The tumour suppressor p53 is a key regulatory protein that prevents proliferation of damaged cells. Under unperturbed conditions, the ubiquitin ligase murine double minute 2 (MDM2) mediates p53 ubiquitination and further degradation by the proteasome. In consequence p53 is present at low levels, but becomes rapidly stabilised and activated in response to a variety of stimuli, such as DNA damage or virus infection. P53 responds to these diverse stresses to regulate the expression of many target genes that induce cell cycle arrest, DNA repair, or apoptosis. The attenuation of p53 interaction with MDM2 is maintained by enzymes catalysing p53 post-translational modifications such as phosphorylation. Casein kinase 1 α (CK1α) is one such enzyme; it stimulates p53 after DNA virus infection. Surprisingly depletion of CK1α using small interfering RNA or inhibition using a CK1 kinase inhibitor activated the transcription factor p53, indicating that p53 steady-state level is controlled by CK1α. Disrupting MDM2-p53 interaction using small molecule Nutlin-3 displayed similar pharmacological properties to the CK1 inhibitor on p53, indicating that the MDM2-CK1α complex co-regulates p53 stability. Indeed co-immunoprecipitation of endogenous CK1α with MDM2 occurred in undamaged cells. CK1α was shown in vitro to directly bind to and phosphorylate MDM2. Therefore it appears that CK1α must be recruited into specific complexes under different conditions, which can influence its substrate selectivity and explain its dual role on the p53 pathway. Apart from CK1, there are few other kinases whose action can directly contribute to the inhibition of p53. A novel pyrazolo-pyridine analogue showing dual activity against CK1 and Checkpoint kinase 1 led to increased p53 activation. These data highlighted the potential value of dual kinase inhibitors as therapeutics in cancer. The dominant protein-protein interface that stabilises the MDM2-CK1α complex was mapped using a peptide-based approach. One CK1α peptide bound strongly to MDM2, it specifically disrupted the protein-protein interaction, and its transfection was able to reduce cancer cell growth. A peptide phage display approach was finally combined with Next-Generation Sequencing to define the change in MDM2 binding motifs when the CK1α peptide or Nutlin-3 is bound, compared to ligand-free MDM2, and thus will help to understand protein-protein interaction network re-wirings which led to cell growth inhibition.

PAWS1/FAM83G, a member of the poorly characterised FAM83 family of proteins that share the conserved domain of unknown function DUF1669, was identified as an interactor of the SMAD1 transcription factor. Because BMP signalling plays a fundamental role during embryogenesis, collaboration with Jim Smith (The Francis Crick Institute, London) led to the discovery that ectopic expression of PAWS1 mRNA in Xenopus embryos leads to a complete duplication of body axis. In similar assays, such a phenotype is associated with either the inhibition of canonical (SMAD4-dependent) BMP signalling or the activation of canonical Wnt/β-catenin signalling. PAWS1 has been reported to modulate only non-canonical BMP signalling and not influence canonical BMP signalling, findings which were also validated in Xenopus embryos and PAWS1-knockout U2OS cells in this thesis. Therefore, focus turned to investigating potential roles of PAWS1 in the regulation of the canonical Wnt/β-catenin signalling pathway. The canonical Wnt/β-catenin signalling pathway plays critical roles during embryogenesis, stem cell self-renewal and in adult tissue homeostasis and is often misregulated in developmental defects, including skin and hair abnormalities, and cancer. In the absence of Wnt signals, sequential phosphorylation of the transcriptional co-factor β-catenin by CK1 and GSK3 results in the ubiquitylation of β-catenin, priming it for degradation via the proteasome. Upon Wnt-activation, β-catenin is stabilised and translocates to the nucleus, where it associates with TCF and LEF and regulates the expression of Wnt-target genes. Although