Biochemistry and Pharmacology - Theses
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ItemNo Preview AvailableInvestigating the cell-specific function of mitochondrial protein import regulators( 2023-06)The paradigms and components of mitochondrial protein import have been well elucidated in fungal organisms such as Saccharomyces cerevisiae. However, the human translocase complexes that facilitate import have diverged from yeast through the evolution of metazoan-specific translocase components. Two pairs of import protein paralogues remain poorly characterised in human cells: Human Tim8a and Tim8b, and, Tim17a and Tim17b. The paralogues are differentially expressed in human tissues, so may inform on novel mechanisms of mitochondrial function in human cell types. The heterogeneity of mitochondria across the human body is in part driven by such tissue specific isoforms. Specialised cells have distinct patterns of mitochondrial activity, and this thesis highlights the contribution of mitochondrial protein import to diverse human cell types. Defective mitochondrial protein import underlies mitochondrial diseases, cancers, and neurodegeneration. Thus, through the study of tissue-specific mitochondrial import proteins, we sought to better understand mitochondrial heterogeneity and how cell-specific defects may manifest the disparate clinical presentations of mitochondrial disease. This thesis investigates the functions of hTim8a, hTim8b, hTim17a and hTim17b in multiple human cell lines using CRISPR/Cas9 gene editing, proteomic analysis, and biochemistry. We find that hTim8 paralogues are not required for protein import, thus have diverged from the function of their yeast homologue. Comparing knock-out cell lines we evidence novel functions in the biogenesis of respiratory Complex IV and show that loss of hTim8 proteins manifests cell-specific perturbations in mitochondrial bioenergetics. Affinity enrichment and complexome profiling reveal that the hTim8 paralogues are required for the integration of the COX2 module of Complex IV in the late stages of assembly. By a similar approach, we find that hTim17a and hTim17b stabilise the human TIM23 complex but either paralogue is sufficient to constitute the translocase. Both hTim17 proteins interact with the same TIM23 subunits, and only hTim17a is degraded under stress but not via its divergent C-terminus. Knock-out of hTim8 and hTim17 proteins across cell lines demonstrate the cell-specific effects of mitochondrial dysfunction on metabolism and cell survival. The cell-specific impacts in our knock-out cell models parallel the tissue-specific manifestation of mitochondrial disease symptoms, offering insight into possible therapeutic paradigms. Overall, our research shows that human protein import is crucial to the heterogeneity of human biology in health and disease.
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ItemTranscriptional regulation and functional differences driven by STAT3 isoforms, STAT3α and STAT3β( 2018)The Signal transducer and activator of transcription 3 (STAT3) protein, a member of the STAT family of transcription factors, plays important roles in the regulation of critical biological processes. STAT3 is best known for its role in the JAK-STAT pathway, mediating the transcriptional regulation of target gene expression following its activation by extracellular signal, and continues to be the subject of intense research efforts due to its essential roles in crucial physiological processes, such as embryonic development and inflammation, as well as its involvement in numerous types of cancer. While the genome-wide DNA-binding and transcriptional regulation activities of the full-length STAT3α have been widely studied, those of a naturally occurring shorter STAT3β spliceform are less understood. Despite having identical DNA-binding domains, the STAT3 spliceforms display significant differences in their transcriptional regulation activities. Furthermore, STAT3α and STAT3β can drive opposing oncogenic and tumour suppression outcomes, respectively. Thus, the major focus of this study is to explore STAT3α- and STAT3β-specific genome-wide DNA-binding, transcriptional regulation of non-coding micro-RNAs (miRNAs), and functional outcomes in cancer cell biology. By employing ChIP-seq and miRNA expression profiling assays, this study presents results revealing that STAT3α and STAT3β display clear differences in genome-wide DNA-binding and regulation of miRNA expression. Bioinformatic analyses of transcription factor binding sites also uncovered the possible roles of co-transcription factors in the distinct STAT3 spliceform-specific transcriptional regulation activities. In addition, a Morpholino-directed splicing modulation approach to drive STAT3 knockdown and STAT3α-to-β splicing switch in cancer cells showed that the STAT3 spliceforms can differentially alter the tumourigenic properties of cancer cells, with STAT3β expression being associated with tumour suppression outcomes by driving the suppression of cell proliferation, survival and migration. Taken together, this study highlights the distinct properties of the STAT3 spliceforms in genome-wide DNA-binding which possibly underlie their different gene transcriptional regulation activities in both protein-coding and non-coding genes, and further provides evidence that STAT3β can drive distinct tumour suppression outcomes.
