A síndrome de McCune Albrigth (SMA) é uma doença genética não hereditária, com incidência estimada entre 1/100.000 e 1/1.000.000 casos/ano. A SMA caracteriza-se clinicamente pela tríade: displasia óssea fibrosa (FD), manchas cutâneas café-com-leite e hiperfunção endócrina tais como: síndrome de Cushing, pseudo-puberdade precoce, hipertiroidismo, acromegalia. O diagnóstico da SMA clássica é usualmente baseado no quadro clínico associado a dosagens hormonais e exames de imagem, principalmente cintilografia do esqueleto. No entanto, quadros atípicos e formas parciais muitas vezes dificultam o diagnóstico preciso da síndrome. O objetivo deste estudo foi padronizar dentre as técnicas de PNA (peptide nucleic acid) e PCT em Tempo Real, para a detecção de polimorfismos de base única (SNPs), a técnica mais sensível para a discriminação das mutações ativadoras da subunidade da proteína G. Para este estudo foram selecionados 32 pacientes, 1 masculino e 31 femininos, com SMA, todos em seguimento no Hospital das Clínicas da Faculdade de Medicina da USP. Como resultado positivo, apresentamos nesse trabalho pela primeira vez o uso do RT-PCR genotipagem na detecção das mutações ativadoras da proteína G, em DNA extraído de tecidos afetados e em leucócitos de sangue periférico, sendo a técnica considerada sensível o suficiente para discriminar de forma simples e rápida as mutações ativadoras da PGs. Sugerimos nesse estudo o uso da técnica de discriminação alélica pelo sistema Taqman. Essa técnica possibilita a detecção destas mutações gsp no sangue periférico mesmo numa baixa porcentagem, uma vez que nem sempre o tecido afetado (gônada, osso, hipófise) é disponível. / The McCune-Albright Syndrome (MAS) is a genetic disease, with incidence estimated at 1/100.000 and 1/1000000 cases per year. MAS is clinically characterized by the triad: bone fibrous dysplasia (FD) café-au-lait skin spots and endocrine hyperfunction, such as: precocious puberty (PP), Cushing's syndrome, hyperthyroidism and acromegaly. The diagnosis of MAS is originally based on clinical characteristics associated with hormonal and imaging studies. However, atypical and partial forms often hamper the accurate diagnosis of the syndrome. For this study we selected 32 patients, 1male and 31 females, all being treated in Hospital das Clínicas, School of Medicine, University of São Paulo. As a positive result, we showed for the first time the use of Real Time PCR/genotyping for the detection of activating mutations of the stimulatory G protein, using blood leucocytes DNA. This technique was sensible and can bring fast results for the patient and the physician, making the diagnosis easier. Our study proposes the use of allelic discrimination by Taqman system, which can be used as a probe that allows the identification of specific genotypes. These techniques could help detect these mutations in peripheral blood when the affected tissue is not available.

