La existencia de largas listas de espera para trasplante hepático de cadáver para tratar la hepatopatía irreversible y el hepatocarcinoma hace necesaria la búsqueda de alternativas como el trasplante hepático de donante vivo. El estudio prequirúrgico de los potenciales candidatos a donante vivo de hígado debe incluir la determinación del volumen hepático que va a trasplantarse, así como la distribución arterial, portal venosa hepática y biliar que presenta el donante. Todo ello debe efectuarse con las técnicas menos agresivas a nuestro alcance. La resonancia magnética (RM) permite evaluar estos parámetros en una sola exploración. En este trabajo se ha intentado demostrar que la RM es la técnica de elección en el estudio prequirúrgico del donante vivo hepático, ya que es segura y fiable en la valoración de la anatomía vascular y biliar, en el estudio del parénquima y en la determinación del volumen hepático de forma que puede utilizarse como única técnica en la valoración global del donante.Se han evaluado 39 donantes vivos de lóbulo derecho hepático consecutivos en los que se obtuvieron estudios dinámicos multifásicos durante la inyección intravenosa de contraste paramagnético, secuencias colangiográficas fuertemente potenciadas en T2, secuencias colangiográficas adicionales tras administrar contraste de excreción biliar y cálculo volumétrico. Los hallazgos vasculares observados en la RM se correlacionaron con los obtenidos en la angiografía y en la cirugía. Los hallazgos de la colangiografía por RM se compararon con los de la colangiografía intraoperatoria. El volumen hepático estimado para el lóbulo derecho hepático en la RM se comparó con el peso de la pieza de hepatectomía derecha en el quirófano.La correlación observada entre el volumen hepático calculado en la RM y el peso del injerto, utilizando el análisis de correlación de Pearson dio un valor de r2 = 0.78, con una sobreestimación por parte de la RM del 18% de media. Las arterias hepática, esplénica, mesentérica y tronco celíaco se identificaron siempre de forma excelente. Las arterias hepáticas derecha e izquierda se observaron de forma suficiente en 74% y 71,7% de los individuos. El patrón de distribución arterial se evaluó siguiendo la clasificación de Michels y la RM clasificó correctamente este patrón en el 92,3% de los individuos.La vena porta y sus ramas lobares y segmentarias se identificaron de forma excelente en el 100% de los individuos. El patrón de distribución venosa portal se evaluó siguiendo la clasificación de Cheng, y los hallazgos en la RM fueron coincidentes con de la angiografía y la cirugía en el 97,4% de los donantes. La prueba exacta de Fisher mostró una asociación significativa entre la RM y la angiografía y cirugía para diferenciar entre patrones normales y variantes en los vasos arteriales y en la vena porta.Las 3 venas hepáticas fueron visualizadas de forma excelente en todos los estudios de RM. La S de la RM para identificar las venas accesorias que requirieron anastomosis adicionales fue del 80%, la E del 100% y la PD del 94,8%.Para evaluar el patrón de distribución biliar se siguió la clasificación de Couinaud. La colangiografía con contraste fue mucho mejor que la obtenida sin contraste, permitiendo la primera identificar el patrón de distribución correcto en 88% de los individuos mientras que la segunda sólo lo hizo en el 57% de ellos.En conclusión, El estudio por RM de los candidatos a donante vivo hepático permite definir con precisión en un único examen los volúmenes lobares y evaluar la anatomía vascular y biliar esenciales para la planificación quirúrgica. La angiografía debe reservarse para aquellas situaciones en las que no se obtenga una representación adecuada de la vascularización arterial hepática. / . TÍTLE:Living donor liver transplantation. Magnetic resonance imaging contribution to liver volume calculation, and vascular and biliary anatomy depiction.SUMMARY:Large waiting lists for cadaveric liver transplantation have lead to the development of live donor liver transplantation (LDLT). Evaluation of potential candidates for donation includes liver volume calculation and arterial, portal, hepatic venous and biliary anatomy assessment. The use