Emergence of proteomics and high-throughput technologies has allowed the identification of protein expression patterns of disease that potentially hold clinical importance in predictive medicine. The analysis of complex data generated by these technologies incorporates the use of computer algorithms for data mining and identification of important protein biomarkers. Such candidate biomarkers can potentially be used for diagnosis, prognosis and monitoring a variety of diseases as well as the prediction of therapy response. Mass spectrometry has been used widely, for the discovery and quantitation of disease associated biomarkers using a variety of samples such as serum and tissue. In particular, matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) has been used to generate proteomic profiles or “fingerprints” from serum to distinguish patients at different clinical stages of disease. Currently, early stage disease is difficult to diagnose in most cancers as current cancer markers have limited sensitivity and specificity. In advanced stage metastatic disease, treatment options are limited, although it is recognised that some patients may benefit from immunotherapy and in particular vaccine therapy. The use of animal models is critical to evaluate the efficacy of immunotherapies and to investigate tumour immunity in general and the mechanisms involved in tumour progression. These models provide an in vivo environment which cannot be reproduced in vitro, which results in more accurate and reliable information on the host response to immunotherapy and the mechanisms involved. The research presented in this thesis has introduced the use of MALDI-TOF MS proteome profiling and bioinformatic analysis, to detect candidate biomarkers of tumour progression and responce to immunotherapy in a CT26 murine model of colorectal carcinoma. Proteomic profiles from serum and tissue were generated by MALDI-TOF MS followed by artificial neural networks (ANNs) analysis of the complex data. The methods used in this study for sample preparation and analysis demonstrates that good quality proteomic data from serum and tissue can be obtained, and that it is possible to generate discriminatory protein profiles that correlate with clinical outcomes. In the first instance, using the CT26 progression model, serum and tissue samples were collected at four time-points from tumour-bearer and control mice, providing the opportunity to assess the tumour proteome changes with in a time-course from tumour initiation and through different stages of growth. Through the analysis of serum and tissue it is possible to classify samples based on their stage of tumour growth and the discriminatory patterns may reveal novel pathways associated with tumour progression. In addition, this study employed two separate mouse models of colon carcinoma immunotherapy (CT26 tumour model), to investigate biomarkers that are associated with therapy response. Using either disabled infectious single cycle-herpes simplex virus (DISC-HSV) or dendritic cell-based vaccination therapy with CTLA-4 and blockade of VEGFR-2 immunotherapy, up to 70% of the treated tumours tend to regress after receiving the immunotherapy (tumour regressors). Therefore, these models of immunotherapy were used to screen and evaluate serum protein and peptide biomarkers for the detection of progressors from regressors by using MALDI-MS coupled with an ANN algorithm. Comprehensive clean-up methods were conducted on the sera prior to MALDI analysis to reduce the complexity of the specimens. A panel of 4 biomarkers associated with response to DISC-HSV therapy was identified and successfully validated using non-mass spectrometry techniques. Furtheremore, discriminatory patterns corresponding to different stages of tumour progression and immunotherapy were identified in the mouse model with DC-based immunotherapy. Moreover, potential markers associated with response to therapy were proposed using this model. The work presented demonstrates a proof-of-principle that the different types of information that can be obtained from animal models can be expanded and applied to human studies.

A common feature of neurodegenerative conditions including Parkinson’s disease (PD) is the presence of intracytoplasmic proteinacious inclusions. In PD these inclusions are called Lewy bodies (LBs) and contain a number of proteins including α-synuclein, ubiquitin and neurofilaments (NFs). NFs, the intermediate filaments expressed in neuronal cells are responsible for the maintenance of axonal structure. Although NFs were the first proteins identified in LBs their role in PD pathogenesis has not been fully explored. The work presented here attempts to address some of the gaps in the current knowledge concerningNF turnover and the role of NFs in PD using the human SH-SY5Y neuroblastoma cell line, commonly used as a cellular model of neurodegeneration. Mitochondrial dysfunction, dopamine (DA) mediated oxidative stress and impaired protein degradation have all been implicated in PD pathogenesis. The complex I inhibitor MPTP and its active metabolite (MPP+) induce Parkinsonism in humans and other primates and have been extensively used as PD mimetics in both cellular and animal models. Addition of specific protease inhibitors in the presence of cycloheximide (an inhibitor of new protein synthesis) revealed that NF-heavy chains are degraded by macroautophagy and cathepsin D, possibly with some involvement of cysteine cathepsin, but not calpain or the ubiquitin proteasome system (UPS). This is in contrast to α-synuclein which was degraded by macroautophagy, the UPS and calpain. Treatment with MPP+ did not increase NF halflife despite a reduction in the activity of the 20S proteasome, cathepsin D and macroautophagy. However an activation of cysteine cathepsin activity was observed with MPP+ treatment, suggesting a role for these cathepsins in NF turnover following complex I inhibition. Inhibition of cysteine cathepsins in MPP+ treated cells resulted in increased cell death suggesting that activation of cysteine cathepsins is protective in this context. Following treatment with 100 μM DA, NF turnover appeared to be reduced, accompanied by a reduction in 20S proteasome activity and macroautophagy but activation of calpain. To allow analysis of the effects of mitochondrial dysfunction on the distribution of NFs in live cells, Green Fluorescent Protein (GFP) tagged NFs were produced. Live imaging of cells treated with a high dose of MPP+ displayed a retraction of axonal processes which occurred within the first 20 - 30 minutes of treatment. Analysis of the rate of retraction of GFP fluorescence and the remaining axonal structure indicated that NF retraction was not an early event in axonal retraction, at least with this concentration of MPP+. The effect of mitochondrial dysfunction and/or proteasome inhibition on protein aggregation and inclusion formation was investigated in differentiated SH-SY5Y cells. Treatment with MPP+, epoxomicin (proteasome inhibitor, epoxo) or MPP+/epoxo resulted in an enrichment of NFs in a detergent insoluble fraction and accumulation of NF-H in a perinuclear location together with p62 and LC3, suggesting the attempted degradation of accumulated NF-H by macroautophagy. Further analysis of GFP-tagged NFs in MPP+ treated cells should assist in the elucidation of the role of NFs in the formation of inclusions and will allow analysis of protein partners during complex I dysfunction and other cellular states linked to neurodegeneration.

