Im Rahmen dieser Arbeit wurde die Rolle des Rezeptors VEGFR-2, Flk-1, im neuronalen Kontext untersucht. In einem ersten Schritt wurde in embryonalen Stammzellen der Maus das fluoreszierende Protein eGFP unter der Kontrolle regulatorischer Sequenzen des flk-1-Promotors, -Enhancers exprimiert. Nach der Differenzierung zu Sphäroiden wurden Endothelzellen nachgewiesen, die sowohl eGFP als auch das zelltypspezifische Oberflächenantigen CD31 ausprägen. Ebenso wurden nach der neuronalen Differenzierung in Gegenwart von Stromazellen eGFP-exprimierende Zellen identifiziert. Diese standen mit Zellen, die das für neuronale Vorläuferzellen charakteristische Protein Nestin ausprägten, in einem räumlichen Zusammenhang. Die Vorgehensweise, die Inaktivierung des flk-1-Gens mit der Differenzierung embryonaler Stammzellen in vitro zu kombinieren, sollte hier die Interpretation des Phänotyps des flk-1-defizienten Mausmodells ermöglichen. Der Rezeptor war während der neuronalen Differenzierung der Stammzellen auf Stromazellen in vitro für die Regulation der Anzahl der Vorläuferzellen essentiell. Ferner spielte der Rezeptor im Rahmen eines weiteren Differenzierungsmodells, das auf der Zugabe relevanter Wachstumsfaktoren beruht, eine instruktive Rolle im Hinblick auf die Identität der Neuronen. Kriterium war hier die differentielle Expression Homeobox-enthaltender Transkriptionsfaktoren. In einem zweiten Schritt wurden mit Hilfe dieses Modells differentiell-exprimierte Gene von Stammzellen des Wildtyps sowie Zellen mit einer Inaktivierung des flk-1-Gens nach der neuronalen Differenzierung durch subtraktive Hybridisierung in Verbindung mit der PCR identifiziert. Tatsächlich wurde das Protein PEA-15 nicht nur differentiell exprimiert sondern auch als Bestandteil des VEGFR-2-vermittelten Signalwegs identifiziert. Die biologischen Funktionen des Proteins PEA-15 wurden durch VEGF-vermittelte Phosphorylierung reguliert. Die Stimulation durch VEGF führte zunächst zu einer Aktivierung des Proteinkinase B-, Akt-Signalwegs. Für die Stimulation des Akt-Signalwegs war die Phosphorylierung der intrazellulären Tyrosinreste Y1052 und Y1057 des Rezeptors essentiell. Damit einhergehend wurde PEA-15 gegenüber der proteasomalen Degradation stabilisiert. Es wurde gezeigt, daß das Protein PEA-15 die Teilungsaktivität von Zellen beeinflusst. Die VEGF- vermittelte Stimulation führte zur Phosphorylierung der Mitogen-aktivierten Proteinkinasen ERK1 und ERK2. Die weitere Phosphorylierung der Substrate dieser Kinasen im Zellkern wurde durch Interaktion mit PEA-15 unterdrückt. Die Regulation des c-fos-Promotors war zugleich Indikator der Inhibition der Phosphorylierung betreffender Substrate sowie der proliferativen Aktivität. Auf diese Weise ist die Phosphorylierung von PEA-15 nach Stimulation durch VEGF für die Selektivität des Flk-1-vermittelten Signalwegs von unmittelbarer Bedeutung. Die Regulation der biologischen Funktion von PEA-15 erklärt die differentielle Ausprägung im Rahmen der neuronalen Differenzierung embryonaler Stammzellen in vitro. So war die Anzahl GFAP- beziehungsweise PEA-15-exprimierender Zellen nach Differenzierung muriner Stammzellen mit einer Inaktivierung des flk-1-Gens deutlich geringer. Die differentielle Expression identifizierter Gene wurde im Mausmodell nach konditionaler Inaktivierung des flk-1-Gens überprüft. Tatsächlich wurde Vimentin in verschiedenen Arealen des Gehirns differentiell ausgeprägt. Ein Zusammenhang zwischen der differentiellen Expression des Proteins PEA-15, der Anzahl GFAP-exprimierender Zellen und der Ausprägung des Rezeptors Flk-1 ergab sich aus der Identifikation einer Zellpopulation in der subgranulären Zone des Gyrus Dentatus. Dort wurde in flk-1-defizienten, adulten Mäusen eine geringere Anzahl GFAP-exprimierender Zellen nachgewiesen. Schließlich wurden sowohl im Cerebellum als auch im Cortex histologische Unterschiede deutlich, die sich im adulten Organismus aus der Inaktivierung des Rezeptors Flk-1 ergeben. Die vorliegende Arbeit zeigt, daß der Rezeptor VEGFR-2, Flk-1, im neuronalen Kontext eine Rolle spielt, die sich nicht ausschließlich auf die Vermittlung eines Schutzmechanismus gegenüber der neuronalen Apoptose beschränkt, sondern auch auf eine Beteiligung an der Neurogenese hinweist. Die Vorgehensweise, mit Hilfe der subtraktiven Hybridisierung Bestandteile Rezeptor-vermittelter Signalwege vor dem Hintergrund der Differenzierung embryonaler Stammzellen zu identifizieren, verdeutlicht die Eignung der Methode auch bei komplexen Zellpopulationen.

