Este estudo teve como objetivo avaliar a eficácia dos cimentos obturadores do sistema de canais radiculares quando submetidos ao desafio ácido em ambiente bucal. Foram utilizadas 55 raízes de incisivos centrais inferiores humanos com comprimento padronizado em 10 mm. Realizou-se o preparo biomecânico dos canais até o instrumento #40.02 e as raízes foram esterilizadas em autoclave. Quarenta e quatro raízes foram obturadas pela técnica de condensação lateral com um dos quatro cimentos de diferentes bases: AH Plus, MTA Fillapex, Sealapex ou Endofill. Nas 11 raízes remanescentes, apenas foi executado o preparo biomecânico dos canais e esterilização (controle negativo). Foram selecionados 11 participantes que atenderam aos critérios de inclusão na pesquisa. Foi realizada a moldagem das arcadas dentais e confecção dos dispositivos acrílicos intra-bucais palatinos com 5 nichos, sendo 4 deles para as raízes correspondentes a cada cimento experimental e 1 nicho para a raiz controle. As raízes foram fixadas com cera e tela para favorecer o acúmulo de biofilme. Durante 14 dias, 11 participantes utilizaram o dispositivo o dia todo e foram orientados a gotejar solução de sacarose 20% sobre as amostras, seis vezes ao dia, simulando alto desafio cariogênico. Após os 14 dias, as raízes foram removidas dos dispositivos, seccionadas em slices e foram realizadas as seguintes análises: perfil de desgaste do material obturador e superfície dentinária antes e após a exposição ao ambiente ácido bucal por microscopia confocal de varredura a laser (MCVL); resistência adesiva (MPa) do material obturador à dentina radicular por teste de push-out e análise qualitativa da morfologia da interface adesiva e desmineralização ao redor do material obturador por MCVL. Os dados do perfil de desgaste foram avaliados pelo teste não-paramétrico de Kruskal-Wallis e teste t (&alpha;=0,05), os dados da resistência adesiva foram avaliados por Análise de Variância a dois critérios (cimentos e terços radiculares) e teste de Tukey (&alpha;=0,05). Verificou-se que não houve diferença estatisticamente significante entre os cimentos (p=0,6190), porém todas as amostras apresentaram desgaste da dentina e material obturador após exposição ao ambiente bucal (p<0,05). As raízes obturadas com o cimento AH Plus apresentaram maior resistência adesiva à dentina (11,40 ± 7,74 a) (p<0,05). Resultados intermediários foram encontrados nas raízes obturadas com o MTA Fillapex (7,22 ± 5,88 ab) e Endofill (7,37 ± 6,75 ab). As raízes obturadas com o Sealapex apresentaram menores valores de resistência adesiva (5,18 ± 4,34 b). Não houve diferença significante para os terços radiculares, nem na interação dos fatores (p>0,05). Houve predomínio de falhas adesivas em dentina nas raízes obturadas com os cimentos AH Plus, MTA Fillapex e Endofill (respectivamente 66%, 75% e 54,2%). Nas raízes obturadas com o Sealapex houve predomínio de falhas mistas (54,2%). Todos os cimentos apresentaram degradação do material obturador e superfície dentinária, além da desmineralização ao redor da obturação, sendo esta última mais intensa nas raízes obturadas com os cimentos Sealapex e Endofill. Nas raízes não obturadas, houve acúmulo intenso de biofilme bacteriano e desmineralização da dentina intrarradicular. Nenhum cimento foi capaz de evitar a degradação da interface adesiva e da dentina. No entanto, nestas situações de alto desafio ácido, os cimentos AH Plus e MTA Fillapex demonstraram desempenho superior aos demais cimentos testados, por apresentarem melhor resistência adesiva do material obturador à dentina, além de degradação e desmineralização ao redor da obturação menos intensa. / This study has the purpose to evaluate the efficacy of root canal sealers following in situ acid challenge. The root canals of 55 human mandibular central incisors with standardized root canals length 10 mm were used. Roots were as submitted to biomechanical preparation up to #40.02 instrument and canals were sterilized in an autoclave. Forty-four roots were filled with one of the four sealers using the lateral condensation technique: AH Plus, Endofill, MTA Fillapex e Sealapex. The remaining 11 roots were only submitted to biomechanical preparation and were sterilized (negative control). Eleven 11 participants that fulfill the inclusion criteria were selected. The impressions of dental arcs were performed and intraoral acrylic devices were done with 5 spaces, being 4 for roots with experimental sealers and 1 for control root. The roots were fixed with wax and screen to promote the accumulation of biofilm. During 14 days, 11 participants were instructed to use the devices every day. Dental biofilm was allowed to accumulate on root and drops of 20% sucrose solution were dripped onto them, simulating a high acidic challenge. After 14 days, the roots were removed from the intraoral devices sectioned in slices and the following analyses