Made available in DSpace on 2016-06-21T13:43:16Z (GMT). No. of bitstreams: 2 833.pdf: 2723781 bytes, checksum: a830784ddefc542106be74ce8aa431fa (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Made available in DSpace on 2016-07-05T22:16:54Z (GMT). No. of bitstreams: 3 833.pdf.txt: 159741 bytes, checksum: 9e6065c4897ceb926534eb7e10453293 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 833.pdf: 2723781 bytes, checksum: a830784ddefc542106be74ce8aa431fa (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / A Yersinia pestis é o agente etiológico da peste, uma doença primária de roedores, transmitida por pulgas infectadas e que pode infectar o homem e outros mamíferos. O objetivo do trabalho foi realizar a tipagem de 63 cepas de Y. pestis de três focos de peste do PE. As cepas foram isoladas de diferentes fontes e períodos. Das 63 cepas, 20 foram isoladas de um epizootia, em agosto de 1967, na Chapada do Araripe-PE. Também foram estudadas oito cepas de Y. pestis isoladas em outros países, cinco cepas de Y. pseudotuberculosis e nove de Y. enterocolitica. Foram utilizados onze VNTRs pela técnica do MLVA. Dos onze VNTRs para as cepas da epizootia apenas um revelou-se polimórfico apresentando diferentes alelos. Os demais VNTRs revelaram-se monomórficos. Entre os onze VNTRs analisados para as 51 cepas de Y. pestis (43 brasileiras e 8 estrageiras) dois se revelaram monomórficos gerando amplicons com 7 e 2 unidades repetitivas (UR). Os outros nove VNTRs analisados revelaram-se polimórficos gerando dois a oito alelos. As cepas de Y. pseudotuberculosis apresentaram-se polimórficas para 10 VNTRs gerando amplicons de tamanhos diversos, o VNTR ms09 foi o único monomórfico gerando um amplicon de 700 pb com 28 UR. Das nove cepas de Y. enterocolitica analisadas com os onze locos, sete apresentaram-se monomórficos com amplicons de 700, 250, 270, 690, 231 e 379 pb. Os outros quatro VNTRs analisados apresentaram um padrão de amplificação polimórfico com amplicons de tamanhos diferentes para o mesmo loco. O padrão de amplificação gerado com as cepas de Y. pestis possibilitou distribui-las em 35 perfis genotípicos. A análise das cepas pelo dendrograma permitiu agrupá-las em cinco clados, onde no clado I ficaram agrupadas a maioria das cepas brasileiras de Y. pestis, as cepas estrangeiras de Y. pestis ficaram agrupadas nos clados II e IV, enquanto que Y. enterocolitica e Y. pseudotuberculosis ficaram nos clados III e V respectivamente. Diante dissso pode-se considerar que o MLVA mostrou-se uma ferramenta útil em estudos filogenéticos e epidemiológicos das cepas brasileiras de Y. pestis, além de estudos intraespecíficos com as espécies de Y. enterocolitica e Y. pseudotuberculosis. As análises revelaram diversidade genética entre as cepas de Y. pestis isoladas de diferentes fontes e períodos e sua continuação poderá gerar dados importantes para estabelecer relações filogenéticas entre as cepas, contribuindo para um melhor entendimento da disseminação e transmissão do agente etiológico da peste na natureza e a dinâmica da epidemiologia no Brasil

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-27T15:32:09Z No. of bitstreams: 1 Arquivototal.pdf: 1846108 bytes, checksum: fb9211d08b5d703a2c5cc6f6f58d918c (MD5) / Made available in DSpace on 2018-04-27T15:32:09Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 1846108 bytes, checksum: fb9211d08b5d703a2c5cc6f6f58d918c (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Plague is a zoonosis whose reservoir system is composed by communities of small mammals in foci that are independent in time and space. The small mammals’ assemblage of a focus is one of the factors that regulates the fluctuations on the transmission cycle through composition variance and population parameters of each species. The comprehension of the structure and dynamics of the reservoir system of a plague focus can be a predictive tool to foresee risk of epizootic events. The present dissertation analysed the Borborema Plateau Plague focus through a zoonosis and community ecology by studying the small mammals that compose the reservoir system. The first chapter demonstrates through theory that Plague is a complex ecological entity, with several models created to explain its dynamics, although the current knowledge has a bias caused by the epidemiological surveillance models. The second chapter performs an analysis of beta-diversity on the focus, with data from a field survey in Alagoa