A vinca (Catharanthus roseus) é uma planta perene, arbustiva, pertencente à família Apocinaceae, cujas folhas e raízes possuem propriedades medicinais. A presença de sintoma de mosaico e deformação foliar em plantas dessa espécie, associados com a presença de partículas alongadas e flexuosas, característica de vírus pertencentes ao gênero Potyvirus, conduziu a estudos complementares para a identificação e caracterização desse vírus. No estudo da gama parcial de hospedeiras foram testadas 28 espécies, envolvendo oito famílias botânicas. Catharanthus roseus e Nicotiana benthamiana apresentaram sintomas de mosaico foliar e Chenopodium amaranticolor e C. quinoa apresentaram lesões locais cloróticas nas folhas inoculadas. A transmissão do vírus com afídeos foi avaliada com as espécies Aphis gossypii, Myzus nicotianae e Toxoptera citricidus. Apenas Aphis gossypii e Myzus nicotianae transmitiram o vírus. O antissoro policlonal produzido contra este potyvirus reagiu com o vírus homólogo e com o Passionfruit woodiness virus (PWV) e Cowpea aphid-borne mosaic virus (CABMV), mas não com o Lettuce mosaic virus (LMV), Papaya ringspot virus - type W (PRSV-W), Potato virus Y (PVY) e Zucchini yellow mosaic virus (ZYMV). O peso molecular da proteína capsidial (CP) foi de aproximadamente 34 kDa. A reação de PCR realizada com os oligonucleotídeos universais de potyvirus e oligonucleotídeos específicos posteriomente confeccionados amplificaram três fragmentos de aproximadamente 0,8, 1,0 e 1,4 Kb, os quais após o seqüênciamento geraram um fragmento de 1654 nucleotídeos (nt) da região 3' terminal do genoma, que inclui parte do gene da replicase viral (Nib), a região codificadora completa do gene da proteína capsidial (CP), seguida de 286 nt da região 3' não traduzida (3'NTR). A identidade da seqüência de nucleotídeos do gene da CP variou de 67,0 a 76,0%, quando comparada com as de outros membros da família Potyviridae. A maior identidade foi com o Omphalodes virus Y (76,0%). A identidade dos aminoácidos deduzidos da proteína capsidial variou de 62,0 a 71,0%, sendo a maior com East Asian Passiflora virus (71%). Para a região não traduzida (3'NTR) a identidade variou de 16,8 a 28,6%. Em conjunto esses dados indicam que este vírus é uma nova espécie dentro do gênero Potyvirus, para o qual se propõe o nome de Vírus do mosaico do Catharanthus (Catharanthus mosaic virus - CatMV). / Catharanthus roseus is known as the common periwinkle or Madagascar periwinkle. It is a perennial, evergreen herb in the family Apocynaceae, which was originally native to the island of Madagascar, although both name and classification are contradictory in some literature. The plants grow up to 80 cm high; have glossy, dark green leaves and bloom during summer. The flowers range from white to hot pink to purple. The species has historically been used in popular medicine to treat a wide assortment of human diseases, as it contains more than 150 useful alkaloids. Plants of C. roseus exhibiting mosaic symptoms followed by malformation of the leaf blades and flower variegation were collected from a garden at the University of São Paulo, School of Agriculture (Piracicaba, State of São Paulo, Brazil). Preliminary electron microscopy exams of negatively stained leaf sap revealed that the symptoms were associated with potyvirus-like particles. The objective of the present work was to obtain further biological, immunological and molecular data to better characterize this species of the genus Potyvirus, family Potyviridae. Of 28 plant species from eight botanical families inoculated mechanically with this potyvirus, only C. roseus and Nicotiana benthamiana developed systemic mosaic, whereas Chenopodium amaranticolor and C. quinoa exhibited only chlorotic local lesions. The virus was transmitted by Aphis gossypii and Myzus nicotianae, but not by Toxoptera citricidus. Polyclonal antiserum raised against this potyvirus reacted with the homologous virus, Passion fruit woodiness virus (PWV) and Cowpea aphid borne mosaic virus (CABMV) in PTA-ELISA. The molecular mass of the coat protein (CP) was approximately 34 kDa. RT-PCR from viral RNA amplified a fragment of approximately 1654 nucleotides (nt) at the 3'-terminal of the viral genome, containing portion of the replicase gene (Nib), the entire CP gene and the 3' untranslated region (3'UTR) (286 nt). When the nucleotide sequence of the CP gene was compared with other members of the Potyviridae family, identities varied from 67.0 to 76.0%. The highest identity was with Omphalodes virus Y. Identity of the deduced amino acid of the CP varied from 62.0 to 71.0%, with the highest for East Asian Passiflora virus. For the 3' UTR, identities varied from 16.8 to 28.6%. The name Catharanthus mosaic virus (CatMV) is proposed for this new potyvirus.

