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Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused by the common deletion of 26-28 contiguous genes in the 7q11.23 region, which poses difficulties to the establishment of genotype-phenotype correlations. The use of mouse models would broader the knowledge of the syndrome, the role of deleted genes, affected pathways and possible treatments. In this thesis project, several mouse models, tissues and cells have been used to define the phenotypes at different levels, the deregulated genes and pathways and to discover modifying elements and novel treatments for the cardiovascular phenotype. In addition, a new binding motif has been described for Gtf2i, a deleted gene encoding a transcription factor with a major role in WB, providing new target genes from deregulated pathways. The obtained results reveal the essential role of mouse models for the study of Williams-Beuren syndrome and provide new treatments options and affected pathways and genes which could be future treatment targets. / La síndrome de Williams-Beuren és una malaltia del neurodesenvolupament causada per una deleció comú d’entre 26 i 28 gens contigus a la regió 7q11.23, dificultant l’establiment de relacions genotip-fenotip. L’ús de models de ratolí pot augmentar el coneixement sobre la malaltia, el paper dels gens delecionats, les vies moleculars afectades i els futurs tractaments. En aquesta tesi s’han usat diversos models de ratolí, les seves cèl·lules i teixits per tal de descriure i definir fenotips, gens i vies moleculars desregulades i per descobrir elements modificadors i nous tractaments. Per últim, s’ha definit un nou motiu d’unió per Gtf2i, uns dels gens delecionats que codifica per un factor de transcripció amb un rol central en la síndrome, proporcionats possible nous gens diana de vies moleculars desregulades. Els resultats obtinguts revelen el paper essencial dels models de ratolí per a l’estudi de la síndrome de Williams-Beuren, proporcionen noves opcions terapèutiques i defineixen nous gens i vies moleculars afectades que podrien suposar noves dianes terapèutiques.

Chez les plantes, le RET (Root Extracellular Trap) est une structure cellulo-moléculaire jouant un rôle central dans la défense racinaire face aux stress abiotiques et biotiques. De nombreuses similitudes de composition ont été observées entre le RET et le NET (Neutrophil Extracellular Trap) du système immunitaire des mammifères, connu pour capturer et tuer certains microorganismes bactériens et fongiques. Le RET est composé de cellules frontières et de leurs sécrétions (composés de haut et de bas poids moléculaire) comprenant des polysaccharides de la paroi cellulaire, des protéoglycannes et des métabolites secondaires. Il contient également des protéines antimicrobiennes et de l'ADN extracellulaire, tout comme le NET. Dans le cadre de mon projet de thèse, nous avons caractérisé la composition moléculaire et la structuration de cette entité de défense chez deux légumineuses, le soja (Glycine max (Merr) L.) et le pois (Pisum sativum L.), par des approches d’imagerie cellulaire photonique et électronique. Nous avons également étudié l’impact du RET du soja sur des pathogènes telluriques, à savoir Phytophthora parasitica et Aphanomyces euteiches. Nous avons ainsi pu mettre en évidence la présence de différents morphotypes de cellules frontières et de mucilage au sein du RET de soja et de pois. Pour la première fois, nous avons montré la présence d’hétéromannanes, de xyloglucane et de cellulose dans le RET, formant une ossature stabilisant le mucilage et reliant les cellules frontières entre elles. Ces polysaccharides structuraux semblent être essentiels à l’intégrité structurale et fonctionnelle du RET. Enfin, nos résultats ont montré que le RET de soja était impliqué dans la défense précoce de la racine contre P. parasitica. Cette étude apporte de nouvelles connaissances relatives à la composition moléculaire et la structure du RET, nous amenant ainsi à comparer le RET à d’autres modèles que le NET des mammifères, tels que les biofilms bactériens et les mucilages de graines. En effet, de nombreuses similitudes existent entre ces différents complexes en termes de composition et de fonctionnement, qui méritent d’être explorer plus en détail dans l’avenir. / In higher plants, the RET (Root Extracellular Trap) is a complex made up of border cells and secretions, released by root tips and believed to play a central role in biotic and abiotic stress tolerance. This structure is quite similar to the Neutrophil Extracellular Trap (NET) known as one of the first lines of defense in mammals, able to trap and kill microbial pathogens. RET secretions consist of high and low-molecular weight compounds including cell wall polysaccharides, proteoglycans and secondary metabolites. They also contain a variety of anti-microbial proteins and extracellular DNA much like the NET. During my thesis work, we investigated the release and morphology of root border cells