O ácido clavulânico é um potente inibidor de β-lactamases utilizado como terapêutico em associação à amoxicilina. O processo industrial de obtenção envolve o cultivo bacteriano em processo descontínuo, enquanto que a sua purificação é realizada, principalmente, por processos de extração líquido-líquido com solventes orgânicos e etapas cromatográficas. Assim, métodos alternativos como a purificação empregando sistemas micelares de duas fases aquosas (SMDFA), os quais oferecem um ambiente hidrofóbico e hidrofílico que possibilita seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, são de grande interesse. Neste trabalho estudou-se a viabilidade da utilização de sistema micelar misto (não-iônico/aniônico) de duas fases aquosas formados pelos tensoativos não-iônico Triton X-114 e aniônico AOT na extração do ácido clavulânico proveniente de cultivo submerso de Streptomyces clavuligerus em processo descontínuo. A estabilidade do ácido clavulânico em relação ao pH, sais e suas concentrações, temperatura e aos tensoativos foi investigada visando limitar as regiões a serem estudadas nas extrações. Determinou-se ainda o ponto névoa dos sistemas Triton X-114/Tampão e Triton X-114/AOT/Tampão em diferentes condições, bem como, estudos de migração da biomolécula para as fases e do tempo necessário para o equilíbrio do sistema. A partir destas investigações iniciais, foram definidos os níveis das variáveis: concentração de AOT (0,50, 0,75 e 1,00 mM), Triton X-114 (1, 3 e 5% m/m), NaCl (0, 0,05 e 0,10 M) e temperatura (24, 26 e 28°C), cujas influências sobre o coeficiente de partição (KAC) e recuperação da biomolécula na fase diluída (nAC) foram investigadas através de um planejamento fatorial. A análise estatística e o modelo matemático obtido a partir dos resultados para KAC revelou ser possível obterem-se KAC≈ 1,65 e nAC≈ 71,58%. No entanto, a análise estatística empregando nAC como resposta apontou condições experimentais desprovidas de significado físico-químico. Em experimentos seriados realizados com Triton X-114 3% m/m, Tampão Mcllavine pH 6,5 e adição gradual de AOT, um aumento da concentração deste tensoativo levou a maiores KAC e Balanço de massa (BMAC)≈ 85%. Desta forma é possível concluir que o sistema micelar misto de duas fases aquosas proporciona baixa perda da biomolécula e partição para a fase diluída, que é uma vantagem devido a menores problemas em uma etapa subsequente de purificação. / Clavulanic acid is a potent inhibitor of β-lactamases that is used as a therapeutic in conjunction with amoxicillin. Its industrial process of production involves bacterial growing in a batch process, while its purification is carried out mainly by process of liquid-liquid extraction with organic solvents and chromatographic steps. Thus, alternative methods like the purification employing two-phase aqueous micellar systems, which offer both hydrophobic and hydrophilic environments to solute species and allow selectivity in partitioning depending on the hydrophobicity of biomolecules, are of great concern. This work studied the feasibility of using two-phase aqueous mixed (nonionic/anionic) micellar systems formed by nonionic surfactant Triton X-114 and anionic surfactant AOT to extract clavulanic acid from broths fermented by Streptomyces clavuligerus in batch process. The stability of clavulanic acid at different values of pH, salts and their concentration, temperature and in the presence of different surfactants was investigated so as to limit the areas to be submitted to further studies. The cloud-point of the systems Triton X-114/Buffer and Triton X-114/AOT/Buffer was also determined under different conditions, and studies were performed on partitioning the biomolecule between the phases and the time needed to balance of the system. From these initial investigations the following levels of the variables were defined: concentration of AOT (0.50, 0.75, and 1.00 mM), Triton X-114 (1, 3 and 5% w/w), NaCl (0, 0.05 and 0.10 M) and temperature (24, 26 and 28°C), whose influences on the partition coefficient (KCA) and yield in the top phase (nCA) were investigated using factorial design. Statistical analysis and the mathematical model derived from the results allowed obtaining KCA≈ 1.65 e nCA≈ 71.58%. However, the statistical analysis using the response nCA indicated experimental conditions devoid of any physico-chemical meaning. In serial experiments performed with Triton X-114 3% w/w, Mcllvaine buffer at 6.5 pH and gradual addition of AOT, an increase in the concentration of anionic surfactant led to higher KCA and mass balance (BMCA≈ 85%). Thus, is possible conclude that the two-phase aqueous mixed micellar system provides low loss and partition to dilute phase, which is an advantage because it implies less problems in a subsequent step of purification.