A literatura corrente possui informações limitadas sobre o projeto da área de troca térmica de tanques com jaqueta, serpentina helicoidal, serpentina espiral e chicana tubular vertical, em operação com fluidos não-Newtonianos. A presente tese teve por objetivo principal analisar a transferência de calor, potência consumida e ampliação de escala em tanques com impulsores mecânicos na agitação de fluidos não-Newtonianos com duas superfícies de transmissão de calor, chicana tubular vertical e serpentina em espiral. O trabalho também visou fornecer métodos de ampliação de escala de tanques com agitação para fluidos não-Newtonianos que sigam o modelo reológico da lei das potências. A unidade experimental contemplou dois tanques de acrílico, com volume de 10 litros e 50 litros, respectivamente, chicanas tubulares verticais e serpentina em espiral. Os impulsores mecânicos utilizados foram o axial com 4 pás inclinadas a 45° e o radial turbina com 6 pás planas. Como fluidos utilizaram-se soluções aquosas de carboximetilcelulose (0,5%, 1,0% e 1,5%), solução aquosa de carbopol 940 (1,5%), solução aquosa de sacarose (50%) e água. Todos os experimentos foram conduzidos em batelada. Com os dados obtidos, empregou-se o uso de regressões para a obtenção da Equação de Nusselt, as quais forneceram valores de coeficiente de determinação ajustados entre 0,83 e 0,89 com Reynolds no intervalo de 20 a 405000, Prandtl na faixa de 4 a 6400 e índice reológico do modelo da lei das potências entre 0,45 e 1,00. Observou-se que no aquecimento realizado com a chicana tubular vertical, o impulsor radial forneceu coeficientes de convecção 20% acima quando comparado com o impulsor axial, entretanto o consumo de potência foi cerca de 66% maior em relação ao impulsor axial. No caso da serpentina espiral, o impulsor axial promoveu coeficientes de convecção por volta de 15% superiores em relação ao impulsor radial com um consumo de potência 65% menor. Desse modo, em processos em que não é necessária uma elevada turbulência, recomenda-se o uso do impulsor axial com a serpentina espiral, porém, se o processo demandar uma turbulência significativa, deve-se usar o impulsor radial com a chicana tubular vertical. Em uma última análise, os modelos não-lineares obtidos para ampliação de escala forneceram erros entre 11% e 20% na predição da rotação no tanque industrial, os quais são válidos para Reynolds modificados de Metzner e Otto (1957) na faixa de 20 a 4000 e para fluidos não-Newtonianos pseudoplásticos com índices reológicos entre 0,45 e 1,00. / Current literature has limited information on the design of the thermal exchange area of tanks with jacket, helical coil, spiral coil and vertical tuber baffle, in operation with non-Newtonian fluids. The main purpose of this thesis was to analyze heat transfer, power consumption and scale-up in tanks with mechanical impellers in the agitation of non-Newtonian fluids with two heat transfer surfaces, vertical tube baffle and spiral coil. The work also aimed to provide methods of scale-up tank scale with agitation for non-Newtonian fluids that follow the rheology model of the law of powers. The experimental unit included two acrylic tanks, with a volume of 10 liters and 50 liters, respectively, vertical tube baffles and spiral coil. The mechanical impellers used were the 45° pitched blade turbine (PBT) and the Rushton turbine (RT). Aqueous solutions of carboxymethylcellulose (0.5%, 1.0% and 1.5%), aqueous solution of carbopol 940 (1.5%), aqueous solution of sucrose (50%) and water were used as fluids. All the experiments were conducted in batch. With the obtained data, we used the regressions to obtain the Nusselt Equation, which provided coefficient of determination values adjusted between 0.83 and 0.89 with Reynolds in the range of 20 to 405000, Prandtl in the range of 4 to 6400 and rheological index of the power law model between 0.45 and 1.00. It was observed that in the heating performed with the vertical tube baffle, the RT provided convection coefficients 20% higher when compared to the axial impeller, however the power consumption was about 66% higher in relation to the PBT. In the case of the spiral coil, the PBT promoted convection coefficients around 15% higher than the RT with 65% lower power consumption. Thus, in processes where high turbulence is not required, it is recommended to use the PBT with the spiral coil, but if the process requires significant turbulence, the RT must be used with the vertical tubular chassis. In a final analysis, the nonlinear models obtained for scaling provided errors between 11% and 20% in the prediction of rotation in the industrial tank, which are valid for Metzner and Otto (1957) modified Reynolds in the range of 20 to 4000 and for non-Newtonian pseudoplastic fluids with rheological indexes between 0.45 and 1.00.

