Made available in DSpace on 2015-04-14T13:09:31Z (GMT). No. of bitstreams: 1 437829.pdf: 3097644 bytes, checksum: 63052063f3fb08648c7ca5a4506262bc (MD5) Previous issue date: 2011-12-16 / The genus Angiostrongylus Kamensky, 1905 belongs to the Phylum Nematode, with round shape as its main feature. Two species have medical importance, A. costaricensis living in mesenteric arteries of wild mice and causing abdominal angiostrongyliasis in human and A. cantonensis which lives in pulmonary arteries of rats and may cause eosinophilic meningoencephalitis in humans. The diagnosis of both diseases is difficult due to absence of parasite in feces in case of the infection by A. costaricensis and seldom detected larvae in the cerebrospinal fluid in case of eosinophilic meningoencephalitis. Several studies have been performed to improve the diagnosis of angiostrongyliasis which should be able to differentiate in a specific and sensitive way among other parasitic infections. The 31kDa antigen has been considered the main antigen for eosinophilic meningoencephalitis diagnosis due to A. cantonensis infection. However this antigen is obtained from crude extracts of the worm by a laborious process of purification with low yielding and insufficient amount for large distribution to other diagnostic centers. In order to improve the serologic diagnostic of angiostrongyliasis and make the antigens widely available the present work aimed to identify new antigenic targets and also characterize the 31kDa antigen for further recombinant production. Besides that, essential molecules for parasite survival were investigated which in the future may be targets for disease treatment. Two sources of antigen from female worms were used: excretion and secretion products (ES) and total extract (TE). In ES, sample antioxidant enzymes activity were detected such as catalase and superoxide dismutase. Also was identified by Western blot and Mass spectrometry (MS), 17 proteins target for disease diagnosis and treatment like hemoglobinases, heat shock proteins and proteases inhibitors. In TE sample antioxidant enzymes as well as glutathione transferases (GST) which is another kind of defense enzyme were also detected. GSTs were purified by affinity chromatography and analyzed by MS. Peptide sequences from this experiment matched with homologous sequences of sigma class GST. In TE samples was possible to characterize the 31kDa and after two-dimensional electrophoresis was shown to be composed of four spots around 4.5 of isoelectric point (pI) and being recognized by sera from patients infected with Angiostrongylus spp. The spots were analyzed by MS and three different proteins were identified: 14-3-3 protein, NAC domain containing protein, and epsilon subunit of the coatomer protein complex isoform 2. The 31kDa antigen was characterized as a glycoprotein through studies of oxidation of carbohydrate where it was observed that the antigenicity of four spots was dependent on sugar residues. The DNA sequences of the antigens were obtained by random sequencing of the genome for 454 platform (Roche) and deposited in Genbank. The data generated in this study contribute significantly to the development of recombinant antigens that may be widely distributed for independent diagnostic validation / O g?nero Angiostrongylus Kamensky, 1905 agrupa animais pertencentes ao filo Nematoda, cuja caracter?stica marcante ? a forma corporal cil?ndrica. Duas esp?cies possuem import?ncia m?dica: A. costaricensis cujo habitat natural s?o as art?rias mesent?ricas de camundongos silvestres e na infec??o humana pode levar ao desenvolvimento de angiostrongil?ase abdominal; e A. cantonensis que habita as art?rias pulmonares de roedores e na infec??o humana pode causar meningoencefalite eosinof?lica. O diagn?stico de ambas as doen?as ? dificultado pela aus?ncia de formas parasit?rias nas fezes, no caso de infec??es por A. costaricensis e raramente encontradas no l?quido cefaloraquidiano no caso de meningoencefalite eosinof?lica. Muitos estudos v?m sendo desenvolvidos para o aprimoramento da detec??o das angiostrongil?ases visando testes que sejam capazes de discernir das diferentes infec??es parasit?rias de forma sens?vel e