O nadolol é um agente bloqueador de receptores β-adrenérgicos empregado principalmente, na \"angina pectoris\", hipertensão, certas arritmias cardíacas e no tratamento do glaucoma (SING, 2006). A ivermectina e a abamectina são fármacos que apresentam ação antiparasitária (SHOOP, 1995). Na presente pesquisa, a cromatografia em fase líquida de alta eficiência foi uma das técnicas estudadas para a quantificação dos enantiômeros do nadolol e dos homólogos presentes na abamectina e ivermectina. A versatilidade desta técnica reside no grande número de fases estacionárias existentes, as quais possibilitam análises, separações e determinações quantitativas de uma ampla gama de compostos com alta eficiência (Aquino Neto e Nunes, 2003). Para identificação dos enantiômeros do nadolol foi utilizado o dicroísmo circular que permite a determinação da configuração absoluta de enantiômeros (LIMA, 1997). Para os enantiômeros do nadolol e dos homólogos presentes na abamectina e na ivermectina também foram realizados testes para desenvolvimento de uma metodologia de quantificação por meio de uma técnica relativamente recente chamada de eletroforese capilar (EC), a qual tem alcançado desde sua introdução um rápido desenvolvimento e ampla aplicação na análise de fármacos em medicamentos (SANTORO, 2000). Para a comprovação da qualidade e segurança dos sistemas computadorizados dos equipamentos de cromatografia em fase líquida de alta eficiência (CLAE) e de eletroforese capilar (EC) foram efetuadas, neste trabalho, as respectivas validações. Após esta validação, pode-se confirmar o correto funcionamento de um software, e suas interações com o hardware, onde devem ser levados em consideração, dentre outros, os aspectos relacionados à infra-estrutura, segurança e manutenção de dados (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2010). As metodologias analíticas desenvolvidas a para quantificação do nadolol, abamectina e ivermectina por cromatografia em fase líquida de alta eficiência foram validadas. A validação analítica deve garantir, por meio de estudos experimentais, que o método atenda às exigências das aplicações analíticas, assegurando a confiabilidade dos resultados. Para tanto, o método deve apresentar especificidade, linearidade, intervalo, precisão, sensibilidade, limite de quantificação e detecção, exatidão, adequados à análise (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2003). Portanto, o objetivo proposto nesta pesquisa é primeiramente a validação dos sistemas computadorizados dos equipamentos de cromatografia em fase líquida de alta eficiência (CLAE) e de eletroforese capilar (EC). Para isto, serão desenvolvidos e validados os métodos analíticos de separação, identificação e quantificação dos enantiômeros do nadolol e dos homólogos presentes na abamectina e na ivermectina, em medicamentos, empregando as técnicas analíticas selecionadas. / Nadolol is a blocking agent with activity in the β -adrenergic receptors. It is mainly used in angina, hypertension, certain heart arrhythmias and in the treatment of glaucoma (SING, 2006). Ivermectin and abamectin are drugs with antiparasitic activity (SHOOP, 1995). In the present research, high performance liquid chromatography is one of the techniques used in the quantification of the enantiomers of nadolol and homologues present in abamectin and ivermectin. The versatility of this technique and the large number of existing stationary phases, enables the separation and quantitative determination of a wide range of compounds with high efficiency (Aquino Neto e Nunes, 2003). For identification of the nadolol enantiomers, circular dichroism was used which allows the determination of the absolute configuration of the enantiomers (LIMA, 1997). Nadolol enantiomers and the homologues present in abamectin and ivermectin will be also quantified by capillary zone electrophoresis (CE), a separation technique relatively recent, which has achieved, since its introduction, a wide application in the analysis of drugs in pharmaceutical preparations (SANTORO, 2000). In order to assure the quality of the analytical results, the computer systems of the liquid chromatograph and capillary electrophoresis equipments, must be validated prior to the analytical methods validation. Computer systems validation is used to verify and confirm the proper operation of softwares, and their interactions with the hardwares, besides the infrastructure, safety and storage of data (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2010). The analytical methodologies developed for quantification of nadolol, abamectin, ivermectin by using high efficiency liquid chromatography and capillary electrophoresis were validated. The analytical methods validation should ensure, through experimental studies, that the method meets the requirements for analytical applications, ensuring the reliability of the results. Parameters like, specificity, linearity, range, accuracy, sensitivity, limits of detection and quantification and accuracy, must be determined (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2003). The objective of this study is to validate the computer systems of the high performance liquid chromatograph and capillary electrophoresis equipments and then to develop and validate analytical methods for separation, identification and quantification of nadolol enantiomers and the homologues of abamectin and ivermectin.

