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1
Dust, Sofia [Verfasser]. "Biochemical characterization, regulation, and inhibition of human transcription kinases CDK12 and CDK13 and human cell cycle-related kinase CDK14 / Sofia Dust." Bonn : Universitäts- und Landesbibliothek Bonn, 2019. http://d-nb.info/1223538028/34.
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Lianga, Noel. "Cdk1 Regulates Anaphase Onset." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31860.
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Cdk1 is an important cell cycle regulator that, in association with different cyclin regulatory subunits, is responsible for signaling important cell cycle events in all eukaryotic cells. In budding yeast, inhibition of Cdk1 by selective deletion of cyclin subunits has been shown to prevent anaphase onset, suggesting that Cdk1 activity is critically important for triggering anaphase onset. In many eukaryotes, Cdk1 has been shown to phosphorylate subunits of the anaphase promoting complex (APC), an E3 ubiquitin ligase which directly signals anaphase onset by triggering the degradation of the anaphase inhibitor securin. It is currently unclear, however, whether the APC is the sole essential substrate of Cdk1 in anaphase onset or if Cdk1 triggers anaphase onset by phosphorylating additional proteins. Eukaryotic Cdk1 is regulated by the Wee1 family of tyrosine kinases and the Cdc25 family of phosphatases which directly oppose Wee1 activity. Wee1 phosphorylation of Cdk1 on a single tyrosine residue inhibits Cdk1 and has been shown to prevent or delay mitotic entry. In this work we sought to further elucidate the mechanism through which Cdk1 regulates anaphase onset. We showed that, in addition to regulating mitotic entry, the budding yeast Wee1 kinase and Cdc25 phosphatase (Swe1 and Mih1 respectively in S. cerevisiae) regulate anaphase onset by modulating Cdk1 activity. Activation of Swe1 delays anaphase onset and cells lacking SWE1 enter anaphase prematurely, demonstrating that Swe1 regulates anaphase onset in unperturbed cell cycles. Deletion of the CDC55 regulatory subunit of PP2A has been shown to bypass cell cycle delays due to Swe1 activation. We showed that this is due, in part, to PP2ACdc55 dephosphorylation of Cdk1 sites on the APC. We have also shown that Cdk1 directly phosphorylates separase, the protease that dissolves sister chromatid linkages upon release from inhibitory securin/separase complexes upon APC-mediated securin degradation. Similar to phosphoregulation of the APC, we showed that Cdk1 phosphorylation of separase is opposed by PP2ACdc55. Phosphoregulation of separase appears to be important for regulation of the separase substrate Slk19 which cooperates with the conserved kinesin-5 Cin8 and microtubule bundling protein Ase1 to regulate spindle elongation at the spindle midzone.
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Chun, Stella Soyoung. "Identification and validation of CDK13 interacting proteins." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43130.
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Cyclin dependent kinases (CDKs) are components of signal transduction pathways that regulate cellular functions by phosphorylation of substrate proteins in response to upstream signals. The kinase domains of CDK12 and CDK13 are most similar to CDK9; CDK9 phosphorylates the C terminal domain (CTD) of RNA Polymerase II in order to stimulate processive transcription elongation. However, while most human CDKs consist of little more than a kinase domain, CDK12 and CDK13 are much larger and have several protein-protein interaction domains suggesting that they could participate within regulatory cascades. They also have a RS domain found in the SR protein family of splicing factors. Consistent with these features CDK12 and CDK13 co-localize with splicing factors and RNA Polymerase II in nuclear speckles. Based on these features CDK12 and CDK13 have been proposed to coordinately regulate splicing and transcription. Consistent with this hypothesis, both kinases phosphorylate the CTD of RNA polymerase II and regulate the alternative splicing of the Adenovirus E1a mini-gene model substrate. CDK12 has been found to interact with the splicing factors PRP19, CDC5L, RBM25, FBP11 and SRP55. Due to the similarity of CDK13 to CDK12, I investigated the interacting partners of CDK13 by immunoprecipitation and mass spectrometry and determined that CDK13 interacts with same splicing factors as CDK12. These interactions were validated by immunoprecipitation – western blot analysis. My results also indicated that PRP19 and CDC5L interact as a complex with CDK13. Therefore, the protein interaction partners of CDK13 and CDK12 suggest functional mechanisms for their ability to regulate splicing. In parallel projects, to begin investigating the functional roles of the kinase domain of CDK12 I constructed and expressed different CDK12 mutants in insect cells and in mammalian cells. Also to investigate the role of the CDK12 mutants and the protein-protein interactions of CDK13 in alternative splicing, I also developed a PCR based E1A mini-gene splicing assay.
