Les matériaux méso-structurés sont des matériaux à porosité contrôlée, issus de synthèses de chimie sol/gel entre micelles de tensioactifs et précurseur inorganique, le plus souvent de silice. Parmi ces matériaux, le SBA-15, formé en milieu super-acide à partir de P123 (un copolymère tribloc) comme tensioactif et de TEOS comme précurseur inorganique de silice, est un des plus étudiés. La synthèse de ce matériau a été étudiée par diffusion des rayons X à petits angles (SAXS) in situ, et a permis de décrire qualitativement et quantitativement les mécanismes d’auto-assemblages entre micelles de tensioactifs et particules silicatées. Spécifiquement, nos résultats ont montré que l’hydrolyse-condensation du TEOS en solution a permis la formation d’oligomères de silice, qui interagissent avec la couronne des micelles sphériques de P123, pour progressivement former des micelles hybrides cylindriques organiques/inorganiques, qui vont ensuite précipiter en une mésophase hybride 2D-hexagonale. Ce modèle de synthèse est appelé « transition sphères-cylindres ». De plus, afin de comprendre les mécanismes de formation du matériau à toutes les échelles, nous avons étudié l’influence des conditions de synthèse sur la morphologie des grains de matériau méso-structurés. En effet, nous avons montré que sans agitation pendant la synthèse, les grains de matériaux présentent une forme d’équilibre qui dépend fortement de la température de synthèse. En fonction de la température, il est donc possible de former des matériaux hybrides de forme grains de riz, bâtons (courts ou longs) ou mêmes sous forme de tores. Nous proposons un modèle théorique qui permet d’expliquer l’ensemble des morphologies observées, en fonction des tensions de surfaces et des énergies de courbures de cristal-liquide 2D-hexagonal lors de la précipitation de la mésophase hybride. Nous appuyons ce modèle théorique sur des études en diffusion des rayons X à très petits angles (USAXS), qui permettent d’étudier la croissance des grains. Grâce à ces études, nous proposons une description très complète de la formation du matériau SBA-15. Fort de ces connaissances, nous avons pu, grâce à de nouvelles études de SAXS in situ, appliquer le modèle de précipitation de type « transition sphères-cylindres » à d’autres matériaux, issus de tensioactifs non-ioniques fluorés, et ainsi expliquer la formation de matériaux dits « bimodaux », c’est-à-dire issus d’un mélange entre P123 et un tensioactif fluoré(Rf₈(EO)₉ et qui présentent deux ordres poreux bien définis. Enfin, nous avons cherché à utiliser nos connaissances pour la fabrication de matériaux innovants, en remplaçant la solution micellaire par des émulsions dopées en nanoparticules d’or (NPs), afin de fabriquer des matériaux hybrides dopés en NPs. / Meso-structured materials are materials with a well-controlled porosity, designed from sol/gel syntheses between surfactant micelles and an inorganic precursor, most often a silica precursor. Among them, the SBA-15 material, formed in super-acid solution from P123 (a tri-block copolymer) as surfactant and TEOS a silica precursor, is one of the most studied. Synthesis of this material has been studied by in situ Small Angle X-Ray Scattering (SAXS), which allowed to describe both qualitatively and quantitatively the self-assembly mechanisms between surfactant micelles and silica particles. In details, our results showed that TEOS hydrolysis-condensation allow the formation of silica oligomers that interact with the corona of the spherical micelles of P123, and progressively the micelles reshape in hybrid organic/inorganic cylindrical micelles. The hybrid micelles, first free in solution, eventually precipitate in a hybrid 2D-hexagonal mesophase. This model of synthesis is called “sphere-to-rod transition”. Moreover, in order to understand the material mechanisms of formation at every length scales, we studied the influence of the synthesis conditions on the morphology of the meso-structured material grains. Indeed, in agreement with a previous study, our results show that if the synthesis is made without stirring, the material grains has an equilibrium shape that strongly depends on the synthesis temperature. By changing the temperature, one can form hybrid materials with a “rice grain” shape, or a (short or long) rod shape, or even a torus shape. We propose a theoretical model to explain all the observed morphologies, model that takes into account surface tensions and curvature energy of the newly formed 2D-hexagonal liquid-crystal at the precipitation of the hybrid mesophase. We measured the nucleation and growth of the grains by Ultra Small Angle X-Ray Scattering (USAXS). Thanks to all these studies, we present a complete description of the formation of the SBA-15 material. Our knowledge were used to describe new in situ SAXS measurements of new materials: our “sphere-to-rod transition” model was able to describe the formation of two materials, form two fluorinated surfactants and allowed to explain the formation of a “bimodal” material, which means a material with two well-defined porous order, that is synthesized from a mix of two surfactant (P123 and (Rf₈(EO)₉). Finally, we tried to use our knowledge to form innovative materials, by replacing the micellar solution as template by an emulsion doped in gold nanoparticles (NPs) to form hybrid materials doped in NPs.

