Phytoplasmas are bacteria with a persistent propagative transmission by insect vectors that generates direct and indirect interactions among them. In order to understand these interactions for maize bushy stunt phytoplasma (MBSP) and the leafhopper vector Dalbulus maidis (Hemiptera: Cicadellidae), two research lines were addressed. The first one aimed to determine the indirect effects of maize infection by MBSP on some biological and behavioral parameters of the vector, whereas a second line investigated direct interactions of the phytoplasma with D. maidis during its movement through the vector body following acquisition from plants, and associated microbiota. Indirect effects were investigated in choice experiments in which alighting and oviposition preferences by D. maidis were compared on healthy vs. MBSP-infected plants with variable incubation time (diseased plants with early and advanced symptoms, or still asymptomatic). Likewise, indirect effect of MBSP on the D. maidis biology was determined in two life table experiments in which the vector was reared on healthy vs. MBSP-infected plants expressing advanced disease symptoms or still asymptomatic. Choice experiments showed that alighting and oviposition preferences of D. maidis on MBSP-infected plants compared to healthy plants depend on the pathogen incubation period in the plant. The leafhopper preferred MBSP-infected plants over healthy ones during the asymptomatic phase of the disease, but rejected infected plants with advanced symptoms. The vector was able to acquire MBSP from asymptomatic infected plants shortly (3 days) after inoculation, but transmission efficiency increased when acquisition occurred at later stages of the pathogen incubation period (≥14 days) in the source plants and the test plants showed disease symptoms faster. These results suggest that MBSP modulates D. maidis preference for asymptomatic infected plants in the early stages of the crop, allowing rapid spread of this pathogen. Maize infection by the phytoplasma had a neutral effect on most life table parameters of D. maidis; a lower net reproductivity rate (Ro) was observed in the cohort reared on MBSP-infected plants with advanced symptoms, which was compensated to some extent by a higher sexual ratio. MBSP acquisition by all vector nymphal stadia was confirmed by PCR, and the pathogen as detected in both male and female reproductive organs. Concerning direct MBSP-vector interactions, transmission electron microscopy analyses showed phytoplasma-like cells in the midgut lumen, microvilli and epithelial cells, suggesting that MBSP enters the epithelium midgut through the microvilli wall. Within the epithelial cells, mitochondria and bacteria-like cells (possibly endosymbionts) were observed together with masses of phythoplasma-like cells. In the hemocoel, phytoplasma-like cells grouped into a matrix were also observed in association with bacteria-like cells similar to those observed in the midgut epithelium. Similar associations were found in the salivary gland. Interestingly, in-situ hybridization (FISH) technique revealed a variation in diversity and abundance of the microbiota in intestine and salivary glands of D. maidis adults over time after MBSP acquisition from plants. Sulcia sp., Cardinium sp. and eubacteria increased their abundance over time, whereas Rickettsia sp. decreased. The frequent association of the vector microbiota with the phytoplasma in some tissues of D. maidis suggests that endosymbiotic bacteria may play some role in MBSP-vector interactions. / Fitoplasmas são bactérias transmitidas de forma persistente propagativa por insetos vetores, havendo interações diretas e indiretas envolvendo tais organismos. Para entender algumas dessas interações no caso do enfezamento vermelho do milho, associado ao \'maize bushy stunt phytoplasma\' (MBPS) e à cigarrinha vetora Dalbulus maidis (Hemiptera: Cicadellidae), foram desenvolvidas duas linhas de trabalho. Na primeira determinou-se o efeito indireto de MBSP no comportamento e biologia do vetor, enquanto que na segunda estudaram-se as interações diretas do fitoplasma com o vetor durante sua movimentação através de órgãos internos e associação com a microbiota do inseto. O efeito indireto no comportamento foi demostrado em testes de escolha nos quais se variou o período de incubação de MBSP na planta (plantas com sintomas iniciais ou avançados da doença, e plantas infectadas assintomáticas) sobre a preferência para pouso e oviposição pelo vetor. O efeito indireto na biologia do D. maidis foi estabelecido em dois experimentos de tabela de vida, alimentando o inseto durante seu ciclo de vida em plantas de milho sadias ou plantas infectadas com o fitoplasma em dois tempos de incubação (plantas com sintomas avançados da doença e plantas infectadas assintomáticas). Os testes de escolha mostraram que a preferência de D. maidis para pouso e oviposição em plantas infectadas por MBSP em relação a plantas sadias depende do período de incubação do patógeno na planta. A cigarrinha preferiu plantas infectadas por MBSP na fase assintomática da doença, mas rejeitou plantas com sintomas avançados. Ocorreu aquisição do fitoplasma pelo vetor em plantas assintomáticas a partir de 3 dias após a inoculação, mas a eficiência de transmissão aumentou após 14 dias de incubação do patógeno na planta-fonte, e diminuiu o tempo para expressão de sintomas nas plantas-teste inoculadas. Os resultados sugerem que MBSP modula a preferência do vetor para plantas infectadas no estágio inicial da cultura de modo a permitir sua rápida disseminação. O efeito da infecção de plantas por MBSP na biologia de D. maidis mostrou ser neutro para a maioria dos parâmetros biológicos estimados; houve menor taxa líquida de reprodução (Ro), que foi compensada por maior razão sexual, para insetos criados em plantas com sintomas avançados. Verificou-se, por PCR, aquisição do fitoplasma por todos os estádios ninfais do vetor e sua presença em órgãos reprodutivos dos adultos. Por microscopia eletrônica de transmissão, observaram-se células do tipo fitoplasma no lúmen, microvilosidades e células epiteliais do mesêntero de D. maidis, sugerindo que o MBSP penetra no epitélio através das microvilosidades. No epitélio intestinal, foram observadas massas de células do tipo fitoplasma próximas a mitocôndrias e células bacterianas, possíveis endossimbiontes. Na hemocele, também foram observadas células do tipo fitoplasma agrupadas numa matriz, em associação com bactérias similares às observadas no intestino. Associações semelhantes foram observadas na glândula salivar. A técnica de FISH revelou uma variação na riqueza e abundância das espécies na da microbiota no mesêntero e glândula salivar de D. maidis em função do tempo após a aquisição de MBSP. A abundância de Sulcia sp., Cardinium sp. e eubacteria aumentou, enquanto que a de Rickettsia sp. decresceu. A associação frequente dessas bactérias com fitoplasma em alguns tecidos de D. maidis sugere que endossimbiontes possam ter um papel nas interações fitoplasma-vetor.

