Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-06-21T18:51:44Z No. of bitstreams: 1 AntonioCarlosSilveiraMarcolinoDissertacao2016.pdf: 6156292 bytes, checksum: d429c57126e7bc2351ea9c3c48c300ab (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-06-21T18:51:54Z (GMT) No. of bitstreams: 1 AntonioCarlosSilveiraMarcolinoDissertacao2016.pdf: 6156292 bytes, checksum: d429c57126e7bc2351ea9c3c48c300ab (MD5) / Made available in DSpace on 2017-06-21T18:51:54Z (GMT). No. of bitstreams: 1 AntonioCarlosSilveiraMarcolinoDissertacao2016.pdf: 6156292 bytes, checksum: d429c57126e7bc2351ea9c3c48c300ab (MD5) Previous issue date: 2016-03-29 / The guanylate cyclase activator 2B, also known as uroguanylin, is part of the guanylin peptide family, which includes peptides such as guanylin and lymphoguanylin. The guanylin peptides are related to sodium absorption inhibition and water secretion induction. Uroguanylin may be related to various pathologies such as chronic renal failure, congestive heart failure and nephrotic syndrome. Uroguanylin mutations have already been associated with essential hypertension. However, there are no studies on the structural changes in the uroguanylin???s protein that used single nucleotide polymorphisms through the use of molecular dynamics simulations. This study used 16 in silico SNP impact prediction tools to evaluate non synonymous SNPs and to select mutations considered as convergent deleterious, which were further analyzed through long time molecular dynamics simulations of 1 microsecond of duration. The results of the molecular dynamics simulations suggest that all SNPs considered as convergent deleterious suffered some kind of structural change, however, four of these nsSNPs have also undergone flexibility changes, possibly resulting in functional changes. / O ativador guanilato ciclase 2B, tamb??m conhecido como uroguanilina, faz parte da fam??lia de pept??deos da guanilina, a qual inclui pept??deos como a guanilina e a linfoguanilina. Os pept??deos da guanilina est??o ligados ?? inibi????o da absor????o de s??dio e indu????o da secre????o de ??gua. A disfun????o da uroguanilina est?? relacionada ao desenvolvimento de v??rias patologias, como insufici??ncia renal cr??nica, insufici??ncia card??aca congestiva e s??ndrome nefr??tica. Muta????es na uroguanilina tamb??m j?? foram associadas ?? hipertens??o essencial. Entretanto, n??o existem estudos sobre a utiliza????o de simula????es por din??mica molecular para avaliar o impacto estrutural causado por nsSNPs presentes no gene codificador da prote??na uroguanilina. Este estudo utilizou 16 ferramentas in silico de predi????o de impacto de nsSNPs para filtrar nsSNPs convergentes delet??rios para posterior an??lise por simula????es por din??mica molecular de longa dura????o (1 microssegundo de dura????o). Os resultados das simula????es por din??mica molecular sugerem que todos os nsSNPs considerados como convergentes delet??rios sofreram algum tipo de altera????o estrutural, por??m, quatro destes SNPs tamb??m sofreram altera????es de flexibilidade, possivelmente resultando em altera????es funcionais.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-11-09T10:26:59Z No. of bitstreams: 1 NicoleBerwangerCobachoDissertacao2017.pdf: 2681393 bytes, checksum: 06c28d06b89b63056ccfd84351dd8d36 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-11-09T11:17:54Z (GMT) No. of bitstreams: 1 NicoleBerwangerCobachoDissertacao2017.pdf: 2681393 bytes, checksum: 06c28d06b89b63056ccfd84351dd8d36 (MD5) / Made available in DSpace on 2017-11-09T11:17:55Z (GMT). No. of bitstreams: 1 NicoleBerwangerCobachoDissertacao2017.pdf: 2681393 bytes, checksum: 06c28d06b89b63056ccfd84351dd8d36 (MD5) Previous issue date: 2017-08-22 / UCB / The resistance of microorganisms to commonly used antibiotics has increased dramatically in recent years, suggesting that we will soon enter a post-antibiotic era where no therapy currently used will be effective in the fighting against infection. Thus, the search and study of new drugs and models of action of compounds that prevent or reduce the development of pathogens is essential. In this context, antimicrobial peptides (PAMs) appear as a new generation of compounds that demonstrate a great therapeutic potential. These molecules present themselves in low concentration in the organism of origin, making their isolation from natural sources an expensive and often impracticable process. In order to obtain a greater quantity of these peptides, recombinant expression via the heterologous system can be considered an efficient alternative in terms of time, cost and productivity. In 2009, Mandal et al. isolated three coconut water (Coco nucifera) peptides, called Cn-AMP1-2 and -3. Among them, Cn-AMP1 presented better activity against bacteria and fungi of medical importance. Subsequently, Lopez-Abarrategui et al. in 2012 isolated peptides with antimicrobial activity from the mollusk Cenchritis muricatus called Cm-p1 and Cm-p2. From these, a series of variant peptides were theoretically proposed in silico and evaluated against Candida albicans and the peptide named Cm-p5 demonstrated, among them, better antifungal activity. In this work, several constructs containing the peptides Cm-p5 and Cn-AMP1 were drawn in tandem and inserted into the pETSUMO (Life Technologies) vector. Genes were expressed in large quantities in strains derived from Escherichia coli BL21 (DE3) and after affinity column purification, they were evaluated, still fused with SUMO protein, against pathogenic microorganisms. However, none of the expressed peptides demonstrated bactericidal activity against the strains evaluated. / A resist??ncia dos microrganismos aos antibi??ticos que s??o comumente utilizados vem aumentando drasticamente nos ??ltimos anos, sugerindo que brevemente, entraremos em uma era p??s-antibi??ticos onde terapias utilizadas atualmente n??o ser??o mais eficientes no combate a infec????es. Desta forma, a procura e o estudo de novas drogas e modelos de a????o de compostos que impe??am ou reduzam o desenvolvimento de pat??genos ?? essencial. Neste contexto, os pept??deos antimicrobianos (PAMs) surgem como uma nova gera????o de compostos que demonstram um grande potencial terap??utico. Essas mol??culas apresentam-se em baixa concentra????o no organismo de origem, tornando o seu isolamento a partir de fontes naturais um processo dispendioso e, por muitas vezes, invi??vel. Para a obten????o de maior quantidade desses pept??deos, a express??o recombinante via sistema heter??logo pode ser considerada uma alternativa eficiente em rela????o ao tempo, custo e produtividade. Em 2009, Mandal e colaboradores isolaram tr??s pept??deos da agua de coco (Coco nucifera), denominados Cn-AMP1 -2 e -3. Dentre eles, o Cn-AMP1 apresentou melhor atividade contra bact??rias e fungos de import??ncia medica. Posteriormente, Lopez-Abarrategui e colaboradores em 2012 isolaram pept??deos com atividade antimicrobiana do molusco Centrichis muricatus denominados Cm-p1 e Cm-p2. A partir destes, uma serie de pept??deos variantes foram teoricamente propostos in silico e avaliados contra C. albicans sendo que o pept??deo nominado o Cm-p5 demonstrou, entre todos, melhor atividade antif??ngica. Neste trabalho, v??rias constru????es contendo os pept??deos Cm-p5 e Cn-AMP1 foram desenhadas em tandem e inseridas no vetor pETSUMO (Life Technologies). Os genes foram expressos em grandes quantidades em cepas derivadas de Escherichia coli BL21(DE3) e ap??s purifica????o em coluna de afinidade, os mesmos foram avaliados, ainda fusionados com a prote??na SUMO, contra microrganismos patog??nicos. No entanto, nenhum dos pept??deos expressos demonstrou atividade bactericida contra as cepas avaliadas.