Pathology - Theses
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ItemTherapeutic implications in genetic prion disease( 2011)Prion diseases are fatal neurodegenerative diseases caused by the misfolding of the prion protein (PrP). Of the three subsets of prion diseases (sporadic, genetic, acquired), genetic prion disease is associated with a mutation of PrP, leading to an increased disposition for the mutant PrP to misfold, and adopt a disease associated conformation (PrPD ). The following project investigated how a mutation of the prion protein, the P102L mutation, affected the disease associated properties of PrP and the impact of this mutation upon therapeutic outcome. The 102L and 101L mutation were introduced into human and mouse PrP respectively and expressed in rabbit kidney epithelial (RK13) cells to further investigate the effect of this mutation on the prion protein, and the outcome of prion infection. The 102L and 101L mutant PrP produced by RK13 cells was found to increase the disease associated characteristics of human and mouse PrP. Mutant PrP was also found to enhance the binding of PrP to glycosaminoglycans, a cofactor potentially involved in conversion of PrPC to PrPD . The human and mouse PrP expressing RK13 cell with or without the 102L/101L mutation were exposed to several human and mouse strains of prions to determine the susceptibility of the cells to prion infection. Prion infection of RK13 cells expressing 102P or 102L human PrP (huPrP) was not observed. RK13 cells expressing 101L mouse PrP (moPrP) were more susceptible to infection from two strains of prions, compared with 101P moPrP expressing RK13 cells. The enhanced susceptibility of 101L moPrP expressing RK13 (moRK13) cells was determined to be due to increased uptake of protease resistant PrP (PrPres ), mediated aberrant glycosaminoglycan binding. The therapeutic efficacy of glycosaminoglycan based therapy was investigated in the 101L model of genetic prion disease to determine if the enhanced binding of 101L moPrP affected the therapeutic action. Investigation of the membrane localisation of 101P and 101L moPrP by subcellular fractionation identified a subtle difference between 101P and 101L moPrP. A reduction in the proportion of 101L moPrP was detected in the lipid raft associated fractions compared with 101P moPrP, suggesting that a population of 101L moPrP is aberrantly located in another membrane domain or intracellular compartment. The results of the study suggest that the enhanced binding of 101L moPrP to glycosaminoglycans mediates the association of 101L PrP in an aberrant membrane domain, and mediates conversion of 101L moPrP to PrPres in spite of treatment with glycosaminoglycan or lipid raft altering therapeutics. The prophylactic treatment of persons found to have pathogenic mutations associated with genetic prion disease provides the best opportunity of intervening in prion disease progression, more-so than sporadic and acquired forms of prion disease which are treatable only after transmission or disease is detected. The results generated in the current study suggest that the generation of PrPres in genetic forms of prion disease occurs in additional, or different cellular sites to other forms of prion disease, where, potentially, current therapeutics have little or no effect. Therefore further testing of potential therapeutics developed for treatment and prevention of prion disease is required before use in genetic forms of prion disease is granted.
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ItemIntegrin β3 as a therapeutic target for breast cancer metastasis to bone( 2011)Breast cancer is the most common cancer in women, with patients currently having high survival rates if the disease is confined to the breast. However, these survival rates drop drastically if the cancer has metastasised to distant sites. Previous studies have shown both tumour and stromal integrin β3 are involved in breast cancer metastasis, yet questions remain regarding its specific role in the process. In particular, whether integrin β3 is essential for metastasis and if so, to which organ and at which stage of the metastatic cascade it is required. Furthermore, the relative contribution of stromal and tumour integrin β3 in tumour growth and metastasis needs to be clarified. To address these questions, the transplantable 4T1 model of breast cancer with spontaneous metastasis was utilised. RNA interference was used to suppress tumour integrin β3 expression in the highly metastatic 4T1.2 and 4T1BM2 cells, and investigate whether tumour integrin β3 is essential for metastasis. Additionally, the contribution of stromal cell populations expressing integrin β3 was investigated in integrin β3 knockout mice. Lastly, the therapeutic potential of targeting integrin β3 was investigated using the disintegrin DisBa-01. Downregulation of tumour integrin β3 expression induced a coordinated decrease in the surface level of the integrin αv subunit. Functional assays revealed integrin αvβ3 dependent decreases in adhesion, migration and MMP9 secretion. In vivo, downregulation of integrin αvβ3 had no affect on primary tumour growth, but significantly reduced spontaneous metastasis to bone, lung and other soft tissues, indicating integrin β3 is required