Esse trabalho de tese descreve a síntese e a caracterização de materiais amorfos e cristalinos na forma de pó e de filmes finos pertencentes ao sistema Er2O3-Y2O3-Al2O3-B2O3. O principal objetivo do trabalho foi o de obter filmes finos amorfos contendo íons Er3+ próximo à composição YAI3(BO3)4 (YAB), visando sua aplicação como amplificador em dispositivos ópticos integrados. Na síntese das amostras foi empregado o método dos precursores poliméricos e o método sol-gel. Uma vez estabelecida às condições de síntese das resinas e de sóis estáveis, diferentes parâmetros foram otimizados visando à deposição de filmes relativamente espessos e amorfos. As propriedades térmicas, estruturais e ópticas de amostras cristalinas ou amorfas, na forma de pó e na forma de filmes finos, foram caracterizadas através das técnicas de análise termogravimétrica, calorimetria exploratória diferencial, difração de raios X, espectroscopia vibracional na região do infravermelho com transformada de Fourier, espectroscopia Raman e microscopia eletrônica de varredura de alta resolução, microscopia de força atômica, espectroscopia \"m-line\", medidas de espectros de luminescência. Baseado na análise térmica e estrutural das amostras na forma de pó, foi possível realizar a síntese de amostras na forma de filmes finos de composição Y1-xErxAl3(BO3)4 que apresentaram características estruturais, microestruturais e ópticas adequadas à aplicação desejada. Através do estudo das propriedades térmicas e estruturais, foi observado que as amostras preparadas através do método dos precursores poliméricos e do método sol-gel apresentam respectivamente uma temperatura de transição vítrea (Tg) à 723 &#176C e à 746 &#176C e uma temperatura de cristalização (Tc) à 814 &#176C e à 830&#176C. O tratamento térmico à aproximadamente 1150 &#176C da amostra amorfa na forma de pó de composição Y0,9Er0,1Al3(BO3)4 contendo boro em excesso levou a obtenção de uma solução sólida cristalina de composição Y0,9Er0,1Al3(BO3)4. No que se refere às propriedades dos filmes finos, as melhores condições de densificação, homogeneidade, eficiência de guiamento, emissão luminescente e tempo de vida foram observadas quando os filmes de composição Y0,9Er0,1Al3(BO3)4 foram tratados entre as temperaturas de Tg e Tc. Os filmes amorfos de composição Y0,9Er0,1Al3(BO3)4 se comportam como guias monomodos, apresentando alta emissão luminescente e um tempo de vida comparável a outros sistemas amorfos a base de boro. Esse comportamento mostra a viabilidade de aplicação desses filmes amorfos como amplificadores ópticos planares. / This work specifies the synthesis and the characterization of amorphous and crystalline powder and thin films materials belonging to the Er2O3-Y2O3-Al2O3-B2O3 system. The main objective of this work was to develop amorphous thin film samples near the YAI3(BO3)4 composition with yttrium partially substituted by erbium Y1-xErxAl3(BO3)4 with the aim of applying these thin films as waveguide amplifiers in integrated optical systems. The polymeric precursor and the sol-gel methods were applied in order to produce such thin films. After the first step where the conditions to obtain very stable resins and sols was established, different parameters were adjusted aiming the deposition of thick and stable thin films. To well define the best conditions to obtain amorphous and dense thin films, the powder samples of the same compositions were first characterized by thermal analysis techniques, X-ray diffraction, IR and Raman spectroscopies, high resolution electron microscopy and atomic force microscopy. From thermal analysis and structural results, it was observed that the samples prepared from the polymeric precursor and sol-gel methods exhibit glass transition temperatures, Tg, at 723 and 746 &#176C and cristallisation temperatures, Tc, at 814 and 830 &#176C, respectively. The heat treatment at around 1150 &#176C of Y0,9Er0,1Al3(BO3)44 powder sample containing a smaller amount of boron in excess produced by both methods lead to the formation of a crystalline