Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2016-10-07T21:30:40Z No. of bitstreams: 1 INCID?NCIA, VARIABILIDADE MOLECULAR DE V?RUS E CULTIVO IN VITRO DE MERISTEMAS DE _20160429093320061.pdf: 1513731 bytes, checksum: b1a69730145b3f5bec766a5adb8b491c (MD5) / Made available in DSpace on 2016-10-07T21:30:40Z (GMT). No. of bitstreams: 1 INCID?NCIA, VARIABILIDADE MOLECULAR DE V?RUS E CULTIVO IN VITRO DE MERISTEMAS DE _20160429093320061.pdf: 1513731 bytes, checksum: b1a69730145b3f5bec766a5adb8b491c (MD5) Previous issue date: 2016-03-21 / Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB / The vine is grown in many countries, the berries being the main product with many uses, such as fresh grapes, juices and wines. The wine industry is an important economic activity in Brazil, highlighting the pole Petrolina, PE / Juazeiro, BA in sub middle of San Francisco Valley (SSFV), as the main producing region of fine table grapes in the country. The multiplication of vine cuttings is done mainly by vegetative propagation, facilitating the spread of pathogens, one of the main sources of the spread of the virus. This study aimed to evaluate the incidence of Grapevine virus A virus (GVA), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 3 (GLRV-3), Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV) by detecting by Double Antibody Sandwich ELISA (DAS-ELISA) in grapevines of SSFV in six municipalities located in the states of Bahia and Pernambuco, as well as analyze the regenerative capacity and in vitro development of 13 different tropical clones of vine obtained by cultivation of apical meristems and axillary buds, and finally, to analyze the variability of the fragments of the protein coat (PC) gene of the virus winding the sheet cultivars of tropical vines of the San Francisco Valley. Of the 490 samples evaluated, 320 are cultivars for table with and without seeds, 94 for wine and 76 rootstocks. Infections caused by viruses were detected in 79.4% of samples. The GLRaV-3 was the most widespread viruses, followed by GVA GFkV, GLRaV-1 and GFLV, respectively. Mixed infection was detected in 153 samples. Cultivars wine, fine table grapes and rootstocks showed total percentage of 93.2% infection, 52.6% and 31.9%, respectively. For the regenerative capacity and development in vitro, we analyzed the percentage of regeneration time and the numbers of buds, roots and leaves produced as well as the length of the internodal segments 30, 60 and 90 days of cultivation in culture medium 'MS' and 'Galzy', respectively. Of the 13 cultivars tested, four were regenerated with a percentage ranging between 7% and 47%. The occurrence of significant differences among cultivars showed different responses over time. 'Cabernet Sauvignon' presented the best regeneration response starting from apical meristem and axillary buds. 'IAC-572' stood out with the highest average height and bud and leaf numbers 30, 60 and 90. To determine the variability of the CP gene, samples of 28 BAG vines cultivars of the Embrapa Semiarid with and without of disease symptoms were analyzed by reverse transcriptase and polymerase chain reaction (RT-PCR). Of all the samples GLRaV -1, -2 and -3, zero, 10 and 19 presented infection, respectively, and eight had mixed infection. There was a high and low identity between isolates obtained from Brazilian and foreign isolated for both viruses, finding genetic divergence of the CP gene in some isolates. 