Fibroblast growth factor 23 (FGF-23) has recently been shown to be involved in phosphate regulation and bone mineralization. This study evaluated the effect of FGF-23 on three human cell lines (Caco-2, HK-2, SaOS-2) representing three different sites of phosphate regulation (small intestine, kidney proximal tubules, and bone, respectively). FGF-23 induced gene expression was studied using Clontech human Atlas glass microarrays containing various assortments of genes and by a custom designed oligo microarray containing specific genes selected for their biological relevance to FGF-23's potential function. FGF-23 induced differential gene expression in all three cell types, suggesting that FGF-23 may be capable of acting on these three primary sites of phosphate regulation. Human small intestine-like endothelial cell line, Caco-2, showed upregulation of several genes including parathyroid hormone receptors 1 and 2. FGF-23 inhibited the expression of water channel transporters aquaporin 5 and 6 in human osteoblast-like SaOS-2 cells while upregulating aquaporin expression in HK-2 cells. Somatostatin receptors 1-4 were identified to be upregulated in the human kidney, HK-2 cell line. Mucin 2, a gene that is linked to abnormal cellular growth, was consistently induced by FGF-23 in all three cell lines. Families of aquaporins, somatostatins, parathyroid hormones, and other identified differentially expressed genes are involved in different signaling pathways that are associated with phosphate and calcium regulation. Selected candidates were analyzed further by real-time RT-PCR. These data support FGF-23 induced regulation of aquaporin 5 mRNA in HK-2 cells and 1-alpha-hydroxylase mRNA in Caco cells. FGF-23 induced changes in mRNA analysis of four additional genes was less than two-fold in triplicate analysis of selected samples. Taken together, these results suggest that each cell type may have responded to FGF-23, but additional validation of the array data set will be required to identify those genes specifically regulated by FGF-23. Further refinement of this data set will undoubtedly uncover additional functions of FGF-23 and may provide valuable insight into designing therapeutic approaches for phosphate specific disorders.

Le cancer de vessie représente la sixième cause de mortalité par cancer en France. Son incidence a augmenté ces 20 dernières années, mais les taux de survie restent inchangés. La carcinogénèse vésicale fait intervenir différents mécanismes moléculaires qui agissent en réseau comme c’est le cas dans de nombreux cancers. Le développement récent de nouveaux traitements prenant spécifiquement pour cible certaines voies de signalisation apportent de nouveaux espoirs thérapeutiques. Nous nous sommes intéressés dans ce travail à trois axes de recherche pour tenter d’identifier, dans les carcinomes urothéliaux, de nouveaux marqueurs pronostiques moléculaires et de nouvelles cibles thérapeutiques potentielles: l’angiogénèse, la voie de signalisation Hedgehog et les microARNs. Nous avons choisi la RT-PCR quantitative en temps réel à grande échelle permettant d’évaluer le niveau d’expression de nombreux gènes, avec une quantification précise et reproductible des transcrits. L’expression de ces gènes a été corrélée aux données de suivi clinique afin d’identifier de nouveaux biomarqueurs moléculaires prédictifs de l’évolution des tumeurs de vessie.Nous avons ainsi pu démontrer que les niveaux d’expression de certains de ces gènes variaient de façon significative dans les tumeurs de vessie, confirmant le rôle de l’angiogénèse dans la carcinogénèse urothéliale, et plus particulièrement de la voie du VEGF, et suggérant une implication majeure de la voie de signalisation Hedgehog et des microARNs. Par ailleurs, nous avons également pu identifier plusieurs biomarqueurs ayant une valeur pronostique en terme de survie globale dans les tumeurs infiltrantes. C’est le cas du VEGF, qui semble être un biomarqueur moléculaire particulièrement intéressant puisqu’il existe des thérapies ciblées spécifiquement dirigées contre ce ligand ou ses récepteurs avec plusieurs essais cliniques actuellement en cours dans le cancer de vessie. C’est également le cas d’une signature moléculaire associant 3 miARNs (miR-9, miR-182 et miR-200b) ayant une valeur péjorative dans les tumeurs infiltrantes, ouvrant la voie vers de nouvelles stratégies thérapeutiques.L’ensemble de ces études confirment l’intérêt majeur d’une meilleure compréhension des bases moléculaires de la carcinogénèse urothéliale