<p>The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall.</p><p>In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models.</p><p>Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models.</p><p>Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.</p>

The physical properties of glycerophospholipids (GPLs) are not only determined by the head group (HG), but also by their fatty acid (FA) chains, which affect their distribution and function within membranes in the cell. Understanding the microheterogenity of lipid membranes on a molecular level requires qualitative and quantitative characterization of individual lipids and identification of their FA moieties. The aim of my study was to introduce the new technology of multiple precursor ion scanning (MPIS) on a QSTAR Pulsar time-of-flight mass spectrometer (QqTOF) to analyze lipids. Detailed information on fatty acid composition of individual GPL molecules could be obtained in parallel with conventional profiling of lipid classes, and this could be done by direct analysis of total lipid extracts. This method was termed Fatty Acid Scanning (FAS) and Head Group Scanning HGS, respectively. In this way the molecular GPL composition of total lipid extracts could be charted in a single analysis accurately and rapidly at a low picomole concentration level. Furthermore, combining FAS and HGS together with ion trap MS3 analysis allowed complete charting of the molecular composition of PCs, including quantification of their positional isomers, thus providing a detailed and comprehensive characterization of molecular composition of the pool of PCs. Development of the Lipid Profiler software allowed full automation and rapid processing of complex data, including identification and quantification of molecular GPLs. This approach was evaluated by preliminary applications. First, the molecular composition of PCs of total lipid extracts of MDCK cells and of human red blood cells (RBC) could accurately be charted. Significant presence of positional isomers was observed increasing the total number of individual PC species close to one hundred. Secondly, the molecular PC and SM species distribution in detergent resistant membranes (DRMs) prepared by Triton X-100 DRMs were analyzed and were found to be enriched in distinct GPLs. The distribution in PCs and SMs of Triton X-100 DRMs of RBC were compared with those of the DRMs of MDCK cells. Finally, combining the use of a 96 well plate and a robotic system demonstrated that these analyses can be automated and analyzed with high throughput. This system we termed Shotgun Lipidomics. Taken together, this mass spectrometric methodology provides rapid and detailed insight into the distribution of the molecular GPLs of membranes and membrane sub-fractions.

Proteolytic processing of the amyloid precursor protein (APP) may lead to the formation of the Abeta peptide, the major constituent of amyloid plaques in Alzheimer`s disease. The full-length APP is a substrate for at least 2 different (alpha and beta) proteases (&amp;quot;secretases&amp;quot;). The beta-secretase, BACE, cleaves APP in the first step of processing leading to the formation of the neurotoxic Abeta. BACE competes for APP with alpha-secretase, which cleaves APP within its Abeta sequence, thus precluding Abeta formation. It is thus important to understand how is the access of the alpha- and beta-secretase to APP regulated and how are the individual activities of these secretases modulated. Both these regulatory mechanisms, access to substrate and direct activity modulation, can be determined by the lipid composition of the membrane. Integral membrane proteins (like APP and BACE), can be viewed as solutes in a two-dimensional liquid membrane, and as such their state, and biological activity, critically depend on the physico-chemical character (fluidity, curvature, surface charge distribution, lateral domain heterogeneity etc.) of the lipid bilayer. These collective membrane properties will influence the activity of embedded membrane proteins. In addition, activity regulation may involve a direct interaction with a specific lipid (cofactor or co-structure function). Interactions of membrane proteins are furthermore affected by lateral domain organization of the membrane. Previous results had suggested that the regulation of the activity of the alpha- and beta-secretases and of their access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have purified recombinant human full-length APP and BACE to homogeneity, and reconstituted them in large (~100nm, LUVs) and giant (10-150microm, GUVs) unilamellar vesicles. Using a soluble peptide substrate mimicking the beta-cleavage site of APP, we have examined the involvement of individual lipid species in modulating BACE activity in LUVs of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1.cerebrosides, 2.anionic glycerophospholipids, 3. cholesterol. Furthermore, we have co-reconstituted APP and BACE together in LUVs and demonstrated that BACE cleaves APP at the correct site, generating the beta-cleaved ectodomain identical to that from cells. We have developed an assay to quantitatively follow the beta-cleavage in proteoliposomes, and we have shown that the rate of cleavage in total brain lipid proteoliposomes is higher than in phosphatidylcholine vesicles. We have also studied partitioning of APP and BACE in GUVs between liquid ordered (lo) and liquid disordered (ld) phases. In this system, significant part of the BACE pool (about 20%) partitions into the lo phase, and its partitioning into lo phase can be further enhanced by cross-linking of membrane components. Only negligible fraction of APP can be found in the lo phase. We continue to study the behavior of co-reconstituted APP and BACE in GUVs The work presented in this thesis has yielded some interesting results and raised further questions. One of the important assignments of this project will in the next stage be the characterization of the impact of membrane domain organization on the beta-cleavage. Different domain arrangements that can be hypothesized in cell membranes can be modeled by varying the degree of phase fragmentation in proteoliposomes comprising reconstituted APP and BACE.