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-06-05T17:52:22Z No. of bitstreams: 1 LianaCostaPereiraDissertacao2014.pdf: 1788731 bytes, checksum: aaf1eef40e98ed7a21c08304f7dce33b (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-06-05T17:54:37Z (GMT) No. of bitstreams: 1 LianaCostaPereiraDissertacao2014.pdf: 1788731 bytes, checksum: aaf1eef40e98ed7a21c08304f7dce33b (MD5) / Made available in DSpace on 2017-06-05T17:54:37Z (GMT). No. of bitstreams: 1 LianaCostaPereiraDissertacao2014.pdf: 1788731 bytes, checksum: aaf1eef40e98ed7a21c08304f7dce33b (MD5) Previous issue date: 2014-05-29 / Viral infections affect every living organism. Health care programs and vaccines have been developed to control and prevent those infections, but there is still the occurrence of severe diseases and with great social-economic importance, whose only alternative is the treatment with antivirals drugs. Those compounds possess different mechanisms of action and are specific for each virus. Nevertheless, with many viruses resistant strains rise, a growing interest in novel antivirals development or in the improvement of existing drugs have been observed. In the last few years, antimicrobial peptides have shown antiviral activities making them candidates for antiviral drugs. The present study aimed to evaluate the possible antiviral activities of synthetic peptides clavanin MO, LL-37, Cn-AMP1 and variants of the Pa-MAP1 against HSV-1 and the Aichivirus replication. The peptides cytotoxic effects against Vero cells were evaluated by MTT method, whereas the clavanin MO, the Pa-MAP 1.8 and Pa-MAP 18br showed more cytotoxicity, with concentrations higher than 15,4 ??M and 12 ??M, respectively. Considering the antivirals assays, it was observed that the peptide Pa-MAP 1 causes 90 % of HSV-1 replication, with a SI higher than 4.8 and a virucidal mechanism of action, but null against the Aichivirus. Futhermore LL- 37 presented 90 % of Aichivirus inhibition, with a SI of 3.4. Others peptides tested showed percentages below 41 %. Future studies are need in order to elucidate which HSV-1 structure does Pa-MAP1 interacts and futher extend to in vivo tests. In summary, this study it is the first one to describe antiviral tests for the treatment of the Aichivirus infection. / As infec????es virais acometem todos os organismos vivos. Para o controle e preven????o dessas infec????es, programas de sa??de p??blica e vacinas t??m sido desenvolvidos, mas ainda h?? a ocorr??ncia de doen??as graves e de grande impacto socioecon??mico cuja ??nica alternativa ?? o tratamento com antivirais. Estes possuem diferentes mecanismos de a????o e s??o espec??ficos para cada tipo viral. Entretanto, o surgimento de variantes resistentes de muitos tipos virais tem levado h?? um crescimento no interesse de desenvolver novos antivirais ou at?? mesmo melhorar os existentes. Nos ??ltimos anos, estudos sobre pept??deos antimicrobianos relatam a atividade antiviral desses compostos, tornando-os candidatos a medicamentos antivirais. O presente estudo teve como objetivo avaliar a poss??vel atividade antiviral dos pept??deos sint??ticos clavanina MO, LL-37, Cn-AMP1 e variantes da Pa-MAP contra o Herpes v??rus humano 1 e Aichiv??rus. O efeito citot??xico dos pept??deos em c??lulas Vero foi avaliado pelo m??todo do MTT, sendo que a clavanina MO, a Pa- MAP 1.8 e Pa-MAP 18br demonstraram maior citotoxicidade em concentra????es maiores que 15,4 ??M, e 12 ??M, respectivamente. Considerando os ensaios antivirais, observou-se que o pept??deo Pa-MAP1 apresentou 90 % de inibi????o da replica????o do HHV-1, com um IS maior que 4,8, sendo que seu mecanismo de a????o foi definido como virucida, por??m contra o Aichiv??rus o PI foi nulo. Al??m disso, LL-37 apresentou 90 % de inibi????o do Aichiv??rus com um IS de 3,4. Os outros pept??deos testados apresentaram percentuais abaixo de 41 % contra ambos os v??rus. Futuros estudos moleculares s??o necess??rios para se determinar com qual estrutura do HHV-1 a Pa-MAP1 interage e para estender os testes para modelos in vivo. Este estudo ?? o primeiro a relatar testes antivirais para o tratamento do Aichiv??rus.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-08-27T11:15:23Z No. of bitstreams: 1 474252 - Texto Completo.pdf: 3311481 bytes, checksum: b67f5f48a8325dd3ae5469933fff90ba (MD5) / Made available in DSpace on 2015-08-27T11:15:23Z (GMT). No. of bitstreams: 1 474252 - Texto Completo.pdf: 3311481 bytes, checksum: b67f5f48a8325dd3ae5469933fff90ba (MD5) Previous issue date: 2015-03-02 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Peptide YY (PYY) belongs to the neuropeptide Y (NPY) family, which also includes the neuropeptide Y (NPY) and pancreatic polipeptide (PP). These substances are biologically active, constituted of 36 aminoacids, and act via G protein coupled receptors. There are four functional subtypes of NPY family receptors in humans, namely Y1, Y2, Y4, and Y5. PYY is secreted by the intestinal L cells, being present in the blood stream in two active forms capable of crossing the blood brain barrier, PYY (1-36) and its cleavage product, PYY (3-36). PYY is a selective agonist for the Y2 receptor (Y2R) and has been identified as a modulator of appetite, promoting satiety sensation in mammals. Y2R are abundant in the brain hippocampus, where these receptors inhibit excitatory synaptic transmission and glutamatergic release when activated by potassium in hippocampal slices. Besides, knockout mice for Y2R present deficits in spatial and non-spatial memory tasks, showing a role of Y2R in learning and memory. The aim of this Master?s dissertation was searching for a better understanding of the interaction of PYY (3-36) and its Y2 receptor in CNS cells. For this purpose the activity of this peptide was investigated on Ca2+ influx in hippocampal cell cultures of Wistar neonate rats. We