Paracoccidioidomicose (PCM), micose sistêmica prevalente na América Latina, é uma doença granulomatosa causada pelo fungo termodimórfico do gênero Paracoccidioides spp. O maior número de casos dessa doença tem sido reportado no Brasil, Colômbia, Venezuela e Argentina. A existência de extensas áreas endêmicas, alto potencial incapacitante devido a fibroses pulmonares, grande quantidade de mortes prematuras, faz com que a PCM seja considerada um grave problema de Saúde Pública. Apesar do tratamento medicamentoso ser relativamente eficiente, o tempo longo de uso das drogas e os efeitos colaterais provocados podem levar a diminuição da adesão ao tratamento diminuindo assim eficácia. O uso de vacinas terapêuticas pode ser uma importante ferramenta no controle da PCM. Uma das modalidades investigadas pelo nosso grupo é a utilização de células dendríticas (DCs) pulsadas com o peptídeo P10 como vacina terapêutica no controle da PCM. Resultados apresentados por nosso grupo, demonstraram que DCs diferenciadas a partir de células de medula óssea de camundongos saudáveis pulsadas com o peptídeo 10 (P10) podem ser utilizadas como adjuvante no tratamento da paracoccidioidomicose experimental em camundongos imunocompetentes e imunossuprimidos. Assim sendo, nossa proposta no presente trabalho foi avaliar se DCs diferenciadas de células de medula óssea (BMDCs) ou de monócitos circulantes (MoDCs) de camundongos (BALB/c) infectados apresentam modulação prévia devido à exposição a antígenos fúngicos, avaliamos in vitro a capacidade das células dendríticas diferenciadas a partir de monócitos circulantes de camundongos infectados com Pb18 estimular proliferação de linfócitos CD8+ e CD4+, analisamos a capacidade de células dendríticas derivadas de monócitos circulantes de camundongos infectados com Pb18 pulsadas com P10 em induzir uma resposta protetora. Nossos resultados obtidos são promissores e reforçam dados já publicados pelo grupo os quais demonstraram a eficiência do peptídeo P10 no tratamento da paracoccidioidomicose experimental; apontam que o P10 é capaz de modular ativamente células dendríticas diferenciadas de monócitos circulantes e diferenciadas de células de medula óssea; demonstram que BMDCs e MoDCs pulsadas ou não com P10 tem a capacidade de estimular proliferação de linfócitos T CD4+ e CD8+; confirmaram que BMDC e MoDCs pulsadas ou não com P10 diminuem a carga fúngica no tecido pulmonar, estimulando um padrão de citocinas misto, porém com predominância de citocinas pró-inflamatórias que incitam resposta imune celular do tipo Th1, que é protetora na PCM. Estes dados foram respaldados com a análise dos cortes histológicos onde os pulmões dos camundongos tratados com BMDCs ou MoDCs pulsadas com P10 apresentaram parênquima pulmonar mais conservado em comparação com os pulmões dos grupos que não receberam tratamento com BMDCs ou MoDCs, com diminuição significativa de células fúngicas viáveis / Paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America, is a granulomatous disease caused by the thermodymorphic fungus of the genus Paracoccidioides spp. The highest number of cases of this disease has been reported in Brazil, Colombia, Venezuela and Argentina. The existence of extensive endemic areas, a high incapacitating potential due to pulmonary fibroses, and a large number of premature deaths, makes the PCM considered a serious public health problem. Although the drug treatment is relatively efficient, the long time of use of the drugs and the side effects provoked can lead to the decrease of the adherence to the treatment thus diminishing their effectiveness. The use of therapeutic vaccines can be an important tool in the control of PCM. One of the modalities investigated by our group is the use of dendritic cells (DCs) pulsed with the peptide P10 as therapeutic vaccine in the control of PCM. Results presented by our group demonstrated that DCs differentiated from bone marrow cells of healthy mice pulsed with peptide 10 (P10) can be used as an adjuvant in the treatment of experimental paracoccidioidomycosis in immunocompetent and immunosuppressed mice. Thus, our proposal in the present study evaluated the capacity of differentiated DCs of bone marrow cells (BMDCs) or circulating monocytes (MoDCs) of infected mice (BALB/c) exhibited previous modulation due to exposure to fungal antigens, we evaluated in vitro the ability of differentiated dendritic cells from circulating monocytes from Pb18 infected mice to stimulate proliferation of CD8+ and CD4+ lymphocytes, we analyzed the ability of dendritic cells derived from circulating monocytes from mice infected with P. brasiliensis pulsed with P10 to induce a protective response. Our results are promising and reinforce data already published by the group which demonstrated the efficiency of the peptide P10 in the treatment of experimental paracoccidioidomycosis; suggest that