Το λεμφαγγειακό σύστημα διαδραματίζει σημαντικό ρόλο στη διατήρηση της ομοιόστασης των ιστών, στην ανοσολογική απόκριση του οργανισμού, στην απορρόφηση των λιπών από τον πεπτικό σωλήνα, και στη διασπορά των καρκινικών κυττάρων. Η πρόσφατη ανακάλυψη ειδικών για τα λεμφαγγειακά ενδοθηλιακά κύτταρα δεικτών και αναπτυξιακών παραγόντων, όπως επίσης και η δημιουργία γενετικών μοντέλων ποντικιών με διαταραγμένη τη λειτουργία του λεμφαγγειακού συστήματος, παρείχαν σημαντικότατες πληροφορίες για τη μοριακή ρύθμιση της εμβρυικής ανάπτυξης του λεμφαγγειακού συστήματος και της φυσιολογίας του. Τα σχετικά πρόσφατα αναγνωρισμένα μοριακά σηματοδοτικά μονοπάτια από τα οποία ρυθμίζεται η λεμφαγγειογένεση επέτρεψαν τη μελέτη της σχετιζόμενης με όγκους λεμφαγγειογένεσης. Οι μελέτες αυτές κατέδειξαν ότι η σχετιζόμενη με τους όγκους λεμφαγγειογένεση αποτελεί σημαντικό στοιχείο της μεταστατικής διαδικασίας, ενώ παράλληλα αναδεικνύουν συνεχώς καινούρια μόρια/σηματοδοτικά μονοπάτια-ρυθμιστές της εν λόγω διαδικασίας. / The lymphatic vascular system plays an important role in the maintenance of fluid homeostasis, in the afferent immune response, in the intestinal lipid uptake and in the metastatic spread of malignant cells. The recent discovery of specific markers and growth factors for lymphatic endothelium and the establishment of genetic mouse models with impairment of lymphatic function have provided novel insights into the molecular control of the lymphatic system in physiology and in embryonic development. Recently, molecular pathways that signal for lymphangiogenesis have been described allowing analyses of tumor lymphangiogenesis to be performed. These studies demonstrate that tumor lymphangiogenesis is a major component of the metastatic process, while at the same time new molecules and transduction pathways are discovered to regulate tumor lymphatics growth.

No description available.

Notre laboratoire a démontré que la capacité proinflammatoire du vascular endothelial growth factor (VEGF-A165) implique la synthèse endothéliale du facteur d’activation plaquettaire (PAF) via l’activation du récepteur tyrosine kinase homodimérique VEGFR-2/R-2. La synthèse du PAF requiert l’activation de la p38 MAPK et p42/44 MAPK qui activent la phospholipase A2 secrétée de type V (sPLA2-V). Nous avons découvert que la synthèse aigue de prostacycline (PGI2) induite par le VEGF-A165 requiert l’activation des récepteurs hétérodimériques VEGFR-1/R-2. L’activation sélective des récepteurs du VEGF peut donc agir comme balance dans la synthèse de facteurs pro-(PAF) et anti-(PGI2) inflammatoire. Cependant, les tyrosines impliquées dans la transphosphorylation de VEGFR-2/R-2 menant à la synthèse du PAF sont inconnues. Par mutagenèse dirigée, nous avons effectué des transfections transitoires de cellules endothéliales avec des plasmides codant pour le VEGFR-2 dont les tyrosines ciblées ont été remplacées de façon séquentielle par une phénylalanine. Un vecteur vide pcDNA a été utilisé comme contrôle négatif. La stimulation des cellules endothéliales de l’aorte bovine (BAEC) transfectées avec le VEGF-A165 (1nM) pendant 15 minutes augmente la synthèse du PAF de 300%, laquelle était similaire dans les BAEC non transfectées. Dans les BAEC transfectées avec les vecteurs pcDNA codant pour les mutations Y801F, Y1059F, Y1175F et Y1214F, nous avons observé une réduction de 54, 73, 68, et 57% respectivement de la synthèse du PAF induite par le VEGF par rapport au pcDNA témoin. Nos résultats apportent un nouvel aperçu sur le mécanisme par lequel le VEGF induit la synthèse du PAF qui est connu pour sa contribution dans l’activité pro-inflammatoire du VEGF. / Vascular endothelial growth factor (VEGF) inflammatory effects require acute platelet-activating factor (PAF) synthesis by endothelial cells (EC). We reported that VEGF-mediated PAF synthesis involves the activation of the