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Efeito da adição do plasma seminal nas mudanças semelhantes à capacitação (Criocapacitação) em espermatozóides criopreservados de eqüinos / Effects of seminal plasma addition has on capacitation-like changes (cryocapacitation) in cryopreserved equine spermatozoaAndrade, André Furugen Cesar de 13 March 2009 (has links)
A criopreservação dos espermatozóides eqüinos gera alterações nas membranas semelhantes às que ocorrem durante o processo de capacitação (criocapacitação). Sabe-se que o plasma seminal possui fatores decapacitantes. Além disso, é citado na literatura a possibilidade melhorar a preservação dos espermatozóides com membrana plasmática íntegra e motilidade, após refrigeração ou criopreservação, quando adicionado plasma seminal de animais que apresentam boa resposta à congelação ao sêmen de animais que apresentam espermatozóides com baixa capacidade de congelação. Assim, o presente experimento verificou se a criopreservação gera modificações semelhantes à capacitação e se a adição ao sêmen pós-descongelação de plasma seminal autólogo ou adquirido de um animal com boa resposta à congelação (homólogo) diminuía estes efeitos. Para isso, foram realizadas 5 colheitas de sêmen de 4 garanhões. O sêmen in natura foi avaliado e então, processado e submetido à congelação. Após a descongelação o sêmen foi novamente avaliado e então dividido em três tratamentos: adicionado de diluidor de criopreservação sem crioprotetor (DIL), deste com 20 % (v/v) de plasma seminal homólogo (PH) ou com 20 % (v/v) de plasma seminal autólogo (PA). Em seguida, o sêmen foi incubado em banho-Maria (37°C) e submetido as avaliações nos três diferentes períodos de amostragem (0, 60 e 120 minutos de incubação). As avaliações do sêmen consistiram na análise computadorizada das características da motilidade (CASA) e nas análises por citometria de fluxo da reação acrossomal, do potencial de membrana mitocondrial, da estabilidade da membrana plasmática, da peroxidação lipídica e na detecção da fosforilação do aminoácido tirosina presente na superfície da célula espermática. As variáveis foram submetidas à análise de variância e ao teste LSD para comparação das médias, ao nível de 5 % de significância. A criopreservação não aumentou a população de células viáveis com membrana acrossômica reagida, nem a população de células viáveis com aumento na desordem da membrana plasmática (capacitadas). Entretanto, a criopreservação levou a uma redução na quantidade de células com potencial de membrana mitocondrial e aumentou a peroxidação das membranas, além de induzir o aumento na fosforilação de proteínas presentes na superfície da célula. A criopreservação ocasionou uma redução de todas as variáveis analisadas pelo CASA, incluindo a população identificada como hiperativada. A adição do plasma seminal, indiferentemente da origem, melhorou a integridade da membrana plasmática, reduziu a população com aumento na desordem da membrana plasmática (capacitados), além de reduzir a quantidade de proteínas fosforiladas na superfície do espermatozóide. Porém esta melhora só foi encontrada nas análises iniciais (tempo 0), com o tempo o plasma seminal mostrou não ser um meio adequado para se manter o sêmen armazenado levando a um prejuízo na maioria das variáveis estudadas. Baseado nos resultados, pode-se sugerir a adição de plasma seminal com o intuito de reduzir os efeitos da criocapacitação. Contudo, ao adicionar o plasma seminal ao sêmen, este deve ser depositado no trato reprodutivo da fêmea o mais breve possível. / Cryopreservation of equine spermatozoa leads to capacitation-like changes (cryocapacitation). Seminal plasma contains decapacitating factors. Moreover, it has been known that addition of seminal plasma from stallions with good post-thaw semen quality to semen of stallions with low post-thaw semen quality preserve progressive motility and membrane integrity better. Therefore, this study was intended to find out whether the cryopreservation process induces capacitation-like changes and if addition of autologous seminal plasma or seminal plasma from stallion with good post-thaw semen quality (homologous) reduces cryocapacitation