Bumble bees and other wild pollinators are crucial to the support of both natural and agricultural ecosystems. However, unprecedented declines of pollinator populations have been observed all over the world, raising concerns of a looming threat to both the human food supply, as well as sustainability of the biodiversity in local ecological niches. Though declines are well described, the cause behind these still evades scientists. Proposed contributors include anthropogenic-mediated environmental stress, including application of xenobiotics for pest control, and increase of pathogen diversity and abundance due to the shipment of infection human-managed colonies. This research examined these theories and attempted to quantify the threats they may pose. Through development of a chronic, oral toxicity experiment, susceptibility of all Bombus impatiens castes to clothianidin exposure was examined. This exposed a substantial increase in vulnerability of male bumble bees to realistic concentrations of neonicotinoid pesticides, highlighting the crucial need to examine all members of wild bumble bee life cycles before determining pesticide regulations. Additionally, sublethal effects on fitness-related foraging behaviors in Bombus impatiens were examined through development of a voluntary task switching assay. The results of this experiment suggest humoral immune stimulation, through pathogenic infection, leads to significant impairment of cognitive flexibility. Taken together, this data suggests that pesticides and pathogens are capable of causing severe detrimental effects, both lethally and sublethally, in wild bumble bees. I hope this data will eventually contribute to reassessment of environmental regulations and establishment of effective conservation strategies in order to sustain the critical populations of wild bumble bees.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-11-07T17:53:28Z No. of bitstreams: 1 StellaMarisdeFreitasLimaTese2018.pdf: 12066835 bytes, checksum: 9ad384b4f040333154c3598b59852583 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-11-07T17:53:52Z (GMT) No. of bitstreams: 1 StellaMarisdeFreitasLimaTese2018.pdf: 12066835 bytes, checksum: 9ad384b4f040333154c3598b59852583 (MD5) / Made available in DSpace on 2018-11-07T17:53:52Z (GMT). No. of bitstreams: 1 StellaMarisdeFreitasLimaTese2018.pdf: 12066835 bytes, checksum: 9ad384b4f040333154c3598b59852583 (MD5) Previous issue date: 2018-08-21 / Failure of endodontic therapy is mainly based on the persistence of the microorganism inside the root canal system, perpetuating periradicular pathologies. The need for endodontic reintervention reduces the success rate and turns the development of new therapies into a necessity. Host defense peptides (HDPs) have a therapeutic potential because of its characteristics as broad spectrum antimicrobial activity, immunomodulatory capacity and repair induction. The present study aimed to demonstrate HDPs HHC-10, LL-37 and synoeca-MP activities compared to the commonly used chemical agents Ca(OH)2, NaOCl and chlorhexidine (CHX) in in vitro and in vivo environments. In vitro analysis involved (1) antimicrobial assays for MIC/MBC/MFC determination, antibiofilm and synergistic activities (against Candida albicans, Enterococcus faecalis and Staphylococcus aureus), (2) hemolytic assay and determination of therapeutic index, (3) immunomodulation assays (from RAW 264.7 monocytes) and (4) osteoclastogenesis assays (from mouse bone marrow cells). In vivo assays were based on periradicular lesions induced and treated in rats with subsequent analysis by radiographic, tomographic and histological examinations. Results demonstrated effective antimicrobial activity of HHC-10 (except antibiofilm activity) and synoeca-MP besides NaOCl and CHX. Higher therapeutic index was observed for peptides (HHC-10 and synoeca-MP) with low or no hemolytic activity. Synergism analysis demonstrated better interaction between synoeca-MP and CHX. Immune assays from monocyte cultures demonstrated a pro-inflammatory activity of LL-37 and synoeca-MP and a pro- and anti-inflammatory effect of HHC-10 and Ca(OH)2. However, none of the agents tested reduced osteoclastogenesis in vitro. In in