P10 is capable of actively modulating differentiated dendritic cells from circulating and differentiated monocytes from bone marrow cells; demonstrate that BMDCs and MoDCs pulsed or not with P10 have the ability to stimulate proliferation of CD4+ and CD8+ T lymphocytes; confirmed that BMDC and MoDCs pulsed or not with P10 decrease the fungal load in lung tissue, stimulating a pattern of mixed cytokines but with a predominance of pro-inflammatory cytokines that stimulate Th1-type cellular immune response that is protective in PCM, these data were supported with analysis of the histological sections where the lungs of the mice treated with BMDCs or MoDCs pulsed or not with P10 showed more conserved pulmonary parenchyma compared to the lungs of the groups that did not receive treatment with BMDCs or MoDCs, with significant decrease of viable fungal cells

Paracoccidioidomicose (PCM) é uma micose sistêmica causada por fungos dimórficos do gênero Paracoccidioides - P. brasiliensis ou P. lutzii. A alta incidência da PCM no Brasil, somada à gravidade que o quadro clínico pode assumir e ainda à ausência de um antifúngico de baixa toxicidade para tratamento de curta duração, motivaram a avaliação de adjuvantes e caracterização dos componentes antigênicos de P. brasiliensis em um modelo imunoterapêutico de PCM. Assim, inicialmente, avaliou-se o efeito de monofosforil lipídeo A (MPLA) de Salmonella minnesota, PAM3CSK4, hidróxido de alumínio e AS04 em camundongos cronicamente infectados com P. brasiliensis. Quando camundongos da linhagem BALB/c foram infectados pela via intratraqueal com 3 × 105 leveduras de P. brasiliensis, tratados no dia 20 após a infecção com um dos adjuvantes e analisados 30 dias após a administração do tratamento, observou-se que MPLA, ao contrário dos outros adjuvantes, induziu (1) uma redução significativa no número de unidades formadoras de colônias (UFC), (2) uma expressiva diminuição no número de granulomas e células fúngicas no tecido pulmonar e (3) uma resposta imunológica protetora (Th1) quando comparado aos animais controle tratados com PBS. Quanto ao rastreamento de antígenos de P. brasiliensis candidatos a agentes terapêuticos, evidenciou-se que as frações proteicas da preparação de exoantígenos (ExoAg) eram as mais promissoras. Dentre essas frações, a que continha o ExoAg majoritário diidrolipoil desidrogenase induziu maior porcentagem de proliferação de linfócitos T CD3+ esplênicos de camundongos infectados e tratados com MPLA. O gene de diidrolipoil desidrogenase foi clonado em vetor de expressão e a proteína recombinante produzida, purificada e utilizada no protocolo terapêutico da PCM experimental. Surpreendentemente, a administração terapêutica de diidrolipoil desidrogenase recombinante induziu uma redução significativa na contagem de UFC dos animais infectados cronicamente com P. brasiliensis, mesmo na ausência de adjuvantes, o que não foi observado quando foi administrada a preparação de ExoAg nos animais. A localização subcelular de diidrolipoil desidrogenase, por microscopia eletrônica de transmissão, utilizando anticorpos policlonais de camundongos contra essa proteína, mostrou que a mesma está localizada na mitocôndria e também no citoplasma. A análise da expressão gênica diferencial de diidrolipoil desidrogenase em P. brasiliensis mostrou que essa proteína é significativamente mais expressa em leveduras em comparação a hifas e formas de transição. Em conclusão, nossos resultados mostram os efeitos benéficos de MPLA na PCM experimental e sugerem que diidrolipoil desidrogenase é um alvo terapêutico potencial para o tratamento da PCM. / Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the dimorphic fungus Paracoccidioides - P. brasiliensis and P. lutzii. The high incidence of PCM in Brazil, added to the severity of the disease and the absence of an antifungal that brings together low toxicity and short-term treatment, led us to evaluate adjuvants and characterize antigenic components of P. brasiliensis that could be used as immunotherapy in an experimental model of PCM. In this work, firstly, we evaluated monophosphoryl lipid A (MPLA) from Salmonella minnesota, PAM3CSK4, aluminum hydroxide and AS04 in mice chronically infected with P. brasiliensis. When mice were infected intratracheally with 3 × 105 P. brasiliensis yeasts, treated with one of the adjuvants or vehicle on day 20 postinfection, and analyzed 30 days after the treatment, we observed that MPLA, unlike other adjuvants, induced (1) a significant reduction in the number of colony forming units (CFU) in the lungs of the mice, (2) an expressive decrease of granulomas and yeast cells in lung tissue, and (3) a more protective immune response (Th1) when compared with the control mice. Concerning to detection of antigens for PCM therapy, we noticed that among the fractions of a preparation of exoantigens (ExoAg), the one