effects. For this, five ejaculates were obtained from each of four mature stallions. Samples of in natura semen were analyzed, and submited to cryopreservation. Post-thawed semen was analyzed again so divided in three treatments: only cryopreservation media without cryoprotectants, cryopreservation media without cryoprotectants added 20 % (v/v) of seminal plasma homologous, and cryopreservation media without cryoprotectants added 20 % (v/v) of seminal plasma autologous. All samples were maintained in water bath (37°C) for 120 minutes, while semen analyzes were assessed at time 0, 60 and 120 min. The assessment of each sample was determined by computer-assisted sperm analyses of motility (CASA) and flow cytometry analyzes of acrosome reaction, mitochondrial potential, increased membrane fluidity, lipid peroxidation and degree of protein tyrosine phosphorylation in sperm surface. The variables were submitted to analysis of variance and LSD test at 5% level of significance. Cryopreservation did not increase viable cells with acrosome reaction, neither viable cells with increased membrane fluidity (capacitated). However, cryopreservation led an decrease of cell with mitochondrial potential and an increased in lipid peroxidation, besides increase tyrosine phosphorylation of the equine sperm surface proteins. The cryopreservation caused a reduction of all variables assessed by CASA, including hyperactivated cells. Seminal plasma addition, regardless of origin, improved integrity of plasma membrane, reduced number of viable cells with increased membrane fluidity, besides decrease phosphorylation of sperm surface proteins. Nevertheless, the improved created only was found in the initial analysis (time 0), seminal plasma shown a harmful effect in maintenance of sperm during incubation. Incubation of sperm in seminal plasma damaged majority of variables assessed. Based on results, seminal plasma could be added to post-thawed semen to decrease the cryocapacitation effects. However, post-thawed semen has to be deposited as soon as possible in female reproductive tract when the seminal plasma is added.
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Estudo das alterações morfo-funcionais de espermatozóides bovinos submetidos à sexagem por meio da técnica de citometria de fluxo / Study of morpho-functions alterations in bovine spermatozoa submitted on sorting through flow cytometry technologyTanno, Priscilla Harumi 14 September 2009 (has links)
O objetivo deste estudo foi avaliar as alterações morfo-funcionais do sêmen bovino submetido à sexagem através da técnica de citometria de fluxo e comparar com o sêmen convencional e a diferença entre as subespécies. O sêmen foi obtido de seis touros, sendo três taurinos (Holandês preto e branco) e três zebuínos (Gir leiteiro), com quatro ejaculados de cada animal (n = 24). Cinco tratamentos foram realizados: sêmen convencional com diluidor L (Lagoa); sêmen convencional com diluidor S (Sexing) e sêmen sexado (nos tempos de zero, três e seis horas após a ejaculação para avaliar se existem alterações de membranas ao longo do tempo). Foram avaliados pela citometria de fluxo quanto à integridade de membrana plasmática e reação acrossomal (PI/FITC-PSA); peroxidação lipídica (C11-BODIPY581/591) e capacitação espermática através de análises da fosforilação da tirosina e aumento da fluidez e desorganização da membrana plasmática (Merocianina 540 e Yo-Pro1). Os efeitos de tratamentos foram avaliados por análises de variância (ANOVA), empregando-se o programa estatístico Stat-View® (SAS Institute, Inc. 1998, Cary, NC, USA). Foram considerados significativos os valores com P<0,05 e com tendência a significância (P<0,10). O sêmen proveniente da subespécie taurina teve um efeito negativo mais significante do que a zebuína na qualidade do sêmen. O sêmen sexado apresentou maior peroxidação lipídica nos espermatozóides com membrana íntegra e a presença de proteínas fosforiladas na superfície da membrana plasmática, fatores que são indicativos de capacitação espermática. Porém, ao contrário do esperado, este aumento não foi acompanhado pela quantidade de células classificadas como capacitadas pela análise de associação de sondas