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Murine Mikrogliakulturen wurden mit Astrozyten-konditioniertem Medium (ACM) in einen deaktivierten Zustand überführt. Dies wurde anhand morphologischer (Grad der Ramifizierung) und immunologischer (Expression von Adhäsionsmolekülen) Parameter verifiziert. Durch den Einsatz von Makrophagen-koloniestimulierenden Faktor (M-CSF), Granulozyten/Makrophagen-koloniestimulierenden Faktor (GM-CSF), transformierenden Wachstumsfaktor beta (TGF-beta) und den gegen sie gerichteten Antikörpern wurde gezeigt, daß alle untersuchten Zytokine in unterschiedlichem Maße an der Deaktivierung der Mikrogliazellen durch ACM beteiligt sind. Außerdem wurde nach Stimulation mit ACM an murinen Mikrogliazellen eine transiente Hochregulation eines Kaliumauswärtsstromes beobachtet Das Auftreten dieses Kalium-stromes nach Inkubation der Mikrogliazellen mit ACM konnte auf die Wirkung von TGF-beta, welches im ACM enthalten ist, zurückgeführt werden. Der durch ACM in deaktivierter Mikroglia induzierte Kaliumkanal entsprach in seinen kinetischen und pharma-kologischen Eigenschaften am ehesten dem klonierten Kanal Kv1.3. Die Kv1.3 Expression durch TGF-beta oder ACM war durch den unspezifischen Proteinkinaseinhibitor H7 unterdrückbar. Diese Ergebnisse zeigen, daß die Expression des Kv1.3 Kanals nicht, wie bisher angenommen, ein Indikator für aktivierte Mikroglia ist. / Murine microglial cultures were deactivated with astrocyte-conditioned medium (ACM). The deactivation process was verified measuring morphological (ramification index) and immunological (expression level of adhesion molecules) parameters. By using macrophage-colony stimulating factor (M-CSF), granulocyte/macrophage-colony stimulating factor (GM-CSF), transforming growth factor beta (TGF-beta) and their corresponding antibodies it was shown, that to a different extent all of these cytokines influence the deactivation process of microglial cells by ACM. ACM treatment of microglial cultures also lead to a transient upregulation of a delayed potassium outward current. This upregulation was due to the impact of TGF-beta contained in ACM. The ACM induced potassium channel resembled in its kinetic and pharmacological properties the cloned Kv1.3 channel. Expression of Kv1.3 in microglial cells by TGF-beta or ACM was inhibited by the unspecific protein kinase inhibitor H7. These results show, that expression of Kv1.3 channels is not a special feature of activated microglia, which has been proposed in recent publications.

L’infection chronique par le virus de l’hépatite C (VHC) conduit au développement de la fibrose et de la cirrhose qui risque d’évoluer vers le carcinome hépatocellulaire (CHC). La protéine de capside du VHC interagit avec de nombreuses protéines de l’hôte et en particulier avec Smad3, protéine majeure de la voie de signalisation du transforming growth factor beta (TGF-Β). Mon travail de thèse consistait à étudier les conséquences biologiques de l’interaction entre la protéine de capside avec la voie de signalisation du TGF-Β. Le VHC présente une grande variabilité génétique et des travaux du laboratoire ont montré l’existence de séquences différentes de protéines de capside du virus entre les régions tumorales (cT) et cirrhotiques (cNT) d’un même sujet. Nous avons montré que ces différentes protéines de capside exprimées dans des hépatocytes orientent les réponses biologiques du TGF-Β vers la promotion tumorale en diminuant l’apoptose et en augmentant la transition épithelio-Mésenchymateuse (TEM) en particulier le variant cT. Cet effet est attribué à la capacité de la protéine de capside de diminuer l’activité transcriptionnelle de Smad3. De plus, les variants de la protéine de capside activent le TGF-Β latent via l’augmentation de l’expression de la trombospondine. L’un des marqueurs classiquement exprimé au cours d’une TEM est l’alpha-Actine musculaire lisse (αSMA). Nous avons montré qu’une autre isoforme, la γSMA, était polymérisée dans les cellules hépatiques développant une TEM. L’expression de γSMA a été retrouvée sur des coupes de CHC et a pu être significativement corrélée à la fois avec des marqueurs de la TEM, des marqueurs progéniteurs et avec l’agressivité de la tumeur.Ce travail apporte une meilleure compréhension du rôle de la protéine de capside dans la fibrose hépatique liée à l’infection virale. En effet, la protéine de capside du VHC agit à la fois de façon autocrine dans les hépatocytes en modulant les réponses du TGF-Β vers la promotion tumorale et de façon paracrine, en affectant l’activation des cellules étoilées en myofibroblastes par le TGF-Β activé. / Chronic HCV infection) may progress to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV core binds several cellular proteins and in particular Smad3, a major protein of transforming growth factor beta (TGF-Β) signalling.. The aim of this study was to determine the implication of HCV core protein in TGF-Β responses. High genetic variability is a characteristic of HCV and it was previously shown that HCV core protein isolated from tumour (cT) or adjacent non-Tumour (cNT) livers displayed different sequences. Both were able to shift TGF-B responses from tumour suppressor to tumour promotor by decreasing hepatocyte