the fundamental steps in Wnt signalling are established, many gaps remain in our understanding of the precise regulation of the pathway. It is demonstrated in this thesis that PAWS1 activates Wnt signalling in both Xenopus embryos and human cells. Furthermore, in PAWS1-knockout U2OS cells Wnt signalling is attenuated. Collectively, these data uncover a role for PAWS1 as a novel regulator of canonical Wnt/β-catenin signalling. In search of molecular mechanisms through which PAWS1 regulates Wnt/β-catenin signalling, a proteomic approach on endogenous PAWS1 revealed the Ser/Thr protein kinase CK1α as a robust PAWS1 interactor. PAWS1 interacts and colocalises with endogenous CK1α. CK1 isoforms are key regulators of Wnt signalling and they phosphorylate many components of the pathway, however their precise regulation in cells, despite being critically important, is poorly understood. After mapping CK1-interaction sites to key residues within the conserved DUF1669 domain of PAWS1, it was possible to demonstrate that the interaction between PAWS1 and CK1α is critical for PAWS1 to activate Wnt signalling in both Xenopus embryos and U2OS cells. Although the phosphorylation of β-catenin on Ser45, which is reported to be phosphorylated by CK1 isoforms, appears to be unaltered by PAWS1-deficiency, the Wnt3A-induced nuclear translocation of β-catenin is slightly inhibited in PAWS1 knockout U2OS cells. It is likely that PAWS1 controls Wnt signalling by directing CK1α to key subcellular locations and substrates upon Wnt stimulation to regulate the nuclear translocation of β-catenin. Consistent with this hypothesis, a global phosphoproteomics analysis of wild type and PAWS1-/- U2OS cells has revealed differential phosphorylation of proteins that may be regulated by the PAWS1:CK1α interaction. Interestingly, PAWS1 appears to control levels of endogenous CK1α protein and vice versa, although the mechanisms by which each achieves this are still unclear. The findings that the DUF1669 domain of PAWS1 interacts with CK1α led to the discovery that all FAM83 members bind to different CK1 isoforms through an identical mechanism. This has led to the hypothesis that FAM83 members serve as anchoring proteins for CK1 isoforms, and in doing so, they regulate CK1 subcellular localisation and substrate accessibility in cells. Regulation of PAWS1 by post-translational modifications remains poorly defined. A proteomic approach identified calcium and calmodulin-dependent kinase isoforms D and G (CaMK2D and CaMK2G) as two novel interactors of PAWS1. CaMK2 enzymes are activated in response to calcium signals to control cytoskeletal rearrangements and cell movement. PAWS1 has been implicated in actin cytoskeletal dynamics and cell migration, through its dynamic interaction with the adapter protein CD2AP at the cell periphery. Here, PAWS1 has been shown to be phosphorylated at Ser356 by CaMK2D in cells and this phosphorylation event is demonstrated to be important for PAWS1-dependent cell migration. Lastly, several PAWS1 mutations have been recently linked with the pathogenesis of skin diseases in dogs and humans. However, how these mutations relate to PAWS1 function in cells and potentially cause the disease phenotypes remain elusive. In this thesis, initial steps have been taken to address the potential impact of these pathogenic mutants on PAWS1 function.

Memoria para optar al Título Profesional de Médico Veterinario / La proteína CK1 es una familia de proteínas quinasa a la que recientemente se la ha relacionado con el desarrollo embrionario. De esta familia se han clonado al menos siete isoformas: CK1α, CK1β, CK1γ1, CK1γ2, CK1γ3, CK1δ y CK1ε. En esta Memoria de Título se realizaron estudios de expresión y función de las isoformas CK1δ1 y CK1δ2 en el desarrollo embrionario del pez cebra (Danio rerio). Para esto, analizamos los patrones de expresión de estos genes mediante RT-PCR e hibridización in situ. Los resultados indican que estas isoformas se expresan de manera similar en cuanto a su temporalidad, ya que ambas se detectan desde la primera hora post