Pathology - Theses
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ItemA comparison of benefits between bilateral cochlear implants and bimodal devices in adults( 2012)Investigations of two-eared hearing benefits have demonstrated advantages for cochlear implant users who wear a hearing aid (HA) or a cochlear implant (CI) in the contralateral ear (termed bimodal devices and bilateral CIs, respectively). However, the comparison of benefits between users of bimodal and bilateral devices across a range of perceptual tasks has not been demonstrated conclusively. The aim of this study was to investigate these benefits to assist in patient counselling and to inform surgical decisions. Participants were unilateral (n=24), bimodal (n=21), and bilateral (n=8) CI users. Three bimodal participants received a second CI during the course of the study and were assessed in the bimodal and bilateral conditions for a within participant comparison of performance. Participants completed tests of speech perception, sound localisation, pitch perception, music appreciation and a larger cohort completed a functional performance questionnaire. Study outcomes showed strong benefit for the use of a contralateral device. Sound localisation benefit was strongly associated with bilateral CI use. Benefit from the contralateral device was seen for speech perception ability in the bimodal and bilateral groups; however, the behavioural technique of facing the poorer ear toward the background noise was the most effective method of improving speech perception in background noise for bimodal device users. The bimodal group showed speech perception benefit with coincident signal and noise, probably due to low-frequency phonetic information provided by the HA. The head-shadow effect was strongly associated with the improvements obtained in noise for bilateral CI users. For pitch perception, bimodal device users with residual hearing even at only the lowest frequencies in the HA ear gained significant benefit. Benefit to pitch perception and subjective benefit for the appreciation of ensemble music was strongly associated with bimodal device use. Benefit for the subjective appreciation and perceived naturalness of speech sounds can result to a comparable extent through use of bimodal and bilateral CIs. The results of the functional performance questionnaire showed that the measured objective two-eared benefits translated into a real-world benefit noticeable to CI users. Bimodal device users with high-frequency residual hearing showed the least benefit for speech perception in quiet and pitch perception ability in the bimodal condition. No other demographic or audiological participant characteristics were strongly associated with benefit, particularly across the range of perceptual tasks. Benefit for performance across the perceptual tasks was variable for individual participants, a commonly reported characteristic of CI users. The results of this study demonstrate that it is inappropriate to attempt to judge whether bimodal or bilateral CI use is better overall. The outcomes show that there are certain auditory skills that tend to be better with the bimodal or bilateral device configuration, and that the optimal configuration will depend on the individual.
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ItemThe contribution of stromal caveolin-1 to breast cancer metastasis( 2012)In a previous study, our group has shown that the expression of caveolin-1 (CAV1) in normal tissue surrounding a primary breast cancer is a powerful prognostic indicator of subsequent metastatic disease. While reports linking expression of CAV1 within breast tumour cells to clinical outcome have not led to any clear conclusions, the finding of a strong positive correlation between loss of CAV1 expression in breast tumour stroma and poor prognosis by our group and others, is novel and exciting as it offers the potential of a reliable prognostic indicator of metastatic disease. These clinical observations have led us to propose the hypothesis that breast tumour cells decrease stromal CAV1 expression, and a reduction in stromal CAV1 expression leads to promotion of tumour growth and metastasis. The aims of this project were to model the progression in breast cancer following stromal CAV1 loss, and determine the underlying breast tumour-stroma paracrine metastasis. Tumours arising from weakly metastatic mammary cell lines 66cl4 and 4T1ch5, grow at a faster or similar rate respectively, when co-injected with CAV1 null mammary fibroblasts. In contrast, co-injection with CAV1 expressing mammary fibroblasts has a suppressive effect on tumour growth. Metastasis to lung was significantly higher in mice with resected primary tumours that arose from co-inoculation of 4T1ch5 cells and CAV1 null mammary fibroblasts, in addition to a significant increase in individual lung tumour nodule size. However no significant difference in immune infiltrate in these resected tumours was observed in preliminary flow cytometry analysis. No significant differences in primary tumour growth or metastasis were observed in human xenograft models. To mimic the phenotype observed in vivo, 3D co-culture assays were developed. Although these assays demonstrated a positive effect of fibroblasts on tumour cell invasion and proliferation, no stromal CAV1 specific effect was observed in response to fibroblast co-culture or conditioned medium. To further understand the consequences of the loss of stromal CAV1, profiling of CAV1 expressing and null mouse mammary fibroblasts was conducted using cytokine arrays and cDNA microarrays. A significant increase in Gas6 and a decrease in RANTES cytokine secretion were observed as a result of CAV1 loss, with no significant changes in their transcript levels. In summary, results from this project demonstrate that stromal CAV1 is an important prognostic factor in breast cancer progression. Based on these findings, a stromal targeted therapy to that restores or substitutes for CAV1 activity in stromal cells, or that targets CAV1 regulated cytokines such as Gas6, may be a viable therapy in the treatment of breast cancer metastasis.
