Sonchus yellow net virus (SYNV), Potato yellow dwarf virus (PYDV) and Lettuce Necrotic yellows virus (LNYV) are members of the Rhabdoviridae family that infect plants. SYNV and PYDV are Nucleorhabdoviruses that replicate in the nuclei of infected cells and LNYV is a Cytorhabdovirus that replicates in the cytoplasm. LNYV and SYNV share a similar genome organization with a gene order of Nucleoprotein (N), Phosphoprotein (P), putative movement protein (Mv), Matrix protein (M), Glycoprotein (G) and Polymerase protein (L). PYDV contains an additional predicted gene between N and P, denoted as X, that has an unknown function. In order to gain insight into the associations of viral proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps using novel plant expression vectors. Sub‐cellular localization was determined by expressing the viral proteins fused to green fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta using bimolecular fluorescence complementation (BiFC). All three viruses showed Mv to be localized to the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N‐P and M‐M interactions are common to all three viruses. Associations unique to only one virus include G‐Mv for SYNV, M‐Mv, M‐G, and N‐M for PYDV and P‐M for LNYV. The cognate N‐P proteins of all three viruses exhibit changes in localization when co‐expressed. To complement the mapping data, we also mapped the functional domains in the glycoproteins of SYNV and LNYV. The truncation of the carboxy terminus has no effect on localization compared to the full‐length protein; the nuclear localization signals (NLSs) present in SYNV‐G do not interact with known importins. These data suggest that although the interactions of the three viruses differ, each protein may have similar functional domains.

Os estudos em reprodução e desenvolvimento das plantas, especialmente voltados ao pistilo, são de grande interesse agronômico, econômico e científico. Em nosso laboratório, recentemente, foi identificado e caracterizado SCI1 (Stigma/style Cell-cycle Inhibitor 1), um inibidor do ciclo celular que atua de forma tecido específica no pistilo de Nicotiana tabacum L. e Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). Foi identificada a proteína NtCDKG;2 (N. tabacum Cyclin-dependent Kinase 2) como parceira de interação de NtSCI1 (N . tabacum SCI1), em um ensaio de pull-down (STRINI, 2014). A literatura aponta que os inibidores de ciclo celular regulam o ciclo através da inibição de CDK, o que sugere que NtSCI1 possa regular o ciclo celular através da inibição de NtCDKG;2. O presente estudo mostra análises detalhadas da localização de GFP-NtCDKG;2 em células epiteliais de N. benthamiana. Verificou-se que a proteína NtCDKG;2 está presente no nucleoplasma e também co-localiza em speckles nucleares. Em cultura de células BY2 expressando GFP-NtCDKG;2 de forma estável, foi observado que, durante a metáfase e anáfase, a proteína NtCDKG;2 está junto ao fuso acromático. Adicionalmente, ensaios de BiFC (Bi-molecular Fluorescence Complementation) realizados neste trabalho mostram que a interação entre as proteínas NtCDKG;2 e NtSCI1 ocorre em uma região localizada na periferia nucleolar, durante a interfase. Também foram identificadas possíveis isoformas de NtCDKG;2. A possibilidade da ocorrência de isoformas sugere que, de maneira análoga à sua homóloga em humanos, as isoformas resultantes de NtCDKG;2 possam atuar em diferentes processos. Em busca de parceiros de interação de NtCDKG;2, para identificar em que vias esta proteína atua, foi realizado um screening de uma biblioteca de cDNAs de estigmas e estiletes de N. tabacum, no sistema de duplo-híbrido em leveduras (Y2H). Através desse ensaio, foram identificados diversos parceiros envolvidos com transcrição e processamento de RNA. Dentre as proteínas identificadas, cuja interação foi confirmada neste trabalho, destaca-se a proteína NtCDKF;1, uma proteína que fosforila o CTD da RNA Polimerase II e, dessa forma, auxilia a transcrição e o splicing cotranscricional (HAJHEIDARI et al., 2012). O presente trabalho mostra também a interação entre NtCDKG;2 e a proteína NtCBP1, uma proteína que possui um papel importante na regulação inicial da transcrição de proteínas mediadoras do crescimento do tubo polínico (LI et al., 2015). xx Adicionalmente, o screening de Y2H possibilitou a identificação da interação entre NtCDKG;2 e NtRanBP1, uma proteína chave na formação do fuso acromático que, em humanos, interage com uma isoforma homóloga a NtCDKG;2, a CDK11p46 (MIKOLAJCZYK et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011). Análises in silico realizadas com a sequência de aminoácidos de NtCDKG;2 apontaram motivos de interação