Made available in DSpace on 2015-03-26T13:51:50Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3209236 bytes, checksum: 730f245d59f62f2daccf53d9d6fb9b93 (MD5) Previous issue date: 2010-07-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Lactic acid bacteria (LAB) can be infected and lysed by an extensive range of bacteriophages, which is recognized as the main cause of failure or slow fermentation in the modern dairy industry. Contaminated fermentation processes affect product quality or even cause its complete loss. Although technological advances have reduced the incidence of these infections, they certainly do not eliminated it. The objective of this work was the isolation, identification and molecular characterization of lytic bacteriophages of Lactococcus lactis from sera from lactic fermentation. For this, serum samples were collected from three different dairies facilities. From one of them, were isolated 17 bacteriophages, which were propagated in L. lactis grown in GM17 medium and molecularly characterized using the techniques of multiplex PCR, DNA restriction profile, protein profile, transmission electron microscopy, cloning and sequencing. The extraction of the genome of bacteriophages isolated and its analysis revealed a strand of DNA of 48 kb for phage LIMG1 and 42 kb for phage LIMG4, while phage protein profile allowed the distinction of four groups, and the differentiation between them was consistent with the sample from which they were isolated. The PCR, coupled to microscopic analysis, confirmed the presence of members of the group 936-type phages, Siphoviridae family, the most abundant in dairy products worldwide. Phages contain isometric heads with 50 nm in diameter and a 180 nm long tail, without basal plate. The amplified sequence of isolate LIMG1 revealed high identity with other members of 936-type phages, although two punctual mutations had differed LIMG1 from the others. / Bactérias do Ácido Láctico (BAL) podem ser infectadas e lisadas por uma extensa faixa de bacteriófagos, o que constitui reconhecidamente a principal causa de fermentação falha ou lenta na indústria de laticínios moderna. Processos fermentativos contaminados prejudicam a qualidade do produto ou causam até mesmo sua perda completa. Embora avanços tecnológicos tenham reduzido a incidência destas infecções, eles certamente não as eliminaram. Assim, o objetivo deste trabalho foi o isolamento, identificação e caracterização molecular de bacteriófagos líticos de Lactococcus lactis a partir de soros provenientes de fermentações lácticas. Para isto, amostras de soro foram coletadas em três diferentes laticínios e, a partir deles, foram isolados 17 bacteriófagos, os quais foram propagados em L. lactis cultivado em meio GM17 e caracterizados molecularmente utilizando-se as técnicas de multiplex PCR, perfil de restrição enzimática de DNA, perfil proteico, microscopia eletrônica de transmissão, clonagem e sequenciamento. A extração do genoma dos bacteriófagos isolados e sua análise revelaram uma fita de DNA de 48 kb para o isolado LIMG1 e 42 kb para o isolado LIMG4, enquanto o perfil de proteínas dos fagos permitiu a distinção de quatro grupos, sendo que a diferenciação entre eles foi condizente com a amostra da qual foram isolados. A técnica de PCR, associada à análise microscópica, confirmou a presença de fagos do grupo 936, família Siphoviridae, o mais abundante em laticínios em todo o mundo. Foram encontrados fagos de cabeça isométrica de 50 nm de diâmetro e caudas longas de cerca de 180 nm de comprimento, sem placa basal. O sequenciamento do genoma do isolado LIMG1 revelou alta identidade com outros representantes do grupo 936, embora tenham sido encontradas duas mutações pontuais que o diferenciaram dos demais.

