Made available in DSpace on 2017-07-24T19:22:02Z (GMT). No. of bitstreams: 1 Luis Figueroa.pdf: 5025387 bytes, checksum: 9330cb3bf6d0af8254b86f433f7a5433 (MD5) Previous issue date: 2015-12-18 / This in vitro study aimed to assess the amount of hydrogen peroxide (H2O2) from different bleaching agents in the pulp chamber when subjected to different bleaching systems. Seventy-two human premolars were used and randomly divided into 12 groupsaccording to the combination of factors: bleaching agents (Opalescence Boost 38% [B] Whiteness HP Maxx 35% [WM] Total Blanc Office 35% [TBO] Whitenesse HP Blue 35% [WB] Lase Peroxide Sensy 35% [LPS] application modes (3 times 15 minutes and 1 time 45 minutes). An additional group of teeth not bleached (control) was added to the study.To assess the amount of hydrogen peroxide in the pulp chamber, all teeth were cut 3mm from the cement enamel junction, the pulp tissue removed and acetate buffer was placed in the pulp chamber. After the application of bleaching agents in accordance with the mode of application to be tested, H2O2 diffusion was measured for UV-visible spectroscopy of H2O2 by reaction with 4-amino-2,3-dimethyl-1-phenyl-3 pirazolin- 5 -one with a wavelength of 510 nm. The optical density of solution was determined and converted to micrograms of H2O2. Average data and standard deviation of the amount of H2O2 detected within the pulp chamber of the factors was significant (p < 0,0001).Overall, there was no difference between the groups when the bleaching gels tested were applied in technique 3 x 15 min (p > 0,05). However, there was significant difference when the gels were applied in technique 1 x 45 min (p < 0,05), and bleaching treatments Opalescence Boost PF 38% Whiteness HP Blue and 35% those who had the lowest values of hydrogen peroxide into the pulp. Based on the study results it is possible to conclude that the application technique for three times 15 minutes there was no difference between the tested bleaching agents, however, when applied the technique once 45 minutes, bleaching agents containing desensitizing agents are alkaline and had the lowest percentages of hydrogen peroxide within the pulp chamber. / Este estudo in vitro teve o objetivo de avaliar a quantidade de peróxido de hidrogênio (H2O2) de diferentes agentes clareadores na câmara pulpar de dentes humanos extraídos quando submetidos a diferentes sistemas clareadores. Setenta e dois prémolares humanos hígidos foram selecionados e divididos em 12 grupos de acordo com a combinação dos fatores: agentes clareadores (Opalescence Boost 38% [OB], Whitenesse HP Maxx 35% [WM], Total Blanc Office 35% [TBO], Whiteness HP Blue 35% [WB], Lase Peroxide Sensy 35% [LPS]) e modos de aplicação (3 vezes de 15 minutos e 1 vez de 45 minutos, em uma sessão). Dois grupos de dentes não clareados (controle) foram adicionados ao estudo. Para avaliar a quantidade de peróxido de hidrogênio na câmara pulpar, todos os dentes foram seccionados 3 mm da junção cemento-esmalte, o tecido pulpar foi removido e um tampão de acetato foi colocado na câmara pulpar. Após a aplicação dos agentes clareadores de acordo com o modo de aplicação a ser testado, a difusão de H2O2 foi mensurada em espectroscopia UV-visível pela reação do H2O2 com 4 –amino-2,3-dimetil-1 fenil–3 pirazolin– 5 – ona com comprimento de onda de 510 nm. A densidade óptica da solução foi determinada e convertida em microgramas de H2O2. Os dados de média e desvio-padrão da quantidade de H2O2 detectadas dentro da câmara pulpar entre os fatores foi significante (p < 0,0001). Em geral, não houve diferença entre os grupos quando os géis clareadores testados foram aplicados na técnica 3 x 15 min (p > 0,05). Contudo, houve significativa diferença quando os géis foram aplicados na técnica 1 x 45 min (p < 0,05), sendo os géis clareadores Opalescecence Boost PF 38% e Whiteness HP Blue 35% os que apresentaram os menores valores de peróxido de hidrogênio dentro da câmara pulpar.Baseado nos resultados do presente estudo é possível concluir que: na técnica de aplicação durante três vezes de 15 minutos não houve nenhuma diferença entre os agentes clareadores testados, entretanto, quando aplicados pela técnica de uma vez 45 minutos, os agentes clareadores que contem agentes dessensibilizantes e são alcalinos tiveram os menores percentuais de peróxido de hidrogênio dentro da câmara pulpar.