Métodos de Microbiologia de anaeróbios estritos e de Biologia Molecular foram empregados para se conhecer a diversidade de microrganismos relacionados à degradação de ascarel em reatores anaeróbios metanogênicos. A avaliação de potencial metanogênico foi realizada para a escolha da melhor condição nutricional, bem como, para a seleção de material suporte e solvente adequado a solubilização do ascarel. Nos ensaios em batelada, a produção de metano foi maior nos reatores contendo etanol (média de 0,22 - 0,46 molCH4/gSTV, 46h). Remoção de 85,6% (86,7 mg/L de PCB em Aroclor 1016 e 1260) foi obtida na condição com espuma de poliuretano, etanol (46g/L) e formiato (680 mg/L). Diferentes solventes e surfactantes, tais como, DMSO, dioxano, ácido acético, ácido fórmico, n-hexano, acetona, etano, metanol, Tween 80, SDS (10%) e Triton X-100 foram avaliados para a solubilização de ascarel. Dentre esses, metanol, Triton X-100 e ácido fórmico foram eleitos para a realização de ensaio em reatores em batelada contendo espuma de poliuretano, com o propósito de avaliar o potencial metanogênico na degradação de PCB. Os valores de produção de metano foram muito semelhantes (0,21 0,38 molCH4/mLgSTV, 45h) nas diferentes condições, no entanto, a remoção de PCB foi maior nos reatores com metanol 790 mg/L (86,6%), ácido Fórmico 600 mg/L (84,5%) e Triton X-100 1% (72,1%). Portanto, a melhor condição foi contemplada para a operação do reator anaeróbio horizontal de leito fixo (RAHLF) no tratamento do ascarel, ou seja, células imobilizadas em espuma de poliuretano, etanol e formiato (como fonte de carbono), Triton X-100 (0,1%) e metanol (como solvente). No RAHLF, a remoção média de matéria orgânica (DQO) foi de 91% para concentração afluente média de 1270 mg/L. A presença de morfologias semelhantes à Methanosarcina e bacilos fluorescentes foi confirmada em exames microscópicos. Na análise filogenética, por meio de PCR/DGGE e seqüenciamento das bandas recortadas, os grupos encontrados foram relacionados aos Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi e Chloroflexi, sendo que neste último estão incluídos representantes relacionados a degradação de PCBs. Dentre as arquéias metanogênicas verificou-se similaridade de 99% e 97% com Methanosaeta sp. e Methanolinea sp., relacionadas com a metanogenese acetoclástica e hidrogenotrófica, respectivamente / Molecular biology and microbiology methods were used to study the microbial communities related to degradation of ascarel at methanogenic conditions in an anaerobic reactor. The methanogenic potential was evaluated to choose the better nutritional condition as well as to select the better support material and the most suitable solvent to favor the solubilization of ascarel. The methane production was higher (0.22 0.46 molCH4/mLgSTV, 46h) in batch reactors containing ethanol (46 g/L) and formate (680 mg/L), the PCB elimination attaining 85.6% (86.7 mg/L de PCB as Aroclor 1016 and 1260) when Polyuretane foam was used as support material. Different solvents, namely DMSO, dioxane, n-hexane, acetic acid, formic acid, acetone, ethane, methanol, and surfactants, such as 10% SDS,, Triton X-100, were evaluated aiming o determine the better condition to solubilize ascarel. According to the results of such experiments, methanol, formic acid and Triton X-100 were selected for carrying out the batch experiments in reactors containing polyurethane foam to evaluate the methane production during the PCBs degradation. Regardless of the operation conditions the methane production rates were similar (0.21 0.38 molCH4/gSTV, 45h), however the elimination of PCB was higher in the reactors containing methanol (790 mg/L), formic acid (600 mg/L) and Triton X-100 (1%). Therefore, the better condition for treating ascarel-containing residues in a bench-scale horizontal-flow immobilized biomass (HAIB) was attained with cells immobilized in polyurethane foam when ethanol and formate were used as carbon sources, and in presence of Triton X-100 and methanol, the average elimination of organic material attaining 91% for affluent concentration of 1270 mg/L. The presence of Methanosarcina and fluorescent rods was confirmed by microscopy analysis. According to the filogenetics analysis, which was carried out by PCR/DGGE and band-sequencing, the Bacteria domain are related to the Filos Proteobacteria, Firmicutes, Spirochaetes, Chlorobi and Chloroflexi, this latter being directly related to the degradation of PCB. Among the methanogenic Archea, a similiraty of 99% and 97% was observed to Methanosaeta sp. and Methanolinea sp. related to acetoclastic and hydrogenthrophic methanogenesis, respectively