com proteína do tipo F-Box, ciclina, CDK, fosfatase, 14-3-3, BRCA1 e indicaram o local provável de interação do complexo CDK-Ciclina com o respectivo inibidor. Foi testada e comprovada a interação entre NtCDKG;2 e a 14-3-3D, por Y2H, uma parceira de NtSCI1. Outra lacuna que precisava ser preenchida é referente à regulação da expressão de NtSCI1. Com este intuito, foram realizadas análises in silico para identificar elementos cis-regulatórios na sequência genômica de NtSCI1. Essas análises indicaram a presença de importantes elementos cis-regulatórios relacionados à identidade meristemática (como WUSCHEL e AINTEGUMENTA), identidade do carpelo (AGAMOUS, BELL) e progressão do ciclo celular (E2F e CDC5). Algumas considerações podem ser feitas associando os resultados obtidos a estudos feitos paralelamente em nosso laboratório: 1) Compilando a localização de NtCDKG;2 em splicing speckles e sua interação com os diferentes parceiros de interação relacionados à transcrição e splicing, sugere-se que NtCDKG;2 também atue nos processos transcricionais e de splicing. 2) Considerando a localização subcelular de NtCDKG;2 durante as diferentes fases do ciclo celular, às análises in silico dessa proteína que identificaram sua possível interação com BRCA1, além da interação confirmada com a proteína NtRanBP1, é possível sugerir que NtCDKG;2 atue, direta ou indiretamente, na organização do fuso acromático de plantas. 3) Propõem-se que NtSCI1 regule a proliferação celular no pistilo através da interação com NtCDKG;2 que se dá no nucléolo das células. Dessa forma, NtSCI1 prenderia NtCDKG;2 no nucléolo e inibiria sua atuação, como na organização do fuso acromático, o que acarretaria inibição da divisão celular. 4) Devido aos motivos cis-regulatórios encontrados na sequência genômica de NtSCI1 e o efeito que a proteína possui desde as fases iniciais do desenvolvimento do pistilo, sugere-se que a expressão desse gene seja regulada por elementos diretamente envolvidos no controle do término do meristema floral e nas vias de desenvolvimento de órgãos florais. / Studies on plant reproduction and development, specifically those related to the pistil, are of great agronomic, economic and scientific interest. In our laboratory, we recently identified and characterized SCI1 (Stigma/style Cell-cycle Inhibitor 1), an inhibitor of the cell cycle which acts tissuespecifically in the pistil of Nicotiana tabacum L. and Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). The NtCDKG;2 (N. tabacum Cyclin-dependent Kinase G; 2) protein was identified as an interaction partner of NtSCI1 (N. tabacum SCI1) in a pulldown assay (STRINI, 2014). The literature suggests that cell cycle inhibitors control the cycle through the inhibition of CDKs, indicating that NtSCI1 might control cell cycle by inhibiting NtCDKG;2. This study shows detailed analysis of GFP-NtCDKG;2 localization in leaf cells of N. benthamiana. The analysis shows that NtCDKG;2 is present in the nucleoplasm and also co-localizes with nuclear speckles. In BY2 cell culture stably expressing GFP-NtCDKG;2, it was observed that NtCDKG;2 is at the achromatic spindle during metaphase and anaphase. Additionally, BiFC (Bimolecular Fluorescence Complementation) assays performed in this study have shown that the interaction of NtCDKG;2 and NtSCI1 occurs in the nucleolar periphery during interphase. Putative isoforms of NtCDKG;2 were also identified. The possible occurrence of these isoforms suggests that, in a similar way to its human homologue, NtCDKG;2 putative isoforms could act in different processes. To identify in which processes this protein could act, a search for NtCDKG;2 interaction partners was performed through the screening of a N. tabacum stigma and style cDNA library in the yeast two-hybrid (Y2H) system. Several partners identified through this assay have roles in RNA transcription and processing. Among the identified partners with interaction confirmed during this work, stands out the NtCDKF;1 protein, a CDK that phosphorylates the RNA polymerase II CTD, and thus, supports transcription and co-transcriptional splicing (HAJHEIDARI et al., 2012). This study also shows the interaction of NtCDKG;2 with NtCBP1, a protein which has an important role in the transcriptional regulation of genes encoding proteins mediating pollen tube growth (LI et al., 2015). Furthermore, the Y2H screening allowed the identification of the interaction of NtCDKG;2 with NtRanBP1, a key protein in the formation of the achromatic spindle which, in humans, interacts with the CDK11p46 isoform (MIKOLAJCZYK xxii et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011), a homologue of NtCDKG;2. In silico analysis