La formation du complexe C3b-H est essentielle au bon fonctionnement de la voie alterne du système du complément puisqu’elle permet d’inhiber la formation du complexe d’attaque membranaire à la surface de nos cellules, préservant l’organisme de toute destruction auto-immune. L’interaction de H avec C3b permet notamment de favoriser l’inactivation protéolytique de C3b par le facteur I, bloquant de ce fait l’activation de la voie alterne. Malgré l’importance du complexe C3b-H, l’interaction entre ces deux protéines à l’échelle moléculaire reste méconnue. En effet, à l’heure actuelle, aucune structure tridimensionnelle de ce complexe n’a pu être obtenue, que ce soit par cristallographie ou par résonance magnétique nucléaire, en raison de sa taille (335 kg.mol-1) et de la flexibilité de H. Cependant, la connaissance de l’organisation de ce complexe est nécessaire à la compréhension de la régulation de la voie alterne et pourrait de plus, se révéler utile quant au développement de thérapies vis-à-vis des maladies auto-immunes tel que le syndrome hémolytique et urémique atypique. Au cours de ces travaux, nous nous sommes donc intéressés à l’étude du complexe C3b-H à l’échelle de l’édifice entier par marquage oxydatif et pontage chimique couplés à la spectrométrie de masse. Ces approches expérimentales nous ont ainsi permis de proposer, à l’échelle de la molécule entière, un modèle tridimensionnel de ce complexe de haut poids moléculaire. / The C3b-H complex plays an essential role in regulation of the complement alternative pathway (AP) because it blocks the attack membrane complex (MAC) formation on our self-surface, thus preserving host from autoimmune disease. The C3b-H interaction leads to the proteolytic inactivation of C3b induced by factor I, thus blocking the MAC formation and consequently AP activation. Despite this importance of C3b-H complex for the innate immunity, the mechanism of interaction between these two molecules at the molecular level remains to be established. Indeed, up to date, no three-dimensional structure of this complex is available, either by X-ray crystallography or nuclear magnetic resonance, due to its large size (335 kg.mol-1) and flexibility of H. Yet, these data are required for a better understanding of AP regulation and, might moreover, be useful for the implementation of therapy against autoimmune disorders. In this study, we investigate the C3b-H complex by using oxidative footprinting and cross-linking coupled with mass spectrometry. Our experimental approaches have been successfully applied, allowing us to propose a three-dimensional model of this high molecular complex.

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The changing climate and damaging effects of CO2 on the environment has led to awareness throughout the construction industry of the need to deliver more sustainable solutions. The use of geosynthetics as a sustainable construction solution was demonstrated by the Waste and Resources Action Programme (WRAP) in a report entitled Sustainable Geosystems in Civil Engineering Applications (WRAP, 2010). The WRAP report presented a series of case studies in which geosynthetic solutions provided both cost and CO2 savings in comparison to non-geosynthetic solutions. However, in what is a huge field the report concentrated on specific areas relative to the calculation methods or on the potential construction applications. This EngD research built on this work by WRAP and aimed to establish a rigorous framework for the comparison of CO2 emissions between geosynthetic and non-geosynthetic solutions. This EngD research reviewed CO2 calculation methodologies and techniques to produce a rigorous framework that could be adopted in comparative CO2 studies between geosynthetic and non-geosynthetic solutions. It was demonstrated on three case studies looking at geosynthetics in the function of containment, drainage, and reinforcement, highlighting the possible CO2 benefits of employing geosynthetics. The development of the case studies and framework highlighted the need for accurate embodied carbon data. There was an absence of geosynthetic specific embodied carbon values in the commonly employed databases. The EngD research sought to address this and through some experimental work in collaboration with geosynthetic manufacturers calculated embodied carbon values for four types of geosynthetics.

<p>Abstract</p><p>Transcription factors control numerous vital processes in the cell through their ability to control gene expression. Dysfunctional regulation by transcription factors lead to disorders and disease. Transcription factors regulate gene expression by binding to DNA sequences (motifs) on the genome and altering chromatin. DNase-seq footprinting is a well-established assay for identification of DNA sequences that bind to transcription factors. We developed computational techniques to analyze footprints and predict transcription factor binding. These transcription factor specific predictive models are able to correct for DNase sequence bias and characterize variation in DNA binding sequence. We found that DNase-seq footprints are able to identify cell-type or condition specific transcription factor activity and may offer information about the type of the interaction between DNA and transcription factor. Our DNase-seq footprint model is able to accurately discover high confidence transcription factor binding sites and discover alternative interactions between transcription factors and DNA. DNase-seq footprints can be used with ChIP-seq data to discover true binding sites and better understand transcription regulation.</p> / Dissertation

