Medicine (St Vincent's) - Theses
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ItemStructural and functional characterisation of LIM kinases( 2012)LIMK1 and LIMK2 constitute a family of serine/threonine protein kinases that serve as important regulators of actin dynamics. They phosphorylate and inactivate the actin depolymerising factor, cofilin, thereby leading to the accumulation of actin filaments. Both LIMKs have been implicated in pathological conditions such as tumour cell invasion and ocular hypertension, where the regulation of actin polymerisation is affected. Several small-molecule LIMK inhibitors have been identified, but the lack of a three-dimensional structure of the kinase domain has hampered structure-based development of these compounds. This thesis describes the expression and purification of the LIMK1 kinase domain for X-ray crystallographic studies to enable rational structure-based optimisation of LIMK inhibitors. The GST-tagged LIMK1 kinase domain was expressed in baculovirus-infected insect cells and purified using ammonium sulphate precipitation prior to glutathione affinity and size-exclusion chromatography. Limited proteolysis of the kinase domain revealed improved domain boundaries, yielding a catalytically active fragment consisting of residues 332-607. The low solubility of the kinase domain in the absence of the GST tag was enhanced by addition of the solubilising agent, NV10. However, crystallisation trials yielded only amorphous precipitates, but no protein crystals. Despite the development of improved algorithms to predict important protein features such as domain boundaries and regions of high entropy, the purification of soluble protein and the crystallisation thereof remain the major bottleneck in structure determination. The recent determination of the crystal structure of the LIMK1 kinase domain in complex with staurosporine has paved the way for co-crystallisation studies using LIMK-specific inhibitors to determine the inhibitor:enzyme interactions and improve the compounds’ potency and substrate specificity. The second part of this thesis investigates the molecular mechanisms leading to the obesity phenotype of LIMK2a knockout mice. Phenotypic characterisation revealed a significant increase in the body mass and fat mass in male LIMK2a knockout mice compared to male wildtype mice. The knockout mice were hyperlipidaemic and showed signs of insulin resistance. Their adipocytes were hypertrophic and showed impaired glucose uptake and lipolysis. The inverse correlation between LIMK2a expression and body mass was also observed in the adipose tissue of obese ob/ob and lean wildtype mice. It was hypothesised that altered actin organisation as a result of lack of LIMK2a may be responsible for the enlargement of adipocytes and possible changes in the adipogenic potential of progenitor cells. In cultured 3T3-L1 pre-adipocytes, the level of LIMK2a mRNA and total LIMK2 protein was transiently up-regulated following stimulation with an adipogenic differentiation cocktail. However, adipogenic differentiation of LIMK2- and LIMK2a-deficient MEFs showed no difference in lipid accumulation or cellular morphology when compared to wildtype MEFs. These results indicate that LIMK2 or LIMK2a are not involved in the adipogenic cell fate decision in MEF cultures. However, it remains to be determined whether the lack of LIMK2a affects the adipogenic potential of mesenchymal stem cells or the morphology of fully differentiated adipocytes derived from these progenitors.