As ligações de hidrogênio (LH), fundamentais na determinação da estrutura da água, proteínas, etc., são muito importantes no reconhecimento molecular e nos mecanismos de reações enzimáticas. A determinação da energia das LHs intramoleculares em proteínas e intermoleculares entre uma proteína e o solvente água, porque fornece informações sobre a estrutura secundária, terciária e quaternária das proteínas. Um método para quantificar e qualificar as LHs foi desenvolvido utilizando critérios de distância, geométricos e energéticos a partir das trajetórias obtidas por simulações de dinâmica molecular. O método foi testado com o monômero de uma fosfolipase A2 homodimérica, sem atividade catalítica, isolada do veneno da Bothrops asper(BaspMT-II). No dímero, a análise das LHs mostrou que elas são também essenciais na manutenção da estrutura quaternária. Essa análise permitiu identificar movimentos do tipo dobradiça acompanhados da formação transitória, na interface dimérica, de LHs controladas pelo triptofano na posição 77. Esses movimentos podem estar associados à ação danosa às membranas, uma vez que podem promover a inserção da região C-terminal na membrana. Estudos prévios mostraram que o peptídeo sintético (3Y codificado pelos aminoácidos 115-129 da BaspMT-II) apresenta atividade bactericida e citolítica. Um outro peptídeo (3W), mutante de 3Y, no qual três resíduos tirosina são substituidos por triptofano, apresenta um aumento do dano às membranas e do efeito miotóxico. Os mecanismos de ação desses peptídeos e as suas estruturas foram estudados por dinâmica molecular, dicroísmo circular (DC), microscopia de fluorescência e monocamadas de Langmuir (Mlang). As adsorções dos peptídeos em monocamadas de ácido dimiristoil fosfatídico (DMPA) e dimiristoilfosfatidilcolina (DMPC) se processam por mecanismos diferentes ocasionados pelas diferentes naturezas físico-químicas dos resíduos tirosina e triptofano. A microscopia de fluorescência acoplada a Mlang de DMPA com 3W adsorvido mostra um aumento da fluidez da monocamada, enquanto que o 3Y modifica os domínios do DMPA para pequenas estruturas circulares. Foram realizadas simulações dos peptídeos 3Y e 3W em meio aquoso e nas regiões interfaciais água/n-hexano e água/bicamadas de DMPC. Os resultados confirmam os obtidos por Mlang, demonstrando que os peptídeos interagem diferentemente com as membranas por adotar conformações alternativas definidas previamente. Essas conformações, diferentes das observadas em meio aquoso, dependem da natureza da interface. As estruturas encontradas no final das simulaçoes corroboram o mecanismo proposto por Mlang, assim como as estruturas sugeridas por DC. Isso sugere que a atividade biológica reduzida do peptídeo 3Y ocorre porque os seus dois resíduos Leu se adsorvem na interface sem penetrá-la. Ao contrário de 3W, os resíduos carregados do peptídeo 3Y não estão localizados corretamente para promover uma interação suficientemente atrativa para permitir a sua inserção na membrana celular. / Hydrogen bonds (HB) are highly important in the determination of the structure of the water and proteins. They also play a important role in molecular recognition and in enzyme reaction mechanisms. The determination of protein/water intermolecular and protein intramolecular HB energies provide information with respect to the formation and stabilization of secondary, tertiary and quaternary protein structure. A method that quantifies and qualifies the properties of HB was developed using distance, geometric and energy criteria as applied to data obtained from the atomic trajectories generated by molecular dynamics simulations. The method was tested with a monomer of a catalytically inactive homodimeric phospholipase A2 from Bothrops asper(BaspMT-II) venom. HBs at dimmer interface are essential for maintaining the quaternary structure, and are highly conserved during hinge-like movements of the dimmer. HB formed by tryptophan residue at position 77 controls this movement. These motions can be associated to the membrane damaging action since they facilitate the insertion of the C-terminus into the cellular membrane. Previous studies have shown that synthetic peptide (3Y, coding the amino acids 115-129 of BaspMT-II ) presents bactericidal and cytolitic activities. A peptide variant ( 3W ), in which tyrosine residues were substituted by tryptophan residues, presents an enhanced membrane damaging activity increased miotoxic effect. The mechanism of action of the peptides and their structures were studied by molecular dynamics simulations, circular dichroism (CD), fluorescence microscopy and Langmuir monolayers (Mlang). The adsorption of the peptides on a monolayer composed of dimiristoyl phosphatidic acid (DMPA) and dimiristoylphosphatidyl choline (DMPC) occurs through different processes due to the differences in the physic-chemical nature of the tyrosine and tryptophan residues. Fluorescence microscopy together with Mlang of DMPA with adsorbed 3W indicates an increase of the membrane fluidity while small circular domains are formed with DMPA. Simulations were conducted with the 3Y and 3W peptides in aqueous media, is a water/n-hexane and water/DMPC bilayers. The results confirm the Mlang results, showing that the peptides interact differently with the membranes by adopting alternative previously defined conformations. These two conformations, both of which are different to those observed in water, are dependent of the nature of the interfaces. The final simulated configurations confirm the mechanism proposed by Mlang and the structures proposed by CD. It is suggest that the reduced biological activity of the 3Y peptide is due to the two Leu residues that only adsorb to the cellular membrane without penetrating the bilayer. In contrast to the 3W peptide, no charged residue is correctly located to promote the interaction and insertion of the 3Y peptide into the membrane.

