L'utilisation du plutonium (Pu(IV) pour des applications militaires et civiles peut engendrer des contaminations internes chez les personnes exposées. Plusieurs voies de contamination sont possibles : par ingestion, par inhalation ou par blessure. En cas d'inhalation, le plutonium se présente le plus souvent sous forme de particules d'oxyde de plutonium qui vont se localiser au sein des alvéoles pulmonaires. Par un mécanisme de phagocytose, les particules sont internalisées par les macrophages de l'épithélium pulmonaire. Actuellement, le seul agent de décorporation administré en cas de contamination au plutonium est le DTPA (l'acide diethylenetriaminepentaacetique). Il est administré en France sous forme de CaNa3-DTPA par injection intraveineuse et est efficace pour les contaminations par ingestion et par blessure. Les nanoparticules fonctionnalisées à base de polymères naturels sont un concept innovant de décorporation du Pu(IV) solubilisé dans les macrophages pulmonaires et ouvrent la voie au développement de nouvelles familles de décorporants. C'est dans ce contexte que deux stratégies ont été développées : des nanoparticules à base de N-trimethyl chitosan fonctionnalisées par le ligand DTPMP (l'acide diéthylènetriaminepentamethylene phosphonique) qui est l'analogue phosphonique du DTPA et des nanoparticules chélatantes à base de -cyclodextrines amphiphiles anioniques. Ce travail a consisté en la synthèse et la caractérisation des nano-objets puis de l'étude de leur complexation avec les actinides (Th/Pu) en utilisant la spectroscopie EXAFS. Et enfin, des tests préliminaires biologiques in vitro ont été réalisés. Les résultats obtenus, avec les nanoparticules à base de chitosan et de DTPMP montrent que les nanoparticules présentent des tailles et une stabilité compatible avec l’application visée. D’autre part, leur affinité pour les actinides (IV) (Th,Pu) est comparable à celle du chélatant de référence, le DTPA. Enfin, les tests, effectués sur deux lignées de macrophages montrent que les nanoparticules sont internalisées très rapidement et que la matrice polysaccharidique semble se dégrader, permettant le relargage du chélateur DTPMP au niveau des sites de rétention du Pu(IV). Cette thèse constitue un travail préliminaire au développement d'une nouvelle famille d’agents décorporants plus ciblés pour une contamination au plutonium par inhalation. / The use of plutonium (Pu(IV) for military and civil applications can lead to internal contamination. There are several possible routes of contamination: ingestion, inhalation or injury. In case of plutonium inhalation, the plutonium forms oxide particles that reach the pulmonary alveoli. Through a phagocytosis mechanism, the particles are internalized by the macrophages of the pulmonary epithelium and continue to exert their toxicity. Currently, the only decorporating agent administered in the event of contamination with plutonium is DTPA (diethylenetriaminepentaacetic acid). in France, it is administered as CaNa3-DTPA by intravenous injection. This standard is effective for contamination by ingestion and injury. However, it is not effective in case of contamination by inhalation. Functionalized nanoparticles based on natural polymers constitute an innovative concept for decorporating Pu(IV) solubilized in pulmonary macrophages and open the way for the development of new families of decorporants. We investigated two strategies: chitosan-based nanoparticles functionalized by the DTPMP (diethylenetriaminepentamethylene phosphonic acid) which is the phosphonic analog of DTPA and self-organized chelating β-cyclodextrin-based nanoparticles. This work was first focused on the synthesis and characterization of the nano-objects and then on the study of their complexation abilities with actinides (Th/Pu) using EXAFS spectroscopy. Finally, preliminary in vitro biological tests were carried out. Our obtained results with DTPMP and chitosan based nanoparticles showed that these aggregates exhibit size and stability compatible with the application. Furthermore, we demonstrate their affinity for the actinides(IV) (Th, Pu) is comparable to the reference DTPA. Finally, in vitro tests realized onto macrophages show that our nanoparticles are rapidly internalized through phagocytosis and that the polysaccharide matrix seems to undergo degradation which allows the DTPMP to be released and targeted right into the sequestration sites of Pu(IV). This work constitutes a first step in the development of new family of decorporating agents with a higher efficiency in case of plutonium contamination through inhalation.

