http://hdl.handle.net/10023/50 2013-05-23T16:33:08Z http://hdl.handle.net/10023/3511 Abstract: Phospholipase C enzymes are important cell signalling enzymes that catalyse the cleavage of phosphatidylinositol 4,5-bisphophate PI(4,5)P₂ into two biologically active second messenger molecules. These are the inositol 1,4,5-trisphosphate which initiates Ca²⁺ release from the endoplasmic reticulum and the diacylglycerol that activates protein kinase C. Although this basic function is shared between the different isoforms, the PLC family encompasses a diverse collection of proteins with various domain structures in addition to the PLC-specific domains. The neuron-specific “6th family” of these enzymes, PLCηs have most recently been identified with two members, PLCη1 and PLCη2. The aim of the thesis is to characterise the PLCη2 variant from several aspects. Firstly, it describes that PLCη2 possesses a high sensitivity towards Ca²⁺. Secondly, it investigates how the Ca²⁺-induced enzymatic activity of PLCη2 is controlled by its different domains. Also it provides evidence that the pleckstrin homology domain targets PLCη2 to membranes by recognising PI(3,4,5)P₃. Moreover, the uniquely structured EF-hand is responsible for the Ca²⁺-sensitivity of the enzyme. Finally, it is demonstrated that the C2 domain is important for activity. The initial biochemical characterisation is followed by the description of a physiological role for PLCη2. It is shown using a neuroblast model that PLCη2 is crucial for neuronal differentiation and neurite growth. Further efforts were made to assess how PLCη2 is responsible for this effect. It was revealed that it might be involved in regulating intracellular Ca²⁺ dynamics, transcriptional activity and actin reorganisation in differentiating neurons. As the functions of PLCη2 are just beginning to come to light, more aspects for future research are also suggested in the thesis. Hopefully, this and the data presented within the thesis will stimulate even greater interest in this fascinating new field of research. 2013-06-28T00:00:00Z Popovics, Petra Phospholipase C enzymes are important cell signalling enzymes that catalyse the cleavage of phosphatidylinositol 4,5-bisphophate PI(4,5)P₂ into two biologically active second messenger molecules. These are the inositol 1,4,5-trisphosphate which initiates Ca²⁺ release from the endoplasmic reticulum and the diacylglycerol that activates protein kinase C. Although this basic function is shared between the different isoforms, the PLC family encompasses a diverse collection of proteins with various domain structures in addition to the PLC-specific domains. The neuron-specific “6th family” of these enzymes, PLCηs have most recently been identified with two members, PLCη1 and PLCη2. The aim of the thesis is to characterise the PLCη2 variant from several aspects. Firstly, it describes that PLCη2 possesses a high sensitivity towards Ca²⁺. Secondly, it investigates how the Ca²⁺-induced enzymatic activity of PLCη2 is controlled by its different domains. Also it provides evidence that the pleckstrin homology domain targets PLCη2 to membranes by recognising PI(3,4,5)P₃. Moreover, the uniquely structured EF-hand is responsible for the Ca²⁺-sensitivity of the enzyme. Finally, it is demonstrated that the C2 domain is important for activity. The initial biochemical characterisation is followed by the description of a physiological role for PLCη2. It is shown using a neuroblast model that PLCη2 is crucial for neuronal differentiation and neurite growth. Further efforts were made to assess how PLCη2 is responsible for this effect. It was revealed that it might be involved in regulating intracellular Ca²⁺ dynamics, transcriptional activity and actin reorganisation in differentiating neurons. As the functions of PLCη2 are just beginning to come to light, more aspects for future research are also suggested in the thesis. Hopefully, this and the data presented within the thesis will stimulate even greater interest in this fascinating new field of research. http://hdl.handle.net/10023/3464 Abstract: Exosomes are small secreted vesicles of endocytic origin with a size range of 50-150 nm. They are secreted by many cell types and display multiple biological functions including immune-activation, immune-suppression, antigen presentation, and the shuttling of mRNA and miRNA, as well as other cargo. We have characterised the exosomes secreted from two breast cancer cell lines, MDA-MB-231 and MCF7. Exosomes secreted from both cell lines display typical markers including ALIX, Tsg101, CD9 and CD63, and were capable of inducing apoptosis of the Jurkat T cell line, indicating the potential immune-suppressive function of such tumour-derived exosomes. To further investigate the biological potential of exosomes, we loaded purified exosomes with gene specific siRNAs using electroporation, and observed the targeted inhibition of both a known component of the exosome pathway, Rab27a, and also the arthritis associated gene ERAP1, demonstrating the potential novel use of exosomes as therapeutic gene delivery vectors. We have also shown that exosomes derived from MDA-MB-231 cells and the parental cells have different lipid composition, as analysed by lipidomics study. Nanoparticle tracking analysis (NTA), which allows the rapid detection of size and concentration of nanoparticles within the size range 10 nm-1000 nm was tested for its ability to accurately measure size and concentration of exosomes and microvesicles under different conditions. NTA was capable of detecting apoptotic vesicles induced by Taxol and Curcumin treatment. Immunodepletion was used to determine the percentage of CD9 and CD63 positive vesicles. Our data suggest that NTA is a useful technique for measuring size and concentration of exosomes and microvesicles. We hypothesized that NTA could assist in the screening of agents that interfere or promote exosome release. NTA was therefore used to detect increases in exosomes secretion induced by Tamoxifen and Thimerosal treatment, and to monitor the inhibition of exosome secretion from MDA-MB-231 breast cancer cells expressing inhibitory RNA targeted for Rab27a, a component of the exosome pathway. Increases in exosome release induced by Tamoxifen and Thimerosal was detected by NTA and a significant reduction in the release of exosomes by inhibition of Rab27a expression was also observed. Treatment with the known exosomal pathway inhibitor DMA also reduced exosome release, establishing the principle of NTA as a screening technique. We further compared the siRNA targeted cells for their ability to migrate, invade and form anchorage-independent colonies, which were all significantly reduced. Supplementation with MDA-MB-231 derived exosomes restored the ability to form colonies, suggesting exosomes may contribute to metastatic lesion formation. These data suggest that the exosomal pathway is a valid target to disrupt the behaviour of tumour cells and NTA can be used to monitor its activity. 2012-11-30T00:00:00Z Zheng, Ying Exosomes are small secreted vesicles of endocytic origin with a size range of 50-150 nm. They are secreted by many cell types and display multiple biological functions including immune-activation, immune-suppression, antigen presentation, and the shuttling of mRNA and miRNA, as well as other cargo. We have characterised the exosomes secreted from two breast cancer cell lines, MDA-MB-231 and MCF7. Exosomes secreted from both cell lines display typical markers including ALIX, Tsg101, CD9 and CD63, and were capable of inducing apoptosis of the Jurkat T cell line, indicating the potential immune-suppressive function of such tumour-derived exosomes. To further investigate the biological potential of exosomes, we loaded purified exosomes with gene specific siRNAs using electroporation, and observed the targeted inhibition of both a known component of the exosome pathway, Rab27a, and also the arthritis associated gene ERAP1, demonstrating the potential novel use of exosomes as therapeutic gene delivery vectors. We have also shown that exosomes derived from MDA-MB-231 cells and the parental cells have different lipid composition, as analysed by lipidomics study. Nanoparticle tracking analysis (NTA), which allows the rapid detection of size and concentration of nanoparticles within the size range 10 nm-1000 nm was tested for its ability to accurately measure size and concentration of exosomes and microvesicles under different conditions. NTA was capable of detecting apoptotic vesicles induced by Taxol and Curcumin treatment. Immunodepletion was used to determine the percentage of CD9 and CD63 positive vesicles. Our data suggest that NTA is a useful technique for measuring size and concentration of exosomes and microvesicles. We hypothesized that NTA could assist in the screening of agents that interfere or promote exosome release. NTA was therefore used to detect increases in exosomes secretion induced by Tamoxifen and Thimerosal treatment, and to monitor the inhibition of exosome secretion from MDA-MB-231 breast cancer cells expressing inhibitory RNA targeted for Rab27a, a component of the exosome pathway. Increases in exosome release induced by Tamoxifen and Thimerosal was detected by NTA and a significant reduction in the release of exosomes by inhibition of Rab27a expression was also observed. Treatment with the known exosomal pathway inhibitor DMA also reduced exosome release, establishing the principle of NTA as a screening technique. We further compared the siRNA targeted cells for their ability to migrate, invade and form anchorage-independent colonies, which were all significantly reduced. Supplementation with MDA-MB-231 derived exosomes restored the ability to form colonies, suggesting exosomes may contribute to metastatic lesion formation. These data suggest that the exosomal pathway is a valid target to disrupt the behaviour of tumour cells and NTA can be used to monitor its activity. http://hdl.handle.net/10023/3213 Abstract: This thesis describes the development of artificial disc technology for the replacement of intervertebral discs in the human lumbar spine. The clinical problem is back pain. There may be a relationship between certain forms of back pain and disc degeneration. The mechanical properties of human intervertebral discs are examined in detail. The genetic basis of disc degeneration is presented. The hypothesis is that such degeneration leads to a loss of normal stiffness in the segments affected leading to abnormal mechanical behaviour which in turn leads to pain. The evidence for this is presented. The development of surgical solutions to relieve back pain, from fusion through first generation mechanical artificial discs to elastomeric designs, is traced. The author‘s personal contributions to this area of knowledge are set out. The appreciation of the requirement for a restoration of physiological stiffness is argued throughout, showing where fusion and first generation discs have not met the clinical aim of pain relief, because they have not restored physiological stiffness. The path to an elastomeric, viscoelastic, polyhydrocarbon, rubber solution in the form of the “Freedom“ disc has filled 17 years of the author‘s academic pursuits. It will be shown that this technology may represent a possible solution to the clinical problem. Failure is part of all new advancement and this too is presented, to show how that has influenced thinking, producing original ideas to overcome these failures. Providing lessons are learned from these failures then our patients in the future will benefit. 