homodimeric tyrosine kinase receptor VEGFR-2/R-2 which is leading to p38 and p42/44 mitogen-activated protein kinases (MAPKs) and secreted group V phospholipase A2 (sPLA2-V) activation. We also reported that VEGF-A165-mediated prostacyclin (PGI2) synthesis requires VEGFR-1/R-2 heterodimeric receptor activation. Selective activation of VEGF receptors can thus act as a balance in the synthesis of pro-(PAF) and anti-(PGI2) inflammatory factors. It is unknown which VEGFR-2 tyrosine phosphorylation site(s) contribute(s) to PAF synthesis. Bovine aortic endothelial cells (BAEC) were transfected with pcDNA vectors encoding for native VEGF receptor-2 (VEGFR-2) cDNA, or tyrosine phosphorylation sites mutated into phenylalanine (Y801F), (Y1059F), (Y1175F), (Y1214F), and an empty pcDNA vector was used as negative control. Treatment of pcDNA-transfected BAEC with VEGF-A165 (1nM) for 15 minutes increased PAF synthesis by 300%, which was similar to VEGF-mediated PAF synthesis in untransfected BAEC. In BAEC transfected with pcDNA vectors encoding mutated Y801F, Y1059F, Y1175F or Y1214F VEGFR-2 cDNA, we observed a marked reduction of VEGF-mediated PAF synthesis by 54, 73, 68 and 57% respectively as compared to pcDNA-transfected BAEC. Our current data provide novel insight on the mechanisms by which VEGF promotes endothelial PAF synthesis which is known to contribute to VEGF pro-inflammatory activities.

Das Monozyten chemotaktische Protein-1 (MCP-1) ist ein CC-Chemokin, das in seiner Rolle als Chemoattraktor auf Monozyten in der Genese von Malignomen eine wesentliche Rolle spielt. Dabei kann es sowohl zur lokalen Tumorabwehr als auch zur Tumorgenese, Tumor-angiogenese und Metastasierung beitragen. Die vorliegende Arbeit untersucht die MCP-1-Inhibition und die Stimulation durch MCP-1 auf die Koloniebildung und die Zytokinexpression von Plattenepithelkarzinomen der Kopf-Hals-Region (HNSCC) im FLAVINO-Assay. Dieser ist ein klonogener, qualitätskontrollierter Ex-vivo-Koloniebildungsassay, der an der Klinik für Hals-Nasen-Ohrenheilkunde der Universität Leipzig etabliert und patentiert wurde und unter flavinschützenden Bedingungen durchgeführt wird. Weiterhin wird die Eignung von MCP-1, Interleukin-6 (IL-6), Interleukin-8 (IL-8) und des Vascular endothelial growth factor (VEGF) als Biomarker in HNSCC, die mithilfe von ELISA in Seren und Kulturüberständen quantifiziert wurden, untersucht. Durch die Stimulation durch MCP-1 und dessen Blockade sowie durch in vivo tolerierbare Konzentrationen von Cisplatin, Docetaxel, Cilengitide und Temsirolimus wurde die Expression der untersuchten Zytokine in den Kulturüberständen der HNSCC unterschiedlich moduliert. Cisplatin und MCP-1 supprimierten die Koloniebildung signifikant, während unter Docetaxel und Temsirolimus eine insignifikante Reduktion und durch Cilengitide eine insignifikante Stimulation der Koloniebildung beobachtet wurde. Die MCP-1-Blockade durch einen Anti-MCP-1-Antikörper führte zu keiner signifikanten Modulation der Koloniebildung. MCP-1 und der Anti-MCP-1-Antikörper senkten die Zytokinexpression, während bis auf Cisplatin alle Zytostatika die Zytokinexpressionen steigerten. Bezüglich der kombinierten Testung der Zytostatika und der MCP-1-Blockade bzw. Stimulation unterschieden sich die Proben, sodass additive, synergistische und antagonistische Effekte resultierten. Da durch MCP-1 gesteuerte tumorassoziierte Makrophagen das Mikromilieu eines Tumors wesentlich beeinflussen, gebührt diesen ebenfalls eine besondere Aufmerksamkeit. In dieser Arbeit wurden unter MCP-1 antitumoröse Effekte beobachtet, sodass weitere klinische Testungen der antitumorösen Wirkung des MCP-1 auf HNSCC lohnenswert erscheinen. Die individuelle Chemoresponse-Testung kann dabei helfen, das biologisch heterogene Verhalten der HNSCC besser zu verstehen. In diesem Sinne wäre die klinische Validierung solcher Testsysteme wertvoll.