containing the protein identified as dihydrolipoyl dehydrogenase induced a high percentage of proliferation of splenic CD3+ T lymphocytes from mice infected and treated with MPLA. The gene of dihydrolipoyl dehydrogenase was cloned into expression plasmid vector, and the recombinant protein produced, purified and used in the therapeutic protocol of experimental PCM. Surprisingly, the therapeutic administration of recombinant dihydrolipoyl dehydrogenase, but not ExoAg preparation, induced a significant reduction in fungal load in the animals chronically infected with P. brasiliensis, even in the absence of adjuvants. Immunogold labeling and transmission electron microscopy revealed that the dihydrolipoyl dehydrogenase is localized in mitochondria and cytoplasm of P. brasiliensis. The differential expression of dihydrolipoyl dehydrogenase gene in P. brasiliensis showed that yeast had an expression more significant than hyphae, with an intermediate level of gene expression in the transitional forms. In conclusion, our results show that MPLA and dihydrolipoyl dehydrogenase are potential therapeutic targets for the treatment of PCM due to their beneficial effects in a therapy model of PCM.

Animals acquire immunity against gastrointestinal (GI) nematode infection depending on their age and continual exposure to larvae, however, expression of this acquired immunity is often penalized during pregnancy and lactating period. This is described as periparturient relaxation in immunity (PPRI), and suggested to have nutritional basis. Although dietary protein has positive effect on immunity against GI nematode infection in mammalian host, we have not fully achieved to characterise the detailed interaction between PPRI and dietary protein. Therefore, this PhD aimed to further investigate this interaction in a well-established Nippostrongylus brasiliensis re-infected lactating rat model. Feeding high protein diet (HP) as opposed to low protein diet (LP) during pregnancy was necessary in maintaining sufficient maternal performances and systemic immune response (Chapter 2 and 3). Accumulation of host’s body protein reserve during pregnancy was significantly higher in HP fed animals compared to LP fed counterparts, which led to improvement in both maternal performances and immunity during the early stage of lactation. However, as lactation period progressed and re-infection of N. brasiliensis took place, importance of current dietary status, rather than the accumulated protein reserve, became evident for maternal performances and immunity. Indeed, animals fed HP during lactation showed significantly heavier pup weight compared to LP fed animals; HP animals showed higher serum immunoglobulin levels and reduced worm burden compared to LP. N. brasiliensis, however, goes through systemic migration, entering host’s skin and migrating to lung parenchyma through blood vessels before reaching the intestine. In Chapter 4, a detailed lung pathology study following N. brasiliensis infection was performed to generate data on the effects of the nematode in the lung of the rat host as such data were scarce. Rat host showed similar lung pathology to that of mice; up-regulation of genes related to type 2 immunity and development of emphysema-like pathology were observed following N. brasiliensis re-infection. In Chapter 5, the effect of dietary protein supplementation on lung and intestinal histology and gene expression analysis was investigated. It was shown that HP fed animals showed higher expression of genes related to type 2 immunity compared to LP in the lung. This effect of protein supplementation in the lung may have contributed to fewer worm burdens in HP fed rats compared to LP in the intestine. Dietary protein supplementation significantly affected the expression of genes related to goblet cells; it resulted in up-regulation of the expression of Retnlb and down-regulation of Agr2 and Tff3 in HP fed animals compared to LP. It is evident that dietary protein is modulating intestinal immunity, and this may be targeted towards specific pathways. In addition, the effect of dietary protein supplementation on immune cell populations of secondary lymphoid organs was analysed. Marked increase in the percentage of macrophage in the spleen and T cell in the mesenteric lymph node was observed following protein supplementation, highlighting the importance of dietary protein on systemic immunity during the parasite infection. These results demonstrate that dietary protein supplementation is effective for improving both maternal performances and immune responses, not only at the intestinal phase but also at the migrating phase, when animal is undergoing PPRI during N. brasiliensis infection. Such information is expected to define strategic utilisation of nutrient supply and to result in development of sustainable parasite control strategies in mammals.