fecundación (h.p.f). CK1δ1 presenta un patrón ubicuo hasta las 8 h.p.f y limitándose después al sistema nervioso central. Por su parte, la expresión de CK1δ2 presenta ubicuidad hasta las 24 h.p.f, restringiéndose posteriormente a la zona cefálica y al primordio de las aletas pectorales. Para analizar la función de ambas isoformas se efectuaron estudios de sobre expresión, mediante la inyección en embriones de pez cebra de los mRNAs codificante para CK1δ1 y CK1δ2, y ensayos de bloqueo de función mediante la inyección en embriones de los mRNA de las formas Dominantes Negativas de cada uno de los genes estudiados. Estos experimentos se realizaron utilizando la técnica de microinyección en embriones en estadío de una célula y posteriormente incubados y observados en distintos estadíos bajo lupa estereoscópica, y clasificados según su fenotipo. Además, se procedió a analizar el patrón de expresión de algunos genes marcadores específicos de desarrollo embrionario, en embriones inyectados con una cantidad conocida de mRNA de CK1δ1 y CK1δ2, con el fin de establecer con mayor precisión los procesos y vías en los que podrían estar involucradas estas isoformas. Los resultados obtenidos con los experimentos anteriores indican que la sobre expresión de ambas isoformas, CK1δ1 y CK1δ2, inducen distintos grados de malformación en cerebro anterior y ojos, siendo el fenotipo principal observado con la inyección de CK1δ1 los embriones sin ojos. El bloqueo de función mediante Dominantes Negativos indujo, en ambas isoformas, embriones dorsalizados y ciclópicos. Se puede concluir, que las isoformas CK1δ1 y CK1δ2 están participando en la formación de los ejes embrionarios en el desarrollo temprano del pez cebra y además en la formación de estructuras del sistema nervioso central, específicamente cerebro anterior y ojos. / FONDECYT N° 1020753 y por Millenium Nucleus in Developmental Biology

Doctor en Ciencias con Mención en Biología Molecular, Celular y Neurociencias / La proteínaquinasa CK1 es una enzima altamente ubícua y conservada en todos los eucariontes estudiados y, particularmente, dentro de los vertebrados. En estos organismos, la enzima existe como una familia de siete miembros genéticamente distintos, denominados CK1#, #, #1, #2, #3, # y #. Hasta el momento se ha determinado que los mensajeros de tres de estas isoformas, #, #1 y #3, sufren procesamiento alternativo, dando lugar así a nuevas variantes. En particular, el mRNA de la isoforma a da origen a 4 subisoformas, definidas por la presencia o ausencia de 2 segmentos adicionales, llamados inserto L e inserto S. El primero se ubica en medio del dominio catalítico de la enzima, en la llamada region “bisagra” y el segundo se encuentra localizado en el extremo C-terminal. La coexistencia de algunas o todas estas variantes se ha descrito anteriormente en rata, pollo y humano y las secuencias de los insertos se encuentran altamente conservadas entre estas especies. En esta Tesis, se clonaron las cuatro variantes de procesamiento de CK1# de pez cebra ( Danio rerio ) y se expresaron tanto en células de E. coli como en células eucarióticas en cultivo. La caracterización bioquímica de las variantes recombinantes expresadas en bacterias determinaron que las que contienen el inserto L tienen una menor afinidad por ATP, al igual que por el inhibidor específico, CKI-7, que compite por el sitio de unión del nucleótido. Estas variantes, además, muestran una mayor actividad hacia #-caseína, aunque no existen diferencias en el valor de la Km aparente entre las cuatro isoformas con respecto a los sustratos proteicos y peptídicos ensayados, #-caseína, fosvitina y el péptido RRKDLHDDEEDEAM SITA, derivado del inhibidor-2 de la proteína fosfatasa 1 (PPI2). Las variantes L también exhiben una mayor termolabilidad a 40°C, comparadas con las que no contienen este inserto. Esta secuencia contiene además un sitio canónico para la fosforilación por PKA y, al ser incubadas con esta enzima, la fosforilación es estimulada en 2 veces para CK1#, 6 veces para #S, 16 veces para #L y aproximadamente 70 veces para #LS. Esto