Browsing by Author "Woodman, Anthony C."
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Item Open Access Computional selection of synthetic antigens for application in diagnostic, vaccine and therapeutic development(Cranfield University, 2006-02) Larcombe, L.; Woodman, Anthony C.This work set out to apply computational tools and biophysics principles to develop a systematic approach to the rational selection of synthetic antigens from appropriate DNA sequences. The wider potential to the rapid development of diagnostics will be demonstrated by application towards a chlamydia-specific immunodiagnostic, and the potential for vaccine and therapeutic development will be discussed.Item Open Access Detection of inappropriate cell proliferation in breast epithelium leading to breast cancer(2004-11) Degan, Simone A.; Woodman, Anthony C.; McCarthy, Keith; Bristol, James B.Breast cancer is predominantly caused by unrestrained cell proliferation. Proliferation is a complex process mediated by a network of signals that converge to a point called the ‘initiation of genome replication’ after which either proliferation or cell death could take place. The minichromosome maintenance (MCM) proteins are located at this point and play a pivotal role in regulating DNA replication. The detection of an aberrant level of such proteins can be of use in early breast cancer diagnosis. The main aim of this thesis was to propose a new system to detect inappropriate cell proliferation in breast epithelium. An in vitro model using cancer cell lines was developed to lay the foundation for subsequent studies employing human breast specimens. The application of the in vitro findings in breast excisions allowed assessing of the specificity and sensitivity of the biomarkers to ascertain slowly proliferating neoplastic cells. The most striking finding of this study was the abnormal presence of the MCM proteins in tumour compared to normal tissues with a typical pattern of expression unique for the histological classification of the lesion. The potential of MCM proteins as indicators of cell proliferation defects was further investigated with association studies with Ki-67, Bcl-2 and ER. MCM consistently identified a higher proportion of proliferating cells compared to Ki-67 suggesting that they are interesting markers of the Gi/S-phase. In fact, the MCM proteins start to co-localise in early Gi whereas Ki-67 is almost absent in this phase. Importantly, MCM proteins could recognise not only the proliferating compartment of the tumour but also those cells with replication potential. Based on these findings, the novel MCM biomarkers can be helpful in identifying both malignant and potentially malignant breast tissues. This feature can be useful in predicting patients at risk of tumour progression.Item Open Access The development of a novel native prothrombin assay for the more efficient management of oral anticoagulation therapy(Cranfield University, 2004-05) Sylvester, Adrian Jose; Woodman, Anthony C.Disturbances of the natural balance between pro-coagulant and anticoagulant systems, due to hereditary or acquired factors may result in haemorrhagic or thrombotic diseases. Currently the INR/ISI coagulation monitoring system, introduced in 1983 by the World Health Organisation (WHO), is that of choice for most anticoagulation management clinics. Patients undergoing anticoagulation therapy must regularly attend specialised outpatient clinics for the close monitoring and maintenance of their INR. The automated laboratory caters for rapid online simultaneous analysis of multiple blood samples, resulting in the calculation of a patient’s INR from the recorded prothrombin time. The insensitivity of the prothrombin time test has been well documented, requiring a reduction in the prothrombin concentration of 45 % prior to the materialisation of clinically significant prothrombin times. The project aims to employ three avenues of biotechnology to aid in the development of an immuno or molecular imprinted polymer (MIP) based anticoagulation assay. The project will utilise computational molecular modelling in an attempt to visualise the tertiary structure of human prothrombin, which will allow the rational selection of antigenic sites for molecular imprinting or antibody production. An aqueous, prothrombin-imprinted homo-polymer was grafted to the gold surface of a surface plasmon resonance biosensor (Biacore). The Biacore allowed the real time monitoring of imprinted polymer binding characteristics to i homogeneous protein solutions. As a direct comparison of two technologies, molecular imprinting and immuno technology, polyclonal antibodies showing specificity towards the same prothrombin antigen were immobilised onto Biacore chips. The imprinted polymer graft and polyclonal antibody based assays recognised homogeneous solutions of prothrombin at a concentration range of 0.01 nM to 14.2 nM and 0.01 nM to 0.5 nM respectively. A randomised preliminary clinical trial was initiated to compare the two assays’ ability to differentiate plasma samples with a variety of INR values. The results thus far show promise for the development of a new anticoagulation assay using molecularly imprinted polymer technology. The ultimate aim for this project is to develop a consistently more accurate point-of-care anticoagulation therapy monitoring kit, incorporating this new technology, which can replace or be used concomitantly with the INR/ISI system currently in use. This thesis raises more questions regarding the efficacy of oral anticoagulation therapy (OACT) and argues for and against the necessity of a novel OACT management assay.Item Open Access Development of medical point-of-care applications for renal medicine and tuberculosis based on electronic nose technology(Cranfield University, 2004) Fend, Reinhard; Woodman, Anthony C.; Bessant, ConradINTRODUCTION: Current clinical diagnostics are based on biochemical, immunological or microbiological methods. However, these methods are operator dependent, time consuming, expensive and require special skills, and are therefore not suitable for point-of-care testing. Recent developments in gas-sensing technology and pattern recognition methods make electronic nose technology an interesting alternative for medical point-of-care devices. METHODS: We applied a gas sensor array based on 14 conducting polymers to monitor haemodialysis in vitro and to detect pulmonary tuberculosis in both culture and sputum. RESULTS and DISCUSSION: The electronic nose is able to distinguish between control blood and “uraemic” blood. Furthermore, the gas sensor array is not only capable of discriminating pre- from post-dialysis blood (97% accuracy) but also can follow the volatile shift occurring during a single haemodialysis session. The electronic nose can be used for both dialysate side and blood-side monitoring of haemodialysis. The pattern observed for post- and pre-dialysis blood might reflect the health status of the patients and can therefore be related to the long-term outcome. Furthermore, the gas sensor array was also able to discriminate between Mycobacterium spp. and other lung pathogens such as Pseudomonas aeruginosa. More importantly the gas sensor array was capable of resolving different Mycobacterium spp. such as Mycobacterium tuberculosis, M. scrofulaceum, and M. avium in both liquid culture and spiked sputum samples. The detection limit for M. tuberculosis in both sputum and liquid culture is 1 x 104 mycobacteria ml-1 and therefore partially fulfils the requirement set by the WHO. The gas sensor array was able to detect culture proven TB with a sensitivity of 89% and a specificity of 91%. CONCLUSIONS: In conclusion, this study has shown the ability of an electronic nose as a point-of-care device in these areas.Item Open Access An investigation into minichromosomal maintenance proteins (MCMs) for the diagnosis of prostate cancer, as a possible alternative to prostate specific antigen (PSA)(Cranfield University, 2005-02) Watkins, Jane Louise; Bailey, Tracey A.; O'Donnell, J. O.; Woodman, Anthony C.The current strategy for the diagnosis of prostate cancer includes serum prostate specific antigen (PSA) measurement. There is however debate into its specificity and sensitivity, so new diagnostic markers are under investigation. Minichromosomal maintenance proteins (MCMs) are potential markers for the diagnosis of neoplasia, as they are involved in cellular replication. The aim of this study is to assess MCM2, 5 and 7 as new diagnostic markers for prostate cancer, to compare the clinical usefulness of PSA and to develop a less invasive technique for diagnosis. PSA specificity was investigated in several human cellular lines, and a clinical study was performed to assess expression in prostatic tissue and blood serum. MCM2, 5 and 7 was investigated by translational and transcriptional means in two prostate cell lines PNT1A and PC-3. In addition, a clinical study was performed to assess the expression of MCM2, 5 and 7 in prostate tissue, urine and blood The results suggest that PSA is not prostate specific, as it is synthesised and secreted by several non-prostatic cell lines. In addition PSA testing does not conclusively indicate neoplastic tissue and serum testing only has 63% sensitivity and 60% specificity in accurately identifying prostate cancer. The in vitro results suggest that the PC-3 cells express less MCM2, 5 and 7 on both the protein and mRNA level compared to the PNT1A cells, suggesting that MCM2, 5 and 7 maybe performing a bigger role than just replication of DNA. The tissue results indicate that there is an increase in MCM2, 5 and 7 epithelial nuclei staining for neoplastic condition compared to BPH. While the clinical study on urine sediment indicates increased MCM2, 5 and 7 staining in prostatic neoplasia compared to BPH and the transcriptional study on MCM5 can identify neoplastic tissue from BPH as 11/12 cancerous patients expressed MCM5 compared to only 3/23 BPH patients. Finally the transcriptional study on the blood samples is inconclusive and need to be repeated These results suggest that serum PSA testing is not ideal for the diagnosis of prostate cancer, that MCM2, 5 and 7 appear to have potential as new diagnostic markers and may aid the histopathologist to allocate Gleason score. Also the MCMs may have potential in the development of a less invasive technique through the use of urine sediment.Item Open Access Investigations of a possible link between age-related macular degeneration and atherosclerosis(Cranfield University, 2005-08) Sivaprasad, Sobha; Bailey, Tracey A.; Chong, Victor N. H.; Woodman, Anthony C.Increasing epidemiological evidence suggests a link between atherosclerosis and age-related macular degeneration (AMD). There are two main hypotheses that explain the link. One hypothesis is that AMD and atherosclerosis are tissue responses to injury. The ‘Response to Injury’ hypothesis defined as ‘abnormal reparative response to chronic, recurrent injurious stimuli’ speculates that AMD and atherosclerosis may be cellular effects of chronic or repetitive risk factors. The second hypothesis suggests that AMD is secondary to vascular insufficiency caused by atherosclerosis. The research in this thesis is an attempt to test these hypotheses to better understand the correlation of AMD and atherosclerosis. Both basic and clinical science approaches are employed. Part one focuses on the role of extracellular matrix (ECM) proteins in the pathogenesis of AMD. Both serum elastin derived peptides (S-EDP) and matrix metalloproteinases (MMP-2 and MMP-9) are raised in subjects with atherosclerosis. The circulating levels of these matrix components were tested in patients with varying degree of severity of AMD and compared to age-matched controls. Both S-EDP and MMP-9 were found to be significantly raised in patients with AMD, while MMP-2 did not correlate with it. S-EDP is elevated in abdominal aortic aneurysm (AAA), a manifestation of atherosclerosis. Since S-EDP correlates with size of both AAA and severity of AMD, this research looked at a possible association between the two diseases but found no significant correlation. The second part of this thesis investigates whether chronic inflammation may explain the co-existence of the two diseases. With the recent finding that Complement Factor H (CFH) is related to AMD, this study focused on the role of complement activation in AMD, speculating that the final common pathway of both diseases may be chronic inflammation. Increased systemic complement activation was found in neovascular AMD, as assessed by the measurement of C3a des Arg. Part three of this thesis tested the second hypothesis that AMD is secondary to the vascular insufficiency caused by atherosclerosis. Both the choroidal blood flow and retinal vessel calibre in patients with asymmetric AMD were studied and no significant changes in ocular haemodynamics were noted. In conclusion, this research favours the concept that atherosclerosis and AMD are parallel responses to chronic, recurrent injurious stimuli, with extracellular matrix remodelling and probably inflammatory response being the common cellular responses. This research did not find any significant ocular haemodynamic changes in subjects with asymmetric AMD.Item Open Access Laser ice scaffolds modeling for tissue engineering(John Wiley & Sons, Ltd, 2005-09-01T00:00:00Z) Meglinski, I. V.; Varejka, M.; Woodman, Anthony C.; Turner, Anthony P. F.; Piletsky, Sergey A.Tissue engineering is one of the most exciting and rapidly growing areas in biomedical engineering that offers vast potential for changing traditional approaches to meeting many pharmaceutics and critical health care needs. Currently the bottle-neck area in this multidisciplinary field appears to be materials and fabrication technology for the design of artificial extracellular matrices/scaffolds that support culturing and growth of new tissue. We have shown that stable relief structures can be created and maintained in the bulk of ice by continuous s canning with computer-guided IR CO2 laser. The optimal laser beam intensity and fluence rate distribution within the ice sample, as well as the rate of scanning were estimated based on the Monte Carlo model utilized physical/optical properties of ice. The results of numerical simulation are agreed well with the observed experimental results of thermo-coupling measurements and obtained microscopic images.Item Open Access Molecular basis for maize as a risk factor for oesophageal cancer in a South African population(Cranfield University, 2007-03) Pink, Ryan; Woodman, Anthony C.; Bailey, Tracey A.; Sammon, A.Throughout the world squamous cell carcinoma of the oesophagus seems to be an increasing problem. There is a huge variation in prevalence globally; locations such as Japan, Iran, China and Finland can have ten times the prevalence compared to other western countries. One place that is hugely affected is Transkei, a 16,000 square mile area of South Africa. Some of the factors proposed to be implicated with squamous cell carcinoma in this region include tobacco smoking, alcohol consumption, bacterial infections and fungal infection of common food crops. In addition, the ‘Sammon theory’ links carcinogenesis in Transkei to the high consumption of maize by the population. Through a chain of reactions it is postulated that a component of maize inhibits the breakdown of growth factors, which have already been implicated in cancer. This study investigates the Transkei population and updates the Sammon theory with current research to predict a theory at a molecular level. This theory is then tested with novel research to show PGE2, shown here in high concentrations in gastric fluid samples, directly increases the proliferation of oesophageal cell lines. Gastric fluid samples from the Transkei population are then shown to have a mitogenic effect on oesophageal cells, supporting a theory that gastric fluid regurgitation commonly found in this population predisposes them to cancer. Further experimentation on the expression of related proteins shows how high PGE2 may increase its own production by increasing COX 2 expression, leading to a positive feedback loop causing constant proliferative stimulation of the oesophageal squamous tissue in the presence of the COX 2 substrate, aracadonic acid. Therefore this thesis suggests that a high maize diet provides the correct conditions for regurgitation of increased concentrations of PGE2 into the oesophagus leading to squamous hyper-proliferation over long periods of time through self stimulated production, which would normally have ceased over a much shorter time if only localised PGE2 was produced through natural restitution.Item Open Access Towards the molecular diagnosis of bladder and colorectal cancer : Analysis of CD44 exon splicing(2002-08) Morgan, Sarah Louise; Woodman, Anthony C.The literature suggests that CD44 has the potential to be used as a non-invasive tumour specific marker for early detection and monitoring. The gene transcript can undergo alternative splicing, leading to the production of a number of isoforms and it has been noted that in neoplastic states this splicing becomes abberant, leading to the production of a number o f variant isoforms. The aim of this study was to analyse CD44 variant expression with the goal o f reaching a platform from which a non- invasive assay for routine clinical use could be produced. In this study, the analysis o f exon junction splicing in bladder cancer has also led to the finding of a tumour specific junction expressed in 64% o f tumours studied. This exon junction (5/11) was found to be the same as that expressed in colorectal cancer. Though mRNA based assays would provide a satisfactory method for exon junction identification, a protein based approach would provide the platform for a more robust assay. Translation of gene expression data led to the design of a short synthetic peptide which overlapped the 5/11 junction, and this was used to produce a novel polyclonal antibody for this transitional epitope. The resulting polyclonal antibody was affinity purified and used in a pilot study of 5/11 expression in bladder and colorectal tumours. In bladder tumours the antibody demonstrated an overall specificity o f 76.5% and an overal sensitivity of 73.1 %, comparable to current commercially available early detection assays. In a Dukes staged colorectal cancer study, no link between stage and grade was noted, but the antibody gave an overall specificity of 76%. Though further analysis is required, it is thought the 5/11 antibody may prove to be a useful tool in the development o f a sensitive and specific assay for the non-invasive detection of bladder and colorectal cancer.