Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Atualmente, a dengue à considerada a arbovirose mais importante do mundo, emergindo em paÃses onde a doenÃa parecia erradicada e ressurgindo em paÃses onde antes estava sob controle. Apesar de todos os esforÃos de pesquisa empreendidos na produÃÃo e desenvolvimento de uma vacina contra a doenÃa, ainda nÃo se dispÃe de uma terapia preventiva eficaz; a Ãnica alternativa de manejo nos dias atuais se dà atravÃs do combate ao Ãnico elo vulnerÃvel, o mosquito vetor Aedes aegypti. Apesar dos programas de manejo das populaÃÃes de vetores disporem de vÃrias opÃÃes de inseticidas sintÃticos, o surgimento de populaÃÃes resistentes apresenta-se como um entrave no controle desse mosquito vetor. AlÃm do monitoramento dessas populaÃÃes resistentes, tornou-se evidente que estudos moleculares podem ser a chave para o manejo sustentÃvel desses vetores. Assim, o presente trabalho teve por objetivo avaliar as alteraÃÃes na expressÃo proteica de larvas de Ae. aegypti tratadas com o larvicida orgÃnico m-pentadecadienil-fenol, um lipÃdeo fenÃlico isolado de sementes de Myracrodruon urundeuva a fim de elucidar os possÃveis mecanismos de detoxificaÃÃo e resposta molecular a esse composto. Para isso foi realizada eletroforese bidimensional (focalizaÃÃo isoelÃtrica seguida de eletroforese em gel de poliacrilamida em condiÃÃes desnaturantes - SDS-PAGE) de larvas de terceiro estÃdio tratadas com m-pentadecadienil-fenol, em concentraÃÃes subletais (CL50 10,16 Âg.mL-1), em comparaÃÃo com um grupo controle nÃo tratado. Treze spots foram identificados como diferencialmente expressos, e doze foram identificados consistentemente em bancos de dados apÃs anÃlise dos espectros obtidos por espectrometria de massas (ESI-Q-ToF). ApÃs a anÃlise das vias nas quais essas proteÃnas estÃo envolvidas, foi proposto que o larvicida m-pentadecadienil-fenol elicita a superexpressÃo de proteÃnas que promovem a formaÃÃo de barreiras mais eficientes, e que uma vez dentro das cÃlulas promove um estresse oxidativo que leva ao aumento do metabolismo de degradaÃÃo lipÃdica, desestabilizaÃÃo da membrana lisossomal, aumento do metabolismo em resposta ao estresse tÃxico e possivelmente resulta em morte celular programada (apoptose). / Currently, dengue fever is considered the most important arboviral disease in the world, emerging in countries where the disease seemed eradicated and reappearing in countries where before was under control. Despite all the efforts to produce and develop a vaccine against the disease, there is not a preventive therapy. Nowadays, the disease management is through the eradication of the mosquito vector Aedes aegypti. Despite the several options of synthetic insecticides available for management programs of vector populations, the emergence of resistant populations is presented as an obstacle in mosquito control vector. In addition to monitoring these resistant populations, it became evident that molecular studies may be the key to the sustainable management of these vectors. Thus, the present study aimed to evaluate the changes in protein expression of Ae. aegypti larvae treated with the organic larvicide m-pentadecadienil-phenol, a phenolic lipid isolated from Myracrodruon urundeuva seeds to elucidate the putative mechanisms of detoxification and molecular response to this compound. For this, two-dimensional electrophoresis (isoelectric focusing followed by electrophoresis in polyacrylamide gel under denaturing conditions - SDS-PAGE) of third-instar larvae treated with m-pentadecadienil-phenol, at sublethal concentrations (LC50 10.16 microg.mL-1) was performed. The results were compared to a non-treated group. Thirteen spots were identified as differentially expressed, and twelve were consistently identified in databases analysis of the spectra obtained by mass spectrometry (ESI-Q-ToF). After analysis of the pathways in which these proteins are involved, it was proposed that the larvicide m-pentadecadienil-phenol elicits the overexpression of proteins that promote the formation of more efficient barriers and that once inside the cells, it promotes oxidative stress which leads to increased lipid degradation metabolism, destabilization of lysosomal membrane, increased metabolism in response to stress due to the moleculeâs toxicity and possibly results in programmed cell death (apoptosis).