13 isolates showed no significant homology with other isolates in the present study and available on GENBANK, to GLRaV-3. / A videira ? cultivada em v?rios pa?ses, sendo as bagas o produto principal com diversos usos, como uvas frescas, sucos e vinhos. A vitivinicultura ? uma atividade econ?mica importante no Brasil, destacando-se o polo Petrolina, PE/Juazeiro, BA no Subm?dio do Vale do S?o Francisco (SVSF), como a principal regi?o produtora de uvas finas de mesa do pa?s. A multiplica??o de mudas de videira ? feita principalmente por propaga??o vegetativa, facilitando a difus?o de pat?genos, sendo uma das principais fontes de dissemina??o dos v?rus. No presente estudo, objetivou-se avaliar a incid?ncia dos v?rus Grapevine virus A (GVA), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 3 (GLRV-3), Grapevine fanleaf virus (GFLV), e Grapevine fleck virus (GFkV), atrav?s da detec??o por Double Antibody Sandwich ELISA (DAS-ELISA), em parreirais do SVSF, em seis munic?pios, localizados nos estados da Bahia e Pernambuco, como tamb?m analisar a capacidade regenerativa e desenvolvimento in vitro de 13 diferentes clones tropicais de videira obtidos pelo cultivo de meristemas apicais e de gemas axilares, e por fim, analisar a variabilidade dos fragmentos do gene da capa proteica (CP) do v?rus do enrolamento da folha de cultivares de videiras tropicais do Vale do S?o Francisco. Das 490 amostras avaliadas, 320 s?o cultivares para mesa com e sem sementes, 94 para vinho e 76 de porta-enxertos. Infec??es causadas por v?rus foram detectadas em 79,4% das amostras. O GLRaV-3 foi o v?rus mais disseminado, seguido por GVA, GFkV, GLRaV-1 e GFLV, respectivamente . Infec??o mista foi detectada em 153 amostras. Cultivares para vinho, uvas finas de mesa e porta-enxertos apresentaram porcentagem total de infec??o de 93,2%, 52,6% e 31,9%, respectivamente. Para a capacidade regenerativa e desenvolvimento in vitro, foram analisadas a porcentagem de regenera??o, altura, e os n?meros de gemas, ra?zes e folhas produzidas, bem como o comprimento dos segmentos internodais, aos 30, 60 e 90 dias de cultivo em meio de cultura ?MS? e ?Galzy?, respectivamente. Das 13 cultivares testadas, quatro foram regeneradas com porcentagem variando entre 7% e 47%. A ocorr?ncia de diferen?as significativas entre as cultivares revelaram respostas distintas ao longo do tempo. ?Cabernet Sauvignon? apresentou a melhor resposta de regenera??o de plantas partindo de meristema apical e de gemas axilares. ?IAC-572? destacou-se com as maiores m?dias de altura e n?meros de gemas e de folhas aos 30, 60 e 90. Para determinar a variabilidade do gene da CP, amostras de 28 cultivares de videiras do BAG da Embrapa Semi?rido com presen?a e aus?ncia dos sintomas da doen?a foram analisadas por Transcriptase Reversa e Rea??o em Cadeia da Polimerase (RT-PCR). De todas as amostras para GLRaV -1, -2 e -3, zero, 10 e 19 apresentaram infec??o, respectivamente, sendo que oito apresentaram infec??o mista. Observou-se alta e baixa identidade entre os isolados obtidos com isolados brasileiros e estrangeiros para ambos os v?rus, encontrando-se diverg?ncia gen?tica do gene da CP em alguns isolados. 13 isolados n?o apresentaram homologia significativa com outros isolados do presente estudo e dispon?veis no GENBANK, para GLRaV-3.

