Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-06T11:37:49Z No. of bitstreams: 1 2016 - Gabriel Alves Landulfo.pdf: 2085230 bytes, checksum: a7f7c364b23211ad81d1168cb1f6db85 (MD5) / Made available in DSpace on 2017-01-06T11:37:49Z (GMT). No. of bitstreams: 1 2016 - Gabriel Alves Landulfo.pdf: 2085230 bytes, checksum: a7f7c364b23211ad81d1168cb1f6db85 (MD5) Previous issue date: 2016-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also aggressive towards humans. It inhabits some countries in the Neotropical region. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to ascertain the main molecules expressed in the gut of argasid ticks after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon. Sixty females were fed and dissected to extract the gut tissue. The transcriptome was obtained through pyrosequencing and the de novo assembly method on mRNA of the gut tissue. We identified 2,235 contigs, of which 1,729 matched database sequences, while 506 did not present any hits. The transcripts were annotated and grouped according to their biological function. Catalytic, binding and transporter activity were the most representative functions, accounting for 780, 709 and 106 contigs, respectively. The transcripts were classified into 31 categories, using both bioinformatics and data curation practices. The most representative categories were, respectively, unknown, catalytic activity and transporter channels. One hundred and three (103) digestives transcritps associated to digestion of proteins (67), carbohydrates (19) and lipid (17) were identified in the transcriptome analysis. Peptidases associated with hemoglobin digestion, such as serine, cysteine, aspartic protease and metalloenzymes, were identified in the gut of the engorged females. Genes associated with transport (hemelipoglycoprotein) and storage (ferritin) of nutrients resulting from hemoglobin digestion, such as heme, were also found in the digestive tract. The presence of a cathepsin O-like cysteine peptidase was recorded in ticks, for the first time. Two thousand and two hundred thirteen (2213) transcripts were deposited to the Transcriptome Shotgun Assembly (TSA) portal of the NCBI.The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. Genes for cathepsin L in O. mimon appear to have diverged from other more common recent ancestors. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on different datasets, such as mitochondrial genome and nuclear rRNA sequences. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity / Ornithodoros mimon ? um carrapato argas?deo parasita de quir?pteras, aves e marsupiais, al?m de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de mol?culas vitais no processo digest?o e na rela??o parasito-hospedeiro, al?m de fornecer tamb?m informa??es sobre a evolu??o dos artr?podes hemat?fagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais mol?culas expressas no intestino de uma esp?cie de carrapato argas?deo ap?s o repasto sangu?neo, atrav?s de uma an?lise transcrit?mica do intestino de f?meas ingurgitadas de O. mimon. Sessenta f?meas foram alimentadas e dissecadas para coleta o tecido intestinal. O RNAm da amostra do intestino foi extra?do, purificado e quantificado. Esse serviu de molde para s?ntese do cDNA, que foi utilizado no pirosequenciamento. O transcritoma foi obtido atrav?s do m?todo de montagem de novo do cDNA do tecido intestinal. Identificou-se 2235 sequ?ncias consensos (contigs) ou transcritos, dos quais 1729 apresentaram similaridade (hit) com sequ?ncias dos bancos de dados, enquanto que 506 n?o tiveram nenhuma similaridade. Os transcritos foram anotados e agrupados conforme as fun??es biol?gicas atribu?das as eles no processo de anota??o g?nica. Atividade catal?tica, ades?o e transporte foram as fun??es mais representativas com 780, 709 e 106 transcritos, respectivamente. Em uma an?lise n?o automatizada, os transcritos foram subcategorizados em 31 categorias. As categorias mais representativas foram desconhecido, atividade catal?tica e transportadores-canais. Identificamos 103 transcritos digestivos associados ? digest??o de prote?nas (67), carboidratos (19) e lip?dios (17). Proteinases das classes serino, ciste?ne, asp?rtica e metalo representaram as enzimas atuantes na digest?o intracelular do constituinte prote?co do repasto sangu?neo. Genes associados com o transporte (hemelipoglicoprote?na) e estocagem (ferritina) dos nutrientes resultantes da digest?o foram encontrados bem expressos no trato digestivo. Registrou-se pela primeira vez a presen?a de uma ciste?na peptidase do tipo catepsina O em carrapatos. Foram depositados no banco de dados g?nico p?blico 2213 transcritos de O. mimon. A an?lise filogen?tica das peptidases revelou que a maioria das proteinases de O. mimon ? pr?xima aos genes codificadores de proteinases de carrapatos. Transcritos de catepsinas L de O. mimon parecem ter divergido de ancestrais recentes diferentes. A infer?ncia filogen?tica baseada em conjunto de dados transcritos hom?logos tem uma resolu??o topol?gica similar a de outros conjuntos de dados, como genoma mitocondrial e sequ?ncias nuclear de RNA riboss?mico (rRNA). Os achados obtidos no presente estudo podem contribuir para compreens?o dos importantes processos dos carrapatos argas?deos, como digest?o, nutri??o e imunidade, al?m de fornecer informa??es sobre a filogenia dos carrapatos.

