Peptídeos antimicrobianos (PAMs) fazem parte de um dos mecanismos da imunidade inata contra infecções. A microplusina é um PAM de 10.204 Da, isolado da hemolinfa livre de células e dos ovos do carrapato Rhipicephalus (Boophilus) microplus. É um PAM aniônico em pH fisiológico, possui seis resíduos de cisteína, com formação de três pontes dissulfeto, além de sete resíduos de histidina concentrados principalmente na sua porção C-terminal. O presente trabalho teve como objetivo investigar o mecanismo de ação antimicrobiana da microplusina. A microplusina recombinante é ativa contra várias bactérias Gram-positivas e fungos, porém não apresenta atividade contra bactérias Gram-negativas. Para avaliar o seu mecanismo de ação, foram utilizados dois modelos: a bactéria Micrococcus luteus e o fungo Cryptococcus neoformans. A microplusina é bacteriostática contra M. luteus e apresenta localização intracelular na bactéria. Além disso, observamos que a microplusina liga cobre e que a adição deste metal ao meio de cultivo reduz sua atividade antibacteriana. Bactérias M. luteus pré-incubadas com microplusina retomam o seu crescimento quando cobre é adicionado ao meio. Estes dados indicam que a atividade da microplusina está relacionada à sua habilidade de depletar cobre do meio extra ou intracelular, sugerindo um efeito nutricional para o peptídeo. A microplusina apresenta estrutura terciária com cinco a-hélices e sua ligação ao cobre não induz mudanças conformacionais. Observou-se que as histidinas 1, 2 e 74 da microplusina podem estar envolvidas na formação de um sítio de ligação ao cobre. Quanto à C. neoformans, verificou-se que a microplusina inibe a melanização do fungo, um fator de virulência catalisado pela lacase, uma enzima cobre-dependente. Entretanto, a microplusina não afeta a atividade da lacase, nem sua expressão gênica. O peptídeo também não inibe a auto-polimerização de substratos fenólicos que levam à melanização. Sendo assim, mais estudos são necessários a fim de avaliar o mecanismo pelo qual a microplusina inibe a melanização. Adicionalmente, a microplusina afeta a viabilidade do fungo e reduz o tamanho de sua cápsula, outro importante fator de virulência. As atividades da microplusina sobre C. neoformans sugerem o seu potencial terapêutico. Experimentos in vivo com modelo murino, mostraram que a microplusina reduz o processo inflamatório e a viabilidade de C. neoformans nos pulmões, indicando que em condições otimizadas, o peptídeo pode atuar no controle de infecções. / Antimicrobial peptides (AMPs) take part of innate immune mechanisms against infections. Microplusin is a 10,204 Da AMP, isolated from cell-free hemolymph and eggs of the tick Rhipicephalus (Boophilus) microplus. It is an anionic AMP at physiological pH, with six cysteine residues forming three disulfide bridges and seven histidine residues clustered mainly at the carboxy end portion. The goal of the present work was investigate the antimicrobial action mechanism of microplusin. Recombinant microplusin is active against Gram-positive bacteria and fungi, however, no activity is detected for Gram-negative bacteria. Two models were used to evaluate the action mechanism of microplusin: the bacteria Micrococcus luteus and the yeast Cryptococcus neoformans. Microplusin is bacteriostatic against M. luteus and its localization is intracellular for these bacteria. Moreover, microplusin binds copper and the addition of this metal into the medium reduces its antibacterial activity. M. luteus bacteria pre-treated with microplusin recover its growth when copper is added. These data indicate that microplusin activity is related to its ability to deplete copper present in the extracellular or intracellular environment, suggesting a nutritional effect. Microplusin presents a tertiary structure with five a-helix and the copper binding does not induce conformation changes. In addition, it was observed that histidines 1, 2 and 74 from microplusin may be involved in the formation of a copper binding site. About C. neoformans, it was verified microplusin inhibits its melanization, a virulence factor catalyzed by laccase, a copper dependent enzyme. However, microplusin does affect neither laccase activity nor its gene expression. The melanization caused by auto-polymerazation of phenolic substrates, is also not inhibited by microplusin. Hence, additional studies are required to evaluate the mechanism by which microplusin inhibits melanization. In addition, microplusin also affects the fungi viability and reduces the capsule size, another important virulence factor.The microplusin activities against C. neoformans suggest its therapeutic potential. In vivo experiments with murine model showed that microplusin reduces the inflammation and the viability of C. neoformans in the lungs, indicating that, in optimized conditions, the peptide may act in the infection control.