Orientador: Áureo Tatsumi Yamada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T04:02:31Z (GMT). No. of bitstreams: 1 Lima_PatriciaDanieleAzevedo_D.pdf: 2840118 bytes, checksum: 470a2cf0b7d63e81fd8f48aba5af44fc (MD5) Previous issue date: 2012 / Resumo: Células Natural Killer uterina (uNK) produzem moléculas angiogênicas e citocinas críticas ao sucesso da gestação , assim como proteínas citolíticas relacionadas à resposta imune inata. Contudo, se as capacidades angiogênicas e citolíticas são provenientes de diferentes subpopulações de células uNK não é conhecido; da mesma forma, estes fenótipos ainda não são estabelecidos. Assim, a proposta inicial deste trabalho foi avaliar...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Angiogenic and cytokine molecules produced by uterine natural killer (uNK) cells are critical for successful pregnancy. Cytolytic proteins are also express by uNK cells. However, it is unknown whether the angiogenic and cytolytic capacities are from different uNK subsets, or the same cells. Thus, we initially proposed to evaluate...Note: The complete abstract is available with the full electronic document / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Submitted by Kenia Bernini (kenia.bernini@ufpel.edu.br) on 2016-09-01T20:07:24Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Alexandre_Pinto_Coelho_ Almeida_Dissertação.pdf: 5180057 bytes, checksum: 40c548ee0b6cb39d2957a93bbe76b6c1 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-05T19:33:13Z (GMT) No. of bitstreams: 2 Alexandre_Pinto_Coelho_ Almeida_Dissertação.pdf: 5180057 bytes, checksum: 40c548ee0b6cb39d2957a93bbe76b6c1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-05T19:33:21Z (GMT) No. of bitstreams: 2 Alexandre_Pinto_Coelho_ Almeida_Dissertação.pdf: 5180057 bytes, checksum: 40c548ee0b6cb39d2957a93bbe76b6c1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-05T19:33:29Z (GMT). No. of bitstreams: 2 Alexandre_Pinto_Coelho_ Almeida_Dissertação.pdf: 5180057 bytes, checksum: 40c548ee0b6cb39d2957a93bbe76b6c1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-05-23 / Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS / O presente estudo é focado nos resultados da análise do material lítico do sítio arqueológico Florestal I, situado na região do Médio Rio Doce, Minas Gerais/Brasil. Através dos resultados das análises do material lítico, discuto as escolhas tecnológicas empregadas para se obter os instrumentos identificados, procurando compreender a execução da cadeia operatória de produção e a funcionalidade destes objetos e de que maneira estas escolhas auxiliam na compreensão da utilização e ocupação do espaço, além de abranger outros aspectos compreendidos dentro da dinâmica social e econômica destes portadores da cerâmica Tupiguarani que habitaram no passado a região do Médio Rio Doce. Paralelamente, apresentamos uma discussão a respeito de como os conceitos de cultura e civilização, advindos do pensamento ocidental, influenciaram de sobremaneira os arcabouços teóricos e metodológicos da pesquisa científica e na construção de um discurso colonialista da qual a arqueologia participou ativamente. / This study is focused on the analysis of the results of the lithic material from the archaeological site Florestal I, located in the Middle Rio Doce region. Through the results of the lithic material analysis, we discuss the technological choices used to obtain the identified tools, trying to understand the implementation of the operative production chain and the functionality of these objects and how these choices help to understand the use and occupation of space and covers other aspects understood within the social and economic dynamics of these Tupiguarani ceramic bearers who lived in the past the region of the Middle Rio Doce. At the same time, we present a discussion of how the concepts of culture and civilization, arising from the Western thought, greatly influenced the theoretical and methodological frameworks of scientific research and construction of a colonialist discourse which archeology participated actively.

<p>Ever since the invention of antibiotics nearly a century ago,the threat of antibiotic resistance has been gradually increasing. As antibiotics are continually prescribed, the rate at which bacteria are becoming resistant to antibiotics is increasing as well. It is projected that antibiotic resistance is one of the largest threats to overall world health, and bacteriophage therapy is one of the leading strategies to combat it. Bacteriophages are viruses that infect and kill specific host bacteria andcan potentially be utilized to kill desired bacteria causing infections that are resistant to antibiotics.</p> <p><br></p> <p>The purpose of this research project is to learn more about the bacteriophage-host interaction through mass spectrometry and bioinformatic tools. This is done through the analysis of proteins and lipids that are produced when the bacteriophage infects the host bacteria. The growth curve of a Passage One From Frozen (P1FF) and a Passage Two From Frozen (P2FF) sample of Mycobacterium smegmatiswas calculated to determine to optimum time for bacteriophage infection. Twobacteriophages were chosen, PotatoSplit and Zalkecks, the Mycobacterium smegmatis samples were infected, samples collected, and mass spectrometry performed. A large portion of this research project is based on the analysis of the proteins and lipids that are produced during each bacteriophage’s infection. Proteomic and lipidomic strategies can be implemented to understand more about the bacteriophage-host interaction and discover any proteins and lipids that are produced at varying timepoints throughout the inoculation process. Bioinformatic tools can then be used to understand the potential functions of each protein or lipid and potential functions or applications of the bacteriophage in general, including the pathogenicity of each bacteriophage.</p> <p><br></p> <p> Determined from proteomic and lipidomic analysis, a list of all proteins and lipids found within each phage infected sample was made. An important trend discovered is that more phage proteins were expressed at later times during the phage infection –Hour 7 and Hour 10, whereas more bacterial proteins were expressed initially –Hour 0 and Hour 3. A case study to investigate the usage of different intensity types produced from mass spectrometry was completed. Overall, it was determined that both the number of phageproteins and bacterial proteins can differ depending on if LFQ or iBAQ intensity type data was used. Correlation between proteins and lipid ontology classes was performed and shows whether groups of lipids are upregulated or downregulated at 14each time point. Understanding the function of lipid ontology groups and the type of regulation provides insight into how the phage or bacteria are potentially using the lipids produced. Some of the main findings include lipids that are involved in bacterial defense mechanisms/energy usage increase over time. Some correlation trends were not consistent across the different bacteriophages, which can be contributed to the different phage life cycles  and therefore different phage-host interactions. Further investigation should be performed to determine the specific biological function of proteins and lipids to confidently make claims about potential applications for each phage. Also, further investigation should be performed to understand if the differences in results between bacteriophage PotatoSplit and Zalkecks are due to the varying life cycles.</p>