L’angiogenèse est la formation de nouveaux vaisseaux sanguins à partir d’un réseau vasculaire existant. C’est un phénomène essentiel pour des processus physiologiques et pathologiques. L’activation des cellules endothéliales est contrôlée par plusieurs facteurs de croissance. Le VEGF et son récepteur le VEGFR-2 ont été prouvés comme étant spécifiques et critiques pour la formation des vaisseaux sanguins alors que Tie2, le récepteur auquel se lie l’Ang-1, est requis aussi bien dans le développement vasculaire que dans l’angiogenèse tumorale. Il est connu que l’activation de Tie2 est nécessaire à la stabilisation finale de la vascularisation en inhibant la perméabilité vasculaire induite par le VEGFR-2. Nous avons premièrement découvert que le facteur de croissance pro-angiogénique, l’Ang-1 contrecarre les effets de perméabilité cellulaire induits par le VEGF en inhibant la production de NO dans les cellules endothéliales. Cet effet inhibiteur de Tie2 intervient directement au niveau de l’activité de l’enzyme eNOS. Suite à l’activation de Tie2 par l’Ang-1, eNOS devient fortement phosphorylé sur la Thr497 après la phosphorylation et l’activation de la PKCζ. Nos résultats suggèrent que l’inhibition, par Tie2, de la perméabilité vasculaire durant l’angiogenèse serait due, en partie, à l’inhibition de la production de NO. Deuxièmement nous avons pu distinguer entre deux modes de migration cellulaire endothéliale induits par l’Ang-1 et le VEGF. À l’opposé du VEGF qui promeut une migration individuelle aléatoire, l’Ang-1 induit une migration collective directionnelle. Dans cette étude, nous avons identifié la β-caténine comme un nouveau partenaire moléculaire de la PKCζ. Cette association de la PKCζ à la β-caténine amène le complexe de polarité Par6-aPKC et le complexe des jonctions d’adhérences cellulaires à interagir ensemble à deux localisations différentes au niveau de la cellule endothéliale. Au niveau des contacts intercellulaires, le complexe PKCζ/β-caténine maintien la cohésion et l’adhésion cellulaire nécessaire pour le processus migratoire collectif. Ce complexe se retrouve aussi au niveau du front migratoire des cellules endothéliales afin d’assurer la directionalité et la persistance de la migration endothéliale en réponse à l’Ang-1. D’une manière intéressante, lors de l’inhibition de la PKCζ ou de la β-caténine on assiste à un changement du mode de migration en réponse à l’Ang-1 qui passe d’une migration directionnelle collective à une migration individuelle aléatoire. Ce dernier mode de migration est similaire à celui observé chez des cellules endothéliales exposées au VEGF. Ces résultats ont été corroborés in vivo par une polarité et une adhésion défectueuses au cours de la vasculogenèse chez le poisson zèbre déficient en PKCζ. En résumé, Ang-1/Tie2 module la signalisation et les réponses biologiques endothéliales déclenchées par le VEGF/VEGFR-2. L’identification des mécanismes moléculaires en aval de ces deux récepteurs, Tie2 et VEGFR-2, et la compréhension des différentes voies de signalisation activées par ces complexes moléculaires nous permettra de mettre la lumière sur des nouvelles cibles thérapeutiques pour le traitement des maladies angiogéniques. / Angiogenesis is the formation of new blood vessels from a pre-existing vascular network. It is an essential mechanism for many physiological and pathological conditions. Also, the general mechanism in both conditions remains the same. VEGF and its receptor VEGFR-2 have been proven to be specific and critical for blood vessel formation. The Angiopoietin-1 receptor, Tie2, is required for vascular development as well as in tumor angiogenesis. It is known that the activation of Tie2 is required for vascular stabilization by inhibiting vascular permeability induced by VEGFR-2. First, we found that the pro-angiogenic growth factor, Ang-1 counteracts the effects of VEGF-induced permeability by inhibiting NO production by endothelial cells. This inhibitory effect of Tie2 acts directly on eNOS activity. Following activation of Tie2 by Ang-1, eNOS becomes highly phosphorylated on the inhibitory site, the Thr497, following PKCζ phosphorylation and activation. Our results suggest that the inhibition by Tie2 of vascular permeability during angiogenesis is due, in part, to the inhibition of NO production. In our second study we distinguished between two types of endothelial cell migration induced by Ang-1 and VEGF. At the opposite of Ang-1 that induced collective and directional cell migration, VEGF