O presente estudo analisou aspectos do ciclo de vida e níveis de metais em Atherinella brasiliensis da praia de Itamambuca, Ubatuba (SP). As coletas foram mensais, nos períodos diurno e noturno, de setembro de 2003 a fevereiro de 2005, em quatro estações na praia e uma no rio Itamambuca, utilizando-se picaré. Os indivíduos migram diariamente para dentro do rio no período noturno. O melhor ajuste ao modelo de von Bertalanffy (CT= 164,85*(1-e-2,16(t+0,0038))) e à longevidade (A0,95= 1,378 ano) foi obtido pelo método de Ford-Walford. O índice de performance de crescimento e a mortalidade variaram de 2,77 a 2,91 e 1,64 a 1,82 ano-1, respectivamente. O valor de L50 estimado para sexos agrupados foi 86,6 mm, e os de RGS, DK e a freqüência de desovantes sugerem desova durante todo o ano, com maior intensidade do final da primavera até início do outono. A desova é parcelada, com alta freqüência e em pequenos lotes. Altas concentrações de Cr, Fe e Zn foram detectadas na musculatura, sendo maiores em indivíduos jovens e na alta temporada turística. Pode-se concluir que o peixe-rei passa todo seu ciclo de vida na praia de Itamambuca, apresenta altas taxas de crescimento e mortalidade natural, e desova ao longo de todo o ano. / The present study analyzed the life history and metal levels of Atherinella brasiliensis from Itamambuca beach, Ubatuba (SP). Surveys were conducted monthly, in diurnal and nocturnal periods, between September 2003 and February 2005, in four stations at the beach and one at the Itamambuca river, using beach seine. Individuals showed daily migration into the river at nocturnal period. The better adjustment to von Bertalanffy?s model (Lt = 164.85*(1-e-2.16(t+0.0038))) and to longevity (A0.95= 1.378 year) was obtained through Ford-Walford method. The value of estimated L50 for grouped gender was 86.6 mm, and the GSR, the DK and the spawning frequency suggest spawning during all year around, with higher intensity in late spring until middle autumn. The high frequency of spawning and the low batch fecundity indicate that the species is a batch spawner. High concentrations of Cr, Fe and Zn were detected in musculature, being higher among young individuals, and during tourist high season. In conclusion, the Brazilian silverside spends its life cycle on the Itamambuca beach, shows high growth and natural mortality rates, and spawning throughout the year.

O presente estudo visou avaliar os possiveis efeitos imunotóxicos e imunoteratogênicos da integerrimina N-óxido presente no resíduo butanólico (RB) do ,Senecio brasiliensis, uma planta hepatotóxica que contém alcalóides pirrolizidínicos (APs). Tem sido relatado o uso de APs como fitoterápicos em todo o mundo e também como contaminantes de pastagens e alimentos, sendo assim responsáveis por intoxicações em animais de produção e humanos; porém, até o momento, não há relatos de sua ação sobre o sistema imune. Assim, após a extração da planta, quantificou-se no RB de Senecio brasiliensis, por meio de análise elementar e de espectro (1H e 13C), cerca de 70% de integerrimina N-óxido. O RB foi administrado a ratos Wistar machos adultos, nas doses de 3, 6 e 9 mg/kg, por gavagem, durante 28 dias. Foram avaliados o ganho de peso, o consumo de alimento, os órgãos linfóides e a fenotipagem de sua células, a atividade de fagócitos, as respostas imunes humoral e celular e atividade proliferativa de linfócitos. Já ratas gestantes, receberam as mesmas doses do RB do 6º ao 20º dia de gestação. No final da gestação, parte das fêmeas foi destinada à cesariana e as restantes prosseguiram à termo até o nascimento, para a análise das proles aos 60 dias de idade. Os fetos provenientes da cesariana foram avaliados quanto ao tamanho, peso, viabilidade e malformações externas e também submetidos à análise óssea. As mães tiveram seu desempenho reprodutivo avaliado. Naquelas proles avaliadas até 60 dias de idade, procedeu-se a avaliação dos órgãos linfóides e dos parâmetros hematológicos. O presente estudo revelou que integerrimina N-óxido promoveu diminuição no ganho de peso corporal de ratos machos adultos, redução na atividade proliferativa das células T e interfência na contagem de células sanguíneas destes animais. Por outro lado, esses mesmos animais não apresentaram diferenças na atividade de fagócitos, na fenotipagem de linfócitos e nas respostas imunes estudadas. Em ratas gestantes, o RB causou hepatotoxidade, diminuição do ganho de peso materno, diminuição no tamanho das placentas. A análise fetal revelou menor ossificação. Quando da avaliação da prole aos 60 dias de idade, não foram observados efeitos no ganho de peso atribuídos ao RB, nos órgãos linfóides, e também parâmetros hematológicos e bioquímicos. Concluindo, os estudos aqui realizados permitem sugerir que as doses baixas de integerrimina N-óxido ora empregadas, não promovem efeitos imunotóxicos significantes, bem como não apresentaram potencial teratogênico, seja por meio das avaliações clássicas de teratologia quanto naqueles protocolos de imunoteratogenicidade. / The aim of this study was to evaluate