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ItemInvestigating novel therapies for osteosarcoma using advanced medical imaging( 2012)Osteosarcoma is the most common primary cancer of bone. Current best treatment consists of chemotherapy and surgery, however many tumours are chemoresistant. Spread of osteosarcoma to the lungs is frequent and is the most common cause of death. This thesis examines the role of pigment epithelium-derived factor (PEDF) in the processes of osteosarcoma growth, invasion and metastasis. In order to evaluate this potential role, both in vitro and in vivo studies have been performed. In vitro studies have examined the biological effects and mechanisms of PEDF. PEDF exhibits a multifaceted ability to inhibit osteosarcoma tumorigenicity. PEDF inhibits proliferation, induces apoptosis and reduces cell cycling of osteosarcoma cells in vitro. Additionally, the metastatic capacity of osteosarcoma cell lines is diminished by PEDF. Osteosarcoma cells treated with PEDF demonstrate an enhanced capacity for adhesion and a reduced ability for invasion through collagen I, the most abundant protein in bone. An established murine model of orthotopic osteosarcoma has been optimised for the evaluation of novel therapeutic agents in vivo. This model allowed for an evaluation of systemically delivered PEDF and PEDF-derived peptides, both as sole treatments and in combination with doxorubicin. Systemic administration of PEDF causes a reduction in both primary tumour volume and pulmonary metastatic disease. PEDF-derived peptides, StVOrth-2 (residues 78-102) and StVOrth-3 (residues 90-114) were also delivered systemically. StVOrth-2 primarily restricts growth of primary osteosarcoma while StVOrth-3 restricts pulmonary metastatic disease. Finally, advanced medical imaging techniques have been applied to this murine model of orthotopic osteosarcoma for the study of tumour growth, invasion and metastasis; in vivo bioluminescent imaging, [18F]-Fluoride-PET, [18F]-FDG-PET and micro-computed tomography provide novel information about this model. Use of these imaging modalities has improved osteosarcoma modelling and allowed closer monitoring of disease progression. This is the first time that in vivo imaging has been used in the assessment of PEDF’s anti-osteosarcoma properties and is a major advance from previously published studies demonstrating direct effects on osteosarcoma growth and metastasis.
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ItemPost-operative Crohn's disease recurrence: clinical and microbiological studies( 2012)Crohn’s disease is a chronic inflammatory bowel disease that causes major morbidity. The cause is unknown but thought to relate to an exaggerated immune response to gut microbiota in genetically susceptible individuals. There is currently no known cure. The majority of patients with Crohn’s disease undergo surgery during their life, and 70 percent of these require a second operation due to disease recurrence. Surgery involves removing the diseased segment and joining the unaffected bowel. Even if all macroscopically involved bowel is removed disease usually recurs at, and proximal to, the anastomosis. Recurrent Crohn’s disease can be identified endoscopically before symptoms develop. However, it is unknown whether intervention based on endoscopic findings of recurrence influences the disease course. Immunosuppressive and antitumour necrosis factor therapy have emerged as effective treatments in the treatment of Crohn’s disease but their optimal usage for the prevention of post-operative recurrence of Crohn’s disease remains to be determined. This thesis encompasses the design and conduct of a clinical study which aimed to determine whether prospective endoscopic monitoring of post-operative patients, with treatment intensification for mucosal recurrence, is superior to the current standard of care based on treatment of symptoms. This thesis also includes a laboratory study, allied to the clinical study, in which the microbiota has been characterised and followed over time, from a starting point of absent mucosal disease at a site of known recurrence. This latter study aimed to establish whether specific changes in gut mucosal microbiota at the anastomosis are associated with disease recurrence. We initially demonstrated in a retrospective cohort study that the rates of endoscopic, clinical, and surgical recurrence are high after surgery and that that approaches to the management of postoperative recurrence have been variable in the past. Although there was no clinical benefit from colonoscopy or step-up therapy in this cohort we concluded that that this lack of benefit related to inconsistent timing of post-operative colonoscopy and a lack of standardised drug intervention in response to the endoscopic findings. We then devised a prospective randomized controlled trial to compare endoscopic monitoring of post-operative patients with treatment step-up for endoscopic recurrence, with standard care. In an analysis of the six month endoscopic outcome of patients with a high risk of recurrence we found that adalimumab was significantly superior to thiopurines in preventing endoscopic recurrence. In the laboratory we simultaneously characterised the mucosa associated microbiota in a sub-group of patients enrolled in the clinical study at the time of surgical resection and at the anastomosis six months post-operatively. We found that patients who developed recurrence or remained in remission could be differentiated on the basis of specific microbiota profiles, both at the time of surgery and six months later. In summary, postoperative recurrence remains an important issue despite the availability of immunosuppressive and anti-TNF drug therapy, when managing Crohn’s disease. This thesis comprises clinical studies focussing on the prevention of disease recurrence, and laboratory studies aimed at determining which bacteria may be implicated in recurrent disease.
