The application of capillary electrophoresis and mass spectrometry to clinical and environmental problems

Using capillary electrophoresis (CE) as a separation technique has allowed analytes, previously difficult to separate by standard methods because they did not conform to requirements for GC or HPLC, to be separated with speed and great efficiency. The only requirements for CE analysis are that the sample is soluble in a liquid matrix and that analytes are present as positive or negative ions whilst in this matrix. This technique has been used here to analyse both clinical and environmental samples, some as cations and others boron-containing complexes as anions. Samples were analysed using a combination of CE alone, mass spectrometry alone and also coupled capillary electrophoresis/electrospray mass spectrometry (CE/ES). Clinically orientated analytes, dipeptides in urine and acylcamitines from blood spots were examined and peaks detected directly via uv absorbance. The environmental samples analysed included those which contained chromophoric or non-chromophoric herbicides as well as those containing diisocyanates. Analytes were either detected in their native form as with the dipeptides and chromophoric herbicides, or more typically after derivatisation to improve their absorbance characteristics. The exception was the non-chromophoric herbicides which were detected via indirect uv. CE was an experimental technique for the analysis of all these compounds, except for the dipeptides, all the others having originally been analysed using HPLC or GC methods. In each case an evaluation of the CE method was performed to determine the suitability of the method. By analysing standards in each case, it was possible to confirm that the technique was suitable for qualitative and quantitative analysis of each class of compound. CE proved to be a viable technique for the separation of all classes of compound dealt with in this thesis. However the method could not be relied upon to confirm the identity of these analytes by their migration time alone. To identify the analytes, experiments were carried out to couple CE with a mass spectrometer. Two techniques of mass spectrometry were used within this thesis, fast atom bombardment and electrospray but only electrospray ionisation mass spectrometry was used to couple to capillary electrophoresis and was the only mass spectrometric technique used to analyse clinical and environmental samples. CE instruments were successfully coupled to an electrospray mass spectrometer which then became the detector. Mass/charge ratio measurements were obtained for each analyte used and these allowed the unambiguous identification of each analyte. Other work involved using CE, ES and FAI3 mass spectrometry, to develop a new technique to detect diol containing compounds. This involved complexing the diol with a boron-containing acid to produce an anion which could then be detected using ES and FAB mass spectrometric methods. This work was viewed as a possible technique for the detection of diol containing lipids found within some body fluids

Geographical and disease influences on intestinal antibodies in man

Gastrointestinal infections are a major cause of mortality and morbidity especially in the developing world. It is nearly a century since it was first observed that antibodies were present in the gut and it is now clear that secreted antibodies in the gut mucosa are crucial for host defence against pathogens. In addition, they may be important in the interaction of the host with commensal bacteria and oral vaccines.The development of a new clinical technique, whole gut lavage, has facilitated the collection of large amounts of gut fluid containing antibodies for the study of immune responses in the Gl tract in health and disease.The aims and related hypotheses of this thesis were: a) To develop an antibody test that could be used as a tool to examine the gut humoral immune response to bacteria of the gut flora. In order to develop the antibody test, various antigens were prepared from the bacterial cell wall and their biological potentials were examined with human and murine cell lines. These tests could be combined with studies of other facets of gut immunity for which methods were already available, in order to explore active immunity and tolerance in the mucosal and systemic compartments. b) To examine and compare humoral immunity in the gut and serum of immunologically normal people from Edinburgh and Dhaka, in order to establish and test the following hypotheses: 1. In view of the probable higher antigenic load from a potentially contaminated environment, there would be evidence of gut damage and inflammation in the healthy people of Dhaka, and thus high levels of IgA, compared to people in Edinburgh. 2. In the developing country, the drive for production of high levels of humoral responses to bacteria would have the additional effect that antibodies to other gut antigens, such as foods, would be absent or of low titre.By studying patients with chronic colitis (inflammatory bowel disease), it might be possible to investigate how chronic gut inflammation affects specific antibodies and to identify the sources of gut antibodies, ie. serum- derived or locally produced. Prompted by the case of a patient with hypereosinophilic syndrome, high gut IgA concentrations and ulcerative colitis, the stimuli for eosinophil migration into the gut and eosinophil activation were examined and possible interaction with the regulation of humoral immunity investigated.My literature review concentrates on a monograph on The gut as an immune organ' and illustrates the important features of intestinal immunoglobulins and antibodies. In the first chapter a section on bacterial structure and antigens has been included and the current knowledge on the regulation of eosinophil migration has been discussed. There is a small section on an appraisal of the whole gut lavage procedure that has been used in this thesis. Chapter 2 includes characteristics of patients and healthy volunteers, laboratory methods and technical development. A pilot study to confirm technical competence and reproducibility of methods is presented in chapter 3. Chapter 4 describes various gut damage and immune parameters in people from Edinburgh and Dhaka. Despite higher antigenic load, no evidence of gut damage, but high IgA, was found in the Dhaka groups. Results of anti-endotoxin and antibacterial antibodies, including those to various core types of E.coli LPS are presented in chapter 5. A section on the purity of the antigens and their biological potential by producing nitric oxide, inducing various cytokines in murine and human cell line have been presented. For the first time it has been found that IgA antibodies against various core types of LPS of E.coli are present in the gut, and their potential in the therapy of sepsis syndrome and for oral vaccine development has been discussed. Chapter 6 describes the of humoral immunity, role of gut bacteria, and cytokines controlling local and systemic immunity and pathogenicity in chronic inflammatory bowel disease. Evidence has been presented in chapter 7 to support the view that the source of IgG antibody detected in the gut of patients with active IBD is locally produced and not serum derived.The drive for production of a high humoral response to bacteria may be responsible for absent or low titre antibodies to other gut antigens, such as food. The antigen-driven bystander suppression or recently described Th1/Th2 paradigm is the purported mechanism for these results. The implications of these results in therapeutics and strategies for oral vaccine against infectious disease have been discussed in chapter 8. The stimuli of eosinophil migration in to the gut and their role in the mucosal inflammation of IBD have been discussed in chapter 9. For the first time it has been shown that the eosinophil specific chemoattractant, eotaxin, is secreted into the gut lumen; this may help in our understanding of the diseases related to eosinophil accumulation.The final chapter is an overview with some speculation on the relationship of the findings to future developments