vésicale débouchant sur l’utilisation rationnelle de nouvelles thérapies ciblées dans le cancer de vessie, avec l’espoir d’en améliorer la prise en charge et l’évolution. / Bladder cancer is the sixth cause of cancer mortality in France and its incidence is increasing since the last 20 years, with no improvement in survival outcomes. Bladder carcinogenesis involves different molecular mechanisms such as in many cancers. The recent development of new targeted therapies targeting signaling pathways provides new therapeutic hopes.In this work, we choose to study three molecular pathways in order to identify new prognostic markers and new therapeutic targets in urothelial carcinoma: angiogenesis, Hedgehog signaling pathway, and microRNAs. Real-time quantitative RT-PCR was performed to measure simultaneously expression levels of several genes with precise and reproductible RNA quantification. Our results were correlated with clinical outcomes to identify new molecular markers associated with bladder cancer evolution and to guide the potential use of targeted therapies.We were able to demonstrate that expression levels of several transcripts differ significantly in bladder tumors as compared to normal bladder and that some of them may have a prognostic implication. This is the case of VEGF, which appears to be an interesting molecular marker since there are targeted therapies specifically targeting the pathway and several ongoing trials in bladder cancer. The Hedgehog pathway also appears to be altered in bladder tumors, with a ligand-dependent activation. Then, we were able to identify several deregulated microRNAs and describe a molecular 3 miRNA-signature (miR-9, miR-182 and miR-200b) having a prognostic value in muscle-invasive bladder tumors. All these studies confirm the major interest of molecular biology and new targeted therapies in the treatment of bladder cancer, with the hope of improving management and evolution.

O vírus da cinomose canina (VCC) é um patógeno viral, altamente, contagioso que pode causar doença sistémica letal, em cães e outros carnívoros em toda parte do mundo. Os cães afetados podem apresentar sintomas gastrentéricos, respitatórios e nervosos. As manifestações clínicas da doença inclue depressão, diarréia, vómito, desidratação, hiperqueratose dos coxins e focinho e espasmos musculares ou paresia de membros pélvicos, a qual pode persistir por longos períodos. Cães infectados, com sintomas clínicos de VCC, foram estudados para detecção do RNA viral pela técnica de PCR e Nested-PCR. Neste estudo, amplificou-se o gene da nucleoproteína (NP) em células mononucleares do sangue periférico (linfócitos), urina e saliva, de cães infectados com VCC, para detectar o genoma do mesmo, por RT-PCR, em diferentes amostras clínicas. A identificação do RNA viral foi concluída com sucesso, pelo método de RT-PCR, utilizando 2 pares de \"primers\" específicos do gene da nucleoproteína (NP). A técnica de RT-PCR, descrita neste etudo, pode ser um sistema de ensaio útil para determinar se cães suspeitos de infecção, por VCC, tenha níveis detectáveis de genes. Os resultados demonstram que a técnica de RT-PCR é exequível para o diagnóstico laboratorial de cinomose canina. / Canine distemper vírus (CDV) is a highly contagious Viral pathogen which may cause lethal systemic in dogs and other carnivores throughout the world. Affected dogs show gastrointestinal and respiratory clinical slgns, and frequently develop clinical signs in the central nervous system (CNS). Clinical manifestations of the disease include depression, progressive loss of weight, dehydration, hyperkeratosis of the foot pads and nose, nervous symptoms and muscular spasms or posterior paralysis which may perslst for long periods. Infected dogs with clinical symptoms for CDV, were by detection of viral RNA by Polymerase Chaln Reaction (PCR) and Nested PCR. In this study,w e determinebdy the RT-PCRth e presenceo f nucleoprotein (NP) gene in peripheral blood mononuclear cells, urine and saliva from dogs infected with CDV. The goals of this study was to detect CDV renome by RT-PCR in different clinical samples. In this study, Identificatlon of NP mRNA was successfully achieved by using the RT-PCR method with two sets of NP gene specific primers. The RT-PCR technique described in thls study, may provide a useful assay system to determine whether the dogs suspected of CDV infection have detectable leveis of CDV genes. The results demonstrate that RT-PCR technique is rapid, sensitivity and specificity for vírus diagnosis.

