Au cours de ce travail de thèse expérimental, nous nous sommes intéressés à l’étude de la localisation nucléaire et nucléolaire de la protéine de la nucléocapside (NC) du VIH-1. Des études antérieures menées au laboratoire avaient mis en évidence une très forte accumulation de la NC dans les nucléoles. Ce compartiment nucléaire est connu pour être ciblé par de nombreux virus afin de promouvoir leur réplication. Des expériences de microscopie électronique avaient révélé la structure complexe du nucléole et montré qu’il est composé de trois sous-compartiments : les centres fibrillaires, le compartiment fibrillaire dense et le compartiment granulaire dans lesquels se déroule la synthèse des ribosomes. Afin de caractériser la localisation de la NC dans ces trois sous-compartiments, nous avons développé une approche de microscopie optique à haute résolution permettant d’obtenir des images à deux couleurs avec une résolution spatiale améliorée. Pour cela, nous avons mis au point un protocole qui permet d’utiliser simultanément une protéine fluorescente photocommutable et un fluorophore organique introduit par immunomarquage. Après avoir minimisé les aberrations optiques et corrigé les dérives mécaniques inhérentes au montage, nous avons visualisé simultanément la localisation de la NC surexprimée dans des cellules HeLa avec des marqueurs spécifiques des trois sous-compartiments nucléolaires (immunomarquage). La microscopie de fluorescence à haute résolution a permis de résoudre pour la première fois les différents compartiments et de montrer que la NC se localise préférentiellement dans le compartiment granulaire. Finalement, des expériences préliminaires avec des cellules vivantes ont permis de mettre en évidence que la NC est transportée de manière active dans le noyau et qu’elle pourrait interagir directement avec des protéines nucléolaires / During this experimental thesis work, we investigated the nuclear and nucleolar localization of the nucleocapsid protein (NC) of HIV-1. Previous studies performed in our laboratory evidenced a strong accumulation of NC in a subnuclear structure called nucleolus. Playing role in multiple cellular processes, nucleolus is often targeted by viruses to promote their replication. Electron microscopy revealed three nucleolar components (fibrillar centers, dense fibrillar component and granular component) associated to specific steps of the ribosome biogenesis. To characterize the distribution of the NC in these three sub-compartments and therefore shed light on the nucleolar localization of NC during the replication cycle, we developed a high-resolution optical microscopy approach. After having minimized the optical aberrations and corrected the mechanical drifts inherent to the imaging setup, the NC-mEos2 fusion protein overexpressed in HeLa cells was visualized simultaneously with immunolabeled nucleolar markers. The use of high-resolution fluorescence microscopy enabled us to resolve for the first time the three nucleolar compartments and to demonstrate the preferential localization of NC in the granular compartment of nucleolus. Finally, preliminary experiments performed with living cells showed that NC is actively transported in the nucleus and therefore may interact directly with nucleolar proteins.

Ce travail présente une étude, par microscopie optique et de fluorescence, de matériaux hybrides, basés sur des monolithes de silice contenant des dérivés du cyano-anthracène : 9,10-dicyano-anthracène (DCA) ou 9,14-dicyano-benzo(b)triphénylène (DBTP), qui sont des photo-sensibilisateurs de l'oxygène singulet. Alors que ces matériaux sont bien caractérisés du point de vue de la photo-oxydation des sulfures sous des conditions hétérogènes par des études macroscopiques, certaines propriétés concernant l'association du photosensibilisateur avec l'absorbant peuvent être masquées, dominées ou encore mal interprétées par uniquement des mesures d'ensemble. Ici, nous combinons la spectroscopie d'ensemble et la microscopie optique et de fluorescence, et développons des protocôles expérimentaux concernant des échantillons solides épais, dans le but d'étudier la distribution spatiale et la mobilité des photosensiblisateurs dans la matrice hôte ainsi que d'analyser les interactions entre ces deux entités. La microscopie optique montre dans tous les cas des inhomogénéités localisées à l'interface du monolithe et attribuées à la formation de bulles pendant la synthèse et une accumulation locale du DBTP. A partir de simulations Monte-Carlo de lancer de rayons, nous développons un protocôle pour corriger les artéfacts de réfraction dans les profils d'intensité de fluorescence en fonction de la profondeur, obtenus par des mesures confocales, pour déterminer la distribution axiale du photosensibilisateur ce qui permet de mettre en évidence une nette augmentation de la concentration en photosensibilisateurs dans les premiers 50—100 m dessous la surface. L'analyse FRAP montre la très lente mobilité de tous