solid solution of the same composition without the presence of secondary phases. These results show that dense, free of cracks, thick and homogeneous thin films could be obtained when they were submitted at a heat treatment in temperatures between Tg and Tc. From the \"m-line\" spectroscopy technique and luminescence measurements we observed that the Y0,9Er0,1Al3(BO3)4 amorphous thin films can be considered as monomode waveguides showing a high luminescence intensities and a lifetime similar to other amorphous borate systems. These results show the potentiality to apply such amorphous thin films as optical planar amplifiers.

Les oligomères solubles de peptide Bêta-amyloïde (A-bêta) apparaissent comme les acteurs majeurs de la perte synaptique précoce observée au cours de la maladie d'Alzheimer. Notre équipe a précédemment montré que ces oligomères de peptide A-bêta activent la phospholipase A2 cytosolique (cPLA2), qui entraîne la libération d'acide arachidonique à partir des phospholipides membranaires. En utilisant un modèle d'injection intra cérébro ventriculaire unique d'une faible quantité de peptide A-bêta, nous avons pu observer que l'inactivation constitutive du gène de la cPLA2 protége les souris KO contre les perturbations mnésiques et empêche la réduction de l'expression de protéines synaptiques au sein de l'hippocampe, ces deux effets délétères étant constatés chez les animaux wild-type. Par la suite, nous avons montré que l'activation des sphingomyélinases, consécutive à l'exposition aux oligomères A-bêta, est indétectable dans des neurones en culture issus de souris KO. Dans ces mêmes neurones KO, nous avons constaté que la phosphorylation de Akt/PKB n'est pas altérée suite à l'exposition des cellules aux oligomères A-bêta. Enfin, nous avons pu mettre en évidence une diminution de l'expression de la protéine précurseur du peptide A-bêta (protéine APP), tant au niveau d'homogénats hippocampiques que de neurones en cultures, issus de souris KO. Néanmoins, des travaux supplémentaires sont requis pour établir le lien exact entre cette réduction de l'expression d'APP et la résistance aux oligomères A-bêta, tant in vitro qu'in vivo. Toutefois, ces résultats soulignent l'implication de la cPLA2 dans la neuro dégénérescence entrainée par les oligomères A-bêta, et font apparaitre cette enzyme comme une cible thérapeutique potentielle pour le traitement de la maladie d'Alzheimer / Soluble beta-amyloid (A-beta) oligomers putatively play a critical role in the early synapse loss and cognitive impairment observed in Alzheimer's disease. We previously demonstrated that A-beta oligomers activate cytosolic phospholipase A2 (cPLA2) which specifically releases arachidonic acid from membrane phospholipids. By using a single A-beta oligomers intra cerebro ventricular injection, we observed that cPLA2 gene suppression prevents both the alterations of cognitive abilities and the reduction of hippocampal synaptic markers levels which are observed in wild type mice. We further demonstrated that the A-beta oligomers-induced sphingomyelinase activation is suppressed and that the phosphorylation of Akt/PKB is preserved in neuronal cells isolated from KO mice. Interestingly, expression of the A-beta precursor protein (APP) is reduced in hippocampus homogenates and neuronal cells from KO mice, but the relationship with the resistance of these mice to the A-beta oligomers toxicity requires further investigation. These results therefore show that cPLA2 plays a key role in the A-beta oligomers-associated neurodegenerative effects, and as such represents a potential therapeutic target for the treatment of Alzheimer's disease