Yo-Pro-1 e Merocianina 540, pois o sêmen convencional apresentou maior população espermática com membrana plasmática íntegra desorganizada (P<0,05). Não houve piora na qualidade do sêmen sexado devido ao tempo de espera do ejaculado (P<0,05). / The objective of this study was to evaluate the morpho-functions alterations in bovine semen submitted on sorting through flow cytometry technology and compare with conventional semen and the difference between the subspecies. Semen was obtained from six bulls, that three were taurine (Holstein Black and White) and three were zebuine (Dairy Gir) with four ejaculates from each animal (n = 24). Five treatments were performed: conventional semen with L (Lagoa) media; conventional semen with S (Sexing) media and sexed semen (zero, three and six hours after ejaculation to evaluate if there were membrane alterations over the time). The treatments were analyzed with flow cytometry as for plasmatic membrane integrity and acrosome reaction (PI/FITC-PSA); lipid peroxidation (C11-BODIPY581/591) and sperm capacitation through protein tyrosine phosphorylation and the increase in plasma membrane fluidity and disorganization (Merocyanine 540 and Yo-Pro-1). The treatments effects were evaluated by analysis of variance (ANOVA) (Stat-View® SAS Institute, Inc. 1998, Cary, NC, USA). Effects were considered significant if the values were P<0.05 and with significant tendency (P<0.10). Semen from the taurine subspecie had a more significant negative effect than zebuine on semen quality. Sexed semen showed more lipid peroxidation in sperm with membrane integrity and the presence of phosphorylated proteins in plasma membrane surface that seemed to be sperm capacitation. However, in the contrary of expected, this increase did not accompany with quantity of cells classified as capacitated by the association probes Yo-Pro-1 e Merocyanine 540 analyse because the conventional semen showed more sperm population with plasma membrane integrity disorganized (P<0.05). There was not worsening in sexed semen quality over the time ejaculate waiting (P<0.05).
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Efeito da adição do plasma seminal nas mudanças semelhantes à capacitação (Criocapacitação) em espermatozóides criopreservados de eqüinos / Effects of seminal plasma addition has on capacitation-like changes (cryocapacitation) in cryopreserved equine spermatozoaAndré Furugen Cesar de Andrade 13 March 2009 (has links)
A criopreservação dos espermatozóides eqüinos gera alterações nas membranas semelhantes às que ocorrem durante o processo de capacitação (criocapacitação). Sabe-se que o plasma seminal possui fatores decapacitantes. Além disso, é citado na literatura a possibilidade melhorar a preservação dos espermatozóides com membrana plasmática íntegra e motilidade, após refrigeração ou criopreservação, quando adicionado plasma seminal de animais que apresentam boa resposta à congelação ao sêmen de animais que apresentam espermatozóides com baixa capacidade de congelação. Assim, o presente experimento verificou se a criopreservação gera modificações semelhantes à capacitação e se a adição ao sêmen pós-descongelação de plasma seminal autólogo ou adquirido de um animal com boa resposta à congelação (homólogo) diminuía estes efeitos. Para isso, foram realizadas 5 colheitas de sêmen de 4 garanhões. O sêmen in natura foi avaliado e então, processado e submetido à congelação. Após a descongelação o sêmen foi novamente avaliado e então dividido em três tratamentos: adicionado de diluidor de criopreservação sem crioprotetor (DIL), deste com 20 % (v/v) de plasma seminal homólogo (PH) ou com 20 % (v/v) de plasma seminal autólogo (PA). Em seguida, o sêmen foi incubado em banho-Maria (37°C) e submetido as avaliações nos três diferentes períodos de amostragem (0, 60 e 120 minutos de incubação). As avaliações do sêmen consistiram na análise computadorizada das características da motilidade (CASA) e nas análises por citometria de fluxo da reação acrossomal, do potencial de membrana mitocondrial, da estabilidade da membrana plasmática, da peroxidação lipídica e na detecção da fosforilação do aminoácido tirosina presente na superfície da célula