Die vorliegende Arbeit befasst sich mit der Untersuchung der mikromagnetischen Domänenstruktur und des gekoppelten magneto-strukturellen Phasenübergangs dünner epitaktischer MnAs-Filme auf GaAs. Im Besonderen wird der Einfluss der Substratorientierung, der Filmdicke und eines externen magnetischen Feldes auf die magnetischen und strukturellen Eigenschaften untersucht. Dabei kommen die komplementären Untersuchungsmethoden AFM (atomic force microscopy) / MFM (magnetic force microscopy) und LEEM (low energy electron microscopy) / XMCDPEEM (X-ray magnetic circular dichroism photoemission electron microscopy) zum Einsatz. Im Zuge des Phasenübergangs erster Ordnung zeigen MnAs Filme auf GaAs (001) und (311)A eine regelmäßige Anordnung ferromagnetischer alpha-MnAs und paramagnetischer beta-MnAs Streifen. Die Breite der Streifen ist eine Funktion der Temperatur, während die Periodizität eine lineare Funktion der Filmdicke ist. Die Domänenstruktur hängt stark von der Breite bzw. dem Abstand der ferromagnetischen Streifen ab, da diese direkt die Formanisotropie bzw. die magnetische Kopplung beeinflussen. Die Domänenstrukturen wird, abhängig von der Zahl der Subdomänen entlang der leichten Magnetisierungsrichtung, klassifiziert, wobei bis zu drei elementare Domänentypen beobachtet werden. Bei MnAs-Filmen die auf der GaAs (111)B Oberfläche gewachsen wurden, führt die Epitaxie zu einem geänderten Spannungszustands des Films, wobei eine erhöhte Phasenübergangstemperatur beobachtet wird. Durch temperaturabhängige XMCDPEEM-, AFM- und MFM-Messungen kann gezeigt werden, daß durch den lokalen Abbau der Verspannung in der Nähe eines Risses die Phasenübergangstemperatur lokal erhöht ist. Um Ummagnetisierungsprozesse auf einer mikroskopischen Skala untersuchen zu können und um den Einfluß eines magnetischen Feldes auf die Domänenstruktur sichtbar zu machen, wurde das temperaturvariable Rastersondenmikroskop um einen variablen Magnetfeldaufbau ergänzt. / This work presents the study of the micromagnetic domain structure and the coupled magneto-structural phase transition of epitaxial MnAs thin films on GaAs. In particular, the influence of substrate orientation, film thickness and external magnetic field on the magnetic and structural properties are investigated, employing the complementary measurement techniques atomic force microscopy (AFM) / magnetic force microscopy (MFM) and low energy electron microscopy (LEEM) / X-ray magnetic circular dichroism photoemission electron microscopy (XMCDPEEM. In the course of the first-order phase transition MnAs films on GaAs (001) and (311)A substrates show a regular array of ferromagnetic alpha- and paramagnetic beta-MnAs stripes. The width of the ferromagnetic stripes are a function of the temperature, whereas the periodicity of the stripe pattern is a function of the film thickness. The domain structure strongly depends on the width and the distance of the ferromagnetic stripes, as it directly affects the shape anisotropy and magnetic coupling, respectively. The domain patterns are classified depending on the number of subdomains along the easy axis direction. Up to three basic domain types can be distinguished. For MnAs films grown on GaAs (111)B, the epitaxy leads to a different strain state of the film, resulting in polygonal ferromagnetic structures embedded in a honeycomb-like paramagnetic network, and a higher phase transition temperature. Using temperature-dependent AFM, MFM and XMCDPEEM it is shown that the local strain relaxation in the vicinity of cracks in the MnAs film results in a locally increased phase transition temperature. In order to study magnetization reversal processes on a microscopic scale, as well as the influence of the magnetic field on the domain structure, a variable-magnetic field set-up is employed.