Submitted by NOALA VICENSOTO MOREIRA MILHAN null (noalinha@gmail.com) on 2017-03-14T15:32:19Z No. of bitstreams: 1 TESE_FINAL_biblioteca_pdf_com ficha..pdf: 9569489 bytes, checksum: 1675ea8facc1f735605660b0b01b8cad (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-20T22:29:35Z (GMT) No. of bitstreams: 1 milhan_nvm_dr_sjc.pdf: 9569489 bytes, checksum: 1675ea8facc1f735605660b0b01b8cad (MD5) / Made available in DSpace on 2017-03-20T22:29:36Z (GMT). No. of bitstreams: 1 milhan_nvm_dr_sjc.pdf: 9569489 bytes, checksum: 1675ea8facc1f735605660b0b01b8cad (MD5) Previous issue date: 2017-01-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O peptídeo LL-37 (catelicidina derivada de humano), é liberado por algumas células humanas e capaz de neutralizar os tecidos com lipopolissacarídeo (LPS), além de atrair células da polpa, e induzir a angiogênese, características que o tornam um possível adjunto para a regeneração do complexo dentino-pulpar. O objetivo desse trabalho foi avaliar in vitro a biocompatibilidade do peptídeo LL-37 nas concentrações de 5 e 10 μg/mL, e sua possível atuação na diferenciação de células-tronco da polpa dentária (DPSC) para odontoblastos- like. Com esse propósito, foram avaliados: (a) a citotoxicidade, pelo teste MTT; (b) a genotoxicidade, através do ensaio do micronúcleo; (c) a produção e quantificação de óxido nítrico; (d) as fases do ciclo celular, por citometria; (e) a expressão de alguns genes associados à formação de tecido mineralizado, através do teste qRT-PCR; (f) o conteúdo de proteína total; (g) a atividade de fosfatase alcalina (ALP); e (h) a produção de sialofosfoproteína dentinária (DSPP), pelo ensaio imunoenzimático ELISA. Foi observado que as concentrações de 5 e 10 μg/mL de LL-37 não foram citotóxicas e ainda aumentaram, em geral, a viabilidade celular (p<0,05), sendo que os maiores valores de absorbância foram observados no 3° dia de contato. As concentrações testadas também não induziram genotoxicidade, após 7 dias de contato, tendo sido genotóxico apenas o grupo controle positivo (EMS) (p<0,05). Ainda, não foi observado diferença estatisticamente significativa na produção de nitrito, pelas células expostas ao LL-37 após 7 dias, em ambas as concentrações. A análise do ciclo celular, evidenciou maior porcentual de células na fase G0/G1, em todos os grupos (p<0,05). Quando estes foram comparados, foi observado maior quantidade de células na fase G0/G1 na concentração de 10 μg/mL de LL- 37 comparada ao grupo controle (p<0,05). Por outro lado, o grupo controle exibiu mais células na fase G2 e em mitose (M) que os grupos tratados com 5 e 10 μg/mL de LL-37 (p<0,05), e mais células na interfase (S) que o grupo tratado com 10 μg/mL de LL-37 (p<0,05). A análise da expressão gênica demonstrou que não houve aumento de expressão dos genes fosfatase alcalina, osteocalcina, osteopontina e Runx2 após tratamento com ambas as concentrações do peptídeo, no 3° dia. Além disso, não foi observado diferença estatisticamente significativa na ALP nos grupos tratados e controle, após 3 e 14 dias, enquanto o conteúdo de proteína total foi maior aos 14 dias nos grupos tratados com LL-37 (p<0,05). Ainda, aos 3 dias, a produção da proteína DSPP foi maior no grupo tratado com 10 μg/mL de LL-37 (p<0,05). Com base nesses resultados, pode-se concluir que o LL-37 é biocompatível nas concentrações testadas