espermática. As variáveis foram submetidas à análise de variância e ao teste LSD para comparação das médias, ao nível de 5 % de significância. A criopreservação não aumentou a população de células viáveis com membrana acrossômica reagida, nem a população de células viáveis com aumento na desordem da membrana plasmática (capacitadas). Entretanto, a criopreservação levou a uma redução na quantidade de células com potencial de membrana mitocondrial e aumentou a peroxidação das membranas, além de induzir o aumento na fosforilação de proteínas presentes na superfície da célula. A criopreservação ocasionou uma redução de todas as variáveis analisadas pelo CASA, incluindo a população identificada como hiperativada. A adição do plasma seminal, indiferentemente da origem, melhorou a integridade da membrana plasmática, reduziu a população com aumento na desordem da membrana plasmática (capacitados), além de reduzir a quantidade de proteínas fosforiladas na superfície do espermatozóide. Porém esta melhora só foi encontrada nas análises iniciais (tempo 0), com o tempo o plasma seminal mostrou não ser um meio adequado para se manter o sêmen armazenado levando a um prejuízo na maioria das variáveis estudadas. Baseado nos resultados, pode-se sugerir a adição de plasma seminal com o intuito de reduzir os efeitos da criocapacitação. Contudo, ao adicionar o plasma seminal ao sêmen, este deve ser depositado no trato reprodutivo da fêmea o mais breve possível. / Cryopreservation of equine spermatozoa leads to capacitation-like changes (cryocapacitation). Seminal plasma contains decapacitating factors. Moreover, it has been known that addition of seminal plasma from stallions with good post-thaw semen quality to semen of stallions with low post-thaw semen quality preserve progressive motility and membrane integrity better. Therefore, this study was intended to find out whether the cryopreservation process induces capacitation-like changes and if addition of autologous seminal plasma or seminal plasma from stallion with good post-thaw semen quality (homologous) reduces cryocapacitation effects. For this, five ejaculates were obtained from each of four mature stallions. Samples of in natura semen were analyzed, and submited to cryopreservation. Post-thawed semen was analyzed again so divided in three treatments: only cryopreservation media without cryoprotectants, cryopreservation media without cryoprotectants added 20 % (v/v) of seminal plasma homologous, and cryopreservation media without cryoprotectants added 20 % (v/v) of seminal plasma autologous. All samples were maintained in water bath (37°C) for 120 minutes, while semen analyzes were assessed at time 0, 60 and 120 min. The assessment of each sample was determined by computer-assisted sperm analyses of motility (CASA) and flow cytometry analyzes of acrosome reaction, mitochondrial potential, increased membrane fluidity, lipid peroxidation and degree of protein tyrosine phosphorylation in sperm surface. The variables were submitted to analysis of variance and LSD test at 5% level of significance. Cryopreservation did not increase viable cells with acrosome reaction, neither viable cells with increased membrane fluidity (capacitated). However, cryopreservation led an decrease of cell with mitochondrial potential and an increased in lipid peroxidation, besides increase tyrosine phosphorylation of the equine sperm surface proteins. The cryopreservation caused a reduction of all variables assessed by CASA, including hyperactivated cells. Seminal plasma addition, regardless of origin, improved integrity of plasma membrane, reduced number of viable cells with increased membrane fluidity, besides decrease phosphorylation of sperm surface proteins. Nevertheless, the improved created only was found in the initial analysis (time 0), seminal plasma shown a harmful effect in maintenance of sperm during incubation. Incubation of sperm in seminal plasma damaged majority of variables assessed. Based on results, seminal plasma could be added to post-thawed semen to decrease the cryocapacitation effects. However, post-thawed semen has to be deposited as soon as possible in female reproductive tract when the seminal plasma is added.