Les paramètres comportementaux sont de plus en plus considérés comme étant des indicateurs sensibles et précoces de toxicité chez les organismes aquatiques. Cependant, l’utilisation du comportement natatoire comme critère de toxicité pour l’analyse de risque environnemental ou pour le contrôle de la qualité de l’eau reste pour l’heure encore limitée. En effet, il est actuellement difficile de quantifier la sensibilité des paramètres comportementaux et d’établir un lien entre les seuils d’effets comportementaux et les effets aigus et subaigus classiquement mesurés dans les tests en écotoxicologie. Dans le but d’améliorer notre compréhension des effets comportementaux des polluants sur Daphnia magna, nous avons développé un nouveau système de mesure du comportement multi-cuves baptisé «Multi-DaphTrack ». Douze substances toxiques, couvrant une large gamme de modes d’action différents ont été testées dans ce système. Un test standard d’immobilisation ainsi que des analyses de comportement dans le toximètre DaphToxI®, ont également été effectués pour chaque substance afin de comparer les seuilsd’effets comportementaux avec les paramètres classiques d’immobilisation. Les résultats des expositions aux différentes substances ont démontré que notre nouveau système d’exposition multi-cuves permet de détecter des effets comportementaux (i.e.,augmentation de la vitesse de nage) significatifs et précoces pour l’ensemble des substances testées et ce, à des concentrations proches de la CE10(48 h) du test aigu d’immobilisation dès la première heure d’exposition. Des profils comportementaux différents ont été observés selon les substances testées (i.e., intensité, temps de latence et durée de l’effet) mais ceux-ci ne sont pas spécifiques d’un mode d’action particulier. Les résultats obtenus avec le toximètre DaphToxI®ont révélé des profils d’effet similaires (i.e.,augmentation de la vitesse de nage) bien que ce système soit globalement moins sensible par rapport au système «Multi-DaphTrack». Pour conclure, notre nouveau système d’exposition multi-cuves «Multi-DaphTrack» est un outil plus sensible et précoce que letest standard d’immobilisation pour l’évaluation de la toxicité de substances chimiques. L’utilisation du système «Multi-DaphTrack» pourrait donc être envisagée, après quelques améliorations et validation supplémentaires, pour l’évaluation de la qualité des masses d’eauet des effluents. / Swimming behavior is increasingly reported as a sensitive and early indicator of toxicant stress in aquatic organisms. However, it remains unclear how to quantify the sensitivity of swimming behavioral endpoints and how to compare these effect thresholds with standard ecotoxicological endpoints used in risk assessment. To date, the systematic assessment of the sensitivity of swimming behavioral endpoints in daphnia is limited because of the restrained test capacity of existing behavioral analysis systems. Hence, we developed a new behavioral analysis multi-cellsystemnamed “Multi-DaphTrack” with a high throughput testing capacity in order to enhance our understanding of swimming behavioral effects in Daphnia magnaunder chemical exposure. Twelve compounds covering different modes oftoxic action were selected and tested in this new system andin a single-cell commercialized biomonitor (DaphToxI®) and with the acute standard test.Our new multi-cellexposure system detectedsignificant and early swimming behavioral effects (i.e.,increase of the average speed) for most of the tested compounds and this, from the first hour of exposure at concentrations near the EC10(48h). Contrastedbehavioral profiles were observed for average speed (i.e., intensity, time of effect onset, effect duration), but no distinct behavioral profiles could be drawn from the chemical mode of action. Despite less sensitive,the DaphToxI®gave similar trends (i.e.,rapid peak increase) compared to our “Multi-DaphTrack” system. To conclude, behavior analysis using our “Multi-DaphTrack”systemcould be used as an alternative or complement to the current acute standard test for toxicity assessment of chemicals. With some additional improvements and validations, it also could be used forquality assessment of waterbodiesand sewages.