nesse trabalho, e ainda aumenta o número de células viáveis, principalmente em período inicial. Além disso, aos 3 dias, na concentração de 10 μg/mL, ele retarda o ciclo celular e aumenta a expressão da proteína DSPP, além de aumentar a síntese proteica aos 14 dias, o que indica que esse peptídeo pode desempenhar algum tipo de função na diferenciação odontoblástica. / The LL-37 peptide (human derived cathelicidin) is released by some human cells and able of neutralizing the tissues that present lipopolysaccharide (LPS), as well as, attracts pulp cells and induces angiogenesis; characteristics that makes it a possible adjunct for regeneration of the dentin-pulp complex. The aim of this study was evaluate in vitro the biocompatibility of LL-37 in the concentrations of 5 and 10 μg/mL, and its possible performance in the differentiation of dental pulp stem cells (DPSC) into odontoblasts-like cells. For this purpose, it was evaluated: (a) the cytotoxicity by MTT assay; (b) the genotoxicity by the micronucleus test; (c) the production and quantification of nitric oxide; (d) the cell cycle, by flow cytometry; (e) the expression of genes associated with the mineralization by qRT-PCR; (f) the total protein content; (g) the alkaline phosphatase activity (ALP); and (h) the production of dentine sialofosfoprotein (DSPP) by indirect enzyme-linked immunosorbent assay (ELISA). It was observed that the concentrations of 5 and 10 μg/ml of LL-37 were not cytotoxic, in addition to they increased, in general, the cell viability (p<0,05). Moreover, higher absorbance values were observed on 3rd day of contact. After 7 days, the tested concentrations also did not induce genotoxicity, (p<0,05); only the positive control group (EMS) was genotoxic (p<0.05). Furthermore, there was not statistical significance in the nitrite production by the cells exposed to LL-37 for 7 days, in both concentrations. The cell cycle test showed higher percentage of cells in the phase G0/G1 in all groups (p<0.05). When they were compared, it was noticied that concentration of 10 ug/ml of LL-37 arrested the cells in G0/G1 compared to the control group (p<0.05). On the other hand, the control group, exhibited higher amount of cells in G2 and mitosis (M) than the others (p<0.05) and also higher number of cells in interfase (S) than the group treated with 10 μg/mL of LL-37 (p<0.05). On the 3rd day, the analysis of gene expression demonstrated no increase in the expression of the genes alkaline phosphatase, osteocalcin, osteopontin and Runx2, after treatment with both peptide concentrations. Furthermore, it was not observed statistical significance in the ALP in the treated and control groups after 3 and 14 days, while total protein content was higher in the groups treated with LL-37, at 14 days (p<0.05). On the 3rd day, the production of DSPP protein was higher in the group treated with 10 μg/mL of LL-37 (p<0.05). Based on these results, it can be concluded that LL-37 is biocompatible at these concentrations and increases the number of viable cells, especially in the initial period. Moreover, on the 3rd day, the concentration of 10 μg/mL arrests the cell cycle, and increases the expression of DSPP protein, in addition to raising the protein content at 14 days, which indicates that this peptide may present some kind of function in the odontoblastic differentiation.

