Les carbènes N-hétérocycliques (NHC) sont très utilisés pour complexer les métaux de transition. Ils quittent rarement ce rôle de ligand ancillaire et trouvent, depuis une vingtaine d’années, des applications en catalyse ou, plus récemment, en chimie médicinale. Dans ce travail, nous discuterons d’une méthode de synthèse douce conduisant à la formation de complexes AgI – NHC via une source d’argent soluble. Cette méthode nous a permis d’obtenir des complexes bien connus mais également d’accéder à une nouvelle série de complexes NHC-Ag-phosphine. Nous présenterons également une nouvelle réaction où des NHC portant une fonction azoture à proximité du carbone du carbène quittent leur rôle de ligand ancillaire et conduisent à la formation d’hétérocycles azotés par cyclisation carbène-nitrène. Cette réaction sera présentée en détail, ainsi que la caractérisation spectroscopique concernant une sous-série de composés fluorescents obtenus par cette méthode. Enfin, nous présenterons une stratégie de post-fonctionnalisation de complexes développée dans notre équipe. Des complexes argent(I)-NHC portant un azoture proches du centre carbénique catalysent leur propre fonctionnalisation. De plus, des complexes de cuivre(I) portant des azotures en position éloignée du centre métallique seront greffés sur des nanoparticules magnétiques pour servir de catalyseur recyclables. / N-heterocyclic carbenes (NHC) are widely used to complex transition metals. They rarely leave their role as ancillary ligand and find, since 20 years, applications in catalysis or, more recently, in medicinal chemistry. In this work, we will discuss a mild synthetic method leading to the formation of AgI – NHC complexes via a soluble silver species. This method allowed us to obtain well known complexes but also to access a new series of NHC-Ag-phosphine complexes. We will also present a new reaction where NHC ligands bearing an azide function close to the carbenic center leave their role as ancillary ligand and lead to the formation of nitrogen rich heterocycles by a carbene-nitrene cyclization. This reaction will be presented in detail, along with the spectroscopic characterization regarding a sub-series of fluorescent compounds obtained by this method. Finally, we will present a post-functionalization strategy of complexes developed in our team. Silver(I)-NHC complexes tagged by an azide close to the carbenic center catalysed their own functionalization. Moreover, copper(I) complexes tagged by an azide function in a distant position from the metallic centre will be grafted on magnetic nanoparticles to act as recyclable catalysts.

O potencial farmacológico de espécies do gênero Lippia no tratamento de infecções é conhecido popularmente, assim foi considerado o estudo da atividade da Lippia salviaefolia nativa do cerrado brasileiro. O objetivo do trabalho foi avaliar a atividade antimicrobiana de extratos de Lippia salviaefolia Cham. e sua citotoxicidade, visando aplicação como conservante natural em formulações cosméticas. O ensaio antimicrobiano foi realizado através do método de difusão em placa e determinação do CMI por de microdiluição em microplacas, utilizando os microrganismos: P. aeruginosa ATCC 9027, S. aureus ATCC 6538, E. coli ATCC 8739, C. albicans ATCC 10231. Desenvolveu-se e avaliou-se a estabilidade acelerada de cremes, geis e xampus contendo o extrato de Lippia salviaefolia. As formulações de melhor desempenho quanto à estabilidade foram submetidas ao teste de eficácia de conservante. Avaliou-se segurança das formulações in vitro, em substituto cutâneo dermo-epidérmico por meio de estudos histológicos e o teste de citotoxicidade em cultura de queratinócitos humanos. No teste de atividade antimicrobiana verificou-se que a fração de acetato de etila e a fração de clorofórmio foram frações mais ativas com CMI de 0,2 % para bactérias e fungos. Os testes de eficácia de conservante das formulações contendo fr. ac. etila de L. salviaefolia apresentaram atividade antimicrobiana para bactérias e C. albicans em creme, gel e xampu, para A. niger apresentaram atividade adequada somente em xampu. No teste de citoxicidade foi verificado segurança em concentrações a partir de 0,05 %. Sendo que na concentração de 0,2 %, correspondente ao CMI, se atinge a faixa de segurança apenas com meia hora de contato. Os resultados sugerem que a fração de acetato de etila da Lippia salviaefolia Cham. pode ser utilizada como conservante natural em produtos cosméticos. / The pharmacological potential of species of the Lippia in the treatment of infections has been known popularly, thus was considered the study of the activity of the native salviaefolia Lippia of the Brazilian Cerrado. The purpose of the work was to