Doctor of Philosophy(PhD) / Particle formation in RAFT-mediated emulsion polymerization has been studied using reaction calorimetry. By measuring the heat flow during controlled feed ab-initio emulsion polymerization in the presence of amphipathic RAFT agents, particle formation by self-assembly of these species could be observed. Two different monomer systems, i.e. styrene and n-butyl acrylate, and various degrees of hydrophobicity of the initial macro-RAFT agents have been studied and compared. The different macro-RAFT agents were synthesized by first forming a hydrophilic block of poly(acrylic acid) that would later on act as the electrosteric stabilizing group for the particles. Subsequently, different lengths of hydrophobic blocks were grown at the reactive end of the poly(acrylic acid) hydrophilic block via the RAFT-mediated controlled radical polymerization, either comprised of n-butyl acrylate or styrene. Two processes govern particle formation: adsorption of macro-RAFT agents onto growing particles and formation of new particles by initiation of micellar aggregates or by homogeneous nucleation. Competition between these processes could be observed when monomers with a relatively high (n-butyl acrylate) or low (styrene) propagation rate coefficient were used. A model describing particle formation has been developed and the results of model calculations are compared with experimental observations. Preliminary modeling results based on a set of reasonable physico-chemical parameters already showed good agreement with the experimental results. Most parameters used have been verified experimentally. The development of the molecular weight distribution of the macro-RAFT agents has been analyzed by different techniques. Quantification of the particle formation process by analytical techniques was difficult, but qualitative insights into the fundamental steps governing the nucleation process have been obtained. The amount of macro-RAFT agents initially involved in particle formation could be determined from the increase of molecular weight. The particle size distribution has been measured by capillary hydrodynamic fractionation, transmission electron microscopy and dynamic light scattering. From the data obtained from these particle-sizing techniques, the number of particles during the reaction could be monitored, leading to an accurate estimate for the particle formation time. Upon implementation of the experimental data obtained for the surface active macro-RAFT systems, the model demonstrated to be very sensitive towards the “headgroup” area of the macro-RAFT species. Three nucleation cases based on the initial surface activity of the macro-RAFT species in the aqueous phase are proposed to explain the deviations from the assumptions of the nucleation model. Even though the macro-RAFT species have a narrow molecular weight distribution, they are nevertheless made up of a distribution of block lengths of polystyrene upon a distribution of block lengths of poly(acrylic acid). The resulting differences in initial surface activity are the most probable reason for the observed differences between model calculations and experimental results for the nucleation time and particle size distribution of the final latex product. With the procedure described above, latexes have been synthesized without using conventional surfactants and the mechanisms involved in the particle formation for these systems have been elucidated. The results of this work enable production of latex systems with well defined molecular mass distributions and narrow particle size distributions. Furthermore, the technique based on the application of amphipathic RAFT agents is promising for the production of complex polymeric materials in emulsion polymerization on a technical scale.

Doctor of Philosophy(PhD) / Particle formation in RAFT-mediated emulsion polymerization has been studied using reaction calorimetry. By measuring the heat flow during controlled feed ab-initio emulsion polymerization in the presence of amphipathic RAFT agents, particle formation by self-assembly of these species could be observed. Two different monomer systems, i.e. styrene and n-butyl acrylate, and various degrees of hydrophobicity of the initial macro-RAFT agents have been studied and compared. The different macro-RAFT agents were synthesized by first forming a hydrophilic block of poly(acrylic acid) that would later on act as the electrosteric stabilizing group for the particles. Subsequently, different lengths of hydrophobic blocks were grown at the reactive end of the poly(acrylic acid) hydrophilic block via the RAFT-mediated controlled radical polymerization, either comprised of n-butyl acrylate or styrene. Two processes govern particle formation: adsorption of macro-RAFT agents onto growing particles and formation of new particles by initiation of micellar aggregates or by homogeneous nucleation. Competition between these processes could be observed when monomers with a relatively high (n-butyl acrylate) or low (styrene) propagation rate coefficient were used. A model describing particle formation has been developed