A hipermetioninemia é uma condição caracterizada por altos níveis de metionina no sangue e em outros tecidos, podendo causar danos neurológicos, hepáticos e musculares. Considerando que a placenta transfere a metionina do sangue materno para a circulação fetal e que pouco se sabe sobre o efeito da hipermetioninemia gestacional sobre o feto em desenvolvimento, o principal objetivo deste trabalho foi desenvolver um modelo animal de hipermetioninemia materna quimicamente induzido em ratas e utilizar o mesmo para investigar parâmetros bioquímicos (estresse oxidativo, atividade da Mg2+-ATPase, atividade e imunoconteúdo da Na+,K+-ATPase, número de neurônios, níveis de neurotrofinas, metabolismo energético, inflamação e apoptose), moleculares (expressão gênica da Na+,K+-ATPase) e histológicos (microscopia eletrônica) nos encéfalos da prole, bem como avaliar tarefas comportamentais (campo aberto, esquiva inibitória e reconhecimento de objetos). Também analisamos parâmetros de estresse oxidativo/nitrosativo no músculo esquelético e parâmetros de dano muscular e inflamação no soro da prole. A hipermetioninemia foi induzida em ratas através de duas injeções subcutâneas diárias de metionina durante todo o período gestacional. Um grupo de ratas recebeu a dose 1 (1,34 μmol/g peso corporal) e outro recebeu a dose 2 (2,68 μmol/g peso corporal). O grupo controle recebeu salina. Após o nascimento, um grupo de filhotes foi eutanasiado no sétimo dia de vida e outro grupo foi eutanasiado aos 21 dias. Ambas as doses aumentaram os níveis encefálicos de metionina das mães e a dose 2 aumentou os níveis de metionina nos encéfalos da prole. Após estabelecer o modelo, a dose 2 de metionina foi escolhida para estudar os efeitos do tratamento sobre a prole. Os testes bioquímicos subsequentes foram realizados nos filhotes de 21 dias, a histologia foi realizada na prole de 21 e 30 dias e os testes comportamentais foram realizados em filhotes de 30 dias. Os resultados demonstraram que a hipermetioninemia materna reduziu a atividade da Na+,K+-ATPase, Mg2+- ATPase, catalase e complexo II/succinato desidrogenase, o conteúdo de sulfidrilas, número de neurônios e níveis de NGF e BDNF, bem como aumentou os níveis de RNAm e imunoconteúdo da Na+,K+-ATPase nos encéfalos dos filhotes. Foram observados também alterações morfológicas, indicativas de degeneração celular nos neurônios da prole, e os testes comportamentais indicaram deficit de memória. Com relação aos danos musculares, houve um aumento na produção de espécies reativas de oxigênio e lipoperoxidação e uma redução do conteúdo de sulfidrilas, atividades das enzimas antioxidantes e nos níveis de nitritos no músculo esquelético da prole. A atividade da creatina cinase foi reduzida e os níveis de ureia e proteína C reativa foram aumentados no soro. Esses resultados foram acompanhados por perda de massa muscular. Tais achados mostraram que a hipermetioninemia gestacional induziu alterações bioquímicas, moleculares e histológicas no encéfalo e bioquímicas no músculo esquelético e soro dos filhotes, as quais podem contribuir para o entendimento dos mecanismos fisiopatológicos envolvidos nos danos neurológicos e musculares causados por essa condição. Ressaltamos a importância do desenvolvimento do referido modelo de hipermetioninemia gestacional que além de ampliar o entendimento da toxicidade de altos níveis metionina, também abriu perspectivas para novos estudos a respeito dos efeitos ocasionados pela exposição ao excesso de metionina devido a uma condição genética ou uma dieta rica em proteína durante a vida pré-natal. / Hypermethioninemia is a condition characterized by elevated levels of methionine in blood and other tissues and may cause neurological, hepatic and muscular damages. Considering that placenta transfers methionine from maternal blood to the fetal circulation and little is known about the effect of gestational hypermetioninemia on the developing fetus, the main objective of this work was to develop a chemically induced animal model of maternal hypermethioninemia in rats and to use it to investigate biochemical (oxidative stress, activity of Mg2+-ATPase, activity and immunocontent of Na+,K+-ATPase, number of neurons, neurotrophins levels, energy metabolism, inflammation, and apoptosis), molecular (gene expression of Na+,K+-ATPase) and histological parameters (electron microscopy) in encephalon of the offspring, as well as evaluate behavioral tasks (open field, inhibitory avoidance and object recognition). We also analyzed oxidative/nitrosative stress parameters in skeletal muscle and parameters of muscle damage and inflammation in serum of the offspring. Hypermethioninemia was induced in rats through two daily subcutaneous injections of methionine throughout the gestational period. A group of pregnant rats received dose 1 (1.34 μmol/g body weight) and the other received dose 2 (2.68 μmol/g body weight). The control group received saline. After birth, a first group of pups was euthanized at the 7th day of life and the second group at the 21st day of life. Both doses 1 and 2 increased methionine levels in the brain of the mother rats and dose 2 increased methionine levels in encephalon of the offspring. After establishing the experimental model, the highest dose of methionine was chosen to study the effects of treatment on offspring. The