Объектом исследования является композитный сорбент, модифицированный оксигидратом железа. Цель работы - синтез композитного неорганического сорбента и изучение его свойств. В работе проводился синтез нового неорганического сорбента с помощью полного факторного эксперимента. Был изучен механизм сорбции ионов фтора и хлора. Изучена устойчивость сорбента к агрессивным средам. Экспериментально определена емкость различных образцов сорбента. / In this paper, research arsenic-containing slimes of neutralization of waste electrolytes was carried out to selectively extract valuable metals (copper, nickel). The method of Atomic Absorption Spectroscopy (AAS), X-ray Diffraction (XRD) was applied. The chemical and phase analysis of multicomponent manmade waster was carried out, the leaching chelating reagent Trilon B was offered and its choice is reasonable. A review of modern methods of processing and laboratory studies were carried out. The processes of leaching of slimes by aqueous solution of Trilon B, purification of the obtained trilonate solution from metal-impurities and solvent regeneration were studied and optimized. Also, in the thesis the optimal scheme of processing of hydroxide sludges was chosen and the ecological and economic justification of the chosen technology of processing was represented.

O presente trabalho teve como objetivo avaliar, por meio da microscopia eletrônica de varredura (MEV), a capacidade de remoção da smear layer dos terços médio e apical do canal radicular utilizando soluções quelantes e desmineralizantes e, quantificar, por meio da espectrometria de absorção atômica com chama, a concentração de íons cálcio presentes nessas soluções após suas utilizações. Vinte e cinco caninos superiores foram preparados pela técnica Free Tip Preparation com 4 instrumentos acima do inicial e irrigados com hipoclorito de sódio 1% a cada troca de instrumento. Os dentes foram distribuídos aleatoriamente em 4 grupos, conforme protocolo utilizado para a irrigação final: G1 - EDTA 15%, G2 - quitosana 0,2%, G3 - ácido cítrico 10%, G4 - ácido acético 1%. O grupo controle (G5) não recebeu irrigação. Foram utilizados 5 mL de cada solução por 3 minutos. Após percorrer toda extensão do canal radicular a solução extravasada pelo forame foi coletada e encaminhada para análise espectrométrica. Os espécimes foram seccionados longitudinalmente e preparados para análise em MEV. As fotomicrografias obtidas foram avaliadas qualitativamente por três examinadores, que atribuíram escores às imagens, conforme a quantidade de smear layer. Os dados obtidos pela MEV foram analisados estatisticamente por meio do Teste de Kruskal-Wallis e Dunn. Para avaliação da espectrometria utilizou-se Tukey-Kramer (one-Way ANOVA). Os resultados mostraram que o EDTA 15%, quitosana 0,2% e ácido cítrico 10% removeram a smear layer de forma semelhante entre si e estatisticamente diferente (p<0,05) do ácido ácético 1% e controle. Não houve diferença na capacidade de limpeza das soluções quando os terços médio e apical foram comparados. A maior concentração de íons cálcio foi observada no grupo do EDTA 15% e quitosana 0,2%, sem diferença entre ambos. O grupo do ácido acético 1% apresentou as menores concentrações e o ácido cítrico 10%, concentrações intermediárias e diferentes estatísticamente dos dois grupos (p<0,01). Concluiu-se que as soluções de EDTA 15%, quitosana 0,2% e ácido cítrico 10% foram eficientes na remoção da smear layer do terço médio e apical do canal radicular. As soluções de EDTA 15% e quitosana 0,2% promoveram o maior efeito desmineralizante, seguidas pelo ácido cítrico 10% e acido acético 1%. / This study aimed to evaluate, by scanning electron microscopy (SEM), the ability to remove the smear layer from the apical and middle thirds of the root canal using chelators and demineralizing solutions, and quantify, by atomic absorption spectrophotometry flame, the concentration of calcium ions present in these solutions after their use. Twenty-five