Multiplexed immunoassays have been explored using the fluorescent and luminescent properties of fluorophores and nanoparticles. Epi-fluorescence microscopy, confocal laser scanning microscopy and programmable array microscopy were used to detect signals from mixtures of conventional organic fluorophores, quantum dots and silica nanoparticles doped with europium, samarium and terbium in single-welled multiplexed immunosorbent assays. Spectral unmixing was investigated using mixtures of fluorophores and cadmium selenide quantum dots. Mixtures of up to four dyes were separated quantitatively using least squares minimisation, with relative standard error ranging from 0.5 to 13 %. Silica nanoparticles doped with luminescent lanthanides were synthesised and used in a model immunoassay system for simultaneous, single-welled detection of human and mouse IgGs. The results indicated the lanthanides are well suited to multiplexed assays, mainly because of their atomic line emission bands. Analytes in a mixture could be quantified with < 5 % error. The multiplexed assay developed was applied to the detection of anti-dengue IgM and IgG in mouse sera, to differentiate primary and secondary dengue infection. The assay traced the kinetics of antibody production for both IgM and IgG with an IgM/IgG ratio of 1. The fluorescencebased methods compared favourably with ELISA results (r2 ≥ 0.8), with results using conventional fluorophores showing the best correlation with ELISA (r2 > 0.98). Differentiation of serotype specific IgGs was also explored but was complicated due to cross reactivity of the antibodies. A model to differentiate cross reactive dengue antigens was applied to the data through fitting parameters of the five parameter logistic equation to the intensity obtained for an assay. Results indicated the family of dengue antigens are too closely related to be distinguished by the method. The model was however successful in differentiating a partially cross reactive system (p > 0.95).

¹⁸F-FDG PET can predict response using both qualitative and quantitative measures. PET Therapy Response Assessor (PETTRA) software was developed to allow users to view and analyse pre- and post- therapy images and compute quantitative measures for predicting response to therapy. Additionally, registration methodology was developed to register pre- and post- therapy PET/CT images. The methodology registers pre- and post- therapy PET/CT scans by registering CT scans using customised rigid and non-rigid registration performed by the Image Registration Toolkit (IRTK). Registration success was assessed using qualitative visual analysis and quantitative landmark analysis on a cohort of 20 lymphoma patients. Landmark analysis results found average misalignment on IRTK of ~10mm for rigid registration and ~6.5mm for non-rigid registration, in comparison with ~40mm with no registration applied. The effect of both rigid and non-rigid registration on transformed images was assessed. While rigid registration transformation caused minimal changes on intensity and tumour volume (&lt;2%), non-rigid transformations caused changes of 11% and 21% respectively. PETTRA software was used to analyse quantitative parameters in 14 patients with mesothelioma and 85 patients with diffuse large B-cell lymphoma (DLBCL). For the 14 patients with mesothelioma, a range of parameters were used to assess response including SUVmax, SUVpeak, tumour volume (TV), total lesion glycolysis (TLG) and intensity volume histogram (IVH) parameters. TV and TLG were obtained using 13 fixed and 9 adapative threshold segmentation methods. Pre-and post- therapy SUVmax, SUVpeak, TV and TLG all showed promise in predicting survival. The comparison between TV and TLG obtained using different segmentation methods was negligible. For the 85 patients with DLBCL, SUVmax, TV and TLG struggled to predict response in patients according to ROC curves.