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ItemA qualitative and quantitative characterisation of CD8+ T cell targets on human beta cells in Type 1 diabetes: perspectives from a proteomic strategy( 2016)Type 1 diabetes (T1D) is a tissue specific autoimmune disease that result from the targeted destruction of the insulin-secreting beta cells in the pancreatic islets. A hallmark of T1D is the infiltration of autoimmune T cells into the islets that is predominantly composed of CD8+ T cells. The strongest genetic association with susceptibility to T1D are to the human leukocyte antigen (HLA) genes, particularly the class II, and to a lesser extent, the class I HLA genes. The class I HLA genes encode for cell surface HLA molecules that constitutively present endogenous antigenic peptides for scrutiny by incoming CD8+ T cells. Autoantigens involved in T1D have been widely characterised, however, little is known about the identities of autoantigenic peptides presented by less studied alleles and the nature of naturally processed and presented CD8+ T cell epitopes that leads to a targeted beta cell destruction as disease unfolds. In this study, the nature of bound peptide antigens (immunopeptidomes) restricted to a panel of T1D associated class I HLA allotypes (HLA-A*01:01, -A*02:01, -A*24:02, - B*08:01 and –B*18:01) were studied in depth using advanced mass spectrometry techniques. First I assessed the immunopeptidomes of C1R cell lines that were individually transfected with each HLA allotype. An extensive class I ligand dataset (up to 18 600 naturally presented peptides) was identified in this study which allowed the extension and clarification of previously reported binding motifs for these class I HLA molecules. We found no differences in the nature of peptide presentation by T1D disease predisposing and protective HLAs. Overall, features (subcellular localisation and biological functions) of source proteins for all five HLA allotypes were similar and we observed that peptide sampling was not affected by source protein masses and lengths. Next, we examined the features of naturally presented autoantigenic peptides by class I HLA allotypes on a panel of surrogate beta cells (class I HLA expressing C1R cells supertransfected with key beta cell autoantigens including PPI, prepro-IAPP, IGRP and ChgA). Within the immunopeptidome, novel PPI, prepro-IAPP and ChgA peptides, alongside previously reported epitopes were identified. Over half of these candidate epitopes were considered as non-canonical ligands, often present in nested sets of overlapping peptides of heterogeneous length with signal sequence derived peptides over-represented in my datasets. More importantly, preliminary tetramer staining experiments performed herein showed that a nested set of HLA-A2 restricted PPI signal sequence derived peptides encompassing the PPI15-24 epitope attracted different populations of CD8+ T cells from patients, highlighting a potential, and novel role of epitope length heterogeneity in T1D pathogenesis. Finally, the characterisation of CD8+ T cell targets was complemented with the first qualitative and quantitative analysis of the class I immunopeptidome of a HLA-A2(+) human islet beta cells. Apart from the identification of naturally processed HLA-I peptides derived from beta cell antigen such as islet cell autoantigen-1, I confirmed the natural presentation of two HLA-A2 restricted PPI epitopes (PPI15-24 and Ins B10-18), and identified two novel, overlapping ChgA peptides. Moreover, a novel HLA-A2 restricted, beta cell specific PTM version of PPI15-24 was also identified to be naturally presented in my study. To our knowledge, this account of a naturally presented PTM peptide represents the first class I HLA restricted PTM identified in T1D. Furthermore, quantitation of the presentation levels of PPI15-24 and Ins B10-18 revealed that PPI signal sequence region derived epitope is more abundantly represented in the beta cell immunopeptidome, suggesting a beta cell specific antigen processing machinery for secretory proteins. The beta cell immunopeptidome study was further complemented with the proteomic coverage of the islet from the same donor. My proteomic coverage represent the largest human islet dataset reported to date, and reveals a robust beta cell specific post translational modifications that are known to be induced by inflammation, including deamidation by TGase2, W→Kyn by IDO, and for the first time confirmed the local presence and generation of spliced peptide species composed of an Ins C-peptide fragment fused to the ChgA derived WE14 peptide. These PTM neoantigens represents a rich source for peptide sampling by both HLA-I and HLA-II molecules in the islets as disease progresses. In summary, this study has provided key insights into the nature of beta cell CD8+ T cell targets that is beginning to lead us to understand the molecular events that culminate in the beta cells being singled out for autoimmune destruction during the pathogenesis of T1D.