subsequent biochemical tests were performed on 21-day-old pups, histological analyses were performed on offspring of 21 and 30 days of age, and behavioral tests were performed on 30-day-old pups. The results demonstrated that maternal hypermethioninemia reduced Na+,K+-ATPase, Mg2+-ATPase, catalase and complex II/succinate dehydrogenase activities, sulfhydryl content, number of neurons and levels of NGF and BDNF, as well as increased levels of mRNA and immunocontent of Na+,K+-ATPase in the brains of the pups. Morphological changes indicative of cellular degeneration were also observed in offspring neurons, and behavioral tests indicated memory deficit. With regard to muscle damage, there was an increase in the production of reactive oxygen species and lipoperoxidation, and a reduction of the sulfhydryl content, antioxidant enzymes activities and in the levels of nitrites in skeletal muscle of the offspring. Creatine kinase activity was reduced and urea and C-reactive protein levels were increased in serum. These results were accompanied by loss of muscle mass. These findings showed that gestational hypermethioninemia induced biochemical, molecular and histological changes in the brain and biochemical changes in skeletal muscle and serum of pups, which may contribute to the understanding of the pathophysiological mechanisms involved in the neurological and muscular damages caused by this condition. We emphasize the importance of the development of this model of gestational hypermetioninemia that, in addition to increasing the understanding of toxicity of high methionine levels, also opened perspectives for new studies regarding the effects caused by exposure to excess methionine due to a genetic condition or a diet rich in protein during prenatal life.

Made available in DSpace on 2014-07-29T15:26:21Z (GMT). No. of bitstreams: 1 Tese Cleyde Barreto Valotto.pdf: 528164 bytes, checksum: 4db4ca9a7a45641fc2cd83c8e861e824 (MD5) Previous issue date: 2009-06-25 / The dengue and yellow fever are caused by Flavivirus, and are an importants problems in public health in the range of tropical and subtropical world. There is only available vaccine for yellow fever. The transmission of both diseases to humans is by the bite of female Aedes aegypti. The vector control is done through the elimination of breeding potential, application of larvicides in collections of water for adults vectors space applications of insecticides. The problems arising from continuous use of chemical insecticides have been the emergence of resistant populations to these products and concern about the environmental damage caused by slow degradation. In search of alternatives for the vector control, studies are being developed with substances of botanical origin, with the aim to control the vectors and preserve the environment. The use of natural insecticides has some advantages when compared to synthetic because they are renewable and have rapid degradation. Moreover, its composition is quite complex, reducing the speed of emergence of resistance and be of low toxicity to mammals, birds and environment. This study presents the morphohistologic and ultrastructural changes of catechist tannin extracted from Magonia pubescens, and the Diterpene extracted from Copaifera reticulata on the larvae of A. aegypti with the objective to evidence the mechanisms of action of these substances. For light microscopy, the 3rd stage larvae were subjected to a solution of catechist tannin 37ppm and the diterpene 9ppm, where they remained for up to 24h. Larvae that reached lethargic state were collected and fixed in 4% in the paraformoldehyde buffer 0.1 M sodium cacodilate pH 7.2, included in resin and the slides stained with hematoxylineosin technique. For electron microscopy, larvae were subjected to the same concentrations of catechist tannin and diterpene, where they remained for 22 h. Larvae that reached lethargic state were collected and dissected and their gut fixed in 2% glutaraldehyde, 2% paraformoldehyde, 3% sucrose in sodium buffer cacodilate 0.1 M pH 7.2 and post fixed in osmium tetroxide at 1% in cacodilate sodium 0.1 M. After this they were contrasted with uranyl acetate 1%, dehydrated, embedded and polymerized. The ultra sections were contrasted with uranyl acetate 3% and lead citrate and were caracterized by eletron microscopy. The changes caused by catechist tannin observed by light 20 and electron microscopy were high vacuolation, absence of cytoplasmic limits, cellular aging, cell disruption, vesicular apical formation with release of cytoplasmic content, increase in intercellular spaces, detachment of cells from the basal lamina and structural change of microvilli. Changes by diterpene by light and electron microscopy were high cytoplasmic vacuolation, cells and nuclei hypertrophy, degeneration of the edge brush, vesicular apical formation with release of cytoplasmic contents of cells, stratification of the epithelium and folds in the peritrophic matrix. / A dengue e a febre amarela urbana são doenças causadas por Flavivirus, constituindo problemas de importância em saúde pública nas faixas tropical e subtropical do mundo. Há apenas vacina disponível para a febre amarela. A transmissão de ambas as doenças ao homem se faz pela picada da fêmea de Aedes aegypti. O controle do vetor é feito através