Orientador: Arício Xavier Linhares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:41:06Z (GMT). No. of bitstreams: 1 Franco_CarolineSiqueira_M.pdf: 1657929 bytes, checksum: 0b27568a919b921d3ae68ae3161f87fd (MD5) Previous issue date: 2014 / Resumo: A febre maculosa brasileira (FMB) é uma doença infecciosa aguda transmitida através da picada de carrapatos infectados com Rickettsia rickettsii Brumpt 1922. O carrapato Amblyomma cajennense Fabricius 1787 (Acari: Ixodidae) é um dos principais vetores no Brasil. Contudo, outras espécies de carrapatos podem ser vetores da doença e parasitar humanos. Neste estudo, procurou-se avaliar a competência vetorial de Ornithodoros mimon Kohls 1969 (Acari: Argasidae) em laboratório após ser infectado experimentalmente com a cepa Taiaçu de Rickettsia rickettsii, utilizando coelhos domésticos como modelo animal. Primeiramente, os carrapatos foram separados em grupos de acordo com seu estágio de desenvolvimento (larva, ninfa 2 e adulto). Na segunda etapa, dois coelhos foram utilizados como grupo tratamento, e foram inoculados com a Rickettsia, e um coelho foi usado como controle. Os carrapatos utilizados foram separados em lotes, de acordo com o coelho e dia em que foram alimentados. Na terceira etapa, novos coelhos sadios foram infestados com os carrapatos usados na segunda etapa, para verificar se houve transmissão. Carrapatos que tiveram contato com a bactéria foram macerados e inoculados em duas cobaias, e uma terceira cobaia foi infestada com carrapatos que foram alimentados em coelho infectado. PCR com os primers gltA e ompA (R. rickettsii), ftsZ (Wolbachia sp.), 28S (eucarioto) foi realizado, para avaliar se os carrapatos se tornaram infectados. Sequenciamento de amostras foram amplificadas com os primers ompA e 28S. Além da Reação de Imunofluorescência Indireta (RIFI), que foi realizada com o soro dos coelhos para verificar a presença de infecção. Os dois coelhos que foram inoculados com Rickettsia rickettsii apresentaram picos de febre. Um dos coelhos apresentou resultado positivo na RIFI e o outro morreu no décimo sexto dia após a inoculação. Os coelhos que foram infestados com carrapatos que foram alimentados em coelho infectado não apresentaram febre e nenhum outro sintoma da doença. As cobaias que foram inoculadas com carrapatos e a cobaia que foi infestada com carrapatos não demonstraram nenhum sintoma da doença. Das amostras amplificadas com o primer gltA apenas uma amostra foi positiva. A PCR das amostras de carrapatos usando o primer ompA não apresentou o resultado esperado, provavelmente devido a presença do DNA da bactéria Wolbachia sp. A sequência gerada, com amostras que foram amplificadas com o primer 28S, foi o DNA do carrapato. A PCR com o primer ftsZ não amplificou nenhum DNA. Portanto, conclui-se que O. mimon não se tornou infectado com a Rickettsia rickettsii, e não representa risco para a saúde pública / Abstract: The Brazilian Spotted Fever (BSF) is an acute infectious disease transmitted by the bite of ticks infected with Rickettsia rickettsii Brumpt 1922. The tick Amblyomma cajennense Fabricius 1787 (Acari: Ixodidae) is one of the main vectors in Brazil. However, other species of ticks can be vectors of this disease and parasitize humans. This study aimed to evaluate the vector competence of Ornithodoros mimon Kohls 1969 (Acari: Argasidae) in the laboratory after being experimentally infected with the Taiaçu strain of Rickettsia rickettsii, using domestic rabbits as the animal models. Firstly, the ticks were separated into groups according to their developmental stage (larva, nymph 2 and adult). Secondly, two rabbits were used as treatment group, and were inoculated with the Rickettsia, and one rabbit was used as control. The ticks were separated into batches according to the rabbit and the day they were fed. New healthy rabbits were infested with the ticks used in the second experiment, to verify the occurrence of transmission. Ticks that had contact with the bacteria were macerated and inoculated into two guinea pigs, and a third guinea pig was infested with ticks that were fed on infected rabbits. PCR with gltA e ompA primers (R. rickettsii), ftsZ (Wolbachia sp.), 28S (eukaryotic) was performed, to assess if the ticks become infected. Sequencing of the samples was amplified with