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ItemTargeting DNA-dependent protein kinase promotes accelerated senescence of irradiated human cancer cells( 2012)Ionizing radiation is a widely used anti-cancer modality. Unfortunately however, relapse rates are high following radiation treatment indicating an urgent need for novel radiosensitizing strategies. Since radiation potently induces DNA double-strand breaks (DSBs), targeting signaling networks involved in DSB repair is a promising approach for enhancing cellular radiosensitivity. In mammalian cells the primary repair mechanism of radiation-induced DSBs is the non-homologous end-joining (NHEJ) pathway, in which DNA-dependent protein kinase (DNA-PK) plays a critical role. As a result, DNA-PK potentially represents an important molecular target for inhibiting DSB repair and enhancing the cytotoxicity of radiation. Using BEZ235, a novel small molecule inhibitor of DNA-PK and phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) currently in clinical trials, the first aim of this thesis was to characterize the effects of inhibiting DNA-PK on tumor radiosensitivity in vitro and in vivo. The second aim of this thesis was to examine the mechanisms through which DNA-PK inhibition improves tumor radiosensitivity as little is known about the mechanisms involved in the radiation-enhancing effects of DNA-PK blockade. BEZ235 was seen to abrogate radiation-induced DSB repair and potently increase the radiosensitivity of H460 and A549 cells, human non-small cell lung cancer (NSCLC) cell lines. BEZ235 also potentiated the anti-tumor activity of ionizing radiation in H460 xenografts. Significantly, radiation enhancement by BEZ235 coincided with a prominent p53-dependent accelerated senescence phenotype characterized by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression and senescence-associated cytokine secretion. Subsequent experiments sought to examine the mechanisms involved in the pro-senescence response of irradiated cells to BEZ235, and specifically whether selective inhibition of DNA-PK is sufficient to promote accelerated senescence after radiation. Significantly, it was shown that specific pharmacological inhibition of DNA-PK but not PI3K or mTORC1 delays DSB repair leading to accelerated senescence after radiation. It was additionally demonstrated that PRKDC knock down using small interfering RNA promotes a striking accelerated senescence phenotype in irradiated cells comparable to that of BEZ235. Collectively, these data establish accelerated senescence as a novel mechanism of radiosensitization induced by DNA-PK blockade and underline the emerging link between unrepaired DSBs and enforcement of p53-dependent accelerated senescence. These data highlight the potential benefits of using DNA-PK blockade to modulate repair of therapeutically-induced DSBs and thereby promote radiation-induced accelerated senescence. In turn, these findings provide a rationale for further pre-clinical and clinical evaluation of DNA-PK inhibitors in combination with anti-cancer agents that induce DSBs or inhibit DSB repair.