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ItemBalancing the knee: factors affecting outcomes after primary total knee replacement( 2012)Arthritis currently affects 15% of the Australian population and is the leading cause of pain and disability in the elderly. Knee joint arthroplasty is a successful way of providing patients with relief from debilitating end-stage arthritic knee pain. Unfortunately 5% of knee replacements will require revision surgery, which is technically and financially demanding with often inferior functional outcomes. Half of all knee revisions can be attributed to a preventable cause such as knee balancing. A balanced knee is defined as having a full range of motion, rectangular, even joint space, symmetrical soft-tissue tension and correct leg alignment. This thesis investigated the role of knee balance in primary knee arthroplasty. It helped establish and improve methods of measuring balance, identified ways to improve balance and investigated the effects of knee balancing on the patient’s functional outcome and quality of life. Chapter 2: The Accuracy of Measured Range of Motion of the Arthritic Knee - Chapter two assessed the validity of retrospective data and helped identify the most appropriate research strategy for the remainder of the thesis. The range of motion of forty-eight patients was assessed using a goniometer and compared to that entered in the patient notes by the examiner during a routine clinical examination, without the examiner being aware. The range of motion of a further twenty patients was subsequently assessed and compared to the findings of the same clinical examiners but this time with the examiner being aware. Accuracy of measurements improved significantly when the examiner was aware. Routinely measured range of motion was found to be inaccurate, implying inaccuracy of retrospective data. Chapter 3: Assessing Alignment - Chapter three compared alignment as measured by long-leg radiographs (LLRs) and computer tomography (CT) to intra-operative navigation measurements in 40 patients undergoing a primary total knee replacement. This study attempted to identify the most accurate way of measuring alignment. Inter-modality correlation proved to be higher when comparing LLRs and CT (>0.893), than when comparing either of these modalities with computer navigation (>0.643 and >0.671 respectively). Further investigation using reformatted computer tomography supported these findings. Given its availability and reduced radiation dose when compared to CT, LLRs should remain the mainstay of measuring the mechanical alignment of the lower limb. Chapter 4: The Joint-Line in Primary Knee Arthroplasty - Chapter four assessed the role of computer-assisted surgery in maintaining the level of the joint-line using a randomized controlled trial comparing computer-assisted to conventional primary total knee arthroplasty. No significant difference was found between computer-assisted and conventional surgery in terms of maintaining the joint line. Patients with depressed joint-lines post-operatively improved the least in terms of functional scores. Change in joint line was found to be related to change in alignment. Change in alignment was found to significantly effect change in joint line and functional scores. Chapter 5: Impact of Joint Distraction on Functional Outcome in Primary Total Knee Arthroplasty - Chapter five assessed over-distraction of the tibio-femoral and analyzed its effects on function and quality of life. Measures of knee joint distraction were devised using long-leg alignment radiographs and lateral radiographs. Seventy-three patients were prospectively recruited and their joint-distraction measured post-operatively. Twelve months follow-up was achieved. Those knees that were over-distracted post-operatively suffered significantly more pain, less flexion and a lower International Knee Society score compared to their counterparts. Chapter 6: A Comparison of Different Surgical Techniques of Primary Knee Arthroplasty - A total knee replacement can be completed using two standard techniques; measured resection and balanced resection. Direct comparisons between these techniques are few. In chapter six, a randomized controlled trial was completed with 103 patients randomized to either one of these techniques. Primary outcomes measured were coronal alignment, femoral component rotation and joint-line maintenance. Secondary outcomes were function and quality-of-life outcomes. A significant difference was found in coronal alignment, with the measured resection technique resulting in an average coronal alignment 1 degree varus to the balanced resection group. No significant difference was found in femoral component rotation or joint-line maintenance. Functional outcomes and quality-of-life were not significantly different between groups at 1 year. Future direction - This thesis hoped to investigate current concepts of knee balancing and introduce new concepts for future researchers. Future research should concentrate on techniques for maintaining the joint line and preventing joint over-distraction. These can involve more specific research into why the joint becomes distracted and hence find ways to prevent it. Research should concentrate on being less observational and more interventional, with more emphasis on tools such as computer navigation, and the newly introduced patient-specific guides. Future research could concentrate on how patient specific guides could be used to improve joint-line maintenance and prevent over-distraction. Larger cohorts would help identify even more minute effectors of knee function post-arthroplasty and cement the novel effectors introduced in this thesis. Longer follow-up of the current cohorts will allow determination of the long-term effects of attributes discussed in this thesis on the longevity of the prosthesis and achieving the ultimate goal of preventing the need for revision surgery.