in soybean (Glycine max (Merr) L.) using light and scanning electron microscopy. The molecular composition of the mucilage was also investigated using immunocytochemistry, anti-cell wall glycan antibodies and confocal microscopy. Immunocytochemistry was also applied to pea (Pisum sativum L.) border cells and secretions to examine the occurrence of specific polysaccharides. We also studied the impact of soybean RET on the soilborne pathogens, Phytophthora parasitica and Aphanomyces euteiches. Our findings showed that root tips of soybean released three border cell morphotypes all of which secreted substantial amounts of mucilage. Immunocytochemical data showed that mucilage was enriched in pectin and the two hemicellulosic polysaccharides xyloglucan and heteromannan. Mucilage also contained cellulose, histone and extracellular DNA. Interestingly, the structural polysaccharides formed a fibrous network surrounding the cells and holding them together, supporting their role in maintaining mucilage architecture and integrity. In addition, we found that xyloglucan and cellulose were also secreted into the mucilage of pea, connecting border cells together. Finally, our findings revealed that RET prevented P. parasitica zoospores from colonizing soybean root tip, by stopping their penetration and inducing their death. Overall the study revealed novel insights into the composition, structure and function of plant RETs. Currently, the RET is much less studied than its mammal counterpart, the NET, but structural and functional similarities exist between these two traps. Interestingly, similarities do also exist between the RET and other important biological complexes, including bacterial biofilms and seed mucilage, that deserve to be further investigated and compared in the context of immunity.

Les levures Candida sont des pathogènes opportunistes responsables d’infections graves chez les patients immunodéprimés. Au cours de ce travail, nous avons développé un modèle cellulaire in vitro pour la caractérisation multiparamétrique des phénotypes d’interaction entre les levures Candida et les macrophages et les neutrophiles, principaux effecteurs de la défense anti-Candida. Il repose sur l’utilisation de marqueurs fluorescents pour le suivi quantitatif de l’interaction en cytométrie en flux et en fluorimétrie. Ce modèle a été validé par la comparaison de l’interaction de trois espèces de levures, C. albicans, C. glabrata et C. lusitaniae, avec des macrophages murins et des neutrophiles humains. Deux stratégies principales de survie des levures à la phagocytose ont été mises en évidence : par la résistance à la phagolyse et la multiplication des levures à l’intérieur des phagocytes jusqu’à leur éclatement, ou par l’évitement de la phagocytose et la multiplication des levures à l’extérieur des phagocytes. L’interprétation des données quantitatives a été confirmée par microscopie à fluorescence et vidéo-microscopie. Afin de mieux comprendre les interactions Candida-phagocytes, nous avons mis au point des outils pour l’analyse fonctionnelle de gènes chez C. lusitaniae. Une stratégie de PCR chevauchante a été développée pour l’obtention de mutants nuls de C. lusitaniae, sans étape de clonage. C’est ainsi que le gène OLE2, codant une Δ9 désaturase d’acides gras potentiellement impliquée dans la biosynthèse de la prostaglandine PGE2, a été invalidé. Le mutant ole2Δ présentait de très nets défauts de filamentation et de reproduction sexuée. Par rapport à une souche sauvage, le mutant ole2∆ était massivement phagocyté par les macrophages, et la survie des phagocytes était plus importante, ce qui suggère un rôle important des lipides insaturés et des oxylipides dans la signalisation cellulaire au cours de l’interaction Candida-phagocytes. Dans la dernière partie de notre travail, nous avons construit une banque de 10 000 mutants de C. lusitaniae par l’intégration aléatoire d’un marqueur dans le génome. Le criblage de cette banque à travers notre modèle cellulaire d’interaction permettra d’identifier de nouveaux gènes impliqués dans l’interaction avec les phagocytes afin de mieux comprendre la physiopathologie des candidoses et de trouver de nouvelles pistes thérapeutiques. / Candida species are opportunistic pathogens causing severe infectious diseases in immunocompromised patients. In this work, we developed a tool for a multi-parameter characterization of the cell interactions between the yeasts Candida and both macrophages and neutrophils, which constitute the main defense against candidiasis. It relies on the labelling of each population with specific fluorescent markers, and on the use of fluorimetry and flow cytometry to assess interactions. The tool has been validated by comparing the interactions of three yeast species C. albicans, C. glabrata and C. lusitaniae, with murine macrophages and human neutrophils. We found that yeasts use two main ways for escaping phagocytosis, which has been confirmed using video-microscopy: either (1) by surviving to phagolysis