of the least noninvasive diagnostic tool for this group of individuals is mandatory. This work tries to demonstrate that magnetic resonance imaging (MRI) is enough in the evaluation of live living donors (LLD) candidates because it is reliable for the vascular and biliary anatomy evaluation and for liver volume calculation. Thirty nine consecutive LLD were studied with MRI. Angiographic and cholangographic sequences, the latest both prior and after biliary enhancing contrast agent administration were performed. MRI vascular findings were correlated with angiographic and surgical data, right lobe liver calculation on MRI was compared to graft weight and MRI cholangiographic findings were correlated with those obtained at intraoperative cholangiography. Simple linear Pearson correlation and Fisher test were used for statistical analyses.A high correlation between right lobe liver calculation on MRI and graft weight (r2 = 0.78) was observed, with an overestimation of about 18%. Hepatic, celiac, splenic and mesenteric arteries, as well as main trunk and lobar and segmental portal branches and the three main hepatic veins were always excellently depicted. Right and left hepatic arteries were sufficiently depicted in 74% and 72% of individuals respectively. Arterial distribution, according to Michel's pattern was correctly assessed in 92.3% and portal venous distribution, according to Cheng's classification was correctly assessed in 97.4% of subjects. MRI sensitivity, specificity and accuracy in depicting accessory hepatic veins requiring additional vascular anastomosis was 80%, 100% and 94,8% respectively.Contrast enhanced MRI cholangiography was better than non-enhanced MRI cholangiography for biliary anatomy assessment. A correct depiction of the biliary distribution in 88% and 57% was respectively achieved, following the Couinaud's classification pattern. In conclusion, MRI evaluation of LLD is reliable for liver volume calculation and vascular and biliary anatomy depiction, and can be used as the only imaging tool prior to surgery. Angiography can be reserved for those individuals with inadequate arterial vascular demonstration on MRI.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Plant virus identification and characterization can be achived by several methods based in the biological, morphological, cytological, serological and molecular virus properties. The molecular properties have been used with frequency for vÃrus identification and characterization and the reverse transcription polymerase chain reaction (RT-PCR) has constituted an efficient and precise method for researches with RNA plant viruses. On the other hand, the enzyme-linked immunosorbent assay (ELISA) is the most used serological method for virus detection in plant tissues. A techncal innovation developed for plant virus identification represents a great technological development and support for plant virus research. A new approach involving virus particle immune precipitation to be used for RNA amplification by RT-PCR named IP-RT-PCR was sucessefuly used for amplification of RNA fragments from five virus species from the genera Comovirus, Cucumovirus, Potyvirus and Sobemovirus. Considering that the immune biological Companies have developed several DAS-ELISA kits, but neither of them produce and commercialize PTA-ELISA kits, a simple and practical PTA-ELISA kit was developed and validated for plant virus detection. The second part of the research had the objective to study, comparatively, the biological, serological and molecular properties of four plant virus isolates obtained from naturally infected Passiflora edulis (PWV-PET and PWV-GUA) and from naturally infected Vigna unguiculata (CABMV-BV and CABMV-FOR) with the objective to elucidate the identity of the causal agent of passion fruit woodiness in Brazil. In host range studies onle Canavalia ensiformes and Macroptilium lathyroides were infected by virus isolates obtained from cowpea and from passion fruit. The isolate PWV-GUA was purified from systemically infected M. lathyroides plants and the virus purified preparation (18.24 mg of virus.ml-1) was used for rabitt immunization for polyclonal antiserum production, which