promoted individual and random cell motility. We identified β-catenin as a new molecular partner of PKCζ. This association of PKCζ with β-catenin brings the Par6-aPKC polarity complex and the adherens junctions complex to interact with each other at two different locations in endothelial cells. PKCζ/β-catenin complex is located specifically at cell-cell contacts to maintain cohesion and cell adhesion necessary for the collective migration process. This complex was located also at the leading edge of endothelial cells during migration to ensure the directionality and the persistence of migration in response to Ang-1. In addition, inhibition of PKCζ or β-catenin switched the migration mode, in response to Ang-1, from directional and collective to a more random and individual cell migration which resembles the type of migration of endothelial cells exposed to VEGF. These results were confirmed in vivo by aberrant cell polarity and cell adhesion defects of tip cell during vascular sprouting of intersegmental vessels in PKCζ deficient zebrafish embryos. In summary, Ang-1/Tie2 modulates endothelial cell signaling and biological responses induced by VEGF/VEGFR-2. The identification of molecular mechanisms involved in the action of these two receptors, VEGFR-2 and Tie2, and the understanding of the different signaling pathways activated by these molecular complexes will allow us to identify new therapeutic targets for the treatment of angiogenic diseases treatment.

Les acides gras polyinsaturés n-3 (acide docosahexaènoïque, DHA et acide eicosapentaènoïque, EPA) sensibilisent les tumeurs mammaires aux agents anticancéreux. Cette sensibilisation implique la régulation de la vascularisation tumorale. En effet, un régime nutritionnel EPA/DHA, associé à une chimiothérapie par le Docétaxel (Taxane) diminue la quantité de vascularisation (effet anti-angiogénique). Une amélioration de la qualité vasculaire est aussi observée par une diminution de la pression du liquide interstitiel, paramètre décrit comme un frein à la délivrance des drogues dans les tumeurs. Cette diminution est associée à une extravasation plus importante du bleu d’Evans, suggérant une meilleure distribution des agents anticancéreux au sein des tumeurs. L’effet antiangiogénique du DHA implique une diminution de la voie de signalisation VEGF/eNOS/NO. Ainsi, l’activation de la NO synthase endothéliale (eNOS) est diminuée dans des cellules endothéliales en culture et dans les tumeurs mammaires. Nos résultats suggèrent que l’apport d'acides gras EPA/DHA aux patients au cours de la chimiothérapie pourrait être une nouvelle approche thérapeutique pour normaliser la vascularisation tumorale et améliorer l’efficacité des traitements anticancéreux. / Polyunsaturated fatty acids n-3 (docosahexaenoic acid, DHA and eicosapentaenoic acid, EPA) sensitize mammary tumors to anticancer drugs. This sensitization involves the regulation of tumor vasculature. Indeed, a nutritional diet with EPA / DHA, in combination with taxane chemotherapy (docetaxel) decreases the vascular quantity (anti-angiogenic effect). An improvement of vascular quality is also observed by a decrease of interstitial fluid pressure, a parameter described as a barrier to drug delivery in tumors. This decrease improves extravasation of Evans blue, suggesting a better distribution of anticancer agents in tumors. The antiangiogenic effect of DHA involves a decrease of signaling pathway VEGF / eNOS / NO. Thus, activation of endothelial NO synthase (eNOS) is decreased in endothelial cells in culture and in mammary tumors. Our results suggest that intake of fatty acids EPA / DHA to patients during chemotherapy could be a new therapeutic approach to normalize tumor vasculature and improve the efficacy of cancer treatments.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Orf virus (ORFV) belongs to the family Poxviridae, subfamily Chordopoxvirinae and genus Parapoxvirus and is the agent of contagious ecthyma, a mucocutaneous disease that affects mainly young sheep and goats and, occasionally, may affect people. The clinical lesions progress through stages of hyperemia, papules, vesicles, pustules, ulcers and proliferative and scabby lesions, located mainly on the labial commissure, lips and nostrils. Variable clinical lesions with different degrees of severity often occur in sheep and goats, and may be associated with host and/or viral genetics. The present study aimed to investigate the phenotype in vivo and to characterize virulence genes of four