Diffusion Tensor MRI can be used to depict the anisotropy of tissue. Translation of this technique to moving objects such as the beating heart has recently become feasible, but remains a challenging task, often leading to high noise levels and limited accuracy. Ultimately, knowledge of the 3D fibre architecture of the myocardium in­ vivo should allow for a better understanding of the cardiac function both in healthy and pathological situations. The main goal of the work presented in this thesis is to overcome the difficulties that such technology presents, by introducing a combination of image process­ ing and analysis approaches. In particular, the characteristic ellipsoidal shape of the left ventricular chamber is used to introduce a shape-based prolate spheroidal coordinate frame that allows for comprehensive, robust and dedicated analysis of diffusion tensor data within the myocardial wall. It is shown that the description of this information is more compact in this coordinate frame. Furthermore, it is demonstrated that the acquisition limitations can be overcome by introducing an approximation scheme based on this coordinate frame. These techniques are tested on ex-vivo datasets to assess their fidelity and sensitivity. Finally, these techniques are applied in-vivo on a group of healthy volunteers, where 2D DTI slices of the LV were acquired at end diastole and end systole, using cardiac dedicated diffusion MR acquisition. Results demonstrate the advantages of using curvilinear coordinates both for the analysis and the approximation of cardiac DTI information. Resulting in-vivo fibre architectures were found to agree with previously reported studies on ex-vivo specimens. The outcome of this work can open the door for clinical appli­ cations and cardiac electrophysiology modelling, and improve the understanding of the left ventricular structure and dynamics.

No description available.

An outline is given of the present state of knowledge regarding the pathogenesis of scrapie agents in mice. Emphasis is laid on the unconventional nature of the disease and its causal agent. A methods section describes the relevant aspects of the special methodology which has been developed in the research institutes where this thesis was written. An investigation of the incubation periods of three different strains of scrapie agent injected at a range of ages from birth to weaning in three strains of inbred mice shows that the undeveloped state of these hosts at birth causes a considerable modification of pathogenesis: in particular, many newborn mice do not develop scrapie after a dose which would always kill weanlings. Reasons are given for suspecting that the relevant undeveloped organ system is the lymphoretioular system, and that some form of scrapie 'inactivation' must also be involved. Attempts to throw light on agent pathogenesis by looking for pharmacological treatments which will change inoubation periods, produced numerous negative results (outlined) and some positive ones (described in full). Using the i.p. route of agent injection and drug treatment in the weeks immediately before and after infection, signifioantly changed inoubation periods were obtained with prednisone acetate, arachis oil, prednisone acetate + cyclophosphamide, and prednisone acetate + peritoneal-oell provocation with thioglyoollate medium. Preliminary positive results using neonatal treatment with 6-hydroxydopamine and adult treatment with phytohaemagglutinin are also desoribed. Evidence is reported for a prolonging of inoubation period of i.o. injected agent by subsequent actinongrcin D injections. Experiments are desoribed in which the peripheral pathogenesis of ME7 sorapie appears to be greatly modified both in terms of incubation period and pattern of lesion distribution in the brain by donor-tissue components. Observations on the histological differences are reported and a number of experiments described which suggest that agent pathogenesis may require specific reactions of an immunological type on the part of the host to donor-specified antigens in the inoculum. It is shown in a large range of agent/host strain combinations that there are early changes in drinking (and in some cases feeding) habits of mice infected with scrapie. Reasons are given for believing that these are due to an upset of normal brain function by the agent the physiological basis of which is close to the primary lesion due to scrapie, i.e. some derangement of the function of the sinc gene or its immediate product (SECTION 2). The possibility that this is an upset in catecholamine function is discussed. A number of experiments and observations ancillary to the main seotions are collected in five appendices. Full discussions are given at the end of each SECTION, while in the Final Discussion an attempt is made to bring all the above observations together and to point the way to further research. Several alternative models of scrapie pathogenesis in peripheral organs are briefly reviewed.

