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.

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.

Avaliou-se a tipificação das fibras musculares e determinou-se parâmetros morfoquantitativos dos efeitos da imobilização por 2 e 4 semanas. Após o período experimental, os músculos (mm.) foram corados por HE e Picro-sírius. Para a tipificação das fibras, utilizou-se as reações da ATPase e NADH-tr; a análise ultraestrutural procedeu-se com microscopia eletrônica de transmissão. Nos resultados, alguns mm. imobilizados (Im) exibiram núcleos centralizados e fibras tipo IIB com formas elusivas. O perimísio destes mm. se espessou, sendo constituído por fibras colágenas tipo III. Os fusos musculares apresentaram espessamento da cápsula. Houve alterações nas linhas Z e modificação de outras ultraestruturas como as mitocôndrias. A área de secção transversa dos mm. Im por 4 semanas foi menor. As fibras do tipo IIB reduziram a área de perfil e aumentaram a densidade, além disso, se ampliou a porcentagem das fibras do tipo IIA. Portanto, a imobilização promoveu alterações nos fusos e esqueleto fibroso muscular, assim como transformou fibras do tipo IIB em IIA. / The effects of muscle immobilization for 2 and 4 weeks were studied by determining morphoquantitative parameters and muscle fiber-typing. After the trial period the muscles (mm.) were subject to HE and Picrosirius stain. The reactions of ATPase and NADH-tr were used for muscles fiber-typing. The ultrastructural analyses were made by transmission electron microscopy. The results showed changes in some of immobilized (Im) mm., it exhibited centralized nuclei cells and type IIB fibers took indefinite forms. The perimisium of Im mm. thicker up, and consisted of type III collagen fibers. Also the muscle spindles capsule thickened. Changes in the Z lines and other ultrastructures such as mitochondria were noticed. The cross-section area of Im mm. for 4 weeks was lower. The type IIB fibers reduced the profile area and density increased. Also the percentage of type IIA fibers expanded. Therefore the immobilization promoted changes in muscle spindles, fibrous skeleton morphology and provide an interconversion of muscle fibers type IIB into IIA.

