A levedura Saccharomyces cerevisiae é o sistema eucariótico com a genética mais conhecida, reconhecido como \"GRAS\", vem sendo proposta como hospedeira para a expressão de genes que codificam produtos de interesse biotecnológico. No Brasil, vários processos industriais empregam linhagens selvagens de S. cerevisiae, incluindo a produção de etanol combustível. A maioria dessas linhagens industriais são mais vigorosas e apresentam crescimento muito mais rápido que as linhagens de laboratório, além de já estarem adaptadas a processos industrias de larga escala. Neste trabalho, foi estabelecido um sistema de transformação genética, que permite a inserção de genes codificadores de proteínas de interesse biotecnológico no genoma de linhagens selvagens haplóides ou de ploidia maior. O sistema de transformação origina de um vetor de clonagem, denominado YlpC, formado por um fragmento do gene CAN1 (permease da L-arginina e do análogo tóxico L-canavanina), contendo um sítio de restrição interno (BstEII), onde se realiza a inserção do cassete de expressão gênica desejado. A digestão do plasmídio resultante, com HindlIlI, causa a liberação do fragmento de DNA linear, composto pelo cassete de expressão flanqueado por seqüências de CAN1. Esse fragmento resultante é destinado à transformação de leveduras. As células recombinantes sofrem interrupção do gene CAN1 selvagem pelo cassete de expressão presente no fragmento de transformação, tornando-as resistentes à Lcanavanina, permitindo assim, a seleção positiva dos clones transformantes. Para análise da eficiência desse sistema a glicoamilase de A. awamori foi utilizada como proteína repórter. O cassete de expressão contendo a sequência sinal e estrutural da glicoamilase de A. awamori sob a regulação do promotor e terminador de transcrição de PGK de S. cerevisiae foi subclonado no vetor YlpC, dando origem ao plasmídio YlpCGC e depois pUCGc. Esses vetores, digeridos com HindlIlI, liberam o fragmento CGC, empregado nas transformações de levedura deste trabalho. Obtivemos sucesso na transformação de linhagens diplóides de laboratório. Análise dos esporos e amplificação de DNA por PCR, demonstrou que o fragmento CGC encontra-se inserido em ambos alelos CAN1 cromossômicos dessas linhagens recombinantes. Das 20 linhagens de levedura industriais, submetidas à transformação com o fragmento CGC, 10 resultaram em clones transformantes, e assim como os clones recombinantes de linhagens de laboratório diplóides, mantêm a informação adicional 100% estáveis. O sistema também se mostrou adequado para a construção de linhagem de levedura diplóide heterozigota CGC+/CGC:, empregada na detecção de substâncias indutoras de recombinação mitótica, que, como é conhecido, são potencialmente carcinogênicas. / The yeast Saccharomyces cerevisiae is the eukaryotic system with the most extensively studied genetics, it is generally recognized as safe, and it has broadly been used as a host system for the expression of heterologous genes of biotechnological interest. In Brazil, the vast majority of industrial processes, which include the production of fuel ethanol, utilize wild-type strains because of their higher resistance to adverse conditions, their adaptation to industrial processes in large scale, and because they exhibit higher growth rates than laboratory strains. In the present work, a genetic transformation system was developed for the chromosomal integration of heterologous genes of commercial interest in both haploid and polyploid industrial strains. This system utilizes an integrative shuttle vector, YIpC, which contains a CAN1 gene fragment (L-arginine permease and L-canavanine toxic analogous), bearing an internar restriction site (BstEII), where the gene expression cassette can be inserted. The resultant plasmid is then digested with HindlIII, releasing a linear DNA fragment containing the expression cassette flanked by CAN1 sequences. Following the introduction of the transforming fragment into yeast cells, the wild-type CAN1 gene is interrupted by the expression cassette, thus allowing positive selection of the recombinant clones by their resistance to the toxic properties of L-canavanive. To analyze the efficiency of this system, glucoamylase of Aspergillus awamori was used as reporter. An expression cassette containing the structural and signal sequences of A. awamori glucoamylase, under the control of the S. cerevisiae PGK1 transcriptional promoter and termination sequences, was subcloned in YIpC to obtain the plasmids YIpCGC and pUCGc. Both vectors, when digested with HindlIII, released a fragment (CGC) which was subsequently used for yeast transformation. Spore analysis and DNA PCR amplification indeed confirmed that the CGC fragment was inserted in both CAN1 chromosomal alleles of transformed diploid laboratory strains. Most importantly, 10 out of 20 industrial yeast strains submitted to transformation with the CGC fragment resulted in recombinant clones and, like observed for the diploid laboratory strains, the additional information was 100% stable. In concluding, this system also seems to be suitable for the construction of diploid heterozygote CGC+/CGC yeast strains, which in turn can be used for the detection of inductor substances of mitotic recombination that, as known, are potentially carcinogenic.

