Medicine (St Vincent's) - Theses

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    Differentiation of dermal papilla cells into a myogenic lineage for the treatment of Duchenne muscular dystrophy
    Rashidi, Mahsa ( 2016)
    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in children. It is due to an X-linked recessive mutation in the dystrophin gene resulting in the lack of, or production of non-functional, dystrophin. Dystrophin is a pivotal part of the dystophin glycoprotein complex, which transfers mechanical force from the extracellular matrix to the cytoskeleton and stabilizes the myofibre structure. DMD is characterized by significant muscle weakness leading to loss of ambulation and respiratory/cardiac failure and death. No specific treatment has been established so far, thus new therapeutic strategies are being sought. The application of stem cells for therapy in DMD is one of the more promising approaches. Multipotent stem cells residing in the hair follicle dermal papilla are highly plastic and are reprogrammable to bone, cartilage, fat, haematopoietic and myogenic differentiation. Dermal papilla cells (DPC) from the hair follicles of skin were specifically the focus of this study. DPC derived from mouse whisker pad were shown to undergo myogenic differentiation when co-cultured with different types of myoblasts, including normal and dystrophic human myoblasts. Lamin A/C staining was used to distinguish DPC- and myoblast-derived myonuclei inside myotubes to enable measuring the contributions of DPC to differentiated myotubes in cocultures. DPC incorporated into myotubes and up-regulated the muscle marker myogenin in coculture with human myoblasts, suggesting that DPC fully underwent myogenic differentiation in these co-cultures. This study also defines new methods for quantifying myogenic reprogramming in vitro. The data obtained with this method suggested that DPC incorporation efficiency was low in all co-cultures and differed significantly between various types of myoblast; however, no significant difference was observed between normal and dystrophic human myoblasts. These encouraging findings suggested that the altered properties of dystrophic myoblasts did not compromise the myogenic differentiation of DPC in vitro, supporting their in vivo application and possible therapeutic potential. This dissertation also presents a comprehensive evaluation of the in vitro effects on DPC myogenic differentiation of different bioactive molecules, including galectin-1, reversine and activation of the Shh signalling pathway via recombinant Shh and purmorphamine. None of the bioactive molecules could induce or increase myogenic differentiation in DPC, but the data generated in this study suggested that the activation of Shh signalling might function in a dose-dependent manner. Weaker Shh signalling promotes differentiation and stronger signalling promotes proliferation. In summary, this dissertation reports that DPC are a readily accessible source of stem cells that can undergo myogenic differentiation in vitro. Thus DPC remain a candidate stem cell type for DMD treatment; however, their differentiation efficiency requires improvement in order to make them suitable for therapeutic applications in muscle wasting disorders. Furthermore, these newly defined methods to quantify myogenic reprogramming in vitro could be used to identify strategies to enhance the myogenic differentiation of DPC and other stem cell types. This dissertation provides an impetus for future studies investigating stem cell treatments for myogenic differentiation.