the possible immunotoxic and immunoteratogenic effects of integerrimine N-oxide content in the butanolic residue (BR) of Senecio brasiliensis, a hepatotoxic plant that contained pyrrolizidine alkaloids (PAs). PAs have been reported as a pasture and food contaminant and as herbal medicine used worldwide and are related to promote poisoning events in livestock and human beings. After the plant extraction, BR residue from Senecio brasiliensis was submitted to elemental and spectral analyses (1H and 13C ) and was found to contain approximately 70% of integerrimine N-oxide by, which was administered to adult male Wistar rats at doses of 3, 6 and 9 mg/kg for 28 days, by gavage. Body weight gain, food consumption, lymphoid organs and their cell phenotype, phagocyte analysis, humoural and cellular immune responses, and lymphocyte proliferation were evaluated. In addition, pregnant rats received the same doses of BR from the 6th to the 20th gestational day. In late pregnancy, some of the dams were destined for cesarean section and the other females followed the pregnancy until the birth for offspring analysis at 60 days old. Fetuses from cesarean section were evaluated for size, weight, viability and external malformations and also subjected to bone analysis. The reproductive performance of females were performed. The present study showed that integerrimine N-oxide could promote in adult male Wistar rats, body weight gain impairment, interference with blood cell counts and a reducing T cell proliferative; however, no differences in phagocyte activities, lymphocytes phenotyping and immune responses here evaluated were observed. In pregnant rats, BR caused hepatotoxicity, decreased body weight gain and reduced placental size. Fetus analysis showed lower ossification. When 60-days old offspring were it was not observed any toxic effects resulted from BR exposition in body weight gain, lymphoid organs, haematological and biochemical parameters of these rats. It is concluded that low doses of integerrimine N-oxide here employed did not produce marked immunotoxic effects and seems to be a teratogenic signs by both methods, the classical evaluations or by immunoteratological protocols.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-22Bitstream added on 2014-06-13T20:44:20Z : No. of bitstreams: 1 miranda_et_dr_arafcf.pdf: 641942 bytes, checksum: 8d27d1b81ac16fe68d939f32e284f53f (MD5) / Universidade Estadual Paulista (UNESP) / Neste trabalho foi realizado um estudo comparativo entre isolados de Pb18 e Pb265, que têm capacidades distintas de adesão às células Hela e Vero, sob influência da temperatura e diversos fatores químicos. Foram também avaliados eventos em células de epitélio pulmonar expostas à gp 43 e células de Pb18, antes e após inoculação animal, representando, respectivamente, isolados menos e mais virulentos. Os resultados mostram que as adesinas fúngicas são termo-lábeis, sugerindo sua natureza protéica. Os açúcares aminados glucosamina e galactosamina foram os mais eficientes para inibir a adesão celular, em relação à manose, glicose e galactose, indicando que esta atividade envolve mecanismos específicos tipo lectina. O estudo com componente da matriz extracelular mostrou que a laminina, assim como seus derivados sintéticos, inibiu a adesão do isolado Pb18, fato não verificado com Pb265, tanto com células Hela, quanto com a linhagem Vero, mas variável sob influência de fatores químicos. As distintas vias de sinalização foram demonstradas pelos eventos Ras-Raf, Rho e AKT verificados, sendo que o isolado menos virulento causou menos estresse citotóxico, comprovado por menor sinal Ras- Raf e AKT de proliferação. Os sinais intensos de proliferação observados nos experimentos com o isolado mais virulento e exposição à gp 43 poderiam ser associados à maior sobrevida do fungo que se internaliza mais facilmente nas células do hospedeiro, dificultando o seu reconhecimento por células do sistema monocítico-fagocitário, antes de causar apoptose, evadir e disseminar-se. A ativação das vias de sobrevida, associada a sinais citosólicos quando o Pb entra na célula epitelial, foi demonstrada de modo inédito neste trabalho. / In this work it was accomplished a comparative study between isolates Pb18 and Pb265, which have distinct capacities of adhesion to the cells Hela and Vero under influence of temperature and several chemical factors. Events in pulmonary epithelial cells exposed to gp 43 and in Pb18 cells, before and after animal inoculation, representing, respectively, isolates less and more virulent were evaluated as well. The results show that the fungal adhesins are term-labels, suggesting its proteical nature. The aminated sugars glucosamine and galactosamine were the most efficient, in comparison with manose, glycose and galactose, in inhibiting the cellular adhesion, indicating that this activity involves specific mechanisms lectin-type. The study with extracellular matrix component indicated that the laminin, as well as its synthetic derivatives, inhibited the adhesion of the isolate Pb18 to the