The Queensland study of melanoma : Environmental and genetic associations (Q-MEGA); Study design, baseline characteristics, and repeatability of phenotype and sun exposure measures

Cutaneous malignant melanoma (CMM) is a major health issue in Queensland, Australia, which has the world’s highest incidence. Recent molecular and epidemiologic studies suggest that CMM arises through multiple etiological pathways involving gene-environment interactions. Understanding the potential mechanisms leading to CMM requires larger studies than those previously conducted. This article describes the design and baseline characteristics of Q-MEGA, the Queensland Study of Melanoma: Environmental and Genetic Associations, which followed up 4 population-based samples of CMM patients in Queensland, including children, adolescents, men aged over 50, and a large sample of adult cases and their families, including twins. Q-MEGA aims to investigate the roles of genetic and environmental factors, and their interaction, in the etiology of melanoma. Three thousand, four hundred and seventy-one participants took part in the follow-up study and were administered a computer-assisted telephone interview in 2002-2005. Updated data on environmental and phenotypic risk factors, and 2777 blood samples were collected from interviewed participants as well as a subset of relatives. This study provides a large and well-described population-based sample of CMM cases with follow-up data. Characteristics of the cases and repeatability of sun exposure and phenotype measures between the baseline and the follow-up surveys, from 6 to 17 years later, are also described.

Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Vibrational spectroscopic studies of laboratory scale polymer melt processing : application to a thermoplastic polyurethane nanocomposite

A laboratory scale twin screw extruder has been interfaced with a near infrared (NIR) spectrometer via a fibre optic link so that NIR spectra can be collected continuously during the small scale experimental melt state processing of polymeric materials. This system can be used to investigate melt state processes such as reactive extrusion, in real time, in order to explore the kinetics and mechanism of the reaction. A further advantage of the system is that it has the capability to measure apparent viscosity simultaneously which gives important additional information about molecular weight changes and polymer degradation during processing. The system was used to study the melt processing of a nanocomposite consisting of a thermoplastic polyurethane and an organically modified layered silicate.

Where the bloody hell does parody fit in Australian copyright law?

This article examines the role of the recently introduced fair dealing exception for the purposes of parody and satire in Australian copyright law. Parody and satire, while central to Australian expression, pose a substantial challenge for copyright policy. The law is asked to strike a delicate balance between an author’s right to exploit their work, the interests of the public in stimulating free speech and critical discussion, the rights of artists who rely on existing material in creating their own expression, and the rights of all artists in their reputation and the integrity of their works. This article highlights the difficulty parodists and satirists have historically faced in Australia and examines the potential of the new fair dealing exceptions to relieve this difficulty. This article concludes that the new exceptions have the potential, if read broadly, not only to bridge the gap between humorous and non-humorous criticism, but also to allow for the use of copyright material to critique figures other than the copyright owner or author, extending to society generally. This article will argue that the new exceptions should be read broadly to further this important policy goal while also being limited in their application so as to prevent mere substitutable uses of copyright material. To achieve these twin goals, I suggest that the primary indication of fairness of an unlicensed parody should be whether or not it adds significant new expression so as not to be substitutable for the original work.

The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model

Conventional clinical therapies are unable to resolve osteochondral defects adequately, hence tissue engineering solutions are sought to address the challenge. A biphasic implant which was seeded with Mesenchymal Stem Cells (MSC) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a Polycaprolactone (PCL) cartilage scaffold and a Polycaprolactone - Tri Calcium Phosphate (PCL - TCP) osseous matrix. Autologous MSC was seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL - collagen electrospun mesh that served as a substitute for periosteal flap in preventing cell leakage. Controls either without implanted MSC or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by the superior Glycosaminoglycan (GAG) maintenance. This positive morphological outcome was supported by a higher relative Young's modulus which indicated functional cartilage restoration. Bone in growth and remodeling occurred in all groups with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover healing was inferior at the patellar groove as compared to the medial condyle and this was attributed to the native biomechanical features.

Characterisation of mesenchymal cells from osteophytes in osteoarthritis

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.

Runx2 overexpression in bone marrow stromal cells accelerates bone formation in critically-sized femoral defects

The repair of large non-unions in long bones remains a significant clinical problem due to high failure rates and limited tissue availability for auto- and allografts. Many cell-based strategies for healing bone defects deliver bone marrow stromal cells to the defect site to take advantage of the inherent osteogenic capacity of this cell type. However, many factors, including donor age and ex vivo expansion of the cells, cause bone marrow stromal cells to lose their differentiation ability. To overcome these limitations, we have genetically engineered bone marrow stromal cells to constitutively overexpress the osteoblast specific transcription factor Runx2. In the present study, we examined Runx2-modified bone marrow stromal cells, delivered via poly(caprolactone) scaffolds loaded with type I collagen meshes, in critically-sized segmental defects in rats compared to unmodified cells, cell-free scaffolds and empty defects. Runx2 expression in bone marrow stromal cells accelerated healing of critically-sized defects compared to unmodified bone marrow stromal cells and defects receiving cell-free treatments. These findings provide an accelerated method for healing large bone defects which may reduce recovery time and the need for external fixation of critically-sized defects.