les photosensibilisateurs et un retour partiel de l'intensité ce qui signifie que les photosensibilisateurs se trouvent dans des régions compartimentées, probablement dues à des contraintes aléatoires du réseau de pores. De plus, l'analyse FLIM montrent des propriétés photo physiques semblables pour le DBTP inclus et greffé ce qui permet d'envisager l'inefficacité de la fonctionnalisation. Ces observations soulignent que les monolithes à base de silice sont des systèmes hors d'équilibre et correspondent à un instantané des inhomogénéités gelées pendant les derniers instants du processus de condensation-hydrolyse des monomères de silice. Enfin, corréler la spectroscopie classique avec nos observations confocales sur différentes formes de DBTP, nous permet d'établir que la bande d'émission de fluorescence non-structurée et fortement déplacée vers le rouge est probablement due à la formation d'excimères. / This work presents an optical and fluorescence microscopy study of hybrid materials based on porous silica monoliths containing derivatives of cyano-anthracene: 9,10-dicyano-anthracene (DCA) or 9,14-dicyano-benzo(b)triphenylene (DBTP), photo-sensitizers of singlet oxygen. While these materials are well known from bulk studies for the efficient photo-oxidation of sulphides under heterogeneous conditions, some characteristics of the association of the photo-sensitizer and the absorbent may be masked, overlooked or otherwise misinterpreted by bulk investigations alone. Here, we combine classical bulk spectroscopy with optical and fluorescence microscopy, and develop experimental protocols for thick solid state samples, to study the spatial distribution and the mobility of the guest in the host matrix, and analyse guest-host interactions. Optical microscopy shows in all cases localised inhomogeneities at monolith interface, ascribed to bubble formation during synthesis; wide-field fluorescence microscopy shows that these features are associated with local accumulation of the larger, more hydrophobic of the two photo-sensitizers, DBTP. Photo-sensitizer lateral distribution at the monolith interface is otherwise homogeneous. Based on Monte Carlo ray-tracing simulations, we develop a protocol for correcting refraction artefacts in measured confocal fluorescence depth profiles, to obtain the photo-sensitizer axial distribution. While it in general exhibits a sharp increase in concentration in the first 50—100 m below the surface compared to the bulk, this layer contributes negligibly to the total content of the monoliths. FRAP analysis shows mobility of the photo-sensitizers in all cases, but with diffusion constants implying months or years to equilibrate the centimetre-sized monoliths. Classical bulk and confocal spectroscopy with FLIM analysis show similar photo-physical properties of DBTP included and grafted. The main effects of funcionalization in this photo-sensitizer are to slow down diffusion and to counter its aggregation. Incomplete FRAP recovery implies photo-sensitizer mobility is compartmented, probably due to random constrictions in the pore network. These observations underline that silica-based monoliths are non-equilibrium systems encapsulating a snapshot of any homogeneities frozen in during the later stages of hydrolysis-condensation of silicate units. Correlating classical bulk spectroscopy with our confocal observations on the different DBTP forms, conclude that its unusual structureless, red-shifted emission is probably due to excimer emission.

Os micro-organismos constituem um grande problema em termos econômicos para a indústria petrolífera. Estes são responsáveis pela produção de substâncias corrosivas e a formação de biofilmes, que causam deterioração dos materiais metálicos. Os principais grupos microbianos presentes em amostras ambientais da indústria do petróleo são as bactérias anaeróbias heterotróficas totais (BANHT) e as bactérias redutoras de sulfato (BRS). Atualmente, a quantificação desses grupos microbianos é realizada através da técnica do Número Mais Provável (NMP) que estima o resultado em aproximadamente 28 dias. Neste trabalho foi otimizada uma metodologia para a microscopia de fluorescência de amostras salinas provenientes de tanques de armazenamento de água/óleo. As condições testadas foram o tipo de óleo de imersão, o tipo de diluente, o volume do corante, o volume da amostra corada e a concentração do fixador (glutaraldeído) numa tentativa de correlacionar com resultados de quantificação de BANHT e BRS através da técnica convencional do NMP. Nesse caso, as células totais foram quantificadas por microscopia de fluorescência utilizando o corante fluorescente laranja de acridina (AO). Verificou-se que houve uma correlação entre os resultados da quantificação de células totais por microscopia de fluorescência e os resultados de BANHT pela técnica do NMP, devido a pouca variação de valores expressos em ambas as quantificações. Entretanto, não foi possível correlacionar os resultados