La migration des éosinophiles dans la muqueuse bronchique est une caractéristique importante de l'asthme. Différentes protéases contribuent à la migration: la MMP-9, l'uPA et la plasmine. Notre groupe a déjà démontré que d'autres protéases sont possiblement impliquées dans le processus. Cette étude vise à documenter l'expression de l'«eosinophil serine protease» (esp)-l par l'éosinophile et à étudier le rôle de l'esp-l dans la migration de l'éosinophile. Les éosinophiles expriment l'esp-l, tant en ARNm qu'en protéine. Les expériences de migration des éosinophiles et les essais enzymatiques n'ont pas permis d'établir que l'esp-l est importante dans la migration. Les sujets normaux et asthmatiques expriment de façon similaire l'esp-l et l'expression de la protéase n'est pas modulée par les médiateurs inflammatoires utilisés. Le rôle de l'esp-l dans la biologie de l'éosinophile demeure encore inconnu. / Migration of eosinophils to bronchial mucosa is a main feature of asthma. Different proteases contribute to migration: MMP-9, uPA and plasmin. Our group has demonstrated that other proteases are possibly involved in this process. This study aims to document the expression of eosinophil serine protease (esp)-l by eosinophils and study the role of esp-1 in eosinophil migration. Eosinophils express esp-1 at mRNA and protein levels. Eosinophil migration experiments and enzymatic assays failed to establish that esp-1 is involved in migration. Eosinophils from normal and asthmatic subjects similarly express esp-1, and the expression of the protease is not modulated by the inflammatory mediators we used. Consequently, the role of esp-1 in eosinophil biology is still unknown.

La bradykinine (BK) est une hormone peptidique libérée dans la circulation à partir des kininogènes. La BK est reconnue dans la littérature scientifique pour sa multitude d'effets pathologiques, mais elle exerce aussi un rôle protecteur dans diverses complications liées à l'ischémie cardiaque et des effets bénéfiques sur la circulation. Les effets potentiellement salutaires de la BK, soit le relâchement de monoxyde d'azote et de plasminogène de type tissulaire entrainant une vasodilatation, semblent être causés par une activation sélective des récepteurs B2 (B2R) endothéliaux. Nous avons émis comme hypothèse qu'il était faisable de stimuler sélectivement et durablement le récepteur B2R pour ses effets bénéfiques en utilisant une variété de ligands (résistants à des peptidases ou types pro-drogues prolongés à partir de la séquence originale de la BK). Nos objectifs spécifiques sont de vérifier l'effet de ceux-ci in vivo en mesurant des paramètres hémodynamiques et leur intensité (fréquence cardiaque, pression artérielle, signal Doppler). Des rats mâles de souche Sprague-Dawley ont été anesthésiés et deux cathéters (un dans la veine jugulaire pour l'administration des drogues en bolus (courbes doses-réponses), et l'autre dans l'artère fémorale pour la mesure des paramètres) ont été implantés. L'injection de BK provoque des épisodes hypotenseurs transitoires de courte durée. La convertase de l'angiotensine (ECA) domine dans le milieu extracellulaire du système vasculaire comme voie de dégradation principale de la BK. En présence d'énalaprilate (inhibiteur de l'ECA), la puissance de l'effet hypotenseur de la BK est augmentée de 15X. Les séquences prolongées en C-terminal régénèrent de la BK via l'action de peptidases spécifiques (clivage). Les pro-drogues libérant BK progressivement via l'activité de peptidases vasculaires ont un profil intéressant comme potentiel traitement de l'hypertension. Le fardeau des effets indésirables de ces stratégies reste à clarifier pour le développement des agonistes du B2R comme médicaments.