apoptosis and increasing epithelial-Mesenchymal transition (EMT). Core cT was more potent than core cNT to promote this effect that was mainly attributed to the capacity of HCV core to alleviate Smad3 activity. Moreover, HCV core protein activated the latent form of TGF-Β through increased thrombospondin expression. It is commonly accepted that αSMA (alpha smooth muscle actin) is a hallmark of EMT. In the current study another SMA isoform, γSMA was found to be polymerized during hepatocyte EMT. γSMA was expressed in HCC tissues and correlated with EMT, stem cell and aggressiveness markers. In conclusion, this work contributed to a better understanding of the HCV core role in hepatitis fibrosis and HCC related to HCV. Indeed, HCV core might act both as an autocrine and paracrine way by modulating TGF-Β responses within hepatocytes and by activating hepatic stellate cells in stromal environment through its capacity to activate TGF-Β.

L'infiltration des mélanomes par les macrophages (TAM) est souvent corrélée à un mauvais pronostic. Cependant, les mécanismes qui régulent le recrutement et la fonction de ces cellules restent encore mal compris. Des études récentes ont montré un rôle majeur de la sphingosine kinase 1 (SK1) tumorale, l'enzyme qui produit la sphingosine-1-phosphate (S1P), dans le remodelage du stroma associé à la tumeur. Le but de ce projet a été d'étudier le rôle de la SK1 tumorale sur le microenvironnement inflammatoire, et en particulier les macrophages, lors du développement des tumeurs mélaniques. In vitro, nous avons montré que l'inhibition de SK1 dans les cellules de mélanome : 1) bloque la migration des macrophages. A l'inverse, la surexpression de cette kinase dans les cellules tumorales stimule la migration des cellules inflammatoires. Cet effet est dépendant de la S1P et de sa fixation sur les récepteurs S1PR présents à la surface des macrophages ; 2) réduit la sécrétion de TGF-ß et 3) stimule la différenciation des macrophages vers un phénotype M1 antitumoral. Ce phénomène n'est pas dépendant de la S1P, ni des S1PRs, mais de la sécrétion de TGF-ß par les cellules tumorales. En effet, la différenciation macrophagique peut-être réversée par l'addition de TGF-ß recombinant dans le milieu de sécrétion des cellules tumorales. In vivo, nos résultats montrent que la greffe orthotopique, i.e. intradermique, de cellules de mélanome murin invalidées pour la SK1 à des souris syngéniques C57BL/6 est associée à une réduction de la croissance tumorale, comparée à des souris ayant reçu des cellules de mélanome contrôles. De plus, l'invalidation de la SK1 tumorale conduit à une augmentation significative de l'expression de cytokines anti-tumorales ainsi qu'à une polarisation Th1 au sein de la tumeur. Ce phénomène s'accompagne d'une réduction du pourcentage de macrophages M2 CD206+MHCIIlow, et à l'inverse, d'une augmentation du pourcentage de macrophages M1 CD206-MHCIIhigh ainsi que de lymphocytes T CD4+ et CD8+ infiltrés dans la tumeur. Ces résultats suggèrent un rôle clé de la SK1 tumorale dans le recrutement et la polarisation des macrophages dans les mélanomes. Ainsi, l'axe SK1/ TGF-ß pourrait constituer une cible thérapeutique prometteuse dans le contrôle de la croissance de cette tumeur. / Melanoma infiltration by macrophages (TAM) is often correlated with poor prognosis. However, the mechanisms that regulate the recruitment and function of these cells remain poorly understood. Recent studies have shown a major role of tumor sphingosine kinase 1 (SK1), the enzyme that produces sphingosine-1-phosphate (S1P), in tumor stroma remodeling. The aim of this project was to investigate the role of tumor SK1 on the inflammatory microenvironment, particularly macrophages, during the development of melanoma. In vitro, we showed that the inhibition of SK1 in melanoma cells: 1) blocks macrophage migration. Conversely, overexpression of this kinase in tumor cells stimulates the migration of inflammatory cells. This effect is dependent on S1P binding to its receptors (S1PR) on the macrophage surface; 2) reduces the secretion of TGF-ß and 3) stimulates macrophage differentiation towards an antitumor M1 phenotype. The latter phenomenon does not depend on S1P nor S1PRs, but on the secretion of TGF-ß by tumor cells. Indeed, macrophage differentiation can be reversed by adding recombinant TGF-ß in the tumor cell-conditioned medium. In vivo, our results showed that orthotopic injection, i.e. intradermal, of murine melanoma cells invalidated for SK1 in C57BL / 6 syngenic mice was associated with a reduction in tumor growth compared to mice having received control melanoma cells. Furthermore, the invalidation of tumor SK1 leads to a significant increase in the expression of anti-tumor cytokines and a Th1 polarization within the tumor. This phenomenon is accompanied by a reduction in the percentage of CD206+MHCIIlow M2 macrophages, and conversely, an increase in the percentage of M1 macrophages CD206-MHCIIhigh as well as CD4+ and CD8+ cells infiltrated into the tumor. These results suggest a key role of tumor SK1 in the recruitment of macrophages and their polarization in melanoma. Thus, the axis SK1 / TGF-ß could be a promising therapeutic target in controlling the growth of this tumor.