2012-11-30T00:00:00Z Ross, Edward R. S. This thesis describes the development of artificial disc technology for the replacement of intervertebral discs in the human lumbar spine. The clinical problem is back pain. There may be a relationship between certain forms of back pain and disc degeneration. The mechanical properties of human intervertebral discs are examined in detail. The genetic basis of disc degeneration is presented. The hypothesis is that such degeneration leads to a loss of normal stiffness in the segments affected leading to abnormal mechanical behaviour which in turn leads to pain. The evidence for this is presented. The development of surgical solutions to relieve back pain, from fusion through first generation mechanical artificial discs to elastomeric designs, is traced. The author‘s personal contributions to this area of knowledge are set out. The appreciation of the requirement for a restoration of physiological stiffness is argued throughout, showing where fusion and first generation discs have not met the clinical aim of pain relief, because they have not restored physiological stiffness. The path to an elastomeric, viscoelastic, polyhydrocarbon, rubber solution in the form of the “Freedom“ disc has filled 17 years of the author‘s academic pursuits. It will be shown that this technology may represent a possible solution to the clinical problem. Failure is part of all new advancement and this too is presented, to show how that has influenced thinking, producing original ideas to overcome these failures. Providing lessons are learned from these failures then our patients in the future will benefit. http://hdl.handle.net/10023/3134 Abstract: Sister chromatid cohesion is essential for the equal distribution of genetic material in mitosis. The cohesin complex plays a central role in the establishment of sister chromatid cohesion. The cohesin complex is a ring shaped structure that encircles sister chromatids prior to the onset of anaphase ensuring equal distribution of genetic material. The DEAD/H DNA helicase ChlR1 is important in the establishment of sister chromatid cohesion. ChlR1 interacts with the cohesin complex and is required for the loading of cohesin onto DNA. Cohesin is loaded onto the DNA during DNA replication. Here I identified a novel interacting partner of ChlR1. The multifunctional DNA binding protein FHL2 was shown to interact with ChlR1 and FHL2 was shown to have a role in sister chromatid cohesion since depletion of FHL2 resulted in abnormal metaphase spreads and reduced centromeric cohesion. These sister chromatid cohesion defects also result in a G₂/M delay. Here I show an additional function of ChlR1 in the repair of DNA damage. ChlR1 was required for the repair of DNA double strand breaks and ChlR1 was recruited to DNA double strand breaks. Furthermore the function of ChlR1 in DNA double strand break repair is S phase specific. This suggests that ChlR1 is important in the homology recombination repair pathway. I also show that ChlR1 is important in DNA replication. Depletion of ChlR1 results in inefficient DNA replication. In addition depletion of ChlR1 results in defects in DNA replication after hydroxyurea treatment. The results in this thesis shed light on novel functions of the DNA helicase ChlR1 in DNA replication and DNA damage repair and the multifunctional DNA binding protein FHL2 in cohesion establishment. 2011-01-01T00:00:00Z Wasson, Christopher Sister chromatid cohesion is essential for the equal distribution of genetic material in mitosis. The cohesin complex plays a central role in the establishment of sister chromatid cohesion. The cohesin complex is a ring shaped structure that encircles sister chromatids prior to the onset of anaphase ensuring equal distribution of genetic material. The DEAD/H DNA helicase ChlR1 is important in the establishment of sister chromatid cohesion. ChlR1 interacts with the cohesin complex and is required for the loading of cohesin onto DNA. Cohesin is loaded onto the DNA during DNA replication. Here I identified a novel interacting partner of ChlR1. The multifunctional DNA binding protein FHL2 was shown to interact with ChlR1 and FHL2 was shown to have a role in sister chromatid cohesion since depletion of FHL2 resulted in abnormal metaphase spreads and reduced centromeric cohesion. These sister chromatid cohesion defects also result in a G₂/M delay. Here I show an additional function of ChlR1 in the repair of DNA damage. ChlR1 was required for the repair of DNA double strand breaks and ChlR1 was recruited to DNA double strand breaks. Furthermore the function of ChlR1 in DNA double strand break repair is S phase specific. This suggests that ChlR1 is important in the homology recombination repair pathway. I also show that ChlR1 is important in DNA replication. Depletion of ChlR1 results in inefficient DNA replication. In addition depletion of ChlR1 results in defects in DNA replication after hydroxyurea treatment. The results in this thesis shed light on novel functions of the DNA helicase ChlR1 in DNA replication and DNA damage repair and the multifunctional DNA binding protein FHL2 in cohesion establishment. http://hdl.handle.net/10023/3104 2012-06-01T00:00:00Z Schmidt, Xenia http://hdl.handle.net/10023/2815 Abstract: The thesis looks in detail at three inter-related aspects of Alison's life. It examines, firstly, his role in the development of Edinburgh's rudimentary 'health' network, achieved through the expansion of the existing medical charity structure and the introduction of a more interventionist and coordinated approach to the city's health problems. It traces, secondly, the development of Alison's social thought - in 1820 he believed that medical and practical relief for the poor could and should be supplied through the voluntary charities and only when that proved unsatisfactory through the poor law, whereas by 1840 he argued that public health should be the responsibility of government and that the excessive increase in poverty and disease in Scotland, which he believed had occurred, was proof that the charitable and legal relief provided was inadequate. Finally, Alison's influence on the passage of Scottish poor law and public health legislation in the 1840s and 1850s is examined - the latter involving an assessment of how far he was responsible for the legislative delay. The poor law debate, 1840-1845, which reveals the forces shaping the reform and the prevailing attitudes to poverty, highlights the challenge which Alison's opinions represented and the resulting turmoil in Scottish social thinking, while his reasons for opposing health legislation, which established London control are of great importance. They reveal differences in the rationale behind, and way in which, the concept of public health was developed in Scotland and England. Unlike Chadwick and his supporters, Alison emphasised poverty amelioration and sanitary reform. Part of the explanation for the differing opinions lay in their respective miasmatic and contagionist theories for fever generation, but it also reflects, perhaps more significantly, the impact of European medical police ideas on Scottish medical opinion - Alison's view of public health closely resembled that of the French hygienists. 1997-01-01T00:00:00Z Martin, Sheonagh M. K. The thesis looks in detail at three inter-related aspects of Alison's life. It examines, firstly, his role in the development of Edinburgh's rudimentary 'health' network, achieved through the expansion of the existing medical charity structure and the introduction of a more interventionist and coordinated approach to the city's health problems. It traces, secondly, the development of Alison's social thought - in 1820 he believed that medical and practical relief for the poor could and should be supplied through the voluntary charities and only when that proved unsatisfactory through the poor law, whereas by 1840 he argued that public health should be the responsibility of government and that the excessive increase in poverty and disease in Scotland, which he believed had occurred, was proof that the charitable and legal relief provided was inadequate. Finally, Alison's influence on the passage of Scottish poor law and public health legislation in the 1840s and 1850s is examined - the latter involving an assessment of how far he was responsible for the legislative delay. The poor law debate, 1840-1845, which reveals the forces shaping the reform and the prevailing attitudes to poverty, highlights the challenge which Alison's opinions represented and the resulting turmoil in Scottish social thinking, while his reasons for opposing health legislation, which established London control are of great importance. They reveal differences in the rationale behind, and way in which, the concept of public health was developed in Scotland and England. Unlike Chadwick and his supporters, Alison emphasised poverty amelioration and sanitary reform. Part of the explanation for the differing opinions lay in their respective miasmatic and contagionist theories for fever generation, but it also reflects, perhaps more significantly, the impact of European medical police ideas on Scottish medical opinion - Alison's view of public health closely resembled