Les biofilms sont des communautés de microorganismes incorporés dans une matrice exo-polymérique complexe. Ils sont reconnus pour jouer un rôle important comme barrière de diffusion dans les systèmes environnementaux et la santé humaine, donnant lieu à une résistance accrue aux antibiotiques et aux désinfectants. Comme le transfert de masse dans un biofilm est principalement dû à la diffusion moléculaire, il est primordial de comprendre les principaux paramètres influençant les flux de diffusion. Dans ce travail, nous avons étudié un biofilm de Pseudomonas fluorescens et deux hydrogels modèles (agarose et alginate) pour lesquels l’autodiffusion (mouvement Brownien) et les coefficients de diffusion mutuels ont été quantifiés. La spectroscopie par corrélation de fluorescence a été utilisée pour mesurer les coefficients d'autodiffusion dans une volume confocal de ca. 1 m3 dans les gels ou les biofilms, tandis que les mesures de diffusion mutuelle ont été faites par cellule de diffusion. En outre, la voltamétrie sur microélectrode a été utilisée pour évaluer le potentiel de Donnan des gels afin de déterminer son impact sur la diffusion. Pour l'hydrogel d'agarose, les observations combinées d'une diminution du coefficient d’autodiffusion et de l’augmentation de la diffusion mutuelle pour une force ionique décroissante ont été attribuées au potentiel de Donnan du gel. Des mesures de l'effet Donnan (différence de -30 mV entre des forces ioniques de 10-4 et 10-1 M) et l'accumulation correspondante d’ions dans l'hydrogel (augmentation d’un facteur de 13 par rapport à la solution) ont indiqué que les interactions électrostatiques peuvent fortement influencer le flux de diffusion de cations, même dans un hydrogel faiblement chargé tel que l'agarose. Curieusement, pour un gel plus chargé comme l'alginate de calcium, la variation de la force ionique et du pH n'a donné lieu qu'à de légères variations de la diffusion de sondes chargées dans l'hydrogel. Ces résultats suggèrent qu’en influençant la diffusion du soluté, l'effet direct des cations sur la structure du gel (compression et/ou gonflement induits) était beaucoup plus efficace que l'effet Donnan. De même, pour un biofilm bactérien, les coefficients d'autodiffusion étaient pratiquement constants sur toute une gamme de force ionique (10-4-10-1 M), aussi bien pour des petits solutés chargés négativement ou positivement (le rapport du coefficient d’autodiffusion dans biofilm sur celui dans la solution, Db/Dw ≈ 85 %) que pour des nanoparticules (Db/Dw≈ 50 %), suggérant que l'effet d'obstruction des biofilms l’emporte sur l'effet de charge. Les résultats de cette étude ont montré que parmi les divers facteurs majeurs qui affectent la diffusion dans un biofilm environnemental oligotrophe (exclusion stérique, interactions électrostatiques et hydrophobes), les effets d'obstruction semblent être les plus importants lorsque l'on tente de comprendre la diffusion du soluté. Alors que les effets de charge ne semblaient pas être importants pour l'autodiffusion de substrats chargés dans l'hydrogel d'alginate ou dans le biofilm bactérien, ils ont joué un rôle clé dans la compréhension de la diffusion à travers l’agarose. L’ensemble de ces résultats devraient être très utiles pour l'évaluation de la biodisponibilité des contaminants traces et des nanoparticules dans l'environnement. / Biofilms are primarily communities of microorganisms embedded in a complex exopolymer matrix. They are thought to play an important role as diffusive barriers in environmental systems and human health, resulting in increased resistance to disinfectants and antibiotics. Since mass transport in a biofilm is primarily due to molecular diffusion, it is critical to understand the main parameters influencing diffusive fluxes in a biofilm. In this thesis, a Pseudomonas fluorescens biofilm and two model hydrogels, (agarose and calcium alginate), were investigated. Both self-diffusion (Brownian motion) and mutual diffusion coefficients were quantified. Fluorescence correlation spectroscopy was used to measure the self-diffusion coefficients in a ca. 1 m3 confocal volume in the gels or biofilms, whereas a diffusion cell setup was employed for mutual diffusion measurements. In addition, microelectrode voltammetry was used to evaluate Donnan potential of the gels in order to determine its impact on diffusion. For the agarose hydrogel, the combined observations of a decreasing self-diffusion coefficient coupled with increasing mutual diffusion as a function of a decreasing ionic strength have been attributed to the gel’s Donnan potential. Measurements of the Donnan effect (difference of -30 mV between ionic strengths of 10-4 and 10-1 M) and the corresponding accumulation of ions in the hydrogel (13x enhancement with respect to the bulk solution) indicated that electrostatic interactions can strongly influence the diffusive flux of cations, even in a weakly charged hydrogel, such as agarose. Somewhat surprisingly, for a more highly charged gel such as calcium alginate, varying ionic strength and pH resulted in only small changes to the diffusion of charged probes in the hydrogel. These