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Every year over two hundred million cases of malaria occur worldwide causing human death and suffering often in the poorest countries. Most people who die from malaria are children under five years of age. Malaria is caused by parasites spread by mosquitoes when they feed on human blood. Currently prevention methods include insecticides and anti-malarial drugs. The problem with both is the increasing resistance towards them by mosquitoes and parasites, respectively. Therefore other approaches need to be investigated to find new solutions to this problem. One such research area is paratransgenesis, the genetic modification of symbiotic microorganisms in the mosquitoes to produce anti-malaria parasite molecules. One bacterium identified as a potential candidate for paratransgenesis is Thorsellia anophelis. When this study started, only one Thorsellia isolate existed in the world. The aim of this study was therefore to retrieve more Thorsellia isolates from Kenyan mosquito and water samples. The samples were screened by PCR followed by bacterial culturing of positive samples, which resulted in 38 new Thorsellia isolates confirmed by DNA sequencing. The isolation of new Thorsellia species enables further investigation of the potential for their use in paratransgenesis with the aim of contributing to the prevention of malaria transmission.

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-03Bitstream added on 2014-06-13T18:41:12Z : No. of bitstreams: 1 caperucci_d_dr_rcla.pdf: 4394663 bytes, checksum: d9193aa7506e45c78a130332f4fb392f (MD5) / Este estudo trouxe a análise morfológica, histológica, ultraestrutural e citoquímica do intestino médio de fêmeas de Amblyomma cajennense e mostrou que esse órgão passa, por grandes modificações à medida que o processo de alimentação progride, tornando-se acentuadas nos indivíduos reinfestados. Do intestino médio de A. cajennense, partem dois pares de divertículos que se ramificam totalizando 8 ramos. Naquelas em jejum a porção anterior do intestino médio é enrugada devido à forte musculatura. Em algumas regiões do intestino médio, estruturas, descritas pela primeira vez, foram denominadas de nódulos, resultado dos espaços presentes entre as fibras musculares ao redor do intestino. Á medida que progridem as infestações a superfície dos divertículos em jejum, semi e ingurgitados de 1ª e 2ª infestações enruga-se extremamente estando nas semi de 3ª infestação menos enrugada e ingurgitadas de 3ª infestação mais lisa. A histologia do intestino médio em jejum mostrou a presença de epitélio pseudo-estratificado com células: generativas e digestivas. Nas semi e ingurgitadas de 1ª infestação e semi de 2ª infestação o epitélio sofre estratificação. Nas semi de 2ª infestação observou-se no epitélio a presença do terceiro tipo celular: a secretora. Nas ingurgitadas de 2ª infestação e nas semi e ingurgitadas de 3ª infestação o epitélio se desorganizou. No lúmen do intestino das semi submetidas à 1ª infestação, observou-se esferas membranosas; com estruturas fortemente coradas no interior. Outras com seu interior preenchido apenas por restos de citoplasma. A desorganização no epitélio intestinal de fêmeas fixadas em hospedeiros reinfestados sugeriu que os anticorpos liberados pelo hospedeiro contra o intestino dos carrapatos tenham agido nestes. A histoquímica detectou pouca proteína no epitélio intestinal daquelas em jejum... / This study showed the morphological, histological, ultrastructural and cytochemical analysis of the medium intestine of Amblyomma cajennense females since this organ goes through significant modifications as the feeding process progresses and these modifications are stronger in reinfested individuals. Two pairs of diverticulum emerge from A. cajennense medium intestine and are divided in eight branches. In the unfed females ones the anterior portion of the medium intestine is wrinkled due to the strong musculature. In some regions of the medium intestine, structures which were described for the first time were called nodules, resulting from the spaces between the muscular fibers around the intestine. As the infestations progress the surface of unfed, semi and engorged females diverticulum are extremely wrinkled, being smooth in the semi of 3rd infestation and smoother in engorged of 3rd infestation. The histology of the unfed medium intestine showed the presence of a pseudostratified epithelium with generative and digestive cells. In semi and engorged of 1st infestation and semi of 2nd infestation the epithelium suffered stratification. In semi of 2nd infestation the presence of a third cellular type was observed: the secretory one. In engorged of 2nd infestation and in semi and engorged of 3rd infestation the epithelium was disorganized. In semi submitted to 1st infestation the intestine lumen presented membranous spheres, with strongly stained structures. Others were filled by rests of cytoplasm. The disorganization of the intestinal epithelium of females fixed in reinfested hosts suggested that the antibodies released by the host against the ticks’ intestine had acted on them. Histochemistry revealed little protein in the intestinal epithelium in the unfed ones, unlike those of first infestation semi and engorged and of second semi, which had the epithelium strongly stained... (Complete abstract click electronic access below)