canines were prepared by Free Tip Preparation Technique with four instruments above the initial and irrigated with 1% sodium hypochlorite in each change of instrument. The teeth were randomly divided into four groups, according to the protocol used for the final irrigation: G1 - EDTA 15%, G2 - 0.2% chitosan, G3 - citric acid 10%, G4 - 1% acetic acid. The control group (G5) did not receive irrigation. We used 5 mL of each solution for 3 minutes. After irrigating the entire length of the root canal through the apical foramen, the solution was collected and sent to spectrometric analysis. The specimens were sectioned longitudinally and prepared for SEM analysis. The photomicrographs were qualitatively evaluated by three observers, who attributed scores to the images, equivalent to the amount of smear layer removal. The data obtained by SEM was statistically analyzed using the Kruskal-Wallis and Dunn tests. To evaluate the spectrometer, the Tukey-Kramer (one-way ANOVA) was used. The results showed that 15% EDTA, 0.2% chitosan and 10% citric acid had a similar smear layer removal with statistically different (p <0.05) when compared to 1% acetic acid and the control group. There was no difference between the solutions cleaning ability when the middle and apical thirds were compared. The highest concentration of calcium ions was observed in 15% EDTA group and 0.2% chitosan, with no statistical difference between them. The 1% acetic acid group had the lowest concentration and the 10% citric acid intermediate concentrations with statistical difference between each other (p <0.01). It can be concluded that the solutions of 15% EDTA, 0.2% chitosan and 10% citric acid were effective in removing the smear layer of the middle and apical thirds of the root canal. 15% EDTA solutions and 0.2% chitosan promoted the greatest effect on the demineralization, followed by 10% citric acid and 1% acetic acid.

O presente trabalho avaliou, por microscopia eletrônica de varredura (MEV), a limpeza das paredes dos canais radiculares instrumentados pelas técnicas rotatória e Self-adjusting file (SAF®) com uso do NaOCl a 1% como solução irrigadora e o uso do EDTA a 15% e da Quitosana 0,20% como soluções quelantes, e quantificou os íons cálcio removidos pelas diferentes soluções irrigadoras por meio de espectrometria de absorção atômica. Trinta dentes caninos inferiores humanos foram divididos em dois grupos (n=15), de acordo com a técnica de instrumentação utilizada: A - instrumentos FlexMaster® usados na técnica Free Tip Preparation, assegurado alargamento mínimo de 200, até o instrumento # 60.02, irrigados com 1 mL de NaOCl 1% em cada troca de instrumento e B - sistema SAF®, com prévio alargamento manual até o instrumento K #20, aplicação da SAF® por 4 minutos, com fluxo de irrigação pelo sistema VATEA de 5mL/min. Os dentes foram divididos em três subgrupos conforme a irrigação final: G1-NaOCl 1%, G2- EDTA 15% e G3 Quitosana 0,20%. Utilizou-se 5mL de cada solução à 1mL/min. A solução extravasada pelo forame foi coletada e submetida à análise espectrométrica. O EDTA 15% apresentou maior capacidade de remoção de íons cálcio do que a Quitosana a 0,20% (p<0,05) e o NaOCl 1% não removeu íons. Os espécimes foram analisados em MEV. As fotomicrografias obtidas foram avaliadas qualitativamente em relação à remoção da camada de smear observada. Não houve diferença entre as instrumentações rotatória e SAF® nos terços dos canais avaliados. Analisadas as interações entre as técnicas utilizadas e aos terços do canal, o EDTA 15% apresentou melhores resultados que a Quitosana 0,20% (p<0,05), sendo ambas as soluções melhores que o NaOCl 1%. Conclui-se que as soluções quelantes testadas foram capazes de remover íons cálcio das paredes dos canais radiculares - com superioridade do EDTA 15% sobre a Quitosana 0,20%, e o NaOCl a 1% não apresentou essa capacidade - e removeram a camada de smear das paredes dos canais radiculares com superioridade do EDTA 15%, sem interferência das