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    The role of Hepatitis B surface antigen in the development of severe liver disease and hepatocellular carcinoma
    Lim, Lucy ( 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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    Therapeutic effects of phosphodiesterase 4 inhibitor roflumilast in chronic kidney disease
    Lau, Xianzhong ( 2016)
    Progressive chronic kidney disease (CKD) is a major health problem in Australia, and its prevalence is increasing due to the growing incidence of diabetes and hypertension. However, current clinical management of CKD, particularly the use of antihypertensive drugs, and glycaemic control for diabetic patients, is only partially effective against preserving renal function and in delaying end stage kidney disease (ESKD). Therefore, there is a need for new approaches to slow or halt the progression of CKD to ESKD. As chronic inflammation and consequently progressive fibrosis are important features of CKD, this thesis explored the therapeutic potential of phosphodiesterase 4 (PDE4) inhibitor, roflumilast (RFL), a drug has been found to broadly inhibit inflammation, in progressive CKD. Firstly, the efficacy of RFL to inhibit TGF-β1 stimulated effects, where TGF-β1 is a major pro-fibrotic cytokine, was tested in vitro in renal cell cultures. RFL exhibited both anti inflammatory and anti-fibrotic properties, diminishing the expression of pro inflammatory cytokine MCP-1 and weakening TGF-β1 induced pro-fibrotic responses. RFL also normalised cell proliferation changes triggered by TGF-β1 stimulation. However, the mechanism of how PDE4 inhibition by RFL led to the attenuation of these TGF-β1 stimulated effects remains inconclusive, and was at least not solely due to the changes in intracellular cAMP levels. The efficacy of RFL was then investigated in an animal model of progressive CKD, the subtotal nephrectomised rat. RFL treatment attenuated renal decline in this rat model by preserving glomerular filtration rate, reducing proteinuria, and attenuating structural deterioration, such as glomerulosclerosis and tubulointerstitial fibrosis. Moreover, RFL indirectly downregulated TGF-β1 mediated response, implying that the therapeutic effects of RFL may be through a simultaneous inhibition of inflammation and fibrosis than merely through the suppression of inflammation and subsequent attenuation of fibrosis. Finally, the localisation of PDE4 isoforms was investigated in the renal cortex. PDE4 isozymes were mainly localised in the cells of the distal tubules of the tubulointerstitium and the glomeruli. In addition, the expression of PDE4A, PDE4B and PDE4C isozymes in the glomerulus were elevated in the diseased kidney, and colocalised with podocytes. RFL treatment was found to reduce the elevated PDE4 expression, which correlated with the preservation of podocyte number, suggesting that PDE4 inhibition may be relevant in preserving podocyte integrity. In conclusion, it was clearly demonstrated in this thesis that RFL treatment was able to reduce inflammatory and fibrotic processes, in vitro and in an animal model of progressive CKD, and preserve podocyte survival, mechanisms which were independent of hemodynamic changes. Instead, these effects presented by RFL might be through the inhibition of inflammation and TGF-β1 stimulated responses, although the exact mechanism of this remains inconclusive in this thesis. Nonetheless, there is strong basis for use of RFL as a novel therapy to delay the progression of CKD to ESKD. Further investigation on PDE4 inhibition in combination with antihypertensive drugs is necessary to determine potential clinical utility of PDE4 inhibitors in treating CKD.
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    Induction of antigen-specific tolerance and development of autoreactive T cells in an experimental model of autoimmune diabetes
    Jhala, Gaurang ( 2016)
    Immune responses to proinsulin initiate anti-islet autoimmunity in non-obese diabetic (NOD) mice and possibly in humans. This results in autoimmune destruction of insulin secreting beta cells leading to type 1 diabetes (T1D). Therapies that bolster immune tolerance to islet antigens are highly desirable, however such approaches have failed to prevent clinical T1D. The major aim of this thesis was to determine a stage of life when antigen-specific tolerance is most effective in preventing anti-islet immune responses. Chapter 2 describes generation and validation of transgenic NOD mice engineered to express islet antigens proinsulin (TIP mice) and IGRP (TII mice) in the antigen presenting cells (APCs) in a tetracycline dependent manner. MHC class II IEα promoter in combination with tet-OFF transactivator induced robust, doxycycline dependent and APC specific expression of proinsulin and IGRP in TIP and TII mice respectively. TIP mice expressing proinsulin did not develop insulitis and were protected from cyclophosphamide-induced diabetes, suggesting that proinsulin expression in TIP mice was sufficient to induce functional antigen-specific tolerance. In chapter 3, we examined the impact of antigen-specific tolerance on the development of autoreactive T cells and spontaneous diabetes by expressing islet antigens proinsulin and IGRP in the APCs during defined periods of life in TIP and TII mice. Our results indicate that tolerance to proinsulin in early life until the weaning period is sufficient to prevent diabetes development in TIP mice. The protection from diabetes was not due to dominant tolerance, but mainly due to a significant reduction in the insulin reactive T cells. Although insulin reactive T cells were not completely absent, the residual autoreactive T cells lacked pathogenic potential. By tracking IGRP reactive T cells in TII mice we demonstrate that IGRP T cells also emerge during early life. These data suggest that early life is a vulnerable period for escape of islet reactive T cells, and that boosting immune tolerance to islet antigens during this time imparts durable protection from islet autoimmunity. Immune tolerance to proinsulin-2 imparts robust protection from autoimmune diabetes in the NOD mice. Whether dampening immune responses to proinsulin-1 would influence diabetes development in NOD mice remains to be investigated. Chapter 4 describes the generation of transgenic NOD mice that express proinsulin-1 in the APCs (TIP-1 mice) in a tetracycline dependent manner. TIP-1 mice displayed a significantly reduced incidence of spontaneous diabetes, which was associated with reduced severity of insulitis and insulin autoantibody development. Antigen experienced proinsulin specific T cells were significantly reduced in number in TIP-1 mice indicating immune tolerance. Although immune response to downstream antigen IGRP was reduced in TIP-1 mice, tolerance to proinsulin-1 was unable to prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, despite being highly conserved to proinsulin-2, tolerance to proinsulin-1 only partially prevents islet-autoimmunity in NOD mice, which suggests an ongoing residual immune response to proinsulin-2 epitopes in TIP-1 mice.