O trigo (Triticum aestivum L.) é uma importante cultura no Brasil. Poucas cultivares são recomendadas para produção do tipo sequeiro no Bioma Cerrado onde a escassez de água limita o rendimento de grãos. Aqui reportamos uma análise de transcriptoma do MGS1 Aliança (cultivar de trigo adaptada ao Cerrado) sob estresse de seca. Um grupo de 4.422 transcritos diferencialmente expressos foi encontrado em raízes e folhas. O número de transcritos reprimidos em raiz (1.102) foi menor que os transcritos induzidos (1.706), enquanto o oposto ocorreu em folhas (1,017 induzidos e 647 reprimidos). O número de transcritos comuns entre ambos órgaõs foi 1.249, enquanto 2.124 foram específicos para raíz e 1.049 específicos para folhas. Análises de RT-qPCR de 35 transcritos selecionados ao acaso revelou uma correlação de 0,78 com os dados de transcriptoma. Os transcritos diferencialmente expressos foram distribuídos por todos os cromossomos e componentes do genoma. O número de transcritos no genoma B foi maior do que nos genomas A e D. Ainda, um grande número de transcritos relacionados à seca foi mapeado nos cromossomos 3B, 5B e 2B. Quando consideramos ambos órgãos, 116 diferentes rotas metabólicas foram alteradas. Uma rota em comum, entre as três mais alteradas em ambos órgãos, foi o metabolismo do amido e da sacarose. A comparação de transcritos derivados de raiz e de folha permite a identificação de transcritos importantes relacionados à respota ao estresse de seca em cada um destes órgãos. Os dados obtidos, também, abrem caminho para o desenvolvimento de futuros marcadores e seleção de genes candidatos ligados à característica. Estes resultados são úteis para o entendimento de rotas metabólicas envolvidas na tolerância à seca em trigo. A informação gerada será usada, a mais longo prazo, para propósitos de transgenia. Para isto, a metodologia de duplo-haploides é desejável e uma primeira investigação sobre a eficiência de protocolo se mostrou necessária. Micrósporos são células gaméticas com capacidade de dar origem a uma nova planta via embriogênese in vitro. Plantas duplo-haploides geradas pela cultura de micrósporos isolados são completamente homozigotas e representam uma importante ferramenta para estudos genéticos e melhoramento de plantas O processo androgenético é desencadeado por diferentes pré-tratamentos de estresse, os quais são empregados para mudar os micrósporos da rota gametofítica para a rota esporofítica. Embora a cultura de micrósporos isolados tenha inúmeras vantagens, importantes limitações tem impedido sua apliação em larga escala. Diferenças genotípicas na resposta androgenética e na formação de plantas albinas ainda constituem desafios. Embora o albinismo seja principalmente uma característica genética, pré-tratamentos e meios de cultura apropriados podem evitar este fenômeno até certo ponto. A resposta androgenética de cinco genótipos de trigo brasileiro foi avaliada no presente estudo. Dois pré-tratamentos foram testados: frio (4°C) e ácido 2-hidroxinicotinico (100 mg/L). O frio foi melhor que o pré-tratamento químico, produzindo mais plantas verdes em quatro de cinco genótipos. Somente dois genótipos brasileiros tratados com ácido 2-hidroxinicotinico produziram plantas, e um deles apenas uma única planta albina. Nossos reultados mostram, também, que o meio semilíquido (contendo 10% de Ficoll) promoveu uma maior resposta androgenética que o meio líquido, aumentando o número de embriões e plantas regeneradas. / Wheat (Triticum aestivum L.) is an important crop cultivated in Brazil. Few cultivars are recommended for rainfed production in the Cerrado Biome where water scarcity limits grain yield. Here we report a transcriptome analysis of MGS1 Aliança (a wheat cultivar adapted to the Cerrado) under drought stress. A set of 4,422 differentially expressed transcripts was found in roots and leaves. The number of down-regulated transcripts in roots (1,102) was lower than the up-regulated transcripts (1,706), while the opposite occurred in leaves (1,017 induced and 647 repressed). The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis of 35 randomly selected transcripts revealed a 0.78 correlation with the transcriptome data. The differentially expressed transcripts were distributed across all chromosomes and component genomes. The number of transcripts on the B genome was greater than on the A and D genomes. Additionally, a greater number of drought related transcripts was mapped on chromosomes 3B, 5B and 5D. When considering both tissues, 116 different metabolic pathways were changed. One common pathway, among the top three changed pathways in both tissues, was starch and sucrose metabolism. The comparison of root- and leaf-derived transcripts allows the identification of