Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

Biomarkers

Introduction – Why do we need ‘biomarkers? Biomarkers of protein oxidation Introduction Major issues/questions Protein carbonyl biomarkers Biochemistry Methods of measurement Storage, stability and limitations in use Protein thiol biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Aliphatic amino acid biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Oxidised Tryptophan Biomarkers Biochemistry Method of measurement Storage, stability and limitations on use Oxidised tyrosine biomarkers Biochemistry Methods of measurement Storage, stability and limitations on use Formation of neoepitopes on oxidised proteins Validation of assays for protein oxidation biomarkers Relationship of protein oxidation to disease Modulation of protein oxidation biomarkers by antioxidants Future perspectives Introduction to lipid peroxidation biomarkers Introduction: biochemistry of lipid peroxidation Malondialdehyde Methods of measurement Storage, stability and limitations on use Conjugated dienes Method of measurement Storage, stability and limitations of use LDL lag phase Method of measurement Storage, stability and limitations of use Hydrocarbon gases Biochemistry Method of measurement Storage, stability and limitations on use Lipofuscin Biochemistry Method of measurement Storage, stability and limitation on use Lipid peroxides Biochemistry Method of measurement Storage, stability and limitations on use Isoprostanes Biochemistry Method of measurement Storage, stability and limitations on use Possible new biomarkers of lipid oxidation Relationship of lipid peroxidation to disease Modulation of lipid peroxidation biomarkers by antioxidants Functional consequences of lipid peroxidation Contribution of dietary intake to lipid peroxidation products Biomarkers of DNA oxidation Introduction Confounding factors Units and terminology Nuclear and mitochondrial DNA damage Lymphocytes as surrogate tissues Measurement of DNA damage with the comet assay Practical details Storage, stability, and limitations of the assay Measurement of DNA base oxidation by HPLC Practical details Storage, stability and limitations of the method Measurement of DNA base oxidation by GC–MS Biochemistry of 8-oxoguanine, adenine and fapy derivatives Methods of measurement Storage, stability and limitations of the method Analysis of guanine oxidation products in urine Method of measurement Limitations and criticisms Immunochemical methods Methods of measurement Storage, stability, and limitations of the assay 32P post-labelling Method of measurement Limitations and criticisms Validation of assays for DNA oxidation Oxo-dGuo in lymphocyte DNA Urinary measurements DNA–aldehyde adducts Biochemistry Method of measurement Products of reactive nitrogen species Endpoints arising from oxidative DNA damage Mutations Chromosome aberrations Micronuclei Site-specific DNA damage Relationship of DNA oxidation to disease Modulation of DNA oxidation biomarkers by antioxidants Direct and indirect effects of oxidative stress: measures of total oxidant/antioxidant levels Visualisation of cellular oxidants Biochemistry: histochemical detection of ROS Method of measurement Limitations, storage and stability Measurement of hydrogen peroxide Biochemistry Methods of measurement Storage, stability and limitations on use Measurement of the ratio of antioxidant/oxidised antioxidant Biochemistry Method of measurement Storage, stability and limitations on use Total antioxidant capacity Biochemistry Terminology Methods of measurement Storage, stability and limitations on use Validation of assays for direct oxidant and antioxidant biomarkers Relationship of oxidant/antioxidant measurement to disease Modulation of oxidant/antioxidant biomarkers by dietary antioxidants Induction of genes in response to oxidative stress Background Measurement of antioxidant responsive genes and proteins Effects of antioxidant intake on the activity of antioxidant enzymes

Oxidiative lipidomics coming of age:advances in analysis of oxidized phospholipids in physiology and pathology

