This thesis describes the design, optimisation and shipboard deployment of a flow injection - chemiluminescence (FI-CL) technique for the determination of labile Cu(II) and total copper (by UV irradiation) in seawater. The operational parameters of the FI manifold in a UHP water sample matrix and the 1,10-phenanthroline CL reaction were rigorously optimised. Interferences to the CL reaction were investigated and the good analytical figures of merit obtained presented. The FI-CL method was modified for the determination of ultra trace levels of Cu(II) in seawater by the incorporation of a new design of micro-column containing 8-hydroxyquinoline (8HQ) resin for in-line matrix separation and preconcentration. Reagent clean-up techniques, blank procedures and a standard addition protocol are detailed. The optimised method is selective for Cu(II) in the linear range 0.1 - 50 nM, with precision of <4% (n=4) for a typical seawater analysis, and a limit of detection (3s) of 25 pM for a loading time of 90 s. The FI-CL analyser was fully automated and then validated by field deployment on the Tamar Estuary, during which its robustness, reliability and stand alone capability were demonstrated. Good accuracy was achieved for a seawater CRM analysed onboard. The near real time Cu data obtained was in good agreement with a comparative voltammetric method. The FI-CL method was further validated by field deployment on the Atlantic Meridional Transect (AMT 9) during which Cu(II) (filtered, acidified (pH 2) HNO3) in the surface waters (<250 m) of the North and South Atlantic (50°N to 50 °S) was mapped. Spatial variation in Cu(II) concentrations was observed (<0.7 to 6.1 nM) through the contrasting biogeochemical provinces encountered that representated coastal, upwelling and oligotrophic regions. Copper (II) enrichments were imposed on a trend of decreasing Cu(Il) concentrations away from European coastal waters (>2.5 nM) to open ocean gyres (< 1 nM). Away from strong input mechanisms, upper water column Cu(II) concentrations were ca. 1.5 nM, being dominated by long range aerosol input mechanisms. Input sources are fingerprinted via correlation with nutrients and hydrographic data, whilst the dominant sinks are active biological uptake and particle reactivity. Cu(II) vertical distributions through the upper mixed layer display strong relationships with chlorophyll α particularly in remote oceanic regimes. An in-line UA photo-oxidation system was constructed and optimised for the digestion of organically complexed Cu to enable near real time determination of total Cu in seawater by FI-CL. It achieved very efficient digestion of DOM (96.3 %) using a short irradiation time (600 s), with good recovery of Cu. Robustness, reproducibilty of irradiation, effective operational life and safety were considerably improved compared to existing systems. DOM rich Tamar Estuary and Celtic Sea samples were in good agreement with Cu(II) results from conventional batch UV digestion and voltammetric detection.

Spinal cord injuries (SCI) are characterized by the inability of mature neurons to regenerate or repair by themselves. In an attempt to overcome the SCI, a novel photo-sensitive cyclic Arg-Gly-Asp-Ser (RGDS) peptide was synthesized using solid phase peptide synthesis (SPSS) to control 3T3 fibroblast cell attachment on hyaluronic acid (HA) hydrogel. The circular RGDS peptide was designed using RGDS sequence labeled with Anp group (3-Na-fmoc-amino-3-(2-nitrophenyl) propionic acid) at the N terminus. The peptide was photo-labile cyclic caged to shelter its bioactivity and UV light was used to make the peptide uncaged. Accuracy of the cyclic caged RGDS peptide was confirmed by high-performance liquid chromatography (HPLC) and mass spectrum (MS). The molecular weight of cyclic caged RGDS peptide was confirmed as 881 by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrum. Stability of the cyclic caged RGDS peptide under various pH conditions was verified by circular dichroism spectroscopy. The bioactivity of cyclic caged and uncaged RGDS peptide was tested by photo-controllable directing cell growth based on cell attachment study, cell counting study, and cell morphology study. Three dimensional model structures of cyclic caged and uncaged RGDS peptides were computed by Hyperchem program. The first order reaction theory of Anp uncaging reaction was confirmed by kinetic study. Bioactivity caging and uncaging property of the peptide was also fully confirmed by cell attachment study. This cyclic caged RGDS peptide would be a promising tool in cell patterning for repairing of SCI.