were conducted: degradation (wear profile) in the dentin-sealer interface subjected to confocal laser scanning microscope (CLSM); bond strength of filling material (MPa) to root canal (push-out test) and qualitative analysis of adhesive interface morphology and demineralization around filling material by CLSM. The wear profile data were assessed by non-parametric Kruskal-Wallis and t-test (&alpha;=0.05), the bond strength were evaluated by two-way ANOVA (cements and root thirds) and Tukey test (&alpha;=0.05). Statistical analyses were set at 5% significance level. Regarding the surface of the wear profile, it was found that there was no significant statistical difference between sealers (p=0.6190), but all samples showed wear of dentine and filling material after exposure to the oral environment (p <0.05). Roots filled with AH Plus sealer showed the higher bond strength to dentin (11.40 ± 7.74 a) (p<0.05). Intermediate results were found in roots with MTA Fillapex (7.22 ± 5.88 ab) and Endofill (7.37 ± 6.75 ab). The worst result was found in roots with Sealapex (5.18 ± 4.34 b). There were no significant differences in root thirds, neither in the interaction of factors (p>0.05). Adhesive failure were predominant in root canals with AH Plus, MTA Fillapex and Endofill (respectively, 66%, 75% e 54.2%). Root canals with Sealapex presented more mixed failure (54.2%). Qualitative morphological analysis showed that all sealers presented dentin demineralization around root canal filling, being greater when using to Sealapex and Endofill sealers. In unfilled roots, there was intense accumulation of bacterial biofilm and demineralization of intraradicular dentin. After the exposure of roots to oral environment for 14 days, it may be concluded that no sealer was able to prevent degradation of the adhesive interface and dentin. However, in these situations of high acid challenge, AH Plus and MTA Fillapex sealers have shown superior performance than other tested sealers for their high adhesive strength of the filling material to dentin, and less intense degradation and demineralization around the root canal filling.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Ions in the soil solution traverse many layers (epidermis, exodermis, central cortex, and endodermis) within the root to reach the stele. The endodermis is present in almost all vascular plants while the exodermis is found only in majority of angiosperm roots tested. The maturation of the exodermis and the death of epidermis alter the plasma membrane surface areas (PMSA) potentially available for ion uptake. Do these changes reduce the ion uptake in proportion to the loss of absorptive surface areas? To answer this question onion (Allium cepa L cv. Wolf) adventitious root segments representing above features: Immature Exodermis Live Epidermis (IEXLEP), Mature Exodermis Live Epidermis (MEXLEP), Mature Exodermis Dead Epidermis (MEXDEP) were excised. Using a compartmental elution technique, radioactive sulphate and phosphate present in various internal compartments were quantified. Quantities of ions moved across the plasma membrane, a summation of quantities in the cytoplasm, ‘vacuole’, and ‘bound’ compartments, indicated that the maturation of the exodermis reduces the uptake of sulphate but not phosphate. In contrast, epidermal death reduced the movement of both ions across the plasma membranes. Although there is a reduction in the available PMSA with the maturation of the exodermis and death of the epidermis, these events do not necessarily reduce the ion movement into the plasma symplast. The endodermal cells of onion roots deposit suberin lamellae as secondary walls. As seen in cross-sections some cells remain without these lamellae and are known as ‘passage cells’. What is the pattern of suberin lamella deposition along the root? Is the suberin lamella a continuous layer? To answer these questions, endodermal layers isolated from onion adventitious roots were used in the present study. These layers were observed using four stains (Sudan Red 7B, Fluorol yellow 088 [Fy], berberine, and Nile red) and three microscopes (compound-white light, compound-epifluorescence and confocal scanning). In differentiating cells with and without suberin lamellae in endodermal layers Sudan Red 7B served the best results for compound-white light microscope, Fy for compound-epifluorescence microscope and Nile for confocal laser scanning microscope (CLSM). Suberin lamellae deposition initiated almost in a random manner; they continued to be deposited resulting in the production of longitudinal files alternating with files with passage cells, and were ultimately deposited in almost all cells at a distance of 255 mm from the tip. The suberin lamellae are perforated with pores, a consistent feature even as far as 285 mm from the tip. These pores may serve as portals for water, ions, and pathogen movement.