Triclosan (TCS) is a personal care product with many fields of application and is of public interest for several years now. Monitoring studies showed that TCS is a ubiquitous chemical in the aquatic environment. Aquatic organisms are exposed to TCS in a broad range of concentrations, from ng L-1 up to lower μg L-1. TCS has a bactericidal effect for various types of gram-positive and gram negative bacteria. TCS targets a specific bacterial fatty acid biosynthetic enzyme, enoyl-[acyl-carrier protein] reductase (Schweizer, 2001). Therefore the terminal reaction in the fatty acid elongation cycle is inhibited (Levy et al., 1999). Although effects on non-target organisms are reported, the Mode of Action (MoA) of TCS is not well examined for those organisms. The aim of this PhD thesis was to investigate effects of TCS on non-target autotrophic organisms at different levels of biological complexity in the aquatic environment. In this thesis microalgae have been found to be very sensitive to TCS. In some cases even higher sensitivities than in bacteria were observed, which is in accordance with published effect data (Harada et al., 2008; Orvos et al., 2002). Similarly to bacteria, high species sensitivity differences were observed for algae (Franz et al., 2008). In bacteria these sensitivity differences can be ascribed to several resistance mechanisms reported in Schweizer (2001). These findings lead to the question about the reasons for species sensitivity differences in algae. A mesocosm study was performed to detect effects of TCS across levels of biological organization and to investigate the impact of sensitivity differences on complex aquatic communities. For that purpose, structural and functional effects parameters were observed.

β-lactam antibiotics have long been a reliable course of treatment for bacterial infections. However, with recent increases in resistance and rising populations of immunocompromised patients new β-lactams have been synthesized and tested. The Turos laboratory has recently discovered novel β-lactams that have a mode of action distinct from penicillin and other β-lactam antibiotics as cell lysis is not observed. In the current investigations, these compounds are shown to also have antifungal properties. The rising incidence and prevalence of invasive fungal infections has become an increasing concern. The most common fungal pathogens involved in these infections are species in the genus Candida. In this study antifungal activity is observed for a wide range of N-methylthio β-lactams against C. albicans, C. tropicalis, C. keyfr, C. glabrata, C. lusitinae, C. utilis, and C. parapsilosis. The structure-activity relationship based on studies of β−lactam derivatives leaving different substituents at various positions on the lactam ring are investigated, and the minimum inhibitory concentration values determined using standard methods. In studies towards understanding the mode of action, the products of the interaction between the drug and fungal cells in a suspension were investigated using nuclear magnetic resonance spectroscopy and transmission electron microscopy. The mode of action of these new lactams seems to be similar to that observed in bacteria, involving transfer of the methylthio group to a cellular thiol.

Submitted by CAROLINE PLACIDI DE BORTOLI null (carubortoli@yahoo.com.br) on 2018-02-22T10:06:56Z No. of bitstreams: 1 Tese_Caroline_Placidi_De_Bortoli.pdf: 20556112 bytes, checksum: 3c463875d34d1f0b87961046250a6dab (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-02-22T12:45:28Z (GMT) No. of bitstreams: 1 debortoli_cp_dr_jabo.pdf: 20556112 bytes, checksum: 3c463875d34d1f0b87961046250a6dab (MD5) / Made available in DSpace on 2018-02-22T12:45:28Z (GMT). No. of bitstreams: 1 debortoli_cp_dr_jabo.pdf: 20556112 bytes, checksum: 3c463875d34d1f0b87961046250a6dab (MD5) Previous issue date: 2018-02-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Plutella xylostella é uma praga de grande importância para as crucíferas em todo o mundo. Embora seja controlada com inseticidas sintéticos e biológicos, ela pode desenvolver resistência rapidamente a uma variedade de inseticidas. Os biopesticidas mais comuns utilizados para controlar P. xylostella são baseados na bactéria entomopatogênica Bacillus thuringiensis. Embora muitos estudos tenham sido realizados com Bt, o modo de ação ainda não é totalmente compreendido. Uma grande diversidade de genes é diferencialmente expressa no intestino médio de insetos resistentes, o que sugere que vários processos celulares podem estar envolvidos na resistência. Descobertas recentes mostraram que as mutações no gene que codifica o transportador ABCC2 são responsáveis pela resistência às toxinas Bt em diferentes espécies de insetos. O objetivo desta pesquisa foi testar a hipótese de que a susceptibilidade de P. xylostella a Bt se correlaciona