da quantificação de células totais com os resultados de BRS por NMP devido à grande variação dos valores de quantificação de BRS. Na microscopia de fluorescência, foi possível, quantificar os micro-organismos em aproximadamente 30 minutos e através das fotografias, verificou-se ainda que as amostras apresentaram-se nítidas e os micro-organismos com uma boa fluorescência / Microbial cells constitute a severe problem, from the economic point of view, for the petroleum industry. They are responsible for the production of corrosive metabolites and for the formation of biofilms, causing deterioration in the surface of metallic materials. The main microbial groups present in environmental samples from the petroleum industry include total anaerobic heterothrophic bacteria (TANHB) and sulfate-reducing bacteria (SRB). Nowadays, the quantification of those microbial groups is performed through the use of the most probable number technique (MPN), providing the final quantification after 28 days. In the present work a new methodology, based on fluorescence microscopy, was optimized on saline samples from water/oil storage tanks. The conditions tested were the type of immersion oil, type of diluent, the volume of the dye, the stained sample volume and concentration of fixative (glutaraldehyde) in order to quantify total cells, in an attempt to correlate with TANHB and SRB quantification through MPN. In that case, total cells were quantified with the help of acrydine orange as fluorescent dye. It could be observed a clear correlation between the results obtained for total cells quantification by fluorescence microscopy and the results obtained for TANHB through MPN technique, due to the negligible differences observed in both quantifications. However, when a correlation with SRB cells was tried results of total cells through fluorescence microscopy did not fit entirely. With the use of fluorescence microscopy, it was possible to quantify microbial cells in around 30 minutes and with the help of photographic reports obtained, it could be observed that the samples were clearly observed and the microbial cells indicated a good fluorescence

Entender os processos em superfície/interface de filmes e seus efeitos sobre as propriedades ópticas e elétricas de materiais orgânicos é de grande importância tecnológica. Esta pesquisa descreve a fabricação e caracterização de filmes poliméricos extremamente finos (espessura <10 nm) e homogêneos compostos por camadas de polímero/polieletrólitos e estruturas com modulação de energia ou poços quânticos. O objetivo principal foi o estudo dos processos de transferência de carga e energia em tais estruturas. Os polímeros luminescentes utilizados foram poli(9,9 dioctilfluoreno) (PFO) poli(p-fenileno vinileno (PPV). O PPV foi obtido a partir do precursor poli(cloreto de tetraidrotiofeno de xililideno) (PTHT). A técnica de deposição denominada deposição camada por camada assistida por spin (SA-LbL) foi utilizada para obtenção dos filmes. Medidas de absorbância confirmaram o crescimento linear das camadas para as interfaces polieletrólito/polieletrólito e polímero/polieletrólito. Com o objetivo de entender a transferência do elétron &pi; do polímero conjugado para o polieletrólito, as configurações das estruturas poliméricas foram alteradas através da deposição de diferentes monocamadas de polieletrólito sobre o filme polimérico. Observamos que os elétrons &pi; foram efetivamente transferidos para os polieletrólitos que possuem alta afinidades eletrônica. Este efeito interfere fortemente na absorção bem como nas características de condução do filme polimérico ultrafino. A absorção é restabelecida após a conversão de PTHT em PPV. Medidas de fotoluminescência (PL) em filmes PFO/PPV resultam em curvas de emissão com picos característicos de ambos os polímeros, o que confirma que a técnica SA-LbL permite a deposição de estruturas poliméricas multicamadas. As várias configurações de filmes obtidas elucidaram os processos de transferência que ocorrem em diferentes interfaces, tais como: mudança da sequencia de deposição do polieletrólito, número de camadas duplas PTHT/DBS e a introdução de camadas separadoras. Além disso, um único poço quântico, ou seja, estruturas formadas por uma camada PPV cercada por barreiras de PFO com 10 nm de espessura foram obtidas. Medidas de absorbâcia, PL e excitação mostraram uma eficiente migração estado excitado da barreira de PFO para o PPV (poço). A homogeneidade da imagem confocal, demonstrou um rigoroso controle da camada de cobertura ao nível de um única monocamada e sem contaminação pelos materiais depositados sequencialmente. A microscopia confocal de fluorescência (CFM) e espectroscopia de fluorescência resolvida no tempo (FLIM) foram utilizadas para caracterizar a dinâmica do exciton e o seu confinamento nos poços quânticos. As medidas de CFM demonstraram que excitons que são gerados na barreira de PFO são eficientemente transferidos para o PPV. Além disso, o tempo