The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall. In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models. Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models. Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.

Alzheimer’s disease is an incurable neurodegenerative disorder linked to the amyloid β (Aβ) peptide, a 38-43 residue peptide. The detailed molecular disease mechanism(s) is (are) unknown, but oligomeric Aβ structures are proposed to be involved. In common for the papers in this thesis is interactions; interactions between Aβ(1-40) and selected molecules and metal ions. The purpose has been to find out more about the structural states that Aβ can adopt, in particular the β-sheet state, which probably is linked to the oligomeric structures. The methods used have been nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopy using Thioflavin T (ThT). Upon addition of SDS/LiDS detergent or Congo red (CR) to Aβ(1-40), the initial random coil/PII-helix state was transformed into β-sheet and, in the case of detergent, a final α-helical state. In contrast to SDS/LiDS and CR, the dimeric Affibody molecule locks monomeric Aβ(1-40) in a β-hairpin state. It was found that by truncating the flexible N-terminal end of the Affibody molecule its affinity to Aβ was improved. The aggregation of Aβ(1-40) was further studied in the presence of a β-cyclodextrin dimer by a kinetic assay using ThT. Although having a weak dissociation constant in the millimolar range, the β-cyclodextrin dimer modified the aggregation pathways of Aβ. Finally Aβ(1-40) was studied in presence of Cu2+ and Zn2+ at physiological and low pH. Cu2+ was observed to maintain its specific binding to Aβ when decreasing the pH to 5.5 while Zn2+ behaved differently. This could be of importance in the Alzheimer’s disease brain in which the environment can become acidic due to inflammation.        In conclusion the results show that Aβ(1-40) is very sensitive to its environment, responding by adopting different conformations and aggregating in aqueous solutions. The β-sheet state is induced by varying molecules with different properties, properties that govern the final Aβ state. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

COSTA, Eveline de Alencar. Avaliação microbiológica e parasitológica nos processos de higienização de alfaces (Lactuca sativa L.) de diferentes cultivos. 2011. 115 f. : Dissertação (mestrado) - Universidade Federal do Ceará, Centro de Ciências Agrárias, Departamento de Tecnologia de Alimentos, Fortaleza-CE, 2011 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-06-03T14:11:52Z No. of bitstreams: 1 20011_dis_eacosta.pdf: 2296033 bytes, checksum: 50ec8fb1765aeda69ae79e6e4e5fb2c5 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-06-03T14:12:05Z (GMT) No. of bitstreams: 1 20011_dis_eacosta.pdf: 2296033 bytes, checksum: 50ec8fb1765aeda69ae79e6e4e5fb2c5 (MD5) / Made available in DSpace on 2016-06-03T14:12:05Z (GMT). No. of bitstreams: 1 20011_dis_eacosta.pdf: 2296033 bytes, checksum: 50ec8fb1765aeda69ae79e6e4e5fb2c5 (MD5) Previous issue date: 2011 / The vegetables sanity that are eaten raw is a relevant factor to health must be ensured by sanitizing with chemicals that have effective action to eliminate, reduce and / or removal of contaminants. The traditional way of cleaning includes washing vegetables under running water followed by immersion in 200ppm chlorine solution and rinse to remove dirt and remnants of the chemical waste. However, some studies indicate that this sanitization does not guarantee the safety of vegetables, for though while reduces the microbial load, it is not efficient in the elimination of some parasitic forms. Therefore, this study aimed to detect microorganisms, protozoa, helminths and arthropods in lettuce (Lactuca sativa L.), curly variety, from conventional farming, organic and hydroponic, in its fresh form and after two methods of cleaning: test (application of detergent and chlorine solution to 200ppm) and traditional (use of only a 200ppm chlorine solution). All lettuce were contaminated