and dividing into the phagosome until phagocytes burst, or (2) by avoiding phagocytosis and dividing outside phagocytes. In order to better understand the cellular and molecular mechanisms involved in Candida-phagocytes interactions, we developed new molecular tools for the functional analysis of genes in C. lusitaniae, notably a two-step cloning-free PCR-based method for the deletion of genes. This method was successfully used for the deletion of OLE2, a gene encoding a Δ9-desaturase of fatty acids, possibly implicated in prostaglandin PGE2 biosynthesis. The ole2Δ mutant exhibited strong defects in both pseudofilamention and sexual mating. During macrophages infection, ole2Δ yeast cells were massively internalized and triggered less phagocytes cell death than the wild type strain, suggesting that unsaturated fatty acids and/or oxylipids could play a role during interaction with phagocytes. Lastly, a bank of 10,000 mutants was constructed in C. lusitaniae by the random integration of a genetic marker in the genome. The screening of this bank through our tool to analyse cellular interactions will be undertaken to gain insights into understanding of the early stages of the infectious process.

Les granulocytes de faible densité (LDGs) sont un sous-type de neutrophiles mis en évidence initialement dans le sang de patients atteints de différentes maladies telles que le lupus érythémateux systémique ou le psoriasis. Cependant, des études rapportent également leur présence chez des individus sains. On connait mal à ce jour les caractéristiques des LDGs, notamment en ce qui a trait à leur profil inflammatoire. De plus, leur biogenèse demeure toujours mal connue. Mes travaux de maîtrise visaient à comparer les propriétés des LDGs à celles des neutrophiles de densité normale (NDNs). Pour ce faire, 8 chevaux atteints d’asthme équin sévère et 11 chevaux sains bien caractérisés ont été sélectionnés pour l’étude et sur lesquels des isolations de NDNs ainsi que des LDGs ont été réalisées. La morphologie des neutrophiles a ensuite été évaluée par microscopie optique. Le contenu en myéloperoxidase, un composant des granules primaires azurophiles des neutrophiles, et la présence de récepteurs du N-formylméthionine-leucyl-phénylalanine (fMLP-R) ont été évalués par cytométrie de flux et immunofluorescence, respectivement. Enfin, la capacité fonctionnelle de ces cellules à produire spontanément des pièges extracellulaires des neutrophiles (NETs) a été étudiée in vitro par microscopie confocale. Les résultats démontrent que le nombre de LDGs est augmenté dans le sang des chevaux asthmatiques lors d'exacerbation de la maladie. De plus, ces cellules présentent une morphologie différente puisqu’elles sont de taille plus petite et contiennent plus de fMLP-R que les NDNs. Le contenu en myéloperoxidase est cependant similaire dans les deux populations de neutrophiles. Enfin, les LDGs produisent plus de NETs, et sont plus sensibles aux stimuli activateurs que les NDNs. Ces caractéristiques sont similaires dans les 2 groupes de chevaux suggérant ainsi que ce sont des propriétés intrinsèques des LDGs et qu’ils représentent une population cellulaire préactivée et qui de plus, est majoritairement mature. Cette étude caractérise et compare pour la première fois les LDGs chez des animaux sains et ceux retrouvés chez des animaux atteints d’une maladie inflammatoire chronique. / Low-density granulocytes (LDGs) are a subset of neutrophils first described in the bloodstream upon pathological conditions. However, several studies also reported the presence of these cells in the blood of healthy patients. Whether LDGs characteristics, especially their enhanced pro-inflammatory profile, are specific to this subset of neutrophils and not related to disease states is unknown. Thus, we sought to compare the properties of LDGs to those of autologous normal-density neutrophils (NDNs), in both health and disease. We studied 8 horses with severe equine asthma and 11 healthy animals. Neutrophil morphology was studied using optical microscopy, and content in myeloperoxidase and N-formylmethionine-leucyl-phenylalanine receptors (fMLP-R) evaluated using flow cytometry and immunofluorescence, respectively. Confocal microscopy was used to determine their functional capacity to spontaneously release neutrophil extracellular traps (NETs) stimulating with phorbol-12-myristate-13-acetate (PMA). LDGs were smaller and contained more fMLP-R than NDNs, but myeloperoxidase content was similar in both populations of neutrophils. They also had an increased capacity to produce NETs, and were more sensitive to activation stimuli. These characteristics were similar in both healthy and diseased horses, suggesting that these are intrinsic properties of LDGs. Furthermore, these results suggest that LDGs represent a population of primed and predominantly mature cells. Our study is the first to characterize LDGs in health, and to compare their characteristics with those of animals with a naturally occurring disease.