O ácido clavulânico é um potente inibidor de β-lactamases utilizado como terapêutico em associação à amoxicilina. O processo industrial de obtenção envolve o cultivo bacteriano em processo descontínuo, enquanto que a sua purificação é realizada, principalmente, por processos de extração líquido-líquido com solventes orgânicos e etapas cromatográficas. Assim, métodos alternativos como a purificação empregando sistemas micelares de duas fases aquosas (SMDFA), os quais oferecem um ambiente hidrofóbico e hidrofílico que possibilita seletividade na partição de biomoléculas de acordo com sua hidrofobicidade, são de grande interesse. Neste trabalho estudou-se a viabilidade da utilização de sistema micelar misto (não-iônico/aniônico) de duas fases aquosas formados pelos tensoativos não-iônico Triton X-114 e aniônico AOT na extração do ácido clavulânico proveniente de cultivo submerso de Streptomyces clavuligerus em processo descontínuo. A estabilidade do ácido clavulânico em relação ao pH, sais e suas concentrações, temperatura e aos tensoativos foi investigada visando limitar as regiões a serem estudadas nas extrações. Determinou-se ainda o ponto névoa dos sistemas Triton X-114/Tampão e Triton X-114/AOT/Tampão em diferentes condições, bem como, estudos de migração da biomolécula para as fases e do tempo necessário para o equilíbrio do sistema. A partir destas investigações iniciais, foram definidos os níveis das variáveis: concentração de AOT (0,50, 0,75 e 1,00 mM), Triton X-114 (1, 3 e 5% m/m), NaCl (0, 0,05 e 0,10 M) e temperatura (24, 26 e 28°C), cujas influências sobre o coeficiente de partição (KAC) e recuperação da biomolécula na fase diluída (nAC) foram investigadas através de um planejamento fatorial. A análise estatística e o modelo matemático obtido a partir dos resultados para KAC revelou ser possível obterem-se KAC≈ 1,65 e nAC≈ 71,58%. No entanto, a análise estatística empregando nAC como resposta apontou condições experimentais desprovidas de significado físico-químico. Em experimentos seriados realizados com Triton X-114 3% m/m, Tampão Mcllavine pH 6,5 e adição gradual de AOT, um aumento da concentração deste tensoativo levou a maiores KAC e Balanço de massa (BMAC)≈ 85%. Desta forma é possível concluir que o sistema micelar misto de duas fases aquosas proporciona baixa perda da biomolécula e partição para a fase diluída, que é uma vantagem devido a menores problemas em uma etapa subsequente de purificação. / Clavulanic acid is a potent inhibitor of β-lactamases that is used as a therapeutic in conjunction with amoxicillin. Its industrial process of production involves bacterial growing in a batch process, while its purification is carried out mainly by process of liquid-liquid extraction with organic solvents and chromatographic steps. Thus, alternative methods like the purification employing two-phase aqueous micellar systems, which offer both hydrophobic and hydrophilic environments to solute species and allow selectivity in partitioning depending on the hydrophobicity of biomolecules, are of great concern. This work studied the feasibility of using two-phase aqueous mixed (nonionic/anionic) micellar systems formed by nonionic surfactant Triton X-114 and anionic surfactant AOT to extract clavulanic acid from broths fermented by Streptomyces clavuligerus in batch process. The stability of clavulanic acid at different values of pH, salts and their concentration, temperature and in the presence of different surfactants was investigated so as to limit the areas to be submitted to further studies. The cloud-point of the systems Triton X-114/Buffer and Triton X-114/AOT/Buffer was also determined under different conditions, and studies were performed on partitioning the biomolecule between the phases and the time needed to balance of the system. From these initial investigations the following levels of the variables were defined: concentration of AOT (0.50, 0.75, and 1.00 mM), Triton X-114 (1, 3 and 5% w/w), NaCl (0, 0.05 and 0.10 M) and temperature (24, 26 and 28°C), whose influences on the partition coefficient (KCA) and yield in the top phase (nCA) were investigated using factorial design. Statistical analysis and the mathematical model derived from the results allowed obtaining KCA≈ 1.65 e nCA≈ 71.58%. However, the statistical analysis using the response nCA indicated experimental conditions devoid of any physico-chemical meaning. In serial experiments performed with Triton X-114 3% w/w, Mcllvaine buffer at 6.5 pH and gradual addition of AOT, an increase in the concentration of anionic surfactant led to higher KCA and mass balance (BMCA≈ 85%). Thus, is possible conclude that the two-phase aqueous mixed micellar system provides low loss and partition to dilute phase, which is an advantage because it implies less problems in a subsequent step of purification.

