Submitted by LIERGE RAMOS null (lrg.ramos@hotmail.com) on 2017-09-06T20:31:40Z No. of bitstreams: 1 Lierge_Ramos_Dissertação_Biotecnologia.2017.pdf: 2068937 bytes, checksum: 107fae4a7bea607b2de9a6fd531de01f (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-11T19:41:07Z (GMT) No. of bitstreams: 1 ramos_l_me_araiq.pdf: 2068937 bytes, checksum: 107fae4a7bea607b2de9a6fd531de01f (MD5) / Made available in DSpace on 2017-09-11T19:41:07Z (GMT). No. of bitstreams: 1 ramos_l_me_araiq.pdf: 2068937 bytes, checksum: 107fae4a7bea607b2de9a6fd531de01f (MD5) Previous issue date: 2017-08-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As hemoglobinas constituem um grupo de proteínas que desempenham um papel vital nos organismos. Suas propriedades intrínsecas, assim como a sua relação estrutura-atividade, envolvem fenômenos tais como a cooperatividade e afinidade por ligantes específicos, como o oxigênio, que estão associados a uma variedade de processos que viabilizam a vida. As hemoproteínas, em especial as hemoglobinas de anelídeos têm sido objeto de estudo de diferentes grupos de pesquisa, devido a sua alta estabilidade oligomérica, resistência à oxidação, alta cooperatividade e afinidade por ligantes específicos, apresentando um alto potencial em aplicações biotecnológicas como, por exemplo, substituto sanguíneo. Estudos sobre a caracterização estrutural e a determinação da estabilidade de hemoproteínas na presença de surfactantes, por meio de várias técnicas como absorção ótica, emissão de fluorescência, CD (Dicroísmo Circular) e espalhamento de luz podem trazer informações sobre esta classe de proteínas, principalmente sobre o mecanismo de oxidação, dissociação e desnaturação. Desta forma, no presente projeto de pesquisa objetivou realizar a caraterização biofísica da hemoglobina extraída de Amynthas gracilis (HbAg) na presença de surfactantes iônicos (SDS e CTAC) nos valores de pH 5,0 e 7,0. Os resultados nos mostram que ambos os surfactantes são capazes de interagir fortemente com a HbAg, sendo que o pH do meio influência diretamente na intensidade da interação proteína-surfactante. O SDS em pH 5,0 interage fortemente com a HbAg formando precipitados de complexo proteína-surfactante, podendo ser observados em baixas concentrações de SDS (0,01 – 0,2 mmolL-1). Enquanto que para o CTAC ocorre uma forte interação entre o surfactante e a HbAg em pH 7,0 em uma faixa de concentração de 0,01 – 0,07 mmolL-1. A formação de agregados nestes sistemas provavelmente ocorre em função do ponto isoelétrico (pI) da HbAg ser ácido (6,0 ±3), assim como o de outras Hb extracelulares, como resultado de uma forte interação eletrostática. As medidas espectroscópicas indicam que com o aumento da concentração dos surfactantes ocorre a ressolubilização dos agregados. Os resultados obtidos neste estudo demonstraram que o SDS e o CTAC promovem o processo de oxidação/dissociação da HbAg em baixas concentrações e que nas concentrações máximas de surfactantes utilizadas neste trabalho o processo de desnaturação da HbAg não é completo. / Hemoglobins are a group of proteins that play a vital role in organisms. Their intrinsic properties, as well as their structure-activity relationship, involve phenomena such as cooperativity and affinity for specific ligands, such as oxygen, which are associated with a variety of processes that make life possible. Hemoproteins, especially hemoglobins of annelids have been studied by different research groups, due to their high oligomeric stability, resistance to oxidation, high cooperativity and affinity for specific ligands, presenting a high potential in biotechnological applications, for example, a blood substitute. Studies on the structural characterization and determination of hemoprotein stability in the presence of surfactants by optical absorption, fluorescence emission, CD and light scattering can bring information about this class of proteins, mainly on the mechanism of dissociation and denaturation. Thus, in the present master's project the main objective was to perform biophysics characterization studies, with the hemoglobin extracted from the annelid of Amynthas gracilis (HbAg) in the presence of ionic surfactants (SDS and CTAC) at pH values 5,0 and 7,0. The results show that both surfactants are capable of interacting strongly with HbAg, and the pH of the medium directly influences the intensity of the protein-surfactant interaction. SDS at pH 5.0 strongly interacts with HbAg forming precipitates of protein-surfactant complex, can be observed with low concentrations of SDS (0.01 - 0.2 mmolL -1). While for CTAC a strong interaction between surfactant and HbAg occurs at pH 7.0 in a concentration range of 0.01-0.07 mmolL-1. The formation of aggregates in these systems probably occurs as a function of the isoelectric point (pI) of HbAg being acid (6.0 ± 3), as well as that of other extracellular Hb, as a result of a strong electrostatic interaction. This study showed that SDS and CTAC promote the oxidation/dissociation process of HbAg at low concentrations and that at the maximum concentrations of surfactants used in this work the denaturing process of HbAg is not complete.