evaluated the influence of the presence of Ca2+ in the extracellular fluid, as well as the involvement of plasma membrane voltage-dependent Ca2+ and Na+ channels, and the influence of intracellular mechanisms related to the endoplasmic reticulum (ER), such as the SERCA pump, the inositol triphosphate (IP3) receptors and the ryanodine receptors (RyRs) in the responses induced by PYY (3-36) on the modulation of the [Ca2+]i, by using blockers specific for these channels. It was observed that the increase of the cytosolic [Ca2+] evoked by PYY (3-36) in hippocampal cells is independent of Ca2+ from the extracellular environment. Using a voltage-dependent Na+ channels blocker it was possible demonstrate that PYY (3-36) action is independent on Na+, suggesting that its activity on hippocampal cells does not induce or does not depend on cellular depolarization. In the experiments using RyRs or SERCA pump blockers it was observed an elevation of Ca2+ influx, that probably occurred due to the activation of SOCC, but with the concomitant presence of the a voltage-dependent Na+ channels blocker, this effect was abolished, suggesting a probable inhibition of SOCC channels in these conditions. In the experiments in the presence of an IP3Rs inhibitor, there was a decrease in cytosolic [Ca2+] evoked by PYY (3-36). The results from our experiments indicate that the action of PYY (3-36) on Ca2+ mobilization is mediated by intracellular receptors of the ER, suggesting that the observed elevation of [Ca2+]i is modulated especially by the activation of the intracellular Ca2+ signalling cascade through IP3 receptors. / O pept?deo YY (PYY) pertence ? fam?lia do neuropept?dio Y que ? composta tamb?m pelo neuropept?dio Y (NPY) e pelo polipept?dio pancre?tico (PP). S?o pept?deos biologicamente ativos, compostos por 36 amino?cidos e atuam via receptores acoplados a prote?na G. Existem quatro subtipos de receptores para a fam?lia do NPY que s?o funcionais em humanos, denominados como: Y1, Y2, Y4 e Y5. O PYY ? secretado no intestino pelas c?lulas L e circula no organismo em duas formas ativas que atravessam a barreira hematoencef?lica, que s?o o PYY (1-36) e sua forma clivada PYY (3-36). O PYY (3-36) ? um agonista seletivo do receptor Y2 e tem sido evidenciado por seu papel como um modulador do apetite, promovendo a sensa??o de saciedade em mam?feros. O hipocampo ? uma regi?o rica em receptores Y2 e j? se demonstrou que a ativa??o destes receptores no hipocampo pode inibir a transmiss?o sin?ptica excitat?ria e a libera??o de glutamato estimulada por pot?ssio em fatias hipocampais. Em camundongos knockout para o receptor Y2 observou-se d?ficits na mem?ria espacial e na mem?ria n?o espacial nestes animais evidenciando tamb?m seu envolvimento na regula??o da fun??o cognitiva associada com o aprendizado e mem?ria. O desenvolvimento desta disserta??o teve por objetivo buscar uma melhor compreens?o da intera??o do PYY (3-36) e seu receptor Y2 em c?lulas do sistema nervoso central. Para isso investigou-se a atividade deste pept?deo no influxo de c?lcio (Ca2+) em c?lulas cultivadas do hipocampo de ratos Wistar neonatos. Avaliou-se a influ?ncia do Ca2+ presente no meio extracelular, bem como o envolvimento dos canais de Ca2+ e Na+ voltagem dependentes presentes na membrana plasm?tica, e a influ?ncia de mecanismos intracelulares presentes no ret?culo endoplasm?tico (RE), como a bomba do ret?culo sarco/endoplasm?tico Ca2+- ATPase (SERCA), os receptores de inositol trifofato (IP3Rs) e os receptores de rianodina (RyRs) nas respostas induzidas pelo PYY (3-36) sobre a modula??o da concentra??o de Ca2+ intracelular ([Ca2+]i), atrav?s da utiliza??o de bloqueadores espec?ficos para estes canais. Foi observado que o aumento da concentra??o de Ca2+ ([Ca2+]) citos?lico evocado pelo PYY (3-36) em c?lulas do hipocampo ? independente do Ca2+ do meio extracelular. E atrav?s da utiliza??o de bloqueadores dos canais de Na+ voltagem dependentes conseguiu-se demonstrar que a a??o do PYY (3-36) ? independente do influxo de Na+, sugerindo que a sua atividade sobre as c?lulas hipocampais n?o induz ou independe da despolariza??o celular. Nos experimentos utilizando bloqueadores dos RyRs ou da bomba SERCA observou-se uma eleva??o do influxo de Ca2+, o que provavelmente ocorreu devido a ativa??o dos canais de c?lcio operados por estoque (SOCC, do ingl?s store-operated calcium channels), por?m na presen?a concomitante de bloqueadores de canais de Na+ voltagem dependentes, este efeito foi bloqueado, sugerindo uma prov?vel inibi??o dos canais SOCC nessas condi??es. Finalmente, na presen?a de inibidores dos IP3Rs, ocorreu uma diminui??o da [Ca2+] citos?lico evocada pelo PYY (3-36) nas c?lulas. Os resultados evidenciam que a a??o do PYY (3-36) sobre a mobiliza??o do Ca2+ ? atrav?s de receptores intracelulares do RE, sugerindo que a eleva??o da [Ca2+]i observada, ? modulada principalmente pela ativa??o da cascata de sinaliza??o do Ca2+ intracelular pelos receptores de IP3.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-06T11:52:23Z No. of bitstreams: 1 AllanPiresdaSilvaDissertacao2017.pdf: 4098983 bytes, checksum: 94ec346f3edc58586d44cec31a8597f4 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-09-06T11:52:34Z (GMT) No. of bitstreams: 1 AllanPiresdaSilvaDissertacao2017.pdf: 4098983 bytes, checksum: 94ec346f3edc58586d44cec31a8597f4 (MD5) / Made available in DSpace on 2017-09-06T11:52:34Z (GMT). No. of bitstreams: 1 AllanPiresdaSilvaDissertacao2017.pdf: 4098983 bytes, checksum: 94ec346f3edc58586d44cec31a8597f4 (MD5) Previous issue date: 2017-03-14 / Currently, various bacteria can be harmful to human health. Moreover, with continued use of antibiotics and development of resistance by these microorganisms, many infections became worrying, with no effective treatments available generating the need for development of other fighting molecules. In this context, the antimicrobial peptides (AMPs) have been proposed as an alternative in the control of infections caused by resistant microorganisms. Despite the variation in sequence levels, AMPs may present high structural conservation in specific families, especially peptides stabilized by disulfide bonds. Canonically, the identification of PAMs is by exploitation of bioactive natural extracts and subsequent analysis and purification thereof. In the post genomics era, in turn, identifying PAMs could be made from databases using molecular modeling of peptides in direct search. In this work were selected AMPs without structure in PDB, from antimicrobial peptide database (APD) (http://aps.unmc.edu/AP/main.php). The sequences were pre-filtered, being selected two AMPs (myticin B and MiAMP-2b) of classes described with modifications in disulfide bonds pattern arrangement. Additionally, the original bank was