da eliminação dos criadouros potenciais, aplicação de larvicidas em coleções de água e, para os adultos, aplicações espaciais de inseticidas. Os problemas originados do uso contínuo de inseticidas químicos foram o aparecimento de populações resistentes a esses produtos e a preocupação com os danos ambientais provocados pela degradação lenta destes produtos. Na busca de alternativas de controle ao vetor, estudos estão sendo desenvolvidos com substâncias de origem botânica, com o intuito de controlar os vetores e preservar o meio ambiente. A utilização dos inseticidas naturais tem algumas vantagens quando comparados aos sintéticos, por serem renováveis e de degradação rápida. Além disso, a sua composição é bastante complexa, diminuindo a velocidade do aparecimento de resistência e é de baixa toxicidade para mamíferos, aves e meio ambiente. Neste trabalho, apresentam-se as alterações morfohistológicas e ultraestruturais do tanino catéquico extraído da Magonia pubescens, e do Diterpeno extraído da Copaifera reticulata sobre as larvas de A. aegypti a fim de evidenciar os mecanismos de ação dessas substâncias. Para a microscopia de luz, larvas de 3° estádio foram submetidas a soluções a 37ppm de tanino catéquico e 9ppm do diterpeno, onde permaneceram por até 24h. As larvas que atingiram estado letárgico foram coletadas e fixadas em paraformaldeído a 4% em tampão cacodilato de sódio 0.1 M pH 7.2, incluídas em resina e as lâminas coradas pela técnica de Hematoxilina-eosina. Para a microscopia eletrônica, as larvas foram submetidas às mesmas concentrações do tanino catéquico e diterpeno, onde permaneceram por 22 h. As larvas que atingiram estado letárgico foram coletadas e dissecadas e seus tubos digestórios fixados em 2% glutaraldeído, 2% paraformaldeído, 3% sacarose em tampão cacodilato de sódio 0.1 M pH 7.2 e pós fixado em tetróxido de ósmio 1% em cacodilato de sódio 0.1M. Em seguida foram contrastados com acetato de uranila a 1%, desidratados, emblocados e polimerizados. Os cortes 18 ultrafinos foram contrastados com acetato de uranila 3% e citrato de chumbo, depois eletromicrografados. As alterações causadas pelo tanino catéquico observadas pela microscopia de luz e eletrônica foram a alta vacuolização, ausência dos limites citoplasmáticos, morte por autofagia, desorganização celular, formação vesicular apical com liberação de conteúdo citoplasmático, aumento do espaço intercelular, desprendimento de células da lâmina basal e mudança estrutural dos microvilos. As alterações pelo diterpeno, pela microscopia de luz e eletrônica foram alta vacuolização citoplasmática, hipertrofia celular e dos núcleos, degeneração do bordo em escova, formação vesicular apical com liberação de conteúdo citoplasmático das células, estratificação do epitélio e dobras na matriz peritrófica.

Este estudo apresenta um painel de dados hematológicos, bioquímicos, morfológicos e citoquímicos para um grupo de serpentes dos gêneros Bothrops e Crotalus mantidas em cativeiro no Instituto Butantan. Para tanto, amostras de sangue foram colhidas da veia coccígea ventral e processadas de acordo com métodos padronizados. Os resultados alcançados foram analisados para determinar diferenças interespecíficas e sexuais. A avaliação citoquímica incluiu Sudan Black B (SBB), Ácido Periódico de Schiff (PAS) e Benzidina Peroxidase (BP). Os resultados hematológicos e bioquímicos mais relevantes indicaram um número significativamente mais alto de basófilos em B. jararaca e níveis mais altos de cálcio nas fêmeas da maioria das espécies. Os azurófilos marcaram positivamente para todas as colorações. A diferenciação entre trombócitos e linfócitos foi facilmente obtida com PAS. Os basófilos marcaram positivamente apenas com PAS em Bothrops alternatus. A marcação em heterófilos variou consideravelmente entre as espécies. Ultra-estruturalmente, os leucócitos foram semelhantes ao já descrito em literatura com algumas variações no tipo dos grânulos dos basófilos e heterófilos. Os grânulos dos basófilos apresentaram-se redondos em sua maioria, com tamanho e densidade variadas, enquanto que os nos heterófilos foram heterogêneos em forma, tamanho e densidade. / This study reports a panel for hematological, biochemical, morphological and cytochemical data for a group of captive snakes belonging to the genus Bothrops and Crotalus mantained at Instituto Butantan, São Paulo, Brazil. Blood samples were collected from ventral coccigeal vein and were processed according to standard protocols. Cytochemical staining including Sudan Black B (SBB), Periodic Acid-Schiff (PAS) and Benzidine peroxidase (BP) were conducted. Blood values were evaluated to determine interspecific and sex differences. We found a significant increase of basophils in Bothrops jararaca and higher levels of calcium in females. Azurophils stained positively for all stains and a differentiation between lymphocytes and thrombocytes was easily obtained with PAS stain. Basophils stained positively only with PAS in Bothrops alternatus. Heterophils staining varied between species. The ultrastructure of leucocytes were similar within described in literature, with some variations on granules of basophils and heterophils. Basophils granules were round, with heterogeneous size and density; whereas, heterphils granules were heterogeneous in shape, size and density.