A hipóxia é capaz de alterar muitos mecanismos bioquímicos nas células endoteliais. Dentre eles, a indução da expressão endotelial de moléculas de adesão, como o fator de von Willebrand (FVW) que, em resposta ao estímulo, é secretado em sua forma mais ativa na interação com as plaquetas, o que pode resultar em trombose. Nas condições fisiológicas, o padrão multimérico do FVW no plasma é essencialmente determinado pela ADAMTS-13 (uma desintegrina e metaloproteinase com domínios trombospondina). Este estudo teve como objetivo verificar se a atividade da enzima ADAMTS-13, assim como as características do FVW relacionáveis a ela, poderiam estar alteradas na presença de hipoxemia comparativamente à condição de oxigenação normal. Este estudo longitudinal envolveu 56 pacientes portadores de cardiopatias congênitas cianogênicas, em idades entre um e sete anos, candidatos ao tratamento cirúrgico. Os pacientes foram avaliados no pré-cirúrgico (basal), no pós-operatório imediato (pós 48 horas) e após 30 dias de cirurgia, e foram divididos em dois grupos (A e B) baseado na saturação periférica de oxigênio (SpO2) no momento pós 30 dias. Foram determinados o antígeno do FVW e a análise das suas subunidades, a atividade da ADAMTS-13 e a presença de inibidores da ADAMTS-13. Os pacientes de ambos os grupos apresentaram aumento significante da SpO2, da concentração antigênica do FVW e da atividade da ADAMTS-13 nos momentos pós 48 horas e pós 30 dias em comparação com o momento pré (basal). As densidades normalizadas da subunidade principal do FVW (225 kDa) e do fragmento de 176 kDa apresentaram tendência ao aumento nos momentos pós 48 horas e pós 30 dias nos dois grupos. A razão entre a atividade da ADAMTS-13 e o FVW estava menor do que 1 no momento pós 48 horas, indicando consumo da enzima; entretanto, no momento pós 30 dias a razão fica 1:1, e o FVW se aproxima dos valores de referência. Verificamos ainda que 29% destes pacientes apresentaram inibidores contra a ADAMTS-13 no momento pré-operatório. Ainda explorando as variáveis SpO2, FVW:Ag, atividade da ADAMTS-13 e a composição das subunidades do FVW, foi feito um estudo de correlação linear entre estas variáveis. Observamos uma baixa correlação entre a enzima ADAMTS-13 e o FVW:Ag, e da enzima com os fragmentos do FVW de 176 e 140 kDa, principalmente no grupo B. No grupo A, esta correlação no momento pós 48 horas mostrou tendência a ser negativa. A maioria dos pacientes apresentou melhoras na saturação periférica de oxigênio. O aumento das variáveis estudadas no pós-operatório imediato pode ter ocorrido em função da cirurgia, que provavelmente ocasionou um quadro de lesão endotelial com inflamação, indicando que pode existir um equilíbrio entre o FVW e a ADAMTS-13 em níveis fisiológicos. Entretanto, este equilíbrio pode ser quebrado quando ocorre aumento do FVW, provavelmente por consumo da enzima. Parece-nos, portanto, que a ADAMTS-13 pode funcionar como um mecanismo de proteção a estes pacientes com tendência à trombose / Hypoxia has been shown to alter several biochemical mechanisms in endothelial cells. In addition, hypoxia induces the endothelial expression of adhesion molecules, including von Willebrand factor (VWF). Increased release of high-molecular-weight VWF multimers is associated with higher risk for thrombotic events. In physiological conditions, the multimeric pattern of plasma VWF is essentially determined by the action of ADAMTS-13 (a desintegrin and metalloprotease with thrombospondin type 1 domains). The aim of this study was to investigate if ADAMTS-13 activity and VWF subunit fragments were altered by hypoxia in cyanotic congenital heart disease. Fiftysix patients (age 1 to 7 years) with cyanotic congenital heart disease admitted to the Heart Institute for heart surgery were included in this longitudinal study. Patients were evaluated before (baseline) corrective surgery, postoperative 48 hours and postoperative 30 days. Patients were classified in two groups (A and B) based on the peripheral oxygen saturation after 30 days surgery. VWF antigenic concentration, VWF subunit composition, ADAMTS-13 activity and presence of ADAMTS-13 inhibitors were determined. Peripheral oxygen saturation, VWF:Ag and ADAMTS-13 activity were all increased significantly in both groups, in postoperative 48 hours and postoperative 30 days in comparison with baseline moment. Normalized density of VWF main subunit (225 kDa) and proteolytic fragment with 176 kDa tended to increase in postoperative 48 hours and postoperative 30 days in both groups. The rate between ADAMTS-13 activity and VWF:Ag was lower than 1 in postoperative 48 hours, an indicating of enzyme consumption; however, in the postoperative 30 days the rate was 1:1 and VWF:Ag values were near those of reference. 29% of patients presented ADAMTS-13 inhibitors at the baseline moment. A study of correlation among variables as peripheral oxygen saturation, VWF:Ag, VWF subunit composition and ADAMTS-13 was done. It was observed that ADAMTS-13 correlated slightly positively with VWF:Ag and with VWF fragments 176 and 140 kDa, mainly in group B; in group A, the correlation at postoperative 48 hours tended to be negative. Most of the patients improved their peripheral oxygen saturation. The increased value of variables observed in postoperative 48 hours can be explained by the endothelial injury and inflammation caused by the surgery itself. This indicates an equilibrium between VWF:Ag and ADAMTS-13 in physiological conditions. However, this equilibrium could disappear when VWF is increased, probably by enzyme consumption. We conclude that ADAMTS-13 can act as a protective mechanism in these patients with thrombotic tendency