of the amino acid sequence of NtCDKG;2 revealed motifs of predicted interaction with F-box proteins, cyclins, CDKs, phosphatases, 14-3-3s, BRCA1, and also pointed the region where the CDK-cyclin complex might interact with its respective inhibitor. The interaction of NtCDKG;2 with 14-3-3D, a known partner of NtSCI1, was tested and confirmed by Y2H. Another gap that needed to be filled is related to the regulation of NtSCI1 expression. To address this issue, in silico analysis to identify cis-regulatory elements was performed in NtSCI1 genomic region. These analyses revealed the presence of important cis-regulatory elements related to meristem identity (such as WUSCHEL and AINTEGUMENTA), carpel identity (AGAMOUS, BELL), and cell cycle progression (E2F and CDC5). Taken together results from this study and parallel studies performed in our laboratory, a few remarks can be made: 1) Taken the localization of NtCDKG;2 in splicing speckles, and its interaction with different proteins involved in transcription and splicing, it is suggested that NtCDKG;2 also has roles on these processes; 2) Considering the subcellular localization of NtCDKG;2 during the different cell cycle phases, the in silico analysis of this protein that predicts its interaction with BRCA1, and the confirmed interaction with NtRanBP1 protein, it is possible to suggest that NtCDKG;2 has a direct or indirect role in the organization of the achromatic spindle in plants; 3) It is proposed that NtSCI1 regulates cell proliferation in the pistil through its interaction with NtCDKG;2, which occurs in the nucleolus. Thus, NtSCI1 could hold NtCDKG;2 in the nucleolus, inhibiting its actions, such as in the organization of the achromatic spindle, resulting in cell division arrest. 4) Due to the cis-regulatory elements found in the genomic sequence of NtSCI1, and the effect of this protein since the initial stages of pistil development, it is suggested that its expression is regulated by elements directly involved in the control of the floral meristem termination and pathways of floral organ development.

Os estudos em reprodução e desenvolvimento das plantas, especialmente voltados ao pistilo, são de grande interesse agronômico, econômico e científico. Em nosso laboratório, recentemente, foi identificado e caracterizado SCI1 (Stigma/style Cell-cycle Inhibitor 1), um inibidor do ciclo celular que atua de forma tecido específica no pistilo de Nicotiana tabacum L. e Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). Foi identificada a proteína NtCDKG;2 (N. tabacum Cyclin-dependent Kinase 2) como parceira de interação de NtSCI1 (N . tabacum SCI1), em um ensaio de pull-down (STRINI, 2014). A literatura aponta que os inibidores de ciclo celular regulam o ciclo através da inibição de CDK, o que sugere que NtSCI1 possa regular o ciclo celular através da inibição de NtCDKG;2. O presente estudo mostra análises detalhadas da localização de GFP-NtCDKG;2 em células epiteliais de N. benthamiana. Verificou-se que a proteína NtCDKG;2 está presente no nucleoplasma e também co-localiza em speckles nucleares. Em cultura de células BY2 expressando GFP-NtCDKG;2 de forma estável, foi observado que, durante a metáfase e anáfase, a proteína NtCDKG;2 está junto ao fuso acromático. Adicionalmente, ensaios de BiFC (Bi-molecular Fluorescence Complementation) realizados neste trabalho mostram que a interação entre as proteínas NtCDKG;2 e NtSCI1 ocorre em uma região localizada na periferia nucleolar, durante a interfase. Também foram identificadas possíveis isoformas de NtCDKG;2. A possibilidade da ocorrência de isoformas sugere que, de maneira análoga à sua homóloga em humanos, as isoformas resultantes de NtCDKG;2 possam atuar em diferentes processos. Em busca de parceiros de interação de NtCDKG;2, para identificar em que vias esta proteína atua, foi realizado um screening de uma biblioteca de cDNAs de estigmas e estiletes de N. tabacum, no sistema de duplo-híbrido em leveduras (Y2H). Através desse ensaio, foram identificados diversos parceiros envolvidos com transcrição e processamento de RNA. Dentre as proteínas identificadas, cuja interação foi confirmada neste trabalho, destaca-se a proteína NtCDKF;1, uma proteína que fosforila o CTD da RNA Polimerase II e, dessa forma, auxilia a transcrição e o splicing cotranscricional (HAJHEIDARI et al., 2012). O presente trabalho mostra também a interação entre NtCDKG;2 e a proteína NtCBP1, uma proteína que possui um papel importante na regulação inicial da transcrição de proteínas mediadoras do crescimento do tubo polínico (LI et al., 2015). xx Adicionalmente, o screening de Y2H possibilitou a identificação da interação entre NtCDKG;2 e