Enquanto a toxidez de manganês (Mn) pode ser um problema comum nas regiões tropicais com predominância de solos ácidos, a deficiência deste nutriente, relatada em diversas espécies vegetais, tem sido atribuída principalmente ao uso excessivo de calcário. O estudo foi desenvolvido em casa-de-vegetação e teve como objetivo avaliar as causas de maior tolerância à toxidez de manganês e susceptibilidade à deficiência. Os cultivares de soja [Glycine max (L.) Merrill] Santa Rosa, IAC-15 e IAC-Foscarin 31 foram desenvolvidos em solução nutritiva (pH 4,97) com cinco níveis de Mn (0,5; 1,0; 2,0; 4,0 e 10,0 µmol/L), nos experimentos de eficiência de absorção e de uso (experimento 1), enquanto que para o estudo de tolerância ao excesso (experimento 2), as doses foram de 2,0; 100,0; 150,0; 200,0 e 250,0 µmol/L. Para as observações de anatomia e ultraestrutura (experimento 3), foram utilizadas três concentrações de Mn na solução nutritiva: 0,5; 2,0 (controle) e 200,0 µmol/L. O delineamento estatístico foi o de blocos completos ao acaso em esquema fatorial 5x3 (cinco doses de Mn e três cultivares), com três repetições. No experimento 1, os sintomas visuais de deficiência manifestaram-se, nos três cultivares, a partir do décimo dia após o início dos tratamentos, no estádio V3. Na condição do fornecimento das duas mais baixas doses, tanto as folhas inferiores quanto as superiores exibiram os sintomas de carência. As faixas de valores de concentração de Mn no tecido associadas à deficiência variaram entre 9 a 16 mg/kg nas folhas superiores e entre 15 a 26 mg/kg nas folhas inferiores. Ocorreu interação significativa entre as doses de Mn e os cultivares de soja para as produções de massa seca da parte aérea, de raízes e total. Para a produção de grãos e pericarpos constatou-se significância para as doses de Mn somente para IAC-15 e Santa Rosa. O cultivar IAC-Foscarin 31 não apresentou resposta às doses de Mn fornecidas, porém, mostrou-se eficiente na utilização de Mn para a produção de grãos. Os índices de eficiência de produção de grãos (IEP) calculados para IAC-Foscarin 31 (Mn-eficiente IEP>1,0), IAC-15 e Santa Rosa (Mn-pouco eficiente 0,5<IEP<1,0) foram, respectivamente, de 1,41; 0,78 e 0,66. Apesar do menor comprimento e superfície total de raízes produzidos pelo cultivar IAC-Foscarin 31, na condição de baixo suprimento de Mn, o acúmulo de Mn nas raízes, nos grãos e nas plantas foi superior ao dos demais cultivares. Os cultivares apresentaram comportamentos distintos na absorção de Mn, sendo o IAC-Foscarin 31 o mais eficiente na absorção (maior Vmax e menores valores de Km e Cmin) e o Santa Rosa o menos eficiente (menor valor de Vmax e maiores valores de Km e Cmin). No experimento 2, os cultivares IAC-15 e Santa Rosa exibiram sintomas visuais de toxidez de Mn. Os teores de Mn nas folhas associados ao excesso foram de 1.000,0 mg/kg. Entretanto, o IAC-15 apresentou maior produção de grãos e de massa seca da parte aérea, acompanhado pelo IAC-Foscarin 31 (intermediário) e o Santa Rosa (menor produção). O maior comprimento total de raízes, as maiores produções de massa seca de raízes e acúmulo de Mn neste tecido conferiram ao IAC-Foscarin 31 maior tolerância ao excesso de Mn na solução. A taxa de crescimento absoluto do sistema radicular, no início do desenvolvimento das plantas, pode conferir maior eficiência de absorção e maior tolerância ao excesso do elemento. As quantidades de Mn absorvidas no simplasto e apoplasto variaram entre os cultivares. A organização das células do mesófilo foliar (células parenquimáticas), do córtex radicular e das ultraestruturas celulares, como os cloroplastos, grãos de amido e glóbulos de lipídios é afetada pelas desordens nutricionais em Mn, principalmente, no cultivar IAC-15 e Santa Rosa. Há evidencias nas diferenças genotípicas entre os cultivares e que vários mecanismos atuam conjuntamente tanto na eficiência de absorção do Mn, em condição de baixa disponibilidade no substrato, quanto na capacidade de tolerar níveis excessivos do metal. / Manganese (Mn) toxicity may be a major constraint for crop production in acid soils of