O carrapato bovino Rhipicephalus (Boophilus) microplus é o principal vetor da riquétsia Anaplasma marginale, o agente etiológico da anaplasmose, uma doença que acomete os rebanhos e causa sérios prejuízos econômicos à pecuária no Brasil. Estabelecemos em nosso laboratório o cultivo da linhagem de células BME26 que são originárias do R. (B.) microplus e também a infecção dessas células por A. marginale, um patógeno que é naturalmente transmitido pelo carrapato. Detectamos que a expressão gênica da defensina e da ixodidina nas células é aumentada frente à infecção por A. marginale, embora nenhuma alteração da expressão gênica da microplusina foi constatada. As células foram expostas a microorganismos inativados por calor e LPS, sendo que a expressão gênica da microplusina é aumentada frente a todos os estímulos. Na exposição das células BME26 com a bactéria Microccocus luteus, a expressão gênica da defensina e da ixodidina não foi alterada e no estimulo com leveduras a expressão gênica da ixodidina foi reprimida. Frente à infecção por A. marginale detectamos, aumento expressão da defensina e ixodidina. Os genes da microplusina e defensina foram silenciadas por RNAi em células infectadas por A. marginale, mas não houve alteração no número de riquétsias / The cattle tick Rhipicephalus (Boophilus) microplus is the main vector of the rickettsia Anaplasma marginale, the etiological agent of anaplasmosis, a disease that affects cattle and causes serious economic losses to the Brazilian cattle industry. We established in our laboratory the embryonic cell culture line BME26 from R. (B.) microplus and infection by A. marginale, a pathogen naturally transmitted by R. (B.) microplus. We verified that defensin and ixodidin gene expression increased in these cells after an infection by A. marginale and no alteration in microplusin gene expression was detected. The BME26 cells were exposed to heat-inactivated microorganims or to LPS, microplusin gene expression increased after all stimuli. After exposure of BME26 cells to Micrococcus luteus, expression levels of defensin and ixodidin did not change and ixodidin gene expression reduced after exposure of these cells to yeast. In the infection by A. marginale we detected defensin and ixodidin gene expression. Also, microplusin and defensin genes were silenced by RNA interference (RNAi) in A. marginale-infected BME26 cells, but we did not observe alteration in the number of MSP4 rickettsias

O carrapato Rhipicephalus microplus é o vetor da bactéria Anaplasma marginale, agente etiológico da anaplasmose bovina. Este trabalho teve como objetivo a caracterização molecular e funcional das vias de sinalização Toll, Imd, Jnk e Jak/Stat do R. microplus. Através de análises in silico, a maioria dos genes que compõe as vias de sinalização de R. microplus foi identificada. Além disso, verificou-se uma modulação negativa da expressão dos genes dessas vias, nas células BME26, pela infecção por A. marginale. Após o silenciamento dos genes codificadores dos fatores de transcrição das vias de sinalização, através da técnica de RNAi, em carrapatos R. microplus, observou-se que o fator de transcrição Relish (Imd) está envolvido no controle da A. marginale, possivelmente através do peptídeo antimicrobiano microplusina. Com este trabalho, ampliamos o conhecimento sobre o sistema imune de carrapatos para compreender melhor a interface vetor-patógeno. / The cattle tick Rhipicephalus microplus is the vector of the bacteria Anaplasma marginale, the etiological agent of bovine anaplasmosis. This study aimed to evaluate the molecular and functional role of the signaling pathways Toll, Imd, Jnk, and Jak/Stat in R. microplus. An in silico analysis allowed the identification of several genes of these signaling pathways. In addition, there was a negative modulation of expression of genes of these pathways in the tick cell line BME26 upon infection with A. marginale. The silencing of some transcription factor genes via RNA interference in ticks indicated that the transcription factor Relish (from the Imd pathway) was involved in the control of bacterial infection, possibly via regulation of the expression of the antimicrobial peptide microplusin. This study helped elucidate the role of the immune system of ticks in the vector-pathogen interface.

