Chlamydia trachomatis is an obligate intracellular bacterium of major human health concern, causing urogential chlamydia infections, lymphogranuloma venereum (LGV) and trachoma. Chlamydia is one of the most common sexually transmitted infections worldwide and can cause infertility. In the first four papers described herein we used a high resolution multilocus sequence typing (MLST) system to investigate the epidemiology of C. trachomatis, and showed that MLST is superior to conventional ompA genotyping with respect to resolution. In the fifth paper we simplified the methodology by developing and validating a multilocus typing (MLT) DNA microarray based on the MLST system. In more detail, MLST analysis of consecutive specimens from 2006 in Örebro County in Sweden, and comparison to specimens from 1999-2000, showed that the new variant C. trachomatis (nvCT) is monoclonal and likely has appeared in recent years. MLST analysis of LGV specimens from men who have sex with men (MSM) showed that the increase of LGV in Europe in the last decade indeed was a clonal outbreak, contrary to the USA where LGV might have been present all along. In the third paper, clinical symptoms could not be correlated with the MLST genotypes, suggesting, together with the combined results of all previous studies, that bacterial factors, if important, need to be understood in the context of host factors. MLST analysis of specimens from a high incidence C. trachomatis area in North Norway revealed interesting epidemiological details concerning unusual genetic variants, the nvCT and MSM, but found no significant difference in genetic diversity compared to two other geographic areas in Norway. Lastly, we developed a MLT array that provides high resolution while being rapid and cost-effective, which makes it an interesting alternative for C. trachomatis genotyping. In conclusion, the MLST system and the MLT array have proven to be useful tools and should now be applied in further investigations to improve our understanding of C. trachomatis epidemiology.

La reproduction sexuée conduit à l'alternance d'une phase haploïde et d'une phase diploïde, dont la durée relative est très variable entre taxons. La proportion du cycle de vie passée en phase haploïde et en phase diploïde a d'importantes conséquences sur de nombreux processus adaptatifs. Cette thèse combine des approches théoriques qui explorent l'effet de facteurs génétiques et écologiques sur l'évolution des cycles de vie, et un travail expérimental sur l'effet de la ploidy sur l'évolution de l'isolement reproducteur entre populations. La partie théorique a consisté à intégrer des composantes écologiques dans des modèles génétiques pour l'évolution des cycles de vie. En particulier, j'ai exploré l'interaction entre la différenciation de niche entre haploïdes et diploïdes (qui favorise le maintien de cycles biphasiques, impliquant le développement des deux phases) et l'effet d'allèles délétères (qui favorisent soit l'haploïdie, soit la diploïdie). Tandis que la différentiation de niche (ou plus simplement, une différence de valeur sélective intrinsèque entre phases) stabilise les cycles intermédiaires, la présence d'allèles délétères conduit souvent à un branchement évolutif, avec la coexistence stable d'allèles codant pour l'haploïdie et la diploïdie. Cependant, des fluctuations temporelles de l'habitat permettent d'empêcher ce branchement et de stabiliser les cycles biphasiques. La partie expérimentale a consisté à comparer la dynamique de l'isolement reproducteur entre petites populations de levure haploïdes et de diploïdes avec de taux de mutations élevés. Les résultats montrent que tandis que les hybrides haploïdes ont une valeur sélective plus faible que leurs parents, les hybrides diploïdes bénéficient du phénomène d'hétérosis en génération F1, et ont encore une valeur sélective plus élevée que leurs parents en génération F2. La variance de la valeur sélective des hybrides était cependant beaucoup plus élevée chez les haploïdes, avec la production de certains génotypes très performants. / Sexual reproduction leads to an alternation between haploid and diploid phases, whose relative length varies widely across taxa. The proportion of the life cycle spent in the haploid and diploid phase has important consequences on a number of adaptive processes. This thesis combines theoretical approaches exploring the effect of genetic and ecological factors on the evolution of life cycles, and experimental work on the effects of ploidy on the evolution of reproductive isolation between populations. The theoretical part consisted in integrating ecological components into genetic models for the evolution of life cycles. In particular, I explored the interplay between niche differentiation between haploids and diploids (known to favour the maintenance of biphasic life cycles, involving development in both phases) and the effect of deleterious alleles (known to favour either haploid or diploid life cycles). While niche differentiation (or more simply intrinsic fitness differences between phases) stabilizes biphasic cycles, the presence of deleterious alleles often lead to evolutionary branching and to the stable coexistence of alleles coding for haploid and diploid cycles. Branching is prevented, however, when temporal environmental fluctuations are included into the model. The experimental part consisted in comparing the dynamics of reproductive isolation between small populations of haploid and diploid yeasts with elevated mutation rate. The results show that while haploid hybrids tend to have a lower fitness than their parents, diploid hybrids benefit from heterosis in the F1 generation, and still have a higher fitness than the diploid homozygous parents in the F2 generation. However, the variance of hybrid fitness was much higher in haploids, with the production of some highly fit genotypes.