Ions in the soil solution traverse many layers (epidermis, exodermis, central cortex, and endodermis) within the root to reach the stele. The endodermis is present in almost all vascular plants while the exodermis is found only in majority of angiosperm roots tested. The maturation of the exodermis and the death of epidermis alter the plasma membrane surface areas (PMSA) potentially available for ion uptake. Do these changes reduce the ion uptake in proportion to the loss of absorptive surface areas? To answer this question onion (Allium cepa L cv. Wolf) adventitious root segments representing above features: Immature Exodermis Live Epidermis (IEXLEP), Mature Exodermis Live Epidermis (MEXLEP), Mature Exodermis Dead Epidermis (MEXDEP) were excised. Using a compartmental elution technique, radioactive sulphate and phosphate present in various internal compartments were quantified. Quantities of ions moved across the plasma membrane, a summation of quantities in the cytoplasm, ‘vacuole’, and ‘bound’ compartments, indicated that the maturation of the exodermis reduces the uptake of sulphate but not phosphate. In contrast, epidermal death reduced the movement of both ions across the plasma membranes. Although there is a reduction in the available PMSA with the maturation of the exodermis and death of the epidermis, these events do not necessarily reduce the ion movement into the plasma symplast. The endodermal cells of onion roots deposit suberin lamellae as secondary walls. As seen in cross-sections some cells remain without these lamellae and are known as ‘passage cells’. What is the pattern of suberin lamella deposition along the root? Is the suberin lamella a continuous layer? To answer these questions, endodermal layers isolated from onion adventitious roots were used in the present study. These layers were observed using four stains (Sudan Red 7B, Fluorol yellow 088 [Fy], berberine, and Nile red) and three microscopes (compound-white light, compound-epifluorescence and confocal scanning). In differentiating cells with and without suberin lamellae in endodermal layers Sudan Red 7B served the best results for compound-white light microscope, Fy for compound-epifluorescence microscope and Nile for confocal laser scanning microscope (CLSM). Suberin lamellae deposition initiated almost in a random manner; they continued to be deposited resulting in the production of longitudinal files alternating with files with passage cells, and were ultimately deposited in almost all cells at a distance of 255 mm from the tip. The suberin lamellae are perforated with pores, a consistent feature even as far as 285 mm from the tip. These pores may serve as portals for water, ions, and pathogen movement.