submitted to STPs identification. PredSTP was used as an additional evaluation. After prefiltering phases, a new potential STP (CRS4C-2b) with a new hypothetical structural topology was modelled by QUARK and simulated at 300 ns molecular dynamics, maintaining the initial structure. The methodology was then applied to identify PAMs in the Zantedeschia aethiopica transcriptome where two new potential PAMs were found that were predicted to be active by CAMP. Thus, the two methodologies developed here can be successfully applied in the identification of new PAMs and in the analysis of the structural diversity of antimicrobial families. / Atualmente, v??rias bact??rias podem ser prejudiciais ?? sa??de humana. Al??m disso, com o uso cont??nuo de antibi??ticos, e desenvolvimento de resist??ncia por parte desses microrganismos, muitas infec????es se tornaram preocupantes, sem tratamentos eficazes dispon??veis gerando a necessidade de desenvolvimento de outras mol??culas de combate. Nesse ??mbito, os pept??deos antimicrobianos (PAMs) t??m sido propostos como uma alternativa no controle de infec????es causadas por microrganismos resistentes. Apesar da variabilidade nas sequ??ncias, os PAMs podem apresentar grande conserva????o estrutural em fam??lias espec??ficas, principalmente em pept??deos estabilizados por pontes dissulfeto. De forma can??nica, a identifica????o de PAMs se d?? pela explora????o de extratos naturais bioativos e posterior an??lise e purifica????o dos mesmos. Na era p??s-gen??mica, por sua vez, a identifica????o de PAMs pode ser feita a partir de bancos de dados utilizando modelagem molecular na busca direta de pept??deos. Nesse trabalho foram selecionados PAMs sem estrutura no PDB, a partir do banco de dados de pept??deos antimicrobianos (APD) (http://aps.unmc.edu/AP/main.php). Desta forma, as sequ??ncias foram pr??-filtradas, sendo selecionados dois PAMs (miticina B e MiAMP-2b) de classes descritas com varia????o na disposi????o ou padr??o de pontes dissulfeto. Al??m disso, o banco original foi submetido ?? identifica????o de STPs. Para tal, o servidor PredSTP foi utilizado como avalia????o adicional. Ao final das etapas de pr??-filtragem, um novo potencial STP (CRS4C-2b) com uma nova topologia estrutural foi modelado pelo QUARK e simulado em din??mica molecular, mantendo a estrutura inicial. A metodologia foi ent??o aplicada para identifica????o de PAMs no transcriptoma de Zantedeschia aethiopica onde foram encontrados dois novos potenciais PAMs que foram preditos como ativos pelo CAMP. Dessa forma, as duas metodologias desenvolvidas aqui podem ser aplicadas com sucesso na identifica????o de novos PAMs e na an??lise de diversidade estrutural de fam??lias antimicrobianas.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-11-08T13:12:56Z No. of bitstreams: 1 AmandaCarolineMarquesSa??deTese2016.pdf: 1482640 bytes, checksum: 98c7088a3903d189fcf2c6a44b4fcdc7 (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-11-09T11:04:49Z (GMT) No. of bitstreams: 1 AmandaCarolineMarquesSa??deTese2016.pdf: 1482640 bytes, checksum: 98c7088a3903d189fcf2c6a44b4fcdc7 (MD5) / Made available in DSpace on 2017-11-09T11:04:49Z (GMT). No. of bitstreams: 1 AmandaCarolineMarquesSa??deTese2016.pdf: 1482640 bytes, checksum: 98c7088a3903d189fcf2c6a44b4fcdc7 (MD5) Previous issue date: 2016-11-01 / Bacteria are becoming resistant to a growing number of conventional antibiotics at a worrisome rate. Therefore, there is an increasing demand for new antimicrobial therapeutics. Antimicrobial peptides (AMPs) are one of promising alternatives for conventional antibiotics and are thought to be less likely to induce resistance. AMPs have been coupled to molecular scaffolds for biomedical applications such as hydrogels for wound dressings and covering implants. Here AMPs were chemically conjugated to the CR4 hydrophilic polymers. The CPF_C1 is a short peptide isolated from Xenopus clivii, the original sequence was modified one called LCPF_C1, aiming to create some distance between the first glycine and the azide added on the N terminus (on the coupled sequence). The conjugation between the AMP and the CR4 polymer used a click chemistry reaction with two steps, dependent of a hetero crosslinker (DBCO???PEG4???NHS Ester). Dynamic light scattering (DLS) and fluorimeter assays were used to evaluate the efficiency of coupling. MALDI-ToF analysis showed 3 molecules of LCPF_C1 peptide for each CR4 polymer. Moreover, microrheology showed changes on hybrids increasing the viscosity. Finally, the compounds were evaluate against four different bacteria: Staphylococcus aureus, Klebsiela pneumoneae carbapenemases (Kpc), Escherichia coli and Pseudomonas aeruginosa. It was possible to observe MIC???s against P. aeruginosa of 11 mM by using the peptide (LCPF_C1) and 55 mM for the original sequence. When the hibrids were compared to the free polymer was not found MIC values against K. pneumoneae (CR-Kp). On the other hand, the hibrids showed three times less activity than the free polymer against P. aeruginosa. No MIC values were found aganst S. aureus. Finally, against E. coli was observed a MIC value of 1000 mM for the free CR4 and 250 mM for the hibrids. On this way, the present work showed the possibility to functionalize biopolymers by using bioactive molecules coupled to biopolimers, changing the physical-chemical characteristics and increasing they applicability against bacterial infections. / Uma taxa alarmante de bact??rias tem se tornado resistente a um grande n??mero de antimicrobianos convencionais. Desta forma, a demanda por novas terapias antimicrobianas tem aumentado proporcionalmente. Assim, o uso de pept??deos antimicrobianos (PAMs) consistem em uma promissora alternativa para antibacterianos convencionais, particularmente podendo ser acoplado a estruturas moleculares para aplica????es biom??dicas, como hidrog??is para curativos e cobertura de implantes. No presente trabalho, PAMs foram quimicamente conjugados ao pol??mero hidrof??lico inspirado nas prote??nas naturais col??geno e seda CR4. O pept??deo CPF_C1 consiste em um pept??deo contendo 17 res??duos de amino??cidos isolado de Xenopus clivii, com atividade comparada ?? bact??rias Gram-negativas e -positivas. Para este estudo, a sequ??ncia original do CPF-C1 foi modificada e intitulada LCPF_C1, objetivando aumentar a dist??ncia entre a primeira glicina e a azida adicionada na por????o N-terminal. A conjuga????o entre o PAM e o pol??mero CR4 foi realizada por meio de click chemistry reaction em dois passos, dependentes do hetero crosslinker (DBCO???PEG4???NHS Ester). Ensaios de espalhamento de luz din??mico (DLS) e fluorimetria foram utilizados para avaliar a efici??ncia de acoplamento e demonstraram a presen??a do pept??deo acoplado ao pol??mero. An??lises de MALDI-ToF