Η αλματώδης πρόοδος που έχει γινει τα τελευταία χρόνια στην αποκωδικοποίηση των σηματοδοτικών μονοπατιών και η σημαντική αποσαφήνιση των κυτταρικών στοιχείων που εμπλέκονται στην γένεση, την ανάπτυξη, την εξάπλωση του καρκίνου, έχει στρέψει ένα σημαντικό κομμάτι της επιστημονικής κοινότητας προς την κατεύθυνση ανακάλυψης χημικών αναστολέων του κυτταρικού κύκλου. Εξέχοντα ρόλο σε αυτή τη διαδικασία παίζουν οι κυκλινο-εξαρτώμενες κινάσες (cyclin dependent kinases, CDKs). Οι CDKs είναι μια οικογένεια πρωτεϊνικών κινασών σερίνης/θρεονίνης, οι οποίες ενεργοποιούνται σε συγκεκριμένα σημεία του κυτταρικού κύκλου. Η απορρύθμισή τους συμβάλλει στην εκδήλωση παθολογικών καταστάσεων όπως νεοπλασιών και νευροεκφυλιστικών παθήσεων. Η παρούσα εργασία είχε σαν στόχο τη δημιουργία μικρών μορίων που θα αποτελέσουν πρόδρομα σταδια για την σύνθεση ενώσεων οι οποίες θα μελετηθούν ως προς την ικανότητα τους να αναστείλουν την δράση διαφόρων πρωτεϊνικών κινασών, μεταξύ άλλων και των κυκλινο-εξαρτώμενων κινάσων (CDKs). Κατέστη δυνατόν να συντεθεί η ένωση 11 η οποία έχει λειτουργικές ομάδες οι οποίες, αν τροποποιηθούν κατάλληλα, μπορούν να μας δώσουν πρόδρομα μόρια για την σύνθεση τελικών ενώσεων. / The rapid progress made in recent years to decode the signaling pathways and important clarification cellular components involved in the genesis, development, spread of cancer, has turned an important part of scientific community towards the discovery of chemical inhibitors of cell cycle. Prominent role in this process played by cyclin-dependent kinases (cyclin dependent kinases, CDKs). The CDKs are a family of protein serine / threonine kinases that are activated in specific parts of the cell cycle. Deregulation contributes to the onset illnesses such as cancer and neurodegenerative diseases This work was aimed at the creation of small molecules that will be precursor stages to synthesize compounds which will be studied in their ability to inhibit the action of various protein kinases including the cyclin-dependent kinases (CDKs). It was possible to synthesize the compound 11 which has functional groups which, if modified properly, can give us precursor molecules for the synthesis of final compounds.

No description available.

Σκοπός της παρούσης εργασίας είναι η στατιστική ανάλυση της σχέσης δομής-δράσης των χημικών ενώσεων που παράγονται από οξινδόλια και πουρίνες πάνω στα σύμπλοκα CDKs/Cyclins και η εξαγωγή συμπερασμάτων πάνω στις σχέσεις αυτές. Για το σκοπό αυτό ανατρέξαμε στην διεθνή βιβλιογραφία για να συλλέξουμε όσες πληροφορίες έχουν δημοσιευτεί έως τώρα για τις δύο αυτές ομάδες ενώσεων. Έπειτα μέσω ειδικών προγραμμάτων έγινε η επεξεργασία τους και η ανάλυση τους. Σκοπός είναι να συσχετίσουμε τη δομή των ενώσεων αυτών με τη δράση και τη βιολογική τους απόκριση πάνω στα σύμπλοκα των CDKs με τις κυκλίνες. / -

Early Xenopus laevis embryonic cell cycles exemplify rapid, non-pathological cell cycles without checkpoint pathways to arrest cell cycles in response to DNA assaults. There is no transcription or apoptosis during these cell cycles, and they continue unperturbed until the 12th cell cycle, marking a period called the midblastula transition (MBT). At the MBT, the embryo undergoes a period of developmental remodeling: gap phases are acquired, zygotic transcription is initiated, and the maternal mRNAs are degraded. After the MBT, checkpoint pathways can be activated in response to unreplicated DNA, and apoptosis initiates when continued embryonic survival is compromised. These studies examine how cell cycle regulation and apoptotic regulation are related. Specifically, the role of two cell cycle components, Chk1 and cyclin A1/Cdk2, during apoptosis was studied during early development of Xenopus embryos. Chk1 is a serine/threonine kinase that inhibits the activity of cyclin-dependent kinases (Cdks) in response to unreplicated DNA. In the pre-MBT embryo, Chk1 is present, but inactive. Injection of mRNA encoding dominant-negative