Genes de plantas que codificam inibidores de enzimas digestivas de insetos têm sido introduzidos em plantas cultivadas visando o controle de pragas. Os inibidores de proteinases estão presentes nos tecidos vegetais, principalmente nas sementes, e atuam em resposta a ataques por herbívoros e patógenos. Inibidores de serino-proteinases (IPs) dos tipos Bowman-Birk e Kunitz isolados de sementes de soja foram inseridos em variedades de cana-de-açúcar para aumentar a resistência à broca Diatraea saccharalis (Fabr.), principal praga desta cultura. Para utilização de plantas geneticamente modificadas contendo inibidores de proteinases é necessário um conhecimento profundo de sua sustentabilidade e segurança ambiental, determinando a estabilidade da característica inserida e os seus efeitos nos organismos não-alvo. O objetivo desta pesquisa foi avaliar os efeitos diretos e indiretos de inibidores de proteinases de soja em organismos não-alvos: um parasitóide larval, Cotesia flavipes (Cam.) (Hymenoptera: Braconidae); um patógeno, Metarhizium anisopliae (Mestch.) Sorokin (Deuteromycotina: Hyphomycetes); um polinizador, Apis mellifera L. (Hymenoptera: Apidae) e um decompositor, Scheloribates praeincisus (Berlese) (Acari: Oribatida: Scheloribatidae); associados à cultura da cana-de-açúcar. O consumo de Kunitz e BBI não afetou a sobrevivência de S. praeincisus. Por outro lado, a ingestão dos inibidores semi-purificados e purificados do tipo Kunitz diminuiu a duração das fases imaturas de S. praeincisus. A ingestão de folhas de cana GM expressando inibidores de proteinases (Kunitz e BBI) não afetou o tempo de desenvolvimento e a sobrevivência dos imaturos deste oribatídeo quando comparada à ingestão de suas isolinhas. Os inibidores de proteinases semi-purificados e purificados não afetaram a duração dos períodos larval e pupal, o peso e número de pupas e percentual de emergência do parasitóide C. flavipes em D. saccharalis. Por outro lado, a proporção de fêmeas em relação a machos de C. flavipes foi maior no tratamento onde as lagartas foram alimentadas com dieta contendo 0,5% de inibidores semi-purificados comparado-se à testemunha. A proporção fêmea:macho foi significativamente maior também quando os parasitóides foram alimentados com o inibidor do tipo Kunitz em relação ao controle e aos parasitóides alimentados com BBI. A adição de 0,5% (p/v) de inibidores de proteinases semi-purificados e 0,05% (p/v) de inibidores purificados do tipo Kunitz nos meios de cultura MC e BDA resultaram em maiores crescimento vegetativo e produção de conídios de M. anisopliae. Os inibidores purificados do tipo BBI não alteraram a esporulação do fungo. Os resultados dos estudos com A. mellifera não foram conclusivos e novas investigações precisam ser conduzidas para esclarecer os potenciais efeitos de inibidores de proteinases em abelhas. De uma forma geral, observou-se que os inibidores de proteinases (Kunitz e BBI) não afetaram negativamente os organismos não-alvo testados. Por outro lado, a ingestão de inibidor do tipo Kunitz alterou positivamente alguns parâmetros biológicos de C. flavipes, M. anisopliae e S. praeincisus. / Genes of plants expressing insect proteinase inhibitors have been introduced into plants for pest control. Proteases inhibitors are present in plant tissues, mainly in seeds, and act in response to predators and pathogens. The Bowman-Birk type and Kunitz type of serine proteases inhibitors (PI) from soybean seeds are been used to increase resistance of sugarcane to Diatraea saccharalis (Fabr.), the most important pest of this crop. The sustainability and environmental safety of PI crops is still unknown. For these reasons, it is necessary to understand the stability and the non-target effects of this new trait. The objective of this study was to evaluate the direct and indirect effects of soybean PI on the following non-target organisms associated to sugarcane: the larval parasitoid, Cotesia flavipes (Cam.) (Hymenoptera: Braconidae); the entomopathogen, Metarhizium anisopliae (Mestch.) Sorokin (Deuteromycotina: Hyphomycetes); the pollinator Apis mellifera L. (Hymenoptera: Apidae) and the soil mite involved in the process of recycling organic matter, Scheloribates praeincisus (Berlese) (Acari: Oribatida: Scheloribatidae). Kunitz and BBI did not affect S. praeincisus survival. On the other hand, Kunitz semi-purified and purified inhibitor ingestion reduced duration of the immature stages of S. praeincisus. Ingestion of GM senescent leaves did not have an effect on mite immatures development time and survival compared to ingestion of its isolines leaves. The semi-purified and purified proteinases inhibitors did not alter either the duration of larval and pupal stages of C. flavipes on D. saccharalis, or weight and number of pupae and parasitoid emergence. In other hand, the parasitism and proportion of female was higher on the treatment where caterpillars were fed with diet containing 0.5% of semi-purified inhibitors, comparing to control. The ratio female:male was significantly higher also when parasitoids were fed to the Kunitz type inhibitor compared to the control and BBI. The addition of 0.5 % (w/v) of semi- purified proteinase inhibitors and 0.05% (w/v) of Kunitz type purified inhibitors on two culture media (CM and PDA), resulted in increase of vegetative growth and production of conidia. BBI type purified inhibitors did not change the fungus sporulation. The results from the studies with A. mellifera were not conclusive and investigations are needed to clarify the potential impact of proteinase inhibitors on A. mellifera. Overall, proteinase inhibitors (Kunitz and BBI) did not negatively affect the non-target organisms tested. Conversely, ingestion of the Kunitz type of proteinase inhibitors altered positively some biological parameters of C. flavipes, M. anisopliae and S. praeincisus.