evaluate the antimicrobial activity of extracts of Lippia salviaefolia Cham. and its citotoxicy aiming the application as natural preservative in cosmetic formulations. The assay antimicrobial was carried through the method of diffusion in plate and determination of the MIC by microdilution, having used the microorganisms: P.aeruginosa ATCC 9027, S. aureus ATCC 6538, E. coli ATCC 8739, C. albicans ATCC 10231. It has developed and evaluated the stability of creams, gels and xampus with the extract of Lippia salviaefolia Cham. The formulations of the best stability performance have submitted to preservative efficacy test. The safe of the formulations has histological evaluated by in vitro. In the test of antimicrobial activity it has verified that the ethyl acetate and the chloroform fraction have been the most active and with CMI of 0.2 % for bacteria and fungus. The tests of effectiveness of preservatives have showed of the formulations contend the ethyl acetate fraction presented antimicrobial activity for bacteria and C. albican in cream, gel and shampoo. On the other hand, against A.niger it has been suitable activity in shampoo. In the citotoxity test, the 0,05 % ethyl acetate fraction has presented interval of security. And in the concentration of 0,2%, corresponding to CMI, safety\'s strip is just reached with half hour of contact. The results have suggested that the ethyl acetate extract of the Lippia salviaefolia Cham. should be used as natural preservative in cosmetic products.

Cette thèse a pour sujet les protocoles de communication peer-to-peer asynchrones. Nous introduisons deux mesures basées sur la théorie de l'information,la Public Information Complexity (PIC) et la Multi-party Information Complexity (MIC), étudions leurs propriétés et leur relation avec d'autres mesures fondamentales en calcul distribué, telles que la communication complexity et la randomness complexity. Nous utilisons ensuite ces deux mesures pour étudier la fonction parité et la fonction disjointness. / This thesis is concerned with the study of multi-party communicationprotocols in the asynchronous message-passing peer-to-peer model. We introducetwo new information measures, the Public Information Complexity(PIC) and the Multi-party Information Complexity (MIC), study their propertiesand how they are related to other fundamental quantities in distributedcomputing such as communication complexity and randomness complexity.We then use these two measures to study the parity function and the disjointness function.

Failures in networks that reside in business environments cause harm to organizations depending on them. Every minute of inoperativety is hurtful and as a network adminstrator you want to minimize the rates of failures as well as the time of inoperation. Therefore, a fruitful network monitoring system is of great interest for such organizations. This bachelor’s thesis is the outcome of a pre-study performed on behalf of MIC Nordic and sought to advice them in the implementation of a new monitoring system. The aim of this study was to investigate how a Network Operation Center (NOC) can be implemented in an existing monitoring environment, to integrate current monitoring systems to a central point for monitoring. This study takes an holitstic approach to network management and the research can be divided into two main categories: Communication between network components and Presentation of information. Our process involves an analysis of the environment of MIC Nordic and an in depth inquiry on the current state of network monitoring and interface design. The study then culminates in the implementation of a prototype. The prototype serves in first hand as a research tool to collect experience and empirical evidence to increase the crediblity of our conclusions. It is also an attempt of demonstrating the complete process behind developing a NOC, that we believe can fill a gap among the previous research in the field. From our results you can expect a prototype with functionality for monitoring network components and a graphical user interface (GUI) for displaying information. The results are designed towards solving the specific network management problem that was given and the environment that it concerns. This pre-study suggests that the best solution for implementing a NOC in the given environment is to use SNMP for communication. From an investigation on how to present network management information in a effective way we propose to follow a user-centered approach and to utilize human perception theory in the design process. The authors recommend further research that maintain the holistic approach but applies more quantitative methods to broaden the scope. / Störningar i