Enhancing in vivo vascularized bone formation by cobalt chloride-treated bone marrow stromal cells in a tissue engineered periosteum model

The periosteum plays an indispensable role in both bone formation and bone defect healing. In this study we constructed an artificial in vitro periosteum by incorporating osteogenic differentiated bone marrow stromal cells (BMSCs) and cobalt chloride (CoCl(2))-treated BMSCs. The engineered periostea were implanted both subcutaneously and into skull bone defects in SCID mice to investigate ectopic and orthotopic osteogenesis and vascularization. After two weeks in subcutaneous and four weeks in bone defect areas, the implanted constructs were assessed for ectopic and orthotopic osteogenesis and vascularization by micro-CT, histomorphometrical and immunohistochemical methods. The results showed that CoCl(2) pre-treated BMSCs induced higher degree of vascularization and enhanced osteogenesis within the implants in both ectopic and orthotopic areas. This study provided a novel approach using BMSCs sourced from the same patient for both osteogenic and pro-angiogenic purposes in constructing tissue engineered periosteum to enhance vascularized osteogenesis.

The osteogenic properties of CaP/silk composite scaffolds

The rationale for the present study was to develop porous CaP/silk composite scaffolds with a CaP-phase distribution and pore architecture better suited to facilitate osteogenic properties of human bone mesenchymal stromal cells (BMSCs) and in vivo bone formation abilities. This was achieved by first preparing CaP/silk hybrid powders which were then incorporated into silk to obtain uniform CaP/silk composite scaffolds, by means of a freeze-drying method. The composition, microstructure and mechanical properties of the CaP/silk composite scaffolds were ascertained by X-ray diffraction (XRD), Fourier transform infrared spectra (FTIR), scanning electron microscope (SEM) and a universal mechanical testing machine. BMSCs were cultured in these scaffolds and cell proliferation analyzed by confocal microscopy and MTS assay. Alkaline phosphatase (ALP) activity and osteogenic gene expression were assayed to determine if osteogenic differentiation had taken place. A calvarial defect model in SCID mice was used to determine the in vivo bone forming ability of the hybrid CaP/silk scaffolds. Our results showed that incorporating the hybrid CaP/silk powders into silk scaffolds improved both pore structure architecture and distribution of CaP powders in the composite scaffolds. By incorporating the CaP phase into silk scaffolds in vitro osteogenic differentiation of BMSCs was enhanced and there was increased in vivo cancellous bone formation. Here we report a method with which to prepare Ca/P composite scaffolds with a pore structure and Ca/P distribution better suited to facilitate BMSC differentiation and bone formation.

From Sydney and Shanghai : Australian and Chinese women writing modernism

In the 1930s and 1940s, Australian women writers published novels, poems, and short stories that pushed the boundaries of their national literary culture. From their position in the Pacific, they entered into a dialogue with a European modernism that they reworked to invigorate their own writing and to make cross-continental connections. My interest in the work of Australian women prose writers of this period stems from an appreciation of the extent of their engagement with interwar modernism (an engagement that is generally under-acknowledged) and the realization that there are commonalities of approach with the ways in which contemporaneous Chinese authors negotiated this transnational cultural traffic. China and Australia, it has been argued, share an imaginative and literal association of many centuries, and this psychic history produces a situation in which ‘Australians feel drawn towards China: they cannot leave it alone.’1 Equally, Chinese exploration of the great southern land began in the fifteenth century, prior to European contact. In recent times, the intensity of Australia’s cultural and commercial connections with Asia has led to a repositioning of the Australian sense of regionalism in general and, in particular, has activated yet another stage in the history of its relationship with China. In this context, the association of Australian and Chinese writing is instructive because the commonalities of approach and areas of interest between certain authors indicate that Australian writers were not alone in either the content or style of their response to European modernism. This recognition, in turn, advances discussions of modernism in Australia and reveals an alternative way of looking at the world from the Pacific Rim through literature. The intent is to examine selective Australian and Chinese authors who are part of this continuous history and whose writing demonstrates common thematic and stylistic features via the vector of modernism. I focus on the 1930s and 1940s because these are the decades in which Australia and China experienced wideranging conflict in the Pacific, and it is significant that war, both forthcoming and actual, features as an ominous soundtrack in the writing of Chinese and Australian women. I argue that, given the immensity of cultural difference between Australia and China, there is an especially interesting juncture in the ways in which the authors interrogate modernist practices and the challenge of modernism. The process in which writing from the Pacific Rim jointly negotiates the twin desires of engaging with European literary form and representing one’s own culture may be seen as what Jessica Berman identifies as a geomodernism, one of the ‘new possible geographies’ of modernism.2 My discussion centres on the work of the Australian women, to which the Chinese material serves as a point of reference, albeit a critical one. The Chinese writing examined here is restricted to authors who wrote at least some material in English and whose work is available in translation.