sugiere que la PKA fosforila a CK1#, en particular dentro de los insertos y especialmente el inserto L. Por otro lado, las cuatro variantes poseían actividad autofosforilativa y tirosinaquinasa. Otro aspecto abordado en esta Tesis fue la fosforilación de NFAT4 por CK1. El trabajo de Zhu y col. (1998) demuestra que este factor de transcripción es fosforilado in vivo por CK1# y que esta fosforilación previene la entrada de NFAT4 al núcleo. En esta publicación se describe un putativo sitio de acoplamiento o “docking” dentro de la secuencia donde interactúa CK1. En nuestro estudio se utilizaron péptidos sintéticos que abarcaban las regiones conservadas A y Z y la región “linker” entre ellas (L). Tanto las variantes de CK1a de pez cebra como CK1 nativa purificada de hígado de rata fosforilaban residuos no canónicos contenidos en la región A2, pero no la Z, a diferencia de lo encontrado por Zhu y col. (1998). Una extensión de residuos acídicos presentes en la región L demostró ser esencial para la fosforilación eficiente del dominio A2, como se pudo determinar por un aumento en la Km de un orden de magnitud provocada por la deleción de la región L o por la sustitución de los residuos acídicos por glicinas o alaninas. Este resultado también difiere de los de Zhu y col., que habían ubicado el sitio “docking” en la región A2. Por otro lado, al reemplazar una de las serinas del extremo N-terminal de la región A2, la Ser177, por fosfoserina, se desencadena un efecto jerárquico que provoca un aumento dramático en la eficiencia de fosforilación del péptido. Estos datos son consistentes con un mecanismo bifásico de fosforilación, en que la región L provee un sitio “docking” funcional que facilita la fosforilación no canónica de la Ser177, la que, consiguientemente, gatilla una cadena de fosforilaciones jerárquicas río debajo de este residuo. Con objeto de realizar estudios sobre el comportamiento in vivo de las variantes de CK1#, se transfectaron células Cos-7 con construcciones de CK1# y CK1#L. Mediante inmunocitoquímica, se determinó que la CK1# se localizaba en forma generalizada dentro de la célula, predominantemente en el citoplasma. CK1#L, en contraste, se localizaba en forma predominante en el núcleo, lo que sugiere que la secuencia PVGKRKR, contenida dentro del inserto L, constituiría una señal de localización nuclear funcional. La variante más larga también exhibió una vida media 4 veces menor que CK1#, siendo de 97 y 400 minutos, respectivamente, como fue determinado por experimentos de pulso y caza en células transfectadas y marcadas metabólicamente con 35 S. Mediante experimentos de RT-PCR utilizando RNA total de embriones y adultos de pez cebra, se determinó que la proporción relativa de los transcritos de las cuatro subisoformas varía a lo largo del desarrollo y en adulto, observándose un aumento relativo de #S y una caída en el nivel de #L entre los estadíos de 1 célula y larva (4-5 días). Además, por hibridación in situ utilizando una sonda general para las cuatro variantes de CK1#, se observó una expresión generalizada de los mensajeros de esta isoforma de CK1 hasta las 24 h de desarrollo y, entre los 2 y 3 días, una localización más específica en la cabeza y el primordio de la aleta pectoral. En síntesis, la presencia del inserto L confiere a la enzima algunas propiedades que la distinguen de las variantes que no contienen este inserto: Altera el sitio de unión a ATP, disminuyendo levemente la afinidad por el nucleótido, disminuye la estabilidad de la enzima, tanto in vivo como in vitro frente a una temperatura de 40°C, aumenta el nivel de fosforilación de la CK1# en presencia de PKA y concentra la enzima en el núcleo, probablemente debido a la secuencia PVGKRKR contenida en el inserto L.

Yes / Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation. / Funded by a biomedical research grant from the Motor Neurone Disease Association (ref Rattray/Apr15/837-791). The Bioimaging Facility microscopes used in this study were purchased with grants from BBSRC, Wellcome Trust and the University of Manchester Strategic Fund.