nätverk som används i företagsmiljöer skapar problem för organisationer som är beroende av dess funktion. Varje minut som nätverket är verkningslöst är ofördelaktigt och som nätverksadministratör så vill du minimera antal störningar och tiden då nätverket är verkningslöst. Ett effektivt nätverksövervaknings system är därf ör av stort intresse f ör organisationer beroende av ett funktionerande nätverk. Den här rapporten är resultatet av en förundersökning som utfördes på uppdrag av MIC Nordic, för att ge en rekommendation om hur ett nytt övervakningssystem för deras nätverk kan implementeras. Målet med studien var att undersöka hur ett Network Operation Center (NOC) kan implementeras i en existerande miljö för att integrera nuvarande övervakningssystem till en central punkt för övervakning. Den här studien tar ett holistiskt grepp om nätverksövervakning och undersökningen kan delas in i två primära kategorier: Kommunikation mellan nätverkskomponenter och Presentation av information. Vår process involverar en analys av MIC Nordics miljö och en djupgående utredning om nätverksövervakning samt gränssnitts design. Studien kulminerar i en implementaion av en prototyp. Prototypen är i f örsta hand ett undersökningsverktyg för att samla på oss erfarenhet och empiriska belägg för att öka trovärdigheten i våra slutsatser. Prototypen utgör även ett försök av författarna att demonstrera den kompletta proceduren av att utveckla en NOC, avsikten är att det kan fylla ett behov bland tidigare avhandligar i ämnet. Resultatet innehåller en prototyp med funktionalitet för att övervaka nätverkskomponenter samt ett grafiskt gränssnitt för att visa informationen. Resultaten är designade mot en lösning som är specifik för problemet som gavs av MIC Nordic och deras miljö. Denna förstudie proponerar att den bästa lösningen för att implementera en NOC i den givna miljön är att använda SNMP för kommunikation. Efter en granskning av hur man kan presentera information som rör nätverksövervakning på ett effektivt sätt så lanserar författarna en användarcentrerad metod som utnyttjar läran om hur människor uppfattar saker och ting. Författarna uppmuntrar vidare undersökningar som bibehåller det holistiska greppet men som applicerar mer kvantitativa metoder för att utöka undersökningens omfattning.

Compostos de amônio quaternário (QACs) têm sido amplamente utilizados como desinfetantes e antissépticos, sendo essenciais na prevenção e controle de infecções bacterianas na medicina humana e veterinária. Embora patógenos prioritários multirresistentes têm sido muito bem caracterizados quanto ao perfil de suscetibilidade e contexto genético da resistência aos antibióticos, dados de resistência aos QACs são limitados. Assim, o objetivo do presente estudo foi avaliar a atividade in vitro dos QACs de uso doméstico e hospitalar [cloreto de benzalcônio (BAC), cloreto de cetilpiridinio (CPC) e brometo de cetiltrimetilamônio (CTAB)], contra patógenos prioritários multirresistentes, identificando os principais genes de resistência associados. Foram estudadas 100 cepas multirresistentes previamente sequenciados usando as plataformas Illumina MiSeq e NextSeq representativas de diferentes hospedeiros (humanos e animais) e fontes (ambientes e alimentos). As cepas foram identificadas como Klebsiella pneumoniae (n= 24), Escherichia coli (n= 30); Pseudomonas aeruginosa (n= 10), Enterobacter spp, (n= 8), Acinetobacter baumannii (n= 11) e Salmonella spp. (n= 17). Genes de resistência aos QACs foram identificados in silico através do alinhamento dos contigs obtidos de cada cepa sequenciada com genes de referência obtidos do GenBank, utilizando o programa Geneious versão 8 (Biomatters Ltd). A identidade de cada gene foi analisada utilizando o programa BLASTx, no qual um critério baseado em ≥90% identidade resultou na identificação dos genes mdfA (77%), qacE (44%), qacEΔ1 (43%), sugE(c) (29%), emrE (21%), qacA (19%), sugE(p) (5%), qacF (7%), qacH (7%) e qacL (7%) em 85 cepas; enquanto que 15 cepas não possuíam nenhum gene de resistência aos QACs. A concentração inibitória mínima (CIM) dos QACs para as 100 cepas foi determinada pelo método de microdiluição em caldo. Os resultados sugeriram que a resistência em patógenos prioritários circulando na interface humano-ambiente-animal não é restrita aos antibióticos, uma vez que a elevada ocorrência de genes qacE, qacEΔ1 e mdfA poderia estar associada com uma redução da suscetibilidade para QACs. Consequentemente, a resistência aos QACs poderia também contribuir para a persistência e adaptação destes patógenos nos seres humanos e outros animais, assim como em ambientes impactados antropogenicamente. / Quaternary ammonium compounds (QACs) have been widely used as disinfectants