Les peptides antimicrobiens (PAMs) cationiques sont des molécules amphipatiques conservées chez une grande diversité d'espèces vivantes. Ils participent ainsi à la défense immunitaire de nombreux organismes incluant les bactéries, les insectes, les plantes et les vertébrés. En plus de leur activité microbicide directe dirigée contre un large spectre de pathogènes, la plupart des PAMs cationiques sont désormais connus pour exercer des fonctions immunomodulatrices sur les réponses innée et adaptative. Notre équipe a récemment découvert et isolé un nouveau PAM à partir du venin de la fourmi Tetramorium bicarinatum, nommé P17. Dans ce travail, nous avons étudié les propriétés immunomodulatrices du P17 sur la réponse immunitaire innée médiée par les macrophages. Nous nous sommes plus particulièrement intéressés à sa capacité à moduler la différenciation de macrophages dérivés de monocytes humains (h-MDM) ainsi que leurs fonctions fongicides associées vis-à-vis d'une levure opportuniste majeure Candida albicans (C. albicans). Nous avons ainsi pu mettre en évidence que le P17 oriente la différenciation des h-MDM vers un phénotype alternatif caractérisé par la surexpression des récepteurs lectine de type C (CLRs) tels que Dectine-1 et le récepteur mannose (MR). De manière intéressante, nous avons mis en évidence que la surexpression de ces deux récepteurs à la surface des h-MDM activés par le P17 nécessite la mobilisation de l'acide arachidonique et la production de leucotriène B4 (LTB4). Nous avons également démontré que ce métabolite de l'AA conduit à l'activation du récepteur nucléaire PPARƴ, facteur clé de l'activation alternative des macrophages et de l'expression des CLRs associée à ce phénotype. Au cours de ce travail, nous avons démontré que les h-MDM polarisés par le P17 présentent une meilleure capacité à éliminer C. albicans. En effet, ces h-MDM activés par le P17 ont une capacité de reconnaissance, par les CLRs Dectine-1 et MR, et de phagocytose de C. albicans augmentée. De plus, l'étude des mécanismes microbicides conduisant à l'élimination de C. albicans révèle que les h-MDM activés par le P17 produisent de fortes quantités d'espèces réactives de l'oxygène (ROS) et d'IL-1ß via l'inflammasome. Ainsi, ce travail met en évidence que l'induction de l'activité fongicide des h-MDM par le P17 est dépendante de l'axe LTB4/ PPARƴ/Dectine-1-MR. Nous avons finalement confirmé ces données in vivo sur un modèle de candidose gastro-intestinale induite chez des souris traitées par voie intra-péritonéale par P17 ou non. Les résultats obtenus ont révélé que les souris traitées par P17 étaient plus résistantes à l'infection gastro-intestinale à C. albicans. La diminution de la charge fongique au niveau du cæcum des souris traitées par le P17 est associée à une meilleure efficacité de leurs macrophages à phagocyter C. albicans, à produire des ROS et à tuer C. albicans. Ainsi, ces résultats identifient le P17 comme un activateur original des propriétés antifongiques des macrophages agissant en aval de la voie permettant l'induction de l'expression des CLRs via PPARƴ. Ces données révèlent pour la première fois l'implication d'un PAM dans le contrôle de la différenciation des macrophages et leurs fonctions microbicides. / Cationic antimicrobial peptides (AMPs) are evolutionary small and amphipatic conserved molecules which are involved in the immune defense of a wide range of organisms, including bacteria, insects, plants and vertebrates. Beside their direct microbicidal activity against pathogens, most of them are known to exert immunomodulatory functions on innate and adaptive immune cells. Here we evaluated the immunomodulatory properties of an original cationic AMP, named P17, discovered and isolated by our team from the ant Tetramorium bicarinatum venom. We have focused on its efficiency to modulate human monocyte-derived macrophages (h-MDM) differentiation and its capacity to provide them an antifungal activity against the main opportunistic yeast Candida albicans (C. albicans). We showed that P17 directed h-MDM polarization toward an alternative phenotype characterized by mannose (MR) and dectin-1 C-type lectin receptors (CLRs) upregulation. Interestingly, we demonstrated that this upregulation of MR and Dectin-1 in P17-treated h-MDM requires AA mobilization and leukotriene B4 (LTB4) synthesis, essential for PPAR activation. We also demonstrated that this AA metabolite led to the PPARƴ nuclear receptor activation which is a key factor of macrophages alternative activation and the associated CLRs expression. In this study, we observed that P17-activated h-MDM exhibited an improved capacity to eliminate C. albicans. Indeed, these P17-polarized macrophages displayed an increased ability to recognize and phacocyte yeasts. Furthermore, the study of microbicidal mechanisms leading to C. albicans clearance revealed that P17-activated h-MDM produced reactive oxygen species (ROS) and inflammasome-dependant IL-1ß in high amounts. These mechanisms induction in P17-polarized h-MDM was dependent on the LTB4/ PPARƴ/Dectin-1-MR axis. Finally, these data were supported by in vivo experiments demonstrating that P17-treated mice infected with C. albicans developed less severe gastrointestinal infection related to a higher efficiency of their macrophages to engulf C. albicans, to produce ROS and to kill yeasts. Altogether, these results identify P17 as an original activator of the fungicidal response of macrophages that acts downstream the pathway leading to CLRs expression through PPARƴ activation.