Os fungos filamentosos acionam mecanismos eficientes para sobrevivência nas diferentes condições ambientais e modulam, por exemplo, a produção de proteínas. A abordagem proteômica tem sido utilizada com sucesso para investigar os mecanismos fisiológicos envolvidos nas adaptações metabólicas desses microrganismos, frente às mudanças de parâmetros físicos e químicos do meio extracelular. No presente trabalho foi avaliado o efeito das variações de pH, fontes de carbono e nitrogênio (aminoácidos e fontes complexas) sobre a produção de peptidases e sobre o proteoma intra- e extracelular do fungo Aspergillus niveus em bioprocessos submersos. As atividades proteolíticas foram avaliadas durante 96 h de cultivo e as melhores induções, entre os estudos comparativos, foram observadas em pH 6, xilose, cisteína e farinha de pena. O efeito dessas variações sobre o proteoma de A. niveus foi avaliado em 96 h de cultivo submerso e, para isso, foram realizados estudos proteômicos nos quais os conteúdos proteicos foram avaliados por eletroforese bidimensional (2-DE); e os spots proteicos selecionados foram identificados por espectrometria de massas (MALDI-TOF/TOF-MS/MS). As proteínas apresentadas no estudo demonstram que as variações de pH, fontes de carbono e nitrogênio influenciaram as proteínas intra- e extracelulares de A. niveus. Os perfis de proteínas mostram que os parâmetros avaliados foram capazes de influenciar a síntese ou degradação de polímeros biológicos e a resposta aos efeitos químicos e/ou disponibilidade de nutrientes do meio extracelular modularam proteínas envolvidas em diversas rotas metabólicas. Além disso, a suplementação com fontes de nitrogênio orgânicas promoveram a indução de enzimas com potencial biotecnológico para aplicação industrial, o que reforça a importância dos estudos proteômicos para a determinação do comportamento de fungos filamentosos frente às condições ambientais / The filamentous fungi activate efficient mechanisms to survive in different environment conditions and protein production modulation may be a tool of these survival adaptation. Proteomic approach has been used as methodology to investigate physiological processes related to these microorganism adaptation after chemical and physical changes in extracelullar medium. In this study, the influence of pH variation, carbon and nitrogen sources (amino acid and complex sources) was analyzed on protease production and in intra- and extracellular proteomics of filamentous fungus Aspergillus niveus during submerged bioprocess. The proteolytic activities were evaluated for 96 hours of incubation and as the best indutors among those studied, there were proteases in pH 6, xylose, cystein and feather meal conditions. The variation effects over A. niveus proteome 96 hours submerged bioprocess was analyzed, the protein contents was separated, evaluated and identified by bidimensional electrophoresys followed by mass spectometry (MALDI-TOF/TOFMS/ MS). The identified proteins showed that bioprocess condition variations could influence on intra- and extracelullar proteins of A. niveus. The protein profiles presented a modulation on synthesis and degradation of biological polymers, and on proteins from metabolic pathway in response to chemical effects and/or extracellular nutrient available. Moreover, the supplementaiton with nitrogen sources promoted the production of enzymes with biotechnological potential on industrial application, highlighting the importance of proteomics to determine the filamentous fungus behavior under environment changes.

The aim of this study was to investigate the molecular interactions of the anti-cancer drug Doxorubicin (DOX) with poly(methacrylic acid) grafted starch nanoparticles (PMAA-g-St). In order to fully understand the DOX/PMAA-g-St system, we conducted in-depth studies on DOX dimer dissociation and DOX/PMAA-g-St binding interactions using various techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence and absorption spectroscopy. Based on our experimental results, we developed a quantitative thermodynamic model with relevant parameters such as dissociation constant, Kd, as well as enthalpy of binding, ΔH, in order to explain DOX/PMAA-g-St interactions. In addition, we also studied the effect of environmental factors such as pH and NaCl on DOX self-association and DOX/PMAA-g-St complex formation. In conclusion, the combination of results obtained from various techniques as well as the multispecies equilibrium model, enables us to interpret quantitatively the data of drug loading onto and release from polymeric nanoparticles.

The aim of this study was to investigate the molecular interactions of the anti-cancer drug Doxorubicin (DOX) with poly(methacrylic acid) grafted starch nanoparticles (PMAA-g-St). In order to fully understand the DOX/PMAA-g-St system, we conducted in-depth studies on DOX dimer dissociation and DOX/PMAA-g-St binding interactions using various techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence and absorption spectroscopy. Based on our experimental results, we developed a quantitative thermodynamic model with relevant parameters such as dissociation constant, Kd, as well as enthalpy of binding, ΔH, in order to explain DOX/PMAA-g-St interactions. In addition, we also studied the effect of environmental factors such as pH and NaCl on DOX self-association and DOX/PMAA-g-St complex formation. In conclusion, the combination of results obtained from various techniques as well as the multispecies equilibrium model, enables us to interpret quantitatively the data of drug loading onto and release from polymeric nanoparticles.