Ma thèse a pour but d'étudier l'impact des molécules immunosuppressives TGFβ et VEGF exprimées par les cellules tumorales dans la tolérance mise en place contre les tumeurs par les lymphocytes T régulateurs (Tregs). Pour cela, j'ai utilisé des lignées tumorales murines de mélanome B16 invalidées pour l'expression de TGFβ ou de VEGF par shRNA. J'ai pu observer que les invalidations induisent de profondes modifications de la réponse immunitaire contre les tumeurs, qui se traduisent par une forte diminution de son bras régulateur et une forte augmentation de son bras effecteur. Ces modifications sont le fait d'évènements très précoces et différents entre le VEGF et le TGFβ. Le ciblage simultané de TGFβ et de VEGF induit alors un rejet spontané des tumeurs chez 40% des animaux inoculés, et leur ciblage additionnel dans la stratégie thérapeutique utilisant les anticorps anti-PD-1 et anti-CTLA-4 induit un effet additif important. Nos résultats montrent donc que le VEGF et le TGFβ exprimés par les tumeurs sont des facteurs importants pour la tolérance immunitaire aux tumeurs et particulièrement pour la mobilisation des Tregs, et qu'il y a un fort rationnel à chercher à combiner leur ciblage avec les différentes stratégies thérapeutiques anti-tumorales existantes. / My thesis aims at studying the impact tumor-derived immunosuppressive molecules TGFβ and VEGF on the dominant tolerance establishment by regulatory T cells (Tregs). For this I used murine B16 melanoma tumor cell lines knocked-down by shRNA for the expression of TGFβ or VEGF. I observed that these silencings induce dramatic changes in the immune response against tumors, which result in a large decrease of its regulatory arm and a strong increase of its effector arm. These changes result from very early mechanisms that differ between VEGF and TGFβ silencings. Accordingly, simultaneous targeting of TGFβ and VEGF induces significant tumor rejection, and their additional targeting in the anti-PD-1 / anti-CTLA-4 therapeutic strategy brings obvious additive effect. Globally, our results show that tumor-derived VEGF and TGFβ are important factors for the mobilization of Tregs and more generally for immune tolerance to tumors, and that there is a strong rational to combine their targeting with the different existing anti-tumor therapies.