O gênero Neoscytalidium está crescentemente sendo associado às infecções superficiais e profundas causadas em pacientes imunocomprometidos e imunocompetentes. O fungo filamentoso Neoscytalidium dimidiatum é um microrganismo saprofítico e fitopatogênico, encontrado no solo e vegetação de regiões de clima tropical e subtropical. N. dimidiatum var. hyalinum e N. dimidiatum estão envolvidos em infecções superficiais de pele e unha sendo que a espécie produtora de melanina, N. dimidiatum, está associado preferencialmente a infecções profundas sugerindo que a espécie variante não produtora de melanina, N. dimidiatum var. hyalinum, pode ser menos virulenta que a pigmentada. Pouco é descrito na literatura sobre as variedades em questão tanto em relação à sensibilidade a drogas antifúngicas e ao tratamento fotodinâmico antimicrobiano (TFDA), bem como a características fisiológicas referentes à tolerância a variações de temperatura e pH. Assim, o presente trabalho teve como objetivo o estudo de isolados clínicos de N. dimidiatum e N. dimidiatum var. hyalinum obtidos de infecções de pele e unha. Neste estudo foram realizadas a identificação molecular, a filogenia multilocus, a verificação in vitro do desenvolvimento em diferentes temperaturas e pHs e a caracterização in vitro do perfil de susceptibilidade a antifúngicos e TFDA com fotossensibilizadores (FSs) fenotiazínicos, bem como os efeitos do TFDA nas biomoléculas dos artroconídios das variedades estudadas. A filogenia multilocus foi realizada pelo sequenciamento de sete diferentes loci onde foi evidenciado polimorfismo na região Internal Transcribed Spacer (ITS) 1 e nos genes ?-Tubulina (TUB), Fator de Alongamento de cadeia 1? (EF1) e Histona H3 (HH3). O agrupamento dos isolados clínicos deste estudo em genótipos distintos permitiu a separação em 6 tipos de sequência (TSs), sendo que o TS5 foi composto apenas pela espécie variante N. dimidiatum var. hyalinum. A análise de desenvolvimento em diferentes temperaturas e pHs demonstrou um tamanho de colônia reduzido para todos os isolados de N. dimidiatum var. hyalinum. Anfotericina B, voriconazol e terbinafina foram os antifúngicos mais eficientes para ambas variedades. Os valores das concentrações inibitórias mínimas (CIMs) encontrados para os derivados azólicos foram baixos para todos os isolados clínicos de N. dimidiatum var. hyalinum. Os isolados clínicos de N. dimidiatum mostraram ser menos sensível ao TFDA com os FSs azul de metileno (MB), novo azul de metileno N (NMBN), azul de toluidina O (TBO) e novo derivado sintético (S137) quando comparado à variedade hialina. NMBN e S137 mostraram maior eficiência para inativação de Neoscytalidium spp. Adicionalmente, no TFDA todos os FSs apresentaram efeito de permeabilidade de membrana plasmática, embora somente NMBN e S137 apresentaram a produção de Malondialdeido (MDA), isto é, causaram a peroxidação lipídica nos artroconídios de N. dimidiatum e da variedade hialina / Neoscytalidium sp. is increasingly being associated with superficial and deep infections in immunocompromised and immunocompetent patients. The filamentous fungus Neoscytalidium dimidiatum is a saprophytic and plant pathogenic microorganism that is found in soil and vegetation of tropical and subtropical regions. N. dimidiatum var. hyalinum and N. dimidiatum are involved in superficial skin and nail infections. The melanin producer N. dimidiatum is preferably associated with deep infections suggesting that the clinical isolate without melanin, N. dimidiatum var. hyalinum, may be less virulent than the pigmented variety. Little is described in the literature regarding the varieties N. dimidiatum and N. dimidiatum var. hyalinum