This research presents advancements in the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS) for the analysis of different chemical classes of surfactants. Chapter 1 provides a brief introduction that summarizes the mechanics and fundamentals of CEC, including instrumentation and applications for CEC-MS. Chapter 2 describes the on-line hyphenation of a packed CEC column with an internally tapered tip coupled to electrospray ionization-mass spectrometry (ESI-MS) and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) for the analysis of betaine-type amphoteric or zwitterionic surfactants (Zwittergent®). The interesting aspects include CEC-MS column manufacture and charaterization, as well as a comparison between the CEC-ACPI-MS and CEC-ESI-MS ionization pattern of zwittergents. In Chapter 3, the CEC-MS of alkyltrimethyl-ammonium ions (ATMA+) with chain length ranging from C1-C18 is optimized using an internally tapered CEC-MS column packed with mixed mode C6/strong cation exchange stationary phase and coupled to an ESI source. In addition, the optimized CEC-ESI-MS protocol is applied for the challenging analysis of commercial sample Arquad S-50 ATMA+ containing cis-trans unsaturated and saturated soyabean fatty acid derivatives. In Chapter 4, a novel CEC-UV method for separation of the various Triton X-100 oligomers is presented. A systematic mobile phase tuning and comparison of monomeric vs. polymeric stationary phases was conducted. In Chapter 5, we present the first application of CEC coupled to MS for analysis of Triton X (TX-) series surfactants. A characterization from the viewpoint of the ion and adduct formation for TX-series nonionic surfactants with a variable number of ethoxy units (n=1.5-16) in the scan mode are first discussed. Next, utilizing the TX-series as model alkylphenolpolyethoxylates (APEOs), a detailed investigation of the chromatographic separation and MS detection are performed followed by analysis of very long chain TX series with n=30-70. In Chapter 6, CEC-MS utilizing full scan positive ion mode of ESI was employed to study the effect of fragmentor voltage on the in-source collision induced dissociation (IS-CID) of several APEO nonionic surfactants. Finally, in Chapter 7, the preparation and characterization of a novel liquid crystalline stationary phase suitable for separation of neutral and charged compounds in packed column CEC is evaluated.

Les membranes modèles représentent un outil indispensable pour l'étude des membranes biologiques, elles ont en effet grandement contribué à leur description. Dans ce travail, nous nous sommes intéressés à l'étude de la coexistence de phases liquide-ordonnée (lo) et liquide-désordonnée (ld) au sein de membranes modèles de type LUV (« Large Unilamellar Vesicle »). Nous avons cherché en premier lieu à mettre au point une méthodologie permettant de détecter la formation de la phase lo et d'estimer quantitativement la fraction membranaire Φo en phase lo dans des LUVs de composition ternaire PC/SM/Chol (phosphatidylcholine / sphingomyéline / cholestérol), capable d'induire une coexistence de phase. Pour cela, les propriétés d'auto-extinction de fluorescence et de distribution sélective en fonction de la phase lipidique d'une sonde fluorescente unique (C12NBD-PC) ont été mises à profit. La deuxième partie de notre travail a été consacrée à l'étude de la solubilisation par le détergent Triton X-100 des membranes de LUVs présentant une coexistence de phase lo/ld. Nous avons cherché à démontrer qu'il était possible d'extraire la fraction membranaire se trouvant strictement en phase lo. Pour cela, les transitions de structure induites par l'interaction du Triton X-100 avec des LUVs à 4°C ont été étudiées par une procédure de séparation par gradient de densité. Nous avons tenté d'évaluer le rapport effectif approprié détergent/lipides nous permettant d'isoler les fractions résistantes correspondant aux domaines en phase lo existant au niveau de la membrane des LUVs avant l'addition de détergent.

In Chapter I is a general description of novel metal complexes which hydrolytically cleave peptides, proteins, DNA, and other biological molecules. These reagents are becoming the more important as potential therapeutic agents. A panel of ligands was investigated for coordination to ZrIV and other metals in groups 4, 5, and 6 to effect the greatest degree of hydrolysis. Chapter II describes a ZrIV complex which is capable of hydrolyzing a 30 amino acid peptide, insulin chain B, with amino acid specificity. Oxidized insulin chain B peptide was hydrolyzed after only 4 h of treatment at pH 7.0 and 60 °C using ZrCl4 in the presence of 4,13-diaza-18-crown-6. MALDI-TOF and ESI LC-MS mass spectra indicated that insulin chain B was hydrolyzed by ZrIV at the Gly8-Ser9, Ser9-His10, and Gly20-Glu21 amide bonds within the oligopeptide. To our surprise, the cysteine sulfonic acid sequences Cys(SO3H)7-Gly8 and Cys(SO3H)19-Gly20 were also cleaved. To the best of our knowledge, this constitutes the first example of metal-assisted hydrolysis of a Cys(SO3H)-Xaa amide bond. This is significant in light of the fact that cysteine sulfonic acid formation in proteins is triggered by oxidative stress and has been associated with amyloid fibril formation, Parkinson’s disease, and other deleterious, physiological processes. Chapter III describes the metal-assisted hydrolysis of sphingomyelin which is a principle phospholipid component of animal cell membranes. The sphingomyelin assays showed evidence of metal-assisted hydrolysis after 20 h of treatment at lysosomal pH 4.8 and cytosolic pH 7.0 at both physiological temperature 37 °C and 60 °C. The metal ion CeIV was the most reactive, followed by ZrIV, and then HfIV. The goal of this work is to develop metal-based reagents to reverse the lethal build-up of sphingomyelin that occurs in lysosomes of patients suffering from Niemann-Pick disease.