Submitted by (edna.saturno@ufrpe.br) on 2016-11-30T13:46:24Z No. of bitstreams: 1 Thiago Jose de Souza Alves.pdf: 713733 bytes, checksum: 897fa22e09fbd143d16a268c4b4fffe9 (MD5) / Made available in DSpace on 2016-11-30T13:46:24Z (GMT). No. of bitstreams: 1 Thiago Jose de Souza Alves.pdf: 713733 bytes, checksum: 897fa22e09fbd143d16a268c4b4fffe9 (MD5) Previous issue date: 2013-07-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The biological control of the boll weevil in Brazil is realized mostly by insect parasitoids, in which Bracon vulgaris Ashmead, a gregarious larval ectoparasitoid, is the most important biocontrol agent responsible for natural reduction of boll weevil populations in cotton ecosystem in the Northeast of Brazil. Even though it has a major importance in cotton production, there is no much data referring to parasitism of A. grandis by B.vulgaris, such as the effect of age, photoperiod and host availability in the parasitism behavior of this braconid wasp, as well as the morphological mechanisms involved in the parasitoid-host interaction. Therefore, this study investigated the morphological components involved in the success of parasitism of boll weevil by B. vulgaris, and the effects of age, photoperiod and host availability in the parasitism behavior of this natural enemy. Through techniques of scanning electron microscopy that the B. vulgaris ovipositor is a multifunctional structure whose composition and morphology are closely related to the role they play, and the parasitoid’s antennae present four types of sensilla (trichoids, basiconical, coeloconical, and placodes) that act as olfactory and gustatory receptors and or express tactile, therm, and hygro-reception functions. Behavioral tests showed that female five days old, the shortest time to host location occurred 8h after light exposure, when parasitoids were most active searching for the host. In relation to the behavioral repertoire of parasitism, B. vulgaris showed the sequence of following behaviors: host search, static, grooming, host acceptance, oviposition and resting. An associative evaluation of the morphology of sensory structures and behavioral studies allowed the better understanding the high rates of parasitism presented by B. vulgaris on A. grandis. / O controle biológico natural do bicudo-do-algodoeiro, no Brasil, é realizado principalmente por parasitoides. Entre eles, Bracon vulgaris Ashmead (Hymenoptera: Braconidae), um ectoparasitoide larval, constitui o principal agente biológico responsável pela redução natural das populações do bicudo no agroecossistema algodoeiro no Nordeste brasileiro. Embora possua importância na cotonicultura, são escassos os dados referentes à ação de parasitismo de B.vulgaris sobre Anthonomus grandis Boh. (Coleoptera: Curculionidae) tais como o efeito da idade, fotoperíodo e disponibilidade de hospedeiro no comportamento de parasitismo deste parasitoide, assim como os mecanismos morfológicos envolvidos nesta interação parasitoide/hospedeiro. Deste modo, este estudo avaliou os componentes sensoriais envolvidos no sucesso do parasitismo de B. vulgaris sobre A. grandis, bem como o efeito da idade, fotoperíodo e disponibilidade de hospedeiro no comportamento de parasitismo. Através de técnicas de microscopia eletrônica de varredura foi verificado que o ovipositor de B. vulgaris é um órgão multifuncional cuja morfologia e composição estão intimamente relacionadas às funções que exerce, e suas antenas apresentaram quatro tipos de sensilas (tricóides, basicônicas, coelocônicas e placóides) que atuam como receptores olfativos e gustativos e ou expressam função tátil, termo e de higrorrecepção. Os testes comportamentais, por sua vez, mostraram que fêmeas de B. vulgaris com cinco dias de idades, quando submetidas às densidades de seis e 12 larvas hospedeiras por parasitoide, apresentaram as maiores taxas de parasitismo, sendo que o menor tempo para localização do hospedeiro ocorreu após 8h de exposição à luz, onde os parasitoides se mostraram mais ativos em busca do hospedeiro. Quanto ao repertório comportamental de parasitismo, B. vulgaris apresentou os seguintes eventos comportamentais em sequência: busca, parada, autolimpeza, aceite, oviposição e repouso. Através da avaliação associativa da morfologia das estruturas sensoriais e de estudos comportamentais, foi possível compreender melhor os altos índices de parasitismo apresentados por B. vulgaris sobre A. grandis.

