Made available in DSpace on 2015-10-21T12:19:30Z (GMT). No. of bitstreams: 2 rodrigo_godoy_ioc_mest_2012.pdf: 4319793 bytes, checksum: 12512310f752b37e87a767cc8d0a9bae (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-05-21 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Phlebotominae representa um dos grupos de insetos de grande importância médica, especialmente para o Brasil, estando associado à transmissão de agentes das leishmanioses tegumentar e visceral. A geração de conhecimentos sobre a morfologia e morfometria de ambos, machos e fêmeas de flebotomíneos vetores de leishmaniose tegumentar americana (LTA), poderão indicar estruturas diagnósticas importantes como subsídios para estudos filogenéticos que permitam avaliar a relação vetor -patógeno no processo de coevolução. Com isso o trabalho teve como objetivo analisar comparativamente as seguintes espécies de flebotomíneos: Nyssomyia intermedia, Ny. neivai, Ny. whitmani, Migonemyia migonei e Bichromomyia flaviscutellata, importantes vetores de Leishmania spp., causadores da LTA no Brasil. As análises morfométricas foram capazes de separar os gêneros. A análise comparativa dos flebotomíneos por morfometria geométrica e clássica, permitiu aprofundar a discussão sobre a posição taxonômica de Ny. intermedia e Ny. neivai, consideradas por alguns autores como espécies crípticas, onde houve diferença significativa em várias das estruturas analisadas, como no tamanho do centroide das asas e a razão entre os filamentos genitais e a ponta dos filamentos genitais. Através das análises de morfometria clássica e do tamanho dos centróides e as formas médias das asas, as espécies de Nyssomyia se apresentam mais próximas feneticamente de Mg. migonei, vetores de Leishmania (Viannia) braziliensis, do que de Bi. flaviscutellata, vetora de Leishmania (Leishmania) amazonensis, o que também corrobora com a separação dos gêneros Nyssomyia e Bichromomyia / Phlebotominae represents one group of insects of medical importance, especially in Brazil being associated with the transmission of agents of cutaneous and visceral leishmanias e s . The generation of knowledge about the morphology and morphometry of both male and female sandfly vectors of American c utaneous l eishmaniasis (ACL) may indicate structures as important diagnostic information for phylogenetic studies to assess the relationship of vector - pathogen coevolution . In this aspect the work was to analyze comparatively the following species of sandflies : Nyss omyia intermedia, Ny. neivai, Ny. whitmani, Migonemyia migonei and Bichromomyia flaviscutellata , important vectors of Leishmania spp ., causing leishmaniasis in Brazil. The morphometric analyzes were able to separate the genders. The comparative analysis of sandflies by classical and geometric morphometr y allowed us to discuss about the taxonomic position of Ny. intermedia and Ny. neivai , considered by some authors as cr yptic species , where significant differences were observed in several structures analyzed as the centroid size of the wings and the ratio between the genital filaments and the tip of the genital filaments . The classical morphometric analysis, size of the centroids of the wings as well as the mean forms of the wings showed Nyssomyia close st phe netically to Mg. migonei , vectors of Leishmania (Viannia) braziliensis , than Bi. flaviscutellata , vector of Leishmania (Leishmania) amazonensis , these results also corroborate with the separation of the genders Nyssomyia and Bichromomyia

Leishmania tropica ist der Auslöser von kutaner Leishmaniose beim Menschen und kommt in Afrika über den Mittleren Osten bis nach Nordindien vor. Mittels Mikrosatellitentypisierung (MLMT) wurde die weltweite Populationsstruktur dieser Spezies und der nahe verwandten Spezies L. aethiopica aufgedeckt, indem sowohl Methoden angewandt wurden, die auf genetischen Distanzen beruhen als auch solche, die auf der Analyse von Allelfrequenzen basieren. Die 195 Stämme von L. tropica sowie die acht Stämme von L. aethiopica gruppierten hauptsächlich gemäß ihrer geographischen Herkunft. Die Stämme von L. aethiopica stellten eine eigene Gruppe dar, die allerdings innerhalb der afrikanischen Stämme von L. tropica gruppierte. Vorläufige Ergebnisse einer genomweiten SNP-Analyse haben die Ergebnisse der Mikrosatellitenanalyse weitgehend bestätigt. Um die Gründe für die hohe genetische Variabilität innerhalb der Spezies L. tropica herauszufinden, wurde eine Funktionelle Klonierung durchgeführt, in der N-methyl-N’-nitro-N-nitrosoguanidin (MNNG) als Indikator für ein funktionierendes oder eingeschränktes Mismatch Repair (MMR)-System eingesetzt wurde. Dafür wurde ein Akzeptorstamm (hohe MNNG-Toleranz) mit einer Cosmidbibliothek, die genomische DNA eines Donorstammes (niedrige MNNG-Toleranz) enthielt, transfiziert. Die erhaltenen Transfektanten wurden dann auf ihre MNNG-Toleranz getestet. Die zeit- und kosteneffiziente Identifizierung von großen Mengen an Sandmücken ist wichtig für Feldstudien, in denen mehrere Tausend Mücken gefangen werden. Hier wird eine multiplexe Technik zur ligationsabhängigen Sondenamplifizierung vorgestellt, die die Identifizierung von Sandmücken-Spezies im Mittleren Osten ermöglicht. Die Spezies Phlebotomus syriacus, P. arabicus und P. papatasi können durch diese Methode mit spezies-spezifischen Sonden eindeutig identifiziert werden. Außerdem kann diese Methode dazu genutzt werden, weitere Spezies zu diskriminieren und auf gepoolte Sandmücken angewandt werden. / Leishmania tropica is the causative agent of human cutaneous leishmaniasis in foci ranging from Africa through the Middle East to northern India. By multilocus microsatellite typing (MLMT), the world-wide population structure of this species and its closely related species L. aethiopica has been revealed applying methods based on both genetic distances and allele frequencies. The 195 strains of L. tropica and eight strains of L. aethiopica largely clustered according to their geographical origins. The strains of L. aethiopica formed a distinct group, although clustering among other African strains of L. tropica. Preliminary data obtained through a whole genome sequencing approach including strains of L. tropica, L. aethiopica and L. major have largely corroborated the results of the MLMT approach. To reveal the reasons for the high genetic variability among strains of L. tropica, a Functional Cloning approach was conducted using N-methyl-N’-nitro-N-nitrosoguanidin (MNNG) as an indicator for a functioning or impaired mismatch repair (MMR) system. The transfectants retrieved from the transfection of an acceptor strain exhibiting high MNNG tolerance with a cosmid library bearing the genomic DNA of a donor strain with a reduced MNNG tolerance were screened for the restored phenotype of the donor strain. The time- and cost-efficient identification of a large amount of sand flies is important since several thousands are caught during field studies. Here, a multiplex ligation-dependent probe amplification approach (MLPA) for the identification of sand flies endemic to the Middle East is introduced. The unambiguous identification of Phlebotomus syriacus, P. arabicus and P. papatasi was possible with this approach using species-specific probes. Furthermore, this technique has the potential to discriminate more species and to be applied to pooled sand fly specimens.