by fecal coliforms, but the hydroponic and organic samples had mean values within the standard stipulated by law, 21±74NMP/g and 24±40 NMP/g, respectively) unlike the conventional (152 ± 286NMP/g). In 100% of the samples of conventional and organic lettuces were not contaminated with Salmonella sp. but 20% of hydroponic samples were contaminated by this pathogen, being classified as unfit for consumption. In parasitological analyses were detected in organisms potentially pathogenic to humans: Giardia sp cysts. (2), eggs of Ascaris sp. (6) and eggs of Taenia sp. (1), with six occurrence in three lettuces in conventional and organic. Giardia sp cysts. (2), eggs of Ascaris sp. (6) and eggs of Taenia sp. (1), with six occurrence in three lettuces in conventional and organic. Yet been detected amoeba cysts and larvae of nematodes morphologically similar to those found in man, but more likely were forms of life free from the earth. Forms were observed in abundance among free-living protozoa, nematodes, helminths, and arthropods (especially those in hydroponic lettuce) served in lettuce dirt, reflecting the composition of culture environments. Evaluating the two processes of sanitization studied it was found that the mean of fecal coliform in the different types of lettuce was lower in the test method than the traditional, with increased efficiency. The stage of pre-washing reduced by 60% the initial contamination of fecal coliform, the detergent wash was effective when compared with the reduction achieved in the next step. The traditional method promoted significant decrease in the initial contamination of fecal coliform bacteria, however, no significant difference when compared with the test method or with the step of washing with detergent alone, proving once again its effectiveness. For removal of the forms of helminths, protozoa and arthropods in general, both methods were effective, showing no significant difference. When analyzed separately the different stages of cleaning procedures, it was found that there were significant for the removal of helminths, with better efficiency of the testing procedure, which may suggest that the cleaning with detergent performed only achieve the same results of detergent and chlorine. Thus, we conclude that test hygienization, both for the removal of helminths as to reduce fecal coliform bacteria, showed greater efficacy. / A sanidade das hortaliças que são consumidas cruas é fator relevante à saúde devendo ser garantida mediante a sanitização com produtos químicos que tenham ação eficaz na eliminação, redução e/ou remoção dos contaminantes presentes. A forma tradicional de higienização de vegetais compreende a lavagem com água corrente seguida de imersão em solução de cloro a 200ppm e enxágüe para remover as sujidades remanescentes e resíduos do produto químico. No entanto, alguns estudos apontam que esta sanitização não garante a inocuidade das hortaliças, pois embora reduza a carga microbiana, não é eficiente na eliminação de algumas formas parasitárias. Diante disso, a presente pesquisa objetivou detectar microrganismos, protozoários, helmintos e artrópodes em alfaces (Lactuca sativa, L.), variedade crespa, provenientes de cultivo convencional, orgânico e hidropônico, na sua forma in natura e após dois métodos de higienização: teste (aplicação do detergente e solução clorada a 200ppm) e tradicional (uso apenas da solução clorada a 200ppm). Todas as alfaces estavam contaminadas por coliformes fecais, porém as amostras orgânicas e hidropônicas apresentaram valores médios dentro do padrão estipulado pela legislação, (21±74NMP/g e 24±40 NMP/g, respectivamente) ao contrário das convencionais (152±286NMP/g). Em 100% das amostras de alfaces convencionais e orgânicas não apresentaram contaminação por Salmonella sp., mas 20% das amostras hidropônicas estavam contaminadas por esse patógeno, sendo classificadas como impróprias para consumo. Nas análises parasitológicas foram detectados organismos potencialmente patogênicos para o homem: cistos Giardia sp. (2), ovos de Ascaris sp. (6) e ovo de