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Heat-labile enterotoxin (LT) is one of the most important toxins produced by enterotoxigenic Escherichia coli (ETEC). It consists of one A subunit (LTA) for intracellular enzymatic activity and five B subunits (LTB) forming a pentamer for binding to host cell receptors. In the last few decades, LT has been extensively studied as a strong immune stimulator, as well as an effective adjuvant with multiple immunomodulatory properties. To understand better the features of LT, we mapped B-cell linear epitopes of the enzymatic A subunit and explored the relationship between these epitopes and the toxicity of LT. Eleven B-cell linear (continuous) epitopes were in silico identified based on online software. In part one of the study, all 11 epitopes were fused into a modified ovalbumin carrier protein respectively. Each recombinant fusion protein was expressed and purified, and was characterized in ELISA and Western Blot using the anti-LT serum. Moreover, each fusion protein was used to immunize mice to determine immune response specific to LT in vivo. A total of eleven epitopes were identified from the LTA subunit. Results showed that anti-LT serum recognized all 11 epitopes, while the mouse immunization study indicated that antibodies derived from epitope 7 (₁₀₅SPHPYEQEVSA₁₁₅) had significantly greater anti-LT antibody titers and neutralized LT enterotoxicity more efficiently than the other epitopes. In part two of the study, to test whether individual epitope plays a role in LT toxicity, 10 epitopes in the A1 domain of LTA subunit were replaced by a foreign peptide respectively and the mutant LTs were examined for enterotoxicity. Data indicated that all these LT mutants showed enterotoxicity abolished. However, these LT mutants formed holotoxin structure and bound to GM1 in vitro. Results from this study indicated that replacement of these LT epitopes did not affect the forming of LT holotoxin structure and the binding to host receptors, indicating LT can serve as a safe vaccine platform to carry foreign antigens. With the immunodominant epitope 7 being kept while other LTA epitopes replaced by epitopes from other ETEC virulence factors, this platform can be used to construct broadly protective multivalent mucosal vaccines against ETEC, and perhaps as a universal platform for vaccines against other enteric diseases.

Spinal cord injuries (SCI) are characterized by the inability of mature neurons to regenerate or repair by themselves. In an attempt to overcome the SCI, a novel photo-sensitive cyclic Arg-Gly-Asp-Ser (RGDS) peptide was synthesized using solid phase peptide synthesis (SPSS) to control 3T3 fibroblast cell attachment on hyaluronic acid (HA) hydrogel. The circular RGDS peptide was designed using RGDS sequence labeled with Anp group (3-Na-fmoc-amino-3-(2-nitrophenyl) propionic acid) at the N terminus. The peptide was photo-labile cyclic caged to shelter its bioactivity and UV light was used to make the peptide uncaged. Accuracy of the cyclic caged RGDS peptide was confirmed by high-performance liquid chromatography (HPLC) and mass spectrum (MS). The molecular weight of cyclic caged RGDS peptide was confirmed as 881 by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrum. Stability of the cyclic caged RGDS peptide under various pH conditions was verified by circular dichroism spectroscopy. The bioactivity of cyclic caged and uncaged RGDS peptide was tested by photo-controllable directing cell growth based on cell attachment study, cell counting study, and cell morphology study. Three dimensional model structures of cyclic caged and uncaged RGDS peptides were computed by Hyperchem program. The first order reaction theory of Anp uncaging reaction was confirmed by kinetic study. Bioactivity caging and uncaging property of the peptide was also fully confirmed by cell attachment study. This cyclic caged RGDS peptide would be a promising tool in cell patterning for repairing of SCI.