cells Vero and Hela, what did not occur with Pb265, but it was variable under influence of chemical factors. The distinct signaling pathways were demonstrated by the Ras-Raf, Rho and AKT events, considering that the less virulent isolate caused minor cytotoxic stress, proved by minor Ras-Raf and AKT proliferation signals. The intense proliferation signals observed in the experiments with more virulent isolate and exposition to gp 43 could be associated to larger time of survival of the fungus that more easily goes inside the host cells, making it difficult to the monocyte-macrophage system cells to recognize it before the apoptosis, the evasion and the dissemination. The Ras-Raf and AKT pathways acted synergetically with the effects of the cell survival. The decrease of the AKT event implied partial loss of the survival signal that intensified, following and in a opposite way, with Ras-Raf decrease. The activation of the survival pathways, associated to citosolic signals when Pb goes inside the epithelial cell, was for the first time demonstrated in this work.

Orientador: Rinaldo Poncio Mendes / Resumo: A paracoccidioidomicose (PCM) é uma doença sistêmica causada por fungos termodimórficos do complexo Paracoccidioides brasiliensis - P. brasiliensis e P. lutzii. Pacientes com PCM revelam comprometimento imune celular específico. Apesar do tratamento efetivo, os fungos quiescentes podem levar à recaída, geralmente tardia, cujo diagnóstico sorológico tem se mostrado pouco sensível. A sensibilidade do teste de imunodifusão é de cerca de 90% em pacientes com PCM não tratados, mas de apenas 45% em casos de recaída. Além disso, o soro de pacientes com PCM causada por P. lutzii, freqüente na região Centro-Oeste do Brasil, não reage com o antígeno clássico obtido do Pb B-339. No seguimento dos pacientes, continua a busca de um critério que dê maior segurança à decisão de se suspender o tratamento. Esses achados mostraram a necessidade de métodos alternativos de diagnóstico e seguimento, como os utilizados em proteômica, que constituiram o presente estudo. Assim, proteínas expressas no soro de pacientes com PCM em atividade - não tratada (causada por P. brasiliensis e por P. lutzii), com recaída da doença (causada por P. brasiliensis) e de controles saudáveis foram identificadas usando-se abordagem proteômica, realizada em quatro etapas - quantificação de proteínas, digestão de proteínas em solução, sequenciamento de peptídeos por espectrometria de massa e análise de dados para identificação de proteínas. Foram identificadas 29 proteínas plasmáticas à admissão, cuja comparação entre o... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

As intoxicações por Senecio spp. estão entre as principais causas de morte no Sul do Brasil, sendo o controle desse gênero de plantas prioritário para a pecuária local. Uma área de 5,5 hectares severamente infestada por Senecio brasiliensis e Senecio madagascariensis foi roçada e submetida ao pastejo por ovinos e bovinos. Uma população total de 28.629 plantas, entre S. brasiliensis (10.122) e S. madagascariensis (18.507) foi virtualmente eliminada em um período de dois anos. O número de ovinos foi mantido em três unidades por hectare, mas variáveis lotações de bovinos foram associadas com a disponibilidade de forragem. As principais práticas de manejo sanitário aplicadas aos ovinos foram administrações de anti-helmínticos e controle de ectoparasitas. Biópsias hepáticas, colhidas antes e após o período de estudo, não revelaram qualquer alteração histopatológica associável com seneciose. Os níveis de desempenho dos ovinos foram acompanhados através do controle da reprodução, produção de lã e ganho de peso. O desempenho produtivo dos ovinos mantidos sob o pastejo da área infestada por Senecio spp. foi similar ao grupo controle e comparável com o observado em rebanhos manejados em sistemas extensivos tradicionais no Sul do Brasil. / Since poisoning by Senecio spp. is one of the main causes of cattle death in southern Brazil, control of these plants is a priority for the local livestock production. After the pasture has been mowed, grazing by sheep and cattle was efficient for controlling Senecio brasiliensis and Senecio madagascariensis populations in a 5.5-hectare area that had long been severely infested with these species. A total of 28,629 plants among S. brasiliensis (Flower of souls - 10,122) and S. madagascariensis (fireweed - 18,507) were almost completely eliminated in a two year period. The number of sheep was kept at 3.0 stock units/ ha, but a variable number of cattle were temporarily stocked according to pasture availability. The major sanitary practices applied to the sheep were anthelmintic administration and ectoparasites control. Liver biopsies taken from sheep and cattle before and after experimental period didn´t reveal any change associable with seneciosis. The performance levels of the senecio-grazing sheep were similar to the control group and comparable to those observed in flocks managed under traditional extensive grazing systems in southern Brazil.