Taenia sp. (1), com seis ocorrências em alfaces convencionais e três em orgânicas. Foram ainda detectados cistos de ameba e larvas de nematódeos morfologicamente análogos aos que se encontra no homem, porém mais provavelmente eram formas de vida livre provenientes da terra. Foram observadas em abundância formas de vida livre entre os protozoários, os helmintos nematódeos, e os artrópodes (estes com destaque nas alfaces hidropônicas) veiculados nas sujidades das alfaces, refletindo a composição dos ambientes de cultivo. Avaliando os dois processos de sanitização estudados constatou-se que a média dos valores de coliforme fecal nos diferentes tipos de alfaces foi menor no método teste do que no tradicional, evidenciando maior eficiência. A etapa da pré-lavagem reduziu em 60% a contaminação inicial de coliformes fecais; a lavagem com detergente foi eficaz quando comparada com a redução obtida na etapa seguinte. O método tradicional promoveu diminuição significativa na contaminação inicial de coliformes fecais, no entanto, não houve diferença significativa quando comparada com o método teste nem com a etapa da lavagem com detergente isoladamente, comprovando mais uma vez a eficácia deste último. Para a remoção das formas de helmintos, protozoários e artrópodes em geral, ambos os métodos foram eficientes, sem apresentarem diferença significativa. Quando foram analisadas separadamente as diferentes etapas dos procedimentos de higienização, constatou-se que houve significância estatística para a remoção de helmintos, com melhor eficiência do procedimento teste, podendo-se sugerir que a higienização realizada apenas com detergente alcançaria os mesmos resultados de detergente e cloro. Deste modo, concluímos que a higienização teste, tanto para a remoção de helmintos como para a redução de coliformes fecais, apresentou maior eficácia.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this research was to evaluate of three grazing systems (GS) with Elephant grass (EG) + Italian ryegrass (IR) + spontaneous growing species (SGS); EG + IR + SGS + Forage peanut (FP); and EG + IR + SGS + Red clover (RC), in order to use the area in rotational grazing during all agricultural year. EG was planted in rows with a distance of 4 m each one of them. In the cool-season, IR was sowed between rows of EG; RC was sowed and the FP who has found established since 2006 was preserved on respectively GS. Experimental design was completely randomized with three treatments (GS), two replicates (paddocks) and independent evaluations (grazing cycles). For evaluation, were used lactating Holstein cows receiving 1% of BW day-1 feed supplement concentrate. The pre and post forage mass parameters, botanical and structural composition, forage production and stocking rate were evaluated. Samples were collected by the handplucking method to analyze the organic matter, mineral matter, crude protein (CP), neutral detergent fiber (NDF), in situ organic matter digestibility (ISOMD), in situ dry matter digestibility (ISDMD) of forage. Eight grazing cycle were performed during the experimental period (357 days). The values of forage production and stocking rate were 15.03, 16.33, 17.03 t ha-1 and 3.57, 3.52 and 3.65 animal unit ha-1, respectively grazing systems. Grazing systems involving grasses and forage legumes in different cycles provide use of the area during the agricultural year in rotational grazing with dairy cattle. SGS increased significantly (P≤0.05) over in GS without forage legume. Higher value of CP, ISOMD and lower of NDF for GS mixed with forage legumes. Considering the herbage mass, stocking rate and nutritive value, the pastures mixed to forage legumes showed better performance. / O objetivo desta pesquisa foi avaliar três sistemas forrageiros (SF) com capim elefante (CE) + azevém (AZ) + espécies de crescimento espontâneo (ECE); CE + AZ + ECE + amendoim forrageiro (AM); e CE + AZ + ECE + trevo vermelho (TV), com o intuito de utilização da área em pastejo rotativo durante todo o ano agrícola. O CE foi estabelecido em linhas afastadas a cada 4 m. No