ORFV isolates recovered from contagious ecthyma outbreaks in Rio Grande do Sul State, Brazil. Twenty sheep, aged 6 and 8 months, were divided into five groups of four animals each and inoculated in the labial commissure with homogenates of scabs (viral titers 105.6TCID50/ml) obtained from different outbreaks: SV269/11, SV252/11, SV581/11 and SV820/10-Canguçu. The animals were evaluated for 30 days in clinical and virological aspects by clinical inspection and swab collection for virus isolation. A clinical score was established for each animal and group. All ORFV inoculated animals developed classical ecthyma contagious lesions, characterized by hyperemia, papules, macules, vesicles, pustules and scabs in varied degrees and duration. SV269/10 and SV820/10-Canguçu isolates induced more severe lesions resulting in higher clinical scores and longer duration of lesions. The animals inoculated with SV581/11 developed milder lesions and clinical scores significantly lower than other groups, but they shed virus for a longer period of time. For genetic analyses, PCR amplification and nucleotide sequencing of three virulence genes (VEGF, VIR and IL-10v) were performed. Deletion and mutations on VEGF and IL-10v amino acid sequence of SV581/11 and SV252/11 isolates were identified. The degree of amino acid identity among ORFV sequences was variable, and the lowest homology was found in the VEGF gene of SV581/11 when compared with the standard strains and other viruses. Thus, the present results showed that SV581/11 and SV252/11 isolates, particularly the former, are less virulent in sheep than SV269/11 and SV820/10-Canguçu. Possibly, the variable phenotypic observed in vivo is due to genetic alterations detected in the analyzed virulence genes. / O vírus da orf (ORFV) pertence à família Poxviridae, subfamília Chordopoxvirinae gênero Parapoxvirus e é o agente etiológico do ectima contagioso, uma doença mucocutânea que afeta principalmente ovinos e caprinos jovens e pode, ocasionalmente, afetar pessoas. Clinicamente, a enfermidade evolui com a formação de áreas hiperêmicas, vesículas, pústulas, úlceras e lesões proliferativas e crostosas sobre a pele dos lábios, comissura labial e narinas. Apresentações clínicas variáveis, com diferentes graus de severidade, ocorrem frequentemente e podem estar associadas a características do hospedeiro e, principalmente, a características genéticas do agente. O presente trabalho teve como objetivo investigar o fenótipo in vivo e caracterizar genes de virulência de quatro amostras de ORFV oriundas de surtos no Estado do Rio Grande do Sul, Brasil. Para isso, foram utilizados vinte ovinos, com idade entre 6 e 8 meses, divididos em cinco grupos de quatro animais cada. Os ovinos foram inoculados na comissura labial com homogeneizados de crostas (títulos virais 105,6 DICC50/ml) obtidas dos surtos (amostras SV269/11, SV252/11, SV581/11 e SV820/10-Canguçu). Os animais foram avaliados durante 30 dias com relação aos aspectos clínicos e virológicos, por inspeção clínica e coleta de suabes das lesões para detecção da excreção viral. As manifestações clínicas foram convertidas em um escore clínico, para cada animal e para os grupos. Os animais inoculados com os as quatro amostras desenvolveram lesões típicas de ectima contagioso, caracterizadas por hiperemia, pápulas, máculas, vesículas e pústulas, e formação de crostas em diferentes graus de intensidade e duração. As amostras SV269/10 e SV820/10-Canguçu induziram lesões mais graves, escores clínicos maiores e maior tempo de duração das lesões. Os animais inoculados com a amostra SV581/11 desenvolveram lesões e escores clínicos significativamente inferiores aos demais grupos, mas excretaram o vírus por período mais longo. Para a caracterização genética, foi realizada a amplificação por PCR e sequenciamento de nucleotídeos de três genes de virulência (VEGF, VIR e IL-10v). Foram identificadas deleções e mutações nas sequências dos genes VEGF e IL-10v das amostras SV581/11 e SV252/11. O grau de identidade de aminoácidos entre as amostras foi variável, sendo que a menor homologia foi encontrada no gene VEGF da amostra SV581/11, quando comparado com os demais vírus e a cepa padrão. Assim, os resultados obtidos demonstram que as amostras SV581/11 e SV252/11, em especial a primeira, foram menos virulentas em ovinos do que as amostras SV269/11 e SV820/10-Canguçu. Possivelmente essa diferença fenotípica observada in vivo seja resultado das alterações genéticas detectadas nos genes de virulência.