NtRanBP1, uma proteína chave na formação do fuso acromático que, em humanos, interage com uma isoforma homóloga a NtCDKG;2, a CDK11p46 (MIKOLAJCZYK et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011). Análises in silico realizadas com a sequência de aminoácidos de NtCDKG;2 apontaram motivos de interação com proteína do tipo F-Box, ciclina, CDK, fosfatase, 14-3-3, BRCA1 e indicaram o local provável de interação do complexo CDK-Ciclina com o respectivo inibidor. Foi testada e comprovada a interação entre NtCDKG;2 e a 14-3-3D, por Y2H, uma parceira de NtSCI1. Outra lacuna que precisava ser preenchida é referente à regulação da expressão de NtSCI1. Com este intuito, foram realizadas análises in silico para identificar elementos cis-regulatórios na sequência genômica de NtSCI1. Essas análises indicaram a presença de importantes elementos cis-regulatórios relacionados à identidade meristemática (como WUSCHEL e AINTEGUMENTA), identidade do carpelo (AGAMOUS, BELL) e progressão do ciclo celular (E2F e CDC5). Algumas considerações podem ser feitas associando os resultados obtidos a estudos feitos paralelamente em nosso laboratório: 1) Compilando a localização de NtCDKG;2 em splicing speckles e sua interação com os diferentes parceiros de interação relacionados à transcrição e splicing, sugere-se que NtCDKG;2 também atue nos processos transcricionais e de splicing. 2) Considerando a localização subcelular de NtCDKG;2 durante as diferentes fases do ciclo celular, às análises in silico dessa proteína que identificaram sua possível interação com BRCA1, além da interação confirmada com a proteína NtRanBP1, é possível sugerir que NtCDKG;2 atue, direta ou indiretamente, na organização do fuso acromático de plantas. 3) Propõem-se que NtSCI1 regule a proliferação celular no pistilo através da interação com NtCDKG;2 que se dá no nucléolo das células. Dessa forma, NtSCI1 prenderia NtCDKG;2 no nucléolo e inibiria sua atuação, como na organização do fuso acromático, o que acarretaria inibição da divisão celular. 4) Devido aos motivos cis-regulatórios encontrados na sequência genômica de NtSCI1 e o efeito que a proteína possui desde as fases iniciais do desenvolvimento do pistilo, sugere-se que a expressão desse gene seja regulada por elementos diretamente envolvidos no controle do término do meristema floral e nas vias de desenvolvimento de órgãos florais. / Studies on plant reproduction and development, specifically those related to the pistil, are of great agronomic, economic and scientific interest. In our laboratory, we recently identified and characterized SCI1 (Stigma/style Cell-cycle Inhibitor 1), an inhibitor of the cell cycle which acts tissuespecifically in the pistil of Nicotiana tabacum L. and Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). The NtCDKG;2 (N. tabacum Cyclin-dependent Kinase G; 2) protein was identified as an interaction partner of NtSCI1 (N. tabacum SCI1) in a pulldown assay (STRINI, 2014). The literature suggests that cell cycle inhibitors control the cycle through the inhibition of CDKs, indicating that NtSCI1 might control cell cycle by inhibiting NtCDKG;2. This study shows detailed analysis of GFP-NtCDKG;2 localization in leaf cells of N. benthamiana. The analysis shows that NtCDKG;2 is present in the nucleoplasm and also co-localizes with nuclear speckles. In BY2 cell culture stably expressing GFP-NtCDKG;2, it was observed that NtCDKG;2 is at the achromatic spindle during metaphase and anaphase. Additionally, BiFC (Bimolecular Fluorescence Complementation) assays performed in this study have shown that the interaction of NtCDKG;2 and NtSCI1 occurs in the nucleolar periphery during interphase. Putative isoforms of NtCDKG;2 were also identified. The possible occurrence of these isoforms suggests that, in a similar way to its human homologue, NtCDKG;2 putative isoforms could act in different processes. To identify in which processes this protein could act, a search for NtCDKG;2 interaction partners was performed through the screening of a N. tabacum stigma and style cDNA library in the yeast two-hybrid (Y2H) system. Several partners identified through this assay have roles in RNA transcription and processing. Among the identified partners with interaction confirmed during this work, stands out the NtCDKF;1 protein, a CDK that phosphorylates the RNA polymerase II CTD, and thus, supports transcription and co-transcriptional splicing (HAJHEIDARI et al., 2012). This study also shows the interaction of NtCDKG;2 with NtCBP1, a protein which has an important role in the transcriptional regulation of genes encoding proteins mediating pollen tube growth (LI et al., 2015). Furthermore, the Y2H screening