the primers ompA and 28S. In addition, the Immunofluorescence Assay (IFA) was performed with serum of rabbits to verify the presence of infection. The two rabbits that were inoculated with Rickettsia rickettsii presented peaks of fever. One rabbit presented a positive result in the IFA and the other died on the sixteenth day after inoculation. The rabbits that were infested with ticks that were fed on infected rabbits, showed no fever and no other symptoms of the disease. The guinea pigs that were inoculated with ticks and the guinea pig that was infested with ticks showed no symptoms of the disease. Of the samples only one of all samples amplified with the primer gltA was positive. The PCR samples of ticks using the primer ompA did not yield the expected result, probably due to the presence of the DNA of the bacteria Wolbachia sp. The sequence generated, with samples that were amplified with the primer 28S, it was the DNA of the tick. PCR performed with the primer ftsZ did not amplify any DNA. Therefore, we concluded that O. mimon did not become infected with R. rickettsii, and do not represent risk to public health / Mestrado / Relações Antrópicas, Meio Ambiente e Parasitologia / Mestra em Biologia Animal

Please read the abstract in the section 00front of this document / Thesis (PhD (Biochemistry))--University of Pretoria, 2002. / Biochemistry / unrestricted

Thesis (M. Sc.)(Biochemistry)--University of Pretoria, 2008. / Includes bibliographical references. Available on the Internet via the World Wide Web.

Thesis (Ph.D.)(Biochemistry)--University of Pretoria, 2005. / Title from opening screen (viewed March 28, 2006). Includes summary. Includes bibliographical references.

Invertebrates do not possess an adaptive immune system, but rely on several mechanisms similar to the innate immune system of mammals. The synthesis and release of a host of potent antimicrobial proteins is an important component of this immune response. The antibacterial activity in the hemolymph of Ornithodoros savignyi is specific for Gram-positive bacteria, and the synthesis and release of the antibacterial factors need to be induced by challenging the ticks with heat-killed Gram-negative bacterial suspensions. The induction of the factors is very rapid, leading to a maximal response within one hour following bacterial challenge. The factors are stable at high temperatures, and were found to be protein in nature. By using reverse phase high performance liquid chromatography, four fractions exhibiting antibacterial activity were identified in the hemolymph of immune challenged ticks. Four antibacterial peptides were isolated from these fractions, and the mass analyses of the peptides indicate that there are at least two different antibacterial peptides present in the hemolymph. The N-terminal amino acid sequence of one of the peptides was determined, and the analysis showed that the peptide has high homology with defensin peptides isolated from other tick species. This led to the putative classification of the peptides as part of the invertebrate defensin family. The presence of lysozyme in O. savignyi was studied using molecular biological methods. Vertebrate and invertebrate lysozyme sequences were used to design a lysozyme-specific primer, which was used to amplify specific DNA products from whole tick cDNA using the polymerase chain reaction (PCR). The conditions for the amplification reaction were optimized, the products of the optimized reaction were cloned into a cloning vector and the nucleotide sequences of the products were determined. The nucleotide sequences were used for similarity searches of sequence databases to determine homology with sequences of known proteins. It is deduced the degenerate primer was not specific for lysozyme and did not playa significant role in the amplification of the PCR products. This method is thus not feasible for the investigation of the lysozyme of O. savignyi. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