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    Hair follicle transplantation: implications for cell migration and wound healing
    Asgari, Azar ( 2016)
    Chronic wounds are a major problem in the health care system. The increasing incidence of obesity and diabetes and longer life expectancy are adding to the burden. Treatment options are currently limited indicating needs for new treatment modalities. Impaired innate healing response is the main characteristic of chronic wounds. In recent years, there has been a focus on stem cell therapy and tissue engineering as an alternative treatment for chronic wounds to replace the damaged tissue with cells that possess a normal injury response. The hair follicle’s continuous cycling and their response to trauma stimulation, indicate a strong regenerative potential. It has been shown that hair follicles participate in wound re-epithelialization and respond to skin injury by promoting skin innervation and vascularization. This dissertation explores the potential of hair follicle transplantation to enhance wound healing. It was hypothesized that hair follicle transplantation can promote healing in skin wounds. Whole hair follicle were isolated and re-established in the wound site where they preserved their structure and function. This approach exploits the normal endogenous function of hair follicle cells to enhance skin wound healing. First, a vibrissae follicle transplantation technique was established and optimised in nude mice. Using donor follicles from GFP transgenic mice made it possible to investigate cell trafficking from transplanted follicles during epidermal and dermal repair. Transplanted vibrissae follicles integrated normally into the host epithelium and ninety percent of grafted vibrissae produced a hair shaft. The GFP labelled cells were confined to the transplanted hair follicles and were not found in the interfollicular epidermis. It was then possible to identify migrating hair follicle cells from recipient inter-follicular epidermis in lineage tracing wound experiments. Healing responses were then compared in excisional wounds with and without adjacent transplanted follicles. My findings re-confirmed that hair follicle cells participated in wound re-epithelialisation. Neighbouring transplanted follicles accelerated wound closure. Wounds adjacent to transplanted follicles also showed four times higher nerve density in the granulation tissue than the wounds without adjacent follicular transplants. There was no significant difference in capillary density and macrophage density in wounds with and without hair transplants. I also conducted a preliminary investigation of incisional wounds perpendicular or parallel to transplanted follicles, to compare hair follicle cell migration from damaged and intact hair follicles. In these experiments, intact hair follicles participated only in the epidermal component of wound healing and cells migrated from transplanted hair follicles to the healing dermis only when the hair follicles were cross-sectioned. The results of this dissertation indicated that re-epithelialization and promotion of innervation were possible mechanisms by which transplanted follicles contributed to wound healing. These findings lay the foundations for using follicular transplantation to promote healing in chronic or neuropathic wounds and peripheral nerve injuries. In addition, damage to hair follicles may be a starting signal for migration and participation of hair follicle cells in the repair of dermis, which may subsequently influence the healing outcome.