Made available in DSpace on 2016-04-28T14:16:25Z (GMT). No. of bitstreams: 1 Dissertacao LUCIANA SCOGNAMIGLIO DE OLIVEIRA.pdf: 460557 bytes, checksum: 1c88574fd94d3d8d5f2830c6629ac2b8 (MD5) Previous issue date: 2006-04-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Esta dissertação tem como objeto de estudo a idéia de precursor atribuída a Monteiro Lobato no que diz respeito à literatura infantil brasileira Partimos do pressuposto de que Lobato não deve ser considerado precursor embora tenha contribuído de forma significativa para a formação da literatura infantil contemporânea O lançamento de seu primeiro livro A menina do nariz arrebitado aconteceu no momento em que os ideais modernistas fervilhavam no Brasil O Movimento Modernista que inicialmente propunha a atualização do país em relação à Europa acabou por negar o passado e toda a influência exercida por Portugal. As mudanças que ocorrem com o conceito de infância na virada do século exerceram uma profunda influência na concepção da obra lobatiana A criança do século XIX não será a mesma do século XX O avanço da medicina a preocupação em relação à higiene o surgimento da penicilina os estudos psicológicos e pedagógicos dão uma nova dimensão e um novo significado à infância A literatura destinada a elas também passa por uma transformação visto que elas deixarão de ser consideradas adultos pequenos para serem vistas apenas como crianças que necessitam de cuidados apropriados à sua condição Dessa maneira tomaremos como base para nosso estudo a idéia da valorização do contexto histórico para propor uma reflexão sobre o papel que Lobato vem ocupando ao longo de diversas gerações tomado como o criador da literatura infantil no Brasil Para isso partiremos principalmente da análise das correspondências de Lobato ao amigo Godofredo Rangel publicadas posteriormente em A Barca de Gleyre

Les procédés de photodégradation, de conversion photocatalytique du CO2 et la technologie des supercondensateurs représentent des options intéressantes pour palier aux problèmes environnementaux et pour apporter des réponses à la crise énergétique. Dans ces trois domaines, les matériaux à base d'oxydes métalliques sont très prometteurs. Cependant, les voies classiques (c'est-à-dire par voies hydrothermales ou de sol-gels) présentent un certain nombre d’inconvénients tels que leur longues durées de préparation et leurs rendements limités. En outre, la mise en oeuvre de nanopoudres suppose une opération de post-filtration dans les procédés de photodégradation et l’utilisation supplémentaire d’un liant dans la confection d’électrodes de supercondensateurs, ce qui non seulement réduit les performances respectives de ces deux procédés mais entrave également leur développement au niveau industriel. Dans cette thèse, les technologies SPPS (Solution Precursor Plasma Spray) et SPFS (Solution Precursor Flame Spray) ont été introduites pour préparer des films d'oxydes métalliques à base de ZnO, en bénéficiant des avantages de rapidité et simplicité de ces techniques de formation de dépôts. Les films ainsi obtenues ont ensuite été testés dans des expériences de photodégradation, de conversion photocatalytique du CO2 et sous forme de supercondensateurs. Tout d'abord, à notre connaissance, c'est la première fois que l'on synthétise directement des nanostructures de ZnO (par exemple des nanotubes ou des nanofils) par des procédés SPPS. Ces films nanostructurés et hiérarchisés présentent non seulement une croissance préférentielle le long du plan cristallin (002), mais contiennent aussi des lacunes d'oxygène dans leurs réseaux. La mise en œuvre de simulations DFT a permis de proposer un mécanisme possible de croissance des nanostructures de ZnO lors de leur synthèse par la voie SPPS. Deuxièmement, divers films “composites”, constitués de ZnO et d’un second oxide “MO” ont également été préparés par SPPS afin de réduire les bandes d'énergie interdites. Dans cette partie de la thèse, nous avons étudié l’effet du rapport molaire MO/ZnO sur la structure des films MO/ZnO obtenus (M = Mn et Cu); nous avons aussi préparé, par cette nouvelle méthode, des films fins CuO/ZnO et CeO2/ZnO ainsi que des films de ZnO “décorés”. Troisièmement, des films mettant en œuvre des structures du type spinelle (tels que les oxydes mixtes ZnFe2O4, NiCo2O4, ZnCo2O4 et Co3O4) ont également été synthétisés et déposés à l’aide des techniques SPPS et SPFS, ceci en raison de leur grand intérêt