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Moreira, Juliana. "Expressão e purificação da quinase dependente de ciclina 13 humana em sistema bacteriano." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-28082014-135313/.
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As quinases dependentes de ciclinas são proteínas que podem ser divididas de acordo com a sua atuação no ciclo celular ou no controle transcricional, elas se tornam ativas em determinadas etapas do ciclo celular dependendo do seu grau de fosforilação e de sua ligação com ciclinas e proteínas inibitórias, e exercem sua função fosforilando outras proteínas envolvidas no ciclo de divisão celular e transcrição influenciando suas atividades, garantindo que cada processo do ciclo ocorra em uma sequência ordenada. A CDK13 faz parte da família de proteínas quinases dependentes de ciclina, pode se ligar a ciclinas do tipo L ou K, regula os eventos de \"splicing\" alternativo, e interage com a proteína Tat do vírus HIV atuando como um possível fator de restrição, sendo que sua superexpressão diminui a produção de algumas proteínas virais suprimindo a produção do vírus. O DNA referente à CDK13 é replicado em células cancerosas, principalmente dos tipos hepático e cólon e reto, sendo um alvo para inibidores para tratamento de câncer. A fim de contribuir para o estudo dessa proteína, o projeto tem como objetivo expressá-la utilizando métodos de tecnologia de DNA recombinante. A sequência de DNA referente à CDK13 foi amplificada pela reação em cadeia da polimerase, após sua purificação, foi inserida no vetor pCR-Blunt e clonada em células de E. coli DH5α competentes. Porém, o DNA não foi liberado pela reação com as enzimas de restrição BamHI e NdeI. As bactérias Rosetta(DE3) transformadas com um plasmídeo sintético e crescidas em meio de auto-indução expressaram a CDK13. Após lise celular e purificação em coluna de Ni2+, a proteína foi detectada por Western Blot. Já as bactérias Rosetta(DE3) transformadas com o plasmídeo sintético modificado (o qual compreende a região do DNA que expressa o bolsão de ligação da CDK13), e induzidas em meio LB expressaram a CDK13, porém não foi possível purificá-la em coluna de afinidade ao Ni2+.The cyclin-dependent kinases are proteins that can be classified by their function in the cell cycle or transcriptional control. They are activated in particular steps of the cell cycle depending on their phosphorylation degree, cyclin binding and inhibitory proteins. They act phosphorylating other proteins involved in the cell cycle and transcriptional control, influencing in their activities, ensuring that each step of the cell cycle occur in an ordered sequence. The CDK13 is one of the cyclin-dependent kinases family member, it can bind to L or K cyclins, regulates the alternative splicing and interact with HIV Tat protein, acting as a possible restriction factor, its overexpression decreases the production of some viral proteins, and suppresses the virus production. The DNA corresponding to CDK13 is replicated in cancer cells, mainly of hepatic and colon rectal types; therefore it is a target for inhibitors for cancer therapy. In order to contribute for the studies of this protein, the goal of the project is to express it using methods of recombinant DNA technology. The DNA sequence corresponding to CDK13 was amplified by polymerase chain reaction, after its purification, it was inserted to pCR-Blunt vector and cloned into E. coli DH5α competent cells. However, the DNA wasn\'t released by the BamHI and NdeI restriction enzymes. The Rosetta(DE3) cells transformed with a synthetic plasmid pET28a::CDK13 and grown in auto-induction media expressed the CDK13. After cell lysis and purification by Ni2+ affinity colum, the protein was identified by Western Blot. However, the Rosetta(DE3) cells transformed with the modified synthetic plasmid (that comprehends the DNA region which expresses the binding pocket region) induced in LB media, expressed the CDK13. Yet, it wasn\'t possible to purify the protein in the Ni2+ affinity column.
