The aim of this thesis was to examine the archeological material from medieval site in Prague and identify human or animal intestinal parasite eggs or cysts. The concetration methods were used for parasite identification and polymerase chain reaction (PCR) was used for detection of parasitic protist DNA. In comparision with the results of most of similar research, the occurrence of parasites was low.

Made available in DSpace on 2011-05-04T12:42:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2010 / Ascaris lumbricoides e Trichuris trichiura são os helmintos intestinais de maior prevalência na população mundial e também no material arqueológico. Porém, na América do Sul pré-colombiana, o encontro de ovos de A. lumbricoides é raro. Recentemente um estudo de diagnóstico paleoparasitológico molecular apontou para um sub-diagnóstico de Ascaris sp. na América do Sul. No registro arqueológico de parasitos intestinais predominam achados de ovos de Trichuris sp. ao invés de Ascaris sp. Isto parece contraditório, em virtude do número de ovos eliminados por cada parasito. Os objetivos desta pesquisa foram: avaliar marcadores moleculares para o diagnóstico de Ascaris sp. e Trichuris sp. em material moderno pela caracterização molecular destes parasitos; caracterizar geneticamente isolados de sítios arqueológicos sul americanos para verificar a real paleodistribuição destes parasitos em uma perspectiva paleoepidemiológica e compará-la com a epidemiologia moderna destas infecções; avaliar os fatores envolvidos na paleodistribuição encontrada. As amostras foram submetidas ao diagnóstico por microscopia óptica, seguida da extração do DNA, PCR e sequenciamento nucleotídico. Na avaliação dos marcadores moleculares, a região ITS1 de Ascaris sp. apresentou variação intra-indivíduo, o que descartou seu uso com fins taxonômicos e diagnósticos. A caracterização molecular dos genes mitocondriais cox1 e nad1 de Ascaris sp. mostrou infecção cruzada de genótipos entre as espécies humana e suína, o que denota a necessidade de monitoramento das populações avaliadas assim como de outras regiões brasileiras para que a infecção não venha a se tornar uma zoonose em potencial no Brasil. Foi possível o diagnóstico molecular de Trichuris sp. pelo gene ribossomal 18S DNA. A análise paleogenética mostrou que há subdiagnóstico para ambas as infecções na América do Sul pré-colombiana. Este é o primeiro diagnóstico paleoparasitológico molecular de T. trichiura em material sul americano. Estes são também os primeiros registros de recuperação de DNA de parasitos intestinais em material de sítio arqueológico do tipo “sambaqui” e também do período colonial brasileiro. Comparando-se a paleoepidemiologia molecular de Ascaris sp. com a epidemiologia molecular moderna foi possível notar que há haplótipos antigos que ainda estão presentes hoje, no entanto a maioria dos haplótipos é característica ao material arqueológico. Observou-se que há haplótipos comuns ao Velho e Novo Mundo, contudo, há também especificidades regionais. Os resultados da análise genética claramente apontam para uma pobre preservação dos ovos no material arqueológico, principalmente de Ascaris sp. Os fatores principais envolvidos nessa paleodistribuição, seriam fatores tafonômicos que proporcionaram a quebra maior de ovos de Ascaris sp. do que de Trichuris sp., e evidências de consumo de plantas vermífugas pelos povos pré-históricos, as quais teriam maior ação sobre Ascaris sp. do que Trichuris sp. / Ascaris lumbricoides and Trichuris trichiura are the intestinal helminths with higher prevalence in the world today as it was in the past. However, in pre-Columbian South America the findings of A. lumbricoides eggs are rare. Recently a study of paleoparasitological molecular diagnosis showed a sub-diagnosis of Ascaris sp. in South America. In the archeological material, eggs of Trichuris sp. are more common compared with Ascaris sp. eggs. This is contradictory taking into account the number of eggs eliminated by each parasite. The aims of this research was: to evaluate molecular markers for Ascaris sp. and Trichuris sp. diagnosis in modern material; genetic characterization of the samples South American archeological sites aiming the paleodistribution of these parasites in a paleoepidemiological perspective; compare results with the modern epidemiology of these infections; evaluate the factors involved in paleodistribution. Extraction of DNA, PCR and nucleotide sequencing were performed after microscopy. In the evaluation of the molecular markers Ascaris sp. ITS1 region showed intra-individual variation. Therefore, this region to taxonomical and diagnoses studies was discarded. With the molecular characterization of Ascaris sp. cox1 and nad1 mitochondrial genes it was possible to identify cross infection of genotypes between human and pig hosts. Results showed that surveillance field works in modern populations are necessary to verify the zoonotic potential of this infection in Brazil. The molecular diagnosis of Trichuris sp. by ribossomal 18S DNA gene was possible. The paleogenetic analysis showed that there is subdiagnosis for both infections in pre-Columbian South America. This is the first paleoparasitological molecular record of T. trichiura in South American samples. These are also the first recovery of DNA of intestinal parasites in "sambaqui" archeological site, and also of the Brazilian colonial period. Molecular paleoepidemiology of Ascaris sp. infection compared with modern molecular epidemiology showed that there are ancient haplotypes still present today. However, most of the haplotypes are characteristic of the archaeological material. It was observed that there are common haplotypes both to the Old World and to the New World, but showing regional specificities. The results of the genetic analysis clearly pointed to a poor preservation of eggs in archeological material, mainly of Ascaris sp. Taphonomy may be the main factor involved in paleodistribution, breaking more eggs of Ascaris sp. than Trichuris sp. Evidences of consumption of vermifuge plants by prehistoric groups should also have influence, as some plants should have more efficacy eliminating Ascaris sp. than Trichuris sp.

