Orientador: Eloi da Silva Feitosa / Banca: Pietro Ciancaglini / Banca: Rosangela Itri / Banca: João Ruggiero Neto / Banca: Vera Aparecida de Oliveira Tiera / Resumo: Estudos de calorimetria diferencial de varredura (DSC) e fluorescência de estado estacionário da sonda Vermelho do Nilo indicam a formação de vesículas de DODAX (X = Cl- ou Br-) em concentrações de surfactantes tão baixas quanto 10 µM. Estas vesículas foram denominadas microvesículas (µV), cuja Tm diminui monotonicamente com a concentração de DODAX até valores de Tm das vesículas tradicionais preparadas em 1.0 mM do surfactante. O efeito do contra-íon (Br- e Cl-) no comportamento termotrópico de fase das vesículas mistas de DODAB-DODAC foi investigado por DSC, condutimetria e espalhamento dinâmico de luz (DLS). Observou-se que a Tm aumenta sigmoidalmente de 45.8 a 48.9 oC com a fração molar de DODAC (xDODAC), com um ponto de inflexão no ponto eqüimolar. A condutividade e o diâmetro hidrodinâmico das vesículas variam muito pouco com xDODAB, indicando que a densidade superficial de carga das vesículas de DODAX é semelhante entre si, e o efeito do contra íon na Tm de DODAX se deve a interações específicas desses contra-íons na interface das vesículas. Medidas de DSC, fluorescência e turbidez de misturas de DODAB-DDAB indicam que as vesículas de DODAB têm maior afinidade por DDAB do que o oposto, resultando na formação de duas populações de vesículas mistas de DDAB-DODAB, com propriedades distintas. Além disso, medidas de fluorescência mostraram que a presença de pequena quantia de DODAB incorporado nas vesículas de DDAB causa um efeito significante na emissão da sonda devido ao aumento do tamanho das vesículas, sugerido por medidas de turbidez. O estudo dos sistemas DODAB/CnTAB/água na concentração total de surfactante igual a 1,0 e 5,0 mM, variando a concentração de CnTAB, e também mantendo a concentração de DODAB fixa em 1,0 mM, revelou uma forte dependência do comprimento da cadeia de hidrocarbonetos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Differential scanning calorimetry (DSC) and fluorescence of the probe Nile Red studies reveal the formation of DODAX vesicles (X = Br- and Cl-) at surfactant concentrations as low as 10 µM. These vesicles were referred to as microvesicles (mV), whose Tm decreases monotonically with increasing DODAX concentration to the value for the ordinary vesicles at 1 mM. The effect of counterion (Br- and Cl-) on the thermotropic phase behavior of mixed DODAB-DODAC vesicles were investigated by differential scanning calorimetry (DSC), conductimetry and dynamic light scattering (DLS). Tm increases sigmoidally from 45.8 to 48.9 oC with DODAC molar fraction (xDODAC), with an inflection point at the equimolarity. The conductivity and the apparent hydrodynamic diameter vary little with xDODAB, indicating that the surface charge density is similar for DODAX, evidencing that the effect of counterion on Tm is due to the counterion specific interactions. DSC, fluorescence and turbidity measurements indicate a higher affinity of DDAB for DODAB vesicles than the reverse, resulting in two populations of mixed DDAB-DODAB vesicles with different properties. Besides, fluorescence measurements show that the presence of a small amount of DODAB in DDAB vesicles causes a pronounced effect on the Nile Red emission, due to the increase in vesicle size, as suggested from turbidity results. The study of DODAB/CnTAB/água systems at 1.0 and 5.0 total surfactant concentration, and varying CnTAB concentrations with constant 1.0 mM DODAB revealed a strong dependence of the chain length n and relative concentration of the surfactante in the properties of mixed DODAB-CnTAB vesicles. This study allowed analyzing the thermotropic phase behavior containing different amount of DODAB, and the mechanism of vesicle-micelle transition with increasing CnTAB concentration, below and above CMC... (Complete abstract click electronic access below) / Doutor