Les adénovirus humains (HAdVs) provoquent généralement une infection bénigne chez l'hôte sain. En revanche, chez les patients immunodéprimés et immunocompétents, ils peuvent causer des infections sévères à létales. Les vecteurs HAdVs sont couramment utilisés dans les domaines de la thérapie génique et de la vaccination. L'immunité pré-existante de l’hôte protège généralement contre les infections de type sauvage mais peut entraîner des effets indésirables lors du relargage des virus tel que l’induction de processus inflammatoire locale ou général. Après l'infection, l'inflammation va principalement entraîner le recrutement de cellules dendritiques (DCs), de monocytes et de neutrophiles. Les DCs ont la capacité unique de présenter l'antigène et d’activer les cellules T, qui, par la suite, aideront les cellules B à produire des anticorps. En plus de leurs activités phagocytaires, des peptides antimicrobiens dérivés des neutrophiles (AMPs) jouent un rôle central dans l'immunité innée. Les AMPs peuvent neutraliser les microbes infectieux et / ou activer différents types de cellules immunitaires. Dans ce contexte, nous avons étudié l'interaction ex vivo entre le facteur hôte (anticorps anti-HADV et AMPs) contre les HAdVs dans les DCs ainsi que le rôle des DCs activés indirectement (indir-DCs) lors de la réponse immunitaire. Nous avons caractérisé le profil des cytokines et des chimiokines sécrétées par les DCs stimulées par différents HAdVs, AMPs et les combinaisons qui en découlent. Enfin, nous avons constaté que l’opsonisation d’HAdV5 par les IgG accroît la capacité de capture antigénique des MoDCs (cellules dendritiques dérivées de monocytes) et induit la mort cellulaire par pyroptose. Je me suis donc concentrée sur les caractéristiques et la fonction des indir-DCs dans l’immunité contre les HAdVs. Afin de mieux comprendre les propriétés et les phénotypes des indir-DCs et DCs activés directement (dir-DCs), nous avons caractérisé leurs profils de maturation, les facteurs influençant cette maturation, et leur capacité fonctionnelle de recruter les leucocytes. Ainsi nous avons pu mettre en évidence que les dir-DCs empêchent le recrutement leucocytaire tandis que les indir-DCs favorisent la migration des monocytes. L’ensemble de ces données contribue à comprendre comment l'immunité pré-existante contre les HAdVs peut impacter l’efficacité des traitements contre les maladies HAdVs ainsi que la conception de vaccins. / Human adenoviruses (HAdV) generally cause mild infection in healthy host, but in immunocompromised and immunocompetent patients, they cause severe on lethal infections. HAdV vector are also commonly used for gene transfer and vaccination. We know that pre-existing immunity can protect from wild type infection and cause adverse effects during vector delivery including local and system inflammation. Following HAdV infection on vector use, inflammation leads to the recruitment of dendritic cells (DCs), monocyte and neutrophils. DCs have the unique ability to present antigens and activate T cells, that subsequently aid B cells to produce antibodies. In addition to their phagocytic activities, neutrophil-derived antimicrobial peptides (AMPs) play a central role in innate immunity. AMPs can kill invading microbes and/or activate various cell types. Here I studied the ex vivo interaction between host factor (anti-HAdV antibodies and AMPs) to HAdV in DCs and the role of indirect-activated DC (indir-DCs) in immune response. I characterized the profile of cytokines and chemokines of DC stimulated with different HAdV, AMPs and their combination. We recently found that IgG-oposonization of HAdV5 increase the update by MoDCs (monoctyes-derived dendritic cells) and induced pyroptotic cell death. I therefore focused on the characteristic and function of indir-DCs in anti-HAdV immunity. To better understand the properties and phenotypes of indir-DCs and direct-activated DCs (dir-DCs), we characterize their maturation profile, the factors influencing their maturation, and the functional ability to recruit leukocyte. We found that dir-DC prevent leucocyte recruitment while indir-DC increases monocyte migration. These data contribute to understand how the pre-existing immunity to HAdV impacts treatment for HAdV diseases and vaccine design.