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Krämer, Thomas. "Gastrointestinale Stromatumoren (GIST) : CD117-Expression und klinischer Verlauf /." Würzburg, 2007. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000253020.
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Dixon-Clarke, Sarah. "Structure and inhibition of novel cyclin-dependent kinases." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:3c6955c9-469a-4f4b-9577-309ccb57b742.
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Protein phosphorylation by members of the cyclin-dependent kinase (CDK) family determines the cell cycle and regulates gene transcription. CDK12 and CDK16 are relatively poorly characterised family members containing atypical domain extensions and represent novel targets for structural studies, as well as cancer drug discovery. In this thesis, I developed protocols to express and purify the human CDK12 kinase domain in complex with its obligate partner, CycK. I solved three distinct crystal structures of the complex providing insights into the structural mechanisms determining CycK assembly and kinase activation. These structures revealed a C-terminal kinase extension that folded flexibly across the active site of CDK12 to potentially gate the binding of the substrate ATP. My structures also identified Cys1039 in the C-terminal extension as the binding site for the first selective covalent inhibitor of CDK12, which has enormous potential as a pharmacological probe to investigate the functions of CDK12 in the DNA damage response and cancer. I also identified rebastinib and dabrafenib as potent, clinically-relevant inhibitors of CDK16 and solved a co-crystal structure that defined the extended type II binding mode of rebastinib. Preliminary trials using these relatively non-selective compounds to inhibit CDK16 in melanoma and medulloblastoma cancer cell lines revealed rebastinib as the more efficacious drug causing loss of cell proliferation in the 1-2 micromolar range. Use of the co-crystal structure to design more selective derivatives would be advantageous to further explore the specific role of CDK16. Finally, I identified a D-type viral cyclin from Kaposi's sarcoma-associated herpesvirus that could bind to the CDK16 kinase domain and interfere with its functional complex with human CycY causing loss of CDK16 activity. These studies provide novel insights into the structural and regulatory mechanisms of two underexplored CDK family subgroups and establish new opportunities for cancer drug development.
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Bondke, Alexander. "Design and synthesis of selective CDK7 inhibitors." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/43965.
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Cyclin-dependent protein kinases (CDKs) have a central role in the regulation of cell proliferation, apoptosis and gene expression. CDK7, in particular, not only regulates the activation of the cell cycle kinases CDK1, CDK2, CDK4 and CDK6, but is also involved in the regulation of transcription as part of the transcription factor TFIIH-complex. While a common feature of cancer is the over-expression of cyclin, there is compelling evidence that CDK2, CDK4 and CDK6 are not essential for the cell cycle, making CDK7 a highly attractive target for anti-cancer drug development. The two pyrazolo[1,5-a]pyrimidine kinase inhibitors BS181 and BS194 were chosen as starting points for the development of a CDK7 selective drug candidate. BS181 is a selective CDK7 inhibitor (IC50: 21 nM) that shows moderate growth inhibition in the MCF7 breast cancer cell line (GI50: 15 μM) as well as in the HCT116 colorectal carcinoma cell line (GI50: 16 μM). The compound suffers from an insufficient oral bioavailability and a poor pharmacokinetic profile. The biological data of the second lead compound BS194 are significantly different: it is a CDK2 pan-inhibitor (IC50: 3 nM) with excellent growth inhibition in MCF7 (GI50: 0.12 μM) and in HCT116 cancer cells (GI50: 0.12 μM). The compound is highly bioavailable and has a good PK profile. The properties of BS181 and BS194 needed to be 'merged' to create a CDK7 selective compound with good overall properties. The multidimensional optimisation of both compounds was driven by iterative circles of computer-aided drug design (CADD), synthesis and biological assessment. Thus, analogues with highly functionalised northern, southern and eastern side chains as well as a novel series of 2,4-diaminopyrimidine kinase inhibitors were designed and subsequently synthesised.