A cirurgia bariátrica confere proteção cardiometabólica à indivíduos obesos, contribuindo para uma redução do risco de mortalidade. No entanto, a extensão do benefício metabólico pode estar sujeita a mudanças no estilo de vida do paciente após a intervenção cirúrgica. Embora o exercício físico pareça melhorar os efeitos da cirurgia na sensibilidade à insulina, o mecanismo de ação subjacente permanece em grande parte sem explicação. Especula-se que mudanças potenciais na matriz extracelular do músculo esquelético (ECM) poderiam estar associadas à melhora da sensibilidade à insulina induzida pelo exercício físico em pacientes pós-bariátricos. Além disso, não se sabe se os benefícios da cirurgia bariátrica sobre a função endotelial, importante marcador precoce de aterosclerose, são sustentáveis sem alterações no estilo de vida, como a inclusão de exercícios físicos. Dessa forma, foram objetivos do presente estudo, investigar os efeitos do exercício físico sobre a sinalização intracelular envolvida na sensibilidade à insulina e remodelamento da matriz extracelular do músculo esquelético (Estudo 1) e sobre a função endotelial da artéria braquial de pacientes submetidos à cirurgia bariátrica (Estudo 2). Sessenta e duas mulheres foram randomizados após a cirurgia bariátrica para um programa de exercícios físicos de 6 meses ou tratamento padrão. No início do estudo, 3 e 9 meses após a cirurgia, a sensibilidade à insulina foi avaliada pelo teste oral de tolerância à glicose (TOTG), análise da função endotelial e amostras de músculo esquelético foram obtidas a partir do vasto lateral. As amostras de músculo esquelético foram submetidas a análises abrangentes, incluindo expressão de genes e proteínas, fenótipo do músculo esquelético, transcriptoma e identificação de novas vias de sinalização celular. O treinamento físico após a cirurgia bariátrica melhorou a sensibilidade à insulina no músculo esquelético. Esta resposta foi mediada por alterações moleculares e fenotípicas na ECM. A cirurgia bariátrica per se foi incapaz de solucionar completamente a resistência à insulina e a expansão da ECM no músculo esquelético. Candidatos relevantes modulados pelo exercício emergiram como alvos terapêuticos para o tratamento da resistência à insulina do músculo esquelético, nomeadamente a via TGF \'beta\' 1 SMAD 2/3 e seu antagonista folistatina. Em resumo, empregamos uma abordagem \"top-down approach\" para fornecer evidências de que a ECM do músculo esquelético desempenha um papel fundamental nos efeitos sobrepostos da cirurgia bariátrica e do exercício físico sobre a sensibilidade à insulina em mulheres obesas. Além disso, este estudo demonstrou que o treinamento físico evitou a reversão da melhora da função endotelial por meio da melhora do padrão de fluxo sanguíneo e redução de marcadores inflamatórios. Em conclusão, ao revelar um novo mecanismo pelo qual o exercício pode contrabalançar a resistência à insulina em pacientes pós-bariátricos (isto é, atenuar a espessura da ECM) e preservar a função endotelial, este estudo endossa que o exercício físico deve ser adotado como relevante medida terapêutica a fim de garantir os melhores resultados cardiometabólicos em pacientes submetidos à cirurgia bariátrica / Bariatric surgery provides cardiometabolic protection to obese individuals, contributing to a reduction in mortality risk. However, the extent of metabolic benefit may be subject to changes in the patient\'s lifestyle after surgical intervention. Although exercise seems to improve the effects of surgery on insulin sensitivity, the underlying mechanism of action remains largely unexplained. It is speculated that potential changes in the skeletal muscle extracellular matrix (ECM) could be associated with improved insulin sensitivity induced by physical exercise in post-bariatric patients. In addition, it is not known whether the benefits of bariatric surgery on endothelial function, an important marker of early atherosclerosis, are sustainable without changes in lifestyle, such as the inclusion of physical exercise. Thus, the aims of the present study were to investigate the effects of exercise on intracellular signaling involved in insulin sensitivity and skeletal muscle ECM remodeling (Study 1) and the effects of exercise on the brachial artery vasodilator response of patients undergoing bariatric surgery (Study 2). Sixty-two women were randomized after bariatric surgery to a 6-month exercise program or standard of treatment. At the beginning of the study, 3 and 9 months after surgery, insulin sensitivity was assessed by the oral glucose tolerance test (OGTT), endothelial function analysis and skeletal muscle samples were obtained from the vastus lateralis. Skeletal muscle samples were subjected to comprehensive analysis, including gene and protein expression, skeletal muscle phenotype, transcriptome and identification of new cell signaling pathways. Exercise training after bariatric surgery improved insulin sensitivity in skeletal muscle. This response was mediated by molecular and phenotypic changes in ECM. Bariatric surgery per se was unable to completely resolve insulin resistance and skeletal muscle ECM expansion. Relevant exercise-modulated candidates emerged as therapeutic targets for the treatment of skeletal muscle insulin resistance, namely the TGFβ1/SMAD 2/3 pathway and its follistatin antagonist. In summary, we employed a \"top-down approach\" to provide evidence that skeletal muscle ECM plays a key role in the overlapping effects of bariatric surgery and exercise on insulin sensitivity in obese women. In addition, this study demonstrated that physical training avoided reversal of endothelial function improvement by improving blood flow pattern and reducing inflammatory markers. In conclusion, in revealing a new mechanism by which exercise can counterbalance insulin resistance in post-bariatric patients (i.e., attenuate ECM thickness) and preserve endothelial function, this study endorses that exercise should be adopted as a relevant therapeutic measure in order to guarantee the best cardiometabolic results in patients undergoing bariatric surgery.