Le dichroïsme circulaire de photoélectrons (PECD) se manifeste par une intense asymétrie avant/arrière dans la distribution angulaire de photoélectrons selon l’axe des photons, lors de la photoionisation d’un énantiomère pur de molécule chirale en phase gazeuse par un rayonnement polarisé circulairement. Cette thèse est consacrée à l’étude de ce phénomène, avec le synchrotron de la ligne DESIRS, sur différents systèmes chiraux allant de molécules libres (composés bromés, alanine) aux agrégats de glycidol et aux complexes formés avec l’eau. Pour ce faire, des mesures expérimentales menées à l’aide du spectromètre double imageur à coïncidence électron/ion DELICIOUS 3 ont été couplées à des calculs théoriques (CMS-Xα). Le chapitre 1 introduit les concepts fondamentaux nécessaires à l’étude de la chiralité en physique et le chapitre 2 présente les méthodes expérimentales et théoriques mises en œuvre. Le chapitre 3 détaille les résultats obtenus sur des composés chiraux halogénés : l’acide bromo-propionique (BPA) et les isomères 1,3- et 1,4-bromo-phényléthylamine (BrPhEtA). La sensibilité du PECD à l’isomérie, de même que le rôle de la localisation de l’orbitale initiale par rapport au centre chiral sont au cœur de cette étude. Le PECD du plus simple acide aminé chiral protéique, l’alanine, produit par chauffage résistif et par thermo-désorption d’aérosol, est présenté au chapitre 4 avec une étude conformationnelle selon la température conduisant à une distribution plausible des conformères. Ces résultats sont ensuite discutés dans le contexte de l’homochiralité de la vie (i.e. le fait que les acides aminés sont tous de type L dans la biosphère), le PECD étant un processus photophysique asymétrique pouvant induire un enrichissement énantiomérique. Le chapitre 5 s’intéresse au PECD sur des systèmes plus complexes : les agrégats de glycidol et les complexes de glycidol et d’eau. Une simple sélection en masse permet de mettre en évidence un effet spectaculaire de l’agrégation sur le PECD observé. DELICIOUS 3 permet ensuite d’éliminer des processus en cascade par une sélection en taille des neutres natifs. / Photoelectron Circular Dichroism (PECD) is observed as a forward/backward asymmetry, with respect to the photon axis, of the photoelectron angular distributions resulting from the circularly polarized light-induced photoionization of gas phase pure enantiomers of chiral species. This thesis is devoted to the study, with the synchrotron of the DESIRS beamline, of this phenomenon on different chiral systems from free molecules (bromine compounds, alanine) to glycidol clusters and water-glycidol complexes. Chapter 1 introduces the fundamental concepts required for the study of chirality in physics and chapter 2 presents the experimental and theoretical methods which have been used. Chapter 3 gives details concerning the results on the halogenated chiral compounds: bromopropionic acid (BPA) and 1,3- and 1,4-bromo-phenylethylamine isomers (BrPhEtA). The sensitivity of PECD to isomerism as well as the role of the localization of the initial orbital of the outgoing electron with respect to the chiral center is at the core of this study. The PECD of the simplest proteic chiral amino acid, alanine, produced by resistive heating and thermodesorption of an aerosol, is introduced in chapter 4 with a conformational study depending on temperature, leading to a plausible conformer distribution. These results are then discussed in the context of homochirality of life (i.e. the fact that only L-amino acids are found in the biosphere) as PECD is an asymmetric photophysical processes which can induce an enantiomeric enrichment. Chapter 5 is focused on PECD in systems of greater complexity: glycidol clusters and water-glycidol complexes. A simple mass selection enables to unravel a spectacular clustering effect on the observed PECD and the use of DELICIOUS 3 enables then to remove any cascading processes thanks to a size selection of nascent species.

Muitas espécies do gênero Aristolochia (Familia Aristolochiaceae) têm sido usadas na medicina tradicional e folclórica como medicamentos e tônicos, as quais demonstravam atividades farmacológicas de interesse clínica e medica como anti-hemorrágica, anti-parasita, antibacteriano, antifúngico, analgésico, antitumoral entre outras. Visando a obtenção de mais informações sobre essas plantas e na procura por substâncias com efeitos antiofídicos, neste trabalho avaliou-se à ação de extratos aquoso, metanólico e de acetato de etila de folhas e caule contra as ações tóxicas da peçonha de Bothrops asper, ambos procedentes do Panamá e contra o efeito miotóxico da peçonha de Bothrops jararacussu e das miotoxinas BthTX-I (isolada de B. jararacussu) e Mtx-II (isolada de B. asper). O extrato das folhas em acetato de etila apresentou a melhor inibição da atividade fosfolipásica da peçonha de B. asper, demonstrando inibição de 45%, 35% e 33% nas proporções de 1:5, 1:10 e 1:30 (m/m), respectivamente. Enquanto que, o extrato de caule em acetato de etila demonstrou maior eficácia na neutralização da atividade coagulante, e, além disso, inibiu 96%, 92% e 87% do edema, da miotoxicidade e hemorragia induzidas pela peçonha de B. asper, respectivamente. Os percentuais diferenciados na neutralização das ações tóxicas da peçonha de Bothrops asper, revelam diferentes perfis do potencial antiofídico de Aristolochia