A levedura Saccharomyces cerevisiae é o sistema eucariótico com a genética mais conhecida, reconhecido como \"GRAS\", vem sendo proposta como hospedeira para a expressão de genes que codificam produtos de interesse biotecnológico. No Brasil, vários processos industriais empregam linhagens selvagens de S. cerevisiae, incluindo a produção de etanol combustível. A maioria dessas linhagens industriais são mais vigorosas e apresentam crescimento muito mais rápido que as linhagens de laboratório, além de já estarem adaptadas a processos industrias de larga escala. Neste trabalho, foi estabelecido um sistema de transformação genética, que permite a inserção de genes codificadores de proteínas de interesse biotecnológico no genoma de linhagens selvagens haplóides ou de ploidia maior. O sistema de transformação origina de um vetor de clonagem, denominado YlpC, formado por um fragmento do gene CAN1 (permease da L-arginina e do análogo tóxico L-canavanina), contendo um sítio de restrição interno (BstEII), onde se realiza a inserção do cassete de expressão gênica desejado. A digestão do plasmídio resultante, com HindlIlI, causa a liberação do fragmento de DNA linear, composto pelo cassete de expressão flanqueado por seqüências de CAN1. Esse fragmento resultante é destinado à transformação de leveduras. As células recombinantes sofrem interrupção do gene CAN1 selvagem pelo cassete de expressão presente no fragmento de transformação, tornando-as resistentes à Lcanavanina, permitindo assim, a seleção positiva dos clones transformantes. Para análise da eficiência desse sistema a glicoamilase de A. awamori foi utilizada como proteína repórter. O cassete de expressão contendo a sequência sinal e estrutural da glicoamilase de A. awamori sob a regulação do promotor e terminador de transcrição de PGK de S. cerevisiae foi subclonado no vetor YlpC, dando origem ao plasmídio YlpCGC e depois pUCGc. Esses vetores, digeridos com HindlIlI, liberam o fragmento CGC, empregado nas transformações de levedura deste trabalho. Obtivemos sucesso na transformação de linhagens diplóides de laboratório. Análise dos esporos e amplificação de DNA por PCR, demonstrou que o fragmento CGC encontra-se inserido em ambos alelos CAN1 cromossômicos dessas linhagens recombinantes. Das 20 linhagens de levedura industriais, submetidas à transformação com o fragmento CGC, 10 resultaram em clones transformantes, e assim como os clones recombinantes de linhagens de laboratório diplóides, mantêm a informação adicional 100% estáveis. O sistema também se mostrou adequado para a construção de linhagem de levedura diplóide heterozigota CGC+/CGC:, empregada na detecção de substâncias indutoras de recombinação mitótica, que, como é conhecido, são potencialmente carcinogênicas. / The yeast Saccharomyces cerevisiae is the eukaryotic system with the most extensively studied genetics, it is generally recognized as safe, and it has broadly been used as a host system for the expression of heterologous genes of biotechnological interest. In Brazil, the vast majority of industrial processes, which include the production of fuel ethanol, utilize wild-type strains because of their higher resistance to adverse conditions, their adaptation to industrial processes in large scale, and because they exhibit higher growth rates than laboratory strains. In the present work, a genetic transformation system was developed for the chromosomal integration of heterologous genes of commercial interest in both haploid and polyploid industrial strains. This system utilizes an integrative shuttle vector, YIpC, which contains a CAN1 gene fragment (L-arginine permease and L-canavanine toxic analogous), bearing an internar restriction site (BstEII), where