The intestinal mucosa is made up of complex secreted mucus layer consist of mainly mucin 2 (MUC2) and antimicrobial components that defend the underlining cellular barrier from intrusion by luminal microbiota and toxins. In inflammatory bowel diseases (IBD), the mucosal integrity is compromised. This can result from a combination of altered host genetics, gut immune responses and environment factors. However, it is the presence of intestinal bacteria that is central to the pathogenesis of IBD. As part of the dynamic gut microbial flora, mucolytic bacteria produce a wide range of glycosidases that are able to remove the outer oligosaccharide chains of MUC2, which allow other luminal bacteria to further degrade the mucin. We hypothesised that increased mucolytic bacteria will cause excessive degradation of the mucus layer, which in turn, allow more luminal bacteria to be in close proximity to the underlining epithelial cells resulting in inflammation. Consistent with our group’s previous semi-quantitative bacterial 16S rRNA gene clone library analysis, we found increased Ruminococcus gnavus in non-inflamed ulcerative colitis (UC) mucosa. R. gnavus was previously isolated by others based on its mucolytic property. In this study, we quantify total mucosa-associated bacteria and mucolytic bacteria, namely, R. gnavus, R. torques, Akkermansia muciniphila and bifidobacteria. We were able to show quantitatively that total mucosa-associated bacteria were increased in IBD. There was also a population shift in the mucosa-associated mucolytic bacteria, which were increased overall. There was significantly more R. gnavus in non-inflamed IBD biopsies. For the first time, we were also able to demonstrate that R. gnavus can degrade human MUC2 in vitro. To examine whether the numerical association of R. gnavus in IBD does have functional influence on intestinal inflammation and Paneth cell antimicrobial peptide gene expression, we fed mice with R. gnavus. Interestingly, R. gnavus feeding did not result in histological or molecular evidence of gut inflammation; however, it was able to specifically induce Paneth cell cryptdins and lysozyme P genes expression in 3 week old, antibiotic pre-treated C57BL/6 mice. This demonstrated that R. gnavus is not a pathogenic bacterium, which will directly cause colitis. However, the increased Paneth cell response suggested the need for host innate defence when R. gnavus is increased. Other than bacterial degradation, altered host genetics will also influence the mucus barrier. There is evidence to suggest that the MUC2 gene is highly unstable and is susceptible to gene copy number variation (CNV). Therefore, we hypothesised that MUC2 CNV is present, which may result in altered oligomerisation of the MUC2 glycoprotein causing endoplasmic reticulum stress of the goblet cells that appears to be characteristic of UC. Currently, our data partly support the presence of MUC2 CNV. However, further investigation is required to verify the MUC2 CNV identity. Only then can a high throughput methodology be designed to screen a large population for any association with IBD.

Due to increasing problems with bacterial resistance development, there is a growing need for identifying new types of antibiotics. Antimicrobial peptides constitute an interesting group of substances for this purpose, since they are believed to act mainly by disrupting the bacterial membrane, which is a fast and non-specific mechanism. In order to understand the details on this action simplified phospholipid model membranes based on liposomes, monolayers and bilayers, were employed in this thesis. By in situ ellipsometry studies on supported lipid bilayers in combination with leakage from liposomes it was found that peptide-induced membrane rupture to a great extent is related to peptide adsorption. The peptide activity and mechanism of action is highly dependent on peptide properties such as length, topology, charge, and hydrophobicity. Electrostatic interactions are crucial for peptide adsorption, whereas α-helix formation is of less importance, demonstrated by the dominating peptide conformation being random coil both in absence and presence of membranes, as investigated by circular dichroism. Comparable effects were observed in both mono- and bilayer systems, showing that formation of transmembrane structures is no prerequisite for membrane rupture by complement-derived peptides. Electrochemical studies on these peptides further demonstrated that hydrophobic interactions facilitate peptide penetration into the membrane, causing defects in close proximity to the peptides, while strong electrostatic interactions arrest the peptide in the headgroup region. Increasing the peptide hydrophobicity, by e.g., tryptophan end-tagging, also increases salt resistance. Good correlations were found between model membrane investigations and antibacterial activity towards both Gram-negative and Gram-positive bacteria, showing that membrane rupture is a key mechanism of action for the peptides investigated. In addition, for all peptides investigated cell toxicity is low.