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Kamkar, Fatemeh. "Pftaire1 (Cyclin Dependent Kinase14): Role and Function in Axonal Outgrowth During the development of the CNS." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32860.
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Cyclin Dependent Kinase (Cdk) family members play a role in CNS development. Cyclin Dependent Kinase 5 (Cdk5) is well known for its fundamental role in neuronal development and axogenesis, as well as, cell death. Other Cdks include Pctaire and Pftaire. Inhibition of Pctaire results in increased axon outgrowth, however, the role and function of Pftaire is unknown. Pftaire1 is a novel member of the Cdk family that was initially detected in a screen for cdc2-like kinases. Unpublished data from our lab reveals that Pftaire1 (Eip63E) deficiency in Drosophila melanogaster results in defects in the axon and neuronal structure of the ventral nerve cord (VNC). In mammals, Pftaire1 is highly, expressed in the CNS. Here, we proposed that Pftaire1 might have a role in axon outgrowth. To investigate the role of Pftaire1 in mammals, the first germline Pftaire1 knockout mice were generated. Considering the severe effects of Eip63E deficiency in Drosophila and the homology between mammalian and fly Pftaire1, CNS defects in the mouse were anticipated. However, to date, no gross abnormalities have been detected in the overall morphology, fertility, life span, or anatomical brain structures of the Pftaire1 deficient mice. This may be due to the presence of other post-mitotic Cdk proteins that are highly similar to Pftaire1. For instance, mammals possess Pftaire (1, and 2), as well as, Pctaire (1, 2, and 3), while Drosophila only possess the Pftaire1 orthologue where the Pftaire2 and Pctaire (1, 2, and 3) are absent. Furthermore, the mice were of mixed background. In spite of this, we demonstrated that Pftaire1 deficient neurons showed increased axon length, in the initial phases of culture. This was confirmed by expression of dominant negative (DN) D228N-Pftaire1 in wild type neurons. Also classification of axons into different ranges, reveals a higher percentage of hyperextended neurites in D228N and Pftaire1 knockout mice. The mechanism by which Pftaire1 controls axon outgrowth is unknown. In this study we show that, Pftaire1 interacts physically with the small GTPase proteins Rac1, Cdc42, and RhoA. Importantly, we showed that Pftaire1 phosphorylates GDP-RhoA on a serine residue. We propose that this regulates RhoA activity, which in turn controls axon outgrowth.
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Dubbury, Sara Jane. "Cdk12 regulates DNA repair Genes by suppressing intronic polyadenylation." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/115596.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis. Vita.
Includes bibliographical references.