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ItemTrophic role of adipose-derived adult stem cells to support tissue engineering( 2012)Tissue engineering has held much promise for patients suffering from irreversibly damaged organs/tissues who are in desperate need for organ transplantation. Despite technological advances in the biochemical engineering of scaffolds to permit efficient vessel growth during tissue development, vascularisation still represents a major limiting factor in the generation of tissues large enough for clinical applications. Human mesenchymal stem cells (MSC) were originally proposed for stem cell therapies in regenerative medicine due to their propensity to differentiate into specific cell types. However, MSC were found to be more supportive of engineering functional tissue constructs through secretion of a spectrum of growth factors and cytokines, termed paracrine factors, which are angiogenic and cytoprotective. In this thesis, it is aimed to compare the paracrine profile of various MSC populations and determine changes in the expression profile when the cells are induced to differentiate down a specific lineage. In addition, an efficient regulatory method that would enhance the paracrine activity of ASC was investigated and translated into an in vivo animal model. In determining the optimal MSC population for promoting angiogenesis through paracrine activity, human MSC isolated form bone marrow (BMSC), adipose tissue (ASC) and dermal sheath (DSC) or papilla (DPC) of hair follicles were compared. While expression of selected paracrine factors, including SDF-1, VEGF-A, VEGF-C, bFGF, HGF, NGF and ANG, exhibited minor differences within MSC populations examined, ASC expressed the highest levels of IGF-1, VEGF-D and IL-8. Furthermore, ASC-derived conditioned medium (ASCCM) induced the strongest response in proliferation, migration and tube formation of human microvascular endothelial cells (HMEC) in vitro. ASC were therefore suggested as a suitable MSC population for angiogenesis-related applications, as not only can a large number of ASC be generated with minimally invasive isolation procedures, ASCCM also demonstrate an advanced capacity to support angiogenesis in vitro. It is further demonstrated in this thesis, that the unique paracrine factor profile of ASC is altered when the cells commit to specific cell lineages, such as adipogenic and osteogenic. Progress through differentiation significantly diminished the expression of potent angiogenic factors, including VEGF-A and bFGF, so it was hypothesised that this may impact negatively on their angiogenic paracrine activity. Therefore, the paracrine activity of ASC is suggested to be a unique characteristic present only when the cells are multipotent. In addition, it is likely that the beneficial angiogenic activity of ASC paracrine factors in regenerative therapies is associated with their “stemness”, and that maintenance of ASC stemness during tissue formation may benefit the outcome through enhanced angiogenesis in vivo. Lastly, hypoxia was examined as an efficient method to enhance the paracrine factor production of ASC, where both VEGF-A and ANG were significantly increased when cells were subjected to conditioning by hypoxia. The angiogenicity of ASCCM was confirmed by implanting polyvinyl alcohol sponge subcutaneously in mice, where the ability of concentrated ASCCM to promote vascularisation in animal implanted sponges was determined by immunohistochemical staining of the endothelial cell specific marker CD31. Moreover, the ASCCM collected from hypoxia-conditioned cells exhibited enhanced vessel infiltration in sponges, which was diminished by neutralising antibodies against VEGF-A and ANG. The model demonstrated that the increased production of VEGF-A and ANG in hypoxia-conditioned ASC in vitro translated to an in vivo effect with biological significance. Collectively, these results illustrated the potential for utilisation of ASCCM alone for angiogenesis-related applications in vivo. In summary, the data presented in this thesis presents ASC as a useful MSC population for enhancing angiogenesis because of their optimal paracrine factor profile. While the paracrine factor expression is unique to multipotent ASC, the angiogenicity of its paracrine activity can be further enhanced through in vitro hypoxia conditioning. The increased production of VEGF-A and ANG contributed to the observed enhancement of blood vessel infiltration in the in vivo sponge implant and provided evidence of the paracrine activity of ASC. The results demonstrated in this thesis therefore suggest the potential of ASCCM as a suitable agent for induction of angiogenesis, which may be incorporated within scaffold materials to increase vascularisation efficiency of the tissue engineering construct.