Metallic and organic coating development for high performance pre-finished steels.

Organic coated steels are extremely popular in the construction industry due to their flexibility, cost, ease of construction and aesthetic nature. However the fact that they can be produced in many colours and finishes in large volumes slightly belies the complexity of the system itself. Many of the components have a degree of environmental sensitivity and therefore with the introduction and implementation of strict environmental legislation constant improvement is needed to keep in line or ahead of such directives. Improvement of the coating system from the substrate up is presented in this thesis. Initial work was undertaken to understand the species that would be released from an organic coating. To this end PVC based model coatings were produced and subjected to natural weathering for one year, the coatings were designed to degrade by addition of a U grade TiO2 to ensure that results were obtained during the period. The leachate species were monitored on a monthly basis and identified using inductively coupled plasma mass spectrometry (ICPMS). From this work it was found that pigmentation has an effect on the degradation of coatings. It was shown that monitoring of metallic run off is important as it provides details on species type, while some species can be identified as markers to photo-degradation of the coating. The comparison of stabilisers historic, present and novel using the same natural weathering method and testing period. Hydrotalcite was identified as a potential novel stabiliser for PVC based coatings. When compared to organotin based stabilisers as used in the past or Barium/Zinc based stabilisers that are currently used Hydrotalcite performed favourably both from a chemical and physical point of view. With regard to improving the coating system as a whole the next phase of work involved the identification of an optimum galvanised coating weight. Samples of Zn/Al galvanised steels with increasing coating weight were subjected to natural weathering for one year and the amount of zinc monitored monthly. Conventional hot dipped galvanised steel and Zalutite coatings were included in the sample field to provide further comparison points. A coating weight of 255 gm-2 was found to be the optimum with respect to the natural weathering, while Zalutite which contains a high aluminium addition performed best overall. This gave rise to the next section of work that involved used of the Scanning Vibrating Electrode Technique (SVET) to characterise the corrosion performance of galvanised coatings with aluminium additions greater than 4.8%. It was found that increasing the aluminium content improved both surface and cut edge corrosion. Under the parameters under which the samples were produced a wholly eutectic structure was achieved at a target addition of 6.1%. The final phase of work was a study on reducing the coating thickness of the high aluminium coatings to see if the improvement in corrosion performance attributed to increased aluminium addition carried across to thinner coatings. The samples were again tested using the SVET. The results suggest that there is some scope for reducing the coating thickness at certain aluminium levels