de decaimento da emissão PFO residual é fortemente reduzido devido a processos de migração concorrentes no poço. O tempo de decaimento de PPV diminui substancialmente para poços com espessuras abaixo de 5 nm como resultado da auto-aniquilação do exciton. Dessa forma, as estruturas de MQW obtidos pela técnica de SA-LbL podem ser usadas para estudar a transferência de energia, efeitos túneis e para a construção de novos dispositivos optoelectrónicos com maior eficiência. / The understanding of surface/interface processes and their effects on optical/electrical properties of organic materials is of strong technological importance. This research describes the fabrication and characterization of extremely thin (thickness <10 nm) and homogeneous multilayered polymeric structures including polymer/poly-electrolyte layers and structures with energy modulation such as quantum well. Our main purpose was the study of charge and energy transfer processes in such energy modulated structures. The luminescent polymers used were Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly(p-phenylenevinylene) (PPV). PPV has been obtained from the poly(xylyliden tetrahydrothiophenium chloride) (PTHT) precursor. The so-called Spin Self-Assembly Layer-by-Layer deposition method (SA-LbL) was utilized to obtain the films. Absorption measurements confirmed the linear growth of layers using for polyelectrolyte/polyelectrolyte and polymer/polyelectrolyte interfaces. In order to understand the &pi;-electron transfer from the conjugated polymer to charged states of the polyelectrolyte, the configurations of the polymeric structures were modified by depositing different polyelectrolyte monolayer on the polymer film. We observed that &pi;-electrons were effectively transferred to polyelectrolytes that have high electron affinities. This effect strongly affects both absorption and conduction features of such very thin polymeric film. The absorption is restored after the conversion of PTHT in PPV. Photoluminescence measurements on PFO/PPV films result in emission curves with characteristic peaks of both polymers, confirming that SA-LbL technique allows deposition of multilayer polymeric structures. The various film configurations elucidates the transfer processes occurring at different interfaces like: change of polyelectrolyte deposition order, number of PTHT/DBS bilayers and introduction of spacers. In addition, Single Quantum Well (SQW), i.e., structures consisted of PPV layer surrounded of 10 nm thick PFO barriers were obtained. Optical absorption, PL and excitation spectroscopy showed an efficient excited state migration from the PFO barrier to the PPV well. The confocal image homogeneity demonstrated the layer coverage control at a monolayer level and without layer intermixing of the sequentially deposited polymeric materials. High resolution Confocal Fluorescence Microscopy (CFM) and Fluorescence Life spectroscopy and Imaging (FLIM) were used to characterize the exciton dynamics and confinement in quantum well. The CFM measurements demonstrated that excitons generated at the PFO barrier are efficiently transferred to the PPV well. Furthermore, the decay time of the residual PFO emission is strongly reduced due to the competing migration process in the well. The decay time of PPV decreases substantially for well thicknesses below 5 nm as a result of exciton self-annihilation. Thus, the MQW structures obtained by SA-LbL technique can be used to study energy transfer, tunneling effects and to build up new optoelectronic devices with greater efficiency.

Entender os processos em superfície/interface de filmes e seus efeitos sobre as propriedades ópticas e elétricas de materiais orgânicos é de grande importância tecnológica. Esta pesquisa descreve a fabricação e caracterização de filmes poliméricos extremamente finos (espessura <10 nm) e homogêneos compostos por camadas de polímero/polieletrólitos e estruturas com modulação de energia ou poços quânticos. O objetivo principal foi o estudo dos processos de transferência de carga e energia em tais estruturas. Os polímeros luminescentes utilizados foram poli(9,9 dioctilfluoreno) (PFO) poli(p-fenileno vinileno (PPV). O PPV foi obtido a partir do precursor poli(cloreto de tetraidrotiofeno de xililideno) (PTHT). A técnica de deposição denominada deposição camada por camada assistida por spin (SA-LbL) foi utilizada para obtenção dos filmes. Medidas de absorbância confirmaram o crescimento linear das camadas para as interfaces polieletrólito/polieletrólito e polímero/polieletrólito. Com o objetivo de entender a transferência do elétron &pi; do polímero conjugado para o polieletrólito, as configurações das estruturas poliméricas foram alteradas através da deposição de diferentes monocamadas de polieletrólito sobre o filme polimérico. Observamos que os elétrons &pi; foram efetivamente transferidos para os polieletrólitos que possuem alta afinidades eletrônica. Este efeito interfere fortemente na absorção bem