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ItemOrgan size control and the SWH pathway in Drosophila melanogaster( 2012)An important aspect of metazoan development is organ size regulation. This process is governed by both extrinsic and intrinsic mechanisms. Amongst the different signalling pathways that regulate organ growth in an intrinsic manner, the recently identified Salvador-Warts-Hippo (SWH) pathway plays a major role. Therefore, a thorough mechanistic understanding of this pathway will not only improve our fundamental knowledge of organ size regulation, but also have important implications for diseases that are caused by deregulation of size control mechanisms, such as cancer. With this incentive, the three aims of my PhD study were to study organ size control by the SWH pathway: aim 1) investigate whether Willin is a functional homologue of Expanded (Ex), aim 2) investigate how the SWH pathway receptor, Fat (Ft) cadherin regulates Ex, and aim 3) use a newly-devised genetic screening approach to identify novel growth regulatory genes. These studies were conducted in the genetically amenable model organism, D. melanogaster that has a high degree of gene conservation with mammals. The SWH pathway is evolutionarily conserved from flies to humans. Therefore, in the first aim, I investigated whether the closest human homologue of Ex, Willin has a conserved role in the SWH pathway. Results from experiments conducted in vivo in D. melanogaster epithelial tissues (imaginal discs) using an inducible willin transgene showed that: 1) Willin has a similar subcellular localisation to Ex; 2) Willin overexpression did not phenocopy Ex overexpression, and 3) Willin could not rescue the growth defect caused by loss of ex. Collectively, these data suggest that the growth-regulatory function of Ex is not conserved in Willin. This most likely can be ascribed to the lack of PPxY motifs in Willin, that are necessary for Ex to regulate tissue growth. The ‘FERM’ domain that is conserved in Ex and Willin is probably responsible for the similar subcellular localisation of these proteins. Prior studies showed that the Ft receptor affects Ex protein levels. This was hypothesised as an important mode of regulation but the mechanism was not explored. Therefore, in the second aim, I investigated whether Ft influences Ex at the translational level in a microRNA (miR) –dependent manner, or by affecting Ex protein stability. Results using miR-sensors, genetic loss-of-function (LoF) and gain-of-function (GoF) experiments together with an in vivo heat pulse chase assay showed that: 1) Ft does not regulate Ex in a miR-dependent manner; 2) Ft regulates Ex protein stability, and 3) Ft regulates Ex stability independent of Dachs (D) and Crumbs (Crb), both of which are known to regulate Ex levels. Ft’s ability to regulate Ex protein stability is likely to be important for growth control as both of these proteins represent independent upstream branches of the SWH pathway. In order to gain a comprehensive understanding of organ size regulation, in the third aim I conducted a mutagenesis screen using the eyFlp; FRT system to identify novel growth regulators on chromosome arm 2R in an apoptosis-resistant background, D. melanogaster Apaf-1-related-killer (dArk). The idea was to identify growth-regulatory genes that are pleiotropic and whose growth-regulatory function would only be uncovered in an apoptosis-resistant background. 39 mutant alleles were recovered that displayed organ overgrowth phenotypes. Complementation test with alleles in known growth regulators showed that two new alleles of hippo (hpo) were identified, as was one allele of Ubiquitin activating enzyme 1 (Uba1). Importantly, a mutant allele (d2D.2) with a cell-autonomous overgrowth phenotype was identified that showed some evidence of genetic interaction with warts (wts), pointing to a possible role in the SWH pathway. Future experiments to characterise these newly identified growth control genes will be informative for our understanding of growth control by cell-autonomous pathways (such as the SWH pathway) as well as growth control by signalling pathways that regulate tissue growth in a non-cell autonomous fashion e.g. Hedgehog (Hh), Wingless (Wg) and Decapentaplegic (Dpp).