and antiseptics, being applied as essential compounds in the prevention and control of bacterial infections in human-and veterinary hospital medicine. Although multiresistant priority pathogens have been well characterized with respect to their susceptibility profile and their genetic context of resistance for antibiotics, studies of resistance to QACs are limited. Thus, the objective of the present study was to evaluate the in vitro activity of QACs [(benzalkonium chloride (BAC), cetylpyridinium chloride (CPC) and cetyltrimethylammonium bromide (CTAB)] for household and hospital use against multiresistant priority pathogens, identifying the main resistance genes associated. A hundred multiresistant isolates (previously sequenced using the Illumina MiSeq and NextSeq platforms), representative of different hosts (humans and animals) and sources (environment and food) were studied. Isolates were identified as Klebsiella pneumoniae (n=24), Escherichia coli (n=30), Pseudomonas aeruginosa (n=10), Enterobacter spp. (n=8), Acinetobacter baumannii (n=11) and Salmonella spp. (n=17). In silico analysis for identification of genes conferring resistance to QACs were performed by aligning the contigs obtained from the strains with reference genes deposited in GenBank, using the Geneious version program (Biomatters Ltd). Similarities were analyzed using the BLASTx online program, considering the alignment criteria based on ≥ 90% identity. The result of these analysis revealed the presence of the following QAC genes: mdfA (77%), qacE (44%), qacEΔ1 (43%), sugE(c) (29%), emrE (21%), qacA (19%), sugE (p) (5%), qacF (7%), qacH (7%) e qacL (7%); while 15 strains showed no resistance genes for QACs. Determination of QACs minimum inhibitory concentration (MIC) for the 100 isolates, by the broth microdilution method. These results suggest that resistance to QACs in priority pathogens, circulating at the human-environment-animal interface, is not restricted to antibiotics, since the high occurrence of genes qacE, qacEΔ1 and mdfA were associated with a reduced susceptibility to QACs. Consequently, resistance to QACs could also contribute to the persistence and adaptation of these pathogens in humans and othes animals, as well as in anthropogenically impacted environments.

Os Mollicutes causam doenças em várias espécies animais de importância econômica, inclusive em bovinos. Neste estudo, foi avaliada por concentração inibitória mínima (CIM) e citometria de fluxo, a atividade de oito agentes antibacterianos (enrofloxacina, ciprofloxacina, gentamicina, claritromicina, cloranfenicol, oxitetraclina, tiamulina e tilosina) contra Ureaplasma diversum. Foram analisadas 24 amostras de isolados de campo oriundas da mucosa genital de fêmeas bovinas. As amostras foram confirmadas por crescimento em caldo, placa e por PCR. Os inóculos foram submetidos à analise de suscetibilidade aos antibióticos pelo método da microdiluição em microplaca e posteriormente analisados pelo citômetro de fluxo a fim de avaliar a atividade antimicrobiana nas células. A claritromicina apresentou os maiores índices de inibição in vitro, sendo a gentamicina considerada o antibiótico de menor espectro de ação nesse estudo. De acordo com as análises do citômetro, a gentamicina apresentou o menor número de células viáveis enquanto a tiamulina apresentou o maior número. Embora haja resultados destoantes entre as técnicas utilizadas, o citômetro de fluxo pode ser utilizado como uma boa ferramenta para auxiliar a avaliação da suscetibilidade desses microrganismos a antibióticos. / The Mollicutes cause disease in several economically important species, including cattle. In this study, was evaluated by minimum inhibitory concentration (MIC) and flow cytometry, the activity of eight antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, clarithromycin, chloramphenicol, oxitetraclina, tiamulin and tylosin) against Ureaplasma diversum. We analyzed 24 samples of field isolates originating from the genital mucosa of cows. The samples were confirmed by growth in broth, plate, and PCR. The inoculations were subjected to analysis of susceptibility to antibiotics by the method of micro-dilution plate and then analyzed by flow cytometry to assess the antimicrobial activity in cells. Clarithromycin showed the highest levels of inhibition in vitro, the antibiotic gentamicin considered lower spectrum of action in this study. According to the analysis of the flow cytometer, gentamicin showed the lowest number of viable cells as tiamulin showed the greatest number. Although there are divergent results between the techniques used, flow cytometry can be used as a good tool even help assess the susceptibility of microorganisms to antibiotics.