The project contributes to the general theme of complex chemical systems and strengthens ties with Innospec, a multi-national chemical company. Sodium lauroyl methyl isethionate (SLMI. Trade name “Iselux”) is a newly developed surfactant with attractive product properties for personal care applications. Little is known about the fundamental surface and solution properties of SLMI, and it is not currently possible to use information on available surfactants to predict phase behaviour. We characterise the solution and liquid crystalline phase behaviour of the SLMI/water system using a combination of optical microscopy, X-ray scattering and differential scanning calorimetry techniques. SLMI is synthesised using a batch process that leads to variable component concentrations. Preliminary studies conducted by Innospec indicate that the presence of particular process components has a significant influence on SLMI formulation rheological properties. We investigate the effects of synthesis-derived components on the rheological properties of the SLMI/sodium {(3-(dodecanoylamino)propyl)(dimethyl)ammonio)}acetate/water system using rheology and light scattering (static and dynamic) techniques. SLMI is often formulated into personal care products on mixing aqueous formulation components. Micelle growth occurs via a mechanistic process that is not understood and the equilibrium viscosity is attained at a time after mixing that ranges from seconds to weeks. Developing an improved understanding of the micelle growth mechanism is of both academic and industrial value. We utilise static light scattering and nuclear magnetic resonance techniques to probe a range of samples in the viscoelastic region of the SLMI/(carboxymethyl)hexadecyldimethyl ammonium hydroxide/water system. Experimental findings improve our current understanding of micelle growth process and provide a platform for future research on non-equilibrium mixing kinetics. In the final section we investigate salt-induced cloud point and precipitation phenomena in the SLMI/salt/water system. The cloud point is commonly observed in surfactant and protein systems by increasing the solution temperature above a critical value, resulting in phase separation of solute-rich and solute-depleted layers. Cloud point induced phase separation may also be prompted by addition of salt. The mechanistic process driving electrolyte-induced cloud point phenomena is not understood. We use a combination of turbidimetry measurements and lightscattering (static and dynamic) techniques to measure cloud point curves andcharacterise micellar behaviour prior to clouding.