como nas características de condução do filme polimérico ultrafino. A absorção é restabelecida após a conversão de PTHT em PPV. Medidas de fotoluminescência (PL) em filmes PFO/PPV resultam em curvas de emissão com picos característicos de ambos os polímeros, o que confirma que a técnica SA-LbL permite a deposição de estruturas poliméricas multicamadas. As várias configurações de filmes obtidas elucidaram os processos de transferência que ocorrem em diferentes interfaces, tais como: mudança da sequencia de deposição do polieletrólito, número de camadas duplas PTHT/DBS e a introdução de camadas separadoras. Além disso, um único poço quântico, ou seja, estruturas formadas por uma camada PPV cercada por barreiras de PFO com 10 nm de espessura foram obtidas. Medidas de absorbâcia, PL e excitação mostraram uma eficiente migração estado excitado da barreira de PFO para o PPV (poço). A homogeneidade da imagem confocal, demonstrou um rigoroso controle da camada de cobertura ao nível de um única monocamada e sem contaminação pelos materiais depositados sequencialmente. A microscopia confocal de fluorescência (CFM) e espectroscopia de fluorescência resolvida no tempo (FLIM) foram utilizadas para caracterizar a dinâmica do exciton e o seu confinamento nos poços quânticos. As medidas de CFM demonstraram que excitons que são gerados na barreira de PFO são eficientemente transferidos para o PPV. Além disso, o tempo de decaimento da emissão PFO residual é fortemente reduzido devido a processos de migração concorrentes no poço. O tempo de decaimento de PPV diminui substancialmente para poços com espessuras abaixo de 5 nm como resultado da auto-aniquilação do exciton. Dessa forma, as estruturas de MQW obtidos pela técnica de SA-LbL podem ser usadas para estudar a transferência de energia, efeitos túneis e para a construção de novos dispositivos optoelectrónicos com maior eficiência. / The understanding of surface/interface processes and their effects on optical/electrical properties of organic materials is of strong technological importance. This research describes the fabrication and characterization of extremely thin (thickness <10 nm) and homogeneous multilayered polymeric structures including polymer/poly-electrolyte layers and structures with energy modulation such as quantum well. Our main purpose was the study of charge and energy transfer processes in such energy modulated structures. The luminescent polymers used were Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly(p-phenylenevinylene) (PPV). PPV has been obtained from the poly(xylyliden tetrahydrothiophenium chloride) (PTHT) precursor. The so-called Spin Self-Assembly Layer-by-Layer deposition method (SA-LbL) was utilized to obtain the films. Absorption measurements confirmed the linear growth of layers using for polyelectrolyte/polyelectrolyte and polymer/polyelectrolyte interfaces. In order to understand the &pi;-electron transfer from the conjugated polymer to charged states of the polyelectrolyte, the configurations of the polymeric structures were modified by depositing different polyelectrolyte monolayer on the polymer film. We observed that &pi;-electrons were effectively transferred to polyelectrolytes that have high electron affinities. This effect strongly affects both absorption and conduction features of such very thin polymeric film. The absorption is restored after the conversion of PTHT in PPV. Photoluminescence measurements on PFO/PPV films result in emission curves with characteristic peaks of both polymers, confirming that SA-LbL technique allows deposition of multilayer polymeric structures. The various film configurations elucidates the transfer processes occurring at different interfaces like: change of polyelectrolyte deposition order, number of PTHT/DBS bilayers and introduction of spacers. In addition, Single Quantum Well (SQW), i.e., structures consisted of PPV layer surrounded of 10 nm thick PFO barriers were obtained. Optical absorption, PL and excitation spectroscopy showed an efficient excited state migration from the PFO barrier to the PPV well. The confocal image homogeneity demonstrated the layer coverage control at a monolayer level and without layer intermixing of the sequentially deposited polymeric materials. High resolution Confocal Fluorescence Microscopy (CFM) and Fluorescence Life spectroscopy and Imaging (FLIM) were used to characterize the exciton dynamics and confinement in quantum well. The CFM measurements demonstrated that excitons generated at the PFO barrier are efficiently transferred to the PPV well. Furthermore, the decay time of the residual PFO emission is strongly reduced due to the competing migration process in the well. The decay time of PPV decreases substantially for well thicknesses below 5 nm as a result of exciton self-annihilation. Thus, the MQW structures obtained by SA-LbL technique can be used to study energy transfer, tunneling effects and to build up new optoelectronic devices with greater efficiency.