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    Investigating the natural history of Barrett’s oesophagus and its progression to oesophageal adenocarcinoma in the setting of improved assessment techniques and endoscopic therapy
    Cameron, Georgina Rachel ( 2016)
    Barrett’s Oesophagus (BE) is a condition whereby the columnar cells that normally line the oesophagus are replaced by an intestinal metaplastic cell type. Over time, these new cells can become dysplastic and ultimately can develop into oesophageal adenocarcinoma (EAC). Dysplasia is generally graded by pathologists as either low or high grade dysplasia, though previously the terms mild, moderate and severe dysplasia, as part of a three tier system, were used. The risk of malignant progression increases with worsening dysplasia. At present, we know that for high grade dysplasia (HGD) the risk of progression is very high and these patients must be treated to prevent the development of cancer. What is not clear from the current literature is how we should be managing low grade dysplasia (LGD) in BE, as malignant progression rates are variably reported and it is unclear whether we should be treating these patients aggressively or whether, in fact, surveillance only is appropriate. Additionally, over the last five to ten years there have been significant advancements in the therapies used to treat and eradicate dysplastic BE endoscopically, most notably with the use of endoscopic resection of early neoplastic lesions and radio frequency ablation of dysplastic BE. While these techniques have been shown to be safe and effective in trial populations, there are limited data on effectiveness in real-world cohorts. Furthermore, data have been lacking regarding the durability of outcomes following treatment. This thesis involved a range of clinical studies that aimed to address the above issues. I studied a cohort of patients who were diagnosed over ten years ago with LGD within BE, and assessed the percentage of those that went on to develop high grade and cancerous lesions without intervention/treatment. The original histology slides from this group were reviewed by two expert gastroenterology pathologists to 1) confirm the original diagnosis of low grade dysplasia and, 2) also grade the dysplasia according to the three tier system. I have shown that while a consensus diagnosis of LGD does appear to confer an increased risk of malignant potential, it is primarily a diagnosis of moderate LGD that accounts for this risk increase. This finding suggests that in addition to BE patients with LGD undergoing expert histological review, consideration should be given to further risk stratifying this group as having mild or moderate dysplasia, as this may influence a clinician’s decision to heighten surveillance or commence endoscopic therapy. In the second study I evaluated whether a Specialised Barrett’s Unit was better able to detect and stage dysplastic Barrett’s Oesophagus when compared with community endoscopists. I found that assessment of these patients at a Barrett’s unit resulted in improved detection of mucosal abnormalities and cancers, that were often missed by community endoscopists. Endoscopic mucosal resection (EMR) of early cancers was a critical step in determining a patient’s appropriateness for endoscopic therapy. These results highlight the importance of adequate assessment by endoscopists with the appropriate training in Barrett’s assessment and technical ability to perform EMR of suspected cancers, prior to embarking on a definitive management course. In a third clinical study, I assessed the safety, effectiveness and durability of combined endoscopic therapy (radiofrequency ablation and endoscopic mucosal resection) in one of Australia’s largest cohorts of patients with dysplastic Barrett’s oesophagus. This group had predominantly advanced histopathology and often complex Barrett’s segments. I found that in this real world cohort, the effectiveness and safety profile are similar to published data but that there was significant risk of recurrence of both Barrett’s and dysplasia. I demonstrated that recurrence appeared to occur more commonly in patients with advanced pre-treatment histology, and frequently at the gastroesophageal junction. This finding has important clinical implications and we advocate careful ongoing surveillance of this region, particularly in patients with prior advanced dysplasia or cancer.