Dans un contexte de réchauffement climatique, la fonction « puits » de carbone (C) des tourbières est susceptible de changer en fonction « source » libérant ainsi de grandes quantités de C dans l’atmosphère sous l’effet d’une dégradation accrue de la matière organique (MO). Ainsi cette thèse se sont attachés à étudier la dynamique de dégradation de la MO labile (MOL) et récalcitrante (MOR) en fonction de 3 facteurs contrôlant (le niveau relatif de la nappe, un réchauffement simulé in situ et des variations saisonnières) dans la tourbière ombrotrophe du Forbonnet (25, Jura). Basés sur des analyses biogéochimiques et moléculaires de (i) la tourbe (50cm d’épaisseur), (ii) la fraction organique extractible à l’eau (WEOM) et (iii) les eaux piézométriques, les résultats acquis montrent une dégradation accrue de la MOL (e.g. polysaccharides et produits de son hydrolyse tels que le fructose,le glucose et le saccharose) et de la MOR sous l’effet d’une diminution du niveau de la nappe. Les conditions hydrologiques affectent également les processus de dégradation (sénescence et un éventuel priming effect) et d’humification de la MO pouvant conditionner le piégeage potentiel de laMOL. Après 13 mois d’incubation, l’effet du réchauffement simulé se traduirait par une hausse de l’évapotranspiration dans la condition la plus humide, là où l’étude de la WEOM indique une moindre dégradation de la MOL en surface contrairement aux niveaux profonds. Enfin, les travaux portant sur la dynamique saisonnière du C des eaux piézométriques soulignent le besoin d’intégrer la notion d’interactions entre facteurs contrôlant le cycle du C dans les tourbières pour comprendre leur réponse aux changements climatiques. / In a global warming context, the carbon (C) "sink" function of peatlands may switch to a source function releasing large amounts of C into the atmosphere as a result of an increased decay of organic matter (OM). Thus, these works have focused on the decay dynamics of labile OM and recalcitrant OM (respectively LOM and ROM) according to 3 controlling factors (water-level changes, an in situ simulated warming and seasonally changes) in the “le Forbonnet” bog (25, Jura). Based on biogeochemical and molecular analyses (i) on peat, (ii) on water extractable OM and (iii) on free waters, results suggest an increased decay of LOM (e.g. polysaccharides and its hydrolysis products such as fructose, glucose and sucrose) and MOR under water level changes. Hydrological conditions also affect decay (senescence and an eventual priming effect) and humification processes that can facilitate LOM capture. After 13 months of incubation, simulated warming would lead to an increase of evapotranspiration in the wettest condition where WEOM study indicates a lower LOM decay on the contrary to the deeper peat levels. Studies on the seasonal dynamic of C on free waters underline the need to integrate interactions between factors controlling the C cycle in peatlands to understand their responses to climate change.

<p>Enterotoxigenic Escherichia coli (ETEC), the leading cause of traveler's diarrhea and childhood mortality due to diarrhea in the developing world, has been shown to secrete heat-labile enterotoxin (LT) in association with outer membrane vesicles. However, studies on the effect of LT have been performed using soluble LT, which is not its physiologically relevant presentation context. The effect of LT associated with vesicles and its trafficking within human intestinal epithelial cells were compared with soluble LT. Cytokine responses and trafficking of standardized samples of soluble LT and vesicle-associated LT were evaluated in polarized intestinal epithelial cells. Using real-time PCR, immunoblotting, and ELISAs, we found that compared to soluble LT, vesicle-bound LT showed delayed kinetics in the activation of LT. Vesicles containing LT or not also produced cytokines through different signaling pathways than soluble LT. We found that this difference in signaling was due to different trafficking within the cell. Interestingly, not all LT associated with vesicles is active within cells. Vesicle-associated LT must bind to the host receptor GM1 in lipid rafts to be active within cells. This suggests that although vesicles can deliver large amounts of LT to a cell, much of the LT would be inactive and not produce a physiological response. To test this hypothesis, we attempted to develop animal models for ETEC-induced diarrhea. Although the models were largely unsuccessful, the mouse model appears promising for determining the physiological response of a host to LT as fluid accumulation was observed in response to vesicles containing LT. The results in this thesis provide further understanding of the mechanism of LT-induced diarrhea and emphasize the importance of study toxins in their natural context.</p> / Dissertation

A dengue é uma arbovirose que ameaça mais de metade da população mundial. No momento existem duas vacinas, administradas por vias parenterais, aprovada ou em estudos clínicos, que podem conferir proteção parcial em regiões endêmicas, mas que ainda não estão sendo administradas em larga escala. Por outro lado, a utilização de vias de administração alternativas, como a via transcutânea (TC), pode ter um impacto importante na eficácia vacinal. A entrega de formulações vacinais pela via TC tem como vantagem a administração segura e não invasiva de antígenos vacinais e a composição celular específica da pele, rica em células apresentadoras de antígenos, como as células de Langerhans; O presente estudo demonstra que a via TC pode induzir respostas sorológicas vírus-específicas de forma eficiente quando utilizada para a administração de vacinas contra DENV baseadas em partículas virais. Portanto, novos estudos devem ser feitos considerando a TC como uma via de administração alternativa às vias parenterais para a administração de vacinas contra a dengue. / Dengue is an arbovirosis that threatens more than half the world\'s population. Currently there are two vaccines, both administered via parenteral routes, approved or in clinical trials, which provide partial protection but are not being administered on a large scale. On the other hand, the use of alternative routes of administration, such as the transcutaneous (TC) route, can have a significant impact on vaccine efficacy. Delivery of vaccine formulations via the TC route has the advantage of a safe and non-invasive administration method of vaccine antigens through the skin, that has a cell composition rich in antigen-presenting cells, such as the Langerhans cells. The present study demonstrates that the TC route can efficiently induce virus-specific serological responses when used for administration of DENV vaccines based on viral particles. Further studies should explore different dengue vaccines using the TC route as an alternative to parenteral administration routes.