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Molecular Analysis Of Hamster Sperm Capacitation: Significance Of Protein Tyrosine PhosphorylationNaveen, Daniel M 06 1900 (has links)
Fertilization is a process that generates the first cell of a new organism. In mammals,
fertilization occurs in the female reproductive tract. The male gametes (spermatozoa) are rendered fertilization-competent only after they undergo capacitation and acrosome reaction (AR). The set of physiological changes, characterised by the acquisition of hyperactivated
motility, that render the spermatozoa fertilization competent is known as capacitation. Using in vitro models, the complex intracellular signaling events mediating this process are still
being understood. This thesis explores the role of protein tyrosine phosphorylation during capacitation using the golden hamster (Mesocricetus auratus) spermatozoa. The knowledge about the molecular components involved in capacitation, apart from enriching our understanding about a basic cellular process could also provide leads in the management of male (in)fertility.
A comprehensive review on the perspectives of male reproduction, spermatogenesis, the
structural features of a spermatozoon and sperm maturation, relevant to the content of the thesis is provided in Chapter-1 (General Introduction). Molecular mediators that initiate capacitation include cAMP, Ca2+and HCO3- ions. These signalling molecules regulate activities of protein kinases and phosphatases, which control the level of protein phosphorylation in spermatozoa. Capacitation-associated increase in protein
phosphorylation, specifically protein tyrosine phosphorylation (PYP) has been demonstrated in a few species such as mouse, rat and human. The unique nature of PYP signaling during sperm capacitation has been exemplified by discoveries of several male germ cell-specific signalling molecules like soluble adenylate cyclase. However,molecular identities of tyrosine-phosphorylated proteins and their functional role during sperm capacitation are yet to be
investigated in detail. In this context, the effect of modulating intracellular levels of signaling molecules upstream of protein phosphorylation was sought using pentoxifylline (PF), a cAMP phosphodiesterase inhibitor. Interestingly, PF-induced capacitation was associated
with an early induction of tyrosine phosphorylation of proteins (45-80 kDa) localized to the mid piece of the sperm tail. Interestingly, the ultrastructural localization of tyrosine-phosphorylated proteins in the sperm tail by immunoelectron microscopy (IEM) revealed most intense immunolabelling in the fibrous sheath, followed by outer dense fibers (ODFs)and the axoneme. Data pertaining to the effect of PF on sperm capacitation and the associated protein-phosphorylation is presented in Chapter-2.
Since PYP was determined to be extremely critical for hyperactivation in spermatozoa, the involvement of protein tyrosine kinases (PTKs) in this process was assessed using a specific PTK inhibitor, tyrphostin A47 (TP-47: EGFR-TK specific). The third chapter deals with the effect of tyrphostins on sperm capacitation and PYP. A dose-dependent inhibition by TP-47 of capacitation and principal piece associated-PYP of ~45-60 kDa proteins was observed. Interestingly, TP-47 treated-spermatozoa exhibited a circular motility pattern; when assessed for kinematic parameters, by computer aided sperm analysis, sperm showed lower values for key kinematic parameters as compared to the controls. While sperm viability in TP-47-
treated samples was not affected, the ATP content reduced towards latter (4-5 h) part of culture as compared to the controls. When spermatozoa were treated with two other PTK
inhibitors, tyrphostin AG1478 (EGFR-TK specific) and tyrphostin AG1296 (PDGFR-TK
specific), they did not show any changes in kinematic parameters or PYP, indicating that the TP-47-effect was compound-specific.
The fourth chapter of this thesis involves the molecular analysis of proteins hypo-tyrosine phosphorylated in the presence of TP-47, which started with the enrichment of sperm flagellar proteins that are tyrosine phosphorylated during capacitation, using various detergents. Detergent extractions established that most tyrosine-phosphorylated proteins were non-membranous in nature, which complemented the IEM data. Therefore, phosphoproteome analysis of the untreated and TP-47-treated sperm samples was performed. For this, protein extracts were subjected to 2D-PAGE-phosphotyrosine
immunoblots. A 51 kDa spot and two 45 kDa spots, corresponding to the hypo-tyrosine
phosphorylated spots, were analyzed by MS/MS. While peptides from the 51 kDa protein
matched with tektin-2 (a microtubular protein), those of the 45 kDa spots matched with ODF-2 protein of the sperm flagellum. Validation of the presence of tektin-2 and ODF-2 protein and their tyrosine-phosphorylated forms on sperm capacitation in the hamster spermatozoa has also been performed.