Resumo: Este estudo trouxe a análise morfológica, histológica, ultraestrutural e citoquímica do intestino médio de fêmeas de Amblyomma cajennense e mostrou que esse órgão passa, por grandes modificações à medida que o processo de alimentação progride, tornando-se acentuadas nos indivíduos reinfestados. Do intestino médio de A. cajennense, partem dois pares de divertículos que se ramificam totalizando 8 ramos. Naquelas em jejum a porção anterior do intestino médio é enrugada devido à forte musculatura. Em algumas regiões do intestino médio, estruturas, descritas pela primeira vez, foram denominadas de nódulos, resultado dos espaços presentes entre as fibras musculares ao redor do intestino. Á medida que progridem as infestações a superfície dos divertículos em jejum, semi e ingurgitados de 1ª e 2ª infestações enruga-se extremamente estando nas semi de 3ª infestação menos enrugada e ingurgitadas de 3ª infestação mais lisa. A histologia do intestino médio em jejum mostrou a presença de epitélio pseudo-estratificado com células: generativas e digestivas. Nas semi e ingurgitadas de 1ª infestação e semi de 2ª infestação o epitélio sofre estratificação. Nas semi de 2ª infestação observou-se no epitélio a presença do terceiro tipo celular: a secretora. Nas ingurgitadas de 2ª infestação e nas semi e ingurgitadas de 3ª infestação o epitélio se desorganizou. No lúmen do intestino das semi submetidas à 1ª infestação, observou-se esferas membranosas; com estruturas fortemente coradas no interior. Outras com seu interior preenchido apenas por restos de citoplasma. A desorganização no epitélio intestinal de fêmeas fixadas em hospedeiros reinfestados sugeriu que os anticorpos liberados pelo hospedeiro contra o intestino dos carrapatos tenham agido nestes. A histoquímica detectou pouca proteína no epitélio intestinal daquelas em jejum... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study showed the morphological, histological, ultrastructural and cytochemical analysis of the medium intestine of Amblyomma cajennense females since this organ goes through significant modifications as the feeding process progresses and these modifications are stronger in reinfested individuals. Two pairs of diverticulum emerge from A. cajennense medium intestine and are divided in eight branches. In the unfed females ones the anterior portion of the medium intestine is wrinkled due to the strong musculature. In some regions of the medium intestine, structures which were described for the first time were called nodules, resulting from the spaces between the muscular fibers around the intestine. As the infestations progress the surface of unfed, semi and engorged females diverticulum are extremely wrinkled, being smooth in the semi of 3rd infestation and smoother in engorged of 3rd infestation. The histology of the unfed medium intestine showed the presence of a pseudostratified epithelium with generative and digestive cells. In semi and engorged of 1st infestation and semi of 2nd infestation the epithelium suffered stratification. In semi of 2nd infestation the presence of a third cellular type was observed: the secretory one. In engorged of 2nd infestation and in semi and engorged of 3rd infestation the epithelium was disorganized. In semi submitted to 1st infestation the intestine lumen presented membranous spheres, with strongly stained structures. Others were filled by rests of cytoplasm. The disorganization of the intestinal epithelium of females fixed in reinfested hosts suggested that the antibodies released by the host against the ticks' intestine had acted on them. Histochemistry revealed little protein in the intestinal epithelium in the unfed ones, unlike those of first infestation semi and engorged and of second semi, which had the epithelium strongly stained... (Complete abstract click electronic access below) / Orientador: Maria Izabel Camargo Mathias / Coorientador: Gervásio Henrique Bechara / Banca: Glaucia Maria Cavasin / Banca: Roberta Cornelio Ferreira Nocelli / Banca: Pablo Henrique Nunes / Banca: Rusleyd Maria Magalhães de Abreu / Doutor