Les organismes vivants produisent des peptides antimicrobiens (PAMs) pour se protéger contre les microbes. La résistance croissante aux antibiotiques nécessite le développement de nouvelles stratégies thérapeutiques et les PAMs sont des candidats prometteurs pour résoudre ce problème. Ils possèdent une activité à large spectre et leur principal mécanisme d'action par perméation de la membrane engendre peu de phénomènes de résistance. Néanmoins, leur faible biodisponibilité empêche leur utilisation. Certaines limitations peuvent être surmontées en développant des mîmes de PAMs qui conservent leur activité mais avec un potentiel thérapeutique accru. Les peptoïdes (oligomères de N-alkylglycine) structurés en hélice cationique amphiphile sont de bons mimes de PAMs. Les peptoïdes sont plus flexibles que les peptides en raison de l'isomérie cis/trans des amides N,N-disubstitués ; cependant la conformation des amides peut être contrôlée par un choix judicieux des chaînes latérales. Le but de cette thèse est d'étudier l'influence de chaînes latérales(hydrophobes ou cationiques) bloquant la conformation des amides en cis et induisant une structure hélicoïdale de type PolyProline I (PPI) robuste, sur l’activité antibactérienne et la sélectivité de peptoïdes. La conception, la synthèse et l’étude conformationnelle de nouveaux oligomères peptoïdes cationiques portant des chaînes latérales de type tert-butyle et/ou triazolium ont été réalisées. Dans un premier temps, la synthèse en solution d'oligomères à base de tert-butyle a été développée puis une stratégie de synthèse en phase solide a été mise en place pour accéder aux oligomères à base de 1,2,3-triazolium. Ensuite, ces nouveaux oligomères ont été évalués pour leur activité vis à vis d’un panel de bactéries Gram-positive et Gram-négative, leur l'activité antibiofilm et leur sélectivité cellulaire. Enfin, pour visualiser les effets des peptoïdes amphiphiles sur les bactéries, une étude de microscopie a été réalisée. / Living organisms produce antimicrobial peptides (AMPs) to protect themselves against microbes.The growing problem of antimicrobial resistance calls for new therapeutic strategies and the natural AMPs have shown ground-breaking potential to address that issue. They show broad-spectrum activity and their main mechanism of action by bacterial cell membrane disruption implies low emergence of resistance which makes them potent candidates for replacing conventional antibiotics. Nevertheless, few hurdles are impeding their use, notably poor bioavailability profile. Some of these limitations can be overcome by developing peptidomimetics of AMPs which exhibit antibacterial activities together with enhanced therapeutic potential. Peptoids (i.e. N-alkyl glycine oligomers) adopting cationic amphipathic helical structures are mostly competent AMP mimetics. From a conformational point of view, peptoids are fundamentally more flexible than peptides primarily due to the cis/trans isomerism of N,N-disubstituted amides but studies in this area have shown that cis amide conformation can be controlled by careful choice of side-chain to set a PolyProline I-type helical structure of peptoids. In this thesis, the genesis of novel amphipathic cationic peptoids carrying cis-directing tert-butyl and/or triazolium-type side-chains and their untapped potential to act against bacteria will be discussed comprehensively. First, the solutionphase synthesis of tert-butyl-based oligomers was developed. Second, novel method of solid-phase submonomer synthesis was optimised to access 1,2,3-triazolium-based oligomers. Then, the synthesised cationic oligomers were evaluated for their antibacterial potential, followed by antibiofilm activity and cell selectivity assays. In the end, to have insights on the mode of action of amphipathic peptoids, microscopy was carried out.