that of the French hygienists. http://hdl.handle.net/10023/2807 Abstract: This study examined the contribution to quality of care of a goal planning technique called Goal Attainment Scaling and its impact upon the quality of life of people with severe mental handicaps and challenging behaviour. The study also seeks to establish the utility of employing Goal Attainment Scaling as a means of evaluating clinical nursing performance, This study essentially aims to bring about changes in the care practices of nurses using a comprehensive staff management procedure. The study was designed as a four phase intervention using a multiple baseline design across three wards in a hospital for people with mental handicaps. Staff on three wards (n = 41) were initially trained over three phases in the use of Goal Attainment Scaling and other procedures. Training was carried out by a combination of workshops and individual tuition which incorporated the use of individualised learning contracts for staff. The fourth phase consisted of establishing weekly meetings to set objectives for staff to achieve that were specifically related to material covered in training. If targets were achieved, staff performance was followed by letters of recognition from managers and by financial donations to ward funds. Dependent measures included frequency of challenging behaviour, quality of staff-resident interaction and engagement, ward activity, residents' adaptive behaviour, staff attitudes and goals set by staff. Results indicate that adaptive behaviour increased by small but statistically significant levels. Levels of challenging display a mixed pattern of results, as do levels of ward activity and quality of interaction, although encouraging trends may be identified. Despite some increases, residents still spend significant amounts of time unoccupied. The number of goals set increased throughout the study, particularly in phase four, data for staff attitudes were not used because of the low compliance rate and changes indicated below. Considerable problems were encountered with turnover of staff and other organisational changes outwith the researcher's control which compromised both the quality of training given to staff and, by virtue of this, the final results. Statistically significant relationships were found to exist between staff turnover and interaction. The implications of this study are discussed and recommendations made for future research. 1992-01-01T00:00:00Z Turnbull, John This study examined the contribution to quality of care of a goal planning technique called Goal Attainment Scaling and its impact upon the quality of life of people with severe mental handicaps and challenging behaviour. The study also seeks to establish the utility of employing Goal Attainment Scaling as a means of evaluating clinical nursing performance, This study essentially aims to bring about changes in the care practices of nurses using a comprehensive staff management procedure. The study was designed as a four phase intervention using a multiple baseline design across three wards in a hospital for people with mental handicaps. Staff on three wards (n = 41) were initially trained over three phases in the use of Goal Attainment Scaling and other procedures. Training was carried out by a combination of workshops and individual tuition which incorporated the use of individualised learning contracts for staff. The fourth phase consisted of establishing weekly meetings to set objectives for staff to achieve that were specifically related to material covered in training. If targets were achieved, staff performance was followed by letters of recognition from managers and by financial donations to ward funds. Dependent measures included frequency of challenging behaviour, quality of staff-resident interaction and engagement, ward activity, residents' adaptive behaviour, staff attitudes and goals set by staff. Results indicate that adaptive behaviour increased by small but statistically significant levels. Levels of challenging display a mixed pattern of results, as do levels of ward activity and quality of interaction, although encouraging trends may be identified. Despite some increases, residents still spend significant amounts of time unoccupied. The number of goals set increased throughout the study, particularly in phase four, data for staff attitudes were not used because of the low compliance rate and changes indicated below. Considerable problems were encountered with turnover of staff and other organisational changes outwith the researcher's control which compromised both the quality of training given to staff and, by virtue of this, the final results. Statistically significant relationships were found to exist between staff turnover and interaction. The implications of this study are discussed and recommendations made for future research. http://hdl.handle.net/10023/2802 Abstract: The central theme or 'red-thread' that I consider in this thesis is the concept of risk as it is perceived by and affects the two sides of the medical encounter -in this instance ethnic Pakistanis and Health Professionals- in Britain. Each side very often perceives risk quite distinctively, relating to the balance between the spiritual and temporal realms. This is particularly germane in matters to do with possible congenital defects within the prenatal realm for the ethnic Pakistani, and predominantly Muslim, side of this encounter. Thus one of the factors considered in this thesis is how senses of Islam impact upon the two sides. By ethnic Pakistanis Islam is seen as central to all life decisions, whilst Health Professionals view Islam with some considerable trepidation, little understanding it or its centrality to the former's decision-making processes. This is particularly significant with regard to attitudes to health and health care. In the initial stages of the project I had thought first cousin marriage (FCM), seen by ethnic Pakistanis as desirable and by Health Professionals as putting ethnic Pakistanis at-risk to be central to the argument, but concluded that concerns around FCM were a 'red herring', merely a trope for the tensions between the two sides -at once both British and at-risk from audit culture. Although no longer central, FCM remains a viable touchstone in consideration of the two sides' perceptions of genetic risk. In this thesis the medical encounter between ethnic Pakistanis and Health Professionals is performed within the realm of the so called New Genetics. Here the respective understandings of the New Genetics are informed by the enculturation processes that shape the two sides' world view. Furthermore, I will agree with Lord Robert Winston's and others' concern that any attempt to eradicate an adaptive genetic mutation, in this instance, thalassaemia, from the gene pool is not only undesirable in the short term, but also that such eradications may have an adverse, and far reaching, effect on whole population groups in the future. The main thrust of my argument is that audit culture not only compounds risk for both sides, but also perpetuates institutional racism within the National Health Service (NHS), by promulgating what I have called the language myth. That is to say that much institutional racism is the unwanted by-product of the NHS's attempts to become more patient centred and its continuing efforts to develop systems of best practice. This professionalisation process within the NHS can be seen to impact most strongly in relation to communication -particularly the claimed language barrier between the two sides. This 'barrier' has worrying policy implications for any meaningful communication between the two sides, notably relating to obtaining informed consent from ethnic Pakistani patients -with a resultant increase in risk for the two sides and clear economic consequences for the NHS. 2005-01-01T00:00:00Z Murphy, Richard The central theme or 'red-thread' that I consider in this thesis is the concept of risk as it is perceived by and affects the two sides of the medical encounter -in this instance ethnic Pakistanis and Health Professionals- in Britain. Each side very often perceives risk quite distinctively, relating to the balance between the spiritual and temporal realms. This is particularly germane in matters to do with possible congenital defects within the prenatal realm for the ethnic Pakistani, and predominantly Muslim, side of this encounter. Thus one of the factors considered in this thesis is how senses of Islam impact upon the two sides. By ethnic Pakistanis Islam is seen as central to all life decisions, whilst Health Professionals view Islam with some considerable trepidation, little understanding it or its centrality to the former's decision-making processes. This is particularly significant with regard to attitudes to health and health care. In the initial stages of the project I had thought first cousin marriage (FCM), seen by ethnic Pakistanis as desirable and by Health Professionals as putting ethnic Pakistanis at-risk to be central to the argument, but concluded that concerns around FCM were a 'red herring', merely a trope for the tensions between the two sides -at once both British and at-risk from audit culture. Although no longer central, FCM remains a viable touchstone in consideration of the two sides' perceptions of genetic risk. In this thesis the medical encounter between ethnic Pakistanis and Health Professionals is performed within the realm of the so called New Genetics. Here the respective understandings of the New Genetics are informed by the enculturation processes that shape the two sides' world view. Furthermore, I will agree with Lord Robert Winston's and others' concern that any attempt to eradicate an adaptive genetic mutation, in this instance, thalassaemia, from the gene pool is not only undesirable in the short term, but also that such eradications may have an adverse, and far reaching, effect on whole population groups in the future. The main thrust of my argument is that audit culture not only compounds risk for both sides, but also perpetuates institutional racism within the National Health Service (NHS), by promulgating what I have called the language myth. That is to say that much institutional racism is the unwanted by-product of the NHS's attempts to become more patient centred and its continuing efforts to develop systems of best practice. This professionalisation process