P2X-Rezeptoren treten aufgrund ihrer Präsenz in verschiedensten Organsystemen des menschlichen Körpers zunehmend in den Mittelpunkt zahlreicher Forschungsansätze. Besonderes Interesse gilt dabei u.a. den P2X2/3-Rezeptoren, da in ihnen ein neuer Angriffspunkt für die Entwicklung von Schmerztherapeutika gesehen wird. Trotz enormer Fortschritte in diesem Bereich, bleiben die Vorgänge und strukturellen Gegebenheiten, die zur Öffnung der Ionenkanäle führen, weiterhin spekulativ. In der vorliegenden Arbeit wurden mithilfe einer Mutagenese einzelne Aminosäuren des hP2X2-Rezeptors, welche sich in geringer Entfernung zueinander zwischen zwei Untereinheiten befanden, durch Cysteine substituiert. Die Auswahl der Aminosäuren erfolgte dabei anhand eines Homologiemodells des hP2X2-Rezeptors und des Aminosäureabgleichs zwischen den hP2X2- und hP2X3-Rezeptoren. Auf diese Weise sollte deren Interaktion über eine mögliche Ausbildung von Disulfidbrücken zwischen zwei Untereinheiten untersucht werden. Die Rezeptorfunktion wurde anschließend mittels der whole-cell patch-clamp-Technik charakterisiert. Der Rezeptor reagierte bei allen untersuchten Varianten mit einem Funktionsverlust, ein spontan öffnender Kanal konnte somit nicht generiert werden. Durch die Kombination der verschiedenen hP2X2-Rezeptor-Cysteinmutanten mit einer hP2X3-Rezeptor-Cysteindoppelmutante, konnte gezeigt werden, dass sich die verschiedenen Untereinheiten im heterotrimeren hP2X2/3-Rezeptor nicht soweit annähern, dass eine Disulfidbrücken-Bildung zwischen den Untereinheiten möglich wird. Es konnte allerdings verdeutlicht werden, dass für die Aktivierung des heterotrimeren hP2X2/3-Rezeptors zwei funktionelle Bindungsstellen zur Kanalaktivierung ausreichen.

Le paludisme est une maladie dévastatrice causée par un parasite du genre Plasmodium. Du fait de la propagation de souches résistantes aux actuels antipaludéens, il est nécessaire de comprendre les fonctions physiologiques essentielles du parasite afin de trouver de nouvelles cibles thérapeutiques. Les transporteurs membranaires sont une classe importante de cibles chez l'homme du fait de leur rôle physiologique essentiel pour la cellule. Cependant, chez les parasite du genre Plasmodium, seulement quelques transporteurs ont été biochimiquement caractérisés. Des études récentes de délétion de gènes dans un model murin ont montrées que l’ATPase de type P4, ou flippase, ATP2 de Plasmodium est essentielle pour le parasite. Chez les Eucaryotes, l’activité de translocation des lipides des ATPases de type P4 est nécessaire pour maintenir l’asymétrie des membranes, un élément clé dans de nombreux processus essentiels comme la formation de vésicules ou l’apoptose. Les flippases forment des complexes hétéromériques avec les protéines de la famille Cdc50 qui sont également trouvées dans le génome de Plasmodium. Pour comprendre le rôle fonctionnel de ces transporteurs putatifs durant l’infection par le parasite, nous avons besoin d’étudier leur mécanisme de transport et d’identifier leur (s) substrat (s). Nous avons entrepris l’expression hétérologue chez Saccharomyces cerevisiae d’ATP2, en complexe avec les sous unités Cdc50, de trois espèces différentes de Plasmodium. Nous avons réussi à co-exprimer l’orthologue ATP2 de P. chabaudi (PcATP2) et les sous unités PcCdc50 correspondantes. Par co-immunoprécipitation et une chromatographie d’exclusion stérique détectée par fluorescence, nous sommes parvenus à identifier la sous unité s’associant à PcATP2 : PcCdc50.1. Nous avons ensuite purifié le complexe PcATP2/PcCdc50.1 en utilisant des nanobodies reconnaissant la GFP fusionnée à l’extrémité C-terminale de PcATP2 et nous avons initié la caractérisation fonctionnelle avec des tests de phosphorylation et d’activité ATPasique. / Malaria is a devastating disease caused by a parasite of the genus Plasmodium. Due to the spread of strains resistant to current antimalarial drugs, it is necessary to understand essential physiological functions of the parasite in order to find new drug targets. Membrane transport proteins are an important class of drug targets in humans, as they perform essential physiological roles of the cell. However, for Plasmodium parasites, just a few membrane transporters have been biochemically described. Recent gene-deletion studies in malaria mouse models have shown that the Plasmodium P4-ATPase, or lipid flippase, ATP2 is essential for the parasite. In eukaryotes, the phospholipid translocation activity of P4-ATPases is needed to maintain the asymmetric distribution of membranes, a key element in many essential processes like vesicle budding or apoptosis. Lipid flippases form heteromeric complexes with members of the Cdc50 protein family, also found in the genomes of Plasmodium parasites. To understand the functional role of these still putative transporters during malaria infection we need to study their transport mechanism and identify their substrate(s). We have conducted the heterologous expression in Saccharomyces cerevisiae of ATP2 in complex with the Cdc50 subunits from three different Plasmodium species. We succeeded to co-express the ATP2 ortholog of P. chabaudi (PcATP2) and the related putative PcCdc50 proteins. By co-immunoprecipitation and Fluorescence-detection Size Exclusion Chromatography, we have managed to identify the Cdc50 β-subunit that associates to PcATP2: PcCdc50.1. We then purified the complex PcATP2/PcCdc50.1 using immobilized nanobodies that recognize the GFP fused at the C-terminal end of PcATP2 and we initiated the functional characterization using ATPase and phosphorylation activity assays.