and the results of model calculations are compared with experimental observations. Preliminary modeling results based on a set of reasonable physico-chemical parameters already showed good agreement with the experimental results. Most parameters used have been verified experimentally. The development of the molecular weight distribution of the macro-RAFT agents has been analyzed by different techniques. Quantification of the particle formation process by analytical techniques was difficult, but qualitative insights into the fundamental steps governing the nucleation process have been obtained. The amount of macro-RAFT agents initially involved in particle formation could be determined from the increase of molecular weight. The particle size distribution has been measured by capillary hydrodynamic fractionation, transmission electron microscopy and dynamic light scattering. From the data obtained from these particle-sizing techniques, the number of particles during the reaction could be monitored, leading to an accurate estimate for the particle formation time. Upon implementation of the experimental data obtained for the surface active macro-RAFT systems, the model demonstrated to be very sensitive towards the “headgroup” area of the macro-RAFT species. Three nucleation cases based on the initial surface activity of the macro-RAFT species in the aqueous phase are proposed to explain the deviations from the assumptions of the nucleation model. Even though the macro-RAFT species have a narrow molecular weight distribution, they are nevertheless made up of a distribution of block lengths of polystyrene upon a distribution of block lengths of poly(acrylic acid). The resulting differences in initial surface activity are the most probable reason for the observed differences between model calculations and experimental results for the nucleation time and particle size distribution of the final latex product. With the procedure described above, latexes have been synthesized without using conventional surfactants and the mechanisms involved in the particle formation for these systems have been elucidated. The results of this work enable production of latex systems with well defined molecular mass distributions and narrow particle size distributions. Furthermore, the technique based on the application of amphipathic RAFT agents is promising for the production of complex polymeric materials in emulsion polymerization on a technical scale.

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2008. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-24T17:43:54Z No. of bitstreams: 1 dissertao marta.pdf: 2219216 bytes, checksum: dabe85cdb78a95658dc970cbd0e37ce1 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-11-09T13:16:33Z (GMT) No. of bitstreams: 1 dissertao marta.pdf: 2219216 bytes, checksum: dabe85cdb78a95658dc970cbd0e37ce1 (MD5) / Made available in DSpace on 2012-11-09T13:16:33Z (GMT). No. of bitstreams: 1 dissertao marta.pdf: 2219216 bytes, checksum: dabe85cdb78a95658dc970cbd0e37ce1 (MD5) Previous issue date: 2008 / Os biossurfactantes são moléculas anfipáticas produzidas por microrganismos, que reduzem tensões superficiais e interfaciais e possuem propriedades de emulsificação. Apresentam vantagens com relação aos surfactantes químicos como a biodegradabilidade e baixa toxicidade, podendo ser aplicados na indústria de alimentos, farmacêutica, cosmética, na biorremediação e na recuperação de petróleo. Os processos biotecnológicos freqüentemente são limitados pelo investimento de capital. A utilização de resíduos agroindustriais para a produção de biossurfactante é importante do ponto de vista econômico, já que a matéria-prima representa grande parte dos custos de obtenção deste bioproduto. Os objetivos do presente trabalho foram selecionar bactérias com potencial para produzir biossurfactante e estudar a produção por bactéria e fungo em diferentes biorreatores, através de cultivos em estado sólido e submerso utilizando resíduos agroindustriais. O trabalho foi dividido em três etapas: 1) seleção de bactérias produtoras de biossurfactante e estudo da cinética dos processos fermentativos, 2) produção de biossurfactantes bacteriano e fúngico em diferentes biorreatores através de cultivo em estado sólido e 3) produção de biossurfactantes bacteriano e fúngico em diferentes biorreatores através de cultivo submerso. Na primeira etapa foram utilizadas 4 culturas de bactérias: cultura pura de Corynebacterium aquaticum, cultura mista contendo Corynebacterium aquaticum e Bacillus sp., cultura mista contendo Corynebacterium sp., Bacillus cereus e Bacillus mycoides e cultura pura de Bacillus subtilis. A cultura pura de Corynebacterium aquaticum foi a que apresentou menor tensão superficial (28,8 mN.m-1) e, em geral, maiores valores para os parâmetros cinéticos, sendo selecionada como produtora de biossurfactante. Resultados da segunda etapa mostraram que as menores tensões superficiais foram encontradas no cultivo de Corynebacterium aquaticum em biorreator de coluna (33,1 mN.m-1) e frasco Erlenmeyer (31,3 mN.m-1). A menor tensão interfacial foi 6,2 mN.m-1, obtida em cultivo