Submitted by (edna.saturno@ufrpe.br) on 2016-12-01T14:58:58Z No. of bitstreams: 1 Franklin Magliano da Cunha.pdf: 1121804 bytes, checksum: 66cbfdb39feb9b059c8f94887174e30c (MD5) / Made available in DSpace on 2016-12-01T14:58:58Z (GMT). No. of bitstreams: 1 Franklin Magliano da Cunha.pdf: 1121804 bytes, checksum: 66cbfdb39feb9b059c8f94887174e30c (MD5) Previous issue date: 2011-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Despite of the Bt formulation and transgenic Bt plants efficiency against several lepidopteran species, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) exhibits low susceptibility to the majority of them. The reasons to this low susceptibility specifically to toxins Cry1Ac and Cry1Ab are not fully clarified. Also, it is possible that species moderately or not susceptible to Cry1Ac can convey the toxin to the third trophic level. This study investigated response of S. frugiperda related to Bt with three hypotheses about its low suscepbility: regenerative cells from midgut are activated by Cry1Ac protein and renovate the epithelial cells (i) in hemolymph they increase the nitric oxide and phenoloxidases as a toxin response (ii) and the tolerance is associated to the qualitative and quantitative variations from hemocytes. We still tested the hypothesis that S. frugiperda fed with Bt-cotton can induce alterations in the ultrastructure and histochemistry of digestive cells, in humoral parameters, hemogram and ultrastructure of hemocytes of Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae), a generalist predator. It was concluded that Cry1Ac in Bt-cotton is not toxic enough to kill the armyworm, but it induce to humoral and cellular alterations as signal of response to xenobiotic. The Cry1Ac toxin ingested by S. frugiperda can expose the third trophic level (P. nigrispinus) and it causes alterations in the distribution of glycogen, lipids and calcium caused by perimicrovillar matrix disorganization. The TEM revealed the presence of spherocrystals that appear to be involved in the defense against the toxin. The humorals parameters and the hemogram of P. nigrispinus are not affected when they are reared with prey S. frugiperda fed daily with leaf Bt-cotton. However it is capable of induces little modifications in granulocytes and plasmocytes. / Apesar da eficiência de formulações Bacillus thuringiensis (Bt) e plantas transgênicas Bt contra vários lepidópteros, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) apresenta baixa suscetibilidade a maioria deles. As razões dessa baixa suscetibilidade especificamente para toxinas Cry1Ac e Cry1Ab não estão totalmente esclarecidas. É possível que espécies moderadamente ou não suscetíveis ao Cry1Ac possam adquirir essa toxina e expô-la ao terceiro nível trófico. Este estudo investigou a resposta de S. frugiperda relacionadas ao Bt baseado em três hipóteses sobre sua baixa suscetibilidade: células regenerativas do intestino médio são ativadas pela toxina Cry1Ac e renovam as células epiteliais (i), na hemolinfa aumentam a produção de óxido nítrico e fenoloxidase como resposta à toxina (ii), e a tolerância está associada a variações qualitativas e quantitativas dos hemócitos. Testamos ainda a hipotese de que o consumo de S. frugiperda alimentadas com algodão Bt pode levar a alterações na ultraestrutura e histoquímica das células digestivas, nos parâmetros humorais, hemograma e ultraestrutura dos hemócitos de Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae). Conclui-se que toxina Cry1Ac no algodão Bt não é suficientemente tóxica para matar as lagartas, mas induz alteração humoral e celular como sinal de resposta a um xenobiótico. A toxina Cry1Ac ingerida por S. frugiperda pode expor o terceiro nível trófico (P. nigrispinus) e ocasionar alterações na distribuição de glicogênio, lipídios e cálcio em decorrência da desorganização da matriz perimicrovilar. O Microscópio Eletrônico de Transmissão (MET) revelou a presença de esferocristais que parecem estar envolvidos na defesa contra toxina. Os parâmetros humorals e o hemograma de P. nigrispinus não são afetados quando são criados com presa S. frugiperda alimentadas diariamente com folhas frescas de algodão Bt. No entanto, é capaz de induzir modificações pequenas em granulócitos e plasmócitos.

Através da meniscectomia no lado esquerdo, seguida ou não de enxerto, objetiva-se verificar as possíveis alterações nas superfícies articulares da ATM. Os animais formaram os grupos: GI (meniscectomia); GII (meniscectomia e enxerto); GIII (somente o acesso cirúrgico) que originaram subgrupos conforme o período pós-operatório (10 e 30 dias) e lado (direito e esquerdo). Nos animais GIII, GId, GIId, a Cm e a superfície articular da fossa mandibular (Fm) estavam organizadas em camadas com o predomínio de fibras colágenas do tipo I. Alterações morfológicas como erosão da Cm e Fm e amplos cistos subcondrais no osso subjacente foram verificadas nos animais GIe e GIIe. Em GIIe, a Cm apresentou um nível razoável de organização sob o enxerto. Fibras colágenas do tipo I constituíam as camadas articular e condroblástica nos animais GIe e GIIe e fibras do tipo III predominaram na camada pré-condroblástica. Amplos espaços vasculares eram evidentes na zona bilaminar dos animais GI e GII. / The morphological features of the temporomandibular joint (TMJ) of rats were studied after meniscectomy in the left side of the joint. The specimens were submitted or not to an interpositional auricular cartilage graft. The animals formed the following groups: GI (meniscectomy); GII (meniscectomy and graft); GIII (sham). The post-operative periods (10, 30 days) and the side allowed to form the subgroups. The articular surface of the TMJ exhibited organized layers (GIII, GId, GIId groups) where the type I collagen fibers arranged in regular nets prevailed. Morphological changes as erosion of the articular surface with large subchondral cysts in the subjacent bone were verified (GIe, GIIe). The condyle\'s surface of GII was regular and structurally preserved under the graft. The type I collagen fibers constituted the articular and chondroblastic layers in the GIe and GIIe animals where the type III collagen fibers were predominant in the prechondroblastic layer. Wide vascular spaces were evident in the bilaminar zone of the GI and GII animals.

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 (&ge;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.