Grande and Areia, municipalities within the focus, with 3640 traps/night of sampling effort; recovery of data from vigilance campaigns from the Health District of Garanhuns in 1981, Museum registries and literature records; obtaining 30 localities with 29 species registered. The analysis indicated high compositional dissimilarity between localities (beta-diversity > 0.9), with dominance of turnover, but confirmed the unity of the small mammal metcommunity in the Borborema Plateau. The findings in this chapter recovered a partition in the small mammal community between the peridomestic and sylvatic habitats, with beta-diversity of 0.517. The analyses weren’t capable of delimitate the focus solely by its reservoir system. The chapter also introduces the concept of metafocus, areas with geographically restricted activity within the whole expansion of the focus, controlled by a series of variables, as a theoretical possibility to explain the focus’ activity pattern. The third chapter reports the epidemiological surveillance action performed in the two municipalities from within the focus, in the state of Paraíba, capturing 45 individuals, obtaining 27 samples for PCR and bacteriology, and 7 samples for serum analysis. With all test results being negative, the quiescence period in the focus is reaffirmed, but the vigilance measures must go on, as foci might reemerge after long innactive periods, and the current situation of epidemiologic transition. / Peste é uma zoonose cujo sistema reservatório é composto por comunidades de pequenos mamíferos em focos independentes no tempo e espaço. A comunidade de pequenos mamíferos tem papel importante na regulação e mediação das flutuações do ciclo de transmissão através de sua composição e parâmetros populacionais das espécies. A compreensão da estrutura e dinâmica da comunidade de pequenos mamíferos de um foco de peste é uma ferramenta preditora para o risco de eventos epizoóticos. A presente dissertação analisou o foco de Peste da Chapada da Borborema por uma perspectiva de ecologia de zoonoses e comunidades, através de seu sistema reservatório visando responder duas perguntas: há partição da comunidade de pequenos mamíferos entre peridomicílio e remanescentes nativos? E, qual é o perfil pestoso dos ambientes peridomiciliar e selvático. O primeiro capítulo demonstra que a Peste pode ser considerada uma entidade ecológica complexa, com diversos modelos para explicar sua dinâmica, embora ainda incompletos, dado o viés amostral causado pela rotina de vigilância. O segundo capítulo realiza uma análise de beta-diversidade no foco; através de coletas em campo em Alagoa Grande e Areia, municípios circunscritos ao foco, com esforço total de 3640 armadilhas/noite; resgate do registro histórico das coletas de pequenos mamíferos na região através de fichas de atividade de vigilância em zoonoses da Regional de Saúde de Garanhuns em 1981, Livros do Tombo de duas coleções e registros da literatura; obtendo 30 localidades com registro de 29 espécies. As coletas em campo e as coletas da Regional de Saúde foram suficientes em amostrar a fauna do local, tendo a riqueza registrada condizente com a projeção do estimador Chao 1. As análises indicaram alta dissimilaridade composicional entre as localidades (betadiversidade > 0.9), com dominância do turnover, mas confirmaram a unidade da metacomunidade do Planalto da Borborema. Os achados do capítulo resgataram uma partição da comunidade de pequenos mamíferos entre o ambiente peridomiciliar e os remanescentes de vegetação nativa, com uma beta-diversidade de 0.517. O foco não é delimitável à partir apenas do sistema reservatório. Apresenta-se também a noção do metafoco, locais de atividade pontual e difusa dentro do território total do foco, com regime controlado por uma combinação de fatores ambientais ótimos, como uma possibilidade teórica para explicar a atividade do foco. O terceiro capítulo faz um inquérito epidemiológico nos dois municípios circunscritos ao foco no estado da Paraíba amostrados no capítulo anterior, capturando 45 indivíduos em campo, dos quais se obteve amostras para análises bacteriológicas e moleculares (27) e sorologia (7). Com todos os resultados negativos, temos a reafirmação do período quiescente do ciclo de transmissão, mas alertando que a quiescência não descarta as medidas de vigilância, dado que os focos podem reemergir, e o panorama epidemiológico mundial alerta para um período de transição zoonótica.