within the NHS can be seen to impact most strongly in relation to communication -particularly the claimed language barrier between the two sides. This 'barrier' has worrying policy implications for any meaningful communication between the two sides, notably relating to obtaining informed consent from ethnic Pakistani patients -with a resultant increase in risk for the two sides and clear economic consequences for the NHS. http://hdl.handle.net/10023/2800 Abstract: The Self-Regulation Model (Leventhal, Nerenz & Steele, 1984) highlights the roles of patients' illness representations, coping, emotional reactions and appraisal of coping in the progression of chronic disease. This thesis incorporates previous literature on adherence, panic-fear and selfmanagement interventions into the model in order to (a) investigate coping with asthma and (b) develop an intervention aimed at improving asthmatic control. New measures of asthmatic control and illness representations of the consequences of having asthma were developed in order to operationalise the model. A cross-sectional study investigated factors influencing asthmatic control in a sample of 35 adult asthma sufferers recruited through a single general practice. Coping was poor, adherence being low and less than 50% of participants reporting current Peak Flow monitoring or medical contact during the previous 12 months. Good coping appeared to be a response to poor asthmatic control, rather than prophylactic. Good asthmatic control was associated with low perceived consequences, recent medical contact, moderate panic-fear and low general avoidance coping. These results imply that asthmatic control may be improved by encouraging sufferers to maintain regular contact with outpatient services and to implement prophylactic coping. Since epidemiological and clinical evidence suggested asthmatic control to be poor in young adults, an intervention was developed to improve asthmatic control in this group by modifying illness representations, coping and panic-fear. The intervention was evaluated in a randomised controlled study of 50 student asthma sufferers identified initially through an epidemiological screening of 2,979 students. It led to increased Preventer medication use and Peak Flow monitoring and decreased distress over the condition. However, the coping process changed and asthmatic control improved even in the control group, perhaps because self-monitoring of asthmatic control for the study constituted a change in coping. This unanticipated result was entirely compatible with the Self-Regulation Model. The thesis dearly demonstrates value of the Self-Regulation Model in understanding asthma self-management and developing clinical interventions. 1995-01-01T00:00:00Z Williams, Julie M. The Self-Regulation Model (Leventhal, Nerenz & Steele, 1984) highlights the roles of patients' illness representations, coping, emotional reactions and appraisal of coping in the progression of chronic disease. This thesis incorporates previous literature on adherence, panic-fear and selfmanagement interventions into the model in order to (a) investigate coping with asthma and (b) develop an intervention aimed at improving asthmatic control. New measures of asthmatic control and illness representations of the consequences of having asthma were developed in order to operationalise the model. A cross-sectional study investigated factors influencing asthmatic control in a sample of 35 adult asthma sufferers recruited through a single general practice. Coping was poor, adherence being low and less than 50% of participants reporting current Peak Flow monitoring or medical contact during the previous 12 months. Good coping appeared to be a response to poor asthmatic control, rather than prophylactic. Good asthmatic control was associated with low perceived consequences, recent medical contact, moderate panic-fear and low general avoidance coping. These results imply that asthmatic control may be improved by encouraging sufferers to maintain regular contact with outpatient services and to implement prophylactic coping. Since epidemiological and clinical evidence suggested asthmatic control to be poor in young adults, an intervention was developed to improve asthmatic control in this group by modifying illness representations, coping and panic-fear. The intervention was evaluated in a randomised controlled study of 50 student asthma sufferers identified initially through an epidemiological screening of 2,979 students. It led to increased Preventer medication use and Peak Flow monitoring and decreased distress over the condition. However, the coping process changed and asthmatic control improved even in the control group, perhaps because self-monitoring of asthmatic control for the study constituted a change in coping. This unanticipated result was entirely compatible with the Self-Regulation Model. The thesis dearly demonstrates value of the Self-Regulation Model in understanding asthma self-management and developing clinical interventions. http://hdl.handle.net/10023/2720 Abstract: The mechanisms which regulate the growth fraction of the haemopoietic stem cell (CFU-S) and granulocyte macrophage progenitor cell (GM-CFC) have been investigated. In normal murine bone marrow (NMBM) a small proportion of the CFU-S are synthesising DNA (-10%). In contrast, in the bone marrow from mice regenerating after treatment with cytotoxic drugs and in developing haemopoietic tissues such as murine fetal liver a large proportion of the CFU-S (-40%) are synthesising DNA. Medium conditioned by normal murine and human bone marrow cells inhibited the proliferation of rapidly cycling CFU-S from regenerating bone marrow. This inhibitor was contained in a 50-100K daltons ultrafiltration fraction. In contra-distinction medium conditioned by human fetal liver cells stimulated the proliferation of CFU-S from NMBM. The stimulator was produced by adherent cells and was contained in a 30-50K daltons ultrafiltration fraction. An alternative assay for the humoral regulators of CFU-S proliferation was developed. Different numbers of haemopoietic cells were injected into lethally irradiated mice. Five days later they were injected with 2iCi of 125IUdR and sacrificed 2 hours later. There was a linear relationship between the log 125IUdR uptake into the spleen and femur and the log cell dose injected. Pre-treatment of haemopoietic cells with an S-phase specific cytotoxic drug resulted in a reduction in the 125IUdR incorporation into the spleen. This enabled the kinetic properties of a haemopoietic stem cell population to be assessed and the humoral 111 factors which modulate the growth fraction of these cells to be investigated. At early stages of gestation (11-14 weeks) in human fetal liver few GM-CFC are synthesising DNA, whereas later in gestation (>14 weeks) a large proportion of GM-CFC are in S-phase, Moore and Williams (1973b). Incubation of NMBM GM-CFC (approx 40% in DNA synthesis) with a supernatant from an early human fetal liver (11-14 weeks) reduced the proportion synthesising DNA to <5%. In contrast, the proportion of murine GM-CFC synthesising DNA was not affected by incubation with a supernatant from a late human fetal liver (>14 weeks). GM-CFC that had been switched out of cycle by incubation with a supernatant from an early gestation human fetal liver were switched back into cycle following incubation with a late human fetal liver supernatant. The inhibitor and stimulator of GM-CFC proliferation were both produced by non-adherent cells and were contained in >100K and 30-50K daltons ultrafiltration fractions repectively. It is likely that changes in the relative levels of a proliferation inhibitor and stimulator throughout gestation might control the proportion of GM-CFC in cycle. 1984-01-01T00:00:00Z Cork, Michael John The mechanisms which regulate the growth fraction of the haemopoietic stem cell (CFU-S) and granulocyte macrophage progenitor cell (GM-CFC) have been investigated. In normal murine bone marrow (NMBM) a small proportion of the CFU-S are synthesising DNA (-10%). In contrast, in the bone marrow from mice regenerating after treatment with cytotoxic drugs and in developing haemopoietic tissues such as murine fetal liver a large proportion of the CFU-S (-40%) are synthesising DNA. Medium conditioned by normal murine and human bone marrow cells inhibited the proliferation of rapidly cycling CFU-S from regenerating bone marrow. This inhibitor was contained in a 50-100K daltons ultrafiltration fraction. In contra-distinction medium conditioned by human fetal liver cells stimulated the proliferation of CFU-S from NMBM. The stimulator was produced by adherent cells and was contained in a 30-50K daltons ultrafiltration fraction. An alternative assay for the humoral regulators of CFU-S proliferation was developed. Different numbers of haemopoietic cells were injected into lethally irradiated mice. Five days later they were injected with 2iCi of 125IUdR and sacrificed 2 hours later. There was a linear relationship between the log 125IUdR uptake into the spleen and femur and the log cell dose injected. Pre-treatment of haemopoietic cells with an S-phase specific cytotoxic drug resulted in a reduction in the 125IUdR incorporation into the spleen. This enabled the kinetic properties of a haemopoietic stem cell population to be assessed and the humoral 111 factors which modulate the growth fraction of these cells to be investigated. At early stages of gestation (11-14 weeks) in human fetal liver few GM-CFC are synthesising DNA, whereas later in gestation (>14 weeks) a large proportion of GM-CFC are in S-phase, Moore and Williams (1973b). Incubation of NMBM GM-CFC (approx 40% in DNA synthesis) with a supernatant from an early human fetal liver (11-14 weeks) reduced the proportion synthesising DNA to <5%. In contrast, the proportion of murine GM-CFC synthesising DNA was not affected by incubation with a supernatant from a late human fetal liver (>14 weeks). GM-CFC that had been switched out of cycle by incubation with a supernatant from an early gestation human fetal liver were switched back into cycle following incubation with a late human fetal liver supernatant. The inhibitor and stimulator of GM-CFC proliferation were both produced by non-adherent cells and were contained in >100K and 30-50K daltons ultrafiltration fractions repectively. It is likely that changes in the relative levels of a