allowed the identification of the interaction of NtCDKG;2 with NtRanBP1, a key protein in the formation of the achromatic spindle which, in humans, interacts with the CDK11p46 isoform (MIKOLAJCZYK xxii et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011), a homologue of NtCDKG;2. In silico analysis of the amino acid sequence of NtCDKG;2 revealed motifs of predicted interaction with F-box proteins, cyclins, CDKs, phosphatases, 14-3-3s, BRCA1, and also pointed the region where the CDK-cyclin complex might interact with its respective inhibitor. The interaction of NtCDKG;2 with 14-3-3D, a known partner of NtSCI1, was tested and confirmed by Y2H. Another gap that needed to be filled is related to the regulation of NtSCI1 expression. To address this issue, in silico analysis to identify cis-regulatory elements was performed in NtSCI1 genomic region. These analyses revealed the presence of important cis-regulatory elements related to meristem identity (such as WUSCHEL and AINTEGUMENTA), carpel identity (AGAMOUS, BELL), and cell cycle progression (E2F and CDC5). Taken together results from this study and parallel studies performed in our laboratory, a few remarks can be made: 1) Taken the localization of NtCDKG;2 in splicing speckles, and its interaction with different proteins involved in transcription and splicing, it is suggested that NtCDKG;2 also has roles on these processes; 2) Considering the subcellular localization of NtCDKG;2 during the different cell cycle phases, the in silico analysis of this protein that predicts its interaction with BRCA1, and the confirmed interaction with NtRanBP1 protein, it is possible to suggest that NtCDKG;2 has a direct or indirect role in the organization of the achromatic spindle in plants; 3) It is proposed that NtSCI1 regulates cell proliferation in the pistil through its interaction with NtCDKG;2, which occurs in the nucleolus. Thus, NtSCI1 could hold NtCDKG;2 in the nucleolus, inhibiting its actions, such as in the organization of the achromatic spindle, resulting in cell division arrest. 4) Due to the cis-regulatory elements found in the genomic sequence of NtSCI1, and the effect of this protein since the initial stages of pistil development, it is suggested that its expression is regulated by elements directly involved in the control of the floral meristem termination and pathways of floral organ development.

Catharanthus roseus est une plante tropicale de la famille des Apocynacées d’intérêt thérapeutique en raison de sa capacité à synthétiser des alcaloïdes indoliques monoterpéniques (AIM) utilisés en chimiothérapie anticancéreuse. La teneur en AIM in planta est très faible notamment en raison d’une haute compartimentalisation cellulaire et subcellulaire de la voie de biosynthèse. Si la compartimentalisation cellulaire était bien caractérisée, très peu de données de localisation subcellulaire in situ étaient disponibles au début de cette thèse. Une connaissance fine de cette compartimentalisation est cependant nécessaire pour identifier les transports inter-compartiment de métabolites intermédiaires, limitant potentiellement le flux métabolique, afin d’améliorer ensuite le rendement de biosynthèse des AIM par ingénierie métabolique. Dans ce contexte nous avons réalisé une étude exhaustive de la localisation subcellulaire des enzymes de cette voie par imagerie GFP dans des cellules de C. roseus transformées par biolistique permettant d’établir un nouveau modèle intégré d’organisation cellulaire et subcellulaire de la biosynthèse des AIM. / Catharanthus roseus is a tropical plant from the Apocynaceae family with a great therapeutic value due to its ability to synthesize monoterpene indole alkaloids (MIA) used in cancer treatment. The yields of these molecules in planta are very low due to a very high level of compartmentation of the biosynthetic pathway at both cellular and subcellular levels. While the cellular compartmentation was widely characterized, very few in situ subcellular localization data were available at the beginning of this PhD. An accurate knowledge of this compartmentation is necessary to identify intermediate metabolites transport events from one compartment to another one, in order to increase the MIA biosynthesis yield by metabolic engineering approaches. In this context we have proceed to the exhaustive study of the subcellular localization of these enzymes by in vivo GFP imaging in C. roseus cells transformed by biolistic. Potential interprotein interactions of these enzymes have also been studied by BiFC. Altogether, our results enabled us to draw an integrated model of the cellular and subcellular organization of MIA biosynthesis in situ.