Peptídeos antimicrobianos, também chamados na literatura médica de AMPs (Antimicrobial Peptides), são agrupamentos de 12 a 50 aminoácidos que têm funções antimicrobianas e imunorregulatórias. Os AMPs são produzidos principalmente nos epitélios, destacando-se o epitélio intestinal, onde participam da homeostase da barreira intestinal. A Pancreatite Aguda (PA) é uma doença com altas taxas de mortalidade e morbidade, na qual frequentemente ocorre translocação de bactérias e endotoxinas para o epitélio intestinal e para a corrente sanguínea. A translocação bacteriana ocorre, principalmente, devido à falência da barreira intestinal. Em idosos, a PA tende a ser uma doença mais grave, porém os mecanismos que justificam essa maior gravidade ainda não são bem compreendidos. O objetivo deste trabalho é fazer um estudo da expressão gênica dos peptídeos antimicrobianos alfa-defensina 5 e 7 e da catelicidina CRAMP em ratos jovens e idosos submetidos a um modelo de Pancreatite Aguda. Ratos jovens e idosos foram submetidos à PA utilizando-se Ácido Taurocólico 2,5% e, após doze horas, foram sacrificados. Foi coletado 1cm do íleo terminal para realização de RTPCR para os genes das alfa-defensinas 5 e 7 e da catelicidina CRAMP. Adicionalmente, foram quantificados os níveis de mRNA das citocinas TNF-alfa e IL10. Através da técnica de Milliplex, foram dosadas no soro as citocinas IFN-y, IP-10, IL-10, IL-6, IL-18, TNF-alfa, IL-1beta e Leptina. Como resultado, detectou-se maior expressão gênica das alfa-defensinas 5 e 7 no grupo de animais idosos, na vigência de insulto pancreático, quando comparado a animais jovens na mesma condição. Com relação à CRAMP, não houve diferença significativa entre os grupos. Igualmente, obteve-se expressão significativamente maior de TNF-alfa no íleo terminal dos ratos idosos com Pancreatite Aguda. Interessantemente, as dosagens de citocinas séricas foram semelhantes entre os grupos, demonstrando que a diferença entre a resposta inflamatória de ratos idosos e jovens é localizada. Peptídeos antimicrobianos, portanto, devem apresentar um papel central na resposta inflamatória decorrente de injúria pancreática / Antimicrobial peptides, also called AMPs, are 12-50 amino acids molecules that have antimicrobial and immunoregulatory properties. AMPs are produced mainly in epithelial cells, and participate in the homeostasis of the intestinal barrier. Acute pancreatitis (AP) is a disease with high mortality and morbidity and presents a worst outcome when bacterial translocation occurs to the intestinal epithelium and to the bloodstream. Bacterial translocation occurs mainly due to the failure of the intestinal barrier. In the elderly, AP has a worst prognosis, but the mechanisms that justify that higher severity, compared to the young, are poorly understood. The objective of this work is to study the gene expression levels of antimicrobial peptides ?-defensins 5 and 7 and the cathelicidin CRAMP in young and elderly rats submitted to acute pancreatitis. Young and old rats received a taurocholic acid 2.5% injection in the pancreatic ductus and after twelve hours, were sacrificed. 1cm of the terminal ileum was collected for detection of AMPs gene expression levels. In addition, RT-PCR was performed to detect TNF-alfa and IL-10 mRNA levels. Using Milliplex technique, serum levels of IFN-gamma, IP-10, IL-10, IL-6, IL-18, TNF-alfa, IL-1beta and Leptin were measured. As a result, we detected significant higher mRNA levels of alfa-defensins 5 and 7 in the elderly (pancreatitis group). CRAMP gene expression was not significantly different between groups. TNF-alfa gene expression levels was significantly higher in old rats (pancreatitis group). Any difference in the cytokines serum levels could be detected when old and young rats in the pancreatitis groups were compared. We conclude that AMPs may play a pivotal role in the development of acute pancreatic injury

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-18Bitstream added on 2014-06-13T19:19:38Z : No. of bitstreams: 1 cabrera_mps_dr_sjrp.pdf: 1625766 bytes, checksum: 6073fec2bef34125e9db4700102a3680 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho estudamos a conformação em ambientes anisotrópicos e a atividade lítica em vesículas aniônicas e zwitteriônicas de um conjunto de peptídeos biologicamente ativos, extraídos de veneno de vespas solitárias, que se caracterizam por usar seus venenos para paralisar as presas com as quais alimentam suas larvas. Esses peptídeos que são desgranuladores de mastócitos, apresentam atividade antimicrobiana e a maioria deles não é hemolítica. Possuem entre 10 e 15 resíduos, são catiônicos, com alta proporção de resíduos carregados e polares, e são lineares e helicoidais em meios miméticos de membranas. Buscamos correlacionar a atividade lítica em vesículas de diferentes composições, analisada em experimentos de fluorimetria, às mudanças conformacionais, induzidas por diferentes ambientes miméticos, monitoradas por dicroísmo circular, complementando com a análise das características físico-químicas como comprimento da cadeia, amidação do terminal-C, carga líquida, influências no macrodipolo da hélice, hidrofobicidade, momento hidrofóbico e ângulo polar. Observamos que estes