espec?fica. O ant?geno de 31kDa ? considerado atualmente o principal ant?geno para o diagn?stico da meningoencefalite eosinof?lica, causada por A. cantonensis, entretanto ? proveniente da purifica??o de extratos brutos do parasito o que acarreta num processo laborioso e dispendioso que em ?ltima an?lise gera quantidades insuficientes para que haja ampla distribui??o e compartilhamento entre os centros de diagn?stico. Com o intuito de aprimorar o diagn?stico sorol?gico das angiostrongil?ases e tornar os ant?genos dispon?veis globalmente o presente trabalho buscou identificar novos alvos antig?nicos e caracterizar o ant?geno de 31kDa para posterior propaga??o de formas recombinantes. Al?m disso, foram estudadas mol?culas que podem ser fundamentais na manuten??o do parasitismo, que futuramente poder?o ser alvos para o tratamento das angiostrongil?ases. Duas fontes de ant?genos a partir de vermes adultos f?meas foram empregadas: produtos de excre??o e secre??o (ES) e extrato bruto (TE). Nos ES foi detectada a atividade de enzimas antioxidantes como catalase e super?xido dismutase e identificadas, por western blot e espectrometria de massas (MS), 17 prote?nas alvo para o diagn?stico e tratamento das angiostrongil?ases dentre elas hemoglobinases, prote?nas de choque t?rmico e inibidores de proteases. Nas amostras de TE al?m da identifica??o de enzimas antioxidantes, estavam presentes glutationas transferases (GST), outra classe de enzimas de defesa. Estas prote?nas foram purificadas por cromatografia de afinidade e analisadas por MS o que revelou sequencias pept?dicas hom?logas a GST de classe sigma. Em TE tamb?m foi poss?vel a caracteriza??o do ant?geno de 31kDa que quando submetido a eletroforese bidimensional mostrou-se ser composto por 4 spots com ponto isoel?trico (pI) em torno de 4,5 sendo reconhecidos pelo soro de pacientes infectados com Angiostrongylus spp. Os spots foram analisados por MS e tr?s diferentes prote?nas foram identificadas: 14-3-3; prote?na com dom?nio NAC e a subunidade ?psilon do coatamero. O ant?geno de 31kDa foi caracterizado como uma glicoprote?na atrav?s de estudos de oxida??o de glic?deos, onde se observou que a antigenicidade dos 4 spots foi dependente de res?duos de a??car. As sequ?ncias de DNA dos ant?genos foram obtidas pelo sequenciamento aleat?rio do genoma pela plataforma 454 (Roche) e depositadas no Genbank. Os dados gerados no presente trabalho contribuem de forma significativa para o desenvolvimento de ant?genos recombinantes que poder?o ser amplamente distribu?dos para valida??o e aplica??o em diagn?stico

Made available in DSpace on 2015-04-14T13:09:35Z (GMT). No. of bitstreams: 1 440746.pdf: 8186310 bytes, checksum: 2ddc0fe8a6260707c7754fc8b9f27f5b (MD5) Previous issue date: 2012-05-07 / The Angiostrongylus genus includes two species that can infect humans, A. cantonensis and A. costaricensis. They may cause infections known as eosinophilic meningitis and abdominal angiostrongyliasis, respectively. In A. costaricensis infection, eggs and larvae are central elements in the inflammatory reactions, which may get worse with death of the worms. The currently available anthelmintics act on the parasite essential metabolic pathways with a killing effect. Therefore, an alternative substance to treat angiostrongyliasis, acting mainly in worms reproduction is necessary. An in vitro study conducted with Schistosoma mansoni showed inhibition of oviposition by Phenanthroline. In another study with Caenorhabditis elegans, it was shown that serotonin increases the egg-laying rate of the female nematode, besides controlling the change of its posture state (rest and activation). Serotonin is a neurotransmitter present in vertebrates and invertebrates. In order to test the effect on egg laying of Angiostrongylus spp., two substances that interfere with serotonin neurotransmission in humans, Buspirone and Pizotifen, were used in an experimental model in vivo. 28 rodents of species Rattus norvegicus were divided into three groups and infected with 100 L3 of A. cantonensis: a control group (untreated) and two groups treated with each substance. The substances were administered as soon as all rodents started releasing larvae, once a day, orally, for 