Le travail s’est articulé autour de la synthèse et de l’évaluation biologique de nouveaux inhibiteurs de protéines kinases dont essentiellement de kinases dépendantes de cyclines (CDKs). La dérégulation de ces kinases est mise en cause dans l’apparition de nombreuses pathologies prolifératives telles que le cancer ou non prolifératives telles que les maladies neurodégénératives. Deux types d’inhibiteurs ont été préparés. La première famille de composés étudiée est la famille des purines, sur laquelle a été introduite des substructures de type aminodiols en position C-2. Ces aminodiols ont été préparés de manière stéréosélective via la réaction de dihydroxylation asymétrique décrite par Sharpless ou à partir de dérivés d’acides aminés. Parmi les inhibiteurs réalisés, plusieurs ont montré une inhibition envers les protéines CDK5, CDK9 et CK1 très supérieure (IC50 de l’ordre de 100 nM) à celle de la (R)-Roscovitine, molécule actuellement en phase clinique II dans plusieurs cancers. Ces inhibitions des cibles enzymatiques s’accompagnent d’un effet antiprolifératif sur plusieurs lignées tumorales. Enfin l’étude d’une des molécules a été complétée par des tests très favorables sur des enzymes de microsomes hépatiques (IC50 > 5 μM). Dans une seconde partie, des imidazo[1,2-b]pyridazines ont été préparées. Une synthèse originale conduisant aux imidazo[1,2-b]pyridazines 3,6,8-trisubstituées a été mise au point. Les produits présentent une inhibition forte envers les protéines kinases telles que CDK5 et CK1 (IC50 < 50 nM). Ils montrent également des propriétés antiprolifératives sur les cellules tumorales SH-SY5Y. Enfin, des imidazo[1,2-b]pyridazines 3,6-disubstituées se révèlent être des inhibiteurs sélectifs de la protéine CLK1 (IC50 < 100 nM). / This thesis focuses on the synthesis and biological evaluation of new kinase inhibitors. Kinase deregulation is associated with numerous diseases such as cancer or neurodegenerative diseases. In the first part of this work, 2,6,9-trisubstituted purines bearing at C-2 position aminodiols derivatives were prepared. Aminodiols were obtained either via Sharpless asymmetric dihydroxylation or by reduction of amino esters. The compounds appeared to be more potent against kinases than (R)-roscovitine which is presently undergoing phase II clinical tests. In particular, inhibition of CK1, CDK5 and CDK9 were observed with IC50 < 200 nM. The compounds prepared showed an antiproliferative effect against tumor cell-lines (SH-SY5Y). Eventually, one of the most promising compounds was assayed against a series of cyt P450 enzymes and did not showed any inhibition (IC50 > 5 μM). The second family of compounds prepared in this work is imidazo[1,2-b]pyridazines. A new route to 3,6,8-trisubstituted imidazo[1,2-b]pyridazines was first developed. These products were shown to be highly potent inhibitors of several kinases such as CDK5 and CK1 (IC50 < 50 nM). The kinase inhibitions were accompanied by antiproliferative activities against tumor cell-lines. Finally, a series of 3,6-disubtituted imidazo[1,2-b]pyridazines was also prepared and appeared to be selective inhibitors of CLK1 (IC50 < 100 nM).

Made available in DSpace on 2014-08-20T13:59:08Z (GMT). No. of bitstreams: 1 tese_maria_da_graca_de_souza_lima.pdf: 4326568 bytes, checksum: 1e4f3676ef163392730daec1cb217796 (MD5) Previous issue date: 2008-07-18 / In Rio Grande do Sul State, the main system for irrigation of rice cultivation is by flooding, can lead to the salinization of soils with inadequate drainage, especially at the coastal region where crops using water from the Laguna dos Patos, which is subject to the salinization by sea water. This is a major environmental problem in the rice production. This survey aimed to examine the expression of enzyme in Oryza sativa L. ssp. indica S. Kato e Oryza sativa L. ssp. japonica S. Kato, grown in different levels of salinity, in order to identify genes involved in tolerance to salinity, based on the assumption that the second subspecies show greater tolerance to salinity. In the experiment were used Oryza sativa L. ssp. japonica S. Kato (BRS Bojuru, IAS 12-9 Formosa and Goyakuman) and Oryza sativa L. ssp. indica S. Kato (BRS-7 Taim, BRS Querência and BRS Atalanta). The seedling was done in plastic trays, containing sand washed as substrate. The seedlings were transferred to greenhouse with 10 days of emergency under temperature 25 °C and humidity 85 % controlled and placed in basins of 15 L containing nutrient solution of Hoagland half strength increased of 0, 25, 50, 75 and 100 mM NaCl. Seedlings were collected at 14, 28, 42 and 56 days after the transfer and immediately stored in ultrafreezer to -70 °C to subsequent analyses. The plant