Chk1 (DN-Chk1) into single-celled embryos results in the initiation of apoptosis after the MBT. The loss of Chk1 function also results in the initiation of additional rapid rounds of DNA replication after the MBT. These results suggest that Chk1 has a required function for the embryo after the MBT, possibly through the regulation of a cyclin/Cdk complex responsible for the apoptotic checkpoint. Cyclin A1 is a maternally provided mRNA that is degraded at the MBT. Prior to the MBT, cyclin A1 complexes exclusively with Cdc2 to regulate mitosis. When embryos are treated with ionizing radiation (IR), cyclin A1 activity and protein level persist after the MBT, and cyclin A1 complexes with Cdk2. When treated with aphidicolin, cyclin A1-associated activity and protein level persists. Injection of cyclin A1/Cdk2 into single-cell embryos results in apoptosis after the MBT; however, inhibition of cyclin A1 expression does not abrogate apoptosis. Therefore, cyclin A1/Cdk2 activity is sufficient, but not required, for the initiation of apoptosis in the early Xenopus embryo. These studies show that Chk1 and cyclin A1/Cdk2 have roles in regulating apoptosis in the post-MBT embryo. / Ph. D.

Mes travaux de recherche portent sur le contrôle du cycle cellulaire chez la levure de fission,  Schizosaccharomyces pombe. Chez S. pombe, cette régulation est assurée principalement par une protéine kinase cycline-dépendante (CDK), nommée Cdc2. Au cours du cycle cellulaire, cette enzyme s’associe avec différentes cyclines (Cig1, Cig2, Puc1 et Cdc13), formant ainsi une variété de complexes CDK-cycline qui confèrent des activités spécifiques à chaque phase du cycle. Cependant, il a été montré que ce réseau complexe peut être simplifié et remplacé par un système minimal, qui consiste en la fusion des deux gènes cdc2 et cdc13, indépendant d’un grand nombre de régulations endogènes. Cette découverte a ainsi permis d'établir un nouveau modèle pour le contrôle du cycle cellulaire eucaryote. Dans ce travail nous avons d’une part, voulu comprendre pourquoi la régulation du cycle cellulaire s’est complexifiée au cours de l’évolution, étant donné qu’une grande partie du circuit endogène semble dispensable. Dans ce but, j’ai investigué les limites du système minimal, quand les cellules sont exposées à différents stress. De manière surprenante, nous avons découvert que la simplification du réseau des CDKs confère aux cellules une résistance au stress réplicative. Nous avons montré que ce phénotype était indépendant de la régulation de l’inhibiteur Rum1 et des points de contrôles. Il résulte plutôt du fait que le cycle cellulaire soit régulé uniquement par Cdc13. Nous avons trouvé que le programme de réplication était inchangé dans les minimale qui présentaient moins de dommage à l’ADN comparé aux cellules sauvages. Nos data suggèrent que l’activité des CDKs associée au cyclines de phase G1/S, représente un moyen alternatif de moduler la réponse au stress. D’autre part, en utilisant le même système dans lequel l’activité des CDKs peut être finement modulée par l’inhibiteur. Nous avons démontré que la transcription périodique des gènes dépendait d’une régulation quantitative par les CDKs. Par conséquence nous proposons le model, l’opposé de ce qui a été suggéré chez la S. cerevisiea. Dans notre model, la progression du cycle cellulaire ainsi que la transcription périodique des gènes sont toutes les deux sous le contrôle de l’activité des CDKs. / The cyclin-dependent protein kinases (CDKs) are at the core of cell cycle control. In fission yeast, cell proliferation is regulated by CDK1/Cdc2 in association with the four cyclins Cdc13, Cig1, Cig2 and Puc1 at different stages of the cell cycle. However, this complex endogenous system can be replaced by a minimal module consisting of a fusion between Cdc2 and Cdc13 in the absence of G1/S cyclins. Surprisingly, this minimal CDK network drives the entire cell cycle in a wild type manner. Since