Algumas espécies de morcegos têm sido reconhecidas como reservatórios naturais de várias famílias virais, desempenhando um importante papel na trasmissão e manutenção desses micro organismos. Devido à descaracterização e fragmentação de habitats naturais, esses mamíferos buscam alternativas de abrigo e alimento, e assim, ficam cada vez mais expostos aos meios antrópicos e em contato com humanos e animais domésticos. Com exceção do vírus rábico, existem poucos trabalhos realizados na detecção de vírus em morcegos no Brasil. Em virtude disso, o presente estudo objetivou a detecção de vírus (circovírus, astrovírus, coronavírus e lyssavírus relacionados ao vírus da raiva) em amostras de órgãos de morcegos do estado do Rio Grande do Sul. Os ácidos nucléicos foram extraídos das amostras de órgãos de morcegos e submetidos à detecão por PCR e RT-PCR. Após a detecção, os fragmentos obtidos foram sequenciados para realizar análise filogenética dos vírus encontrados. Ao total foram analisadas 108 amostras de diferentes espécies e localidades, das quais dez foram positivas para circovírus, seis para coronavírus e 25 para astrovírus, este último sendo o primeiro registro do vírus em morcegos para o Brasil. Todas as amostras foram negativas para lyssavírus relacionados ao vírus da raiva. Análises filogenéticas revelaram que as sequências de circovírus agruparam em ambos os gêneros Circovirus e Cyclovirus, coronavírus no gênero Alphacoronavirus em dois clados diferentes e astrovírus no gênero Mamastrovirus junto com outros astrovírus de morcegos, o qual formam um clado separado dos outros mamíferos. Os resultados demonstram uma diversidade genética entre os vírus encontrados em diferentes espécies de morcegos, que possuem dietas alimentares e habitats distintos. / Some bat species have been recognized as natural reservoirs of several viral families, playing an important role in the transmission and maintaining of these micoorganism. Due to mischaracterization and fragmentation of natural habitats, these mammals seek shelter alternatives and food, and thus are increasingly exposed to anthropism, which make the contact with humans and domestic animals closer. With the exception of the rabies virus, there are few studies on the detection of viruses in bats in Brazil. Therefore, the present study aimed the detection of viruses (circovirus, astrovirus, coronavirus and rabies-related virus) in bats organs samples from Rio Grande do Sul state. Nucleic acids were extracted from bat organs samples and submitted to detection by PCR and RT-PCR. After detection, the obtained fragments were sequenced to perform phylogenetic analysis of the viruses found. From a total of 108 samples analyzed of different species and locations, ten were positive for circoviruses, six for coronaviruse and 25 for astrovirus, which was the first report of this virus in bats in Brazil. All samples were negative for rabies-related virus. Phylogenetic analyzes revealed that the sequences of circoviruses grouped in both Circovirus and Cyclovirus genus, coronaviruses in Alphacoronavirus genus in two different clades and astroviruses in Mamastrovirus genus along with other bats astrovirus, which form a separate clade from other mammals. Results demonstrate a genetic diversity among viruses found in different species of bats, which have different diets and habitats.

Orientador: Luís Guilherme de Oliveira / Banca: Simone Maria Massami Kitamura Martins / Banca: Lizandra Amoroso / Resumo: A infecção persistente ao vírus da diarreia viral bovina (BVDV) pode viabilizar a disseminação do vírus no rebanho, assim como interferir no controle da infecção. Ao mesmo tempo, em suínos, a presença de soropositivos para BVDV pode causar transtornos aos inquéritos sorológicos para a peste suína clássica (PSC). Este trabalho teve como objetivo determinar a condição de persistentemente infectado em leitões nascidos de porcas infectadas experimentalmente pelo vírus da diarreia viral bovina. Foram selecionadas seis porcas prenhes para este estudo que foram divididas em dois grupos, sendo um grupo inoculado com BVDV-2 (G1; n=4) aos 45 dias de gestação, e um grupo controle (G2; n=2). Foram realizadas avaliações clínicas nas porcas diariamente. Os neonatos foram monitorados durante 35 dias, em que foram realizadas avaliações clínicas rotineiras e colheita de suabes nasal dos leitões e de amostras de sangue venoso das porcas e dos leitões para obtenção de sangue total e soro a cada 72 horas. Foram realizados testes de RT-PCR para diagnóstico direto, e virusneutralização para avaliação sorológica. As porcas apresentaram soroconversão entre o 17ºdia pós-infecção (dpi) e o 22ºdpi, mas não foi detectada viremia. Nenhum leitão apresentou títulos de anticorpos ou viremia ao nascimento. Não ocorreu a