Made available in DSpace on 2014-12-17T15:18:10Z (GMT). No. of bitstreams: 1 MariaAGD.pdf: 3831855 bytes, checksum: 0abe0a10e359f22bab7ed834fa8e296a (MD5) Previous issue date: 2009-02-26 / Sugarcane is one of the most important products of the world and Brazil is responsible for 25 % of the world production. One problem of this culture at northeast of Brazil is the early flowering. In our laboratory, it has been made before four subtractive libraries using early and late flowering genotypes in order to identify messages related to the flowering process. In this work, two cDNAs were chosen to make in silico analysis and overexpression constructs. Another approach to understand the flowering process in sugarcane was to use proteomic tools. First, the protocol for protein extraction using apical meristem was set up. After that, these proteins were separated on two bidimensional gels. It was possible to observe some difference for some regions of these gels as well as some proteins that can be found in all conditions. The next step, spots will be isolated and sequence on MS spectrometry in order to understand this physiological process in sugarcane / A cana-de-a??car ? uma das mais importantes culturas mundiais e atualmente o Brasil representa um dos maiores produtores de cana-de-a??car no ranque mundial. Sabendo-se da import?ncia da cana-de-a??car nos dias atuais, principalmente em rela??o ao biocombust?vel e do problema causado pela flora??o precoce a esta cultura na regi?o Nordeste, foi realizada uma an?lise in silico de dois cDNAs:, 14-3-3 like protein e Protein kinase C inhibitor-like (PKCI), envolvidos no processo de flora??o da cana-de-a??car, utilizando ferramentas gen?micas. Foi escolhido o cDNA PKCI para a constru??o de cassetes de super-express?o de modo a ser caracterizado o papel deste cDNA no processo de flora??o. Outra abordagem utilizada nesse trabalho foi de analisar prote?nas totais de ?pices meristem?ticos de variedades precoce e tardia em g?is uni e bidimensionais. Os resultados mostraram que existem algumas prote?nas que podem ser caracter?sticas de uma das variedades, e em outras foi observado uma express?o diferencial

Estresses abióticos como seca, alta salinidade e baixas temperaturas, afetam o crescimento e a produtividade em culturas de interesse comercial como o feijoeiro comum. Proteínas DREB (Dehydration Responsive Element Binding) são fatores de transcrição que regulam genes específicos envolvidos na tolerância ao estresse abiótico. Para determinar como as plantas toleram condições ambientais adversas, variedades tolerantes, biologia molecular e bioinformática podem ser aplicadas para identificar e caracterizar genes que controlam mecanismos de adaptação a estresses. Baseado nas informações disponíveis nos bancos de dados públicos, a sequência da Orf completa do gene Phvul.009G029600.1| PACid:27146455 contendo 1062 pb foi encontrada e usada para o desenho dos primers e para o sequenciamento. A nova sequência é muito similar ao AtRAP2.4 e foi nomeada como PvDREB6A, segundo a análise filogenética. Ferramentas de predição mostraram que a sequência apresenta 354 aminoácidos e possui uma cópia do domínio AP2, que se dobra em uma estrutura com três ?-folhas e uma ?-hélice apresentando resíduos importantes e motivos específicos de reconhecimento e de ligação ao DNA. Além disso, um peptídeo trânsito foi detectado na porção N-terminal com um sítio de clivagem no resíduo 52. A interação deste fator de transcrição com seu domínio de ligação ao DNA foi validada por Electro Mobility Shift Assay (EMSA). A localização subcelular da proteína foi realizada e expressão da Green Fluorescent Proteín (GFP) foi detectada no núcleo. A transformação genética para a superexpressão do gene PvDREB6A em plantas de Arabidopsis thaliana Columbia-0 e mutantes nocaute para o gene AtRAP2.4 (Salk_020767C) foi realizada. Quatro eventos com cópia única e melhor expressão do gene PvDREB6A denominados Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/ pFEC2.1 #19.7 e Salk_020767C/ pFEC2.1 #23.7, foram selecionados. O evento Salk_020767C/pFEC2.1 #23.7 mostrou melhor expressão do gene PvDREB6A e foi visualizado sob luz UV. A análise funcional revelou que as plantas transgênicas submetidas ao déficit hídrico, à alta salinidade e ao frio, apresentaram maior