peptídeos apresentam intensa atividade em membranas modelo, interagem preferencialmente com bicamadas aniônicas, e sua atividade lítica acontece de modo cooperativo tanto em vesículas aniônicas como nas zwitteriônicas. Com exceção de Anoplin, todos os peptídeos com ação antimicrobiana apresentam curvas de dose-resposta que mostram uma dependência sigmoidal com a concentração do peptídeo. Isso sugere que esses peptídeos se acumulam na superfície da vesícula até atingir uma concentração crítica, além da qual o vazamento aumenta cooperativamente. De uma forma geral os peptídeos mais eficientes como antimicrobianos, são também aqueles caracterizados pela maior eficiência em permeabilizar vesículas aniônicas do tipo PCPG 7030 e por baixas razões limite P/L. / Solitary wasps use their venoms to paralise prays to feed their larvae. A set of biologically active peptides, obtained from these venoms, have been investigated in relation to the conformational changes they undergo in anisotropic environments and their lytic activity on zwitterionic and anionic vesicles. These peptides are mast cell degranulators, present antimicrobial activities and most of them are not hemolytic. They are cationic, their chain length are 10 to 15 residues long, with high hydrophilic / hydrophobic ratio; they are linear and helical in membrane mimetic environments. We searched correlation between the lytic activity in vesicles of different compositions, monitored in fluorimetric experiments, and conformational changes, induced by varied mimetic media, monitored by circular dichroism. The results have been also correlated with peptides' physical-chemical parameters such as chain length, amidated or carboxylated C-terminal, net charge, influences on the helix macrodipole, hydrophobicity, hydrophobic moment and polar angle. We observed that these peptides present intense activity on model membranes, they interact preferentially with anionic bilayers, and their lytic activity is a cooperative process either in anionic or in zwitterionic vesicles. Exception made to Anoplin, all the other peptides that have antimicrobial activity present in their dose-response curves a sigmoidal dependence with the peptide concentration. This fact suggests that these peptides accumulate on the vesicles surface until they reach a threshold concentration, beyond which leakage increases cooperatively. As a general rule, the most efficient antimicrobial peptides are also those characterized by efficient permeabilization of anionic vesicles, namely PCPG 7030 and by small threshold P/L ratios.

Orientador: José Roberto Ruggiero / Banca: Pedro Geraldo Pascutti / Banca: José Maria Pires / Banca: Alexandre Suman de Araújo / Banca: Sabrina Thais Broggio Costa / Resumo: Peptídeos antimicrobianos são moléculas biologicamente ativas que, geralmente, tem as membranas fosfolipídicas como alvo primário. Resultados de diferentes técnicas experimentais têm sugerido que esses peptídeos permeabilizam as membranas pela formação de poros. Parte dos peptídeos caracterizados apresentam especificidade de disrupção para membranas de bactérias, em detrimento das membranas dos hospedeiros. Essa característica tem atraído a atenção da comunidade científica internacional, porque indica que estas moléculas podem ser modelos para o desenvolvimento de novos antibióticos, portanto o entendimento do mecanismo de ação, ou seja, do mecanismo de formação de poro, tem extrema importância. Simulações por Dinâmica Molecular foram produzidas para investigarmos o impacto que peptídeos antimicrobianos da família Mastoparano tem sobre membranas lipídicas modelo. Dois cenários foram explorados: (i) de baixa concentração peptídeo/lipídeo, P/L=1/128, que consistia de simulações fine-grained das interações de um peptídeo com uma bicamada pura de 128 lipídeos aniônicos (POPG) ou zwiteriônicos (POPC); (ii) de alta concentração, P/L=1/21, que abordava as interações de seis peptídeos com uma bicamada mista de 128 lipídeos POPC/POPG (1/1) usando uma modelagem coarse-grained. Tomando o peptídeo MP1 como caso paradigmático, verificamos que em baixo P/L é possível sugerir que sua característica seletiva surge da capacidade de coordenar e perturbar maior número de lipídeos em membrana aniônica comparada à neutra. Essa capacidade fica acentuada nas simulações com membrana mista, onde a atração dos lipídeos aniônicos pelos peptídeos catiônicos guiou a separação local e a formação de domínios de lipídeos aniônicos, o que facilitou o afinamento local da membrana e a formação de poro transmembrânico. Esses achados ajudam a explicar como peptídeos / Abstract: Antimicrobial peptides are biologically active molecules that, usually, have the phospholipid membranes as a primary target. Results from different experimental techniques have suggested these peptides permeabilize membranes by the pore formation. Part of the characterized peptides have specificity of disruption for bacterial membranes, instead of host membrane. This feature has attracted the attention of the international scientific community, because it indicates that these molecules can be models for the development of novel antibiotics, so understanding the mechanism of action, ie, the mechanism of pore formation, is extremely