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    Investigating the effects of downstream parathyroid hormone (PTH) targets on bone strength and quality
    Vrahnas, Christina ( 2016)
    The only currently approved anabolic therapy that increases bone mass and reduces fracture risk in patients with osteoporosis is parathyroid hormone (PTH). To investigate how PTH increases bone mass and improves bone strength, I have investigated the function of two gene products regulated by PTH in osteoblasts on bone structure and strength (gp130 and ephrinB2). Gp130 deletion in late osteoblasts/osteocytes resulted in greater femoral dimensions indicating a role for osteocytic gp130 in maintaining bone width. Although the bending load required to fracture these bones was not altered, they showed a significant reduction in material strength which was associated with a greater proportion of disorganised woven bone suggesting that the increased bone width may have been a compensatory mechanism for poor bone quality. This indicated that gp130 in late osteoblasts/osteocytes maintains the material strength of the cortical bone matrix collagen production and the deposition of organised lamellar bone. The role of another PTH downstream target, ephrinB2, in controlling bone strength and quality was investigated in both osteoblasts and osteocytes. Work conducted prior to my PhD showed that specific deletion of ephrinB2 within the entire osteoblast lineage in 12-week old female mice caused osteoblast apoptosis and delayed initiation of bone mineralisation, indicating a role for ephrinB2 in osteoblasts that promotes mineralisation by preventing apoptosis. During my PhD, I found that mice lacking ephrinB2 in the osteoblast lineage had slender bones which were more compliant when assessed by mechanical testing. Despite their greater bending ability, their bones were more fragile but they exhibited no change in bone composition measured by synchrotron-based Fourier transform infrared microscopy (sFTIRM). This suggested that the previously observed delay in the initiation of mineralisation caused by ephrinB2-deficient osteoblasts may have resulted in the fragile bone phenotype. Since ephrinB2 is expressed throughout the entire osteoblast differentiation pathway, I next sought to determine the role of ephrinB2 in late osteoblasts/osteocytes in 12-week old female mice. Mice lacking ephrinB2 in osteocytes showed an opposing strength phenotype compared to the earlier deletion of ephrinB2 mentioned above. Their bones were more brittle due to greater mineral deposition and parallel stretching/compression of the collagen fibres detected by sFTIRM, highlighting stage-specific roles of ephrinB2 during osteoblast differentiation that regulate bone size and material composition. Correlation analysis revealed that the relationships between collagen fibre alignment and bending strength and between mineral composition and material toughness that normally exist in control mice were lost in bones from mice lacking ephrinB2 in late osteoblasts/osteocytes. This demonstrated that ephrinB2 within late osteoblasts/osteocytes restrains mineral deposition to maintain bone strength. I next investigated the trabecular bone structure in these mice and found that deletion of ephrinB2 in late osteoblasts/osteocytes resulted in greater trabecular bone containing enlarged osteoclast size observed in vivo suggesting impaired osteoclast function. This may also relate to greater carbonate content within the bone mineral which requires further investigation. Finally, I assessed whether osteocytic ephrinB2 is required for the anabolic action of PTH. In contrast to impaired anabolic effect of PTH in mice lacking ephrinB2 within the entire osteoblast lineage, PTH treatment in mice lacking ephrinB2 in late osteoblasts/osteocytes did not significantly impair the anabolic action of PTH on trabecular and cortical bone formation. This led me to use the sFTIRM technique to analyse bone composition in vehicle and PTH-treated bones from control mice. This showed that bone deposited during PTH treatment undergoes a normal process of collagen maturation and mineral accrual. In conclusion, downstream targets of PTH, gp130 and ephrinB2 play distinct roles in regulating bone size, strength and quality. This highlights that osteoblasts utilise these proteins during the formation of bone matrix and the initiation of its mineralisation. In contrast, osteocytes maintain the quality and composition of bone matrix and mineral by regulating mineral accrual and collagen arrangement. Understanding how specific genes expressed in these cells can control bone strength, coupled with the sFTIRM technique developed during my PhD, can provide a new way for investigating different causes of bone fragility. Current diagnostic tests cannot fully predict risk of fracture and treatment options are not patient specific and are limited in their use. In the long term, this study may allow specific targeting of cell types to increase bone mass and improve bone quality which could lead to the development of different personalised treatments for bone fragility.