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ItemCoordinated inhibition of RNA Polymerase I and the PI3K/AKT/mTOR signalling pathway targets ribosome biogenesis and ribosome function to provide a novel and potent therapeutic approach to treat MYC driven cancer( 2014)The growth and proliferation of both normal and malignant cells is dependent on the sustained synthesis of new proteins by ribosomes. Ribosome biogenesis is the tightly regulated multi-step process through which new functional ribosomes are assembled from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs). Importantly the synthesis of ribosomes and ribosomal components is highly coordinated by transcriptional networks and signalling pathways that are drive proliferative growth. These networks and pathways are frequently dysregulated in human cancer, underpinning the near ubiquitous dysregulation of ribosome biogenesis observed in transformed cells. The hyperactivation of ribosome biogenesis and protein translation in cancer cells, in particular those driven by dysregulated c-MYC activity, suggests that they will be vulnerable to therapeutic strategies that target these essential growth processes. This thesis investigates the hypotheses that i) the PI3K/AKT/mTOR signalling pathway plays a critical role in the promotion of ribosome biogenesis and ribosome function in MYC-driven cancer cells and; ii) that the coordinated targeting of ribosome biogenesis and ribosome function will cooperate to improve therapeutic outcomes in B-lymphoma driven by c-MYC over-expression. Signalling mediated by the protein kinase AKT was demonstrated to be critical for ribosome biogenesis via control of both rRNA and RP synthesis, in MYCdriven B-lymphoma cells. Disruption of these essential cellular growth processes following the targeted inhibition of AKT signalling resulted in the delay of disease progression in mice with transplanted MYC-driven Blymphoma. Furthermore, disruption of Pol I transcription mediated by the targeted blockade of the PI3K/AKT/mTOR signalling pathway was linked to the induction of B-lymphoma cell apoptosis. Importantly the potency of different inhibitors in suppressing the PI3K/AKT/mTOR signalling pathway correlated with their potency in reducing the rate of Pol I transcription and cell viability. Selective inhibition of Pol I transcription mediated B-lymphoma cell death via the nucleolar stress response pathway driving the accumulation of free RPs, increased RP-MDM2 binding and robust activation of the p53 pathway. Surprising B-lymphoma cell death following PI3K/AKT/mTOR pathway inhibition was not mediated via this canonical response pathway. PI3K/AKT/mTOR-inhibitor mediated apoptosis was instead achieved via the selective up-regulation of the intrinsic apoptotic machinery, specifically the accumulation of BMF, a pro-apoptotic BH3-only protein encoded by an IRES containing mRNA, the translation of which is promoted following inhibition of PI3K/AKT/mTOR-regulated CAP-dependent protein translation. Consistent with their ability to induce B-lymphoma cell death via distinct pro-apoptotic mechanisms, targeted inhibitors of the PI3K/AKT/mTOR signalling and Pol I transcription combined to enhance B-lymphoma cell apoptosis and extend the survival of mice with transplanted MYC-driven B-lymphoma. Thus, the coordinated inhibition of ribosome biogenesis (rRNA synthesis) and ribosome function (mTORC1-driven protein translation) cooperated to kill MYC-driven lymphoma cells as well as to significantly delay disease relapse and extend survival in a mouse model of MYC-driven malignancy. This work thereby identifies targeting of the ribosome at multiple points as a novel potential therapeutic approach for cancer treatment.