As toxinas termo-lábeis (LT) produzidas por linhagens de Escherichia coli enterotoxigênica (ETEC) são adjuvantes com um importante foco em pesquisas acadêmicas e aplicadas, entretanto pouco é conhecido sobre a capacidade desses adjuvantes em induzir diferentes padrões de glicosilação em anticorpos IgG. Desta forma, o objetivo do presente trabalho foi analisar a capacidade dos adjuvantes LT1 e seus derivados atóxicos (LT1-K63 e LT1-B) influenciarem na qualidade da resposta imunológica humoral, particularmente quanto aos perfis de glicosilação dos anticorpos IgG EIII-específicos. A administração das LTs proporcionaram a potencialização da resposta de anticorpos antígeno-específicos, modularam o perfil de subclasses de IgG, melhoraram a capacidade de neutralização viral e induziram diferentes perfis de glicosilação presentes na estrutura dos anticorpos induzidos. Os resultados obtidos demonstram a importância dos adjuvantes no perfil de glicosilação de anticorpos antígeno-específicos e sugerem a influência da glicosilação na funcionalidade desses anticorpos. / The heat-labile toxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) are an important adjuvants to focus on academic and applied research, however little is known about the ability of these adjuvants to induce different patterns of glycosylation in IgG antibodies. Thus, the aim of this study was to analyze the ability of the adjuvants LT1 and its nontoxic derivative (LT1-K63 and LT1-B) influence on the quality of the humoral immune response, particularly regarding the glycosylation profiles of the EIII-specific IgG antibodies. The management of transmission lines provided potentiation response of antigen-specific antibodies modulate the profile of IgG subclasses, improved ability to induce virus neutralizing and different glycosylation profiles of in the antibodies purified IgG. The results demonstrate the importance of adjuvants in glycosylation of antigen-specific antibody profile and suggest the influence of glycosylation on the functionality of these antibodies.

O processo de compostagem é considerado um dos principais métodos sustentáveis de tratamento de rejeitos orgânicos provenientes dos centros urbanos. Sua importância reside em sua capacidade de sanitizar o material, estabilizar sua fração orgânica e ainda disponibilizar um produto final com características físico-químicas e biológicas que promoverão uma melhoria no sistema produtivo quando aplicado ao solo. Todavia, são vários os fatores que influenciam o desenvolvimento desse processo e exigem um equilíbrio tal que nos casos em que esse tratamento de resíduo é aplicado, muitas vezes o processo é incompleto e o produto final não apresenta as características adequadas de sanitização ou de promoção da qualidade do solo. Portanto, o presente estudo objetivou avaliar o efeito da apliação de inoculantes no desenvolvimento do processo de compostagem e a influência dessa aplicação nas características do produto final, sob a hipótese de que a adição de compostos biológicos energéticos aos resíduos promove a ação decompositora e potencializa o processo da compostagem. Para isso foram montadas leiras de compostagem com material orgânico produzido nos centros urbanos. Foram avaliados os seguintes tratamentos: inoculante usado em compostagem industrial, inoculante desenvolvido pelos pesquisadores e controle (pilhas de controle sem aplicação de inoculantes). Todas as leiras de compostagem foram avaliadas quando à sua temperatura, umidade e densidade, seu pH e conteúdo de macronutrientes, sua atividade microbiana, sua qualidade da matéria orgânica e emissão de gases do efeito estufa. Os dados obtidos demonstraram que o perfil da temperatura das leiras ao longo do processo acompanha o comportamento da atividade microbiológica. Os inoculantes aplicados não alteraram as condições físico-químicas do processo quando comparados ao controle. Foi observado um acúmulo de carbono na menor fração da matéria orgânica, indicando formação de substâncias recalcitrantes pela decomposição. A análise da emissão de gases do efeito estufa demonstrou que esse fator pode ser influenciado pelos manejos realizados sobre a leira de compostagem. Constatou-se que o uso de inoculantes não alterou significativamente nenhum dos parâmetros avaliados quando comparado ao controle, sendo que seu uso não promoveu melhorias na qualidade do produto final, nem tampouco se mostrou essencial ao bom desenvolvimento do processo de compostagem quando se trata resíduos orgânicos, que apresentam