demonstraram 3 mol??culas do pept??deo LCPF-C1 para cada mol??cula do pol??mero CR4. Al??m disso, dados de microrreologia demonstraram mudan??as nos h??bridos como aumento de viscosidade. Finalmente, os compostos foram avaliados contra quatro bact??rias diferentes: Staphylococcus aureus, K. pneumoneae carbapenemase (Kpc), Escherichia coli e Pseudomonas aeruginosa. Foi poss??vel observar para P. aeruginosa MICs de 11 mM utilizando o pept??deo (LCPF_C1) e 55 mM para a sequ??ncia original. Quando comparados aos h??bridos em rela????o a atividade do pol??mero livre n??o foi encontrado valor de MIC contra K. pneumoneae (CR-Kp). Por outro lado, os h??bridos demonstraram um MIC cerca de tr??s vezes menor que o pol??mero livre contra P. aeruginosa. Nenhum valor de MIC foi encontrado contra S. aureus. Finalmente, contra E. coli observou-se MIC de 1000 mM para o pol??mero CR4 e 250 mM para os h??bridos. Desta forma o presente trabalho demonstrou a possibilidade de funcionalizar biopol??meros por meio do acoplamento de mol??culas bioativas, alterando suas caracter??sticas f??sico-qu??micas e aumentando sua aplicabilidade contra infec????es bacterianas.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2016-05-19T19:42:39Z No. of bitstreams: 1 TES_PAULA_JULIANA_BRIZUELA_DE_SEADI_PEREIRA_COMPLETO.pdf: 4958315 bytes, checksum: 38858f11b57077cc60813c0fa01390dd (MD5) / Made available in DSpace on 2016-05-19T19:42:39Z (GMT). No. of bitstreams: 1 TES_PAULA_JULIANA_BRIZUELA_DE_SEADI_PEREIRA_COMPLETO.pdf: 4958315 bytes, checksum: 38858f11b57077cc60813c0fa01390dd (MD5) Previous issue date: 2016-01-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The gastrin-releasing peptide (GRP) and its receptor (GRPR) were recently identified as specific components of pruritus, suggesting an important pharmacological potential for this system; however, the mechanisms underlying GRP/GRPR activity remain undefined. Herein, we evaluated GRPR localization and downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization. Expression profile revealed that PI3K? is downstream of the GRP/GRPR axis, observed by GRP-induced Akt phosphorylation in ex vivo naive mouse spinal cords and in GRPR transiently expressing HEK293 cells. However, ex vivo mouse spinal cord stimulation by GRP failed to induce the activation of MAP-kinases, namely ERK 1/2 and p38. Intrathecal GRP administration led to intense scratching behavior, an effect significantly reduced by either GRPR antagonism or the PI3K? inhibition. We assessed whether the GRP/GRPR system is involved in chronic itching using the dry skin model and found that GRPR blockade or PI3K? inhibition reversed the scratching. We also found that p-Akt and GRPR are co-expressed in the spinal cord and in DRG neurons from dry skin mice, and that p-Akt levels are increased in these animals, an effect prevented by GRPR blockade. The intradermal injection of GRP also triggers scratching behavior, which was significantly decreased after treatment with PI3K? inhibitor. The itching response was also induced by the intrathecal injection of an Akt activator. Altogether, these findings are highly suggestive that GRPR is expressed by the peripheral and central terminals of DRG nociceptive afferents, which transmit itch via the PI3K?/Akt pathway. / O pept?deo liberador de gastrina (GRP) e seu receptor (GRPR) foram recentemente identificados como componentes espec?ficos do prurido, com importante potencial farmacol?gico; no entanto, os mecanismos intracelulares que medeiam a atividade do sistema GRP/GRPR permanecem desconhecidos. O presente estudo avaliou a localiza??o do GRPR e as vias de sinaliza??o downstream ativadas por este receptor. Os dados obtidos mostram que o GRP ativa diretamente neur?nios de pequeno di?metro do DRG, sens?veis a capsaicina, um efeito demonstrado pelo fluxo transit?rio de c?lcio e pela despolariza??o da membrana. O perfil de express?o observado sugere que a PI3K? ? ativada downstream ao sistema GRP/GRPR, como indicado pela fosforila??o de Akt (marcador de atividade de PI3K?) induzida por GRP, em medulas de camundongos ex vivo e, em c?lulas HEK293 transfectadas com GRPR. Contudo, a aplica??o de GRP em medulas de camundongos, ex vivo, n?o parece depender da ativa??o das MAP-quinases, ERK1/2 ou p38. A inje??o intratecal de GRP leva a um comportamento de co?ar intenso, que ? significativamente reduzido por antagonistas de GRPR ou pela inibi??o farmacol?gica de PI3K?. Para avaliar se o sistema GRP/GRPR estaria envolvido no prurido cr?nico, foi empregado o modelo de pele seca em camundongos. Neste paradigma experimental, o bloqueio de GRPR ou de PI3K? reverteu o comportamento de co?ar. Os dados obtidos tamb?m mostram que p-Akt e GRPR est?o co-localizados na medula espinhal e em neur?nios do DRG de animais com pele seca e, que os n?veis de p-Akt est?o aumentados nesses animais, um efeito que foi prevenido pelo bloqueio de GRPR. A inje??o intrad?rmica de GRP tamb?m induziu o comportamento de co?ar, que foi significativamente reduzido ap?s o tratamento com inibidor de PI3K?. Finalmente, foi demonstrado que a inje??o intratecal de um ativador de Akt foi capaz de induzir coceira em camundongos. O conjunto de resultados obtidos sugere que o GRPR ? expresso por terminais perif?ricos e centrais de neur?nios nociceptivos do DRG, transmitindo o prurido atrav?s da ativa??o da via PI3K?/Akt.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-06-09T16:27:04Z No. of bitstreams: 1 ISABELE DA COSTA ANGELO.pdf: 4140152 bytes, checksum: bbd35489ea24b040be77df36cd797987 (MD5) / Made available in DSpace on 2016-06-09T16:27:32Z (GMT). No. of bitstreams: 1 ISABELE DA COSTA ANGELO.pdf: 4140152 bytes, checksum: bbd35489ea24b040be77df36cd797987 (MD5) Previous issue date: 2011-03-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The study evaluated the protein and lipid profiles of Rhipicephalus microplus engorged females after infection by Metarhizium anisopliae, Beauveria bassiana and Fusarium oxysporum. The treatments were immersion or inoculation of conidial suspension in R. microplus. The hemolymph was collected 24 and 48 hours after treatment. The cell-free hemolymph was separated of hemocytes by centrifugation and hemocytes resuspended in phosphate buffer pH 7.2. The amount of total protein was determined in both fractions of hemolymph and hemocytes were quantified. The cell-free hemolymph was filtered through a 100 kDa and 10 kDa membranes, and analyzed by electrophoresis and liquid chromatography (HPLC). The proteome of cell-free hemolymph (treatment by injection) was evaluated by 2DPAGE. Changes were observed in amount total protein and the amount of hemocytes, but no difference was observed in the electrophoretic profile (1D-PAGE) of the cell-free hemolymph. In haemocytes, the entomopathogens