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ItemCharacterisation of susceptibility to Listeria monocytogenes infection in the non-obese diabetic (NOD) mouse( 2012)The immune system has evolved the ability to prevent infection by a wide range of pathogens while maintaining tolerance to self-tissues. Due to the strong selective pressure imposed by microbial pathogens, susceptibility to infection can be modulated by a large number of genetic loci. It is postulated that, at least in some cases, allelic variants for particular loci confer increased resistance to pathogens while simultaneously increasing the risk of developing autoimmune diseases. A number of recently discovered disease loci appear to act as “genetic pivot points” between pathogen defence and autoimmune pathogenesis. It is anticipated that characterising these loci will provide novel insights regarding the interplay between immune and autoimmune responses, as well as reveal potential therapeutic targets for treating both infectious and autoimmune diseases. Murine models provide a complementary approach to human studies for investigating genetic and cellular mechanisms that underlie susceptibility to infectious and autoimmune diseases. The non-obese diabetic (NOD) mouse strain is one of the best-characterised models of type 1 diabetes (T1D), an autoimmune disease caused by the destruction of insulin-producing pancreatic β cells. Similar to humans, predisposition to T1D is attributed to multiple genetic loci in NOD mice. Intriguingly, non-diabetogenic mouse strains can also harbour diabetogenic alleles for some T1D susceptibility loci. Using congenic mice, our laboratory confirmed that non-diabetogenic C57BL/6 (B6) mice harbour a diabetogenic allele for a T1D susceptibility locus on chromosome (Chr) 13, termed Idd14. Coincidentally, the Idd14 locus overlapped with Listr2, a proposed susceptibility locus for Listeria monocytogenes, which is an extensively studied intracellular bacterial pathogen. Notably, B6 mice are resistant to L. monocytogenes infection, whereas NOD mice are reported as susceptible. We therefore hypothesised that allelic variation for this interval, which increases T1D risk, would confer resistance against L. monocytogenes in NOD mice. The goal of this study was to investigate the biological and genetic effects of allelic variation for Idd14/Listr2 in NOD mice for L. monocytogenes infection and T1D. Towards this goal, the first aim was to investigate the immunological basis of susceptibility to L. monocytogenes in NOD mice. In comparison to infected B6 mice, infected NOD mice exhibited reduced antigen-specific CD8+ T-cell responses, which was associated with apparent deficiencies in dendritic cells. Infected NOD mice also exhibited exacerbated neutrophilia, a potential compensatory mechanism in susceptible hosts. The second aim was to determine the role of neutrophils during L. monocytogenes infection. Specific depletion of neutrophils impaired bacterial clearance in susceptible NOD mice, but not in resistant B6 mice, demonstrating that neutrophils are essential for controlling L. monocytogenes infection in susceptible hosts. In addition, NOD macrophages exhibited impaired antimicrobial function. These results indicate that neutrophils may compensate for deficient T-cell and macrophage responses to ensure host survival during L. monocytogenes infection. The third aim was to confirm the Listr2 locus using congenic NOD mouse strains, which harbour different B6-derived Chr 13 interval, and determine if the refined intervals for Listr2 and Idd14 continue to co-localise. Concurrently, it was found that the Idd14 locus could be dissected into two sub-loci, Idd14.1 and Idd14.2. Listr2 was confirmed and co-localised with Idd14.1 to an overlapping interval (~ 18 Mb) on Chr 13. This co-localisation raises the intriguing possibility that allele variation for the same gene(s) within this interval affects both infection and autoimmune disease susceptibility.
