Medicine (Austin & Northern Health) - Theses
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ItemThe evolution of mammalian noncoding RNAs and their expression in development and immunity( 2007-07)The traditional view of the genome is based on the dogma that genetic information flows from DNA to RNA to protein. Genes have essentially been synonymous with proteins, with RNA viewed primarily as an intermediate template for protein translation. Intriguingly, only ~2% of the human genome encodes proteins, and the number of protein-coding genes (~20,000) is similar between humans and the simple nematode worm.I have been involved with the analysis of a large-scale transcriptome study, intiated by the RIKEN Genomic Sciences Centre in Japan. As part of this work, it was discovered that the genome carries instructions for tens of thousands of “non protein-coding RNAs” (ncRNAs)(please refer to Appendix A to view the original articles that appeared in Science). The significance of these ncRNAs remains a matter of intense interest and debate. Some have argued that these ncRNAs are simply transcriptional noise, while others have suggested that they comprise a critical regulatory system, which directs the complex patterns of gene expression that underlie differentiation and development.To investigate this further, I have conducted a series of studies that explore the expression and evolution of ncRNAs in mammals. Firstly, I established a comprehensive, on-line database of ncRNAs. This collection provides information on more than twenty thousand ncRNAs, and has proven a valuable resource for ncRNA studies. Secondly, I have systematically analyzed the conservation of known functional ncRNA subsets. I found that small ncRNAs (microRNAs and snoRNAs) were well-conserved similar to protein-coding sequences, whereas longer functional ncRNAs were not. These results indicate that long ncRNAs are evolving more rapidly than other functional genomic elements, and suggest that many of the recently-discovered ncRNAs – most of which are long and of unknown significance – might still be functional, despite having poor sequence conservation. Thirdly, I have shown that many ncRNA
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ItemThe effect of laser induced thermal ablation on liver tumours( 2005-11)Laser thermal ablation (LTA) is an in situ ablative technique that induces heat destruction of liver tumours. Despite increasing clinical use of LTA, reports of long-term outcomes and limitation of treatment in specific cohorts of patients with liver tumours are lacking. In addition, the mechanisms of action of therapy have not been fully elucidated. This study highlights the long-term clinical results and limitations of LTA in the treatment of a cohort of patients with unresectable colorectal liver metastases and examines the mechanisms of action of thermal ablative injury in a murine model.
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ItemFamily studies of epilepsy with simple and complex inheritance( 2002)The epilepsies are a group of disorders where genetic factors have been long known to play an important role. Genetic research in the last decade has led to the discovery of several mutated genes in some uncommon human epilepsies with autosomal dominant inheritance. The search of genes for epilepsies with complex inheritance has however been less successful. The general aim of this thesis was to study the genetics of epilepsies with simple and complex inheritance, using families with multiple affected family members. Accurate phenotyping was a strategy I used to try and minimise the problem of genetic heterogeneity. The study involved a long interview with all available living family members to collect clinical and genealogical information. Detailed electro-clinical characterisation of each individual's epilepsy syndrome was obtained. Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) was studied as a prototype of simple inheritance. Detailed clinical analysis of 5 ADNFLE families revealed some new clinical features including seizures triggered by sound or movement, and precipitated by fever, extreme tiredness or stress. In order to help distinguishing NFLE from parasomnias, I developed a motor activity scoring system in sleep (MAISS). In most cases this confirmed the clinical and or electrographic diagnosis. Molecular genetic analysis of 5 families and 11 sporadic cases by our collaborators revealed 2 new mutations in the genes coding for α4 and β2 subunits of the acetylcholine receptors (CHRNA4 and CHRNB2). A de novo CHRNA4 mutation was found in the proband and her second son in family A. This is the first de novo mutation found in epilepsy suggesting also that sporadic cases could have a genetic aetiology and such patients should be tested for the known mutations. A new mutation in CHRNB2 was found in family B, the mutation was present in family members with ADNFLE but was not found in family members with parasomnias defined by their MAISS score. The finding does not support the hypothesis of a common genetic aetiology between ADNFLE and parasomnias. Idiopathic generalized epilepsies (IGEs) are the most common and important group of epilepsies with complex inheritance. An exceptionally large family with 35 affected individuals was studied. The phenotypes included childhood absence epilepsy (CAE), febrile seizures (FS) and generalized epilepsy with febrile seizures plus (GEFS+). Molecular genetic analysis showed that affected individuals had a mutation in the gene coding for the γ2 subunit of the GABAA receptor (GABRG2). The structure of the pedigree suggested that, in this family FS were inherited in an autosomal dominant fashion and caused by GABRG2 mutation. However, clinical genetic analysis