reduced the amount of serpins, while F. oxysporum caused increased. In 2D-PAGE variations were observed in both expression and presence/absence of protein between the groups. The cell-free hemolymph antimicrobial activity was tested against Escherichia coli and Staphylococcus aureus and against the fungus used in the treatment of engorged ticks. The hemolymph collected 48 hours after the immersion treatment with B. bassiana apresented activity anti-B. bassiana with 48 hours of evaluation. This hemolymph was subjected to Superose column to HPLC and peak was collected and analyzed on the analytical column C18. The fractions were collected from the C18 and its apresented activity anti-B. bassiana, but showed no activity against Candida albicans. These fractions were analyzed by Maldi-Tof and most of them had in common an ion with m/z 1,119.5; however, other ions may be involved with this activity antimicrobial. The lipids present in cell-free hemolymph, in the hemocyte and fat body were extracted and analyzed by thin layer chromatography (TLC) or HPTLC for neutral lipids and phospholipids. The classes of neutral lipids in the cell-free hemolymph were cholesterol ester, cholesterol (CHO) and fatty acids (FA), which have varied depending on the fungus used, type of treatment and observation time. Phospholipids found were phosphatidylcholine and phosphatidylethanolamine, its were not significantly altered after the fungal infection. In hemocytes, the same classes of lipids were found and B. bassiana modified phospholipids, while M. anisopliae s.l. altered FA and CHO. The fat body showed, in addition to these classes of neutral lipids, the triglycerides, which increased significantly 48 hours after inoculation with M. anisopliae s.l. The lipase activity in fat body was measured and it was demonstrated that increased activity 48 hours after inoculation, mainly in the group inoculated with Metarhizium. Therefore, the results showed alterations related to the proteins expression in the hemocytes and the cell-free hemolymph after inoculation with fungi, immunosuppression of hemocytes and antimicrobial peptides induction after infection with B. bassiana, besides changes in the lipid profile of R. microplus after infection. However, further studies are necessary to understand these changes. / O trabalho avaliou os perfis prot?ico e lip?dico de f?meas ingurgitadas de Rhipicephalus microplus ap?s infec??o com Metarhizium anisopliae s.l., Beauveria bassiana ou Fusarium oxysporum. Os tratamentos foram imers?o ou inocula??o da suspens?o conidial em R. microplus. A hemolinfa foi coletada 24 e 48 horas ap?s os tratamentos. O plasma foi separado dos hem?citos por centrifuga??o e os hem?citos resuspensos em tamp?o fosfato pH 7,2. A concentra??o de prote?na total foi determinada em ambas as fra??es da hemolinfa e os hem?citos quantificados. O plasma da hemolinfa foi filtrado em membrana de 100 kDa e 10 kDa, sendo analisados por eletroforese e cromatografia l?quida de alta efici?ncia (CLAE). O proteoma do plasma da hemolinfa (tratamento por inocula??o) foi avaliado por gel 2D. Foram observadas varia??es na quantidade de prote?na total de ambas as fra??es da hemolinfa, na quantidade de hem?citos bem como na intensidade de prote?nas/pept?deos expressos no plasma da hemolinfa. Nos hem?citos, os entomopat?genos reduziram a quantidade de serpinas, enquanto F. oxysporum causou aumento. No gel 2D foram observadas varia??es na express?o bem como na aus?ncia/presen?a de prote?nas entre os grupos. O plasma da hemolinfa teve sua atividade antimicrobiana testada contra Escherichia coli e Staphylococcus aureus e contra o fungo utilizado no tratamento das f?meas ingurgitadas. A hemolinfa coletada 48 horas ap?s o tratamento por imers?o com B. bassiana apresentou atividade anti-B. bassiana com 48 horas de avalia??o. Esta hemolinfa foi submetida ? coluna Superose de CLAE e o pico coletado analisado na coluna anal?tica C18. As fra??es coletadas da C18 apresentaram atividade anti-B. bassiana, por?m n?o apresentaram atividade contra Candida albicans. Essas fra??es foram analisadas por Maldi-Tof e a maioria delas apresentou um ?on com raz?o m/z 1.119,5; no entanto, outros ?ons podem estar envolvidos com essa atividade antimicrobiana. Os lip?deos presentes no plasma da hemolinfa, nos hem?citos e no corpo gorduroso foram extra?dos e analisados por cromatografia em camada delgada (CCD) ou CCD de alta performance para lip?deos neutros e fosfolip?deos. As classes de lip?deos neutros encontradas no plasma da hemolinfa foram colesterol-?ster, colesterol (CHO) e ?cidos graxos (AG), que sofreram altera??es em fun??o do fungo utilizado, tipo de tratamento e tempo de observa??o. Os fosfolip?deos encontrados foram fosfatidilcolina e fosfatidiletanolamina, que n?o foram significativamente alterados ap?s a infec??o f?ngica. Nos hem?citos, as mesmas classes de lip?deos foram encontradas e B. bassiana alterou os fosfolip?deos, enquanto M. anisopliae s.l. alterou os AG e CHO. O corpo gorduroso apresentou, al?m destas classes de lip?deos neutros, o triacilglicerol, que aumentou significativamente 48 horas ap?s a inocula??o com M. anisopliae s.l. A atividade lipase no corpo gorduroso foi mensurada, sendo evidenciado um aumento 48 horas ap?s a inocula??o, principalmente no grupo inoculado com Metarhizium. Portanto, os resultados demonstraram altera??es na express?o de prote?nas no plasma da hemolinfa e nos hem?citos ap?s inocula??o com os fungos, imunossupress?o dos hem?citos, indu??o de pept?deos com atividade antimicrobiana ap?s infec??o com B. bassiana, al?m de altera??es no perfil lip?dico de R. microplus ap?s infec??o. No entanto, maiores estudos s?o necess?rios para o entendimento dessas altera??es.

Submitted by Kelson Anthony de Menezes (kelson@ucb.br) on 2016-12-19T17:56:31Z No. of bitstreams: 1 SuzanaMeiraRibeiroTese2014.pdf: 4980549 bytes, checksum: df9c1eaadc1ba65bf7c17235d91dc3d7 (MD5) / Made available in DSpace on 2016-12-19T17:56:31Z (GMT). No. of bitstreams: 1 SuzanaMeiraRibeiroTese2014.pdf: 4980549 bytes, checksum: df9c1eaadc1ba65bf7c17235d91dc3d7 (MD5) Previous issue date: 2014-05-19 / Multi-drug resistant Klebsiella pneumoniae that produce the enzyme K. pneumoniae carbapenemase (KPC) are becoming a common cause of infections in health care centers. Furthermore, Klebsiella can develop multicellular biofilms, which lead to elevated adaptive antibiotic resistance. Here, it was described the antimicrobial and anti-biofilm activities of synthetic cationic peptides against K. pneumoniae strains susceptible and carbapenens resistant through in vitro and in vivo assays. By using static microplate assays, it was observed that the concentration of the peptides IDR-1018, DJK-5 and DJK-6 required to prevent biofilm formation by these clinical isolates was below the minimum inhibitory concentration (MIC) of these peptides. Flow cell experiments confirmed the anti-biofilm activity of the peptides against 2 day-old biofilms of different KPC producing isolates and, in some cases, the peptides induced biofilm cell death. Combinations of DJK-6 together with ??