A combined attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR)-atomic force microscopy (AFM) platform was used to visualize and characterize membranolytic protein- and peptide-membrane interactions, allowing spectroscopic details to be correlated with structural features. Modifications to a previous combined platform permitted IR results for physiologically-relevant protein or peptide concentrations as well as provided nanometer-resolution height data for AFM. This combination provides greater insight than individual techniques alone. The interactions of hemolytic sticholysin proteins on a model red blood cell membrane showed evidence of conformational changes associated with a membrane-induced organization. In addition, the examination of a de novo cationic antimicrobial peptide on a model bacterial membrane showed that the peptide adopted a helical structure upon interaction with the membrane, and also provided evidence of membrane disruption and peptide aggregation. These results demonstrate that ATR-FTIR-AFM can be a powerful tool for understanding protein- and peptide-membrane interactions.

A combined attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR)-atomic force microscopy (AFM) platform was used to visualize and characterize membranolytic protein- and peptide-membrane interactions, allowing spectroscopic details to be correlated with structural features. Modifications to a previous combined platform permitted IR results for physiologically-relevant protein or peptide concentrations as well as provided nanometer-resolution height data for AFM. This combination provides greater insight than individual techniques alone. The interactions of hemolytic sticholysin proteins on a model red blood cell membrane showed evidence of conformational changes associated with a membrane-induced organization. In addition, the examination of a de novo cationic antimicrobial peptide on a model bacterial membrane showed that the peptide adopted a helical structure upon interaction with the membrane, and also provided evidence of membrane disruption and peptide aggregation. These results demonstrate that ATR-FTIR-AFM can be a powerful tool for understanding protein- and peptide-membrane interactions.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-09-20T20:31:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriele_azevedo_cardoso.pdf: 4324631 bytes, checksum: 3efd70a64b9fe1de1234371d61299990 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-10-09T13:35:54Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriele_azevedo_cardoso.pdf: 4324631 bytes, checksum: 3efd70a64b9fe1de1234371d61299990 (MD5) / Made available in DSpace on 2017-10-09T13:35:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriele_azevedo_cardoso.pdf: 4324631 bytes, checksum: 3efd70a64b9fe1de1234371d61299990 (MD5) Previous issue date: 2017 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG) / A necessidade de desenvolvimento de novos agentes antimicrobianos cresce ? medida que se torna maior a resist?ncia de microrganismos aos antibi?ticos usualmente empregados. Nesse sentido, os pept?deos antimicrobianos (PAMs) surgem como uma excelente alternativa para o desenvolvimento de novos antibi?ticos. O presente trabalho prop?s a s?ntese do pept?deo antimicrobiano LyeTx-I para estudos de mecanismo de a??o em membranas bacterianas, empregando diferentes t?cnicas biof?sicas e estruturais. O pept?deo LyeTx-I, composto por 24 res?duos de amino?cidos, foi isolado pela primeira vez do veneno de aracn?deos da esp?cie Lycosa erythrognata. Utilizando como t?cnicas principais a ITC e a RMN para obten??o de par?metros cin?ticos, termodin?micos e da intera??o pept?deo-membrana, foi poss?vel avaliar a rela??o estrutura e atividade do pept?deo LyeTx-I. Foram utilizadas ainda, t?cnicas complementares de CD, extravasamento de CF, fluoresc?ncia de Trp, DLS e, potencial zeta para obter informa??es adicionais acerca do modo de intera??o do pept?deo. Observou-se a predomin?ncia de conforma??o helicoidal do pept?deo LyeTx-I, tanto em meios biomim?ticos zwitteri?nicos, quanto em meios ani?nicos. Em meios ani?nicos, observou-se maior conte?do de ?