Proteolytic processes are often mediated by highly orchestrated cascades, through which protease enzymes function coordinately to ensure a stepwise activation. This thesis presents experimental data which supports and complements the previously postulated mechanism of KLK (kallikrein-related protease) activation through proteolytic cascades. Further examination of the seminal KLK cascade has revealed several of its key (patho) physiological roles in human reproductive system. Multiple members of the seminal KLK cascade, in particular KLK14, were shown to play a pivotal role in regulating semen liquefaction. The cascade was further shown to be tightly regulated through a series of highly orchestrated feedback loops, to prevent deleterious effects due to aberrant protease activation. Accordingly, a strong association was observed between the expression level of several seminal KLKs, delayed liquefaction, and other markers of semen quality, including semen hyperviscosity. Furthermore, a strong association was found between delayed liquefaction and abnormal sperm motility. Therefore, dysregulated KLK expressions and/or activities were proposed as an underlying cause of male subfertility. Finally, this thesis has provided initial insights into a novel potential function of multiple members of the seminal KLK cascade in activation of the key immune-deviating agent, TGFβ1, in seminal plasma. TGFβ1 activation is postulated to be mediated directly through complete fragmentation or indirectly through partial cleavage and conformational changes of the LAP propeptide motif of the latent TGFβ1. KLK- mediate proteolytic cleavage of the TGFβ1 binding protein, LTBP1, is also suggested as a potential physiological mechanism for release of the membrane-bound latent TGFβ1. Overall, the data provided here may suggest a common regulatory mechanism, involved co-temporally in the two key processes of semen liquefaction and immune-suppression. This might be critical in protecting motile sperms following their release from semen coagulum. Understanding KLK-mediated proteolytic events in seminal plasma can shed light not only on the physiological role of this family of enzymes, but also on some of causes of male subfertility. Accordingly, therapeutic induction of this cascade may be utilized to supplement the current clinical treatment of male subfertility. Conversely, targeted inhibition of key components of the cascade may have potential pharmaceutical utility as a novel topical contraceptive strategy.

Thèse de doctorat : Sciences Agronomiques : INPL : 2008. / Titre provenant de l'écran-titre.

Proteolytic processes are often mediated by highly orchestrated cascades, through which protease enzymes function coordinately to ensure a stepwise activation. This thesis presents experimental data which supports and complements the previously postulated mechanism of KLK (kallikrein-related protease) activation through proteolytic cascades. Further examination of the seminal KLK cascade has revealed several of its key (patho) physiological roles in human reproductive system. Multiple members of the seminal KLK cascade, in particular KLK14, were shown to play a pivotal role in regulating semen liquefaction. The cascade was further shown to be tightly regulated through a series of highly orchestrated feedback loops, to prevent deleterious effects due to aberrant protease activation. Accordingly, a strong association was observed between the expression level of several seminal KLKs, delayed liquefaction, and other markers of semen quality, including semen hyperviscosity. Furthermore, a strong association was found between delayed liquefaction and abnormal sperm motility. Therefore, dysregulated KLK expressions and/or activities were proposed as an underlying cause of male subfertility. Finally, this thesis has provided initial insights into a novel potential function of multiple members of the seminal KLK cascade in activation of the key immune-deviating agent, TGFβ1, in seminal plasma. TGFβ1 activation is postulated to be mediated directly through complete fragmentation or indirectly through partial cleavage and conformational changes of the LAP propeptide motif of the latent TGFβ1. KLK- mediate proteolytic cleavage of the TGFβ1 binding protein, LTBP1, is also suggested as a potential physiological mechanism for release of the membrane-bound latent TGFβ1. Overall, the data provided here may suggest a common regulatory mechanism, involved co-temporally in the two key processes of semen liquefaction and immune-suppression. This might be critical in protecting motile sperms following their release from semen coagulum. Understanding KLK-mediated proteolytic events in seminal plasma can shed light not only on the physiological role of this family of enzymes, but also on some of causes of male subfertility. Accordingly, therapeutic induction of this cascade may be utilized to supplement the current clinical treatment of male subfertility. Conversely, targeted inhibition of key components of the cascade may have potential pharmaceutical utility as a novel topical contraceptive strategy.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2001. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2001. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

