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Rôle de la SNARE Memb11 comme « récepteur » de la GTPase Arf1 à l’appareil de Golgi chez Arabidopsis thaliana / Role of the SNARE Memb11 as "receptor" of the GTPase Arf1 at the Golgi apparatus of Arabidopsis thalianaMarais, Claire-Line 16 December 2013 (has links)
Les protéines SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) sont essentielles pour la fusion membranaire. J'ai étudié chez Arabidopsis thaliana la SNARE Memb11 de l’appareil de Golgi qui intervient au début de la voie sécrétoire à l'interface Réticulum endoplasmique (RE)-appareil de Golgi. Dans les cellules de mammifères, l'orthologue de Memb11 (Membrine) est un « récepteur » potentiel de la GTPase Arf1 à la membrane golgienne. Cette dernière est impliquée dans le recrutement de la machinerie COPI nécessaire au transport rétrograde de l'appareil de Golgi vers le RE. Le but de ce travail était de déterminer si Memb11 pouvait interagir avec Arf1 dans les cellules végétales. Des anticorps dirigés contre la partie cytosolique de Memb11 ont été obtenus et ont été utilisés sur tissus végétaux pour réaliser des immunomarquages en microscopie électronique à transmission et des immunoprécipitations sur extraits de plantes. Il a été démontré que Memb11 est située au niveau de la membrane cis-golgienne et qu'elle co-immunoprécipite avec Arf1, suggérant ainsi que Arf1 peut interagir avec Memb11. J'ai confirmé l'interaction de Memb11 et Arf1 au niveau de l'appareil de Golgi par des expériences de BiFC (Bimolecular Fluorescence Complementation) in vivo. Cette interaction est spécifique puisque ni Memb12 (90% d'identité avec Memb11) ni Sec22 interagissent avec Arf1. Grâce à une approche de bioinformatique structurale, j'ai déterminé les régions de Memb11 (différentes de Memb12) qui pourraient être critiques pour l'interaction et j’ai commencé à tester in vivo les mutants correspondants par BiFC. En outre, des expériences d’immunoprécipitations avec des protéines recombinantes produites in vitro suggèrent que la forme d’Arf1 liée au GTP interagit avec Memb11. / The SNARE proteins (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) are critical for membrane fusion in the secretory pathway. I have studied the Golgi SNARE Memb11 in Arabidopsis thaliana cells. Memb11 is involved at the ER-Golgi interface. In mammalian cells, the ortholog of Memb11 (Membrin) is the potential “receptor” of the GTPase Arf1 in the Golgi membrane. This protein is involved for the recruitment of the COPI machinery, required for retrograde transport from the Golgi to the ER. The aim of this work was to determine whether Memb11 can interact with Arf1 in plant cells. Antibodies against the cytosolic part of Memb11 were obtained and were applied on plant tissues to perform immunolabeling by transmission electron microscopy and immunoprecipitation (IP) studies. It has been shown that Memb11 is located at the cis-Golgi and that it co-immunoprecipated with Arf1, suggesting that Arf1 may interact with Memb11. I confirmed the interaction of Memb11 and Arf1 at the Golgi by in vivo BiFC (Bimolecular Fluorescence Complementation) experiments. This interaction was specific since neither Memb12 (90% identity with Memb11) nor Sec22 interacted with ARF1. Thanks to a structural bioinformatic approach, I determined the regions in Memb11 (different from Memb12) that could be critical for the interaction and started to test corresponding mutants in vivo by BiFC. In addition, IP experiments with recombinant proteins produced in vitro suggest that the GTP-bound form of ARF1 interacts with Memb11.
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Aspectos clínico-laboratoriais na evolução de pacientes com deficiência de imunoglobulina A diagnosticados na infância e de seus familiares / Clinical and laboratorial features from patients with IgA deficiency diagnosed in childhood and from their relativesFahl, Kristine 24 June 2008 (has links)
A deficiência de imunoglobulina A (DIgA) é a imunodeficiência primária mais comum com prevalência de 1: 223 a 1:1000 em estudos epidemiológcos, sendo menos freqüente em populações asiáticas. As manifestações clínicas variam desde indivíduos assintomáticos até aqueles com manifestações atópicas, auto-imunes ou infecciosas. Entre estas últimas destacam-se os acometimentos dos tratos respiratório e digestório. Associação com outras imunodeficiências primárias tem sido reportada, em especial a imunodeficiência comum variável e a deficiência de subclasses de imunoglobulina G. Os objetivos deste estudo foram a avaliação dos pacientes com DIgA, diagnosticados na infância, após a segunda década da vida e a avaliação diagnóstica de seus familiares quanto aos aspectos clínicos e laboratoriais. A metodologia utilizou, para o diagnóstico de DIgA, o critério adotado pelo ESID/PAGID (1999). Foi realizada a avaliação das concentrações de imunoglobulina A(IgA) e imunoglobulina M (IgM), na saliva e a pesquisa de auto-anticorpos séricos (Anti-tireóide, FR,FAN) nos pacientes e familiares com DIgA. Realizou-se uma curva de distribuição de IgM salivar em indivíduos normais para comparação com os pacientes DIgA. Os resultados mostraram a avaliação dos 34 pacientes (19 do sexo feminino) com deficiência de imunoglobulina A (DIgA) com idade superior a 10 anos (variação: 10 à 52 anos), sendo 27 deles diagnosticados na infância e 7 familiares dos 62, que responderam à convocação. Considerando-se todos os indivíduos com DIgA, processos infecciosos (de repetição ou graves) ocorreram em 91,2%, manifestações atópicas em 58,8% e auto-imunidade em 52,9%. Manifestações clínicas de auto-imunidade foram observadas em 14/18 indivíduos, sendo que sete foram diagnosticados após 10 anos de idade, por ocasião da realização da pesquisa. Auto-anticorpos foram observados em 10 pacientes, sendo quatro pacientes assintomáticos. Fator reumatóide não foi detectado nesta casuística. Tireoidopatias (seis casos) e artropatias (quatro casos) foram as manifestações clínicas auto-imunes mais observadas. As concentrações de IgM salivar mostraramse elevadas em todos, exceto cinco casos. A comparação dessas concentrações nos grupos com e sem auto-imunidade não mostrou diferença significante (p= 0,48). As conclusões desta pesquisa mostraram os processos infecciosos como as manifestações clínicas mais freqüentes nos pacientes com DIgA, observando-se, porém, uma