período hibernal fez-se o estabelecimento do AZ entre as linhas do CE; o TV foi semeado e o AM que já se encontrava estabelecido desde 2006, foi preservado, considerando os respectivos tratamentos. O delineamento experimental foi o inteiramente casualizado, com três tratamentos (SF), duas repetições (piquetes) e avaliações independentes (ciclos de pastejo). Para avaliação foram utilizadas vacas da raça Holandesa em lactação, que receberam suplementação alimentar com concentrado à razão de 1% do peso corporal dia-1. Foram avaliados parâmetros de massa de forragem pré e pós pastejo, composições botânica e estrutural, produção de forragem e taxa de lotação. Para analisar a matéria orgânica, matéria mineral, a fibra em detergente neutro (FDN), a proteína bruta (PB), a digestibilidade in situ da matéria seca (DISMS) e a digestibilidade in situ da matéria orgânica (DISMO) da forragem, foram coletadas amostras de pastejo simulado. Durante o período experimental (357 dias) foram efetuados oito ciclos de pastejo. Os valores de produção de forragem e de taxa de lotação foram de 15,03; 16,33; 17,03 t ha-1 e 3,57; 3,52 e 3,65 UA ha-1, respectivamente para os sistemas forrageiros. Sistemas forrageiros, envolvendo gramíneas e leguminosas de diferentes ciclos, proporcionam a utilização da área durante todo o ano agrícola em pastejo rotativo com bovinos leiteiros. Houve aumento significativo (P≤0,05) para ECE na pastagem sem leguminosa. Foram observados maiores valores de PB, DISMO e menores de FDN para os SF consorciados com leguminosas. Considerando a massa de forragem, a taxa de lotação e o valor nutritivo, as pastagens consorciadas com leguminosas forrageiras apresentaram melhor desempenho.

Made available in DSpace on 2014-07-29T15:13:45Z (GMT). No. of bitstreams: 1 Tese parte1 Fabricia Rocha Chaves Miotto.pdf: 8322893 bytes, checksum: e402e3e9bcec9054d69df88cb3b48403 (MD5) Previous issue date: 2011-08-18 / This study aimed to evaluate intake, apparent digestibility of nutrients and nitrogen balance (NB) of diets containing babassu mesocarp bran (BMB) in replacement of elephant grass silage (0, 21, 38, 62 and 78%). Twenty sheep were placed in metabolism crates and allotted to a completely randomized design with four replicates. Were collected and recorded food intake and excretion of feces and urine. The intakes of dry matter (DM), organic matter (OM), crude protein (CP), ash, neutral detergent fibrous (NDF), acid detergent fibrous (ADF), hemicellulose and total carbohydrates were not affected by experimental diets. Change was observed in the intakes of non fibrous carbohydrates (NFC) and reduced intake of ether extract (EE) as % of live weight (%LW) and grams per unit of metabolic weight (g/UMW). The total digestible nutrients TDN intake had a quadratic response with maximum points at 37.3 and 37.9% of BMB when expressed in % LW and g/UMW, respectively. The apparent digestibilities of OM, NDF, ADF, CP and NFC reduced to the extent that the BMB has increased in the diets. The digestibility of DM, EE, and NFC had a quadratic response, where the maximum digestibility of DM was estimated at 10% BMB. TDN decreased 0.28% for each percentage point of inclusion in the diets of BMB. The BMB use reduces the fibrous fractions digestibility, but increases the NFC and TDN intake, indicating that this byproduct can be used as an energy source for ruminant diets. Note: Thesis w/seven chapters. Presented summary and abstract of the 2nd chapter / Objetivou-se avaliar consumo, digestibilidade aparente de nutrientes e balanço de nitrogênio (BN) de dietas contendo farelo de mesocarpo de babaçu (FMB) em substituição à silagem de capim elefante (0; 21; 38; 62 e 78%). Utilizaram-se 20 ovinos alojados em gaiolas metabólicas individuais e distribuídos em delineamento inteiramente casualisado com quatro repetições. Foram coletados e registrados o consumo de alimentos e excreções de fezes e urina. Os consumos de matéria seca (MS), matéria orgânica (MO), proteína brutua (PB), cinza, fibra em detergente neutro (FDN), fibra em detergente ácido (FDA), hemicelulose