Les insectes présentent une étonnante diversité morphologique dans les organes de vol, et cette évolution a conduit à leur rayonnement au sein du règne animal. L'une des modifications les plus frappantes est la transformation des ailes postérieures en structures d'équilibrage très réduites, appelées haltères. Des travaux pionniers chez la drosophile ont montré que la spécification des haltères est sous le contrôle du gène Hox Ultrabithorax (Ubx). En revanche, la formation des ailes antérieures est décrite pour être indépendante des gènes Hox, mais cette observation est controversée chez d’autres insectes. Au cours de mon doctorat, j'ai réexaminé le rôle des gènes Hox pour la spécification des organes du vol chez la drosophile. Mes travaux montrent que la protéine Hox Antennapedia (Antp) est exprimée à un niveau faible dans des cellules spécifiques du primordium de la marge et est nécessaire à la formation correcte de l’aile adulte. De manière étonnante, Antp peut également fonctionner comme Ubx et former un haltère quand la protéine est exprimée à des niveaux similaires à ceux de Ubx. Ainsi, la formation d’organes de vol divergents chez la drosophile est directement contrôlée par une dose spécifique de protéine Hox et non par une protéine Hox spécifique. Les gènes Hox sont intrinsèquement liés à l'évolution de la diversité morphologique chez les animaux. Par conséquent, le rôle de la dose des protéines Hox a également été testé d'un point de vue évolutif parmi plusieurs lignages d'insectes. Les résultats montrent que la dose de protéines Hox est à peu près la même entre les primordia antérieur et postérieur d’un insecte à quatre ailes comme Bombyx mori. Dans l’ensemble, mes résultats démontrent que la spécification des organes de vol n’est pas un programme Hox-indépendant et que la variation de la dose des protéines Hox est un moyen de modifier la taille et la forme de l’aile, pouvant ultimement aboutir à la création d’un tout nouvel organe d’équilibrage au cours de l’évolution des insectes. Enfin, au cours de mon doctorat, j'ai également participé à plusieurs projets parallèles visant à identifier et à caractériser le rôle des nouveaux cofacteurs des protéines Hox dans différents contextes développementaux, notamment la spécification de l’haltère et la répression de l'autophagie. Ces travaux s'appuient en partie sur la complémentation de fluorescence bimoléculaire (BiFC), une méthode que nous avons récemment couplée à la panoplie d'outils génétiques de la drosophile pour réaliser des criblages d'interactions protéine-protéine à grande échelle in vivo. / Insects display an astonishing array of diversity in flight appendage morphologies and theirevolution led to the catalyzed radiation of insects in the animal kingdom. The first definite modellinking the Hox genes to morphological evolution was demonstrated in Drosophila. One of themost striking modifications is the transformation of hindwings into highly reduced balancingstructures called halteres. Work in Drosophila established that the specification of halteres isunder the control of a single Hox gene, Ultrabithorax (Ubx). In contrast, the formation of forewingshas been described to be Hox-independent. During my Ph.D., I reconsidered the role of Hox genesfor flight appendage specification in Drosophila. I show that the Hox protein Antennapedia (Antp)is expressed at a low level in specific cells of the wing blade primordium and required for theproper formation of the adult wing. Moreover, Antp works like Ubx to form a haltere whenexpressed in the levels of Ubx. Thus, the formation of divergent flight organs in Drosophila is notdependent on a specific Hox protein but on a specific Hox dose.Hox genes are intrinsically linked to the evolution of morphological diversity in animals.Therefore the role of the Hox dose was also tested from an evolutionary point of view amongseveral insect lineages. Results show that the Hox dose is for example pretty much the samebetween the forewing and hindwing primordia of the four-wing insect species Bombyx mori.Altogether, my results demonstrate that the specification of flight appendages is not aHox-independent developmental program and that the variation in the Hox dose is a way tomodify the wing size and shape, ultimately leading to a completely new balancing organ duringinsect evolution.