Les biocapteurs sont des moyens d’analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d’outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d’un pouvoir de reconnaissance pour un analyte ou un groupe d’analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique. Dans ce travail, nous nous sommes intéressés au développement de différents biocapteurs se basant sur l'immobilisation d'enzymes ou de bactéries sur des microélectrodes en vue d’une détection électrochimique. Nous avons montré les potentialités d’application de deux biocapteurs conductimétriques à base de protéinase K ou de protéinase K et de pronase à la détection des modifications de conformation de la myoglobine et de l’albumine de sérum bovin au cours de leur relargage à partir de microsphères de poly (ε-caprolactone). Nous avons également mis au point un biocapteur conductimétrique à base de catalase et d’alcool oxydase pour une détection rapide et sensible des alcools ainsi que deux biocapteurs à catalase pour la détection impédimétrique et conductimétrique du cyanure et l’étude des interactions catalase-cyanure. Nous avons enfin élaboré des biocapteurs bactériens à base de Pseudomonas putida F1 pour la détection du trichloroéthylène dans les eaux souterraines. Pour cela, une voie originale d’immobilisation des cellules, basée sur la fonctionnalisation du transducteur à l’aide de couches autoassemblées et d’anticorps, ainsi que l’utilisation de nanotubes de carbone, a été explorée / The development of biosensors is an expanding research area. Indeed, biosensors are rapid, selective and cost-effective analytical tools which find applications in various fields (environment, health, food,…). They are constituted of a sensitive biological element (antibody, enzyme, microorganism, DNA…), which can selectively recognize one analyte or a group of analytes, associated to an electrochemical, optical or thermal transducer. In this work, we developed different biosensors based on enzymes or bacteria immobilised onto microelectrodes in view of electrochemical detection. First, we demonstrated the potentialities of two conductometric biosensors based on proteinase K or proteinase K and pronase for the detection of myoglobin and bovine serum albumine conformation changes during their release from poly (ε-caprolactone) microspheres. Then, we elaborated a bi-enzymatic conductometric biosensor with catalase and alcohol oxidase as sensing elements, for a rapid and sensitive detection of alcohols. Catalase impedimetric and conductometric biosensors were also developed for cyanide detection and used for the study of catalase-cyanide interactions. Finally, we prepared Pseudomonas putida F1 whole cell biosensors for the determination of trichloroethylene in groundwaters. For that, an original route, including the functionalisation of the transducer with a self-assembled-monolayer and antibodies, and the use of single-wall carbon nanotubes, was investigated for cell immobilisation

La maladie d'Alzheimer (AD) est un désordre neurodégénératif progressif et la forme la plus commune de démence. A l’heure actuelle, il n'y a aucun remède et la maladie est toujours fatale. Une des caractéristiques histopathologiques de l'AD est la présence de dépôts protéiques, les plaques amyloïdes, dans le cerveau. Ces plaques sont formées par les peptides amyloïdes β (Aβ) de 40 et 42 résidus, qui sont les produits de clivage par des protéases de la protéine précurseur de l’amyloïde (l'APP). L'élucidation de certains des processus clés dans la cause et le développement de l'AD est une étape cruciale pour le développement de traitements nouveaux et efficaces.<p><p>Les propriétés conformationnelles du segment transmembranaire (TM) de l’APP peuvent affecter sa protéolyse par la γ-sécrétase. Ces propriétés ne sont pas encore clairement établies. Afin de comprendre le rôle des variations structurelles du TM dans le traitement de l'APP, des détails structurels des peptides APP_TM4K, chimiquement synthétisés, ont été étudiés dans la bicouche lipidique en utilisant la réflexion totale atténuée par spectroscopie infrarouge à transformée de Fourier (ATR-FTIR) et la résonance magnétique nucléaire à l’état solide (ssNMR). Tandis que la structure secondaire globale du peptide APP_TM4K est hélicoidale, une hétérogénéité conformationnelle et orientée a été observée pour le site de clivage γ et, dans une plus faible mesure, pour le site de clivage ζ. Ces variabilités conformationnelles autour des sites de clivage γ et ζ peuvent avoir des implications importantes dans le mécanisme de clivage et donc dans la production d’Aβ. Il a été aussi démontré que la dernière glycine dans le motif de dimérisation GxxxG est transmembranaire. Ceci peut impliquer que la dimérisation via ce motif pourrait servir d’ancrage et conférer une orientation transmembranaire stable au segment transmembranaire de l’APP.