sprucei. Um dos componentes bioativos foi isolado do extrato de caule desta planta por CLAE, e a caracterização química, por ressonância magnética nuclear, demonstrou ser o ácido aristolóquio que inibiu a atividade miotóxica das peçonhas de B. jararacussu e de B. asper em 80% e 85% e assim como a atividade miotóxica da BthTX-I e Mtx-II em 64% e 60%, respectivamente. A atividade hemolítica indireta da peçonha de B. asper foi inibida em 43% pelo o ácido aristolóquio. A análise dos espectros de dicroísmo circular e os estudos de interação por modelagem molecular sugerem que o ácido aristolóquio forma um complexo com a Mtx-II de B. asper inibindo sua atividade. A ligação do ácido aristoloquio com as miotoxinas (MjTX-1, BthTX-II) modificou a forma e a intensidade dos espectros de dicroísmo circular da miotoxina e induziu alterações na porcentagem dos diversos domínios que constituem a estrutura secundária desta miotoxina. Os resultados obtidos confirmam que os extratos de A. sprucei possuem propriedades antiofídicas e sugerem a necessidade de aprofundar estudos que permitam utilizar com segurança os extratos e o principio ativo isolado como suplementos dos antisoros para aumentar a eficácia na neutralização dos efeitos tóxicos locais da peçonha das serpentes. / A lot of species of genus Aristolochia (Familia Aristolochiacheae) have been used in traditional medicine and folk, such as medicaments and tonics, which show pharmacological activities of clinic and medical interest, like antihemorragic, antiparasitic, antibacterial, antifungic, analgesic, antitumoral between others. Expecting to get more information about these plants and in the search by substances with antiophidic effects, in this work was evaluated the action of aqueous, metanolic and ethyl acetate extracts from leaves and stems of Aristolochia sprucei against the toxic action of Bothrops asper venom, both native from Panamá and against the myotoxic effect of Bothrops jararacussu venom and BthTX1 (isolated from B. jararacussu) and Mtx-II (isolated from Bothorps asper). The leaves extracts in ethyl acetate showed the best inhibition registered of PLA2 activity of venom de B. asper showing inhibition of 45 %, 35 % and 33 %, in proportion (m/m) of 1:5, 1:10 and 1:30 respectively. As regards to stem extract in ethyl acetate, it showed high efficacy in neutralization of coagulant activity, besides It inhibited 96 %, 92 % and 87 % of edema, myotoxicity and hemorrhage induced by B. asper venom, respectively. One of bioactives components was isolated from stem extract of this plant by CLAE and the chemical characterization by nuclear magnetic resonance, this showed that the compound is the aristolochic acid. This compound inhibited the myotoxic activity of B. jararacussu and B. asper venom in 80 % and 85 %, so like myotoxic activity of BthTx-I and MTx-II in 64 % and 60 % respectively. The indirect hemolytic activity of B. asper venom was inhibited in 43 % by the aristolochic acid. The analyze of spectrum of circular dichroism and the studies of interaction by molecular modelagem suggest that the aristolochic acid forms a complex 1:1 with the miotoxin inhibiting their activity. The joint of aristolochic acid with the miotoxins (MjTX-1, BthTx-II) changes the way and the intensity of spectra from dichroism circular of miotoxin and It induced alteration in percentage of several domains that constitute a secondary structure from this toxin. The results obtained confirm that the extracts of A. sprucei have antiophidic properties and it suggest the necessity of deepen studies that allow to use with safety the extracts and the isolated active principle, like antiserum supplements to increase the efficacy in the neutralization of local toxics effects of snakes venoms.

Cette thèse est dédiée à l’étude et l’exploitation des propriétés remarquables de la distribution angulaire des photoélectrons dans le référentiel moléculaire (MFPAD pour Molecular Frame Photoelectron Angular Distribution). Cette observable sensible permet de caractériser au meilleur niveau à la fois les processus de photoionisation (PI) de petites molécules, par la détermination des modules et phases relatives des éléments de matrice dipolaire, et l’état de polarisation du rayonnement ionisant, entièrement encodé dans son expression sous la forme des paramètres de Stokes (s1, s2, s3), qui constituent les deux axes du travail présenté. La mesure des MFPADs combine une technique de spectroscopie tridimensionnelle en coïncidence des vecteurs vitesse du photoélectron et du photoion issus de la même réaction de photoionisation dissociative (PID) à l’utilisation de différentes sources de lumière : le rayonnement synchrotron d’une part, sur les lignes DESIRS et PLEIADES (SOLEIL), et le rayonnement harmonique (HHG) généré par l’interaction d’un champ laser intense sur une cible gazeuse d’autre part, sur la plateforme PLFA (SLIC, groupe Attophysique du LIDyL, CEA Saclay).La première partie de la thèse est consacrée à la caractérisation complète de l’état de polarisation d’un rayonnement incident. Pour cela, une méthode originale de « polarimétrie moléculaire » est introduite et qualifiée sur la ligne DESIRS équipée d’un polarimètre optique VUV étalon. Mise en œuvre pour déterminer complètement l’ellipse de polarisation du rayonnement HHG produit dans différentes configurations sur la source secondaire