Universiẗat, Diss., 2004--Münster (Westfalen).

Les paramyxovirus respiratoires humains sont des virus responsables d'infections chez les jeunes enfants, les personnes âgées et les patients immuno déprimés. Ces virus possèdent deux glycoprotéines à la surface de leur enveloppe, jouant un rôle dans l'entrée du virus dans la cellule cible. La glycoprotéine d’attachement (HN, G ou H) permet l’attachement du virus à son récepteur cellulaire et, dans le cas de HN, celle-ci est suspectée d’activer la seconde glycoprotéine, la protéinede fusion (F). Cette dernière réalise la fusion entre l'enveloppe du virus et la membrane cellulaire.Le mécanisme par lequel la protéine HN "active" la protéine F reste mal caractérisé, malgré la détermination récente de leurs structures en cristallographie. Plusieurs modèles sont actuellement proposés. Ce travail de thèse s’est focalisé principalement sur les glycoprotéines d’enveloppe des virus parainfluenza humain de type 2 (hPIV-2) et parainfluenza de type 5 (PIV-5), ainsi que sur la glycoprotéine de fusion du métapneumovirus humain (hMPV). La première partie de ce projet a consisté à caractériser une mutation retrouvée sur la protéine F de souches circulantes hPIV-2. Cette étude a notamment souligné l’importance de la sous-unité F2 dans la régulation de la fusion membranaire. Puis, dans un second temps, l’une des étapes du mécanisme d’entrée du métapneumovirus a été étudiée : la fusion membranaire induite par la glycoprotéine F. Il a été démontré qu’il était possible dans une certaine mesure, et par une approche de mutagenèse combinatoire, de dissocier les caractéristiques de F hMPV et ainsi de pouvoir mieux les étudier. Ce travail d’ingénierie de la glycoprotéine F hMPV s’est également inscrit dans un objectif de recherche appliquée afin de contribuer au développement de nouveaux outils prophylactiques et thérapeutiques. Cette perspective thérapeutique de F PIV-5 a été évaluée dans le cadre d’un vecteur oncolytique basé sur l’adénovirus de type 5 (AdV-5). L’expression de cette glycoprotéine hyperfusogène a ainsi contribué à un effet cytotoxique amplifié des vecteurs armés in vitro ainsiqu’en modèle animal immunocompétent. / Human respiratory paramyxoviruses are responsible for infectious diseases and hospitalisations among infants, children, elderly and the immunocompromised. These viruses harbour two glycoproteins implicated in virus entry into the cell. The attachment glycoprotein (HN,G or H) is implicated the virus attachment on a target cell receptor, and HN is also suspected to activate the second glycoprotein, the fusion protein (F). This latter glycoprotein can perform the fusion between the cellular membrane and the viral envelope. The mechanism of activation of the F protein, is not well-defined, even with the structural characterisation for some viruses studied inthis thesis. This thesis work is focussed on the viral glycoprotein of parainfluenza virus type 2 (hPIV-2), parainfluenza virus type 5 (PIV-5), and the fusion glycoprotein of human Metapneumovirus (hMPV).The first part of this project was the characterization of a mutation observed in the F protein natural variants of hPIV-2. This work highlights the importance of the F2 subunit of F in the fusion regulation. A second part of the project consisted of the study of the mechanism of F hMPV entry into the cell, induced by F glycoprotein. This work showed that it was possible to dissociate the characteristics of the F glycoprotein, in order to allow a better understanding of these characteristics. This engineering work on the F protein was used to understand the basic science but could also be used in the development of therapeutic tools.The therapeutic use of F PIV-5 was also evaluated in an oncolytic vector based on adenovirus type 5 (AdV-5). Its expression in tumours showed a highly cytotoxic activity for the target cells in vivo, but also in vitro on immunocompetent rodents.

No description available.