Pheroid is a patented system comprising of a unique submicron emulsion type formulation. Pheroid vesicles consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful molecules. The aim of this study was to show that a modulation of components and parameters is necessary to obtain the optimum formula to be used in pharmaceutical preparations. Non-optimal or non-predictable stability properties of emulsions can be limiting for the applications of emulsions (Bjerregaard et al., 2001:23). Careful consideration was given to the apparatus used during the processing along with the ratios of the various components added to the formulation and the storage conditions of the Pheroid vesicles. A preliminary study was performed to optimize the most accurate processing parameters during emulsification. The effect of emulsification rate and time, the temperature of the aqueous phase, the number of days the water phase were gassed, the concentration of the surfactant, cremophor® RH 40, used and the concentration of Vitamin F Ethyl Ester CLR added to the oil phase of the o/w emulsion has been studied. Quantification of the mean particle size, zeta potential, turbidity, pH and current values were used to characterize the emulsions. The samples were characterised after 1, 2, 3, 7, 14, 21 and 28 days of storage. The emulsions were also characterised with confocal laser scanning microscopy (CLSM) to measure the number and size and size distribution of the vesicles. After determination of the processing variables influencing the emulsion stability an accelerated stability test was conducted on a final formula. In the present study, accelerated stability testing employing elevated temperatures and relative humidity were used with good accuracy to predict long-term stability of an o/w emulsion kept at both 5 and 25 OC with 60 % relative humidity and 40 OC with 75 % relative humidity. The results of the stability tests were presented in histograms of the physical properties 24 hours, 1 month, 2 months and 3 months after preparation of the emulsion. It was concluded that Pheroid vesicles demonstrate much potential as a drug delivery system. The high stability of this formula allows its use in a wide variety of applications in the pharmaceutical industry. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Pheroid is a patented system comprising of a unique submicron emulsion type formulation. Pheroid vesicles consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful molecules. The aim of this study was to show that a modulation of components and parameters is necessary to obtain the optimum formula to be used in pharmaceutical preparations. Non-optimal or non-predictable stability properties of emulsions can be limiting for the applications of emulsions (Bjerregaard et al., 2001:23). Careful consideration was given to the apparatus used during the processing along with the ratios of the various components added to the formulation and the storage conditions of the Pheroid vesicles. A preliminary study was performed to optimize the most accurate processing parameters during emulsification. The effect of emulsification rate and time, the temperature of the aqueous phase, the number of days the water phase were gassed, the concentration of the surfactant, cremophor® RH 40, used and the concentration of Vitamin F Ethyl Ester CLR added to the oil phase of the o/w emulsion has been studied. Quantification of the mean particle size, zeta potential, turbidity, pH and current values were used to characterize the emulsions. The samples were characterised after 1, 2, 3, 7, 14, 21 and 28 days of storage. The emulsions were also characterised with confocal laser scanning microscopy (CLSM) to measure the number and size and size distribution of the vesicles. After determination of the processing variables influencing the emulsion stability an accelerated stability test was conducted on a final formula. In the present study, accelerated stability testing employing elevated temperatures and relative humidity were used with good accuracy to predict long-term stability of an o/w emulsion kept at both 5 and 25 OC with 60 % relative humidity and 40 OC with 75 % relative humidity. The results of the stability tests were presented in histograms of the physical properties 24 hours, 1 month, 2 months and 3 months after preparation of the emulsion. It was concluded that Pheroid vesicles demonstrate much potential as a drug delivery system. The high stability of this formula allows its use in a wide variety of applications in the pharmaceutical industry. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Este estudo teve como objetivo avaliar a eficácia dos cimentos obturadores do sistema de canais radiculares quando submetidos ao desafio ácido em ambiente bucal. Foram utilizadas 55 raízes de incisivos centrais inferiores humanos com comprimento padronizado em 10 mm. Realizou-se o preparo biomecânico dos canais até o instrumento #40.02 e as raízes foram esterilizadas em autoclave. Quarenta e quatro raízes foram obturadas pela técnica de condensação lateral com um dos quatro cimentos de diferentes bases: AH Plus, MTA Fillapex, Sealapex ou Endofill. Nas 11 raízes remanescentes, apenas foi executado o preparo biomecânico dos canais e esterilização (controle negativo). Foram selecionados 11 participantes que atenderam aos critérios de inclusão na pesquisa. Foi realizada a moldagem das arcadas dentais e confecção dos dispositivos acrílicos intra-bucais palatinos com 5 nichos, sendo 4 deles para as raízes correspondentes a cada cimento experimental e 1 nicho para a raiz controle. As raízes foram fixadas com cera e tela para favorecer o acúmulo de biofilme. Durante 14 dias, 11 participantes utilizaram o dispositivo o dia todo e foram orientados a gotejar solução de sacarose 20% sobre as amostras, seis