The data presented within this thesis shows the generation and characterisation of a complete-, macrophage/granulocyte- and T cell-specific IFNγR2 deficient mouse mutant. This mutant mouse is a valuable tool in dissecting the mechanism of action of the pleiotrophic cytokine IFNγ.The global mutant mouse was tested in three models in vivo - DSS induced colitis, Trichuris muris infection and EAE. The aim of the DSS-induced colitis model was to test the role of IFNγ in the innate immune system and, despite previous reports demonstrating IFNγ deficient mice are protected from DSS-colitis, our IFNγR2 deficient mice displayed equal or more severe colitis than control mice. We hypothesise that this discrepancy is due to differences in the gut microbiota.The Trichuris muris model was utilised as a method of examining the role of IFNγ in the adaptive immune system. The complete IFNγR2 mutant was resistant to a low dose T. muris infection; however, neither the T cell specific nor the macrophage/granulocyte specific mutant duplicated the resistant phenotype observed in the global knock-out mice. Analysis of a double conditional T cell and macrophage/granulocyte specific IFNγR2 mutant produced inconsistent results. Initial experiments suggested that, in combination, these deficiencies are sufficient to duplicate the resistant phenotype observed in the global mutant mice, but this was not reproducible.The final in vivo model that we used to analyse IFNγR2 mutant mice was EAE. This model was chosen as, for a long time, the mechanism of action and the involvement of IFNγ in EAE has been a matter of uncertainty. These results demonstrated that global IFNγR2 mutant mice demonstrate an atypical phenotype, with no signs of recovery. In contrast, control mice develop classical EAE symptoms with almost complete recovery prior to the termination of the experiment. The IFNγ receptor mutant mouse generated will be of great value to the scientific community as IFNγ has been demonstrated to play a role in multiple diseases and this tool allows the mechanism of action of this cytokine to be unravelled.

De nombreuses espèces de rongeurs montrent des variations cycliques de leurs densités. Ces cycles ont un rôle important dans l'émergence de zoonoses en augmentant les contacts entre l'Homme et l'animal. Cette thèse s'inscrit dans le domaine de l'écologie de la santé qui étudie les interactions entre santé humaine, santé animale et dynamique des écosystèmes. Dans ce cadre, j'ai étudié la communauté de parasites d'une communauté de rongeurs à dynamique cyclique afin d'identifier les réservoirs d'agents de zoonoses et les parasites potentiellement impliqués dans les cycles de cette communauté. Je me suis intéressée à trois espèces de campagnols et à deux espèces de mulots en Franche-Comté. Les résultats et les suivis épizootiologiques réalisés permettent d'inférer les facteurs de risques biotiques et abiotiques associés à l'émergence de ces zoonoses.<br />Trois agents de zoonoses sont présents : deux hantavirus (virus Puumala et Tula) et le virus Cowpox. La dispersion et le comportement social des rongeurs sont importants pour la transmission de ces virus spécifiques (hantavirus) et non spécifique (virus Cowpox). Ces virus sont majoritairement trouvés en milieu forestier. Les communautés de parasites détectées en forêt et en prairie sont différentes. Les infestations par des helminthes sont plus nombreuses en prairie qu'en forêt. Une étude immunogénétique montre l'existence d'allèles de susceptibilité et de résistance aux agents de zoonoses étudiés. Des helminthes ou des acariens pourraient aussi intervenir dans l'infection par ces virus. Un de ces helminthes pourrait être impliqué dans la dynamique de ses hôtes. Mes travaux expérimentaux et de modélisation montrent l'impact du nématode non spécifique Trichuris arvicolae sur la reproduction du campagnol des champs et son rôle régulateur pour les populations d'arvicolinés. <br />Cette thèse contribue à montrer l'importance de la biodiversité et de l'écologie des communautés pour évaluer et gérer les risques pour l'Homme vis-à-vis de zoonoses.