During transcription, cyclin-dependent kinases (CDKs) dynamically phosphorylate the C-terminal domain (CTD) of RNA Polymerase II (RNAPII) to recruit factors that coordinate transcription and mRNA biogenesis. Cdk12 phosphorylates Serine 2 (Ser2) of the RNAPII CTD, a modification associated with the regulation of transcription elongation, splicing, and cleavage/polyadenylation. Unlike other transcriptional CDKs that regulate most expressed genes, Cdk12 depletion abrogates the expression of homologous recombination (HR) genes relatively specifically, suppressing the HR DNA damage repair pathway and sensitizing cells to genotoxic stresses that cause replication fork collapse, such as Parp1 inhibitors. The proposed role for Cdk12 in regulating HR is clinically significant for two reasons. First, Cdk12 loss-of-function mutations populate high-grade serous ovarian carcinoma and castration-resistant prostate tumors raising the possibility that Cdk12 mutational status may predict the effectiveness of chemotherapeutics that target HR-deficient tumors. Second, readily available small molecule inhibitors of Cdk12 induce sensitization of HR-competent tumors to Parp1 inhibitors in vivo raising the possibility that inhibitors against Cdk12 could be used as chemotherapeutics. Despite this growing clinical interest, the mechanism behind Cdk12's regulation of HR genes remains unknown. Here we show that Cdk12 suppresses intronic polyadenylation (IPA) and that this mechanism explains the exquisite sensitivity of HR genes to Cdk12 loss. We find that Cdk12 globally enhances transcription elongation rate to kinetically suppress IPA events. Many HR genes harbor multiple IPA sites per gene, and the cumulative effect of these sites accounts for the increased sensitivity of HR genes to Cdk12. Finally, we find evidence that Cdk12 LOF mutations and deletions cause upregulation of IPA sites in HR genes in human tumors. Our results define the mechanism by which Cdk12 regulates transcription, mRNA biogenesis, and the HR pathway. This work clarifies the biological function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumor biomarker.
by Sara Jane Dubbury.
Ph. D.
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Maino, Marcelo Marafon. "Expressão imunoistoquímica de CD117 no carcinoma epidermóide de esôfago." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2008. http://hdl.handle.net/10183/53132.
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Objetivo: Investigar a expressão imunoistoquímica de CD117 em um grupo de pacientes com carcinoma epidermóide de esôfago Pacientes e Métodos: Vinte e sete pacientes com carcinoma epidermóide de esôfago submetidos à ressecção cirúrgica no Hospital de Clínicas de Porto Alegre da Universidade Federal do Rio Grande do Sul foram avaliados para imunoreatividade do CD117. Como grupo controle, foram utilizadas biópsias de mucosa esofágica de dez indivíduos saudáveis. A avaliação imunoistoquímica dos tecidos foi realizada com anticorpo monoclonal anti-CD117 (DAKO). Resultados: Foram avaliados 21 (78%) homens e 6 (12%) mulheres com idade média de 58 anos (36 a 77). A maioria dos pacientes apresentava estadiamento TNM IIb ou III, e a sobrevida média foi de 21 meses (2 a 72). A reação imunoistoquímica em membrana produzida pelo anticorpo anti-CD117 foi considerada positiva em 4 dos 27 dos casos analisados (15%) . Conclusões: Esses achados sugerem que CD117 deve ser investigado como marcador para o carcinoma epidermóide de esôfago. Estudos adicionais são necessários em outras amostras populacionais, para melhor definir o papel do CD117 nesses tumores.Aim: To investigate the CD117 expression in specimens of patients with squamous cell carcinoma of the esophagus (SCCE). Methods: A pilot study was performed for CD177 immunoreactivity, using a monoclonal antibody against CD117 (DAKO), on 27 esophageal squamous cell carcinoma specimens from patients who underwent surgical resection at the Hospital de Clínicas de Porto Alegre University Hospital, Faculty of Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. As a control group, specimens of esophageal mucosa obtained from 10 healthy subjects were also studied. Results: Twenty-one (78%) males and six (12%) females with median (sd) age of 58 (8) years, ranging from 36 to 77 years. Most of the patients were of TNM stage IIb or III and mean overall survival was 21 (2 to 72) months. Cytoplasmic membrane CD117 immunoreactivity was demonstrated in only 4 (15%) out of 27 tumors and in none of the controls (0%). Conclusions: These results suggest that the decreased expression of CD117 may be due to lack of control of the cell cycle in SCCE. Additional studies are needed to better define the role of the CD117 in such tumors.