com o nível de expressão dos componentes desse regulador de estresse. Os níveis de expressão dos genes ALP, APN, CDKAL 1, MAP4K4 e ABCC2 foi comparado utilizando qPCR, entre populações suscetíveis e resistentes de P. xylostella. A investigação da sequência de DNA do cDNA do ABCC2 foi realizada, por PCR e sequenciamento, para testar a hipótese de que a susceptibilidade de P. xylostella a Bt se correlaciona com mutações no gene ABCC2. Foram realizados retrocruzamentos entre populações suscetíveis e resistentes e cruzamentos de complementação entre populações resistentes. Nossa pesquisa demonstrou que não há padrões na expressão dos genes testados demonstrando nenhuma associação entre expressão e resistência/susceptibilidade. No entanto, ao investigar a sequência do gene ABCC2, encontrou-se uma mutação no gene da população brasileira resistente, que poderia ser responsável pela causa da resistência da população estudada neste trabalho. Os ensaios de retrocruzamentos não confirmaram que a resistência foi devida à supressão de 1 pb encontrada, no entanto, os ensaios complementares indicaram que a população brasileira compartilha um alelo de resistência com a população havaiana resistente. / Plutella xylostella is a major insect pest of cruciferous crops worldwide. Although controlled with both synthetic and biological insecticides it can rapidly evolve resistance to a variety of insecticides. The most common biopesticides used to control P. xylostella are based on the entomopathogenic bacterium Bacillus thuringiensis. Although many studies have been performed on Bt, the mode of action is still not fully understood. A wide diversity of genes are differentially expressed in the midgut of resistant insects, this suggests that a variety of cell processes may be involved in the preservation of resistance. Recent discoveries have shown that mutations in the gene encoding an ABCC2 transporter are responsible for resistance to Bt toxins in various different insect species. The aim of this research was to test the hypothesis that the susceptibility of P. xylostella to Bt correlates with the level of expression of components of this putative stress-response regulon. The level of expression of ALP, APN, CDKAL 1, MAP4K4 and ABCC2 genes were compared using qPCR, between susceptible and resistant P. xylostella populations. The investigation of the DNA sequence of ABCC2 cDNA was performed, through PCR and sequencing, to test the hypothesis that the susceptibility of P. xylostella to Bt correlates with mutations on the ABCC2 gene. Backcrosses between susceptible and resistant populations and complementation cross between resistant populations were performed. Our research demonstrated that there were no patterns in the expression of the genes tested demonstrating no association between expression and resistance/susceptibility. However, when investigating the ABCC2 gene sequence, a mutation in the gene of the Brazilian resistant population was found, which could be responsible for the resistance of the Brazilian population studied in this research. Backcrossing assays didn’t confirm that the resistance was due to the 1bp deletion found, however complementation assays indicated that the resistant Brazilian population shares a resistance allele with the resistant Hawaiian population. / FAPESP: 2015/05891-6 / FAPESP: 2016/04868-3 / CNPq: 140916/2014-8 / CNPq: 166510/2017-3

In view of the global spread of multiresistant bacteria and the occurrence of panresistant bacteria, there is an urgent need for antimicrobials with novel modes of action. A promising class is antimicrobial peptides (AMPs), including them proline-rich AMPs (PrAMPs), which target the 70S ribosome to inhibit protein translation. Here, we present a new designer peptide, Api805, combining the N- and C-terminal sequences of PrAMPs Api137 and drosocin, respectively. Api805 was similarly active against two Escherichia coli B strains but was inactive against E. coli K12 strain BW25113. These different activities could not be explained by the dissociation constants measured for 70S ribosome preparations from E. coli K12 and B strains. Mutations in the SbmA transporter that PrAMPs use to pass the inner membrane or proteolytic degradation of Api805 by lysate proteases could not explain this either. Interestingly, Api805 seems not to bind to the known binding sites of PrAMPs at the 70S ribosome and inhibited in vitro protein translation, independent of release factors, most likely using a “multimodal effect”. Interestingly, Api805 entered the E. coli B strain Rosetta faster and at larger quantities than the E. coli K-12 strain BW25113, which may be related to the different LPS core structure. In conclusion, slight structural changes in PrAMPs significantly altered their binding sites and mechanisms of action, allowing for the design of different antibiotic classes.