proliferation inhibitor and stimulator throughout gestation might control the proportion of GM-CFC in cycle. http://hdl.handle.net/10023/2673 Abstract: The pedunculopontine tegmental nucleus (PPTg) lies within the pontomesencephalon and contains cholinergic and non-cholinergic neurones. It has extensive afferent and efferent connections throughout the brain. Early research suggested a role for the PPTg in the mediation of locomotor activity, and it was believed to form the major substrate of the electrophysiologically identified mesencephalic locomotor region (rviLR). Studies using selective excitotoxic lesions of the PPTg demonstrated that it has no role in the mediation of spontaneous or nucleus accumbens-induced (NAcc) locomotion. However evidence has suggested that the cuneiform nucleus (CNF) and not the PPTg is the main locus of the .MLR. The effects of bilateral ibotenate CNF lesions on spontaneous and amphetamine-induced locomotion stimulated from the NAcc were therefore investigated. CNF lesions had no effect on either type of locomotor activity. Bilateral ibotenate lesions of the PPTg have been shown to influence the expression of orofacial stereotypies following administration of systemic amphetamine. Oral stereotypies can be elicited reliably by direct stimulation of the ventrolateral caudate-putamen (VLCP). This thesis sought to clarify the role of the PPTg in the mediation of oral stereotypies, by combining bilateral ibotenate lesions of the PPTg with direct microinjection of amphetamine into the VLCP. Lesions of the PPTg caused a shift in the dose response curve to amphetamine resulting in an increase in the incidence and intensity of oro facial stereotypies at lower doses. Thus the PPTg appears to have inhibitory control over the expression of orofacial behaviors. It is hypothesised that while neither the PPTg nor the CNF have a role in the mediation of locomotor activity per se they may provide an integrative functional role, which influences motor outflow. The role of the CNF in the transmission of nociception and a role for the PPTg in the mediation of striatal outflow is discussed. 1996-01-01T00:00:00Z Allen, Laura F. The pedunculopontine tegmental nucleus (PPTg) lies within the pontomesencephalon and contains cholinergic and non-cholinergic neurones. It has extensive afferent and efferent connections throughout the brain. Early research suggested a role for the PPTg in the mediation of locomotor activity, and it was believed to form the major substrate of the electrophysiologically identified mesencephalic locomotor region (rviLR). Studies using selective excitotoxic lesions of the PPTg demonstrated that it has no role in the mediation of spontaneous or nucleus accumbens-induced (NAcc) locomotion. However evidence has suggested that the cuneiform nucleus (CNF) and not the PPTg is the main locus of the .MLR. The effects of bilateral ibotenate CNF lesions on spontaneous and amphetamine-induced locomotion stimulated from the NAcc were therefore investigated. CNF lesions had no effect on either type of locomotor activity. Bilateral ibotenate lesions of the PPTg have been shown to influence the expression of orofacial stereotypies following administration of systemic amphetamine. Oral stereotypies can be elicited reliably by direct stimulation of the ventrolateral caudate-putamen (VLCP). This thesis sought to clarify the role of the PPTg in the mediation of oral stereotypies, by combining bilateral ibotenate lesions of the PPTg with direct microinjection of amphetamine into the VLCP. Lesions of the PPTg caused a shift in the dose response curve to amphetamine resulting in an increase in the incidence and intensity of oro facial stereotypies at lower doses. Thus the PPTg appears to have inhibitory control over the expression of orofacial behaviors. It is hypothesised that while neither the PPTg nor the CNF have a role in the mediation of locomotor activity per se they may provide an integrative functional role, which influences motor outflow. The role of the CNF in the transmission of nociception and a role for the PPTg in the mediation of striatal outflow is discussed. http://hdl.handle.net/10023/2663 Abstract: In order to increase the number of older adults physically active enough to obtain the health benefits of exercise, inactive individuals must firstly be identified, and attention must then be focused on determinants of exercise amenable to change. This study set out to develop self-complete questionnaires for assessing activity status, and perceived benefits of, and barriers to, physical activity. Of 1456 questionnaires sent out to a random sample of adults aged 55-74 a usable return rate of 37.6% (n=548) was achieved. A principal components analysis of the benefits of physical activity revealed five factors (physical performance, social, weight control, enjoyment, and psychological), and of the barriers to physical activity, also five factors (opportunities, physical exertion, time, limiting health, and support). Alpha internal consistency coefficients for the 10 factors ranged from 0.64 to 0.92, and test-retest reliability coefficients from 0.56 to 0.87. A series of one-way ANOVAs revealed that, with the exception of the benefit weight control, there was a significant gradation in factor scores between active and inactive subjects as classified by 4-, 9-, and 5- point activity classification methods. Validity of the activity classifications was assessed in a subsample of 86 subjects against measures of strength, flexibility, aerobic fitness and objectively measured physical activity. Active and inactive subjects classified using the 4- and 9-point questionnaires differed significantly in 1-mile walk time and energy expenditure estimated by a Caltrac accelerometer. The 5-point questionnaire did not appear able to differentiate active and inactive subjects. Test-retest reliability of the questionnaires ranged from 0.62 to 0.73. The questionnaire developed from this work for measuring perceived benefits and barriers of older adults can be used in either practical or research settings. Further work is required to determine the accuracy of the physical activity questionnaires in identifying low-active individuals in the population. 1997-01-01T00:00:00Z Montgomery, Alan A. In order to increase the number of older adults physically active enough to obtain the health benefits of exercise, inactive individuals must firstly be identified, and attention must then be focused on determinants of exercise amenable to change. This study set out to develop self-complete questionnaires for assessing activity status, and perceived benefits of, and barriers to, physical activity. Of 1456 questionnaires sent out to a random sample of adults aged 55-74 a usable return rate of 37.6% (n=548) was achieved. A principal components analysis of the benefits of physical activity revealed five factors (physical performance, social, weight control, enjoyment, and psychological), and of the barriers to physical activity, also five factors (opportunities, physical exertion, time, limiting health, and support). Alpha internal consistency coefficients for the 10 factors ranged from 0.64 to 0.92, and test-retest reliability coefficients from 0.56 to 0.87. A series of one-way ANOVAs revealed that, with the exception of the benefit weight control, there was a significant gradation in factor scores between active and inactive subjects as classified by 4-, 9-, and 5- point activity classification methods. Validity of the activity classifications was assessed in a subsample of 86 subjects against measures of strength, flexibility, aerobic fitness and objectively measured physical activity. Active and inactive subjects classified using the 4- and 9-point questionnaires differed significantly in 1-mile walk time and energy expenditure estimated by a Caltrac accelerometer. The 5-point questionnaire did not appear able to differentiate active and inactive subjects. Test-retest reliability of the questionnaires ranged from 0.62 to 0.73. The questionnaire developed from this work for measuring perceived benefits and barriers of older adults can be used in either practical or research settings. Further work is required to determine the accuracy of the physical activity questionnaires in identifying low-active individuals in the population. http://hdl.handle.net/10023/900 Abstract: Cervical cancer is a major cause of death in women and is strongly associated with infection by human papillomavirus (HPV). Integration of HPV is thought to form a key step in the formation of cancer, and is thought to involve the upregulation of HPV E6 and E7 due to the loss of E2 transcriptional control. Leptomycin B (LMB), a nuclear export inhibitor, has previously been shown to induce apoptosis in HPV-containing cancer cell lines and HPV 16 E7 or E6/E7 transduced primary keratinocytes, but not in normal cells. This thesis shows that LMB can induce apoptosis and a reduction in the colony survival of derivatives of the W12 cell line that contain HPV 16 in either episomal or integrated form. The HPV genome status, including variations in viral integration type, appears to influence the cumulative and temporal pattern of LMB-induced apoptosis. The effects of LMB were also apparent in cells grown in organotypic raft culture, with differences in behaviour again apparent between cells containing episomal and integrated HPV. As previously noted, treatment with LMB was associated with increased expression of the cell regulators p53 and p21; however, the induction of apoptosis was not dependent upon transcriptionally active p53. It is therefore likely that induction and mediation of LMB-induced apoptosis occurs via alternative, currently unidentified, pathways. These findings suggest that LMB can induce apoptosis in keratinocytes containing HPV 16 in either episomal or integrated form, with genome status and potentially lesion grade likely to influence the response of HPV-associated anogenital lesions to LMB treatment. 