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-01-26T17:52:57Z No. of bitstreams: 1 BrenoGustavoPorfirioBezerra_DISSERT.pdf: 1680172 bytes, checksum: 0f4f20935e8e24b12e505645455bea08 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-01-28T19:06:56Z (GMT) No. of bitstreams: 1 BrenoGustavoPorfirioBezerra_DISSERT.pdf: 1680172 bytes, checksum: 0f4f20935e8e24b12e505645455bea08 (MD5) / Made available in DSpace on 2016-01-28T19:06:56Z (GMT). No. of bitstreams: 1 BrenoGustavoPorfirioBezerra_DISSERT.pdf: 1680172 bytes, checksum: 0f4f20935e8e24b12e505645455bea08 (MD5) Previous issue date: 2014-11-03 / A ?gua produzida representa um grande problema associado com a atividade de extra??o de ?leo bruto. O monitoramento dos n?veis de metais nos res?duos ? constante e requer a utiliza??o de t?cnicas anal?ticas sens?veis. No entanto, a determina??o de elementos tra?o muitas vezes pode exigir uma etapa de pr?-concentra??o. O objetivo deste trabalho foi desenvolver um m?todo anal?tico simples e r?pido para a extra??o e pr?-concentra??o baseada no fen?meno de extra??o no ponto nuvem para a determina??o do Cd, Pb e Tl em ?gua produzida amostras por Espectrometria de Absor??o de alta resolu??o com fonte continua e atomiza??o em forno de grafite. Um planejamento Box Behnken foi usado para obter a condi??o ideal de extra??o dos analitos. Os fatores avaliados foram: concentra??o do agente complexante (o,odietilditilfosfato am?nio, DDTP), a concentra??o do ?cido clor?drico e concentra??o do surfactante (Triton X -114). A condi??o ideal de extra??o foi obtida com: 0,6% m v -1 DDTP, HCl 0,3 mol L-1 e 0,2% m v -1 de Triton X - 114 para o Pb; 0,7% m v -1 DDTP, HCl 0,8 mol L-1 e 0,2% m v -1 Triton X-114 para Cd. Para o Tl foi evidenciado que melhor condi??o de extra??o se d? com aus?ncia de DDTP, as condi??es de extra??o foram ent?o HCl 1,0 mol L-1 e 1,0% m v -1 de Triton X - 114. Os limites de detec??o para o m?todo proposto, foram 0,02 ?g L-1 , 0,004 ?g L-1 e 0,06 ?g L-1 para o Pb, Cd e Tl, respectivamente. Os fatores de enriquecimento foram superiores a 10 vezes. O m?todo foi aplicado para a ?gua produzida da bacia Potiguar, e testes de adi??o e recupera??o foram realizados, e valores ficaram entre 81% e 120%. A precis?o foi expressa com desvio padr?o relativo (RSD) foi inferior a 5% / Produced water is a major problem associated with the crude oil extraction activity. The monitoring of the levels of metals in the waste is constant and requires the use of sensitive analytical techniques. However, the determination of trace elements can often require a pre-concentration step. The objective of this study was to develop a simple and rapid analytical method for the extraction and pre-concentration based on extraction phenomenon cloud point for the determination of Cd, Pb and Tl in produced water samples by spectrometry of high resolution Absorption source continues and atomization graphite furnace. The Box Behnken design was used to obtain the optimal condition of extraction of analytes. The factors were evaluated: concentration of complexing agent (o,o-dietilditilfosfato ammonium, DDTP), the concentration of hydrochloric acid and concentration of surfactant (Triton X -114). The optimal condition obtained through extraction was: 0,6% m v-1 DDTP, HCl 0,3 mol L-1 and 0,2% m v-1 of Triton X - 114 for Pb; 0,7% m v-1 DDTP, HCl 0,8 mol L-1 and 0,2% m v-1 Triton X-114 for Cd. For Tl was evidenced that best extraction condition occurs with no DDTP, the extraction conditions were HCl 1,0 mol L-1 e 1,0% m v-1 de Triton X - 114. The limits of detection for the proposed method were 0,005 ?g L-1 , 0,03 ?g L-1 and 0,09 ?g L-1 to Cd, Pb and Tl, Respectively. Enrichment factors Were greater than 10 times. The method was applied to the water produced in the Potiguar basin, and addition and recovery tests were performed, and values were between 81% and 120%. The precision was expressed with relative standard deviation (RSD) is less than 5%