In addition to detailing the role of PYP in hamster sperm capacitation, this study revealed the identities of a few of these proteins, whose tyrosine phosphorylated status could be critical for optimal sperm flagellar bending, required for sperm hyperactivation. By understanding causes that lead to altered sperm function, for example, as observed with hamster spermatozoa, new insights could be achieved into molecular regulatory mechanisms
that govern sperm function in clinical cases of non-obstructive male infertility in the human.
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Estudo das alterações morfo-funcionais de espermatozóides bovinos submetidos à sexagem por meio da técnica de citometria de fluxo / Study of morpho-functions alterations in bovine spermatozoa submitted on sorting through flow cytometry technologyPriscilla Harumi Tanno 14 September 2009 (has links)
O objetivo deste estudo foi avaliar as alterações morfo-funcionais do sêmen bovino submetido à sexagem através da técnica de citometria de fluxo e comparar com o sêmen convencional e a diferença entre as subespécies. O sêmen foi obtido de seis touros, sendo três taurinos (Holandês preto e branco) e três zebuínos (Gir leiteiro), com quatro ejaculados de cada animal (n = 24). Cinco tratamentos foram realizados: sêmen convencional com diluidor L (Lagoa); sêmen convencional com diluidor S (Sexing) e sêmen sexado (nos tempos de zero, três e seis horas após a ejaculação para avaliar se existem alterações de membranas ao longo do tempo). Foram avaliados pela citometria de fluxo quanto à integridade de membrana plasmática e reação acrossomal (PI/FITC-PSA); peroxidação lipídica (C11-BODIPY581/591) e capacitação espermática através de análises da fosforilação da tirosina e aumento da fluidez e desorganização da membrana plasmática (Merocianina 540 e Yo-Pro1). Os efeitos de tratamentos foram avaliados por análises de variância (ANOVA), empregando-se o programa estatístico Stat-View® (SAS Institute, Inc. 1998, Cary, NC, USA). Foram considerados significativos os valores com P<0,05 e com tendência a significância (P<0,10). O sêmen proveniente da subespécie taurina teve um efeito negativo mais significante do que a zebuína na qualidade do sêmen. O sêmen sexado apresentou maior peroxidação lipídica nos espermatozóides com membrana íntegra e a presença de proteínas fosforiladas na superfície da membrana plasmática, fatores que são indicativos de capacitação espermática. Porém, ao contrário do esperado, este aumento não foi acompanhado pela quantidade de células classificadas como capacitadas pela análise de associação de sondas Yo-Pro-1 e Merocianina 540, pois o sêmen convencional apresentou maior população espermática com membrana plasmática íntegra desorganizada (P<0,05). Não houve piora na qualidade do sêmen sexado devido ao tempo de espera do ejaculado (P<0,05). / The objective of this study was to evaluate the morpho-functions alterations in bovine semen submitted on sorting through flow cytometry technology and compare with conventional semen and the difference between the subspecies. Semen was obtained from six bulls, that three were taurine (Holstein Black and White) and three were zebuine (Dairy Gir) with four ejaculates from each animal (n = 24). Five treatments were performed: conventional semen with L (Lagoa) media; conventional semen with S (Sexing) media and sexed semen (zero, three and six hours after ejaculation to evaluate if there were membrane alterations over the time). The treatments were analyzed with flow cytometry as for plasmatic membrane integrity and acrosome reaction (PI/FITC-PSA); lipid peroxidation (C11-BODIPY581/591) and sperm capacitation through protein tyrosine phosphorylation and the increase in plasma membrane fluidity and disorganization (Merocyanine 540 and Yo-Pro-1). The treatments effects were evaluated by analysis of variance (ANOVA) (Stat-View® SAS Institute, Inc. 1998, Cary, NC, USA). Effects were considered significant if the values were P<0.05 and with significant tendency (P<0.10). Semen from the taurine subspecie had a more significant negative effect than zebuine on semen quality. Sexed semen showed more lipid peroxidation in sperm with membrane integrity and the presence of phosphorylated proteins in plasma membrane surface that seemed to be sperm capacitation. However, in the contrary of expected, this increase did not accompany with quantity of cells classified as capacitated by the association probes Yo-Pro-1 e Merocyanine 540 analyse because the conventional semen showed more sperm population with plasma membrane integrity disorganized (P<0.05). There was not worsening in sexed semen quality over the time ejaculate waiting (P<0.05).