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ItemInvestigating the effects of STAT1 expression on cytokine crosstalk and anti-tumour responses( 2012)IFNs are immunoregulatory cytokines with important roles in anti-viral and anti-tumour responses. The two major classes of IFN, type I which includes IFNα and IFNβ and type II IFN which consists solely of IFNγ, bind to distinct receptors and canonically trigger the phosphorylation of signal transducers and activators of transcription (STAT) 1. In addition to the canonical signalling pathways, both type I and type II IFNs can activate STAT1 independent signalling responses. Despite signalling through distinct receptors it is evident that the functions of type I and II IFNs are linked. Pre-treatment of cells with low levels of type I IFNs leads to more potent type II IFN response and interestingly loss of the type I IFN receptor results in diminished type II IFN responses. In the absence of tonic IFNα/β, cells have low basal expression of STAT1, and other signalling intermediaries, and we hypothesised that this may underpin the decreased response to IFNγ in these cells. As STAT1 is the canonical signal transducer activated following IFNγ receptor binding, we proposed that reconstitution of cells lacking tonic IFNβ signalling with STAT1 would restore IFNγ induced signalling. Data from microarray analysis of IFNγ treated wild type, Ifnar1-/- and empty vector transduced and STAT1-reconstituted Ifnar1-/- cells was used to identify biological pathways in which STAT1 reconstitution was most able to rescue IFNγ-mediated gene regulation. We confirmed that IFNγ-induced expression of genes critical for MHC class I antigen presentation was attenuated in response to IFNγ in Ifnar1-/- cells compared to wild type cells. Furthermore, the IFNγ-dependent induction of these genes achieved wild type levels in the STAT1-reconstituted in the Ifnar1-/- cells. In addition to the effects on IFNγ responses, the diminished activation of STAT1 following IL6 treatment in cells lacking tonic IFNβ signalling also achieved wild type levels in STAT1-reconstituted Ifnar1-/- cells. The studies described herein demonstrate that the regulation of the basal expression of signalling intermediaries, including transcription factors, by tonic IFNαβ signalling, is a mechanism for crosstalk between cytokines especially the type I and type II IFNs. The contribution of STAT1-independent signalling to IFN-dependent biological responses is currently poorly defined however; they may include anti-viral and anti-proliferative responses. IFNs are important mediators of anti-tumour responses with direct and indirect actions of IFNγ being associated with the inhibition of tumour progression. Therefore we sought to determine whether STAT1-independent signalling contributes the direct anti-tumour actions of IFNγ. Growth of Stat1-deficient tumours was accelerated in the absence of IFNγ responses however deficiency of Ifnγ or Ifngr expression resulted in equivalent growth suggesting a lack of direct anti-tumour effects of IFNγ on Stat1-deficient tumours. The STAT1-expressing tumours displayed a similar pattern of IFN responsiveness in vivo as the Stat1-deficient suggesting that IFNγ mediated response of the host to tumours derived in the absence of STAT1 rather than directly affecting the tumour. Finally, the ability of the immune system to inhibit tumour growth is often modulated by interaction with tumour cells. In this study we examined the effects of STAT1 expression within tumour cells on anti-tumour immune responses. Similar to previous reports, these studies confirmed a role for the immune system in inhibiting the progression of Stat1-deficient tumours. NK cells were the principle cytotoxic lymphocytes involved in the immunosurveillance of Stat1-deficient tumours however we also discovered a role for CD8+ T cells. Reconstitution of STAT1 into Stat1-deficient tumours rescued their ability to regulate MHC class I expression however it did not affect direct tumour lysis by NK cells in vitro. Moreover, although STAT1-reconstitution of the Stat1-deficient tumour reduced the requirement for NK cells in the anti-tumour response in vivo, CD8+ T cells and NK cells were both still involved in the immunosurveillance of the STAT1-expressing tumour. Therefore, in addition to the effects regulation of STAT1 expression has on cytokine crosstalk, this research has established that anti-tumour immune responses can be shaped by the regulation of STAT1 expression within tumour cells.
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ItemDeciphering the molecular and biological processes that mediate histone deacetylase inhibitor-induced thrombocytopenia( 2012)Somatic mutations in a variety of genes involved in cell cycle, signalling, differentiation, proliferation and apoptotic pathways can lead to the initiation and progression of malignancy. In addition, multiple ‘epigenetic’ changes have been described, mediated by enzymes linked to DNA packaging and transcription, which are not characterised by changes in the primary sequence of the DNA yet influence the pattern of gene expression. The histone deacetylase inhibitors (HDACi) are new anti-cancer drugs which induce chromatin remodelling by a net increase in histone acetylation, altering gene expression as well as influencing the function of non-histone proteins by direct