facilidade de decomposição. / The composting process is considered one of the main sustainable methods for treatment of the organic waste from urban centers. Its importance lies in its ability to sanitize the material, stabilize its organic fraction and also provide a final product with biological and physicochemical characteristics that will promote an improvement in the productive system when apllied to the soil. However, the factors that influence the development of this process are so many and require such an equilibrium that in cases where this residue treatment methodology is applied, the process is often incomplete and the final product does not have the appropriate sanitation or characteristics that promote soil quality. Therefore, the present study aimed to evaluate the effect of inoculant application on the development of the composting process and the influence of this application on the final product\'s characteristics. For this, composting piles were assembled with organic material produced in urban centers and applied an inoculant used in industrial composting, another one developed by the researchers and control cells were mantained without applications. All compost beds were evaluated for thier temperature, humidity and density, their pH and macronutrient content were measured, along with its microbial activity, quality of organic matter and the greenhouse gas emissions. The data obtained showed that the profile of the temperature of the piles during the process followed the behavior of the microbiological activity. The inoculants applied did not alter the physicochemical conditions of the process when compared to the control. It was observed a carbon accumulation in the smallest fraction of the organic matter, indicating the formation of recalcitrant substances by the decomposition. The analysis of greenhouse gas emissions showed that the management of the composting pile could influence this factor. It was found that the use of inoculants did not significantly alter any of the evaluated parameters when compared to control, and its use did not promote improvements in the final product\'s quality, nor was it essential for the proper development of ther composting process when dealing with organic urban waste, which is easly decomposed.

As toxinas LT são expressas por linhagens de Escherichia coli enterotoxigênicas e possuem marcantes efeitos adjuvantes. No entanto, não se conhece se a exposição prévia à toxina afeta sua atividade adjuvante durante imunizações subsequentes. Assim, o presente estudo avaliou o impacto da imunidade pré-existente nas propriedades inflamatórias e adjuvantes da LT quando inoculada pela via subcutânea. Como antígeno modelo, empregou-se a proteína NS1 do vírus dengue e duas abordagens experimentais distintas: (i) incubação in vitro de LT com anticorpos anti-LT e com o receptor gangliosídio (GM1) antes da administração em camundongos não imunizados em conjunto com a proteína NS1 do vírus dengue; (ii) imunização com NS1 coadministrada à LT em camundongos previamente expostos à LT. Foram avaliados os efeitos inflamatórios locais e os efeitos adjuvantes por meio da resposta imunológica humoral anti-NS1. Nossos resultados indicam que a imunidade prévia contra a LT não afeta seu potencial inflamatório e atividade adjuvante. Além disto, a exposição ao receptor GM1 reduziu as reações inflamatórias locais sem, no entanto, reduzir os efeitos adjuvantes de LT. / LT toxins are expressed by enterotoxigenic Escherichia coli strains and display strong adjuvant effects. Nonetheless, the impact of preexisting immunity on the on LT adjuvant activities is still unknown. Thus, the present study evaluated the impact of pre-existing immunity in the inflammatory and adjuvant properties of LT after subcutaneous administration. the NS1 of dengue virus was employed as a model antigen and two experimental approaches were evaluated: (i) in vitro incubation of LT with LT-specific antibodies and the ganglyoside receptor (GM1) before administration to naïve mice in combination with NS1; (ii) immunization with NS1 co-administered with LT in mice previously exposed to LT. The local inflammatory effects induced by LT were evaluated as wel as the adjuvant effects by means of NS1-specific humoral response. Our results indicate that the LT pre-existing immunity does not affect the inflammatory and adjuvant activities of the toxin. In addition, exposure to GM1 reduced the local inflammatory reactions without affecting the toxin adjuvant effects.