taxa de sobrevivência. Plantas transgênicas superexpressando o gene PvDREB6A apresentaram menor taxa de desidratação e de vazamento de eletrólitos quando submetidas a estresses abióticos. Uma análise da expressão de genes relacionados à tolerância foi conduzida. A quantificação revelou que a expressão de 18 genes: AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, relacionados a tolerância a seca, sal e frio foram up-regulated devido à superexpressão do gene PvDREB6A de feijoeiro nas plantas transgênicas / Abiotic stresses like drought, high salinity and low temperatures affect growth and productivity in crops of economic interest such as common bean. DREB (Dehydration Responsive Element Binding) proteins are transcription factors that activate specific genes involved in tolerance to abiotic stress. To generate new information on the research for drought and other abiotic stresses, tolerant varieties, molecular biology and bioinformatics can be applied to identify and characterize genes that control plant defense and adaptation mechanisms to water deprivation, to excessive salt and to high/low temperature. Based on public databases, a common bean DREB sequence was found and an in silico study was carried out. A complete Orf sequence Phvul.009G029600.1 |PACid:27146455 containing 1062 bp was found and used for primer design and sequencing. The new sequence was very similar to AtRAP2.4 and named as PvDREB6A, according to phylogenetic analysis. Prediction tools showed that the deduced 354 aa sequence has one copy of the AP2 domain, folding in a three ?-sheets and one ?-helix structure, and presenting important residues and motifs for DNA contacting and binding specificity. In addition, a chloroplast transit peptide was detected at the N-terminal region with cleavage site in the 52 residue. Binding activity of this transcription factor was validated by Electro Mobility Shift Assay (EMSA). Subcellular localization was verified by transient expression of PvDREB6A::GFP in Nicotiana benthamiana and the expression of GFP was detected at the nucleus. Genetic transformation for overexpression of PvDREB6A gene in Arabidopsis thaliana wild type and knockout mutant for AtRAP2.4 gene was conducted. Four single copy events with better expression of the PvDREB6A named Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/pFEC2.1 #19.7 and Salk_020767C/pFEC2.1 #23.7 were selected. The event Salk_020767C/pFEC2.1 #23.7 showed the best expression of PvDREB6A and was visualized under UV light. Functional analysis, revealed that transgenic plants under water deficit, high salt, and cold showed higher survival rate. Transgenic plants overexpressing the PvDREB6A exhibited lower water loss rate and electrolyte leakage rate under abiotic stress. A gene expression analysis with tolerant-related genes was conduted. The quantification revealed that 18 genes, AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, related to drought, salt and cold tolerance, were up-regulated due to the overexpression of PvDREB6A from common bean in the transgenic plants

Made available in DSpace on 2014-08-20T13:32:57Z (GMT). No. of bitstreams: 1 dissertacao_lara_dias.pdf: 2572863 bytes, checksum: 41207696775098ff734edf871864bf12 (MD5) Previous issue date: 2011-02-14 / The current challenges in plant breeding are to maximize the productivity of major crop species and to create means for exploring novel crop environments. One of these environments is the lowland hydromorphic soils that are proper for the irrigated rice crop. Adapting other crops to this environment could reduce the incidence of diseases, pests and weeds, therefore benefiting from a crop rotation system. When a plant is exposed to abiotic stresses, it has to cope with environmental changes through physiological and anatomic changes that need quick gene expression responses, i.e., changes in active/silenced status as well as in the rates of transcription. Cis-acting regulatory elements have straight relationship with transcription factors (TF) in complex signaling networks. This TF binding sites (cis-elements) are the functional DNA elements that influence temporal and spatial transcriptional activity. We investigated possible patterns of sequences that can be inferred about the mechanisms that plants use to develop under flooding