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ItemPhospho-regulation of yeast Rad53 checkpoint functions( 2012)The budding yeast Mec1(ATR) and Rad53(Chk2) kinases play crucial roles in S phase checkpoint responses to exogenous genotoxic agents, but their functions in the replication of undamaged DNA are poorly understood. Here I show that Mec1-dependent phosphorylation of the N-terminal Rad53 SQ/TQ cluster domain 1 (SCD1), but not Rad53 kinase activity, is required for appropriate regulation of cellular deoxyribonucleotide triphosphate (dNTP) levels during normal S phase. In the SCD1 phospho-site mutant rad53-4AQ, lower dNTP pools lead to increased spontaneous replication fork arrest and constitutive checkpoint activation, all of which can be suppressed by higher dNTP levels via deletion of the ribonucleotide reductase (RNR) inhibitor Sml1. In rad53-K227A mutants, which lack Rad53 kinase activity, endogenous dNTP regulation and cell survival depend on the presence of at least two adjacent SCD1 phosphorylation sites that promote activation of the Dun1 kinase and subsequent Sml1 degradation, suggesting that a scaffold function of Rad53 for direct Mec1-dependent activation of Dun1 may be sufficient for replication of undamaged chromosomes. Thus, the essential RAD53 function during physiological DNA replication may encompass both the regulation of endogenous dNTP levels in a novel non-catalytic scaffolding mechanism and compensatory kinase-dependent recovery from replication stress if dNTP regulation fails. Mec1-dependent phosphorylation of the Rad53-SCD1 also plays a key role in Rad53 kinase activation in response to DNA damage. The data presented here demonstrate that threonine 8 within the SCD1 is the preferred priming phosphorylation site for Rad53 auto-activation, which, in contrast to the auto-activation of Chk2-like kinases in other organisms, does not seem to involve the N-terminal FHA1 domain. Altogether, this study advances the functional understanding of the essential functions and activation mechanisms of checkpoint kinases.
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ItemThe role of the coronary vasculature and myocardium in the pathogenesis of diabetic cardiomyopathy( 2012)The prevalence of diabetes is increasing worldwide. This poses a significant threat to human health, as diabetes is associated with an increased risk of mortality due to cardiovascular disease. In particular, diabetic patients develop diabetic cardiomyopathy (DCM), characterised by impaired cardiac muscle contraction and relaxation, leading to left ventricle (LV) muscle stiffness and congestive heart failure. Previous studies suggest that changes in the coronary vasculature and cardiac subcellular function may account for the progression to DCM, however as yet this has not been assessed in vivo. Synchrotron radiation (SR) now makes possible novel imaging and diffraction techniques, to investigate the role these mechanisms play in the early development of DCM, where clinical intervention is most efficacious. To assess coronary function in vivo we validated the use of SR imaging to detect and quantify regional differences in resistance microvessel calibre. In type 1 diabetic rodents we found that although endothelium-dependent and –independent vasodilatory responses in individual coronary vessels are preserved, following inhibition of NO and PGI2 production, there is evidence of localised focal and segmental constrictions. This demonstrates, for the first time, localised coronary microvascular endothelial dysfunction in early-stage type 1 diabets (T1D). Contributing to this diabetic coronary impairment is the RhoA/Rho-kinase (ROCK) pathway, which had previously been shown to play a role in endothelial dysfunction and coronary vasospasm. Our data further support a role for ROCK in early diabetic coronary dysfunction, as following nitric oxide synthase/cyclooxygenase blockade, ROCK inhibition greatly reduced regional segmental constrictions and completely alleviated persistent focal stenoses in diabetic animals. Together, these findings provide strong evidence that early vascular dysfunction may contribute to the development of DCM. In addition, although characterised by global cardiac impairment, the role subcellular changes in the sarcomere play in DCM progression is not known. SR, as a source for small-angle X-ray diffraction, allows the assessment of cardiomyocyte cross-bridge dynamics (CB) and myosin interfilament lattice spacing in situ and in real time. Using SR, our data shows that in early T1D, CB dynamics are abnormal in the beating hearts and this is directly related to impaired LV function. The change in CB dynamics is caused by myosin head displacement from actin filaments, but notably is not related to estimated sarcomere length or myofilament order. SR X-ray diffraction thus provides a robust method to assess cardiac CB dynamics in situ and for the first time we provide evidence that impairment in the regulation of myosin head extension in T1D hearts contributes to DCM. Currently 85-90% of diabetics have T2D and it is therefore critical that these coronary microvascular and cardiac subcellular impairments in T1D are explored in T2D. As such, rodent models which account for the environmental factors important in the human development of