<p><p>Le peptide amyloïde β est directement lié à la maladie d’Alzheimer. Partant de sa forme monomérique, l’Aβ s'agrège pour produire en final des fibrilles et aussi de manière transitoire toute une gamme d'oligomères, ces derniers étant la plupart neurotoxiques. Une dérégulation de l’homéostasie du Ca2+ dans le cerveau vieillissant et dans des troubles neurodégénératifs joue un rôle crucial dans de nombreux processus et contribue au dysfonctionnement et à la mort cellulaire. Nous avons postulé que le calcium peut permettre ou accélérer l'accumulation d'Aβ. Le modèle d'accumulation d'Aβ (1-40) et celui d'Aβ (1-40) E22G, un peptide amyloïde portant la mutation arctique qui cause une apparition prématurée de la maladie, ont été comparé. Nous avons constaté qu'en présence de Ca2+, l’Aβ (1-40) forme de préférence des oligomères semblables à ceux formés par l’Aβ (1-40) E22G avec ou sans Ca2+, tandis qu'en absence de Ca2+ l'Aβ (1-40) s’agrège sous forme de fibrilles. Les ressemblances morphologiques entre oligomères ont été confirmées par microscopie de force atomique. La distribution des oligomères et des fibrilles dans des échantillons différents a été détectée par électrophorèse sur gel suivie d’une analyse par Western blot, dont les résultats ont été confirmés par des expériences de fluorescence à la thioflavine T. Dans les échantillons sans Ca2+, l’ATR-FTIR révèle la conversion des oligomères en feuillets β antiparallèles en la conformation caractéristique des fibrilles en feuillets β parallèles. En général, ces résultats nous ont ameré à conclure que les ions calcium stimulent la formation d'oligomères d'Aβ (1-40), qui sont impliqués dans la pathogénèse d'AD.<p><p>Malgré les progrès énormes obtenus dans la compréhension de la maladie (AD), il reste un défi majeur, celui du développement de médicaments nouveaux et efficaces. Afin d’obtenir des éclaircissements sur le mécanisme d'action de deux nouveaux puissants modulateurs de la γ-sécrétase - le benzyl-carprofen et le sulfonyl-carprofen dans la bicouche lipidique, la technique de RMN à l’état solide a été employée. Précédemment, les dérivés du carprofen ont été localisés dans des membranes de lipides par des expériences de diffusion (scattering) des neutrons. Les contraintes déterminées à partir des expériences de ssNMR ont permis d’affiner leurs positions et d’obtenir une orientation précise dans la double couche lipidique. Ces résultats combinés indiquent que le mécanisme probable de modulation du clivage par la γ-sécrétase est une interaction directe des carprofènes avec le domaine TM de l’APP. Une telle interaction, empêcherait à la formation de dimères d'APP, dimérisation nécessaire au clivage séquentiel par la γ-sécrétase, diminuant ou réduisant ainsi énormément la production d’Aβ, tout particulièrement d’Aβ42.<p><p>Les résultats de ce travail apporte de nouvelles informations sur les processus clés impliqués dans l'AD; Production de l'Aβ à partir de l'APP, formation des oligomères d'Aβ et mécanisme d'action potentiel de molécules thérapeutiques. Nous pensons que ces résultats pourront permettre une meilleure compréhension de la maladie et pourront aider dans la conception de nouveaux médicaments contre cette maladie.<p><p>Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. There is no cure and the disease is fatal. One of the characteristic histopathological markers of AD is the presence of proteinaceous deposits, amyloid plaques, in the brain. These plaques are formed by the amyloid β-peptides (Aβ) 40- and 42-residue-long, which are protease cleavage products of the amyloid precursor protein (APP). Elucidation of some of the key processes in the cause and the development of AD is crucial for the development of new and efficient treatments.<p><p>Conformational properties of the transmembrane (TM) segment of APP may affect its proteolytic processing by γ-secretase. These properties have not been definitely established. In addressing the role of structural variations of the TM sequence in APP processing, structural details of the chemically synthesized APP_TM4K peptides within the membrane bilayers were studied using Attenuated total reflection Fourier transform spectroscopy (ATR-FTIR) and solid-state nuclear magnetic resonance (ssNMR) techniques. While the overall secondary structure of the APP_TM4K peptide is an α-helix, conformational and orientational heterogeneity was observed for the γ-cleavage site and, to a smaller extent, for the ζ-cleavage site. Evidence for the conformational variability around γ- and ζ-cleavage sites may have important implications for the cleavage mechanism and hence for the Aβ production. It was also found that the last glycine within the sequence of GxxxG motifs is in the transmembrane orientation, implying that dimerization via these motifs may act as an anchor, confining the TM dimer to the stable transmembrane orientation. <p><p>Amyloid β-peptide is directly linked to AD. Starting from its monomeric form, Aβ aggregates into fibrils and / or oligomers, the latter being the most neurotoxic. Dysregulation of Ca2+ homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. The aggregation pattern of Aβ(1-40) and of Aβ(1-40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease, were compared. We found that in the presence of Ca2+, Aβ(1-40) preferentially formed oligomers similar to those formed by Aβ(1-40)E22G with or without added Ca2+, whereas in the absence of added Ca2+ the Aβ(1-40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy (AFM) imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results which were further supported by thioflavin T fluorescence experiments. In the samples without Ca2+, Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1-40), that have been implicated in the pathogenesis of AD. <p><p>Despite the tremendous progress in understanding AD, there remains the challenge of the development of new and efficient drugs. In order to shed light onto the mechanism of action of two new potent γ-secretase modulators -- benzyl-carprofen and sulfonyl-carprofen within lipid bilayers, ssNMR technique was employed. Using neutron scattering experiments it was previously found that sulfonyl-carprofen and benzyl-carprofen partition into the headgroup region of the lipid bilayer. The orientational constraints derived from the ssNMR experiments refined their position into precise orientation. Combined, these results indicate that carprofen-derivatives can directly interact with the region of APP that mediates dimerization. Such interaction, would interfere with proper APP-dimer formation, which is necessary for the sequential cleavage by γ-secretase, diminishing or greatly reducing Aβ42 production.