10 days, at a concentration of 0.03 mg / mL each. In this period rodent feces were collected daily for counting the number of L1 and after 10 days the animals were euthanized for collection of the worms. The average number of larvae released in feces was 37,934 by the Control group, 10,658 by the Buspirone group and 6,658 by the Pizotifen group. The worms were counted and separated by sex: in the Control group 59 females and 40 males were obtained; in Buspirone group 86 females and 41 males were found; and in the Pizotifen group 83 females and 64 males were counted. The comparison of data from Control and Experimental groups was statistically analyzed by ANOVA and no significant difference was observed. Females were measured using a millimetric eyepiece installed in a stereomicroscope. The ANOVA analysis resulted in a significant difference between Control and Pizotifen, which had an average size of 18 mm, compared with to average size of 19 mm of Control, indicating that Pizotifen would have some effect in the nematodes development, but not affecting their reproduction. These results indicate that the search for alternative drugs that act on egg laying needs a better understanding of the pathways that regulate the reproductive system of parasitic organisms. / O g?nero Angiostrongylus engloba duas esp?cies A. cantonensis e A. costaricensis, que podem infectar o ser humano e causar infec??es conhecidas como meningite eosinof?lica e angiostrongil?ase abdominal, respectivamente. Na infec??o por A. costaricensis, ovos e larvas s?o elementos centrais nas rea??es inflamat?rias podendo haver o agravamento dessas les?es, pela morte dos vermes. Os anti-helm?nticos atualmente dispon?veis, atuam em vias metab?licas essenciais ao parasito, culminando com a morte dos vermes. Portanto, uma droga alternativa para o tratamento das angiostrongil?ases, que atue principalmente na reprodu??o dos vermes, se torna necess?ria. Um estudo in vitro realizado com Schistosoma mansoni demonstrou a inibi??o da oviposi??o pela fenantrolina. Em outro estudo com Caenorhabditis elegans foi demonstrado que a serotonina estimula o aumento da taxa de ovos liberados pela f?mea do nemat?deo, al?m de controlar a altera??o do seu estado de postura (repouso e ativa??o). A serotonina ? um neurotransmissor presente tanto em vertebrados como em invertebrados. Com o objetivo de testar o efeito na oviposi??o de Angiostrongylus cantonensis, duas subst?ncias que interferem na neurotransmiss?o da serotonina em humanos, Buspirona e Pizotifeno, foram utilizadas em modelo experimental in vivo. 28 roedores da esp?cie Rattus norvegicus foram divididos em 3 grupos e infectados com 100 L3 de A. cantonensis: um grupo controle (n?o tratado) e 2 grupos tratados com cada subst?ncia. As subst?ncias foram administradas a partir do momento em que todos os roedores iniciaram a larvipostura, uma vez ao dia por via oral, durante 10 dias, numa concentra??o de 0,03 mg/mL cada. Neste per?odo as fezes dos roedores foram recolhidas diariamente para a contagem do n?mero de L1 eliminadas e, ap?s os 10 dias, os animais foram eutanasiados para coleta dos vermes. A m?dia de larvas eliminadas nas fezes para o grupo controle foi 37.934, para o grupo Buspirona 10.658 e para o grupo Pizotifeno 6.658. Os vermes foram contados e separados pelo sexo: no grupo controle foram obtidas 59 f?meas e 40 machos; no grupo Buspirona foram encontradas 86 f?meas e 41 machos e no grupo Pizotifeno 83 f?meas e 64 machos. A compara??o dos dados dos grupos experimentais e do controle foram analisadas estatisticamente pelo teste ANOVA e nenhuma diferen?a significativa foi verificada. As f?meas foram medidas atrav?s de uma ocular milimetrada instalada em um estereomicrosc?pio. A an?lise foi feita tamb?m pelo teste ANOVA e resultou numa diferen?a significativa entre o grupo controle e o Pizotifeno, no qual teve um tamanho m?dio de 18 mm, em compara??o com o tamanho m?dio do controle de 19 mm, indicando que o Pizotifeno poderia ter algum efeito no desenvolvimento dos nemat?deos, por?m n?o afetando a reprodu??o. Estes resultados indicam que para a procura de drogas alternativas, que atuem na oviposi??o, ? necess?rio uma melhor compreens?o das vias reguladoras do sistema reprodutivo dos organismos parasitos.