a number of aspects of cell cycle control in fission yeast appear to be dispensable, we asked why similarly simplified circuits were not selected over the complex endogenous network during evolution. This led us to investigate the limits of such minimal systems, in particular when challenged by different stresses. Unexpectedly, we uncovered that simplification of the CDK network confers resistance to replication stress. We showed that this phenotype is independent from the CDK inhibitor Rum1 and the existing checkpoint pathways. It solely relies on operating the entire cell cycle with a single cyclin, Cdc13, and is associated with reduced genome instability when replication is challenged. However, it is not the consequence of changes in replication organisation along the chromosomes. Our data suggest that G1/S cyclin-associated Cdc2 activity may represent an alternative as yet unknown means of modulating cellular response to DNA stress. We also took advantage of a derivative of the minimal cell cycle network, in which Cdc2 is made sensitive to specific chemical inhibition. As a result, CDK activity can be externally modulated and cell cycle phases can be precisely controlled. Using this system, we re-visited the interplay between CDK and periodic transcription, a highly conserved process that is critical for proper cell proliferation. In contrast with previous studies in budding yeast, we demonstrate that periodic transcription in fission yeast is not independent from cell cycle progression. On the contrary, our work reveals that cell cycle transcriptional oscillations rely on quantitative changes in CDK activity levels. We therefore propose a new model, in which cell cycle progression and periodic transcription are intimately coupled through their common dependency on a unique input, namely CDK activity levels.

Os estudos de modelagem molecular das interações entre ligantes baseado em oxindóis (isaepy, isapn, [Cu(isapn)]², isaenim e o SU9516) e as proteínas kinases dependentes de ciclina (CDK1 e CDK2) são apresentados neste trabalho. Uma inibição na atividade da CDK1 e CDK2, que catalisam a fosforilação de grupos específicos em proteínas, tem implicações na indução da apoptose celular. O objetivo é tentar determinar qual destes ligantes potencializa a inibição da síntese de ATP (adenosina trifosfato) em ADP (adenosina difosfato) no sítio ativo da CDK1 e CDK2 para, desta forma, induzir a apoptose de células cancerígenas. Os estudos realizados neste trabalho indicam que dentre os ligantes analisados, o isaepy e o isapn obtiveram melhores resultados de estabilidade e ligações de hidrogênio entre aminoácidos dentro do sítio. Analisamos a influência do íon Cu no aumento da eficácia do isapn na atividade inibitória (complexo [Cu(isapn)]²) e comparamos os resultados obtidos dos estudos do isapn e [Cu(isapn)]², quando inseridos no sítio de ligação do ATP da CDK1, com medidas de eletroforese em gel. Verificamos que os nossos resultados foram corroborados com as medidas de eletroforese. Também discutimos os resultados de cálculos de acoplamento hiperfino para o Cu no [Cu(isapn)]² em diferentes ambientes químicos e fizemos a comparação destes resultados com medidas de EPR. Desta forma, conseguimos verificar o ambiente químico do íon Cu e um aumento da estabilidade do isapn dentro do sítio estudado com a inserção do íon Cu. Este trabalho visa contribuir para a síntese de novos ligantes que aumentem a eficácia da inibição da síntese de ATP em ADP nas CDKs e também para a minimização dos custos através da diminuição da realização de experimentos que se baseiam em métodos de tentativa e erro. / Molecular modeling studies of the interaction of oxindol based ligands (isaepy, isapn [Cu(isapn)]²,isaenim and SU9516) with Cyclin Dependent Kinases proteins (CDK1 and CDK2) are presented here. CDK1 and CDK2 catalyze the phosphorylation of specific groups in proteins and inhibition of its activities implies in induction of