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Nouveaux régulateurs de la signalisation TLR2-NF-kB / New regulators of pathway TLR2-NF-kBRossi, Anne-Lise 29 November 2013 (has links)
L’invasion de l’hôte par un pathogène induit l’activation séquentielle des réponses immunitaires innées et adaptatives. La reconnaissance du pathogène par des récepteurs tels que les récepteurs de type Toll (TLRs) initie la réponse innée qui repose sur l’activation des lignées myéloïdes, la production de cytokines, de chémokines et de médiateurs pro-inflammatoires qui contribuent à l’éradication du pathogène. L’amplification incontrôlée de la réaction inflammatoire est délétère pour l’organisme. Afin de mieux comprendre les mécanismes de régulation des réponses dépendant de TLR2, récepteur impliqué dans la reconnaissance de bactéries, parasites ou champignons, nous avons étudié la composition des complexes multimoléculaires d’activation au sein des radeaux lipidiques. En utilisant des approches protéomiques complémentaires, nous avons mis en évidence le rôle de la Src kinase Lyn et de la déshydrogénase IMPDHII après engagement des hétérodimères TLR2/TLR1 ou TLR2/TLR6. La tyrosine kinase Lyn est indispensable à l’activation de NF-kB après engagement de TLR2 et agit en phosphorylant la sous-unité p110 de la PI3-kinase (PI3-K). IMPDHII, cible de l’acide mycophénolique, est un régulateur négatif de la voie TLR2-NF-kB. IMPDHII interagit avec SHP1 pour inhiber la phosphorylation sur tyrosine de p85α, la sous-unité régulatrice de PI3-K, et prévenir la transactivation de NF-kB. Enfin, nous avons étudié le rôle d’un polymorphisme de IMPDHII dans la gravité du choc septique. Ces travaux affinent la compréhension de la régulation de la réponse dépendant de TLR2 et permettent d’identifier de nouvelles cibles thérapeutiques pour la prise en charge des infections graves. / The host invasion by pathogen results in an adaptive immune response subsequent to innate immunity. Pathogens recognition by receptors as Toll Like Receptors (TLRs) initiates activation of innate immune myeloid synchronized with cytokines, chemokines and pro-inflammatory mediators production that contribute to pathogen eradication. Exuberant inflammation should lead to acute organ dysfunction and death. To understand mechanisms of TLR2-dependent response regulation, we studied composition of multi-molecular complexes in lipid rafts. TLR2 recognizes bacterial, parasitic and fungal agents. Using complementary proteomic approaches, we show the roles of Lyn Src kinase and IMPDHII dehydrogenase following involvement of TLR2/TLR1 or TLR2/TLR6. Lyn tyrosine kinase is indispensable in TLR2-dependent activation of NF-kB and phosphorylates p110 subunit of PI3-Kinase (PI3-K). IMPDHII, the mycophenolic acid substrate, is a negative regulator of TLR2-NF-kB pathway. IMPDHII acts on SHP1 to inhibit tyrosine phosphorylation on p85α, regulatory subunit of PI3-K, and prevent NF-kB transactivation. Finally, by a translational approach, we investigate the role of a genetic polymorphism of IMPDHII in the septic shock outcome. These findings strengthen the knowledge of mechanisms in TLR2-dependent control and should predict immunomodulation strategies.