results suggested that the direct effect of the cations on gel structure (due to an induced swelling or compression) was much more effective than the Donnan effect when influencing solute diffusion. Similarly, for a bacterial biofilm, self-diffusion coefficients were virtually constant across a range of examined ionic strengths (10-4-10-1 M) for both negatively and positively charged small solutes (Db/Dw≈85%) and nanoparticles (Db/Dw≈50%), suggesting that the obstruction effect of the biofilms again overwhelmed the charge effect. The results of this work indicated that among the various major factors affecting diffusion in an oligotrophic environmental biofilm (steric exclusion, hydrophobic and electrostatic interactions), obstruction effects appeared to be the most important when attempting to understand the solute diffusion. While charge effects did not appear to be important to the self-diffusion of charged substrates in the alginate hydrogel or bacterial biofilm, they were key to understanding diffusion through another gel, with numerous biomedical and environmental applications, i.e. agarose. These results should be extremely useful when evaluating the bioavailability of the trace contaminants and nanoparticles in the environment.

Os amarelos estão associados a fitoplasmas, procariotos pertencentes à classe Mollicutes que não possuem parede celular e habitam o floema de plantas. Os danos causados pelas doenças de etiologia fitoplasmática são relevantes e podem ocorrer em diversas espécies economicamente importantes. Em videira, pesquisas realizadas em várias partes do mundo têm relatado a presença das doenças do tipo "amarelo", porém, no Brasil, estas doenças ainda não foram relatadas para esta cultura. Em vinhedos comerciais localizados nos Estados de São Paulo e Paraná têm sido observadas plantas com sintomas semelhantes àqueles provocados por fitoplasmas em outros países. Estes sintomas têm sido caracterizados por amarelecimento e ou avermelhamento foliar, necrose do limbo e rachaduras nas nervuras principais. Com o objetivo de detectar e identificar molecularmente fitoplasmas associados a estes tipos de sintomas, folhas e ramos foram amostrados a partir de plantas sintomática e assintomáticas. A detecção foi conduzida com PCR duplo usando-se os iniciadores R16 mF1/mR2 ou P1/P7 na primeira reação e R16 F2n/R2 na segunda reação. A identificação foi realizada através de PCR duplo com iniciadores específicos e análises de RFLP com as enzimas de restrição AluI, RsaI, KpnI, MseI, HhaI, HpaII, HinfI e MboI. Em 23 plantas amostradas, fitoplasmas foram detectados em 10 delas, através da amplificação do 16S rDNA, visualizado em gel de agarose na forma de bandas de 1,2Kb. A identificação por PCR demonstrou que os fitoplasmas associados ao amarelo da videira pertenciam aos grupos 16SrI e 16SrIII. As análises dos perfis eletroforéticos obtidos com o uso da técnica de RFLP revelaram a presença de fitoplasmas afiliados ao subgrupo 16SrI-B. A constatação de fitoplasmas pertencentes a estes dois grupos nas plantas amostradas demonstraram a ocorrência da doença conhecida como amarelo da videira nos Estados de São Paulo e Paraná. As pesquisas desenvolvidas neste trabalho vêm contribuir para aumentar os conhecimentos sobre o papel e a diversidade dos fitoplasmas no agroecossistema brasileiro. / Yellows diseases are associated with phytoplasmas, wall-less prokaryotes, inhabitant of phloem vessels. Damage caused by these diseases are relevant for some important cultivated botanical species. Grapevine yellows diseases have been observed in several areas of the world, but in Brazil the presence of these diseases had not been reported yet. In vineyards located in São Paulo and Paraná States, plants exhibiting symptoms similar those observed in grapevines from other countries have been observed. The symptoms were characterized by yellowing or redding of leaf blade and ribs, leaf blade necrosis and main ribs fissures. In order to detect and identify phytoplasmas associated with those kind of symptoms, leaves and stems were sampled from symptomatic and asymptomatic plants. The phytoplasma detection was conducted with nested PCR using the primer pairs R16mF1/mR2 or P1/P7 for first reaction and 16 F2n/R2 for second reaction. The identification was carried out by nested PCR with group-specifc primer pairs and RFLP analyses with enzymes AluI, RsaI, KpnI, MseI, HhaI, HpaII, HinfI and MboI. From a total of 23 samples analysed, phytoplasmas were detected in 10 of them, through amplification of the 16S rDNA, visualized through a 1.2Kb band in agarose gel. The identification by PCR demonstrated that phytoplasmas associated with grapevine yellow belong to 16SrI and 16SrIII groups. Analyses of electrophoretic profiles revealed the presence of phytoplasmas affiliated to 16SrI-B subgroup. The presence of phytoplasmas belonging to these two groups in the sampled plants demonstrated the occurrence of yellow disease in grapevine in São Paulo and Paraná States. The investigation conducted in the present work contributed to the knowledgement of the role and the diversity of phytoplasmas in Brasilian ecosystem.