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-24T13:11:26Z No. of bitstreams: 2 Dissertação - Ana Paula de Toledo Vieira - 2012.pdf: 10699833 bytes, checksum: 2e25915716db787ecacbd31491c5309a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-24T14:02:12Z (GMT) No. of bitstreams: 2 Dissertação - Ana Paula de Toledo Vieira - 2012.pdf: 10699833 bytes, checksum: 2e25915716db787ecacbd31491c5309a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-24T14:02:12Z (GMT). No. of bitstreams: 2 Dissertação - Ana Paula de Toledo Vieira - 2012.pdf: 10699833 bytes, checksum: 2e25915716db787ecacbd31491c5309a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-02-27 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Nematoda Lagochilascaris minor is the etiologic agent of lagochilascaríase in human beings. Until today, no one knows the natural host of this helminth and Brazil registers about 90% of the cases of lagochilascaríase world-wide human being. The life cycle of L. minor was described using an experimental model in mice and domestic cat, behaving as an intermediate host and definitive host respectively. The objective of this study was to evaluate the histology of the adult males of this helminth worms, using light microscopy. These specimens were removed after necropsy of cats kept in their cages cataloged in male and female worms, and the adult male specimens were fixed in formalin solution 10 % hot, AFA and 4% paraformaldehyde in sodium cacodylate buffer 0 1 M, pH 7.2. After the different fixations, the samples were dehydrated in a gradual series of ethanol in different concentrations of alcohol levels, and included in historesin cut with 3μm –thick microtome, with horizontal and transverse serial sections. The sections were stained with Hematoxylin & Eosin (HE), Carmin, Giemsa and Toluidine Blue. The best fixation was achieved with the use of AFA and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.2 and the best staining was obtained from the use of the dye HE, which showed histological structures of adult male worms L. minor loop as testicular, seminal vesicle, ejaculatory duct, sperm and spicule. It was also observed structures of the digestive system, muscular system, cuticle, wing side and lateral line. The sections were photomicrographed using photomicroscope with the help of the program Motic Images Plus 2.0, selected and analyzed. / O nematoda Lagochilascaris minor é o agente etiológico da lagochilascaríase em humanos. Até hoje, não se conhece o hospedeiro natural deste helminto e o Brasil registra cerca de 90% dos casos da lagochilascaríase humana mundial. O ciclo evolutivo de L. minor foi descrito utilizando um modelo experimental com camundongo e gato doméstico, comportando-se como hospedeiro intermediário e hospedeiro definitivo, respectivamente. O objetivo deste trabalho foi avaliar a histologia dos vermes adultos machos deste helminto, utilizando microscopia de luz. Estes exemplares foram retirados após necropsia de gatos mantidos em biotério, catalogados em vermes machos e fêmeas, sendo que os exemplares machos adultos foram fixados em solução de Formalina a 10% a quente, AFA e Paraformaldeído a 4% em tampão cacodilato de sódio 0,1 M pH 7,2. Após as diferentes fixações, as amostras foram desidratadas em uma série gradual de etanol em diferentes concentrações de teores alcoólicos, incluídas em historesina e cortadas com 3μm de espessura em micrótomo, com secções seriadas horizontais e transversais. Os cortes foram corados com Hematoxilina & Eosina (HE), Carmim, Giemsa e Azul de Toluidina. A melhores fixações foram alcançadas com o uso de AFA e Paraformaldeído a 4% em tampão cacodilato sódio 0,1 M pH 7,2 e a melhor coloração obteve-se a partir da utilização do corante HE, que evidenciou estruturas histológicas dos vermes adultos machos de L. minor como alça testicular, vesícula seminal, ducto ejaculatório, espermatozoides e espículo. Observou-se também estruturas do sistema digestório, sistema muscular, cutícula, asa lateral e linha lateral. As imagens dos cortes foram realizadas utilizando um fotomicroscópio com o auxílio do programa Motic Images Plus 2.0, selecionadas e analisadas.