the gene expression cassette can be inserted. The resultant plasmid is then digested with HindlIII, releasing a linear DNA fragment containing the expression cassette flanked by CAN1 sequences. Following the introduction of the transforming fragment into yeast cells, the wild-type CAN1 gene is interrupted by the expression cassette, thus allowing positive selection of the recombinant clones by their resistance to the toxic properties of L-canavanive. To analyze the efficiency of this system, glucoamylase of Aspergillus awamori was used as reporter. An expression cassette containing the structural and signal sequences of A. awamori glucoamylase, under the control of the S. cerevisiae PGK1 transcriptional promoter and termination sequences, was subcloned in YIpC to obtain the plasmids YIpCGC and pUCGc. Both vectors, when digested with HindlIII, released a fragment (CGC) which was subsequently used for yeast transformation. Spore analysis and DNA PCR amplification indeed confirmed that the CGC fragment was inserted in both CAN1 chromosomal alleles of transformed diploid laboratory strains. Most importantly, 10 out of 20 industrial yeast strains submitted to transformation with the CGC fragment resulted in recombinant clones and, like observed for the diploid laboratory strains, the additional information was 100% stable. In concluding, this system also seems to be suitable for the construction of diploid heterozygote CGC+/CGC yeast strains, which in turn can be used for the detection of inductor substances of mitotic recombination that, as known, are potentially carcinogenic.

Le régulateur endogène de la calcineurine 1 (RCAN1) est exprimé dans les neurones, cependant son rôle dans la régulation de la transmission et de la plasticité synaptique est mal connu. De manière intéressante, plusieurs études dans les cellules cardiaques et les levures montrent une double régulation de la calcineurine 1 (CaN1) par RCAN1. Il est décrit qu'en fonction de son état de phosphorylation par la kinase glycogène synthase 3β (GSK3β), RCAN1 réprime la CaN1 à l'état déphosphorylé, mais il faciliterait son activité à l'état phosphorylé. La régulation de la CaN1 par RCAN1 phosphorylé n'a encore jamais été étudiée dans les neurones et pourrait mettre en relation deux acteurs majeurs de la dépression à long terme (LTD), à savoir la CaN1 et la GSK3β. Par ailleurs une étude récente a montré que RCAN1 peut également être phosphorylé par la protéine kinase A (PKA), une kinase essentielle dans la mise en place de la potentialisation à long terme (LTP), entrainant ainsi une augmentation de l’inhibition de la CaN1 par RCAN1. Dans les neurones, RCAN1 pourrait donc potentiellement réguler à la fois la LTP et la LTD dépendamment de son état de phosphorylation. Mes travaux visent à élucider si RCAN1 est capable de réguler la transmission et de la plasticité synaptique en fonction de son état de phosphorylation et si son action dépend de la CaN1. Afin de déterminer le rôle de RCAN1 dans ces processus, une combinaison de techniques de biologie moléculaire, d’électrophysiologie et d'imagerie a été employée. Nous avons généré des mutations ponctuelles de RCAN1 sauvage de manière à rendre RCAN1 non phosphorylable par la GSK3β ou la PKA. L’expression virale de RCAN1 et de ses différents mutants dans des cultures primaires de neurones d’hippocampe a révélé que RCAN1, dans ses versions sauvage et mutées, est localisé au niveau des épines dendritiques, suggérant une possible fonction de RCAN1 à la synapse. De manière à déterminer les effets de RCAN1 sur la transmission et la plasticité synaptique, j’ai exprimé de manière virale RCAN1 et ses différents mutants dans des tranches organotypiques