vivo endodontic treatment conditions, HHC-10, synoeca-MP and Ca(OH)2 significantly reduced periapical lesions, although only peptides were able to reduce inflammation in histological analysis. Therefore, HHC-10 and synoeca-MP demonstrated in vitro and in vivo potential for application in endodontic therapy. However, further researches are needed regarding the mechanism of action of HDPs and the possible therapeutic formulations for future analysis in animal models. / O insucesso da terapia endodôntica está baseado na persistência de microrganismos principalmente no interior do sistema de canais radiculares, perpetuando as patologias perirradiculares associadas à tal infecção. A necessidade de reintervenção endodôntica reduz o percentual de sucesso e, portanto, o surgimento de novas terapias é necessário. Dessa forma, peptídeos de defesa do hospedeiro (PDHs) possuem potencial terapêutico associado às suas características de atividade antimicrobiana de amplo espectro, capacidade imunomodulatória e indução de reparo. O presente estudo objetivou demonstrar a atividade dos PDHs HHC-10, LL-37 e synoeca-MP comparado aos agentes químicos comumente utilizados Ca(OH)2, NaOCl e clorexidina (CHX) em ambientes in vitro e in vivo. Para tanto, foram realizadas análises in vitro envolvendo: (1) ensaios antimicrobianos com determinação de MIC/MBC/MFC, atividade antibiofilme e atividade sinérgica (contra Candida albicans, Enterococcus faecalis e Staphylococcus aureus), (2) ensaio hemolítico e determinação do índice terapêutico, (3) ensaios de imunomodulação (a partir de monócitos RAW 264.7) e (4) ensaios osteoclastogênese (a partir de células da medula óssea de camundongos). Os ensaios in vivo foram conduzidos a partir das lesões perirradiculares induzidas e tratadas em ratos com subsequente análise por exames radiográficos, tomográficos e histológicos. Assim, os resultados demonstraram efetiva atividade antimicrobiana dos peptídeos HHC-10 (exceto atividade antibiofilme) e synoeca-MP além do NaOCl e CHX. Observou-se maiores índices terapêuticos para os peptídeos (HHC-10 e synoeca-MP) com baixa ou nenhuma atividade hemolítica. As análises de interação demonstraram combinação sinérgica necessitando de menor concentração a partir da interação entre synoeca-MP e CHX. A partir da cultura de monócitos, observou-se uma atividade pró-inflamatória a partir dos peptídeos LL-37 e synoeca-MP e um efeito pró- e anti-inflamatório do peptídeo HHC-10 e do Ca(OH)2. No entanto, nenhum dos agentes testados reduziram a osteoclastogênese in vitro. Já em condições de tratamento endodôntico in vivo, os PDHs HHC-10, synoeca-MP e o Ca(OH)2 reduziram significativamente lesões periapicais, apesar de somente os peptídeos serem capazes de reduzir inflamação em análises histológicas. Portanto, os peptídeos HHC-10 e synoeca-MP demonstraram potencial in vitro e in vivo para aplicação na terapia endodôntica. No entanto, faz-se necessário maiores investigações sobre o mecanismo de ação dos mesmos e possíveis formulações terapêuticas para futuros testes em modelos animais.

Orientador: Leandro Mantovani de Castro / Resumo: A Olivancillaria urceus é uma das espécies de gastrópode marinho mais abundante na região subtropical da costa sudeste brasileira, frequentemente capturada na pesca de arrasto de fundo do camarão sete barbas. Nos últimos anos, a investigação química em moluscos marinhos têm revelado informações sobre uma variedade de compostos de interesse clínico, em especial os peptídeos, apresentando propriedades farmacológicas distintas, como por exemplo, ação antimicrobiana. Neste contexto, o objetivo deste trabalho foi o isolamento e caracterização de peptídeos que existam naturalmente nos tecidos do gastrópode marinho Olivancillaria urceus utilizando-se de um método específico de extração, seguido de fracionamento por cromatografia líquida em coluna C18, ensaio de atividade antimicrobiana contra os microorganismos: M. luteus, E. coli e C. albicans, e identificação das frações com atividade por espectrometria de massas. Nossas análises identificaram uma fração do extrato peptídico, denominada 18-M-3, com atividade antimicrobiana contra a bactéria Micrococcus luteus, apresentando uma concentração média