Aufgrund des wachsenden Interesses an der Systembiologie werden zunehmend mathematische Modelle in Kombination mit Experimenten für die Analyse von Stoffwechselnetzwerken, Genregulationsnetzwerken und zellulären Signalweiterleitungswegen verwendet. Diese Dissertationsschrift benutzt die mathematische Modellierung und kinetische Untersuchungsmethoden zum Studium von zelluären Signalwegen, insbesondere des Netzwerkes zur Festlegung der Rezeptorlokalisation und des Tumorwachstumsfaktor-beta-Signalweges. Ergänzend wurde ein Computerwerkzeug (SBML-PET) entwickelt, das die Modellentwicklung unterstützt und der Parameterschätzung dient. Mit diesem Werkzeug kann man Modelle bearbeiten, die in der Systems Biology Markup Language (SBML) formuliert sind. In dieser Arbeit wird ein quantitatives mathematisches Modell benutzt, um die Signalantwort in unterschiedlichen Rezeptorlokalisationsnetzwerken in Abhängigkeit von der Ligandenanzahl und der Zelldichte zu untersuchen. Die rechnergestützte Analyse des Modells hat ergeben, dass der Zustand eines Rezeptorlokalisationsnetzwerkes potenziell eine sigmoide Abhängigkeit von dem Verhältnis zwischen Ligandenanzahl und Oberflächenrezeptoranzahl pro Zelle zeigen. Dieses Verhältnis ist die entscheidende Kontrollgröße der Signalantwort in Rezeptorlokalisationsnetzwerken. Mit Hilfe des SBML-PET Software-Paketes haben wir eine Modellierungsmethode mit Randbedingungen vorgeschlagen, um ein umfangreiches mathematisches Modell für den Smad-abh?ngigen TGF-beta Signalweg zu erstellen und dessen Parameter aus experimentellen Daten unter Berücksichtigung qualitativer Nebenbedingungen zu fitten. Die Ergebnisse der kinetischen Untersuchung dieses Modells legen nahe, dass die Signalantwort auf einen TGF-beta-Reiz durch die Balance zwischen clathrin-abhängier Endozytose und clathrin-unabhängiger Endozytose reguliert wird. / With growing interests in systems biology, mathematical models, paired with experiments, have been widely used for the studies on metabolic networks, gene regulatory networks and cellular signaling pathways. This dissertation employs the mathematical modeling and kinetic analysis method to study cellular signaling pathways, in particular, the receptor trafficking network and TGF-beta signaling pathway. On the other hand, a systems biology markup language (SBML) based parameter estimation tool (SBML-PET), was developed for facilitating the modeling process. A quantitative mathematical model is employed to investigate signal responses in different receptor trafficking networks by simultaneous perturbations of the ligand concentration and cell density. The computational analysis of the model revealed that receptor trafficking networks have potentially sigmoid responses to the ratio between ligand number and surface receptor number per cell, which is a key factor to control the signaling responses in receptor trafficking networks. Using the SBML-PET software package, we proposed a constraint-based modeling method to build a comprehensive mathematical model for the Smad dependent TGF-beta signaling pathway by fitting the experimental data and incorporating the qualitative constraints from the experimental analysis. Kinetic analysis results indicate that the signal response to TGF-beta is regulated by the balance between clathrin dependent endocytosis and non-clathrin mediated endocytosis.

Hintergrund. L-Arginin spielt eine komplexe Rolle in der renalen Matrixexpansion, eingeschlossen der endogene Stoffwechsel der Aminosäure in Stickoxid (NO), Polyamine, L-Prolin und Agmatin. Bei Ratten mit einer induzierten Anti-Thy1-Glomerulonephritis (GN) ist gezeigt worden, dass die diätetische Gabe von L-Arginin die Überproduktion von transforming growth factor (TGF)-beta sowie die Matrixakkumulation limitieren konnte. Die vorliegende Studie testet die Hypothese, dass die günstigen Effekte auf die Überexpression von TGF-beta in vivo durch die Generierung von NO vermittelt wird. Methoden. Einen Tag nach Induktion einer Anti-Thy-1-Glomerulonephritis wurden männliche Wistar Ratten, die mit normal proteinhaltigen Futter ernährt worden sind, in