acetylation. These changes mediate multiple biological responses such as tumour cell apoptosis and cell cycle arrest, as well as inhibition of angiogenesis and modulation of innate and adaptive immunity. Two structurally different HDACi, panobinostat and romidepsin have had success in clinical trials, particularly in the treatment of cutaneous T-cell lymphoma (CTCL). Although very well tolerated by patients, both cause severe reductions in blood platelet numbers, called thrombocytopenia. Platelets are vital for the prevention of bleeding via the closure of any vessel wall defect. Thrombocytopenia therefore compromises the use of these highly effective novel drugs, particularly in combination treatment strategies. This study examined the pathophysiology of HDACi-induced thrombocytopenia and showed this clinical problem could be circumvented, potentially allowing more patients to receive treatment. Following the finding that HDACi induced dose-dependant reductions in platelet number in C57BL/6 mice, susequent platelet production and half life studies demonstrated that this was not due to HDACi reducing platelet half life or inducing platelet apoptosis. These results were in accordance with mice with genetic ablation of pro-apoptotic molecules also becoming thrombocytopenic post HDACi. Pro-platelet assays in primary cultured megakarycytes confirmed a marked reduction in pro-platelet formation following HDACi treatment. Thrombopoietin (TPO)-mimetics are clinically developed growth factors, which increase megakaryocyte production of platelets. TPO-mimetics given to HDACi treated mice prevented thrombocytopenia occurring in both wild type and tumour bearing settings, a result which may allow patients to continue to receive HDACi despite thrombocytopenia in the future. Decreased pro-platelet formation following HDACi was shown associated with an increase in phosphorylated myosin light chain (pMLC) both in-vitro and in vivo. HDACi increase pMLC levels by causing the proteasomal degradation of the cytoskeletal remodelling Rho-GTPase proteins CDC42, Rac1 and RhoA. Immuno-precipitation studies demonstrated HDACi treatment resulted in the the dissociation of GTPases dissociate from their chaperone protein, Rho-GDI, causing their rapid, transient activation and subsequent degradation. Confocal microscopy confirmed the specific degradation of non-plasma membrane bound Rho-GTPases. Ultimately, exhaustion of the available pool of the Rho-GTPases occurs, resulting in a reduction of both the activated and total fractions available to the cell. HDACi are known to acetylate the heat shock protein (HSP)-90, releasing its chaperone proteins for degradation. Using immuno-precipitation studies, this thesis shows HSP90 to co-chaperone the Rho-GTPases with Rho-GDI, explaining why inhibition of HSP90 recapitulates the effects of HDACi, specifically dissociation of Rho-GTPases from Rho-GDI followed by their degradation, increasing pMLC and reducing pro-platelet formation. The data therefore suggests HDACi-induced acetylation of HSP90, and potentially Rho-GDI may be responsible for causing the clinical problem of thrombocytopenia.
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ItemThe role of c-Fms and GM-Csf in intestinal biology( 2012)Csf-1 and GM-CsF are important growth factors required for proliferation, cell fate specification and differentiation of hematopoietic stem cell and progenitors. Their role in immunity, inflammation and progression of both hematopoietic and non-hematopoietic cancers has been extensively investigated. Despite their known role in inflammation and the presence of the receptors for Csf-1 (c-Fms) and GM-Csf (GMCsfR) on small intestinal epithelial cells, there is little evidence for their contribution to the gastrointestinal homeostasis. Recent investigation however confirmed that the complete loss of c-Fms can lead to an alteration of intestinal proliferation and differentiation. However, it was not clear whether this effect was direct or indirectly mediated by tissue macrophages that also express Csf-1 and c-Fms. Furthermore, the role of GM-Csf has not been investigated previously in terms of small intestine and colon homeostasis. In this thesis, I explored the role of c-Fms and GM-Csf in intestinal biology. The first aim of this study was to directly examine the effect of loss of c-Fms in small intestine using organoid cultures and a tamoxifen inducible, adult intestine specific c-fms conditional knock-out mouse model. The expression pattern of c-Fms in SI epithelium was investigated. The intrinsic deficiency of c-Fms produced a reduction in organoid forming efficiency which was associated with down-regulation of intestinal stem cell gene expression at the mRNA level. In vivo loss of c-Fms produced a decrease in Paneth and enteroendocrine cell numbers as well as a positional