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-03-22T19:38:42Z No. of bitstreams: 1 FranciscoCanindeDeSousaJunior_TESE.pdf: 1665223 bytes, checksum: a3c9c1fa573d5e42243d0808e537d798 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-03-28T19:44:15Z (GMT) No. of bitstreams: 1 FranciscoCanindeDeSousaJunior_TESE.pdf: 1665223 bytes, checksum: a3c9c1fa573d5e42243d0808e537d798 (MD5) / Made available in DSpace on 2016-03-28T19:44:15Z (GMT). No. of bitstreams: 1 FranciscoCanindeDeSousaJunior_TESE.pdf: 1665223 bytes, checksum: a3c9c1fa573d5e42243d0808e537d798 (MD5) Previous issue date: 2015-02-27 / O crescente interesse e aplica??es dos produtos biotecnol?gicos v?m aumentando o desenvolvimento de novos processos de recupera??o e purifica??o de prote?nas. A adsor??o em leito expandido (ALE) tem se destacado como uma t?cnica promissora para essa finalidade, pois combina em uma ?nica opera??o as etapas de clarifica??o, concentra??o e purifica??o da prote?na alvo, reduzindo assim tempo e custos de opera??o. Neste contexto, o objetivo desta tese foi avaliar a recupera??o e purifica??o do ant?geno 503 de Leishmania i. chagasi expresso em E. coli M15 e a remo??o de endotoxina por ALE. Na primeira etapa do trabalho foram realizados ensaios em taques agitados sob a forma de dois planejamentos experimentais, para definir as condi??es ?timas de adsor??o e elui??o do ant?geno na resina Streamline chelating. Nos ensaios de adsor??o usando o leito na forma expandida empregou-se uma coluna de 2,6 cm de di?metro por 30,0 cm de altura, acoplada a uma bomba perist?ltica. Na segunda etapa do trabalho, avaliou-se a remo??o de endotoxina durante o processo de recupera??o do ant?geno, empregando o tensoativo n?oi?nico triton X-114 na etapa de lavagem da ALE. Na terceira etapa, buscou-se elaborar um modelo matem?tico capaz de prever as curvas de ruptura do ant?geno 503 em coluna na forma expandida. Os resultados do planejamento experimental para adsor??o do ant?geno 503 mostraram o pH 8,0 e a concentra??o de NaCl de 2,4 M como melhores condi??es de adsor??o. No segundo planejamento, o ?nico fator significativo para elui??o foi a concentra??o de imidazol, definida em 600 mM. A isoterma de adsor??o do ant?geno 503 mostrou bom ajuste ao modelo de Langmuir (R=0,98) e os valores de qmax (capacidade m?xima de adsor??o) e Kd (constante de equil?brio) estimados foram de 1,95 mg/g e 0,34 mg/mL, respectivamente. Atrav?s dos testes de purifica??o diretamente do homogeneizado n?o clarificado obteve-se uma recupera??o de 59,2% da prote?na de interesse e um fator de purifica??o de 6,0. A adi??o do tensoativo n?o-i?nico Triton X-114 ? etapa de lavagem da ALE proporcionou altos valores (>99%) de remo??o do LPS inicialmente presente nas amostras para todas as condi??es estudadas. O modelo matem?tico obtido para descrever a curva de ruptura do ant?geno 503 na resina Streamline Chelanting em leito expandido apresentou bom ajuste aos dados experimentais, tanto para etapa de estimativa de par?metros quanto para de valida??o. O modelo validado foi utilizado na otimiza??o das efici?ncias, obtendo-se os valores m?ximos de efici?ncia do processo e efici?ncia da coluna de 89,2% e 75,9%, respectivamente. Portanto, ALE mostrou ser uma alternativa eficiente na recupera??o da prote?na-alvo e remo??o de endotoxina a partir de um extrato de E. coli n?o clarificado em apenas uma etapa. / The growing interest and applications of biotechnology products have increased the development of new processes for recovery and purification of proteins. The expanded bed adsorption (EBA) has emerged as a promising technique for this purpose. It combines into one operation the steps of clarification, concentration and purification of the target molecule. Hence, the method reduces the time and the cost of operation. In this context, this thesis aim was to evaluate the recovery and purification of 503 antigen of Leishmania i. chagasi expressed in E. coli M15 and endotoxin removal by EBA. In the first step of this study, batch experiments were carried out using two experimental designs to define the optimal adsorption and elution conditions of 503 antigen onto Streamline chelating resin. For adsorption assays, using expanded bed, it was used a column of 2.6 cm in diameter by 30.0 cm in height coupled to a peristaltic pump. In the second step of study, the removal of endotoxin during antigen recovery process was evaluated employing the non-ionic surfactant Triton X-114 in the washing step ALE. In the third step, we sought developing a mathematical model able to predict the 503 antigen breakthrough curves in expanded mode. The experimental design results to adsorption showed the pH 8.0 and the NaCl concentration of 2.4 M as the optimum adsorption condition. In the second design, the only significant factor for elution was the concentration of imidazole, which was taken at 600 mM. The adsorption isotherm of the 503 antigen showed a good fit to the Langmuir model (R = 0.98) and values for qmax (maximum adsorption capacity) and Kd (equilibrium constant) estimated were 1.95 mg/g and 0.34 mg/mL, respectively. Purification tests directly from unclarified feedstock showed a recovery of 59.2% of the target protein and a purification factor of 6.0. The addition of the non-ionic surfactant Triton X-114 to the washing step of EBA led to high levels (> 99%) of LPS removal initially present in the samples for all conditions tested. The mathematical model obtained to describe the 503 antigen breakthrough curves in Streamline Chelanting resin in expanded mode showed a good fit for both parameter estimation and validation steps. The validated model was used to optimize the efficiencies, achieving maximum values of the process and of the column efficiencies of 89.2% and 75.9%, respectively. Therefore, EBA is an efficient alternative for the recovery of the target protein and removal of endotoxin from an E. coli unclarified feedstock in just one step.