2008-01-01T00:00:00Z Jolly, Carol E Cervical cancer is a major cause of death in women and is strongly associated with infection by human papillomavirus (HPV). Integration of HPV is thought to form a key step in the formation of cancer, and is thought to involve the upregulation of HPV E6 and E7 due to the loss of E2 transcriptional control. Leptomycin B (LMB), a nuclear export inhibitor, has previously been shown to induce apoptosis in HPV-containing cancer cell lines and HPV 16 E7 or E6/E7 transduced primary keratinocytes, but not in normal cells. This thesis shows that LMB can induce apoptosis and a reduction in the colony survival of derivatives of the W12 cell line that contain HPV 16 in either episomal or integrated form. The HPV genome status, including variations in viral integration type, appears to influence the cumulative and temporal pattern of LMB-induced apoptosis. The effects of LMB were also apparent in cells grown in organotypic raft culture, with differences in behaviour again apparent between cells containing episomal and integrated HPV. As previously noted, treatment with LMB was associated with increased expression of the cell regulators p53 and p21; however, the induction of apoptosis was not dependent upon transcriptionally active p53. It is therefore likely that induction and mediation of LMB-induced apoptosis occurs via alternative, currently unidentified, pathways. These findings suggest that LMB can induce apoptosis in keratinocytes containing HPV 16 in either episomal or integrated form, with genome status and potentially lesion grade likely to influence the response of HPV-associated anogenital lesions to LMB treatment. http://hdl.handle.net/10023/873 Abstract: Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model. 2010-06-01T00:00:00Z Terry, Samantha Y.A. Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model. http://hdl.handle.net/10023/863 Abstract: A key effect of human papillomavirus (HPV) infection is to disrupt the normal cell cycle in order to subvert the cellular DNA replication machinery. Morphologically, condylomata induced by high and low risk HPV types cannot be distinguished and many studies have shown that the pattern of viral gene expression is similar in condylomata caused by both high risk and low risk HPV types. Detailed morphological study of cell cycle protein expression has not previously been performed on condylomata infected with low risk HPV types. The findings presented suggest that the mechanisms employed by low risk HPV6 or HPV11 to subvert cellular functions in condylomata acuminata are similar to those employed by high risk HPVs, with the exception of cyclin D1 and p53 protein over-expression. The differences in p53 expression and cyclin D1 expression seen between high and low risk HPV infection, reflect the known differences between high and low risk types and are in agreement with the known differences between high risk and low risk E6 and E7 proteins. PHK transduction studies demonstrated HPV E6 and E7 induce changes in cell cycle protein expression and that there are differences in cell cycle abrogation between HPV6 and HPV16. Disruption of the p53-MDM2 interaction can lead to activation of the p53 pathway. HPV infected lesions almost always contain wild-type p53. The binding of HPV E6 to p53, and its subsequent targeting for degradation, prevents activation of the p53 pathway in HPV infected cells. Cells over expressing HPV genes were treated with Nutlin-3, a MDM2-small molecule antagonist. The findings presented suggest treatment with Nutlin-3 induces cell cycle arrest in cells expressing HPV16 E7 and HPV6 E6 and HPV6 E7. This suggests a potential role for Nutlin-3 in the treatment of HPV infected cells. 2010-07-01T00:00:00Z Lyman, Rachel C. A key effect of human papillomavirus (HPV) infection is to disrupt the normal cell cycle in order to subvert the cellular DNA replication machinery. Morphologically, condylomata induced by high and low risk HPV types cannot be distinguished and many studies have shown that the pattern of viral gene expression is similar in condylomata caused by both high risk and low risk HPV types. Detailed morphological study of cell cycle protein expression has not previously been performed on condylomata infected with low risk HPV types. The findings presented suggest that the mechanisms employed by low risk HPV6 or HPV11 to subvert cellular functions in condylomata acuminata are similar to those employed by high risk HPVs, with the exception of cyclin D1 and p53 protein over-expression. The differences in p53 expression and cyclin D1 expression seen between high and low risk HPV infection, reflect the known differences between high and low risk types and are in agreement with the known differences between high risk and low risk E6 and E7 proteins. PHK transduction studies demonstrated HPV E6 and E7 induce changes in cell cycle protein expression and that there are differences in cell cycle abrogation between HPV6 and HPV16. Disruption of the p53-MDM2 interaction can lead to activation of the p53 pathway. HPV infected lesions almost always contain wild-type p53. The binding of HPV E6 to p53, and its subsequent targeting for degradation, prevents activation of the p53 pathway in HPV infected cells. Cells over expressing HPV genes were treated with Nutlin-3, a MDM2-small molecule antagonist. The findings presented suggest treatment with Nutlin-3 induces cell cycle arrest in cells expressing HPV16 E7 and HPV6 E6 and HPV6 E7. This suggests a potential role for Nutlin-3 in the treatment of HPV infected cells. http://hdl.handle.net/10023/855 Abstract: A genetically determined high level of intrinsic normal tissue radiosensitivity may account for the 5% of patients who experience unexpectedly severe normal tissue side effects following radiotherapy. The pre-treatment identification of these individuals by a diagnostic test or “predictive assay “ may allow appropriate modification of treatment plans and improve the therapeutic index of radiotherapy. Results from studies of cell-based assays measuring the response of a single cell type taken from patients to in vitro irradiation have been inconsistent, leading to the opinion of many that they are of no value in the prediction of normal tissue radiosensitivity. A systematic review of the literature presented here, however, suggests that poor methodology of study design often with inadequate control for those factors other than normal tissue radiosensitivity which influence radiotherapy toxicity and lack of reporting of assay precision means that it is difficult to form any conclusions, positive or negative about the diagnostic accuracy of the cell-based assays studied so far. Analysis of individual patient data extracted from these studies suggests that at least some of these assays may possess some discriminatory value. This finding justified an attempt to develop a novel cell-based assay based on the kinetics of radiation-induced .H2AX in peripheral blood lymphocytes. Assay failure rate was high and intra- and inter-sample assay reproducibility was poor for quantification by microscopy but were better for flow cytometric analysis. A study of 8 volunteers, however, demonstrated that intra-individual variation was higher than inter-individual variation in assay results, strongly suggesting that poor assay reproducibility due to technical or biological factors may limit the assay’s potential to identify radiosensitive individuals. This suspicion needs to be confirmed in a clinical study of patients of known radiosensitivity. As blood sample storage conditions affect assay results these will need to be standardized to prevent confounding of results. 2008-01-01T00:00:00Z Brown, Emma Jane Hay A genetically determined high level of intrinsic normal tissue radiosensitivity may account for the 5% of patients who experience unexpectedly severe normal tissue side effects following radiotherapy. The pre-treatment identification of these individuals by a diagnostic test or “predictive assay “ may allow appropriate modification of treatment plans and improve the therapeutic index of radiotherapy. Results from studies of cell-based assays measuring the response of a single cell type taken from patients to in vitro irradiation have been inconsistent, leading to the opinion of many that they are of no value in the prediction of normal tissue radiosensitivity. A systematic review of the literature presented here, however, suggests that poor methodology of study design often with inadequate control for those factors other than normal tissue radiosensitivity which influence radiotherapy toxicity and lack of reporting of assay precision means that it is difficult to form any conclusions, positive or negative about the diagnostic accuracy of the cell-based assays studied so far. Analysis of individual patient data extracted from these studies suggests that at least some of these assays may possess some discriminatory value. This finding justified an attempt to develop a novel cell-based assay based on the kinetics of radiation-induced .H2AX in peripheral blood lymphocytes. Assay failure rate was high and intra- and inter-sample assay reproducibility was poor for quantification by microscopy but were better for flow cytometric analysis. A study of 8 volunteers, however, demonstrated that intra-individual variation was higher than inter-individual variation in assay results, strongly suggesting that poor assay reproducibility due to technical or biological factors may limit the assay’s potential to identify radiosensitive individuals. This suspicion needs to be confirmed in a clinical study of patients of known radiosensitivity. As blood sample storage conditions affect assay results these will need to be standardized to prevent confounding of results. http://hdl.handle.net/10023/834 Abstract: The MHC class I allele, HLA-B27, is strongly associated with a group of inflammatory arthritic conditions collectively known as spondyloarthropathies (SpA). Ankylosing spondylitis (AS) shows the strongest association with 90-95 % of patients being HLA-B27 positive. The relationship between HLA-B27 and SpA has been known for over 30 years, however despite ongoing research, the reason for this association has not yet been elucidated. In more recent years, research has focused on intrinsic properties of the HLA-B27 allele, in particular its propensity to misfold, forming homodimers. It has been proposed that these homodimers could be associated with the disease process through the activation of an ER stress response known as the unfolded protein response (UPR), or through aberrant recognition at the cell surface. We have investigated whether the expression of HLA-B27 is associated with the activation of the UPR. We have studied the expression of BiP, and the cleavage of XBP1 and ATF6 using stable and transiently expressing