transmissão transplacentária do vírus, portanto, não foi possível observar animais PI. / Abstract: The persistently infection to bovine viral diarrhea virus (BVDV) can enable the spread of virus in the herd, as well as interfere in the control of infection. Concurrently, the presence of seropositive pigs may interfere with serological surveys for classical swine fever (CSF). This project aimed to determine the condition of persistently infected in piglets born from gilts infected with bovine viral diarrhea. BVDV-2 was inoculated in four pregnant gilts (G1; n=4), and a placebo was administered in two gilts, which were the control group (G2; n=2). Clinical evaluations were daily performed in the gilts. The newborns were monitored during 35 days, with clinical evaluation and whole blood, serum and nasal swabs sampling every 72 hours. RT-PCR and virus neutralization tests (VN) were performed. The gilts presented seroconversion between 17º dpi and 22ºdpi, but no viremia was detected. No piglets presented antibody titers or viremia at birth. Transplacental transmission of the virus did not occur, therefore, PI animals could not be observed. / Mestre

O vírus da diarreia viral bovina (BVDV) é um dos principais patógenos virais dos bovinos e sua infecção causa importantes perdas na produção dos rebanhos afetados. O presente trabalho contém os estudos realizados para esclarecer determinados aspectos da epidemiologia e da detecção da infecção causada pelo BVDV no Estado do Rio Grande do Sul, Brasil. O primeiro artigo consistiu de um estudo transversal onde os níveis de anticorpos contra o BVDV em amostras de tanque de leite que foram correlacionados com os aspectos produtivos dos rebanhos. As amostras e as informações foram coletadas de 300 criações escolhidas aleatoriamente entre 1.656 associados de uma cooperativa de leite na região central do Estado do Rio Grande do Sul e que não utilizaram vacina contra BVDV nos últimos 12 meses anteriores ao experimento. Dessas, 80 foram consideradas positivas para BVDV pela detecção de anticorpos contra vírus em níveis superiores ao ponto de corte do teste de ELISA comercial utilizado. A prevalência aparente estimada foi de 26,7% e, usando um intervalo de confiança de 95%, apresentou uma variação entre 22 e 31%. Os parâmetros de produção mensal de leite, densidade de bovinos (relação vacas/hectare), tipo de armazenamento do leite e origem do sêmen não apresentaram associação com a infecção pelo BVDV, mas o aumento do número total de animais por rebanho foi correlacionado positivamente com a infecção. A prevalência estimada foi considerada baixa para uma população de rebanhos, criados em sistema semi-intensivo de produção de leite, que não usava medidas específicas de controle contra a infecção. A hipótese proposta no presente artigo é que o pequeno número de animais por rebanho está favorecendo a uma provável limpeza ou erradicação da infecção nas criações e que a prevalência encontrada deve corresponder ao ponto de equilíbrio entre a pressão de infecção e a limpeza da infecção destes rebanhos. A baixa prevalência estimada pode ser uma condição favorável para iniciar um programa de controle para BVDV na população estudada. No segundo artigo, um teste da transcrição reversa seguida da reação em cadeia da polimerase (RT-PCR) foi estabelecido para detecção do vírus nas amostras de tanque de leite dos rebanhos suspeitos de infecção ativa pelo BVDV identificados no primeiro artigo. O teste foi capaz de detectar até 0,001 TCID50 de BVDV por mL de leite. Esses resultados demonstraram uma sensibilidade analítica 100 ou 1000 vezes superior aos testes de RTPCR convencionais descritos anteriormente para leite e são semelhantes aos resultados obtidos em testes de RT-PCR em tempo real para BVDV. Na análise das amostras de campo, o teste detectou RNA viral em dois rebanhos entre os 59 analisados. Esses resultados comprovaram a capacidade do teste para identificar amostras naturalmente infectadas e a presença de vacas adultas produtivas e virêmicas em 3% dos rebanhos suspeitos de infecção. Este teste é uma alternativa rápida e sensível para monitorar a infecção pelo vírus em rebanhos leiteiros, a partir de amostras coletivas. O terceiro artigo tratou da detecção do BVDV em carrapatos Rhipicephalus (Boophilus) microplus alimentados em bovino persistentemente infectado (PI). Seu objetivo foi investigar o papel do carrapato bovino R. microplus na transmissão do BVDV. O RNA viral foi detectado nas teleóginas alimentadas no bovino PI, mas não pode ser identificado em sua progênie (ovos e larvas). Esses resultados sugeriram não haver transmissão transovariana do BVDV nos carrapatos. Por outro lado, a infestação experimental de um segundo bovino com as larvas oriundas das teleóginas contaminadas com BVDV não