The purpose of this work was to study the effects of reactive oxygen species (ROS) on DNA and to investigate the relevance of ROS-induced DNA damage in systemic lupus erythematosus (SLE). Using model systems of ROS generation, it was found that DNA was damaged by ROS at all levels of its structure, causing strand breaks, base modifications and conformational changes. Hydrogen peroxide, a ROS generated during inflammation in vivo, produced a characteristic type of site-specific damage dependent on the DNA-bound metal ion catalysis of its degradation. 8-hydroxydeoxyguanosine (8OHDG), a modified DNA base, was used as a marker of oxidative damage to investigate the role of DNA damage in the aetiopathogenesis of SLE. Excretion of this adduct was detected in normal urine and is believed to arise from normal oxidative metabolic processes. In patients with active rheumatoid arthritis, this level of 8OHDG excretion was significantly elevated. In contrast, in SLE patients with inflammatory activity, 8OHDG was undetectable in the urine. Investigation of the mechanism responsible for this showed that SLE cells had aberrant removal of 8OHDG from DNA following oxidative stress in vitro compared to normal cells, and that ROS-denatured DNA accumulated in circulating immune complexes associated with the disease. SLE is also characterised by circulating anti-DNA antibodies. These antibodies were found to bind better to ROS-DNA than to native double-stranded DNA. Furthermore, ROS-DNA was able to stimulate lymphocytes to produce anti-DNA antibodies. The pattern of DNA damage seen in SLE patients was typical of that induced by hydrogen peroxide in vitro. This suggests that inflammation generates ROS which cause DNA damage. As a result of defective repair within cells, ROS-DNA is released into the circulation following cell death which can form complexes with anti-DNA antibodies. In addition, the ROS-DNA can stimulate further anti-DNA antibody production by acting directly on cells thus perpetuating the disease process and contributing to immune complex deposition, a deleterious manifestation of the disease process.

Natural killer (NK) cells reconstitute rapidly following allogeneic stem cell transplantation (allo-SCT) at a time when alloreactive T cell immunity is being established. Important differences are seen in the patterns of reconstitution between T cell deplete, T cell replete and umbilical cord stem cell transplants. 82 patients who received T cell-deplete allo-SCT were studied to determine the functional and transcriptional profile of the reconstituting NK cells and to assess the relationship with clinical outcome. NK cells at day 14 (D14-NK) were donor-derived, intensely proliferating and expressed chemokine receptors targeted to lymphoid and peripheral tissue. Spontaneous production of the immunoregulatory cytokine IL-10 was observed in over 70% of cells and transcription of cytokines and growth factors was augmented. D14-NK cell number was inversely correlated with the incidence of grade II-IV acute graft versus host disease (GVHD). These findings reveal that robust reconstitution of immunoregulatory NK cells by day 14 after allo-SCT is an important determinant of clinical outcome and suggest NK cells may suppress development of the T cell-mediated alloreactive immune response through production of IL-10.

Microbial lab-based evolution is a technique to study evolutionary theory. It is a method which can provide insights into the ability of a microbe to adapt to a biological process such as low pH. To investigate pathways that could lead to an acid resistant phenotype in E. coli, we evolved six independent lines or populations of E. coli K-12 MG1655 by iterative growth and dilution experiments for approximately 740 generations at pH 4.5. Clones isolated from evolved populations were significantly fitter than the ancestor at pH 4.5. Five of the six evolved strains had acquired an identical mutation in rpoA, and mutations in cytR in addition to other mutations. PCR analysis of the fossil record of the evolved populations showed that the arcA mutations always arose first followed by the rpoA mutations. Investigating the genetic basis of adaptation showed that the mutations in arcA were loss of function in nature and conferred caused an intermediate increase in fitness. Transcriptional analysis showed a global change in their transcriptional signatures with significant upregulation of the arcA regulon. Our study showed that loss of function of ArcA caused an increase in the RpoS activity of the acid evolved strains leading to a general stress resistant phenotype.

Hodgkin lymphoma (HL) is a malignant lymphoma that is diagnosed mostly in young adults, and is the second most common malignancy to affect this age group. This disease is subdivided into two entities with different aetiologies: classical HL (cHL) (~95% of cases) and nodular lymphocyte-predominant HL. In Europe, ~82% of young adults with cHL are non-Epstein-Barr virus associated and epidemiological studies have suggested that a common infectious agent may play a key role in the aetiology of these cases. The molecular biology of HL is not well understood, primarily due to the low number of Hodgkin and Reed-Sternberg (HRS) cells present within these tumours. However, recently developed techniques for the selection and micromanipulation of single HRS cells from tumours, and the development of molecular cytogenetic techniques (i.e. array-comparative genomic hybridisation (CGH)) are overcoming these difficulties. To investigate a potential candidate virus, DNA samples from cHL biopsies were screened for the measles virus (MV) and polyomaviruses (PyV), using immunohistochemistry and highly sensitive PCR assays. Chromosomal imbalances in six well-established cHL-derived cell lines and a cHL case were analysed by array-CGH. To obtain sufficient DNA for array-CGH from the cHL case, single HRS cells were isolated using laser microdissection. DNA was extracted then amplified by degenerate oligonucleotide primer polymerase chain reaction. MV and PyV genomes were not detected within cHL biopsies. Recurrent chromosomal imbalances were confirmed within the cHL-derived cell lines and cHL case, in addition to several novel imbalances. This is the first time that a cHL case has been analysed by array-CGH.