about the sensitivity to antifungal drugs and photodynamic antimicrobial chemotherapy (PACT), and the physiological characteristics related to the growth at different temperature and pH. Therefore, the present study aimed to investigate clinical isolates of N. dimidiatum and N. dimidiatum var. hyalinum obtained from skin and nail infections. Here in this study were performed the molecular identification, multilocus phylogeny, the in vitro characterization of development at different temperatures and pHs, and the characterization of in vitro susceptibility to antifungal agents and PACT with phenotiazinium photosensitizers (PSs), as well the PACT effects on the biomolecules of the Neoscytalidium spp. arthroconidia. The multilocus phylogeny was performed by sequencing seven different loci where polymorphism were identified in the Internal Transcribed Spacer (ITS) 1 region of rDNA and in the genes ?-tubulin (TUB), elongation factor 1? (EF1) and Histone H3 (HH3). The grouping of the clinical isolates from this study in different genotypes allowed the clustering in 6 sequence types (ST), in which the ST5 was composed exclusively by all N dimidiatum var. hyalinum isolates from this study. The analysis of development at different temperatures and pHs showed a reduced colony size for all N. dimidiatum var. hyalinum isolates. Amphotericin B, voriconazole and terbinafine were the most effective antifungal for both varieties. The minimal inhibitory concentrations (MICs) values found for the azoles derivatives were low for all N. dimidiatum var. hyalinum isolates. N. dimidiatum clinical isolates have shown to be less sensitive to PACT with the PS methylene blue (MB), new methylene blue (NMBN), toluidine blue O (TBO) and new synthetic derivative (S137) when compared to hyaline variety. NMBN and S137 have shown more effectiveness for the inactivation of Neoscytalidium spp. Additionally, all PS in PACT have caused plasma membrane permeability, although only NMBN and S137 showed the production of malondialdehyde (MDA), i.e., caused lipid peroxidation in both N. dimidiatum and hyaline variety

A diversidade genética dos isolados de Chromobacterium sp. foi avaliada por Multilocus sequence analysis com base nas análises dos genes conservados rpoA, lepA, gyrB, fusA e rRNA 16S. A análise do gene rRNA 16S e MLSA agrupou os isolados no gênero Chromobacterium, entretanto, cinco dos isolados estão distantes filogeneticamente das linhagens tipo, C. violaceum e C. subtsugae, sugerindo duas novas espécies. Os extratos brutos, obtidos por soxhlet, dos isolados de Chromobacterium sp., testados em ensaios in vitro em células tumorais humanas, apresentaram atividades antitumorais potenciais e seletivas para determinadas linhagens celulares. Os extratos brutos e frações foram analisados por HPLC-DAD para avaliação da presença ou ausência da violaceína. Além disso, outros metábólitos secundários que não a violaceína podem estar relacionados à atividade antitumoral. / The genetic diversity of strains of Chromobacterium sp was evaluated by Multilocus sequence analysis (MLSA) based on the analysis of conserved genes rpoA, recA, lepA, gyrB, fusA and rRNA 16S. The analysis of 16S ribosomal RNA gene grouped all isolates in the genus Chromobacterium, however, the five isolates are phylogenetically distant of type strain C. violaceum and C. subtsugae, suggesting new species. The crude extracts of the isolates from Chromobacterium sp., obtained by soxlhlet, evaluated in vitro tests on human tumor cells, showed potential and selective antitumor activities for certain cell lines. In addition, other secondary metabolites than violacein may be related with antitumor activity.