resultou em manifestação clínica e o animal foi negativo no teste de RT-PCR. O experimento demonstrou que o carrapato R. microplus pode ser contaminado com BVDV durante o repasto sanguíneo, reforçando a idéia de que vetores hematófagos possam estar envolvidos na disseminação da doença e merecem mais investigações. / Bovine viral diarrhea virus (BVDV) is one of the main pathogenic agents in cattle and the infection with the agent causes important production losses in the affected herds. The present work contains studies carried out to clarify some aspects of the epidemiology and detection of the infection caused by BVDV in the State of Rio Grande do Sul, Brazil. The first paper consisted of a transversal study, where antibody levels against BVDV in samples of bulk milk tank were correlated with some aspects of milk production in the herds. Samples and information were collected from 300 herds randomly chosen between 1656 associates of a dairy cooperative in the region of the central area of the state of Rio Grande do Sul, Brazil, that were not using routine vaccination against BVDV in the last 12 months previous to the experiment. Among them, 80 were considered positive to BVDV by detection of antibodies against the virus in levels above the cut off point of the commercial ELISA test used. The apparent estimated prevalence was 26.7% and, using a confidence interval of 95%, showed a range between 22 and 31%. The parameters of average milk production, bovine density (relationship cattle/hectare), milk storing process and origin of the semen were not significantly associated with BVDV infection, but the increase in the total number of cows in the herds was positively correlated with the infection. The prevalence was considered low for a population of herds raised in a semi-intensive milk production system that was not using specific measures to control the viral infection. The hypothesis proposed in the present study is that the small number of animals present in the analyzed herds would favors a likely virus clearance in the herd and that the prevalence found would correspond to a point of balance between the pressure of infection and the virus clearance. The low prevalence detected can be a favorable condition to launch a control program for BVDV in the studied population. In the second paper, a reverse transcription polymerase chain reaction (RT-PCR) was established for BVDV detection in bulk tank milk samples of the herds suspected of active infection with BVDV in the first paper. The test detected up to 0.001 TCID50 of BVDV per mL of milk. These results demonstrated an analytical sensitivity 100 to 1000 times higher than conventional RT-PCR tests described previously for milk and are similar to results obtained by the use of real time RT-PCR already published. In the analysis of field samples, the test detected viral RNA in 2 out of 59 analyzed herds. These results demonstrated the relationship between the ability of the test to identify samples naturally infected and the presence of viremic adult cows in 3% of the herds suspected of the infection. This test represents a fast and sensitive alternative for monitoring virus infection in dairy herds, through the analysis of collective samples. The third article dealt with the detection of BVDV in the tick Rhipicephalus (Boophilus) microplus fed in a bovine persistently infected (PI). The objective was to investigate the role of the cattle tick R. microplus in the transmission of BVDV. Viral RNA was detected in adult females fed on the PI bovine, but could not be identified in its progeny (eggs and larvae). These results suggest that there is no transovarial transmission of BVDV in ticks. On the other side, experimental infestation of a second bovine with larvae derived from adult cattle contaminated with BVDV did not result in clinical manifestations and the animal was negative in the RT-PCR test. The study demonstrated that the tick R. microplus can be contaminated with BVDV during blood feeding, strengthening the idea that haematophagous vectors could be involved in the dissemination of the disease and would deserve further consideration.