Made available in DSpace on 2014-12-17T15:32:18Z (GMT). No. of bitstreams: 1 FelipeRM.pdf: 2960586 bytes, checksum: f36dd0983baaaf9fe7c3bb48e095490e (MD5) Previous issue date: 2010-02-12 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Central giant cell lesion (CGCL) and peripheral giant cell lesion (PGCL) of the jaws have a distinct clinical behavior, although they share histopathologic features. It is still unclear whether these clinical differences are supported by a distinct pattern of immunoexpression of markers for multinucleated giant cells (GC) and mononuclear cells (MC). The purpose of this study was to compare the immunohistochemical expression of VEGF, MMP-9 in CG and MC and measure the vascularization by vWF to check whether there are differences in expression of these biomarkers between CGCL and PGCL. Paraffin wax blocks of 20 cases of LCCG and 20 LPCG were retrieved. MMP-9 immunoreactivity was greater in the CM of PGCL compared to VEGF (p<0.05). VEGF expression was greater in the CM of CGCL compared to PGCL (p<0.05) and it was greater in the overall expression of CGCL compared to PGCL (p<0.05). Vascularity was quantified by microvascular counting (MVC). MVC was greater in the PGCL compared CGCL (p<0.05). MMP-9 showed a greater tendency of expression in CGCL, though was not significant (p>0.05). We tested correlation between the proteins studied in each group and found a significant negative correlation between VEGF and vWF in CGCL (p<0.05). These results suggest that there are differences in the expression of VEGF in CM and overall expression between the lesions, although no statistically significant difference in the overall expression of the MMP-9. Then, there was a trend in increased expression of MMP-9 and VEGF in CGCL, possibly by the involvement of both proteins in osteoclastogenesis. Additionally, the results of this study indicate a higher degree of vascularization in PGCL compared to CGCL, fact that can be directly linked to the reactive nature of the PGCL, where the inflammatory process with its rich angiogenesis contributes significantly to these findings. / Les?es centrais (LCCG) e perif?ricas de c?lulas gigantes (LPCG) dos maxilares possuem um comportamento cl?nico distinto, embora compartilhem caracter?sticas histopatol?gicas semelhantes. Ainda ? obscuro se essas diferen?as cl?nicas s?o apoiadas por um padr?o distinto de imunoexpress? o de marcadores para c?lulas gigantes multinucleadas (CG) e mononucleadas (CM). O escopo do presente trabalho foi realizar um estudo imuno-histoqu?mico comparativo, analisando quantitativamente c?lulas gigantes multinucleadas e mononucleadas imunorreativas ? MMP-9 e ao VEGF e mensurar a vasculariza??o atrav?s do FvW para verificar se h? ou n?o diferen?as de express?o desses biomarcadores entre as LCCG e LPCG. Foram selecionados 20 casos de LCCG e 20 de LPCG emblocados em parafina. Constatou-se diferen?a significativa (p<0.05) em rela??o ? imunorreatividade na CM para MMP-9 e VEGF nas LPCG, sendo a MMP-9 mais expressa. O VEGF foi mais expresso nas CM das LCCG em rela??o ?s LPCG (p<0.05), assim como sua express?o global (p<0.05). A MMP-9 apresentou uma tend?ncia maior de express?o nas LCCG, embora n?o significativa estatisticamente (p>0.05). Na mensura??o dos vasos atrav?s da contagem microvascular (MVC), verificou-se maior MVC nas LPCG do que nas LCCG (p<0.05). Testou-se correla??o entre as prote?nas estudadas em cada grupo de les?es e constatou-se uma correla??o negativa significativa entre VEGF e FvW nas LCCG (p<0.05). Diante dos achados deste estudo, observa-se que h? diferen?a na express?o do VEGF nas CM, bem como na express?o global entre as les?es. Observou-se uma tend?ncia na maior express?o da MMP-9 nas LCCG, embora n?o significativa estatisticamente. Dessa forma, sugere-se que a maior express?o de ambas as prote?nas nas LCCG esteja mais relacionada possivelmente com a osteoclastog?nese. Adicionalmente, os resultados do presente estudo apontam um maior grau de vasculariza??o nas LPCG quando comparadas com as LCCG, fato este que pode estar relacionado diretamente com a natureza reacional das primeiras, em que o processo inflamat?rio com sua rica angiog?nese contribui sobremaneira para estes achados.