vezes ao dia, simulando alto desafio cariogênico. Após os 14 dias, as raízes foram removidas dos dispositivos, seccionadas em slices e foram realizadas as seguintes análises: perfil de desgaste do material obturador e superfície dentinária antes e após a exposição ao ambiente ácido bucal por microscopia confocal de varredura a laser (MCVL); resistência adesiva (MPa) do material obturador à dentina radicular por teste de push-out e análise qualitativa da morfologia da interface adesiva e desmineralização ao redor do material obturador por MCVL. Os dados do perfil de desgaste foram avaliados pelo teste não-paramétrico de Kruskal-Wallis e teste t (&alpha;=0,05), os dados da resistência adesiva foram avaliados por Análise de Variância a dois critérios (cimentos e terços radiculares) e teste de Tukey (&alpha;=0,05). Verificou-se que não houve diferença estatisticamente significante entre os cimentos (p=0,6190), porém todas as amostras apresentaram desgaste da dentina e material obturador após exposição ao ambiente bucal (p<0,05). As raízes obturadas com o cimento AH Plus apresentaram maior resistência adesiva à dentina (11,40 ± 7,74 a) (p<0,05). Resultados intermediários foram encontrados nas raízes obturadas com o MTA Fillapex (7,22 ± 5,88 ab) e Endofill (7,37 ± 6,75 ab). As raízes obturadas com o Sealapex apresentaram menores valores de resistência adesiva (5,18 ± 4,34 b). Não houve diferença significante para os terços radiculares, nem na interação dos fatores (p>0,05). Houve predomínio de falhas adesivas em dentina nas raízes obturadas com os cimentos AH Plus, MTA Fillapex e Endofill (respectivamente 66%, 75% e 54,2%). Nas raízes obturadas com o Sealapex houve predomínio de falhas mistas (54,2%). Todos os cimentos apresentaram degradação do material obturador e superfície dentinária, além da desmineralização ao redor da obturação, sendo esta última mais intensa nas raízes obturadas com os cimentos Sealapex e Endofill. Nas raízes não obturadas, houve acúmulo intenso de biofilme bacteriano e desmineralização da dentina intrarradicular. Nenhum cimento foi capaz de evitar a degradação da interface adesiva e da dentina. No entanto, nestas situações de alto desafio ácido, os cimentos AH Plus e MTA Fillapex demonstraram desempenho superior aos demais cimentos testados, por apresentarem melhor resistência adesiva do material obturador à dentina, além de degradação e desmineralização ao redor da obturação menos intensa. / This study has the purpose to evaluate the efficacy of root canal sealers following in situ acid challenge. The root canals of 55 human mandibular central incisors with standardized root canals length 10 mm were used. Roots were as submitted to biomechanical preparation up to #40.02 instrument and canals were sterilized in an autoclave. Forty-four roots were filled with one of the four sealers using the lateral condensation technique: AH Plus, Endofill, MTA Fillapex e Sealapex. The remaining 11 roots were only submitted to biomechanical preparation and were sterilized (negative control). Eleven 11 participants that fulfill the inclusion criteria were selected. The impressions of dental arcs were performed and intraoral acrylic devices were done with 5 spaces, being 4 for roots with experimental sealers and 1 for control root. The roots were fixed with wax and screen to promote the accumulation of biofilm. During 14 days, 11 participants were instructed to use the devices every day. Dental biofilm was allowed to accumulate on root and drops of 20% sucrose solution were dripped onto them, simulating a high acidic challenge. After 14 days, the roots were removed from the intraoral devices sectioned in slices and the following analyses were conducted: degradation (wear profile) in the dentin-sealer interface subjected to confocal laser scanning microscope (CLSM); bond strength of filling material (MPa) to root canal (push-out test) and qualitative analysis of adhesive interface morphology and demineralization around filling material by CLSM. The wear profile data were assessed by non-parametric Kruskal-Wallis and t-test (&alpha;=0.05), the bond strength were evaluated by two-way ANOVA (cements and root thirds) and Tukey test (&alpha;=0.05). Statistical analyses were set at 5% significance level. Regarding the surface of the wear profile, it was found that there was no significant statistical difference between sealers (p=0.6190), but all samples showed wear of dentine and filling material after exposure to the oral environment (p <0.05). Roots filled with AH Plus sealer showed the higher bond strength to dentin (11.40 ± 7.74 a) (p<0.05). Intermediate results were found in roots with MTA Fillapex (7.22 ± 5.88 ab) and Endofill (7.37 ± 6.75 ab). The worst result was found in roots with Sealapex (5.18 ± 4.34 b). There were no significant differences in root thirds, neither in the interaction of factors (p>0.05). Adhesive failure were predominant in root canals with AH Plus, MTA Fillapex and Endofill (respectively, 66%, 75% e 54.2%). Root canals with Sealapex presented more mixed failure (54.2%). Qualitative morphological analysis showed that all sealers presented dentin demineralization around root canal filling, being greater when using to Sealapex and Endofill sealers. In unfilled roots, there was intense accumulation of bacterial biofilm and demineralization of intraradicular dentin. After the exposure of roots to oral environment for 14 days, it may be concluded that no sealer was able to prevent degradation of the adhesive interface and dentin. However, in these situations of high acid challenge, AH Plus and MTA Fillapex sealers have shown superior performance than other tested sealers for their high adhesive strength of the filling material to dentin, and less intense degradation and demineralization around the root canal filling.