tissues were macerated and placed in tubes eppendorf with extractor solution of Scandálios. The electrophoresis was performed in 7% of polyacrylamide gels placed in vertical vats. The bands were revealed for several enzymes systems: superoxide dismutase, peroxidase, catalase, esterase, xvi glutamate dehydrogenase, alcohol dehydrogenase, fosfoglucose isomerase, malate dehydrogenase, málica enzyme, alpha amylase and glucose-6-phosphate dehydrogenase. Through the search in silico, conducted with the National Center for Biotechnology Information identified the genes AY785147 - SOS and AF319481 - CK1 involved in the salinity tolerance. The detection of the gene was the extraction of DNA using the method CTAB 2%, followed by reactions of PCR thermocycler held on through the use of primers also drawn in silico. The products of amplification were detected by agarose gel electrophoresis of 1.5%. The view of the DNA stained with bromide etídio was made on ultraviolet light and scanned images. The expression of enzymes involved in the mechanisms of tolerance to salt stress is greater in O. sativa ssp. japonica. Fragment of SOS1 gene was found in all cultivars, except for BRS Atalanta. CK1 gene is present in all cultivars evaluated. It allows to conclude that enzyme systems were more expressed in cultivars O. sativa ssp. japonica, in the leaves and the 14 DAT, featuring bands more intense as the increase of salinity. The expression of enzymes involved in the mechanisms of tolerance to salt stress is greater in O. sativa ssp. japonica and the genes studied are present in both subspecies. / No Rio Grande do Sul, o principal sistema de irrigação da cultura do arroz é por inundação, podendo conduzir à salinização os solos com drenagem inadequada, especialmente as lavouras da região litorânea que utilizam a água da Laguna dos Patos, que está sujeita à salinização pela entrada do mar quando é baixo o nível da referida Laguna, tornando-se uma das maiores limitações ambientais na produção de arroz. Esta pesquisa teve como objetivos analisar a expressão enzimática de cultivares de Oryza sativa L. ssp. indica S. Kato e Oryza sativa L. ssp. japonica S. Kato, crescidas em diferentes níveis de salinidade e detectar genes envolvidos com a tolerância à salinidade, com base na hipótese de que as cultivares da Segunda subespécie apresentam maior tolerância à salinidade. No experimento foram utilizadas as cultivares BRS Bojuru, IAS 12-9 Formosa e Goyakuman pertencentes à O. sativa ssp. japonica e as cultivares de O. sativa ssp. indica BRS-7 Taim, BRS Querência e BRS Atalanta. As plântulas de arroz com 10 dias após a emergência (DAE) foram transferidas para casa de vegetação com temperatura e umidade controlada e crescidas em bacias de 15 L, contendo solução nutritiva de Hoagland meia força acrescida de 0, 25, 50, 75 e 100 mM de NaCl. As plântulas foram coletadas aos 14, 28, 42 e 56 dias após a transferência (DAT) e imediatamente armazenadas em ultrafreezer à -70 °C para posterior anáxiv -lises. Os tecidos vegetais foram macerados e colocados em tubos eppendorf com solução extratora de Scandálios. A eletroforese foi realizada em géis de poliacrilamida 7% colocados em cubas eletroforéticas verticais. As bandas foram reveladas para os sistemas enzimáticos superóxido dismutase, peroxidase, catalase, esterase, glutamato desidrogenase, álcool desidrogenase, fosfoglucose isomerase, malato desidrogenase, enzima málica, alfa amilase e glucose-6-fosfato desidrogenase. Por intermédio de pesquisa in silico, realizada junto ao National Center for Biotechnology Information foram identificados os genes AY785147 SOS e AF319481 - CK1, envolvidos na tolerância a salinidade. A detecção dos genes consistiu da extração de DNA genômico segundo o método CTAB 2%, seguido de reações de PCR realizadas em termociclador mediante a utilização dos primers desenhados também in silico. Os produtos da amplificação foram detectados por eletroforese em gel de agarose 1,5%. A visualização do DNA corado com brometo de etídio foi feita sobre iluminação ultravioleta e as imagens digitalizadas. A expressão das enzimas envolvidas nos mecanismos de tolerância ao estresse salino é maior em O. sativa ssp. japonica. Fragmento do gene SOS1 foi encontrado em todas cultivares, com exceção da BRS Atalanta e o gene CK1 está presente em todas as cultivares avaliadas. Conclui-se que os sistemas enzimáticos são mais expressos nas cultivares de O. sativa ssp. japonica, nas folhas e aos 14 DAT, apresentando bandas mais intensas conforme o aumento da salinidade. A expressão das enzimas envolvidas nos mecanismos de tolerância ao estresse salino é maior em O. sativa ssp. japonica e os genes estudados estão presentes nas duas subespécies.