The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on maturation, internalization, function or/and pharmacology. Bimolecular fluorescence complementation technique (BiFC) / is an innovative approach based on the reassembly of protein fragments which directly report interactions. In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. The target proteins which are going to be tested in terms of interaction are modified with the non-functional fragments, to produce the fusion proteins. The interaction between two target proteins, in this study Ste2p receptors which are alpha pheromone receptors from Saccharomyces cerevisiae, enable the fragments to come in a close proximity and reassemble. After reassembly, EGFP regains its fluorescent function which provides a direct read-out for the detection of interaction. Further studies are required to determine subcellular localization of the interaction. Moreover, by using the fusion protein partners constructed in this study, effects of agonist/antagonist binding and post-translational modifications such as glycosylation and phosphorylation can be examined. Apart from all, optimized conditions for BiFC technique will guide for revealing new protein-protein interactions.

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), a life-threatening disease primarily affecting the lungs that infects about one third of the world's population and causes 1.3 million deaths annually. It is estimated that TB has been infecting humans for around 70,000 years and has killed more people than any other infectious disease. The highly effective, persistent, and multifaceted virulence strategies that have allowed Mtb to continue to spread and thrive for so long are still poorly understood at the molecular level. This lack of knowledge contributes to ongoing challenges to curing TB. Although drugs capable of killing Mtb exist, even strains that are susceptible to these drugs remain so difficult to treat that stringent six- to nine-month courses of four-drug cocktails are required. Practical difficulties in administering full treatments and patient noncompliance have contributed to a rise in drug-resistant TB cases globally. To combat this increasing world health problem, new antibiotic treatments that kill Mtb and drug-resistant Mtb more effectively via new mechanisms of action are necessary. Discovering these antibiotics expediently requires that innovative Mtb-specific drug-screening assays are developed. An ideal and innovative TB drug screening method would target validated protein-protein interactions (PPI) essential to Mtb's pathogenesis and would be performed on whole Mtb cells under relevant in vivo-like conditions. This project focused on engineering several tools relevant to creating an ideal TB drug screen. A protein fragment complementation assay capable of studying PPI of the TB gyrase complex was created, and this assay was assessed for future HTS applications. To streamline the readout, this assay was re-engineered to include green fluorescent protein.

No description available.

A biologia da reprodução de plantas é um campo de grande interesse, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes). O pistilo é o órgão reprodutivo feminino, composto de estigma, estilete e ovário. Devido à importância central do pistilo no sucesso da reprodução de plantas, faz-se necessário um melhor conhecimento dos genes e processos que regulam seu desenvolvimento e funcionamento. Estudos comparativos da expressão gênica nos órgãos vegetativos e reprodutivos de Nicotiana tabacum revelaram genes de expressão preferencial nos órgãos reprodutivos, entre eles alguns codificando proteínas de função ainda desconhecida. Um destes genes foi caracterizado e denominado SCI1 (Stigma/style Cell-cycle Inhibitor 1), por apresentar um papel importante no desenvolvimento do estigma/estilete, atuando como um inibidor de ciclo celular tecido-específico (DePaoli et al., 2011). O presente trabalho teve como objetivo estudar os mecanismos moleculares pelos quais NtSCI1 regula o ciclo celular, investigando seus parceiros de interação. Em um ensaio de pull-down, utilizando-se extrato proteico nuclear de estigmas/estiletes de N. tabacum, vários putativos reguladores de ciclo celular foram identificados, sendo a interação entre NtSCI1 e NtCDKG;2 confirmada por BiFC e localizada no nucléolo. Uma biblioteca de cDNAs de estigmas/estiletes de N. tabacum, no sistema de duplo-híbrido de levedura, foi construída com sucesso. O screening desta biblioteca, utilizando BD-NtSCI1 como \"isca\", permitiu a identificação de vários parceiros de interação com NtSCI1, entre eles: uma helicase de RNA