Ce travail a pour but de développer des méthodes de détection de locus affectant les caractères quantitatifs, appelés QTL, à partir de l'information disponible sur des caractères corrélés et/ou des positions liées, chez les animaux d'élevage.<br />Les méthodologies ont été dans un premier temps caractérisées pour leurs puissances et leurs précisions d'estimation des paramètres (positions et effets des QTL) à partir de données simulées. Nous avons développé d'une part des méthodes multivariées, extrapolées de techniques décrites pour l'analyse de données issues de croisements entre populations supposées génétiquement fixées, et d'autre part des méthodes synthétiques univariées, développées à l'occasion de ce travail. Ces dernières méthodes permettent de synthétiser l'information due à la présence du (des) QTL déterminant plusieurs caractères dans une unique variable, combinaison linéaire des caractères. Le nombre de paramètres à estimer est ainsi indépendant du nombre de caractères étudiés, permettant de réduire fortement les temps de calcul par rapport aux méthodes multivariées. La stratégie retenue repose sur des techniques d'analyse discriminante. Pour chaque vecteur de positions testé, des groupes de descendants sont créés en fonction de la probabilité que les individus aient reçu l'un ou l'autre haplotype de leur père. Les matrices de (co)variance génétique et résiduelle spécifiques de la présence du (des) QTL peuvent alors être estimées. La transformation linéaire permet de maximiser le rapport de ces deux variabilités.<br />Les méthodes basées sur l'analyse de variables synthétiques permettent en général d'obtenir des résultats équivalents, voire meilleurs, que les stratégies multivariées. Seule l'estimation des effets des QTL et de la corrélation résiduelle entre les caractères reste inaccessible par ces méthodes. Une stratégie itérative basée sur l'analyse de variables synthétiques pour la sélection des caractères et des régions chromosomiques à analyser par les méthodes multivariées est proposée. Par ailleurs, nous avons quantité les apports des méthodologies multidimensionnelles pour la cartographie des QTL par rapport aux méthodes unidimensionnelles. Dans la majorité des cas, la puissance et la précision d'estimation des paramètres sont nettement améliorées. De plus, nous avons pu montrer qu'un QTL pléiotrope peut être discriminé de deux QTL liés, s'ils sont relativement distants.<br />Ces méthodologies ont été appliquées à la détection de QTL déterminant cinq caractères de composition corporelle chez le porc sur le chromosome 7. Deux groupes de QTL déterminant des types de gras différents, le gras interne et le gras externe, ont ainsi été discriminés. Pour chacun de ces groupes, les analyses multiQTL ont permis d'identifier au moins deux régions chromosomiques distinctes déterminant les caractères.

<p>Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.</p>

Made available in DSpace on 2014-12-22T16:37:26Z (GMT). No. of bitstreams: 2 maria_carvalho_ipec_mest_2012.pdf: 3003635 bytes, checksum: 510838807146764c9ee0f462f0cf0ea7 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014-10-07 / Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas, Rio de Janeiro, RJ, Brasil / Dentre as espécies de Candida não-albicans, Candida parapsilosis vem emergindo como importante patógeno de infecções fúngicas invasivas com disseminação hematogênica nas últimas décadas em diferentes continentes, principalmente, na Europa e na América Latina. C. parapsilosis foi considerada por muito tempo um complexo de três grupos distintos nomeados I, II e III. Tavanti et al (2005), baseado na tipagem da sequência multilocus (MLST), propôs o reconhecimento dos grupos II e III, como duas novas espécies: C. orthopsilosis e C. metapsilosis, respectivamente, mantendo o grupo I como C. parapsilosis stricto sensu. Até agora só tem sido possível distinguir essas três espécies por análise molecular. Métodos comerciais para testes de susceptibilidade aos antifúngicos vêm sendo utilizados para avaliar o comportamento de Candida spp. frente às drogas de uso clínico, incluindo o Etest® e o sistema Vitek® 2. Neste trabalho, estes dois métodos foram comparados ao método de referência de microdiluição em caldo pelo CLSI (M27-A3) para determinar a susceptibilidade in vitro de isolados clínicos do complexo psilosis aos fármacos antifúngicos anfotericina B, fluconazol, voriconazol e itraconazol. Um total de 53 isolados do complexo psilosis oriundos de hemoculturas obtidas de pacientes hospitalizados no município do Rio de Janeiro, entre 1998 e 2006, associados a episódios de fungemia, foram analisados Cinquenta e um isolados foram discriminados pela PCR, utilizando primers espécie-específicos, e dois isolados, pelo sequenciamento, sendo caracterizados como C. parapsilosis stricto