cancer cells apoptosis. The goal is to determine which ligands increase the inhibition of ATP (adenosine triphosphate) into ADP (adenosine diphosphate) synthesis which occurs inside the CDK1 and CDK2 active site. We analyze the influence of the Cu ion on increasing the inhibitory activity in isapn ([Cu(isapn)]² metal complex). Comparisons between the results obtained from studies of the isapn and [Cu(isapn)]² inserted into the ATP binding site of CDK1 with measurements of gel electrophoresis were performed. The hyperfine coupling at Cu ion in [Cu(isapn)]² in different chemical environments are here obtained and the results are compared with EPR measurements. This work aims to contribute to the development of new ligands which increase the inhibition of the synthesis of ATP into ADP in the CDKs moreover we aim to assist in the reduction of the costs of measurements that are based on trial and error aproaches.

Os estudos de modelagem molecular das interações entre ligantes baseado em oxindóis (isaepy, isapn, [Cu(isapn)]², isaenim e o SU9516) e as proteínas kinases dependentes de ciclina (CDK1 e CDK2) são apresentados neste trabalho. Uma inibição na atividade da CDK1 e CDK2, que catalisam a fosforilação de grupos específicos em proteínas, tem implicações na indução da apoptose celular. O objetivo é tentar determinar qual destes ligantes potencializa a inibição da síntese de ATP (adenosina trifosfato) em ADP (adenosina difosfato) no sítio ativo da CDK1 e CDK2 para, desta forma, induzir a apoptose de células cancerígenas. Os estudos realizados neste trabalho indicam que dentre os ligantes analisados, o isaepy e o isapn obtiveram melhores resultados de estabilidade e ligações de hidrogênio entre aminoácidos dentro do sítio. Analisamos a influência do íon Cu no aumento da eficácia do isapn na atividade inibitória (complexo [Cu(isapn)]²) e comparamos os resultados obtidos dos estudos do isapn e [Cu(isapn)]², quando inseridos no sítio de ligação do ATP da CDK1, com medidas de eletroforese em gel. Verificamos que os nossos resultados foram corroborados com as medidas de eletroforese. Também discutimos os resultados de cálculos de acoplamento hiperfino para o Cu no [Cu(isapn)]² em diferentes ambientes químicos e fizemos a comparação destes resultados com medidas de EPR. Desta forma, conseguimos verificar o ambiente químico do íon Cu e um aumento da estabilidade do isapn dentro do sítio estudado com a inserção do íon Cu. Este trabalho visa contribuir para a síntese de novos ligantes que aumentem a eficácia da inibição da síntese de ATP em ADP nas CDKs e também para a minimização dos custos através da diminuição da realização de experimentos que se baseiam em métodos de tentativa e erro. / Molecular modeling studies of the interaction of oxindol based ligands (isaepy, isapn [Cu(isapn)]²,isaenim and SU9516) with Cyclin Dependent Kinases proteins (CDK1 and CDK2) are presented here. CDK1 and CDK2 catalyze the phosphorylation of specific groups in proteins and inhibition of its activities implies in induction of cancer cells apoptosis. The goal is to determine which ligands increase the inhibition of ATP (adenosine triphosphate) into ADP (adenosine diphosphate) synthesis which occurs inside the CDK1 and CDK2 active site. We analyze the influence of the Cu ion on increasing the inhibitory activity in isapn ([Cu(isapn)]² metal complex). Comparisons between the results obtained from studies of the isapn and [Cu(isapn)]² inserted into the ATP binding site of CDK1 with measurements of gel electrophoresis were performed. The hyperfine coupling at Cu ion in [Cu(isapn)]² in different chemical environments are here obtained and the results are compared with EPR measurements. This work aims to contribute to the development of new ligands which increase the inhibition of the synthesis of ATP into ADP in the CDKs moreover we aim to assist in the reduction of the costs of measurements that are based on trial and error aproaches.