XUV PLFA, elle conduit à des résultats originaux incluant notamment la distinction entre les composantes circulaire et non polarisée du rayonnement étudié.La seconde partie porte sur la PID des molécules H2, D2 et HD induite à résonance avec les états doublement excités Q1 et Q2 par une lumière polarisée circulairement. Dans cette région énergétique (30-35 eV) où l’ionisation directe, l’autoionisation et la dissociation sont en compétition sur une échelle de quelques femtosecondes, l’excitation photonique donne lieu à une dynamique couplée électron-noyau ultrarapide. Les asymétries remarquables observées dans le dichroïsme circulaire dans le référentiel moléculaire, comparées aux calculs quantiques du groupe de F. Martín (UAM, Madrid), constituent une sonde sensible des interférences quantiques entre des chemins réactionnels indiscernables impliquant des cœurs ioniques de symétrie u/g différente. / This is thesis is dedicated to the study and the use of the remarkable properties of the molecular frame photoelectron angular distribution (MFPAD). This observable is a very sensitive probe of both the photoionization (PI) processes in small molecules, through the determination of the magnitudes and relative phases of the dipole matrix elements, and the polarization state of the ionizing light, which is entirely encoded in the MFPAD in terms of the Stokes parameters (s1, s2, s3). MFPAD measurements take advantage of dissociative photoionization (DPI) processes by combining an electron-ion 3D momentum spectroscopy technique with the use of different radiation facilities: SOLEIL synchrotron (DESIRS and PLEIADES beamlines) and the XUV PLFA beamline (SLIC, LIDyL Attophysics group, CEA Saclay) based on the interaction of a strong laser field with a gaseous target called high harmonic generation (HHG).The first part of the thesis is devoted to the complete characterization of the polarization state of an incoming radiation. In this context, an original “molecular polarimetry” method is introduced and demonstrated by comparison with a VUV optical polarimeter available on the DESIRS beamline. Using this method to determine the full polarization ellipse of HHG radiation generated in different conditions on the XUV PLFA facility leads to original results that include the challenging disentanglement of the circular and unpolarized components of the studied radiation.The second part deals with the study of DPI of the H2, D2 and HD molecules induced by circularly polarized light at resonance with the doubly excited states Q1 and Q2. In this energy region (30-35 eV) where direct ionization, autoionization and dissociation compete on a femtosecond timescale, the photonic excitation gives rise to complex ultrafast electronic and nuclear coupled dynamics. The remarkable asymmetries observed in the circular dichroism in the molecular frame, compared to quantum calculations of the group of F. Martín (UAM, Madrid), constitute a very sensitive probe of the quantum interferences between indistinguishable DPI channels involving ionic states of different u/g symmetry.

L'objectif principal de cette thèse est l’étude spectroscopique de molécules ou de complexes portant plusieurs centres chiraux en phase gazeuse, pour comprendre les effets de la stéréochimie sur leurs propriétés structurales. Des alcaloïdes dérivés de la Cinchonine ont été introduits intacts en phase gazeuse par ablation laser. Ils ont été étudiés en combinant un jet supersonique avec de la spectroscopie laser. Les deux pseudo-énantiomères Quinine et Quinidine ont montré des spectres électroniques et vibrationnels similaires, en accord avec leur structure similaire. Leurs propriétés en solution diffèrent davantage, comme le montrent les expériences de dichroïsme circulaire vibrationnel (VCD). Cette différence est encore plus marquée dans l’Hydroquinine et l’Hydroquinidine. Enfin la reconnaissance chirale a été étudiée dans des complexes ioniques dans un piège à ions. La stabilité des complexes formés entre S-camphre et les R et S-Alanine protonées indique une préférence homochirale. Cependant, l'énergie d'interaction calculée ainsi que les spectres IRMPD dans la région des empreintes digitales sont identiques. Le rôle des conformères plus hauts en énergie dans la reconnaissance chirale a été discuté. / The main objective of this thesis is a spectroscopic study of molecules or complexes bearing multiple chiral centres in the gas phase, to understand the effects of stereochemistry on their structural properties. Neutral cinchona alkaloids have been introduced intact in gas phase by laser-ablation. They have been studied by combining supersonic expansion with laser spectroscopy. The two pseudo-enantiomers Quinine and Quinidine show similar electronic and vibrational spectra, in line with similar structure. Their properties in solution differ more, as shown by Vibrational Circular Dichroism (VCD) experiments. This difference is further enhanced in Hydroquinine and Hydroquinidine. Lastly chiral recognition has been studied in ionic complexes in an ion trap. A homochiral preference has been shown in the stability of the complexes formed between S-Camphor and R and S protonated Alanine. However, the calculated interaction energy as well as the IRMPD spectrum in the fingerprint region are identical. The role of higher energy conformers in chiral recognition has been discussed.