Yersinia pestis, agent causal de la peste, persiste dans la nature maintenu par un cycle enzootique dans des foyers conduisant à la réémergence de la maladie. En Afrique du Nord, où une réémergence a eu lieu après des années de ‘silence’, nous avons répertorié les différents épisodes ainsi que le nombre de cas en sur six pays à compter de 1940 en mettant en évidence l’importation de la maladie et un mode de contamination négligé, la transmission par voie orale. Une étude en Algérie sur 237 micromammifères confirme deux foyers et en revèle trois nouveaux porteurs d’un nouveau génotype (MST) de biotype Orientalis. Apodemus sylvaticus est par la même ajouté à la liste des rongeurs pestiférés. La projection des foyers de peste ainsi actualisés sur une carte géographique et écologique met en évidence la proximité des foyers de peste aux points d’eau saumâtre. Une étude statistique a confirmé une corrélation significative entre foyer de peste/eau salée à une proximité minimale <3 km en comparaison à des zones d’eau douce. Des échantillons environnementaux salés ont permis l’isolement d’une souche Y. pestis Algeria 3. Cette découverte confortée par l’observation expérimentale de la résistance de Y. pestis à un milieu hyper salé à 150g/L NaCl se traduisant par un protéome spécifique en réponse à ce stress avec une forme d’adaptation de type forme L de la bactérie dans ce type d’environnement. Notre travail éclaire de façon originale un facteur méconnu de persistance tellurique de Y. pestis, conditionnant la réémergence de la peste dans des foyers séculaires au Maghreb contrairement aux rivages Nord de la Méditerranée où la peste autochtone a disparu depuis un siècle. / Yersinia pestis, the causal agent of plague, persists in nature maintained by an enzootic cycle in foci leading to the re-emergence of the disease. In North Africa, where re-emergence took place after years of 'silence', we have listed the various episodes and the number of cases in six countries from 1940 onwards, highlighting the importation of the disease and A method of neglected contamination, oral transmission. A study in Algeria on 237 micromammals confirms two foci and reveals three new carriers of a new genotype (MST) of orientalis biotype. Apodemus sylvaticus is by the same added to the list of plague rodents. The projection of the plague foci thus updated on a geographical and ecological map highlights the proximity of plague foci to brackish water points. A statistical study confirmed a significant correlation between plague / salt water at a minimal proximity <3 km compared to freshwater areas. Saline environmental samples allowed the isolation of a Y. pestis Algeria 3 strain. This discovery was confirmed by the experimental observation of the resistance of Y. pestis to a hyper-saline medium at 150 g / L NaCl resulting in a specific proteome In response to this stress with an adaptation form of form L of the bacterium in this type of environment. Our work illuminates in an original way an unknown factor of telluric persistence of Y. pestis, conditioning the re-emergence of the plague in secular centers in the Maghreb unlike the northern shores of the Mediterranean where the indigenous plague has disappeared for a century.

Après trois pandémies majeures responsables de millions de morts, la peste n'a pas encore disparu. Cette maladie est causée par la bactérie Yersinia pestis, dont les mécanismes de virulence sont encore mal compris. Le suivi d'infection de la peste bubonique chez la souris, méthode classique pour étudier le processus infectieux, requiert beaucoup d'animaux et de temps pour obtenir des résultats significatifs. L'imagerie in vivo et en temps réel par bioluminescence permet de suivre la progression du pathogène au cours du processus infectieux en observant les animaux de façon non invasive. Nous avons transformé la souche virulente CO92 avec le plasmide pEm7-luxCDABE et confirmé la production de bioluminescence in vitro et in vivo. Nous avons pu quantifier la charge bactérienne dans plusieurs organes colonisés sans sacrifier l'animal et établir le schéma de progression de la bactérie au cours de la maladie. Après formation d'un foyer infectieux au site d'injection, la colonisation du ganglion lymphatique inguinal drainant ce site a été observée. Nous avons démontré que la bactérie suit un trajet direct du ganglion lymphatique inguinal au ganglion lymphatique axillaire. L'étape suivante est la colonisation des organes filtrant le sang, puis survient la septicémie dans les phases terminales de la mort. Nous avons établi que la forte variabilité dans le processus infectieux était due au temps pendant lequel la bactérie était contenue au site d'injection. À partir du moment où les ganglions lymphatiques sont colonisés, la cinétique de progression est à la fois régulière et rapide ; la septicémie survient dans les deux jours, suivie de près par la mort.