A biologia da reprodução sexual de plantas é um campo de pesquisa de grande importância, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes), oriundas do desenvolvimento de partes do pistilo fertilizado. Em Nicotiana tabacum, identificou-se um gene específico de estigma/estilete, SCI1 (Stigma/style Cell-cycle Inhibitor 1), que atua na inibição da proliferação celular (DePaoli et al., 2011). Através de ensaios de pull-down, verificou-se a interação da proteína SCI1 com uma proteína quinase dependente de ciclina (CDK) (Strini, dados não publicados). Este trabalho visou à caracterização dessa nova CDK, ortóloga da CDKG;2 de Arabidopsis. A sequência correspondente de N. tabacum (NtCDKG;2) foi amplificada por PCR, a partir de cDNAs de estigmas/estiletes, clonada e sequenciada, o que permitiu a confirmação de sua identidade. A expressão de NtCDKG;2 foi analisada nos diferentes órgãos vegetativos e reprodutivos, por qRT-PCR, o que evidenciou um perfil de expressão ubíqua. Ao estudar o perfil de expressão desse gene nos estigmas/estiletes dos doze estádios de desenvolvimento floral de N. tabacum, observa-se que NtCDKG;2 é mais expresso nos estádios tardios do desenvolvimento em direção à antese, indicando uma função importante de sua proteína ao final do desenvolvimento do pistilo. Análises de expressão de NtCDKG;2 em estigmas/estiletes, de plantas de N. tabacum com produção aumentada do hormônio auxina no pistilo, sugerem que NtCDKG;2 é regulado transcricionalmente por esse hormônio. A expressão transiente da proteína de fusão NtCDKG;2-GFP, em folhas de N. tabacum, evidenciou a localização nuclear da proteína em estudo. Também foram geradas plantas transgênicas estáveis com superexpressão e com silenciamento por RNAi de NtCDKG;2. Apesar dos altos níveis de transcritos de NtCDKG;2 nas plantas de superexpressão e dos baixos níveis nas plantas silenciadas, não foram observadas alterações fenotípicas macroscópicas nessas plantas. Adicionalmente, obteve-se a expressão da proteína NtCDKG;2, fusionada a uma tag de histidina em sua porção N-terminal, em células de Escherichia coliBL21(DE3)CodonPlusRP. Através dos estudos realizados neste trabalho e análises conjuntas da literatura, é possível propor que NtCDKG;2 codifique uma proteína que está envolvida no controle do ciclo celular nos estigmas/estiletes de N. tabacum. / The biology of plant sexual reproduction is a research field of great importance, since most of the food consumed by humans is composed of plant reproductive parts (fruits and seeds), originated by the development of fertilized pistil parts. In Nicotiana tabacum, it was identified a stigma/style-specific gene, SCI1 (Stigma/style Cell-cycle Inhibitor 1), which acts in the inhibition of cell proliferation (DePaoli et al., 2011). Through pull down assays, the interaction of the SCI1 protein with a cyclin-dependent protein kinase (CDK) was verified (Strini, unpublished). This work aimed the characterization of this new CDK, orthologous to the Arabidopsis CDKG;2. The N. tabacum corresponding sequence (NtCDKG;2) was PCR amplified, from stigmas/styles cDNAs, cloned and sequenced, which allowed the confirmation of its identity. The NtCDKG;2 expression was analyzed in the different vegetative and reproductive organs, by qRT-PCR, evidentiating an ubiquitous expression pattern. Studying the expression pattern of this gene in stigmas/styles of the twelve stages of N. tabacum flower development, it was observed that NtCDKG;2 is more expressed at the later developmental stages towards anthesis, indicating an important function of its protein in the end of pistil development. NtCDKG;2 expression analyses in stigmas/styles of N. tabacum plants with an enhanced auxin production in the pistil suggest that NtCDKG;2 is transcriptionally regulated by this hormone. The transient expression of the fusion protein NtCDKG;2-GFP, in N. tabacum leaves, evidentiated the nuclear localization of the studied protein. Stable transgenic plants overexpressing and silencing NtCDKG;2 by RNAi were also generated. Despite the high transcript levels in the plants overexpressing NtCDKG;2 and the low transcript levels in the silencing plants, macroscopic phenotypic alterations were not observed on these plants. Additionally, the expression of the NtCDKG;2 protein, with a histidine tag fused in its N-terminal, was obtained in Escherichia coli BL21(DE3)CodonPlusRP cells. Through studies performed on this work and literature analyses, it is possible to propose that NtCDKG;2 encodes a protein that is involved in the control of cell cycle at the N. tabacum stigmas/styles.