Hevea brasiliensis est la seule source commerciale du caoutchouc naturel. La maladie CLF (Corynespora Leaf Fall) provoquée par le champignon Corynespora cassiicola est une pathologie foliaire de l’hévéa importante en Asie et en Afrique. Les épidémies sont liées au développement de nouvelles variétés clonales très sensibles. Une méthode conductimétrique de phénotypage de la réponse de l’hévéa aux exsudats de C. cassiicola (filtrats de culture ou toxine purifiée) a été développée et caractérisée. Ce test toxinique, sans contact direct avec le champignon lui-même, peut être mis en œuvre sans risque sur les plantations. Après application de gouttes d’exsudats sur des folioles détachées d’un génotype d’hévéa, deux mesures de conductivité C1 et C2 sont réalisées avant et après autoclavage, et le pourcentage de fuite d’électrolyte dû au traitement, %FE = 100*C1/C2, est calculé. L’influence de différents facteurs tels que le stade foliaire et la durée d’incubation, a été précisée. Une corrélation positive a été trouvée entre les réponses au test toxinique et à l’inoculation par des conidies du champignon. La pertinence de la correction de C1 par C2 a été analysée.Deux études du test toxinique ont été réalisées pour une série de clones d’hévéa, d’une part en France avec des plantes en pots cultivées en serre, et d’autre part en Côte d’Ivoire, dans les conditions normalement envisagées pour ce test, avec des folioles provenant de jardins à bois de greffe. La répétabilité entre ces deux études est imparfaite mais néanmoins satisfaisante. Le critère %FE est apparu plus sensible et plus précis que la mesure de la surface de nécrose induite (SN). Une variabilité importante de la sensibilité des clones et de l’agressivité des exsudats a été observée, avec l’existence d’interactions. L’analyse du déterminisme génétique de la réponse au test toxinique a été menée par détection de QTL sur deux populations de la famille biparentale d’hévéa PB260 x RRIM600, analysées sur deux sites en Côte d’Ivoire. Une carte génétique a été construite au préalable avec 308 marqueurs SSR. Le phénotypage des deux populations a révélé un déterminisme polygénique, sans effet monolocus majeur, avec, pour l’ensemble des exsudats fongiques étudiés, 13 QTL répartis sur 10 groupes de liaison et présentant des pourcentages d’explication R2 variant de 10 à 20 %. Sur les deux sites, deux QTL ont été identifiés conjointement pour le filtrat CCP et pour la toxine Cas1 purifiée à partir du filtrat CCP, démontrant l’importance de Cas1 pour la toxicité de ce filtrat. Des corrélations positives entre certains filtrats ont été observées, avec des profils de réponse similaires et des QTL communsLe test toxinique pourrait remplacer avantageusement la méthode d’inoculation conidienne. Cependant, la valeur prédictive du test pour la sensibilité des clones d’hévéa à la maladie CLF en plantation reste à caractériser. Une meilleure connaissance de l’épidémiologie du pathosystème H. brasiliensis x C. cassiicola contribuerait à l’objectif de sélection, et plus largement à la conception de méthodes de lutte ou d’évitement. / Hevea brasiliensis is the only commercial source of natural rubber. The CLF (Corynespora Leaf Fall) disease caused by the fungus Corynespora cassiicola is an important foliar pathology of the rubber tree in Asia and Africa. The disease progression is related with the development of new highly sensitive clonal varieties. A phenotyping method based on the measurement of electrolyte leakage in response to C. cassiicola exudates (culture filtrates or purified toxin) was developed and characterized. This test, without direct contact with the fungus itself, can be implemented without risk for plantations. After application of exudate samples on detached leaflets, conductivity is measured before (C1) and after (C2) autoclaving, and the percentage of electrolyte leakage %FE=100*C1/C2 is calculated. The influence of different factors such as leaf stage or the duration of incubation has been clarified. A positive correlation was found between the responses to the toxin test and to conidial inoculation. The pertinence of