Coral reefs are one of the most economically important ecosystems on the planet. Despite their great contribution to the world economy, anthropogenic influence via carbon dioxide emissions is leading to unprecedented changes with concerns about subsequent negative impacts on reefs. Surface ocean pH has dropped 0.1 units in the past century; in spite of this rapid shift in oceanic chemistry, it is unclear if individual species or life stages of Caribbean stony corals will be more sensitive to ocean acidification (OA). Examined is the relationship between CO2-induced seawater acidification, net calcification, photosynthesis, and respiration in three model Caribbean coral species: Orbicella faveolata, Montastraea cavernosa, and Dichocoenia stokesi, under near ambient (465 ± 5.52 ppm), and high (1451 ± 6.51 ppm) CO2 conditions. A species specific response was observed for net calcification; D. stokesi and M. cavernosa displayed a significant reduction in CaCO3 secreted under OA conditions, while O. faveolata fragments showed no significant difference. At the cellular level, transmission electron micrographs verified that all species and treatments were actively calcifying. Skeletal crystals nucleated by O. faveolata in the high CO2 treatments were statistically longer relative to controls. These results suggest that the addition of CO2 may shift the overall energy budget, causing a modification of skeletal aragonite crystal structures, rather than inhibiting skeletal crystal formation. Consequential to this energy shift, Orbicella faveolata belongs in the category of Scleractinian corals that exhibit a lower sensitivity to ocean acidification.

Corals combine photosynthesis and calcification in an intricate and delicately balanced relationship to form large biomineralized structures that are dominant features of tropical coastlines worldwide. Coral reefs have great scientific and economic importance but have recently experienced widespread decline attributed to increasing anthropogenic pressure on reef systems. Physical damage events, such as ship groundings, when coupled with existing nutrient stress and changing global climate present a poor outlook for successful natural recovery of reef communities. The main goal of the proposed research is to better understand how environmental factors, both local and global, affect the coral holobiont and influence overall coral fitness. The target species of this research, Montastraea cavernosa and Porites astreoides, are important and widespread Caribbean reef-builders. While it has been shown that nutrient and pCO2 stress affect coral growth and calcification, study of specific effects on coral tissue and reproductive success has not received significant attention in the literature despite considerable current interest. This study addresses this data gap in quantitatively examining the effect of elevated nutrients and pCO2 on 1) P. astreoides recruit survivorship, development, early calcification, and symbiotic zooxanthellae morphology; 2) M. cavernosa and P. astreoides wound regeneration, tissue characteristics over time at the histological and ultrastructural level, and trends in symbiotic zooxanthellae morphology; and 3) survival, growth and histological/ultrastructural characteristics of M. cavernosa and P. astreoides fragments transplanted to the field and in the laboratory. Histological and ultrastructural observations from corals transplanted to the field are then compared to ex-situ laboratory experimental corals. In the fleshy and large-polyped faviid M. cavernosa, healing of a linear wound was characterized by granulation of new tissue across the wound site, facilitated by coalescent granular amoebocytes. The wound healing strategy of this species appears to progress with wound closure and re-epithelialization before calcification resumes, as actively calcifying calicodermis was generally not observed at the healing front. Tissue regeneration in the small-polyped P. astreoides was characterized by formation of multiple islands of eosinophilic healing fronts along the depth of the wound track, and an accumulation of granular amoebocyte cells in regenerating tissue. The wound healing strategy of this species appeared to result in re-epithelialization of exposed body wall without necessarily closing the wound. Elevated pCO2 significantly reduced survivorship in P. astreoides recruits, and both nutrient enrichment and elevated pCO2 significantly reduced wound regeneration rate in M. cavernosa and P. astreoides. In both species, phosphate enrichment had the greatest deleterious effect on wound repair. A significant application of this study is the identification of possible zooxanthellar morphological indices of elevated nutrients and ocean acidification. The similarity in starch, lipid and uric acid accumulation patterns in Symbiodinium sp. from P. astreoides recruits and coral fragments of both species indicate a correlation between these anthropogenic stressors and the intracellular accumulation of excess carbon and nitrogen by the symbiont. Zooxanthellar carbon accumulation, in the form of starch and/or lipid, was the greatest under elevated nitrate. Zooxanthellar nitrogen accumulation, in the form of uric acid, was the greatest under elevated CO2. Comparison of zooxanthellar metrics between the field corals (P. astreoides, and M. cavernosa) and ex-situ corals and recruits indicated that carbon accumulation in Symbiodinium from field corals was consistently significantly less than in the ex-situ experimental P. astreoides recruits and M. cavernosa fragments exposed to elevated nitrate. This indicates that the field corals were likely not exposed to elevated nitrate at the time of collection. Both M. cavernosa and P. astreoides adults in the field accumulated significantly less uric acid than their counterparts in the tissue repair experiment, indicating that the field corals were exposed to higher pH and lower CO2 than the ex-situ corals. These results suggest that the field corals were not exposed to nutrient concentration profiles similar to the experimental treatments, particularly elevated nitrate. However, histological metrics indicated that the transplanted corals were subjected to increasing sedimentation stress over time. Overall, nitrate was found to affect recruits and adults on a similar scale, while phosphate and pCO2 affected carbon and nitrogen storage more in recruits compared to adults. While nutrients and pCO2 had no mechanistic effect on regeneration at histological level, ultrastructural metrics indicate an impact on the mutualistic energy exchange between the symbiotic partners, partially decoupling symbiosis. Effects were generally found to be greater in P. astreoides compared to M. cavernosa, and the unique life history strategy of the subject species and differences in their endosymbiont physiology reveal distinct responses to elevated nutrients and pCO2. Although the laboratory findings were not necessarily applicable to field observations, they provide insight into factors that may influence fragment success in the field. Quantitative assessment of the effect of elevated nutrients and pCO2 is thus useful in management decisions involving water quality standards, and is essential in the prediction of future coral condition and resilience.