Estudos em nosso laboratório caracterizaram camundongos B10.A como susceptíveis e camundongos A/J como resistentes à infecção pulmonar pelo P. brasiliensis. Para investigar o papel das células PMN na paracoccidioidomicose (PCM) pulmonar, camundongos B10.A e A/J foram depletados destas células através da inoculação in vivo por via intraperitoneal (i.p.) do anticorpo monoclonal anti-células PMN e infectados pela via intratraqueal (i.t.) com um milhão de leveduras viáveis. Camundongos-controle receberam doses equivalentes de IgG normal de rato. A depleção de granulócitos diminuiu o tempo de sobrevida dos animais B10.A, mas não dos animais A/J. Quando comparados com os animais não depletados, camundongos resistentes apresentaram aumento da carga fúngica no pulmão somente no dia 7 pós-infecção. Ao contrário, camundongos susceptíveis depletados de PMN apresentaram números mais elevados de células leveduriformes no pulmão, fígado e baço nos dias 7, 15, 30 e 120 pós-infecção, com relação aos seus grupos-controle tratados com IgG. A depleção dos granulócitos, entretanto, não alterou as reações de hipersensibilidade do tipo tardio (HTT) desenvolvidas por ambas as linhagens de animais. Considerando a resposta imune humoral, a depleção de células PMN levou à maior produção de anticorpos específicos em animais B10.A (Ig Total, IgG1, IgA e IgG3) e em animais A/J (Ig Total, IgG2a, IgG2b e IgG3). A depleção também alterou o padrão de citocinas pulmonares. Nos animais B10.A-tratados foram encontradas concentrações mais elevadas de IL-12 aos 15 dias e de IL-4 aos 120 dias pós-infecção, em comparação aos animais controle. Níveis de IL-12 significativamente mais altos foram detectados no grupo de animais A/J-depletados aos 7 e 120 dias e o IFN-γ foi detectado em níveis mais elevados em todo o curso da doença. Então, a depleção de PMN induz níveis mais altos de anticorpos e um ambiente mais pró-inflamatório no local da infecção. De acordo com esses dados, pudemos verificar que os neutrófilos são células importantes na defesa do hospedeiro à infecção pelo P.brasiliensis. Entretanto, o efeito protetor desta população celular depende do patrimônio genético do hospedeiro e é mais marcante na linhagem susceptível de camundongos. Neste trabalho também investigamos o efeito da depleção in vivo de leucócitos PMN na imunidade adquirida e protetora desenvolvida pela pré-imunização de animais B10.A. Assim, os camundongos foram previamente imunizados pela via s.c., depletados ou não de células PMN e desafiados i.t. com 1 milhão de células leveduriformes. Não foram detectadas diferenças significativas na contagem de fungos viáveis do pulmão, fígado e baço, entre os grupos imunizados tratados ou não com o AcM anti-PMN. A depleção não alterou a produção de anticorpos específicos, porém aumentou significativamente a síntese de IL-3, bem como a reatividade de HTT. Portanto, nossos resultados mostraram que, diferentemente da imunidade natural, os leucócitos PMN não exercem um papel protetor na fase adquirida da resposta imune à infecção com o P.brasiliensis. / Previous studies in our laboratory defined susceptible (B10.A) and resistant (A/J) mice to pulmonary P.brasiliensis infection. To investigate the role of PMN cells in pulmonary PCM, resistant and susceptible mice were depleted in vivo of these cells by intraperitoneal (i.p.) injection of a granulocyte-depleting monoclonal antibody and infected intratracheally (i.t) with one million yeast cells. Control mice received equivalent doses of normal rat IgG. PMN depletion decreased survival times of B10.A, but not of A/J infected mice. When compared with the non-depleted counterparts, resistant mice presented increased fungal loads in the lung only at day 7 after infection. On the contrary, PMN-depleted susceptible mice presented higher number of yeast cells in the lung, liver and spleen at days 7, 15, 30 and 120 after infection than their IgG-treated