Mes travaux de thèse portent sur l’application de la théorie de l’échantillonnagecompressif (Compressed Sensing ou Compressive Sampling, CS) à la microscopie defluorescence, domaine en constante évolution et outil privilégié de la recherche fondamentaleen biologie. La récente théorie du CS a démontré que pour des signauxparticuliers, dits parcimonieux, il est possible de réduire la fréquence d’échantillonnagede l’information à une valeur bien plus faible que ne le prédit la théorie classiquede l’échantillonnage. La théorie du CS stipule qu’il est possible de reconstruireun signal, sans perte d’information, à partir de mesures aléatoires fortement incomplèteset/ou corrompues de ce signal à la seule condition que celui-ci présente unestructure parcimonieuse.Nous avons développé une approche expérimentale inédite de la théorie du CSà la microscopie de fluorescence, domaine où les signaux sont naturellement parcimonieux.La méthode est basée sur l’association d’une illumination dynamiquestructurée à champs large et d’une détection rapide à point unique. Cette modalitépermet d’inclure l’étape de compression pendant l’acquisition. En outre, nous avonsmontré que l’introduction de dimensions supplémentaires (2D+couleur) augmentela redondance du signal, qui peut être pleinement exploitée par le CS afin d’atteindredes taux de compression très importants.Dans la continuité de ces travaux, nous nous sommes intéressés à une autre applicationdu CS à la microscopie de super résolution, par localisation de moléculesindividuelles (PALM/STORM). Ces nouvelles techniques de microscopie de fluorescenceont permis de s’affranchir de la limite de diffraction pour atteindre des résolutionsnanométriques. Nous avons exploré la possibilité d’exploiter le CS pour réduiredrastiquement les temps d’acquisition et de traitement.Mots clefs : échantillonnage compressif, microscopie de fluorescence, parcimonie,microscopie de super résolution, redondance, traitement du signal, localisation demolécules uniques, bio-imagerie / My PhD work deals with the application of Compressed Sensing (or CompressiveSampling, CS) in fluorescence microscopy as a powerful toolkit for fundamental biologicalresearch. The recent mathematical theory of CS has demonstrated that, for aparticular type of signal, called sparse, it is possible to reduce the sampling frequencyto rates well below that which the sampling theorem classically requires. Its centralresult states it is possible to losslessly reconstruct a signal from highly incompleteand/or inaccurate measurements if the original signal possesses a sparse representation.We developed a unique experimental approach of a CS implementation in fluorescencemicroscopy, where most signals are naturally sparse. Our CS microscopecombines dynamic structured wide-field illumination with fast and sensitive singlepointfluorescence detection. In this scheme, the compression is directly integratedin the measurement process. Additionally, we showed that introducing extra dimensions(2D+color) results in extreme redundancy that is fully exploited by CS to greatlyincrease compression ratios.The second purpose of this thesis is another appealing application of CS forsuper-resolution microscopy using single molecule localization techniques (e.g.PALM/STORM). This new powerful tool has allowed to break the diffraction barrierdown to nanometric resolutions. We explored the possibility of using CS to drasticallyreduce acquisition and processing times.

L'exocytose régulée, est un processus qui permet la communication entre les cellules à travers la sécrétion des hormones et des neurotransmetteurs. Dans les neurones et les cellules neuroendocrines, l'exocytose est strictement contrôlée par des signaux extracellulaires tels que le potentiel trans-membranaire et la fixation des ligands sur des récepteurs. Des progrès substantiels ont été effectués afin de comprendre le mécanisme moléculaire de l'exocytose. Les composants majeurs de la machinerie de sécrétion ont été dévoilés. Maintenant, la question qui émerge concerne le rôle de la plateforme de protéines qui semble avoir une action coordonnée entre chaque protéine. Dans le cas de la famille des annexines, qui est bien connue pour son action dans l'exocytose, leurs modes d'interactions séquentielles ou concertées avec d'autres protéines ainsi que leurs effets régulateurs sur l'exocytose ne sont pas encore bien établis. Des résultats précédents indiquent que l'Annexine A6 (AnxA6) affecte l'homéostasie du calcium et la sécrétion de la dopamine à partir des cellules PC12, utilisées comme un modèle cellulaire de neurosécrétion (Podszywalow Bartnicka et al., 2010). Afin de déterminer l'effet inhibiteur de l'AnxA6 sur l'exocytose de la dopamine, nous cherchons des partenaires moléculaires de l'AnxA6 dans les cellules PC12. Nous faisons l'hypothèse que l'AnxA6 interagit avec la PLD1, une enzyme active dans l'étape de la fusion des vésicules avec la membrane plasmique. En utilisant la microscopie confocale et la microscopie à onde évanescente, nous avons trouvé que l'isoforme 1 de l'AnxA6 et la PLD1 sont tous les deux recrutés sur la surface des vésicules au cours de la stimulation des cellules PC12. AnxA6 inhibait l'activité de la PLD comme indiqué par notre méthode d'analyse enzymatique au moyen de la spectroscopie infrarouge. En conclusion, nous proposons que l'AnxA6 n'est pas seulement impliquée dans la réorganisation des membranes par ses capacités à se lier avec des phospholipides négativement chargés et avec le cholestérol, mais elle influence également l'activité de la PLD1, changeant la composition lipidique des membranes / The regulated exocytosis is a key process allowing cell-cell