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ItemStructure and function of AMPK: subunit interactions of the AMPK heterotrimeric complex( 2007-12)AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes.
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ItemDefining the in vivo kinetics of mutant Huntingtin aggregation in a Drosophila model( 2013)Huntington’s Disease is caused by a polyQ expansion of ≥ 35Q in exon 1 of the IT15 gene which encodes the protein Huntingtin. Repeat length is inversely correlative with the age of HD onset and expanded polyQ can misfold to form amyloid-fibrils in vitro, which are thought to be a major component of inclusions found in vivo. HD includes a variety of symptoms of which the most damaging is progressive striatal neurodegeneration that leads to death. Although early studies implicated inclusions as the toxic species, more recently inclusions have been suggested to have no correlation with toxicity and have even given neuroprotective attributes. Thus specific links between toxicity and the various stages of aggregation are controversial, however there is a general consensus that aggregation plays some part in pathogenesis. Several techniques have recently been developed to visualise aggregation intermediates in vivo, however this area remains in its infancy and more studies are necessary to gain insight into in vivo aggregation mechanisms. In this thesis, we systematically trial the expression of fluorescently-tagged Huntingtin exon 1 (HttCFP and HttGFP) in various Drosophila tissues, including the larval eye imaginal disc, larval salivary glands and the pro-thoracic gland to monitor aggregate formation in vivo. Consistent with toxicity, expression of Huntingtin exon 1 with pathogenic polyQ expansion lengths using a pan-neural driver reduced survival in adult flies. Importantly we have developed an in vivo system for monitoring progressive inclusion formation by Htt46QGFP over time using the adult Drosophila eye. The Hatters laboratory had previously used fluorescence detection analytical ultracentrifugation (FDS) to characterise the heterogeneity of aggregates formed in a Neuro2a cell culture system. We sought to similarly use FDS to characterise the size distribution of aggregates in adult Drosophila. We found that this was not possible due to significant differences in size between the in vivo aggregates and those from transient transfection of neural cell lines. Thus we showed for the first time that inclusions formed in this Drosophila system are smaller than inclusions formed in a Neuro2a cell culture system. To characterise these in vivo aggregates and form a comparison with those formed in culture, we subsequently developed novel sucrose gradient centrifugation and confocal microscopy coupled with digital quantification methods to quantitatively describe the aggregate size distribution of Drosophila aggregates. Using sucrose gradient centrifugation, as flies aged we observed decreased monomer corresponding with increased Htt46QGFP aggregates in Drosophila lysate, which correlated with increases in inclusion load in adult eyes. This is the first time that in vivo aggregation has been systematically characterised over time using such methods. Additionally, we observed that Htt46QGFP and Htt97QGFP adult Drosophila eye inclusions appeared to have a maximum physical size, that sedimented to 1.2–1.4 M in the sucrose gradient, and were invariably under 4 µm in size. Again, this marks the first time that a physical limit in inclusion size has been described in any in vivo system. Furthermore, the systems developed in this thesis provide a basis for the future examination links between aggregation stages, toxicity and the effect of genetic modifiers and therapeutic targets on aggregation in vivo.