-h?lice, bem como maior constante de intera??o, enquanto que em presen?a de ambientes zwitteri?nicas foram observadas menor helicidade e constante de intera??o. Os dados termodin?micos, obtidos para ambos os meios, mostraram que o processo de intera??o pept?deo-membrana ? dirigido principalmente pela componente entr?pica, uma vez que a componente ent?lpica ? menor. Os dados estruturais e termodin?micos foram coerentes com os demais estudos biof?sicos. Foi observada a partir da an?lise de extravasamento de CF maior capacidade de forma??o de poros no meio ani?nico. Os dados de fluoresc?ncia intr?nseca de Trp e de supress?o de fluoresc?ncia por acrilamida mostraram maior mudan?a de ambiente qu?mico para apolar, do res?duo de Trp-2, quando em presen?a de meio biomim?tico ani?nico. Dessa forma, o pept?deo apresenta maior capacidade de permeabilizar a membrana ani?nica. Al?m disso, o estudo comparativo entre os meios zwitteri?nicos e ani?nicos, permitiu verificar que, embora a intera??o eletrost?tica seja importante para a intera??o pept?deo-membrana, a permeabiliza??o do LyeTx-I na membrana fosfolip?dica ? fundamental para a lise celular. Dessa forma, este estudo mostra que o pept?deo LyeTx-I apresenta elevada prefer?ncia por intera??o com bicamadas fosfolip?dicas ani?nicas, o que faz dele um potencial agente bactericida. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The demand for the development of new antimicrobial agents increases in line with the resistance of microorganisms to the antibiotics usually employed. In this sense, antimicrobial peptides (AMPs) appear as an alternative to the classical antibiotics. The present work proposed the synthesis of the antimicrobial peptide LyeTx-I for studies of mechanism of action in bacterial membranes using a set of biophysical and structural techniques. LyeTx-I peptide is composed of 24 amino acid residues and was isolated for the first time from the venom of the Lycosa erythrognata arachnid species. In order to evaluate the structure-activity relationship of the LyeTx-I, we have employed ITC and NMR as main techniques to obtain the kinetic, thermodynamic and structural parameters of the peptide-membrane interaction. Complementary measurements of CD, CF extravasation, Trp fluorescence, DLS and zeta potential were also used as additional information about the mode of action of the peptide. The ?-helical conformation of the LyeTx-I peptide was observed either in presence of zwitterionic and anionic biomimetic media. Nevertheless, a higher ?-helix content and interaction constant was observed for LyeTx-I in all anionic media when compared to the zwitterionic environments. The thermodynamic data gathered in both media, showed that the peptide-membrane interaction is driven mainly by the entropic contributions, since the enthalpic component is smaller. The structural and thermodynamic data were consistent with the complementary biophysical experiments. It was observed from the CF extravasation a greater capacity of pore formation in the anionic medium. Intrinsic Trp fluorescence showed also a greater change of the residue of Trp-2 to the apolar chemical environment in the presence of anionic biomimetic medium. In this way, the peptide presents a higher capacity to permeabilize the anionic membrane. In addition, the comparative study between the zwitterionic and anionic media, reveals that, although the electrostatic interaction is important to the peptide-membrane interaction, the permeabilization of the LyeTx-I peptide in the phospholipid membrane is fundamental for the cellular lysis. Finally, the study clearly shows the high preference of LyeTx-I for interacting anionic phospholipid bilayers, which makes it a potential bactericidal agent.