-lactam antibiotics, including the carbapenem meropenem, also prevented planktonic growth and biofilm formation of KPC producing strain 1825971. Interestingly, the peptide DJK-6 was able to enhance, by at least 16-fold, the ability of meropenem to eradicate pre-formed biofilms formed by this strain. Although, this combination between meropenem, DJK-6 was effective in vitro, any reduction of bacterial load was observed in murine lung model of infection. Similarly, the peptides HHC-10, IDR-1018 e IDR-1002 was effective in vitro, but ineffective in vivo. The results in vivo, using the peptides HHC-10 and IDR-1018 after infection were contrasting (significant reduction or non-reduction of bacterial load) between two models of lung infection. Others approaches using the peptides IDR-1018 prophylactically or the peptide IDR-1002 (effective in inhibit bacterial growth) before the induction of the infection, as a preventive approach or IDR-1002 (peptide that reduced the bacterial counts in vitro) after infection, also was unable to reduce the bacterial load in lungs of mice. Despite ineffective in acute model of lung infection, the use of cationic peptides, such DJK-6, to potentiate the activity of ??-lactams including meropenem, represents a promising strategy to prevent biofilm formation or eliminate existent biofilms both in medical devices and in body surfaces. / Klebsiella pneumoniae resistente a m??ltiplos antibi??ticos, que produzem a enzima K. pneumoniae carbapenemase (KPC), est??o se tornando uma causa comum de infec????es em centros de cuidado a sa??de. Al??m disso, K. pneumoniae pode desenvolver biofilmes multicelulares, que levam o aumento da resist??ncia adaptativa a antibi??ticos. Aqui foi descrito a atividade antimicrobiana e antibiofilme de pept??deos cati??nicos sint??ticos contra isolados de K. pneumoniae suscept??vel e resistente a carbapenens em ensaios in vitro e in vivo. Usando ensaios de microplaca est??tico, foi observado que a concentra????o dos pept??deos IDR-1018, DJK-5 e DJK-6, requerida para prevenir a forma????o de biofilmes de isolados resistentes foi abaixo da concentra????o inibit??ria m??nima (CIM) desses pept??deos. Experimentos de fluxo de c??lula confirmaram a atividade antibiofilme dos pept??deos contra biofilmes pr??-formados de diferentes isolados de K. pneumoniae produtores de KPC e, em alguns casos, os pept??deos induziram morte celular. Combina????es de DJK-6 e antibi??ticos ??-lact??micos, incluindo o carbapenem meropenem, tamb??m preveniram o crescimento planct??nico e a forma????o de biofilmes do isolado produtor de KPC 1825971. Interessantemente, o pept??deo DJK-6 foi capaz de aumentar, em pelo menos 16 vezes, a habilidade de meropenem erradicar biofilmes pr??-formados desse isolado. Embora, essa combina????o tenha sido efetiva in vitro, nenhuma redu????o da carga bacteriana foi observada em modelo murino de infec????o pulmonar por K. pneumoniae. Os resultados dos pept??deos HHC-10 e IDR-1018 (os quais inibiram o crescimento bacteriano in vitro a concentra????o que apresentou baixa citotoxicidade) foram contrastantes entre os dois modelos de infec????o pulmonar (significando redu????o ou n??o redu????o da carga bacteriana). Outras abordagens utilizando os pept??deos IDR-1018 profilaticamente ou o pept??deo IDR-1002 (efetivo em inibir o crescimento bacteriano in vitro) ap??s a infec????o, tamb??m foram incapazes de reduzir a carga bacteriana em pulm??es de camundongos. Apesar da inefetividade em modelo agudo de infec????o pulmonar, o uso de pept??deos cati??nicos, tal como DJK-6, para potencializar a atividade de ??-lact??micos, incluindo meropenem, representa uma estrat??gia promissora para prevenir a forma????o de biofilmes ou eliminar biofilmes existentes tanto em equipamentos m??dicos quanto em superf??cies do corpo.

Data de aprova??o ausente. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2016-12-21T16:01:47Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) isabela_pereira_gomes.pdf: 4600043 bytes, checksum: 3419d89b2ca19457442da4e144781e1b (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-01-21T11:24:06Z (GMT) No. of bitstreams: 2 license_rdf: 9 bytes, checksum: 42dd12a06de379d3ffa39b67dc9c7aff (MD5) isabela_pereira_gomes.pdf: 4600043 bytes, checksum: 3419d89b2ca19457442da4e144781e1b (MD5) / Made available in DSpace on 2017-01-21T11:24:06Z (GMT). No. of bitstreams: 2 license_rdf: 9 bytes, checksum: 42dd12a06de379d3ffa39b67dc9c7aff (MD5) isabela_pereira_gomes.pdf: 4600043 bytes, checksum: 3419d89b2ca19457442da4e144781e1b (MD5) Previous issue date: 2016 / 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) / Micro-organismos resistentes a antibi?ticos v?m atingindo ?ndices elevados nos ?ltimos anos, agravando problemas de sa?de p?blica e econ?micos. O impacto na sociedade devido ? resist?ncia de bact?rias aos antibi?ticos convencionais tem mobilizado a pesquisa para o desenvolvimento de novas drogas. Nesse contexto, surgem os pept?deos antimicrobianos, que s?o mol?culas efetoras do sistema imune inato e podem ser encontrados em praticamente todos os seres vivos. Para prote??o contra os micro-organismos presentes em seu habitat, a pele de anuros apresenta um verdadeiro arsenal qu?mico, constitu?do, em sua maioria, de diferentes classes de pept?deos. Dentre esses, os antimicrobianos s?o considerados os mais avan?ados participantes do sistema imunol?gico. V?rios estudos sugerem que os pept?deos antimicrobianos podem atuar por diferentes mecanismo como miceliza??o, forma??o de poros na superf?cie da membrana ou, ainda, pelo ataque a algum alvo intracelular. Embora a maioria dos modelos existentes proponham que pept?deos antimicrobianos exercem suas atividades biol?gicas por meio da intera??o com a membrana bacteriana, o mecanismo de a??o dessas mol?culas ainda n?o ? completamente compreendido. Dessa forma, o presente estudo tem como objetivo obter informa??es estruturais, conformacionais e de intera??o em ambientes que mimetizam membranas biol?gicas, do pept?deo Hylaseptina P1 (HSP-1), composto por 14 res?duos de amino?cidos e isolado do anuro da esp?cie Hyla punctata visando o entendimento do seu mecanismo de a??o. Neste trabalho, o pept?deo HSP-1 foi sintetizado manualmente atrav?s da s?ntese de pept?deos em fase s?lida via estrat?gia Fmoc. As prefer?ncias conformacionais do pept?deo em meios micelares de dodecilsulfato de s?dio (SDS), dodecilfosfocolina (DPC) e ves?culas fosfolip?dicas de 1-palmitoil-2-oleilfosfatidilcolina (PC) e 1-palmitoil-2-oleoil-fosfatidilglicerol (PG) foram avaliadas por Dicro?smo Circular. Observou-se que em meio aquoso HSP-1 adota estrutura desenovelada, enquanto que, em meios micelares e ves?culas fosfolip?dicas, apresentou conforma??o ?