Within the last decade, pork consumption has steadily increased and continues to be the most consumed meat globally. However, pathogenic bacterial strains resistant to antibiotics have also been increasingly found in pig farms, animals, and the environment. Bacterial food poisoning cases due to Salmonella spp. and Escherichia coli (E. coli) O157:H7 appear to be linked with a variety of pork products. The meat industry has recognized that research is needed to combat the multi-drug resistance in foodborne pathogens with alternative methods of control. This study evaluated the effects of both E. coli- and Salmonella-specific lytic bacteriophages and lactic acid (LA) on E. coli O157:H7, Salmonella Enteritidis, Salmonella Montevideo, and Salmonella Heidelberg growth in raw pork loins ready for marination. The efficacy of the treatments was determined after 1 h of application and marination. Lytic bacteriophage 5% significantly (PSalmonella spp. population by 2.30 log CFU/cm2 when compared with the initial surface attachment. Moreover, the combined treatment of LA 2.5% + phage 5% significantly (PSalmonella population by 2.35 log CFU/cm2 after 1 hour of attachment. In the post-tenderization surface samples, the combination of both phage and LA showed (PP>0.05) when analyzing the translocation of Salmonella spp. on pork loins. Similar treatment efficacy results were observed in the application of E. coli O157:H7 on pork. Following antimicrobial treatments, both control and treated loin samples were enumerated after 1 h at 4°C. Both the lytic bacteriophage 5% and the combination of lytic bacteriophage 5% with lactic acid 2.5% had a significant reduction of E. coli O157:H7 on surface attachment after 1 h of treatment application. Lytic bacteriophage 5% and Lactic acid 2.5% significantly (P < 0.05) reduced the surface bacterial population by 1.89 log CFU/cm2. Lytic bacteriophage 5% alone significantly (P < 0.05) reduced the surface bacterial population by 1.90 log CFU/cm2 when compared with the initial surface attachment groups. Moreover, in the post-tenderization surface samples, lytic bacteriophage 5% and the combination of lytic bacteriophage 5% with lactic acid 2.5% were the only treatments that had a significant reduction (P < 0.05) when compared with the control group. Interestingly, lactic acid 2.5% was the only treatment that had a significant reduction (P < 0.05) of 0.76 log CFU/cm2 when analyzing the translocation of pathogens on pork chops.

Yes / KSHV is the causative agent of commonly fatal malignancies of immuno-compromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic lifecycle – viral latency and the productive lytic cycle, and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein, replication and transcription activator (RTA), is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts on cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied SILAC-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B, ARID3B) whose expression was enhanced by RTA and that relocalised to replication compartments upon lytic reactivation. We also show that siRNA knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation.