Addressing issues in sparseness, ecological bias and formulation of the adjacency matrix in Bayesian spatio-temporal analysis of disease counts

The main objective of this PhD was to further develop Bayesian spatio-temporal models (specifically the Conditional Autoregressive (CAR) class of models), for the analysis of sparse disease outcomes such as birth defects. The motivation for the thesis arose from problems encountered when analyzing a large birth defect registry in New South Wales. The specific components and related research objectives of the thesis were developed from gaps in the literature on current formulations of the CAR model, and health service planning requirements. Data from a large probabilistically-linked database from 1990 to 2004, consisting of fields from two separate registries: the Birth Defect Registry (BDR) and Midwives Data Collection (MDC) were used in the analyses in this thesis. The main objective was split into smaller goals. The first goal was to determine how the specification of the neighbourhood weight matrix will affect the smoothing properties of the CAR model, and this is the focus of chapter 6. Secondly, I hoped to evaluate the usefulness of incorporating a zero-inflated Poisson (ZIP) component as well as a shared-component model in terms of modeling a sparse outcome, and this is carried out in chapter 7. The third goal was to identify optimal sampling and sample size schemes designed to select individual level data for a hybrid ecological spatial model, and this is done in chapter 8. Finally, I wanted to put together the earlier improvements to the CAR model, and along with demographic projections, provide forecasts for birth defects at the SLA level. Chapter 9 describes how this is done. For the first objective, I examined a series of neighbourhood weight matrices, and showed how smoothing the relative risk estimates according to similarity by an important covariate (i.e. maternal age) helped improve the model’s ability to recover the underlying risk, as compared to the traditional adjacency (specifically the Queen) method of applying weights. Next, to address the sparseness and excess zeros commonly encountered in the analysis of rare outcomes such as birth defects, I compared a few models, including an extension of the usual Poisson model to encompass excess zeros in the data. This was achieved via a mixture model, which also encompassed the shared component model to improve on the estimation of sparse counts through borrowing strength across a shared component (e.g. latent risk factor/s) with the referent outcome (caesarean section was used in this example). Using the Deviance Information Criteria (DIC), I showed how the proposed model performed better than the usual models, but only when both outcomes shared a strong spatial correlation. The next objective involved identifying the optimal sampling and sample size strategy for incorporating individual-level data with areal covariates in a hybrid study design. I performed extensive simulation studies, evaluating thirteen different sampling schemes along with variations in sample size. This was done in the context of an ecological regression model that incorporated spatial correlation in the outcomes, as well as accommodating both individual and areal measures of covariates. Using the Average Mean Squared Error (AMSE), I showed how a simple random sample of 20% of the SLAs, followed by selecting all cases in the SLAs chosen, along with an equal number of controls, provided the lowest AMSE. The final objective involved combining the improved spatio-temporal CAR model with population (i.e. women) forecasts, to provide 30-year annual estimates of birth defects at the Statistical Local Area (SLA) level in New South Wales, Australia. The projections were illustrated using sixteen different SLAs, representing the various areal measures of socio-economic status and remoteness. A sensitivity analysis of the assumptions used in the projection was also undertaken. By the end of the thesis, I will show how challenges in the spatial analysis of rare diseases such as birth defects can be addressed, by specifically formulating the neighbourhood weight matrix to smooth according to a key covariate (i.e. maternal age), incorporating a ZIP component to model excess zeros in outcomes and borrowing strength from a referent outcome (i.e. caesarean counts). An efficient strategy to sample individual-level data and sample size considerations for rare disease will also be presented. Finally, projections in birth defect categories at the SLA level will be made.