LEITE, Hugo de Brito. Aplicação de peptidases laticíferas para produção de queijo coalho vegetariano. 2016. 84 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Aline Mendes (alinemendes.ufc@gmail.com) on 2016-08-22T20:42:34Z No. of bitstreams: 1 2016_dis_hbleite.pdf: 2941503 bytes, checksum: b83be07efbcbd2c1388a3e3876a5a22a (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-08-23T18:27:15Z (GMT) No. of bitstreams: 1 2016_dis_hbleite.pdf: 2941503 bytes, checksum: b83be07efbcbd2c1388a3e3876a5a22a (MD5) / Made available in DSpace on 2016-08-23T18:27:15Z (GMT). No. of bitstreams: 1 2016_dis_hbleite.pdf: 2941503 bytes, checksum: b83be07efbcbd2c1388a3e3876a5a22a (MD5) Previous issue date: 2016 / Peptidases are enzymes capable of performing cleavage of peptide bonds of other proteins and peptides. These enzymes exhibit a broad spectrum of applications because they are used from the food industry to the leather and skin processing and drug formulation. Peptidases also have been used in the dairy industry for the cheese production. The coagulation of milk caseins is the main step in the production of the cheese curds. Rennet or renin are consist of chymosin and when added to the milk they cause the first step of forming cheese, the coagulation. Due to restrictions of food intake using animal rennet because of eating habits (vegetarianism) or religious reasons (Judaism and Islamism) the search for coagulating enzymes alternatives to rennet has intensified. The present work aimed to evaluate the caseinolytic and coagulant activities of the milk of five different latex protein extracts for the production of cheese. The latex fractions obtained were dissolved in Tris-HCl 50 mM pH 6.5 buffer and submitted to total proteolytic activity assays with different activators, milk coagulation, total casein hydrolysis, and of κ-casein over time by SDS-PAGE on 15% polyacrylamide gels, establishing a protocol for cheese curd production and detecting peptidases after the production of cheese. Only the fractions of C. procera, C. grandiflora, and C. papaya showed proteolytic activity and milk coagulation activity. These peptidases were activated by DTT and L-cysteine. The milk coagulation time by latex peptidases is dose dependent with 60 µg being the optimum amount to coagulate 2 mL of milk. C. procera and C. grandiflora showed specific activity of milk coagulation close to the commercial rennet of animal origin. High concentrations of NaCl and CaCl2 did not affect the proteolytic activity and coagulation of milk. However, the pre-incubation of these samples at 75º C for 10 minutes completely eliminated their activities. Both proteolytic fractions proved to be capable of hydrolyzing κ-casein and producing peptides of 16 KDa similarly the commercial rennet. The cheeses generated by the latex peptidases had nice flavor, firm texture, scent and yield similar to cheeses produced with commercial chymosin. Also, no proteolytic activity was detected after the production of cheese. This way, the peptidases from C. procera and C. grandiflora can be used as an alternative to chymosin in the production of cheese curds. / Peptidases são enzimas capazes de realizar a clivagem de ligações peptídicas de outras proteínas e peptídeos. Essas enzimas apresentam um amplo espectro de aplicações, pois são utilizadas desde a indústria alimentícia até mesmo no processamento de couro e pele e formulações de medicamentos. Peptidases também têm sido utilizadas na indústria de laticínios para a produção de queijos. A coagulação das caseínas do leite é a principal etapa na produção de queijos do tipo coalho. O coalho ou renina é composto por quimosina e quando adicionado ao leite produz a primeira etapa de formação do queijo, a coagulação. Devido a restrições de consumo de alimentos que utilizem o coalho animal por razões de hábitos alimentares (vegetarianismo) ou por razões religiosas (judaísmo e islamismo) a busca por enzimas coagulantes alternativas à quimosina tem intensificado. O presente trabalho objetivou avaliar as atividades caseinolítica e coagulante do leite de cinco diferentes extratos de proteínas laticíferas para a produção de queijo. As frações laticíferas obtidas foram dissolvidas em tampão Tris-HCl 50 mM pH 6,5 e submetidas aos seguintes ensaios: atividade proteolítica total, atividade proteolítica com diferentes ativadores, atividade de coagulação do leite, avaliação da hidrólise da caseína total por SDS-PAGE e avaliação da κ-caseína ao longo do tempo através de SDS-PAGE em géis de poliacrilamida 15%, estabelecimento de um protocolo de produção do queijo tipo coalho e detecção de peptidases após a produção do queijo. Somente as frações de C. procera, C. grandiflora e C. papaya exibiram atividade proteolítica e atividade coagulação do leite, estas peptidases foram ativadas por DTT e L-cisteína. O tempo de coagulação do leite pelas peptidases laticíferas foi dose-dependente, sendo 60 µg a quantidade ideal para coagular 2 mL de leite. C. procera e C. grandiflora apresentaram atividade específica de coagulação do leite idênticas ao coalho comercial de origem animal. Altas concentrações de NaCl e CaCl2 não afetaram a atividade proteolítica e de coagulação do leite. Entretanto, a pré-incubação dessas amostras a 75º C por 10 minutos eliminou completamente suas atividades. Ambas as frações proteolítica demonstraram-se capazes de hidrolisar a κ-caseína e produzir peptídeos de 16 KDa de maneira idêntica ao coalho comercial. Os queijos gerados com as peptidases laticíferas apresentaram sabor agradável, textura firme, aroma e rendimento similares aos queijos produzidos com a quimosina comercial. Também não foi detectada atividade proteolítica após a produção do queijo. Dessa forma, as peptidases de C. procera e C. grandiflora podem ser utilizadas como alternativa a quimosina na produção de queijo coalho.