La peste est une zoonose touchant principalement les rongeurs et de façon accidentelle l'Homme. L'agent responsable de la peste bubonique et pulmonaire est la bactérie Gram négative Yersinia pestis. Les mécanismes qui permettent à l'hôte de résister ou non à cette infection sont mal connus. Nous avons montré que certaines lignées consanguines de souris, comme C57BL/6J, meurent après l'injection sous cutanée de 100 bactéries d'une souche virulente (CO92) alors que d'autres, comme SEG/Pas, dérivée de Mus spretus, résistent. Un croisement en retour entre ces deux lignées a permis d'identifier, sur les chromosomes 3, 4 et 6, trois QTLs contrôlant le taux de survie. Les deux premiers ont été retrouvés uniquement chez les femelles alors que celui du chromosome 6 est commun aux deux sexes. Des souris congéniques portant le chromosome 6 de SEG dans un fonds génétique C57BL/6J ont été produites. Après infection, elles meurent dans les mêmes proportions que les souris C57BL/6, mais un peu plus tardivement. Des souris bi- et tri-congéniques sont en cours de production pour tester l'effet des autres QTLs. Nous avons testé une collection de 55 lignées recombinantes congéniques interspécifiques entre SEG/Pas et C57BL/6J. Plusieurs lignées ont montré des différences de taux ou de durée de survie significatives par rapport à C57BL/6J. L'étude de la lignée 120G, qui meurt plus rapidement que C57BL/6J, suggère que la région proximale du chromosome 6 serait responsable de ce phénotype. Nous avons ainsi montré que le contrôle génétique de la résistance à la peste chez les souris SEG/Pas est complexe, et identifié plusieurs régions génomiques qui jouent un rôle important dans ce phénotype.

A vast number of studies have been completed on the virulence determinants of Yersinia spp.; however, the focus of many of these studies has been on the virulence plasmid and the plasmid-encoded Type three secretion system. Nevertheless, many chromosomal genes whose products are directly involved in virulence have also been identified. Some of these critical virulence determinants are outer membrane proteins. Outer membrane proteins of Gram-negative bacteria often have important physiological roles; however, some have also been found to be important for pathogenesis. In this thesis, we investigated two Yersinia. pestis outer membrane proteins, Ail and OmpA, and their roles in virulence. We provide evidence that Y. pestis Ail is a highly expressed outer membrane protein that is absolutely essential for Y. pestis to resist the killing action of the complement system present in human blood and tissues, as well as the blood and tissues of other mammalian hosts. Furthermore, Ail was important for virulence in a Y. pestis-Canorhabditis elegans model of infection.The work in this thesis also provided the first evidence that another surface-exposed outer membrane protein, termed OmpA, is required for both Yersinia pseudotuberculosis and Y. pestis to survive and proliferate intracellularly in macrophages. Finally, we provide evidence that Y. pestis has a functional small RNA MicA that controls the expression of OmpA. This is the first demonstration of sRNA-mediated regulation of a Yersinia virulence factor. This work has paved the way for future studies on the role of outer membrane proteins in virulence, particularly the role of Ail and OmpA.

<p>Lymph nodes are organs of efficiency. Once activated, they essentially function to optimize and accelerate the production of the adaptive immune response, which has the potential to determine survival of the host during an initial infection and protect against repeated infections, should specific and appropriate immunological memory be sufficiently induced. We now have an understanding of the fundamental structure of lymph nodes and many of the interactions that occur within them throughout this process. Yet, lymph nodes are dynamic and malleable organs and much remains to be investigated with regards to their responses to various types of challenges. In this work, we examined multiple inflammatory scenarios and sought to understand the complex ways that lymph nodes can be externally targeted to impact immunity. First, we outline a novel mechanism of cellular communication, where cytokine messages from the periphery are delivered to draining lymph nodes during inflammation. These signals are sent as particles, released by mast cells, and demonstrate the ability of the infected tissue to communicate to lymph nodes and shape their responses. Based on these interactions, we also explored the ability to therapeutically or prophylactically modulate lymph node function, using bioengineered particles based on mast cell granules, containing encapsulated cytokines. When we used these particles as a vaccine adjuvant, we were able to polarize adaptive immune responses, such as to promote a Th1 phenotype, or enhance a specific attribute of the immune response, such as the production of high avidity antibodies. We then explore three examples of lymph node-targeting pathogens: Salmonella typhimurium, Yersinia pestis and Dengue virus. Each of these pathogens has a well-characterized lifecycle including colonization of draining lymph node tissue. In the case of S. typhimurim, we report that the virulence this pathogen depends on a specific shut down of the chemotactic signals in the lymph node that are required to maintain appropriate cellular localization within it. Our results demonstrate that these architecture changes allow S. typhimurim to target the adaptive immune process in lymph nodes and contribute to its spread in vivo and lethality to the host. With Y. pestis, similar targeting of cellular trafficking pathways occurs through the modulation of chemokine expression. Y. pestis appears to use the host's