Latent variable models in statistical genetics

Understanding the complexities that are involved in the genetics of multifactorial diseases is still a monumental task. In addition to environmental factors that can influence the risk of disease, there is also a number of other complicating factors. Genetic variants associated with age of disease onset may be different from those variants associated with overall risk of disease, and variants may be located in positions that are not consistent with the traditional protein coding genetic paradigm. Latent Variable Models are well suited for the analysis of genetic data. A latent variable is one that we do not directly observe, but which is believed to exist or is included for computational or analytic convenience in a model. This thesis presents a mixture of methodological developments utilising latent variables, and results from case studies in genetic epidemiology and comparative genomics. Epidemiological studies have identified a number of environmental risk factors for appendicitis, but the disease aetiology of this oft thought useless vestige remains largely a mystery. The effects of smoking on other gastrointestinal disorders are well documented, and in light of this, the thesis investigates the association between smoking and appendicitis through the use of latent variables. By utilising data from a large Australian twin study questionnaire as both cohort and case-control, evidence is found for the association between tobacco smoking and appendicitis. Twin and family studies have also found evidence for the role of heredity in the risk of appendicitis. Results from previous studies are extended here to estimate the heritability of age-at-onset and account for the eect of smoking. This thesis presents a novel approach for performing a genome-wide variance components linkage analysis on transformed residuals from a Cox regression. This method finds evidence for a dierent subset of genes responsible for variation in age at onset than those associated with overall risk of appendicitis. Motivated by increasing evidence of functional activity in regions of the genome once thought of as evolutionary graveyards, this thesis develops a generalisation to the Bayesian multiple changepoint model on aligned DNA sequences for more than two species. This sensitive technique is applied to evaluating the distributions of evolutionary rates, with the finding that they are much more complex than previously apparent. We show strong evidence for at least 9 well-resolved evolutionary rate classes in an alignment of four Drosophila species and at least 7 classes in an alignment of four mammals, including human. A pattern of enrichment and depletion of genic regions in the profiled segments suggests they are functionally significant, and most likely consist of various functional classes. Furthermore, a method of incorporating alignment characteristics representative of function such as GC content and type of mutation into the segmentation model is developed within this thesis. Evidence of fine-structured segmental variation is presented.

Bioactive mesopore-glass microspheres with controllable protein-delivery properties by biomimetic surface modification

Microsphere systems with the ideal properties for bone regeneration need to be bioactive, and at the same time possess the capacity for controlled protein/drug-delivery; however, the current crop of microsphere system fails to fulfill these properties. The aim of this study was to develop a novel protein-delivery system of bioactive mesoporous glass (MBG) microspheres by a biomimetic method through controlling the density of apatite on the surface of microspheres, for potential bone tissue regeneration. MBG microspheres were prepared by using the method of alginate cross-linking with Ca2+ ions. The cellular bioactivity of MBG microspheres was evaluated by investigating the proliferation and attachment of bone marrow stromal cell (BMSC). The loading efficiency and release kinetics of bovine serum albumin (BSA) on MBG microspheres were investigated after coprecipitating with biomimetic apatite in simulated body fluids (SBF). The results showed that MBG microspheres supported BMSC attachment and the Si containing ionic products from MBG microspheres stimulated BMSCs proliferation. The density of apatite on MBG microspheres increased with the length of soaking time in SBF. BSA-loading efficiency of MBG was significantly enhanced by co-precipitating with apatite. Furthermore, the loading efficiency and release kinetics of BSA could be controlled by controlling the density of apatite formed on MBG microspheres. Our results suggest that MBG microspheres are a promising protein-delivery system as a filling material for bone defect healing and regeneration.

Sustained release and osteogenic potential of heparan sulfate-doped fibrin glue scaffolds within a rat cranial model

This paper explores the potential therapeutic role of the naturally occurring sugar heparan sulfate (HS) for the augmentation of bone repair. Scaffolds comprising fibrin glue loaded with 5 lg of embryonically derived HS were assessed, firstly as a release-reservoir, and secondly as a scaffold to stimulate bone regeneration in a critical size rat cranial defect. We show HS-loaded scaffolds have a uniform distribution of HS, which was readily released with a typical burst phase, quickly followed by a prolonged delivery lasting several days. Importantly, the released HS contributed to improved wound healing over a 3-month period as determined by microcomputed tomography (lCT) scanning, histology, histomorphometry, and PCR for osteogenic markers. In all cases, only minimal healing was observed after 1 and 3 months in the absence of HS. In contrast, marked healing was observed by 3 months following HS treatment, with nearly full closure of the defect site. PCR analysis showed significant increases in the gene expression of the osteogenic markers Runx2, alkaline phosphatase, and osteopontin in the heparin sulfate group compared with controls. These results further emphasize the important role HS plays in augmenting wound healing, and its successful delivery in a hydrogel provides a novel alternative to autologous bone graft and growth factorbased therapies.

Studies of the radiation chemistry and grafting of a fluoropolymer

The radiation chemistry and the grafting of a fluoropolymer, poly(tetrafluoroethylene-coperfluoropropyl vinyl ether) (PFA), was investigated with the aim of developing a highly stable grafted support for use in solid phase organic chemistry (SPOC). A radiation-induced grafting method was used whereby the PFA was exposed to ionizing radiation to form free radicals capable of initiating graft copolymerization of styrene. To fully investigate this process, both the radiation chemistry of PFA and the grafting of styrene to PFA were examined. Radiation alone was found to have a detrimental effect on PFA when irradiated at 303 K. This was evident from the loss in the mechanical properties due to chain scission reactions. This meant that when radiation was used for the grafting reactions, the total radiation dose needed to be kept as low as possible. The radicals produced when PFA was exposed to radiation were examined using electron spin resonance spectroscopy. Both main-chain (–CF2–C.F–CF2-) and end-chain (–CF2–C.F2) radicals were identified. The stability of the majority of the main-chain radicals when the polymer was heated above the glass transition temperature suggested that they were present mainly in the crystalline regions of the polymer, while the end-chain radicals were predominately located in the amorphous regions. The radical yield at 77 K was lower than the radical yield at 303 K suggesting that cage recombination at low temperatures inhibited free radicals from stabilizing. High-speed MAS 19F NMR was used to identify the non-volatile products after irradiation of PFA over a wide temperature range. The major products observed over the irradiation temperature 303 to 633 K included new saturated chain ends, short fluoromethyl side chains in both the amorphous and crystalline regions, and long branch points. The proportion of the radiolytic products shifted from mainly chain scission products at low irradiation temperatures to extensive branching at higher irradiation temperatures. Calculations of G values revealed that net crosslinking only occurred when PFA was irradiated in the melt. Minor products after irradiation at elevated temperatures included internal and terminal double bonds and CF3 groups adjacent to double bonds. The volatile products after irradiation at 303 K included tetrafluoromethane (CF4) and oxygen-containing species from loss of the perfluoropropyl ether side chains of PFA as identified by mass spectrometry and FTIR spectroscopy. The chemical changes induced by radiation exposure were accompanied by changes in the thermal properties of the polymer. Changes in the crystallinity and thermal stability of PFA after irradiation were examined using DSC and TGA techniques. The equilibrium melting temperature of untreated PFA was 599 K as determined using a method of extrapolation of the melting temperatures of imperfectly formed crystals. After low temperature irradiation, radiation- induced crystallization was prevalent due to scission of strained tie molecules, loss of perfluoropropyl ether side chains, and lowering of the molecular weight which promoted chain alignment and hence higher crystallinity. After irradiation at high temperatures, the presence of short and long branches hindered crystallization, lowering the overall crystallinity. The thermal stability of the PFA decreased with increasing radiation dose and temperature due to the introduction of defect groups. Styrene was graft copolymerized to PFA using -radiation as the initiation source with the aim of preparing a graft copolymer suitable as a support for SPOC. Various grafting conditions were studied, such as the total dose, dose rate, solvent effects and addition of nitroxides to create “living” graft chains. The effect of dose rate was examined when grafting styrene vapour to PFA using the simultaneous grafting method. The initial rate of grafting was found to be independent of the dose rate which implied that the reaction was diffusion controlled. When the styrene was dissolved in various solvents for the grafting reaction, the graft yield was strongly dependent of the type and concentration of the solvent used. The greatest graft yield was observed when the solvent swelled the grafted layers and the substrate. Microprobe Raman spectroscopy was used to map the penetration of the graft into the substrate. The grafted layer was found to contain both poly(styrene) (PS) and PFA and became thicker with increasing radiation dose and graft yield which showed that grafting began at the surface and progressively penetrated the substrate as the grafted layer was swollen. The molecular weight of the grafted PS was estimated by measuring the molecular weight of the non-covalently bonded homopolymer formed in the grafted layers using SEC. The molecular weight of the occluded homopolymer was an order of magnitude greater than the free homopolymer formed in the surrounding solution suggesting that the high viscosity in the grafted regions led to long PS grafts. When a nitroxide mediated free radical polymerization was used, grafting occurred within the substrate and not on the surface due to diffusion of styrene into the substrate at the high temperatures needed for the reaction to proceed. Loading tests were used to measure the capacity of the PS graft to be functionialized with aminomethyl groups then further derivatized. These loading tests showed that samples grafted in a solution of styrene and methanol had superior loading capacity over samples graft using other solvents due to the shallow penetration and hence better accessibility of the graft when methanol was used as a solvent.