sensu (75,4%), C. orthopsilosis (20,8%) e C. metapsilosis (3,8%). Os testes de susceptibilidade aos antifúngicos indicaram que a maioria dos isolados de C. parapsilosis stricto sensu foi sensível a todos os fármacos testados. Entretanto, um único isolado de C. parapsilosis stricto sensu apresentou uma CIM = 2 [g/mL para a anfotericina B. Três isolados de C. orthopsilosis apresentaram CIM entre 2 e 8 [g/mL para o fluconazol pelo Etest® e Vitek® 2 e CIM entre 0,19 e 0,25 [g/mL para o itraconazol pelo Etest®. Os isolados de C. metapsilosis foram sensíveis a todos os fármacos testados. A concordância essencial entre Etest® ou Vitek® 2 com CLSI foi excelente (100%), exceto para o itraconazol (90,9%). Por outro lado, a concordância categórica foi 72,7% para o itraconazol pelo Etest® e 100% para os outros fármacos por ambos os métodos. Para anfotericina B, a concordância categórica foi de 100% para o Etest® e de 97,5% pelo Vitek® 2 em relação ao CLSI. Este estudo reforça a importância dos métodos Etest® e Vitek® 2 que podem ser empregados nos laboratórios rotineiros de microbiologia clínica para monitorar e detectar diferenças no perfil de susceptibilidade aos antifúngicos dos isolados de C. parapsilosis stricto sensu, C. orthopsilosis e C. metapsilosis / Among non-albicans Candida species, Candida parapsilosis has emerged as an important agent of invasive fungal infections, and several cases associated with fungemia have been reported worldwide in last decade, mostly in Europe and Latin America. For many years, C. parapsilosis has been characterized as a complex composed of three genetically distinct groups (groups I, II, and III). Tavanti et al. (2005) based on multilocus sequence typing (MLST) technique, proposed the recognition of groups II and III as two different species: C. orthopsilosis and C. metapsilosis, respectively, maintaining the group I as C. parapsilosis sensu stricto. Up to now only has been possible to distinguish these three species just by molecular analysis. Commercial antifungal susceptibility methods including Etest® and Vitek® 2 system have been used to test the antifungal susceptibility of Candida spp. In this study, these methods were compared to the CLSI broth microdilution (BMD) reference method to determine in vitro susceptibility of clinical C. parapsilosis complex isolates to amphotericin B, fluconazole, voriconazole, and itraconazole. A total of 53 C. parapsilosis complex isolates from blood cultures obtained of patients who were hospitalized in the city of Rio de Janeiro between 1998 and 2006 associated with episodes of fungemia were analysed. Fifty-one isolates were discriminated by PCR using species-specific primers and two isolates by sequencing, being characterized as C. parapsilosis sensu stricto (75.4%), C. orthopsilosis (20.8%), and C. metapsilosis (3.8%). Antifungal susceptibility tests indicated that most of C. parapsilosis sensu stricto isolates were susceptible to all tested drugs. However, a single C. parapsilosis sensu stricto isolate presented MIC = 2 [g/ml for amphotericin B. Three C. orthopsilosis isolates showed MIC between 2-8 [g/ml for fluconazole by Vitek® 2 and MIC between 0.19-0.25 [g/ml for itraconazole by Etest®. C. metapsilosis isolates were susceptible to all tested drugs. The essential agreement between the Etest® or Vitek® 2 with the CLSI BMD for all drugs was excellent (100%), except for itraconazole (90.9%). The categorical agreement was 72.7% for itraconazole by Etest®, 97.5% for amphotericin B by Vitek® 2, and 100% for the other drugs by both methods compared with CLSI BMD. This study reinforces the importance of Etest® and Vitek® 2 methods in routine clinical microbiologycal laboratories to survey and detect differences in the profile of the antifungal susceptibility of C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis isolates.

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Rio de Janeiro, RJ, Brasil / De acordo com a Organização Mundial da Saúde (OMS), as Doenças Tropicais Negligenciadas (DTNs), como a doença de Chagas, tripanossomíase africana, entre outras, afetam cerca de 1 bilhão de pessoas no mundo. Apesar do impacto causado por estas doenças, as mesmas continuam sendo pouco estudadas. O reino Protista, ao qual os agentes etiológicos dessas doenças pertencem, incluem espécies de importância veterinária que causam muitas perdas para a indústria pecuária. Com os avanços da genética molecular, técnicas como a genotipagem estão sendo usado cada vez mais utilizadas para o estudo de protozoários parasitos. Genes ortólogos conservados em protozoários podem ser utilizados como loci para genotipagem desses protozoários, de modo a obter uma melhor caracterização interespecífica. Neste estudo, foram utilizadas técnicas de PCR singleplex e multiplex para a genotipagem de 16 espécies de