tropical countries. Nevertheless, deficiency has been recognized as a worldwide nutritional disorder frequently related to excessive lime. A greenhouse experiment was carried out with the objective of evaluating the efficiency of uptake of manganese and use in three-soybean cultivars [Glycine max (L.) Merrill] as well as their tolerance to manganese excess. Cultivars (Santa Rosa, IAC-15 and IAC-Foscarin 31) were evaluated under five rates of Mn (0.5; 1.0; 2.0; 4.0 and 10.0 µmol/L) in the nutrient solution (pH = 4.97) with respect to efficiency of uptake and of use. Other experiment was carried out with the objective of evaluating tolerance to manganese in the same cultivars when grown at five doses (2.0; 100.0; 150.0; 200.0 and 250.0 µmol/L). For anatomical and ultrastructures studies the Mn doses tested were 0.5; 2.0 (control) and 200.0 µmol/L. The experimental design was a 5x3 factorial of five Mn doses and three cultivars, set in a randomized block, with three replications. In experiment 1, visual symptoms of Mn deficiency (in both upper and lower leaves at the two lowest rates), started at the 10th day after transplanting of the three cultivars, at the V3 vegetative growth stage. The average of Mn concentration in the deficient upper leaves was close to 9 to 16 mg/kg, ranging from 15 to 26 mg/kg in the lower ones. Results showed significant interaction of Mn rates and cultivars with the dry matter of the tops, roots and total. Mn rates promoted increase in grain and pericarps yield only on IAC-15 and Santa Rosa cultivars. IAC-Foscarin 31 did not show grain yield responses to Mn supply. However, the indices (grain yield efficiency index) calculated for IAC-Foscarin 31, IAC-15 and Santa Rosa were, respectively, of 1.41; 0.78 and 0.66. They were therefore classified as Mn-Efficient (IEP>1.0) and of Mn-intermediate efficiency use (0.5<IEP<1.0). Although IAC-Foscarin 31 presented the lowest total root length and total root surface of the three cultivars even at low Mn doses, it had higher amount of Mn in roots, grains and whole plant than the other cultivars. The cultivars had distinct behavior in absorption of Mn. IAC-Foscarin 31 was more efficient. It showed higher Vmax and lower Km and Cmin than Santa Rosa and IAC-15. In experiment 2, IAC-15 and Santa Rosa leaves showed visual symptoms of Mn toxicity. The average Mn concentration in these leaf blades was close to 1,000.0 mg/kg. Nevertheless, IAC-15 had grain and dry matter in tops higher than IAC-Foscarin 31 (intermediate yield) and Santa Rosa. The highest total root length and root dry matter yield as well as root Mn content in IAC-Foscarin 31 contributes to explain differences in tolerance to Mn excess. The root absolute growth rate, at beginning of vegetative growth, may confer Mn uptake efficiency and tolerance as well. Total, symplastic and apoplastic Mn absorption varied among the three soybean genotypes. The effects of manganese deficiency and toxicity on the morphology of the roots (cortex, stele and vascular cylinder) and leaves (mesophyll tissues) and ultrastructure, like chloroplasts and its starch grains and lipid globules showed distinct degrees for the cultivars, being IAC-15 and Santa Rosa more affected. There are clear evidences of genotypics differences among the cultivars and that joint mechanisms are involved in the efficiency of absorption of low Mn, in the substrate, as well as in the tolerance to high levels.

<p>Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release <i>in vivo</i> and <i>ex vivo</i> during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure.</p><p>In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO.</p><p>The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM.</p><p>ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.</p>

Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release in vivo and ex vivo during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure. In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO. The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM. ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.