Natural products have been a significant source of new drugs, especially in treating cancer, infectious diseases, hypertension, and neurological disorders. Although many natural metabolites have been screened and yielded pharmaceutically important drugs, many potential sources of natural product drug therapies still need to be investigated, including lichens. Lichens are symbiotic systems consisting of a filamentous fungus and a photosynthetic partner (an eukaryotic alga and/or cyanobacterium). Lichens produce an impressive variety of unique secondary compounds and have been used as ingredients in folk medicines for centuries. Demonstrated biological roles based on lichen chemistry include: antibiotics, anti-proliferative, antioxidants, anti-HIV, anti-cancer, immunomodulation, and anti-protozoans. Although North America is home to an impressive variety of lichen species, there is limited research to examine the biological potentials of these lichens. The core goal of this dissertation research has been to investigate some of the biological roles including, antibiotic, antioxidant, and anticancer potentials using lichen crude extracts and their metabolites collected from various locations in the United States. Antibiotic screening of crude extracts of 36 lichen species demonstrated inhibitory effects against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Methicillin-resistant S. aureus (MRSA). Generally, acetone extractions were found to be more effective than methanol extractions. It has also been shown that L. vulpina extract was bacteriocidal against MRSA with a relatively slow kill rate that disrupts cell membrane integrity and cell division as possible modes of action. Antioxidant screening of extracts from 11 lichen species, using the Oxygen Radical Absorbance Capacity (ORAC) assay, showed that lichen extracts inhibited the oxidative degradation of the fluorescent molecule (fluorescein-sodium salt) by the oxygen free radical initiator AAPH (2,2'-azobis(2-aminidopropane) dihydrochloride Acetone extracts as well as pure compounds from lichen species showed cytotoxic effects against Burkitt's lymphoma (Raji) cells and a colon cancer cell line (HT29 and SW620). They decreased proliferation, arrested cell cycle at various stages and force the cell to undergo apoptosis. The tested extracts or pure compounds were not toxic to normal cells. In colon cancer apoptosis took place independent of casapase-3. The results of this dissertation showed that lichen compounds merits for further investigation.

A utilização de toxinas Cry de Bacillus thuringiensis (Bt) no controle de lepidópteros-praga, principalmente em áreas onde a estratégia de refúgio não é regulamentada, facilita a evolução da resistência em populações de pragas-alvo. Há três relatos de resistência à campo para Spodoptera frugiperda (J.E. Smith), dois para a toxina Cry1F e um para Cry1Ab. No Brasil, ocorrem populações resistentes à Cry1F e Cry1Ab. Esse trabalho foi voltado à identificação do mecanismo de resistência de uma população de S. frugiperda à toxina Cry1F, baseando-se nas hipóteses existentes para explicar o modo de ação de toxinas Cry. Uma dessas hipóteses é baseada na formação de poros na membrana do epitélio intestinal, enquanto a outra na transdução de sinal intracelular e ativação do processo de morte celular. Para a identificação do mecanismo de resistência de S. frugiperda à toxina Cry1F, o receptor caderina de linhagens suscetível (SUS) e resistente (RES) foi caracterizado, bem como realizado estudos de expressão gênica diferencial comparativa pela análise do transcritoma dessas linhagens. Estudos de expressão gênica diferencial comparativa também foram realizados pela análise do transcritoma do intestino de lagartas de linhagens SUS e resistente isogênica (RESiso), para a identificação do mecanismo molecular de resistência à toxina Cry1F. A caracterização do transcrito do gene caderina das linhagens suscetível, resistente e resistente isogênica revelou diferenças na composição de aminoácidos da proteína caderina predita entre as linhagens suscetível e resistente à toxina Cry1F nos domínios de repetição CR5, CR6 e CR10 e no domínio C-terminal. Também foi verificado que das mutações encontradas na linhagem RES, apenas as mutações da região C-terminal foram fixadas na linhagem RESiso. A análise comparativa do transcritoma de linhagens SUS e RES indicou a maior