shift of proliferating cells towards the bottom of crypts previously occupied by Paneth cells. The second aim of this study was to examine the effects of Csf-1 and IL-34 as well as the intestinal mitogens Wnt3a and R-spondin1 on proliferation in immortalized colon cell lines. Furthermore, I explored the hypothesis that CD24+ colon cells provide a niche to colonic stem cells with the underlying rationale that CD24+ Paneth cells are known to contribute to the maintenance of SI stem cells. Finally, the effect of intestine specific loss of c-Fms in adult colon was investigated. It was found that only Csf-1 or R-spondin1 alone can stimulate colon epithelial growth and when combined together this stimulatory effect was further increased. CD24+ colon cells were enriched for goblet cells and a cell population in which Lgr-5 expression was relatively high. The expression of CD24 expression was surprisingly not affected by the loss c-Fms in colon crypts. In contrast to the global c-fms knock-out study, intestine specific inactivation of c-fms led to unimpaired epithelial cell proliferation and differentiation at least over 4 to 8 weeks. The last aim of my thesis was to explore the role of GM-Csf in SI and colon homeostasis through the use of mice lacking of GM-Csf expression. I found that the loss of GM-Csf resulted in a dramatic reduction of intestinal stem cell and proliferation gene expression in gm-csf -/- female mice. Interestingly this was not the case in gm-csf -/- male mice. In addition, intestinal proliferation and differentiation were not altered by the loss of GM-Csf in female mice. These data emphasize the importance of sex related differences in the regulation of intestinal stem cells in mice. Collectively, these data suggest that c-Fms signaling is important for SI homeostasis by mediating Paneth and enteroendocrine cell differentiation and promoting stem cell function. The function of c-Fms might be dispensable for colon epithelial cell differentiation in adult mice or perhaps the absence of c-Fms might be compensated for by other essential factors released by the stroma such as macrophages or myofibroblasts. Moreover, c-Fms activation by Csf-1 may co-operate with Wnt signaling to promote colon cell proliferation. In addition, my work has unraveled a potential role for GM-Csf in sex specific transcriptional regulation of intestinal stem cell and proliferation genes. Overall, my thesis studies have advanced our understanding of the role of c-Fms and GM-Csf in intestinal biology.
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ItemAn assay to screen for mutant p53 gain of function using high content imaging( 2012)The p53 tumour suppressor gene is mutated in approximately half of all human cancers. These mutations not only inactivate the growth inhibitory functions of wild type p53 but certain mutations also confer additional oncogenic properties. These gained functions may contribute to the increased growth rate, resistance to apoptosis, reduced chemosensitivity and increased invasiveness of mutant p53 bearing tumours. For the purposes of discovering novel mediators of mutant p53 gain of function, a multi-parameter assay was developed for future use in a high throughput siRNA screen. Mutant p53 expressing cells were demonstrated to display an increased migratory phenotype in vitro compared to p53 null cells. A wound healing endpoint assay was adapted to a 96 well format using automated liquid handling and image capture. An image analysis algorithm was designed to accurately measure cell migration in a high throughput manner. Performing a low throughput pilot screen of a panel of siRNAs demonstrated that the general migration machinery was necessary for migration. However, knockdown of known components of the mutant p53 pathway failed to provide the robustness necessary for a high throughput screen. p53 protein stability is known to be regulated at multiple levels involving a complex network of feedback loops. Unlike the case with wtp53, the regulation of mutant p53 is only partially understood. A high content assay for mutant p53 stability was developed. The immunofluorescent staining protocol was adapted to a 384 well assay and an algorithm was designed to accurately measure pixel intensity in the nucleus and cytoplasm. A statistically significant and robust downregulation of mutant p53 was measured with siRNA against mutant p53. A pilot screen in endogenous mutant p53 cell lines demonstrated a sufficiently large assay window to identify siRNAs that reduce mutant p53 stability. A genome wide siRNA screen for genes that reduce mutant p53 could uncover novel therapeutic targets, which will enable the design of new molecularly targeted therapeutics. Drugs able to impede the action of mutant p53 will be relevant to a significant proportion of human cancers.