stress. This search for possible homologies between the various cis-elements would lead us to performed interactive analyses about how plants use their molecular mechanisms responding to abiotic stresses. Online databases were searched, looking for genes previously described in literature which are expressed in response to flooding in Oryza sativa, Arabidopsis thaliana and their homologous in Glycine max and Zea mays. The 1.0 Kb upstream portion of each gene was extracted and analyzed in silico. Besides, all the promoters of these four species were subjected to a tool for searching for novel signals, intending to find new motif patterns. Our in silico analysis shows that from 259 cis elements found in PLACE for all promoters of Arabidopsis and rice, 12 of them are common to both species, and are distinguished by having high frequency. Using the MEME program two consensus motifs could be found among the species Oryza sativa and Zea mays. These could represent new cis elements patterns, because they had relatively high occurrences in the gene promoters and they are related to conserved sequences in monocots. The analysis here presented shows important points for future studies related to the waterlogging stress and unmasking molecular tolerance mechanisms to this typical stress. From the data generated, it will be possible to direct experiments on genetic transformation with target genes and/or cis elements in order to attribute some characteristic in plants, such as those found in rice, so they can develop in an environment with O2 deprivation. / Os atuais desafios no melhoramento de plantas são maximizar a produtividade das principais espécies cultivadas e criar meios para explorar uma variedade de ambientes de cultivo. Um desses ambientes são os solos hidromórficos de planícies alagadas. A adaptação de outras culturas a este ambiente poderia reduzir a incidência de doenças, pragas e plantas daninhas, se beneficiando de um sistema de rotação de culturas. Quando uma planta é exposta a estresses abióticos ela tem que lidar com as alterações ambientais através de mudanças fisiológicas e anatômicas, as quais necessitam de rápidas mudanças na expressão gênica, ou seja, no seu estado inicial, bem como nas taxas de transcrição. Elementos regulatórios de ação cis têm relação direta com fatores de transcrição (FT) em complexas redes de sinalização. Estes sítios de ligação de FTs são elementos funcionais de DNA que influenciam a atividade transcricional de forma temporal e espacial. Neste trabalho, foram investigados possíveis padrões de seqüências que se possam inferir sobre os mecanismos que as plantas utilizam para se desenvolver na condição do alagamento. Essa busca por possíveis homologias entre os vários elementos cis permite realizar análises interativas sobre como as plantas utilizam seus mecanismos moleculares para responder a estresses abióticos. Bancos de dados online foram utilizados, na busca de genes previamente descritos em literatura e que são expressos em resposta ao alagamento em Oryza sativa, Arabidopsis thaliana e seus homólogos em Glycine max e Zea mays. A porção de 1,0 Kb a montante de cada gene foi extraída e analisada in silico. Além disso, todos os promotores das quatro espécies foram submetidos a busca por uma variedade de sinais, com a intenção de encontrar novos padrões de motivos de DNA. Esta análise mostrou que, dos 259 elementos cis encontrados em promotores de Arabidopsis e arroz, 12 deles são comuns a ambas as espécies e se distinguem do restante por terem alta freqüência. Utilizando o programa MEME dois motivos consenso foram encontrados entre as espécies Oryza sativa e Zea mays. Estes podem representar novos padrões de elementos cis, pois apresentaram ocorrências relativamente elevada nos promotores de genes e são relacionados com seqüências conservadas em monocotiledôneas. A análise aqui apresentada mostra pontos importantes para futuros estudos relacionados ao estresse por alagamento e auxilia na descoberta de mecanismos moleculares de tolerância ao alagamento nas plantas. A partir dos dados gerados, será possível direcionar experimentos de transformação genética a fim de atribuir alguma característica às plantas, tais como aqueles encontrados no arroz, para que possam se desenvolver em um ambiente com privação de O2.