expressão de genes relacionados à metabolização de xenobióticos, como as monoxigenases do citocromo P450, glutationa-S-transferases e carboxilcolinesterases, em lagartas resistentes, mas não foram encontradas diferenças na expressão de receptores Cry, como aminopeptidase N e fosfatase alcalina. Porém, caderina foi superexpressa e o transportador ABCg5 teve expressão reduzida na linhagem RES. O ABCg5 foi indicado como o provável mecanismo de resistência dessa linhagem à toxina Cry1F, juntamente com o aumento da capacidade de detoxificação relatada. A análise comparativa do transcritoma de linhagens SUS e RESiso produziu resultados semelhantes à análise anterior quanto ao padrão de expressão de enzimas de detoxificação, mas nesse caso foi observada redução da transcrição de caderina na linhagem RESiso em relação à SUS. A análise da linhagem isogênica também indicou alteração na expressão de transportadores ABC na linhagem RESiso; porém, para o transportador ABCb1. A análise comparativa do transcritoma de linhagens SUS e RESiso corroborou a participação do sistema de detoxificação e acrescentou a redução na expressão do receptor caderina como mecanismo de resistência dessa população à toxina Cry1F, assim como a de transportadores ABC, apesar do transportador ABCg5 não ter sido identificado nessa análise comparativa. / The broad use of Cry toxins from Bacillus thuringiensis (Bt) to control lepidopteran pests, particularly where refuge strategies are not legalized or implemented, has facilitated the evolution of resistance of pest populations. There are three records of resistance of Spodoptera frugiperda (J.E Smith) in field condition to Bt toxins so far, two of them to Cry1F and one to Cry1Ab toxins. In Brazil, field-evolved resistance of S. frugiperda has been recorded for both toxins. Thus, we aimed to identify the mechanisms associated to the resistance of S. frugiperda to Cry1F toxin based on the two concurrent hypotheses on the mode of action of Cry toxins. One of such hypotheses is based on the potential of Cry toxins to form pores in the membrane of the gut epithelium, while the other is based on the production of an intracellular transduction signal and the activation of the process of cell death. To identify the resistance mechanisms of S. frugiperda to Cry1F toxin, we characterized the transcript of the cadherin receptor of susceptible (SUS) and resistant (RES) strains of S. frugiperda to search for mutations and performed a comparative analysis of the transcriptome from SUS and RES strains. We also analyzed the transcriptome from the gut of SUS and isogenic resistant strains (RESiso) in order to identify the molecular mechanisms associated to the resistance of S. frugiperda to Cry1F. The characterization of cadherin receptor in SUS, Res and REsiso strains showed differences in the amino acid composition of the repeated domains CR5, CR6 and CR10 and in the C-terminal domain. Only mutations occurring on C-terminal of the RES strain were maintained in the RESiso strain. The comparative transcriptome between SUS and RES strains indicated a higher expression of genes related to the detoxification process, such as cytochrome P450s, glutathione-S-transferases and carboxylcholinesterases in the RES strain, while no differences in the expression of Cry receptors, such aminopeptidade N and alkaline phosphatase, were observed. However, transcriptomic analysis indicated up-regulation of cadherin and down-regulation of the ABCg5 transporter was down-regulated in the RES strain. We propose that ABCg5 is one of the mechanisms involved in S. frugiperda resistance to Cry1F, together with the increased detoxification activity observed. Analysis of the gut transcriptome from SUS and RESiso yielded similar results regarding the differential expression of detoxifying enzymes, but on this case cadherin was down-regulated in RESiso as compared to the SUS strain. Down-regulation of ABC transporters in the RESiso strain was also observed, but for the ABCb1 transporter. Analyses of the transcriptome of SUS and RESiso strains also indicated the resistance of S. frugiperda to Cry1F is related to an increased transcription of detoxifying enzymes and a reduced transcription of the cadherin receptor. Our data also demonstrates the resistance is due to the existence of adequate constitutive levels of transcription of genes that respond to the intoxication with Cry1F.