A laserterapia tem sido muito utilizada como tratamento alternativo em pacientes com dores crônicas relacionadas às disfunções temporomandibulares. Isso se deve aos efeitos: analgésico, antiinflamatório, miorrelaxante, de redução da fadiga durante as contrações tetânicas, aumento da força de mordida e diminuição da dor orofacial. Embora sejam observados resultados clínicos, ainda não é bem compreendido o seu efeito em nível celular. Assim, este estudo tem como objetivo analisar os efeitos das diferentes densidades (doses) de irradiação do laser de baixa intensidade (LLLI), em nível celular, sobre o músculo masseter de ratos Wistar. Os animais foram alocados aleatoriamente em 6 grupos (n=10), receberam 10 irradiações do laser (GaAlAs,780nm, 5mW e spot 0,04cm&sup2;) sobre o músculo masseter esquerdo variando a densidade de energia (I. 0; II. 0,5; III. 1,0; IV. 2,5; V. 5,0 e VI. 20 J/cm&sup2;). Após as 10 irradiações os músculos masseteres foram obtidos dos animais sob anestesia para análises: 1. Histoenzimológicas para atividade da nicotinamida adenina dinucleotídeo diaforase(NADH), succinato desidrogenase (SDH) e adenosina trifosfatase (ATPase), 2. Microscopia de luz (HE), 3. Microscopia eletrônica de transmissão e 4. Imunohistoquímica para fator de crescimento do endotélio vascular (VEGF) e o receptor 2 para VEGF (VEGFR-2). A atividade do NADH nos grupos IV, V e VI (30±1,26; 33,47±2,15; 31,67±1,77 - fibras intermediárias) apresentou um aumento significativo (p>0,05) no metabolismo oxidativo em relação aos demais grupos. Na atividade do SDH, o aumento foi discreto, com aumento significativo (p>0,05), apenas no grupo V (32,2±1,61 fibras intermediárias), com o padrão de aumento metabólico muito parecido nas reações de NADH e SDH. A atividade da ATPase não revelou diferenças entre os grupos tanto em meio ácido como no alcalino. A microscopia de luz revelou fibras musculares arredondadas e núcleos periféricos achatados, os quais tornaram mais arredondados com as densidades maiores de energia. Ultraestruturalmente as irradiações com as maiores densidades de energia revelaram mitocôndrias de tamanhos e formas variadas e cisternas do retículo sarcoplasmático dilatadas entre as miofibrilas. As análises qualitativas mostraram um padrão de aumento a expressão do VEGF e VEGFR-2 proporcionais à densidade de energia do laser usada. Conclui-se que o laser com densidades maiores foi capaz de aumentar o metabolismo oxidativo, sem alterar a capacidade contrátil, aumentar o volume do núcleo, modificar a ultraestrutura das fibras musculares e as expressões do VEGF e VEGFR-2. / The laser therapy has been widely used as an alternative treatment in patients with chronic pain related to temporomandibular disorders. This is due to the effects: analgesic, anti inflammatory, muscle relaxant, reducing fatigue during tetanic contractions, increased bite strength and decrease in orofacial pain. Although clinical results are observed, is not well understood its effect on the cellular level. This study aims to analyze the effects of different densities (doses) irradiation of low level laser therapy (LLLI) on cellular level, on the masseter muscle of rats. The animals were randomly assigned to 6 groups (n=10), received 10 laser irradiation (GaAlAs, 780nm, 5mW spot and 0.04 cm&sup2;) on the left masseter muscle by varying the energy density (I. 0; II. 0.5; III. 1.0; IV. 2.5; V. 5.0 and VI. 20 J/cm&sup2;). After 10 irradiations the masseter muscles were obtained from animals under anesthesia for analysis: 1. Histoenzimologic for nicotinamide adenine dinucleotide (NADH), succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase), 2. Light microscopy (HE), 3. Transmission electron microscopy and 4. Immunohistochemistry for vascular endothelial growth factor (VEGF) and receptor 2 for VEGF (VEGFR-2). The activity of NADH in groups IV, V and VI (30±1,26; 33,47±2,15; 31,67±1,77 - intermediate fiber) increased significantly (p> 0.05) in oxidative metabolism in relation to other groups. The activity of SDH showed a slight increase, only the group V (32,2±1,61 intermediate fiber) increased significantly (p> 0.05), but the pattern of metabolic increase was very similar in both reactions. The ATPase activity showed no differences between groups nor in acid or alkaline. The qualitative analysis showed a pattern of increased expression of VEGF and VEGFR-2 directly proportional to the energy density of laser. Light microscopy showed rounded muscle fibers and peripheral flattened nuclei, which become more rounded with the highest energy densities. Ultrastructurally the irradiation with higher energy densities showed mitochondria of different sizes and shapes and dilated cisterns of sarcoplasmic reticulum between the myofibrils. It is concluded that higher densities of laser was able to increase the oxidative metabolism without altering the contractile capacity, increasing nuclei volume, modify ultrastructure of muscle fibers and the expressions of VEGF and VEGFR-2.