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

Baculoviruses are lytic insect viruses. Upon internalisation, the viral genome orchestrates a sequential expression process ultimately leading to lysis of the infected cell. Release of progeny capable of infecting other cells during the process completes the infection cycle. Studies of the infection cycle in cell culture are typically conducted by synchronous infection, i.e. near simultaneous infection of all cells, by means of high virus concentrations. The behaviour of the synchronously infected culture, such as the timing of onset of progeny release, is considered representative for the infection progression within individual cells. In reality, however, the synchronously infected culture only reflects the average behaviour of all infected cells. The infection progresses in individual cells display large variability; this is most obvious in the observation that within the same culture some cells undergo cell lysis at two days post infection while others remain viable up to four days post infection. Such variabilities or asynchronies observed in synchronously infected culture is the topic of this thesis. Using a simple phenomenological model, it is demonstrated that cell death and associated intracellular product release is adequately described assuming that the waiting time from infection to cell death follows a Gaussian distribution with a mean of 59 hours post infection (hpi) and a standard deviation of 15hpi. Unlike other deterministic models developed over the last decade (Licari and Bailey 1992; Nielsen 2000), this stochastic model does not make the biologically inconsistent assumption that cells continue to be metabolically active following loss of membrane integrity. While elegant in its simplicity, the model provides no explanation for the underlying stochasticity. Investigations into the cause of this dispersion of cell death highlighted further asynchronies in the specific recombinant protein yield, in viral DNA content, in virus budding rate, and in cell volume increase instead of clarifying the issue. A modelling framework developed by Licari & Bailey (1992) and later Hu & Bentley (2000) incorporates the number of infectious particles each individual cell receives as a possible source of the dispersions in the host cell responses. However, this was found NOT to be the cause of the observed asynchronies under non-substrate limiting conditions. The timing of cell death, cell volume increase, recombinant product yield, viral DNA content, and virus budding rate is identical in Sf9 cell cultures infected at multiplicities of infection of ~5, ~15, and ~45 infectious particles per cell. Cell cycle variation has previously been suggested as a possible cause for observed asynchronies in baculovirus infections (Brown and Faulkner, 1975). The cell cycle phase is indirectly linked to the cell volume, because a G_2-phase cell prior to division is inherently twice the cell volume of a G_1- phase cell after cell division. By the same logic, it is also apparent that a G_2-phase cell possesses twice the number of ribosomes of a G_1-phase cell and thus a doubled protein production capacity. The effect of the cell cycle or cell volume on the baculovirus infection was determined by splitting an exponentially growing Sf9 cell culture into 5 cell size dependent fractions by centrifugal elutriation. The subsequent infection of these fractions showed (1) no dependency of the timing of cell lysis and cell volume increase and (2) approximately twofold increase of a) recombinant protein yield, b) viral DNA concentration, and c) budded virus yield. The recombinant protein yield showed a strong proportionality to the initial cell volume and the total RNA concentration during the late phase of the infection. As argued in chapter 6, these proportionalities suggest that the observed differences in the responses of the cell fractions to the baculovirus infection are more likely caused by the difference in the protein production capacity than by cell cycle specific molecules. This investigation gave also reason to speculate that infected cells cannot progress beyond the G_2/M phase, and cell cycle progression continues undisturbed until ~8hpi when all cellular DNA replication appears to cease. Resuspended, infected Sf9 cells synchronised by centrifugal elutriation showed an identical cell cycle distribution as the non-infected control cultures for the first ~8hpi; G_1 and G_2/M phase cell proportions remained unchanged, whereas S phase cells progress to G_2/M phase. Subsequently, the non-infected control cells resumed normal cycling whereas all infected cells remained at the same cell cycle phase from 8 to 11hpi. The initial cell cycle arrests in G_2/M phase in both infected and non-infected cultures were caused through medium exchange.

Bacteriophages are viruses that specifically infect bacteria. The bactericidal nature of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise them in the fight against bacterial infections and antibiotic resistant bacteria in medical settings. More recently, the potential applications of bacteriophages for biocontrol in the agrifood and environmental sectors have been investigated in an attempt to develop ‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages over conventional methods of controlling pathogenic bacteria, such as high host specificity, the ability to self-replicate, and the ability to evolve with their hosts. However, more research is needed to optimise the parameters for phage applications, including the impact of environmental conditions on lysis efficiency, multiplicity of infection, and to significantly minimise the emergence of bacterial resistance to phages. Temperature plays a key role in every biological activity in nature. It is also assumed that temperature has an effect on phage lysis efficiency. A comprehensive study of it and how it affects both the host cells and their corresponding phages is crucial to ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness of the three different phages belonging to the family of the Myoviridea that were isolated and purified from a single water sample taken from a brook receiving treated wastewater. We used the multiplicity of infection of 1 in all of our study in this project. Temperature was found to have a significant impact on phage-mediated lysis efficiency. Both the temperature of incubation of the phage-bacteria mixture (incubation temperature) and the temperature history of bacterial hosts were found to have profound effects on plaque sizes as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C compared to 30 or 37°C. Phages were suggested to be well adapted to the environment where they were isolated from with general implications for use in biological disinfection. Furthermore, the temperature history of the bacteria (prior to phage encounter) was found to have a modulating effect on their susceptibility to lysis. A second part of this study compared the performance of the three phages in regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle to the success of bacteriophages applications. The use of multiple phages is typically recommended and has proven better than the use of a single phage. However, the bestway to perform phage treatment is still very unclear. This study therefore compared simultaneous addition of multiple phages (in form of a cocktail) with the sequential addition of the individual phages at different time points in trying to delay the emergence of bacterial resistance. The data obtained from this work suggest that lysis effectiveness can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use of a single phage with short time maximal lysis efficiency proved most efficient, while the simultaneous addition of phages in the form of a cocktail was most successful strategy in our study. Addition of selected phages sequentially can be normalized in such a way that is just as effective as a cocktail. A third part of this thesis looked into the susceptibility of bacteria that had undergone sublethal disinfection. We addressed the question whether bacteria subjected to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The chlorine treatments indicated the development of a phage-insensitive phenotype for a critical chlorine dose in the transition zone between live and dead. The remaining live (and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage- resistant phenotype may have serious consequences for the application of phages on foods or water that have previously undergone a weak disinfection regime.

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