Na cascavel Crotalus durissus terrificus ocorre o armazenamento de longo prazo do esperma (LTSS), no trato genital da fêmea, durante o intervalo entre a cópula (outono) e a ovulação (primavera). Peptídeos e peptidases estão entre os principais componentes que influenciam a atividade espermática em mamíferos. O presente estudo objetivou caracterizar a presença de peptidases em C. d. terrificus, com esta função reconhecida em mamíferos e/ou que clivam peptídeos atuantes nesta função, bem como avaliar a hipótese do envolvimento destas peptidases na preservação dos espermatozóides desta serpente. O aspecto morfo-funcional dos espermatozóides foi comparado na presença dos peptídeos angiotensina II (AngII), arginina vasotocina (AVT), bradicinina (BK), peptídeo promotor da fecundação (FPP) e hormônio liberador de tireotrofina (TRH). Caracterizamos os efeitos de agentes quelante, tiol-redutor, cofator e inibidores, bem como dos peptídeos supracitados, sobre as atividades enzimáticas relacionadas, quais sejam: aminopeptidases ácida (APA), básica (APB), alanil sensível (APN-PS) e insensível à puromicina (APN-PI), cistil (CAP), dipeptidil-peptidase IV (DPPIV), piroglutamil tipo 1 (PAP-I) e prolil-imino- (PIP), bem como da prolil endopeptidase (POP), em frações solúvel (FS) e/ou de membrana solubilizada (FM) do sêmen proveniente do ducto deferente, assim como deste próprio tecido e dos tecidos uterino e vaginal de C. d. terrificus, ou seja, tecidos por onde passam ou armazenam-se os espermatozóides. Verificamos a variação sazonal destas mesmas atividades, nestes tecidos, incluindo o sêmen armazenado no útero durante o LTSS. O sêmen coletado do ducto deferente foi fracionado para avaliação da distribuição destas atividades peptidásicas. Nossos dados mostraram características de liquefação do sêmen e movimento dos espermatozóides da cascavel que os diferenciam do padrão humano. FPP com cálcio e BK melhoraram a preservação da viabilidade espermática, similarmente ao que ocorre em mamíferos. Em todos os tecidos e no sêmen as atividades APB, PIP e POP foram detectadas apenas da FS, enquanto as demais estão presentes em FS e FM, seguindo um padrão de distribuição observado na maioria dos tecidos de mamíferos. Amastatina e bestatina inibiram APB e APN, enquanto a diprotina A foi o inibidor mais eficiente para a DPPIV em FM. PAP-I e PIP foram inibidas, respectivamente, por bestatina e puromicina. Este perfil de inibição também é similar ao encontrado para as aminopeptidases em tecidos de mamíferos. Todas as atividades peptidásicas foram influenciadas por algum dos peptídeos estudados, sugerindo que tais peptídeos são substratos potenciais e/ou moduladores destas atividades na cascavel. As atividades APB e APN foram caracterizadas como metalopeptidases. APB, CAP e DPPIV foram inibidas por MnCl2. CAP e PAP-I foram caracterizadas como enzimas sulfidril-dependentes. As atividades APB, APN-PI e APN-PS predominaram, em relação às demais peptidases, em todas as estações e na maioria dos tecidos e no sêmen, sugerindo sua maior relevância na fisiologia reprodutiva da C. d. terrificus. Os níveis de todas as atividades peptidásicas estudadas variaram sazonalmente, sugerindo que sua ação moduladora sobre peptídeos susceptíveis está integrada ao ciclo reprodutivo desta serpente. O fracionamento do sêmen do ducto deferente revelou a presença de fluido seminal e espermatozóides, bem como de uma estrutura prostassoma-símile, até então somente identificada em mamíferos. Em todas estas frações há atividade peptidásica, predominando a APN-PI no prostassoma-símile e no fluido seminal, e APN-PS e APN-PI na FS e FM dos espermatozóides, caracterizando um envolvimento com a redução da motilidade espermática, tal qual ocorre em mamíferos. Concluimos que as atividades peptidásicas estudadas apresentam características sazonais e tecido-específicas que sugerem uma atuação relevante na preservação dos espermatozóides de