técnicas de instrumentação avaliadas rotatória e SAF®. / The present study evaluated, by scanning electron microscopy (SEM), the cleanness of the root canal walls instrumented by rotary and the self-adjusting file (SAF®) techniques using 1% NaOCl as an irrigating solution and 15% EDTA, 0.20% Chitosan as chelator solutions, and quantified the calcium ions removed by the different irrigants with atomic absorption spectrometry. Thirty human mandibular canine teeth were divided into 2 groups (n=15) according to the instrumentation technique used: A- FlexMaster® instruments used with the Free tip Preparation, assuring a minimal enlargement of 200m, up to a #60 .02 instrument, irrigated with 1mL of 1% NaOCl between each instrument; and B- SAF® system, with previous enlargement to a type K #20 manual instrument, application of SAF during 4 minutes, with VATEA irrigation system at a flow of 5mL/min. The teeth were divided into three subgroups according to the final irrigation protocol: G1- 1% NaOCl, G2- 15% EDTA and G3- 0.20 % Chitosan. Five mL of solution were used at 1mL/min. The solution extruded thru the foramen was collected and submitted to spectrometric analysis. 15% EDTA showed higher calcium ion removal capacity then 0.20% Chitosan (p<0.05) and the 1% NaOCl did not remove ions. The specimens were evaluated by means of SEM. The photomicrographs obtained were qualitatively evaluated in terms of smear layer removal. There was no significant difference between the rotary and SAF® instrumentation systems in the evaluated canal thirds. The interaction techniques used and canals thirds showed that the 15% EDTA revealed better results than the 0.02% Chitosan (p<0.05), and both were better than 1% NaOCl. It can be concluded that the tested chelating solutions removed calcium ions from the root canals walls with better results for 15% EDTA than 0.20% Chitosan; 1% NaOCl did not present this capacity - and removed the smear layer form the root canal walls with better results for 15% EDTA, with no difference between the evaluated techniques rotary and SAF®.

INTRODUÇÃO: Há poucos modelos experimentais de doença óssea adinâmica (DOA) e os mecanismos fisiopatológicos dessa doença não são completamente compreendidos. Além disso, os efeitos dos quelantes de fósforo (P) na DOA têm sido pouco estudados. Este estudo objetivou estabelecer um modelo de DOA e avaliar os efeitos da terapia com carbonato de cálcio (Ca) e carbonato de sevelamer (sevelamer) sobre os distúrbios do metabolismo mineral e ósseo da doença renal crônica (DMO-DRC), no modelo estabelecido. MÉTODOS: Experimento 1: A DOA e a DRC foram induzidas por nefrectomia 5/6 (Nx) e paratireoidectomia (PTx) em ratos Wistar, que após a cirurgia foram divididos em 2 grupos: Nx+PTx e sham (sham Nx+PTx). Experimento 2: ratos Wistar foram submetidos à Nx e à PTx e após a cirurgia, foram divididos em outros 2 grupos: Nx+PTx+Ca (CaCO3 a 3%); Nx+PTx+Sev (sevelamer a 3%). A dieta de todos os animais após a cirurgia foi rica em P (1,2%) à base de grãos, exceto o grupo sham, que recebeu dieta padrão com 0,6% de P. Após oito semanas, os animais foram sacrificados. Foram realizadas análises bioquímicas, ósseas e de calcificação vascular. RESULTADOS: Experimento 1: A Nx e a PTx foram efetivas, confirmadas pela elevação da creatinina, com diminuição do clearance de creatinina e dos níveis de cálcio iônico, nos animais Nx+PTx comparados aos animais sham. O modelo foi eficaz na indução da DOA, confirmada pela diminuição do turnover ósseo nos animais Nx+PTx, comparados ao grupo sham. Experimento 2: A terapia com quelantes de P não alterou o P sérico, mas reduziu a fração de excreção de P (FeP). A diminuição dos níveis de FGF-23 e PTH nos animais Nx+PTx foram independentes da terapia com quelantes e não houve diferença nos valores entre os grupos. A esclerostina sérica não foi diferente entre os grupos, mas os animais Nx+PTx+Sev apresentaram menor expressão gênica de SOST e menor taxa de apoptose de osteócitos