The heterogeneity of salivary gland neoplasms have made classification and prognostication of these tumours sometimes difficult, and the in­troduction of techniques, such as enzyme and carbohydrate histochemis­try and electron microscopy have only to a certain extent increased our knowledge in these respects. In the present study immunohistochemical methods have been used to identify intermediate filament proteins (IFP) in normal fetal and adult parotid glands, as well as in salivary neo­plasms. The intermediate filaments (IF) make up the cytoskeleton in eucaryotic cells. Epithelial tissue contains IF composed of different cytokeratins (CK 1-19) whilst mesenchymal tissue generally contains IF composed of vimentin, and the IFP pattern is very stable even during cell transformation. It would thus be possible to further clarify the histogenesis of salivary neoplasms by identifying IFP, in addition the IFP pattern would probably be useful in tumour typing. Furthermore, ultrastructural cytochemical studies, microspectorphotometry on nuclear DNA as well as enzyme secretory studies of certain tumour types were carried out, in order to further characterize the biology of salivary neoplasms. The immunohistochemical investigations showed that in normal parotid tissue, the different cell types differed in IFP expression: acinar cells express mainly CK 18 and myoepithelial cells mainly CK 17 and 19, whilst duct cells contained a broad range of CK. Vimentin could in ad­dition to CK be detected in myoepithelial cells and basal cells of ex­cretory ducts. Fetal parotid cells showed a similar CK pattern as mature duct cells. In addition, vimentin could be found in some basal cells of the terminal tubules of the fetal glands. Salivary neoplasms could be divided into three types with regard to their IFP pattern:  Acinic cell carcinomas showed a CK-pattern similar to normal acinar cells but a co-expression of CK and vimentin was present in some cells.  Adenoid cystic carcinomas, mixed tumours and basal cell adenomas showed a CK-pattern of normal duct or myoepithelial cells. The peri­pheral cells were also vimentin positive. 3. Mucoepidermoid carcinomas and adenocarcinomas had a similar CK-pattern as duct cells, and no tu­mour cells contained vimentin. This indicates that typing of IFP may be useful for subgrouping of salivary neoplasms. By stereological measurements, the cells of acinic cell carcinomas were found to be very similar to normal parotid acinar cells. Furthermore, they contained amylase and after stimulation by norepiphrine a secre­tory response was induced, with a rise in intracellular cAMP as well as a release of amylase. By single cell measurements of nuclear DNA con­tent, no difference was found between acinic cell carcinomas with de­finite metastasis and those without recurrence, both in paraffin sec­tions and cytological smears. / digitalisering@umu.se