The aim of this in vitro study was to analyse the performance of CAD/CAM resin-based composites for the fabrication of long-term temporary fixed dental prostheses (FDP) and to compare it to other commercially available alternative materials regarding its long-term stability. Four CAD/CAM materials [Structur CAD (SC), VITA CAD-Temp (CT), Grandio disc (GD), and Lava Esthetic (LE)] and two direct RBCs [(Structur 3 (S3) and LuxaCrown (LC)] were used to fabricate three-unit FDPs. 10/20 FDPs were subjected to thermal cycling and mechanical loading by chewing simulation and 10/20 FDPs were stored in distilled water. Two FDPs of each material were forwarded to additional image diagnostics prior and after chewing simulation. Fracture loads were measured and data were statistically analysed. SC is suitable for use as a long-term temporary (two years) three-unit FDP. In comparison to CT, SC featured significantly higher breaking forces (SC > 800 N; CT < 600 N) and the surface wear of the antagonists was (significantly) lower and the abrasion of the FDP was similar. The high breaking forces (1100–1327 N) of GD and the small difference compared to LE regarding flexural strength showed that the material might be used for the fabrication of three-unit FDPs. With the exception of S3, all analysed direct or indirect materials are suitable for the fabrication of temporary FDPs.

Un papier bioactif est obtenu par la modification d’un papier en y immobilisant une ou plusieurs biomolécules. La recherche et le développement de papiers bioactifs est en plein essor car le papier est un substrat peu dispendieux qui est déjà d’usage très répandu à travers le monde. Bien que les papiers bioactifs n’aient pas connus de succès commercial depuis la mise en marche de bandelettes mesurant le taux de glucose dans les années cinquante, de nombreux groupes de recherche travaillent à immobiliser des biomolécules sur le papier pour obtenir un papier bioactif qui est abordable et possède une bonne durée de vie. Contrairement à la glucose oxidase, l’enzyme utilisée sur ces bandelettes, la majorité des biomolécules sont très fragiles et perdent leur activité très rapidement lorsqu’immobilisées sur des papiers. Le développement de nouveaux papiers bioactifs pouvant détecter des substances d’intérêt ou même désactiver des pathogènes dépend donc de découverte de nouvelles techniques d’immobilisation des biomolécules permettant de maintenir leur activité tout en étant applicable dans la chaîne de production actuelle des papiers fins. Le but de cette thèse est de développer une technique d’immobilisation efficace et versatile, permettant de protéger l’activité de biomolécules incorporées sur des papiers. La microencapsulation a été choisie comme technique d’immobilisation car elle permet d’enfermer de grandes quantités de biomolécules à l’intérieur d’une sphère poreuse permettant leur protection. Pour cette étude, le polymère poly(éthylènediimine) a été choisi afin de générer la paroi des microcapsules. Les enzymes laccase et glucose oxidase, dont les propriétés sont bien établies, seront utilisées comme biomolécules test. Dans un premier temps, deux procédures d’encapsulation ont été développées puis étudiées. La méthode par émulsion produit des microcapsules de plus petits diamètres que la méthode par encapsulation utilisant un encapsulateur, bien que cette dernière offre une meilleure efficacité d’encapsulation. Par la suite, l’effet de la procédure d’encapsulation sur l’activité enzymatique et la stabilité thermique des enzymes a été étudié à cause de l’importance du maintien de l’activité sur le développement d’une plateforme d’immobilisation. L’effet de la nature du polymère utilisé pour la fabrication des capsules sur la conformation de l’enzyme a été étudié pour la première fois. Finalement, l’applicabilité des microcapsules de poly(éthylèneimine) dans la confection de papiers bioactifs a été démontré par le biais de trois prototypes. Un papier réagissant au glucose a été obtenu en immobilisant des microcapsules contenant l’enzyme glucose oxidase. Un papier sensible à l’enzyme neuraminidase pour la détection de la vaginose bactérienne avec une plus grande stabilité durant l’entreposage a été fait en encapsulant les réactifs colorimétriques dans des capsules de poly(éthylèneimine). L’utilisation de microcapsules pour l’immobilisation d’anticorps a également été étudiée. Les avancées au