<p><p>Results obtained during this work shed new light onto some of the key processes in AD: Aβ production from APP, formation of Aβ oligomers and insights into the mechanism of action of potential therapeutics. We believe that these results will promote a better understanding of the disease and will help in future drug design.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les produits laitiers (fromages, yaourts, laits fermentés, …) représentent 10% de notre alimentation. Ces produits sont appréciés pour leur flaveur, leur texture et leurs bénéfices sur la santé. Ces caractéristiques sont dues, entre autres, aux peptides contenus dans ces produits, qui sont issus de la coupure des protéines du lait par des enzymes qui sont présentes dans le lait, ajoutées lors de la transformation ou provenant des bactéries. Comprendre et maîtriser l’élaboration des produits laitiers est donc essentiel pour améliorer et développer les aliments futurs. La fabrication des produits laitiers fermentés tels que le fromage commence par la coupure, ou protéolyse, des protéines majeures du lait (les caséines) grâce à différentes enzymes appelées protéases. Certaines sont attachées à la surface des bactéries lactiques qui sont utilisées au début de la fabrication du fromage, telles que Streptococcus thermophilus ou Lactobacillus helveticus. S. thermophilus possède une protéase de paroi, PrtS, déjà bien étudiée contrairement à L. helveticus qui possède quatre protéases de paroi, PrtH, PrtH2, PrtH3 et PrtH4, qui n’ont pas été jusqu’à présent étudiées séparément. L’objectif de ce travail de thèse est de mieux comprendre les propriétés protéolytiques de L. helveticus et S. thermophilus en déterminant comment leurs protéases participent à la protéolyse des caséines en matrice laitière. Deux stratégies ont été mises en place : exprimer une des protéases de L. helveticus dans S. thermophilus afin de l’étudier de façon indépendante et étudier l’activité de chacune des protéases chez des souches qui n’expriment qu’un seul gène de protéase de paroi. D’une part, un outil de sécrétion hétérologue fonctionnel a été mis au point chez S. thermophilus LMD-9. D’autre part, les protéases étudiées présentaient une activité caséinolytique supérieure à pH 5,2 qu’à pH 7,5 et dégradaient préférentiellement la caséine β. Enfin, PrtH3 de L. helveticus semble présenter la plus forte activité caséinolytique / Dairy products (cheeses, yoghurt, fermented milk…) represent 10% of our food. These products are appreciated thanks to their flavor, their texture and their health benefits. These characteristics are due to various compounds like peptides which come from the cutting of milk protein by enzymes. These enzymes come from the milk, are added during the fabrication or come from bacteria. To understand and to master the dairy products fabrication is essential to develop the food of tomorrow. The fabrication of fermented dairy products as cheese begins with the cutting or proteolysis of caseins, the major milk proteins, thanks to enzymes called proteinases. Some of them are attached at the surface of lactic acid bacteria which are used at the beginning of the cheese fabrication, as Streptococcus thermophilus or Lactobacillus helveticus. S. thermophilus possess one cell envelop proteinase, PrtS, already well-studied unlike L. helveticus which possess four cell envelop proteinases, PrtH, PrtH2, PrtH3 and PrtH4, which were not studied separately yet. The aim of this thesis work is to better understand the proteolytic properties of L. helveticus and S. thermophilus and identify how their proteinases adjust the hydrolysis profile of caseins in a dairy matrix.Two strategies were selected: to express one of the proteinases of L. helveticus in S. thermophilus to study it independently from the others and study each proteinase in strains which only express one proteinase gene

Indiana University-Purdue University Indianapolis (IUPUI) / Histones are highly basic proteins which when digested by trypsin are hard to analyze using mass spectrometry. Because histones are basic nuclear proteins, a nuclei prep followed by acid extraction is the best purification strategy to increase overall abundance of purified histones. Blocking the lysine residues and cleaving with trypsin is a useful technique to increase detection of histone peptides using MudPIT. In particular, carbamylation and propionylation are the best two methods to block lysine residues. Using both propionylation and carbamylation along with no treatment has been shown to increase the identification of unmodified and modified histone peptides when coupled with MudPIT analysis.

Made available in DSpace on 2015-07-22T20:50:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-05-25. Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 1 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 2 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5) Publico-12837b.pdf: 2043061 bytes, checksum: 4edc37b57c73112ae7353868a78233ca (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:12Z : No. of bitstreams: 3 Publico-12837a.pdf: 1258726 bytes, checksum: a7e0f280ec3b88f2f775c2b325704cd7 (MD5) Publico-12837b.pdf: 2043061 bytes, checksum: 4edc37b57c73112ae7353868a78233ca (MD5) Publico-12837c.pdf: 1773766 bytes, checksum: 56addbd5601cb4e7336afb640ed56fe8 (MD5) / Objetivo: O prolapso de órgãos pélvicos (POP) resulta da falha na sustentação do assoalho pélvico, e anormalidades do tecido conjuntivo podem estar envolvidas na etiologia e/ou na progressão da disfunção. Analisar-se-á a expressão diferencial de genes e de proteínas que participam da biossíntese do colágeno e da elastina: lisil oxidases (LOXs), fibulina-5, fibrilinas-1 e -2 e pró-colágeno C proteinase (PCP/BMP1), no tecido vaginal de mulheres sem e com POP acentuado consoante seu estado hormonal. Casuística e Métodos: Durante a histerectomia total, biópsias de parede vaginal anterior foram obtidas de mulheres caucasianas na pré-menopausa (fase proliferativa do ciclo menstrual) e na pós-menopausa com POP acentuado (POPQ estadio III e IV), e de controles assintomáticas (POPQ 0). RNAm e proteínas totais foram extraídos usando Trizol e RIPA Buffer, e os genes e proteínas de interesse quantificados por RT-PCR em tempo real e Imunobloting, respectivamente. As seguintes análises comparativas foram realizadas: (1) expressão dos genes e das proteínas da família LOX (LOX e LOXL1-4), fibulina-5 e fibrilinas- 1 e -2 em pacientes na pré-menopausa com e sem POP; (2) expressão do gene e da proteína PCP/BMP1 em pacientes na pré- e pós-menopausa com POP, e respectivos controles. Os testes de Wilcoxon signed-rank e Fisher foram usados para as análises estatísticas (p<0.05). Resultados: Obtivemos amostras de 15 pacientes e 11 controles na pré-menopausa para o estudo (1), e 39 pacientes na pré-menopausa (POP=23 e Controle=16) e 18 na pósmenopausa (POP=13 e Controle=5) para o estudo (2). A partir das análises, observamos (1) diminuição significativa na expressão dos genes LOX, LOXL1 e LOXL3, bem como nas proteínas LOX e LOXL3 no tecido vaginal de pacientes POP na pré-menopausa comparadas com mulheres assintomáticas (p<0.05); (2) hipoexpressão do gene PCP/BMP1 nos tecidos vaginais de mulheres com POP acentuado comparadas com controles, tanto na prémenopausa como na pós-menopausa (ambos p=0.01); redução significativa das isoformas 130kDa, 92,5kDa e 82,5kDa da PCP/BMP1 no tecido vaginal de pacientes na pósmenopausa (p=0.01), bem como hiperexpressão da isoforma 130kDa nas mulheres com POP acentuado na pré-menopausa (p=0.009), comparadas com as respectivas controles. Conclusão: As expressões das enzimas LOXs e pró-colágeno C proteinase estão alteradas no tecido vaginal de mulheres com POP, e são moduladas pelo estado hormonal. A alteração na regulação destas enzimas, envolvidas na biossíntese da matriz extracelular, pode contribuir para deficiente síntese do tecido conjuntivo e do suporte vaginal, e estar envolvida no desenvolvimento do POP. / Objective: Pelvic organ prolapse (POP) results from the failure of pelvic floor support, and connective tissue abnormalities may be involved in the etiology and/or progression of the dysfunction. We aimed to analyze the differential expression of genes and proteins related to the collagen and elastin biogenesis: lysyl oxidases (LOXs), fibulin-5, fibrillin -1 and -2, and procollagen C proteinase (PCP/BMP1) in vaginal tissue of women without and with advanced POP controlled by hormonal status. Materials and Methods: During total hysterectomy, anterior vaginal wall biopsies were obtained from Caucasian premenopausal women (proliferative phase of menstrual cycle) and postmenopausal women with severe POP (POPQ stage III and IV) and asymptomatic controls (POPQ 0). Total mRNA and protein were extracted using Trizol and RIPA buffer, and the genes and proteins of interest were quantified by real-time RT-PCR and Immunoblotting, respectively. The following analysis were performed: (1) expression of LOX family genes and proteins (LOX and LOXL1-4), fibulin-5, fibrillin-1 and -2 in premenopausal women with and without POP; (2) PCP/BMP1 gene and protein expression in vaginal tissue of pre- and postmenopausal POP women, and respective controls. Wilcoxon signed-rank and Fisher tests were used for statistical analysis (p<0.05). Results: Samples from 15 premenopausal