communication through the release of hormone and neurotransmitters. In neurons and neuroendocrine cells, it is strictly controlled by extracellular signal such as transmembrane potential and ligand bindings to receptors. Substantial progress has been made to understand the molecular mechanism of exocytosis. Major components of secretory machinery have been brought to light. Now the emergent question concerns the role of scaffolding proteins that are thought to coordinate the action of each other. In the case of annexin family well known to be involved in exocytosis, their modes of –sequential or concerted- interactions with other proteins, and their regulatory effects on exocytosis are not very well established. Previous findings indicated that Annexin A6 (AnxA6) affected calcium homeostasis and dopamine secretion from PC12 cells, used as cellular model of neurosecretion (Podszywalow-Bartnicka et al., 2010). To determine the inhibitory effect of AnxA6 on exocytosis of dopamine, we were looking for molecular partners of AnxA6 in PC12 cells. We hypothesized that AnxA6 interacts with phospholipase D1 (PLD1), an enzyme involved in the fusion step. By using confocal microscopy and total internal reflection fluorescence microscopy, we found that isoform 1 of AnxA6 and Phospholipase D1 are both recruited on the surface of vesicles upon stimulation of PC12 cells. AnxA6 inhibited phospholipase D activity as revealed by our enzymatic assay based on infrared spectroscopy. To conclude, we propose that AnxA6 is not only implicated in membrane organization by its capacity to bind to negative charged phospholipids and to cholesterol, but AnxA6 is also affecting PLD1 activity, changing membrane lipids composition

<p>The blood-brain barrier restricts the access of many compounds, including therapeutic agents, to the brain. Several human studies indicate that nasal administration of hydrophilic compounds, such as peptides, can bypass the blood-brain barrier. The aims of this thesis were to develop and refine models for this direct nose-to-brain transfer.</p><p>In a mouse model, [³H]-dopamine was given as a unilateral nasal dose. The resulting radioactivity in the ipsilateral olfactory bulb was significantly higher than that in the contralateral bulb and peaked at 4 h. Tape section autoradiography showed that the radioactivity was concentrated in the olfactory nerve layer and the glomerular layer of the olfactory bulb. The olfactory transfer of dopamine was also studied <i>in vitro</i>. At a lower donor concentration, the mucosal-to-serosal dopamine permeability was higher than the serosal-to-mucosal permeability, but at a higher concentration, the permeability coefficients were similar. Together, these results suggest that the olfactory transfer of dopamine has an active component.</p><p>Olfactory transfer of fluorescein-labeled dextran through the epithelium and deeper tissues was studied in a rat model, which enabled visualization of the transfer using fluorescence microscopy. Although the epithelial transfer appeared to be mainly intracellular, transfer in the following deeper tissues was extracellular. Without altering the route of uptake, a gellan gum formulation enhanced the uptake of fluorescein dextran. The enhancing effect was considered likely to be the result of an increased residence time in the nasal cavity.</p><p>In conclusion, dopamine and fluorescein-labeled dextran were identified as suitable model compounds for the study of olfactory drug transfer mechanisms and the influence of drug formulation. Two new <i>in vitro</i> models of olfactory transfer were compared. Also, a rat model, which enabled the visualization of the entire nose-to-brain transfer, was developed.</p>

The blood-brain barrier restricts the access of many compounds, including therapeutic agents, to the brain. Several human studies indicate that nasal administration of hydrophilic compounds, such as peptides, can bypass the blood-brain barrier. The aims of this thesis were to develop and refine models for this direct nose-to-brain transfer. In a mouse model, [3H]-dopamine was given as a unilateral nasal dose. The resulting radioactivity in the ipsilateral olfactory bulb was significantly higher than that in the contralateral bulb and peaked at 4 h. Tape section autoradiography showed that the radioactivity was concentrated in the olfactory nerve layer and the glomerular layer of the olfactory bulb. The olfactory transfer of dopamine was also studied in vitro. At a lower donor concentration, the mucosal-to-serosal dopamine permeability was higher than the serosal-to-mucosal permeability, but at a higher concentration, the permeability coefficients were similar. Together, these results suggest that the olfactory transfer of dopamine has an active component. Olfactory transfer of fluorescein-labeled dextran through the epithelium and deeper tissues was studied in a rat model, which enabled visualization of the transfer using fluorescence microscopy. Although the epithelial transfer appeared to be mainly intracellular, transfer in the following deeper tissues was extracellular. Without altering the route of uptake, a gellan gum formulation enhanced the uptake of fluorescein dextran. The enhancing effect was considered likely to be the result of an increased residence time in the nasal cavity. In conclusion, dopamine and fluorescein-labeled dextran were identified as suitable model compounds for the study of olfactory drug transfer mechanisms and the influence of drug formulation. Two new in vitro models of olfactory transfer were compared. Also, a rat model, which enabled the visualization of the entire nose-to-brain transfer, was developed.