Introduction Squamous cell carcinoma of the auricle has an unusually high rate of lymph node metastases when compared to similar tumours at other sites. The lymph nodes affected are close to the base of the skull and in the neck. Development of metastasis carries a poor prognosis and most patients will subsequently die of failure of loco-regional control. Despite the likelihood of a poor outcome nothing can be done for patients prior to development of metastasis, as the risk of spread is not sufficiently high to warrant intervention in all patients. They are therefore treated with a ‘wait and see policy’ and only offered treatment once clinical evidence of metastatic spread is detected. This thesis sets out to examine what can be done, at the time of initial presentation with an auricular squamous cell carcinoma to identify patients who would benefit from treatment to the regional lymph node basins. Materials and Methods The thesis is divided into four separate studies. A systematic review examines the evidence available to date, an anatomical study examines the lymphatic drainage of the auricle in cadavers, a sentinel lymph node biopsy study examines the use of this technique to identify early tumour spread and a retrospective analysis of cases of auricular squamous cell carcinoma in our unit examines histopathological prognostic indictors of metastatic spread. Results The systematic review found that these tumours have a metastatic rate of about 11%. Patients developing metastasis usually die from failure of loco-regional control. Depth of tumour invasion, tumour size and mode of invasion seem to be potential indicators of metastatic risk. There is a strong argument for prophylactic intervention to the regional lymph nodes but there is no consensus of opinion as to when this should be carried out The anatomical study comprised 5 cadaveric dissections. They showed that the first echelon nodes draining the auricle lie in the superficial parotid gland, post-auricular/ mastoid nodal group and level II of the neck. There are anastamotic pathways around the mastoid and post-auricular nodes that could permit embolic tumour cells to bypass them. Five lymphatic pathways draining the auricle are described and some of these lie on the lateral and anterior surfaces of the mastoid bone and traverse the insertion of sternocleidomastoid. 28 cases of auricular squamous cell carcinoma were enrolled for sentinel lymph node biopsy. None of them were found to have any metastatic spread. One case showed non-viable tumour cells in a lymph node. There was a high incidence of complications (14%) directly related to the sentinel node biopsy procedure. The retrospective analysis identified 229 cases of auricular squamous cell carcinoma treated in our unit from 1992 - 2004. 212 of these cases had the primary pathology available for analysis. 24 (of 212) patients developed metastasis. 17 patients died as a result of their disease usually due to failure of control at the regional lymph node basin. Primary tumours with a depth of invasion greater than 8mm have metastatic rate of 56%. Tumours with a depth of invasion between 2-8mm and evidence of cartilage destruction, lymphovascular invasion or a non-cohesive invasive front have 24% metastatic rate. Tumours outwith these high-risk groups did not metastasise. Conclusions Elective lymph node dissections of the superficial parotid gland, post-auricular/mastoid and level II nodes should be considered in patients with primary auricular squamous cell carcinomas with a depth of invasion >8mm or a depth of invasion between 2 - 8 mm and evidence of cartilage destruction, lymphatic invasion or a non-cohesive invasive front. This should ideally be done as part of an observational study to evaluate the cost / benefit ratio for these patients. The neck dissection must clear the mastoid bone to a sub-periosteal level on its anterior and lateral surfaces. This will require the removal of the upper portion of sternocleidomastoid. Sentinel lymph node biopsy requires further study to evaluate it as a method for early detection of metastatic spread in auricular squamous cell carcinoma. This could be done as part of an observational study of elective neck dissections.

This study was designed to identify the processes which underlie pain in symptomatic diverticular disease (SDD). Our hypothesis was that a spectrum of both peripheral and central pathologies were involved, with those that had a more peripheral problem having abdominal symptoms only while those with multiple symptoms throughout the body, having an altered central pain processing. The first study examining the brain response to cutaneous pain using functional magnetic resonance imaging (fMRI) has supported this hypothesis. Although a statistically significant difference in sensory pain threshold was not demonstrated between the groups, fMRI imaging has shown greater emotional processing during pain and reduced anticipatory inhibitory responses in the high somatising symptomatic diverticular disease (HSDD) groups. However this is not as clear cut as we had anticipated which may be due to subject selection and demonstrate a spectrum of mixed peripheral and central changes as well as those with only peripheral or central components. In the second part we performed a randomized placebo controlled study of mesalazine 3gm versus placebo. Mesalazine significantly reduced expression of many genes associated with inflammation in SDD patients. A reduction in the median number of hours of pain per week was seen. The study was not designed to allow intention to treat analysis but has shown promising results which will need to be consolidated with future large scale studies. Both these studies support a tailored approach to SDD patient treatment based on the underlying pain process which can be both central and peripheral. The Patient health questionnaire 12(PHQ12) may be one simple measure of doing this, but again needs to be confirmed with further larger studies.