During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are causative agents of Lyme disease and human granulocytic anaplasmosis. Their common vector in Europe are the ticks from the genus Ixodes. In our work, we focused on interaction of innate immune cells with the causative agent of Lyme diseases, that are insubstitutable in their function in the early phase of the disease. Anaplasma phagocytophilum is hard to cultivate, the only possibility is to cultivate it in cell cultures. Successful cultivation of Anaplasma phagocytophilum acquired from patients in our geographic area is crucial for following experiments and for diagnostics too. In our experiments, we used validated cell cultures of HL-60 cells, canine monocytes DH82 and murine monocytes P388D1. During our studies of interaction of the causative agent of Lyme diseases with cells, we used two strains of different species Borrelia. Borrelia garinii M192 and Borrelia burgdorferi sensu stricto B31. These strains vary in virulence. The strain M192 is virulent, but the strain B31 lost its virulence by passages. We specialised in study of morphological changes using light microscopy (observation of dyed and fixed preparates and observation in dark field), eventually by transmision electron microscopy. During our experiments, we concluded that HL-60...

A esquistossomose é uma doença tropical negligenciada que atinge cerca de 200 milhões de pessoas em todo o mundo, abrangindo a América, a África, as Antilhas, o Oriente Médio e Próximo, além do Sudeste Asiático. A espécie encontrada no Brasil é a Schistosoma mansoni, onde se tem como tratamento típico a administração do Praziquantel ou da Oxamniquina. No entanto, sua característica de infecção se associa a saneamento básico precário e baixos padrões sócio-econômicos, de maneira que a reinfecção de doentes apresenta altas taxas de ocorrência, o que motiva a busca por fármacos ou vacinas antihelmíticas que superem esta dificuldade. Neste trabalho são utilizadas técnicas proteômicas para a identificação de proteínas que estejam potencialmente envolvidas na diferenciação entre os sexos, na interação entre parasitas de diferentes sexos ou com o hospedeiro. São estudadas preparações de amostras de sincício e vermes inteiros adultos machos e fêmeas por eletroforese bidimensional e frações de baixo peso molecular de sincício de vermes adultos machos e fêmeas por gel-LC. A expressão diferencial de proteínas de sincício investigada por gel-LC foi avaliada por análise estatítica, sendo detectadas 5 proteínas mais abundantes em machos e 2 em fêmeas, além de 6 proteínas identificadas somente em machos e 21 somente em fêmeas. Estas informações de expressão diferencial possibilitam a investigação dos recursos de sobrevivência e reprodução desenvolvidos evolutivamente por estes parasitas. / Schistosomiasis is a neglected tropical disease that affects approximately 200 million people around the world, occurring in America, Africa, the Antilles, Middle East and Near East, besides Southeast Asia. The species found in Brazil is Schistosoma mansoni, the typical treatment being administration of either Praziquantel or Oxamniquine. Although, the infection characteristics of this disease is associated with poor sanitation and hardened socio-economic conditions, resulting in high reinfection rates, which motivates the search for antihelmintic drugs and vaccines that overcome this situation. In this study proteomics techniques are used in the search of proteins potencially involved in the differentiation of individuals of both sexes, in the interactions between them and between the worms and the host. Samples of worm syncytium and adult whole worms of both male and female are studied by two-dimentional electrophoresis, while low molecular weight syncytium proteins from male and female adult worms were investigated by gel-LC. The differential protein expression in the syncytium investigated by gel-LC was analyzed statistically, being detected 5 proteins most abundant in males, and 2 in females, while 6 were identified solely on males and 21 on females. The information concerning protein differential expression allows the investigation of survival strategies developed evolutionarily by these parasites.