que os outros grupos. Ambos os quelantes de P diminuíram a expressão gênica do Dickkopf-1 e do fator de crescimento ?1 (TGF-?1). Os animais Nx+PTx+Ca apresentaram maior superficie de reabsorção e maior conteúdo de Ca do ventrículo esquerdo (VE) que os animais Nx+PTx, enquanto os animais Nx+PTx+Sev mostraram diminuição do conteúdo de Ca de VE, comparado aos demais grupos. CONCLUSÕES: o modelo experimental desenvolvido é útil para o estudo da DRC com DOA. A FeP parece ser parâmetro mais fidedigno que o P sérico para avaliar o poder dos quelantes de P. A diminuição do FGF-23 esteve relacionada à diminuição dos níveis de PTH e à hipocalcemia. Os animais tratados com Ca apresentaram sobrecarga desse elemento, traduzida por maior calciúria, maior conteúdo de cálcio de VE e maior superfície de reabsorção óssea. Os mecanismos subjacentes à ação do sevelamer na diminuição da expressão da SOST foram independentes do PTH, do P séricos, da função renal e da expressão gênica de TGF-?1. Mais estudos são necessários para melhor compreensão desses mecanismos / INTRODUCTION: There are few experimental models of adynamic bone disease (ABD) and the pathophysiology of this disease is not fully understood. In addition, the effects of different phosphate (P) binders on ABD have not been evaluated. This study aimed to establish a model of ABD and evaluate the effects of therapy with calcium carbonate (Ca) and sevelamer carbonate (sevelamer) on disorders of bone and mineral metabolism in chronic kidney disease (CKD-MBD), on the established model. METHODS: Experiment 1: ABD and CKD were induced by 5/6 nephrectomy (Nx) and parathyroidectomy (PTx) in Wistar rats, which after surgery, were divided into 2 groups: Nx+PTx and sham (sham Nx+PTx). Experiment 2: Wistar rats underwent Nx and PTx and after surgery were divided into 2 more groups: Nx+PTx+Ca(3% Ca-treated) and Nx+PTx+Sev (3% Sev-treated). All animals were fed a high P (1.2%), grain-based diet, except the sham group which was fed a standard P (0,6%) diet. After 8 weeks, the animals were sacrificed. Biochemical, bone and vascular calcification analyses were performed. RESULTS: Experiment 1: Nx and PTx were effective, confirmed by higher creatinina with decreased creatinine clearance and decreased ionized calcium levels respectively, in Nx+PTx animals compared to sham animals. The model was effective in inducing ABD confirmed by decreased bone turnover in animals Nx+PTx compared to sham group. Phosphate binders administration did not change serum P, but decreased the fractional excretion of phosphate (FeP) in treated animals. FGF-23 and PTH levels were reduced in all Nx+PTx animals independent of the therapy with P binders and these levels were not different among groups. Serum sclerostin was not different among groups, however, Nx+PTx+Sev animals had lower SOST gene expression and lower osteocytes apoptotic rate than the other animals. Both P binders decreased Dickkopf-1 and transforming growing factor ?1 (TGF-?1) gene expression. Nx+PTx+Ca animals showed higher eroded surface and higher left ventricle (LV) calcium content than Nx+PTx animals, whereas Nx+PTx+Sev animals showed a decrease in LV calcium content, compared to the other groups. CONCLUSIONS: This experimental model is useful to study CKD with ABD. The FeP seems to be a more reliable parameter than serum P to evaluate the effectiveness of P binders. Decreased FGF-23 levels were related to decreased PTH levels and hypocalcemia. Ca-treated animals showed Ca overload, as seen by higher calciuria, higher LV calcium content and higher eroded surface. The underlying mechanisms involved in sevelamer actions of decreased SOST expression were independent of PTH, serum P, renal function and TGF-?1 gene expression. Further studies are needed to a better understanding of these mechanisms