e carboidratos totais não foram influenciados pelas dietas experimentais. Foi observada alteração nos consumos de carboidratos não fibrosos (CNF) e redução nos consumos de extrato etéreo (EE) em % do PV e em gramas por unidade de tamanho metabólico (g/UTM). O consumo de nutrientes digestíveis totais (NDT) teve comportamento quadrático com pontos de máximo em 37,3 e 37,9% de FMB expressos em %PV e g/UTM, respectivamente. As digestibilidades aparentes da MO, FDN, FDA, PB e CNF reduziram na medida em que o FMB aumentou nas dietas. A digestibilidade da MS, EE e CNF tiveram comportamento quadrático, em que a máxima digestibilidade da MS foi estimada com 10% de FMB. O NDT reduziu 0,28% para cada ponto percentual de inclusão de FMB nas dietas. O uso do FMB reduz a digestibilidade de frações fibrosas, contudo aumenta o consumo de CNF e o consumo de NDT, o que indica que esse subproduto pode ser utilizado como fonte energética para dietas de ruminantes. OBS: Tese c/ sete capítulos. Apresentado resumo e abstract do 2° capítulo.

INTRODUÇÃO: A limpeza prévia de endoscópios rígidos (ER) seguida de desinfecção de nível intermediário com álcool etílico a 70% p/v após o exame de endoscopia nasal é uma prática adotada em muitos serviços de otorrinolaringologia. A literatura atual, no entanto, recomenda a esterilização ou desinfecção de alto nível como o método de descontaminação mais aceito para produtos para saúde classificados como semicríticos. No entanto, há que se fazer distinção entre equipamentos de alta complexidade e sua invasividade como os endoscópios flexíveis com lumens longos e estreitos utilizados na endoscopia digestiva, daqueles de conformação simples sem lumens de baixa invasividade como os endoscópios rígidos utilizados em otorrinolaringologia. OBJETIVO: Avaliar a segurança da desinfecção de nível intermediário com álcool etílico 70% p/v, após limpeza prévia dos endoscópios rígidos utilizados em procedimentos clínicos de endoscopia nasal considerando a carga microbiológica recuperada após o uso. MÉTODO: Imediatamente após a realização do exame, uma gaze úmida foi utilizada para o arraste da carga biológica do endoscópio rígido, gerando as amostras do Controle Positivo e, após a aplicação do POP, um novo arraste para constituir as amostras do Grupo Experimental. Estas gazes foram inicialmente submetidas à sonicação e agitação imersas em soro fisiológico e em seguida a solução foi submetida a uma técnica de extração de carga microbiológica por filtragem por meio de uma Membrana de Celulose de 0,22um de poro que foi, em seguida, semeada nos meios de ágar Sangue, Chocolate, Sabouraud, Löwenstein-Jensen e Tioglicolato. Estes meios ficaram incubados em estufa a 37ºC ± 2ºC e avaliados, no máximo, até por 60 dias conforme o perfil de crescimento dos diferentes microrganismos de interesse; foram analisados de maneira quantitativa e qualitativa para identificação e classificação dos micro-organismos recuperados após as semeaduras. RESULTADO: Os resultados da análise estatística evidenciaram diferença significativa entre Controle Positivo e Grupo Experimental quando comparados em relação à presença de Streptococcus coagulase negativa (p < 0,001), Bacillus spp (p < 0,001) e Staphylococcus aureus (p=0,001). No Controle Positivo, foram encontradas presença desses micro-organismos respectivamente na seguinte frequência: 63,2%, 28,9% e 28,9%, enquanto que, no Grupo Experimental, não foi houve recuperação microbiana alguma. CONCLUSÃO: Os resultados desta pesquisa demonstram a eficiência, na prática diária, da desinfecção de nível intermediário dos endoscópios utilizados na otorrinolaringologia por meio da fricção com álcool etílico 70% p/v por 90 segundos, com protocolo de limpeza prévia / INTRODUCTION: Prior cleaning of rigid endoscopes (REs) followed by intermediate-level disinfection with 70% ethyl alcohol (w/v) after nasal endoscopy is a common practice in many otolaryngology services. Current literature, in turn, recommends high-level sterilization or disinfection as the most accepted decontamination method for health products classified