protozoários de três diferentes gêneros: Trypanosoma spp., Leishmania spp. (classe Kinetoplastea) e Plasmodium spp. (filo Apicomplexa). Como metodologia de biologia molecular foram utilizados PCR e sequenciamento, bem como em bioinformática, onde foi utilizado filogenia molecular. Para esta finalidade, foram desenhados 39 pares de iniciadores degenerados e usados com o DNA genômico de espécies de protozoários de dois grupos: filo Apicomplexa e classe Kinetoplastea, em seguida, as reações de PCR foram realizadas a fim de padronizar as reações para um PCR multiplex. Além disso, os seguintes parasitos de importância para a saúde pública: T. cruzi, Leishmania braziliensis, L. amazonensis, L. guyanensis, Plasmodium falciparum e P. Vivax, foram utilizados neste trabalho A partir dos 39 pares de iniciadores, 9 pares foram selecionados compreendendo 7 loci para o presente estudo: metionil-tRNA sintetase, leucil-tRNA sintetase, seril-tRNA sintetase, subunidade U5 snRNP do spliceossomo, mismatch repair ATPase, triosefosfato isomerase e GTP-binding protein. Os testes a partir de diluições seriadas dos DNAs de Trypanosoma spp., Leishmania spp. e Plasmodium spp., onde mostraram uma sensibilidade de até 1 pg/\03BCL de DNA (para o locus Leucyl-tRNA synthetase em Trypanosoma e Leishmania). A reação inicial de PCR singleplex padronizada foi eficiente para todos os loci, o que proporcionou o desenho da reação de PCR multiplex, onde o melhor resultado foi a reação número 5, a qual foi utilizada um conjunto de iniciadores dos genes GTP-binding protein, U5 snRNP spliceosome subunit e Leucyl-tRNA synthetase, que quando integrados permitiram a genotipagem multilocus de tripanossomatídeos dos gêneros Leishmania spp. e Trypanosoma spp. A metodologia aplicada para a genotipagem destas espécies de parasitas foi bem-sucedida, e pode-se destacar após a observação de conjunto de resultados que o melhor locus em termos de especificidade, sensibilidade e fidelidade aos dados encontrados na literatura, foi Leucyl-tRNA synthetase, que apresentou resultados satisfatórios em todas as análises / According to the World Health Organization (WHO), Neglected Tropical Diseases (NTDs), examples being Chagas disease, African trypanosomiasis, among others, affect about 1 billion people in the world. However, they are little studied, despite its great impact on health. Also, they include species of veterinary importance that cause great harm to the livestock industry. The kingdom Protista, containing etiological agents of NTDs, they include species of veterinary importance and that cause great harm to the livestock industry. With advances in molecular genetics, techniques such as genotyping are being used increasingly used for the study of parasitic protozoans. Orthologous genes conserved in protozoa can be used as loci for genotyping, in order to obtain a better interspecific characterization. In this study, we used singleplex and multiplex PCR techniques for genotyping of 16 species of three different genus of protozoans: Trypanosoma spp., Leishmania spp. (class Kinetoplastea) e Plasmodium spp. (phylum Apicomplexa). As molecular biology methods were used PCR and sequencing of PCR fragments, as well as bioinformatics, using molecular phylogeny. For this purpose, were designed 39 degenerated primers for species of 2 groups: phylum Apicomplexa and class Kinetoplastea, then, the PCR reactions were performed with the aim of standardizing multiplex PCR reactions. Moreover, parasites of importance to public health such as Trypanosoma cruzi, Leishmania braziliensis, L. amazonensis, L. guyanensis, Plasmodium falciparum and P. vivax were used in this study Based on the 39 pairs of primers, 9 pairs were selected comprising 7 loci for this study: methionyl-tRNA synthetase, leucyl-tRNA synthetase, seryl-tRNA synthetase, subunit U5 snRNP the spliceosome, mismatch repair ATPase, triosephosphate isomerase, and GTP- binding protein. The tests from serial dilutions of DNA from Trypanosoma spp., Leishmania spp. and Plasmodium spp., which showed a sensitivity of up to 1 pg/\03BCL DNA (for Leucyl-tRNA synthetase locus on Trypanosoma and Leishmania). The initial standardized singleplex PCR reaction was efficient for all loci, which resulted in the design of multiplex PCR reaction, where the best result was the number 5 reaction which was used a set of primers of the GTP-binding protein genes, U5 snRNP spliceosome subunit and Leucyl-tRNA synthetase, which when integrated allowed genotyping trypanosomatides multilocus of the genus Leishmania spp. and Trypanosoma spp. The methodology used for genotyping of these parasite species was successful, and can highlight after observation of the result set that the best locus in terms of specificity, sensitivity and fidelity to data found in literature, was LeucyltRNA synthetase, which showed satisfactory results in all analyzes