O presente trabalho avaliou, por microscopia eletrônica de varredura (MEV), a limpeza das paredes dos canais radiculares instrumentados pelas técnicas rotatória e Self-adjusting file (SAF®) com uso do NaOCl a 1% como solução irrigadora e o uso do EDTA a 15% e da Quitosana 0,20% como soluções quelantes, e quantificou os íons cálcio removidos pelas diferentes soluções irrigadoras por meio de espectrometria de absorção atômica. Trinta dentes caninos inferiores humanos foram divididos em dois grupos (n=15), de acordo com a técnica de instrumentação utilizada: A - instrumentos FlexMaster® usados na técnica Free Tip Preparation, assegurado alargamento mínimo de 200, até o instrumento # 60.02, irrigados com 1 mL de NaOCl 1% em cada troca de instrumento e B - sistema SAF®, com prévio alargamento manual até o instrumento K #20, aplicação da SAF® por 4 minutos, com fluxo de irrigação pelo sistema VATEA de 5mL/min. Os dentes foram divididos em três subgrupos conforme a irrigação final: G1-NaOCl 1%, G2- EDTA 15% e G3 Quitosana 0,20%. Utilizou-se 5mL de cada solução à 1mL/min. A solução extravasada pelo forame foi coletada e submetida à análise espectrométrica. O EDTA 15% apresentou maior capacidade de remoção de íons cálcio do que a Quitosana a 0,20% (p<0,05) e o NaOCl 1% não removeu íons. Os espécimes foram analisados em MEV. As fotomicrografias obtidas foram avaliadas qualitativamente em relação à remoção da camada de smear observada. Não houve diferença entre as instrumentações rotatória e SAF® nos terços dos canais avaliados. Analisadas as interações entre as técnicas utilizadas e aos terços do canal, o EDTA 15% apresentou melhores resultados que a Quitosana 0,20% (p<0,05), sendo ambas as soluções melhores que o NaOCl 1%. Conclui-se que as soluções quelantes testadas foram capazes de remover íons cálcio das paredes dos canais radiculares - com superioridade do EDTA 15% sobre a Quitosana 0,20%, e o NaOCl a 1% não apresentou essa capacidade - e removeram a camada de smear das paredes dos canais radiculares com superioridade do EDTA 15%, sem interferência das técnicas de instrumentação avaliadas rotatória e SAF®. / The present study evaluated, by scanning electron microscopy (SEM), the cleanness of the root canal walls instrumented by rotary and the self-adjusting file (SAF®) techniques using 1% NaOCl as an irrigating solution and 15% EDTA, 0.20% Chitosan as chelator solutions, and quantified the calcium ions removed by the different irrigants with atomic absorption spectrometry. Thirty human mandibular canine teeth were divided into 2 groups (n=15) according to the instrumentation technique used: A- FlexMaster® instruments used with the Free tip Preparation, assuring a minimal enlargement of 200m, up to a #60 .02 instrument, irrigated with 1mL of 1% NaOCl between each instrument; and B- SAF® system, with previous enlargement to a type K #20 manual instrument, application of SAF during 4 minutes, with VATEA irrigation system at a flow of 5mL/min. The teeth were divided into three subgroups according to the final irrigation protocol: G1- 1% NaOCl, G2- 15% EDTA and G3- 0.20 % Chitosan. Five mL of solution were used at 1mL/min. The solution extruded thru the foramen was collected and submitted to spectrometric analysis. 15% EDTA showed higher calcium ion removal capacity then 0.20% Chitosan (p<0.05) and the 1% NaOCl did not remove ions. The specimens were evaluated by means of SEM. The photomicrographs obtained were qualitatively evaluated in terms of smear layer removal. There was no significant difference between the rotary and SAF® instrumentation systems in the evaluated canal thirds. The interaction techniques used and canals thirds showed that the 15% EDTA revealed better results than the 0.02% Chitosan (p<0.05), and both were better than 1% NaOCl. It can be concluded that the tested chelating solutions removed calcium ions from the root canals walls with better results for 15% EDTA than 0.20% Chitosan; 1% NaOCl did not present this capacity - and removed the smear layer form the root canal walls with better results for 15% EDTA, with no difference between the evaluated techniques rotary and SAF®.