La thérapie des tumeurs cérébrales invasives demeure aujourd'hui un véritable challenge en dépit des progrès réalisés en neurochirurgie, radiothérapie et chimiothérapie ces vingt dernières années. Les tumeurs solides sont des systèmes dynamiques présentant une forte hétérogénéité de structure et de vascularisation et qui varie d'une tumeur à une autre. La stratégie de délivrance des drogues dans ces tissus néoplasiques (cérébraux ou non) s'est révélée être un point critique dans le succès d'une chimiothérapie, notamment en raison de l'existence de mécanismes physiologiques intratumoraux s'opposant à une délivrance efficace dans l'ensemble du tissu tumoral. L'administration locale convective (Convection-Enhanced Delivery, CED) s'est avérée être une technique prometteuse pour distribuer par voie intracérébrale des volumes cliniquement significatifs d'agents thérapeutiques à des concentrations interstitielles élevées tout en réduisant les risques de toxicité systémique. Ce travail a porté sur l'étude de la distribution CED de traceurs in vitro pendant injection et in vivo en post-injection par deux modalités d'imagerie (imagerie par RMN et tomographie quantitative par rayonnement synchrotron). Les données in vitro ont été corrélées à un modèle numérique d'écoulement et de transport du traceur. Finalement, ces résultats sont discutés à la lumière des éléments prédictifs de la littérature. A ce jour, la compréhension et l'optimisation de la distribution CED de drogues in vivo pendant et après infusion restent une étape critique dans le traitement des tumeurs cérébrales et nécessitent davantage d'efforts.

<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.

Les biofilms sont des communautés de microorganismes incorporés dans une matrice exo-polymérique complexe. Ils sont reconnus pour jouer un rôle important comme barrière de diffusion dans les systèmes environnementaux et la santé humaine, donnant lieu à une résistance accrue aux antibiotiques et aux désinfectants. Comme le transfert de masse dans un biofilm est principalement dû à la diffusion moléculaire, il est primordial de comprendre les principaux paramètres influençant les flux de diffusion. Dans ce travail, nous avons étudié un biofilm de Pseudomonas fluorescens et deux hydrogels modèles (agarose et alginate) pour lesquels l’autodiffusion (mouvement Brownien) et les coefficients de diffusion mutuels ont été quantifiés. La spectroscopie par corrélation de fluorescence a été utilisée pour mesurer les coefficients d'autodiffusion dans une volume confocal de ca. 1 m3 dans les gels ou les biofilms, tandis que les mesures de diffusion mutuelle ont été faites par cellule de diffusion. En outre, la voltamétrie sur microélectrode a été utilisée pour évaluer le potentiel de Donnan des gels afin de déterminer son impact sur la diffusion. Pour l'hydrogel d'agarose, les observations combinées d'une diminution du coefficient d’autodiffusion et de l’augmentation de la diffusion mutuelle pour une force ionique décroissante ont été attribuées au potentiel de Donnan du gel. Des mesures de l'effet Donnan (différence de -30 mV entre des forces ioniques de 10-4 et 10-1 M) et l'accumulation correspondante d’ions dans l'hydrogel (augmentation d’un facteur de 13 par rapport à la solution) ont indiqué que les interactions électrostatiques peuvent fortement influencer le flux de diffusion de cations, même dans un hydrogel faiblement chargé tel que l'agarose. Curieusement, pour un gel plus chargé comme l'alginate de calcium, la variation de la force ionique et du pH n'a donné lieu qu'à de légères variations de la diffusion de sondes chargées dans l'hydrogel. Ces résultats suggèrent qu’en influençant la diffusion du soluté, l'effet direct des cations sur la structure du gel (compression et/ou gonflement induits) était beaucoup plus efficace que l'effet Donnan. De même, pour un biofilm bactérien, les coefficients d'autodiffusion étaient pratiquement constants sur toute une gamme de force ionique (10-4-10-1 M), aussi bien pour des petits solutés chargés négativement ou positivement (le rapport du coefficient d’autodiffusion dans biofilm sur celui dans la solution, Db/Dw ≈ 85 %) que pour des nanoparticules (Db/Dw≈ 50 %), suggérant que l'effet d'obstruction des biofilms l’emporte sur l'effet de charge. Les résultats de cette étude ont montré que parmi les divers facteurs majeurs qui affectent la diffusion dans un