O nadolol é um agente bloqueador de receptores β-adrenérgicos empregado principalmente, na \"angina pectoris\", hipertensão, certas arritmias cardíacas e no tratamento do glaucoma (SING, 2006). A ivermectina e a abamectina são fármacos que apresentam ação antiparasitária (SHOOP, 1995). Na presente pesquisa, a cromatografia em fase líquida de alta eficiência foi uma das técnicas estudadas para a quantificação dos enantiômeros do nadolol e dos homólogos presentes na abamectina e ivermectina. A versatilidade desta técnica reside no grande número de fases estacionárias existentes, as quais possibilitam análises, separações e determinações quantitativas de uma ampla gama de compostos com alta eficiência (Aquino Neto e Nunes, 2003). Para identificação dos enantiômeros do nadolol foi utilizado o dicroísmo circular que permite a determinação da configuração absoluta de enantiômeros (LIMA, 1997). Para os enantiômeros do nadolol e dos homólogos presentes na abamectina e na ivermectina também foram realizados testes para desenvolvimento de uma metodologia de quantificação por meio de uma técnica relativamente recente chamada de eletroforese capilar (EC), a qual tem alcançado desde sua introdução um rápido desenvolvimento e ampla aplicação na análise de fármacos em medicamentos (SANTORO, 2000). Para a comprovação da qualidade e segurança dos sistemas computadorizados dos equipamentos de cromatografia em fase líquida de alta eficiência (CLAE) e de eletroforese capilar (EC) foram efetuadas, neste trabalho, as respectivas validações. Após esta validação, pode-se confirmar o correto funcionamento de um software, e suas interações com o hardware, onde devem ser levados em consideração, dentre outros, os aspectos relacionados à infra-estrutura, segurança e manutenção de dados (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2010). As metodologias analíticas desenvolvidas a para quantificação do nadolol, abamectina e ivermectina por cromatografia em fase líquida de alta eficiência foram validadas. A validação analítica deve garantir, por meio de estudos experimentais, que o método atenda às exigências das aplicações analíticas, assegurando a confiabilidade dos resultados. Para tanto, o método deve apresentar especificidade, linearidade, intervalo, precisão, sensibilidade, limite de quantificação e detecção, exatidão, adequados à análise (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2003). Portanto, o objetivo proposto nesta pesquisa é primeiramente a validação dos sistemas computadorizados dos equipamentos de cromatografia em fase líquida de alta eficiência (CLAE) e de eletroforese capilar (EC). Para isto, serão desenvolvidos e validados os métodos analíticos de separação, identificação e quantificação dos enantiômeros do nadolol e dos homólogos presentes na abamectina e na ivermectina, em medicamentos, empregando as técnicas analíticas selecionadas. / Nadolol is a blocking agent with activity in the β -adrenergic receptors. It is mainly used in angina, hypertension, certain heart arrhythmias and in the treatment of glaucoma (SING, 2006). Ivermectin and abamectin are drugs with antiparasitic activity (SHOOP, 1995). In the present research, high performance liquid chromatography is one of the techniques used in the quantification of the enantiomers of nadolol and homologues present in abamectin and ivermectin. The versatility of this technique and the large number of existing stationary phases, enables the separation and quantitative determination of a wide range of compounds with high efficiency (Aquino Neto e Nunes, 2003). For identification of the nadolol enantiomers, circular dichroism was used which allows the determination of the absolute configuration of the enantiomers (LIMA, 1997). Nadolol enantiomers and the homologues present in abamectin and ivermectin will be also quantified by capillary zone electrophoresis (CE), a separation technique relatively recent, which has achieved, since its introduction, a wide application in the analysis of drugs in pharmaceutical preparations (SANTORO, 2000). In order to assure the quality of the analytical results, the computer systems of the liquid chromatograph and capillary electrophoresis equipments, must be validated prior to the analytical methods validation. Computer systems validation is used to verify and confirm the proper operation of softwares, and their interactions with the hardwares, besides the infrastructure, safety and storage of data (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2010). The analytical methodologies developed for quantification of nadolol, abamectin, ivermectin by using high efficiency liquid chromatography and capillary electrophoresis were validated. The analytical methods validation should ensure, through experimental studies, that the method meets the requirements for analytical applications, ensuring the reliability of the results. Parameters like, specificity, linearity, range, accuracy, sensitivity, limits of detection and quantification and accuracy, must be determined (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2003). The objective of this study is to validate the computer systems of the high performance liquid chromatograph and capillary electrophoresis equipments and then to develop and validate analytical methods for separation, identification and quantification of nadolol enantiomers and the homologues of abamectin and ivermectin.