de 2,18 mg/ml. A análise por espectrometria revelou tratar-se de uma fração complexa, constituída principalmente por um conjunto de peptídeos descritos pela primeira vez, fragmentos de proteínas citosólicas e nucleares conservados em diferentes espécies de moluscos. Estes resultados abrem novas perspectivas sobre o potencial farmacológico de extratos O. urceus. / Abstract: Olivancillaria urceus is one of the most abundant marine gastropod species in the subtropical region of the Brazilian southeast coast, often caught in shrimp trawling. In recent years, chemical research in marine molluscs has revealed information on a variety of compounds of clinical interest, especially peptides, exhibiting distinct pharmacological properties, such as antimicrobial action. In this context, the aim of this work was the isolation and characterization of peptides that exist naturally in the tissues of the marine gastropod O. urceus using a specific extraction method, followed by liquid chromatography fractionation on C18 column, antimicrobial activity tests against microorganisms M. luteus, E. coli e C. albicans, and identification of the fractions with activity by mass spectrometry of. Our analyzes identified a fraction of the peptide extract, designated 18-M-3, with antimicrobial activity against the bacterium Micrococus luteus, presenting an average concentration of 2.18 mg / ml of peptides. Spectrometric analysis revealed a complex fraction consisting mainly of a set peptides, fragments of cytosolic and nuclear proteins conserved in different species of mollusks. These results open new perspectives about the pharmacological potential of O. urceus extracts. / Mestre

Os insetos desenvolveram um sistema imune eficiente contra parasitas e patógenos, que compreende a resposta celular e a humoral. Os mecanismos celulares envolvem a fagocitose e a encapsulação pelos hemócitos, enquanto que as respostas humorais incluem a ativação da Profenoloxidase, e a síntese pelo corpo gorduroso dos peptídeos antimicrobianos, que são liberados na hemolinfa. Duas vias de sinalização intracelular, Toll e Imd, controlam a expressão dos genes codificadores dos peptídeos antimicrobianos. A análise do Genoma da abelha Apis mellifera permitiu a identificação dos genes dessas vias. No entanto, pouco se conhece do mecanismo de resposta imune nessas abelhas. Desta maneira, nos propusemos analisar a transcrição de genes efetores da resposta imune (abaecina, hymenoptaecina, defensina, transferrina, profenoloxidase), assim como os genes integrantes das vias de sinalização, tais como os genes de reconhecimento de microorganismos (PGRP, GNBP) e ainda, os de sinalização (cactus, relish, dorsal 1-B). Avaliamos também possíveis proteínas implicadas na resposta imune, como as proteínas de estocagem Vitelogenina, Hexamerina 70a, Lipoforina I/II e Lipoforina III. Finalmente, analisamos o efeito da nutrição e do envelhecimento sobre a imunidade em abelhas. Para análise da expressão dos genes das vias de sinalização, as abelhas foram infectadas com bactérias Serratia marcescens ou Micrococcus luteus por injeção ou via alimentação. A infecção com esses microorganismos provocou a transcrição de peptídeos antimicrobianos e de transferrina em altas quantidades após 3 e 12 horas de tratamento, além da alteração na quantidade de transcritos de outros genes. O papel dos genes profenoloxidase e dorsal na imunidade, descritos como codificadores de importantes proteínas em outros insetos, foi avaliado através da metodologia de silenciamento gênico por RNA de interferência. Observamos a diminuição da transcrição do gene alvo, mostrando a eficiência da metodologia. No entanto, a simples injeção de um RNA de fita dupla foi capaz de ativar o sistema imune de abelhas. Este efeito contribuiu para a dificuldade de atribuição do papel da Profenoloxidase na imunidade de abelhas. Contudo, os resultados de silenciamento de dorsal e suas isoformas, nos levaram a considerar que dorsal 1-A ou dorsal 2 participam da via de sinalização intracelular para produção de peptídeos antimicrobianos, principalmente de defensina. Em relação às proteínas de estocagem, tanto a quantidade de transcritos quanto de proteínas diminui após infecção com