as semi-critical. However, it is necessary to distinguish highly complex equipment according to their invasiveness, e.g., flexible endoscopes with long and narrow lumens used in digestive endoscopy and those with a simple conformation without lumens of low invasiveness, such as rigid endoscopes used in otorhinolaryngology. OBJECTIVE: To evaluate the safety of intermediate-level disinfection with 70% ethyl alcohol (w/v) after cleaning of REs used in clinical procedures of nasal endoscopy considering the microbiological load recovered after use. METHOD: Immediately after the test, a wet gauze was used to drag the biological load from the RE, generating positive control samples; after applying POP, dragging was carried out again to generate samples of the experimental group. These gasses were initially subjected to sonication and shaking while immersed in physiological saline; the solution was then subjected to the microbiological loading technique by filtration through a 0.22-um pore cellulose membrane and then cultivated on blood, chocolate, Sabouraud, Löwenstein-Jensen, and thioglycolate agar media. These media were incubated at 37ºC ± 2ºC and evaluated for up to 60 days, according to the growth profile of the different microorganisms of interest. A quantitative and qualitative analysis was performed for the identification and classification of microorganisms recovered after cultivation. RESULTS: The results of statistical analysis showed a significant difference between the positive control and experimental groups for the presence of coagulase-negative Streptococcus (p < 0.001), Bacillus spp (p < 0.001), and Staphylococcus aureus (p=0.001). In the positive control group, these microorganisms were found in the following proportions: 63.2%, 28.9%, and 28.9%, respectively, whereas in the experimental group, no microorganisms were recovered. CONCLUSION: The results of this study demonstrate the efficiency of the daily practice of intermediate-level disinfection of endoscopes used in otorhinolaryngology by means of treatment with 70% ethyl alcohol (w/v) for 90 seconds, using a previous cleaning protocol

L’ingénierie des protéines fonctionnelles est un processus d’amélioration des propriétés physiques ou catalytiques d’enzyme au travers d’approches rationnelles et d'évolution dirigée, aussi bien que la combinaison des deux méthodes. Malgré le progrès de la modélisation moléculaire des protéines, les méthodes de prédiction restent aléatoires et un grand nombre de variantes restent à tester. De ce fait, le développement et l’utilisation d’un système de criblage d’activité de protéines à très haut débit, comme la microfluidique en gouttes, est indispensable. Cette thèse de doctorat présente trois projets d’évolution dirigée de protéines en trois approches différentes avec expression d’enzyme in vitro et in vivo. Les plateformes microfluidiques ont été développées et validées pour chaque projet. De plus, plusieurs banques de variants ont été criblées avec, dans certains cas, isolement de molécules 5-10 fois que le clone parental. / This work describes the development of high-throughput droplet microfluidic platforms fine-tuned for protein of interest and their employment in directed evolution experiments. When not available, fluorogenic assay for monitoring desired enzyme activity (-ies) in droplets was developed. Moreover, the in vivo expression allowed the successive integration of microfluidic modules on the same chip. After a couple of evolution rounds the initial retro-aldolase variant was significantly improved. In other project, to meet industrial requirements a high-throughput screening platform for protease evolution in detergent has been assembled and validated. Two evolution rounds showed the accumulation of a certain pool of beneficial mutations over the selection rounds. The research described in this work highlighted that in vitro expression systems are sensitive to the amount of supplied DNA and reaction conditions. This observation led to the development of a multistep completely in vitro microfluidic platform.