Uma bacteriocina produzida por uma cepa de Lactobacillus sake 2a, isolada de lingüiça frescal comercial foi purificada, caracterizada e seu modo de ação foi estudado com o objetivo de ser utilizada como bioconservante em alimentos. A bacteriocina apresentou efeito bactericida contra Listeria monocytogenes em meio de cultura. Algumas cepas como Listeria welshimeri, Listeria seeligeri e Listeria inócua foram sensíveis, mas Staphylococcus aureus e Escherichia coli O157H7 foram resistentes a esta bacteriocina. O melhor meio de cultura para a sua produção foi o meio MRS à 25 ou 30°C durante a fase logarítmica de crescimento, obtendo-se 450 UA/ml de bacteriocina. Observou-se estabilidade a 121° C por 15 minutos. A bacteriocina foi purificada 71186 vezes pela técnica de adsorção/desorção de proteínas seguida de cromatografia de troca iônica Mono S, com rendimento de 3%. O peso molecular estimado foi de 3 a 6 kDa, determinado por eletroforese em gel de poliacrilamida-tricina. Nos estudos do mecanismo de ação, a bacteriocina dissipou o potencial de membrana, o gradiente de pH e reduziu de 80% os níveis de ATP intracelular não alterando os níveis de ATP extracelular. Bacteriocina 2a, nas concentrações de 28, 60 e 114 UA/ml, também causou efluxo de 3, 30 e 100% de carboxifluoresceina dos lipossomos construídos com lipídios de membrana de Listeria monocytogenes Scott A, respectivamente. Os resultados indicam que a bacteriocina 2a atua formando poros na membrana citoplasmática de células sensíveis não necessitando de um receptor específico. Conclui-se também que a bacteriocina é um bioconservante em potencial, podendo ser usada no controle de Listeria monocytogenes em alimentos. / Bacteriocin 2a produced by Lactobacillus sake 2a isolated from a Brazilian meat product (lingüiça) was purified, characterized and its mecanism of action was studied. The bacteriocin 2a showed bactericidal effect against Listeria monocytogens, Listeria welshimeri, Listeria seeligeri and Listeria inocua but it did not have an effect against Staphylococcus aureus e Escherichia coli O157H7 strains. The highest concentration of bacteriocin 2a (450 AU/ml) was found in MRS medium incubated at 25 or 30°C and its production was at its maximum towards logaritmic growth. It was stable at 121° C for 15 minutes. Bacteriocin 2a was purified 71186 fold by salt extraction from Lactobacillus sake cells, followed by cation exchange chromatography using Mono S column. It has an estimated molecular mass of 3-6kDa. In mechanistic studies of action against Listeria monocytogenes Scott A, bacteriocin 2a dissipated the pH gradient, the ΔΨ and reduced the ATP internal concentration by 80% with no detectable increase in the external ATP concentration. Bacteriocin 2a concentration of 28, 60 and 114 AU/ml, also mediated carboxyfluorescein efflux of 3, 30 and 100% from liposomes made from lipids extracted from Listeria monocytogenes Scott A, respectively. These data indicate that bacteriocin 2a forms pores in the citoplasmatic membrane of target cells similarly Class II bacteriocins. In addiction it can be use as a potential anti-microbial against Listeria monocytogenes in food.