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Büntemeyer, Tjark-Ole [Verfasser]. "Funktionelle Charakterisierung von CDK11 im Nierenzellkarzinom / Tjark-Ole Büntemeyer." Mainz : Universitätsbibliothek Mainz, 2020. http://d-nb.info/1214326390/34.
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Meschini, Elisa. "Purine-based dual inhibitors of CDK2 and CDK7." Thesis, University of Newcastle Upon Tyne, 2011. http://hdl.handle.net/10443/1363.
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Cyclin-Dependent Kinases (CDKs) play a fundamental role in eukaryotic cell cycle progression, particularly at cell cycle checkpoints, and are therefore important targets for anticancer drug discovery. Activation of CDK2 in complex with Cyclin A regulates entry into S phase of the eukaryotic cell cycle. CDK7, a dual-function enzyme, acts both as a CDK-Activating Kinase (CAK) and as a component of the general transcription factor TFIIH. However, experiments with MAT1-knockdown mice have shown that cell cycle arrest by CAK inhibition would not be detrimental for transcriptional activity in non-dividing cells, as CDK9 in complex with Cyclin T can perform transcriptional duties in the absence of TFIIH. Previous studies have resulted in the identification of NU6102 (1, IC50 mM = 0.005 (CDK2), 4.4 (CDK7)) as a potent and selective CDK2 inhibitor, and NU6247 (2, IC50 mM = 0.12 (CDK2), 0.23 (CDK7)) as an equipotent CDK2/7 inhibitor. It was shown that the sulfonamide group of 1 confers potency and selectivity for CDK2, whereas the pendant piperazinyl substituent of 2 diminishes CDK2 actvity whilst improving activity versus CDK7. S NH N N NH N O O O 2 N N AccordinglyAs part of the work described in the present thesis, sulfonamide 3 (IC50 mM = 0.012 (CDK2), 0.67 (CDK7)) was synthesised and found to be is a potent CDK2 inhibitor, but with some CDK7-inhibitory activity (IC50 mM = 0.012 (CDK2), 0.67 (CDK7)). Further elaboration of the side-chain function has enabled the development of structure-activity relationships (SARs), and the identification of purines (e.g. 4, IC50 mM = 2.6 (CDK2), 0.56 (CDK7)) exhibiting some selectivity for CDK7, albeit with a loss of potency. 4 Subsequent SAR studies conducted on 2 have enabled the following observations to be made: firstly, the purine 6-cyclohexylmethoxy substituent is necessary for activity, with the corresponding 6-unsubstituted purine (5, IC50 mM = 46.9 (CDK2), 20.8 (CDK7)) exhibiting a 100-fold loss of potency against both CDK2 and CDK7. A terminal basic group (e.g. piperazinyl in 2) is required for activity, as replacement by a cyclohexyl substituent results in loss of activity against both kinases (6, 11% inhibition at 10 mM (CDK2), 13% inhibition at 100 mM (CDK7)). The sulfone linker is not a prerequisite for CDK7 activity, with the simple alkylpiperazine derivative (7, IC50 mM = 0.48 (CDK2), 0.51 (CDK7)) exhibiting comparable potency and selectivity. Finally, there appears to be some opportunity for expansion into the gatekeeper pocket of CDK7 by introducing small substituents at the purine C-8 position, with the potential for selectivity over CDK2 (8, 47% inhibition at 100 mM (CDK2), IC50 = 5 mM (CDK7)). Isolation and biological evaluation of the vinyl sulfone 9, an intermediate in the synthesis of 2, indicated a time-dependent inhibition of CDK2, suggesting that 9 is an irreversible inhibitor of CDK2. This would be the first reported irreversible inhibitor of a cyclin-dependent kinase, and therefore the activity of the compound against CDK2 was investigated using protein crystallography and site-directed mutagenesis 5 techniques. From these studies, encouraging evidence has emerged that 9 acts as an irreversible inhibitor of CDK2, covalently binding to a lysine residue within the ATP-binding pocket.