DCM are required. We conducted a comprehensive characterisation of cardiac function and structure in diet-induced rodent models of obesity, insulin resistance and T2D, and uncovered mild systolic dysfunction in fructose fed and mild diastolic dysfunction in high fat fed rodents. Furthermore, we demonstrated mild contractile dysfunction in high fat fed low dose streptozotocin rodents. The characterisation of only mild cardiac dysfunction, in spite of the lengthy time course used, suggests further refinement is required to achieve more robust DCM models. In summary, through the validation of novel SR imaging and diffraction techniques our data has confirmed a role for coronary microvascular dysfunction, via the ROCK pathway and cardiac subcellular impairment, via reduced myosin head extension, in the development of DCM. In addition, further studies investigating rodent models of T2D and DCM are required. These findings provide a strong basis for the future development of novel therapies aimed at preventing and/or reversing the decline in cardiac function associated with diabetes.
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ItemNovel synthetic flavonoids in acute and chronic heart and kidney diseases( 2012)Cardiovascular disease remains one of the leading causes of death worldwide despite the dramatic reduction in mortality rates over the past two decades due to significant advancement in medical and public health interventions. Diabetes exacerbates the complex interaction of cardiovascular risk factors, resulting in the greater incidence of heart failure among individuals with diabetes when compared to those without diabetes. The relative risk of cardiovascular mortality is further increased when individuals with diabetes are diagnosed with the comorbidity of established chronic kidney disease. Therefore, there is a continuous need for the development of novel therapeutic strategies to prevent the progression of cardiac and renal dysfunction in the presence or absence of diabetes, since the protective effects from the routine use of current pharmacotherapy for the management of elevated blood glucose, high blood pressure and abnormal blood lipid profiles remain limited and controversial. The focus of this thesis is to further elucidate the pathophysiological roles of oxidative stress and inflammation in the disease progression and to explore the therapeutic potential of novel synthetic flavonoids, DiOHF and NP202, in the prevention of acute and chronic heart and kidney diseases. Cardiovascular disease remains one of the leading causes of death worldwide despite the dramatic reduction in mortality rates over the past two decades due to significant advancement in medical and public health interventions. Diabetes exacerbates the complex interaction of cardiovascular risk factors, resulting in the greater incidence of heart failure among individuals with diabetes when compared to those without diabetes. The relative risk of cardiovascular mortality is further increased when individuals with diabetes are diagnosed with the comorbidity of established chronic kidney disease. Therefore, there is a continuous need for the development of novel therapeutic strategies to prevent the progression of cardiac and renal dysfunction in the presence or absence of diabetes, since the protective effects from the routine use of current pharmacotherapy for the management of elevated blood glucose, high blood pressure and abnormal blood lipid profiles remain limited and controversial. The focus of this thesis is to further elucidate the pathophysiological roles of oxidative stress and inflammation in the disease progression and to explore the therapeutic potential of novel synthetic flavonoids, DiOHF and NP202, in the prevention of acute and chronic heart and kidney diseases. The manifestation of acute myocardial infarction remains a major contributor to the subsequent development of heart failure despite the successful implementation of coronary reperfusion strategies. There has been substantial evidence showing that the dynamic progression of LV contractile dysfunction persisted up to several weeks following the restoration of coronary blood flow. The oral administration of NP202 during the reperfusion period in an experimental model of AMI resulted in the sustained improvement of LV contractile function in association with the reduction in the accumulation of inflammatory cells in the infarct zone of the heart even after seven days following the induction of AMI. This thesis has provided a strong basis for further therapeutic advancement of synthetic flavonoids as novel pharmacological agents to prevent the progression of heart and kidney diseases in the absence and presence of diabetes. There are clear indications that the modulation of oxidative stress and inflammation in the presence of synthetic flavonoids could be responsible for the prevention of the disease progression. The comprehensive understanding of the pharmacokinetics and oral bioavailability of the synthetic flavonoids is necessary for further preclinical evaluation of these promising therapeutic interventions. The enhancement of the biological actions of the synthetic flavonoids in the oral formulation would thus enable the translation of the basic research for potential clinical utility.