bactérias, indicando que estas proteínas estão envolvidas de alguma forma no processo de imunidade em abelhas. Além disso, consumo de alimentos ricos em proteína aumentou os níveis de transcritos das proteínas de estocagem, o que muito provavelmente favorece a manutenção da capacidade de resposta imune de abelhas. O efeito do envelhecimento no declínio da imunidade foi analisado em abelhas nutridoras (novas) e forrageiras (velhas) de uma colônia típica. Além disso, foram utilizadas abelhas de uma colônia single-cohort, que eram de uma mesma idade, mas algumas eram nutridoras, enquanto outras eram forrageiras. Todas as abelhas, independentemente da idade ou comportamento, foram capazes de ativar o sistema imune após infecção pela bactéria S. marcescens. No entanto, as abelhas com o comportamento de forrageira, independentemente da idade, sempre foram mais susceptíveis a infecções que as nutridoras. Este fato se deve, muito provavelmente, às diferenças fisiológicas entre essas abelhas, que proporciona às nutridoras maior competência à sobrevivência. / Insects have developed an efficient immune system against parasites and pathogens, which is comprised of both cellular and humoral responses. The cellular mechanisms involve phagocytosis and encapsulation by hemocytes, whereas the humoral responses include activation of prophenoloxidase and synthesis of antimicrobial peptides by the fat body, which are released into the hemolymph. Two signaling pathways, Toll and Imd, control the expression of genes encoding antimicrobial peptides. Genome-wide analyses of the honey bee, Apis mellifera, have identified predicted genes for these signaling pathways. However, immune response mechanisms in honey bees were not yet in depth studied. We analyzed the transcription of effector genes (abaecin, hymenoptaecin, defensin, transferin, prophenoloxidase), as well as other immune genes, such as pathogen recognition genes (PGRP, GNBP) and signaling genes (cactus, relish, dorsal 1- B). We also investigated the role of the storage proteins Vitellogenin, Hexamerin 70a, Lipophorin I/II and Lipophorin III in the honey bee immunity. Finally, we analyzed the effect of nutrition and aging on honey bee immunity. Gene expression of signaling pathway components was assessed in honey bees that had been infected with the bacteria Serratia marcescens or Micrococcus luteus through injection or oral challenge. Honey bees infected with these microorganisms had strong up-regulation of antimicrobial peptide genes and of transferin, and also other changes in transcript abundance after 3 and 12 hours of challenge. The roles of prophenoloxidase and dorsal in the immune response, described as genes encoding important proteins in other insects, were also investigated. In this case we used RNA interference (RNAi) to silence the expression of these genes. RNAi efficiently silenced the target genes. However, injection of doublestranded RNA in honey bees induced a reaction by the immune system. This made it difficult to determine the role of prophenoloxidase in honey bee immunity. Yet, silencing of dorsal and its isoforms led us to consider dorsal 1-A or dorsal 2 as members of the signaling pathways that produce antimicrobial peptides, especially defensin. The abundance of storage proteins transcripts and proteins was lower in infected bees than in controls, giving evidence that these proteins participate in the immune process in honey bees. Moreover, protein consumption caused up-regulation of genes encoding storage proteins, which may favor the maintenace of the immune response capacity. The effect of aging on decline in immunity was analyzed in (young) nurse bees and (old) foragers from normal free-flying colony. We also examined bees from a single-cohort colony, in which all individuals were at the same age; but some were nursing, while others were foraging. All the bees, independent of age or behavior, were able to activate the immune system after infection with S. marcescens. However, foragers, independent of age, were always more susceptible to infections than were nurse bees. This is probably due to physiological differences between bees, which confers to the nurses more competence to survivorship.