Mycoplasma hominis est une bactérie opportuniste qui peut être responsable d’infections du tractus urogénital, d’infections néonatales ou d’infections disséminées notamment chez les patients immunodéprimés. La membrane des mycoplasmes constitue l’interface d’interaction directe avec le milieu extérieur en raison de l’absence de paroi. Cette membrane contient de nombreuses lipoprotéines qui ont le pouvoir d’activer des cellules dendritiques humaines (hDCs), d’induire la production de cytokines et de polariser le système immunitaire adaptatif. Nous avons étudié l’interaction de M. hominis PG21 avec les hDCs en nous penchant d’une part sur la fraction du mycoplasme qui active les hDCs et d’autre part sur la réponse immunitaire innée des hDCs. Apres avoir déterminé les lipoprotéines contenues dans un extrait TX-114 de M. hominis PG21, nous avons enrichi en lipoprotéines bioactives une fraction de vésicules membranaires du mycoplasme par une double extraction utilisant deux détergents non dénaturants, le Sarkosyl puis le Triton X-114. Apres séparation par SDS-PAGE, nous avons identifié vingt lipoprotéines qui pourraient entrainer la sécrétion d’IL-23 par les hDCs, notamment la lipoprotéine MHO_4720. Un lipopeptide synthétique correspondant à la fraction N-terminale de MHO_4720 est capable de stimuler les hDCs. En analysant les variations transcriptionnelles des gènes codant pour les 48 lipoprotéines de M. hominis PG21 par qRT-PCR, nous avons également déterminé que 21 lipoprotéines sont surexprimées après 4h ou 24h de contact entre le mycoplasme et les hDCs. Enfin, la réponse cellulaire a été évaluée par PCR array et ELISA. Nous avons observé l’activation d’inflammasome(s) par la mise en évidence de la production d’IL-1β dépendant de la caspase 5. / Mycoplasma hominis is involved in urogenital tract infections, neonatal infections or disseminated infections particularly in immunocompromised patients. Mycoplasmas have no cell wall and their membrane is the main interface mediating the interaction between the mycoplasma and its environment. Lipoproteins that are anchored to the extracellular side of the plasma membrane are known to induce the maturation of human dendritic cells (hDCs), to stimulate the pro-inflammatory cytokine production by hDCs and to polarize the adaptive immune system. We studied the interaction of M. hominis PG21 with hDCs in order to assess the lipoproteins that can induce the stimulation of hDCs, to determine the lipoproteins that are regulated upon interaction of the mycoplasma with the host cell and to evaluate the innate host cell response. Using a double extraction strategy with two non-denaturing detergents, Sarkosyl then Triton X-114, and separation by SDS-PAGE, we found that 20 lipoproteins may induce the secretion of IL-23 by the hDCs, especially the MHO_4720 lipoprotein. We showed that a synthetic lipopeptide corresponding to the N-terminus part of the MHO_4720 lipoprotein can stimulate the hDCs in a dose-dependent manner. Using qRT-PCR for the evaluation of the transcriptional regulation of the 48 lipoprotein-coding genes of M. hominis PG21, we also determined that 21 lipoproteins were upregulated upon 4h and 24h of contact of M. hominis with hDCs. Finally, the hDC innate immune response was evaluated by PCR array and ELISA. We observed a caspase 5-dependent production of IL- 1β corresponding to the activation of an inflammasome.