biofilm environnemental oligotrophe (exclusion stérique, interactions électrostatiques et hydrophobes), les effets d'obstruction semblent être les plus importants lorsque l'on tente de comprendre la diffusion du soluté. Alors que les effets de charge ne semblaient pas être importants pour l'autodiffusion de substrats chargés dans l'hydrogel d'alginate ou dans le biofilm bactérien, ils ont joué un rôle clé dans la compréhension de la diffusion à travers l’agarose. L’ensemble de ces résultats devraient être très utiles pour l'évaluation de la biodisponibilité des contaminants traces et des nanoparticules dans l'environnement. / Biofilms are primarily communities of microorganisms embedded in a complex exopolymer matrix. They are thought to play an important role as diffusive barriers in environmental systems and human health, resulting in increased resistance to disinfectants and antibiotics. Since mass transport in a biofilm is primarily due to molecular diffusion, it is critical to understand the main parameters influencing diffusive fluxes in a biofilm. In this thesis, a Pseudomonas fluorescens biofilm and two model hydrogels, (agarose and calcium alginate), were investigated. Both self-diffusion (Brownian motion) and mutual diffusion coefficients were quantified. Fluorescence correlation spectroscopy was used to measure the self-diffusion coefficients in a ca. 1 m3 confocal volume in the gels or biofilms, whereas a diffusion cell setup was employed for mutual diffusion measurements. In addition, microelectrode voltammetry was used to evaluate Donnan potential of the gels in order to determine its impact on diffusion. For the agarose hydrogel, the combined observations of a decreasing self-diffusion coefficient coupled with increasing mutual diffusion as a function of a decreasing ionic strength have been attributed to the gel’s Donnan potential. Measurements of the Donnan effect (difference of -30 mV between ionic strengths of 10-4 and 10-1 M) and the corresponding accumulation of ions in the hydrogel (13x enhancement with respect to the bulk solution) indicated that electrostatic interactions can strongly influence the diffusive flux of cations, even in a weakly charged hydrogel, such as agarose. Somewhat surprisingly, for a more highly charged gel such as calcium alginate, varying ionic strength and pH resulted in only small changes to the diffusion of charged probes in the hydrogel. These results suggested that the direct effect of the cations on gel structure (due to an induced swelling or compression) was much more effective than the Donnan effect when influencing solute diffusion. Similarly, for a bacterial biofilm, self-diffusion coefficients were virtually constant across a range of examined ionic strengths (10-4-10-1 M) for both negatively and positively charged small solutes (Db/Dw≈85%) and nanoparticles (Db/Dw≈50%), suggesting that the obstruction effect of the biofilms again overwhelmed the charge effect. The results of this work indicated that among the various major factors affecting diffusion in an oligotrophic environmental biofilm (steric exclusion, hydrophobic and electrostatic interactions), obstruction effects appeared to be the most important when attempting to understand the solute diffusion. While charge effects did not appear to be important to the self-diffusion of charged substrates in the alginate hydrogel or bacterial biofilm, they were key to understanding diffusion through another gel, with numerous biomedical and environmental applications, i.e. agarose. These results should be extremely useful when evaluating the bioavailability of the trace contaminants and nanoparticles in the environment.

The aim of this work was a research of the genetic variability of natural populations of Russian wheat aphid Diuraphis noxia (Aphididae) by means of microsatellite markers and markers for EPIC-PCR. First goal was to introduce the methods and optimise them for Diuraphis noxia. In the follow-up pilot study, specimens from 47 lines representing 12 populations from all over the world were analysed. Having used microsatellite markers, I proved expected variability among individual populations and within them. The highest genetic variability was detected between Chile and Algeria using markers for cytochrome C in EPIC-PCR. These findings can be used for further studies of the genetic variability of the aphid Diuraphis noxia.