relevante presença de auto-imunidade nestes pacientes quando reavaliados após a segunda década de vida. Este fato alerta para a necessidade de avaliação rotineira de fenômenos auto- imunes nestes pacientes, durante seguimento. As concentrações de IgM salivar foram semelhantes em pacientes com DIgA com e sem auto-imunidade. Auto-anticorpos foram detectados independentemente da presença de sintomatologia clínica, sendo os mais encontrados aqueles relacionados à tireóide. Os familiares de primeiro grau dos pacientes com DIgA devem ser avaliados, tanto para diagnóstico de imunodeficiências como para detecção de fenômenos auto-imunes, permitindo assim o diagnóstico e abordagens precoces de ambas condições. / IgA deficiency (IgAD) is the most frequent primary immunodeficiency. Its prevalence varies from 1:223 to 1:1000 in epidemiological studies, but in asiatic populations it is uncommon. The clinical manifestations of IgAD are spectral, ranging from asymptomatic patients to recurrent infections, allergic symptoms and/or autoimmunity conditions. The most common infections frequently associated to IgAD involve respiratory and gastrointestinal tracts. The association with other primary immunodeficiency such as common variable immunodeficiency and immunoglobulin G subclasses deficiency has been reported. The aim of this study is to describe clinical and laboratorial evolution regarding autoimmune manifestations in IgAD patients diagnosed during the first years of life and after the second decade and their relatives diagnosed during this study. Laboratorial data included immunoglobulins A and M levels in saliva and auto-antibody screening in immunoglobulin A deficient relatives and patient. The criterion adopted for IgAD diagnosis was defined by Pan-American Group for Immunodeficiency and European Society for Immunodeficiency. The results showed 34 (19 female) immunoglobulin A deficient patients (IgAD) over 10 years of age (range: 10-52 years), 27 of them diagnosed during their childhood, and seven adults detected among 62 screened relatives. Recurrent infections were diagnosed in 91.2% of cases, atopic manifestations in 58.8% and autoimmune clinical manifestation in 52.9%. Autoimmune clinical manifestations were observed in 14 of our 18 IgA deficient patients and relatives with autoimmunity phenomena and seven of them diagnosed only over 10 years old during the study. Auto-antibodies were observed in (10/18) of patients and relatives, with four of them (asymptomatic) showing only the presence of auto-antibodies. Thyroid and osteo-articular involvement (six and four cases, respectively) were the most frequent clinical autoimmune manifestations. The rheumatoid factor was not detected in this casuistic. Auto-antibodies had no statistical difference among patients with or without autoimmunity phenomena. High salivary IgM concentrations were detected in all IgA deficient patients and relatives, except five cases. The comparison of these concentrations in the groups with and without autoimmunity did not show significant difference (p=.0, 48). In conclusion, recurrent infections were the most frequent clinical manifestations of IgA deficient patients and also autoimmune diseases, after the second decade of life. This fact at this series reinforced the necessity of active search for autoimmune conditions diagnosis in these patients. IgM levels showed no statistical difference among IgA deficient patients and relatives with or without autoimmunity. Auto-antibodies, mainly anti-thyroid antibodies, were detected in patients independently of autoimmunity clinical manifestation presence. This study showed the importance of the first degree relatives of IgA deficient patients evaluation, focusing as immunodeficiency as autoimmune disease, permiting an earlier diagnosis of both conditions and an adequate approach to optimize the clinical management and improvement of quality of life of IgAD patients.
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Aquisição passiva de anticorpos protetores reativos com Bordetella pertussis pelo recém-nascido via transferência placentária e aleitamento materno / Passive acquisition of protective antibodies reactive with Bordetella pertussis by the newborn via placental transfer and breastfeedingFaria, Camila Cristina Quinello Gomes de 15 March 2010 (has links)
Atualmente, a coqueluche representa um crescente problema de saúde pública em países desenvolvidos. Embora ainda não existam evidências de um aumento do número de casos de coqueluche no nosso país, não se pode descartar a hipótese de uma futura re-emergência da doença, pois dados epidemiológicos de algumas regiões revelam um aumento da incidência, indicando que provavelmente ocorra uma baixa notificação de novos casos ao Ministério da Saúde. A maioria dos casos ainda ocorre em lactentes menores de seis meses de idade, ou seja, crianças ainda não completamente imunizadas. Diversos trabalhos demonstraram a aquisição de anticorpos IgG reativos com Bordetella pertussis pelo recém-nascido através da passagem transplacentária, mas a partir dos dois meses de vida, observa-se um declínio substancial do título destes anticorpos. Neste caso, outro modo de conferir proteção ao neonato é através da transmissão de anticorpos IgA específicos pelo aleitamento materno, que poderia suprir a falta de anticorpos IgG até que o esquema de vacinação esteja completo. Os objetivos deste trabalho foram analisar a transferência passiva de anticorpos IgG e IgA anti-B. pertussis para o recémnascido a termo e investigar a habilidade destes anticorpos em neutralizar a patogenicidade bacteriana em um modelo experimental in vivo utilizando camundongos desafiados por via intracerebral com B. pertussis viável. Foram coletadas 40 amostras pareadas de sangue materno, de cordão umbilical e de colostro. Foram demonstrados títulos equivalentes de anticorpos IgG anti-B. pertussis entre as amostras de soro materno e de cordão (medianas de 1:225 e 1:265, respectivamente) com taxa de transferência de 118%. Foram observados títulos variáveis de anticorpos IgA específicos nas amostras de colostro materno com mediana de 1:74. O Immunoblotting realizado com extrato bruto de B. pertussis e Pools de soro materno, de soro de cordão e de colostro com alto e baixo título de anticorpos específicos revelou um perfil de reconhecimento idêntico entre os Pools de soro materno e dos