controls. PMN cells depletion, however, did not alter the DTH reaction developed by both mouse strains. Regarding humoral immune response, PMN cells depletion caused increased production of specific antibodies in B10.A (Total Ig, IgG1, IgA and IgG3) and A/J (Total Ig, IgG2a, IgG2b and IgG3) mice. Levels of pulmonary cytokines were also altered after PMN depletion. B10.A-treated mice presented increased levels of IL-12 and IL-4 at days 15 and 120 post-infection, respective/y. In A/J-depleted mice, augmented levels of IL-12 were detected at days 7 and 120 after infection; IFN-γ, however, was produced in higher levels during whole course of infection. Thus, PMN depletion induces higher levels of specific antibodies and enhanced pro-inflammatory milieu at the site of infection. As a whole, our data on PMN depletion at the onset of infection showed that neutrophils are important cells in host defense to P.brasiliensis infection. However, the effect of PMN depletion depends on the genetic background of the host and has a more pronounced effect in the susceptible strain of mice. We have also assessed the effect of in vivo depletion of the leukocytes on the acquired phase of immunity developed by B10.A mice previously immunized by the subcutaneous (s.c.) route were depleted or not of PMN cells and challenged i.t. with one million yeast cells. No differences were detected in the CFU counts in the lung, liver and spleen between untreated and PMN depleted vaccinated mice. PMN depletion also did not alter the production of specific antibodies but enhanced IL-3 synthesis as well as DTH reactivity. In conclusion, our results showed that, differently from natural immunity, PMN cells do not play a protective role in the acquired phase of immune response to P.brasiliensis infection.

O recrutamento de neutrófilos para o sítio da infecção é um evento crucial para o combate aos microrganismos e sobrevivência na sepse. A migração destes polimorfonucleares é dirigida através de um gradiente quimiotático por meio do reconhecimento de quimiocinas por receptores acoplados a proteína G (GPCRs), os quais são regulados por quinases específicas (GRKs). Estudos prévios demonstraram que na sepse ocorre uma falência na migração de neutrófilos para o foco infeccioso em função da dessensibilização de receptores quimiotáticos via GRKs induzida pela ativação de receptores toll-like (TLRs), TLR2 e TLR4. Apesar de a ausência de TLR9 em células dendriticas ter sido relacionada a maior sobrevivência de camundongos sépticos, o papel do TLR9 atuando diretamente em neutrófilos não foi avaliado. Objetivando preencher esta lacuna, propôs-se avaliar o papel direto de TLR9 na falência de migração de neutrófilos na sepse. Os camundongos TLR9-/- apresentaram maior sobrevivência a sepse polimicrobiana avaliada por meio do modelo de ligadura e perfuração do ceco (CLP). A deficiência de TLR9 também acarretou em aumento na migração de neutrófilos para o foco da infecção, menor seqüestro de neutrófilos no pulmão, bem como, menor número de bactérias no lavado peritoneal e sangue. A ativação de TLR9 por oligodeoxinucleotídeo contendo o dinucleotídeo CpG não metilado (ODN CpG) nos neutrófilos reduziu a quimiotaxia destes em direção a quimiocina CXCL2 e expressão do receptor quimiotático CXCR2. Além disso, neutrófilos estimulados com ODN CpG apresentaram aumento na expressão da quinase tipo 2 relacionada a receptores acoplados a proteína G (GRK2). Dessa forma, a ativação de TLR9 em neutrófilos circulantes no sangue é prejudicial na sepse por reduzir a quimiotaxia destes para o foco da infecção ao induzir a dessensibilização de CXCR2 via GRK2. / The recruitment of neutrophils to the site of infection is a crucial event for combating the microorganisms and survival on sepsis. The neutrophil migration is directed by a chemotactic gradient through the recognition of chemokines by G protein-coupled receptors (GPCRs), which are