Le complexe d'espèces Fusarium solani regroupe des champignons phytopathogènes également impliqués en pathologie humaine dans des infections parfois profondes et souvent de mauvais pronostic. Dans un premier temps, une méthode de MLST, s'appuyant sur 5 gènes de ménage a donc été développée. Validée sur 51 isolats épidémiologiquement distincts, cette méthode stable et reproductible présente un pouvoir discriminant de 99,1 %. Après comparaison à la technique de référence utilisée en phylogénie, un schéma consensus à 8 loci a été proposé. Dans un second temps, une étude de la sensibilité de ce pathogène à l'amphotéricine B et au voriconazole a été menée par deux techniques d'évaluation des CMI : microdilution CLSI M38-A2 et bandelettes E-test. Devant le paradoxe entre une sensibilité diminuée in vitro au voriconazole et la recommandation de cette molécule pour le traitement curatif de la fusariose humaine, des mécanismes de résistance ont été exploré. L'hypothèse d'un phénomène d'efflux n'a pas été retenue alors que celle d'une modification de la cible, la 14 alpha stérol déméthylase, peut être envisagée après la description de différentes mutations pour les isoformes CYP51A, B et C / Fusarium solani species complex includes phytopathogenic fungi also involved in human infections with poor prognosis. Firstly, MLST method, based on five housekeeping genes has been developed. This method has been validated on 51 isolates epidemiologically distinct, and has been shown to be stable and reproducible and provides a discriminating power of 99.1%. After comparison with the reference technique used in phylogeny, a consensus method with 8 loci has been proposed. Secondly, a study of the susceptibility to amphotericin B and voriconazole has been conducted with two MIC determination methods : CLSI M38-A2 microdilution and E-test. The paradox between decreased susceptibility to voriconazole in vitro and recommendation of this molecule for the curative treatment of Fusarium infections has lead to the exploration of resistance mechanisms. The hypothesis of an efflux phenomenon has not been retained whereas a change in the target, the sterol 14 alpha demethylase may be considered following the description of different mutations on proteins CYP51A, B and C

Gradientech AB is a company that develops a new ultra-rapid system for antibiotic susceptibility testing and their product will hopefully be used in health care for more efficient diagnosis of sepsis. Today the product has difficulties with the analysis of polymicrobial tests. For our project, the task is therefore to examine the market and the demand for the ability to detect polymicrobial infections. Our project is meant to provide ideas for improvements on their current method for the feasibility to detect several types of bacteria in the same test. Through literature studies, we have received an overview of how optical methods using image analysis can work and how they could be implemented in Gradientech's product. Image analysis can make the identification of bacteria possible. Interesting objects in the image can be distinguished from each other with filters that handle, for example, size, shape and light intensity. A potential improvement in the classification of bacteria by optical methods may be to test several different angles and wavelengths. This is to get more training data for machine learning and thus more accurately detect and analyze samples. The results from literature studies and interviews with clinicians, a microbiologist, and professor in image analysis have led to a conclusion that techniques that contribute to a decrease of antimicrobial resistance will be crucial for the future. In Sweden, the development of resistant bacteria is pretty low, but in many other parts of the world, there is a different reality. The product’s greatest potential will probably be in countries with a high incidence of resistant bacteria and with educated personnel available to use the product. Ultra-rapid systems for AST can contribute to saving lives by making sure that the right type of antibiotics is given directly. Detection and identification of polymicrobial samples in sepsis are also relevant, but it will not contribute to reduced antibiotic use since the patient's health will always be prioritized as of now. Finally, we reached the conclusion that identification of polymicrobial tests is not a necessary feature for the success of the product. This is above all due to the mass spectrometry method MALDI-TOF, a technique that is already well-established in health care systems. It can quickly and cheaply identify bacteria in clinical samples.