SOUZA, Terezinha Maria de. Alterações na expressão proteica de larvas de Aedes aegypti após intoxicação com o larvicida m-pentadecadienil-fenol isolado de sementes de Myracrodruon urundeuva. 2013. 104 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2013. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-13T12:32:02Z No. of bitstreams: 1 2013_dis_tmsouza.pdf: 6716791 bytes, checksum: 61605b4532a8159ed57140ee2fa4bc4d (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-14T23:13:00Z (GMT) No. of bitstreams: 1 2013_dis_tmsouza.pdf: 6716791 bytes, checksum: 61605b4532a8159ed57140ee2fa4bc4d (MD5) / Made available in DSpace on 2016-07-14T23:13:00Z (GMT). No. of bitstreams: 1 2013_dis_tmsouza.pdf: 6716791 bytes, checksum: 61605b4532a8159ed57140ee2fa4bc4d (MD5) Previous issue date: 2013 / Currently, dengue fever is considered the most important arboviral disease in the world, emerging in countries where the disease seemed eradicated and reappearing in countries where before was under control. Despite all the efforts to produce and develop a vaccine against the disease, there is not a preventive therapy. Nowadays, the disease management is through the eradication of the mosquito vector Aedes aegypti. Despite the several options of synthetic insecticides available for management programs of vector populations, the emergence of resistant populations is presented as an obstacle in mosquito control vector. In addition to monitoring these resistant populations, it became evident that molecular studies may be the key to the sustainable management of these vectors. Thus, the present study aimed to evaluate the changes in protein expression of Ae. aegypti larvae treated with the organic larvicide m-pentadecadienil-phenol, a phenolic lipid isolated from Myracrodruon urundeuva seeds to elucidate the putative mechanisms of detoxification and molecular response to this compound. For this, two-dimensional electrophoresis (isoelectric focusing followed by electrophoresis in polyacrylamide gel under denaturing conditions - SDS-PAGE) of third-instar larvae treated with m-pentadecadienil-phenol, at sublethal concentrations (LC50 10.16 microg.mL-1) was performed. The results were compared to a non-treated group. Thirteen spots were identified as differentially expressed, and twelve were consistently identified in databases analysis of the spectra obtained by mass spectrometry (ESI-Q-ToF). After analysis of the pathways in which these proteins are involved, it was proposed that the larvicide m-pentadecadienil-phenol elicits the overexpression of proteins that promote the formation of more efficient barriers and that once inside the cells, it promotes oxidative stress which leads to increased lipid degradation metabolism, destabilization of lysosomal membrane, increased metabolism in response to stress due to the molecule’s toxicity and possibly results in programmed cell death (apoptosis). / Atualmente, a dengue é considerada a arbovirose mais importante do mundo, emergindo em países onde a doença parecia erradicada e ressurgindo em países onde antes estava sob controle. Apesar de todos os esforços de pesquisa empreendidos na produção e desenvolvimento de uma vacina contra a doença, ainda não se dispõe de uma terapia preventiva eficaz; a única alternativa de manejo nos dias atuais se dá através do combate ao único elo vulnerável, o mosquito vetor Aedes aegypti. Apesar dos programas de manejo das populações de vetores disporem de várias opções de inseticidas sintéticos, o surgimento de populações resistentes apresenta-se como um entrave no controle desse mosquito vetor. Além do monitoramento dessas populações resistentes, tornou-se evidente que estudos moleculares podem ser a chave para o manejo sustentável desses vetores. Assim, o presente trabalho teve por objetivo avaliar as alterações na expressão proteica de larvas de Ae. aegypti tratadas com o larvicida orgânico m-pentadecadienil-fenol, um lipídeo fenólico isolado de sementes de Myracrodruon urundeuva a fim de elucidar os possíveis mecanismos de detoxificação e resposta molecular a esse composto. Para isso foi realizada eletroforese bidimensional (focalização isoelétrica seguida de eletroforese em gel de poliacrilamida em condições desnaturantes - SDS-PAGE) de larvas de terceiro estádio tratadas com m-pentadecadienil-fenol, em concentrações subletais (CL50 10,16 µg.mL-1), em comparação com um grupo controle não tratado. Treze spots foram identificados como diferencialmente expressos, e doze foram identificados consistentemente em bancos de dados após análise dos espectros obtidos por espectrometria de massas (ESI-Q-ToF). Após a análise das vias nas quais essas proteínas estão envolvidas, foi proposto que o larvicida m-pentadecadienil-fenol elicita a superexpressão de proteínas que promovem a formação de barreiras mais eficientes, e que uma vez dentro das células promove um estresse oxidativo que leva ao aumento do metabolismo de degradação lipídica, desestabilização da membrana lisossomal, aumento do metabolismo em resposta ao estresse tóxico e possivelmente resulta em morte celular programada (apoptose).