Background: The pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) is one of the world's most important pests of cotton. Insecticide sprays and transgenic cotton producing toxins of the bacterium Bacillus thuringiensis (Bt) are currently used to manage this pest. Bt toxins kill susceptible insects by specifically binding to and destroying midgut cells, but they are not toxic to most other organisms. Pink bollworm is useful as a model for understanding insect responses to Bt toxins, yet advances in understanding at the molecular level have been limited because basic genomic information is lacking for this cosmopolitan pest. Here, we have sequenced, de novo assembled and annotated a comprehensive larval midgut transcriptome from a susceptible strain of pink bollworm. Findings: A de novo transcriptome assembly for the midgut of P. gossypiella was generated containing 46,458 transcripts (average length of 770 bp) derived from 39,874 unigenes. The size of the transcriptome is similar to published midgut transcriptomes of other Lepidoptera and includes up to 91 % annotated contigs. The dataset is publicly available in NCBI and GigaDB as a resource for researchers. Conclusions: Foundational knowledge of protein-coding genes from the pink bollworm midgut is critical for understanding how this important insect pest functions. The transcriptome data presented here represent the first large-scale molecular resource for this species, and may be used for deciphering relevant midgut proteins critical for xenobiotic detoxification, nutrient digestion and allocation, as well as for the discovery of protein receptors important for Bt intoxication.

No description available.

Orientador: Paulo Eduardo Martins Ribolla / Banca: Henrique M. C. da Silveira / Banca: Alexandre A. Peixoto / Resumo: Estudos recentes vêm elucidando a importância de uma diversidade de proteínas do intestino médio de insetos vetores, tanto nos processos de digestão como em respostas imunológicas e interações parasita-hospedeiro. Este trabalho teve como objetivo analisar a expressão de genes do intestino médio de Anopheles aquasalis, um importante vetor de malária no Brasil, a partir de clones sequenciados de bibliotecas de cDNA de machos e fêmeas alimentados apenas com sacarose. Nas fêmeas, pôde-se notar a grande predominância de serino proteases, proteínas ligantes de quitina e fatores relacionados à imunidade. Os machos também apresentaram diversos peptídeos de defesa imune, porém apenas uma protease digestiva e uma glicosidase. Alguns genes foram selecionados das bibliotecas para estudo de suas expressões durante a vida de An. aquasalis. Tripsina 1, peritrofina 1 e quinurenina 3-monooxigense tiveram seus níveis de expressão aumentados 6h após a ingestão de sangue, analisados através de qRT-PCR. No entanto, o silenciamento desses genes não resultou em alterações na longevidade de fêmeas adulta. O gene da serpina foi expresso em todas as fases do desenvolvimento do mosquito, exceto em ovos; e o gene da cecropina foi expresso em trato digestivo e carcaça de machos e fêmeas, principalmente após alimentação de açúcar ou sangue. Considerando que a ingestão de alimentos é a principal porta de entrada a microorganismos durante a vida adulta destes mosquitos, a presença de diversos produtos antimicrobianos, bem como a precoce expressão de peritrofina, outra proteína relacionada com a proteção do trato digestivo, mostrou que An. aquasalis está bem preparado imunologicamente contra esses microorganismos. Esta proteção está envolvida com o hábito alimentar desta espécie e pode também estar associada à sua baixa capacidade vetorial com relação aos plasmódios. / Abstract: The importance of midgut proteins of Anopheles aquasalis has been elucidated both in digestion process as in immune responses and parasite-host interactions. This project targeted to analyze the midgut genes expression from An. aquasalis, an important malaria vector in Brazil, selecting clones from midgut cDNA library of female and male mosquitoes fed only on sugar. Serine proteases were predominant in females besides chitin binding proteins and immunity factors. Male mosquitoes also showed immune defense peptides, however only one digestive protease and one glucosidase. Some genes were selected from these libraries to expression study during mosquito development stages. Trypsin 1, peritrophin 1 and kynurenine 3-monoxygenase expression were up regulated at the midgut 6h after blood feeding, analyzed by real time PCR. Nevertheless, the gene silencing did not change the survivorship of adult females. Serpin gene was expressed in all mosquito development stages but eggs; cecropin gene was expressed in midgut and carcass from male and female, mainly after sugar or blood feeding. Considering the alimentation is the main entrance way of pathogens, the presence of antimicrobial peptides as the early peritrophin expression showed that An. aquasalis is immunologically adapted against these microorganisms. This protection is involved in feeding behavior of this specie and can be also related to its low Plasmodium vector capacity. / Mestre