respectivos neonatos. Os Pools de colostro apresentaram, em seu perfil de reconhecimento, diferentes intensidades que variaram de acordo com os títulos de anticorpos IgA específicos. No desafio intracerebral com B. pertussis, embora todos os Pools de soro materno, de cordão e de colostro tenham apresentado capacidade significativa de neutralizar a patogenia bacteriana quando comparados ao controle positivo, os Pools com alto título de anticorpos revelaram maior capacidade neutralizante. Os Pools de soro e colostro absorvidos com B. pertussis e, portanto, sem anticorpos IgG e IgA específicos, protegeram 30% dos animais testados e anticorpos IgG purificados, apresentando alto título de anticorpos anti-B. pertussis (1:2.560), protegeram 65% dos camundongos. Nossos dados confirmaram a transferência de anticorpos reativos com B. pertussis para o neonato via placenta e aleitamento materno e sua eficácia na neutralização da patogênese bacteriana, o que pode proteger a criança contra infecções respiratórias causadas por Bordetella pertussis. / Pertussis is currently considered an important public health problem in developed countries. Although there is no evidence of an increase in the number of pertussis cases in our country, can not rule out the hypothesis of a future re-emergence of disease, as epidemiological data from some regions show an increase in incidence, indicating that probably there is a low report of new cases to the public health authorities. Most cases still occurs in infants under six months of age, i.e. children not fully immunized. Several works have demonstrated the acquisition of IgG antibodies reactive with Bordetella pertussis by the newborn through placental transfer, but by age of two months it was observed a substantial decay of titers these antibodies. In this case, another way to confer protection the neonate is through the transmission of IgA antibodies via breast-feeding, which could supply the lack of IgG antibodies until the vaccination schedule will be completed. The aims of this work were to analyze the passive transfer of IgG and IgA anti-B. pertussis antibodies to term newborns and to investigate the ability of these antibodies to neutralize the bacterial pathogenicity in an experimental model in vivo using mice intracerebrally challenged with viable B. pertussis. It was collected 40 paired samples of maternal blood, cord umbilical blood and colostrum. Equivalent titers of anti-pertussis IgG antibodies were demonstrated between maternal and cord serum samples (medians of 1:225 and 1:265, respectively) with transfer rate of 118%. It was observed variable specific IgA titers in maternal colostra with a median of 1:74. Immunoblotting performed with B. pertussis crude extract and Pools of maternal serum, cord serum and colostrum with high and low specific antibody titers revealed an identical recognition profile between paired maternal and newborn serum Pools. Colostrum Pools presented, in their recognition profile, different intensities that varied according to specific IgA antibody titers. In the intracerebral challenge with B. pertussis, although all maternal and cord serum and colostrum Pools presented a significant bacterial neutralizing ability when compared with positive control group, Pools with high antibody titers revealed higher neutralizing capacity. Serum and colostrum Pools absorbed with B. pertussis and, thus, without specific IgG and IgA antibodies, protected 30% of the animals tested and purified IgG antibodies, presenting a high anti-pertussis antibody titer (1:2,560), protected 65% of the mice. Our data confirmed the transfer of antibodies reactive with B. pertussis to the neonate via placenta and breast-feeding and their effectiveness in bacterial pathogenesis neutralization, which could protect infants against respiratory infections caused by Bordetella pertussis.
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Sistemas subterrâneos de Asteraceae do Cerrado paulista: abordagens anatômica, ecológica e reprodutiva / Underground systems of Asteraceae from the São Paulo state Cerrado: anatomical, ecological and reproductive approachesCury, Graziela 30 September 2008 (has links)
A presença de sistemas subterrâneos em plantas do Cerrado é uma característica que há algum tempo vem atraindo a atenção de pesquisadores. Suas funções adaptativas e ecológicas já são bem conhecidas, mas devido à diversidade de tipos a denominação desses órgãos nem sempre é aplicada corretamente, já que muitas vezes são realizados apenas estudos morfológicos que se mostram insuficientes. Portanto, análises anatômicas representam uma ferramenta indispensável na correta aplicação terminológica dessas estruturas. A família Asteraceae, muito bem representada no Cerrado paulista, apresenta grande quantidade de espécies que possuem sistemas subterrâneos espessados com capacidade gemífera e que acumulam frutanos como fonte de reservas. Essas características permitem a sobrevivência das plantas nesse ecossistema garantindo a regeneração da parte aérea que é eliminada devido a episódios de fogo ou seca prolongada, fenômenos comuns no Cerrado, ressaltando a importância desses órgãos subterrâneos na contribuição para o banco de gemas. Em sistemas subterrâneos espessados de Asteraceae, apesar de haver poucos estudos que enfoquem este assunto, observa-se a ocorrência de estruturas secretoras com valor diagnóstico que auxiliam estudos taxonômicos da família e possuem importante papel ecológico, já que constituem estratégia de defesa contra herbivoria. No entanto, devido à realização de análises incompletas verificada na literatura, muitas vezes os espaços secretores internos são genericamente denominados como canais, quando em observações em secções longitudinais, verifica-se que na verdade não são estruturas alongadas ou sua formação ocorre de maneira diversa. Alguns sistemas subterrâneos permitem a propagação vegetativa das plantas e podem constituir importante estratégia adaptativa no Cerrado. A relação entre a taxa germinativa e a capacidade de propagar-se vegetativamente explica os diferentes resultados obtidos em ensaios de germinação de sementes de espécies que possuem ou não a capacidade de reprodução clonal através de suas estruturas subterrâneas. O conjunto de informações obtidas, não só contribui para ampliar o conhecimento da flora do Cerrado, mas também fornece argumentos que