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    Mass spectrometry based examination of ADMA/DDAH modification in diabetes and cardiac disease
    Garlick, John ( 2016)
    Nitric Oxide (NO) is an important mediator in the prevention of monocyte recruitment and endothelial dysfunction, which drive the development of atherosclerosis. Asymmetric Dimethylarginine (ADMA) but not its isomer Symmetric Dimethylarignine (SDMA) has been identified as a circulating inhibitor of Nitric Oxide Synthase (NOS), with submiromolar increases of ADMA previously been demonstrated associated with an increase in cardiovascular disease risk. Currently ADMA has been measured with low reproducibility in most analytical techniques other than the gold standard HPLC-MS/MS. This work shows the separation that can be achieved with the emerging technique of Ion Mobility Spectrometry coupled with traditional mass spectrometry. Also using a more traditional HPLC based separation we were able to develop a mass spectrometry based measurement of metabolites ADMA, SDMA, Arginine, Ornithine and Citrulline critical in the NOS/ADMA pathway and the Urea cycle. This assay was used to measure changes across cardiac surgery within the circulation as well as generation from the myocardium. We demonstrated our technique had high reproducibility (>7% CV for ADMA) and showed that ADMA but not SDMA significantly decreases after cardiac surgery. This was the first such study to perform measurements of ADMA and SDMA alongside Urea cycle metabolites and with clinical data also collected will provide the foundation to further work. Our assay was also used in a cell culture model whereby DDAH the enzyme responsible for the majority (80%) of ADMA metabolism has previously been shown to have increased expression and activity with high glucose treatment. Our results demonstrate that ADMA is highly regulated compared to SDMA and the change with high glucose treatment does not correspond to a decrease in intracellular ADMA concentration or from the treatment media. Also tested was the efficacy of the proton pump inhibitor Omeprazole which has been recently demonstrated to inhibit DDAH in endothelial cells. This work demonstrated that Omeprazole does not inhibit DDAH within our HepG2 cell culture model in low or high glucose concentrations.
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    Influence of electrical stimulation on cardiac differentiation of human induced pluripotent stem cells
    Hernández de Santiago, Héctor Damián ( 2016)
    The possibility of remodelling human cardiac tissue raises hopes that in the future we may be able to fully restore cardiac function after myocardial infarction. In addition, the ability of human induced pluripotent stem cells (iPSCs) to differentiate into bona fide cardiomyocytes provides a platform for cardiac disease modelling, drug discovery and pharmacological safety testing of new drugs. One of the major limitations for the use of cardiomyocytes derived from iPSCs is that they resemble foetal cardiomyocytes and are immature, both morphologically and functionally. Considering that the developing heart grows in an electric field, I hypothesised that electrical stimulation might affect cardiogenesis of human iPSCs. The overall aim of this PhD thesis is to investigate whether appropriate electrical stimulation can promote cardiac differentiation and maturation of cardiomyocytes derived from human iPSCs. A custom made electrical stimulator was developed to investigate the effect of brief electrical stimulation on cardiac differentiation of iPSCs. Embryoid bodies (EBs) generated from human iPSCs were electrically stimulated for a few minutes with an electric field of 65 or 200 mV/mm at 1 Hz frequency. Brief electrical stimulation for 5 minutes significantly increased the percentage of beating EBs when compared to the non- electrically stimulated contros after 14 days after re-plating them (11.7 ± 2.3% vs. 4.9 ± 1.4% in control, p<0.05). This cardiogenic effect was not observed in EBs electrically stimulated for longer (10-15 minutes) or shorter (1.5 minutes) durations, suggesting 5 minutes of electrical stimulation is not only sufficient to promote cardiac differentiation, but also optimal. In addition, electrically stimulated EBs displayed increased gene expression of cardiac transcription factors (TBX4 and NKX2.5) and cardiac contractile proteins (α-actinin, cardiac troponin-T, myosin heavy chain beta isoform and myosin light chain atrial isoform), compared to the non-electrically stimulated group. Furthermore, only beating EBs from the electrically stimulated group were responsive to the chronotopic agents, carbachol and isoprenaline. In a separate study, brief electrical stimulation did not further enhance the cardiogenic effect of growth factors and small molecules (a combination of trichostatin A, activin A and BMP4 treatment during EB formation). To test the effect of long-term electrical stimulation on cardiomyocytes derived from human iPSCs, immature cardiomyocytes at day 27 post-differentiation were subjected to continuous electrical stimulation at 200 mV/mm for 7 days. The long-term electrical stimulation significantly increased the percentage of cardiomyocytes with organized sarcomeres (39 ± 8% vs. 23 ± 11% in control, p<0.05). Electrical stimulation also promoted alignment of cardiomyocytes in parallel to the electric field (10 ± 1% vs. 6 ± 2% in control, p<0.05) and decreased the circularity index (0.69 ± 0.02 vs. 0.74 ± 0.02 in control, p<0.05). The latter suggested that electrically stimulated cardiomyocytes develop a more rod-like morphology. In addition, a novel rat tissue engineering chamber with an integrated electrical stimulator and electrodes has been developed to allow in vivo electrical stimulation of a vascularised cardiac tissue constructed with human cardiomyocytes derived from iPSCs, but technical issues prevented advancing this approach very far. In conclusion, in this thesis I showed that brief electrical stimulation modestly enhances cardiac differentiation of human iPSCs and longer term continuous electrical stimulation promotes maturation of cardiomyocytes derived from human iPSCs. Mature cardiomyocytes recapitulate better the pathophysiological function of the human heart for more accurate disease modelling and drug testing, as well as providing a superior substrate for cardiac regeneration and repair by tissue engineering in the future.