Dans cette thèse, j'ai essayé d'identifier le mode d'action de composés biscationiques récemment synthétisés et leurs interactions avec les parasites résistants à la chloroquine. Les activités de divers représentants des composés ammonium bisquaternaire, des amidines alkyl (provenant du groupe de professeur Vial, Montpellier, France), des amidines bisbenzyl (provenant du groupe Chimie de l'Université de Liverpool, Royaume-Uni) ont été comparés à la chloroquine et la pentamidine. Leurs potentiels de résistance croisée avec la chloroquine ont également été étudiés. Dans ce but, deux lignées cellulaires modifiées génétiquement par échange allélique, C3Dd2 et C2GCO3 furent utilisées.Parmi les amidines bisbenzyl, une série de composés appartenant aux guanidines, thiazoles et triazoles ont été criblés pour leur activité contre des souches résistantes et sensibles à la chloroquine chez Plasmodium falciparum. Une hypersensibilité significative est observée pour les amidines bisbenzyl parmi les isolats affichant un PfCRT mutant. Aucune différence n'est observée pour les composés provenant du groupe Vial. Pour comprendre le mode d'action et le rôle de PfCRT, j'ai réalisé des expériences de fixation compétitives (competitive binding') et de cristallisation d'hème. Tous les composés ont montré à différents degrés des interactions avec l'hème, cependant il fut observé que leur activité ne corrélait pas avec l'inhibition de la cristallisation d'hème. Une des raisons possibles à cela est que les différences structurales peuvent jouer un rôle important dans le transport du composé. De plus, j'ai étudié l'effet du pH sur l'activité des composés en utilisant les lignées cellulaires modifiées génétiquement par échange allélique afin d'observer l'effet du gradient de proton sur le transport de la chloroquine et de la pentamidine. Des différences significatives de l'activité de la chloroquine furent observées chez les deux souches. Malgré les valeurs de pKa élevées pour la pentamidine, il y avait une différence significative dans la sensibilité pour ce composé chez les souches quand le pH a changé.Car les diamidines requièrent des transporteurs pour traverser les barrières membranaires et qu'un possible transporteur de choline a été caractérisé chez Plasmodium falciparum, j'ai également réalisé des études initiales sur la caractérisation moléculaire de ce transporteur. Un gène qui encode une protéine chez P. falciparum avec une similarité significative aux eucaryotes supérieurs fut identifié en utilisant des analyses bioinformatiques et fut employé dans une transformation et des études analyses fonctionnelles.En conclusion, ce travail suggère qu'il est possible d'utiliser de nouveaux amidines bisbenzyl pour cibler spécifiquement les souches résistantes à la quinoléine chez Plasmodium falciparum, arborant des allèles de PfCRT mutantes. En adhérant à cette hypothèse et sachant que les deux classes de composés fixent la même cible non parasitaire (soit l'hème), il serait possible de créer rationnellement une combinaison de composés quinoléine / diamidine. Ainsi, les souches résistantes à un des deux composés seraient plus sensibles à l'autre partenaire, retardant ainsi l'apparition de résistance. / In this thesis I have attempted to subject the issues of mode of action of recently synthesized bis cationic compounds and their possible interactions with chloroquine resistance. Antimalarial activities of representatives of various bis quarternary ammonium compounds, alkyl amidines (received from Dr.Vial group, Montpellier) and of bisbenzyl amidines (received from Chemistry group, Liverpool) activity have been investigated with chloroquine and pentamidine and looked for cross resistance with chloroquine. For this purpose two genetically modified allelically exchanged cell lines C3Dd2 and C2GCO3 modified on the chloroquine resistance-related PfCRT (P.falciparum chloroquine Resistance Transporter) gene were used. Among the benzyl amidines, a significant hypersensitivity tobis benzyl amidines was observed among the isolates bearing the mutant PfCRT. No such difference is observed for the bisalkyl amidines. To understand the mode of action and role of PfCRT, competitive binding assay to heme (which may mediate the well-known cellular accumulation of the compounds) and effect on heme crystallization assays (which is involved in the toxic effect against the intracellular parasite) were performed. All these compounds were shown to interact with heme in various degrees. Their activity was observed not to be correlating with heme crystallization inhibition. This is likely due to the structural differences between the compound which discriminate the compounds in the transport of the compound to the parasite and their mechanism of antimalarial activities. In addition I have studied the effect of pH on the pharmacological activity of the drugs using allelically exchanged genetically modified cell lines (for PfCRT) to characterize the importance of proton gradient on the transport of chloroquine and pentamidine to the intracellular parasite. Significant difference (reduced antimalarial activity with increased pH) in the activity of chloroquine was observed for both the strains. Despite of the high pKa values for pentamidine, there was significant difference in the sensitivity of the strains to this compound, when the pH is changed. As both the diamidines and choline analogs require transporters to cross the membrane barriers and enter the parasite where they accumulate I have also performed initial studies on the molecular characterization of a potential carrier in P.falciparum. Using basic bioinformatic tools, a gene encoding a P.falciparum protein with significant similarity to higher eukaryotes choline transporter was identified and preliminary work for its functional analysis was performed. In conclusion, this work establishes substantial differences between the various classes of bis-cationic compounds essentially (based on benzamidine and choline-analogs alkylkamidine series) concerning their interaction with the infected erythrocyte and their antimalarial activity. The series are diffentallly affected by the PfCRT mutation and the chloroquine resistance. Results suggest that it may be possible to use novel bisbenzyl amidines to specifically target quinoline resistant Plasmodium falciparum malaria, harbouring mutant pfcrt alleles. Taking this idea further and since both classes of compound target the same non-parasite target (heme), it may even be possible to rationally design a quinoline / diamidine drug combination, in which isolates resistant to one partner drug become more sensitive to the other partner, thus delaying the onset of resistance.

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