C1 correction by C2 was analyzed.Two toxin test studies have been carried out for a set of rubber clones, in France, with potted plants in greenhouse, and also in Ivory Coast, under the conditions normally considered for this test, with leaflets from budwood gardens. Repeatability between these two experiments was not perfect but nevertheless satisfying. The %FE character was found more sensitive and accurate than surface necrosis (SN) measurement. A significant variability in clone susceptibility and treatment virulence was observed, with clone x treatment interactions. The genetic determinism of sensitivity to C. cassiicola exudates was analyzed using the toxin test, by QTL detection on two populations of the biparental rubber family PB260 x RRIM600, in two field trials in Ivory Coast. A genetic map was previously built, using 308 SSR markers. Phenotyping of the two progeny populations revealed a polygenic determinism, without major monolocus effect. Over all the fungal exudates, 13 QTL distributed over 10 different linkage groups were identified, with percentages of explanation R2 varying from 10 to 20 %. On both sites, two QTL have been identified jointly for both the culture filtrate CCP and the toxin Cas1 purified from CCP. Positive correlations have been observed between some of the filtrates, with similar response profiles and common QTL.The toxin test could usefully replace the conidial inoculation method. However, the value of the test for predicting rubber clones susceptibility to the CLF disease in field conditions remains to be better characterized. Better understanding of the epidemiology of the H. brasiliensis x C. cassiicola pathosystem would contribute to the breeding objective, and more broadly to the design of control or avoidance methods.

Submitted by Alberto Vieira (martins_vieira@ibest.com.br) on 2018-01-19T14:15:15Z No. of bitstreams: 1 574810.pdf: 7112385 bytes, checksum: 6f6499c9ffa59ef9d17afb323e949d4f (MD5) / Made available in DSpace on 2018-01-19T14:15:15Z (GMT). No. of bitstreams: 1 574810.pdf: 7112385 bytes, checksum: 6f6499c9ffa59ef9d17afb323e949d4f (MD5) Previous issue date: 2000-11-20 / CAPES / Com o intuito de estimar a produção secundária de Emerita brasiliensis e Excirolana braziliensis, na Prainha (RJ), foram realizadas coletas mensais durante o período de junho de 1993 a maio de 1995. Foram estabelecidos três transectos na zona entre-marés, perpendiculares à linha d' água, cada qual dividido em cinco estratos, paralelos a linha d' água. De cada estrato foram retiradas quatro réplicas com um amestrador metálico de 0,04m2. A expectativa de vida das fêmeas de Emerita brasiliensis foi calculada em 10,3 e 8,0 meses, respectivamente, para o primeiro (93/94) e segundo anos (94/95). Para os machos este parâmetro foi estimado em tomo de 9 meses para os dois anos de estudo. A produção foi calculada utilizando-se os métodos de Hynes e de Crisp 3A, sendo estimada entre 15,589 e 12,378g(PSLC)m^-2a^-1 , para o primeiro ano amostral, e entre 53,326 e 47,534g(PSLC)m^-2a^-1 , para o segundo ano, sendo a produção do segundo ano, significativamente superior a do primeiro ano. Emerita brasiliensis apresentou... / With the aim of estimating the secondary production of Emerita brasiliensis and Excirolana braziliensis, at Prainha Beach (RJ), monthly samplings were performed in the period from June 1993 to May 1995. Three transects were established in the intertidal zone, perpendicular to the waterline, each divided into five strata parallel to the waterline. From each level, four replicates were taken using a 0.04 m2 metal sampler. The life span of Emerita brasiliensis females was calculated in 10.3 and 8.0 months for the first (93/94) and second (94/95) years, respectively. For the males this parameter was estimated around 9 months for the two years of study. The production was calculated by the methods of Hynes and Crisp 3A, and was estimated between 15.589 and 12.378g(AFDW)m^-2a^-1 for the first sample year, and between 53.326 and 47.534g(AFDW)m^-2a^-1 for the second year, which was significantly higher than the first year. For Emerita brasiliensis...