Campylobacter jejuni is the main cause of bacterial diarrhoeal illness in developed countries, with ~7000 cases being reported each year in Sweden. C. jejuni has received growing attention since it’s recognition as a human pathogen in the 1970s, but its epidemiology is complex and much still remains unknown. There are several potential reservoirs for C. jejuni, including environmental sources as water and soil, wild and domesticated animals, particularly poultry, but also other livestock and pets. In this study 348 Swedish C. jejuni isolates from the year 2000 from humans (n = 164) and broilers (n = 184) were characterized with multilocus sequence typing (MLST) with the aim of comparing the population structures and diversity of C. jejuni between isolates from the two hosts. MLST is a method for characterization of bacterial isolates that indexes the variation in DNA sequence of multiple protein encoding housekeeping genes. A secondary aim in this study was to compare populations of C. jejuni from 11 subgroups of isolates based on location of the sampling. The overlap between the populations was analyzed numerically based on genotypes detected and with analysis of phylogeny, gene flow and molecular variation. It was shown that the population structure of C. jejuni isolates from broilers and humans show a high degree of similarity, supporting broilers as an important source of human infection. However, even though the population structure of human and broiler C. jejuni were almost genetically indistinguishable other sources of C. jejuni infections in humans cannot be ruled out since the same genotypes can be found in other sources as well. Analysis of the 11 subgroups suggested that there may be a difference in populations infecting humans in different Swedish regions, and between populations of C. jejuni in broilers from different slaughterhouses. But this could be a result of chance since most of the subgroups were small. Future studies to improve the understanding of C. jejuni epidemiology, for which MLST has proven itself as a valid method, is important to develop control strategies to prevent infection with this common cause of diarrhoeal illness.

Orientador: Suzete Aparecida Lanza Destéfano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T14:37:32Z (GMT). No. of bitstreams: 1 Tonin_MarianaFerreira_D.pdf: 2371085 bytes, checksum: d7c915d664efeec24397b855164c65b9 (MD5) Previous issue date: 2012 / Resumo: Espécies pertencentes ao gênero Xanthomonas são responsáveis por doenças que podem causar grandes perdas econômicas em diversas culturas. Esses fitopatógenos têm sido objeto de diversos estudos taxonômicos resultando em significativas alterações na classificação em nível inter e infraespecífico. O presente estudo teve por objetivo caracterizar taxonomicamente bactérias do gênero envolvendo: (1) diferenciação e análises filogenéticas de espécies do gênero Xanthomonas; (2) esclarecimento da posição taxonômica de linhagens classificadas como Xanthomonas sp.; (3) diferenciação de patovares da espécie X. campestris; (4) desenvolvimento de primers específicos para X. campestris, X. translucens, X. cucurbitae e X. melonis. O par de primers rpoB2F/rpoB3R, desenhado a partir de sequências do gene rpoB, foi empregado em experimentos de amplificas;ao utilizando-se DNAs de 26 espécies do gênero Xanthomonas e os produtos de amplificas;ao (800 pb) foram digeridos com diversas endonucleases. Os perfis de restrição obtidos com a utilização da enzima Hae III permitiram a diferenciação da maioria das espécies, incluindo os patógenos de mesmo hospedeiro como X. albilineans e X. sacchari (patogênicas à cana-de-açúcar); X. cucurbitae . e X. melonis (patogênicas ao melão); X. vesicatoria, X. gardneri e X. euvesicatoria/X. perforans (patogênicas ao tomateiro). Ainda, os produtos de amplificação do gene rpoB foram seqüenciados e a árvore filogenética construída a partir destas sequências também possibilitou a diferenciação das espécies do gênero, indicando que o gene rpoB pode ser considerado um marcador molecular eficiente para o estudo das relações filogenéticas de espécies do gênero Xanthomonas. Os DNAs de linhagens classificadas como Xanthomonas sp. também foram analisados por meio de experimentos de amplificação com primers específicos para a espécie X. axonopodis, de hibridizas;ao DNA-DNA, e analise de multilocus utilizando-se sequências dos genes rpoB, rpoA, atpA, recA e região espaçadora 16S-23S DNAr. Os resultados mostraram que as linhagens de X. sp. pv. viticola, X. sp. pv. betae e X. sp. pv. paulliniae pertencem a espécie X. axonopodis, entretanto, não foi possível definir a posição taxonômica das linhagens de X. sp. pv. arracaciae, X. sp. pv. esculenti e X. sp. pv. eucalypti, embora várias ferramentas