C.d. terrificus. / In the rattlesnake Crotalus durissus terrificus occurs the long-term sperm storage (LTSS), in the female tract, during the interval between mating (autumn) and ovulation (spring). Peptides and peptidases are among the main components that influence the spermatic activity in mammals. The present study aimed to characterize the presence of peptidases in C. d. terrificus, that are well-recognized to exert this function and/or that have the ability to hydrolyze peptides that exert this function in mammals, as well to evaluate whether these peptidases are related to the preservation of spermatozoids in this snake. The morphological and functional characteristics of spermatozoids were compared in the presence of angiotensin II (AngII), arginine-vasotocin (AVT), bradykinin (BK), fertilization promoting peptide (FPP) and thyrotropin-releasing hormone (TRH). We have checked the effect of chelating and thiol-reducing agents, cofactor and inhibitors, as well the effect of aforementionated peptides on related enzyme activities such as acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive alanyl (APN-PI), cystyl (CAP), pyroglutamyl type 1 (PAP-I) and prolyl-imino (PIP) aminopeptidases, and dipeptidyl-peptidase IV (DPPIV), as well as on prolyl endopeptidase (POP), in soluble (FS) and/or solubilized membrane-bound (FM) fractions of semen from vas deferens, of this own tissue, and vagina and uterus tissues of C. d. terrificus, i.e. tissues where spermatozoids pass through or where they are stored. The seasonal variation of these peptidase activities, in all tissues, including the semen stored in uterus during the LTSS, were evaluated. The semen from vas deferens was fractioned in order to know the distribution of these peptidase activities. The features of seminal liquefaction and movement of spermatozoids were different between the rattlesnake and human. Similar to mammals, FPP plus calcium and BK improved the preservation of the viability of spermatozoids from C. d. terrificus. In all tissues and semen, the APB, PIP and POP activities were detected only in FS, while others peptidases were present in FS and FM, following a similar pattern of distribution usually observed in mammalian tissues. Amastatin and bestatin inhibited APB and APN activities, while diprotin A was the most efficient inhibitor of DPPIV in FM. PAP-I and PIP activities were inhibited by bestatin and puromycin, respectively. This inhibition profile was similar to that of mammalian tissues. All peptidase activities were influenced at least by one of the peptides under study, suggesting these peptides as potential substrates and/or modulators for these peptidases of the rattlesnake. The APB and APN activities were characterized as metallopeptidases. APB, CAP and DPPIV were inhibited by MnCl2. CAP and PAP-I were characterized as sulfhydryl-dependent enzymes. The APB, APN-PI and APN-PS activities were predominant, in relation to the other examined peptidases, in all seasons and in most tissues and semen, suggesting their great relevance in the reproductive physiology of the C. d. terrificus. The levels of all studied peptidase activities were seasonally variable, suggesting that their modulator actions on susceptible peptides are integrated to the reproductive cycle of this snake. The fractionation of C. d. terrificus semen revealed the presence of seminal fluid and spermatozoids, as well a prostasome-like structure, until then identified only in mammals. In all of these fractions, there are peptidase activities, predominating the APN-PI in prostasome and seminal fluid, and the APN-PS and APN-PI in FS and FM of spermatozoids, suggesting their involvement in the reduction of the spermatic mobility, such as in mammals. In conclusion, the studied peptidase activities present seasonal and tissue-specific characteristics, which suggest a relevant role in the preservation of the spermatozoids of C. d. terrificus.