showed a title of 1:128,000 in PTA-ELISA. The electrophoresis analysis of the purified virus showed a unique capsidial protein with 34 kDA. Plant virus interaction studies in C. ensiformis indicated unilateral cross protection between PWV-GUA and CABMV-FOR. On the other hand, the isolate PWV-PET did not cross protect passion fruit plants against PWV-GUA. Filogenetic analysis of nucleotiode sequencies from cDNA fragments corresponding to coat protein (CP) genes amplified by IP-RT-PCR from the genomic virus isolates compared with virus sequencies from the Genbank grouped according to the host specifities. Based on the biological, serological and mainly molecular results, the virus isolates studied were classified into two biotypes: Biotype CABMC-C (Cowpea) to include isolates obtained from cowpea that do not infect passion fruit, and biotype CABMV-P (Passion fruit) to include the virus isolates responsible for the passion fruit woodiness in Brazil. / A identificaÃÃo e a caracterizaÃÃo de vÃrus de planta podem ser realizadas por vÃrios mÃtodos envolvendo propriedades morfolÃgicas, biolÃgicas, citolÃgicas, moleculares e sorolÃgicas. As tÃcnicas moleculares tÃm sido usadas com frequencia para identificaÃÃo e caracterizaÃÃo de vÃrus, e a tÃcnica de âreverse transcription polymerase chain reactionâ (RT-PCR) tem se constituÃdo em mÃtodo eficiente e preciso para pesquisas com vÃrus de planta com genoma de RNA. De outra parte, a tÃcnica de enzyme-linked immunosorbent assay (ELISA) constitui o mÃtodo sorolÃgico mais usado para detecÃÃo de vÃrus em tecidos vegetais. Uma inovaÃÃo tecnolÃgica desenvolvida nesta pesquisa para diagnose de vÃrus de planta representa grande avanÃo tecnolÃgico e suporte para pesquisa em virologia vegetal. A inovaÃÃo envolvendo a imunoprecipitaÃÃo (IP) de partÃculas de vÃrus para uso na RT-PCR denominada de IP-RT-PCR foi usada com sucesso para amplificaÃÃo de fragmentos de RNA de cinco espÃcies de vÃrus dos gÃneros Comovirus, Cucumovirus, Potyvirus e Sobemovirus. Considerando que kits de DAS-ELISA tÃm sido produzidos e comercializados por companhias de imunobiologicos, mas nenhuma companhia produz kits de PTA-ELISA, um kit simples e prÃtico de PTA-ELISA foi desenvolvido e validado para detecÃÃo de vÃrus de planta. A segunda etapa da pesquisa teve como objetivo estudar as propriedades biolÃgicas, sorolÃgicas e moleculares de isolados de vÃrus do gÃnero Potyvirus obtidos de maracujazeiro (Passiflora edulis) (PWV-PET e PWV-GUA) e isolados de Cowpea aphid-borne mosaic virus (CABMV-FOR e CABMV-BV) obtidos de feijoeiro caupi (Vigna unguiculata), visando elucidar a identidade do agente causal do endurecimento dos frutos do maracujazeiro no Brasil. Em estudos de gama de plantas hospedeiras, somente Canavalia ensiformis e Macroptilium lathyroides foram infetadas por isolados obtidos de maracujazeiro e de feijoeiro caupi. O PWV-GUA foi purificado a partir de plantas de M. lathyroides sistemicamente infetadas e a preparaÃÃo viral purificada (18,24 mg de vÃrus.ml-1) foi usada para imunizaÃÃo de coelho com a produÃÃo de antissoro policlonal com tÃtulo de 1:128.000 em PTA-ELISA. AnÃlise eletroforÃtica da preparaÃÃo viral purificada revelou uma Ãnica proteÃna capisidial com peso molecular de 34 kDa. Experimentos de interaÃÃo entre os isolados virais em C. ensiformis indicaram proteÃÃo unilateral entre PWV-GUA e CABMV-FOR. De outra parte, o isolado PWV-PET nÃo protegeu plantas de maracujazeiro contra a super infecÃÃo de PWV-GUA. AnÃlises filogenÃticas das seqÃÃncias dos fragmentos de cDNA correspondentes Ãs capas protÃicas (CP), amplificados a partir dos genomas dos isolados virais de maracujazeiro e de feijoeiro caupi por IP-RT-PCR, agruparam-se com as seqÃÃncias de isolados virais de referidas culturas depositadas no GenBank, apresentando um agrupamento em funÃÃo da especificidade de hospedeiros. Com base nos resultados dos estudos biolÃgicos, sorolÃgicos e, sobretudo moleculares, os isolados virais estudados foram classificados em dois biÃtipos: BiÃtipo CABMV-C (Cowpea) incluindo os isolados obtidos de feijoeiro caupi e biÃtipo CABMV-P (Passion fruit) para incluir os isolados responsÃveis pelo endurecimento dos frutos do maracujazeiro no Brasil.