The identification of the source of a raised total alkaline phosphatase activity in plasma, by the measurement of individual ALP forms, is of clinical value although many of the methods available for this purpose are complex, imprecise and non-specific. This thesis has validated, and in some cases modified, available methods for the measurement of the main forms of alkaline phosphatase (ALP; EC 3.1.3.1) in plasma: liver, bone, intestinal and high-molecular-mass ALP. The following methods were selected on the basis of their reliability and specificity: polyacrylamide gel electrophoresis, with densitometric scanning, for liver and bone ALP, an enzyme-linked immunosorbent assay (ELISA) for intestinal ALP and ion-exchange chromatography for high-molccular-mass ALP. These methods were then used to quantify individual ALP forms in specific disease groups and compare activities to those found in healthy adults. The diseases studied (diabetes mellitus and hyperthryoidism) were those where the source of the raised total ALP activity has not been clearly established. Intestinal ALP activity was found to be an important source of the raised total ALP in diabetics of blood group B and O who were secretors. Abnormalities in liver ALP were also present, but these were found mainly in type 2 diabetics. Bone ALP and, to a lesser extent, liver ALP contributed to the raised total ALP activity in patients with hyper-thyroidism. These abnormalities were not present in euthyroid patients who were previously hyperthyroid, indicating that they were a temporary feature of the thyrotoxic state. Specific diseases (chronic renal failure and obstructive liver disease) where the measurement of an individual ALP form was likely to be of more value than total ALP measurement were also studied. In patients with chronic renal failure maintained on haemodialysis, measurements in plasma of activities of total ALP and gammaglutamyl-transferase identified bone abnormalities in most patients. However, measurement of bone ALP was essential in those patients (16% of the group) who had co-existent liver disease. In patients with obstructive liver disease, the measurement of plasma total ALP activity was of no value in determining the cause of obstruction (intra- or extrahepatic). In these patients, however, intestinal ALP activity, when measured by ELISA and related to blood group/secretor status category, showed absolute specificity for intrahepatic obstruction.

The component forms of three hydrolytic enzymes, N-acetyl-P-D-hexosaminidase, a-D-glucosidase and S-D-glucosidase, were studied in certain tissues, cultured cells and fluids. The possibility that hexosaminidases A and B had a common subunit and differed by a subunit, which was also a constituent of the neutral component, hexosaminidase C, was investigated and it was found that hexosaminidase C was unlikely to be related to either of the two major hexosaminidase components. The properties of another minor component, hexosaminidase S, suggested that this may contain the unique subunit of hexosaminidase A. In addition to the a-glucosidase component deficient in Pompe's disease, three other apparently genetically unrelated components were identified. One of these, which had a neutral pH optimum and little activity at pH 4.0 occurred in most tissues, but was very labile. The others, one found in amniotic fluid and the other in kidney and leucocytes, although having neutral pH optima, had considerable activity at pH 4.0, thereby interfering with the diagnosis of Pompe's disease. The enzyme found to be deficient in Gaucher's disease was associated with the P-glucosidase activity eluting in the void volume of Sephadex G-150. Two other more neutral components were detected, one of which was also in the void volume and the other, only present in some livers and in spleen, was of lower molecular weight. Neither of these components was deficient in either of the two cases of neuronopathic Gaucher's disease studied. Methods were developed for detecting enzyme deficiencies when there were other components with hydrolytic activity towards the artificial substrate in the tissue being used. The IEAE-cellulose batch method was the most useful for detecting hexosaminidase A. Interfering a-glucosidase components were removed by isoelectric precipitation at pH 5.0 or the activity of the acid a-glucosidase was determined indirectly using equations derived on the basis of the relative susceptibility of components to inhibitors. Non-specific O-glucosidases residual in Gaucher tissues could be removed by preincubation in 50RK-sodium chloride buffered at pH 4.0.

In an attempt to produce a quantitative biochemical assay which would assist in the detection of individuals heterozygous and homozygous for the cystic fibrosis (CF) gene, two general approaches were adopted. In the first, attempts were made to produce an antiserum specific for the cystic fibrosis factor (CFF), a substance known to be present in the serum and secreted by fibroblasts of both CF patients and heterozygotes. Three types of material were used for immunisation schedules - serum from CF homozygotes, the IgG fraction of CF serum and partially purified medium from CF fibroblasts. Both normal and neonatally tolerised rabbits were used in these experiments. The antisera produced were tested by immunoprecipitation, immunofluorescent and radiolabelling techniques. Immunoprecipitation and immunofluorescence showed that, after absorption, the antisera produced from fibroblast medium had specificity for CF samples. Sadiolabelling experiments indicated that the absorbed antisera produced from serum and the IgG fraction of serum also gave specific reactions with CF samples. However, all the antisera produced were too weak, but if suitable methods for fractionation and concentration of the antisera or production of stronger antisera could be perfected, one or more of these methods may prove suitable for the development of quantitative assays for CFF. The second approach involved an investigation of a claim made by Hosli, Erickson and Vogt (1976), that alkaline phosphatase is induced in CF fibroblasts by the addition of Tamm-Horsfall urinary glycoprotein. Alkaline phosphatase, alpha-glucosidase and hexosaminidase activities were measured in a series of normal and CF fibroblast lines, both before and after incubation of the cells with Tamm-Horsfall protein. No significant induction of the enzyme activities could be found in CF or normal control cell lines. It was therefore concluded that this method does not constitute a realistic approach to the development of methods for the diagnosis of homozygous and heterozygous CF individuals.