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-03-05T13:21:28Z No. of bitstreams: 1 BRUNO_LOPES_ABBADI_TES.pdf: 4934736 bytes, checksum: 58e4c3c86d9fb96bb1476ae3c18bfdf0 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-03-06T16:15:19Z (GMT) No. of bitstreams: 1 BRUNO_LOPES_ABBADI_TES.pdf: 4934736 bytes, checksum: 58e4c3c86d9fb96bb1476ae3c18bfdf0 (MD5) / Made available in DSpace on 2018-03-06T16:21:42Z (GMT). No. of bitstreams: 1 BRUNO_LOPES_ABBADI_TES.pdf: 4934736 bytes, checksum: 58e4c3c86d9fb96bb1476ae3c18bfdf0 (MD5) Previous issue date: 2018-01-19 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Epidemiologic data regarding tuberculosis (TB) show that there is still a high burden of this disease worldwide. In addition, the emergence of drug-resistant strains imposes a new threat in preventing TB spread. Therefore, it is pivotal to continuously find new candidates for drug development. In the Chapter 2 of this thesis, the compound IQG-607 is presented, which is a metal complex that has been reported as a promising anti-TB molecule against isoniazid (INH)-resistant strains of M. tuberculosis. Previous studies suggested that the compound inhibits both the wild-type NADH-dependent trans-2-enoyl-[ACP] reductase (InhA) enzyme and some of its structural mutants in the absence of NAD+ or NADH and without requiring KatG enzyme. IQG-607 has also shown a favorable toxicological profile in vivo, with a considerable lesser toxicity compared to INH. However, there is still a gap regarding the activity of IQG-607 against strains carrying mutations in the katG gene, which are the most common genetic alterations in clinical isolates resistant to INH. Therefore, this study focused in elucidating the mechanism of resistance (MOR) of the Mycobacterium tuberculosis, the main causative agent of TB, to compound IQG-607. First the minimum inhibitory concentration (MIC) of IQG-607 was established against eight multi-drug resistant (MDR) clinical isolates, which were resistant to our compound. Then spontaneous mutants were selected using high concentrations of compound in 7H10 agar medium, and their whole genomes were sequenced; the results revealed alterations in the katG gene. A laboratory strain carrying the mutant katG(S315T) gene was developed to assess the effect of this single mutation in the compound activity both by MIC determination and by a macrophage infection model. Results showed that this mutation was indeed sufficient to confer resistance to IQG-607. Finally, the resistance observed for a strain expressing a mutant InhA(S94A) protein suggested that IQG-607 has this enzyme as its molecular target. In the Chapter 3, two new compounds, called Labio-16 and Labio-17, are presented, which were previously selected to interact and inhibit the InhA enzyme and that had already shown to be active against M. tuberculosis H37Rv strain. A set of experiments were conducted to elucidate their mechanism of action (MOA) and to understand the MOR of the M. tuberculosis against them, similar to those carried for studying IQG-607. So far, results suggested that the InhA is not the molecular target of these compounds. Other experiments are undergoing in our laboratory to evaluate in a murine model of TB infection their potential as anti-TB drug candidates. / Os dados epidemiol?gicos relacionados ? tuberculose (TB) indicam que ainda existe uma carga elevada desta doen?a no mundo todo. Al?m disso, o surgimento de cepas resistentes aos f?rmacos imp?e uma nova amea?a na preven??o da propaga??o da TB. Portanto, ? fundamental buscar continuamente novos candidatos para o desenvolvimento de medicamentos. No Cap?tulo 2 desta tese ? apresentado o composto IQG-607, que ? um complexo met?lico que tem sido reportado como uma mol?cula anti-TB promissora contra cepas de M. tuberculosis resistentes ? isoniazida (INH). Estudos pr?vios sugeriram que o composto inibe a enzima selvagem trans-2-enoil-[ACP] redutase dependente de NADH (InhA) e algumas das suas mutantes estruturais, na aus?ncia de NAD+ ou NADH e sem necessitar da enzima KatG. O IQG-607 tamb?m mostrou um perfil toxicol?gico favor?vel in vivo, com uma menor toxicidade