tenham sido utilizadas. Assim, somente estudos complementares deverão ser conduzidos visando esclarecer a classificação dessas linhagens. Nesse estudo também foram analisadas as espécies X. campestris, X. translucens, X. cucurbitae e X. melonis. Visando a diferenciação dos patovares da espécie X. campestris, os DNAs destas linhagens foram submetidos a experimentos de PCR-RFLP do gene rpoB e as digestões duplas utilizando-se Cfo I/Mbo I. Os resultados permitiram a diferenciação dos patovares X.c. pv. raphani, X.c. pv. barbariae, X.c. pv. incanae, X.c. pv. armoraciae e X.c. pv. campestris/ X.c. pv. aberrans. Ainda, os dados obtidos nas amilises do gene rpoB permitiram o desenvolvimento de primers específicos para as espécies X. campestris, patogênicas as crucíferas (rpoB2F/xcamR) e X. translucens, patogênicas a gramíneas e cereais (trans1F/trans2R). Alem disso, amilises de sequências da região espaçadora 16S-23S DNAr possibilitaram o desenho do par de primers mecF/mecR, especifico para X. cucurbitae e X. melonis, espécies patogênicas ao melão. A especificidade destes primers foi confirmada em experimentos de amplificação utilizando-se DNAs de algumas bactérias de diferentes gêneros isoladas de mesma espécie de plantas hospedeiras. O nível de sensibilidade da técnica de PCR utilizando-se os primers desenvolvidos foi de 0,1 pg para rpoB/xcam, de 0,01 ng para trans1F/trans2R e de 1 pg para mecF/mecR / Abstract: Xanthomonas species are responsible for diseases causing economic losses in many crops. These phytopathogens have been subject of several taxonomic studies resulting in significant changes at interespecific or infraspecific level. This study aimed the taxonomic characterization of Xanthomonas species including: (1) differentiation and phylogenetic analysis of Xanthomonas species; (2) clarification of taxonomic position of strains classified as Xanthomonas sp.; (3) differentiation of the pathovars of X. campestris; ( 4) development of specific primers for X. campestris, X. translucens, X. cucurbitae and X. melonis. The rpoB2F/rpoB3R primers, designed from rpoB gene sequences, were employed in amplification experiments using DNAs from 26 species of Xanthomonas genus and the products (800 bp) were digested with different restriction enzymes. Profiles using Hae III allowed to differentiate the most species of the genus, including pathogens the affect the same plant host as X. albilineans e X. sacchari (pathogenic to sugarcane); X. cucurbitae e X. melonis (pathogenic to melon); X. vesicatoria, X. gardneri and X. euvesicatoria/X. perforans (pathogenic to tomato). Amplification products of the rpoB gene were sequenced and the phylpgenetic tree constructed from these sequences also allowed the differentiation of the Xanthomonas species, indicating that the rpoB gene can be used as an efficient molecular marker for phylogenetic relationships studies within the Xanthomonas genus. DNA of the strains classified as Xanthomonas sp. were also analysed by the amplification experiments using specific primers for X. axonopodis species, DNA-DNA hybridization and multilocus sequence analysis of the genes rpoB, rpoA, atpA, recA and intergenic spacer region 16S-23S rDNA. Results showed that the strains of X. sp. pv. viticola, X. sp. pv. betae and X. sp. pv. paulliniae belong to X. axonopodis species, however, it was not possible to define the taxonomic position of X. sp. pv. arracaciae, X. sp. pv. esculenti and X. sp. pv. eucalypti, although different approaches have been used. Thus, further studies should be conducted in order to clarify their classification. In this study X. campestris, X. translucens, X. cucurbitae and X. melonis were also analyzed. In order to differentiate the pathovars of the X. campestris specie, the DNAs of these strains were submitted to PCR-RFLP analysis of the rpoB gene and the double digestions using Cfo I/Mbo I allowed the differentiation of the pathovars X. c. pv. raphani, X.c. pv. barbariae, X.c. pv. incanae, X.c. pv. armoraciae and X.c. pv. campestris/ X.c. pv. aberrans. Also, the data obtained in the rpoB gene analysis allowed the development of specific primers for the species X. campestris, pathogenic to crucifers (rpoB2F/xcamR) and X. translucens, pathogenic to grasses and cereals (trans1F/trans2R). Furthermore, intergenic spacer 16S-23S rDNA sequences analysis enabled the development of the mecF/mecR primers, specific for X cucurbitae and X melonis, both pathogenic to melon. The specificity of these primers was confirmed by amplification experiments using DNAs from bacteria belonging to different genera pathogenic to the same plant hosts. Sensitivity level of the PCR technique using the primers developed was 0,1 pg for rpoB/xcam, 0,01 ng for trans1F/trans2R and 1 pg for mecF/mecR / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular