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Tierärztl. Hochsch., Diss., 2004--Hannover.

Orientadores: José Dirceu Ribeiro, Edson Antunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T08:13:24Z (GMT). No. of bitstreams: 1 Grotta_MilenaBaptistella_D.pdf: 2330139 bytes, checksum: 5a69a2a222d3f7028574efcf65a49676 (MD5) Previous issue date: 2011 / Resumo: Introdução: A prevalência da obesidade e da asma tem aumentado muito durante as últimas décadas. Investigações atuais sugerem que a obesidade está associada à asma em numerosos estudos. Os mecanismos pelos quais a obesidade interfere nos sintomas da asma ainda são controversos. Objetivo: avaliar o efeito da obesidade na atividade eosinofílica através da quimiotaxia e adesão em crianças e adolescentes asmáticos e não asmáticos em associação a dosagem das adipocinas séricas. Método: Incluídos 32 asmáticos obesos (AO) e não obesos (ANO), 5 não asmáticos obesos (NAO) e 5 não asmáticos não obesos (NANO). Colhido sangue periférico para isolamento de eosinófilos através do gradiente de Percolll seguido de separação imunomagnética. Para quimiotaxia utilizada câmara de microquimiotaxia com 48 poços em triplicata com MEM (espontânea), eotaxina, PAF (fator de ativação plaquetária) e RANTES (regulated on activation, normal T cell expressed and secreted). Leitura através de filtro com contagem em microscópio óptico. A adesão foi realizada com placas de fibronectina em triplicata com MEM e eotaxina. Leitura através da absorbância das amostras desconhecidas com as da curva padrão de células. Feito dosagem de leptina e adiponectina por ELISA. Resultado: A quimiotaxia espontânea foi maior entre os AO e ANO (p=0,02) e entre os AO e NANO (p=0,03). Com eotaxina houve aumento para o grupo AO em relação aos ANO e aos NANO, e do grupo dos NAO em relação aos ANO e NANO (p<0,0001). Para o PAF, o grupo AO mostrou-se maior do que NANO (p=0,02). O RANTES apresentou significância entre os NAO em relação aos ANO e NANO (p=0,01). Na adesão espontânea dos eosinófilos a fibronectina, não foi encontrado relevância entre os grupos. Com eotaxina, houve maior adesão para os AO em relação aos NANO (p=0,04). Maior concentração de leptina no grupo dos AO e NAO (p=0,0001). Não foi encontrado diferença entre os grupos para adiponectina total. Conclusão: Este é o primeiro estudo a demonstrar uma maior atividade eosinofílica (quimiotaxia e adesão) em crianças e adolescentes obesos asmáticos atópicos em relação aos não obesos e aos voluntários saudáveis com associação ao aumento de leptina sérica / Abstract: Background: The prevalence of obesity and asthma has increased over the past several decades. Recent investigations suggest relationship between asthma and obesity in many studies, but the mechanisms are unclear. The aim of this study was evaluate the obesity effect in eosinophil activity by chemotaxis and adhesion in asthmatic and non-asthmatic children and adolescents in association with serum adipokines measurement Method: 32 asthmatic obese (AO) and asthmatic non obese (ANO), 5 non asthmatic obese (NAO) and 5 non asthmatic non obese (NANO) were included. Peripheral blood was collected and eosinophils were purified using a Percoll gradient followed by immunomagnetic cell separator. Chemotaxis was performed with microchemotaxis chamber in triplicate with MEM (spontaneous), eotaxin, PAF (platelet-activating factor) and RANTES (regulated on activation, normal T cell expressed and secreted). The measurement was done by optical microscope count. The adhesion was performed by fibronectin plates in triplicate with MEM and eotaxin. Eosinophilic adhesion was calculated by comparison between absorbance of unknown samples with the standard curve. Leptin and adiponectin were quantified by ELISA. Results: spontaneous chemotaxis was higher between AO and ANO (p=0.02) and between AO and NANO (p=0.03).With eotaxin, the increased was between AO and ANO and between AO and NANO, and between ANO and NANO (p<0.0001).With PAF, AO was higher than NANO (p=0.02). RANTES was increased among NAO and ANO and NANO (p=0.01). In spontaneous adhesion, there was no difference between groups. With eotaxin, the adhesion was higher between AO and NANO (p=0.04). Leptin was higher in AO and NAO than the others (p=0.0001). There was no difference among groups for total adiponectin. Conclusion: This is the first study that showed higher eosinophilic activity (chemotaxis and adhesion) in atopic obese asthmatic children and adolescents in relationship to asthmatic non obese and healthy volunteers / Doutorado / Saude da Criança e do Adolescente / Doutor em Saude da Criança e do Adolescente