The aim of this thesis was to search for and identify human and/or animal intestinal parasite eggs in archeological material from medieval period in Brno and Prague. Concentration methods - sedimentation and flotation were used for parasite identification. Polymerase chain reaction (PCR) was used for the detection of parasite DNA. The results were put into context with other data published in similar studies and research.

IL-1 is a pro-inflammatory cytokine which play an important role in the activation and regulation of host defence and immune responses to inflammation or injury. IL-1 is able to bind and activate IL1-RI and IL1-RII, which are found on many cells types. The role of the IL-1 signalling in the deployment of Th cell subsets, especially Th17 cells is well known. However, the specific cells which are responsible for the expression of IL-1 signalling in the immune defense and in the development of the Th cell lineage in response to infection, is still largely unclear. Therefore in this thesis, IL1-RI conditional knockout mice specifically in hematopoietic cells (IL1-RI vaviCre+) were generated. Using IL1-RI vaviCre+ mice in comparison with IL1-RI global knockout mice (IL1-RI-/-) would determine whether the expression IL-1 signalling from hematopoietic cells is responsible for the immune defense and in the development of the Th1, Th2 and Th17 cells against gastrointestinal helminth Trichuris muris (T.muris) infections. The generation of IL1-RI vaviCre+ mice have been investigated at the genomic and proteomic level in order to confirm that the Il1-rI gene is inactivated in hematopoietic cells. The characterisation of IL1-RI vaviCre + mice at the genomic level confirmed that the Il1-rI gene was obliterated successfully. At protein level the characterisation of IL1- RI vaviCre + mice confirmed that IL1-RI was dysfunctional in hematopoietic cells. Additionally, the development of the immune cells was investigated in IL1-RI vaviCre + and IL1-RI-/- mice. Our findings demonstrated that the lymphocyte development was not affected by the deletion of the IL1- RI gene. This data indicated that IL1- RI vaviCre + and IL1-RI-/- mice are vital in vivo models. In high dose infection, both IL1-RI vaviCre + and IL1-RI -/- mice were able to clear the infections due to their ability to generate a Th2 response. Both IL1-RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris were susceptible to infections and showed high levels of Th1 cytokines. Thus, we hypothesised that IL1-RI signalling in hematopoietic cells was not required for worm expulsion and the generation of Th2 and Th1 response. Interestingly, low dose T.muris infection showed a clear reduction in the Th17 cytokines IL22 and IL17 in both IL1-RI vaviCre + and IL1-RI -/- mice, suggesting that IL-1 signalling expressed from hematopoietic cells is responsible for the development of Th17 cells and secretion of IL17 and IL22. IL1- RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris also showed an increase in inflammation in the colon and decreased of goblet cell hyperplasia. It is well known that IL22 plays an important role in preventing tissue damage and repair. Thus, in this study IL22 global knockout mice (IL22 -/-) were used to determine if the change in crypt lengths and goblet cell hyperplasia in IL1-RI vaviCre + and IL1-RI -/- was due to an absence of IL22. Our finding showed that IL22 -/- mice infected with low dose of T.muris had increased crypt length and a reduction in goblet cells. The similar phenotype in crypt length and goblet cell hyperplasia between IL22 -/-, IL1-RI vaviCre + and IL1-RI -/- mice suggested that a lack of IL22 in IL1-RI vaviCre + and IL1-RI -/- mice is responsible for the change in mice phenotype. It also provides more evidence for the role of IL-1 signaling in hematopoietic cells in the generation of Th17 cells and in the production of its cytokine IL22.IL1-RII is an inhibitor of IL1-RI, thus, in this study IL1-RII global knockout mice (IL1-RII -/-) mice was used in comparison with IL1-RI -/- mice to verify the role of IL-1 signaling in the development of Th17 cells. Our finding showed an overexpression of IL17 and IL22 in IL1-RII -/- compared with IL1-RI -/- mice and a higher level of IL17 in IL1-RII -/- mice compared with IL1-RII flox/flox mice. This data confirmed that IL-1 signaling is important for the development of Th17 cells and the production of its cytokine IL17 and IL22.