bacteriano em biorreator de coluna. Os cultivos com o fungo Aspergillus fumigatus em biorreator de coluna e frasco Erlenmeyer apresentaram maiores atividades emulsificantes óleo em água (291,8 UE e 327,9 UE, respectivamente). A atividade emulsificante água em óleo foi incrementada nos cultivos de Aspergillus fumigatus e pela utilização do biorreator de coluna, atingindo 60,4 UE. Na terceira etapa verificou-se que as menores tensões superficiais foram encontradas no cultivo de Corynebacterium aquaticum em biorreator de mistura (28,6 mN.m-1) e frasco Erlenmeyer (29,1 mN.m-1). As menores tensões interfaciais foram obtidas nos cultivos bacterianos (3,0 mN.m-1). A maior atividade emulsificante óleo em água foi 232,0 UE, obtida no cultivo com a bactéria Corynebacterium aquaticum em biorreator de mistura. Os cultivos bacterianos em biorreator de mistura e frasco Erlenmeyer apresentaram maiores atividades emulsificantes água em óleo (37,9 UE e 36,1 UE, respectivamente). Logo, o biossurfactante produzido pela bactéria foi capaz de reduzir tensões e promover a formação de emulsões tanto em meio sólido quanto em meio líquido, enquanto o fungo mostrou-se apto a produzir biossurfactante com alta capacidade emulsificante em meio sólido. Constatou-se a viabilidade de produção de biossurfactante a partir de resíduos agroindustriais, o que torna o processo mais econômico, obtendo-se um bioproduto de valor agregado a partir de substratos de baixo custo, além de contribuir com o meio ambiente pela diminuição da poluição e desequilíbrio gerados pela emissão desses resíduos. / Biosurfactants are amphipathic molecules produced by microorganisms that reduce surface and interfacial tensions and possess emulsification properties. They present advantages with relationship to the chemical surfactants as the biodegradability and low toxicity, and can be applied in the food, pharmaceutical and cosmetic industry, in the bioremediation and in the recovery of petroleum. The biotechnology processes are frequently limited by the capital investment. The use of agroindustrial wastes for the biosurfactant production is important from the economical point of view, since the raw material represents great part of the costs in the obtaining of this bioproduct. The purposes of this study were to select bacteria with potential to produce biosurfactant and to study the production by bacteria and fungus in different bioreactors, through solid-state and submerged cultivations using agroindustrial wastes. The work was divided in three stages: 1) selection of bacteria for biosurfactant production and fermentation process kinetics study, 2) bacterial and fungic biosurfactants production in different bioreactors through solid-state cultivation and 3) bacterial and fungic biosurfactants production in different bioreactors through submerged cultivation. In the first stage 4 bacterial strains were used: pure culture of Corynebacterium aquaticum, mixed culture containing Corynebacterium aquaticum and Bacillus sp., mixed culture containing Corynebacterium sp., Bacillus cereus and Bacillus mycoides and pure culture of Bacillus subtilis. The pure culture of Corynebacterium aquaticum presented the smallest surface tension (28.8 mN.m-1) and, in general, larger kinetic parameters, being selected as biosurfactant producer. Results of the second stage showed that the smallest surface tensions were found in the cultivation of Corynebacterium aquaticum in column bioreactor (33.1 mN.m-1) and Erlenmeyer flask (31.3 mN.m-1). The smallest interfacial tension was 6.2 mN.m-1, obtained in bacterial cultivation in column bioreactor. The cultivations with the fungus Aspergillus fumigatus in column bioreactor and Erlenmeyer flask presented larger emulsifying activities oil in water (291.8 UE and 327.9 UE, respectively). The emulsifying activity water in oil was increased in the cultivations of Aspergillus fumigatus and for the use of the column bioreactor, reaching 60.4 UE. In the third stage it was verified that the smallest surface tensions were found in the cultivation of Corynebacterium aquaticum in mixture bioreactor (28.6 mN.m-1) and Erlenmeyer flask (29.1 mN.m-1). The smallest interfacial tensions were obtained in the bacterial cultivations (3.0 mN.m-1). The largest emulsifying activity oil in water was 232.0 UE, obtained in the cultivation with the bacteria Corynebacterium aquaticum in mixture bioreactor. The bacterial cultivations in mixture bioreactor and Erlenmeyer flask presented larger emulsifying activities water in oil (37.9 UE and 36.1 UE, respectively). Therefore, the biosurfactant produced by the bacteria was able to reduce tensions and to promote emulsions formation in solid and liquid medium, while the fungus was able to produce biosurfactant with high emulsifying capacity in solid medium. The viability of biosurfactant production from agroindustrial wastes was verified, what makes the process more economical, with the obtaining of a value joined bioproduct from low cost substrates, besides contributing with the environment for the decrease of the pollution and unbalance generated by the emission of those residues.