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-03-27T18:48:03Z No. of bitstreams: 1 2017 - Tays Araujo Camilo.pdf: 3555895 bytes, checksum: 8b6818596f8654186472296bc3b528aa (MD5) / Made available in DSpace on 2018-03-27T18:48:03Z (GMT). No. of bitstreams: 1 2017 - Tays Araujo Camilo.pdf: 3555895 bytes, checksum: 8b6818596f8654186472296bc3b528aa (MD5) Previous issue date: 2017-03-07 / CNPq / Infectious Bronchitis Virus (vBIG) is a highly contagious viral agent among the species Gallus gallus, being considered one of the pathologies that most affects small and lare world poultry producers. Vaccination as a form of prevention to this disease, has not been effective, due to the great genetic variability found. As in the world, in Brazil there are already several studies characterizing the genetic variability found in this virus. In Rio de Janeiro, poultry farming suffers from the disease, but there is no recent study to analyze the genetic variability of the strains found, as a way of supporting epidemiological studies that may help in the future, in the choice of Protocols. The objective of this study was to characterize the S1 gene of the Chicken Infectious Bronchitis virus (vBIG) in chickens farms in the southern region of Rio de Janeiro, as well as to evaluate the genetic variability of the S1 gene in a BIG outbreak on a farm Of laying hens of the northern region of. Tissue samples were collected from animals presenting BIG-compatible clinical signs, such as weight loss, respiratory problems and low productivity. Samples of collected tissues were stored in solution of RNA Later and formalin 10% buffered for molecular and histopathological diagnoses, respectively. In order to perform the molecular analyzes, RNA extraction techniques, as well as RT-PCR (Reverse Transcriptase, Polymerase Chain Reaction), real-time PCR (RT-qPCR), Nested PCR (RT-nPCR), and Sequencing of some samples collected. For the histopathological analyzes, cleavage and preparation of microscopy slides were performed. The results obtained from the sequencing were compared to the sequence of the Ma5 vaccine strain as reference strain, in addition to other sequences deposited on GenBank, exhibiting a variable identity percentage of 72.41% to 100%. Among the samples from each farm, there was variability, even within the same bird. All sequences analyzed were described as genotype 1, belonging to strains 1 and 11; In the comparison of amino acids, there were changes in the hypervariable regions of the virus in the sequences classified as BR-1, being able to explain the low cross-protection that have been occurring in Brazilian plants. These results demonstrated the importance of virus identification, since it becomes an important tool in epidemiological surveillance and in the elaboration of efficient vaccine protocols. / O v?rus da Bronquite Infecciosa das Galinhas (vBIG) ? um agente viral altamente contagioso entre a esp?cie Gallus gallus, sendo considerado uma das patologias que mais acomete a avicultura mundial. A vacina??o como forma de preven??o a esta doen?a, n?o tem sido eficaz, devido a grande variabilidade gen?tica encontrada. Assim como no mundo, no Brasil tamb?m j? existem diversos estudos caracterizando a variabilidade gen?tica encontrada neste v?rus. No Rio de Janeiro, a cria??o av?cola sofre com a doen?a, por?m ainda n?o existe um estudo recente com o objetivo de analisar a variabilidade gen?tica das cepas encontradas, como uma forma de apoio a estudos epidemiol?gicos, que possam auxiliar no futuro, na escolha de protocolos vacinais mais adequados. Este estudo teve como objetivo realizar a caracteriza??o molecular do gene S1 do v?rus da Bronquite Infecciosa das Galinhas (vBIG) em granjas de frangos de corte na regi?o Sul fluminense, como tamb?m avaliar a variabilidade gen?tica do gene S1 em um surto de BIG em uma granja de poedeiras da regi?o Norte fluminense. Foram realizadas coletas de tecidos dos animais que apresentavam sinais cl?nicos compat?veis com BIG, como perda de peso, problemas respirat?rios e baixa produtividade. Amostras de tecidos coletados foram armazenadas em solu??o de RNA Later e formalina 10% tamponada para os diagn?sticos molecular e histopatol?gico, respectivamente. Para realiza??o das an?lises moleculares foram utilizadas t?cnicas de extra??o de RNA, bem como RT-PCR (?Reverse Transcriptase, Polymerase Chain Reaction?), PCR em tempo real (RT-qPCR), ?Nested? PCR (RT-nPCR), e sequenciamento de algumas amostras coletadas. Para as an?lises histopatol?gicas foram realizadas clivagem e elabora??o de l?minas de microscopia. Os resultados obtidos do sequenciamento foram comparados a sequ?ncia da cepa vacinal Ma5, como cepa refer?ncia, al?m de outras sequencias depositadas no GenBank, apresentando um percentual de identidade vari?vel de 72,41% a 100%. Dentre as amostras de cada granja, houve variabilidade, at? mesmo dentro de uma mesma ave. Todas as sequencias analisadas foram descritas como gen?tipo 1, pertencentes as linhagens 1 e 11; Na compara??o de amino?cidos, houve mudan?as nas regi?es hipervari?veis do v?rus nas sequ?ncias classificadas como BR-1, podendo explicar a baixa prote??o-cruzada que v?m ocorrendo nos plant?is brasileiro. Estes resultados demonstraram a import?ncia da identifica??o g?nica do v?rus, pois se torna uma ferramenta importante na vigil?ncia epidemiol?gica e em elabora??es de protocolos vacinais que sejam eficientes.