This thesis aims to provide a geographical contribution to the understanding of disease causation, primarily through the development of causal models of chronic disease mortality incorporating both the physical and social environments. The overwhelming impression of previous research in this field is one of conflicting findings. For example, studies examining the relationship between disease and water hardness have found positive relationships, negative relationships and no relationship whatsoever. It is contended that this failure to replicate results is a direct consequence of applying an unsuitable 'confirmatory' approach to the quantitative analysis of geographical data. It is argued also that it is necessary to adopt a more appropriate statistical methodology, that of 'exploratory' statistics, before progress can be made. After an exegesis of the exploratory approach, the commonly used technique of multiple regression is given an exploratory interpretation. Each of the assumptions of this technique is discussed, and attention focuses on the effects of breaking the assumptions and on methods of detecting and overcoming the resultant problems. This exposition is illustrated by the re-analysis of previous studies, and it is demonstrated that inappropriate methods have led some researchers to inferential error. Finally in this methodological part of the research, an examination of the analysis of ratios is undertaken; here too it is suggested that the inappropriate analysis of death rates has resulted in some researchers making incorrect inferences. The empirical aspects of the thesis centre on the analysis of mortality variations in England and Wales. A critical appraisal of previous studies of the relationship between disease and water hardness is undertaken, and it is concluded that quantitative techniques have been poorly applied. Exploratory data analysis is then employed to develop models accounting for geographical variations in mortality experienced by the County Boroughs of England and Wales. In contrast to previous studies that have analysed these variations, no strong relationship is found between disease and water hardness. Moreover, an examination of the mortality experiences of Boroughs whose water supply has changed substantially over time also results in the conclusion that the effects of water hardness have been overestimated. Finally, the study examines the difficult problem of drawing inferences from aggregate data. Although it is concluded that much work remains to be undertaken, it is again argued that the exploratory approach may allow progress to towards the solution of this problem and, consequently, some guidelines for further research are outlined.

Nystagmus is a disorder of eye movement characterised by irregular, uncontrolled and repetitive eye movements. It can occur in a broad spectrum of clinical situations and diseases or it may occur in isolation and an inherited disorder. Surprisingly little is known about the underlying mechanisms of ocular-motor control. Similarly, the pathophysiological mechanisms underpinning nystagmus is also poorly understood. By studying pedigrees in whom nystagmus seems to be inherited as an isolated trait (Congenital Idiopathic Nystagmus), it may be possible to identify some of the genetic causes of this disorder and subsequently understand the pathophysiology. This thesis describes a molecular genetic study of congenital nystagmus. A clinical phenotyping study is followed by linkage analysis and positional cloning. A novel nystagmus gene is investigated in a large cohort of Congenital Idiopathic Nystagmus (CIN) patients and X-inactivation studies are performed. Subsequently, cell culture and RT-PCR work is performed to study expression of this gene. Additionally a pedigree with an atypical congenital nystagmus disorder is investigated and a new mutation within a known cerebellar disease gene is identified. This work contributed to the identification of the first gene for Congenital Idiopathic Nystagmus (CIN). The first detailed temporal expression study of the FRMD7 nystagmus gene was also performed in this study which has directed further studies into the pathogenesis of CIN. Identification of a new mutation in the CACNA1A gene in a pedigree with nystagmus and subtle cerebellar signs has lead to the consideration of this gene in patients who present to hospital with isolated atypical nystagmus.

No description available.

Over the last few years, there has been an increase in the clinical methods targeting the human tracheobronchial tree, both for therapeutic and diagnostic purposes. For these methods to be effective, a good understanding of the lung structure is necessary. This knowledge can be attained through the use of medical imaging protocols such as CT and MRI, and can in turn be used to predict aerosol deposition for particles employed for inhalation therapy via the simultaneous use of radionuclide imaging. However, due to limitations imposed by the technologies currently available, not enough information can be gathered in-vivo about the respiratory tract. Consequently, widespread use of anatomical models of the lung is being made by clinicians in order to enable them to fill this gap in information. The thesis is concerned with the improvement of such models and the introduction of new, more advanced ones in an effort to accurately describe the human lung using mathematical and physical principles. A method is developed for improving the Conceptual Model constructed in the Nuclear Medicine Department of Southampton General Hospital by incorporating to it real, patient-specific data obtained through CT imaging. A model of the bronchopulmonary segments of the lung is also created and an atlas that can be used for the identification of these sub-structures in any lung space is formed. An algorithm for the generation of a fully-descriptive 3D model of the airway tree is then designed and implemented, the morphometry of which is assessed to confirm that it is a realistic representation of the target organ. The deterministic algorithm reveals the 3D geometry and orientation of the lung airways, thus enabling aerosol deposition and flow-pattern studies to be performed in a comprehensive way in previously inaccessible regions of the lung.