Pathophysiologische und therapeutische Bedeutung der a1- und a2-Untereinheiten des GABAA-Rezeptors für Dystonien: Untersuchungen im dtsz Hamstermodell

No / Trafficking motifs present in the intracellular regions of ion channels affect their subcellular location within neurons. The mechanisms that control trafficking to dendrites of central neurons have been identified, but it is not fully understood how channels are localized to the soma. We have now identified a motif within the calcium-activated potassium channel K(Ca)2.1 (SK1) that results in somatic localization. Transfection of hippocampal neurons with K(Ca)2.1 subunits causes expression of functional channels in only the soma and proximal processes. By contrast, expressed K(Ca)2.3 subunits are located throughout the processes of transfected neurons. Point mutation of K(Ca)2.1 within this novel motif to mimic a sequence present in the C-terminus of K(Ca)2.3 causes expression of K(Ca)2.1 subunits throughout the processes. We also demonstrate that blocking of clathrin-mediated endocytosis causes K(Ca)2.1 subunit expression to mimic that of the mutated subunit. The role of this novel motif is therefore not to directly target trafficking of the channel to subcellular compartments, but to regulate channel location by subjecting it to rapid clathrin-mediated endocytosis.

Les glucocorticoïdes sont les médicaments les plus efficaces pour le contrôle de l'obstruction respiratoire chez les chevaux atteints du souffle, et de l'asthme humain. Toutefois, les neutrophiles persistent dans les voies respiratoires suite à ce traitement. Nous avons précédemment rapporté que les neutrophiles sanguins humains et équins sont sensibles à l'action des glucocorticoïdes. Comme elle contribue à l'insensibilité des cellules épithéliales pulmonaires humaines aux glucocorticoïdes, nous avons émis l'hypothèse que l'IL-17 a un effet similaire sur les neutrophiles et qu’elle contribue à leur persistance dans les voies respiratoires asthmatiques. Objectifs : Évaluer 1. L’expression des deux sous-unités du récepteur de l’IL-17 (l'IL-17RA/IL-17RC) chez les neutrophiles équins hautement purifiés. 2. Si l'IL-17 active directement les neutrophiles et si cette réponse est sensible à l'action des glucocorticoïdes. 3. L'effet de l'IL-17 sur la viabilité et l'apoptose des neutrophiles. Résultats: 1. Les neutrophiles expriment l’IL-17RA/IL-17RC aux niveaux translationnel et protéique. 2. L’IL-17 induit une activation sélective des neutrophiles (surrégulation de l’IL-8), qui n’est pas atténuée par dexaméthasone et 3. l’IL-17 augmente la viabilité des neutrophiles stimulés (LPS) par une diminution de l'apoptose. Nos résultats indiquent que l'IL-17 active directement le neutrophile équin, et que l’augmentation de l’IL-8 (puissant chimioatractant des neutrophiles) qui en résulte n’est pas contrôlée par la dexaméthasone. L'IL-17 pourrait aussi contribuer à la persistance de neutrophiles dans les voies respiratoires chez les chevaux atteints du souffle, en diminuant l'apoptose. / Glucocorticoids are the most potent drugs for the control of the airway obstruction present in heaves and asthma, although the airway neutrophilia persist with these agents. It was previously reported that human and equine’s peripheral blood neutrophils are sensitive to glucocorticoid. Interleukin-17 (IL-17) is a cytokine contributing to glucocorticoid insensitivity airway epithelial cells in human. We therefore hypothesized that IL-17 has a similar effect on neutrophils and contributes to the persistence of these cells in the airways in response to glucocorticoid therapy. In this study, we evaluated: 1. The expression of both subunits of IL-17 receptor (IL-17RA/ IL-17RC) in highly purified neutrophils. 