Le projet de ma thèse a consisté à étudier chez les nouveau-nés la réponse des cellules épithéliales intestinales (IEC) en microARN (miR) et en peptides antimicrobiens (PAM) dans le contexte de l’infection par Cryptosporidium parvum. Ce protozoaire monoxène se développe uniquement dans les IEC et affecte plus particulièrement les nouveau-nés et les individus immunodéprimés. Nous avons étudié la réponse en miR dans les IEC après infection par C. parvum. La comparaison des réponses obtenues dans les IEC in vitro et in vivo nous a permis de montrer que l’expression du miR-181d-5p est diminuée durant l’infection et que cette diminution d’expression lèverait l’inhibition de l’expression des facteurs anti-apoptotiques OPG et BCL2, favorisant la survie du parasite dans les IEC. Nous avons également caractérisé l’impact de C. parvum sur l’expression des PAM chez le nouveau-né et leur rôle durant l’infection. Nous avons mis en évidence que l’infection entraine une forte modification de l’expression des PAM. Notre attention a été retenue par la diminution, surprenante, de l’expression de la chimiokine antimicrobienne CCL20 et de la cathélicidine CRAMP au cours de l’infection. Pour ces deux peptides, nous avons montré qu’une administration aux souriceaux réduisait significativement la charge parasitaire. Nous avons pu montrer que la protection induite par ces deux molécules antimicrobiennes résultait de leur activité parasiticide sur C. parvum. Ainsi, leur diminution d’expression semble être favorable au développement du parasite et nous avons suggéré qu’elle puisse être induite par le parasite pour échapper à cette activité parasiticide avec notamment la modulation de certains miR. / The aim of my thesis was to study in the mouse model, the intestinal epithelial cell (IEC) response during neonatal Cryptosporidium parvum infection with a focus on microRNAs (miR) and antimicrobial peptides (AMP) response. C. parvum is a protozoan parasite that affects preferentially newborn, young or immunocompromised adult and completes its life cycle only in IECs. In a first part, we studied the expression of miRs in IEC during C. parvum infection. We compared the responses between in vitro infected IEC and IECs purified from infected neonatal mice and observed a decrease of miR-181d-5p expression. This reduced expression of miR-181d-5p was associated with an upregulation of the mRNA coding for two putative targets OPG and BCL2 which are anti-apoptotic agents that may favor parasite survival in IEC. This functional relation between miR-181d-5p and OPG was next demonstrated by using reporter dual-luciferase assay. In a second part of my thesis, we characterized the AMP expression profile and studied their role during C. parvum infection in neonates. We showed that infection up-regulates a broad expression of AMP except for CCL20 and CRAMP cathelicidin for which mRNA expression was decreased. We next choose to focus our work on these two molecules and reported that administration of CCL20 and CRAMP to infected neonatal mice significantly reduced the number of parasites in the intestine through a direct killing activity on free stages of the parasite. As the decreased expression of these two AMPs during infection seems to favor the development of the parasite, this could be an escape mechanism developed by C. parvum that may occur through the modulation of miR.

Peptídeos antimicrobianos são vistos como alternativas promissoras a serem empregadas pela iindústria farmacêutica no controle de infecções causadas por microrganismos, como também na indústria alimentícia, onde podem desempenhar papéis como conservantes naturais de alimentos. Plantaricina149 é um membro deste grupo, sendo composto por 22 resíduos de aminoácidos, com natureza catiônica e atividade inibitória sobre algumas bactérias patogênicas. Neste trabalho, foram sintetizados diferentes peptídeos análogos à Plantaricina149 para investigar suas ações sobre microrganismos (bactérias e fungos), a fim de correlacionar estes estudos com a ação lítica do peptídeo em modelos de membrana diversos (monocamadas e vesículas fosfolipídicas). A interação de Plantaricina149 com estes sistemas foi monitorada pelas espectroscopias de dicroísmo circular e fluorescência, ensaios de tensão superficial, calorimetria e ressonância plasmônica de superfície, e mostrou ser altamente específica para superfícies fosfolipídicas que apresentam densidade de cargas negativas, tais como a membrana celular de bactérias. A interação eletrostática inicial que se estabelece entre o peptídeo e os fosfolipídios é de extrema importância, sendo capaz de induzir uma estruturação helicoidal na região C-terminal do peptídeo, enquanto