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Chan, Wai-man Vivian. "Functional characterization of liver intestine-cadherin (CDH17) in hepatocellular carcinoma." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B37424671.
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Chan, Wai-man Vivian, and 陳慧雯. "Functional characterization of liver intestine-cadherin (CDH17) in hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B38891116.
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Aldridge, Roland Christopher Lochore. "Investigating the influence of CDK11 in developmental and cancer phenotypes." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31527.
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Cyclin-Dependent Kinase 11 (CDK11) is a serine/threonine kinase encoded at human locus 1p36.3 by two paralogous genes CDK11A and CDK11B. CDK11 has diverse roles in the regulation of transcription, splicing, apoptosis and mitosis. In proliferating cells, two predominant isoforms are expressed: CDK11p58 and CDK11p110. CDK11p110 is expressed throughout the cell cycle and regulates transcription and splicing. CDK11p58 is expressed at mitosis via IRES-dependent translation; it mediates mitotic progression and faithful chromosome segregation. Loss of Cdk11 in murine models causes early embryonic lethality, demonstrating that CDK11 is essential for normal development. Furthermore, dysregulated CDK11 expression is associated with numerous late-onset disease states, indicating its importance in adult life. In cancer, abnormal expression of CDK11 correlates with poor prognosis in a variety of tumours. Moreover, deletion of the chromosomal region 1p36.3, containing the CDK11 locus, is frequently observed in cancer and has recently been identified in a case of the development disorder, Cornelia de Lange Syndrome (CdLS). This thesis aimed to examine the functions of CDK11 and the impact of their dysregulation in cancer and developmental phenotypes. The initial aim was to investigate the novel role for CDK11 in regulating autophagy in cancer cells; CDK11 depletion causes a marked autophagy phenotype, with accumulation of autophagy protein LC3. I demonstrate that this CDK11-mediated autophagy occurs as a consequence of mitotic dysregulation. Subsequently, I examined the role of autophagy following aberrant mitosis and chromosome missegregation. I show that autophagy is important in the maintenance of aneuploid karyotypes, with loss of autophagy impairing the survival of aneuploid cell populations. I then investigated the effects of CDK11 in regulating cancer cell motility and determined that CDK11 depletion retards cancer cell migration. However, I was unable to identify any failure in cell adhesion or cell polarization to explain this migration phenotype. Subsequently, I interrogated the CDK11 interactome to further characterize the mechanisms through which CDK11 regulates both novel and established functions. This work indicated the involvement of the distinct CDK11 isoforms in pathways that have not previously been reported. This included the interaction of CDK11p110 with ribosomal and spliceosomal proteins during mitosis and the interaction of CDK11p58 with spliceosomal and proteosomal constituents also during mitosis. These findings may provide the foundation for further study. Finally I describe work undertaken to sequence the CDK11 locus in a cohort of CdLS patients, with no known causative genetic mutation, to investigate CDK11A/CDK11B as candidate disease-associated genes. Although no causative mutation in CDK11A or CDK11B was identifying, sequencing of this region indicated NCBI and UCSC genome assemblies of this locus were inaccurate due to the genomic duplication. This has been confirmed by others and corrected in the most recent genome assemblies.
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Santos, Níkolas Paparidis Ferreira dos. "Obtenção das quinases dependentes de ciclinas CDK9 e CDK11 humanas utilizando um sistema bacteriano de expressão (E. coli)." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-16062015-091524/.