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ItemThe regulation of SnRK1 from Arabidopsis thaliana( 2013)An intrinsic characteristic of all life is the ability to regulate the organism’s internal energy status despite sometimes wildly fluctuating conditions in the external environment. SnRK1 (SNF1-related kinase 1) is the plant orthologue of an evolutionarily conserved, energy-sensing, eukaryotic protein kinase family, including SNF1 from yeast and AMPK from mammals, that plays a major role in the regulation of both cellular and systemic energy homeostasis. The family is functional as heterotrimeric holoenzymes comprising catalytic α¬ and regulatory β and γ subunits, each consisting of several functional domains and each of which may exist in multiple isoforms giving rise to various isoenzymes. Plants contain genes encoding isoforms for each of these subunit types, some of which display unique domain architecture not found outside the plant kingdom. Whilst SNF1 and AMPK have been extensively studied, relatively little is known of how the SnRK1 holoenzyme from plants is regulated and how the various subunit isoforms and their respective functional domains contribute to this regulation. This thesis addresses several aspects of SnRK1 regulation by using biochemical, molecular, and genetic techniques. The present work has described SnRK1 from the model plant Arabidopsis thaliana as a heterotrimeric complex in vitro, existing in six isoenzyme combinations, comprising both classical-type SNF1/AMPK subunits and non-typical plant isoforms, these subunit interactions being confirmed in vivo, with the plant-specific βγ determined to be the sole γ-type subunit. SnRK1, like SNF1 and AMPK, is only minimally active as individual catalytic subunits indicating that SnRK1 is functional as a heterotrimer. Whilst phosphorylation of the α-subunit T-loop threonine is critical for activity this residue is resistant to dephosphorylation by phosphoprotein phosphatases signifying a major divergence in mode of regulation in comparison to SNF1 and AMPK. In addition, whilst the β-subunit carbohydrate-binding modules of AMPK bind glycogen, those of SnRK1 unexpectedly do not associate with glycogen, starch, or a vast array of oligosaccharides and plant-derived polysaccharides. Genetic and molecular studies focused on the β2 subunit. Analysis of T-DNA insertion mutations in this gene suggest that β2, whilst not drastically impacting plant phenotype under normal growth condition, does reduce the plant’s extent of growth. When the dark period was extended, however, the β2 mutants appeared to suffer from a starch rationing phenotype, not being able to conserve transient leaf starch as in wild-type plants. Using the GUS reporter system, β2 gene expression was observed strongly in vascular tissue, some floral tissue, and the floral organ abscission zone. Finally, in accordance with SnRK1’s role as a central integrator of metabolism, the β2 protein was observed to be localised mainly to the cytoplasm and nucleus by way of fusion to the yellow fluorescent protein. This thesis has contributed to the growing body of knowledge regarding SnRK1 with several of the findings prompting modification to assumptions regarding the regulation of the enzyme. These findings indicate that the central metabolic regulator, whilst conserving many features, is regulated in a considerably different manner to that of its yeast and mammalian orthologues, likely reflecting the unique metabolism of the plant life-style.
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ItemTargeted analysis of antigen presentation using mass spectrometry( 2012)The immune system constantly samples the cellular proteome through the process of antigen presentation. T cells specifically recognize cellular peptides bound to the molecules encoded by the major histocompatibility complex (MHC) that are expressed on the surface of virtually all nucleated cells. In type 1 diabetes (T1D), the immune system mistakenly recognises peptides derived from normal cellular (self) proteins, triggering an attack against the insulin secreting pancreatic β cells, leading to β cell loss and insulin deficiency. Recognition of MHC-bound peptides is critical for both the initiation and progression of the disease. Identifying the peptide epitopes targeted during diabetes remains a critical step in defining the molecular basis of the disease. Human patient testing in combination with the use of the Non-Obese Diabetic (NOD) mouse which develops spontaneous autoimmune diabetes has lead to the characterisation of multiple antigens that are recognised by autoreactive lymphocytes. However, the inherent complexities of antigen processing and presentation, the polygenic nature of the disease, the extensive polymorphism of the MHC and the technical hurdles in working with T cells have made epitope discovery and quantitation challenging. This study examined the peptides selected for presentation by MHC class I molecules on the surface of β cells under normal and inflammatory conditions by mass spectrometry and compared these to those peptides found on the surface of primary tissue (spleen and thymus) from NOD mice. Over 2000 MHC-bound peptides, 1100 of these presented by β cells grown under normal conditions or following exposure to IFNγ, were identified. These include sequences from a number of known autoantigens, however, many previously characterised T