justifiquem a sua conservação. / It has some time that the presence of underground systems in plants from Cerrado is a feature that has been attracting the researchers attention. Their ecological and adaptive functions are already well-known, but due to the diversity of underground organs types the denomination of these organs is not always correctly applied, since many times only morphological studies are performed what have shown unsatisfactory. Therefore anatomical analyses represent an essential tool to the correct terminological application of these structures. The family Asteraceae, very well represented in the Cerrado of the São Paulo state, exhibit great amount of species that possess bud bearing thickened underground systems that accumulate fructans of the inulin type as reserve compounds. These features allow the plants survival in this ecosystem assuring the regeneration of the aerial part that is eliminated due to fire episodes or extended dry, common phenomenon in Cerrado, reinforcing in this way the importance of these underground organs in the contribution to the bud bank. In thickened underground systems of Asteraceae in spite of having few studies which focus this subject, it is observed the occurrence of secretory structures with diagnosis value that aid taxonomic studies of the family and possess important ecological role since they constitute protection strategy against herbivory. Nevertheless due to the achievement of incomplete analysis as verified in the literature, many times the internal secretory spaces are generically named as canals, while in longitudinal sections observations it is verified that they are not actually elongated structures or their formation occurs of diverse way. Some underground systems allow the vegetative propagation of the plants and they can constitute important adaptation strategy in Cerrado. The relation between the germination rates and the vegetative propagation capacity explains the different results obtained in seed germination experiments of species that possess or not clonal reproduction capacity through their underground structures. The whole of information obtained contribute to extend the knowledge about the Cerrado flora and also provides arguments that justify its conservation.
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Aktivität Ubiquitin-konjugierender Enzyme an den RING-Ligasen des ERAD-SystemsBagola, Katrin 05 June 2012 (has links)
Fehlerhafte sekretorische Proteine werden über einen speziellen Abbauweg, die ER-assoziierte Proteindegradation (ERAD), mit Lysin48-verknüpften Ubiquitinketten polyubiquitiniert und dem proteolytischen Abbau am 26S Proteasom zugeführt. In der Hefe Saccharomyces cerevisiae bilden die beiden ER-membranständigen RING-Ubiquitinligasen Hrd1 und Doa10 zentrale Komponenten im Ubiquitinierungsprozess. Das lösliche zytosolische Ubiquitin-konjugierende Enzym Ubc7, welches mit beiden Ligasen bei der Polyubiquitinierung von Substratproteinen zusammenwirkt, wird über den membranverankerten Co-Faktor Cue1 an die ER-Membran rekrutiert. Die in dieser Arbeit dargestellten Ergebnisse belegen zwei weitere Funktionen für Cue1 im Ubiquitinierungsprozess: Die Bindung von Ubc7 an einen carboxyterminalen Bereich in Cue1 führt zur Stimulation der Ubiquitinierungsaktivität von Ubc7 mit den RING-Ligasen. Darüber hinaus bewirkt die Ubiquitin-bindende CUE-Domäne in Cue1 eine Steigerung der Länge der Ubiquitinketten und deren Syntheserate, was zum effektiven Abbau einiger ER-membrangebundener Substratproteine beiträgt. Die durch Ubc7 synthetisierten Lysin48-verknüpften Ubiquitinketten werden in Abhängigkeit eines schleifenförmigen sauren Bereichs in Ubc7 gebildet. Entfernen dieses Bereichs resultiert im Abbruch der Ubiquitinierung nach Konjugation eines Monoubiquitins auf dem Substrat. An der Hrd1-Ligase werden durch Ubc7 polyubiquitinierte Proteine umgehend zum Proteasom transferiert. Für den Doa10-abhängigen Substratabbau ist die Funktion eines weiteren Ubiquitin-konjugierenden Enzyms, Ubc6, notwendig. Die hier gezeigten Daten weisen auf eine Ubc6-abhängige Verknüpfung von Ubiquitinmolekülen in einer Lysin11-abhängigen Weise hin. Eine Inhibition der Synthese Lysin11-verknüpfter Ubiquitinketten hatte jedoch keinen Effekt auf den Abbau von Substratproteinen. Stattdessen wurde der Abbau von Ubc6 selbst durch Unterbindung der Bildung Lysin27-verknüpfter Ubiquitinketten verhindert. / Aberrant secretory proteins are removed from the cell in a process termed „endoplasmic reticulum-associated protein degradation" (ERAD), as it screens the endoplasmic reticulum for unwanted polypeptides and triggers their elimination via the 26S proteasome. To this end, client proteins of the ERAD pathway are polyubiquitinated with lysine48-linked ubiquitin chains at the ER membrane. Two ER membrane-integrated RING ubiquitin ligases, Hrd1 and Doa10, constitute central components of the ubiquitination machinery in Saccharomyces cerevisiae. To polyubiquitinate substrate proteins, both ligases interact with the ubiquitin-conjugating enzyme Ubc7. Since Ubc7 itself is a soluble cytosolic protein, it is recruited to the ER-membrane by is anchoring factor Cue1. Results in this study reveal two additional functions of Cue1 in the ubiquitination reaction: First, binding of Ubc7 to the Cue1-carboxyterminus stimulates the ubiquitin chain formation by Ubc7 and the ligases. Second, the CUE domain within Cue1 increases the chain length and accelerates the synthesis of the polyubiquitin chain, which results in efficient degradation of certain substrate proteins. Formation of lysine48-linked ubiquitin chains by Ubc7 depends on an acidic loop within Ubc7. Deletion of this structure leads to inhibition of ubiquitin chain elongation after the initial substrate monoubiquitination. Client proteins, ubiquitinated by Ubc7 and Hrd1, are immediately transferred to the proteasome. For Doa10-dependent substrate degradation, the activity of another ubiquitin-conjugating enzyme, Ubc6, is required. Data shown here indicate a function of Ubc6 in the formation of lysine11-linked polyubiquitin, since mutation of this lysine residue resulted in the prevention of ubiquitin chain synthesis. However, expression of this ubiquitin mutant had no effect on substrate degradation. Moreover, the proteolysis of Ubc6 itself is inhibited by prevention of lysin27-linked polyubiquitin chain formation.