for metastasis to multiple sites. Surprisingly, unlike studies in other tumour types, integrin β3 suppression in 4T1.2 cells did not impact on experimental breast cancer metastasis to lung or bone, suggesting it is acting at an early stage of metastasis. Unlike reports in other models, primary tumour growth was not affected by the loss of stromal integrin β3, indicating orthotopic growth of breast tumours is not dependent on the expression of integrin β3 in stromal cell populations. However, metastasis to bone, but not lung, was reduced in integrin β3 null mice. These observations allow the reconciliation of previous studies that reported conflicting conclusions with regard to the role of stromal integrin β3 in tumour growth and metastasis. In vitro treatment of 4T1BM2 cells with DisBa-01 achieved similar results to down-regulation of the protein by shRNA, suppressing integrin αvβ3 dependent adhesion, proliferation, migration and MMP9 secretion. Unfortunately, these promising results did not translate to effects on tumour growth and metastasis in vivo, and neither experimental nor spontaneous metastasis were suppressed by DisBa-01 treatment under the conditions tested. However, the results obtained provide valuable information with regards to future protocol design. This study demonstrates that tumour expressed integrin β3 is essential for spontaneous breast cancer metastasis to multiple organs, and provides evidence the protein acts at an early stage in the process. It also supports the role of stromal integrin β3 in metastasis to bone, but not to lung. Taken together, these data suggest both tumour and stromal integrin β3 are potential therapeutic targets, with tumour integrin β3 contributing to the process to a greater degree.
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ItemUsing DNA methylation to study benign breast and breast tumours( 2011)Breast cancer is a heterogeneous disease comprising of numerous distinct entities that have different biological and clinical behaviours. Clinical diagnosis and accurate prognosis are not infrequently difficult to make, leading to over or under treatment. Although improvements in survival have been observed over the last 10 years, a substantial number of women die from the disease. A better understanding of the molecular mechanisms and signalling pathways that are involved in breast cancer development (e.g. the hypoxia pathway) as well as a deeper appreciation of the role of epigenetic alterations through DNA methylation, will greatly improve our combative efforts against breast cancer. The aim of this thesis is to assess DNA methylation of candidate genes in breast cancer with the goal of identifying biologically important pathways of hypoxia-inducible factor 1 (HIF-1) and to find novel diagnostic and prognostic markers. HIF-1 is the master regulator of cellular oxygen homeostasis that is induced by hypoxia. DNA methylation assessment was performed on the genes that regulate HIF-1 to understand the link between DNA methylation changes in cancer and the altered response to hypoxia. However, promoter methylation of a panel of HIF-1α regulating genes (VHL, CUL2, TCEB1, TCEB2, RBX1, PHD1, PHD2, PHD3, FIH, SDHA, SDHB, SDHC, SDHD, FH and SIAH2) was not present in breast cancers. This showed that promoter methylation is unlikely to be an important mechanism in stabilising HIF-1 in breast cancers through the down-regulation of the expression of these genes. Nevertheless, the study has demonstrated that the loss of expression of CITED4, a negative regulator of HIF-1, in breast cancer is in some cases due to DNA methylation that leads to increased HIF-1α activity and its downstream genes. Demethylation and histone modification was able to reactivate CITED4 gene expression in the methylated cell lines and lead to changes in tumour behaviour. The data of the study also suggests that histone modification may be the alternative epigenetic mechanism that regulates CITED4 expression. The distinction between fibroadenomas and benign phyllodes, which are both fibroepithelial tumours of the breast, can be difficult with no specific features that can reliably distinguish these neoplasms. I have demonstrated that the strong methylation of RASSF1A and/or TWIST1 was associated with phyllodes tumours. TWIST1 methylation is also correlated with increasing malignancy in phyllodes tumours. Hence, RASSF1A and TWIST1 methylation, and potentially other markers such as APC and WIF1, may be useful clinically for distinguishing phyllodes tumours from fibroadenomas. Additionally, the absence of frequent methylation in fibroadenomas supports a non-neoplastic origin. DNA methylation profiling and array comparative genomic hybridisation have been demonstrated as a valuable diagnostic tool in differentiating newly arisen tumours for ‘true’ relapses from new independent tumours. DNA methylation profiling results showed that ipsilateral tumours have a higher chance to be a recurrent tumour, whereas contralateral tumours were mostly a de novo tumour. Overall, this thesis have demonstrated that DNA methylation is an important mechanism in understanding the depth of hypoxia in breast cancer and has the potential to be a useful diagnostic tool in helping to answer some clinical questions in breast cancer.