No description available.

A classe de proteínas das MEGs (codificadas por genes de micro-éxon) presente em Schistosoma mansoni, ganhou evidência após a publicação do genoma deste parasita, principalmente por ser majoritariamente secretada, estando em contato direito com moléculas do hospedeiro, e possuir alta taxa de variação, o que poderia ter relação com um possível um mecanismo de evasão do sistema imune. Assim, foram escolhidas proteínas da classe das MEGs, todas com predição de hélice anfipática em sua estrutura, para investigação neste trabalho. Hélices anfipáticas são amplamente descritas na literatura como tendo alta propensão à interação com membranas celulares e interessantes funções fisiológicas. O objetivo deste projeto foi estudar a dinâmica de interação com membranas e estabelecer possíveis indícios de função biológica das proteínas MEG-24 e MEG-27 a partir de técnicas biofísicas. Optou-se por trabalhar com essas proteínas produzidas a partir da síntese química. Utilizando as técnicas de CD, Fluorescência e DLS, observou-se que a presença de miméticos de membrana induzem o enovelamento e o aumento da estabilidade térmica de MEG-27, e interferem no seu estado oligomérico. Para MEG-24, também foi observado aumento da estabilidade térmica e influência no estado oligomérico na presença de sistemas miméticos de membrana. Utilizando OCD, inferiu-se que MEG-27 possivelmente interage com a superfície da membrana, a passo que MEG-24 se insere na bicamada. Adicionalmente, ambas foram capazes de interferir na dinâmica de vesículas, conforme observado pelo ensaio de vazamento de calceína e DSC. Utilizando células de eritrócito e um modelo de membrana a partir dessas células (ghosts) foram realizados ensaios biológicos, DSC e EPR. A capacidade de MEG-24 e MEG-27 realizarem hemólise e hemoaglutinação, respectivamente, reitera o potencial de interação destas proteínas com membranas biológicas. Os resultados de DSC apontaram que ambas interferem nas transições das proteínas de membrana embebidas na bicamada de ghosts de eritrócitos. Por EPR, notou-se que MEG-27 tem maior efeito na dinâmica de lipídios em detrimento a MEG-24, possivelmente devido a um mecanismo de acomodação envolvendo domínios raft e a diferença de orientação de interação entre ambas as proteínas. A expressão de MEG-27 exclusivamente na região da glândula do esôfago em vermes adultos verificada por WISH, sugere que a mesma deva entrar em contato com células recém ingeridas pelo parasita. Não foi observada atividade antimicrobiana para ambas e não foram encontrados parceiros de interação proteínas pela técnica de Duplo-híbrido para MEG-27. Concluiu-se que os peptídeos estudados interagem com membranas biológicas, podendo ter papéis importantes na interface de interação parasito-hospedeiro. / The class of MEGs proteins (coded by micro-exon genes) present in Schistosoma mansoni, drawn attention after the parasite genome publication. This proteins are mostly secreted, being in direct contact with host molecules and with a high rate of variation, which could be a mechanism of Imune system evasion. Thus, proteins of the MEGs class, all with amphipathic prediction in their structure, were chosen for investigation in this work. Amphipathic helices are widely described in the literature with high propensity to interact with membranes and, consequently, probability of related biological function. The objective of this project was to study the interaction dynamics with membranes and to establish possible indications of biological function of MEG-24 and MEG-27 proteins from biophysical techniques. We chose to work with these proteins produced by chemical synthesis. Using CD, Fluorescence and DLS techniques, it was observed that the presence of membrane mimetics induced the folding and increased thermal stability of MEG-27, and interfered with its oligomeric state. For MEG-24, an increase in thermal stability and influence on the oligomeric state was also observed when in the presence of membrane mimetic systems. The results of OCD suggest that MEG-27 possibly interacts with the membrane surface, whereas MEG-24 is inserted into the bilayer. Both were able to interfere with vesicle dynamics, as observed by the Leakage and DSC tests. Using a more realistic membrane model from erythrocyte cells, biological assays, DSC and EPR were performed. The ability of MEG-24 and MEG-27 to perform hemolysis and hemagglutination, respectively, provides evidence of the potential for interaction of these proteins with biological membranes. The DSC results indicated that both interfere with the transitions of the membrane proteins embedded in the bilayer of erythrocyte ghosts. By EPR, it was noted that MEG-27 has a greater effect on lipid dynamics than MEG-24, possibly due to an accommodation mechanism involving raft domains and the difference in orientation of interaction between both proteins. The expression of MEG-27 exclusively in the region of the esophagus gland in adult worms verified by WISH suggests that it should come into contact with cells just ingested by the parasite. No antimicrobial activity was observed for both and no protein interaction partners were found by the MEG-27 double-hybrid technique. It was concluded that the studied peptides interact with biological membranes and may have important roles in the parasite-host interaction interface.

Face à l’émergence de pathogènes multi-résistants aux antibiotiques classiques, et au développement des armes biologiques, la découverte de nouveaux agents antimicrobiens reste un enjeu majeur de santé public. Dans ce contexte, la conception d’oligomères peptidomimétiques, capables de mimer le caractère amphiphile et la structure en hélice des peptides antimicrobiens naturels, effecteurs clés de l’immunité innée, offre d’intéressantes perspectives. Il a été établi que des foldamères à base d’urées amphipathiques, structurés en hélice-2,5, possédaient une forte activité bactéricide contre Bacillus anthracis, bactérie considérée comme une arme biologique potentielle. En vue d’optimiser l’activité anthracidale et la sélectivité in vitro de la première génération de composés, une étude relation structure-activité a été initiée en réalisant une série de modifications (i.e. séquence primaire, longueur et squelette de l’oligourée). Des oligomères originaux possédant des motifs isostères de type thiourée et guanidine ont ainsi été préparés en solution puis sur support solide. Des études conformationnelles approfondies soulignèrent que seule l’insertion de lien thiourée à proximité du dipôle négatif était bien tolérée par l’hélice-2,5. Parallèlement, les études in vivo ont montré une forte stabilité des oligourées avec une accumulation sélective dans le rein ainsi qu’une protection partielle des souris contre l’infection systémique par Bacillus anthracis. Enfin l’étude de l’interaction de ces oligourées avec des membranes lipidiques modèles a confirmé leur capacité à perturber les membranes et a mis en avant des mécanismes d’action différents selon le type de lipides utilisés. / The increasing antibiotic resistance among pathogens and the emergence of biological weapons have highlighted the urgent need of new antimicrobial agents. In this context, the design of peptidomimetics as urea-based foldamers, capable of mimicking the amphiphilic character and conformation of natural antimicrobial peptides, key effector molecules of innate immunity, offers new prospects. It has been previously established that amphipathic oligourea 2.5-helices have a strong bactericidal activity against Bacillus anthracis, bacteria considered as a potential biological warfare agent. Based on these results and with the aim of optimizing the potency and selectivity in vitro of the first generation of compounds, a structure-activity relationship study was carried out by performing series of modifications on a lead compound (i.e. side-chain replacement, size and backbone modifications). Among them, new series oligomers incorporating isosteric substitutions such as thiourea and guanidine moieties were prepared in solution then on solid support. Interestingly, the conformational studies revealed that only the insertion of thiourea linkage near the negative end of the helix dipole was well-tolerated by the 2.5-helix. Concurrently, in vivo studies highlighted a strong stability of the lead oligourea with a selective accumulation in the kidneys as well as a partial protection of the mice after systemic infection by Bacillus anthracis. Finally, biophysical interaction studies of selected oligoureas with model membranes confirmed their capacity to disturb membranes and showed different mechanisms of action depending on the lipid composition.

Les gouttelettes lipidiques (GL) sont des organites intracellulaire qui jouent un rôle central dans le métabolisme des lipides. Elles sont également impliquées dans des maladies telles que l'obésité ou le diabète. Les GL ont une structure unique : une monocouche de phospholipides (PL) qui entoure un cœur de lipide neutre composé de triglycérides (TAG) et d'esters de cholestérol (CE). Certaines protéines sont recrutées sur les GL mais également à la surface d'autres organites, alors que d'autres protéines ciblent spécifiquement la surface des GL. Il a été montré que quelques une de ces protéines seraient sensibles à une haute tension de surface, soit une augmentation de l'aire par lipide, dans des GL reconstituées. Comment les propriétés de surface d'une GL diffèrent d'une membrane ? Comment la surface d'une GL répond à l'augmentation de la tension de surface ?Comment les protéines interagissent avec la surface des GL ? Nous avons réalisé des simulations de dynamique moléculaire atome-unifié de système tricouche qui mime la surface d'une GL afin de caractériser les propriétés de surface de cet organite. Plusieurs simulations ont été effectuées à différentes tension de surface en augmentant l'aire par lipide. Les propriétés de surface ont été caractérisées en terme de défauts de \textit{packing} (i.e. vides interfaciaux à l'interface membrane/eau). Aucune différence n'a été observé avec une bicouche à l'équilibre. Cependant, la tension de surface promeut l'insertion de lipides neutres dans la monocouche et augmente significativement les défauts de \textit{packing}. Des simulations préliminaires sur l'interaction d'une protéine modèle, la périlipine 4, qui se lie aux GLs \textit{in vivo} via une longue hélice amphipathique 11/3 ont été faites. Les premiers résultats montrent que la protéine adopte une structure plus flexible dans une interface huile/eau que dans une interface membrane/eau. Des essais de dimérisation montrent que la répartition des résidus chargés serait importante pour le processus d'oligomérisation. Pris globalement, ces résultats apportent une compréhension moléculaire quantitative sur l'effet de la tension de surface sur la monocouche de GL et des résultats préliminaires sur l'interaction protéine/GL. Notre travail constitue une première étape vers la description du comportement et de la structure des propriétés de surface des GL et peut être utile à la compréhension du ciblage protéique vers la surface de GL. / Lipid droplets (LD) are intracellular organelles that have a central role in lipid metabolism andimplication in diseases such as obesity and diabetes. LDs have a unique architecture: aphospholipid (PL) monolayer that surrounds a neutral lipid core composed of triacylglycerols (TAG)and cholesteryl esters (CE). Some proteins are recruited both to LDs and to other cellularorganelles, whereas others are targeted specifically to the surface of LDs. It has been shown thatsome of these proteins could be sensitive to a high surface tension (ST), increase in the area perlipid, in reconstituted LD. How do surface properties differ between a membrane and an LD? Howdoes the LD surface respond to an increase in ST? How do proteins interact with LDs? Weperformed united-atom molecular dynamics simulations on trilayer systems that mimic the LDsurface to investigate the surface properties of this organelle. Several simulations were performedat different ST by increasing the area per lipid. Surface properties were characterized in terms ofpacking defects (i.e interfacial voids at the membrane-water interface). No difference was observedwith a bilayer at equilibrium. However, high ST promoted the insertion of neutral lipids into themonolayer and a significant increase of packing defects. Preliminary simulations has been done oninteraction of a model protein called perilipin 4, which binds to LDs \textit{in vivo} using a long 11/3amphipathic helix. The first results show that the protein adopts a more flexible conformation on oilwaterinterface than in bilayer-water interface. Attempts of dimerisation show that the localization ofthe charged residues may be involved in the oligomerisation process. Taken together, our resultsprovide a quantitative molecular understanding of how ST affects the LD surface and preliminaryresults on protein-LD interaction. Our work constitutes a first step towards characterizing thebehavior and structure of LD surface properties and will be useful for a better understanding onhow some specific proteins are targeted to LD.

Short interfering RNAs (siRNAs) are 21-23 nucleotide-long double-stranded RNA molecules that can trigger the RNA interference (RNAi). RNAi is a post-transcriptional gene silencing process whereby siRNAs induce the sequence-specific degradation of complementary messenger RNA (mRNA). Despite their promising therapeutic capabilities, siRNA-based strategies suffer from enzymatic degradation and poor cellular uptake. Several carrier-based approaches have been employed to enhance the stability and efficiency of siRNA delivery. Considering their safety, efficiency, and targeting capabilities, peptide-based delivery systems have shown great promise for overcoming the main obstacles in siRNA therapeutic delivery. Peptides are versatile and easily designed to incorporate a number of specific attributes required for efficient siRNA delivery. This thesis focuses on the design, characterization and utilization of a new class of amphipathic peptides for siRNA delivery. The study includes: (i) designing amphipathic, amino acid pairing peptide sequences for siRNA delivery, (ii) siRNA delivery experiments in vitro to evaluate transfection efficacy of the designed peptides, (iii) physicochemical characterization of the interaction between promising peptides and siRNA, and (iv) identifying internalization pathway and kinetics of a promising peptide, C6M1. The peptide C6, an 18-mer amphipathic, amino acid pairing peptide, was designed as an siRNA delivery carrier by incorporating three types of amino acids, i.e., arginine, leucine, and tryptophan. This peptide adopted a helical structure upon co-assembling with siRNA. The C6-siRNA co-assembly showed a size distribution between 50 and 250 nm, confirmed by dynamic light scattering and atomic force microscopy. The C6-siRNA interaction enthalpy and stoichiometry were 8.8 kJ.mol-1 and 6.5, respectively, obtained by isothermal titration calorimetry. A minimum C6:siRNA molar ratio of 10:1 was required to form stable co-assemblies/complexes, indicated by agarose gel shift assay and fluorescence spectroscopy. C6 showed lower toxicity and higher efficiency in cellular uptake of siRNA, compared with Lipofectamine 2000, a lipid-based positive control. Fluorescence microscopy images confirmed the localization of C6-siRNA complexes in the cytoplasm. In order to enhance the solubility and delivery efficiency further, a modified peptide, C6M1, was designed by replacing three leucine with tryptophan residues in the C6 sequence. The fluorescence assay confirmed that the sequence mutation significantly increased the solubility of C6M1. C6M1 adapted a stable helical structure in saline or upon interaction with siRNA. The toxicity assay showed lower toxicity of C6M1 with an IC50 (the concentration of peptide at 50% cell viability) of 22 ??M, compared with C6 with that of 12 ??M. Naked siRNA was completely degraded after 4 h incubation in 50% serum, while the siRNA in complex with C6M1 was preserved even after 24 h. Western blotting showed a significant decrease in GAPDH protein contents (75%) in CHO-K1 Chinese hamster ovary cells, 48 h after treatment with C6M1-GAPDH siRNA complexes. The interaction of C6M1-siRNA complexes with cell surface and the mechanisms involved in the internalization of the complex in different size ranges were studied. Heparin and chlorate treatments revealed that the electrostatic interaction of the C6M1-siRNA complex with heparan sulphate proteoglycans at the cell surface is required to trigger the uptake process. Using endocytic inhibitors, it was found that small C6M1-siRNA complexes (mean ~155 nm) mainly enter CHO-K1 cells through an energy-independent mechanism, most likely involving direct translocation. In contrast, large complexes (mean ~460 nm) internalize the cells mainly through a lipid raft-dependant macropinocytosis. The integrity of the cytoskeletal components also showed significant impact on the efficient internalization of the C6M1-siRNA complex. The kinetics experiments confirmed the fast internalization of small complexes (with uptake half-time of 25 min) in comparison to large complexes (70 min). This work provides essential information for peptide design and characterization in the development of amphipathic peptide-based siRNA delivery.