2. The gene expression of proinflammatory cytokines in neutrophils to assess their activation by IL-17 and their effect in these cells treated with glucocorticoids. 3. The effects of IL-17 on the viability and apoptosis in neutrophils were performed. Results showed that 1. Equine neutrophils express IL-17RA and IL-17RC at transcriptional at protein level. 2. IL-17 induces a selective activation in neutrophils, which results in an upregulation of IL-8, not attenuated by dexamethasone and finally, 3. IL-17 increases the viability of neutrophils stimulated with LPS by a decrease in apoptosis. Our results indicate that IL-17 directly activates neutrophils to upregulate IL-8, a potent neutrophils chemoattractant, and that this response is not prevented by glucocorticoids. They also suggest a possible role of IL-17 in the persistence of neutrophils in the asthmatic airways by decreasing apoptosis.

No description available.

A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation in relation to osteogenesis and relevant pathology. aBSCs may be different from their embryonic or neonatal counterparts, and are influenced by species-/age-specific and other factors. Mice heterozygous for a null allele of prkar1a (Prkar1a+/-, a gene encoding for cyclic adenosine mono-phosphate (cAMP)-dependent regulatory subunit of protein kinase A (PKA), developed bone lesions that resembled fibrous dysplasia (FD) originated from cAMP-responsive osteogenic cells. Prkar1a +/- mice were crossed with mice heterozygous for catalytic subunit Calpha (Prkaca+/-), the main PKA activity-mediating molecule and generated mouse model with double heterozygosity for prkar1a and prkaca (Prkar1a +/-Prkaca+/-). Unexpectedly, Prkar1a+/-Prkaca+/- mice developed a large number of osseous lesions starting at 2--3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of tail vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions were fibroblast- and FD-like, and almost always originated from an area proximal to the growth plate and adjacent to endosteal surface of the periosteum; they expanded gradually in the bone marrow space. These cells expressed osteogenic cell markers, showed higher PKA activity that was mostly type II (PKA-II) and display an alternate pattern of catalytic subunit expression, and surprisingly possessed higher cAMP levels. In addition, markers of bone synthesis and lysis were increased. Gene expression profiling not only confirmed an early (progenitor) osteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial (MET) transition and increased Wnt signaling, particularly the brachyury expression. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by PKA-II and altered Calpha activity, generating the only available germline mutant mouse model of a disorder that has similarities to human FD. Along with previous data, these studies also suggest that the effects of cAMP signaling on osteogenesis and stromal cell maintenance and proliferation in mice are age-, bone-, site- but also PKA-type and catalytic subunit-specific. / Parts of the work have been published in Proceedings of the National Academy of Sciences of the United States of America 2010; 107(19):8683--8. / Tsang, Kit Man. / Advisers: Constantine A. Stratakas; Kwak-Pui Fung. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

No description available.