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ItemOcular pathology: a compilation of research articles( 2011)These articles comprise publications in the field of ocular pathology over 15 years, with particular interest in lymphomas of ocular adnexal and intra-ocular sites; ocular impression cytology and its application in the diagnosis and management of ocular surface squamous neoplasia; extraocular muscle in ageing and disease; diode laser photocoagulation of ciliary body in intractable glaucoma; rare and unusual ocular infections and tumours and early giant cell arteritis.
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ItemConformational interchange in the KirBac3.1 K+ channel investigated by X-ray crystallography( 2011)Potassium channels are ion-selective pores that facilitate the passive transport of K+ across the cell membrane. The flow of potassium through the pore is switchable, a process known as gating. Two regions of the potassium channel have been previously implicated in direct control of conduction—the intracellular aperture, defined by the crossing point of the helices that line the pore, and the selectivity filter, the narrow, carbonyl-lined section of the conduction pathway near the extracellular mouth of the channel, which enables the selective and efficient conduction of potassium ions. Historically, the bundle-crossing has been characterised as the primary gate to ion conduction, and structural studies of the prokaryotic potassium channels KcsA and MthK support a model in which the pore-lining inner helix regulates conduction by bending at a flexible “glycine hinge” in order to enlarge the intracellular aperture of the transmembrane pore. The selectivity filter has long been implicated in the time-dependent rundown of Kv channel activity known as C-type inactivation. It is only recently, however, that functional studies of some channel families, such as the KCa and BK potassium channels, have proposed a role for the selectivity filter as the primary gate to ion conduction. Structural comparisons of channels from different families (such as MthK and KcsA) suffer from the limitation that conformational changes corresponding to physiological functional states cannot be readily discerned from those which are specific attributes of the family. In this thesis, we present a structural investigation of conformational changes in a single type of potassium channel, KirBac 3.1, accomplished by X-ray crystallographic analysis of the crystalline protein in multiple crystal forms and conformational states. This analysis reveals a conformational change consisting of a rotation of the cytoplasmic assembly about the molecular axis of the channel, correlated with specific changes in the selectivity filter configuration of the channel that are indicative of channel inactivation. Kir K+ channels such as KirBac3.1 are blocked by intracellular polyamines, producing the inwardly biased current characteristic of inward rectifiers. In this study, we have identified two polyamine binding sites within KirBac3.1, one “shallow” site within the cytoplasmic assembly, at the interface between subunits, and the other deep within the transmembrane pore, on the molecular axis of the channel. We have also identified a novel ligand-binding pocket within the cytoplasmic assembly of KirBac3.1 that appears to be conserved in the available structures of eukaryotic Kir channels. In KirBac3.1, this pocket serendipitously binds components of the crystallant mixture, namely Fos-choline detergents and an oxidised derivative of the alkylphosphine reducing agent, TCEP.
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ItemInvestigating the functional role of the amyloid precursor protein’s copper binding domain( 2011)Alzheimer’s disease is a progressive and eventually fatal neurodegenerative disorder characterized by specific proteins deposition in the brain: amyloid plaques and tau tangles. While age represents the major risk factor for AD, the mechanisms triggering the pathology remain unclear. According to the amyloid cascade hypothesis, accumulation of the main component of the amyloid plaques, the β-amyloid (Aβ) peptide, is crucial for the onset of the disease. This phenomenon is mainly accounted for by mis-metabolism of the Amyloid Precursor Protein (APP) from which Aβ is derived and by low Aβ clearance. Copper dyshomeostasis, which has been reported in AD, contributes to Aβ accumulation by influencing APP metabolism. Conversely, APP regulates copper homeostasis, a process in which imbalances can lead to oxidative stress and inflammatory response. This study investigated the relationship between APP and copper in terms of APP-mediated copper homeostasis and toxicity, as well as copper-mediated APP metabolism. The role of the APP copper binding domain (CuBD), and in particular the copper binding site histidine residues, was examined with the results revealing that the domain mediates APP metabolism and structure stability. This study has significantly contributed to the understanding of APP CuBD in modulating APP metabolism and stability, and highlights the potential of this domain as a novel therapeutic target in AD.