pour les applications susmentionnées. Il a été constaté que les structures obtenues sont très sensibles aux rapports MO/ZnO et que les morphologies de surface dépendent davantage des paramètres d'injection des solutions. En outre, la puissance de la torche joue un rôle plus critique dans la synthèse in situ de la phase spinelle binaire que la température de préchauffage du substrat. Enfin, la voie de préparation par SPPS favorise la formation de dépôts de textures floconneuses, notamment dans le cas des films de NiCo2O4 et Co3O4, tandis que des particules de formes sphériques ont été plutôt observées dans les échantillons préparés par la voie SPFS... / The fields of research dealing with photodegradation, photocatalytic conversion of CO2 and supercapacitors are important to address environmental problems and respond to the energy crisis. Metal oxides are promising materials in these three domains. However, the conventional routes (i.e. hydrothermal, sol-gel) suffer from major deficiencies, namely their multi-step natures, their long preparation duration and small-scaled yields. Moreover, the usage of nanopowders implies a post-filtration operation at the end of the photodegradation processes and requires an additional binder in supercapacitor electrodes. In this thesis, “Solution Precursor Plasma Spray” (SPPS) and “Solution Precursor Flame Spray” (SPFS) technologies have been introduced to develop metal oxide films in view of the three aforementioned applications, benefiting from the facility and rapidity advantages of this one-step process.Firstly, to our best knowledge, it is the first time that films composed by ZnO nanostructures (e.g. nanorods, nanowires) are directly synthesized via a SPPS process. These hierarchical ZnO nanostructured films not only exhibit preferential orientation growth along the (002) crystal plane, but also feature in-situ oxygen vacancies. As a result, a possible growth mechanism of ZnO nanostructures via SPPS route was proposed.Secondly, various metal oxides composite films containing ZnO and a second metal oxide were also prepared by SPPS in an effort to narrow the energy bandgaps. In this work, not only the effect of the molar CuO/ZnO and MnO/ZnO ratio was investigated, but also laminated CuO/ZnO and CeO2/ZnO films and CuO, Co3O4 and Fe2O3 decorated ZnO nanorods films were pioneeringly deposited via this novel route.Thirdly, films involving spinel-type materials (including ZnFe2O4, NiCo2O4, ZnCo2O4 and Co3O4) were also synthesized and deposited by the SPPS and SPFS technologies, owing to their high-interest in the aforementioned applications. We found that the phase compositions are more sensitive to the Fe/Zn and Ni/Co ratios and that the surface morphologies are more dependent on the patterns of the solution injection. In addition, the power of the torch plays a more critical role on the in-situ synthesis of binary spinel phase. Besides, the SPPS route promotes the formation of flake-like particles both in the NiCo2O4 and Co3O4 films, while sphere-like particles were observed in the SPFS-prepared samples.Finally, some as-prepared films were selected to evaluate their performances within the three applications. On the one hand, Orange II was successfully (100%) degraded within 2h under UV irradiation and about 85% was removed within 6h under visible light irradiation. On the other hand, Co3O4 samples exhibited specific capacitances up to 1190 F g−1 with a retention capacity of 136% after 2500 cycles at a 20 mV/s scanning rate in 2 M KOH electrolyte. Finally, when using ZnCo2O4 as photocatalyst, CO2 was converted into CO by visible light irradiation with a maximum turnover number as high as 61.38 and a selectivity as high as 90.5 %.Overall, this work not only improves the performances of the three studied processes thanks to the use of novel, fast preparation methods, but also suggests that “Solution Precursor Thermal Spray” should be a highly promising technology for further, alternative functional applications that involve finely structured metal oxides film.