important transcripts related to water stress response in each of these tissues. It also paves the way for future marker development and selection of candidate genes linked to that trait. These results are useful for understanding the metabolic pathways involved in wheat drought response. The information generated will be used for transgenic wheat purposes. For this the doubled-haploid method is desirable and an investigation about the protocol eficiency is needed. Microspores are gametic cells with capacity to give rise to a new plant via in vitro embryogenesis. Doubled haploid plants generated by isolated microspore culture are completely homozygous and represent an important tool for plant genetics and breeding research. This process is triggered by different stress pretreatments, which are employed to switch microspores from gametophytic to a sporophytic pathway. Although isolated microspore culture has innumerous advantages, important limitations have prevented its application on a large scale. Genotypic differences in androgenic response and the formation of albino plants remain great challenges. Although albinism is a major genetic characteristic, appropriated pretreatments and culture medium can avoid this phenomenon to some extent. The androgenic response of five Brazilian wheat genotypes was evaluated in the present study. Two pretreatments were tested: cold (4°C) and 2-hydroxynicotinic acid (100 mg/L). Cold was better than chemical pretreatment, producing more green plants in four out of five genotypes. Only two Brazilian genotypes treated with 2-hydroxynicotinic acid produced plants, and one of them produced a single albino plant. Our results also show that semi-liquid medium (containing 10% Ficoll) promoted a higher androgenic response than did liquid medium, increasing the number of embryos and regenerated plants.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O Brasil à o segundo maior produtor mundial de soja, destacando-se por sua multiplicidade de uso. Entretanto, perdas na produtividade de seus grÃos em campo sÃo ainda considerÃveis, particularmente oriundas das doenÃas causadas por fungos. Diante desse obstÃculo, emerge a necessidade de busca de molÃculas naturais capazes de inibir o progresso dessas doenÃas, sem causar impactos ambientais. Dentre as molÃculas presentes na soja, com potencial de uso para essa finalidade, destaca-se a toxina da soja (SBTX), uma proteÃna isolada de sementes, composta por duas subunidades (17 e 27 kDa) e ativa contra fungos fitopatogÃnicos. Esse trabalho teve como objetivos verificar a localizaÃÃo tecidual da SBTX em cotilÃdones de sementes maduras, bem como avaliar seu perfil de expressÃo gÃnica ao longo do desenvolvimento da soja e, tambÃm, em resposta ao tratamento com elicitores de defesa vegetal. Sementes de soja foram cultivadas em casa de vegetaÃÃo e, ao longo do desenvolvimento da planta, diferentes tecidos vegetais coletados. Em adiÃÃo, folhas primÃrias da soja foram tratadas com Ãcido salicÃlico (AS) ou inoculadas com esporos do fungo Cercospora kikuchii (CK) e coletadas em diferentes tempos apÃs os tratamentos. Iniciadores foram desenhados com base nas sequÃncias NH2-terminal das subunidades de SBTX e a expressÃo gÃnica foi avaliada pela tÃcnica de RT-PCR quantitativa. A localizaÃÃo de SBTX em sementes foi avaliada por imunohistoquÃmica, usando anti-SBTX. Transcritos dos genes SBTX17 e SBTX27 foram detectados em todos os tecidos vegetais coletados, porÃm seus nÃveis de expressÃo foram diferenciados. NÃveis mais elevados de transcritos para ambas as subunidades da SBTX foram detectados em sementes maduras, cotilÃdones e folhas unifoliadas. Nos cotilÃdones, SBTX foi encontrada na epiderme. InduÃÃo da expressÃo de transcritos da SBTX ocorreu em ambos os tratamentos, porÃm essa resposta se manifestou mais rÃpida (a partir de 6 h) com CK ao invÃs de AS (a partir de 12 h). Praticamente, em todas as anÃlises, transcritos do gene SBTX27 prevaleceram em relaÃÃo Ãqueles do SBTX17. A presenÃa constitutiva e ubÃqua de transcritos dos genes da SBTX ao longo do desenvolvimento da planta, a induÃÃo da expressÃo desses genes por elicitores de resposta de defesa e a localizaÃÃo da toxina na superfÃcie dos cotilÃdones validam o papel de defesa atribuÃdo a SBTX, suscitando a possibilidade de uso dessa proteÃna na produÃÃo de soja resistente ao ataque de fungos de relevÃncia agronÃmica. / Brazil is the second major global soybean producer, whose magnitude is due to its use multiples. However, losses in productivity of soybean grains in the field are still significant, especially those caused by pathogenic fungi. In view of this obstacle, it is important to search natural molecules able to inhibiting the progress of fungal diseases in an