The thesis explores the Jewish view of Jesus Christ of Geza Vermes concept and its possible influence on the Jewish-Christian dialogue. This Jewish researcher is known primarily as a popularist of the Dead Sea Scrolls, which were translated by him into English for the first time. Admittedly he is significant in the field of Jesus Studies. We present the author's perception of Jesus according to his life and work with focus on selected New Testament passages. Furthermore we focus on Messianic Judaism as a potential bridge in the Jewish-Christian dialogue, and discuss the conteporary state of these relations. A research survey was also developed on the subject of the dialogue. Finally, we summarize and evaluate the contribution of Geza Vermes' work, with emphasis on its use in the Jewish-Christian dialogue, with the support of our own research based on Czech experts,that complements the acquired piece of knowledge.

Why has the resurrection once again become the centre point of a new storm brewing in both popular and academic culture? Because of the combination of a realisation of death, and of human beings’ need to interpret its (death’s) mysteries; a question innate to the human experience. In a fear-filled world where war, terrorism, and economic collapse bring the question of death (and the afterlife) to the fore, people are asking – perhaps more than ever – what happens after we die. This popular fascination with the end, with death, and with what (if anything) lies beyond it, has also influenced the theme and the direction of academic work in the theological field. For this reason an informed analysis of the resurrection debate has become necessary – a process of analysing the different strata of understanding as it relates to current resurrection research. Any consideration given to gender or power, birth or burial, money or food is made in an effort to situate the debates being studied. Could a reason for these still varied conclusions on the subject be that those writing on it are not equipped for the task of analysing and interpreting history and historical method? In order to be able to begin answering this question, one of this study's main objectives is to learn and apply the approach of historians – outside of the community of Biblical scholars – to the question of whether Jesus of Nazareth rose from the dead; thus providing interaction with philosophers of history related to hermeneutical and methodological considerations. The method proposed here is a combination of historiography and an ethics of understanding, with the use of Correspondence theory (in which history is described as knowable, and some hypotheses as truer than others in a correspondence sense). This study wants to address both the different questions and analyses of the debate by asking: What if we see things differently? What if we were to ask a different set of questions? In order for this to be possible, we need to develop an ethics of interpretation – instead of asking the expected questions, this study aims to ask: What interests and frameworks inform the questions we ask and the way in which we interpret our sources? How does scholarship echo (and even participate in) contemporary public discourses about Christian identity? These questions will be attended to through three intersecting practices – critical reflexivity, complemented by the use of the two related practices of textual re-reading and public debate. However, these are not methodical steps in a linear progression, they are mutually interacting practices that draw on each other; raising new possibilities for the way in which we historically reconstruct the Jesus movement, allowing us to enter into the public debate about Jesus and eschatology in a way that takes the ethical possibilities and consequences of our reconstructions of Christian origins and identity seriously. For, though fragmentary and broken human words may be, they nevertheless possess a capacity to function as the medium through which God is able to disclose himself. Copyright / Dissertation (MTh)--University of Pretoria, 2010. / New Testament Studies / unrestricted