niveau de la plateforme d’immobilisation de biomolécules par microencapsulation qui ont été réalisées lors de cette thèse permettront de mieux comprendre l’effet des réactifs impliqués dans la procédure de microencapsulation sur la stabilité, l’activité et la conformation des biomolécules. Les résultats obtenus démontrent que la plateforme d’immobilisation développée peut être appliquée pour la confection de nouveaux papiers bioactifs. / Biosensing paper attracts increasing attention due to its benefits of being simple, visible, portable and useful for detecting various contaminants, pathogens and toxins. While there has been no bioactive paper commercialized since glucose paper strips developed in the fifties, many research groups are working to immobilize biomolecules on paper to achieve a bioactive paper that is affordable and has good shelf life. The goal of this research is to develop some highly useful bioactive paper that could, for example, measure blood glucose, or immediately detect and simultaneously deactivate pathogens such as neuraminidase and E.coli. Previously, bioactive paper was produced either through physically absorbing biorecognition elements or printing bio-ink onto paper substrate. Our methodology for fabrication of bioactive paper strips is compatible with existing paper making process and includes three procedures: the fabrication of microcapsules, enzyme or antibody microencapsulation, immobilization of enzymes or antibody-entrapped microcapsules into paper pulp. The first step, in fabricating of bioactive paper strips is to produce biocompatible and inexpensive microcapsules with suitable parameters. To do so, two types of microencapsulation methods were compared; the emulsion method and the vibration nozzle method accomplished with an encapsulator. The parameters for producing optimal microcapsules with both methods were studied. Factors that affect their diameter, wall thickness, shell pore size, encapsulation efficiency and membrane compositions were also discussed. By comparison, microcapsules prepared with poly(ethyleneimine) (PEI) by the emulsion method exhibit properties that were more suitable for enzyme encapsulation and paper making process, whereas the microcapsules prepared by the vibration nozzle method were too big to be immobilized within paper pulp, and had lower encapsulation efficiency, enzymatic activity and productivity. Thus the emulsion method was chosen for subsequent experiments such as enzyme and antibody microencapsulation and bacterial vaginosis (BV) paper preparation. Microcapsules made by the emulsion method were semi-permeable in that the diffusion of substrate and product molecules were allowed freely across the membranes but the encapsulated enzymes would be retained inside. Glucose oxidase from Aspergillus niger (GOx) and laccase from Trametes versicolor (TvL) microcapsules showed high encapsulation efficiency, but the encapsulation process caused a severe decrease in the specific activities of both enzymes. Results from circular dichroism (CD) studies, fluorescence properties, enzymatic activities of free enzymes and Michaelis-Menten behavior demonstrated that the Vmax decrease for GOx was due to the restriction of diffusion across microcapsule membranes with pore size less than 5 nm. The microencapsulation process improved the thermal stability of GOx but decreased that of laccase. Bioactive papers were fabricated either by incorporating microcapsules containing different enzymes or empty microcapsules soaked in substrate and enhancer solution into the paper pulp during the sheet making process. Both the GOx and the BV paper strips underwent a color change in the presence of glucose and potassium iodide, and sialidase from Clostridium perfringens respectively. Some preliminary studies on antibody sensitized microcapsules, in which antibody was either encapsulated within the PEI microcapsules or conjugated to its membranes, were also performed. Our objective was to establish an enzyme immobilization platform based on microencapsulation techniques for paper based biosensors. Even though our current studies only focused on the microencapsulation of two enzymes, TvL and GOx, as well as the bioactive paper preparation, a similar approach can be applied to other enzymes. We believe that this immobilization method can potentially be employed for bioactive paper preparation on an industrial scale.