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Die Funktion der HRD-Ubiquitinligase bei der Protein- Dislokation aus dem Endoplasmatischen RetikulumMehnert, Martin 13 May 2013 (has links)
Fehlgefaltete Proteine des sekretorischen Weges werden aus dem Endoplasmatischen Retikulum (ER) in das Zytosol transportiert und dort durch das Ubiquitin-Proteasom-System abgebaut. Dieser Qualitätskontrollmechanismus wird als Endoplasmatisches Retikulum-assoziierte Proteindegradation bezeichnet (ERAD). In der Bäckerhefe Saccharomyces cerevisiae stellt die HRD-Ubiquitinligase eine zentrale Komponente dieses Abbausystems dar. Eine Untereinheit dieses Multienzymkomplexes ist das ER-ständige Membranprotein Der1, das über den Faktor Usa1 an die Ubiquitinligase Hrd1 rekrutiert wird und ausschließlich für den Abbau löslicher luminaler ERAD-Substrate notwendig ist. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der C-Terminus von Der1 die Interaktion zu Usa1 und damit die Rekrutierung des Proteins zur HRD-Ligase vermittelt. Usa1 wirkt nicht nur als Rekrutierungsfaktor, sondern induziert auch die Der1-Oligomerisierung. Punktmutationen in den Transmembrandomänen von Der1 beeinträchtigen die Dislokation luminaler Substratproteine aus dem ER. Um weitere Hinweise für eine Beteiligung von Der1 beim Substrattransport zu erhalten, wurde die Methode des zielgerichtetem in vivo photocrosslinking für Der1 angewendet. Hierbei wurden bestimmte Positionen von Der1 mit dem photoreaktiven Aminosäureanalogon p-Benzoylphenylalanin markiert, was die Ausbildung von Quervernetzungen von Der1 zu Interaktionspartnern nach einer UV-Bestrahlung ermöglichte. Schließlich konnte auf diese Weise eine räumliche Nähe der luminal exponierten Bereiche von Der1 zum Substratrezeptor Hrd3 gezeigt werden, während die Transmembransegmente Quervernetzungen zu Hrd1 ausbildeten. Beide Bereiche von Der1 konnten zudem mit einem luminalen ERAD-Substrat quervernetzt werden. Anhand dieser Ergebnisse wurde somit erstmals eine direkte Beteiligung von Der1 insbesondere in den ersten Schritten der Substratdislokation gezeigt, was eine Funktion von Der1 als zentrale Komponente des Exportkomplexes nahelegt. / Newly synthesized proteins of the secretory pathway are subjected to an efficient quality control system in the endoplasmic reticulum. In order to prevent a harmful aggregation misfolded proteins are exported via a largely unknown mechanism into the cytosol and degraded by the ubiquitin-proteasome system in a process termed ER associated degradation (ERAD). In the yeast Saccharomyces cerevisiae the HRD-ligase constitutes a central component of ERAD. A subunit of this multi-enzyme complex is the small multispanning membrane protein Der1, which is exclusively required for the degradation of misfolded ER luminal proteins but dispensable for the turnover of membrane-bound substrates. In this study a short conserved motif in the cytosolic carboxyterminus of Der1 was identified that mediates the binding to the HRD-ligase. Moreover, co-immunoprecipitation experiments show that Der1 forms oligomers, which relies on its assembly into the degradation complex. Mutations in the transmembrane domains of Der1 block the export of soluble proteins across the ER-membrane. To further investigate the function of Der1 in substrate dislocation an in vivo site-specific photocrosslinking approach was applied. Various positions of Der1 were labelled with the photoreactive amino acid analogue p-benzoyl-phenylalanine followed by UV irradiation of living cells expressing these Der1 constructs. The crosslinking experiments reveal a spatial proximity of ER luminal exposed parts of Der1 to the substrate receptor Hrd3. By contrast, the membrane-embedded domains of Der1 reside adjacent to the ubiquitin ligase Hrd1. Intriguingly, both regions also form crosslinks to a client protein. In summary the data of this work imply that multimeric Der1 initiates the export of aberrant polypeptides from the ER-lumen by threading such molecules into the ER-membrane and routing them to Hrd1 for ubiquitylation.