Les interactions intimes et répétées entre hôtes et parasites peuvent engendrer la spécialisation d’un parasite à son hôte, grâce à des adaptations comportementales, morphologiques et/ou génétiques, combinées avec un flux de gènes limité. C’est un processus clef car il participe à l’évolution de la biodiversité parasitaire et peut ainsi permettre de mieux comprendre l’émergence d’organismes pathogènes. Encore peu étudié, une spécialisation d’hôtes a néanmoins été démontré lors de précédentes études chez deux espèces de tiques nidicoles : chez Ixodes uriae une tique dure, parasite des oiseaux marins coloniaux en zone arctique, et dans un complexe de tiques molles Ornithodoros capensis sensu lato, parasitant aussi de nombreuses espèces d’oiseaux marins, mais cette fois-ci en zones tempérées et tropicales. Ces deux espèces sont vectrices d’une grande diversité d’agents pathogènes incluant des virus, des bactéries et des protozoaires. Cependant, les facteurs impliqués dans le phénomène de spécialisation d’hôte restent inconnus. Dans ce cadre, le but de ma thèse était donc de déterminer 1) si l’évolution des divergences en fonction des hôtes est toujours accompagnée par les mêmes changements phénotypiques et 2) si ces changements pourraient permettre d’identifier les facteurs de sélection sous-jacents. Dans ce contexte, des campagnes d’échantillonnage de tiques ont été menées durant la période de reproduction des hôtes oiseaux dans les différentes zones de leur répartition et nous avons réalisé des analyses morphométriques, basées sur l’utilisation de landmarks et de contours sur chaque individu tique et des analyses phylogénétiques et génétiques des populations sur les mêmes individus. L’ensemble de ces résultats suggère la présence de convergences morphologiques au sein de ces systèmes et souligne un rôle de la sélection dans ce processus de divergence. En effet, les caractéristiques écologiques des hôtes mais aussi le micro-habitat exercent des pressions sélectives importantes dans ces deux systèmes pouvant être à l’origine de la divergence observée entre les populations. De plus, les caractéristiques biologiques de chaque espèce de tiques, telle que la capacité de dispersion, entrent également en jeu et peuvent fortement modifier l’épidémiologie des agents infectieux dont elles sont vectrices.Mots clés : Argasidae, écologie de la transmission, évolution convergente, interactions hôte-parasite, Ixodidae, oiseaux marins. / Intimate and repeated interactions between hosts and parasites can lead to parasite specialization to a given host via behavioral, morphological and/or genetic adaptations that act in combination with restricted gene flow. Specialization is a key process leading to the generation of parasite biodiversity and can help us understand the emergence of pathogenic organisms. Although little studied, host specialization has already been demonstrated to occur in previous studies of two nidicolous tick species: Ixodes uriae a hard tick parasitizing colonial seabirds in polar regions, and soft ticks of the complex Ornithodoros capensis sensu lato, that also exploit colonial seabirds, but this time in temperate and tropical zones. Both of these species act as vector to a wide variety of pathogenic organisms, including viruses, bacteria and protozoa. However, the factors involved in host specialization remain unknown. In this context, the aim of my thesis was to determine 1) whether the evolution of host specialization is always accompanied by the same phenotypic changes and 2) whether these changes could help to identify the selective factors that influence this phenomenon. In this context, tick collections were conducted during the breeding period of the host birds in different areas of their distribution and morphometric analyses, based on landmark and contour methods, were performed on each individual tick. Phylogenetic and population genetic analyses were also carried out using the same individuals. Overall, the results demonstrate that morphological convergence occurs within these systems, highlighting the role of selection in the divergence process. Indeed, the ecological characteristics of the hosts, but also their micro-habitat, may exert significant selective pressures on ticks and may cause the observed divergence among populations. Likewise, the biological characteristics of each tick species, particularly in relation to dispersal capacity, may also come into play and will greatly modify the epidemiology of associated infectious agents.Keywords: Argasidae, convergent evolution, host-parasite interactions, Ixodidae, transmission ecology, seabirds.