cell lines. We have also investigated the formation of non-B27 homodimers using a human cell line stably expressing HLA-B8, and finally we have studied the expression of homodimers in exosomes, small immunomodulatory vesicles released from numerous cell types. The results presented here lead us to conclude that in vitro studies of the UPR are complicated, prone to a number of technical issues, and may therefore not be appropriate for gaining information that would be of significant use when comparing to the real disease scenario. Our data suggest that non-B27 dimers may be strongly influenced by both the overexpression of MHC class I heavy chains and also the redox environment within the cell. We have isolated a novel fully folded, beta-2m-associated, MHC class I homodimer in exosomes and have detected a novel HLA-A and HLA-B mixed heavy chain dimer. Our results suggest that these dimers form through interactions between the cysteine residues in the cytoplasmic tail and that these dimers form in exosomes because they contain lower levels of the important antioxidant glutathione when compared to whole cells. Together, these results define a new MHC class I structure present on exosomes at significant levels, which could potentially influence immune recognition by both antigen-specific T cell receptors and NK family receptors. The data also poses questions about whether these novel structures, when they involve HLA-B27, could influence the pathogenesis of spondyloarthropathies. Description: Electronic version excludes material for which permission has not been granted by the rights holder 2009-11-01T00:00:00Z Lynch, Sarah Janice The MHC class I allele, HLA-B27, is strongly associated with a group of inflammatory arthritic conditions collectively known as spondyloarthropathies (SpA). Ankylosing spondylitis (AS) shows the strongest association with 90-95 % of patients being HLA-B27 positive. The relationship between HLA-B27 and SpA has been known for over 30 years, however despite ongoing research, the reason for this association has not yet been elucidated. In more recent years, research has focused on intrinsic properties of the HLA-B27 allele, in particular its propensity to misfold, forming homodimers. It has been proposed that these homodimers could be associated with the disease process through the activation of an ER stress response known as the unfolded protein response (UPR), or through aberrant recognition at the cell surface. We have investigated whether the expression of HLA-B27 is associated with the activation of the UPR. We have studied the expression of BiP, and the cleavage of XBP1 and ATF6 using stable and transiently expressing cell lines. We have also investigated the formation of non-B27 homodimers using a human cell line stably expressing HLA-B8, and finally we have studied the expression of homodimers in exosomes, small immunomodulatory vesicles released from numerous cell types. The results presented here lead us to conclude that in vitro studies of the UPR are complicated, prone to a number of technical issues, and may therefore not be appropriate for gaining information that would be of significant use when comparing to the real disease scenario. Our data suggest that non-B27 dimers may be strongly influenced by both the overexpression of MHC class I heavy chains and also the redox environment within the cell. We have isolated a novel fully folded, beta-2m-associated, MHC class I homodimer in exosomes and have detected a novel HLA-A and HLA-B mixed heavy chain dimer. Our results suggest that these dimers form through interactions between the cysteine residues in the cytoplasmic tail and that these dimers form in exosomes because they contain lower levels of the important antioxidant glutathione when compared to whole cells. Together, these results define a new MHC class I structure present on exosomes at significant levels, which could potentially influence immune recognition by both antigen-specific T cell receptors and NK family receptors. The data also poses questions about whether these novel structures, when they involve HLA-B27, could influence the pathogenesis of spondyloarthropathies. http://hdl.handle.net/10023/755 Abstract: Prostate cell lines were derived from two regions of prostate tissue from the same patient. The objective was to produce cell lines (as a useful in vitro model) from these two different regions which exhibit different properties for carcinoma development. The tissue was obtained from patients suffering from benign prostate hyperplasia undergoing trans-urethral resection. Tissue was taken from the deep (peripheral) and superficial (peri-urethral) areas. The cells were immortalised by transduction with constructs over expressing the cdk4 and hTERT genes. These cell lines were then characterised for their cellular phenotypes utilized for radiation transformation studies and utilized to investigate the role of plant derived polyphenols on normal and tumour cells. The cell line from the superficial region (P21s) was treated to fractionated doses of gamma radiation and a transformed cloned cell line was derived (P21s 40Gy (clone-a)). The cell line from the deep region (P21d) was found to consist of a mixed population of abnormal cells and a transformed cloned cell line was derived from it (P21d 0Gy (clone-a). In an attempt to obtain a normal P21d cell line cloned cell lines from early passage P21d cells were established. All seven cloned lines were abnormal with an average of 80 chromosomes per cell, invasive using a Matrigel assay and produced anchorage independent colonies. All cell lines were fully characterised with immunocytochemistry, chromosome analysis, invasion assays, and anchorage independent colony formation. P21s expressed basal cell markers (cytokeratin 5 (CK5) and 14), were positive for stem cell markers (prostate specific stem cell antigen PSCA, CK6), positive for p16, p63 and telomerase expression and negative for c-Myc expression. P21s was not invasive in a Matrigel assay and did not produce anchorage independent colony formation. P21d and P21d 0Gy (clone-a) also expressed CK5, CK14, PSCA, CK6, and telomerase but not p16 or p63 and showed an increase in expression of nuclear c-Myc, highly invasive and produced anchorage independent colonies. P21s 40Gy (clone-a) expressed CK5, CK14, PSCA, CK6, telomerase and p63, produced anchorage independent colonies, and was weakly positive for c-Myc expression. Spectral karyotyping analysis (SKY) showed P21s had a normal chromosome complement except an additional chromosome 20 whereas the P21s 40Gy (clone-a), P21d and P21d 0Gy (clone-a) cell lines had an abnormal chromosome complement with P21d and P21d 0Gy (clone-a) cell lines expressing multiple copies of every chromosome including loss of the Y chromosome. These results were echoed in the single nucleotide polymorphism chip (SNP) results which showed P21s as normal but P21d and P21d 0Gy (clone-a) to have large deletion and amplification regions that correlated with the SKY analysis. No differential cytotoxic response was noted between normal and abnormal cell lines including prostatic carcinoma cell lines LNCaP and PC-3 following treatment with strawberry polyphenol compounds. Most reports of a cytotoxic response to tumour cells in the literature did not compare the response to normal cells and used established cell lines. Human lymphocytes were also tested and all compounds were toxic in high doses. Polyphenol and ellagitannin rich polyphenol fractions were very cytotoxic and the anthocyanin rich fraction less toxic. In contrast to the lack of a direct differential cytotoxic effect, plant polyphenols did produce a protective effect to a carcinogenic insult. However a protective effect was noted via micronucleus assay with 3 hour incubation with the polyphenol rich fraction prior to radiation treatment. Finally, the expression and association of metabolic enzymes within the cells cytosol were investigated. The P21s cells were found to express both isoforms of LDH and so thought to be able to metabolise anaerobically and aerobically. P21d and P21d 0Gy (clone-a) cells were found to only express one isoform in the complex and so it was assumed that these cells favoured anaerobic metabolism of ATP in correlation to the Warburg effect. c-Myc association with compounds in the cell cytosol of P21s cells existed whereas, abnormal cells lost this association along with up-regulation of c-Myc expression and down stream targets of c-Myc in the nuclei. Thus these newly established human prostate cell lines provide a useful model system for investigating the biology of the prostate and prostate cancer. 2009-06-26T00:00:00Z Weaver, Jennifer Prostate cell lines were derived from two regions of prostate tissue from the same patient. The objective was to produce cell lines (as a useful in vitro model) from these two different regions which exhibit different properties for carcinoma development. The tissue was obtained from patients suffering from benign prostate hyperplasia undergoing trans-urethral resection. Tissue was taken from the deep (peripheral) and superficial (peri-urethral) areas. The cells were immortalised by transduction with constructs over expressing the cdk4 and hTERT genes. These cell lines were then characterised for their cellular phenotypes utilized for radiation transformation studies and utilized to investigate the role of plant derived polyphenols on normal and tumour cells. The cell line from the superficial region (P21s) was treated to fractionated doses of gamma radiation and a transformed cloned cell line was derived (P21s 40Gy (clone-a)). The cell line from the deep region (P21d) was found to consist of a mixed population of abnormal cells and a transformed cloned cell line was derived from it (P21d 0Gy (clone-a). In an attempt to obtain a normal P21d cell line cloned cell lines from early passage P21d cells were established. All seven cloned lines were abnormal with an average of 80 chromosomes per cell, invasive using a Matrigel assay and produced anchorage independent colonies. All cell lines were fully characterised with immunocytochemistry, chromosome analysis, invasion assays, and anchorage independent colony formation. P21s expressed basal cell markers (cytokeratin 5 (CK5) and 14), were positive for stem cell markers (prostate specific stem cell antigen PSCA, CK6), positive for p16, p63 and telomerase expression and