CD34 and its relatives, Podocalyxin and Endoglycan, comprise of a family of surface sialomucins expressed by hematopoietic stem/progenitor cells, and vascular endothelia. Recent data suggest that they serve as either pro- or anti-adhesion molecules depending on their cellular context and their post-translational modifications. We were interested in identifying Podocalyxin ligands and their cellular distribution and understanding the role of these factors in signaling, adhesion and migration. Using both a lambda phage screen assay and mass spectrometry, we identified the Na⁺/H⁺ exchanger regulatory factor-i (NHERF-l) as a selective ligand for Podocalyxin and Endoglycan but not for the closely related CD34. Furthermore, we showed that NHERF-1 is expressed by all, lineage⁻, Sca-1⁺ and c-kit⁺ (LSK) cells, which are known to express Podocalyxin and have long-term repopulating characteristics of hematopoietic stem cells. In addition, upon IL-3 stimulation of a factor dependent cell line (FDC-P 1) these proteins re-localize and co-localize in an asymmetrical pattern. By using a lentiviral based shRNA system to silence Podocalyxin and NHERF- i proteins, we observed that migration across stromal monolayer towards a CXCL12 and SCF gradient is significantly impeded in cells that lack Podocalyxin but not NHERF-1. Following in vitro stimulation with a combination of CXCL12 and SCF we observed that Podocalyxin co-associates with CXCR4. Furthermore, cells lacking Podocalyxin have decreased phospho-AKT, a key signaling molecule downstream of c-kit and CXCR4 receptors. Taken together, our data supports the conclusion that Podocalyxin co-association with CXCR4 modulates downstream signaling to efficiently regulate HSC homing. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum. We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACA-mediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.

The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.

L'axe formé par la chimiokine CXCL12 et son récepteur CXCR4 est conservé chez les vertébrés où il joue un rôle important dans l'embryogenèse et la vie adulte, régule de nombreux processus des réponses immunitaires grâce à ses fonctions dans la migration cellulaire, la survie et la prolifération.En outre, cet axe est impliqué dans les processus pathologiques tels que les cancers (croissance et métastase) et immunodéficiences ainsi que des dysfonctionnements (par exemple l'expression dérégulée, polymorphismes ou mutations) et est également détourné par certains agents pathogènes (par exemple le virus de l'immunodéficience humaine, virus du papillome humain).Un grand groupe de travail est consacré à cette paire comme cible thérapeutique, mais seulement un composé (à savoir Plérixafor) a atteint l'approbation pour une utilisation clinique faisant le potentiel de cet axe comme cible de médicament encore inexploré.Bien que cet axe est l'objet d'un grand intérêt, des questions demeurent quant aux déterminants structurels impliqués dans l'interaction CXCL12/CXCR4.Cependant, la structure récemment résolue par diffraction de CXCR4 a donné quelque indice au sujet de ces questions, et au­ delà, la possible stoichiométrie entre CXCL12 et CXCR4.Plusieurs éléments de preuve appuient le concept que les formes CXCR4 homo- et hétéro- oligomères (qui peut contribuer à la diversité des fonctions de récepteur), telles que la structure de diffraction, le gain de fonction d'un récepteur CXCR4 mutant responsable du syndrome WHIM et la modulation allostérique des fonctions de CXCR4 par CXCR7 (ACKR3), le second récepteur de CXCL12. La possibilité de former des oligomères ouvre de nombreuses questions en matière de CXCL12 et ses interactions avec CXCR4 et CXCR7/ACKR3. La stoichiométrie de cette interaction reste une question ouverte, comme le récepteur est capable de former des oligomères avec