Master of Science / Department of Entomology / Marcelo Ramalho-Ortigao / Sand flies (Diptera:Psychodidae) are vectors of parasites of the genus Leishmania transmitted to suitable vertebrate host during blood feeding. For blood feeding arthropods, including sand flies, blood meal digestion requires the secretion of inhibitory molecules, such as Kazal-type serine proteinase inhibitors that are involved in preventing the blood from coagulating within the mouthparts and the midgut. Previous studies have identified such molecules in mosquitoes, ticks, and triatomine bugs. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). We are interested in the role of these proteins as inhibitors of coagulation cascades, in addition to their potential effects on blood digestion in P. papatasi. Ppkzl1 is similar to thrombin and trypsin inhibitors in triatomines and mosquitoes and Ppkzl2 is similar to Kazal-type inhibitors in mosquitoes with unknown function. Analyses of expression profiles indicated that although both transcripts are expressed prior to blood feeding in the midgut of P. papatasi they are tightly regulated by the blood meal. Reverse genetics studies using RNAi-targeted knockdown of PpKzl1 and PpKzl2 by dsRNA injection did not result in a detectable effect on mRNA expression levels. Thus, we expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess activity against various serine proteinases. Recombinant PpKzl2 inhibited chymotrypsin at nanomolar levels and also inhibited thrombin and trypsin at micromolar levels, suggesting that native PpKzl2 is an active serine proteinase inhibitor and may regulate digestive enzymes and thrombin in the midgut. Leishmania development within the sand fly midgut is faced with several barriers that can severely impact the parasites. For transmission to occur, parasites must be able to overcome these barriers including digestive proteinases, escape from the peritrophic matrix, and midgut attachment. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sand fly midgut. Thus, targeting serine proteinase inhibitors may provide a new strategy to prevent transmission of Leishmania.

Esse trabalho investigou as principais carboidrases já descritas em Lepidoptera e principalmente as enzimas relacionadas com hidrólise de sacarose presentes na hemolinfa e no intestino do sexto instar larval de Lonomia obliqua. Avaliou também o efeito das diferentes dietas Eriobotrya japonica, Psidium guajava, Mangifera indica e Alchornea ssp sobre as atividades das carboidrases presentes no sistema circulatório e no sistema digestório dessa lagarta. As pectinases e celulases foram identificadas somente no intestino, porém a amilase, a α-glucosidase, a β-glucosidase, a α-fucosidase e a β-fructosidase foram detectadas também na hemolinfa dessa lagarta. Os géis nativos e os pHs ótimos indicam que há distintas carboidrases na hemolinfa e no intestino de Lonomia obliqua. Alterações nas dietas afetam somente as carboidrases intestinais. A sacarose das folhas foi hidrolisada pela ação conjunta da α-glucosidase e da β-frucosidase. A investigação das carboidrases de Lonomia obliqua contribuirão para o desenvolvimento de medidas de controle desse inseto. / This work investigated the main carbohydrases already described in Lepidoptera and especially the enzymes present in the hemolymph and intestine of the 6th instar of Lonomia obliqua related to the hydrolysis of sucrose. It has also evaluated the effect of different diets Alchornea ssp, Eriobotrya japonica, Mangifera indica and Psidium guajava, on the activities of the carbohydrases present in the circulatory and digestive system of this caterpillar. Pectinases and cellulases were identified in the midgut only, but amylase, α-glucosidase, β-glucosidase, α-fucosidase and β-fructosidase were detected in both hemolymph and midgut of this caterpillar. Native gels and effect of pH indicated that there are distinct carboidrases in the hemolymph and the midgut of Lonomia obliqua. Diet change have affected the digestive carbohydrases only. The sucrose in the leaves was hydrolyzed by the combined action of α-glucosidase and β-frucosidase. The investigation of the carboidrases of Lonomia obliqua can contribute to the development of control strategies to this insect.