Orientador: Fábio Cesar Gozzo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-16T13:20:07Z (GMT). No. of bitstreams: 1 Pilau_EduardoJorge_D.pdf: 5051807 bytes, checksum: 376ee3a4968e0ab254d7cda0cccd2819 (MD5) Previous issue date: 2010 / Resumo: A espectrometria de massas (MS) consiste no estudo dos íons em fase gasosa, tendo como uma das suas principais aplicações a caracterização estrutural de moléculas. Desde o início da técnica, ela vem sendo empregada na elucidação estrutural de um grande número de moléculas orgânicas e é hoje uma das principais ferramentas de caracterização estrutural. Dentro os métodos baseado em MS para o estudo de estruturas superiores de proteínas desenvolvidos recentemente estão aqueles conhecidos como footprinting e ligação cruzada (cross-linking). Nesta tese, estudos foram realizados para o desenvolvimento, melhorias e otimização de técnicas de footprinting e ligação cruzada acopladas à espectrometria de massas para o estudo estrutural de proteínas. Em footprinting, foram desenvolvidas fontes alternativas de radiação UV para geração de radicais ¿OH a partir da fotólise do H2O2 para mapeamento da superfície exposta ao solvente de proteínas, bem como o desenvolvimento de uma técnica mais apropriada de quantificação de peptídeos oxidados. Os resultados obtidos demonstraram que as fontes propostas apresentaram boa eficiência no processo de fotólise do H2O2 Na técnica de ligação cruzada, foram realizados estudos fundamentais a respeito da reatividade dos Agentes de Ligação Cruzadas (ALC¿s) tanto com relação à especificidade dos ALC¿s frente às cadeias laterais dos aminoácidos quanto à cinética da reação com proteínas e reação competitiva de hidrólise. Nos experimentos realizados foi possível verificar a reatividade do ALC frente a diferentes grupos nucleofilicos presentes nas cadeias laterais dos resíduois de aminoacido bem como ser suscetivel a mudança de reatividade em virtude do pH utilizado. / Abstract: Mass spectrometry (MS) study ions in the gas phase, and one of its main applications is the structural characterization of molecules. Since the beginning of the technique, it has been used in structural study of a large number of organic molecules and is now a major tool for structural characterization. Among the MSbased methods for the study of higher structures of proteins are the newly developed methods of footprinting and cross-linking. In this thesis, studies were undertaken for the development, improvement and optimization of, footprinting and cross-linking coupled with mass spectrometry for the structural analysis of proteins. In footprinting, we developed alternative sources of UV radiation to generate ¿OH radicals from the photolysis of H2O2 to map the solvent exposed surface of proteins as well as the development of a more appropriated technique for quantification of oxidized peptides. The results showed that the proposed sources showed good efficiency in the process of photolysis of H2O2. For cross-linking, fundamental studies were carried out concerning the reactivity of the cross-linkers regarding both the specificity of ALC in respect to amino acid side chains and the kinetics of reaction with proteins as well as hydrolysis reaction. In the experiments it was possible to verify the reactivity of ALC against different nucleophilic groups present in side chains of amino acid residues as well as being susceptible to change in reactivity due to the pH used. / Doutorado / Quimica Analitica / Doutor em Ciências

In the South West of England tourism provides an extremely important form of economic revenue, with 92 million nights spent in the region, generating over £9 billion in visitor spending and 11% of the total workforce employed either directly or indirectly in the sector. However this additional seasonal influx of visitors inevitably places a strain on the natural environment, built resources, infrastructure and communities. In order to readdress the balance tourism as a sector needs to be more sustainable and the emphasis for change is now placed on the individual. Social marketing has been used successfully to encourage behaviour change in the health sector, and is beginning to be recognised for its potential in encouraging sustainable behaviour, but has never been specifically applied in a tourism context. Therefore this research evaluates the potential of applying a social marketing methodology to encourage sustainable behaviour amongst tourists in two case study areas in South West England. Social marketing focusses on changing behaviour by understanding individual perceptions of the barriers to and motivations for behaviour. A social methodology then works to segment individuals into groups that share similar attitudes and beliefs, those groups identified as most likely to respond, are targeted with an intervention to encourage behaviour change. This research identified the perceived and actual barriers to (cost, time, convenience), and motivations for sustainable tourist behaviour among participants from the case study areas and identified three distinct clusters of tourists, one of which was identified as suitable for targeting with a social marketing intervention. This research also revealed that even those most committed to range of sustainable behaviours in the home environment do not continue this behaviour when in the holiday environment. A further dimension was added to this research by exploring the use of an ecological footprint calculator (REAP for Tourism) to quantify the environmental impact of individual tourists and to explore whether pro-environmental attitudes and behaviour equate to lower environmental impact.