Les cathepsines à cystéine (CP) participent à la dégradation du tissu bronchique ainsi qu’à l’inactivation de protéines de l’immunité innée lors de maladies inflammatoires. Lors de la mucoviscidose (CF), on observe un déficit de la protéine du surfactant pulmonaire SP-A, qui participe à la défense antimicrobienne. Nous avons caractérisé les CP dans des expectorations CF et analysé leur capacité à hydrolyser le SP-A. La balance CP/inhibiteurs est déséquilibrée en faveur des CP et la cathepsine B participe à ce déséquilibre en hydrolysant leurs inhibiteurs, les kininogènes. Cependant, les CP ne sont pas des marqueurs de colonisation par Pseudomonas aeruginosa. De plus, la cathepsine S clive sélectivement SP-A dans son site de liaison aux sucres et aux lipides, induisant la perte de ses propriétés antibactériennes et d’agrégation, contribuant au déficit de la défense innée, à la perte d’homéostasie du surfactant et à l’exacerbation de l’inflammation au cours de la mucoviscidose. / Cysteine cathepsins (CP) that are implicated in bronchial tissue injuries and inactivation of antibacterial proteins emerge as key players in pulmonary inflammations. A decrease of pulmonary surfactant protein SP-A which is involved in innate host defence has been reported in patients suffering from cystic fibrosis (CF). We characterized sputum CP and their ability to hydrolyze SP-A. There is an imbalance CP/inhibitor tipped in favor of CP proteolytic activities. Furthermore, cathepsin B, which is able to degrade major plasma CP inhibitor, kininogens, favors this imbalance. However, CP are not biomarkers of colonization by Pseudomonas aeruginosa. Moreover, Cat S cleaves SP-A specifically in its lectin-like domain (CRD) that conducts to the loss of antibacterial and aggregation properties. So, CP, especially cathepsin S, participate to the deficiency of innate immunity, surfactant homeostasis defect and to the exacerbation of inflammatory response in cystic fibrosis.

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This work was performed as a preliminary transdermal drug delivery (TDD) study to investigate the diffusion characteristics and effects of skin surfactants in vitro of four active ingredients on a poly(dimethyl siloxane) polycarbonate copolymer membrane. A Franz-type diffusion cell and various receptor solutions were used. The adhesive used was comprised of a polyisobutylene-based pressure sensitive adhesive manufactured by Adhesives Research Inc. High performance liquid chromatography was used to analyze the diffusion characteristics of these systems. In addition, the effects of two skin surfactants (sodium lauryl sulfate and dimethyl sulfoxide) on the adhesive were also investigated. Results from peel testing and thermal analysis showed that the peel strength, glass transition, and softening temperature of the adhesive was greatly reduced with the addition of the surfactants. / Master of Engineering

This study examined several structure-property features of flexible polyurethane foams that are important aspects of foam production. AFM and WAXS were used to demonstrate the existence, for the first time in typical polyurethane foam systems, of lamellae-like polyurea structures ca. 0.2 mm long and ca 5-10 nm across. Aggregations of these lamellae-like hard domains may be the polyurea balls typically observed via TEM. Diethanolamine, a widely used cross-linking agent in molded foams, was shown to disrupt ordering in the polyurea hard domains and alter the interconnectivity of hard domains by preventing the formation of lamellae-like structures. These changes were shown to lead to softening of the foam. Copolymer polyol is frequently applied as reinforcing filler in foams. It was found that a common method of adding this component alters the hard segment/soft segment (HS/SS) ratio, thus increasing the load bearing capacity of the foam. It was observed in this report that at constant HS/SS ratio, the copolymer polyol only increased load bearing under humid conditions. It was also shown that the collapse of the cellular structure of a foam prior to the point of urea precipitation alters the aggregation behavior of the hard domains and alters solid-state properties. Surfactant is thus suggested to play a secondary role in the development of the hard domains by maintaining the cellular structure in the foam as the phase separation occurs and at least until the polyurethane foam has more fully organized hard segment domains. It was found that cure temperature could be manipulated to predictably change interdomain spacings and hydrogen bond development in the polymer. Curing above 100°C was found to lower hard segment content for plaques of the same formulation possibly as a result of water and isocyanate vaporization. Apart from polyurethane materials, structure-property relationships were examined in cast blends based on poly(tetrafluoroethylene) (PTFE) and the terpolymer poly(tetrafluoroethylene-co-vinylidene fluoride-co-hexafluoropropylene) (FKM). This revealed that tensile and dynamic moduli could be predictably altered by controlling the degree of FKM cross-linking or by varying PTFE content in the film. Inducing PTFE fibrillation was found to yield higher modulus films without increasing PTFE content. / Ph. D.

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