Significance: Oxidized phospholipids are now well-recognized as markers of biological oxidative stress and bioactive molecules with both pro-inflammatory and anti-inflammatory effects. While analytical methods continue to be developed for studies of generic lipid oxidation, mass spectrometry (MS) has underpinned the advances in knowledge of specific oxidized phospholipids by allowing their identification and characterization, and is responsible for the expansion of oxidative lipidomics. Recent Advances: Studies of oxidized phospholipids in biological samples, both from animal models and clinical samples, have been facilitated by the recent improvements in MS, especially targeted routines that depend on the fragmentation pattern of the parent molecular ion and improved resolution and mass accuracy. MS can be used to identify selectively individual compounds or groups of compounds with common features, which greatly improves the sensitivity and specificity of detection. Application of these methods have enabled important advances in understanding the mechanisms of inflammatory diseases such as atherosclerosis, steatohepatitis, leprosy and cystic fibrosis, and offer potential for developing biomarkers of molecular aspects of the diseases. Critical Issues and Future Directions: The future in this field will depend on development of improved MS technologies, such as ion mobility, novel enrichment methods and databases and software for data analysis, owing to the very large amount of data generated in these experiments. Imaging of oxidized phospholipids in tissue MS is an additional exciting direction emerging that can be expected to advance understanding of physiology and disease.

Aspects of developing foundry classification schemes particularly for cost-estimating

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What determines the molecular composition of abnormal protein aggregates in neurodegenerative disease?

Abnormal protein aggregates, in the form of either extracellular plaques or intracellular inclusions, are an important pathological feature of the majority of neurodegenerative disorders. The major molecular constituents of these lesions, viz., beta-amyloid (Abeta), tau, and alpha-synuclein, have played a defining role in the diagnosis and classification of disease and in studies of pathogenesis. The molecular composition of a protein aggregate, however, is often complex and could be the direct or indirect consequence of a pathogenic gene mutation, be the result of cell degeneration, or reflect the acquisition of new substances by diffusion and molecular binding to existing proteins. This review examines the molecular composition of the major protein aggregates found in the neurodegenerative diseases including the Abeta and prion protein (PrP) plaques found in Alzheimer's disease (AD) and prion disease, respectively, and the cellular inclusions found in the tauopathies and synucleinopathies. The data suggest that the molecular constituents of a protein aggregate do not directly cause cell death but are largely the consequence of cell degeneration or are acquired during the disease process. These findings are discussed in relation to diagnosis and to studies of to disease pathogenesis.

Structure, dynamics, and energetics of siRNA-cationic vector complexation:a molecular dynamics study

The design and synthesis of safe and efficient nonviral vectors for gene delivery has attracted significant attention in recent years. Previous experiments have revealed that the charge density of a polycation (the carrier) plays a crucial role in complexation and the release of the gene from the complex in the cytosol. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with six cationic carrier systems of varying charge and surface topology. The simulations reveal detailed molecular-level pictures of the structures and dynamics of the RNA-polycation complexes. Estimates for the binding free energy indicate that electrostatic contributions are dominant followed by van der Waals interactions. The binding free energy between the 8(+)polymers and the RNA is found to be larger than that of the 4(+)polymers, in general agreement with previously published data. Because reliable binding free energies provide an effective index of the ability of the polycationic carrier to bind the nucleic acid and also carry implications for the process of gene release within the cytosol, these novel simulations have the potential to provide us with a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance the rational design of nonviral gene delivery systems.

Structure and dynamics of multiple cationic vectors-siRNA complexation by all-atomic molecular dynamics simulations

Understanding the molecular mechanism of gene condensation is a key component to rationalizing gene delivery phenomena, including functional properties such as the stability of the gene-vector complex and the intracellular release of the gene. In this work, we adopt an atomistic molecular dynamics simulation approach to study the complexation of short strand duplex RNA with four cationic carrier systems of varying charge and surface topology at different charge ratios. At lower charge ratios, polymers bind quite effectively to siRNA, while at high charge ratios, the complexes are saturated and there are free polymers that are unable to associate with RNA. We also observed reduced fluctuations in RNA structures when complexed with multiple polymers in solution as compared to both free siRNA in water and the single polymer complexes. These novel simulations provide a much better understanding of key mechanistic aspects of gene-polycation complexation and thereby advance progress toward rational design of nonviral gene delivery systems.