die folgenden Gruppen zugeordnet worden: (1) normale Kontrollen; (2) GN; (3) GN-Arg [plus 500 mg L-Arginin/die]; (4) GN-Arg-NAME [plus 500 mg L-Arginin/die und 75 mg/die des NO-Synthase-Inhibitors Nitro-L-Arginin-Methyl-Ester (L-NAME) im Trinkwasser] und (5) GN-Molsi [plus 10 mg/die des NO-Donors Molsidomin]. In Versuchsprotokoll 1 wurde die Behandlung bis Tag 7, in Protokoll 2 bis Tag 12 nach Induktion der Glomerulonephritis durchgeführt. Analysiert wurden die Daten des systolischen Blutdrucks, die histologische glomeruläre Matrixexpansion, die Proteinurie sowie die glomeruläre mRNA- und Protein-Expression des Schlüsselfibrogens TGF-beta, des Matrixproteins Fibronektin und des Protease-Inhibitors Plasminogen-Aktivator-Inhibitor Typ 1(PAI-1). Ergebnisse. Die Blutdruckwerte zeigten sich normal in unbehandelten Anti-Thy-1-Tieren und nicht signifikant beeinflusst durch eine der Behandlungen. Verglichen mit unbehandelten, nephritischen Ratten, reduzierten die Gabe von L-Arginin sowie von Molsidomin signifikant die glomeruläre TGF-beta Überexpression und auch in ähnlichem Mass in beiden Studienprotokollen. Die günstigen Effekte von L-Arginin wurden durch gleichzeitige Blockade der NOS-Synthese mit L-NAME aufgehoben. Die glomeruläre Matrixakkumulation, Fibronektin und PAI-1 mRNA- und Protein-Expression folgten eng der TGF-beta-Expression. Die Proteinurie wurde durch keine der Behandlungen signifikant beeinflusst.Schlussfolgerung. Die vorliegende Studie zeigt, dass die antifibrotischen Effekte der Aminosäure L-Arginin in der normotensiven Anti-Thy-1-Glomerulonephritis der Ratte hauptsächlich über die endogene Produktion von NO vermittelt werden. Diese Daten lassen vermuten, dass NO in vivo die TGF-beta-Überexpression auf einem blutdruck-unabhängigen Weg limitiert und dass NO-Donoren sich günstig in der Behandlung von menschlichen fibrotischen Nierenerkrankungen auswirken könnten. / Background. L-arginine olays a complex role in renal matrix expansion, involving endogenous metabolism into nitric oxide (NO), polyamines, L-prolin and agmatine. Supplementing dietary L-arginine intake has been shown to limit transforming growth factor (TGF)-beta1 overproduction and matrix accumulation in rats with induced anti-thy1 glomerulonephritis (GN). The present study tests the hypothesis that this beneficial effect on in vivo TGF-beta overproduction is mediated via the generation of NO. Methods. One day after inducion of anti-thy1 GN, male wistar rats fed a normal protein diet were assigned to the following groups: (1) normal controls; (2) GN; (3) GN-Arg [plus 500 mg L-arginine/day]; (4) GN-Arg-NAME [plus 500 mg L-arginine/day and 75 mg/day of the NO synthase inhibitor nitro-L-arginine-methyl ester (L-NAME) in the drinking water] and (5) GN-Molsi [plus 10 mg/day of the NO donor molsidomine]. In protocol 1, treatment lasted until day 7 and in protocol 2 until day 12 after disease induction, respectively. Analysis included systolic blood pressure, proteinuria, a glomerular histologic matrix score and the glomerular mRNA and protein expression of the key fibrogen TGF-beta1, the matrix protein fibronectin and the protease inhibitor plasminogen activator inhibitor type 1 (PAI-1). Results. Blood pressure was normal in untreated anti-thy1 animals and not significantly affected by any of the treatments. Compared to nephritic rats, administration of both L-arginine and molsidomine reduced glomerular TGF-beta1 overexpression significantly and to a similar degree in both protocols, while the beneficial effect of L-arginine was abolished by concomitant NO synthesis inhibition. Glomerular matrix accumulation, fibronectin and PAI-1 mRNA and protein expression cloxely followed the expression of TGF-beta1. Proteinuria was not significantly affected by any treatment. Conclusion. The present study shows that L-arginine´s antifibrotic action in normothensive anti-thy1 GN is mainly mediated by endogenous production of NO. The data suggest that NO limits in vivo TGF-beta overexpression in a pressur-independent manner and that NO donors may be of benefit in the treatment of human fibrotic renal disease.