Le cancer constitue l’une des principales causes de mortalité, et représente, de ce fait aujourd’hui un problème de santé publique majeur. Depuis quelques années, les alcaloïdes marins représentent une source d’inspiration importante pour les chimistes en vue d’obtenir de nouveaux médicaments anticancéreux. Dans cette optique, des recherches effectuées au sein de notre laboratoire ont fait état de la synthèse d’analogues de ces alcaloïdes possédant une structure tris aromatique. Nous avons développé des molécules originale analogues de ces alcaloïdes disposant d’un hétérocycle central (1,2,4-triazine et imidazo[1,2-b][1,2,4,5]tétrazine) sur lequel sont greffés deux noyaux phényles diversement substitués. L’obtention de ces composés a également été l’occasion de développer de nouvelles méthodologies de synthèse. Ainsi une nouvelle réaction de type Buchwald-Hartwig sur des méthylsulfanyl-1,2,4-triazines a pu être mise au point ainsi qu’une méthode de CH arylation palladocatalysée sur le noyau imidazo[1,2-b][1,2,4,5]tétrazine. Une partie est aussi consacrée aux réactions multicomposants de type Groebke-Blackburn. Différentes évaluations pharmacologiques ont été réalisées, notamment des tests d’inhibition sur différentes kinases et de cytotoxicité sur diverses lignées cellulaires cancéreuses humaines. / Cancer, one of the leading causes of death, represents today a major public health problem. Over the last few years, marine alkaloids represent a source of inspiration for chemists in order to obtain new anticancer drugs. For this purpose, as a part of our laboratory researches, analogues of marine alkaloids were synthesized possessing a tris-aromatic structure. We developed originals analogs of these alacaloïds formed by a central heterocycle core (1,2,4-triazine et imidazo[1,2-b][1,2,4,5]tetrazine) on wich is graft two arylic moiety variously substituted. Obtaining these compounds was also an opportunity to develop news synthetic methodologies. So a new Buchwald-Hartwig reaction type based on methylsulfanyl-1,2,4-triazines has been perfect, as palladocatalyzed CH arylation pathway on imidazo[1,2-b][1,2,4,5]tetrazine. A part is devoted to Groebke-Blackburn multicomponant reaction. Various pharmacological analyses were carried out in particular with inhibition of various kinases and cytotoxicity evaluation on various human cancer cell lines.