Lors de la prise d'un repas sanguin par le moustique Aedes (Ae) aegypti, le virus de la dengue (DENV) est transmis à l'homme avec la salive du moustique. Ce mélange complexe est en partie déposé dans le compartiment cutané extravasculaire lors de la piqûre de moustique. Il est donc important de prendre en compte la triade virus-vecteur-hôte vertébré dans les mécanismes de transmission de ce virus à l'hôte vertébré. Des analyses de génomique et protéomique des glandes salivaires d'Ae. Aegypti infectées ou non par le DENV nous ont permis de mettre en évidence dans les glandes salivaires de moustiques infectés, la surexpression d'un gène codant pour un peptide antimicrobien (PAM) cationique. Nous avons démontré, que ce PAM possède une activité antibactérienne et antivirale contre les virus de la dengue et du chikungunya. Nos travaux soulignent l'importance chez les invertébrés, du compartiment « glandes salivaires » dans la réponse immunitaire innée du moustique. Nous avons également démontré que les kératinocytes humains sont des cellules permissives pour le DENV et que l'infection de ces cellules stimule la réponse immunitaire innée antivirale. Nos travaux démontrent que des extraits de glandes salivaires d'Ae. Aegypti augmentent l'infection du virus de la dengue dans les kératinocytes humains. Nous avons par la suite identifié une protéine salivaire d'Ae. Aegypti (34kDa), qui augmente l'infection du DENV en supprimant la production d'interféron. L'ensemble de ces travaux ont permis de contribuer aux connaissances sur la transmission du DENV, mais aussi d'identifier de nouvelles cibles potentielles pour le contrôle de la réplication virale. / Dengue virus (DENV) transmission is initiated when a blood-feeding Aedes (Ae) aegypti mosquito injects saliva, together with the virus, into the epidermis of its mammalian host. Studies of DENV should, therefore, take into account the triad virus-vector-vertebrate host. We have used functional genomic and proteomic analyses, of the salivary glands of female Ae. Aegypti, to demonstrate that this compartment harbors a potent immune response against DENV, represented by the production of an antimicrobial peptide (AMP). This AMP was found to exert, in addition to its anti-bacterial activity, an anti-viral activity against DENV and Chikungunya. Our data demonstrate, for the first time, the permissiveness of human epidermal keratinocytes to DENV infection. Remarkably, DENV replication in keratinocytes contributes to the establishment of anti-viral innate immunity that might occur shortly after the mosquito bite. To investigate the role of Ae. aegypti saliva in DENV transmission to man, primary human keratinocytes were infected with DENV in the presence of Ae. aegypti salivary gland extract. We show that Ae. aegypti saliva enhances dengue virus infection of human keratinocytes by suppressing innate immune responses. Furthermore, we have found a 34-kDa protein, in the saliva of Ae.aegypti, that strongly enhances DENV replication by suppressing type-I IFN production. This study provides new insights into the role of Ae. aegypti salivary glands and saliva in DENV transmission. The data presented here provide novel targets for the control of DENV replication in mammalian hosts.

Antimicrobial peptides (AMPs) are small proteins produced by epithelial surfaces, which have broad-spectrum antimicrobial and immunomodulatory activities. In the lung, skin and alimentary tract AMPs are known to be important in infectious and inflammatory conditions. Far less is known regarding the role of AMPs within the female reproductive tract, but as infection and inflammation are causes of preterm labour, AMPs may have a key function in maintain and protecting pregnancy. The major groups of human AMPs include the human beta defensins (HBDs), two antileukoproteinases (secretory leukocyte protease inhibitor (SLPI) and Trappin-2/Elafin), and the human cathelicidin hCAP18/LL-37, with several studies identifying their presence at sites throughout the reproductive tract. The cervix in pregnancy is positioned between the upper genital tract containing the developing fetus and the lower tract where infections usually arise. I hypothesise that AMPs are fundamental to mucosal immune defence of the cervix in pregnancy, preventing ascending infection and excessive inflammation that can cause preterm labour. This thesis focused on the human cathelicidin hCAP18/LL-37 and its role within the cervix and vagina. The aims of this thesis were to; investigate the inflammatory effects of LL-37 from cervical and vaginal derived epithelial cells and determine the pathways and receptors in which LL-37 may elicit its effects