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ItemMechanisms of action of histone deacetylase inhibitors( 2011)The opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in tightly regulating gene expression through regulated chromatin remodelling. It is now well-established that alterations in epigenetic regulation are commonplace in haematological malignancies and solid tumours and HDACs have been identifi ed as promising therapeutic targets for the reversal of aberrant epigenetic states associated with cancer. Structurally diverse HDAC inhibitors (HDACi) have been developed that can bind to HDACs, inhibit their enzymatic activity and induce histone acetylation. In addition to the acetylation of histones, HDACi can also cause the acetylation of non-histone proteins which might contribute to the anticancer effects of these compounds. For example, the degradation of Bcr-Abl following HDACi-mediated hyperacetylation of the molecular chaperone HSP90 has been proposed to be a major effector mechanism of HDACi. HDACi can induce a range of biological responses including cellular differentiation, suppression of angiogenesis, modulation of anti-tumour immunity and induction of tumour cell apoptosis. Moreover, HDACi can inhibit cell proliferation by blocking progression through the G1 or G2/M phases of the cell cycle and upregulate p53- independent expression of p21waf1/cip1. Although the mechanisms of action of HDACi remain largely unknown, the therapeutic effects of HDACi treatment can be affected by p21waf1/cip1 expression which infl uences the decision of a tumour cell to undergo cell cycle arrest or apoptosis. Thus, it may be a combination of many events that is important for the overall therapeutic activities of these compounds. Most HDACi currently being tested in the clinic, have broad inhibitory activity against the eleven class I, II and IV HDACs. While isoform-specifi c HDACi are currently in development, whether or not these inhibitors will have lower toxicity profi les while maintaining biological anti-cancer activity remains to be determined. In this study, we utilized the recently developed HDACi MRLB-223 that selectively inhibits recombinant HDAC1 and 2 at low nanomolar concentrations to determine if specifi cally targeting certain class I HDACs would result in similar induction of apoptosis and therapeutic effi cacy as seen using and the broad spectrum HDACi vorinostat. While MRLB-223 could induce apoptosis in B cell lymphomas derived from Eμ-myc transgenic mice and provided a therapeutic benefi t to mice bearing Eμ-myc lymphomas, the kinetics of histone hyperacetylation and tumour cell apoptosis induced by this compound was delayed, and its potency in vivo was inferior to that observed using vorinostat. In addition, MRLB-223, vorinostat, and the class I specifi c inhibitor romidepsin, were all capable of killing the IL-3-dependent FDCP1 mouse myeloid cell line that was engineered to grow independently of IL-3 through forced expression of the HSP90 client protein Bcr-Abl. All three inhibitors induced degradation of Bcr-Abl and hyperacetylation of HSP90. The degradation of Bcr-Abl was caspase dependent, and occurred as a downstream consequence of tumour cell apoptosis. Furthermore, we demonstrated that p21waf1/cip1 did not have tumour suppressor function as shown by the loss of CDKN1A in our Eμ-myc transgenic mouse model. Knockout of CDKN1A did not sensitise the lymphoma cells to HDACi-induced apoptosis, nor did it prevent HDACi-induced cell cycle arrest. These results challenge some the current dogma regarding the molecular events that underpin the anti-tumour activities of HDACi, and indicates that many of the current mechanistic models may be too simple. This research provides important insight into the mechanisms of action of small molecules with diverse HDAC-inhibitory specifi cities and provides evidence that: (i) selective inhibition of HDACs 1 and 2 may not be the optimal strategy for the clinical development of more potent and tolerable HDACi; (ii) degradation of HSP90 client proteins via the inhibition of HDAC6 is not a major effector mechanism of these agents, and (iii) HDACi-induced apoptosis and cell cycle arrest can still occur in the absence of p21waf1/cip1.