environmental friendly practice. Among the soybean molecules which could be used for this purpose, the soybean toxin (SBTX) stands out. SBTX is a protein composed of two subunits (17 and 27 kDa) isolated from seeds with in vitro activity against phytopathogenic fungi. The present study aimed to verify the SBTX tissue localization in soybean seed cotyledons, as well as to evaluate the gene expression profile of two SBTX subunits, both in different stages of plant development and in response to treatment with plant defense elicitors. Soybean seeds were grown in a greenhouse and plant tissues harvested at different days. In addition, soybean primary leaves were treated with salicylic acid (SA) or inoculated with the Cercospora kikuchiii (CK) spores and harvested at different times after the treatments. Based on the N-terminal sequences of the SBTX subunits, primers were designed and their gene expression evaluated by quantitative real-time PCR technique. SBTX tissue localization was performed by immunohistochemistry using anti-SBTX. Transcripts for both SBTX subunits were detected in all plant tissues, predominantly in cotyledons and unifoliate leaves in the early stages of their development, as well as in mature seeds. SBTX was found in the epidermis of the cotyledons. Transcripts were detected for both genes SBTX17 e SBTX27 in all tissues collected, but their expression levels were different. The highest transcript levels for both SBTX subunits were found in mature seeds, cotyledons and unifoliate leaves. In cotyledons, SBTX was found in the epidermis. Leaves treated with elicitors showed induction of the corresponding 17 and 27 kDa subunit transcripts, however this response was earlier in the CK treatment (from 6 h) compared to AS treatment (from 12 h). In almost all analyses, the highest transcript levels were found for the 27 kDa subunit. The ubiquitous and constitutive gene expression during plant development, the induction of gene expression by defense response elicitors and the localization on the surface of cotyledons support the role of SBTX in plant defense and its use to produce fungal-resistant transgenic soybean plants.

O vírus da cinomose canina (VCC) é um patógeno viral, altamente, contagioso que pode causar doença sistémica letal, em cães e outros carnívoros em toda parte do mundo. Os cães afetados podem apresentar sintomas gastrentéricos, respitatórios e nervosos. As manifestações clínicas da doença inclue depressão, diarréia, vómito, desidratação, hiperqueratose dos coxins e focinho e espasmos musculares ou paresia de membros pélvicos, a qual pode persistir por longos períodos. Cães infectados, com sintomas clínicos de VCC, foram estudados para detecção do RNA viral pela técnica de PCR e Nested-PCR. Neste estudo, amplificou-se o gene da nucleoproteína (NP) em células mononucleares do sangue periférico (linfócitos), urina e saliva, de cães infectados com VCC, para detectar o genoma do mesmo, por RT-PCR, em diferentes amostras clínicas. A identificação do RNA viral foi concluída com sucesso, pelo método de RT-PCR, utilizando 2 pares de \"primers\" específicos do gene da nucleoproteína (NP). A técnica de RT-PCR, descrita neste etudo, pode ser um sistema de ensaio útil para determinar se cães suspeitos de infecção, por VCC, tenha níveis detectáveis de genes. Os resultados demonstram que a técnica de RT-PCR é exequível para o diagnóstico laboratorial de cinomose canina. / Canine distemper vírus (CDV) is a highly contagious Viral pathogen which may cause lethal systemic in dogs and other carnivores throughout the world. Affected dogs show gastrointestinal and respiratory clinical slgns, and frequently develop clinical signs in the central nervous system (CNS). Clinical manifestations of the disease include depression, progressive loss of weight, dehydration, hyperkeratosis of the foot pads and nose, nervous symptoms and muscular spasms or posterior paralysis which may perslst for long periods. Infected dogs with clinical symptoms for CDV, were by detection of viral RNA by Polymerase Chaln Reaction (PCR) and Nested PCR. In this study,w e determinebdy the RT-PCRth e presenceo f nucleoprotein (NP) gene in peripheral blood mononuclear cells, urine and saliva from dogs infected with CDV. The goals of this study was to detect CDV renome by RT-PCR in different clinical samples. In this study, Identificatlon of NP mRNA was successfully achieved by using the RT-PCR method with two sets of NP gene specific primers. The RT-PCR technique described in thls study, may provide a useful assay system to determine whether the dogs suspected of CDV infection have detectable leveis of CDV genes. The results demonstrate that RT-PCR technique is rapid, sensitivity and specificity for vírus diagnosis.