Lectinas são proteínas que possuem pelo menos um domínio não catalítico que se liga reversível e especificamente a um monossacarídeo ou oligossacarídeo. A capacidade de ligação a diferentes tipos de açúcares torna essas moléculas ferramentas úteis no estudo de diversos processos celulares específicos. Embora as lectinas de plantas sejam amplamente estudadas, aquelas referentes à família Apocynaceae ainda são pouco exploradas. Resultados prévios obtidos pelo nosso grupo de pesquisa mostraram que extratos brutos de súber do caule da apocinácea Tabernaemontana hystrix Steud apresenta atividade hemaglutinante. Além de aglutinar eritrócitos do sistema ABO, a putativa aglutinina foi capaz de estimular a síntese de RNAm de IL-6 e TGF- beta em células esplênicas de camundongos. À vista disso, no presente projeto tivemos como objetivo identificar, caracterizar bioquimicamente e avaliar o possível potencial imunoestimulador da aglutinina de T. hystrix. Os extratos de T. hystrix obtidos por meio da farinha de raspas do súber apresentaram atividade hemaglutinante, o que não foi observado no extrato do caule destituído de súber e no extraído das folhas. Para comprovar que se tratava da atividade observada anteriormente, obtivemos a inibição da hemaglutinação com a glicoproteína fetuína, mas não houve inibição por monossacarídeos. Foi determinado um protocolo de isolamento da hemaglutinina com precipitação do extrato do súber com sulfato de amônio, cuja atividade foi recuperada no material precipitado na faixa de 30 a 60% de saturação, seguido de cromatografias sequenciais por (1) interação hidrofóbica (HiTrap Octyl), (2) troca catiônica (HiTrap SP), (3) fase reversa (EC Nucleosil C18) e (4) afinidade (Blue Sepharose). Nessas colunas a atividade foi recuperada do (1) material não retido e dos eluatos (2 e 4) com 1M e 0,5M de NaCl, respectivamente, e (3) 83% de acetonitrila. Esse protocolo produziu uma preparação homogênea contendo um peptídeo cuja análise eletrofóretica revelou massa molecular (MM) aproximada de 3kDa e concentração hemaglutinante mínima de 50g/mL. A fim de determinar se esse peptídeo formava estrutura quaternária (dímeros, tetrâmetros, etc.), característica da maioria das lectinas de plantas, submeteu-se a preparação a uma eletroforese em gel nativo (PAGE), não sendo observadas mudanças na MM do peptídeo e nem a presença de outras moléculas com MM maiores que pudessem estar associadas a ele, o que sugere que a aglutinina de T. hystrix (denominado aqui de Tabhys) é um peptídeo de MM aproximada de 3kDa. O fato da heveína, um dos peptídeos lectínicos com atividade antifúngica mais estudado, ter especificidade por quitina nos motivou a tentar o isolamento do peptídeo em coluna desse polissacarídeo. Observou-se atividade hemaglutinante e presença de peptídeo com MM de 3kDa no material eluído com Ácido acético a 0,1M da coluna de quitina. Curiosamente, nenhuma de nossas preparações foram capazes de inibir o crescimento do fungo Trichophyton rubrum. O peptídeo purificado foi testado quanto a sua capacidade em induzir a proliferação celular e a produção de citocinas em células esplênicas murinas. Os resultados dos ensaios de RT-PCR em tempo real e citometria de fluxo demonstraram que o a aglutinina de T. hystrix não foi capaz de estimular a proliferação de linfócitos, entretanto, induziu o aumento de mensagem para a citocina TGF-beta, cujo pico de produção ocorreu em célula estimuladas com 37ng/mL. Neste estudo, relatamos a presença de um peptídeo no extrato de T. hystrix com atividade hemaglutinante, o que é relativamente raro e novo. Devido a isso, este estudo pode proporcionar novas perspectivas e paradigmas nos estudos das lectinas a nível molecular e estrutural. / Lectins are proteins that have at least one non-catalytic domain that binds specifically and reversibly to a monosaccharide or oligosaccharide. This ability to bind to different types of sugars makes these molecules useful tools in the study of various specific cellular processes. Although the plant lectins are widely studied, those belong to Apocynaceae family are still little explored. Previous results obtained by our research group showed that bark crude extracts from Tabernaemontana hystrix Steud (Apocynaceae) had hemagglutination activity. Besides to agglutinate erythrocytes from ABO blood group system, the putative agglutinin induced the synthesis of IL-6 and TGF-beta mRNA in mouse spleen cells. Here we aim to identify, characterize biochemically and evaluate the possible immunostimulatory potential of T. hystrix agglutinin. The haemagglutination activity was obtained from crude extracts of bark flour, but not of flours of stems without bark and leaves. The activity of the bark extract was similar to that from the previous study, since the haemagglutination was inhibited by the glycoprotein fetuin, but not by monosaccharides. An isolation protocol was determined by using ammonium sulfate precipitation, with haemagglutination activity recovered in the range of 30-60% of saturation, and sequential chromatography procedures: (1) hydrophobic interaction (HiTrap Octyl), (2) cation-exchange (HiTrap SP), (3) reverse phase (EC Nucleosil) and (4) affinity (BlueSepharose) chromatography. From these columns the activity was recovered in the (1) unbound material, and eluates (2 and 4) with 1M and 0,5M of NaCl, respectively, and (3) 83% acetonitrile. On the basis of electrophoresis analysis, the protocol produced a preparation comprised of only band corresponding a peptide with molecular weight (MW) of about 3-kDa, with minimum haemagglutination concentration of 50g/ml. To determine if this molecule arrangement had a quaternary structure arrangement, a feature of most known lectins, we submitted the preparation to a native electrophoresis. Because there was neither change in migration pattern nor presence of molecules of higher molecular mass, we suggested that T. hystrix peptide (Tabhys) is a peptide with MW of about 3-kDa. Since hevein, which is a most studied lectin-like peptide with antifungal activity, binds specifically to chitin, we performed an affinity chromatography in the chitin column with bark extract. We observed haemagglutination activity and the presence of peptide with MW of 3-kDa in the material bound to column and eluted with 0,1M acetic acid. Curiously, this peptide was not able to inhibit the growth of the fungus Trichophyton rubrum. Thereafter, when the purified peptide was used to stimulate murine spleen cells, we detected the expression of TGF-beta message, with a peak production obtained in cell stimulated with 37 ng/mL of Tabhys. In the current study, we isolated a peptide from crude extract of T. hystrix bark with haemagglutination activity, providing new perspectives in molecular and structural researches of peptide lectins.