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炭素と窒素の栄養バランス応答における緑藻のタンパク質リン酸化酵素TAR1の機能新川, はるか 23 July 2019 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22021号 / 生博第418号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 福澤 秀哉, 教授 河内 孝之, 教授 荒木 崇 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM
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Biologický význam tyrozínové fosforylace v SH3 doméně proteinu CAS / The biological importance of CAS SH3 domain tyrosine phosphorylationJanoštiak, Radoslav January 2010 (has links)
Protein CAS is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes. It is a multidomain adaptor protein, which serves as a scaffold for assembly of signalling complexes which are important for migration and invasiveness of Src-transformed cells. A novel phosphorylation site in N-terminal SH3 domain was identified - tyrosine 12 located on binding surface of CAS SH3 domain. To study biological importance of tyrosine 12 phosphorylation, non-phosphorylable (Y12F) and phosphomimicking ( Y12E) mutant of CAS were prepared. We found that phosphomimicking mutation Y12E leads to decreased interaction of CAS SH domain with kinase FAK a phosphatase PTP-PEST and also reduce tyrosine phosphorylation of FAK. Using GFP-tagged CAS protein, we show that Y12E mutation caused delocalization of CAS from focal adhesion but has no effect on localization of CAS to podosome-type adhesion. Non-phosphorylable mutation Y12F cause hyperphosphorylation of CAS substrate domain and decrease turnover of focal adhesion and associated cell migration of mouse embryonal fibroblasts (MEFs) independent to integrin singalling. Analogically to migration, CAS Y12F decrease invasiveness of Src-transformed MEF. The results of this diploma thesis show that phosphorylation of Tyr12 in CAS SH3 domain is...
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Effect of nanoparticles on human cells from healthy individuals and patients with respiratory diseasesOsman, Ilham F. January 2010 (has links)
Ever increasing applications of nanomaterials (materials with one or more dimension less than 100 nm) has raised awareness of their potential genotoxicity. They have unique physico-chemical properties and so could have unpredictable effects. Zinc oxide (ZnO) and titanium dioxide (TiO2) are widely used in a number of commercial products. There are published studies indicating that some forms of these compounds may be photo-clastogenic in mammalian cells. What has not been investigated before is the effect of nanoparticles from these compounds in human germ cells. Thus the present study has examined their effects in the presence and absence of UV light in human sperm and compared responses to those obtained with human lymphocytes using the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range) was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV and simultaneous irradiation with UV. The studies do provide some evidence that there are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity. The cytotoxic and genotoxic potentials of ZnO and TiO2 as well as their effect on phosphotyrosine expression, were examined in the human epithelial cervical carcinoma cells (Hela cells). This was done to try and determine the underlying molecular events resulting from their exposure to ZnO and TiO2 nanoparticles occurring at the same time as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations were reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects of TiO2 nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were compared with those in healthy individuals using genotoxic endpoints to determine whether there are any differences in sensitivity to nano-chemical insult between the patient and control groups. The results have shown concentration dependent genotoxic effects of TiO2 in both respiratory patient and control groups in the Comet assay and an increasing pattern of cytogenetic damage measured in the micronucleus assay without being statistically significant except when compared with the untreated controls of healthy individuals. Furthermore, modulation of ras p21 expression was investigated. Regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels that showed modulation as the result of nanoparticle treatment. Results have suggested that both ZnO and TiO2 nanoparticles can be genotoxic over a range of concentrations without either photoa-ctivation or being cytotoxic.
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The role of CD5 in T lymphocyte activationLacey, Erica January 2011 (has links)
No description available.
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