important. Molecular dynamics simulations were performed to investigate the impact of antimicrobial peptides from the Mastoparano family have on model lipid membranes. Two scenarios were explored: (i) of low peptide/lipid concentration, P/L=1/128, which consisted of fine-grained simulations of the interactions of a peptide with a pure bilayer of 128 anionic (POPG) or zwitterionic (POPC) lipids; (ii) of high concentration, P/L=1/21, which addressed the interactions of six peptides with a mixed bilayer of 128 POPC/POPG (1/1) lipids, using a coarse-grained modeling. Taking the MP1 peptide as a paradigmatic case, we found that in low P/L is possible to suggest that its selective feature arises of its ability to coordinate and disturb large number of lipids in the anionic membrane compared to neutral one. This ability is accentuated in simulations with mixed membrane, where the attraction of the anionic lipids by the cationic peptides led to the local segregation and formation of POPG lipid domains, which facilitated the local thinning of the membrane and the formation of transmembrane pore. These findings help to explain how short peptides, such as MP1, are able of forming pores in a membrane whose thickness is larger than the length of the peptide / Doutor

La moule de Méditerranée Mytilus galloprovincialis (Mollusque Bivalve), est un animal important tant au niveau écologique qu'économique. Comme tous les invertébrés, elle ne possède qu'un système d'immunité innée pour lutter contre les infections. Cependant, étant constamment exposée à une grande variété de microorganismes invasifs et potentiellement pathogènes, et existant depuis plus de 500 millions d'années, son système immunitaire paraît très efficace. C'est afin de mieux comprendre comment celui-ci fonctionne, que ces travaux concernant la structure des peptides impliqués dans la réponse immunitaire, ainsi que leur régulation, ont été entrepris. Ils ont permis (i) de déterminer que le niveau d'expression constitutive des gènes liés à l'immunité, ainsi que la nature et l'intensité de la régulation de leur expression, sont fortement dépendant de la saison et de l'origine géographique des moules ; (ii) de confirmer le rôle essentiel de la structure tridimensionnelle des peptides antimicrobiens (AMP) dans les activités biologiques ; (iii) de déterminer la structure complète de la mytimycine, peptide strictement antifongique, ainsi que de la cytokine MIF ; (iv) de confirmer l'existence d'un fort polymorphisme des ARNm codant les molécules effectrices de l'immunité au niveau individuel, intra et inter-populationnel ; (v) de déterminer que les niveaux d'expression des gènes liés à l'immunité dépendent des microorganismes injectés, ce qui suggère une reconnaissance/réponse spécifique; (vi) de démontrer l'existence d'une voie de transmission du signal fonctionnelle depuis des récepteurs membranaires de type Toll (TLR) jusqu'au facteur NF-κB, mais pas d'une voie de type IMD. Ainsi, la réponse immunitaire innée de la moule apparait extrêmement complexe, mettant en jeux des effecteurs polymorphes dont l'expression est modulée en fonction de la saison, de l'origine géographique et de façon spécifique en réponse à différents microorganismes. Par contre, la transcription de leurs gènes pourrait être sous le contrôle d'une seule voie de transmission du signal. / The Mediterranean mussel, Mytilus galloprovincialis (bivalve, mollusc), is an ecologically and economically essential animal. As other invertebrates, it possesses only an innate immune system to protect itself against infections. However, constantly exposed to a large variety of invasive and potentially pathogen microorganisms, and existing since more than 500 million years, its immune system seems very effective. To improve our understanding on such a system, present works were made concerning the structure and expression regulation of peptides involved in the immune response. They allowed (i) to determine that the constitutive expression levels of genes linked to immunity, as well as the nature and intensity of their expression regulation, are strongly dependent on the season and on the geographical origin of mussels; (ii) to confirm the crucial role of the three-dimensional structure of antimicrobial peptides (AMP) in biological activities; (iii) to determine the complete structure of mytimycine, a strictly antifungal peptide, as well as of cytokine MIF; (iv) to confirm the existence of an extended polymorphism of mRNA coding for the molecular effectors of immunity in individuals, within and between populations; (v) to determine that expression levels of genes linked to immunity are strongly dependent to the injected microorganisms, suggesting a specific recognition/ response; (vi) to demonstrate the existence of a functional signalling pathway from Toll-like receptors (TLR) to NF-κB factor, but not of an IMD-like pathway. In conclusion, the immune response of the mussel appeared extremely complex, involving polymorphic effectors expressed differently according to the season, the geographical origin, and specifically in response to different microorganisms. On the other hand, their gene transcription could be under the control of only one signal transmission pathway.