<p>Induction of vulval fates in the C. elegans hermaphrodite is mediated by a conserved RTK/Ras/MAP kinase signalling pathway, in which the core components can be placed into a linear genetic and biochemical pathway. However, the events that occur downstream of this pathway are not yet well understood. This thesis describes studies on three genes, lin-1, lin-25 and sur-2 that function genetically downstream of the RTK/Ras/MAP kinase pathway in vulva induction. lin-1 encodes an ETS protein that appears to be a direct target of the RTK/Ras/MAP kinase pathway during the induction of vulval fates. To understand more in detail how Ras signalling in C. elegans affects cell fate specification we have analysed the effects of lin-1 mutations on various Ras-mediated cell fate specification events. Our results show that lin-1, besides its function in vulval induction, functions in most other Ras-mediated cell fate specification events in C. elegans, and that lin-1 appears to have a negative function in a majority of these events. Two other genes, lin-25 and sur-2, also function genetically downstream of the RTK/Ras/MAP kinase pathway during induction of vulval fates. Previously, two different models have been proposed for the function of these genes (I) that they function together with a gene in the homeotic cluster to specify the identity of the vulval precursor cells or (II) that they constitute components of the RTK/Ras/MAP kinase signalling pathway. To help clarify the role of lin-25 and sur-2, we have caried out studies of the effects of lin-25 and sur-2 mutations on other cells in the worm in which the RTK/Ras/MAP kinase pathway functions. The results exclude the possibility that lin-25 and sur-2 solely function in vulva induction and suggest that the two genes are intimately involved in Ras-mediated signalling. In addition we show that the major focus for lin-25 during vulval induction is in the vulva precursor cells themselves. Furthermore, results presented here suggest that LIN-25 and SUR-2 function together in the same process in the cell. We show here by both genetic and immunological experiments that LIN-25 is associated with Mediator in C. elegans, a multiprotein complex required for transcriptional regulation. Taken together, these results suggest that lin-25 and sur-2 function in regulating transcription of genes in response to Ras signalling.</p>

<p>Neuroendocrine tumors (NET´s) are often characterized by overproduction of peptide hormones. In spite of pronounced clinical symptoms, the tumor lesions can be small and difficult to detect. The general aim of this thesis was to investigate, in vitro and in vivo, some of the potential monoamine pathways present in NET´s, using radiolabeled tracers for positron emission tomography (PET), with the intention to explore the value of PET-imaging in the management of NET´s.</p><p>We used the 11C-labeled serotonin precursor 5-hydroxy tryptophan (HTP) as the tracer for imaging of NET´s. More than 95% of the subjects displayed a high tracer uptake on PET and tumor detection rate with PET was higher in >50% of the patients compared both to computed tomography (CT) and somatostatin receptor scintigraphy (SRS). The primary tumor was imaged by PET in 84% (16/19), compared to 47% and 42% for SRS and CT, respectively. Tumor visibility was better with PET due to a higher tumor-to-background ratio and a better spatial resolution. There was a strong correlation (r = .907) between changes in urinary-5-hydroxy indole acetic acid and changes in transport rate of 11C-5-HTP during treatment, indicating the use of PET in treatment monitoring of NET´s. </p><p>Pretreatment with carbidopa decreased the urinary radioactivity concentration four-fold and significantly (p<0.001) increased the tumor tracer uptake. This greatly improved image interpretation and tumor lesion detection.