Made available in DSpace on 2014-08-20T13:42:10Z (GMT). No. of bitstreams: 1 Dissertacao_Joceani_Dal_Cero.pdf: 1522635 bytes, checksum: 6a5a31f6f6667ef5f524442ba0bb1e72 (MD5) Previous issue date: 2010-02-26 / Regulatory molecules, such as transcription factors, have been thoroughly investigated, especially in hormone-mediated responses that involve gene expression modulation. Frequently, the main determinant of gene expression is its transcriptional rate. Thus, molecular mechanisms underlying transcription regulation have become an important topic in genetic studies of ethylene signaling. The present work aimed to investigate the ERF (Ethylene Response Factor) family employing bioinformatic tools, integrating publicly available datasets from the model species Arabidopsis thaliana and phylogenetic analyses to help elucidating the biological roles of the family in apple. The preliminary survey of the ERF sequences in Malus has provided basic information to be incorporated in further studies of the functional role of ERFs in this perennial species. Expression analyses of MdERF1 and MdERF in apple fruits suggest that other factors, besides ethylene, are involved in their transcriptional regulation in Malus. The second chapter reports the investigation of the transcriptional profiling of those ERF genes in response to pathogen attack, using a biological assay of in vitro propagated plants inoculated with the fungus Venturia inaequalis (apple scab disease). The study has provided evidences of the involvement of MdERF1 in eliciting the plant response; whereas, MdERF2 does not appear to be participate in the pathogenesis. / Moléculas que participam dos processos regulatórios, como os fatores de transcrição, têm recebido atenção especial, pois uma das principais ações dos estímulos hormonais é a modulação da expressão gênica. Como a taxa de transcrição de um gene é o maior determinante da sua expressão, os mecanismos moleculares pelos quais a transcrição gênica é regulada têm se tornado um dos tópicos principais de estudos em genética molecular envolvendo o hormônio etileno. O objetivo deste trabalho foi realizar análises de bioinformática para a família ERF (Ethylene Response Factors), integrar bases de dados existentes na internet no modelo Arabidopsis, bem como análise filogenética que permitam avaliar os papéis dos diferentes membros da família. Este levantamento preliminar das seqüências ERF em Malus forneceu informação básica para estudos posteriores mais aprofundados, com relação aos mecanismos moleculares da família nesta importante cultura perene. A análise da expressão de MdERF1 e MdERF2 em frutos de maçã indica que outros fatores além do etileno estão envolvidos na regulação da transcrição dos ERF em Malus. O segundo capítulo refere-se à resposta dos ERF frente ao ataque de patógenos. Para isso, foram infectadas plantas de macieira provenientes de cutivo in vitro com o fungo Venturia inaequalis (sarna da maçã). As evidências desses estudos sugerem o envolvimento do gene MdERF1 no processo de patogênese, enquanto que o gene MdERF2 parece não estar envolvido no processo.

En 2015, la récidive tumorale et les métastases du cancer du sein demeurent une cause importante de décès à travers le monde. Toutefois, ces cancers sont souvent hétérogènes car en dépit d’un phénotype similaire, l’évolution clinique et la réponse au traitement peuvent varier considérablement. Il y a donc un intérêt évident à identifier et à caractériser de nouveaux biomarqueurs pour permettre classer les tumeurs mammaires dans des sous-groupes plus homogènes. Notre hypothèse est que chaque cancer mammaire possède des caractéristiques distinctes au plan des altérations du génome et des profils d’expression géniques et que ces changements se traduisent cliniquement par une prédisposition à former des métastases ou à répondre ou non à la chimiothérapie et aux thérapies ciblées. Dans le cadre de nos travaux, nous nous sommes intéressés aux sous-types agressifs de tumeurs mammaires et notamment les cancers de type triple négatif. Nous avons aussi tenté d’identifier des marqueurs capables de distinguer l’une de l’autre les tumeurs de type luminal A et luminal B. Pour ce faire, nous avons d’abord utilisé une stratégie in silico à partir de données publiques (micro-puces d’ADN et séquençage de l’ARN). Nous avons ensuite construit sept micro-matrices tissulaires (TMA) provenant de tissus mammaires normaux et tumoraux fixés à la formaline et enrobés en paraffine. Ces outils nous ont permis d’évaluer par immunohistochimie les niveaux d’expression différentielle des marqueurs suivants : ANXA1, MMP-9, DP103 et MCM2. Ceux-ci ont été comparés aux marqueurs usuels du cancer du sein (ER, PR, HER2, CK5/6 et FOXA1) et corrélés aux données cliniques (survie globale et métastase). Nos résultats indiquent que ces nouveaux marqueurs jouent un rôle important dans l’évolution clinique défavorable des tumeurs de haut grade. Dans un premier article nous avons montré que l’expression d’ANXA1 est dérégulée dans les cancers