Peptídeos antimicrobianos, também chamados na literatura médica de AMPs (Antimicrobial Peptides), são agrupamentos de 12 a 50 aminoácidos que têm funções antimicrobianas e imunorregulatórias. Os AMPs são produzidos principalmente nos epitélios, destacando-se o epitélio intestinal, onde participam da homeostase da barreira intestinal. A Pancreatite Aguda (PA) é uma doença com altas taxas de mortalidade e morbidade, na qual frequentemente ocorre translocação de bactérias e endotoxinas para o epitélio intestinal e para a corrente sanguínea. A translocação bacteriana ocorre, principalmente, devido à falência da barreira intestinal. Em idosos, a PA tende a ser uma doença mais grave, porém os mecanismos que justificam essa maior gravidade ainda não são bem compreendidos. O objetivo deste trabalho é fazer um estudo da expressão gênica dos peptídeos antimicrobianos alfa-defensina 5 e 7 e da catelicidina CRAMP em ratos jovens e idosos submetidos a um modelo de Pancreatite Aguda. Ratos jovens e idosos foram submetidos à PA utilizando-se Ácido Taurocólico 2,5% e, após doze horas, foram sacrificados. Foi coletado 1cm do íleo terminal para realização de RTPCR para os genes das alfa-defensinas 5 e 7 e da catelicidina CRAMP. Adicionalmente, foram quantificados os níveis de mRNA das citocinas TNF-alfa e IL10. Através da técnica de Milliplex, foram dosadas no soro as citocinas IFN-y, IP-10, IL-10, IL-6, IL-18, TNF-alfa, IL-1beta e Leptina. Como resultado, detectou-se maior expressão gênica das alfa-defensinas 5 e 7 no grupo de animais idosos, na vigência de insulto pancreático, quando comparado a animais jovens na mesma condição. Com relação à CRAMP, não houve diferença significativa entre os grupos. Igualmente, obteve-se expressão significativamente maior de TNF-alfa no íleo terminal dos ratos idosos com Pancreatite Aguda. Interessantemente, as dosagens de citocinas séricas foram semelhantes entre os grupos, demonstrando que a diferença entre a resposta inflamatória de ratos idosos e jovens é localizada. Peptídeos antimicrobianos, portanto, devem apresentar um papel central na resposta inflamatória decorrente de injúria pancreática / Antimicrobial peptides, also called AMPs, are 12-50 amino acids molecules that have antimicrobial and immunoregulatory properties. AMPs are produced mainly in epithelial cells, and participate in the homeostasis of the intestinal barrier. Acute pancreatitis (AP) is a disease with high mortality and morbidity and presents a worst outcome when bacterial translocation occurs to the intestinal epithelium and to the bloodstream. Bacterial translocation occurs mainly due to the failure of the intestinal barrier. In the elderly, AP has a worst prognosis, but the mechanisms that justify that higher severity, compared to the young, are poorly understood. The objective of this work is to study the gene expression levels of antimicrobial peptides ?-defensins 5 and 7 and the cathelicidin CRAMP in young and elderly rats submitted to acute pancreatitis. Young and old rats received a taurocholic acid 2.5% injection in the pancreatic ductus and after twelve hours, were sacrificed. 1cm of the terminal ileum was collected for detection of AMPs gene expression levels. In addition, RT-PCR was performed to detect TNF-alfa and IL-10 mRNA levels. Using Milliplex technique, serum levels of IFN-gamma, IP-10, IL-10, IL-6, IL-18, TNF-alfa, IL-1beta and Leptin were measured. As a result, we detected significant higher mRNA levels of alfa-defensins 5 and 7 in the elderly (pancreatitis group). CRAMP gene expression was not significantly different between groups. TNF-alfa gene expression levels was significantly higher in old rats (pancreatitis group). Any difference in the cytokines serum levels could be detected when old and young rats in the pancreatitis groups were compared. We conclude that AMPs may play a pivotal role in the development of acute pancreatic injury