DEAD-BOX, a proteína 14-3-3D2, dois fatores de transcrição (HOMEOBOX-22 e STOREKEEPER), um fator de splicing portador do domínio SWAP, uma quinase de adenosina e uma transposase. As interações entre NtSCI1 e os três primeiros parceiros citados já foram confirmadas por BiFC (observadas no núcleo e nucléolo) e a interação entre NtSCI1 e Nt14-3-3D2 foi confirmada também por co-imunoprecipitação. O envolvimento de NtSCI1 com a regulação do ciclo celular foi corroborado pela interação entre NtSCI1 e a proteína NtCICLINA-L1 (subunidade regulatória de CDKG;2), confirmada por duplo-híbrido e por BiFC, no nucléolo. A interação entre NtSCI1 e NtCICLINA-RELATED também foi confirmada por BiFC. Para entender a dinâmica de NtSCI1 no nucléolo, foi estudada a localização subcelular da proteína de fusão NtSCI1-GFP durante as fases do ciclo celular. NtSCI1-GFP foi observada no nucléolo de células BY-2 em interfase e prófase, desaparecendo na metáfase e anáfase e reaparecendo no nucléolo no final da telófase, mostrando que a presença de NtSCI1 na célula é controlada pelo ciclo celular. A construção de uma primeira versão do interactoma de NtSCI1 mostrou seu envolvimento direto e indireto com proteínas relacionadas ao metabolismo de RNAs, controle da transcrição e regulação do ciclo celular. Estes resultados sugerem que NtSCI1 possa atuar no controle do ciclo celular de forma não canônica, por meio de múltiplos processos paralelos que interconectam aspectos da regulação da transcrição e o processamento de RNAs com o controle do ciclo celular. / The biology of plant reproduction is a field of great interest, since most of the food consumed by humans is composed of reproductive parts of plants (fruits and seeds). The pistil is the female reproductive organ, composed of stigma, style and ovary. Due to the central importance of the pistil in the success of plant reproduction, a better knowledge of the genes and processes that regulate pistil development and function is necessary. Comparative studies of gene expression in vegetative and reproductive organs of Nicotiana tabacum have revealed genes preferentially expressed in the reproductive organs, among them some encoding proteins of unknown function. One of these genes was characterized and denominated SCI1 (Stigma/style Cell-cycle Inhibitor 1), since it has an important role in stigma/style development, acting as a tissue-specific cell-cycle inhibitor (DePaoli et al., 2011). The objective of the present work was to study the molecular mechanisms through which NtSCI1 regulates the cell cycle investigating its interaction partners. In a pull-down assay, using nuclear protein extracts from N. tabacum stigmas/styles, several putative cell cycle regulators were identified. Among them, the interaction between NtSCI1 and NtCDKG;2 was confirmed by BiFC and localized in the nucleolus. A N. tabacum stigma/style cDNA library in the yeast two-hybrid system was successfully constructed. The screening of this library, using BD-NtSCI1 as bait, allowed the identification of several NtSCI1 interaction partners, among them: a DEAD-BOX RNA helicase; the 14-3-3D2 protein; two transcription factors (HOMEOBOX-22 and STOREKEEPER); a splicing factor containing a SWAP domain; an adenosine kinase; and a transposase. The interactions between NtSCI1 and the first three mentioned partners have already been confirmed by BiFC (observed in the nucleus and nucleolus) and the interaction between NtSCI1 and Nt14-3-3D2 was also wconfirmed by co-immunoprecipitation. The NtSCI1 involvement in cell cycle regulation was corroborated by the interaction between NtSCI1 and the NtCYCLIN-L1 (a regulatory subunit of CDKG;2), which was confirmed by two-hybrid and BiFC in the nucleolus. The interaction between NtSCI1 and NtCYCLIN-RELATED was also confirmed by BiFC. To understand the dynamics of NtSCI1 in the nucleolus, the subcellular localization of the fusion protein NtSCI1-GFP was studied during the different cell cycle phases. NtSCI1-GFP was observed in the nucleolus of BY-2 cells at interphase and prophase, disappearing at metaphase and anaphase and reappearing in the nucleolus at the end of telophase, showing that NtSCI1 presence in the cell is controlled by the cell cycle. The construction of the first version of NtSCI1 interactome showed its direct and indirect involvement with proteins related to RNA metabolism, transcription control and cell cycle regulation. These results suggest that NtSCI1 may act in cell cycle control in a non-canonical way, through multiple parallel processes interconnecting aspects of transcription regulation, RNA processing and cell cycle control.

No description available.