-h?lice. A estrutura tridimensional foi estudada na presen?a de micelas de SDS e DPC por Espectroscopia de Resson?ncia Magn?tica Nuclear em solu??o, revelando que, em micelas de SDS o pept?deo HSP-1 exibe conforma??o helicoidal mais prolongada do que em micelas de DPC, no qual foi poss?vel verificar pequena dobra da h?lice na regi?o N-terminal da cadeia pept?dica, sugerindo maior inser??o do pept?deo em micelas zwiteri?nicas. O efeito da adi??o de pept?deo no tamanho e na carga superficial de ves?culas fosfolip?dicas de PC e PC:PG (3:1) foi investigado por medidas de espalhamento de luz din?mica e Potencial Zeta. Verificou-se que a adi??o de HSP-1 resulta no aumento do di?metro hidrodin?mico (?Dh ? 20 nm) e aumento do potencial Zeta (?? ? 15 mV) de ves?culas unilamelares (LUV?s) de PC:PG, indicando intera??o eletrost?tica com a superf?cie das ves?culas. Quando ves?culas de PC foram tituladas com HSP-1, foi observada maior altera??o no di?metro hidrodin?mico das LUVs (?Dh ? 22 nm), enquanto que n?o se observaram altera??es significativas nos valores de potencial Zeta (?? ? 0 mV), sendo indicativo de uma maior inser??o do pept?deo em membranas zwiteri?nicas. Conforme observado nos resultados de Calorimetria de Titula??o Isot?rmica, a intera??o com ves?culas zwiteri?nicas foi da ordem de 102 maior do que o valor encontrado para intera??o com ves?culas negativas. Contudo, os dados termodin?micos revelaram a predomin?ncia de intera??es eletrost?ticas de HSP-1 com ves?culas de PC:PG e hidrof?bicas com ves?culas de PG. Finalmente, a capacidade de forma??o de poros de HSP-1 foi examinada por meio de medidas de extravasamento de carboxifluoresce?na (CF) em ves?culas de PC e PC:PG. Em PC-LUVs observou-se uma maior intensidade de fluoresc?ncia em compara??o com PC:PG-LUVs, sugerindo uma maior atividade l?tica do pept?deo em ves?culas zwiteri?nicas. A porcentagem m?xima de CF liberada foi superior em LUVs-PC (65%) quando comparado a LUVs de PC:PG (50%), confirmando que HSP-1 tem maior capacidade de permeabiliza??o em meio zwiteri?nico. Os dados de extravasamento demostraram tamb?m menor cin?tica de libera??o de CF em PC-LUVs, condizente com um mecanismo no qual h? maior inser??o do pept?deo na interface da bicamada lip?dica, e maior cin?tica de libera??o de CF em PC:PG-LUVs, de acordo com um mecanismo tipo carpete, o qual envolve apenas intera??o superficial do pept?deo com a membrana. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The resistance of microorganisms to antibiotics have reached high levels in recent years, exacerbating public health and economic problems. The impact of bacterial resistance on the global society has mobilized the research community for the development and new drugs discovery. In this context, antimicrobial peptides appear as an alternative antibiotics class, which are effector molecules of the innate immune system in almost every living beings. In order to protect against microorganisms present in their habitat the skin of frogs presents a real chemical arsenal consisting mostly of different peptides classes, which are considered the most advanced participants of the immune system. Several studies suggest that antimicrobial peptides may act by different models such as micellization, pore formation in membrane surface or also intracellular target attack. Although most of models propose that antimicrobial peptides exert their biological activities by interacting with the bacterial membrane, the mechanism of action is not still fully understood. In order to study the action mechanism of antimicrobial peptide Hylaseptina P1 (HSP-1), composed of 14 amino acid residues and isolated from Hyla punctate species, this work presents conformational and thermodynamic analysis of peptide-membrane interaction in biomimetic environments. In this work, the HSP-1 peptide was synthesized manually by solid phase peptide synthesis using Fmoc strategy. The conformational preferences of peptide were evaluated by Circular dichroism in sodium dodecylsulfate (SDS) and dodecilfosfocolina (DPC) micellar media or 1-palmitoyl-2-oleilfosfatidilcolina (PC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (PG) phospholipid vesicles. A random conformation was observed to HSP-1 in aqueous medium while in micellar or phospholipid vesicles media HSP-1 showed a predominant ?-helix conformation. The three dimensional structure were obtained by solution Nuclear Magnetic Resonance in the presence of SDS and DPC micelles, revealing that HSP-1 exhibits a longer helical conformation in SDS than DPC micelles. In zwiterionic medium could be verified a subtle bend at the N-terminus, suggesting a partial insertion of the peptide in DPC micelles. The effects of adding peptide in size or surface charge of PC and PC:PG phospholipid vesicles were investigated by Dynamic Light Scattering and Zeta Potential measurements. The addition of HSP-1 to PC:PG-LUVs results in increasing of both hydrodynamic diameter (?Dh ? 20 nm) and zeta potential (?? ? 15 mV), indicating an electrostatic interaction with the surface vesicles. On the other hand, when PC-LUVs were titrated with HSP-1 a greater change in hydrodynamic diameter (?Dh ? 22 nm), no significant variations were observed in superficial charge, indicating a partial insertion of the peptide in zwitterionic PC membranes. In addition, the thermodynamic studies carried out by isothermal titration calorimetry showed a peptide-membrane interaction approximately 102 higher with PC-LUVs when compared to negative PC:PG-LUVs. Nevertheless, whereas the greater enthalpic contribution in PC:PG-LUVs revealed the predominance of electrostatic interactions with HSP-1, in PC vesicles predominate hydrophobic interactions. Finally, the ability of poring formation of HSP-1 was examined by carboxyfluorescein (CF) release from PC and PC:PG vesicles. It was observed a higher fluorescence intensity in PC-LUVs compared to PC:PG-LUVs, suggesting a greater lytic activity of the peptide in zwiterionic vesicles. The maximum percentage of CF released was approximately 65% to PC-LUVs and 50% to PC:PG-LUVs, confirming a greater membrane permeabilization in zwiterionic medium. Furthermore, this study has also demonstrated a lesser kinetics of CF leakage in PC-LUVs, consistent with a mechanism in which there is greater insertion of the peptide in the lipid bilayer interface. On the other hand, the higher kinetics of CF leakage in PC:PG?LUVs is in accordance with a carpet-like mechanism, which involve only superficial interaction between peptide and membrane.