Wood fibres can be used as reinforcement in plastics for load carrying purposes. Some advantages compared with conventional man-made fibres are that wood fibres come from a renewable resource, have high specific stiffness and strength, are generally less hazardous to health, biodegradable, and can be manufactured at low cost and high volumes. A clear disadvantage with cellulose-based materials for structural use is their dimensional instability in humid environments. The hygroelastic properties are of high importance in materials development of improved wood-fibre composites. This work deals with the stiffness and hygroexpansion of wood fibres for composite materials. The long-term aim is to design engineered wood fibre composites based on better basic knowledge of wood fibres. Mechanistic models have been used to link the fibrous microstructure with macroscopic composite engineering properties. The properties have been characterized experimentally for various wood-fibre composites and their fibre-mat preforms, by means of curvature measurements at various levels of relative humidity, as well as tensile and compressive tests. From these test results and microstructural characterization, the longitudinal Young’s modulus and transverse coefficient of hygroexpansion of wood fibres were identified by inverse modelling. Some effects of various pulp processes and fibre modifications on the elastic properties of the fibre were observed, illustrating how the mixed experimental-modelling approaches can be used in more efficient materials screening and selection. An improved micromechanical analysis for wood-fibre composites has been presented. The model is more appropriate to combine with laminate analogy, to link fibre properties on the microscale to the macroscopic composite properties and vice versa. It also offers the possibility to include the effects of ultrastructure since it can account for an arbitrary number of phases. An approach to model ultrastructure-fibre property relations has been demonstrated. It includes analytical modelling of multilayered cylindrical fibres as well as finite element modelling of fibres with irregular geometry characterized with microscopy. Both approaches are useful and could be combined with experiments to reveal insights that can pave way for a firmer link between the wood fibre ultrastructure and wood fibre properties. / QC 20100914

The objective of my thesis was to develop novel techniques and methods to probe the dielectric properties of biomembranes in air and their natural environment - liquid solution. The dielectric constant ε(r) of biomembranes is a parameter especially important in cell electrophysiology as it ultimately determines the ion membrane permeability, the membrane potential formation or the action potential propagation velocity, among others. However, no technique is able to provide this quantity with the required nanoscale spatial resolution and in electrolyte solution. AFM is an extremely versatile tool to investigate electric properties at the nanoscale, and hence constitutes a good candidate technique to approach the quantification of the nanoscale dielectric properties of biomembranes. Although a few AFM techniques exist, capable of investigating polarization properties, it remains difficult to extract quantitative values of εr from the measurements, especially in liquid environment. One reason for this is on the instrumental side, since for studies at the nanoscale very small quantities have to be measured, that can be easily overwhelmed by electronic noise as it maybe for example the case in current sensing based techniques. Electrostatic Force sensing techniques may in principle have an advantage here, since the used cantilevers for force detection are extremely sensitive and naturally, undesired nonlocal electrical signals from the cantilever are suppressed. Another important aspect is attributed to a lack of sufficiently precise quantitative models to relate measured force with the dielectric constant value of the sample. Indeed, for measurements on insulating substrates like mica or glass that are sometimes required for biological samples, still no quantitative model is available. Moreover, successful measurements of dielectric properties in liquid media, that is fundamental for the functionality of some biological samples, has not been shown until now. As consequence of the existing limitations for quantitative dielectric imaging the objectives of this work were to extend the quantitative capabilities of Electrostatic Force Microscopy to image the dielectric constant of biomembranes with nanoscale spatial resolution. In particular, the three objectives I addressed in the work are: 1. To evaluate the possibility to perform quantitative dielectric measurement on biomembranes on metallic substrates and in air with Electrostatic Force Microscopy that may offer higher precision with respect to current sensing techniques. 2. To extend the applicability of quantitative dielectric measurement to the case of thick insulating substrates in order to facilitate its use with biomembranes that cannot be prepared on metallic substrates. 3. To develop a setup for dielectric imaging in liquid environment based either on current detection or on the principles of electrostatic force microscopy. Finally to perform nanoscale dielectric measurements on bio-membranes in their natural liquid environment. How each of these objectives could be reached is detailed in my thesis. / El objetivo de mi tesis era desarrollar nuevas técnicas y métodos para medir las propiedades dieléctricas de biomembranas en aire y en su medio natural, es decir, en solución líquida. La constante dieléctrica (εr) de las biomembramas es un parámetro especialmente importante en la electrofisiología celular, ya que fundamentalmente determina la permeabilidad iónica de la membrana, la formación del potencial de membrana o la velocidad de propagación del potencial de acción, entre otros. El AFM es una herramienta extremadamente versátil para investigar propiedades eléctricas a nanoescala, y por ello constituye una buena técnica candidata para la cuantificación de las propiedades dieléctricas de las biomembranas a nanoescala. Aunque existen algunas técnicas basadas en el AFM capaces de investigar las propiedades de polarización, continúa siendo difícil extraer valores cuantitativos de εr de las medidas, especialmente en medio líquido. Una de las razones radica en la parte instrumental, ya que para los estudios en la nanoescala tienen que medirse cantidades muy pequeñas, y este proceso puede verse entorpecido por ruido electrónico como puede ser el caso, por ejemplo, en las técnicas basadas en la detección de corriente eléctrica. En principio, las técnicas de detección de fuerza electroestática disponen aquí de una ventaja, ya que las sondas utilizadas para la detección de la fuerza son extremadamente sensibles y naturalmente se suprimen señales eléctricas no locales y no deseadas. Otro aspecto importante se atribuye a la falta de modelos cuantitativos suficientemente precisos para relacionar la fuerza medida con el valor de la constante dieléctrica de la muestra. En realidad, para realizar medidas en sustratos aislantes como son la mica o el vidrio, que a veces son necesarios para muestras biológicas, todavía no se dispone de ningún modelo cuantitativo. Por otra parte, hasta ahora no se han publicado medidas de propiedades dieléctricas en medio líquido, que para algunas muestras biológicas es fundamental para mantener la funcionalidad. Como consecuencia de las limitaciones existentes de las medidas dieléctricas, el objetivo de este trabajo fue extender las capacidades cuantitativas de la Microscopía de Fuerzas Eletroestáticas para hacer imágenes dieléctricas de biomembranas con resolución espacial a nanoescala en substratos conductores, aislantes y en medio líquido.