This project examined multiple aspects of the reproductive biology of the wetland invasive species, purple loosestrife (<i>Lythrum salicaria</i> L.), in central Saskatchewan. An examination of insect taxa visiting the three floral morphs of <i>Tristylous</i> L. <i>salicaria</i>, as well as a ranking of the pollination efficiency of individual insect species, an apparent first for L salicaria, was undertaken. Surface features of the floral nectary of L. <i>salicaria</i>, as well as floral nectar secretion dynamics, were also investigated. This project also re-visited some of the previous work done on this invasive species, including various floral organ morphometrics in relation to heterostyly, and aspects of the tristylous breeding system including self-fertilization, and fertilization potential of both illegitimate pollination and legitimate pollination.<p>The trimorphic nature of the sexual floral organs of L. <i>salicaria</i> were well defined in Saskatchewan. Significant differences in length (long-, intermediate- and short-style lengths) exist between all three floral morphs. Lengths of the staminal filaments (long, intermediate, and short) were also significantly different. Also the floral nectary in L. <i>salicaria</i> is located in a depression formed at the interface of the hypanthium and the gynoecium. Several stomata are located at regular intervals along the nectary surface, and may constitute the escape route for floral nectar. No morphological differences in nectary structure were apparent among the three floral morphs.<p>Nectar secretion dynamics of L. <i>salicaria</i> were examined between the three floral morphs throughout two summer days in 2006. Peak average nectar volumes and nectar sugar quantities were detected at 3:00 pm, and, interestingly, no significant differences were detected between floral morphs, in accordance with nectary morphology. The estimated secretion rates for L. <i>salicaria</i> ranged from 61 83 µg of nectar sugar per flower per hour.<p>Hand-pollination experiments carried out over the summers of 2006 and 2007 at three field sites in and around Saskatoon have verified the strong self-incompatibility in the breeding system of this tristylous species. Intramorph pollination, using illegitimate pollen, did not result in fertilisation, whereas legitimate hand-pollination experiments yielded multiple pollen tubes at the style base, without exception.<p><i>Lythrum salicaria</i> in central Saskatchewan was visited by several bee taxa including honeybees (<i>Apis mellifera</i> L.), bumblebees (Bombus spp.), leafcutter bees (Megachile spp.), and sweat bees (Lasioglossum spp.). A single visit by <i>Anthophora furcata</i> (Panzer) was also recorded in 2007. Generally, bee visits led to high levels of pollination success as determined by fluorescence microscopy of pollen tubes following single insect visits to previously-unvisited flowers. However, most visits by hoverflies (Syrphidae) were non-pollinating. Visits by Pieris rapae (L.), yellowjacket wasps (Vespidae) and some non-syrphid flies (Diptera) also yielded no pollen tubes at the style base.<p>A study of the ultrastructure and development of the floral nectary of the purple loosestrife cultivar Morden Gleam (<i>Lythrum virgatum</i> L. x L. alatum Pursh.) showed that starch build up in pre-secretory nectary tissues declined throughout secretion, and is virtually absent in post-secretory nectary tissues. The lack of a direct vascular supply to the floral nectary suggests that the starch breakdown products likely make up most of the floral nectar carbohydrates. Surface features of the floral nectary in Morden Gleam closely resembled those of L. salicaria, located in the valley formed between the hypanthium and gynoecium. Nectary stomata, occasionally in pairs, likely serve as outlets for nectar in this cultivar.