suggested that whilst this mutation contributed to the CAE and GEFS+ phenotypes, it was likely that other genes were also involved. A second linkage analysis inclusive of only subjects with CAE disclosed a number of putative loci that could contain genes contributing to the absence epilepsy phenotype. I also studied 55 small families with IGEs that were more representative of IGEs found in the community. The clinical genetic analysis of the families showed a relatively homogeneous pattern of syndrome distribution within families. The phenotypic concordance with the proband was higher in first degree relatives compared to more distant relatives. The family analysis showed that the two recognized absence epilepsy syndromes were genetically closely related whilst juvenile myoclonic epilepsy was more distinct. The GABRG2 mutation was not found in any of these families. Two families with biparental IGEs were studied. The affected offspring of the 2 families had epilepsy phenotypes with characteristics of both parents' epilepsies. Finally, of 121 consecutive patients with new-onset IGE, 28% had seizures beginning at age 20 years or later (adult-onset). Similar to the classical IGE with age of onset before 20 years (as described by the ILAE classification), patients with adult-onset IGE could also be divided in subgroups depending on the predominant seizure types including absences, tonic-clonic and myoclonic seizures. Pedigree analysis of the 121 IGE patients and the comparison of the proportion of affected relatives between classical and adult-onset IGEs patients suggested a predominant genetic aetiology in both. In some pedigrees classical and late-onset IGEs patients co-occurred suggesting that they might share genetic determinants. Therefore adult-onset IGEs should be included in studies were genes for IGEs are sought. In conclusion the data from the large family in which a GABRG2 mutation was found, from the small multiplex families including biparental families and from patients with adult-onset IGEs all underscore the genetic basis of IGEs. Moreover, the clinical analyses, with distinct identifiable subsyndromes, is more suggestive of an oligogenic model of inheritance, involving few genes, rather than a polygenic model where many genes of small effect contribute to the individual phenotype.
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ItemComparing the costs of stroke subtypes: a cost of illness study( 2000)Worldwide, stroke is the second leading cause of death and one of the most important causes of disability. Stroke is responsible for about 4% of the total costs of disease in Australia. Apart from this study, there are no data about the patterns of health care and community resource use among distinct subtypes of stroke patients in Australia. The role of informal carers in the care of stroke survivors has not previously been investigated, and, with the exception of this study, there are no Australian data about the costs personally incurred by patients and their carers for stroke-related medical and community services. A community-based stroke incidence study conforming to 'ideal' methodology was conducted in urban Melbourne (population 133,816) during 1996 and 1997. All registered cases (380 events in 352 persons) were classified into distinct stroke subtypes. The crude annual incidence rate (first-ever strokes) was 206 per 100,000 per year. Details of stroke-related resource use during the first 12 months after stroke were obtained for 165 cases. Methods included face-to-face interviews conducted by research nurses with patients and carers. An incidence-based cost-of-illness model was developed which linked subtype-specific incidence rates, mortality rates and resource use data. The total first year costs and the present value of total lifetime costs for all first-ever-in-a-lifetime stroke cases occurring in Australia in 1997 were estimated to be $A541.3 million and $A1.3 billion respectively. The average total costs during the first year and the average present value of total costs over a lifetime following first-ever-in-a-Iifetime stroke were $18,483/case and $43,565/case respectively. The estimated total cost of inpatient rehabilitation for all first-ever-in-a-lifetime stroke cases occurring in Australia in 1997 was $A150 million. This is similar to the total cost of hospitalisation for acute stroke care and approximately twice that of a previous estimate. The 'time costs' of informal care provided by relatives and friends represented about 4% of total first year costs and 14% of total lifetime costs for first-ever-in-a-lifetime strokes. Virtually all surviving stroke cases reported 'out of pocket' costs as a consequence of stroke. The largest costs were for home modifications, private nursing care and aids and equipment. A clear relationship between stroke subtype and cost was demonstrated. During the first year after stroke, although the average cost per case was similar for cerebral infarction and intracerebral haemorrhage, large cost differences were demonstrated between subtypes of cerebral infarction. The most expensive infarct subtype was Total Anterior Cerebral Infarction (TACI) and the least expensive was Lacunar Infarction (LACI). On average, cases of TACI costed 1.8 times as much as cases of LACI. Over a lifetime, on average, cases of intracerebral haemorrhage costed 1.7 times as much as cases of cerebral infarction and cases of LACI cost two-thirds as much as TACI. This is the first comprehensive description of the patterns and costs of stroke care during the first year after stroke in Australia. Given the magnitude of the costs of stroke rehabilitation in this study, formal evaluation of the cost-effectiveness of these services needs to become a research priority.