La présente Thèse représente une importante contribution à la connaissance de l’ultrastructure de la spermiogenèse et du spermatozoïde des Digènes. Nous présentons des nouvelles données spermatologiques concernant huit espèces appartenant à sept familles et superfamilles de Digènes. Ils s’agissent de Brachycoelium salamandrae (Gorgoderoidea : Brachycoeliidae), Diplodiscus subclavatus (Paramphistomoidea : Diplodiscidae), Elstia stossichianum et Wardula capitellata (Microscaphidioidea : Mesometridae), Euryhelmis squamula (Opisthorchioidea : Heterophyidae), Mediogonimus jourdanei (Microphalloidea : Prosthogonimidae), Robphildollfusium fractum (Lepocreadioidea : Gyliauchenidae), et Rubenstrema exasperatum (Plagiorchioidea : Omphalometridae). Les résultats obtenus sur la spermiogenèse et/ou le spermatozoïde sont comparés avec le reste de Digènes. De plus, nous discutons de l’importance de certains caractères ultrastructuraux pour une meilleure compréhension des relations phylogénétiques chez les Digènes. La spermiogenèse chez les Digènes est assez homogène. Cependant, il y a certains aspects comme la variabilité du nombre de bandes du corps intercentriolaire et l’angle de rotation flagellaire qui méritent une attention particulière. Concernant le spermatozoïde, nous avons proposé, après une analyse précise des données spermatologiques disponibles, sept modèles de spermatozoïde chez les Digènes. Les caractères du spermatozoïde les plus utiles pour l’établissement de ces modèles sont : (a) le type d’axonème/es (patron 9+‘1’ des Trepaxonemata, 9+‘1’ spécial et 9+0), (b) l’extrémité antérieure du spermatozoïde (avec un ou deux axonèmes), (c) la présence/absence de l’association « expansion latérale+ornementations externes+microtubules corticaux » ou « ornementations externes+microtubules corticaux », (d) la présence ou l’absence d’ornementations externes et leur localisation sur le spermatozoïde, (e) le patron des microtubules corticaux parallèles, absents ou disposés en un ou deux champs, et (f) le caractère terminal dans l’extrémité postérieure du spermatozoïde (noyau, axonème ou microtubules corticaux). D’autres caractères comme les corps épineux ou les boutons cytoplasmiques ornementés peuvent avoir une importance particulière pour certains taxons. La récente description des corps épineux suppose une reconsidération de certaines études anciennes où ces structures peuvent être mal interprétés ou considérés comme des artéfacts. Les boutons cytoplasmiques ornementes sont un nouveau caractère trouvé chez les Mesometridae (E. stossichianum et W. capitellata). Les spermatozoïdes des espèces étudiées dans cette thèse appartiennent aux modèles 1, 2 ou 3. Le modèle 1, caractérisé par la présence de l’association « expansion latérale+ornementations externes+microtubules corticaux », est présent chez Diplodiscus subclavatus, Elstia stossichianum et Wardula capitellata. De plus, la plupart des Echinostomatoidea, Paramphistomoidea et Pronocephaloidea présentent ce modèle. Le modèle 2 est caractérisé par l’association « ornementations externes+microtubules corticaux » et par une extrémité postérieure du spermatozoïde contenant le noyau. Il est présent chez Brachycoelium salamandrae, Robphildollfusium fractum et Rubenstrema exasperatum. Finalement, Euryhelmis squamula et Mediogonimus jourdanei présentent un spermatozoïde du modèle 3 comme chez certains Opisthorchioidea et certaines familles telles que les Allocreadiidae, Deropristidae, Lepocreadiidae et Troglotrematidae. Le modèle 3 diffère du modèle 2 par l’extrémité postérieure contenant un axonème. Le modèle 4 est similaire aux modèles 2 et 3, mais l’extrémité postérieure contient seulement des microtubules corticaux. Il est présent chez les Opecoelidae et Opistholebetidae. Le modèle 5 exhibe un seul champ (ventral) de microtubules corticaux et ne présente pas l’association « ornementations externes+microtubules corticaux ». Il est observé chez les Faustulidae, Hemiuridae et les Lecithasteridae. Le modèle 6 est observé chez les schistosomes. Ils présentent un spermatozoïde aberrant, non filiforme, avec un seul axonème du type 9+‘1’ spécial. Finalement, le modèle 7, caractérisé par la présence d’axonèmes du type 9+0 et par l’absence de microtubules corticaux, est établit seulement pour Didymozoon sp. / The present Thesis represents a contribution to the knowledge about the ultrastructure of spermiogenesis and of the mature spermatozoon of digeneans. We present for the first time spermatological data concerning eight species belonging to seven families and superfamilies: Brachycoelium salamandrae (Gorgoderoidea: Brachycoeliidae), Diplodiscus subclavatus (Paramphistomoidea: Diplodiscidae), Elstia stossichianum and Wardula capitellata (Microscaphidioidea: Mesometridae), Euryhelmis squamula (Opisthorchioidea: Heterophyidae), Mediogonimus jourdanei (Microphalloidea: Prosthogonimidae), Robphildollphusium fractum (Lepocreadioidea: Gyliauchenidae), and Rubenstrema exasperatum (Plagiorchioidea: Omphalometridae). A global analysis of the spermatological characteristics of these species and those already described in the Digenea, allow us to establish seven sperm models within digeneans. The importance of several ultrastructural characteristics, combined with the proposed models are discussed in order to contribute to a better understanding of digenean phylogenetic relationships.