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ItemNatural killer cells in the graft versus host and graft versus tumour responses( 2012)Since their description as agents of natural cytotoxicity more than 30 years ago, natural killer (NK) cells have generated great interest due to their ability to kill tumour cells in vitro in the absence of prior encounter or stimulation and without the need for antigen presentation. Animal studies have shown that depletion of NK cells leads to enhanced tumour growth, and these observations led to efforts to harness the anti-tumour effect of adoptively transferred “lymphokine activated killer” (LAK) cells some 20 years ago. Although LAK cells ultimately failed to deliver on their preclinical promise, exciting results in the field of haploidentical bone marrow transplantation then led to a resurgence of interest in the application of NK cell alloreactivity for the treatment of malignancy. Unlike T cells, allogeneic NK cells do not cause graft-versus-host disease (GVHD), providing further reassurance that NK cells could be safely transferred from allogeneic MHC-mismatched donors into patients. Indeed, recent efforts to re-infuse ex-vivo expanded autologous or allogeneic NK cells into patients with malignancy have been shown to be feasible and safe. However, conclusive evidence of clinical efficacy is still lacking. The main aim of this dissertation was to explore the barriers to successful NK cell anti-tumour immunotherapy, by tracing the fate of NK cells adoptively transferred into mice. A systematic approach was employed to interrogate each of the requisite steps in successful cellular therapy: Trafficking to tumour, accumulation, target recognition through cognate receptors, engagement of effector functions, and persistence of effector cells until all target cells are eliminated. Using several NK-sensitive mouse tumour models, in Chapter 3 NK cells were shown to be unable to mediate regression of established tumours or improve the survival of tumour-bearing mice, despite robust homing and accumulation within the tumour. This surprising observation implied that NK cells had become dysfunctional after transfer, and in Chapter 4 the transferred NK cells were shown to have down-regulated expression of activating receptors and reduced cytokine production and cytotoxic capacities. A molecular mechanism for this observation was provided by highlighting a concomitant down-regulation of the canonical NK cell transcription factor Eomesodermin, with partial correction of the dysfunctional phenotype after overexpression of this transcription factor in transferred NK cells. The acquired dysfunction was dependent on proliferation, which could be induced by homeostatic expansion or by exposure to tumour. Due to similarities with the process of CD8+ T cell exhaustion, this novel observation was termed “NK cell exhaustion”. Subsequently, adoptively transferred NK cells were found to exhibit anti- T cell immunity functions, impairing the function of tumour-specific T cells (Chapter 5) and mediating a reduction in T cell-mediated GVHD (Chapter 6), and suggesting that under certain conditions the role of NK cells switches from a predominantly inflammatory to a mostly immunoregulatory function, arguably a form of functional plasticity. These findings have clear implications for the field of NK cellular immunotherapy, explore important concepts in immunology, and will hopefully contribute to a future systematic evaluation of the transcriptional programs involved in NK cell exhaustion thus leading to a better understanding of the requirements for successful NK cellular immunotherapy.