de type triple-négatif et aussi, dans une certaine mesure, dans les tumeurs HER2+. Nous croyons qu’ANXA1 permet de mieux comprendre le processus d’hétérogénéité tumorale et facilite l’identification des tumeurs de haut grade. Nous proposons également qu’ d’ANXA1 stimule la transition épithélio-mésenchymateuse (EMT) et la formation des métastases. Dans un second temps, nous avons montré que les niveaux d’expression de MMP-9 reflètent la différenciation cellulaire et corrèlent avec les sous-types de cancers mammaires ayant un mauvais pronostic. Nous estimons que MMP-9 permet de mieux comprendre et d’identifier les tumeurs mammaires à haut risque. De fait, la surexpression de MMP-9 est associée à une augmentation des métastases, une récidive précoce et une diminution de la survie globale. Dans le cadre d’un troisième article, nous avons montré que la surexpression du marqueur de prolifération MCM2 s’observe dans les cancers triple-négatifs, HER2+ et Luminal B par comparaison aux cancers luminal A (p< 0.0001). Nos résultats suggèrent qu’en utilisant un seuil de 40% de noyaux marqués, nous pourrions distinguer l’une de l’autre les tumeurs de type luminal A et luminal B. Cela dit, avant de pouvoir envisager l’utilisation de ce marqueur en clinique, une étude de validation sur une nouvelle cohorte de patientes s’impose. En somme, les résultats de nos travaux suggèrent qu’ANXA1, MMP-9 et MCM2 sont des marqueurs intéressants pour mieux comprendre les mécanismes physiopathologiques impliqués dans la progression tumorale et le développement des métastases. À terme, ces nouveaux marqueurs pourraient être utilisés seuls ou en combinaison avec d’autres gènes candidats pour permettre le développement de trousses « multigènes » ou d’essais protéomiques multiplex pour prédire l’évolution clinique des cancers mammaires. / In 2015, breast cancer remains a leading cause of death among women worldwide due to relapse and metastases. However, mammary tumors are known to be heterogeneous in terms of their clinical course and response to treatment, despite a seemingly similar phenotype. There is therefore an obvious need to identify and characterize new biomarkers of progression in breast cancers so that each tumor can be properly classified. Our hypothesis is that each breast cancer has its own set of genomic abnormalities or altered pattern of gene expression that can explain the aggressiveness of each tumor, its ability to metastasize and its response to chemotherapeutic agents or other forms of targeted therapies. In this study, our aim is to identify and characterize new biomarkers with prognostic value in aggressive subsets of breast cancer focusing primarily on triple-negative tumors and luminal B breast cancer. To achieve those aims, we conducted an in silico search from public databases of DNA microchip and RNA sequencing data. We next constructed seven tissue microarrays (TMA) using paraffin blocks from human breast cancer along with normal breast to examine the differential expression of new putative markers: ANXA1, MMP-9, DP103 and MCM2. Expression levels measured by immunohistochemistry were then compared to other conventional markers of breast cancer (ER, PR, HER2, Ki-67, CK 5/6, FOXA1) and correlated with clinical data (overall survival and metastasis). By comparing the relative expression of these markers in human breast tumors we were able to pinpoint the important role of ANXA1, MMP-9, DP103, and MCM2 in aggressive tumor subtypes recognized for their poor clinical course. Firstly, we have shown that ANXA1 expression is severely deregulated in high-grade breast cancers including triple-negative and, to some extent, HER2-positive breast cancers. In addition, our results also indicated a possible role of ANXA1 in regulating EMT and breast cancer cell metastasis. Secondly, expression of MMP-9 was found to mirror the degree of tumor differentiation and to correlate with breast cancers of unfavorable outcome. This implies that MMP-9 can help better characterize the biology of breast carcinoma and to identify subgroups of high-risk breast tumors. In fact, we found that high levels of MMP-9 in tumors were associated with increased metastatic dissemination, early relapse and reduced survival. Thirdly, we demonstrated that MCM2 is overexpressed in triple-negative, HER2 positive and luminal B breast cancer in comparison to luminal A breast cancer (p-value < 0.0001). Our findings support the notion that MCM2 can be used to distinguish luminal A from luminal B breast cancer based on a 40% index cut-point. However, an independent validation cohort is needed to confirm the clinical utility of MCM2. Lastly, our results suggest that ANXA1, MMP-9 and MCM2 are valuable genes/proteins candidate that can help better understand the mechanisms involved in tumor progression and metastasis. One may also envisage their use, alone or in combination with other genes, in the development of a multi-gene panel or multiplex proteomic assay to predict clinical outcome and guide therapeutic decisions.