cellular trafficking pathways to spread to lymph nodes in two distinct waves, first exploiting dendritic cell movement to lymph nodes and then enhancing monocyte chemoattractants to replicate within monocytes in draining lymph nodes. These processes also promote bacterial spread in vivo and we further demonstrate that blocking monocyte chemotaxis can prolong the host's survival. In the third example of pathogen challenge, we report for the first time that mast cells can contribute functionally to immunosurveillance for viral pathogen, here, promoting cellular trafficking of innate immune cells, including NK cells, and limiting the spread of virus to draining lymph nodes. For each of these three examples of lymph node targeting by microbial pathogens, we provide data that modulation of cellular trafficking to and within lymph nodes can drastically influence the nature of the adaptive immune response and, therefore, the appropriateness of that response for meeting a unique infectious challenge. Cumulatively this work highlights that a balance exists between host and pathogen-driven modulation of lymph nodes, a key aspect of which is movement of cells within and into this organ. Cytokine and chemokine pathways are an area of vulnerability for the host when faced with host-adapted pathogens, yet the lymph node's underlying plasticity and the observation that slight modulations can be beneficial or detrimental to immunity also suggests the targeting of these pathways with therapeutic intentions and during vaccine design.</p> / Dissertation

This thesis addresses gaps that exist in the theory and knowledge of ancient DNA (aDNA). Much of the underlying basis of the field has been neglected in the excitement that followed the first aDNA studies. Therefore the results of many studies have been based on untested assumptions about the nature of post mortem DNA damage, sample preservation, contamination, and the efficacy of sample decontamination techniques. The validity of such results is questionable if the assumptions prove false. Hydrolytic post mortem DNA damage may modify recovered aDNA sequences. This thesis reports new insights into the biochemical basis of, predisposition of certain sequences and nucleotide positions towards, and subsequent effects of, such damage. Parallels of post mortem damage with in vivo mutation also enable insights into DNA sequence evolution. The long-term survival of DNA, and contamination of samples with exogenous DNA are two related problems characteristic to aDNA. The survival of endogenous DNA within bone, teeth and hair samples, the susceptibility of such samples to contamination, and the efficacy of decontamination techniques used to remedy such problems are investigated. The results highlight serious flaws in using bone and teeth as a DNA source. In contrast, the results demonstrate that hair may present a valuable DNA source for future studies. Numerous studies have reported the retrieval of ancient pathogen DNA from human samples. Analyses of the DNA content within teeth extracted from putative victims of the 2^nd plague argue that such studies are at great risk from DNA degradation, and contamination arising due to environmental microorganisms. An extrapolation of these results using basic physical and chemical theory is used to evaluate the potential survival of aDNA in ancient Egyptian remains. This suggests that positive results from such samples are unlikely.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 6, 2008). Includes bibliographical references.

The lcrF gene of Yersinia pestis, the etiological agent of plague, encodes a transcription activator responsible for inducing expression of several virulence-related proteins (Yops) in response to temperature. The mechanism of this thermoregulation was investigated. Using a yopE::lacZ reporter fusion, lcrF-mediated thermal regulation was observed in Y. pestis and Escherichia coli. The lcrF gene was sequenced, the 30.8 kDa. LcrF protein identified and purified, and LcrF-dependent yopE-specific DNA binding activity was detected. A sequence similarity search revealed that LcrF exhibits 98% homology to VirF of Yersinia enterocolitica and significant homology to the carboxy termini of other members of the AraC family of transcription activators. During localization studies, a significant proportion of LcrF was found associated with the membrane fraction in E. coli. However, pulse-chase experiments indicated that this result is an artifact of fractionation. lcrF-mediated thermal induction of the yopE::lacZ reporter fusion remains intact in a Shigella flexneri virR mutant. The virR mutation is known to affect thermal induction of Shigellavirulence genes, which are also controlled by an activator in the AraC family. As a first step toward identifying the temperature-sensitive step in the regulation of yop expression, lcrF::lacZ transcriptional fusions were constructed and analyzed in Y. pestis and E. coli. The activity of the fusions was not affected by the native pCD1 virulence plasmid, an intact lcrF gene, or temperature. Thus, induction of lcrF transcription is not essential for temperature-dependent activation of yopE transcription. To confirm these results, attempts were made to identify both the native lcrF message in Y. pestis, and a lcrF-lacZ hybrid message in Y. pestis and E. coli. These attempts were unsuccessful. Examination of LcrF protein production revealed temperature-dependent expression in Y. pestis. Surprisingly, high-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at both 26 and 37°C, suggesting that translation rate or message degradation is thermally controlled. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37°C in E. coli indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented.