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1
Lioutas, Antonio 1980. "Aurora A kinase function during anaphase." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/97290.
Full textAbstract:
Aurora A (AurA) is an important mitotic kinase mainly studied for its
involvement in cell cycle progression, centrosome maturation,
mitotic spindle pole organization and bipolar spindle formation. It
localizes to duplicated centrosomes and spindle microtubules (MTs)
during mitosis where it regulates various factors participating in
metaphase spindle formation. AurA is degraded late in mitosis
suggesting that it might also have a function in anaphase. In this
study we focused in understanding AurA function during anaphase
in two different experimental systems.
First, we kept AurA active in cycled Xenopus egg extracts and found
that MTs maintained their mitotic organization longer throughout
mitotic exit. We also observed chromosome segregation defects and
problematic nuclear envelope formation. These observations
indicate that AurA activity needs to be down-regulated for the
transition from metaphase back to interphase.
To get insights into the role of AurA during metaphase-anaphase
transition we initially asked whether its kinase activity is still
necessary for the maintenance of the metaphase spindle. We saw
that the inhibition of AurA kinase activity in metaphase resulted to a
collapse of the established metaphase spindle in HeLa cells.
Indicating that AurA activity is necessary for the metaphase spindle
maintenance.
Then, we looked whether AurA kinase activity is still necessary
during anaphase. We inhibited AurA at the onset of anaphase in
Hela cells and found that anaphase spindles were smaller. We also
observed that the MT structure responsible for anaphase spindle
elongation, the central spindle, was defectively assembled and
organized. Moreover, in cells where AurA was inhibited segregation
of chromosomes was defective. These results indicate that AurA
kinase activity is necessary for anaphase spindle elongation, central
spindle assembly and organization and chromosome segregation.
To understand further how AurA regulates anaphase spindle
formation we looked known AurA substrates. We depleted TACC3,
a known AurA substrate involved in MT formation earlier in mitosis
and observed that TACC3 depletion phenocopied AurA inhibition.
This indicates that TACC3 has a function in MT organization and
chromosome segregation during anaphase and this function could
possibly be regulated by AurA.
In this study we have demonstrated that AurA activity is essential for
metaphase spindle maintenance. We also found that during
anaphase when AurA is either maintained active or inhibited MT
organization is greatly affected and chromosome segregation is
defective. Suggesting that AurA activity needs to be tightly controlled
during anaphase for a correct completion of mitosis.Aurora A (AurA) es una quinasa mitótica importante que se ha estudiado principalmente en su papel durante la progresión del ciclo celular, la maduración del centrosoma, la organización y la formación del polo y del huso mitótico. Durante la mitosis, AurA se localiza en los centrosomas duplicados y en los microtúbulos (MTs) del huso y se ha observado que regula varios factores que participan en la formación del huso mitótico. AurA se degrada al final de la mitosis indicando que pueda tener una función durante la anafase. En este estudio nos hemos centrado en la comprensión de la función de AurA durante la anafase en dos sistemas experimentales diferentes. En primer lugar, utilizando extractos de huevos de Xenopus hemos mantenido AurA activa durante la transición de metafase a anafase y hemos visto que los MTs del huso mitótico mantienen su organización durante más tiempo. También hemos observado que cuando AurA se mantiene activa existen defectos en la segregación cromosómica y la formación de la membrana nuclear. Esto indica que la actividad de AurA tiene un papel regulador sobre los MTs y la chromatina durante la transición de la metafase a la interfase. Para entender cual es la función de AurA durante la transición de metafase a anafase primero hemos estudiado si la actividad de la quinasa es necesaria para el mantenimiento del huso mitótico. Hemos visto que la inhibición de la actividad quinasa AurA resultó en el colapso del huso durante la metafase en células HeLa. Esto indica que la actividad de AurA es necesaria para el mantenimiento del huso mitótico de metafase. A continuación hemos analizamos si la actividad quinasa de AurA sigue siendo necesaria para la anafase. Para ello hemos inhibido AurA en células Hela al inicio de la anafase. En estas condiciones los husos de la anafase son más pequeños y la estructura de los MTs responsable del alargamiento del huso mitótico durante la anafase, el huso central, se organiza defectuosamente. Además, se encontraron errores durante la segregación de los cromosomas. Estos resultados indican que la actividad quinasa de AurA es necesaria para el alargamiento del huso durante la anafase y la organización y segregación cromosómica. Para entender el mecanismo de la función de AurA durante la anafase hemos estudiado a sustratos de AurA. Al estudiar TACC3 , un sustrato conocido de AurA que participa en la formación de MTs en las fase iniciales de la mitosis hemos encontrado que su eliminación de células HeLa produce el mismo fenotipo que la inhibición de AurA. Esto indica que TACC3 tiene una función en la organización de MT y la segregación de cromosomas durante la anafase y que esta función podría estar regulada por la quinasa AurA. En este estudio hemos demostrado que la actividad quinasa de AurA es esencial para el mantenimiento del huso mitótico. También hemos encontrado que durante la anafase cuando la quinasa AurA se mantiene activa o se inhibe la organización de los MTs del huso mitótico se ve muy afectada y los cromosomas se segregan defectuosamente. Por tanto los resultados de este estudio indican que la actividad quinasa de AurA está estrechamente controlada durante la anafase para el correcto cumplimiento de la mitosis.
2
Dittmann, Linda [Verfasser]. "Expressionsanalyse von Aurora-Kinase A, Aurora-Kinase B, Repp 86, Cyclin-Dependent-Kinase 1 und Cyclin-Dependent-Kinase 2 bei Mamma- und Ovarialkarzinomen / Linda Dittmann." Kiel : Universitätsbibliothek Kiel, 2014. http://d-nb.info/1062536061/34.
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3
Douglas, M. E. "Regulation of cytokinesis by aurora B kinase." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598609.
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In the first part of this thesis, I identify 14-3-3 protein as an interaction partner of an Aurora-phosphorylated region of MKLP1, the kinesin component of centralspindlin. I show that MKLP1 contains a highly conserved amino acid motif that encompasses a known Aurora B phosphorylation site, S708, and binds 14-3-3 when phosphorylated at a conserved serine, S710. Aurora-mediated phosphorylation of S708 inhibits binding of 14-3-3 to MKLP1. Given that S708-phosphorylated MKLP1 (which cannot bind 14-3-3) becomes highly enriched at the central spindle during anaphase, whereas S710-phosphorylated MKLP1 (which can bind 14-3-3) does not, these results suggest that Aurora B regulates centralspindlin by controlling the binding and release of 14-3-3 at different subcellular structures during cell division. Finally, I investigate whether regulated binding of MKLP1 to 14-3-3 is required for centralspindlin function and cytokinesis. I show that the sole function of Aurora-mediated S708 phosphorylation is the removal of 14-3-3 that is bound to MKLP1, and that this is required for stable binding of MKLP1 to the central spindle. 14-3-3 is necessary and sufficient to prevent the assembly of centralspindlin into higher order multimers that are known to be required for centralspindlin to bind and bundle microtubules. Collectively, these findings show that Aurora B regulates cytokinesis by releasing MKLP1 from 14-3-3 protein, enabling centralspindlin to assemble into higher order multimers that bind and bundle microtubules into the central spindle.
4
Khan, Jabbar. "Relationship between aurora kinase-C and cancer." Rennes 1, 2011. http://www.theses.fr/2011REN1S082.
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Aurora kinases belong to a conserved family of serine/threonine kinases key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved mainly in mitosis while Aurora-C is expressed during spermatogenesis and oogenesis and is involved in meiosis. Aurora-C is hardly detectable in normal somatic cells. However all three kinases are overexpressed in many cancer lines. Aurora-A possesses an oncogenic activity while Aurora-B does not. Here we investigated whether Aurora-C possesses such an oncogenic activity. We report that overexpression of Aurora-C induces abnormal cell division resulting in centrosome amplification and multinucleation in both transiently transfected cells and in stable cell lines. Only stable NIH3T3 cell clones overexpressing active Aurora-C formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3T3 stable cell lines overexpressing Aurora-C induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumour aggressiveness was positively correlated with the rate of kinase activity, making Aurora-C a potential anti-cancer therapeutic targetLes kinases Aurora appartiennent à une famille très conservée de sérine/thréonine kinases. Ces kinases sont des régulateurs clés de la progression dans le cycle cellulaire. Aurora-A et Aurora-B sont exprimées dans les cellules somatiques et sont impliquées principalement en mitose. Aurora-C, en revanche, est exprimée au cours de la spermatogenèse et de l’oogenèse et est impliquée en méiose. Aurora-C est à peine détectable dans les cellules somatiques normales. Ces trois kinases cependant sont surexprimées dans de nombreuses lignées issues de cellules cancéreuses. Aurora-A est un oncogène alors que Aurora-B ne l’est pas. Dans ce travail, nous cherchons à déterminer si Aurora-C présente aussi une activité oncogénique. Nous rapportons que la surexpression d’Aurora-C induit une division cellulaire anormale résultant en une amplification des centrosomes et une multinucléation à la fois dans les cellules transfectées transitoirement et dans les lignées stables surexprimant Aurora-C. Seuls les clones stables de cellules NIH3T3 surexprimant Aurora-C active sont capable de former des colonies en agar mou, indiquant qu’un gain d’activité d’Aurora-C est suffisant pour transformer des cellules. De plus, nous rapportons que les clones stables de cellules NIH3T3 surexprimant Aurora-C active sont capable d’induire une formation de tumeur quand ces cellules sont injectées dans des souris immuno-compromises, démontrant l’activité oncogénique d’Aurora-C enzymatiquement active. Finalement, nous trouvons que l’aggressivité des tumeurs est positivement corrélée au taux d’activité kinase d’Aurora-C. Nos travaux posent Aurora-C en tant que bonne cible potentielle thérapeutique pour le traitement de cancers
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Hoang, Thi My Nhung. "Survivine et Aurora B kinase, deux cibles potentielles des drogues anti-mitotiques ; identification d’une nouvelle classe d’inhibiteurs des aurora kinases." Grenoble 1, 2008. http://www.theses.fr/2008GRE10007.
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Le complexe passager joue un rôle clé en mitose: contrôlant à la fois la ségrégation des chromosomes, la tension du fuseau, l'entrée en anaphase et la cytodirèse. Le complexe est composé de quatre protéines: INCENP, la kinase B, Survivine et Boréaline. Sachant que la protéine Survivine est phosphorylée par Aurora B et qu'elle a un role p sein du complexe, nous avons étudié un mutant mimant sa phosphorylation: Survivine TlI7E. La phosphoryla Survivine est nécessaire à la transition Métaphase! Anaphase. Le mutant Survivine TIl 7E est faiblement centromères en métaphase et agit comme un dominant négatif de la cytodirèse, empêchant la séparation de cellules filles. Lors de la recherche d'inhibiteurs des Aurora kinases, nous avons identifié une nouvelle classe de molécu inhibent la phosphorylation de l'histone H3 et le point de contrôle du fuseau. Ces molécules prévienr prolifération des cellules tumorales. Ces composés sont des outils intéressants pour étudier la fonction du COI passager et représentent un nouveau motif moléculaire pour le développement de drogues anti-cancéreuses. Survivine et Aurora B kinase dont l'expression est restreinte à la mitose sont deux cibles pour de nouvelles thérapies anti-mitotiquesThe chromosomal passenger complex (CPe) plays a key role in mitosis : controlling both chromosome segre spindle tension, anaphase onset and cytokinesis. The complex is composed offour proteins : INCENP, Aurora B 1 Survivin and Borealin. Taking into account that Survivin is phosphorylated by Aurora Band has a pivotaI role complex, we have studied the phosphomimetic mutant SurvivinTl 17E. Survivin phosphorylation is requir anaphase onset and the phospho-mutant is poorly linked to centromere. Moreover it exhibits a dominant 11( function in cytokinesis, preventing abscission. Ln a search for Aurora kinase inhibitors we have identified a new class of Aurora B kinase inhibitors that pl Histone H3 phosphorylation, impairs mitotic spindle checkpoint. Moreover these molecules prevent tum! proliferation. These inhibitors are interesting too]s for understanding CPC function and represent a new lead development of anti-cancer drugs. Survivin and Aurora B kinase, which are expressed exclusively in mitosis, are thus two druggable targets for ne\ mitotic therapies
6
Diallo, Alghassimou. "Identification d’une nouvelle fonction de la protéine kinase Aurora-A." Thesis, Rennes 1, 2013. http://www.theses.fr/2013REN1S111/document.
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Les protéines kinases « Aurora » sont des régulatrices clés du cycle cellulaire. Alors que l'activité de Aurora-A est requise en début de la mitose, Aurora-B et -C sont nécessaires pour la fin de mitose. Toute perturbation de leur activité peut conduire au processus de tumorisation. Plus spécifiquement, Aurora-A se comporte à la fois comme oncogène et suppresseur de tumeur. Par conséquent, la connaissance du rôle de Aurora-A durant cycle cellulaire est essentielle. Toutefois, peu d'études ont exploré, jusque là, le rôle de Aurora-A dans les phases tardives de la mitose. En fait, l'inhibition de Aurora-A conduit à l'arrêt du cycle rendant impossible de voir ce qui se passe au delà de la transition métaphase/anaphase. Néanmoins, en combinant le couple génétique chimique et inhibiteur spécifique, j'ai pu identifier une nouvelle fonction de la kinase Aurora-A liée au checkpoint (SAC). En effet, mes résultats montrent que l'inhibition de l'activité de Aurora-A induit un défaut de congression et une chute de l'index mitotique. Ce résultat paradoxal suggère un défaut du SAC. J'ai donc pu montré que cette inhibition outrepassait le SAC en perturbant la localisation aux kinétochores de Mad2 et BubR1. Cependant, ma tentative pour sauver le phénotype du SAC par le mutant S19D-P150Glued n'a pas réussi malgré que le mutant S19AP150Glued se soit comporté comme un vrai dominant négatif. Enfin, j'ai pu montré que l'activité de Aurora-A est requise pour maintenir le SAC actif durant la prométaphaseProtein kinases "Aurora " are the key regulators of the cell cycle. While the activity of Aurora-A is required at the beginning of mitosis, Aurora-B and -C are required for the end of mitosis. Any disruption of their activity can lead to process tumorisation. Specifically, Aurora-A acts as both oncogene and tumor suppressor. Therefore, knowledge of the role of Aurora-A is essential for cell cycle. However, few studies have explored so far, the role of Aurora-A in the late stages of mitosis. In fact, inhibition of Aurora-A leads to cell cycle arrest making it impossible to see what happens beyond the transition metaphase / anaphase. However, by combining chemical genetics couple and specific inhibitor, I have identified a new function of Aurora-A kinase -related checkpoint (SAC). Indeed, my results show that inhibition of the activity of Aurora-A induces a congression defect and the mitotic index decrease. This paradoxical result suggests a defect in the SAC. So I have shown that this inhibition was beyond the SAC disrupting kinetochore localization of Mad2 and BubR1. However, my attempt to rescue the phenotype of the SAC by the S19D-P150Glued mutant failed despite the fact that S19A-P150Glued mutant behaved like a true negative dominant. Finally, I have shown that the activity of Aurora-A is required to maintain the active SAC during prometaphase
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Kotwaliwale, Chitra V. "Regulation and functions of the Ipl1/aurora protein kinase /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/5081.
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Noujaim, Michael. "The role of microtubules in Aurora-B's kinase activity." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107846.
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Cell division is the process by which a single cell divides into two daughter cells. Each of these two daughter cells must inherit one complete copy of the genome. If not, cancer or cell-death occurs. Not surprisingly, then, life has evolved mechanisms for ensuring that cell division is error-free. The mechanisms, broadly termed "checkpoints," prevent cell division from occurring if mistakes arise or if each daughter cell were not to inherit one copy of the genome. A major molecule involved in regulating the cell division checkpoint is called Aurora-B. Aurora-B can be thought of as a police officer--Aurora-B stops the cell division process whenever mistakes arise and corrects them before allowing cell division to continue. Unfortunately, if Aurora-B itself becomes faulty or error-prone, the police officer is off-duty, and a greater number of errors occur during cell division. For this reason, Aurora-B is commonly found to be aberrant in tumor cells. Consequently, Aurora-B has recently emerged as a main target for a vast range of novel anti-cancer drugs. In order to divide the genome properly, the cell builds a special mechanical scaffold, reminiscent of the scaffolds at construction sites, called the mitotic spindle. The building blocks of this scaffold are called microtubules. Microtubules are also a target of anti-cancer therapies. Aurora-B interacts with these microtubules in order to perform its role properly. Although significant, this interaction remains elusive for the most part. Here we provide experimental evidence for the importance of these interactions at the spindle midzone during anaphase. Using in vitro kinase assays we show that microtubules sequester Aurora-B's activity to ensure the efficiency with which crucial microtubule-associated substrates are phosphorylated at the spindle midzone. Thus, allowing the unhindered progression of anaphase and the completion of cytokinesis.La division cellulaire est le processus par lequel une cellule se divise en deux cellules filles. Chacune de ces cellules filles doivent hériter d'une copie complète du génome. Sinon, cela mène à la mort cellulaire ou au cancer. Ainsi, il n'est pas surprenant que la vie a fait évoluer des mécanismes pour s'assurer que la division cellulaire est dénuée d'erreurs. Ces mécanismes, appelés « points de contrôle », empêchent la division cellulaire de se produire si des erreurs apparaissent ou si chaque cellule fille n'a pas hérité d'une copie complète du génome. Une molécule exerçant un rôle majeur dans la régulation du point de contrôle du cycle cellulaire est appelée Aurora-B. Son rôle est en quelque sorte analogue à celui d'un officier de police – Aurora-B arrête la division cellulaire dès que des erreurs surviennent et les corrige avant de permettre à la division cellulaire de continuer. Malheureusement, si Aurora-B devient défectueux ou sujet à des erreurs, l'officier de police est hors-service, alors un plus grand nombre d'erreurs peuvent survenir pendant la division cellulaire. Pour cette raison, Aurora-B est fréquemment identifié comme étant aberrant dans les cellules tumorales. Conséquemment, Aurora-B a récemment émergé en tant que cible principale pour une vaste gamme de nouveaux médicaments anti-cancer. Afin de diviser le génome correctement, la cellule construit un échafaudage mécanique spécial, qui rappelle les échafaudages retrouvés dans les chantiers de construction, qu'on appelle le fuseau mitotique. Les morceaux constituant cet échafaudage sont appelés microtubules. Les microtubules sont aussi une cible de certaines thérapies anti-cancer. Aurora-B interagit avec ces microtubules afin d'exécuter son rôle correctement. Malgré le caractère significatif de cette interaction, elle demeure nébuleuse en bonne partie. Nous fournissons donc ici des preuves expérimentales de l'importance de ces interactions dans la zone médiane du fuseau pendant l'anaphase. En utilisant des essais kinase in vitro, nous montrons que les microtubules séquestrent l'activité d'Aurora-B afin d'assurer l'efficacité avec laquelle les substrats associés aux microtubules sont phosphorylés dans la zone médiane du fuseau. Ainsi, cela permet une progression sans entrave de l'anaphase et la fin de la cytocinèse.
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Janecek, Matej. "Inhibiting the interactions between Aurora kinase A and TPX2." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708828.
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He, Lili. "Molecular Mechanism of Aurora-A Kinase in Human Oncogenesis." [Tampa, Fla] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002582.
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Gavard, Olivia. "Modélisation et analyse d’un interactome de la kinase humaine Aurora A." Thesis, Rennes 1, 2015. http://www.theses.fr/2015REN1S160/document.
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La kinase Aurora A est une protéine essentielle au cycle cellulaire et plus particulièrement lors de la mitose. En effet, Aurora A est nécessaire à l'entrée en mitose et joue un rôle dans la maturation des centrosomes. Elle participe à l'assemblage du fuseau mitotique et est nécessaire à la réussite de la cytodiérèse. Elle est également nécessaire à l'égale répartition des mitochondries dans les cellules filles et joue un rôle dans l'épissage alternatif des ARNm de facteurs apoptotiques. Au-delà de ses fonctions mitotiques, plusieurs études récentes indiquent qu'Aurora A présente des fonctions supplémentaires dans les cellules en interphase. Elle est notamment essentielle au désassemblage du cil primaire et joue un rôle dans la dynamique des microtubules et la migration cellulaire. Enfin, une dérégulation de son expression, de sa stabilité et/ou de son activité perturbe le déroulement du cycle cellulaire ce qui conduit à la transformation des cellules et favorise l'apparition de cancers. Ses fonctions normales ainsi que ses fonctions lors de la carcinogenèse sont conduites à travers les nombreux partenaires protéiques qui entrent en interaction avec elle. Ils modulent son activité, sa localisation et sa stabilité. En retour Aurora A phosphoryle un bon nombre d'entre eux régulant ainsi leur activité, localisation et stabilité. Cependant, l'analyse des interactions déjà connues d'Aurora A ne permet pas d'expliquer tous les phénotypes observés lors de sa dérégulation. Afin de mieux comprendre les fonctions d'Aurora A, les mécanismes qui la régulent et mettre en évidence ses multiples rôles au sein de la cellule, j'ai construit puis analysé un interactome d'Aurora A généré à partir d'une méthode de purification d'affinité couplée à la spectrométrie de masse en tandem. J'ai identifié 477 partenaires potentiels dont 180 présentant une forte probabilité d'être des partenaires directs de la kinase. L'analyse bioinformatique approfondie de cet interactome a permis de révéler les partenaires associés à des mécanismes liés à la mitochondrie et l'épissage des ARN messagers mettant en évidence une implication potentielle d'Aurora A dans ces mécanismes. Pour valider cet interactome, j'ai choisi d'étudier plus précisément deux partenaires identifiés dans cette étude : les protéines WDR62 et CEP97. J'ai montré que ces deux partenaires co-localisent avec Aurora A et sont phosphorylés par la kinase. Ainsi, ce travail de thèse a permis de mettre en évidence un nombre important de nouveaux partenaires d'Aurora A associés à de nouvelles fonctions. L'étude de ces nouvelles fonctions liées aux mitochondries et à l'épissage des ARN, constitue deux nouveaux projets actuellement menés par des collaborateurs au sein de notre institutThe kinase Aurora A is an essential mitotic cell cycle protein. Aurora A is necessary for mitotic entry and for the maturation and separation of centrosomes. It participates in mitotic spindle assembly and chromosome biorientation, and it is essential for the completion of cytokinesis. Furthermore, Aurora A activity is necessary for the equal distribution of mitochondria to daughter cells and, through its role in the alternative splicing of mRNA of apoptotic factors, it provides a link between cell cycle control and apoptosis. Beyond its mitotic functions, several recent studies suggest that Aurora A is also important during interphase. Notably, it influences microtubule dynamics, promotes cell migration and polarity control and is essential for primary cilia disassembly. Reflecting the fact that Aurora A is found to be up-regulated in many cancers, deregulation of Aurora A activity can result in an aberrant cell cycle, ultimately leading to malignant transformation of cells. The crucial regulation of Aurora A’s numerous functions is achieved through its interaction with several protein partners, which modulate its activity, localisation and stability. Aurora A in turn phosporylates a number of them, thus regulating their activity, localisation and stability. However, the known interactions of Aurora A cannot explain all the phenotypes that have been described of its deregulation.To better understand the functions of Aurora A, the regulation mechanisms governing it, and to expose its multiple roles in the cell, I have built and analysed an Aurora A interactome using tandem affinity purification coupled with mass spectrometry. This resulted in the identification of 477 potential interacting partners, of which, 180 were determined to have a high probability of interacting directly with the kinase.In-depth bioinformatic analysis of this interactome has revealed the associated partners to be related to mitochondria and mRNA splicing, highlighting the potential involvement of Aurora A in these mechanisms. To validate the interactome, two of the proteins identified in this study, WDR62 and CEP97, were examined in detail. Here I show that these two proteins colocalise with Aurora A, and are phosphorylated by the kinase.WDR62 is implicated in microcephaly and is deregulated in certain cancers. I have shown that Aurora A phosphorylates WDR62 during mitosis, and that this phosphorylation is necessary for its localisation to the centrosomes. CEP97 is a poorly charactarised protein of the primary cilium, abnormalities of which are associated with ciliopathies. I have shown that Aurora A phosphorylates CEP97 in vitro, and that the inhibition of Aurora A activity in vivo perturbs the localisation of CEP97 to cilia and centrosomes.This study has identified a number of new Aurora A-interacting proteins, implicating the kinase with novel functions. These functions, related to mitochondria and mRNA splicing have opened up a new area for further investigation
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Kufer, Thomas. "Regulation der humanen Aurora-A Kinase und Identifikation potentieller Interaktionspartner." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-19608.
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Scrittori, Laetitia. "Etude fonctionnelle des kinase Aurora au cours de la mitose." Université Joseph Fourier (Grenoble), 2004. http://www.theses.fr/2004GRE10044.
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La mitose est un processus qui assure la répartition équilibrée de l'information génétique au cours de la division cellulaire. Les différentes étapes de ce processus sont finalement régulées et toute anomalie est susceptible d'entraîner un phénotype d'aneuploi͏̈de et de mener au cancer. Les sérine/thréonine kinases Aurora, présentes dans diverses organismes, sont une nouvelle famille de kinases oncogéniques impliquées dans de nombreux processus mitotiques. Leur mode de régulation et leur fonction sont encore mal caractérisés. Chez l'humain, il existe trois homologues : Aurora-A, Aurora-B, Aurora-C. Aurora-A participe à la fonction centromèrique. Aurora-B est impliquée dans l'assemblage du fuseau mitotique, la régulation du point de contrôle du fuseau mitotique et la cytodiérèse. Cette kinase est engagée au sein d'un complexe de protéines passagères avec les protéines INCENP et Survivine, ce qui lui permet d'adopter un profil de localisation itinérant au cours de la mitose, où elle phosphoryle séquentiellement différents substrats. Afin de déterminer le domaine d'Aurora-B impliqué dans sa localisation de protéine passagère, j'ai réalisé l'étude fonctionnelle de la kinase Aurora-B humaine au cours de la mitose par une approche récente et originale : la "pseudo génétique" en cellules humaines. Il apparaît que le domaine catalytique d'Aurora B, très conservé chez les Aurora, est responsable du profil de localisation spécifique de cette protéine passagère en mitoseMitosis is the process by which the equilibrated repartition of the genetic information is ensured during the cellular division. The different steps of this process are tightly regulated and each anomaly can lead to aneuploidy and cancer. The serine/thhreonine Aurora kinases, which are present in many organisms, are a new family of oncogenic kinases implicated in many mitotic processes. Their regulation modes and functions are poorly characterised. There are three homologues in human : Aurora-A, Aurora-B, Aurora-C. Aurora-A participates in the centrometric function. Aurora-B is implicated in the assembly of the mitotic spindle, the regulation of the mitotic spindle checkpoint and cytokinesis. This kinase is engaged in a complex of passenger proteins with the INCENP and surviving proteins, in a way that permit it to adopt a localisation of itinerant protein during mitosis, where it sequentially phosphorylates many substrates. In order to determine the Aurora-B domain involved in its passenger protein localisation, we realised the functional study of the Aurora-B kinase during mitosis by a new approach : the "pseudo-genetic" in human cells. It appears that the catalytic domain of Aurora-B, highly conserved among the Aurora kinases, is responsive localisation profile of this passenger protein during mitosis
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Rojanala, Sangeeta. "AURORA-A, A POTENTIAL TARGET IN PANCREATIC CANCER AND ITS STRUCTURAL ROLE IN LOCALIZATION TO THE CENTROSOMES." Diss., The University of Arizona, 2005. http://hdl.handle.net/10150/194492.
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Aurora-A kinase is overexpressed in many human cancers and leads to centrosome amplification resulting in multipolar spindles, chromosome segregation defects and aneuploidy. Aurora-A belongs to a family of serine/threonine mitotic kinases involved in centrosome separation, duplication and maturation as well as in bipolar spindle assembly. In this work, we demonstrate that Aurora-A is both amplified and overexpressed in human pancreatic cancer cell lines, with a 2-5 fold increase in gene copy number and a 3-4 fold increase in protein levels compared to controls. Aurora-A is also amplified and overexpressed in pancreatic cancers taken directly from patients. An immunohistochemistry of tissues taken directly from patients demonstrated an overexpression of Aurora-A in 26 of 28 pancreatic cancers compared to 0 of 18 normal pancreas samples. Antisense nucleotides specifically targeted at Aurora-A arrest the cell cycle in pancreatic cancer cells, indicating the potential of Aurora-A as a therapeutic target in pancreatic cancer. To understand the role of Aurora-A at the centrosome, we investigated the mechanism of how Aurora-A is targeted to the centrosome. We used deletion fragment analysis of Aurora-A to identify a specific region that is required to localize to the centrosome. We also show that subcellular localization of Aurora-A is independent of its intrinisic kinase activity and its phosphorylation states. These results show that Aurora-A is targeted to the centrosome by a mechanism that does not require its kinase activity and phosphorylation of T288 and T287. Furthermore, the region containing the catalytic domain, 131-333, is sufficient to localize Aurora-A to the centrosome.
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Portier, Nathan Charles. "A role for aurora A kinase in timely nuclear envelope breakdown." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3270970.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed April 9, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Gavard, Olivia. "Modélisation et analyse de l'interactome de la kinase humaine Aurora A." Doctoral thesis, Université Laval, 2016. http://hdl.handle.net/20.500.11794/26685.
Full textAbstract:
La kinase Aurora A est une protéine essentielle au cycle cellulaire et plus particulièrement lors de la mitose. En effet, Aurora A est nécessaire dès l’entrée en mitose et régule sa progression. Elle joue un rôle dans la maturation et la séparation des centrosomes. Elle participe à l’assemblage du fuseau mitotique et du fuseau central pour le rassemblement et l’orientation des chromosomes. Enfin elle est nécessaire à la réussite de la cytodiérèse. Elle est également nécessaire à l’égale répartition des mitochondries dans les cellules filles et joue un rôle dans l’épissage alternatif des ARNm de facteurs apoptotiques. Au delà de ses fonctions mitotiques, plusieurs études récentes indiquent qu’Aurora A présente des fonctions supplémentaires dans les cellules en interphase. Elle est notamment essentielle au désassemblage du cil primaire et joue un rôle dans la dynamique des microtubules et la migration cellulaire. Enfin, une dérégulation de son expression, de sa stabilité et/ou de son activité perturbe le déroulement du cycle cellulaire ce qui conduit à la transformation des cellules et favorise l’apparition de cancers. Ses fonctions normales ainsi que ses fonctions lors de la carcinogenèse sont conduites à travers les nombreux partenaires protéiques qui entrent en interaction avec elle. Ils modulent son activité, sa localisation et sa stabilité. En retour Aurora A phosphoryle un bon nombre d’entre eux régulant ainsi leur activité, localisation et stabilité. Cependant, l’analyse des interactions déjà connues d’Aurora A ne permet pas d’expliquer tous les phénotypes observés lors de sa dérégulation. Afin de mieux comprendre les fonctions d’Aurora A, les mécanismes qui la régulent et mettre en évidence ses multiples rôles au sein de la cellule, j’ai construit puis analysé un interactome d’Aurora A généré à partir d’une méthode de purification d’affinité couplée à la spectrométrie de masse en tandem. J’ai identifié 477 partenaires potentiels dont 180 présentant une forte probabilité d’être des partenaires directs de la kinase. L’analyse bioinformatique approfondie de cet interactome a permis de révéler les partenaires associés à des mécanismes liés à la mitochondrie et l’épissage des ARN messagers mettant en évidence une implication potentielle d’Aurora A dans ces mécanismes. Pour valider cet interactome, j’ai choisi d’étudier plus précisément deux partenaires identifiés dans cette étude : les protéines WDR62 et CEP97. J’ai montré que ces deux partenaires co-localisent avec Aurora A et sont phosphorylés par la kinase. WDR62 est impliquée dans la microcéphalie et est dérégulée dans certains cancers. J’ai montré qu’Aurora A phosphoryle WDR62 en mitose et que cette phosphorylation est nécessaire à sa localisation aux centrosomes. CEP97 est une protéine du cil primaire encore peu caractérisée et des anomalies du cil primaire sont associées aux ciliopathies. Or l’activité d’Aurora A est nécessaire au désassemblage du cil primaire. J’ai montré qu’Aurora A phosphoryle in vitro CEP97 et que l’inhibition de l’activité d’Aurora A dans les cellules perturbe la localisation de CEP97 au niveau des cils et des centrosomes. Ainsi, ce travail de thèse a permis de mettre en évidence un nombre important de nouveaux partenaires d’Aurora A associés à de nouvelles fonctions. L’étude de ces nouvelles fonctions liées aux mitochondries et à l’épissage des ARN, constitue deux nouveaux projets actuellement menés par des collaborateurs au sein de notre institut.The serine-threonine kinase Aurora A is an essential mitotic cell cycle protein. Aurora A is necessary for mitotic entry and for the maturation and separation of centrosomes. It participates in mitotic spindle assembly and chromosome biorientation, and it is essential for the completion of cytokinesis. Furthermore, Aurora A activity is necessary for the equal distribution of mitochondria to daughter cells and, through its role in the alternative splicing of mRNA of apoptotic factors, it provides a link between cell cycle control and apoptosis. Beyond its mitotic functions, several recent studies suggest that Aurora A is also important during interphase. Notably, it influences microtubule dynamics, promotes cell migration and polarity control and is essential for primary cilia disassembly. Reflecting the fact that Aurora A is found to be up-regulated in many cancers, deregulation of Aurora A activity can result in an aberrant cell cycle, ultimately leading to malignant transformation of cells. The crucial regulation of Aurora A’s numerous functions is achieved through its interaction with several protein partners, which modulate its activity, localisation and stability. Aurora A in turn phosporylates a number of them, thus regulating their activity, localisation and stability. However, the known interactions of Aurora A cannot explain all the phenotypes that have been described of its deregulation. To better understand the functions of Aurora A, the regulation mechanisms governing it, and to expose its multiple roles in the cell, I have built and analysed an Aurora A interactome using tandem affinity purification coupled with mass spectrometry. This resulted in the identification of 477 potential interacting partners, of which, 180 were determined to have a high probability of interacting directly with the kinase. In-depth bioinformatic analysis of this interactome has revealed the associated partners to be related to mitochondria and mRNA splicing, highlighting the potential involvement of Aurora A in these mechanisms. To validate the interactome, two of the proteins identified in this study, WDR62 and CEP97, were examined in detail. Here I show that these two proteins colocalise with Aurora A, and are phosphorylated by the kinase. WDR62 is implicated in microcephaly and is deregulated in certain cancers. I have shown that Aurora A phosphorylates WDR62 during mitosis, and that this phosphorylation is necessary for its localisation to the centrosomes. CEP97 is a poorly charactarised protein of the primary cilium, abnormalities of which are associated with ciliopathies. I have shown that Aurora A phosphorylates CEP97 in vitro, and that the inhibition of Aurora A activity in vivo perturbs the localisation of CEP97 to cilia and centrosomes. This study has identified a number of new Aurora A-interacting proteins, implicating the kinase with novel functions. These functions, related to mitochondria and mRNA splicing have opened up a new area for further investigation.
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Ridgway, Ellen. "Investigation into the role of Aurora A kinase activity during mitosis." Thesis, University of Manchester, 2010. https://www.research.manchester.ac.uk/portal/en/theses/investigation-into-the-role-of-aurora-a-kinase-activity-during-mitosis(58d806c8-8393-4bab-b91d-dcf6f58c0012).html.
Full textAbstract:
Aurora A is an important mitotic regulator that has been found to be up-regulated in a variety oftumours provoking a great deal of attention and the development of a number of small moleculeAurora kinase inhibitors. Most of these inhibitors though have predominantly targeted Aurora B,meaning that our understanding of the role of the kinase activity of Aurora A is comparatively lesswell developed.MLN8054 however, is a small molecule inhibitor that has been reported in vitro to have a highdegree of specificity towards Aurora A activity. In this thesis, I show in vivo that MLN8054 can beused to specifically inhibit Aurora A activity, and exploit this quality to probe the role of Aurora Aactivity in human cells. I was consequently able to show that Aurora A activity not only has a clearrole in spindle formation, where it is required for the determination of K-fibre length and in thedegree of centrosome separation, but also in the regulation of microtubule organisation. Despite thespindle deformities seen after inhibiting Aurora A activity, the majority of HeLa and DLD-1 cellswere still able to form bipolar spindles capable of attaching to kinetochores. These spindlestructures did not however, assert normal levels of force through the kinetochores, and cells wereconsequently unable to efficiently align their chromosomes, causing significant delays to mitoticprogression. Cells were still able to divide in the absence of Aurora A activity, although thedetection of segregation defects and aneuploid progeny indicates a role for Aurora A activity in thefaithful segregation of the genetic material. Importantly however, Aurora A activity was not foundto have a prominent role in the spindle assembly checkpoint.Increasing the potency of Aurora A inhibition by using a drug-resistant cell line confirmed theobservations made in HeLa and DLD-1 cells, emphasising that although Aurora A activity isrequired for spindle assembly, cells can still activate the spindle checkpoint and divide in itsabsence. I therefore propose that Aurora A activity is required for the formation of normal spindlestructures capable of efficiently aligning and evenly dividing chromosomes during cell division.These roles were attributed in part to the kinase activity of Aurora A in the regulation of TACC3and chTOG localisation on the spindle and centrosomes.Interestingly however, Aurora A activity did not appear to be required for spindle assembly in nontransformedcells, which were able to more efficiently align their chromosomes and dividefollowing Aurora A inhibition than the cancer cell lines. Furthermore, the non-transformed cellsaccumulated with 2N DNA after longer-term Aurora A inhibition, as opposed to the cancer celllines, which exhibited profound aneuploidy following the equivalent treatment. This finding isencouraging, as consistent with recently published reports, it indicates that Aurora A inhibitionmay be successfully used in order to specifically target cancer cells.
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Mohan, Pooja. "Modification of RHAMM and TPX2 optimizes Aurora kinase A (AURKA) inhibition in malignant peripheral nerve sheath tumours." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44345.
Full textAbstract:
Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatments as they often resist chemotherapies and have high rates of disease recurrence. Published analysis of copy number variation identified hemizygous loss of Hyaluronan Mediated Motility Receptor (HMMR, encodes RHAMM) in half of the examined high-grade MPNST, but not in benign neurofibromas or low grade tumours. RHAMM is a molecular brake for the mitotic kinase Aurora A (AURKA), so this loss of HMMR in high-grade MPNST may cause tumours to rely on AURKA activity and sensitizes them to aurora kinase inhibitors (AKI).
Three MPNST cell-lines were profiled for the expression and activity of AURKA, as well as their responses to three AKI. The sensitivity of cell-lines with amplification of AURKA was reliant upon kinase activity, which correlated with the expression of the regulatory gene products TPX2 and RHAMM. Silencing of RHAMM, but not TPX2, increased AURKA activity and sensitized MPNST cells to AKI. All three AKIs reduced kinase activity in a dose-dependent manner, and AKI treatment induced cellular responses such as apoptosis, endoreduplication and cellular senescence. Additionally, two primary human MPNSTs grown in vivo as xenotransplants were treated with the AURKA-specific inhibitor MLN8237. Treatment resulted in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulated in stabilized disease.
The MPNST cell-line S462 has a population of tumorigenic stem-like cells that can be grown in sphere culture. AURKA activity was critical to the propagation and self-renewal of sphere-enriched MPNST stem-like cells. AKI treatment significantly reduced the formation of spheroids, attenuated the self-renewal of spheroid forming cells, and promoted their differentiation. Silencing of TPX2 decreased AURKA activity, while silencing of RHAMM was sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST, and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of RHAMM and TPX2.
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Chowath, Rashmi. "Role of Aurora kinase in Medulloblastoma development with correlation to MYCN activity." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-255237.
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Brain tumors are abnormal tissue masses found, either malignant or benign in nature. Medulloblastoma is a brain tumor subtype found to arise in the hind region of the brain, which is highly malignant and has poor long term prospects in general. On the basis of the driving force behind the tumor, medulloblastoma is further subgrouped into 4 categories: WNT; SHH; Group 3 and Group 4 tumors. Group 3 tumors show a high expression of N-Myc protein which is seen in certain types of cancerous cells. The cell cycle is regulated at several checkpoints by cyclin/cdk inhibitors. The primary cilium is an organelle found on the cellular surface, which has functions in cell growth, differentiation and neurogenesis. Aurora kinase is a protein kinase involved in the regulation and maintainence of the cilium. Often the cilium gets deleted from the cellular surface in tumors coupled with an increase in the kinase level inside the cells. Hence aurora kinase is found to be a viable target for therapy. Aurora kinase is also involved in stabilizing the MYCN gene by protecting it from degradation. In this project, the primary cilum was studied in neural stem cells and followed by study of its presence on tumor cells in culture. The gene involved in cilium development i.e. Kif3a was mutated and its aggressive nature was compared with that of the tumor cells. Aurora kinase was commonly found to be over-expressed in both the tumors and the mutants whereas N-Myc over-expression was seen only in tumors. Experiments suggest that cilia repression in Kif3a mutants takes place via an aurora kinase dependent pathway.
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Grundy, Martin. "Activity of the aurora kinase B inhibitor AZD1152 in acute myeloid leukaemia." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12758/.
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Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies including those of leukaemic origin. Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells whose prognosis is particularly poor and where standard induction therapy has changed little over the past thirty years. This thesis evaluated the effects of AZD1152-hQPA (barasertib-hQPA), a highly selective inhibitor of aurora-B kinase, in AML cell lines and primary samples. Inhibition of phospho-Histone H3 (pHH3) on serine 10 can be used as a biomarker for AZD1152-hQPA activity and an assay was optimized to measure pHH3 in our cell lines and primary samples. AZD1152-hQPA inhibited pHH3 in our cell lines resulting in polyploid cells, apoptosis, and cell death, irrespective of cellular p53 status. Over-expression of the ATP-binding cassette (ABC) drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in AML. A cell line which over-expresses Pgp was developed by selecting for daunorubicin (DNR) resistance in OCI-AML3 cells. Pgp and also BCRP expressing AML cell lines were found to be resistant to AZD1152-hQPA and it was found that AZD1152-hQPA is effluxed by these transporters. pHH3 inhibition by low dose AZD1152-hQPA was seen in all of the primary samples tested with Pgp and BCRP positive samples being less sensitive. However, 50% inhibition of pHH3 by AZD1152-hQPA was achieved in 94.6% of these samples. The FLT3-ITD-expressing MOLM-13 and MV4-11 cell lines were particularly sensitive to AZD1152-hQPA. Internal tandem duplications (ITDs) within the FLT3 tyrosine kinase receptor are found in approximately 25% of AML patients and are associated with a poor prognosis. It was demonstrated that AZD1152-hQPA directly targets phosphorylated FLT3 in the FLT3-ITD cell lines along with inhibiting its downstream target pSTAT5. FLT3-ITD primary samples were particularly sensitive to clonogenic inhibition and pSTAT5 down-regulation after treatment with AZD1152-hQPA compared with FLT3 wild-type (WT) samples.
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Wilson, Alastair. "Disrupting the inhibitory Kappa B kinase (IKK)-Aurora A axis in cancer." Thesis, University of Strathclyde, 2013. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=22402.
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The IKK complex is an essential regulator of the NF-κB- signalling pathway. It is comprised of three sub-units; catalytic subunits IKKα and IKKβ and the catalytically inactive scaffolding protein IKKγ/NF-κB Essential Modulator (NEMO). The complex can be assembled in multiple conformations as either hetero- or homo-dimers of IKKα/β with or without the scaffolding protein IKKγ. This regulates NF-κB signalling and related cellular inflammatory responses that are often constitutively active in many cancer cells. Beyond regulation of NF-κB signalling a number of other potential IKK substrates have been proposed including the mitotic kinase Aurora A, one of three serine/threonine kinases of the Aurora kinase family that mediate mitotic progression in cells. Aurora A kinase specifically has several roles in the cell cycle, regulating spindle formation/attachment, centrosome maturation and cytokinesis, and has been observed to be overexpressed in several cancers. Several studies have linked IKK complex to Aurora A signalling, as either a substrate of IKKα (Prajapati et al, 2006) or as a target for IKKβ-mediated βTRCP-regulated degradation (Irelan et al, 2007) both linked to cell cycle progression independent of NF-κB signalling. Aurora A kinase has however also been linked to NF-κB signalling through the phosphorylation and degradation of IκBα in primary breast tumours in a subgroup of patients exhibiting Aurora A gene amplification (Briassouli et al, 2007). Prostate cancer is a significant worldwide health issue and is the 2nd most prevalent cancer in males and 5th most prevalent cancer overall with over 900,000 new cases diagnosed in 2008 (Ferlay et al, 2010). The current therapies treating prostate cancer are far from ideal and have significant side effects. Both Aurora A and NF-κB-related kinases have been shown to be over-expressed or constitutively active in prostate cancer and therefore the suggested novel interaction may serve as a potential mechanism for intervention in cancer. This study aimed to validate IKK-Aurora A interactions and potentially identify novel targets for the development of therapeutics in prostate cancer. Using recombinant protein methodologies the direct interaction of IKKα and IKKβ with Aurora A were confirmed. Utilising peptide array technology, it has been possible to further elucidate the nature of the interaction between Aurora A and IKKα/β. The interaction of Aurora A and IKKα/β was mapped to two key regions of the IKKs, the kinase domain and the NEMO binding domain. The NEMO-Binding Domain is a conserved hexapeptide sequence, L-D-W-S-W-L, across both IKKα and IKKβ mediating the interaction with the scaffolding protein IKKγ/NEMO. This highlighted a novel role of the NBD as a multi-protein docking site. Binding of these proteins was further confirmed endogenously in a cellular setting, by means of co-immunoprecipitation and also the kinetic profiles of these interactions were characterised using recombinant proteins and Surface Plasmon Resonance. Thereafter Cell Permeable Peptides (CPPs) based on the IKK NBD were utilised as pharmacological tools, to examine potential competitive perturbation of Aurora A-IKK interactions mediated by the NBD in prostate cancer cells. This identified in a cellular setting treatment with NBD CPPs resulted in the inhibition of Aurora A phosphorylation and induced Aurora A degradation, which correlated with cell cycle arrest. It was also suggested through molecular modeling that the IKK NBD peptides could bind to the Aurora A protein, potentially at sites engaged by TPX2, an activation accessory protein for Aurora A. This purported mechanism may account for the observed IKK NBD peptide-mediated de-phosphorylation and subsequent degradation of Aurora A. Therefore, this study has identified the NEMO binding d omain as a potential multi-protein binding site and has identified novel functionality of the IKK NBD cell permeable peptides. This may represent a novel target for the intervention of IKK-mediated regulation of the cell cycle and serve as the basis for the development of novel peptidomimetics and related small molecules for the treatment of Prostate cancer.
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Frangini, Alberto. "Characterization of the role of the Aurora B kinase in quiescent lymphocytes." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11597.
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The role of the Aurora B kinase in mediating cellular functions outside mitosis was investigated. A SILAC-based approach was used to show that Aurora B interacts with proteins implicated in gene regulation and chromatin organization in G1 activated B cells. Among these interactors, I identified the PRC1 component Ring1B as a new partner of Aurora B. Both proteins are bound to the promoters of highly expressed genes in resting B and T lymphocytes. Quiescent resting B cells require the maintenance of a transcriptional programme that keeps the cells viable and ready to proliferate upon encounter with a specific antigen. Aurora B and Ring1B physically co-occupy the same promoters in resting B cells but, upon activation, Aurora B is replaced by MSK1 on its target genes, whereas Ring1B binding is preserved. Analysis of the active promoters in resting B cells shows that other PRC1 components, such as Cbx7 and Bmi1, and the histone deubiquitinase USP16, together with Aurora B and Ring1B, bind almost exclusively to the regulatory elements of active promoters and are not found at repressed genes. Binding of PRC2 components and deposition of the H3K27me3 and H2Aubq marks are largely confined to silent genes. By employing conditional knockout mouse models, I showed that removal of either Aurora B or Ring1B results in a global reduction in the binding of unphosphorylated and serine 5-phosphorylated RNA Polymerase II to active promoters. This phenomenon is also accompanied by a reduction in the production of transcripts from Aurora B and Ring1B target genes and reduced cell viability. Aurora B is also required to maintain high levels of phosphorylated H3S28 and low levels of the repressive H2Aubq mark at active promoters. These results identify a new role for both Aurora B and Ring1B in the regulation of active transcription in quiescent lymphocytes.
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Jetton, Neal. "Trypanosoma brucei Aurora kinase-1 Function, turnover and potential as a therapeutic target /." Ann Arbor, Mich. : ProQuest, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3244452.
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Thesis (Ph.D. in Molecular and Cell Biology)--S.M.U., 2007.Title from PDF title page (viewed Mar. 18, 2008). Source: Dissertation Abstracts International, Volume: 67-12, Section: B, page: 6890. Adviser: Lawrence S. Ruben. Includes bibliographical references.
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Sizaire, Florian. "Développement d’un criblage automatisé de l’activité kinase d’un biosenseur Aurora A par FLIM." Thesis, Rennes 1, 2019. http://www.theses.fr/2019REN1B033.
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La surexpression d’Aurora A est un marquer majeur de certains cancers épithéliaux. Ce gène code pour la kinase multifonctionnelle Aurora A et son activation est requise pour l’entrée et la progression vers la mitose. Jusqu'à présent, aucun inhibiteur de cet oncogène n'a été approuvé par la FDA et il est donc primordial d'identifier de nouvelles molécules. Notre équipe a développé un biosenseur FRET (Forster Resonance Energy Transfer) pour l’activité kinase d’Aurora A, constitué de la kinase entière flanquée de deux fluorophores, une GFP et une mCherry. Le changement de conformation d’Aurora A lorsqu’elle est activée rapproche les fluorophores et augmente l’efficacité du FRET. Il est ainsi possible de suivre l’activation d’Aurora A dans les cellules vivantes exprimant le biosenseur à des niveaux endogènes. Nous pouvons mesurer le FRET en utilisant la technique de FLIM (Fluorescence Lifetime Imaging Microscopy) grâce à un microscope développé dans l’équipe et appelé fastFLIM. Mes travaux de thèse ont consisté à développer une stratégie de criblage robuste et automatisée en combinant les capacités du fastFLIM et le biosenseur d’activité d’Aurora A. Cette stratégie basée sur une automatisation des acquisitions et de l’analyse de données a permis de cribler une banque de molécules en plaque 96 puits afin de trouver de potentielles inhibiteurs de l’activité kinase d’Aurora A. De plus, j’ai participé à la validation du biosenseur pour un suivi de l’activité kinase dans des cellules vivantes en montrant que les variations de FRET mesurées correspondent bien à l’état de phosphorylation d’Aurora A sur le résidu Thréonine 288, marqueur de son activation. Enfin, j’ai participé à l’élaboration de nouvelles techniques de microscopie pour suivre l’activité du biosenseur. Pour cela, j’ai utilisé un biosenseur de type homoFRET avec l’enjeu de pouvoir utiliser plusieurs biosenseurs dans un contexte multiplex. J’ai aussi utilisé la technique de 2c-FCCS (2-colors Fluorescence Cross Correlation Spectroscopy) sur le biosenseur Aurora A afin de pouvoir mesurer le FRET dans des régions où celui-ci est faiblement exprimant et dont la mesure de durée de vie de fluorescence n’est pas possible par le FLIM. Ainsi, mes travaux de thèse s’inscrivent dans la tendance à développer une microscopie quantitative et autonome avec comme enjeu d’apporter un grande nombre de données phénotypiquesOverexpression of Aurora A is a major marker of some epithelial cancers. This gene encodes the multifunctional Aurora A kinase and its activation is required for entry and progression to mitosis. So far, no inhibitor of this oncogene has been approved by the FDA and it is therefore essential to identify new molecules. Our team developed a Forster Resonance Energy Transfer (FRET) biosensor for Aurora A kinase activity, consisting of the entire kinase flanked by two fluorophores, a GFP and a mCherry. The conformational change of Aurora A when it is activated brings the fluorophores closer and increases FRET efficiency. It is thus possible to follow the activation of Aurora A in living cells expressing the biosensor at endogenous levels. We can measure FRET using FLIM (Fluorescence Lifetime Imaging Microscopy) technique using a microscope developed in the team called fastFLIM. My thesis work consisted of developing a robust and automated screening strategy by combining the capabilities of fastFLIM and the Aurora A activity biosensor. This strategy based on automation of acquisitions and data analysis allowed to screen a library of 96-well plate molecules for potential inhibitors of Aurora A kinase activity. In addition, I participated in the validation of the biosensor for kinase activity monitoring in living cells, showing that the FRET variations measured correspond to the phosphorylation state of Aurora A on the Threonine 288 residue, a marker of its activation. Finally, I participated in the development of new microscopy techniques to monitor the activity of the biosensor. For that, I used a homoFRET biosensor with the challenge of being able to use several biosensors in a multiplex context. I also used the 2c-FCCS (2-color Fluorescence Cross Correlation Spectroscopy) technique on the Aurora A biosensor to measure FRET in regions where it is weakly expressing and whose lifetime measurement of Fluorescence is not possible by FLIM. Thus, my thesis work is part of the trend to develop a quantitative and autonomous microscopy with the challenge of providing a large number of phenotypic data
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Loury, Romain. "Régulation de la phosphorylation mitotique de l'histone H3 par la kinase Aurora-B." Université Louis Pasteur (Strasbourg) (1971-2008), 2003. https://publication-theses.unistra.fr/public/theses_doctorat/2003/LOURY_Romain_2003.pdf.
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La condensation mitotique des chromosomes s'accompagne d'une phosphorylation du résidu sérine 10 de l'histone H3. Nos résultats démontrent qu'il existe une parfaite corrélation spatio-temporelle entre cette modification et la localisation d'Aurora-B, membre de la famille de kinases mitotiques Ipl1/Aurora. De plus, Aurora-B phosphoryle de manière spécifique la sérine de l'histone H3, avec laquelle elle interagit. Cette série de résultats nous ont permis de conclure à l'implication directe d'Aurora-B, acteur primordial du processus de ségrégation des chromosomes, dans la phosphorylation mitotique de l'histone H3. Au terme d'une discussion de ces résultats, menée de manière comparative à d'autres travaux publiés dans ce domaine, plusieurs hypothèses relatives au rôle de cette modification mitotique seront émisesMitotic phosphorylation of histone H3 is directly linked to chromosome condensation. The results presented in this manuscript show a perfect spatio-temporal correlation between this modification and the localisation of Aurora-B, member of the Ipl1/Aurora family of mitotic kinases. Moreover, Aurora-B specifically phosphorylates histone H3 on serine 10, and interacts with it. These results led us to conclude that Aurora-B, a major regulator of chromosome segregation, is directly implicated in mitotic phosphorylation of histone H3. After discussion of these results, different hypotheses concerning the role of this modification will be proposed
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Amoussou, Nathalie. "Conception, synthèse, extraction phytochimique et étude biologique d'inhibiteurs potentiels de kinases à visées anticancéreuse." Thesis, Nantes, 2017. http://www.theses.fr/2017NANT1003.
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Les protéines kinases constituent un groupe d’enzymes jouant un rôle essentiel dans la physiologie cellulaire. La kinase Haspin est une sérine/thréonine kinase appartenant au groupe des protéines kinases atypiques. A ce jour, son seul substrat connu est l’histone H3. Elle joue un rôle important au cours de la mitose notamment dans la cohésion des chromosomes. Du fait de ce rôle primordial, la kinase Haspin constitue une cible de choix dans la thérapie anticancéreuse. Ce travail a pour objectif principal la valorisation des structures d’origine naturelle dans une application antiproliférative ou immunomodulatrice potentielle. Elle est organisée en deux parties: -la première partie est constituée d’un travail de pharmacomodulation d’une structure connue pour améliorer ses caractéristiques physico-chimiques ainsi que son activité biologique. Le modèle utilisé, une structure 8-amino-4-oxo-3,4-dihydro-5H-pyridazino[4,5- b]indole, est inspiré du scaffold fourni par les grossularines A et B. Les nouveaux analogues synthétisés ont été testés sur un panel de kinases et sur deux lignées cancéreuses. -la deuxième partie repose sur l’étude d’extraits éthanoliques de cinq plantes de la pharmacopée traditionnelle béninoise : Amaranthus spinosus, Momordica charantia, Boerhaavia erecta, Boerhaavia diffusa et Combretum paniculatum. Les tests phytochimiques réalisés ont permis d’identifier les différents groupes de métabolites contenus dans ces plantes. Un test de toxicité aiguë a aussi été réalisé avec ces extraits. Après fractionnement de l’extrait total de Combretum paniculatum, les différentes fractions ont été testées sur un panel de kinasesProteins kinases constitute a large group of enzymes that catalyze that play an essential role in the several cellular processes. Haspin, a serine/threonine kinase, is often classified as an atypical member of eukaryotic protein kinase family. The only substrate of Haspin identified to date is histone H3. Haspin plays an important role during mitosis and appears to be vital for maintaining chromosome cohesion. Due to it important role during mitosis, Haspin may have therapeutic utility in treating cancer. The aim of this work was to identify and valorize natural product as effective anti-cancer agents. This work is divided in two parts: - Firstly, we modulated a known structure to improve physico-chemical characteristics as well as biological activity. 8-amino-4-oxo-3,4-dihydro-5H-pyridazino[4,5- b]indole scaffold based on the grossularines A and B structure was designed. These new analogs were tested against many kinases and also evaluated against two human cancer cell lines. - In the second part, we studied five Beninese traditional medicine plants extracts: Amaranthus spinosus, Momordica charantia, Boerhaavia erecta, Boerhaavia diffusa and Combretum paniculatum. Phytochemical studies revealed that these plants have several active constituents. Acute toxicity studies were performed with plants extracts. After fractionation of the total extract of Combretum paniculatum, the various fractions were tested against many kinases
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Pinsky, Benjamin Alan. "Characterization of the Ipl1/Aurora protein kinase in chromosome segregation and the spindle checkpoint /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/5028.
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Larsson, Anna. "Histone modification regulated bysuppressor of Zeste 12 and Ipl1 : Aurora-like kinase in Drosophila." Licentiate thesis, Umeå University, Department of Molecular Biology (Faculty of Science and Technology), 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-36214.
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Polycomb group (PcG) proteins are a group of genome wide silencers that are crucial for many processes during the development. In Drosophila PcG proteins are organised into four different complexes: PRC1, PRC2, PhoRC and PR-DUB. PRC2 consists of four core proteins: Su(z)12, E(z), Esc and Nurf. E(z) is the only known proteins with a known function, it possess a methyltransferase activity that specifically methylates lysine 27 on histone 3 (H3K27). A novel PcG gene was identified in 2001 in screen for modifiers of zeste-white interaction. This gene suppressed zeste’s repression of white and gave it the name Suppressor of zeste 12 (Su(z)12). The Su(z)12 gene is alternatively spliced into two transcripts; a 4.1 kb mRNA called Su(z)12A and a 3.7 kb mRNA called Su(z)12B. These transcripts are translated into two isoforms; a 95 kDa Su(z)12A protein and 100 kDa Su(z)12B protein. These isoforms show a sequence similarity of 95% and the only difference is the C-terminal end. During development these two isoforms are present at different levels. Interaction of the two isoforms with the other core components in PRC2 showed that only Su(z)12B interacts with Nurf. Also the two isoforms showed interaction with each other with the exception of a single copy of Su(z)12A that couldn´t interact with Su(z)12B. Overexpression of Su(z)12B in vivo caused lethality and homeotic transformations. Aurora kinases belong to a conserved family of serine/threonine kinases that are important for many processes in mitotsis, such as spindle formation, chromosomal segregation and cytokinesis. Aurora kinases are overexpressed in many human cancers and inhibitors of Aurora A and Aurora B has shown to inhibit growth and induce apoptosis. There are three Aurora kinases in vertebrates; Aurora A, Aurora B and Aurora C and although they are highly similar, they have different roles and location during mitosis. Aurora B is a chromosomal passenger protein and forms the chromosomal passenger complex with INCENP, Survivin and Borealin. Depletion of Aurora B causes severe effects in mitosis and lead to large cells with several nuclei and polyploidy. The Drosophila homologue of Aurora B is called IpI1-like-Aurora kinase (ial). The c-Myc transcription factor, or its relatives N-Myc and L-Myc, are also overexpressed in many, if not all human cancers. Drosophila has only one Myc protein, dMyc, which is encoded by the diminutive (dm) locus. In Drosophila, dMyc is mostly associated with size and growth regulation and depletion of dm results in endoreplication and growth arrest in early development. Previous work has shown that mammalian c-Myc induces Aurora A and Aurora B kinases. When Myc-driven lymphomas are treated with Aurora B inhibitors, cells are accumulated in G2/M phase and apoptosis is induced. Here we show that these conserved proteins have a potential connection in Drosophila as well since knockdown of ial causes severe phenotypes and leads to larger cells. When ial is knocked down or when dMyc is overexpressed the flies become smaller. Interestingly however transgenic flies which overexpress dMyc and knock down ial exhibit a different pheontype - the flies become bigger. This showing evidence that a relationship between Myc and Aurora B is evolutionary conserved down to Drosophila.
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Deretic, Jovana. "Identifying new shared substrates of Aurora kinases at the mitotic apparatus." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31137.
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Aurora A and B are the major kinases that control key events in mitosis, such as centrosome function, spindle assembly, chromosome segregation and cytokinesis, through phosphorylation of multiple proteins. These kinases share identical consensus target motifs, so the substrate specificity is determined by distinctive sub-cellular localization of the Auroras. Many proteins have been identified as targets of either Aurora A, or Aurora B, or both kinases by mass spectrometry studies. However, only a few of the identified phosphorylation sites in these targets have a characterized function in vivo. Therefore, the molecular mechanisms underlying the regulation of certain mitotic events by Aurora kinases remain unclear. The objective of my work was to develop a tool for identifying new substrates of both Aurora kinases. More specifically, I aimed to identify the molecular targets of Aurora A at the kinetochores, and determine how Aurora A contributes to the error correction near spindle poles. I first demonstrated that the outer kinetochore protein HEC1/Ndc80, phosphorylated by Aurora B at kinetochores, can also be phosphorylated by Aurora A close to the centrosomes (Chapter 2). My finding showed that Aurora kinases can share substrates in the cells and revealed the mechanism by which Aurora A contributes to the error-correction near spindle poles. To identify and characterise novel substrates of Aurora kinases, I developed a bioinformatic approach in collaboration with the Centre Bioinformatician, Alastair Kerr. This bioinformatic method uses the Auroras’ shared consensus motifs combined with several parameters that control the substrate specificity of Aurora kinases. I tested the phosphorylation of the chosen candidates in vitro using radiolabelled kinase assays. In my study, five proteins were validated - SPICE1, TTLL4, AHCTF1, CLASP2 and an uncharacterized protein KIAA1468 - as in vitro substrates of Aurora A and Aurora B kinases (Chapter 3). I then focussed on the Aurora kinases-dependent regulation of spindle and centriole-associated protein, SPICE1, in cells (Chapter 4). Using either site-directed mutagenesis of SPICE1 or inhibition of Aurora kinases with small molecule inhibitors, I found that the predicted phosphorylation of the SPICE1 C terminus had the function in cells of directing the SPICE1 localization on the spindle MTs. My results demonstrate the high accuracy of this genome-wide bioinformatics approach. By complementing mass spectrometry studies, here lies a potential for the identification of other unknown substrates, which is important for the general understanding of how Aurora kinases regulate the mitotic apparatus.
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Vaufrey, Lucie. "Étude du rôle de la kinase Aurora-A dans le développement de la larve et du cerveau de Drosophila melanogaster." Thesis, Rennes 1, 2017. http://www.theses.fr/2017REN1B021/document.
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Aurora-A (AurA) est une sérine/thréonine kinase jouant un rôle majeur dans le cycle cellulaire. Elle est connue pour son rôle oncogène et les compagnies pharmaceutiques développent des inhibiteurs ciblant son activité kinase. Cependant, il a été montré chez différentes espèces qu’Aurora-A possède des rôles indépendants de son activité kinase et agit également comme suppresseur de tumeur quand son activité kinase est altérée. Ceci pose donc un problème dans le développement des inhibiteurs car cibler l’activité kinase d’Aurora-A pour traiter le cancer pourrait mener à l’effet inverse. Pour résoudre ce dilemme, j’ai étudié en détail les phénotypes de mutants AurA nul et hypomorphe chez Drosophila melanogaster. J’ai étudié à la fois les défauts de développement en me basant sur le temps de pupation des larves et le rôle de suppresseur de tumeur en me basant sur les neuroblastes du cerveau central. Dans ce modèle, une caractéristique des suppresseurs de tumeur est leur capacité à induire la formation de neuroblastes supplémentaires dans le cerveau central conduisant à une surcroissance du cerveau. Chez les mutants AurA, la taille du cerveau est plus petite jusqu’à 96h de développement larvaire. Cependant, la pupation arrivant normalement entre 96h et 120h de développement larvaire est retardée chez le mutant et les larves ont une taille plus importante. Chez les mutants en retard de pupation le cerveau devient plus gros que ceux du contrôle. Le cerveau des mutants AurA a une importante augmentation du nombre de cellules positives pour Deadpan, un marqueur spécifique des neuroblastes et ce, avant que le cerveau des mutants AurA devienne plus grand que celui du contrôle. De plus, les disques imaginaux d’ailes et la glande annulaire sont clairement plus petits que ceux du contrôle à 96h de développement larvaire et les larves mutantes atteignent les stades L2 et L3 plus tôt. En conclusion, les mutants AurA montrent 1) une avance dans leur développement précoce certainement reliée au défaut de croissance de la glande annulaire ; 2) un retard de pupation ressemblant à celui observé en cas de défauts dans la voie de l’ecdysone, certainement dû à des défauts de croissance des disques imaginaux d’ailes ; 3) une surcroissance du cerveau à mettre en lien à la fois avec une augmentation du nombre de pseudo-neuroblastes et avec le retard de pupationAurora-A (AurA) is a major kinase playing various roles in cell cycle. It’s a well-known oncogene and companies are developing drugs inhibiting its kinase activity. However, it has been shown in different species that AurA can have a kinase independent role or act as a tumor suppressor when its kinase activity is altered. This represents a problem for drugs development as inhibiting AurA kinase activity only could lead to life threatening phenotypes. To address this dilemma, we carefully deciphered phenotypes of AurA null and AurA hypomorph mutants in Drosophila melanogaster using the pupation as readout for development timing and larval central brain neuroblasts as model for tumorigenic study. One readout to define a tumor suppressor in this model is a brain overgrowth phenotype associated to central brain neuroblasts over-proliferation. In AurA mutants, brain size appears slightly smaller until 96h of larval development. However, pupation occurring normally between 96 and 120h of larval development is delayed in AurA mutants and larvae have an increased size. In this “delayed” mutant larvae, brains are eventually bigger than wild-type controls. Furthermore, AurA mutant central brains show a huge increased number of cells positive for deadpan, a marker of neuroblast identity, even before the appearance of brain over-growth phenotype. Additionally, wing discs and ring glands are clearly smaller in AurA mutants at 96h compared to control and mutant larvae reach L2 and L3 developmental stage earlier than control. In conclusion, AurA mutants have: 1) a precocious developmental advance certainly related to ring gland growth defect; 2) a pupation delay which resembles Ecdysone pathway timing defects certainly due to wing discs growth defect; 3) an enlarged brains phenotype due to an increased of the number of neuroblast-like cells and the pupation delay
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Bruderer, Roland Marc. "Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17828.
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Davidas, Axelle. "Dissection des fonctions mitotiques de la kinase Aurora B par CALI (Chromophore-Assisted Light Inactivation)." Phd thesis, Université de Grenoble, 2012. http://tel.archives-ouvertes.fr/tel-00903744.
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La kinase Aurora B appartient au complexe des protéines passagères. Ce complexe est impliqué dans la régulation de la condensation, constriction et ségrégation des chromosomes, ainsi que dans la cytokinèse. Son rôle est donc crucial pour prévenir la formation de cellules cancéreuses. Cependant, l'étude de la fonction précise d'Aurora B dans chacune des phases de la mitose est limitée par la durée de celle-ci, et par le manque de spécificité des inhibiteurs existants. Nous avons donc développé une stratégie basée sur la photo-inactivation de la kinase par le chromophore Killer-Red, fusionné à la protéine. L'émission locale de ROS après irradiation, permet alors la photo-inactivation spécifique et temporelle d'Aurora B. La photo-inactivation d'Aurora B avant anaphase aboutie soit à un arrêt de la mitose, soit à la régression du sillon de division, provoquée par l'entrée en anaphase en présence de chromosomes retardés. La photo-inactivation d'Aurora B en début d'anaphase a pour conséquence la régression du sillon de division en cytokinèse ; apportant la première indication directe de l'implication d'Aurora B dans le fuseau mitotique en cytodiérèse. De façon surprenante, la photo-inactivation de la kinase au niveau du corps résiduel, après constriction du sillon de division, n'affecte pas l'abscission. La photo-inactivation d'Aurora B n'affecte pas la localisation des autres membres du complexe des protéines passagères, indiquant que la kinase n'est pas impliquée dans la dynamique du complexe. Les résultats obtenus montrent sans aucun doute l'implication d'Aurora B dans chacune des phases de la mitose, suggérant que la phosphorylation par Aurora B de ses substrats permet le controle de la division cellulaire.
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Scalabrini, Luiza Coimbra. "O papel da quinase Aurora A na biologia das células iniciadoras de turmor pulmonares com mutação em KRAS." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-11042017-082202/.
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Mutações ativadoras no gene KRAS são prevalentes em cancer de pulmão e a as vias de sinalização de RAS estão aumentadas em células iniciadoras de tumor (CITs), que são definidas como células autorrenováveis capazes de iniciar a formação tumoral, sustentar o crescimento tumoral e promover a disseminação tumoral. Entretanto, terapias direcionadas a RAS não foram efetivas até hoje e a identificação de alvos de KRAS que contribuam para o fenótipo oncogênico é necessária. Como a quinase Aurora A (AURKA) já foi implicada, tanto na oncogênese induzida por KRAS, quanto em promover a função das CITs, nós hipotetizamos que a inibição das vias de AURKA seria detrimental para a função de CITs pulmonares portadoras de KRAS oncogênica, desta forma diminuindo o comportamento maligno do câncer de pulmão. Para avaliar a função das CITs, nós usamos ensaios de crescimento de tumoresferas que permitem o crescimento seletivo de CITs in vitro. As linhagens pulmonares positivas para KRAS H358 e A549 formaram tumoresferas em cultura de baixa aderência e, quando comparadas às linhagens parentais, às células oriundas de tumoresferas apresentaram maior capacidade clonogênica in vitro e maior tumorigenicidade in vivo. Além disso, uma análise por qPCR revelou que as células oriundas de tumoresferas possuem expressão aumentada de fatores de células tronco, uma característica de CITs. Em seguida, nós inibimos a AURKA nas linhagens pulmonares positivas para KRAS H358 e A549 por interferência de RNA (RNAi) ou com um inibidor das quinases Aurora (AI II). A inibição de AURKA diminuiu a formação de tumoresferas e o crescimento destas em culturas seriadas, além de reduzir a capacidade clonogênica das células oriundas de tumoresferas. Estes resultados indicam que a AURKA é importante para a autorrenovação e a oncogenicidade de CITs, e que a AURKA induz o fenótipo tronco-tumoral, o que é corroborado pelo achado de que a inibição de AURKA nas tumoresferas reduz a expressão de fatores de célula tronco. Um destes fatores regulados por AURKA é o marcador de superfície de célula tronco CD24. De fato, quando comparadas às células cultivadas de forma aderente, as células oriundas de tumoresferas apresentam maior número de células positivas para CD24 (CD24+) e estes números são reduzidos pelo tratamento com AI II. Finalmente, nós purificamos células H358 CD24+ por citometria de fluxo e mostramos que, quando comparadas às células negativas para CD24, as células CD24+ apresentam maior capacidade de formar tumoresferas em culturas seriadas, e o tratamento com AI II inibe preferencialmente a capacidade de células CD24+ de formarem tumoresferas. Nossos resultados sugerem que uma terapia baseada na inibição de AURKA pode reduzir o número e função de CITs pulmonares portadoras de KRAS oncogênica e, portanto, pode representar uma estratégia terapêutica atraente para reduzir a recidiva e metástase no câncer de pulmão induzido por KRAS.Activating mutations in KRAS are prevalent in lung cancer and RAS sinaling is enhanced in cancer initiating cells (CICs), which are defined as self-renewing tumor cells able to initiate tumor formation, sustain tumor growth and drive tumor dissemination. However, therapies targeted to oncogenic RAS have been ineffective to date and identification of KRAS targets that impinge on the oncogenic phenotype is warranted. Because Aurora kinase A (AURKA) has been implicated both in RAS oncogenesis and in promoting CIC function, we hypothesized that targeting AURKA pathways would impair KRAS-positive lung CIC function, thereby decreasing lung cancer malignant behavior. To evaluate CIC function, we used tumorsphere assays that allow selective growth of CICs in vitro. KRAS positive lung cancer H358 and A549 cells formed tumorspheres under low attachment conditions, and, when compared to the parental cell lines, sphere-forming cells had increased clonogenic ability in vitro and increased tumorigenicity in vivo. In addition, qPCR analysis revealed that tumorsphere cells displayed increased expression of stem cell factors, a hallmark of CICs. Next, we targeted AURKA in KRAS positive lung cancer H358 and A549 cells by RNA interference (RNAi) or with an Aurora inhibitor (AI II). AURKA targeting decreased tumorsphere formation and growth in serial cultures and reduced clonogenic growth of tumorsphere-forming cells. These results indicate that AURKA is important for CIC selfrenewal and oncogenicity and that AURKA induces a CIC phenotype, which is further underscored by the finding that AURKA targeting in tumorspheres decreases expression of stem cell factors. One such factor shown to be regulated by AURKA is the stem cell surface marker CD24. In fact, when compared to adherent cultures, A549 and H358 tumorspheres display increased numbers of CD24-positive (CD24+) cells and these numbers are reduced by AI II treatment. Finally we purified H358 CD24+cells by flow cytometry and showed that, when compared to CD24-negative cells, CD24+ cells have increased ability to form tumorspheres in serial cultures, and AI II treatment preferentially reduced the ability of CD24+ cells to form tumorspheres. Our results suggest that AURKA inhibition therapy can reduce the number and function of KRAS-positive lung CICs, and, therefore might be an attractive therapeutic strategy to reduce recurrence and metastasis in KRAS-induced lung cancer.
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Cherier, Julia. "La kinase Aurora A comme nouvelle cible des agents chimiothérapeutiques Application aux traitements des cancers colorectaux." Phd thesis, Université d'Angers, 2008. http://tel.archives-ouvertes.fr/tel-00449418.
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L'entrée des cellules en mitose se caractérise par l'augmentation d'expression de la kinase Aurora A. Nous avons caractérisé l'effet du sn38, métabolite actif de l'irinotecan utilisé dans le traitement du cancer colorectal sur l'expression de cette kinase. En absence de traitement, le facteur de transcription c-myc se lie au promoteur d'Aurora A et l'active. Cependant, le traitement des cellules avec du sn38 induit l'inhibition de c-myc et son absence du promoteur d'Aurora A s'accompagnant de l'induction du processus de sénescence. Nous avons également étudié l'effet de l'oncogène Ras sur la kinase Aurora A. Cet oncogène induit une diminution d'expression d'Aurora A par une modulation de l'activité des cofacteurs transcriptionnels de c-myc. En résumé, nos résultats indiquent qu' Aurora A est une cible des traitements de chimiothérapie et des mécanismes de contrôles oncogéniques. Le blocage de son expression constitue certainement une protection contre le développement tumoral.
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Cueva, Sabrina Dias Leite. "Análise Citogenética Clássica e Molecular para os Genes Aurora Cinase A e B em Células Hematopoéticas e Mesenquimais da Medula Óssea de Pacientes Portadores de Síndrome Mielodisplásica." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-23042013-084113/.
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A síndrome mielodisplásica (SMD) é uma doença hematológica heterogênea, caracterizada por hematopoese anormal, displasia e instabilidade genômica, portanto, a análise citogenética é determinante no diagnóstico, prognóstico e acompanhamento evolutivo da doença. Considerando que as células hematopoéticas (CHs) e as estromais mesenquimais multipotentes (CTMs) estão em estreita associação, estudos que visem à caracterização destas poderão contribuir para elucidar os mecanismos que governam a progressão tumoral e identificar novos alvos terapêuticos. Objetivo: Caracterizar e comparar as CHs e CTMs derivadas de pacientes através da citogenética convencional e molecular para os genes aurora cinase A e B. Avaliar as propriedades biológicas das CTMs derivadas de SMD e controles saudáveis. Métodos: o estudo iniciou-se com a avaliação clinica de 25 pacientes e 8 controles saudáveis doo HCFMRP-USP e HAC-Jaú. Em seguida, foi realizada a análise cariótipica das CHs e CTMs da medula óssea pelo bandamento G e por FISH para os genes aurora A e B e o perfil imunofenotípico, bem como potencial de diferenciação em adipócito e osteócito das CTMs de pacientes portadores de SMD e controles saudáveis. Resultados: A avaliação clínica mostrou plaquetopenia (76%), neutropenia (100%), hemoglobina baixa (16%). A análise citogenética das CHs revelou cariótipo alterado em 13 pacientes (52%), com cariótipo complexo resultando em alterações numéricas e estruturais. Ao contrário, nas CTMs, o cariótipo se mostrou alterado em sete pacientes (28%) e um padrão de menor complexidade, apenas quatro pacientes apresentaram alterações nas duas populações celulares, porém, diferentes. Foram encontradas apenas alterações numéricas (sendo 86% monossomia e 14% ganho de cromossomo). As CHs e CTMs dos controles apresentaram cariótipos 100% normais. Na análise de FISH não foi evidenciada amplificação dos genes AURKA e AURKB. As CTMs dos pacientes e controles apresentaram-se semelhantes quanto à morfologia e potencial de diferenciação. Entretanto, as CTMs de pacientes mostraram-se alteradas para dois antígenos de superfície, CD90 e CD146, os quais mostraram níveis de expressão mais elevados nas amostras dos pacientes (p= 0,04, p = 0,001 respectivamente). Conclusão: Observou-se que as CTMs se encontram alteradas embora em menor frequência e diferindo das alterações encontradas nas CHs. Esses dados sugerem que as CTMs devem exercer importante papel na progressão tumoral e devem ser consideradas como alvos na busca de novas terapias e melhor esclarecimento dos mecanismos que governam a progressão tumoral. Apesar de não ter evidenciado amplificação dos genes AURKA e AURKB em SMD, estudos futuros que visem avaliar o nível de expressão dessas enzimas em pacientes portadores ou não de alterações citogenéticas poderão contribuir para a compreensão do envolvimento ou não desse gene com a evolução da doença. Além disso, não foi evidenciada associação de anemia profunda e citogenética alterada.The myelodysplastic syndrome (MDS) is a heterogeneous hematologic disease characterized by abnormal hematopoiesis, dysplasia and genomic instability, therefore, cytogenetic analysis is crucial in the diagnosis, prognosis and monitoring of disease evolution. Whereas hematopoietic cells (CHs) and stromal multipotent mesenchymal (MSCs) are in close association studies aimed at the characterization of these may help to elucidate the mechanisms that govern tumor progression and identify novel therapeutic targets. Objective: To characterize and compare the CHs and MSCs derived from patients by conventional cytogenetics and molecular genes aurora kinase A and B. To evaluate the biological properties of MSCs derived from MDS and healthy controls. Methods: The study began with the clinical evaluation of 25 patients and eight healthy controls HCFMRP dooUSP and CH-Jau. Next, we performed a karyotypic analysis of CHs and MSCs from bone marrow by G-banding and FISH for aurora A and B genes and immunophenotypic profile and potential to differentiate into adipocytes and osteocytes of MSCs in patients with MDS and controls healthy. Results: The clinical evaluation showed thrombocytopenia (76%), neutropenia (100%), low hemoglobin (16%). The cytogenetic analysis revealed karyotype of CHs changed in 13 patients (52%), resulting in complex karyotype with numerical and structural changes. In contrast, in MSC, the karyotype was abnormal in seven patients (28%) and a pattern of lower complexity, only four patients had changes in both cell populations, however, different. Were found only numerical changes (monosomy being 86% and 14% gain in chromosome). The CHs and MSCs controls showed 100% normal karyotypes. In FISH analysis there was no evidence of gene amplification and AURKA AURKB. The MSCs of patients and controls were similar regarding the morphology and differentiation potential. However, the CTMs of patients proved to be changed to two surface antigens, CD90 and CD146, which showed higher expression levels in samples of patients (p = 0.04, p = 0.001 respectively). Conclusion: Furthermore, it was observed that the MSCs are changed although less frequently and differing from changes found in CHs. These data suggest that MSCs should play an important role in tumor progression and should be considered as targets in the search for new therapies and better explain the mechanisms that govern tumor progression. Although not shown AURKA amplification of genes in MDS and AURKB, future studies aimed at assessing the level of expression of these enzymes in patients with or without cytogenetic alterations may contribute to the understanding of the involvement or not of this gene with the disease. This study can not associate with profound anemia cytogenetic changes.
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Horn, Virginie. "Mise en évidence et caractérisation d'une interaction fonctionnelle entre la kinase Aurora-A et la phosphatase PP2A." Université Joseph Fourier (Grenoble), 2005. http://www.theses.fr/2005GRE10046.
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Le déroulement de la mitose est très étroitement contrôlé par une succession de réactions enzymatiques en particulier, celles catalysées par de nombreuses protéines kinases et phosphatases. Plus précisément, la sérine/thréonine kinase mitotique Aurora-A est essentielle à ces processus car elle participe à la régulation de la transition G2/M, du cycle des centrosomes, du fuseau mitotique et de la ségrégation des centrosomes. Aurora-A est activée grâce à son interaction avec d'autres protéines telles que TPX2 et AJUBA et son activité kinase est modulée par la phosphorylation de sites spécifiques. Par ailleurs, il a été récemment montré in vitro que la dégradation de Aurora-A par la voie du protéasome est induite par la déphosphorylation d'un résidu très conservé : la sérine 51. Ceci suggère qu'une phosphatase induit la protéolyse de Aurora-A par déphosphorylation du résidu S51. Dans cette étude, nous avons montré que la phosphatase PP2A et la kinase Aurora-A sont co-localisées dans les centrosomes des cellules mammifères et interagissent au sein d'un même complexe. De plus, l'inhibition pharmacologique de l'activité de PP2A ou l'inhibition de son expression conduisent à stabiliser Aurora-A in vivo. Ces résultats indiquent que PP2A contrôle la dégradation de Aurora-A in vivo. Enfin, Nous avons confirmé in vivo dans des cellules mammifères que la phosphorylation du résidu S51 protège Aurora-A de la dégradationMitosis progression is tightly controlled by a succession of enzymatic reactions, including those catalyzed by numerous protein kinases and phosphatases. More specifically, the mitotic serine/threonine kinase Aurora-A is required in these processes as it is involved in the regulation of the G2/M transition, centrosome cycle, mitotic spindle and chromosomes segregation. Aurora-A is activated by interacting with other proteins such as TPX2 or AJUBA and its kinase activity is modulated by the phosphorylation at specific sites. Besides, it has been recently shown in vitro that Aurora-A degradation by the proteasome pathway is induced by the dephosphorylation of a highly conserved residue: serine 51. In this study, we have shown that the phosphatase PP2A and the kinase Aurora-A are co-localized in centrosomes and are interacting within the same complex. Moreover, the pharmacological inhibition of PP2A activity or the inhibition of its expression both led to Aurora-A stabilization in vivo. These results indicate that PP2A controls Aurora-A degradation in vivo. Finally, we confirmed in vivo in mammalian cells that phosphorylation of the S51 residue prevents the degradation of Aurora-A
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Mattei, Jean-Camille. "Evaluation de l'activité anti-tumorale de thérapeutiques ciblées dans les sarcomes : implication des Aurora kinases et de CHK1." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM5070.
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Les sarcomes sont des cancers rares touchant toutes les zones du corps humain, caractérisés par une grande diversité de nature, de comportement clinique et de réponse aux thérapies existantes, certains étant de bon pronostic, d’autres très difficilement curables.Leur traitement de référence est la chirurgie ; la radiothérapie et les protocoles de chimiothérapie n’ayant que peu évolué lors des 30 dernières années.Récemment des caractéristiques génétiques leur étant propres ont été découvertes, prédictives de leur agressivité et contre lesquelles il est possible de diriger des drogues spécifiques pouvant améliorer le pronostic et diminuer les effets secondaires des thérapies conventionnelles.C’est sur l’inhibition d’Aurora Kinase A et B et CHK1 que s’est focalisé ce travail avec le test des effets de deux nouvelles drogues sur 9 types de cellules cancéreuses sarcomateuses avec des résultats très prometteurs, qu’il conviendra de conforter par d’autres expériences, notamment sur l’animalSarcomas are rare cancers, which may arise in all parts of human body. They are characterized by great diversity in their nature, clinical behavior and response to existing therapeutics. Some are of good prognosis and others hard to cure.Their treatment essentially relies on surgery and radiotherapy or chemotherapy haven’t know major breakthrough over the last 3 decades.Recently new genetics abnormalities linked to sarcomas have been discovered. Their analysis can predict their aggressiveness and it is now possible to develop targeted therapies against them. This could help improving cancer prognosis and/or limiting conventional drugs adverse effects.Our work focused on Aurora Kinase A and B and CHK1 inhibition, testing the effects of 2 new drugs on 9 types of sarcoma cells with promising results, which we will confort by other experiments, including on the animal
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Juillet, Charlotte. "Conception, synthèse et évaluation pharmacologique d’analogues simplifiés de métabolites marins, inhibiteurs de la kinase Aurora B, à visée anticancéreuse." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASF019.
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Ce manuscrit porte sur la conception, la synthèse et l’évaluation biologique d’analogues de l’oroïdine, monomère de la benzosceptrine C. Ces molécules sont issues de la famille des pyrrole-2-aminoimidazoles, isolées d’éponges marines. La stratégie de simplification et de diversification structurale a conduit à l’identification d’un hit sélectif, inhibiteur de la kinase Aurora B. Celle-ci joue un rôle essentiel dans la division cellulaire et son inhibition conduit à des anomalies mitotiques sévères. De plus, elle est surexprimée dans de nombreux cancers, en faisant une cible thérapeutique de choix en oncologie. L’objectif du projet, à l’interface entre la chimie et la biologie, était de synthétiser un composé chef de file pouvant conduire à un candidat médicament à visée anticancéreuse. Le squelette du hit est constitué de trois parties : une partie 4,5-dibromopyrrole, une partie imidazo[1,2-a]pyrimidine et enfin un linker alcyne. Le travail de chimie médicinal s’est articulé autour de ces trois sites de modulation structurale, qui ont été successivement modifiés et font l’objet des chapitres II à IV, après le chapitre I dédié à l’introduction. Finalement, quatre-vingt deux dérivés ont été synthétisés et évalués in vitro sur Aurora B et un panel de kinases impliquées dans différentes pathologies. Plusieurs analogues se sont avérés très actifs, avec des IC50 allant jusqu’à 34 nM, soit 150 fois plus actifs que le hit initial. Le dernier chapitre porte sur l’étude du mode d’action des inhibiteurs les plus actifs. Des études de cinétiques enzymatiques ont mis en évidence un mode d’inhibition non-ATP compétitif, jusqu’alors jamais décrit pour Aurora B. Par ailleurs, des expériences d’immunomarquage ont permis d’évaluer et de quantifier les effets du meilleur composé sur les cellules traitées, montrant des résultats cohérents avec l’inhibition d’Aurora B. Enfin, des études de modélisation moléculaire avec le meilleur inhibiteur nous ont permis de situer le site de fixation potentiel de nos inhibiteurs afin de poursuivre les pharmacomodulations et l’étude SAR. Le manuscript se termine par une conclusion générale et des perspectives qui proposent des pistes pour optimiser les propriétés physicochimiques et pharmacocinétiques afin d’améliorer les propriétés nécessaires à un candidat médicamentThis manuscript describes the design, synthesis and biological evaluation of oroidin analogs. Oroidin is a monomer of benzosceptrin C, belonging to the pyrrole-2-aminoimidazole family, isolated from marine sponges. The simplification and structural diversification approaches led us to the identification of a non-natural hit displaying selective inhibitory activity against the kinase Aurora B. This kinase plays a key role in cell division and its inhibition leads to severe mitotic abnormalities. Aurora B is found to be up-regulated in many human cancers, indicating that this kinase is a cancer-relevant target. The objective of the study at the interface between chemistry and biology is to optimize the discovered hit into a lead. The hit scaffold is divided in three parts: the 4,5-dibromopyrrole, the imidazo[1,2-a]pyrimidine and the alkyne moieties. After the first introductive chapter, chapters II to IV are dedicated to the pharmacomodulations of each part. We finally managed to synthesize eighty-two analogs for in vitro evaluations toward Aurora B and a panel of kinases involved in diverse human pathologies. Several compounds were found to be very active with IC50 down to 34 nM, displaying a 150-fold higher activity than the initial hit. The last chapter discusses the mode of action of the most active inhibitors from the hit expansion. The enzymatic kinetic assays revealed an uncommon mode of action with allosteric inhibitors (type IV) of Aurora B. Immunostaining experiments highlighted the typical effects of Aurora B inhibition in treated cells as well as its quantification. At last, molecular docking study with the best inhibitor showed the most probable allosteric binding pocket of Aurora B, providing crucial support in hit-to-lead optimization. In conclusion and perspectives, the efforts to be pursued in order to improve physicochemical and pharmacokinetic properties in the lead-to-candidate process are pointed
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Romé, Pierre. "Identification de nouveaux partenaires de la protéine kinase aurora A au cours de la mitose chez Drosophila melanogaster." Rennes 1, 2011. http://www.theses.fr/2011REN1S105.
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La mitose est une étape fondamentale du cycle cellulaire à l’issue de laquelle la cellule mèretransmet son matériel génétique de façon équitable aux deux cellules filles. Pour ce faire, lacellule nucléée des microtubules qui s’organisent en structure bipolaire complexe appelée lefuseau mitotique. L’assemblage de ce fuseau de division est très finement régulé par denombreuses protéines. Parmi celles-ci, la sérine-thréonine kinase Aurora A possède des rôlesmitotiques cruciaux et sa dérégulation entraîne de nombreuses perturbations dansl’assemblage du fuseau. L’objectif de ma thèse a été d’identifier de nouveaux partenaires d’Aurora A au cours de lamitose chez la drosophile afin de mieux comprendre les fonctions mitotiques de cette kinase. Nous avons, dans un premier temps, caractérisé l’interaction physique et fonctionnelle quiexiste entre Aurora A et la sous-unité p150glued du complexe dynéine-dynactine (DDC). LeDDC est un complexe moteur requis pour l’assemblage du fuseau mitotique. Nous avonsdémontré que la phosphorégulation de ce complexe par Aurora A était nécessaire pour limiterl’accumulation de celui-ci sur le fuseau, une régulation qui apparaît importante pourl’assemblage du fuseau mitotique. Par la suite, nous avons identifié la kinésine NCD comme étant un nouveau partenaired’Aurora A. NCD est également régulée par Aurora A qui semble limiter l’accumulation decette kinésine sur le fuseau mitotique. Nos études révèlent une nouvelle fonction mitotique de la kinase Aurora A dans la régulationdu recrutement de certains moteurs moléculaires sur les microtubules, un événement quisemble essentiel lors de l’assemblage correct du fuseau mitotique. Mots clés : division cellulaire, centrosome, microtubules, fuseau mitotique, protéine kinase,protéine associée aux microtubules, kinésine, dynéineMitosis is a key step of the cell cycle by which the mother cell transmits equally its owngenetic material to the daughter cells. To do so, the cell nucleates microtubules, whichorganise a complex bipolar structure called the mitotic spindle. Mitotic spindle assembly istightly regulated by several proteins included the serine-threonine kinase Aurora A. Aurora Aassures different roles during mitotis an its deregulation leads to many defect in spindleassembly. In this context, the goal of my thesis was to identify new partners of Aurora A during mitosisin Drosophila in order to better understand the mitotic role of this kinase. We first characterised the physical and functional interaction between Aurora A and p150glued,a subunit of the dynein-dynactin complex (DDC). DDC is a motor complex required duringspindle assembly. We showed that phosphoregulation of DDC by Aurora A was required tolimit the accumulation of this complex on the spindle, an important regulation during spindleassembly. Secondly, we identified the kinesin NCD as a new partner of Aurora A. NCD is also regulatedby Aurora A which seems to limit the accumulation of the kinesin on the mitotic spindle. Our study reveals a new mitotic function for Aurora A kinase in the limitation of molecularmotors recruitment, an event which appear required during mitotic spindle assembly. Key Words: cell division, centrosome, microtubules, mitotic spindle, protein kinase,microtubules associated protein, kinesin, dynein
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Souza, Felipe Canto de. "Envolvimento das Aurora-quinases e DIDO na instabilidade cromossômica na leucemia linfoide crônica." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17154/tde-30032017-112908/.
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Durante a divisão celular as Aurora-quinases (AURKA e AURKB) participam da formação e controle das fibras do fuso mitótico enquanto as isoformas proteicas (DIDO1, DIDO2 e DIDO3), originadas do splicing alternativo do gene DIDO, auxiliam na junção dos microtúbulos aos cinetócoros. Portanto, ambas são relevantes na regulação do ciclo celular. Interessantemente, a superexpressão (ou o ganho de função) das AURKs ou a baixa expressão (ou perda de função) das isoformas de DIDO estão ambos associados com amplificação dos centrossomos e à instabilidade cromossômica (CIN), com consequente aneuploidia. Dentre as doenças hematológicas com registros de CIN, a leucemia linfoide crônica (LLC) pode apresentar amplificação dos centrossomos e alteração nos níveis de expressão das AURKs acarretando aneuplodias. Apesar disso, não existem estudos avaliando a potencial associação destes genes com CIN na LLC. Avaliando seus níveis de expressão gênica em amostras de LLC de pacientes com ou sem aberrações cromossômicas, mostramos que o aumento dos níveis de AURKA e AURKB e, inversamente, a redução dos níveis das variantes de DIDO, são significativamente associados com ganhos cromossômicos e com aumento da contagem de glóbulos brancos (WBC). Claramente, amostras de LLC sem qualquer anormalidade citogenética apresentam níveis de expressão semelhantes às amostras que contêm aberrações não-numéricas. O achado de que níveis de expressão de AURKs e variantes de DIDO são completamente opostos, mostrando um padrão discreto de inter-relação, levou-nos a investigar o potencial mecanismo regulatório por trás disso. Tendo em vista que outros, anteriormente, mostraram que o cluster oncogênico miR-17~92 é significativamente hiper-regulado em células de pacientes com LLC purificadas expressando genes IGHV não mutados (em comparação com células mutadas de pacientes) e, que o miR-17 é expresso em níveis significativamente mais elevados em células IGHV não mutadas ou ZAP-70 positivas (mau prognóstico geralmente associada à CIN), resolvemos investigar o potencial de regulação negativa dos microRNAs deste cluster sobre as variantes de DIDO. Além disso, com base no mecanismo regulatório já descrito pelo qual a superexpressão de AURKA induz a transcrição do cluster miR-17~92, mediada por E2F1 (com uma correlação entre as expressões de ambas as proteínas em diferentes tipos de câncer), decidimos investigar este eixo regulatório em LLC. Notavelmente, todas as variantes de DIDO apresentam-se preditas como fortes alvos de vários microRNAs deste cluster oncogênico. Mostramos, então, que amostras de LLC com baixa expressão de DIDO, além dos já mencionados níveis elevados de AURK, exibiram níveis significativamente mais elevados do fator de transcrição E2F1 e de seu alvo transcricional, o transcrito primário do miR-17~92 (MIR17HG). Além disso, por meio do uso da linhagem de celular NTERA-2, como modelo experimental, mostramos que o siRNA nocaute para AURKA (nos níveis transcricional e proteico, como confirmado por qPCR e western blot) é acompanhada por uma significativa redução de E2F1 e também de MIR17HG. Ainda, a transfecção de células NTERA-2 com sintéticos microRNAs miméticos do cluster miR-17~92 (ou seja, 19a-miR, miR-20a e miR-92a) resultou em uma clara e significativa redução dos níveis de transcrição de todas as variantes de DIDO. Por fim, a inibição do siRNA especifico para a variante DIDO3 (mas não às outras variantes) levou a uma redução significativa dos níveis de transcrição de todas as variantes de DIDO, indicando um mecanismo adicional contribuindo para a downregulação dos transcritos de DIDO. Ao todo, nossos resultados demonstram a existência de um potencial mecanismo regulatório interconectado entre AURK e DIDO, associado à CIN e maior contagem de WBC na LLC. Mais importante, os níveis de expressão elevada de AURKs e os baixos níveis associados das variantes de DIDO são especificamente relacionados com anormalidades citogenéticas apresentando ganhos cromossomais, com destaque para o mecanismo celular específico, subjacente à CIN, observado neste grupo distinto LLC. Dado o papel central da CIN na gênese e progressão do câncer, esses achados provavelmente terão um impacto importante no prognóstico ou tratamento da LLC.During cell cycle division Aurora kinases (AURKA and AURKB) participate in the formation and control of mitotic spindle fibers, while, protein isoforms (DIDO1, DIDO2 and DIDO3), derived by alternative splicing of the DIDO gene, assist at the junction of microtubules to kinetochores. Thus, both are relevant to cell cycle maintenance. Interestingly, overexpression (or gain of function) of AURKs or low expression (or loss of function of DIDO) are both associated with centrosomal amplification and chromosomal instability (CIN), leading to aneuploidy. Among hematological diseases with CIN records, chronic lymphocytic leukemia (CLL) can display centrosome amplification and changes in AURKs expression levels leading to aneuploidy. Despite this, there are no studies evaluating the potential association of these genes with CIN in CLL. By evaluating their gene expression levels in CLL samples from patients with or without chromosomal aberrations, we show that increased levels of AURKA and AURKB and, conversely, reduced levels of DIDO variants, are both significantly associated with chromosomal gains and with increased white blood cell (WBC) counts. Clearly, CLL samples without any cytogenetic abnormality had expression levels similar to samples mostly harboring non-numerical aberrations. The finding that the expression levels of AURKs and DIDO variants are completely opposed, showing a discrete inter-related pattern, led us to investigate the potential regulatory mechanism behind this. Given that other have previously shown that the oncogenic miR-17~92 cluster is significantly upregulated in purified CLL patient cells expressing unmutated IGHV genes (as compared to mutated patient cells), and that miR-17 is expressed at significantly higher levels in unmutated or ZAP-70 high cases (bad prognostic cases generally associated with chromosomal instability), we investigated the potential negative regulation of DIDO variants by microRNAs from this cluster. In addition, based on the already described regulatory mechanism by which AURKA overexpression induces the E2F1-mediated transcription upregulation of the miR-17~92 cluster (with an observed expression correlation of both proteins in cancer specimens); we decided to investigate this regulatory axis in CLL. Notably, we found that all DIDO variants are predicted to be heavily targeted by several miRs of this oncogenic cluster. We show that CLL samples with low DIDO expression, in addition to the already mentioned AURK high levels, displayed significant higher levels of the transcription factor E2F1 and of its transcriptional target, the miR-17~92 primary transcript (MIR17HG). Moreover, by using the NTERA-2 cell line as a model, we show that siRNA knockdown of AURKA (at the transcript and protein level, as confirmed by qPCR and western blot) is accompanied by a striking significant reduction of E2F1 and also of MIR17HG. Furthermore, transfection of NTERA-2 cells with synthetic mimics of the miR-17~92 cluster (namely, miR-19a, miR-20a and miR-92a) results in a clear and significant reduction in the transcript levels of all DIDO variants. Finally, specific siRNA inhibition of the DIDO3 variant (but not the others) led to a significant reduction in the transcript levels of all DIDO variants, indicating an additional mechanism contributing to the downregulation of DIDO transcripts. Altogether, our results demonstrate the existence of a potential interconnected regulatory mechanism between AURK and DIDO, associated with CIN and higher WBC counts in CLL. More importantly, the high expression levels of AURKs and the associated low levels of DIDO variants are specifically associated with cytogenetic abnormalities presenting chromosomal gains, highlighting the specific cellular mechanism underlying the CIN observed in this distinct CLL group. Given the central role of CIN in cancer genesis and progression, these findings will likely have an important impact on prognosis or treatment of CLL.
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Koch, André [Verfasser], and Silke [Akademischer Betreuer] Hauf. "Identification of Aurora Kinase Substrates in Schizosaccharomyces pombe by Chemical Genetics and Quantitative Phosphoproteomics / André Koch ; Betreuer: Silke Hauf." Tübingen : Universitätsbibliothek Tübingen, 2012. http://d-nb.info/1162843071/34.
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Patel, Anisha Anilkumar. "Mechanisms of EPS8-mediated oncogenesis." VCU Scholars Compass, 2007. http://hdl.handle.net/10156/1148.
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Bonet, Caroline. "Contrôle de la sénescence des cellules de mélanome : implication de la kinase Aurora B et du facteur de transcription MITF." Nice, 2012. http://www.theses.fr/2012NICE4100.
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Le mélanome est une tumeur hautement agressive, dont l’incidence est en forte augmentation depuis ces dernières décennies, ce qui en fait un important problème de santé publique. Le fort potentiel métastatique du mélanome implique une éradication chirurgicale précoce afin d’éviter la dissémination des cellules tumorales qui deviennent alors hautement résistantes à tous types de traitements. Grâce à une meilleure compréhension des mécanismes moléculaires de pathogénie, des nouveaux traitements de thérapie ciblée ont pour la première fois permis d’augmenter la survie des patients atteints de mélanome métastatique. Cependant, malgré l’espoir énorme que suscitent ces traitements, il y a encore de nombreux patients qui ne répondent pas et la guérison parfois incomplète est souvent suivie de rechutes fatales. Dans une première partie de ma thèse, j’ai montré que la kinase Aurora B est contrôlée par la voie MAPK/ERK, dérégulée dans 80% des cas de mélanomes, et que son inhibition par un nouvel inhibiteur plus spécifique de cette kinase conduit à la mise en place d’un programme de sénescence et à la mort des cellules de mélanome par de la catastrophe mitotique. Le Vémurafénib qui inhibe la forme oncogénique BRAFV600E, mutée dans 50% des cas de mélanomes, augmente la survie des individus atteints de mélanomes métastatiques mais ce traitement est suivi de rechutes très rapides. L’enjeu actuel est donc de trouver de nouvelles cibles afin d’éviter les résistances et de prolonger les effets du Vémurafénib. J’ai montré que les mélanomes résistants au Vémurafénib sont sensibles à la mort cellulaire induite par l’inhibiteur d’Aurora B. Ainsi, la kinase Aurora B semble être une cible prometteuse dans le traitement du mélanome et également chez les patients développant des résistances au Vémurafénib. Dans une deuxième partie de la ma thèse je me suis intéressée au facteur de transcription MITF. MITF, dont l’isoforme M est restreinte au lignage mélanocytaire est un facteur de transcription qui joue un rôle important dans la tumorigenèse. Nous avons montré au laboratoire que l’invalidation de MITF par la technique de l’interférence à l’ARN engage une voie de réponse de dommage de l’ADN et la mise en place d’un programme de sénescence cellulaire qui peut être considéré comme une barrière anti-tumorale. Cette étude a révélé comment MITF contrôle la prolifération et supprime la sénescence des cellules mélanocytaires. De plus, le laboratoire a identifié une mutation de MITF qui change le glutamate en position 318 en lysine (E318K). Cette mutation affecte un site de sumoylation et prédispose au mélanome et au cancer du rein. Ce mutant de MITF contrôle un répertoire de gènes différent de la forme sauvage. L’objectif de mon travail a été de mieux comprendre comment le mutant exerce ces activités pro-tumorales en focalisant mes recherches sur le contrôle du programme de sénescence. Dans ce contexte, j’ai montré que ce mutant de MITF induit un dépassemen de la sénescence induite par des agents chimiothérapeutiques (AZD1152, Hydroxyurée, Fotémustine) mais également par l’oncogène BRAF. La sénescence induite dans les cellules contrôle a été caractérisée par l’augmentation de l’activité de la β-galactosidase, des dommages de l’ADN et une augmentation des marqueurs de la sénescence, phénomènes absents dans les cellules exprimant la mutation. De plus, j’ai montré que les cellules exprimant la forme mutée de MITF surexpriment le facteur de transcription FOXM1 et que l’inhibition de FOXM1 restaure la sénescence induite par l’AZD1152. Ces résultats confirment le rôle de MITF dans le contrôle de la sénescence dans les cellules mélanocytaires et montrent que la forme mutée E318K de MITF entraîne un dépassement du programme de sénescence et favorise le développement du mélanomeMetastatic melanoma is an aggressive tumor with almost no effective treatment options. Therefore, a better understanding of the molecular mechanisms underlying melanoma disease will be essential for new advances in melanoma therapy. I showed that the kinase Aurora B is regulated by the MAPK/ERK signaling pathway, deregulated in 80% of melanoma. I showed that Aurora B inhibition triggers senescence entry, characterized by a growth arrest and the death of melanoma cells. Vemurafenib, an inhibitor of BRAF mutated melanoma which occurs in approximately 50% of cases, increases the overall survival of individuals but this treatment induces resistance. In this context, I showed that melanoma cells resistant to Vemurafenib’s effect are sensitive to the Aurora B inhibitor. Collectively, my results indicate that the inhibition of the kinase Aurora B might be a promising strategy for the treatment of metastatic melanoma. On the other hand, my project aims to investigate the role of the transcription factor MITF. We have shown that the invalidation of MITF triggers senescence. In addition, we have identified a mutation in MITF that affects a sumoylation site and predisposes to melanoma. The objective was to understand the molecular mechanisms by which this mutant of MITF exerts its pro-tumoral activity. I showed that MITF prevents senescence entry of melanoma cells mediated by different pro-oncogenic drugs and oncogenes and in several melanoma cell lines. My results confirm the role of MITF in controlling senescence in melanocyte ells, and indicate how MITF, that overrides the senescence, could favor melanoma development
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Hu, Yanmei, Jiantao Zhang, Rami Musharrafieh, Raymond Hau, Chunlong Ma, and Jun Wang. "Chemical Genomics Approach Leads to the Identification of Hesperadin, an Aurora B Kinase Inhibitor, as a Broad-Spectrum Influenza Antiviral." MDPI AG, 2017. http://hdl.handle.net/10150/626106.
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Influenza viruses are respiratory pathogens that are responsible for annual influenza epidemics and sporadic influenza pandemics. Oseltamivir (Tamiflu((R))) is currently the only FDA-approved oral drug that is available for the prevention and treatment of influenza virus infection. However, its narrow therapeutic window, coupled with the increasing incidence of drug resistance, calls for the next generation of influenza antivirals. In this study, we discovered hesperadin, an aurora B kinase inhibitor, as a broad-spectrum influenza antiviral through forward chemical genomics screening. Hesperadin inhibits multiple human clinical isolates of influenza A and B viruses with single to submicromolar efficacy, including oseltamivir-resistant strains. Mechanistic studies revealed that hesperadin inhibits the early stage of viral replication by delaying the nuclear entry of viral ribonucleoprotein complex, thereby inhibiting viral RNA transcription and translation as well as viral protein synthesis. Moreover, a combination of hesperadin with oseltamivir shows synergistic antiviral activity, therefore hesperadin can be used either alone to treat infections by oseltamivir-resistant influenza viruses or used in combination with oseltamivir to delay resistance evolution among oseltamivir-sensitive strains. In summary, the discovery of hesperadin as a broad-spectrum influenza antiviral offers an alternative to combat future influenza epidemics and pandemics.
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Maton, Gilliane. "Etude de la régulation de la protéine kinase Aurora-A au cours de la maturation méiotique de l'ovocyte de xénope." Paris 11, 2004. http://www.theses.fr/2004PA11TO43.
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Venoux, Magali. "ASAP, une nouvelle protèine du fuseau mitotique : étude d'un partenaire, la kinase Aurora-A et implication durant le cycle cellulaire." Montpellier 1, 2008. http://www.theses.fr/2008MON13507.
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Le laboratoire a caractérisé une nouvelle protéine associée aux microtubules (MAP) interphasique et au fuseau mitotique, appelée ASAP (ASter Associated Protein) ou MAP9. La dérégulation de son expression entraîne de sévères défauts mitotiques : fuseaux anormaux, retard de la progression mitotique avec des défauts de congression et de ségrégation des chromosomes aboutissant à une cytokinèse défectueuse, des cellules aneuploïdes et à la mort cellulaire. ASAP est donc nécessaire à l’assemblage du fuseau et au bon déroulement de la mitose. Les mécanismes contrôlant la mitose et l’assemblage du fuseau sont très finement régulés par phosphorylation assurées par plusieurs familles de kinases parmi lesquelles la kinase Aurora-A qui recrute et phosphoryle de nombreuses MAPs. Mon projet principal de recherche consistait à déterminer le lien fonctionnel entre ASAP et Aurora-A. J’ai montré qu’ASAP était phosphorylée par Aurora-A, caractérisé le site majeur de cette phosphorylation in vivo sur la sérine 625 et montré que cette phosphorylation était essentielle à l’assemblage du fuseau mitotique et à la progression correcte de la mitose. Par ailleurs, la déplétion d’Aurora-A induit la dégradation d’ASAP par le protéasome suggérant que cette interaction et/ou phosphorylation est nécessaire à la stabilisation d’ASAP. Ce travail a ainsi permis de caractériser le premier partenaire d’ASAP, de confirmer son rôle crucial au cours de la mitose et d’en déterminer les premiers mécanismes moléculaires. Enfin, la caractérisation de l’orthologue de souris et de son expression tissulaire m’a permis de valider le modèle murin comme modèle d’étude de différentes fonctions biologiques d’ASAP.
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Berthezene, Julien. "Rôles de la kinase Aurora et de la condensine dans la dynamique des télomères en méiose chez la levure fissipare Schizosaccharomyces pombe." Thesis, Toulouse 3, 2021. http://www.theses.fr/2021TOU30008.
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La ségrégation des chromosomes est une étape critique de la division cellulaire. Une transmission erronée des chromosomes conduit à l'aneuploïdie, caractéristique des cancers. En méiose, ces erreurs engendrent de lourdes pathologies congénitales telles que les trisomies, dues à la transmission directe des altérations chromosomiques à la descendance. Les télomères jouent un rôle important dans la ségrégation des chromosomes en mitose ainsi que dans les étapes préliminaires aux divisions méiotiques. Cette thèse est consacrée à la compréhension des mécanismes prenant place aux télomères pour garantir une ségrégation correcte des chromosomes en méiose. Le travail effectué durant cette thèse montre qu'un processus dynamique de séparation des télomères prend place au cours de la méiose chez la levure à fission Schizosaccharomyces pombe. Les télomères se séparent en étapes, à partir du regroupement majeur trouvé en prophase I pour finalement aboutir à une séparation complète à l'issue de la méiose II. Les étapes de ce processus prenant place en méiose I sont dépendantes de la kinase Aurora aux télomères et du complexe Condensine. Cependant, les télomères ainsi que les bras des chromosomes sont capables de se séparer au cours de la méiose II en l'absence d'activité d'Aurora, malgré des défauts d'attachement des chromosomes et l'absence de Condensine. Ces travaux montrent l'importance des mécanismes qui permettent la séparation des bras des chromosomes et illustre les différences fondamentales entre la méiose I et la méiose II mais aussi celles qui existent entre la méiose II et la mitoseChromosome segregation is a highly critical stage of cell division. Indeed, mistakes in chromosome transmission lead to aneuploidy, a hallmark of cancer. Chromosome segregation errors during meiosis cause particularly detrimental congenital diseases (e.g. human trisomies), as resulting chromosome alterations impact the entire offspring genome. Several studies report that chromosome ends, telomeres, play important roles both in accurate mitotic segregation of chromosome arms and in stages priming meiotic divisions. This thesis is committed to the comprehension of mechanisms that operate at telomeres in order to secure proper meiotic chromosome segregation. Work conducted during this thesis uncover a dynamic telomere separation process during meiosis of fission yeast Schizosaccharomyces pombe. Telomeres separate in stages, from the major telomere cluster of prophase I up to a complete separation by the end of meiosis II. The meiosis I stages of this process are dependent on Aurora kinase at telomeres and on Condensin complex. However, telomeres and chromosome arms are able to separate during meiosis II in the absence of Aurora function, despite erroneous chromosome attachments and lack of Condensin. This work highlights the importance of the mechanisms that allow chromosome arm separation and illustrates the fundamental differences between meiosis I and meiosis II but also between meiosis II and mitosis
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Islam, Shariful, Eric Vick, Bryan Huber, Carla Morales, Catherine Spier, Laurence Cooke, Eric Weterings, and Daruka Mahadevan. "Co-targeting aurora kinase with PD-L1 and PI3K abrogates immune checkpoint mediated proliferation in peripheral T-cell lymphoma: a novel therapeutic strategy." IMPACT JOURNALS LLC, 2017. http://hdl.handle.net/10150/627065.
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Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate similar to 30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4(+)/CD8(+) cells, we hypothesized that Program Death Ligand-1 (PDL1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3K alpha (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (similar to 9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-kappa B expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) similar to 30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI > 90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.
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Sirot, Fabienne. "Dynamique de localisation de la kinase mitotique Aurora-A et caractérisation de la protéine passagère TD-60 au cours de la mitose." Phd thesis, Université Joseph Fourier (Grenoble), 2005. http://tel.archives-ouvertes.fr/tel-00011481.
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De nombreuses kinases participent au bon déroulement de chaque étape du cycle cellulaire. Chez les eucaryotes supérieurs, les kinases Aurora-A et Aurora-B, structuralement très proches, exercent des rôles fondamentaux durant la mitose. Aurora-A est une protéine localisée au niveau des centrosomes, impliquée dans le cycle de division du centrosome et la formation du fuseau mitotique. Aurora-B est une protéine passagère localisée sur les centromères et qui migre, en anaphase, sur le sillon de division et se concentre en cytocinèse sur le corps résiduel. Aurora-B est responsable de la phosphorylation massive, en mitose, du résidu Serine 10 de l'histone H3. Par un système de pseudo-génétique, j'ai ciblé, dans l'extrémité amino-terminale de Aurora-A, le domaine responsable de sa localisation centrosomique. Ces expériences ont montré que les domaines catalytiques de Aurora-A et Aurora-B possèdent tous deux un signal de localisation centromérique. Mais, à l'inverse de Aurora-B, le domaine catalytique de Aurora-A ne se transfert pas des centromères vers le sillon de division en anaphase. Ces travaux montrent également que Aurora-A est capable d'assurer une partie des fonctions mitotiques de Aurora-B. J'ai par ailleurs identifié et cloné la séquence de la protéine passagère TD-60 de Xenopus laevis. J'ai exprimé des domaines protéiques de la protéine xTD60, afin de générer un anticorps spécifique de TD-60 de xénope. Des expériences de co-sédimentation de complexes protéiques d'extraits mitotiques d'œufs de xénope et des expériences d'immunolocalisation cellulaire nous permettent d'envisager pour xTD-60 des fonctions plus larges que celles attribuées aux protéines passagères.
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Mooser, Daniela [Verfasser], Christian [Akademischer Betreuer] [Gutachter] Peschel, and Florian C. [Gutachter] Bassermann. "Untersuchungen zur Resistenz gegenüber Aurora Kinase Inhibitoren in Myc-induzierten Lymphomen / Daniela Mooser ; Gutachter: Florian C. Bassermann, Christian Peschel ; Betreuer: Christian Peschel." München : Universitätsbibliothek der TU München, 2017. http://d-nb.info/1127728504/34.
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