regulated by specific kinases (GRKs). Previous studies have shown a failure of neutrophil migration into infectious focus on sepsis due to chemotactic receptor desensitization via GRKs induced by activation of toll- like receptors (TLRs), TLR2 and TLR4. Despite the absence of activation of TLR9 in dendritic cells have been related to increase survival of septic mice, the role of TLR9 acting directly on neutrophils was not evaluated. We proposed to verify the direct role of TLR9 in the failure of neutrophil migration on sepsis. The TLR9 knockout mice (TLR9-/-) showed high survival to polymicrobial sepsis using cecal ligation and puncture model (CLP). TLR9-/- mice had high neutrophil migration to the focus of infection, low neutrophil sequestration in the lung, as well as, few bacteria in the peritoneal exudates and blood. The activation of TLR9 by oligodeoxinucleotide containing unmethylated dinucleotide CpG (CpG ODN) in neutrophils also reduced chemotaxis toward CXCL2 and the expression of chemokine receptor CXCR2. In addition, neutrophils stimulated with CpG ODN showed increased expression of kinase-related G protein-coupled receptor type 2 (GRK2). Thus, the activation of TLR9 in blood circulating neutrophils is harmful on sepsis by reducing their chemotaxis into the site of the infection by inducing CXCR2 desensitization via GRK2.

In der vorliegenden Arbeit wurde eine Beeinflussung der Aktivität der humanen neutrophilen Elastase (EC 3.4.21.37) durch wässrige und Dichlormethan-Extrakte von 15 Basidiomyceten festgestellt. Durch aktivitätsgeleitete Fraktionierung (mehrfache SC, GC-MS) der Dichlormethan-Extrakte von Heterobasidion annosum (Fr.) Bref. und Lactarius deterrimus Grög. wurden Fraktionen freier langkettiger Fettsäuren als ein wirksames Prinzip der Elastase-Hemmung und auch der Kollagenase-Hemmung (Clostridium histolyticum Kollagenase, EC 3.4.24.3) isoliert und identifiziert. Das Screening von 17 freien langkettigen Fettsäuren zeigte, dass einfach ungesättigte Fettsäuren eine stärkere Hemmung der Elastase-Aktivität bewirkten als ihre gesättigten bzw. mehrfach ungesättigten Homologa: Ölsäure (C18:1 cis-9): IC50 5µM; Stearin-(C18:0), Linolsäure (C18:2 cis-9,12): IC50 10µM; alpha- (C18:3 cis-9,12,15), gamma-Linolensäure (C18:3 cis-6,9,12): IC50 15µM. Inhibitorisch am stärksten wirksam war Erucasäur! e (C22:1 cis-13): IC50 450nM. Für Kollagenase wurde hingegen gezeigt, dass die gesättigten Fettsäuren eine erheblich stärkere Hemmaktivität als ihre ungesättigten Homologa aufwiesen. Aktivste Verbindungen waren Palmitin- (C16:0), Heptadecan- (C17:0), Stearin- und Nonadecansäure (C19:0) mit IC50-Werten von 20-45µM. Die Untersuchung von 9 ausgewählten Fettsäuren bezüglich der Hemmung der Aktivität der MMP-9 (EC 3.4.24.35) zeigte als aktivste Verbindungen Palmitolein- (16:1 cis-9), alpha- und gamma-Linolensäure. Die wirksamen Konzentrationen (250µM) lagen jedoch sehr hoch. Zytotoxizitätsuntersuchungen (ECV-304) der Extrakte von H. annosum und L. deterrimus sowie der freien Fettsäuren schlossen sich ebenso wie Untersuchungen zur Proteaseaktivität der Zelllinien ECV-304, MCF-7 und MDA-MB 231 an. Die Proteaseaktivität der Zellen nahm in der Reihenfolge ECV-304 < MCF-7 < MDA-MB 231 zu. Die einzig untersuchte Fettsäure gamma-Linolensäure zeigte keine reproduzierbare Beeinflussung d! er Proteaseaktivität. / In the present paper it was established that the activity of humane neutrophil elastase (EC 3.4.21.37) is affected by aqueous and dichloromethane extracts of 15 basidiomycetes. Bioassay-guided fractionation (repeated CC, GC-MS) of dichloromethane extracts of Heterobasidion annosum (Fr.) Bref. and Lactarius deterrimus Grög. led to isolation and identification of fractions of free fatty acids as one active principle of elastase inhibition as well as collagenase inhibition (Clostridium histolyticum collagenase, EC 3.4.24.3). By testing 17 free fatty acids for elastase