Nous avons étudié la stabilisation de microbulles d’air par les tensioactifs semi-fluorés CF3(CF2)n-1(CH2)mOP(O)(OH)2 (FnHmPhos, où n = 8, 10, m = 2) et des nanoparticules magnétiques d’oxyde de fer et de ferrite de cobalt. Ces nouveaux objets combinant propriétés acoustiques et magnétiques pourraient servir d’agent de contraste à la fois en échographie et en imagerie IRM. Nous avons étudié des propriétés physico-chimiques des tensioactifs en solution et à l’interface aireau. Nous avons montré par tensiométrie dynamique et par des études de films de Langmuir que les tensioactifs F8H2Phos et F10H2Phos forment des monocouches dont le module élastique est très élevé, ce qui favorise la stabilisation de bulles de petite taille ainsi que de bulles non-sphériques. Nous avons proposé une approche de dégonflement/gonflement contrôlé en faisant varier la pression dans la bulle par la température et nous avons étudié le froissement de la paroi des bulles pendant le dégonflement. Des microbulles portant des nanoparticules ont été obtenues dans une suspension des nanoparticules d’oxyde de fer ou de ferrite de cobalt en présence de F8H2Phos ou F10H2Phos, en déstabilisant la suspension par NaCl, ce qui permet de diminuer la barrière électrostatique entre les nanoparticules et la surface des bulles et de favoriser l’adsorption. Les microbulles obtenues sont stables pendant plus d’une semaine grâce à la paroi rigide des nanoparticules. / The stabilization of air microbubbles by semi-fluorinated surfactants CF3(CF2)n-1(CH2)mOP(O)(OH)2 (FnHmPhos, where n = 8, 10, m = 2) and iron oxide nanoparticles was studied in order to design novel microbubbles that possess both acoustical and magnetic properties. Such microbubbles have potential applications as dual constrast agents by enhancing the signals from both ultrasonography and MRI imaging. We have studied physic-chemical properties of the surfactants in solution and at water-air interface. We have demonstrated by dynamic tensiometry and studies of Langmuir monolayers that F8H2Phos and F10H2Phos form highly elastic interfacial films that are favorable for long-term stabilization of small bubbles and bubbles of non-spherical shape. We designed an approach of temperature-controlled pumping/shrinking of the microbubbles and demonstrated the correlation of the collapse structures of the bubble shell with microbubble stability. The stabilization of microbubbles by iron oxide or cobalt ferrite nanoparticles was achieved by destabilizing the nanoparticle suspension in the presence of F8H2Phos or F10H2Phos. Under these conditions the nanoparticles were adsorbed onto the surface of the microbubbles forming a rigid shell which enhanced their stability and lengthened their lifetime to greater than one week.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-09T09:57:22Z No. of bitstreams: 2 Tese - Filipe Augusto Sales Gomes - 2017.pdf: 1914139 bytes, checksum: da7b6d8f638ab8b24c9e26f881b99673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-09T09:57:51Z (GMT) No. of bitstreams: 2 Tese - Filipe Augusto Sales Gomes - 2017.pdf: 1914139 bytes, checksum: da7b6d8f638ab8b24c9e26f881b99673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-09T09:57:51Z (GMT). No. of bitstreams: 2 Tese - Filipe Augusto Sales Gomes - 2017.pdf: 1914139 bytes, checksum: da7b6d8f638ab8b24c9e26f881b99673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Chitosan based materials are a class in development because they possess excellent characteristics for use in the biomedical sciences. Regarding chitosan suture, few studies have been performed and no studies have evaluated its response to systemic inflammation and mechanical resistance. Therefore, the aim of this thesis is to analyze the possible systemic inflammatory reactions in adult rats subjected to laparorrhaphy with