A biologia da reprodução sexual de plantas é um campo de pesquisa de grande importância, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes), oriundas do desenvolvimento de partes do pistilo fertilizado. Em Nicotiana tabacum, identificou-se um gene específico de estigma/estilete, SCI1 (Stigma/style Cell-cycle Inhibitor 1), que atua na inibição da proliferação celular (DePaoli et al., 2011). Através de ensaios de pull-down, verificou-se a interação da proteína SCI1 com uma proteína quinase dependente de ciclina (CDK) (Strini, dados não publicados). Este trabalho visou à caracterização dessa nova CDK, ortóloga da CDKG;2 de Arabidopsis. A sequência correspondente de N. tabacum (NtCDKG;2) foi amplificada por PCR, a partir de cDNAs de estigmas/estiletes, clonada e sequenciada, o que permitiu a confirmação de sua identidade. A expressão de NtCDKG;2 foi analisada nos diferentes órgãos vegetativos e reprodutivos, por qRT-PCR, o que evidenciou um perfil de expressão ubíqua. Ao estudar o perfil de expressão desse gene nos estigmas/estiletes dos doze estádios de desenvolvimento floral de N. tabacum, observa-se que NtCDKG;2 é mais expresso nos estádios tardios do desenvolvimento em direção à antese, indicando uma função importante de sua proteína ao final do desenvolvimento do pistilo. Análises de expressão de NtCDKG;2 em estigmas/estiletes, de plantas de N. tabacum com produção aumentada do hormônio auxina no pistilo, sugerem que NtCDKG;2 é regulado transcricionalmente por esse hormônio. A expressão transiente da proteína de fusão NtCDKG;2-GFP, em folhas de N. tabacum, evidenciou a localização nuclear da proteína em estudo. Também foram geradas plantas transgênicas estáveis com superexpressão e com silenciamento por RNAi de NtCDKG;2. Apesar dos altos níveis de transcritos de NtCDKG;2 nas plantas de superexpressão e dos baixos níveis nas plantas silenciadas, não foram observadas alterações fenotípicas macroscópicas nessas plantas. Adicionalmente, obteve-se a expressão da proteína NtCDKG;2, fusionada a uma tag de histidina em sua porção N-terminal, em células de Escherichia coliBL21(DE3)CodonPlusRP. Através dos estudos realizados neste trabalho e análises conjuntas da literatura, é possível propor que NtCDKG;2 codifique uma proteína que está envolvida no controle do ciclo celular nos estigmas/estiletes de N. tabacum. / The biology of plant sexual reproduction is a research field of great importance, since most of the food consumed by humans is composed of plant reproductive parts (fruits and seeds), originated by the development of fertilized pistil parts. In Nicotiana tabacum, it was identified a stigma/style-specific gene, SCI1 (Stigma/style Cell-cycle Inhibitor 1), which acts in the inhibition of cell proliferation (DePaoli et al., 2011). Through pull down assays, the interaction of the SCI1 protein with a cyclin-dependent protein kinase (CDK) was verified (Strini, unpublished). This work aimed the characterization of this new CDK, orthologous to the Arabidopsis CDKG;2. The N. tabacum corresponding sequence (NtCDKG;2) was PCR amplified, from stigmas/styles cDNAs, cloned and sequenced, which allowed the confirmation of its identity. The NtCDKG;2 expression was analyzed in the different vegetative and reproductive organs, by qRT-PCR, evidentiating an ubiquitous expression pattern. Studying the expression pattern of this gene in stigmas/styles of the twelve stages of N. tabacum flower development, it was observed that NtCDKG;2 is more expressed at the later developmental stages towards anthesis, indicating an important function of its protein in the end of pistil development. NtCDKG;2 expression analyses in stigmas/styles of N. tabacum plants with an enhanced auxin production in the pistil suggest that NtCDKG;2 is transcriptionally regulated by this hormone. The transient expression of the fusion protein NtCDKG;2-GFP, in N. tabacum leaves, evidentiated the nuclear localization of the studied protein. Stable transgenic plants overexpressing and silencing NtCDKG;2 by RNAi were also generated. Despite the high transcript levels in the plants overexpressing NtCDKG;2 and the low transcript levels in the silencing plants, macroscopic phenotypic alterations were not observed on these plants. Additionally, the expression of the NtCDKG;2 protein, with a histidine tag fused in its N-terminal, was obtained in Escherichia coli BL21(DE3)CodonPlusRP cells. Through studies performed on this work and literature analyses, it is possible to propose that NtCDKG;2 encodes a protein that is involved in the control of cell cycle at the N. tabacum stigmas/styles.