No description available.

Chez les végétaux supérieurs, lembryogenèse est une phase clé du développement au cours de laquelle lembryon établit les principales structures de la future plante. La compréhension des processus moléculaires et physiologiques menant à la formation de la graine est donc dun intérêt agronomique majeur. Chez Phaseolus la caractérisation moléculaire de lembryogenèse permet de mieux comprendre les mécanismes du développement embryonnaire et de son dysfonctionnement observé chez les hybrides interspécifiques. Cette thèse sinscrit dans ce cadre et vise à identifier et caractériser des gènes clés impliqués dans le développement de l'embryon chez Phaseolus. Des hybridations interspécifiques ont été réalisées entre lespèce P.vulgaris L. (cultivar NI637) utilisée comme parent mâle et lespèce P. coccineus L. (cultivar NI16) utilisée comme parent femelle. Des analyses ont aussi été effectuées sur un mutant obtenu par mutagenèse chimique à l'EMS (Ethyl Méthyl Sulfonate) de graines de la variété BAT93 de P.vulgaris. Une étude histologique comparative a permis de suivre la dynamique de lembryogenèse du haricot commun à partir dembryons prélevés 3 à 12 jours après la pollinisation et provenant de plantes normales et déficients dans la production de graines. Les embryons de P. vulgaris se développent plus rapidement par rapport à ceux issus du mutant EMS. Ces derniers présentent des anomalies au niveau de lembryon et du suspenseur. La caractérisation fonctionnelle de deux gènes candidats MIPS (myo-inositol phosphate synthase) et Sus (sucrose synthase) a été réalisée par RT-PCR quantitative et hybridation in situ suite à une étude spatio-temporelle dexpression de ces deux gènes candidats au cours de développement embryonnaire chez Phaseolus. Lanalyse du profil dexpression de ces deux gènes montre quils sont exprimés différemment au niveau des tissus de lembryon et du suspenseur. Lanalyse in silico nous a permis de sélectionner 22 gènes candidats dont nous avons vérifié l'expression au cours de développement de la graine chez Phaseolus. Des variations au niveau de la méthylation de lADN ont été déterminées chez les hybrides interspécifiques comparativement à leurs parents. La technique de lHSS a permis disoler des fragments dADNs complémentaires différemment exprimés au cours de développement de la graine chez Phaseolus. Lanalyse des séquences de ces ADNs complémentaires montre quils codent pour plusieurs protéines intervenant dans le développement cellulaire et embryonnaire, en particulier le "storage protein activator" (SPA), le "pentatricopeptide repeat-containing protein" (PPR) et lacetyl-CoA carboxylase (ACCase). La caractérisation de ces différents gènes exprimés au cours du développement de la graine, fournit de nouveaux outils susceptibles de mettre en évidence des mécanismes de dysfonctionnement embryonnaire chez le genre Phaseolus.