Spatial aspects of MRSA epidemiology:A case study using stochastic simulation, kernel estimation and SaTScan

The identification of disease clusters in space or space-time is of vital importance for public health policy and action. In the case of methicillin-resistant Staphylococcus aureus (MRSA), it is particularly important to distinguish between community and health care-associated infections, and to identify reservoirs of infection. 832 cases of MRSA in the West Midlands (UK) were tested for clustering and evidence of community transmission, after being geo-located to the centroids of UK unit postcodes (postal areas roughly equivalent to Zip+4 zip code areas). An age-stratified analysis was also carried out at the coarser spatial resolution of UK Census Output Areas. Stochastic simulation and kernel density estimation were combined to identify significant local clusters of MRSA (p<0.025), which were supported by SaTScan spatial and spatio-temporal scan. In order to investigate local sampling effort, a spatial 'random labelling' approach was used, with MRSA as cases and MSSA (methicillin-sensitive S. aureus) as controls. Heavy sampling in general was a response to MRSA outbreaks, which in turn appeared to be associated with medical care environments. The significance of clusters identified by kernel estimation was independently supported by information on the locations and client groups of nursing homes, and by preliminary molecular typing of isolates. In the absence of occupational/ lifestyle data on patients, the assumption was made that an individual's location and consequent risk is adequately represented by their residential postcode. The problems of this assumption are discussed, with recommendations for future data collection.

Present perspectives on the automated classification of the G-protein coupled receptors (GPCRs) at the protein sequence level

The G-protein coupled receptors--or GPCRs--comprise simultaneously one of the largest and one of the most multi-functional protein families known to modern-day molecular bioscience. From a drug discovery and pharmaceutical industry perspective, the GPCRs constitute one of the most commercially and economically important groups of proteins known. The GPCRs undertake numerous vital metabolic functions and interact with a hugely diverse range of small and large ligands. Many different methodologies have been developed to efficiently and accurately classify the GPCRs. These range from motif-based techniques to machine learning as well as a variety of alignment-free techniques based on the physiochemical properties of sequences. We review here the available methodologies for the classification of GPCRs. Part of this work focuses on how we have tried to build the intrinsically hierarchical nature of sequence relations, implicit within the family, into an adaptive approach to classification. Importantly, we also allude to some of the key innate problems in developing an effective approach to classifying the GPCRs: the lack of sequence similarity between the six classes that comprise the GPCR family and the low sequence similarity to other family members evinced by many newly revealed members of the family.

Some aspects of the interaction of humic substances with metal ions

Humic substances are the major organic constituents of soils and sediments. They are heterogeneous, polyfunctional, polydisperse, macromolecular and have no accurately known chemical structure. Their interactions with radionuclides are particularly important since they provide leaching mechanisms from disposal sites. The central theme to this research is the interaction of heavy metal actinide analogues with humic materials. Studies described focus on selected aspects of the characteristics and properties of humic substances. Some novel approaches to experiments and data analysis are pursued. Several humic substances are studied; all but one are humic acids, and those used most extensively were obtained commercially. Some routine characterisation techniques are applied to samples in the first instance. Humic substances are coloured, but their ultra-violet and visible absorption spectra are featureless. Yet, they fluoresce over a wide range of wavelengths. Enhanced fluorescence in the presence of luminescent europium(III) ions is explained by energy transfer from irradiated humic acid to the metal ion in a photophysical model. Nuclear magnetic resonance spectroscopy is applied to the study of humic acids and their complexes with heavy metals. Proton and carbon-13 NMR provides some structural and functionality information; Paramagnetic lanthanide ions affect these spectra. Some heavy metals are studied as NMR nuclei, but measurements are restricted by their sensitivity. A humic acid is fractionated yielding a broad molecular weight distribution. Electrophoretic mobilities and particle radii determined by Laser Doppler Electrophoretic Light Scattering are sensitive to the conditions of the supporting media, and the concentration and particle size distribution of humic substances. In potentiometric titrations of humate dispersions, the organic matter responds slowly and the mineral acid addition is buffered. Proton concentration data is modelled and a mechanism is proposed involving two key stages, both resulting in proton release after some conformational changes.