chitosan suture by means of the electrophoretic analysis of polyacrylamide gel (SDS-PAGE) and its relation with the hematological findings and biochemical. In addition, investigate the mechanical resistance of the abdominal wall, the probable local inflammatory changes and the formation of collagen, through the tensiometric and histopathological analyzes. To that end, 54 adult rats (Rattus norvegicus) were divided into three groups, basel group (without surgery), chitosan and polydioxanone (gold standard), evaluated at three, seven, 14 and 21 days after laparorrhaphy. The result was that the chitosan and polydiaxonone wires have similar behaviors in the systemic and local inflammatory response, although the polydioxanone thread has a more intense response. In the evaluation of resistance, they also presented similar results, and with polydioxanone, resistance was better on the twenty-first day, with the conclusion that chitosan suture, despite the small number of researches, is a promising thread. With this, perspectives are opened for the use of chitosan suture in surgeries in veterinary medicine. However, as this is a new product, other studies should be carried out, even in different areas, as well as in other species / tissues and at different evaluation periods. / Os materiais à base de quitosana são uma classe em desenvolvimento por possuírem excelentes características para utilização nas ciências biomédicas. Em relação ao fio de quitosana, poucas pesquisas foram realizadas e não há estudos que avaliaram sua resposta à inflamação sistêmica e à resistência mecânica. Diante disso, a presente tese tem por objetivo analisar, em ratas adultas submetidas à laparorrafia com fio de quitosana, as possíveis reações inflamatórias sistêmicas, por meio da análise eletroforética em gel de poliacrilamida (SDS- PAGE) e sua relação com os achados hematológicos e bioquímicos. Além disso, investigar a resistência mecânica da parede abdominal, as prováveis alterações inflamatórias locais e a formação do colágeno, por meio das análises tensiométrica e histopatológica. Para tal, 54 ratas adultas (Rattus norvegicus) foram divididas em três grupos, basal (sem cirurgia), quitosana e polidioxanona (padrão ouro), avaliados com três, sete, 14 e 21 dias pós- laparorrafia. O resultado foi que os fios de quitosana e polidiaxonona possuem comportamentos semelhantes na resposta inflamatória sistêmica e local, apesar do fio de polidioxanona ter resposta mais intensa. Na avaliação de resistência, também apresentaram resultados semelhantes, sendo que com a polidioxanona, a resistência foi melhor no vigésimo primeiro dia, tendo como conclusão que o fio de quitosana, apesar do pequeno número de pesquisas realizadas, é um fio promissor. Com isso, abrem-se perspectivas para a utilização do fio de quitosana em cirurgias na medicina veterinária. Todavia, por tratar-se de um novo produto, outros estudos devem ser realizados, mesmo que em áreas distintas, bem como, em outras espécies/ tecidos e em diferentes períodos de avaliação.

Hyaluronová kyselina je v současné době velmi významná biomolekula používaná v medicíně a kosmetice, a její výzkum je důležitý pro další budoucí použití. Zaměření této práce je studování povrchových vlastností hyaluronové kyseliny, jejích hydrofobních derivátů a roztoků SDS pro srovnání, za použití tenziometrie. Tyto sloučeniny byly zkoumány ve vodě a v roztocích ve formě sodné soli. Sledované vzorky byly měřeny dvěmi metodami v různých koncentračních rozmezích při laboratorní teplotě pomocí nového BPA-800P bublinového tenziometeru. Byly navrženy takové experimenty, aby se zjistily využitelné možnosti tohoto přístroje pro další výzkum. Výsledky práce ukazují rozdílnosti jednotlivých povrchově aktivních látek v závislosti na jejich koncentraci a použitém prostředí. Hyaluronová kyselina nevykazuje povrchovou aktivitu, zatímco její deriváty a SDS ano.

Ce manuscrit décrit la synthèse et l’étude physico-chimique de tensioactifs innovants utilisés comme outils biochimiques pour le maintien et la cristallisation de protéines membranaires en solution aqueuse. Un premier chapitre présente les moyens techniques actuels à disposition pour la manipulation et l’étude des protéines membranaires ainsi que les problèmes rencontrés concernant leur inactivation et les alternatives actuelles. Une seconde partie décrit la synthèse d’un lipide hémifluoré possédant un ligand métallique spécifique, qui a été utilisé pour la formation d’un film de Langmuir. Les propriétés du film lipidique (stabilité et fluidité) ont été étudiées et des essais de cristallisation 2D suivant le concept interfacial ont été réalisés sur une protéine recombinante SUR1 « his tag » solubilisée dans des micelles de détergents hydrocarbonés. Le troisième chapitre aborde la notion d’amphiphilie faciale et décrit la synthèse de tensioactifs glucosidiques par « click chemistry » basés sur corps aromatique central. La persubsitution sélective de têtes hydrophiles sur les positions 1,3,5 et de parties hydrophobes sur les positions 2,4,6 apporte une amphiphilie aux molécules via une ségrégation faciale. Enfin, le dernier chapitre est dédié à l’étude du comportement et des propriétés physico-chimiques des tripodes amphiphiles faciaux en solution aqueuse grâce à différentes techniques : tensiométrie, diffusion de la lumière, CPLH,... / This thesis deals with the synthesis and the physico-chemical study of new surfactants used as tools for holding membrane proteins in aqueous media. A first part presents the existing methods that allow the manipulation of the membrane proteins and describes the current issues encountered which lead to its denaturation. In a second chapter, the synthesis of a hemifluorinated lipid with a specific ligand is presented in order to form a film of Langmuir. The stability and fluidity of the monolayer lipid is monitored and used in experiments of cristallisation 2D following the interfacial concept on the recombinant membrane protein SUR1 « his tag » keep soluble in water with hydrocarbonated detergents. The third part defines the term associated to facial amphiphile and presents a synthesis by « click chemistry » of glucosidic surfactants with an aromatic core persubstitued. The alternated and selective substitution on 1,3,5 and 2,4,6 positions of the aromatic ring by respectively hydrophilic heads and hydrophobic tails induces a facial segregation. The last chapter concerns the study of facial amphiphiles behavior and its physico-chemical properties in aqueous solution by using several methods : tensiometry, dynamic diffusion light scattering, HPLC,...

Ce mémoire rapporte les études de protéines amyloïdes en interaction avec des membranes modèle afin d’établir une relation structure toxicité. Nous avons choisi différents modèles membranaires (monocouches, bicouches) de composition lipidique et charges différentes et utilisé différentes méthodes physico-chimiques afin de caractériser les interactions des protéines amyloïdes avec les membranes.Nous avons montré l’importance de la contribution électrostatique dans les interactions de la protéine amyloïde HET-s (218-289) et ses mutants avec les membranes modèles.L’ellipsométrie a démontré que les mutants toxiques de HET-s (218-289) (M8, WT.Y1Y2V2) perturbentfortement les monocouches lipidiques à l’interface air-eau. La structure riche en feuillets β antiparallèles des protéines àl’interface air-eau et dans l’interaction avec les monocouches de lipides a été démontrée par la spectroscopie PMIRRAS (Polarization Modulation – Infrared Reflection Absorption Spectroscopy). Nous avons établie que l’interface air-eau peut modifier l’agrégation des protéines amyloïdes. A l’aide de la spectroscopie de fluorescence, la spectroscopie PWR (Plasmon-Waveguided Resonance) et la spectroscopie ATR-FTIR (Attenuated Total Reflection – Fourier Transform Infrared), nous avons mis en évidence que la protéine toxique M8 adopte une structure riche en feuillets β antiparallèles en altérant fortement l’intégrité des bicouches lipidiques. Au contraire, la protéine non toxique WT se structure en feuillets β parallèles dans ces interactions et elle ne perturbe pas l’homogénéité des membranes. La toxicité de la protéine M8 semble liée à son organisation différente et à sa capacité à réorganiser les membranes.Nos résultats confortent également l’hypothèse de la toxicité des oligomères amyloïdes.Une étude sur la fabrication d’une cellule microfluidique pour la séparation de différents types d’autoassemblage afin de les détecter et de les étudier en interaction avec des liposomes par spectroscopie infrarouge est présentée. Une cellule microfluidique de CaF2 de 8 μm d’épaisseur de canaux est obtenue et est utilisée pour la détection d’une protéine de test. / This manuscript reports the studies of amyloid proteins in interaction with membrane models in order to establish their structure-toxicity relationship.Membrane models (monolayer, bilayer) of different charge and lipid composition were used. We used various physico chemical methods to characterize the interaction of these amyloid proteins with membranes.We showed the importance of the electrostatic contribution in the interactions of the amyloid protein HET-s(218-289) and its mutants with model membranes.Ellipsometry showed that the toxic mutants of HET-s (218-289) (M8, WT.Y1Y2V2) strongly disturbed thelipid monolayers at the air-water interface. The structure rich in antiparallel β sheets of auto-assembled proteins at theair-water interface and in interaction with lipid monolayers at the air-water interface has been demonstrated by the PMIRRAS spectroscopy (Polarization Modulation - Infrared Reflection Absorption Spectroscopy). We established that theair-water interface can change the aggregation properties of amyloid proteins.By using fluorescence spectroscopy, PWR spectroscopy (Plasmon Resonance-Waveguided spectroscopy) and ATR-FTIR spectroscopy (Attenuated Total Reflection - Fourier Transform Infrared spectroscopy), we found that thetoxic protein (M8) adopted a structure rich in antiparallel β sheets greatly altered the integrity of lipid bilayers. Incontrast, the protein non-toxic (WT) organized in a structure rich in parallel β sheets in these interactions and it did notdisturb the homogeneity of the membranes. The toxicity of the protein M8 appears to be related to its differentorganization and its ability to rearrange membranes.Our results also support the hypothesis of the toxicity of amyloid oligomers.A study on the fabrication of a microfluidic cell for the separation of different aggregation states of amyloidproteins in order to detect these assemblies and to study their interaction with liposomes by infrared spectroscopy is presented. A CaF2 microfluidic cell with channels of 8 μm of thickness was obtained and was used for the detection of atested protein.

Dans le but d’approfondir les connaissances fondamentales entre la structure des polymères associatifs intramoléculaires (polysavons) et leurs propriétés physico-chimiques en milieux aqueux, trois nouvelles familles de polymères amphiphiles cationiques ont été synthétisées par deux méthodes complémentaires permettant une grande variabilité de structure. Les polymères obtenus sont des poly(méth)acrylamides en peigne avec des groupes latéraux de type ammonium quaternaire portant une chaîne alkyle de taille variable. Une étude du comportement physico-chimique de ces polymères en solution, par viscosimétrie et spectroscopie de fluorescence avec deux sondes aux caractéristiques complémentaires, montre qu’ils présentent des propriétés de polysavons qui varient progressivement avec la structure des polymères amphiphiles étudiés, notamment la longueur de la chaîne alkyle latérale, la taille de l’espaceur entre les deux sites polaires amide et ammonium quaternaire et la masse molaire moyenne en nombre. En parallèle, la tensiométrie a montré que ces polyamphiphiles ont une très faible activité à l’interface eau/air confirmant la prédominance de l’effet hydrophobe, alors que les modèles moléculaires correspondants présentent d’excellentes propriétés tensio-actives. Des films de Langmuir ont ensuite été réalisés dans le cadre de la première étude de cette importance sur des polyamphiphiles cationiques. Dans ce domaine également, la grande variabilité de structure des polymères a permis des observations originales et de dégager de nouvelles relations entre la structure du polymère et les caractéristiques des isothermes de compression obtenues / In order to improve the fundamental knowledge of the relationships between the chemical structure of intramolecular associative polymers (polysoaps) and their physical chemical properties in aqueous media, three new families of cationic amphiphilic polymers were obtained by complementary methods offering great structure variability. The corresponding polymers were comb poly(meth)acrylamides with pendant ammonium groups with alkyl side chains of variable lengths. A first investigation of their physical chemical behaviour in aqueous solutions, by viscometry and fluorescence spectrometry with two complementary fluorescent probes, showed that they displayed polysoap properties which varied progressively with their chemical features, in particular the length of the alkyl side chain, the size of the spacer between the two polar amide and ammonium groups and the polymer molecular weight. Tensiometry confirmed the prevailing of the hydrophobic effect by showing that these polymers displayed a very weak activity at the water/air interface although the corresponding molecular models showed excellent tensio-active properties. Langmuir’s films were eventually obtained in the first study of this importance on cationic amphiphilic polymers. Here again, the great structural variability enabled original observations and new structure/properties relationships were obtained for the corresponding compression isotherms

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