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Aspectos clínico-laboratoriais na evolução de pacientes com deficiência de imunoglobulina A diagnosticados na infância e de seus familiares / Clinical and laboratorial features from patients with IgA deficiency diagnosed in childhood and from their relativesKristine Fahl 24 June 2008 (has links)
A deficiência de imunoglobulina A (DIgA) é a imunodeficiência primária mais comum com prevalência de 1: 223 a 1:1000 em estudos epidemiológcos, sendo menos freqüente em populações asiáticas. As manifestações clínicas variam desde indivíduos assintomáticos até aqueles com manifestações atópicas, auto-imunes ou infecciosas. Entre estas últimas destacam-se os acometimentos dos tratos respiratório e digestório. Associação com outras imunodeficiências primárias tem sido reportada, em especial a imunodeficiência comum variável e a deficiência de subclasses de imunoglobulina G. Os objetivos deste estudo foram a avaliação dos pacientes com DIgA, diagnosticados na infância, após a segunda década da vida e a avaliação diagnóstica de seus familiares quanto aos aspectos clínicos e laboratoriais. A metodologia utilizou, para o diagnóstico de DIgA, o critério adotado pelo ESID/PAGID (1999). Foi realizada a avaliação das concentrações de imunoglobulina A(IgA) e imunoglobulina M (IgM), na saliva e a pesquisa de auto-anticorpos séricos (Anti-tireóide, FR,FAN) nos pacientes e familiares com DIgA. Realizou-se uma curva de distribuição de IgM salivar em indivíduos normais para comparação com os pacientes DIgA. Os resultados mostraram a avaliação dos 34 pacientes (19 do sexo feminino) com deficiência de imunoglobulina A (DIgA) com idade superior a 10 anos (variação: 10 à 52 anos), sendo 27 deles diagnosticados na infância e 7 familiares dos 62, que responderam à convocação. Considerando-se todos os indivíduos com DIgA, processos infecciosos (de repetição ou graves) ocorreram em 91,2%, manifestações atópicas em 58,8% e auto-imunidade em 52,9%. Manifestações clínicas de auto-imunidade foram observadas em 14/18 indivíduos, sendo que sete foram diagnosticados após 10 anos de idade, por ocasião da realização da pesquisa. Auto-anticorpos foram observados em 10 pacientes, sendo quatro pacientes assintomáticos. Fator reumatóide não foi detectado nesta casuística. Tireoidopatias (seis casos) e artropatias (quatro casos) foram as manifestações clínicas auto-imunes mais observadas. As concentrações de IgM salivar mostraramse elevadas em todos, exceto cinco casos. A comparação dessas concentrações nos grupos com e sem auto-imunidade não mostrou diferença significante (p= 0,48). As conclusões desta pesquisa mostraram os processos infecciosos como as manifestações clínicas mais freqüentes nos pacientes com DIgA, observando-se, porém, uma relevante presença de auto-imunidade nestes pacientes quando reavaliados após a segunda década de vida. Este fato alerta para a necessidade de avaliação rotineira de fenômenos auto- imunes nestes pacientes, durante seguimento. As concentrações de IgM salivar foram semelhantes em pacientes com DIgA com e sem auto-imunidade. Auto-anticorpos foram detectados independentemente da presença de sintomatologia clínica, sendo os mais encontrados aqueles relacionados à tireóide. Os familiares de primeiro grau dos pacientes com DIgA devem ser avaliados, tanto para diagnóstico de imunodeficiências como para detecção de fenômenos auto-imunes, permitindo assim o diagnóstico e abordagens precoces de ambas condições. / IgA deficiency (IgAD) is the most frequent primary immunodeficiency. Its prevalence varies from 1:223 to 1:1000 in epidemiological studies, but in asiatic populations it is uncommon. The clinical manifestations of IgAD are spectral, ranging from asymptomatic patients to recurrent infections, allergic symptoms and/or autoimmunity conditions. The most common infections frequently associated to IgAD involve respiratory and gastrointestinal tracts. The association with other primary immunodeficiency such as common variable immunodeficiency and immunoglobulin G subclasses deficiency has been reported. The aim of this study is to describe clinical and laboratorial evolution regarding autoimmune manifestations in IgAD patients diagnosed during the first years of life and after the second decade and their relatives diagnosed during this study. Laboratorial data included immunoglobulins A and M levels in saliva and auto-antibody screening in immunoglobulin A deficient relatives and patient. The criterion adopted for IgAD diagnosis was defined by Pan-American Group for Immunodeficiency and European Society for Immunodeficiency. The results showed 34 (19 female) immunoglobulin A deficient patients (IgAD) over 10 years of age (range: 10-52 years), 27 of them diagnosed during their childhood, and seven adults detected among 62 screened relatives. Recurrent infections were diagnosed in 91.2% of cases, atopic manifestations in 58.8% and autoimmune clinical manifestation in 52.9%. Autoimmune clinical manifestations were observed in 14 of our 18 IgA deficient patients and relatives with autoimmunity phenomena and seven of them diagnosed only over 10 years old during the study. Auto-antibodies were observed in (10/18) of patients and relatives, with four of them (asymptomatic) showing only the presence of auto-antibodies. Thyroid and osteo-articular involvement (six and four cases, respectively) were the most frequent clinical autoimmune manifestations. The rheumatoid factor was not detected in this casuistic. Auto-antibodies had no statistical difference among patients with or without autoimmunity phenomena. High salivary IgM concentrations were detected in all IgA deficient patients and relatives, except five cases. The comparison of these concentrations in the groups with and without autoimmunity did not show significant difference (p=.0, 48). In conclusion, recurrent infections were the most frequent clinical manifestations of IgA deficient patients and also autoimmune diseases, after the second decade of life. This fact at this series reinforced the necessity of active search for autoimmune conditions diagnosis in these patients. IgM levels showed no statistical difference among IgA deficient patients and relatives with or without autoimmunity. Auto-antibodies, mainly anti-thyroid antibodies, were detected in patients independently of autoimmunity clinical manifestation presence. This study showed the importance of the first degree relatives of IgA deficient patients evaluation, focusing as immunodeficiency as autoimmune disease, permiting an earlier diagnosis of both conditions and an adequate approach to optimize the clinical management and improvement of quality of life of IgAD patients.