S100A12 porcina é um membro da família das proteínas S100, um grupo de pequenas proteínas ligantes de cálcio caracterizado pela presença de dois motivos “EF-hand". Estas proteínas estão envolvidas em diversos eventos celulares, como a regulação da fosforilação protéica, atividade enzimática, tamponamento de Ca+2, processos inflamatórios e a polimerização de filamentos intermediários. Adicionalmente, algumas dessas proteínas podem ligar Zn+2, o qual pode afetar a ligação do íon Ca+2, particularmente para as proteínas S100. Neste trabalho, a seqüência gênica que codifica a proteína S100A12 porcina foi obtida por meio da construção de um gene sintético usando códons preferenciais para E.coli, permitindo a produção recombinante de grandes quantidades da proteína. Um estudo termodinâmico da estabilidade estrutural foi realizado, assim como a interação da proteína recombinante com íons divalentes usando técnicas de dicroísmo circular (CD) e fluorescência extrínseca. A desnaturação e renaturação induzidas por uréia ou temperatura indicam que se trata de um processo reversível e que a ligação dos íons Zn+2 e ou Ca+2 à rS100A12 aumenta sua estabilidade. A interação da sonda ANS com a proteína na presença de seus ligantes expõe superfícies hidrofóbicas podendo assim facilitar sua interação com macromoléculas alvo. Analisados em conjunto, os resultados obtidos indicam que S100A12 porcina é capaz de assumir diferentes conformações as quais podem estar correlacionadas com sua função fisiológica. / Porcine S100A12 is a member of S100 family, a small acidic calcium-binding proteins group characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events as the regulation of protein phosphorylation, enzymatic activity, Ca+2 homeostasis, inflammatory processes and intermediate filament polymerization. In addition, some of these proteins can bind Zn+2, which can affect the binding of Ca+2 particularly to S100 proteins. In this study, the gene sequence encoding S100A12 was obtained by the synthetic gene approach using E. coli codon bias allowing the recombinant production of large amounts of the protein. We report here a thermodynamic study on the structural stability of this recombinant protein and its interaction with divalent ions using circular dichroism and extrinsic fluorescence. The folding/unfolding induced by urea or temperature indicated a reversible process and the binding of Zn+2 or Zn+2 and Ca+2 to S100A12 increasing its stability. The interaction of the ANS probe with the protein in the ligant presence can lead to exposition of hydrofobic regions allowing its interaction with target macromolecules. Taken together, the results indicated that porcine S100A12 may assume different conformations that could be correlated to its physiological function.

Les huiles essentielles sont connues pour leur richesse en molécules chirales. L'identification et la caractérisation de ces différentes molécules en termes de la configuration absolue des énantiomères majoritaires représente un important pas dans la compréhension des actions thérapeutiques des huiles essentielles. Dans le cadre de cette thèse, une étude est réalisée sur la composition chimique des huiles essentielles de quelques plantes aromatiques du Sahara algérien (l'Artemisia herba-alba, le Bubonium graveolens et l'Artemisia arborescens) ayant un usage fréquent dans la pharmacopée traditionnelle. Différentes techniques analytiques chromatographiques telles que la CG-SM classique et chirale, la CLHP chirale et spectroscopiques comme l'IR et le VCD seront mises en œuvre afin d’étudier les constituants chiraux de ces HE pour la connaissance de leurs signatures chiroptiques qui peuvent être des paramètres essentiels pour leurs caractérisations. En associant la performance du traitement chimiométrique, la fiabilité des techniques spectroscopiques et le potentiel discriminant de la signature chirale, nous avons développé des outils de caractérisation, de contrôle qualité et de traçabilité des HE. Les configurations absolues de la (-)-α-thujone, la (+)-β-thujone, l'acetate de (-)-cis-chrysanthenyl, le (+)-oxocyclonerolidol et l'acetate de (-)-cis-acetoxychrysanthenyl ont été obtenues par la comparaison des spectres VCD expérimentaux et calculés et on a montré qu'on peut utiliser le VCD pour l'étude et la modélisation des matrices complexes. / Essential oils are known for their richness in Chiral molecules. Identification and characterization of these different molecules in terms of absolute configuration of the majors enantiomers represents an important step in the understanding of the therapeutic actions of essential oils. In this thesis, a study was done to investigate the chemical composition of the essential oils of some aromatic plants of the Algerian Sahara (Artemisia herba-alba, Bubonium graveolens and Artemisia arborescens) frequently used in the traditional pharmacopoeia. Different chromatographic analytical techniques such as classic and chiral GC-MS, chiral HPLC and spectroscopic techniques as IR and VCD will be implemented to study the chiral constituents in these EO for the knowledge of their chiroptical signatures which can be essential parameters for their characterizations. Combining chemometrics processing performance, reliability of spectroscopic techniques and potential discriminating chiral signature, we have developed tools for the characterization, quality control and traceability of EO. Absolute configuration of (-)-α-thujone, (+)-β-thujone, (-)-cis-chrysanthenyl acetate, (+)-oxocyclonerolidol and (-)-cis-acetoxychrysanthenyl acetate were obtained by comparison of calculated and experimental VCD spectra and we demonstrated that VCD can be used for the study and modeling of complex matrices.