A classe de proteínas das MEGs (codificadas por genes de micro-éxon) presente em Schistosoma mansoni, ganhou evidência após a publicação do genoma deste parasita, principalmente por ser majoritariamente secretada, estando em contato direito com moléculas do hospedeiro, e possuir alta taxa de variação, o que poderia ter relação com um possível um mecanismo de evasão do sistema imune. Assim, foram escolhidas proteínas da classe das MEGs, todas com predição de hélice anfipática em sua estrutura, para investigação neste trabalho. Hélices anfipáticas são amplamente descritas na literatura como tendo alta propensão à interação com membranas celulares e interessantes funções fisiológicas. O objetivo deste projeto foi estudar a dinâmica de interação com membranas e estabelecer possíveis indícios de função biológica das proteínas MEG-24 e MEG-27 a partir de técnicas biofísicas. Optou-se por trabalhar com essas proteínas produzidas a partir da síntese química. Utilizando as técnicas de CD, Fluorescência e DLS, observou-se que a presença de miméticos de membrana induzem o enovelamento e o aumento da estabilidade térmica de MEG-27, e interferem no seu estado oligomérico. Para MEG-24, também foi observado aumento da estabilidade térmica e influência no estado oligomérico na presença de sistemas miméticos de membrana. Utilizando OCD, inferiu-se que MEG-27 possivelmente interage com a superfície da membrana, a passo que MEG-24 se insere na bicamada. Adicionalmente, ambas foram capazes de interferir na dinâmica de vesículas, conforme observado pelo ensaio de vazamento de calceína e DSC. Utilizando células de eritrócito e um modelo de membrana a partir dessas células (ghosts) foram realizados ensaios biológicos, DSC e EPR. A capacidade de MEG-24 e MEG-27 realizarem hemólise e hemoaglutinação, respectivamente, reitera o potencial de interação destas proteínas com membranas biológicas. Os resultados de DSC apontaram que ambas interferem nas transições das proteínas de membrana embebidas na bicamada de ghosts de eritrócitos. Por EPR, notou-se que MEG-27 tem maior efeito na dinâmica de lipídios em detrimento a MEG-24, possivelmente devido a um mecanismo de acomodação envolvendo domínios raft e a diferença de orientação de interação entre ambas as proteínas. A expressão de MEG-27 exclusivamente na região da glândula do esôfago em vermes adultos verificada por WISH, sugere que a mesma deva entrar em contato com células recém ingeridas pelo parasita. Não foi observada atividade antimicrobiana para ambas e não foram encontrados parceiros de interação proteínas pela técnica de Duplo-híbrido para MEG-27. Concluiu-se que os peptídeos estudados interagem com membranas biológicas, podendo ter papéis importantes na interface de interação parasito-hospedeiro. / The class of MEGs proteins (coded by micro-exon genes) present in Schistosoma mansoni, drawn attention after the parasite genome publication. This proteins are mostly secreted, being in direct contact with host molecules and with a high rate of variation, which could be a mechanism of Imune system evasion. Thus, proteins of the MEGs class, all with amphipathic prediction in their structure, were chosen for investigation in this work. Amphipathic helices are widely described in the literature with high propensity to interact with membranes and, consequently, probability of related biological function. The objective of this project was to study the interaction dynamics with membranes and to establish possible indications of biological function of MEG-24 and MEG-27 proteins from biophysical techniques. We chose to work with these proteins produced by chemical synthesis. Using CD, Fluorescence and DLS techniques, it was observed that the presence of membrane mimetics induced the folding and increased thermal stability of MEG-27, and interfered with its oligomeric state. For MEG-24, an increase in thermal stability and influence on the oligomeric state was also observed when in the presence of membrane mimetic systems. The results of OCD suggest that MEG-27 possibly interacts with the membrane surface, whereas MEG-24 is inserted into the bilayer. Both were able to interfere with vesicle dynamics, as observed by the Leakage and DSC tests. Using a more realistic membrane model from erythrocyte cells, biological assays, DSC and EPR were performed. The ability of MEG-24 and MEG-27 to perform hemolysis and hemagglutination, respectively, provides evidence of the potential for interaction of these proteins with biological membranes. The DSC results indicated that both interfere with the transitions of the membrane proteins embedded in the bilayer of erythrocyte ghosts. By EPR, it was noted that MEG-27 has a greater effect on lipid dynamics than MEG-24, possibly due to an accommodation mechanism involving raft domains and the difference in orientation of interaction between both proteins. The expression of MEG-27 exclusively in the region of the esophagus gland in adult worms verified by WISH suggests that it should come into contact with cells just ingested by the parasite. No antimicrobial activity was observed for both and no protein interaction partners were found by the MEG-27 double-hybrid technique. It was concluded that the studied peptides interact with biological membranes and may have important roles in the parasite-host interaction interface.

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