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Morfoanatomia, tricomas glandulares e fitoquímica de Lomatozona artemisiifolia Baker (ASTERACEAE - EUPATORIEAE) - uma planta endêmica do Cerrado de Goiás / Morfoanatomy, Trichomes Glandular and Phytochemistry of Lomatozona artemisiifolia Baker (ASTERACEAE-EUPATORIEAE)- A Plant of Endemic of Cerrado of GoiásTrindade, Luma Mota Palmeira 30 September 2013 (has links)
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Previous issue date: 2013-09-30 / Morphoanatomy, trichomes glandular and phytochemistry of Lomatozona artemisiifolia Baker (Asteraceae - Eupatorieae) - a plant of endemic of Cerrado of Goiás - The Asteraceae family is widely distributed, having 24,000 species and genera 1600-1700, constituting one of the largest families of phanerogams. In the Cerrado, among phanerogams, is the second largest family in number of species. Among the numerous species of Asteraceae is present Lomatozona artemisiifolia Baker, belongs to the tribe Eupatorieae. The objective of this study was to describe anatomically aerial vegetative organs of this species, characterize the glandular trichomes that occur in these vegetative organs and inflorescences, determine the composition of the oil and perform the phytochemical study of the species, as there is no work on this species which is endemic to the Cerrado. For anatomical study were used apexes, samples of completely expanded leaves and stems. The phytochemical screening was performed with aerial vegetative organs and oil extraction was performed with fresh plant material (aerial vegetative organs). The epidermis is uniseriate covered with a thin cuticle striated and mesophyll is dorsiventral. The stem has uniseriate epidermis, cortex and with angular collenchyma lamellar secretory ducts associated with the cortical parenchyma, sclerenchyma fibers forming ice outside the phloem bundles are collateral. Is the presence of eight types of glandular trichomes on the leaf and stem. We identified the presence of traces of alkaloids, coumarins, traces of digitalis glycosides, saponins, flavonoids and glycosides. In oil extraction were identificandos 21 compounds, the major compounds are γ-muuroleno about 28.81%, the β-germacrene with 18.23%, 12.48% caryophyllene, δ-elemene 8.61%, 7.55% δ-carene, β-ocimene 5.94%. / Morfoanatomia, tricomas glandulares e fitoquímica de Lomatozona artemisiifolia Baker (Asteraceae - Eupatorieae) – Uma planta endêmica do cerrado de Goiás - A família Asteraceae é amplamente distribuída, possuindo 24.000 espécies e 1.600 a 1700 gêneros, constituindo uma das maiores famílias de fanerógamas. No Cerrado, dentre as fanerógamas, é a segunda maior família em quantidade de espécies. Dentre as inúmeras espécies de Asteraceae está presente Lomatozona artemisiifolia Baker, pertence à tribo Eupatorieae. O objetivo do presente estudo foi descrever anatomicamente os órgãos vegetativos aéreos desta espécie, caracterizar os tricomas glandulares que ocorrem nestes órgãos vegetativos e nas inflorescências, determinar a composição do óleo essencial e realizar o estudo fitoquímico da espécie, já que não há trabalhos com esta espécie que é endêmica do Cerrado. Para o estudo anatômico foram utilizados ápices caulinares, amostras de folhas completamente expandidas e de caules. A prospecção fitoquímica foi realizada com órgãos aéreos vegetativos e a extração do óleo foi realizada com material vegetal fresco (órgãos aéreos vegetativos). A epiderme é uniestratificada recobertas por cutícula delgada estriada e o mesofilo é dorsiventral. O caule possui epiderme uniestratificada, córtex com colênquima angular e lamelar, ductos secretores associados ao parênquima cortical, fibras esclerenquimáticas formando calotas externas ao floema, os feixes são colaterais. Ocorre a presença de oito tipos de tricomas glandulares na lâmina foliar e caule. Foram identificadas a presença de traços de alcalóides, cumarinas, heterosídeos flavonóides e saponinas. Na extração do óleo foram identificandos 21 compostos, os majoritários foram γ- muuroleno com cerca de 28.81%, o β-germacreno com 18,23%, cariofileno 12,48%, δ-elemeno 8,61%, δ-careno 7,55%, β-ocimeno 5,94%.
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Aquisição passiva de anticorpos protetores reativos com Bordetella pertussis pelo recém-nascido via transferência placentária e aleitamento materno / Passive acquisition of protective antibodies reactive with Bordetella pertussis by the newborn via placental transfer and breastfeedingCamila Cristina Quinello Gomes de Faria 15 March 2010 (has links)
Atualmente, a coqueluche representa um crescente problema de saúde pública em países desenvolvidos. Embora ainda não existam evidências de um aumento do número de casos de coqueluche no nosso país, não se pode descartar a hipótese de uma futura re-emergência da doença, pois dados epidemiológicos de algumas regiões revelam um aumento da incidência, indicando que provavelmente ocorra uma baixa notificação de novos casos ao Ministério da Saúde. A maioria dos casos ainda ocorre em lactentes menores de seis meses de idade, ou seja, crianças ainda não completamente imunizadas. Diversos trabalhos demonstraram a aquisição de anticorpos IgG reativos com Bordetella pertussis pelo recém-nascido através da passagem transplacentária, mas a partir dos dois meses de vida, observa-se um declínio substancial do título destes anticorpos. Neste caso, outro modo de conferir proteção ao neonato é através da transmissão de anticorpos IgA específicos pelo aleitamento materno, que poderia suprir a falta de anticorpos IgG até que o esquema de vacinação esteja completo. Os objetivos deste trabalho foram analisar a transferência passiva de anticorpos IgG e IgA anti-B. pertussis para o recémnascido a termo e investigar a habilidade destes anticorpos em neutralizar a patogenicidade bacteriana em um modelo experimental in vivo utilizando camundongos desafiados por via intracerebral com B. pertussis viável. Foram coletadas 40 amostras pareadas de sangue materno, de cordão umbilical e de colostro. Foram demonstrados títulos equivalentes de anticorpos IgG anti-B. pertussis entre as amostras de soro materno e de cordão (medianas de 1:225 e 1:265, respectivamente) com taxa de transferência de 118%. Foram observados títulos variáveis de anticorpos IgA específicos nas amostras de colostro materno com mediana de 1:74. O Immunoblotting realizado com extrato bruto de B. pertussis e Pools de soro materno, de soro de cordão e de colostro com alto e baixo título de anticorpos específicos revelou um perfil de reconhecimento idêntico entre os Pools de soro materno e dos respectivos neonatos. Os Pools de colostro apresentaram, em seu perfil de reconhecimento, diferentes intensidades que variaram de acordo com os títulos