La profondeur possible d’imagerie en laser-scanning microscopie est limitée non seulement par la distance de travail des lentilles de objectifs mais également par la dégradation de l’image causée par une atténuation et une diffraction de la lumière passant à travers l’échantillon. Afin d’étendre cette limite, il est possible, soit de retourner le spécimen pour enregistrer les images depuis chaque côté, or couper progressivement la partie supérieure de l’échantillon au fur et à mesure de l‘acquisition. Les différentes images prises de l’une de ces manières doivent ensuite être combinées pour générer un volume unique. Cependant, des mouvements de l’échantillon durant les procédures d’acquisition engendrent un décalage non seulement sur en translation selon les axes x, y et z mais également en rotation autour de ces même axes, rendant la fusion entres ces multiples images difficile. Nous avons développé une nouvelle approche appelée 2D-SIFT-in-3D-Space utilisant les SIFT (scale Invariant Feature Transform) pour atteindre un recalage robuste en trois dimensions de deux images. Notre méthode recale les images en corrigeant séparément les translations et rotations sur les trois axes grâce à l’extraction et l’association de caractéristiques stables de leurs coupes transversales bidimensionnelles. Pour évaluer la qualité du recalage, nous avons également développé un simulateur d’images de laser-scanning microscopie qui génère une paire d’images 3D virtuelle dans laquelle le niveau de bruit et les angles de rotations entre les angles de rotation sont contrôlés avec des paramètres connus. Pour une concaténation précise et naturelle de deux images, nous avons également développé un module permettant une compensation progressive de la luminosité et du contraste en fonction de la distance à la surface de l’échantillon. Ces outils ont été utilisés avec succès pour l’obtention d’images tridimensionnelles de haute résolution du cerveau de la mouche Drosophila melanogaster, particulièrement des neurones dopaminergiques, octopaminergiques et de leurs synapses. Ces neurones monoamines sont particulièrement important pour le fonctionnement du cerveau et une étude de leur réseau et connectivité est nécessaire pour comprendre leurs interactions. Si une évolution de leur connectivité au cours du temps n’a pas pu être démontrée via l’analyse de la répartition des sites synaptiques, l’étude suggère cependant que l’inactivation de l’un de ces types de neurones entraine des changements drastiques dans le réseau neuronal. / Although laser scanning microscopy is a powerful tool for obtaining thin optical sections, the possible depth of imaging is limited by the working distance of the microscope objective but also by the image degradation caused by the attenuation of both excitation laser beam and the light emitted from the fluorescence-labeled objects. Several workaround techniques have been employed to overcome this problem, such as recording the images from both sides of the sample, or by progressively cutting off the sample surface. The different views must then be combined in a unique volume. However, a straightforward concatenation is often not possible, because the small rotations that occur during the acquisition procedure, not only in translation along x, y and z axes but also in rotation around those axis, making the fusion uneasy. To address this problem we implemented a new algorithm called 2D-SIFT-in-3D-Space using SIFT (scale Invariant Feature Transform) to achieve a robust registration of big image stacks. Our method register the images fixing separately rotations and translations around the three axes using the extraction and matching of stable features in 2D cross-sections. In order to evaluate the registration quality, we created a simulator that generates artificial images that mimic laser scanning image stacks to make a mock pair of image stacks one of which is made from the same stack with the other but is rotated arbitrarily with known angles and filtered with a known noise. For a precise and natural-looking concatenation of the two images, we also developed a module progressively correcting the sample brightness and contrast depending on the sample surface. Those tools we successfully used to generate tridimensional high resolution images of the fly Drosophila melanogaster brain, in particular, its octopaminergic and dopaminergic neurons and their synapses. Those monoamine neurons appear to be determinant in the correct operating of the central nervous system and a precise and systematic analysis of their evolution and interaction is necessary to understand its mechanisms. If an evolution over time could not be highlighted through the pre-synaptic sites analysis, our study suggests however that the inactivation of one of these neuron types triggers drastic changes in the neural network.