negative for c-Myc expression. P21s was not invasive in a Matrigel assay and did not produce anchorage independent colony formation. P21d and P21d 0Gy (clone-a) also expressed CK5, CK14, PSCA, CK6, and telomerase but not p16 or p63 and showed an increase in expression of nuclear c-Myc, highly invasive and produced anchorage independent colonies. P21s 40Gy (clone-a) expressed CK5, CK14, PSCA, CK6, telomerase and p63, produced anchorage independent colonies, and was weakly positive for c-Myc expression. Spectral karyotyping analysis (SKY) showed P21s had a normal chromosome complement except an additional chromosome 20 whereas the P21s 40Gy (clone-a), P21d and P21d 0Gy (clone-a) cell lines had an abnormal chromosome complement with P21d and P21d 0Gy (clone-a) cell lines expressing multiple copies of every chromosome including loss of the Y chromosome. These results were echoed in the single nucleotide polymorphism chip (SNP) results which showed P21s as normal but P21d and P21d 0Gy (clone-a) to have large deletion and amplification regions that correlated with the SKY analysis. No differential cytotoxic response was noted between normal and abnormal cell lines including prostatic carcinoma cell lines LNCaP and PC-3 following treatment with strawberry polyphenol compounds. Most reports of a cytotoxic response to tumour cells in the literature did not compare the response to normal cells and used established cell lines. Human lymphocytes were also tested and all compounds were toxic in high doses. Polyphenol and ellagitannin rich polyphenol fractions were very cytotoxic and the anthocyanin rich fraction less toxic. In contrast to the lack of a direct differential cytotoxic effect, plant polyphenols did produce a protective effect to a carcinogenic insult. However a protective effect was noted via micronucleus assay with 3 hour incubation with the polyphenol rich fraction prior to radiation treatment. Finally, the expression and association of metabolic enzymes within the cells cytosol were investigated. The P21s cells were found to express both isoforms of LDH and so thought to be able to metabolise anaerobically and aerobically. P21d and P21d 0Gy (clone-a) cells were found to only express one isoform in the complex and so it was assumed that these cells favoured anaerobic metabolism of ATP in correlation to the Warburg effect. c-Myc association with compounds in the cell cytosol of P21s cells existed whereas, abnormal cells lost this association along with up-regulation of c-Myc expression and down stream targets of c-Myc in the nuclei. Thus these newly established human prostate cell lines provide a useful model system for investigating the biology of the prostate and prostate cancer. http://hdl.handle.net/10023/442 Abstract: Currently the use of adenoviruses in cancer gene therapy is limited by efficient delivery of the virus into the tumour cells, detargeting of the virus from the liver, and the efficient spread of the virus within the tumour. Rapid and easy modification of adenoviruses enables expression of different genes from the genome of an oncolytic virus. I developed a system where the E1-pIX region of the adenovirus 5 genome could be mutated via recombination of a recipient virus with the deleted E1-pIX region flanked by a loxP and an attB-site and an “addback” plasmid with the mutated E1-pIX region flanked by a loxP and an attP-site. The recipient virus was found not to be producible even on a pIX-complementing cell line. The pIX was further modified by fusing GFP, FCU1 and MMP7 to the C-terminus with a 2A sequence that enables the ribosome to skip one specific peptide bond enabling the expression of genes flanking this sequence. Two different 2A sequences were used: FMDV 2A (F2A) and PTV-1 2A (P2A). The pIX-P2A-GFP expressing virus was found to have similar heat stability, CPE, burst size and plaque size characteristics as the parental virus, whereas the pIX-F2A-GFP expressing virus was found to have reduced heat stability, CPE, burst size and smaller plaque size. The viruses expressing FCU1 and MMP7 were found only to be producible on a pIX-complementing cell line due to the low expression of pIX from these constructs. I concluded that 2A sequences can be used in the context of adenoviruses but optimisation of the sequence may be needed depending on the fusion partners. 2008-01-01T00:00:00Z Kallioinen, Susanna E. Currently the use of adenoviruses in cancer gene therapy is limited by efficient delivery of the virus into the tumour cells, detargeting of the virus from the liver, and the efficient spread of the virus within the tumour. Rapid and easy modification of adenoviruses enables expression of different genes from the genome of an oncolytic virus. I developed a system where the E1-pIX region of the adenovirus 5 genome could be mutated via recombination of a recipient virus with the deleted E1-pIX region flanked by a loxP and an attB-site and an “addback” plasmid with the mutated E1-pIX region flanked by a loxP and an attP-site. The recipient virus was found not to be producible even on a pIX-complementing cell line. The pIX was further modified by fusing GFP, FCU1 and MMP7 to the C-terminus with a 2A sequence that enables the ribosome to skip one specific peptide bond enabling the expression of genes flanking this sequence. Two different 2A sequences were used: FMDV 2A (F2A) and PTV-1 2A (P2A). The pIX-P2A-GFP expressing virus was found to have similar heat stability, CPE, burst size and plaque size characteristics as the parental virus, whereas the pIX-F2A-GFP expressing virus was found to have reduced heat stability, CPE, burst size and smaller plaque size. The viruses expressing FCU1 and MMP7 were found only to be producible on a pIX-complementing cell line due to the low expression of pIX from these constructs. I concluded that 2A sequences can be used in the context of adenoviruses but optimisation of the sequence may be needed depending on the fusion partners. http://hdl.handle.net/10023/222 Abstract: Isoflavones are biologically active plant chemicals (phytoestrogens) which are ordinarily present in human diets. There is considerable research interest in their potential to prevent or treat several chronic diseases. Biomarkers can demonstrate compliance during dietary interventions and validate associations between intake of isoflavones and health outcomes. The objectives of this study were to validate 24-hour urine collections, timed spot urine samples and timed plasma samples as biomarkers of isoflavone intake up to 165mg/day. Healthy volunteers (20 women and 11 men) consumed 55mg/d, 110mg/d or 165mg/d soy isoflavones or placebo for seven consecutive days in a randomised, double-blind, crossover study. Timed blood samples, timed spot urine samples (taken in the afternoon, 5-7 hours after consuming the isoflavone supplement) and 24-hour urines were obtained at baseline and during each intervention. Isoflavone content of the samples was assayed by liquid chromatography and mass spectrometry. 24-hour urines were validated by percentage PABA recovery. The relationship between daily isoflavone intake and 24-hour urinary isoflavone excretion was: y = 6.63132xº•⁷⁴²¹ ≡ x = (y ÷ 6.63132)¹•³⁴⁷⁵ where y = isoflavone excretion in μg/24h and x = isoflavone intake in μg/24h r² = 0.86; p < 0.001; n = 109 samples from 31 volunteers. The relationship between daily isoflavone intake and plasma isoflavone concentration was: y = (3.3543x10⁻³)xº•⁴⁸⁸⁹ ≡ x = (y ÷ 3.3543x10⁻³)²•º⁴⁵⁴ where y = plasma isoflavone in μg/ml and x = isoflavone intake in μg/24h r² = 0.61; p < 0.001; n = 100 samples from 30 volunteers. The relationship between daily isoflavone intake and spot urine isoflavone concentration was: y = (2.0324x10⁻³)xº•⁸ºº⁹ ≡ x = (y ÷ 2.0324x10⁻³)¹•²⁴⁸⁶ where y = isoflavone excretion in μg/ml and x = isoflavone intake in μg/24h r² = 0.69; p < 0.001; n = 143 samples from 31 volunteers. It was concluded that 24-hour urine collections, timed plasma samples and timed spot urine samples are valid biomarkers of isoflavone intakes up to 165mg/day. A curvilinear relationship was defined between a) isoflavone dose and bioavailability in plasma and b) isoflavone dose and 24-hour urinary excretion. 2007-06-01T00:00:00Z Mackinnon, L. Jay Isoflavones are biologically active plant chemicals (phytoestrogens) which are ordinarily present in human diets. There is considerable research interest in their potential to prevent or treat several chronic diseases. Biomarkers can demonstrate compliance during dietary interventions and validate associations between intake of isoflavones and health outcomes. The objectives of this study were to validate 24-hour urine collections, timed spot urine samples and timed plasma samples as biomarkers of isoflavone intake up to 165mg/day. Healthy volunteers (20 women and 11 men) consumed 55mg/d, 110mg/d or 165mg/d soy isoflavones or placebo for seven consecutive days in a randomised, double-blind, crossover study. Timed blood samples, timed spot urine samples (taken in the afternoon, 5-7 hours after consuming the isoflavone supplement) and 24-hour urines were obtained at baseline and during each intervention. Isoflavone content of the samples was assayed by liquid chromatography and mass spectrometry. 24-hour urines were validated by percentage PABA recovery. The relationship between daily isoflavone intake and 24-hour urinary isoflavone excretion was: y = 6.63132xº•⁷⁴²¹ ≡ x = (y ÷ 6.63132)¹•³⁴⁷⁵ where y = isoflavone excretion in μg/24h and x = isoflavone intake in μg/24h r² = 0.86; p < 0.001; n = 109 samples from 31 volunteers. The relationship between daily isoflavone intake and plasma isoflavone concentration was: y = (3.3543x10⁻³)xº•⁴⁸⁸⁹ ≡ x = (y ÷ 3.3543x10⁻³)²•º⁴⁵⁴ where y = plasma isoflavone in μg/ml and x = isoflavone intake in μg/24h r² = 0.61; p < 0.001; n = 100 samples from 30 volunteers. The relationship between daily isoflavone intake and spot urine isoflavone concentration was: y = (2.0324x10⁻³)xº•⁸ºº⁹ ≡ x = (y ÷ 2.0324x10⁻³)¹•²⁴⁸⁶ where y = isoflavone excretion in μg/ml and x = isoflavone intake in μg/24h r² = 0.69; p < 0.001; n = 143 samples from 31 volunteers. It was concluded that 24-hour urine collections, timed plasma samples and timed spot urine samples are valid biomarkers of isoflavone intakes up to 165mg/day. A curvilinear relationship was defined between a) isoflavone dose and bioavailability in plasma and b) isoflavone dose and 24-hour urinary excretion.