de anticorpos IgA específicos. No desafio intracerebral com B. pertussis, embora todos os Pools de soro materno, de cordão e de colostro tenham apresentado capacidade significativa de neutralizar a patogenia bacteriana quando comparados ao controle positivo, os Pools com alto título de anticorpos revelaram maior capacidade neutralizante. Os Pools de soro e colostro absorvidos com B. pertussis e, portanto, sem anticorpos IgG e IgA específicos, protegeram 30% dos animais testados e anticorpos IgG purificados, apresentando alto título de anticorpos anti-B. pertussis (1:2.560), protegeram 65% dos camundongos. Nossos dados confirmaram a transferência de anticorpos reativos com B. pertussis para o neonato via placenta e aleitamento materno e sua eficácia na neutralização da patogênese bacteriana, o que pode proteger a criança contra infecções respiratórias causadas por Bordetella pertussis. / Pertussis is currently considered an important public health problem in developed countries. Although there is no evidence of an increase in the number of pertussis cases in our country, can not rule out the hypothesis of a future re-emergence of disease, as epidemiological data from some regions show an increase in incidence, indicating that probably there is a low report of new cases to the public health authorities. Most cases still occurs in infants under six months of age, i.e. children not fully immunized. Several works have demonstrated the acquisition of IgG antibodies reactive with Bordetella pertussis by the newborn through placental transfer, but by age of two months it was observed a substantial decay of titers these antibodies. In this case, another way to confer protection the neonate is through the transmission of IgA antibodies via breast-feeding, which could supply the lack of IgG antibodies until the vaccination schedule will be completed. The aims of this work were to analyze the passive transfer of IgG and IgA anti-B. pertussis antibodies to term newborns and to investigate the ability of these antibodies to neutralize the bacterial pathogenicity in an experimental model in vivo using mice intracerebrally challenged with viable B. pertussis. It was collected 40 paired samples of maternal blood, cord umbilical blood and colostrum. Equivalent titers of anti-pertussis IgG antibodies were demonstrated between maternal and cord serum samples (medians of 1:225 and 1:265, respectively) with transfer rate of 118%. It was observed variable specific IgA titers in maternal colostra with a median of 1:74. Immunoblotting performed with B. pertussis crude extract and Pools of maternal serum, cord serum and colostrum with high and low specific antibody titers revealed an identical recognition profile between paired maternal and newborn serum Pools. Colostrum Pools presented, in their recognition profile, different intensities that varied according to specific IgA antibody titers. In the intracerebral challenge with B. pertussis, although all maternal and cord serum and colostrum Pools presented a significant bacterial neutralizing ability when compared with positive control group, Pools with high antibody titers revealed higher neutralizing capacity. Serum and colostrum Pools absorbed with B. pertussis and, thus, without specific IgG and IgA antibodies, protected 30% of the animals tested and purified IgG antibodies, presenting a high anti-pertussis antibody titer (1:2,560), protected 65% of the mice. Our data confirmed the transfer of antibodies reactive with B. pertussis to the neonate via placenta and breast-feeding and their effectiveness in bacterial pathogenesis neutralization, which could protect infants against respiratory infections caused by Bordetella pertussis.
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The effect of bovine colostrum supplementation on levels of secretory immunoglobulin-A (S-IgA) in saliva of elite atheletes, non-exercising controls and non-exercising older adults : a project [i.e. thesis] completed as fulfilment of the requirements of a doctoral thesis in Clinical Nutrition, Massey University, Albany Campus, New ZealandCrooks, Christine January 2007 (has links)
Secretory immunoglobulin-A (S-IgA) in saliva may reflect levels of immune defence at other mucosal sites. Reduced levels of salivary S-IgA have been associated with an increased risk for upper respiratory symptoms (URS) in athletes. Previously, the consumption of a nutrition supplement, bovine colostrum (BC) by distance runners, was shown to significantly increase levels of salivary S-IgA compared to baseline; however the mechanism was not known. The immunomodulatory effect of BC is investigated further in these current studies. Twenty-five swimmers (12 males [M], 13 females [F], age 14-23 years) training at an elite level, 28 lightly-exercising students (9M, 19F, age 18-27 years), and 45 healthy older adults (20M, 20F, age 65-76 years), consumed a supplement of either BC or placebo for ten weeks. Saliva samples were collected at baseline, weekly for four weeks during supplementation and post-supplementation. Blood samples were collected at baseline, monthly during supplementation and post-supplementation. No significant changes were seen in levels of S-IgA in either BC or placebo groups within any of the cohorts. There was a trend towards a significant difference in URS reportage between BC and placebo groups in the swimmers cohort, but not in the students or older adults. There was also a trend towards a difference in the number of swimmers reporting URS. Fewer numbers of swimmers consuming BC reported URS compared the placebo (P=0.062) after consuming BC for four weeks compared to those consuming the placebo. Post-exercise plasma cortisol results were significantly reduced in the BC subgroup compared to the placebo (P=0.004). These results do not support the findings of previous intervention studies investigating the immunomodulatory effect of BC in athletes. However the reduced reportage of URS, among swimmers consuming the BC supplement, suggested there was some benefit to their health. A possible explanation is that BC has impacted on non-infectious causes of URS. Growth factors present in BC may enhance intestinal repair which could be advantageous to athletes recovering from bouts of prolonged intensive exercise. The effect of gastrointestinal disturbances on local and systemic immunity may be minimised which benefits immune protection. However an inconsistent effect of BC supplementation on immune protection in athletes means further research is still required. In these studies there was no benefit to immune protection in the student or older adult cohort. Further investigation into the safety of BC for all population groups is still required.
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