Genes homeobox, vitais para muitos aspectos relacionados com crescimento e diferenciação celular, têm sido descritos desregulados em alguns cânceres. Seu papel na carcinogênese, principalmente de carcinomas epidermóides de boca, permanence pouco claro e pobremente caracterizado. Desse modo, esse estudo objetivou avaliar, em cultura de células, o perfil de expressão de seis genes homeobox (ASH2L, HOXA7, HHEX, PKNOX1, PITX1, TGIF) selecionados dentre aqueles previamente identificados no Projeto Genoma Câncer de Cabeça e Pescoço (2001) sob estímulo de EGF e TGF-beta1. Para tal, linhagens celulares de carcinoma epidermóide de cabeça e pescoço primário (HN6) e metastático (HN31) e uma linhagem não-tumoral (HaCat) foram cultivadas sob condições-padrão. Após a confecção dos cDNAs de cada linhagem, por meio de RT-PCR, os transcritos foram amplificados e quantificados pela técnica de PCR em tempo real. Os dados foram normalizados com o gene HPRT e a quantificação relativa foi realizada seguindo o método do delta Ct. De acordo com os resultados foi possível verificar que o EGF produziu uma modulação variável da expressão dos genes avaliados em todas as linhagens celulares, enquanto que, em geral, o TGF-beta1 foi capaz de aumentar significantemente (ANOVA, p<0,05) a expressão dos transcritos de 5 genes homeobox (HOXA7, HHEX, PKNOX1, PITX1, TGIF). Particularmente transcritos dos genes PITX1 e TGIF foram signicantemente mais expressos nas linhagens tumorais (HN6 e HN31) frente à linhagem não-tumoral quando tratados com TGF-beta1. Desse modo, sugere-se que os genes homeobox estudados desempenhem diferentes funções na carcinoma epidermóide de boca, e que, especialmente PITX1 e TGIF atuem como oncogenes inibindo a resposta anti-proliferativa dependente de TGF-beta e levando a progressão tumoral. / Homeobox genes, vital to many aspects related with cellular growth and differentiation, had been described as deregulated in some cancers. Their role in carcinogenesis, mainly oral squamous cell carcinomas, remains unclear and poorly characterized. Thus, this study had the purpose to evaluate, in cell cultures, the expression profile of six homeobox genes (ASH2L, HOXA7, HHEX, PKNOX1, PITX1, TGIF) selected among genes previously identified in the Head and Neck Cancer Genoma Project (2001), under stimulation with EGF and TGF-beta1. Oral squamous cell carcinoma cell lines from primary tumour (HN6) and from methastasis (HN31), and a non-tumoral cell line (HaCat) were cultured under standard procedures. CDNAs were obtained by RT-PCR and the transcripts were amplified and quantified by real-time PCR. Data were normalized by HPRT gene and the relative quantification was made by the delta Ct method. According to the results, it was possible to observe that EGF produced a variable modulation of the analyzed genes, in all cell lines. Generally, TGF-beta1 was able to significantly increase (ANOVA, p<0,05) the expression of the transcripts of 5 homeobox genes (HOXA7, HHEX, PKNOX1, PITX1, TGIF). Transcripts of PITX1 and TGIF genes were particularly more expressed in the tumoral cell lines (HN6 e HN31), when compared to the non-tumoral cell line, when treated with TGF-beta1. It is suggested that the studied homeobox genes play different roles in oral squamous cell carcinoma and that, especially the PITX1 and TGIF act as oncogenes, inhibitting the TGF-dependent anti-proliferative response, leading to tumour progression.