La BSP1, principale protéine du plasma séminal bovin, interagit avec les membranes des spermatozoïdes et joue un rôle crucial dans les événements qui conduisent à la fécondité des spermatozoïdes, lors du phénomène de la capacitation. Le but de cette recherche est d’investiguer la nature de ces interactions. Ce travail vise à démontrer l’influence des lipides qui composent les membranes sur l’action de la protéine BSP1. À l’aide de la fluorescence intrinsèque de la protéine, l’affinité de la protéine a été caractérisée pour quatre systèmes lipidiques. Les résultats montrent que la composition lipidique affecte significativement l'affinité de la protéine pour les membranes. Nous avons observé l'ordre suivant : 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphoéthanolamine (POPE) ≈ POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phospho-L-sérine (POPS) > POPC/cholestérol. La protéine interagit préférentiellement avec POPC. La présence de POPE, POPS, ou cholestérol dans la membrane diminue systématiquement l’affinité. Il est connu que la présence de POPE ou cholestérol augmente l’empilement des lipides dans les membranes. Cet effet de condensation des chaînes pourrait être défavorable à l’insertion de la partie hydrophobe de la protéine dans les membranes et réduire ainsi l'affinité. La diminution de l’affinité de la protéine induite par la présence de POPS, un lipide chargé négativement, pourrait être associée aux interactions électrostatiques répulsives car la protéine porte une charge globale négative. La littérature mentionne que la BSP1 extrait sélectivement les phospholipides de type choline et le cholestérol lors de son association avec les membranes de spermatozoïdes. Un efflux lipidique est aussi observé avec des membranes modèles. Nous avons désiré caractériser la « solubilisation » des membranes par la BSP1, par diffusion dynamique de la lumière. Comme étape préliminaire, nous avons étudié comment le détergent Triton X-100 solubilise les membranes en utilisant cette technique. Les mesures démontrent que la composition lipidique des membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3- [phospho-rac-(1-glycérol)] (POPG)) n’affecte pas le mécanisme général de solubilisation/reconstitution des membranes modèles. Il a été montré qu'il existe trois régions lors des processus de solubilisation pour les différents systèmes lipidiques : i) le détergent se distribue dans les membranes, ii) une coexistence de membranes saturées en détergents et de micelles mixtes de phospholipides/Triton X-100 et iii) exclusivement des micelles mixtes de phospholipides/Triton X-100. Nos résultats montrent que la forme conique de POPE augmente la résistance des membranes à la solubilisation. La présence de POPG, apportant une charge négative à l’interface des membranes, n’induit aucun changement aux processus de solubilisation/reconstitution des membranes par Triton X-100. La diffusion dynamique de la lumière a également permis d’observer si la protéine BSP1 induit des modifications morphologiques des membranes suite à son interaction avec les membranes de POPC. Nos observations n'ont montré aucune variation significative de la taille des particules lors du titrage des vésicules de POPC par la protéine, sur une gamme de rapport molaire de POPC/BSP1 variant de 20 à 0.6. Avec des compositions aussi différentes, on suppose une transition des vésicules saturées en protéine à des complexes de protéine avec un peu de lipides. Cependant, il semble impossible avec la diffusion dynamique de la lumière de différencier ces particules. / BSP1, the main protein in bovine seminal plasma, interacts with sperm membranes and plays a crucial role in events that lead to sperm fertility, during the capacitation. The purpose of this research is to investigate the nature of these interactions. This work aims to demonstrate the influence of the lipids that compose membranes on the action of the BSP1 protein. Using the intrinsic fluorescence of the protein, the affinity of the protein was characterized for four lipid systems. The results show that the lipid composition significantly affects the affinity of the protein for membranes. We observed the following order: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) ≈ POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) > POPC/cholesterol. The protein interacts preferentially with POPC. The presence of POPE, POPS, or cholesterol in membranes decreases systematically the affinity. It is established that the presence of POPE or cholesterol increases the packing of lipids in membranes. This condensation effect could be detrimental to the insertion of the hydrophobic part of the protein into the membranes and reduces, as a consequence, the affinity. The decrease in protein affinity induced by the presence of POPS, a negatively charged lipid, could be associated with repulsive electrostatic interactions as the protein global charge is negative. The literature mentions that BSP1 selectively extracts choline phospholipids and cholesterol when combined with sperm membranes. A lipid efflux is also observed with model membranes. We characterized membrane "solubilisation" by BSP1, using dynamic light scattering. As a preliminary step, we studied how Triton X-100 detergent solubilizes membranes using this technique. The measurements showed that the lipid composition of the membranes does not affect the general solubilization/reconstitution mechanism of the model membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1-rac-glycerol) (POPG)). It is known that three different regions exist during the solubilization process for the different lipid systems: i) the detergent is distributed in the membranes, ii) a coexistence of membranes saturated with detergents and mixed phospholipid/Triton X-100 micelles and iii) exclusively mixed phospholipid/Triton X-100 micelles. Our results show that the conical shape of POPE increases the resistance of the membranes to solubilization. The presence of POPG, bringing a negative charge at the membrane interface, does not induce any change in solubilization/reconstitution processes. Dynamic light scattering also made it possible to observe if the BSP1 protein induces morphological changes in the membranes following its interaction with POPC membranes. Our observations showed no significant variation in particle size during the titration of POPC vesicles by the protein, over a molar ratio range of POPC/BSP1 from 20 to 0.6. Considering such different compositions, a transition from vesicles saturated with protein to protein complexes with some lipids is assumed. However, it appeared impossible with dynamic light scattering to differentiate these particles.