patients and 11 controls were obtained for study (1); 39 premenopausal (POP=23 and Control=16) and 18 postmenopausal women samples (POP=13 and Control=5) for study (2). We observed: (1) significant decrease in expression of LOX, LOXL1 and LOXL3 genes, as well as LOX and LOXL3 proteins in vaginal tissue of premenopausal POP patients compared with asymptomatic women (p<0.05); (2) PCP/BMP1 gene downregulation in the vagina of women with severe POP compared with controls, in both premenopausal and postmenopausal phase (both p=0.01); significant reduction of 130 kDa, 92.5 kDa and 82.5 kDa PCP/BMP1 isoforms in vaginal tissue of postmenopausal patients (p=0.01), and 130 kDa isoform upregulation in premenopausal women with severe POP (p=0.009), compared with their respective controls. Conclusion: The expression of LOXs enzymes and PCP/BMP1 are altered in vaginal tissue of women with severe POP, and are modulated by hormonal status. Dysregulation of these enzymes involved in the extracellular matrix biogenesis may contribute to impaired tissue and vaginal support, and may be involved in POP development. / TEDE / BV UNIFESP: Teses e dissertações

INTRODUÇÃO: A prevalência de asma tem crescido e a maioria dos pacientes com asma grave não obtém o controle total dos sintomas com as terapias disponíveis, fazendo-se necessária a busca por novas alternativas terapêuticas. Inibidores de proteinases têm sido estudados como tratamento de processos inflamatórios, dentre eles o Enterolobium contortisiliquum Tripsin Inhibitor (EcTI) OBJETIVO: Avaliar se o inibidor de proteinase EcTI modula a hiperresponsividade brônquica à metacolina, inflamação, remodelamento e estresse oxidativo nas vias aéreas e septos alveolares em um modelo experimental de inflamação pulmonar alérgica crônica. MÉTODOS: Vinte e quatro camundongos Balb/c machos, entre seis e sete semanas de vida, pesando em media 25 g foram divididos em quatro grupos: C (controle), OVA (sensibilizados com ovalbumina, 50 ug intraperironeal (i.p) nos dias 0 e 14 e desafiados nos dias 22, 24, 26, 28); C+EC (controle tratados com EcTI (2 mg/kg/i.p) nos dias 22 a 28); OVA+EC (sensibilizados e desafiados com ovalbumina e também tratados com EcTI (2 mg/kg -i.p) nos dias 22 a 28). No dia 29, foram realizadas realizadas: (i) hiperresponsividade à metacolina e obtidas as respostas máximas de resistência e elastância do sistema respiratório; (ii) análise histopatológica do pulmão para quantificação de eosinófilos, fibras colágenas e elásticas nas vias aéreas (VA) e nos septos alveolares (SA); e (iii) imunohistoquímica para quantificação de células positivas para IFN-y, IL-4, IL-5, IL-13, MMP-9, TIMP-1, TGF-beta, iNOS, NF-kB e fração de volume de isoprostano nas VA e nos SA. Uma semana após o dia 29 foi realizada a técnica de anafilaxia cutanea passiva(PCA) para quantificar IgE e IgG1. A significância foi considerada quando p < 0,05. RESULTADOS: Houve aumento de todos os parâmetros avaliados no grupo OVA em relação ao grupo controle (p < 0,05). Houve atenuação da resposta máxima de Rrs e Ers no grupo OVA+EC comparado as grupo OVA (p < 0,05). O tratamento com EcTI nos animais sensibilizados atenuou o número de eosinófilos, células positivas para IL-4, IL-5, IL-13,IFN-y, iNOS, MMP-9, TIMP-1, NF-kB e TGF-beta e fração de volume de isoprostano, fibras colágenas e elásticas nas vias aéreas e nos séptos alveolares quando comparado ao grupo OVA (p < 0,05).Houve reaçao de PCA nos animais sensibilizados com ovalbumina. CONCLUSÃO: EcTI atenuou a hiperresponsividade brônquica, a inflamação, o remodelamento e o estresse oxidativo nesse modelo experimental de inflamação pulmonar alérgica crônica. Embora sejam necessários mais estudos, esse inibidor pode ser considerado uma futura ferramenta farmacológica para o tratamento de asma / BACKGROUND: The number of cases of asthma has grown in recent decades. People who have severe asthma are likely to have more attacks and are at greater risk of a fatal attack, which propose to keep up global attention and keep approaching for advances in asthma care. Proteinase inhibitors of vegetable origin have been studied as a modulator of inflammatory responses and diseases. Among these inhibitors is Enterolobium contortisiliquum Trypsin Inhibitor (EcTI). AIMS: To evaluate the effects of EcTI in pulmonary mechanical, eosinophilic recruitment, inflammatory cytokines, remodeling of extracellular matrix and oxidative stressin an experimental model of chronic allergic pulmonary inflammation. METHODS: Twenty-four young adult male pathogen-free mice BALB/c (6-7 weeks old, 25-30g) were divided into 4 groups: C (control), OVA (sensitized with ovalbumin, 50 ug intraperitoneal (i.p), on days 0 and 14 and