L'intensification de l'agriculture s'est accompagnée d'une utilisation importante de pesticides qui a généré une pollution généralisée des sols et des eaux, problème environnemental majeur et actuel. Sous la pression de sélection liée à l'usage régulier de pesticides (molécules xénobiotiques) des bactéries du sol se sont adaptées à ces molécules et ont acquis la capacité de les utiliser comme source nutritive et donc de les dégrader. La biodégradation constitue un service écologique majeur fournit par le sol, puisqu'elle est à la base des capacités épuratrices du sol et au-delà, de la résilience des écosystèmes. Le sol étant également un grand réservoir de biodiversité, ces bactéries dégradantes sont sous contrôle de différentes interactions biotiques et notamment celles impliquant les lombriciens, qualifiés d'organismes ingénieurs des sols de par leur action de bioturbation. Grâce à un développement méthodologique important et novateur (RT-qPCR, SIP-ARN), nous avons étudié l'impact de la bioturbation du sol par la macrofaune lombricienne sur les communautés bactériennes du sol intervenant dans la biodégradation de molécules xénobiotiques. L'atrazine a été utilisée comme molécule modèle à double titre : d'un point de vu fondamental, son utilisation pendant plus de 50 ans en France a permis aux bactéries du sol de s'adapter et au titre de l'actualité, bien qu'elle soit interdite en France depuis 2003, il s'agit toujours du principal polluant retrouvé dans les eaux souterraines et de surface. Nous avons analysé par une double approche quantitative et qualitative l'impact de la bioturbation du sol par les lombriciens sur l'abondance, l'activité et la diversité des bactéries indigènes du sol et sur celles dégradant l'atrazine. Nous avons mis en évidence que : (i) la digestion du sol par les lombriciens stimule l'activité d'une partie des bactéries du sol mais qu'une autre fraction ne résiste pas au passage dans le tube digestif des vers, (ii) la bioturbation du sol par les lombriciens génère des ‘hot spot' pour l'activité de dégradation de l'atrazine. Ainsi dans les parois de galeries les bactéries dégradantes sont sélectionnées, la dissipation de l'atrazine est rapide et les premiers acteurs du processus de dégradation ont été identifiés, (iii) la dégradation accélérée de l'atrazine dépend d'espèces bactériennes clés interagissant au sein de consortia dégradants, ainsi la diversité des bactéries dégradantes n'est pas corrélée à la fonction de dégradation. L'ensemble des résultats obtenus nous montre également que l'impact de la bioturbation par la macrofaune lombricienne sur l'activité de dégradation dépend des propriétés physico-chimiques et biologiques initiales du sol. L'ensemble de ces connaissances présente un intérêt dans un contexte de bioremédiation in situ des sols pollués puisque les lombriciens constituent une grande part de la macrofaune dans nos sols tempérés et qu'ils modifient significativement les bactéries dégradant l'atrazine au sein des microsites de sols qu'ils génèrent.

Les travaux développés dans ce mémoire se situent dans le cadre de l'analyse et de la conception des systèmes temps réel. Nous proposons une approche d'aide à la translation entre analyse fonctionnelle et analyse orientée objet. L'utilité d'une telle démarche ainsi que les différents travaux effectués dans ce domaine sont d'abord étudiés. Ensuite, nous exposons les différentes étapes de la démarche. Son originalité découle de l'utilisation des réseaux de Petri comme moyen de translation. D'abord, les spécifications fonctionnelles (de type SA-RT) sont décrites par réseaux de Petri. Les composantes conservatives de ces derniers sont alors utilisées pour identifier et définir les objets. Le formalisme HOOD/PNO est adopté pour la spécification des objets. L'approche proposée peut s'appuyer sur l'aspect statique des spécifications ou sur l'aspect dynamique. Il est également possible d'adopter une approche mixte. Les différentes étapes de la démarche et les approches possibles sont illustrées par des exemples significatifs. Nous étudions également les limites de la démarche proposée, ses parties automatisables et la possibilité de l'utiliser avec d'autres techniques. Par ailleurs, nous nous intéressons à la possibilité d'exploiter les résultats obtenus dans des objectifs d'évaluation des spécifications fonctionnelles. L'apport de certaines extensions temporelles des réseaux de Petri est mis en évidence.