</p><p>A screening for expression of monoamine pathways in NET´s revealed a high in vitro binding of the monoamineoxidase-A ligand harmine to tumor sections. PET examinations with 11C-harmine could visualize tumors in all patients, including non-functioning endocrine pancreatic tumors (EPT´s).</p><p>Finally, the in vitro turnover and in vivo distribution of the amino acids glutamate, glutamine and aspartate was investigated. Limited uptake in vivo indicates the lack of utility for these substances as PET-tracers for imaging and characterization of NET´s. </p>

<p>The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall.</p><p>In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models.</p><p>Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models.</p><p>Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.</p>

Induction of vulval fates in the C. elegans hermaphrodite is mediated by a conserved RTK/Ras/MAP kinase signalling pathway, in which the core components can be placed into a linear genetic and biochemical pathway. However, the events that occur downstream of this pathway are not yet well understood. This thesis describes studies on three genes, lin-1, lin-25 and sur-2 that function genetically downstream of the RTK/Ras/MAP kinase pathway in vulva induction. lin-1 encodes an ETS protein that appears to be a direct target of the RTK/Ras/MAP kinase pathway during the induction of vulval fates. To understand more in detail how Ras signalling in C. elegans affects cell fate specification we have analysed the effects of lin-1 mutations on various Ras-mediated cell fate specification events. Our results show that lin-1, besides its function in vulval induction, functions in most other Ras-mediated cell fate specification events in C. elegans, and that lin-1 appears to have a negative function in a majority of these events. Two other genes, lin-25 and sur-2, also function genetically downstream of the RTK/Ras/MAP kinase pathway during induction of vulval fates. Previously, two different models have been proposed for the function of these genes (I) that they function together with a gene in the homeotic cluster to specify the identity of the vulval precursor cells or (II) that they constitute components of the RTK/Ras/MAP kinase signalling pathway. To help clarify the role of lin-25 and sur-2, we have caried out studies of the effects of lin-25 and sur-2 mutations on other cells in the worm in which the RTK/Ras/MAP kinase pathway functions. The results exclude the possibility that lin-25 and sur-2 solely function in vulva induction and suggest that the two genes are intimately involved in Ras-mediated signalling. In addition we show that the major focus for lin-25 during vulval induction is in the vulva precursor cells themselves. Furthermore, results presented here suggest that LIN-25 and SUR-2 function together in the same process in the cell. We show here by both genetic and immunological experiments that LIN-25 is associated with Mediator in C. elegans, a multiprotein complex required for transcriptional regulation. Taken together, these results suggest that lin-25 and sur-2 function in regulating transcription of genes in response to Ras signalling.