le même récepteur ou autre récepteurs, en particulier CXCR7/ACKR3. Ce récepteur, connu comme scavenger, n'a pas de structure résolue et son mécanisme d'interaction avec CXCL12 reste inconnu.Afin d'étudier les interactions CXCL12/CXCR4/CXCR7, nous avons appliqué plusieurs techniques de modélisation moléculaire tels que peptid-peptide docking et simulations de dynamique moléculaire.Objets du projet ont étés : la résolution des possibles formes stoichiométriques de l'interaction CXCR4/CXCL12 (modélisation moléculaire, docking et dynamique); la modélisation de la structure du récepteur CXCR7/ACKR3 et son interaction avec CXCL12 (homology modeling), avec caractérisation des domaines et des résidus clef de l'activation des pathways de signalisation en aval du récepteur (mutants CXCR7/ACKR3); l'étude et la caractérisation de nouveaux outils innovants pour la détection de l'oligomerisation de ces récepteurs en conditions endogènes. (Nanobodies, HTRF)Les résultats du premier objectif ont été publiés en janvier 2016 : PMID 26813575.La modélisation de CXCR7/ACKR3 nous a permit de générer plusieurs mutants du récepteur pour tester nos hypothèses sur l’activation.Les nanobodies caractérisés pour CXCR4 seront utilisé dans une deuxième étude pour l’identification des formes oligomériques du récepteur sur tissus et cellules. / The axis formed by the chemokine CXCL12 and its receptor CXCR4 is conserved in vertebrates where it plays an important role in embryogenesis and adult life, regulates many processes of immune responses through its functions in cell migration, survival and proliferation.In addition, this axis is involved in pathological processes such as cancers (growth and metastasis) and immune deficiencies and malfunctions (eg deregulated expression, mutations or polymorphisms) and is also hijacked by certain pathogens (eg HIV, human papilloma virus).A large working group is dedicated to this pair as a therapeutic target, but only a compound (ie Plerixafor) achieved approval for clinical use by the potential of this area as a drug target unexplored.Although this axis is the subject of great interest, questions remain about the structural determinants involved in CXCL12 / CXCR4 interaction.However, the recently resolved diffraction structure of CXCR4 gave some clue about these questions, and beyond possible stoichiometry between CXCL12 and CXCR4.Several lines of evidence support the concept that forms CXCR4 homo- and hetero-oligomers (which can contribute to the diversity of the receptor functions), as shown in the diffraction structure, the gain function of a mutant CXCR4 receptor responsible for the syndrome WHIM and allosteric modulation of CXCR4 functions by CXCR7 (ACKR3), the second receptor of the chemokine CXCL12. The ability to form oligomers opens many issues of CXCL12 and its interaction with CXCR4 and CXCR7 / ACKR3.The stoichiometry of this interaction still remains an open question, as the receptor is capable to form oligomers with the same receptor or other receptors, particularly CXCR7 / ACKR3. This receptor, known as scavenger, has not solved structure and the mechanism of interaction with CXCL12 is unknown.To study the interactions CXCL12 / CXCR4 / CXCR7, we applied several molecular modeling techniques such as peptide-peptide docking and molecular dynamics simulations.Objectives of this project were: the resolution of the different stoichiometric forms for the interaction of CXCR4 and CXCL12 (molecular modeling, docking and dynamic); modeling the CXCR7 / ACKR3 receptor structure and its interaction with CXCL12 (homology modeling), with the characterization of domains and residues key in the activation of downstream signaling pathways of the receptor (CXCR7 / ACKR3 mutants); the study and characterization of new innovative tools for the detection of oligomerization of these receptors in endogenous conditions. (Nanobodies, HTRF)The results of the first objective were published in January 2016: PMID 26813575.Modeling of CXCR7 / ACKR3 allowed us to generate several mutants of the receptor to test our hypothesis about the activation pathways.Nanobodies were fully characterized for CXCR4 to be used in a second study to identify oligomeric forms of the receptor in tissues and cells.