Task classification and decision processes in monitoring behaviour

Task classification is introduced as a method for the evaluation of monitoring behaviour in different task situations. On the basis of an analysis of different monitoring tasks, a task classification system comprising four task 'dimensions' is proposed. The perceptual speed and flexibility of closure categories, which are identified with signal discrimination type, comprise the principal dimension in this taxonomy, the others being sense modality, the time course of events, and source complexity. It is also proposed that decision theory provides the most complete method for the analysis of performance in monitoring tasks. Several different aspects of decision theory in relation to monitoring behaviour are described. A method is also outlined whereby both accuracy and latency measures of performance may be analysed within the same decision theory framework. Eight experiments and an organizational study are reported. The results show that a distinction can be made between the perceptual efficiency (sensitivity) of a monitor and his criterial level of response, and that in most monitoring situations, there is no decrement in efficiency over the work period, but an increase in the strictness of the response criterion. The range of tasks exhibiting either or both of these performance trends can be specified within the task classification system. In particular, it is shown that a sensitivity decrement is only obtained for 'speed' tasks with a high stimulation rate. A distinctive feature of 'speed' tasks is that target detection requires the discrimination of a change in a stimulus relative to preceding stimuli, whereas in 'closure' tasks, the information required for the discrimination of targets is presented at the same point In time. In the final study, the specification of tasks yielding sensitivity decrements is shown to be consistent with a task classification analysis of the monitoring literature. It is also demonstrated that the signal type dimension has a major influence on the consistency of individual differences in performance in different tasks. The results provide an empirical validation for the 'speed' and 'closure' categories, and suggest that individual differences are not completely task specific but are dependent on the demands common to different tasks. Task classification is therefore shovn to enable improved generalizations to be made of the factors affecting 1) performance trends over time, and 2) the consistencv of performance in different tasks. A decision theory analysis of response latencies is shown to support the view that criterion shifts are obtained in some tasks, while sensitivity shifts are obtained in others. The results of a psychophysiological study also suggest that evoked potential latency measures may provide temporal correlates of criterion shifts in monitoring tasks. Among other results, the finding that the latencies of negative responses do not increase over time is taken to invalidate arousal-based theories of performance trends over a work period. An interpretation in terms of expectancy, however, provides a more reliable explanation of criterion shifts. Although the mechanisms underlying the sensitivity decrement are not completely clear, the results rule out 'unitary' theories such as observing response and coupling theory. It is suggested that an interpretation in terms of the memory data limitations on information processing provides the most parsimonious explanation of all the results in the literature relating to sensitivity decrement. Task classification therefore enables the refinement and selection of theories of monitoring behaviour in terms of their reliability in generalizing predictions to a wide range of tasks. It is thus concluded that task classification and decision theory provide a reliable basis for the assessment and analysis of monitoring behaviour in different task situations.

An evaluation of certain aspects of an occupational health service

This thesis has been concerned with obtaining evidence to explore the proposition that the provision of occupational health services as arranged at the present time represents a misallocation of resources. The research has been undertaken within the occupational health service of a large Midlands food factory. As the research progressed it became evident that questions were being raised about the nature and scope of occupational health as well as the contribution, in combating danger at work, that occupational health services can make to the health and safety team. These questions have been scrutinized in depth, as they are clearly important, and a resolution of the problem of the definition of occupational health has been proposed. I have taken the approach of attempting to identify specific objectives or benefits of occupational health activities so that it is possible to assess how far these objectives are being achieved. I have looked at three aspects of occupational health; audiometry, physiotherapy and pre-employment medical examinations as these activities embody crucial concepts which are common to all activities in an occupational health programme. A three category classification of occupational health activities is proposed such that the three activities provide examples within each category. These are called personnel therapy, personnel input screening and personnel throughput screening. I conclude that I have not shown audiometry to be cost-effective. My observations of the physiotherapy service lead me to support the suggestion that there is a decline in sickness absence rates due to physiotherapy in industry. With pre-employment medical examinations I have shown that the service is product safety oriented and that benefits are extremely difficult to identify. In regard to the three services studied, in the one factory investigated, and because of the immeasurability of certain activities, I find support for the proposition that the mix of occupational health services as provided at the present time represents a misallocation of resources.