a região Nterminal contribui com as interações hidrofóbicas necessárias para a penetração do peptídeo nas camadas fosfolipídicas levando a ruptura das mesmas. De forma semelhante, a atividade antimicrobiana de Plantaricina149a (e alguns de seus análogos) também mostrou ser resultado das interações das duas regiões da molécula, e foi afetada com a retirada ou modificação da região N-terminal do peptídeo. Com a deleção desta região, o peptídeo passou a ter somente ação bacteriostática sobre Staphylococcus aureus e Pseudomonas aeruginosa, perdendo a capacidade bactericida. / Antimicrobial peptides are seen as promising alternatives to be employed in pharmaceutical industry for controlling infections caused by microorganisms, and also in food industry, where they can play roles as natural food preservatives. Plantaricina149 is a member of this group, constituted of 22 amino acid residues, cationic in nature and presenting inhibitory activity against some pathogenic bacteria. In this work, different Plantaricina149 analog peptides were synthesized to investigate their action against microorganisms (bacteria and fungi), with the aim of correlating these studies with the lytic action of the peptide on several membrane models (phospholipid monolayers and vesicles). The Plantaricina149 interaction with these systems was monitored by circular dichroism and fluorescence spectroscopies, surface tension assays, calorimetry and surface plasmon resonance, and showed to be highly specific to phospholipid surfaces that present negative charge density, such as the bacteria cell membrane. The initial peptide-phospholipids electrostatic interaction is extremely important, and it is capable of inducing a helical structure in the peptide C-terminal region, while the Nterminal region contributes with the hydrophobic interactions needed to the peptide penetration in the phospholipid layers and to the disruption of them. Similarly, the Plantaricina149 antimicrobial activity has also proved to be a result of the interactions from the two regions of the molecule, and it was strongly affected by the removal or modification of the peptide N-terminal region. Promoting the deletion of this region has left the peptide only with a bacteriostatic action against Staphylococcus aureus and Pseudomonas aeruginosa, removing its bactericide ability.

Dermicidina (DCD) é um gene mapeado no cromossomo 12, lócus 12q13.1, e codifica uma proteína de 110 aminoácidos, que sofre um processamento proteolítico, gerando peptídeos ativos. O peptídeo C-terminal (DCD-1L) de 48 aminoácidos tem uma carga -2, e exerce função antibacteriana e antifúngica, e o peptídeo C-terminal splice variante, denominado DCD-SV de 59 aminoácidos, tem carga neutra, e suas propriedades ainda não foram estabelecidas. Neste trabalho são apresentados os resultados da expressão, purificação e sequenciamento da DCD nativa produzida em E. coli BL21 transformada com o vetor pAE-DCD. Na segunda parte são descritas as análises físico-químicas e bioquímicas da interação dos peptídeos sintéticos DCD-1L e DCD-SV com vesículas lipídicas gigantes e vesículas unilamelar grandes sintetizadas com palmitoil-oleoil-fosfatidilcolina. As preferenciais estruturais dos peptídeos foram investigadas por espectroscopia de Dicroísmo Circular. Nossos resultados sugerem que a DCD-SV tem alta propensão para adotar uma estrutura helicoidal permitindo sua inserção e oligomerização em membranas biomiméticas, e possível formação de canais de condutância molecular. / Dermicidin (DCD) is mapped a gene on chromosome 12, locus 12q1.13 whose 110 amino acids protein is proteolytically processed to N and C-terminal peptides. The 48-amino acid C-terminal peptide (DCD-1L) has -2 net charges and display antibacterial and antifungal properties and the 59-amino acid splice variant C-terminal peptide (DCD-SV) has neutral net charge; however, its structure and biological function are unknown. Here we show the results of expression, purification and amino acid sequencing of recombinant DCD protein produced in E.coli transformed with pAE-DCD vector. We also describe the results of physical-chemical and biochemical analyses showing the visible differences between the interactions of DCD-1LL and DCD-SV synthetic peptides with giant unilamellar vesicles and large unilamellar vesciles made of palmitoyl-oleoyl phosphatidylcholine, used as biomimetic membranes. The structural preferences of peptides were analyzed by circular dichroism spectroscopy. Our results suggest that DCD-SV peptide has higher propensity to adopt helicoidal structure enabling it to insert into mimetic membranes, undergo oligomerization and formation of conductance channel.