d'hippocampes de rat et analysé leurs effets par enregistrement en ‘‘patch-clamp’’ en configuration de cellule entière. J’ai pu observer que le blocage du site de phosphorylation de RCAN1 par la GSK3β entraînait une augmentation de la transmission synaptique ainsi qu’un blocage de la LTD. De plus j’ai démontré que la LTP été bloquée lorsque la PKA ne pouvait pas phosphoryler RCAN1. Enfin nous avons pu déterminer que ces différents effets de RCAN1 sur la transmission et la plasticité synaptique étaient dépendants de la CaN1. Nous avons donc démontré une cascade d’évènements et mis en évidence le rôle clé de RCAN1 dans la régulation de la LTP et de la LTD. Nous proposons donc que RCAN1 permet de moduler la transmission et la plasticité synaptique en fonction de son état de phosphorylation par la GSK3β et la PKA en agissant sur la CaN1, en étant un effecteur de la GSK3β lors de l’induction de la LTD ainsi qu’un effecteur de la PKA lors de l’induction de la LTP. / The endogenous regulator of calcineurin 1 (RCAN1) is expressed in neurons, nevertheless its role in the regulation of synaptic transmission and plasticity is not well understood. Interestingly, several studies in cardiac cells and yeasts show that RCAN1 is able to inhibit or activate CaN1 depending on its phosphorylation state by glycogen synthase kinase 3β (GSK3β). RCAN1 is able to inhibit CaN1 when it is not phosphorylated by GSK3β and able to activate it in its phosphorylated state. The regulation of CaN1 by phosphorylated RCAN1 has never been studied in neurons although it could provide a critical link between two major actors of long-term depression (LTD), CaN1 and GSK3β. Furthermore, a recent study revealed that RCAN1 can also be phosphorylated by protein kinase A (PKA), a kinase involved in regulating long-term potentiation (LTP), leading to an increase of CaN1 inhibition by RCAN1. Thus, in neurons, the differential phosphorylation of RCAN1 could potentially regulate both LTP and LTD. My work therefore investigates how, depending on its phosphorylation state, RCAN1 affects synaptic transmission and plasticity and if this occurs via a direct action on CaN1. In order to determine the role of RCAN1 phosphorylation in synaptic plasticity, a combination of molecular biology, imaging and electrophysiology was used. We generated point mutations of wild type RCAN1 in order to obtain two non-phosphorylable forms of RCAN1: one that couldn’t be phosphorylated by GSK3β, and another one that PKA could not phosphorylate. Viral expression of RCAN1 and its phosphorylation deficient mutants in dissociated hippocampal cultures revealed that they are localized within dendritic spines, hinting at a synaptic function of RCAN1. To determine the effects of RCAN1 on synaptic transmission and plasticity, I virally expressed RCAN1 and the phosphorylation deficient mutants of RCAN1 in rat organotypic hippocampal slice cultures and analyzed their effects on synaptic plasticity by whole cell ‘‘patch-clamp’’ recordings. I observed that the blockade of the GSK3β phosphorylation site in RCAN1 increased synaptic transmission and blocked LTD induction. Furthermore, I demonstrate that LTP was blocked when PKA was unable to phosphorylate RCAN1. Finally, I determined that these distinct effects of RCAN1 on synaptic transmission and plasticity were directly dependent on CaN1. I thus define a cascade of events as well as demonstrate the key role of RCAN1 in the regulation of both LTP and LTD. Based on my results, I propose that iv RCAN1 modulates synaptic transmission and plasticity according to its phosphorylation states by GSK3β and PKA, via its direct action on CaN1, being an effector of both GSK3β during LTD and PKA during LTP induction.