A esquistossomose é uma doença tropical negligenciada que atinge cerca de 200 milhões de pessoas em todo o mundo, abrangindo a América, a África, as Antilhas, o Oriente Médio e Próximo, além do Sudeste Asiático. A espécie encontrada no Brasil é a Schistosoma mansoni, onde se tem como tratamento típico a administração do Praziquantel ou da Oxamniquina. No entanto, sua característica de infecção se associa a saneamento básico precário e baixos padrões sócio-econômicos, de maneira que a reinfecção de doentes apresenta altas taxas de ocorrência, o que motiva a busca por fármacos ou vacinas antihelmíticas que superem esta dificuldade. Neste trabalho são utilizadas técnicas proteômicas para a identificação de proteínas que estejam potencialmente envolvidas na diferenciação entre os sexos, na interação entre parasitas de diferentes sexos ou com o hospedeiro. São estudadas preparações de amostras de sincício e vermes inteiros adultos machos e fêmeas por eletroforese bidimensional e frações de baixo peso molecular de sincício de vermes adultos machos e fêmeas por gel-LC. A expressão diferencial de proteínas de sincício investigada por gel-LC foi avaliada por análise estatítica, sendo detectadas 5 proteínas mais abundantes em machos e 2 em fêmeas, além de 6 proteínas identificadas somente em machos e 21 somente em fêmeas. Estas informações de expressão diferencial possibilitam a investigação dos recursos de sobrevivência e reprodução desenvolvidos evolutivamente por estes parasitas. / Schistosomiasis is a neglected tropical disease that affects approximately 200 million people around the world, occurring in America, Africa, the Antilles, Middle East and Near East, besides Southeast Asia. The species found in Brazil is Schistosoma mansoni, the typical treatment being administration of either Praziquantel or Oxamniquine. Although, the infection characteristics of this disease is associated with poor sanitation and hardened socio-economic conditions, resulting in high reinfection rates, which motivates the search for antihelmintic drugs and vaccines that overcome this situation. In this study proteomics techniques are used in the search of proteins potencially involved in the differentiation of individuals of both sexes, in the interactions between them and between the worms and the host. Samples of worm syncytium and adult whole worms of both male and female are studied by two-dimentional electrophoresis, while low molecular weight syncytium proteins from male and female adult worms were investigated by gel-LC. The differential protein expression in the syncytium investigated by gel-LC was analyzed statistically, being detected 5 proteins most abundant in males, and 2 in females, while 6 were identified solely on males and 21 on females. The information concerning protein differential expression allows the investigation of survival strategies developed evolutionarily by these parasites.

During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.

No description available.

Le mouvement masticatoire est généré et coordonné par un générateur de patron central (GPC) situé au niveau du pont. Plusieurs résultats antérieurs de notre laboratoire soutiennent que le réseau de neurones à l’origine de la rythmogénèse est situé dans le noyau sensoriel principal du nerf trijumeau (NVsnpr). Ces mêmes expériences révèlent que des diminutions de la concentration calcique extracellulaire ([Ca2+]e) tiennent une place importante dans la génération des bouffées de décharges des neurones de cette région. Notre laboratoire tente de vérifier si la contribution des astrocytes à l’homéostasie de la concentration calcique extracellulaire est impliquée dans la genèse du rythme. Cette étude a pour but la caractérisation spatiale du syncytium astrocytaire au sein du NVsnpr dorsal et l’étude de l’effet de la [Ca2+]e sur les propriétés astrocytaires électrophysiologiques et de connectivité. Nous avons utilisés pour ce faire la technique d’enregistrement par patch-clamp sur une préparation en tranche de tronc cérébral de rat. Nous démontrons ici que la diminution de la [Ca2+]e n’affecte pas les propriétés électrophysiologiques astrocytaires, mais induit une augmentation de la taille du syncytium. De plus, nous établissons l’existence au sein du NVsnpr dorsal d’une organisation anatomofonctionnelle du réseau astrocytaire calquée sur l’organisation neuronale. / The masticatory movement is generated and coordinated by a central pattern generator (CPG) located in the pons. Previous results from our laboratory suggest that the neural network responsible for its rythmogenesis is located in the trigeminal main sensory nucleus (NVsnpr). Moreover, results indicate that in this region, decrease in extracellular calcium concentration ([Ca2+]e) plays an important role in genarating burst. One of our laboratory's goal is to assess if the contribution of astrocytes to the extracellular calcium concentration homeostasis is involved in the genesis of the mastication rhythm. With this study, we characterized the astrocyte syncytium within the NVsnpr and measured the effect of [Ca2+]e on the astrocytes electrophysiology and their networks. A patch-clamp recording technique in conjunction with a rat brain stem slice preparation was used. We demonstrate that a decrease in [Ca2+]e does not affect the electrophysiological properties of astrocytes but induces an increase in the size of the syncytium. We also report the existence, within the dorsal NVsnpr, of an anatomofunctional organization between neurons and astrocytes.