and how production may be regulated; investigate the role of CRAMP in a mouse model of preterm birth; and determine the production of AMPs by the pregnant cervix whilst investigating the relationship between AMP concentrations in cervicovaginal secretions and preterm labour. The inflammatory effect of LL-37 was investigated using cell lines derived from endocervical, ectocervical and vaginal epithelium. The study of these cell lines suggests divergent responses of cervical and vaginal epithelial cells. LL-37 mediated induction of IL-8 and IL-6 production from endocervical epithelial cells was observed in a dose-dependent and time-dependent manner, whilst ectocervical and vaginal cells also respond to treatment with LL-37 through IL-8 and IL-6 production. To determine a possible mechanism of action of LL-37 on IL-8 and IL-6 in the three cell lines, inhibitors against MAPK cascades, ERK, p38 MAPK and JNK, and known LL-37 receptors were investigated. In endocervical cells LL-37 mediated IL-8 occurs via activation of unidentified GPCRs, whilst in ectocervical cells this effect on IL‐8 and IL-6 is via the activation of ERK and p38 MAPK cascades. The mechanism by which LL-37 induces IL-8 secretion in vaginal epithelial cells remains unknown. Expression of LL-37 was shown to be mediated by vitamin D3 in vitro in cervical and vaginal epithelial cells. However when this relationship was investigated in vivo, using matched serum and cervicovaginal secretions from woman at early pregnancy, no correlation was observed between circulating vitamin D and cervicovaginal or circulating hCAP18/LL-37. However, the majority of women in this study reported with insufficient levels of vitamin D, which may effect the relationship observed with hCAP18/LL-37. Using a mouse model of LPS-induced preterm labour, to mimic the presence of intrauterine infection bacterial infection, I aimed to characterise the role of CRAMP, the mouse orthologue of hCAP18/LL-37, in the lower inflammatory and immune response that results in preterm labour. Wild type C57Bl/6J mice receiving an intrauterine injection of LPS deliver prematurely, within 24 hours of injection. However mice deficient in CRAMP (Camp -/-) receiving an intrauterine injection of LPS deliver significantly later and have a non-significant increase in pup survival compared to wild type C57Bl/6J mice. Cervical tissue collected post partum showed no difference in inflammatory markers between wild type C57Bl/6J and Camp -/- mice, however there was increased expression of the neutrophil chemoattractant marker, Cxcl5, and the neutrophil marker, Ngp in Camp -/- mice. In the lower genital tract, levels of antimicrobial peptides were determined in samples of cervicovaginal secretions collected from pregnant women. AMPs, hCAP18/LL-37, HBD-2 and SLPI were found in cervicovaginal secretions, and levels of hCAP18/LL-37 were increased in women with the common vaginal infection bacterial vaginosis. However no relationship was identified between the concentration of AMPs and preterm birth in this study. This work has shown that the lower genital tract, where infections that are associated with preterm labour originate, expresses the human cathelicidin hCAP18/LL-37. It may play an important role in modulating the immune response to invading infection associated with preterm labour. Further investigation of these responses may increase understanding of the physiology and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-06-07Bitstream added on 2014-06-13T19:40:48Z : No. of bitstreams: 1 lopesfilho_fc_dr_sjrp_parcial.pdf: 183424 bytes, checksum: 8601f6f72a7635a3c9ada79092a5873d (MD5) Bitstreams deleted on 2015-06-25T13:01:06Z: lopesfilho_fc_dr_sjrp_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:24Z : No. of bitstreams: 1 000694954_20160706.pdf: 183130 bytes, checksum: 1526b9f9e1347a4fb71fe218102cf0ba (MD5) Bitstreams deleted on 2016-07-25T13:17:36Z: 000694954_20160706.pdf,. Added 1 bitstream(s) on 2016-07-25T13:18:45Z : No. of bitstreams: 1 000694954.pdf: 1071220 bytes, checksum: ead8820e5de7c1e29fdd2ec0459005b1 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / 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 / 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