em compara??o ? INH. No entanto, ainda existe uma lacuna em rela??o ? atividade do IQG-607 contra cepas que carregam muta??es no gene katG, as quais s?o as altera??es gen?ticas mais comuns em isolados cl?nicos resistentes ? INH. Sendo assim, este estudo focou em elucidar o mecanismo de resist?ncia (MOR) do Mycobacterium tuberculosis, o principal agente causador da TB, ao composto IQG-607. Primeiramente, a concentra??o inibit?ria m?nima (MIC) do IQG-607 foi estabelecida contra oito isolados cl?nicos multirresistentes a f?rmacos (MDR), os quais foram resistentes ao nosso composto. Ent?o, mutantes espont?neos foram selecionados, usando-se altas concentra??es do composto em meio ?gar 7H10, e seus genomas completos foram sequenciados; os resultados revelaram altera??es no gene katG. Uma cepa laboratorial, carregando o gene katG(S315T) mutante, foi desenvolvida para acessar o efeito desta ?nica muta??o na atividade do composto, atrav?s da determina??o de MIC e por meio de um modelo de infec??o de macr?fagos. Os resultados mostraram que essa muta??o de fato foi suficiente para conferir resist?ncia ao IQG-607. Finalmente, a resist?ncia observada para a cepa que expressa a prote?na InhA(S94A) mutante sugeriu que o IQG-607 tem esta enzima como seu alvo molecular. No Cap?tulo 3, dois novos compostos, denominados Labio-16 e Labio-17, s?o apresentados, os quais foram previamente selecionados para interagir e inibir a enzima InhA, e que j? tinham mostrado ser ativos contra a cepa H37Rv de M. tuberculosis. Um conjunto de experimentos foi conduzido para elucidar os seus mecanismos de a??o (MOA) e para compreender o MOR do M. tuberculosis contra eles, similar ?quele usado para estudar o IQG-607. At? o momento, os resultados sugerem que a InhA n?o ? o alvo molecular desses compostos. Outros experimentos est?o em andamento em nosso laborat?rio, para avaliar em um modelo murino da infec??o da TB os seus potenciais como candidatos a f?rmacos anti-TB.

Os Mollicutes causam doenças em várias espécies animais de importância econômica, inclusive em bovinos. Neste estudo, foi avaliada por concentração inibitória mínima (CIM) e citometria de fluxo, a atividade de oito agentes antibacterianos (enrofloxacina, ciprofloxacina, gentamicina, claritromicina, cloranfenicol, oxitetraclina, tiamulina e tilosina) contra Ureaplasma diversum. Foram analisadas 24 amostras de isolados de campo oriundas da mucosa genital de fêmeas bovinas. As amostras foram confirmadas por crescimento em caldo, placa e por PCR. Os inóculos foram submetidos à analise de suscetibilidade aos antibióticos pelo método da microdiluição em microplaca e posteriormente analisados pelo citômetro de fluxo a fim de avaliar a atividade antimicrobiana nas células. A claritromicina apresentou os maiores índices de inibição in vitro, sendo a gentamicina considerada o antibiótico de menor espectro de ação nesse estudo. De acordo com as análises do citômetro, a gentamicina apresentou o menor número de células viáveis enquanto a tiamulina apresentou o maior número. Embora haja resultados destoantes entre as técnicas utilizadas, o citômetro de fluxo pode ser utilizado como uma boa ferramenta para auxiliar a avaliação da suscetibilidade desses microrganismos a antibióticos. / The Mollicutes cause disease in several economically important species, including cattle. In this study, was evaluated by minimum inhibitory concentration (MIC) and flow cytometry, the activity of eight antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, clarithromycin, chloramphenicol, oxitetraclina, tiamulin and tylosin) against Ureaplasma diversum. We analyzed 24 samples of field isolates originating from the genital mucosa of cows. The samples were confirmed by growth in broth, plate, and PCR. The inoculations were subjected to analysis of susceptibility to antibiotics by the method of micro-dilution plate and then analyzed by flow cytometry to assess the antimicrobial activity in cells. Clarithromycin showed the highest levels of inhibition in vitro, the antibiotic gentamicin considered lower spectrum of action in this study. According to the analysis of the flow cytometer, gentamicin showed the lowest number of viable cells as tiamulin showed the greatest number. Although there are divergent results between the techniques used, flow cytometry can be used as a good tool even help assess the susceptibility of microorganisms to antibiotics.