inhibition it was shown that the inhibition rate of unsaturated acids was much higher than the rate of the saturated ones: oleic acid (C18:1 cis-9): IC50 5µM; stearic acid (C18:0), linoleic acid (C18:2 cis-9,12): IC50 10µM; linolenic acid (C18:3 cis-9,12,15), gamma-linolenic acid (C18:3 cis-6,9,12): IC50 15µM. The highly active erucic acid with an IC50 value of 450nM is remarkable. As a result for collagenase we can assume that the saturated fatty acids were more potent than the unsaturated ones. Palmitic acid (C16:0), heptadecanoic acid (C17:0), stearic acid, and nonadecanoic acid (C19:0) were the most potent fatty acids with IC50 values of 20-45µM. 9 selected fatty acids were investigated for their ability to inhibit the activity of MMP-9 (EC 3.4.24.35). Palmitoleic acid (16:1 cis-9), linolenic acid, and gamma-linolenic acid were the most potent fatty acids but their inhibiting concentrations were very high (250µM). Investigation of cytotoxicity of the extracts of H. annosum, L. deterrimus, and free fatty acids as well as investigation of protease activity of ECV-304, MCF-7, and MDA-MB 231 cells followed. Protease activity of cells increased in the following manner: ECV-304 < MCF-7 < MDA-MB 231. The only investigated fatty acid gamma-linolenic acid did not influence protease activity reproducibly.

Os neutrófilos são os leucócitos circulantes mais abundantes. Apesar de serem importantes no combate às infecções, o intenso recrutamento e a consequente ativação dessas células levam à liberação de mediadores inflamatórios que estão relacionados com o agravamento do quadro clínico de inúmeras doenças. Dessa forma, nos últimos anos tem-se intensificado a procura por substâncias terapêuticas que minimizem os danos teciduais ocasionados pela infiltração neutrofílica. Estudos prévios do grupo de pesquisa mostraram que as 3-fenilcumarinas, que constituem uma classe de produtos naturais de origem vegetal, são substâncias promissoras como moduladores do metabolismo oxidativo de neutrófilos. Em continuidade a essas investigações, o derivado sintético 3-fenilcumarínico 6,7-diidroxi-3-[3\',4\'- metilenodioxifenil]-cumarina (C13) foi selecionado, por apresentar o grupo substituinte ortodiidroxi nas posições C-6 e C-7 do esqueleto cumarínico e a metilenodioxila ligada ao anel fenílico, no intuito de avaliar o seu efeito nas demais funções efetoras dos neutrófilos, como também em um modelo animal de inflamação articular. Foi avaliado o efeito modulatório in vitro da C13 sobre a quimiotaxia, a produção de ERO e interação com o sítio ativo da mieloperoxidase (MPO), a fagocitose de imunocomplexos (IC), a desgranulação da enzima elastase, a atividade microbicida sobre Candida albicans, a mobilização e o influxo de cálcio, a polimerização do citoesqueleto, e a formação e liberação das NETs em neutrófilos humanos. A C13 inibiu, de forma dependente da concentração, o metabolismo oxidativo de neutrófilos estimulados via receptores de IgG (Fc?R) e/ou de complemento (CR), utilizando-se diferentes tipos de IC e foi capaz de interagir com o sítio ativo da MPO. Nas concentrações próximas da necessária para inibir 50% do metabolismo oxidativo (~ 2µmol/L), a C13 não interferiu nas demais funções efetoras dos neutrófilos avaliadas. No entanto, na maior concentração avaliada (20 µmol/L), a C13 inibiu cerca de: 30% da capacidade das células de migrar a favor dos agentes quimiotáticos n-formil-metionil-leucil-fenilalanina (fMLP) e leucotrieno B4; 70% da fagocitose de IC mediada por CR; 40% e 80% da desgranulação estimulada por fMLP e IC imobilizados, respectivamente; 50% do processo de formação e liberação das NETs. Esta concentração de C13 também interferiu na polimerização do citoesqueleto de actina, mas não inibiu: a quimiotaxia de neutrófilos frente à interleucina (IL) 8; a capacidade fagocítica de IC estimulada via Fc?R e Fc?R+CR; e a atividade microbicida sobre C. albicans. Na segunda parte deste