensity exercise protocol were carried out to examine the effect of one bout of exercise on glucose tolerance. Additional Western blot experiments were also performed. There was no difference in body, heart, fat pad weights and food intake between the groups. gys1-KO mice presented postprandial hyperglycaemia and subsequent hyperinsulinaemia, especially when challenged with a glucose load, indicative of impaired glucose tolerance. Under basal turnover conditions, there was no difference in fasting plasma glucose and insulin levels between the groups, while the Basal Rate of glucose disappearance (RD) was lower in the gys1-KO mice. Under both states of insulin-stimulated conditions, gys1-KO mice showed reduced RD and decreased skeletal-muscle and brown adipose tissue glucose uptake, suggestive of peripheral insulin resistance. gys1-KO mice also showed decreased exercise capacity, as revealed by maximal speed and maximal workload as well as reduced endurance capacity. The mechanistic studies showed reduction in hexokinase II and glucose-6-phosphate (G-6-P) levels, increased basal, insulin-stimulated and post-exercise plasma lactate levels, insulin-stimulated muscle lactate levels, as well as no difference in liver lactate, glycogen phosphorylase, GLUT-4 mRNA and protein and G-6-P isomerase protein levels. The current dissertation demonstrates that a muscle-specific gys1 deletion in adult mice results in glucose intolerance due to peripheral insulin resistance and reduced muscle glucose uptake, as well as impaired exercise and endurance capacity. The data support an important role for muscle glycogen synthase in glucose metabolism and exercise capacity.
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ItemDetection and neuromuscular response to respiratory loading in obstructive sleep apnoea( 2016)Obstructive sleep apnoea (OSA) is characterised by the repetitive narrowing or collapse of the upper airway during sleep. While anatomy is considered important in OSA pathogenesis it has been recognised that other factors must be involved. This thesis aimed to examine whether (i) sensory detection, and (ii) upper airway neuromuscular compensation, were impaired in awake OSA patients, in response to small negative pressure respiratory loads, close to the conscious detection threshold. It was reasoned that this may be important in OSA pathogenesis, as failure to detect and respond to minor airway patency threat may lead to worsening collapse which is difficult to remedy. Sensory detection was measured using the early P1 component of the respiratory related evoked potential (RREP). The RREP is the average cortical response to multiple presentations of the same respiratory stimulus and the P1 component is thought to represent arrival of somatosensory information at the cortex. The neuromuscular response was measured with genioglossus muscle intramuscular electromyogram (EMGgg). No significant differences were found between control and OSA participants in the threshold or the sensitivity of relationship between P1 amplitude and stimulus intensity. Also, there were no significant differences between control and OSA participants in the threshold or the sensitivity of the relationship between EMGgg amplitude and stimulus intensity. These results do not support the concept that a neuromuscular response deficit or a sensory detection deficit contribute to OSA pathogenesis. However, the neuromuscular results were confounded by a counterintuitive genioglossus suppression observed in a significant proportion of participants. This response was not more commonly observed in OSA participants and may be related to the centrally mediated protective reflex inhibition seen in other inspiratory muscles in response to sudden onset negative pressure. A methodological study examined the impact of using two abbreviated signal montages, typically used in portable polysomnography (PSG) during OSA diagnosis, on sleep and cortical arousal scoring, compared to a standard reference montage. The results demonstrated that abbreviated signal montages may result in underestimation of the arousal index and poorer precision in sleep scoring, with potential consequences for OSA diagnosis. The results guided thesis methodology and have implications for future PSG standards. A further experiment aimed to improve understanding of respiratory stimuli sensory detection. From prior sensory detection studies it has been hypothesised that there is a subcortical gating mechanism, preventing arrival of afferent information at the cortex. This concept was examined by comparing RREPs produced by consciously detected vs. undetected loads, near the detection threshold. Results obtained from ten healthy males suggested that load-related sensory information may reach the somatosensory cortex for sub-threshold loads. The findings argue against subcortical gating of somatosensory information.