The goal of this body of research was to elucidate the mechanism by which CD4+ T cells provide help for CD8+ cytotoxic T lymphocyte (CTL) responses in different immunization types. The establishment of diseases, such as chronic infections and cancers, is attributed to severe loss of or dysfunctions of CD4+ T cells. Even in acute infections, CD4+ T cell deficiency leads to poor memory responses. While the role of CD4+ T cells is being increasingly appreciated in these diseases, the timing and nature of CD4+ T help and associated molecular mechanisms are not completely understood. Growing evidence suggests that, depending on the type of infections or immunizations, the requirements of CD4+ T cells can vary for optimal CD8+ CTL responses. In order to understand the modulatory effects of CD4+ T cells for optimal CD8+ CTL responses, two distinct immunization types were chosen. These include: 1) non-inflammatory dendritic cell (DC) immunization, which fails to provide inflammatory/danger signals; and 2) inflammatory adenovirus (AdV) immunization, which provides profound inflammatory/danger signals. This allowed us to study CD4+ T cell’s participation under different inflammatory conditions. The studies described in Chapters 2 and 3 of this thesis were performed to further understand the concept of how CD4+ T cells mediate optimal CD8+ CTL responses. This has been called the “new dynamic model of CD4+ T helper – antigen (Ag)-presenting cells (Th-APCs),” proposed in 2005 by our laboratory. The study described in Chapter 2 shows that Th-APCs participate not only in augmenting CTL-mediated immune responses, perhaps during early phase, but also in regulating cellular immunity, perhaps during a later phase. Through enhanced IL-2, CD80 and CD40L singnaling, and weaker peptideMHC I (pMHC) signaling, Th-APCs stimulated naïve CD8+ T cells to differentiate into effector CTLs, capable of developing into, central memory CTLs. Th-APC-stimulated CD4+ T cells behaved like Th cells in function, augmenting the overall magnitude of CTL responses. In contrast, Th-APCs were able to kill DCs and other Th-APCs, predominantly through perforin-mediated pathway. The experiments described in Chapter 3 revealed a novel co-operative role of cognate Th-CTL interactions, contrary to previously known immune-regulatory mechanisms among Th-Th or CTL-CTL interactions. In our experiments, Th cells, via CD40L, IL-2, and acquired pMHC-I signaling, enhanced CTL survival and transition into functional memory CTLs. Moreover, RT-PCR, flow cytometry and western blot analysis demonstrate that increased survival of Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of FasL and TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (NFATc1, Bcl-10, Casp-3, Casp-4, Casp-7) genes/ molecules. Finally, helped CTLs were also able to induce protection against highly metastasizing tumor challenge, explaining why memory CTLs generated under cognate Th1’s help show survival and recall advantages. The studies in Chapter 4 showed how the precursor frequency (PF) of CD8+ T cells impacts CD4+ T helper requirements for functional CTL responses. At endogenous PF, CD4+ T helper signals were necessary for both primary and memory CTL responses. At increased PF, CD4+ T help, and its CD40L but not IL-2 signal became dispensable for primary CTL responses. In contrast, memory CTL responses required CD4+ T cell signals, largely in the form of IL-2 and CD40L. Thus, these results could impact the development of novel immunotherapy against cancers, since their efficacy would be determined in part by CD4+ T help and CD8+ T cell PF. Finally, the study showed the importance of CD4+ T cells for multiple phases of AdV transgene product-specific CTL responses. These include: a) cognate CD4+ T cells enhanced CTL responses via IL-2 and CD40L signaling during primary, maintenance and memory phases; b) polyclonal CD4+ T environment enhanced the survival of AdV-specific CTL survival, partially explaining protracted CTL contraction phase; and c) during the recall phase, the CD4+ T environment, particularly memory CD4+ T cells, considerably enhanced not only helped, but also unhelped, memory CTL expansion. Thus, these results suggest the participation of both cognate and polyclonal CD4+ T cells for multiple phases of AdV-specific CTLs. Taken together, the current work delineated the critical roles of CD4+ T cells in different stages of CTL responses and in the development of anti-tumor immunity. The results presented here will significantly advance our current understanding of immunity to cancers, autoimmunity and chronic infections, since pathogenesis of these diseases is largely determined by CD4+ T helper functions. As most immunization procedures use the principle that is based on functions of memory cells, the knowledge gained from this work will also have a major impact on designing vaccines against intractable diseases, including cancers and chronic infections. Moreover, in advanced tumors, vaccines developed using this knowledge may act synergistically with other cancer treatments such as irradiation, chemotherapy and microsurgery, minimizing their side effects and prolonging the lives of patients.