trabalho, foi avaliada a toxicidade da C13 sobre neutrófilos mantidos em condições de cultura celular por 6 e 12 h. Nos períodos avaliados, essa substância não alterou a viabilidade dos neutrófilos. Por último, a C13 foi incorporada em vesículas lipossomais e sua atividade biológica foi avaliada em ratos Wistar com inflamação articular induzida por zimosan. O tratamento dos animais com a C13 lipossomal (1mg/Kg) reduziu a formação do edema e a infiltração de leucócitos e neutrófilos na sinóvia inflamada, mas não alterou a concentração sinovial das citocinas inflamatórias fator de necrose tumoral ?, IL-1? e IL-6. Portanto, o derivado 3-fenilcumarínico C13 pode servir de protótipo para o desenvolvimento de novos agentes terapêuticos com aplicação no tratamento de doenças onde há intensa participação dos neutrófilos. / Neutrophils are the most abundant circulating leukocytes. Although neutrophils are important to fight against infections, the massive recruitment and consequent activation of these cells result in the release of inflammatory mediators that are associated with worsening of the clinical condition in many diseases. In this sense, the search for new therapeutic compounds that minimize tissue damage caused by neutrophil infiltration has been intensified in the past few years. Previous studies have demonstrated that 3-phenylcoumarins, a class of plantderived natural compounds, are promising modulators of neutrophil oxidative metabolism. To continue these investigations, we selected the 3-phenylcoumarin derivative 6,7-dihydroxy-3- [3?,4?-methylenedioxyphenyl]-coumarin (C13), bearing the 6,7-dihydroxyl and the 3?,4?- methylenedioxyl groups, to further assess its effects on the other effector functions of neutrophils, as well as on in an animal model of articular inflammation. We examined the in vitro modulator effect of C13 on the human neutrophil chemotaxis, production of ROS and interaction with active site of myeloperoxidase (MPO), phagocytosis of immune complexes (IC), degranulation, killing of Candida albicans, calcium mobilization and influx, cytoskeleton polymerization, and the formation and release of NETs. C13 inhibited, in a concentration-dependent manner, the neutrophil ROS generation elicited via IgG (Fc?R) and/ or complement (CR) receptors using different types of IC and it interacted with active site of MPO. At concentrations near to that required to suppress the ROS production by 50% (~ 2µmol/L), C13 did not modulate the other neutrophil effector functions assessed. However, at the highest concentration tested (20 µmol/L), C13 inhibited nearly: 30% of the cell ability to migrate towards n-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4; 70% of CR-mediated phagocytosis of IC; 40% and 80% of cell degranulation triggered by fMLP and immobilized IC, respectively; and 50% of the NETs formation and release process. Such C13 concentration also interfered in the actin cytoskeleton polymerization, but it did not suppress the neutrophil: chemotaxis towards interleukin (IL) 8; capacity to phagocytose IC elicited via Fc?R and Fc?R+CR; and killing of C. albicans. In the second part of this study, we examined whether C13 was toxic towards neutrophils maintained in culture for 6 and 12 h. At the time points assessed, this compound did not change the viability of neutrophils. Finally, we incorporated C13 into liposomes and examined its biological activity in Wistar rats of zymosan-induced articular inflammation. Treatment of animals with liposomal C13 reduced edema formation, and leukocyte and neutrophil infiltration in the inflamed synovia, but it did not change the synovial concentration of the inflammatory cytokines tumor necrosis factor ?, IL-1?, and IL-6. Therefore, the 3-phenylcoumarin derivative C13 can be a prototype for the development of novel therapeutic agents to be used in the treatment of diseases where neutrophils have intense participation.