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ItemHypoxia and angiogenesis in renal cell carcinoma( 2009)Hypoxia is one of the hallmarks of cancer. It was first postulated to occur in solid tumours by Thomlinson and Gray in 1955.1 The presence of hypoxia has been demonstrated in different types of solid tumours.2 Intratumoral hypoxia is caused by the lack of functional blood vessels in proliferating tumour tissue, resulting in low intratumoral oxygen concentrations. If hypoxia is severe or prolonged, cell death occurs.3 Malignant cells can undergo genetic and adaptive changes that allow them to escape from dying of oxygen deprivation. These changes are associated with a more aggressive malignant phenotype 4,5 conferring resistance to radiation 6,7 and chemotherapeutic agents.3,8,9 Hence hypoxia is known to be a key factor responsible for tumour resistance in humans. Invasive polarographic oxygen sensor measurements have demonstrated hypoxia in solid tumours and it is generally defined to occur at an oxygen tension less than ten mmHg.10 Perhaps of more importance is that hypoxia has been demonstrated to be a prognostic indicator for local control after treatment with radiotherapy in glioma, head and neck and cervical cancers.11-13 It has also been able to predict for survival and the presence of distant metastases in soft tissue sarcomas.14 Finally, the significance of hypoxia in the activation and induction of functional molecules such as hypoxia inducible factors (HIFs) and VEGF, the modulation of gene expression (e.g. carbonic anhydrase IX), increased proto-oncogene levels, activation of nuclear factors and accumulation of other proteins (e.g. TP53) although progressing, is yet to be defined.15,16 Thus, it is of clinical interest to understand the levels of hypoxia and numbers of hypoxic cell populations in tumours, particularly those resistant to radiation and chemotherapy. In doing so clinicians and researchers may formulate more accurate prognostic information and develop treatments targeting hypoxic cells. Renal cell carcinoma (RCC) is a tumour resistant to radiation and chemotherapy that is yet to have its oxygen status investigated. Although the “gold standard” of oxygen tension measurement is the Polarographic Oxygen Sensor (POS or Eppendorf pO2 histograph), non-invasive means of measuring oxygen status via imaging, immunohistochemistry or serum tumour markers are more practical. As highlighted by Menon and Fraker, it is imperative that reliable, globally usable, and technically simplistic methods be developed to yield a consistent, comprehensive, and reliable profile of tumour oxygenation. Until newer more reliable techniques are developed, existing independent techniques or appropriate combinations of techniques should be optimized and validated using known endpoints in tumour oxygenation status and/or treatment outcomes.17 Hanahan and Weinberg 18 surmised that the field of cancer research has largely been guided by a reductionist focus on cancer cells and the genes within them- a focus that has produced an extraordinary body of knowledge. Looking forward in time, they believe that progress in cancer research would come from regarding tumours as complex tissues in which mutant cancer cells have conscripted and subverted normal cell types (endothelial cells, immune cells, fibroblasts) to serve as active collaborators in their neoplastic agenda. The interactions between the genetically altered malignant cells and these supporting coconspirators will prove critical to understanding cancer pathogenesis and to the development of novel, effective therapies.18 Essentially, the background outlined here not only highlights the core aim of this thesis: to better understand the oxygen status of renal cell carcinoma and the relationship of this to angiogenesis so that better targeted therapies may be pursued in the future; but it also places this research in the context of the future proposed by Hanahan and Weinberg,18 by clearly focusing on collaborators in the neoplastic agenda, rather than just tumour cells themselves, to better understand RCC.