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ItemThe role of involved node radiotherapy (INRT) as a new application of radiotherapy in the management of limited stage lymphomas( 2011)Radiotherapy is well recognised as an effective treatment modality for Hodgkin lymphoma (HL), follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), and is routinely incorporated into the treatment strategies for favourable-risk, limited stage disease to optimise local control and thereby improve progression-free survival. However, long-term survivors are at risk of late radiation-induced toxicities, including the risk of second malignancy. The rationale of involved node radiotherapy (INRT) is to reduce the size of the radiotherapy field to cover only the original sites of lymphoma, with the aim of lowering the incidence of radiation-induced toxicities whilst maintaining high rates of local lymphoma control. Reducing the radiotherapy field size lowers the radiation exposure to organs at risk, and this is likely to be associated with a lower incidence of radiation-induced toxicities. However, in practice it is difficult to quantify the risk of late toxicities due to the long latency period and the large numbers of patients required to detect the less common effects. Therefore, reducing the radiation dose parameters of organs at risk is proposed as a surrogate end point for lowered risks of late, radiotherapy-induced toxicities. The aims of this research are to assess the patterns of failure and survival rates of patients treated with INRT for favourable-risk, limited stage lymphoma (HL, FL, DLBCL), compared to patients receiving larger, conventional radiotherapy fields. For each lymphoma group, a population-based, retrospective study is performed to analyse these endpoints. Additionally, a radiotherapy planning study is performed on 10 randomly selected female patients with supra-diaphragmatic HL, to quantify the dosimetric advantages of INRT compared to larger conventional radiotherapy fields, on organs at risk of late toxicities. The results of these studies demonstrate that INRT fields can safely replace larger, conventional radiotherapy fields in patients with limited stage HL, FL and DLBCL, without detrimental effects on prognosis. Furthermore, supra-diaphragmatic INRT fields reduce the radiation exposure to lungs, breasts and thyroid, and may thereby reduce the risk of second malignancy in these organs. In conclusion, replacing conventional radiotherapy fields with smaller INRT fields is likely to improve the therapeutic ratio in long-term survivors of favourable-risk, limited stage HL, FL or DLBCL.
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ItemEvaluation of high resolution melting methodology for biomarker studies in non-small cell lung cancer( 2011)The adoption of molecularly targeted therapies in non-small cell lung cancer (NSCLC) has demonstrated the clinical efficacy of treating individual patients based on tumour molecular defects, emphasising the necessity of accurate and reliable methodology for molecular biomarker studies. High resolution melting (HRM) is a fast, sensitive, and cost-effective methodology that can screen both genetic and epigenetic biomarkers. Using HRM-based assays developed as part of this thesis, testing for a range of genetic and epigenetic biomarkers that have a predictive role for various therapies in NSCLC has been studied with a focus on the problems associated with diagnostic material. Mutational profiling of seven selected genes in the EGFR signalling pathway (EGFR, HER2, KRAS, BRAF, PIK3CA, and AKT1/3) was performed in a panel of 200 NSCLC samples that had been sent for diagnostic EGFR testing. Gain-of-function mutations were detected in 54% of the NSCLC samples. All 75 EGFR mutations that were previously identified by Sanger sequencing were detected by HRM analysis. Forty nine per cent of the NSCLC tumours harboured either EGFR exon 18-21 mutations (36.5%) or KRAS codon 12 and 13 mutations (12.5%). Somatic PIK3CA (3.5%), BRAF (3%), and AKT1 (0.5%) mutations were less frequently detected. EGFR, KRAS, and BRAF mutations were mutually exclusive whereas concomitant PIK3CA and EGFR mutations were detected in four NSCLC tumours. Limited copy number (LCN)-HRM was developed for screening and detection of low levels of mutations present below the sensitivity of Sanger sequencing as the limited sensitivity of this method is a particular problem in lung cancer diagnostics. The analytic sensitivity of HRM (5%) was shown to be greater than of Sanger sequencing (10%), demonstrating that low levels of mutations (5-10%) was detectable by HRM only. When clinical samples showing the discordant results between Sanger sequencing and HRM were tested by LCN-HRM, both low levels of true mutations and PCR artefacts were detected. Template-mediated PCR artefacts, mainly transitional changes (G>A and C>T), were prevalent in DNA extracted from formalin-fixed paraffin-embedded tissues. STK11 mutations were screened by HRM in a panel of tumours from 195 NSCLC patients who were surgically treated in combination with platinum-based chemotherapies. A total of 27 non-synonymous STK11 mutations (14%) were detected, including 9 nonsense, 5 frame-shift, 6 missense, and 7 splice site variants. The majority of STK11 mutations (93%) were detected in the kinase domain. STK11 mutations were significantly associated with worse progression free survival and overall survival of the NSCLC patients. The frequency of promoter region methylation in two sets of genes, DNA repair genes and selected genes previously associated with recurrence after surgery, was examined in 56 N1 tumours using methylation sensitive (MS)-HRM. DNA methylation was not detected in BRCA1, MLH1, and XPC, contrary to previous reports in the literature. Methylation was detected at a low frequency in ERCC1 (2%) and RAD23B (2%). Modest levels of MGMT methylation was seen in more individuals (12.5%) and was significantly associated with the ‘T’ allele of the MGMT promoter rs16906252 SNP (P<0.0001). A high frequency of methylation in the APC, CDH13, RASSF1A, and p16 genes was confirmed in NSCLC tumours, giving an overall methylation frequency of 25%, 50%, 32%, and 25% respectively. A strong association between KRAS mutation and CDH13 methylation was also detected. In conclusion, HRM is a sensitive and accurate methodology that can be used in a range of molecular diagnostic tests for genetic and epigenetic biomarkers. When used in combination with sequencing, it is suitable for routine diagnostic testing for molecularly tailored therapies for NSCLC patients.