Le sujet de ma thèse était de décrire sur le plan moléculaire le processus d'embryogenèse somatique chez la vigne. Pour cela, les étapes-clés d'entrée et de sortie du cycle d'embryogenèse secondaire ont été caractérisées par l'analyse de l'expression de quelques gènes impliqués dans le développement ou la défense, en particulier les gènes PR10.Grâce à l'exploitation de la séquence complète du génome de Vitis vinifèra disponible sur le site du Genoscope, j'ai pu caractériser exhaustivement la famille multigènique des PR10. Celle-ci est composée de 17 séquences disposées en tandem et formant un cluster compact sur le chromosome 5, dont 3 pseudogènes et au moins 13 séquences transcrites. L'expression de 10 de ces gènes a d'abord été analysée par RT-PCR semi-quantitative dans différents organes de la plante et dans des tissus traités au 2,4-D. Elle suggère une diversification fonctionnelle marquée. De plus, le niveau d'expression de plusieurs gènes PR10 est élevé dans les cals embryogènes, suggérant qu'ils pourraient jouer un rôle lors de l'embryogenèse somatique. L'étude de l'expression des gènes PR10 par RT-PCR quantitative en temps réel dans différents tissus ayant montré une capacité embryogénique variable lorsqu'ils sont soumis à un traitement par le 2,4-D met en évidence que le niveau d'expression varie entre les gènes et selon les tissus. L'expression de certains gènes est fortement induite par le 2,4-D dans les tissus à capacité embryogénique et seulement faiblement dans les tissus ne donnant jamais d'embryons somatiques, ce qui suggère fortement que ceux-ci pourraient être des marqueurs de la capacité embryogénique chez la vigne. / The objective of my work was to analyse the somatic embryogenesis process of Vitis vinifera at a molecular scale. Thus, the expression of genes implied in development or defence, especially PR10 genes, was monitored during the key-steps of entrance and exit of secondary somatic embryogenesis. The complete sequence of the Vitis vinifera genome available on the Genoscope website allowed the exhaustive characterization of the PR10 multigene family, which is constituted by 17 sequences localised on a tandem array on the chromosome 5. Among these 17 sequences, 3 are pseudogenesand at !east 13 are transcribed sequences. The expression of 10 PR10 genes was first monitored in various grapevine tissues and in tissues after 2,4-D treatment using semi-quantitative RT-PCR. The results suggest a strong functional diversification. Moreover, the expression of several PR10 genes is high in embryogenic calli, suggesting that these genes could intervene in somatic embryogenesis. The expression of PR10 genes was also monitored in tissues showing different somatic embryogenic capabilities under 2,4-D treatment using quantitative RT-PCR. The results show that regulation of PR10 genes is dependent of the gene and tissue considered. Moreover, the expression of some genes is highly induced by 2,4-D treatment in tissues having embryogenic capability, white it is only weakly induced in tissues having no embryogenic capability, suggesting that these gene could be markers of embryogenic capability in grapevine.

<p>Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression <i>in vivo</i>, i.e. to accomplish <i>in vivo</i> hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. </p><p>This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced <i>in vivo</i> imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents.</p><p>The present study aimed to investigate ⁷⁶Br- and ⁶⁸Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-<i>O</i>-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of <i>in vivo</i> hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.</p>

Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno-sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S-VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels comparable to those from kidney cortex, while VEGF165 was lower. In PRCC and ChRCC, VEGF189 levels correlated inversely with advancing tumour stage, and in PRCC, VEGF165 levels correlated inversely with increasing tumour size. VEGF189 was an independent prognostic factor for survival in patients with PRCC. Conclusions. S-VEGF has a stronger association to progression in RCC than platelet count. CRCC showed high levels of VEGF mRNA isoforms and VEGF-R1 mRNA compared to PRCC. VEGF mRNA isoforms expression decreased with advancing stage. IHC demonstrated VEGF expression in cell cytoplasm related to tumour growth, particular in CRCC. Different VEGF isoform patterns were found in different RCC types. Protein VEGF189 expression was associated with tumour stage and was an independent prognostic factor in PRCC. Protein VEGF165 expression was generally low and had no prognostic value. The trend for decreasing levels of VEGF isoforms in advanced tumour stages may indicate that angiogenic activity is an early event in tumour growth induced by VEGF, but that during later tumour progression the role of VEGF is less clear.

Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression in vivo, i.e. to accomplish in vivo hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced in vivo imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents. The present study aimed to investigate 76Br- and 68Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-O-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of in vivo hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.

Worldwide obesity is an increasing problem. Apart from the fact that obesity greatly  impairs the health, quality and length of life for the affected individuals, it is also has the  potential to become a major socioeconomic problem in a near future. However preventive  actions require an understanding of the cause. Before the psychological influence on  eating can be evaluated a profound understanding of the biological regulatory system and  how this interacts with the food consumed is required. On the assumption that food  consumption is regulated by interplay between food and genes, the food itself may  influence the genes that regulate consumption, hence change the expression levels of the  genes regulating food intake.     To evaluate the interplay between food and gene expression, the project contained several  parts, reflecting different aspects of the area of research. The feeding studies had in  common that they were initial trials in a larger project. The results of these will be  evaluated and used in combination with further studies.     The mice typed for food preference illustrate the complexity of the feeding regulatory  system by pointing out the differences between individuals even in a relatively small  group of animals. Mice in general like food high in fat and here the animals that showed a  preference for sugar also showed a significant increase in their intake of chow. Since  chow consists mainly of carbohydrates the results might indicate a preference not for  sucrose in particular but for carbohydrates in general. The effect this may have on other  studies is still unclear as further studies are needed to determine whether the difference  may be the result of an innate genetic difference.      Leucine has been previously shown to reduce the total caloric intake. When given in  combination with palatable food the addition of Leucine primarily reduced the intake of  chow. From a dietary perspective this would translate to a preference to sweets and fast  food at the expense of food with more nutritious content.     The RT-PCR analysis’s gives clues to how the energy regulatory circuitry responds to the  intake of selected macronutrients. When it comes to gene expression there is a significant  effect of macronutrients on the gene expression levels. The common theme for many of  the genes tested seems to be down regulation of satiety signals, as if to support over  feeding on palatable diets and in many cases sucrose in particular.     The intake of macronutrients such as sugar or fat has been showed to have an effect on  the feeding regulatory circuitry, demonstrated by the change in gene expression levels.   The response to said macronutrients is site specific which is clearly shown both by RTPCR analysis of samples from different parts of the brain, such as the brainstem or  hypothalamus, and by immunohistochemistry of selected areas. The  immunohistochemistry also confirms that the novel Oxytocin receptor-antagonist, who is  injected IP, actually passes over the blood-brain barrier and has an actual affect on the  regions of interest. The areas affected by the antagonist can be visualized and identified  through the staining of active sites.