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    Osteoblastic EphrinB2 in normal and parathyroid hormone (PTH)-stimulated bone formation.
    Takyar, Miralireza ( 2016)
    Bone remodelling renews the skeleton through cycles of bone resorption by osteoclasts and formation by osteoblasts. Two interacting members of the Eph tyrosine kinase family, EphrinB2 and EphB4, are expressed by osteoblasts and their expression of EphrinB2 is stimulated by parathyroid hormone (PTH), the only approved anabolic therapy for osteoporosis. I investigated the roles of the ephrinB2/EphB4 interaction in osteoblast lineage cells in bone remodelling and mediating anabolic actions of PTH. I began by assessing the effects of EphB4 overexpression in mouse osteoblasts in vivo and how this affected anabolic PTH. While PTH induced increases in osteoblast and osteoid parameters in both wild-type and EphB4-overexpressing mice, these changes were not significantly different between the two groups. However, we observed some evidence of a suppressed osteoclast response to PTH, and a slight augmentation of the anabolic action of PTH on bone volume in female mice only. Lack of consistent overexpression of EphB4 in these mice limited our ability to gain further conclusions. Next, I investigated the effects of pharmacological blockade of ephrinB2/EphB4 interaction in vivo in the presence and absence of anabolic PTH. A previously described pharmacologic inhibitor of this interaction, sEphB4, was used alone or in combination with anabolic PTH. In both settings, sEphB4 treatment increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1a1, and PTH receptor [PTHR1]) without causing significantly higher bone formation rate or late markers of osteoblast/osteocyte differentiation. In the presence of PTH, sEphB4 treatment significantly increased osteoclast formation and converted the anabolic effect of PTH to a catabolic effect. This effect on osteoclasts was recapitulated in vitro in co-cultures, suggesting that it is a cell-autonomous effect. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis. To assess the role of ephrinB2 signalling in the osteoblast lineage in normal bone formation, I analyzed bone remodelling in mice that lacked osteoblastic ephrinB2. For this purpose, an osteoblast-specific genetic deletion mouse model was used. These mice showed a delay in bone mineralisation along with lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis. Additionally, osteoblastic support of osteoclast formation was defective in these mice and in osteoblasts cultured from these mice. Finally, I assessed how lack of osteoblastic ephrinB2 signalling affected anabolic PTH effects by treating the above-mentioned mice with anabolic PTH. This experiment showed that osteoblastic ephrinB2 is required, not only for physiological bone remodelling, but also for effects of anabolic PTH in causing higher osteoblast number, surface and osteoid production. This requirement appears to be due to the control ephrinB2 may have over osteoid deposition and mineralisation in trabecular and cortical bone. I conclude that ephrinB2, through its binding partner EphB4, promotes the late stages of osteoblast maturation that are required for mineralisation, and supports osteoclast formation. These functions are essential for the full anabolic action of PTH, such that blockade of signalling events downstream of the ephrinB2:EphB4 interaction turns anabolic action of PTH into a catabolic effect. Given the current interest in targeting the ephrinB2/EphB4 interaction in antiangiogenic and anticancer therapies, my findings show that targeting EphB4 may have less skeletal side effects than blocking both sides of this interaction. Enhancing ephrinB2 signalling may be a viable strategy for conditions where more bone formation is desired such as osteoporosis, osteogenesis imperfecta, and fracture healing.