cell epitopes in the NOD mouse were not observed. This suggested that many of these peptides were present at very low levels. I therefore sought to develop a more sensitive approach to target analysis towards these species. A novel approach was developed to detect known epitopes and directly quantitate their presentation levels. This methodology involved combining the peptide-MHC complex immunoaffinity purification used for the β cell repertoire analysis followed by interrogation of the peptide sample using a mass spectrometry-based technique called multiple reaction monitoring (MRM). This approach incorporates epitope specific information to target the analysis towards the peptide of choice leading to large gains in sensitivity and specificity. When used in combination with an isotopic peptide standard (AQUA peptide) more accurate quantitation can be achieved. I initially tested and optimised the method by quantitating the ovalbumin derived SIINFEKL epitope from cells that express physiological levels of this antigen (EG-7 cells). In vitro quantitation of SIINFEKL (728 copies per cell) from EG-7 cells and in vivo quantitation of cross-presented SIINFEKL in mouse spleen (4 fmol) were successfully demonstrated. The multiplexing ability of the quantitative workflow was also demonstrated by simultaneous monitoring of 9 peptides purified from antigen presenting cells. The robust and absolute quantitative nature of this newly proposed workflow in addition to its ability to multiplex, allowing for high-throughput analysis of multiple epitopes in a single sample, are superior to existing cell based epitope quantitation techniques. The quantitative workflow was applied in the study of an immunodominant H-2Kd-restricted epitope IGRP206-214. Quantitation of IGRP206-214 revealed low level presentation (~25 complexes/cell) on NIT-1 cells following IFNγ treatment compared with the simultaneous presentation of the endogenously processed H-2Kd-restricted peptide JAK-1355-363 (~15000 copies per cell). The potential to perform quantitative studies of disease related epitope presentation in vivo was also further demonstrated using pancreatic lymph nodes and IGRP206-214. These studies foreshadow future large scale epitope presentation and vaccine efficacy studies from live organisms and tissues. Ultimately, the application of modern mass spectrometry-based workflows will progress our efforts and success in immunological therapy of diabetes and other diseases.
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ItemThe reversibility of amyloid fibril formation( 2009)The aggregation of misfolded proteins into amyloid fibrils is implicated in the pathogenesis of several human degenerative diseases, including Alzheimer’s, Parkinson’s and Type II diabetes. Links between the deposition of amyloid fibrils and the progression of these diseases are poorly understood, with much of the current research focused on monomer misfolding and subsequent assembly of oligomers and mature fibrils. This project examines the formation of human apolipoprotein (apo) C-II amyloid fibrils, with a focus on the stability and reversibility of amyloid fibril assembly. The initial stages of the project were to develop a model for apoC-II amyloid fibril formation. This was achieved by analysis of the concentration dependent kinetics of apoC-II amyloid fibril formation, and correlation of these data with the final size distribution of the fibrils, determined by sedimentation velocity experiments. On the basis of these studies, a new reversible model for apoC-II amyloid fibril formation is proposed that includes fibril breaking and re-joining as integral parts of the assembly mechanism. The model was tested by rigorous experimentation, with antibody-labelling transmission electron microscopy providing direct evidence for spontaneous fibril breaking and re-joining. The development of this model for apoC-II fibril assembly provided the foundation for experiments to investigate factors that promote, inhibit or reverse amyloid fibril formation. Factors that were considered include a molecular chaperone protein, αB-crystallin, and a chemical modification, methionine oxidation. Investigations on the effect of αB-crystallin revealed that the inhibition of apoC-II fibril formation occurs by two distinct mechanisms: transient interaction with monomer preventing oligomerisation, and binding to mature fibrils, which inhibits fibril elongation. Studies on the effect of methionine oxidation on apoC-II fibril formation showed that both the assembly and stability of the fibrils was affected by this modification. ApoC-II contains two methionine residues (Met-9 and Met-60), and upon oxidation of these residues fibril formation was inhibited. In addition, the treatment of pre-formed fibrils with hydrogen peroxide caused dissociation of the fibrils via the oxidation of Met-60, located with the fibril core structural region. The final chapter details the development of antibodies that specifically recognise the conformation of apoC-II amyloid fibrils, which provide the foundation for future studies to examine the role that apoC-II amyloid fibrils play in disease. Overall, this thesis reveals the dynamic and reversible nature of amyloid fibril formation. New insight is also obtained of the general stability of amyloid fibrils and the processes that may regulate their formation, persistence and disease pathogenesis in vivo.