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ItemIroning out the involvement of tau protein in neurodegenerative diseases( 2012)Tau protein has been extensively implicated in Alzheimer’s disease (AD), Parkinson’s disease (PD), and other neurodegenerative diseases which exhibit tau depositions, termed tauopathies. Brain iron accumulation is a cooccurred pathological feature of many tauopathies and hypothesized to contribute to neurodegeneration by engendering oxidative stress. It is currently unknown what, if any, link exists between tau and iron accumulation in these diseases. The aims of this thesis were to understand, 1) how does tau participate in neurodegeneration; 2) whether tau is involved in brain iron homeostasis; and 3) whether this putative interaction contributes to neurodegeneration in the tauopathies. The normal function of tau has remained elusive, partly owing to the fact that tau knockout mice (tau KO) have been reported to be viable and fertile without behavioural deficits or neurodegeneration. These mice, however, have not extensively been investigated older than 7-months of age. Therefore, an analysis of aged (12-24 months) tau KO mice was undertaken in this thesis. Aged tau KO mice exhibited features of dementia (reduction of brain wet weight, neural cortical atrophy and cognitive impairment) and parkinsonism (L-DOPA responsive motor disability, neuron loss in substantia nigra [SN], striatal dopamine reduction and dopaminergic terminal atrophy). These observations may have implications for AD, which have been reported to exhibit reduced soluble tau in affected regions, and also PD, which was shown to have a similar reduction in this thesis. Iron accumulation was observed in the brains of aged tau KO mice. This specific brain-iron accumulation was prevented by chronic, orally administration of clioquinol, a moderate iron chelator previously reported to prevent MPTP-induced parkinsonism. When administration commenced before disease onset (6-months-of-age), chronic iron chelation prevented the onset of disease phenotype and neuronal loss in tau KO mice at advanced age. Likewise, treatment with clioquinol after the onset of disease (12-months-of-age) prevented further atrophy, and ameliorated behavioural disability. Therefore, tau depletion (as also observed in AD and PD) may engender pro−oxidant neuronal Fe2+ elevation preceding neurodegeneration. The mechanism of tau-induced iron accumulation was investigated in this thesis, which revealed a previously overlooked functional interaction between tau and the AD-implicated, amyloid precursor protein (APP). Immature APP undergoes several post-translational modifications before it is transported to the cell surface where it functions as an iron-export ferroxidase. Deletion of tau decreased the presence of mature APP presented on the cell surface which prevented the efficient neural export of iron. Disrupted trafficking of APP could therefore explain iron accumulation in the tau KO mouse, and in diseases exhibiting soluble tau reduction. Tau reduction may also be pharmacologically induced by the cation, lithium, which is a drug for bipolar disorder. In this thesis, lithium chloride caused intracellular iron accumulation and did not affect copper or zinc. This accumulation was abolished when treated to neurons that lacked tau or APP, demonstrating that lithium-induced iron accumulation was mediated by disruption to the tau-APP axis presented in this thesis. Lithium treatment has been previously associated with Parkinsonian side-effects; in this thesis, oral administration of lithium (in an upper-therapeutic range) to background mice caused brain-iron accumulation accompanying Parkinsonian neurodegeneration (motor disability, neuronal loss in SN, dopamine reduction in striatum). Given these striking findings, an MRI-analysis of iron in the brains of individuals who were treated with lithium (for three months) was performed, which revealed evidence of reversible iron elevation in selected regions upon treatment. This thesis, which investigated the function of tau and its loss-of-function phenotype, concluded that the reduction of soluble tau primes neurons for age-dependent neurodegeneration by decreasing APP-mediated iron export. Therefore, strategies that maintain tau solubility, or reduce iron content, may be promising therapeutic strategies for diseases featuring soluble tau loss such as AD and PD.
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ItemTumour-specific mutant protein targets in colorectal cancers with microsatellite instability( 2012)Background: Frameshift mutations in microsatellite instability high (MSI-High) colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD), but nonsense transcripts with a coding microsatellite (cMS) in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay. Therefore, NMD-resistant transcripts are a likely source of expressed mutant proteins in MSI-High tumours. Methods: Using antibodies to the conserved N-termini of predicted mutant proteins, MSI-High colorectal cancer cell lines were analysed by immunoprecipitation and Western blot experiments for examples of naturally expressed mutant proteins arising from frameshift mutations in cMS. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA) knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines. Mutated genes were assessed for predicted T cell epitopes using an in silico approach to identify the most promising immunological targets. Results: Mutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK), but not NMD-sensitive transcripts (BAX, CASP5, MSH3). Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12) were found to be mutated in at least 5 of 11 (45%) of the MSI-High cell lines tested. A set of eleven mutant proteins was predicted to include T cell epitopes for 99% of MSI-High colorectal cancer patients, with an average of four mutant protein targets expected per tumour. Conclusions: NMD-resistant transcripts give rise to expressed mutant proteins in MSI-High colon cancer cells. These mutant proteins may have diagnostic and therapeutic applications in familial and sporadic MSI-High tumours. A set of commonly expressed MSI-derived mutant proteins could serve as cancer specific immune targets in a vaccine targeting MSI-High colonic tumours.