Os amarelos estão associados a fitoplasmas, procariotos pertencentes à classe Mollicutes que não possuem parede celular e habitam o floema de plantas. Os danos causados pelas doenças de etiologia fitoplasmática são relevantes e podem ocorrer em diversas espécies economicamente importantes. Em videira, pesquisas realizadas em várias partes do mundo têm relatado a presença das doenças do tipo “amarelo”, porém, no Brasil, estas doenças ainda não foram relatadas para esta cultura. Em vinhedos comerciais localizados nos Estados de São Paulo e Paraná têm sido observadas plantas com sintomas semelhantes àqueles provocados por fitoplasmas em outros países. Estes sintomas têm sido caracterizados por amarelecimento e ou avermelhamento foliar, necrose do limbo e rachaduras nas nervuras principais. Com o objetivo de detectar e identificar molecularmente fitoplasmas associados a estes tipos de sintomas, folhas e ramos foram amostrados a partir de plantas sintomática e assintomáticas. A detecção foi conduzida com PCR duplo usando-se os iniciadores R16 mF1/mR2 ou P1/P7 na primeira reação e R16 F2n/R2 na segunda reação. A identificação foi realizada através de PCR duplo com iniciadores específicos e análises de RFLP com as enzimas de restrição AluI, RsaI, KpnI, MseI, HhaI, HpaII, HinfI e MboI. Em 23 plantas amostradas, fitoplasmas foram detectados em 10 delas, através da amplificação do 16S rDNA, visualizado em gel de agarose na forma de bandas de 1,2Kb. A identificação por PCR demonstrou que os fitoplasmas associados ao amarelo da videira pertenciam aos grupos 16SrI e 16SrIII. As análises dos perfis eletroforéticos obtidos com o uso da técnica de RFLP revelaram a presença de fitoplasmas afiliados ao subgrupo 16SrI-B. A constatação de fitoplasmas pertencentes a estes dois grupos nas plantas amostradas demonstraram a ocorrência da doença conhecida como amarelo da videira nos Estados de São Paulo e Paraná. As pesquisas desenvolvidas neste trabalho vêm contribuir para aumentar os conhecimentos sobre o papel e a diversidade dos fitoplasmas no agroecossistema brasileiro. / Yellows diseases are associated with phytoplasmas, wall-less prokaryotes, inhabitant of phloem vessels. Damage caused by these diseases are relevant for some important cultivated botanical species. Grapevine yellows diseases have been observed in several areas of the world, but in Brazil the presence of these diseases had not been reported yet. In vineyards located in São Paulo and Paraná States, plants exhibiting symptoms similar those observed in grapevines from other countries have been observed. The symptoms were characterized by yellowing or redding of leaf blade and ribs, leaf blade necrosis and main ribs fissures. In order to detect and identify phytoplasmas associated with those kind of symptoms, leaves and stems were sampled from symptomatic and asymptomatic plants. The phytoplasma detection was conducted with nested PCR using the primer pairs R16mF1/mR2 or P1/P7 for first reaction and 16 F2n/R2 for second reaction. The identification was carried out by nested PCR with group-specifc primer pairs and RFLP analyses with enzymes AluI, RsaI, KpnI, MseI, HhaI, HpaII, HinfI and MboI. From a total of 23 samples analysed, phytoplasmas were detected in 10 of them, through amplification of the 16S rDNA, visualized through a 1.2Kb band in agarose gel. The identification by PCR demonstrated that phytoplasmas associated with grapevine yellow belong to 16SrI and 16SrIII groups. Analyses of electrophoretic profiles revealed the presence of phytoplasmas affiliated to 16SrI-B subgroup. The presence of phytoplasmas belonging to these two groups in the sampled plants demonstrated the occurrence of yellow disease in grapevine in São Paulo and Paraná States. The investigation conducted in the present work contributed to the knowledgement of the role and the diversity of phytoplasmas in Brasilian ecosystem.

In our work we have studied the ultrasonic imaging systems and their use in ophthalmology, especially with the device Nidek 4000. We described ophthalmological examination methods. In addition, we are using the simulation program Field II. It simulated eye phantom and created his B-scan and biometry, where we compared the effects of different central frequency ultrasonic probes and different speeds of sound in the resulting values. We also created phantoms using agarose gel and materials of different properties. On phantoms, we studied the effect of the velocity of ultrasound in measurement results, effect of the concentration of the agarose gel to the velocity of sound. And we created phantoms simulating the human eye. Measurement protocol was created for use in teaching.

This master thesis is engaged in problematics of creation and compensation of geometric distortions in 1D agarose electrophoresis. This master thesis analyze the problematics of cause of these distortions and summarize the theory needed for compensation of these distortions. Based on acquired theory and created electrophoretic phantoms, the master thesis contains several suggestions for compensation of incurred distortions. These suggestions are recreated into functions, which are connected into a functional user interface for gel image analysis and geometric distortions compensation.