Generation and Applications of Extra-Terrestrial Environments on Earth

This book has been prepared under the auspice of the European Low Gravity Research Association (ELGRA). The main task of ELGRA is to foster the scientific community in Europe and beyond in conducting gravity and space-related research.This publication is dedicated to the science community, and especially to the next generation of scientists and engineers interested in space research and in the means to use Earth to reproduce the space environment. ELGRA provides a comprehensive description of space conditions and the means that have been developed on Earth to perform space environmental and (micro-) gravity related research. .The book covers ground-based research instruments and environments for both life and physical sciences research. It discusses the opportunities and limitations of protocols and instruments to compensate gravity or simulate microgravity, such as clinostats, random positioning machines, levitating magnets, electric fields, vibrations, tail suspension or head down tilt, as well as centrifuges for hyper-g studies. Other space environmental conditions are addressed too, like cosmic radiation or Mars atmospheric and soil properties to be replicated and simulated on Earth. Future long duration of manned missions, personal well-being and crew interaction are major issues dealt with

Good research practices

In this dissertation, entitled “Good Research Practices”, I examine research practices and reform ideas aiming to combat the crisis of confidence in psychology (Pashler & Wagenmakers, 2012). I do so through theoretical contributions and empirical work, propose practical guidelines for researchers, and demonstrate how principles of good research can be conveyed to students. The research methods and statistical practices I present facilitate the adherence to the following three principles: (1) respect the empirical cycle; (2) acknowledge uncertainty; and (3) enrich statistical models with theoretical knowledge

Analytical and computational methods towards a metabolic model of ageing in Caenorhabditis elegans

Human life expectancy is increasing globally. This has major socioeconomic implications, but also raises scientific questions about the biological bases of ageing and longevity. Research on appropriate model organisms, such as the nematode worm Caenorhabditis elegans, is a key component of answering these questions. Ageing is a complex phenomenon, with both environmental and genetic influences. Metabolomics, the analysis of all small molecules within a biological system, offers the ability to integrate these complex factors to help understand the role of metabolism in ageing. This thesis addresses the current lack of methods for C. elegans metabolite analysis, with a particular focus on combining analytical and computational approaches. As a first essential step, C. elegans metabolite extraction protocols for NMR, GC-MS and LC-MS based analysis were optimized. Several methods to improve the coverage, automatic annotation and data analysis steps of NMR and GC-MS are proposed. Next, stable isotope labelling was explored as a tool for C. elegans metabolomics. An automated stable isotope based workflow was developed, which identifies all biological, non-redundant features within a LC-MS acquisition and annotates them with molecular compositions. This demonstrated that the vast majority (> 99.5%) of detected features inside LC-MS metabolomics experiments are not of biological origin or redundant. This stable isotope workflow was then used to compare the metabolism of 24 different C. elegans mutant strains from different pathways (e.g. insulin signalling, TOR pathway, neuronal signalling), with differing levels of lifespan extension compared to wild-type worms. The biologically relevant features (metabolites) were detected and annotated, and compared across the mutants. Some metabolites were correlated with longevity across the mutant set, in particular, glycerophospholipids. This led to the formulation of a hypothesis, that lifespan extension in C. elegans requires increased activity of common downstream longevity effector mechanisms (autophagy, and mitochondrial biogenesis), that also involve subcellular compartmentation and hence membrane formation. This results in the alterations in lipid metabolism detected here.Open Acces

Computational analysis of innate and adaptive immune responses

Both innate and adaptive immune processes rely on the activation of differentiated haematopoietic stem cell lineages to affect an appropriate response to pathogens. This thesis employs a largely network biology focused approach to better understand the specificity of immune cell responses in two distinct cases of pathogenic challenge. In the context of adaptive immunity, I studied the transcriptional responses of T cells during Graft-versus-Host Disease (GvHD). GvHD represents one of the major complications to arise following allogeneic hematopoietic stem cell transplantation and yet why only particular organs are damaged as a result of this pathology is still unclear. To investigate whether key GvHD transcriptional signatures seen in effector CD8+ T cells compared to naïve T cells are triggered in target organs or the secondary lymphoid organs, a module-based association test was developed to combine the output of gene clustering algorithms with that of differential expression analysis. This methodology significantly aided the identification of skin specific effector T cell transcriptional programs believed to drive murine GvHD pathogenesis at this site. Turning to the innate immune response, I investigated the transcriptional profiles of resting and activated macrophages in the setting of Tuberculosis (TB), the second leading cause of death from infectious disease worldwide. Regression-based analyses and clustering of macrophage expression data provided insight into the variations in gene expression profiles of naïve macrophages compared to those infected with Mycobacterium tuberculosis (MTB) or a vaccine strain of mycobacteria (BCG). The availability of genotype data as part of the macrophage dataset facilitated an expression quantitative trait loci (eQTL) study which highlighted a novel association between the cytoskeleton gene BCAR1 and TB risk, together with a previously undescribed trans-eQTL module specific to MTB infected macrophages. Potential genetic variants impacting expression of the aforementioned GvHD specific T cell transcriptional signatures were additionally investigated using external trans-eQTL datasets