Medicine (St Vincent's) - Theses
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ItemRegulation of bone and fat cells by zinc-finger protein- 467(University of Melbourne, 2010)
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ItemCalcitonin inhibition of parathyroid hormone anabolic action(University of Melbourne, 2009)
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ItemFunctional interactions between c-MYC and mTORC1 in preclinical models of haemopoietic malignancy(University of Melbourne, 2008)
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ItemThe doctor as moral agent, with reference to the distinction between killing and "letting die"( 2007-07)In the bioethics literature, arguments about the nature of the distinction between killing and “letting die” seem irresolvable. There is a disparity between the dominant (consequentialist) opinion on this issue and that of the medical profession. No previous studies have investigated how doctors who work with the dying understand the distinction in the medical context. The aim of my research was to explore the moral reasoning of these clinicians in relation to this question. A focused ethnographic study involved thirty Melbourne doctors (thirteen palliative care physicians, nine oncologists, six intensivists, and two advocates of physician-assisted suicide) of whom eighteen were male and twelve female, with an age range from 31 to 77 years. Half had a religious belief (Jewish or Christian) and half were atheist/agnostic. (For complete abstract open document)
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ItemFactors influencing cryopreserved allograft heart valve degeneration( 2006)Heart valve replacement is becoming more commonplace in developed nations. Despite this the ideal valve prosthesis has not been found. The allograft valve has been used for over 40 years and remains an important prosthesis with many advantages. However, like other biological valve prosthesis, they have a finite durability. The causes of allograft valve degeneration are still unknown. The study aims to identify factors associated with cryopreserved allograft valve degeneration. Knowledge of such factors will improve our understanding of the potential causes and mechanisms of allograft heart valve degeneration. (For complete abstract open document)
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ItemSmall animal models of Gal-mediated and xenograft rejection( 2004-11)Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.
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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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