Depuis la mise en évidence de l’existence des protéines kinases vers la fin des années 1950 cette famille d’enzymes s’est vu attribuer d’importants rôles dans divers mécanismes pathologiques notamment dans des processus de cancérisations. Plus récemment ces enzymes ont été identifiées comme potentiellement impliquées dans d’autres types de maladies telles que les maladies neurodégénératives.Deux projets de recherche seront présentés. Le premier projet expose la conception et la synthèse de nouveaux composés tricycliques de la famille des pyrido[3,4-g]quinazolines. Les propriétés inhibitrices de kinases des premiers dérivés ont été évaluées sur un panel de cinq kinases (CDK5, CK1, GSK3, CLK1 and DYRK1A) connues pour leurs implications dans la maladie d’Alzheimer. L’intérêt de ces nouveaux squelettes tricycliques comme inhibiteurs de kinases a été validé par des activités inhibitrices nanomolaire à l’encontre des kinases DYRK1A et CLK1. D’autre part l’obtention de structures co-crystallographiques d’interaction de deux dérivés avec le site ATP de la kinase CLK1 a permis de rationnaliser la substitution du motif pyrido[3,4-g]quinazoline. Le second projet présente le développement d’un nouveau dérivé de la staurosporine aglycone (K252c) dans lequel la partie lactame a été remplacée par un noyau pyrazole. Une étude préliminaire des propriétés biologiques de l’indolopyrazolocarbazole obtenu met en avant une cytotoxicité, du même ordre de grandeur que K252c, contre les lignées cellulaires K562 (leucémie humaine) et HCT116 (carcinome du colon). En revanche, le composé chef de file s’est révélé être un faible inhibiteur de cibles connues de K252c, les isoformes α and γ de la protéine kinase C et présente un bon potentiel inhibiteur des kinases Pim 1-3. Ce nouveau chemotype pourrait être un inhibiteur de kinases prometteur. / In 1950’s protein kinases were found to play a critical role in cell signaling, rising strong research potential for this enzyme family. Initially investigated for their implications in cancerogenesis they were more recently found to be involved in a wide variety of diseases including neurodegenerative pathologies. Herein will be presented two research projects that offer bright new perspectives for the inhibition of kinases involved whether in neurodegenerative diseases or cancers.First, the design and synthesis of new pyrido[3,4-g]quinazoline derivatives will be described as well as their protein kinase inhibitory potencies toward five CMGC family members (CDK5, CK1, GSK3, CLK1 and DYRK1A) that are known to play a potential role in Alzheimer’s disease. The interest for this original tricyclic heteroaromatic scaffold as modulators of CLK1/ DYRK1A activity was validated by nanomolar potencies. CLK1 co-crystal structures with two inhibitors revealed the binding mode of these compounds within the ATP-binding pocket and led to the synthesis of new diversely substituted pyrido[3,4-g]quinazolines.Then the synthesis of a new derivative of the staurosporine aglycon (K252c), in which the lactam ring was replaced by a pyrazole moiety, will be depicted. The resulting indolopyrazolocarbazole inhibited Pim isoforms 1–3 whereas it did not impair the activity of two known targets of K252c, protein kinase C isoforms α and γ . The lead compound exhibited same cytotoxic activity as K252c toward both human leukemia and colon carcinoma cell lines (K562 and HCT116), strongly suggesting that this new scaffold deserves further investigations for treatment of malignancies associated with kinases activities.