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ItemThe role of perforin in human health and disease( 2011)Cytotoxic lymphocytes (CL) are key effectors of the immune system responsible for the elimination of intracellular pathogen-infected and transformed cancerous cells. This occurs predominantly through the perforin-dependent granule exocytosis pathway, where perforin pores formed in the target cell membrane allow the delivery of pro-apoptotic serine proteases, granzymes, into the cytoplasm of the target cell and the initiation of apoptosis. However, despite a critical role of perforin in CL biology, the physiological role of perforin in human health and disease states is not fully understood. In humans, perforin deficiency is most commonly associated with an autosomal recessive disorder, Type 2 Familial Haemophagocytic Lymphohistiocytosis (FHL), the manifestations of which suggest a role for perforin in maintaining immune homeostasis. However, unlike studies in perforin deficient mice, no evidence has yet linked defective perforin cytotoxicity with cancer susceptibility in humans. Here, a putative role for perforin in the immune surveillance of human cancers was investigated. Although most patients with bi-allelic perforin mutations develop FHL in early infancy, some individuals have been reported to develop an atypical delayed late-onset of disease. Perforin function was examined in these individuals, using surrogate assays developed in rat basophilic leukaemia cells and mouse cytotoxic T lymphocytes (CTLs). Accordingly, it was found that perforin dysfunction was associated with an increased susceptibility to haematological malignancies, providing the most compelling evidence to date for a role for perforin and thus CLs in human cancer immune surveillance. To account for the delayed age of disease onset in these patients, it was postulated that partial CL cytotoxicity protected from overwhelming FHL in early childhood. Novel assays were developed in both mouse and human natural killer (NK) cells to compare the effects of early- and late-onset disease-associated perforin mutations on CL cytotoxicity. Whilst perforin alleles associated with early-onset FHL were completely detrimental to NK cell cytotoxicity, perforin alleles associated with atypical late-onset disease retained partial activity in NK cells. By contrast, almost all these mutations were completely detrimental to CTL cytotoxicity. Hence, it appears that residual NK cell cytotoxicity, rather than CTL cytotoxicity, is responsible for protecting against overwhelming FHL in early infancy, supporting a predominant role for the loss of NK cell cytotoxicity in the pathogenesis of FHL. Furthermore, these studies revealed that partial perforin deficiency is caused by protein misfolding, providing a rationale for the development of specific drug therapies aimed at stabilising perforin structure. Finally, the case studies of two patients with clinical suspicion of FHL were presented. Sequence analysis of the patients’ perforin genes was performed in order to confirm diagnosis, resulting in the identification of two novel perforin mutations. The molecular basis of perforin dysfunction was analysed in each case using the functional assays generated in previous chapters, in combination with structural information extrapolated from the recently resolved crystal structure of mouse perforin. These studies revealed valuable insight into the role of perforin and CLs in disease pathology, including the discovery of the gain of glycosylation as a pathogenic mechanism common to perforin mutations associated with early-onset disease. In summary, the body of work presented in this thesis highlights the absolute importance of functional perforin in maintaining human health. It has also widened the range of available diagnostic assays and provided possible novel approaches for managing diseases associated with perforin deficiency.