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ItemPathways to phenotypes in melanoma: regulation of plasticity and treatment escape( 2016)Melanoma exhibits a wide range of biological behaviours, many of which recapitulate melanocytic developmental processes. Alterations in melanoma cell behaviour and phenotype contribute to melanoma progression, metastasis, and influence the efficacy of drug therapies. This study examines how complex regulatory molecular networks differ between melanoma phenotypes and distinct biological states. Through the use of whole-cell transcriptomic data, the relationships between gene expression and microRNA expression in melanoma were described. Coupling between seemingly distinct phenotypes such as pigmentation and migration/invasion was shown to be frequently linked to the core melanocytic transcription factor MITF, but may involve transcriptional regulatory mechanisms driven by other factors, such as SOX10. Differential expression analysis identified a preliminary tumour cell specific gene set including several inflammatory and immune-related genes, indicative of brisk lymphocytic infiltration of melanoma tumours. A novel Systems Analysis workflow was developed to integrate gene and microRNA expression data, incorporating phenotypic annotation data to identify putative regulatory interactions of relevance to the phenotype(s) under study. Validation of this workflow using the candidate microRNA miR-29b-3p confirmed the utility of this approach in directing experimental resources, and identified a novel role for miR-29b-3p in regulating melanoma invasiveness. The specific scenario of sensitivity and/or resistance to BRAF-targeted therapy in melanomas harbouring activating BRAF V600 mutations was used to explore the adaptive changes that occur under treatment. The molecular pathway alterations in drug-resistant melanoma cells at gene level, and at microRNA level were identified, demonstrating that dysregulation of genes and of microRNAs act as an additive strategy to maximise potential regulation of the proteome. Furthermore, the finding of significantly heterogeneous and non-recurrent changes in gene and, particularly, microRNA abundance led to the proposal of a multi-state model of drug resistance and plasticity-like resensitisation. Several key microRNA species, including miR-155-5p and the miR-199a~miR-214 cluster, were implicated as direct modulators of BRAF inhibitor sensitivity. Lastly, off-target effects of BRAF mutation-selective inhibitors were examined in two unique settings of paradoxical MAPK pathway activation. The first involved a KRAS mutated colon cancer, whilst the second involved a haematological malignancy – chronic myeloid leukaemia – characterised by similar MAPK pathway activation but a substantially more diverse array of activated parallel signalling pathways. The contrasting clinical outcomes in these two cases were used to highlight the sensitive dependence on cellular context when determining the outcome of an otherwise identical pharmacological manipulation. A synthetically lethal combination of BRAF and MEK inhibitors in chronic myeloid leukaemia was demonstrated both pre-clinically and clinically, arguing for further clinical evaluation of this strategy in CML and potentially other cancer types. Together, this work provides a firm basis for understanding and evaluating the complex microRNA-gene interaction networks active in melanoma cells across diverse biological states and phenotypes. Key candidate microRNAs and target genes have been identified for further study as potential therapeutic targets in melanoma. In parallel, the complexity of signal pathway manipulation has been demonstrated in the context of paradoxical MAPK pathway activation, arguing strongly for further clinical evaluation of synthetic lethality as a therapeutic strategy.
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ItemImpact of a pregnancy-associated protein (PAPPA) on melanoma: a link between pregnancy and progression of cancer( 2016)Melanoma is the most common cancer diagnosed in pregnant women, and an aggressive course with poorer outcomes is commonly described during pregnancy or shortly after childbirth. The underlying mechanisms for this are not understood. This thesis reports that migration, invasiveness and progression of melanoma are promoted by pregnancy-associated plasma protein-A (PAPPA), a pregnancy-associated metalloproteinase produced by the placenta that increases the bioavailability of insulin-like growth factor (IGF)-1 by cleaving it from a circulating complex formed with IGF binding protein-4. The results presented in this thesis confirm that PAPPA is widely expressed by metastatic melanoma tumours and is elevated in melanoma cells exhibiting mesenchymal, invasive and label-retaining phenotypes. Notably, inhibition of PAPPA significantly reduced invasion and migration of melanoma cells in vitro and in the avian embryonic neural crest model in vivo. Treatment with PAPPA-enriched pregnancy serum enhanced the motility of melanoma cells in vitro. Furthermore, this thesis reports that IGF-1 can induce the phenotypic and functional effects of epithelial-mesenchymal transition (EMT) in melanoma cells. This study establishes a clear relationship between the pregnancy-associated protein PAPPA, melanoma, and functional effects mediated through IGF-1, providing a plausible mechanism for accelerated progression of melanoma during pregnancy. This opens the possibility of therapeutic targeting of the PAPPA/IGF-1 axis.