<p>Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. </p><p>Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd³⁺ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. </p><p>The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.</p>

Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd3+ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.

L'oligomérisation enzymatique de la rutine et esculine a donné lieu à cinq fractions d'oligomères de masse moléculaire moyenne entre 2127,42 et 8331,85 g/mol pour la rutine et 688,12 et 6973 g/mol pour l'esculine. L'analyse de ces fractions par FTIR montre que les fractions d'oligorutines sont obtenues à travers des liaisons C-C, C-O et C=O. Les fractions d'oligoesculines sont obtenues à travers des liaisons C-C. Une meilleure solubilité des oligorutines et des oligoesculines dans l'eau et une plus faible solubilité de ces oligomères dans l'éthanol comparé à leurs monomères a été mis en évidence. Une diminution de l'activité antiradicalaire vis-à-vis de DPPH., ABTS+. et OH. proportionnelle à la masse moléculaire moyenne des fractions d'oligorutines a été observé, contrairement aux fractions d'oligoesculines qui montrent un important pouvoir chélateur de ces mêmes radicaux comparé à leurs monomère. Une augmentation du pouvoir chélateur de fer, inhibiteur de la xanthine oxydase, réducteur du cuivre (CUPRAC), de l'activité antigénotoxique, ainsi que de l'activité stimulatrice de la prolifération des splénocytes, et des lymphocytes (B et T) proportionnelle au degré d'oligomérisation des oligomères étudiées a été noté. L'effet des fractions d'oligorutines et oligoesculines étudiées sur les macrophages en suivant la production de monoxyde d'azote (NO) montre un pouvoir anti-inflammatoire comparé à leurs monomères. L'étude de l'activité lysosomale induite par les fractions d'oligorutine révèle un pouvoir immunostimulateur proportionnelle à la masse moléculaire moyenne des oligorutines, et inversement proportionnelle à celle-ci pour les oligoesculines / Rutin and esculin have been polymerized by laccase. Five fractions with between 2127.42 and 8331.85 g/mol for oligorutins, and between 688.12 and 6973 g/mol for oligoesculins, were obtained. Fourier transformed infrared analysis showed that oligorutins were formed through C-C, C-O and C=O linkages, while oligoesculins were obtained through C-C linkages. Oligorutins and oligoesculins show a higher solubility in water and a lower solubility in ethanol compared to their monomers. The oligomerization of rutin decrease its antiradical capacity, while oligoesculin fractions demonstrated a high antiradical activity compared to monomeric esculin. Oligomer fractions showed a better iron chelating power, xanthine oxidase inhibition, copper reducing power (CUPRAC), antigenotoxic activity, and splenocytes stimulator activity compared to their monomers. Oligorutin and oligoesculin exhibited an important anti-inflammatory capacity through the nitric oxide inhibition. Moreover, oligorutin fractions demonstrated an immunostimulatory effect proportional to their degree of oligomerization, while oligoesculin fractions showed an immunostimulatory effect inversely proportional to their degree of oligomerization

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