challenged with ova 1%, on days 22, 24, 26, 28); C+EC (control treated with EcTI- 2 mg/kg/i.p. from days 22 to 28); OVA+EC (sensitized and challenged with ovalbumin and treated with EcTI (2 mg/kg/i.p) from days 22 to 28). At day 29, we performed: (i) Bronchial hyperresponsiveness to methacholine and obtained the maximum response of resistance (Rrs) and elastance (Ers) of the respiratory system; (ii) lung histopathological analysis by morphometry to quantify eosinophils, collagen and elastic fibers volume fraction in airways; and (iii) immunohistochemistry to quantify IFN-y, IL-4, IL-5, IL-13, MMP-9, TIMP-1, TGF-, iNOS, NF-kB positive cells and isoprostane volume fraction in airways. One week after the day 29 we performed PCA technique to quantify IgE and IgG1 antibodies. Significance was considered at p < 0.05. RESULTS: The EcTI treatment in the ovalbumin-sensitized animals attenuated the maximal response of resistance and elastance of respiratory system after methacholine, the number of eosinophils, IL-4, IL-5, IL-13, IFN-y, NF-kB and iNOS-positive cells, isoprostane, elastic, collagen volume fraction, MMP-9, TIMP-1 and TGF-beta-positive cells compared to OVA group (p < 0.05). PCA was positive in sensitized animals. CONCLUSION: EcTI attenuates bronchial hyperresponsiveness, inflammation, remodeling and oxidative stress activation in this experimental asthma mice model. Although more studies are needed this inhibitor may be considered a future pharmacological tool for the treatment of asthma

Orientador: Raghuvir Krishnaswamy Arni / Banca: Adélia Cristina Oliveira Cintra / Banca: Patrick Jack Spencer / Abstract: Toxins isolated from vemos have been used as molecular tools to understand many physiological processes. The enzymes isolated from the venoms of Crotalus and Bothrops species interfere with the control and balance of the hemostatic system (PEREZ et al., 1996) and thus, the determination of their structures is potentially very important. These enzymes are serine proteinases that are similar to tyrpsin in their specificity but are generally referred to as thrombin-like enzymes due to their ability to cleave fibrinogen. The principal aim of this project was to isolate and characterize snake venom poteins that inetefere with the control and regulation of the hemostatic system in quantities and purity required for structural studies. Gel filtration, ion-exchange and HPLC chromatographic techniques were used to isolate convulxin, crotoxin, giroxin and crotamine, the principle components from the venoms of Crotalus durissus collineatus and Crotalus durissus terrificus and the serine and metalo proteinases from the venom of Bothrops jararaca. The purity of the samples was evaluated by SDS-PAGE and the specific activity of the samples was determined. Crystallization experiments were then carried out. / Mestre

INTRODUÇÃO: A prevalência de asma tem crescido e a maioria dos pacientes com asma grave não obtém o controle total dos sintomas com as terapias disponíveis, fazendo-se necessária a busca por novas alternativas terapêuticas. Inibidores de proteinases têm sido estudados como tratamento de processos inflamatórios, dentre eles o Enterolobium contortisiliquum Tripsin Inhibitor (EcTI) OBJETIVO: Avaliar se o inibidor de proteinase EcTI modula a hiperresponsividade brônquica à metacolina, inflamação, remodelamento e estresse oxidativo nas vias aéreas e septos alveolares em um modelo experimental de inflamação pulmonar alérgica crônica. MÉTODOS: Vinte e quatro camundongos Balb/c machos, entre seis e sete semanas de vida, pesando em media 25 g foram divididos em quatro grupos: C (controle), OVA (sensibilizados com ovalbumina, 50 ug intraperironeal (i.p) nos dias 0 e 14 e desafiados nos dias 22, 24, 26, 28); C+EC (controle tratados com EcTI (2 mg/kg/i.p) nos dias 22 a 28); OVA+EC (sensibilizados e desafiados com ovalbumina e também tratados com EcTI (2 mg/kg -i.p) nos dias 22 a 28). No dia 29, foram realizadas realizadas: (i) hiperresponsividade à metacolina e obtidas as respostas máximas de resistência e elastância do sistema respiratório; (ii) análise histopatológica do pulmão para quantificação de eosinófilos, fibras colágenas e elásticas nas vias aéreas (VA) e nos septos alveolares (SA); e (iii) imunohistoquímica para quantificação de células positivas para IFN-y, IL-4, IL-5, IL-13, MMP-9, TIMP-1, TGF-beta, iNOS, NF-kB e fração de volume de isoprostano nas VA e nos SA. Uma semana após o dia 29 foi realizada a técnica de anafilaxia cutanea passiva(PCA) para quantificar IgE e IgG1. A significância foi considerada quando p < 0,05. RESULTADOS: Houve aumento de todos os parâmetros avaliados no grupo OVA em relação ao grupo controle (p < 0,05). Houve