Em Triatoma infestans ainda não há pesquisas de isolamento de Peptídeos Antimicrobianas e como têm contato muitos microrganismos acreditamos que eles produzem esses componentes antimicrobianos. Neste estudo identificamos quatro peptídeos antimicrobianos principais. Os Tin-TK-I e Tin-TK-II que são similares com Taquicininas de insetos, não são hemolíticos e ativos contra três bactérias e três fungos, e são capazes de lisar membranas. Tin-TK-I é degradado por aminopeptidases e tem estrutura randômica. Tin-TK-II é degradado por carboxipeptidases e tem estrutura de hélice 310. A Triastina que é similar a uma proteína cuticular de T. infestans, é ativa contra duas bactérias e três fungos, não hemolítica, forma uma hélice 310, lisa membranas, e sofre ação de carboxi e aminopeptidases. E o Triatogênio que é similar ao fibrinopeptideo-A humano, e possui um análogo. É ativo contra três bactérias e seis fungos, e seu análogo contra três bactérias e três fungos. Não são hemolíticos, formam poros em membranas, formam alfa-hélices e são degradados por aminopeptidases. / Regarding new researches with Antimicrobial Peptides in triatomines, none have been developed with T. infestans, and due to its survival in a highly infectious habitat, we believe that it products antimicrobial molecules. On this study, four main AMPs have been identified. Tin-TK-I and II that were similar to Tachykinin-like proteins from insects, they were not hemolytic, and were active against three bacteria and three fungi on the antimicrobial assay. Tin-TK-I presents a random structure and was degraded by aminopeptidases, as Tin-TK-II presents a helix 310 structure and was affected by carboxipeptidases. Both of them can disrupt negatively charged membranes. Triastin that was similar to a cuticular protein from T. infestans, was active against two bacteria and three fungi, had no hemolytic activity, presents a helix 310 structure and can disrupt negatively charged membranes. Triatogen was similar to the human fibrinopeptide A, and it has an analogue. Triatogen was active against three bacteria and six fungi, and its analogue was active against three bacteria and three fungi. They were not active against human erythrocytes, both were degraded by aminopeptidases. They can generate pores on membranes and both have alfa-helix structure.

Peptídeos antimicrobianos são importantes componentes do sistema imune de vertebrados e invertebrados. Neste trabalho purificamos e caracterizamos quatro moléculas presentes na hemolinfa da aranha Acanthoscurria gomesiana: 1) theraphosinina, peptídeo de 4052,5 Da purificado do plasma, apresenta atividade anti-Micrococcus luteus e não apresenta similaridade com outros peptídeos. A partir dos hemócitos foram purificados: 2) mygalomorphina, um peptídeo de 415,9 Da com atividade anti-Escherichia coli. Sua atividade está relacionada à produção de H2O2 pois é inibida por catalase; 3) gomesina, um peptídeo de 2270,4 Da que apresenta alta similaridade com taquiplesinas e protegrinas. Apresenta amplo espectro de atividade contra bactérias, leveduras, fungos e Leishmania; 4) acanthoscurrina, um peptídeo rico em glicina, que apresenta duas isoformas com 10132,4 e 10249,1Da. Este peptídeo tem atividade contra E. coli e Candida albicans e apresenta grande similaridade com proteinas antifúngicas de insetos e também com proteínas relacionadas com a defesa em plantas. / Antimicrobial peptides are important components of the vertebrates and invertebrates immune system. In this work we purified and characterized four molecules from Acanthoscurria gomesiana spider hemolimph: 1) theraphosinin, a 4,052.5 Da peptide purified from plasma with anti-Micrococcus luteus activity. It does not show similarity with any other invertebrate immune peptides. From the hemocytes three peptides have been purified: 2) mygalomorphin, a peptide with 415.9 Da, which shows anti-Escherichia coli activity. This activity is inhibited by catalase, therefore it may be, related to the H2O2 production; 3) gomesin, a peptide with 2,270.4 Da, that shows high similarity with tachyplesins and protegrins. It have large activity spectrum against bacteria, yeast, fungi and Leishmania; 4) acanthoscurrin, a glycine-rich peptide that shows two isoforms of 10,132.4 and 10,249.1 Da. This peptide has activity against E. coli and Candida albicans and shows high similarity with antifungal proteins of insects and plants defense proteins.

The inhibitory effects on Escherichia coli ATPase activity by two venom peptides, lasioglossin II and mastoparan B. Membrane bound F1FO ATP synthase was isolated from E. coli strain pBWU13.4/DK8 and treated with varied concentrations of lasioglossin II and mastoparan B. Lasioglossin II caused very low inhibition of ATPase activity, but the inhibition profile of mastoparan B was suggestive of an interesting biological effect. A relatively shorter total length, a smaller net positive charge, and a reduced amphipathic character of both peptides, as compared to previously tested antimicrobial peptides, may account for the limited degree of inhibition observed in the present study.