atenuação da resposta máxima de Rrs e Ers no grupo OVA+EC comparado as grupo OVA (p < 0,05). O tratamento com EcTI nos animais sensibilizados atenuou o número de eosinófilos, células positivas para IL-4, IL-5, IL-13,IFN-y, iNOS, MMP-9, TIMP-1, NF-kB e TGF-beta e fração de volume de isoprostano, fibras colágenas e elásticas nas vias aéreas e nos séptos alveolares quando comparado ao grupo OVA (p < 0,05).Houve reaçao de PCA nos animais sensibilizados com ovalbumina. CONCLUSÃO: EcTI atenuou a hiperresponsividade brônquica, a inflamação, o remodelamento e o estresse oxidativo nesse modelo experimental de inflamação pulmonar alérgica crônica. Embora sejam necessários mais estudos, esse inibidor pode ser considerado uma futura ferramenta farmacológica para o tratamento de asma / BACKGROUND: The number of cases of asthma has grown in recent decades. People who have severe asthma are likely to have more attacks and are at greater risk of a fatal attack, which propose to keep up global attention and keep approaching for advances in asthma care. Proteinase inhibitors of vegetable origin have been studied as a modulator of inflammatory responses and diseases. Among these inhibitors is Enterolobium contortisiliquum Trypsin Inhibitor (EcTI). AIMS: To evaluate the effects of EcTI in pulmonary mechanical, eosinophilic recruitment, inflammatory cytokines, remodeling of extracellular matrix and oxidative stressin an experimental model of chronic allergic pulmonary inflammation. METHODS: Twenty-four young adult male pathogen-free mice BALB/c (6-7 weeks old, 25-30g) were divided into 4 groups: C (control), OVA (sensitized with ovalbumin, 50 ug intraperitoneal (i.p), on days 0 and 14 and challenged with ova 1%, on days 22, 24, 26, 28); C+EC (control treated with EcTI- 2 mg/kg/i.p. from days 22 to 28); OVA+EC (sensitized and challenged with ovalbumin and treated with EcTI (2 mg/kg/i.p) from days 22 to 28). At day 29, we performed: (i) Bronchial hyperresponsiveness to methacholine and obtained the maximum response of resistance (Rrs) and elastance (Ers) of the respiratory system; (ii) lung histopathological analysis by morphometry to quantify eosinophils, collagen and elastic fibers volume fraction in airways; and (iii) immunohistochemistry to quantify IFN-y, IL-4, IL-5, IL-13, MMP-9, TIMP-1, TGF-, iNOS, NF-kB positive cells and isoprostane volume fraction in airways. One week after the day 29 we performed PCA technique to quantify IgE and IgG1 antibodies. Significance was considered at p < 0.05. RESULTS: The EcTI treatment in the ovalbumin-sensitized animals attenuated the maximal response of resistance and elastance of respiratory system after methacholine, the number of eosinophils, IL-4, IL-5, IL-13, IFN-y, NF-kB and iNOS-positive cells, isoprostane, elastic, collagen volume fraction, MMP-9, TIMP-1 and TGF-beta-positive cells compared to OVA group (p < 0.05). PCA was positive in sensitized animals. CONCLUSION: EcTI attenuates bronchial hyperresponsiveness, inflammation, remodeling and oxidative stress activation in this experimental asthma mice model. Although more studies are needed this inhibitor may be considered a future pharmacological tool for the treatment of asthma

Made available in DSpace on 2014-06-11T19:22:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-08Bitstream added on 2014-06-13T20:10:06Z : No. of bitstreams: 1 oliveira_dgl_me_sjrp.pdf: 2024541 bytes, checksum: 9e617f882421a4dd2f2cc715da6fb79f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Toxins isolated from vemos have been used as molecular tools to understand many physiological processes. The enzymes isolated from the venoms of Crotalus and Bothrops species interfere with the control and balance of the hemostatic system (PEREZ et al., 1996) and thus, the determination of their structures is potentially very important. These enzymes are serine proteinases that are similar to tyrpsin in their specificity but are generally referred to as thrombin-like enzymes due to their ability to cleave fibrinogen. The principal aim of this project was to isolate and characterize snake venom poteins that inetefere with the control and regulation of the hemostatic system in quantities and purity required for structural studies. Gel filtration, ion-exchange and HPLC chromatographic techniques were used to isolate convulxin, crotoxin, giroxin and crotamine, the principle components from the venoms of Crotalus durissus collineatus and Crotalus durissus terrificus and the serine and metalo proteinases from the venom of Bothrops jararaca. The purity of the samples was evaluated by SDS-PAGE and the specific activity of the samples was determined. Crystallization experiments were then carried out.