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As CDKs 9 e 11 fazem parte da subfamília de CDKs transcricionais, e portanto desempenham papéis de controle na dinâmica atividade de síntese e processamento do RNA mensageiro pela RNA Polimerase II. Devido à sua capacidade de modular individualmente a atividade dos complexos de transcrição da RNAP II, estas quinases assumem uma grande importância para a regulação da expressão gênica em células eucarióticas. Casos de desregulação da atividade de CDKs transcricionais têm sido frequentemente relacionados a diversas patologias humanas graves, incluindo vários tipos de câncer e também a AIDS (em razão do papel essencial desempenhado pela CDK9 na replicação do HIV. O objetivo deste trabalho é a obtenção das CDKs humanas 9 e 11 através da expressão heteróloga em Escherichia coli, buscando o aperfeiçoamento de métodos para produzir estas enzimas em quantidade e pureza suficientes para aplicação em estudos estruturais. A utilização de um sistema bacteriano oferece muitas vantagens práticas, como a simplicidade da técnica, baixo custo, altos níveis de expressão, e elevado rendimento na purificação do produto. No entanto, a obtenção de quinases humanas ativas a partir de E. coli sempre representou um desafio experimental, devido aos problemas comumente associados à expressão heteróloga. Os resultados apresentados aqui demonstram a possibilidade de se obter a CDK9 humana enzimaticamente ativa pelo reenovelamento in vitro da proteína expressa pela bactéria na forma de corpos de inclusão, após etapas de solubilização e purificação em condições desnaturantes. Por outro lado, a CDK11 humana só pôde ser obtida em uma versão encurtada, consistindo apenas no seu domínio quinase, devido à forte inibição que um trecho N-terminal da sequência mostrou exercer sobre a expressão da proteína. Assim, este trabalho fornece exemplos de como é possível superar algumas das adversidades comuns da expressão heteróloga a fim de se obter CDKs humanas ativas empregando o sistema bacteriano.CDK9 and CDK11 are members of the subfamily of transcriptional CDKs and therefore play central roles in the control of the dynamic activity of messenger RNA synthesis and processing by RNA polymerase II. Because of their ability to individually modulate the activity of RNAP II transcription complexes, these kinases are of great importance for the regulation of gene expression in eukaryotic cells. Several cases of deregulation of the transcriptional CDKs have been linked to important human diseases, including various types of cancer and also AIDS (due to the essential role of CDK9 in HIV replication). The objective of this work is to obtain human CDK9 and CDK11 heterologously expressed in Escherichia coli, seeking improved methods to produce these enzymes in quantity and purity suitable for structural studies. A bacterial system offers many practical advantages to protein production, such as technical simplicity, low costs, high levels of expression and high purification yield. However, it has always been an experimental challenge to obtain active human kinases from E. coli, because of the problems commonly associated with heterologous expression. The results presented here demonstrate the possibility of obtaining the enzymatically active human CDK9 by in vitro refolding of the protein expressed as bacterial inclusion bodies, after its solubilization and purification under denaturing conditions. Human CDK11, on the other hand, could only be obtained in a shortened form consisting just of its kinase domain, due to the strong inhibition that an N-terminal stretch exerts on the protein\'s own expression. Therefore, this work provides examples of how it is possible to overcome some of the common adversities of heterologous expression in order to obtain active human CDKs through the bacterial system.
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Zeng, Fanli. "Novel Modes of Regulation of Cyclin Dependent Kinase Cdk1." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/133357.
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Les quinases dependents de ciclina (CDKs) dirigeixen la progressió del cicle cel·lular a
les cèl·lules eucariotes. A l’organisme eucariota model Saccharomyces cerevisiae
(llevat de gemmació) una sola quinasa dependent de ciclina, Cdk1, és essencial i
suficient per dirigir el cicle cel·lular. Unida alternativament a ciclines de fase G1, S i
G2/M, Cdk1 regula els programes transcripcionals del cicle cel·lular, la replicació i
segregació dels cromosomes, la dinàmica del fus mitòtic, el creixement cel·lular polar,
la morfogènesi, etc. La desregulació de l’activitat CDK promou la proliferació
descontrolada i la inestabilitat genòmica.
Donada la seva funció essencial en la progressió del cicle cel·lular, Cdk1 és
estretament regulada per proteïnes associades (les ciclines, Cks1, i inhibidors de les
CDKs –CKIs) i per modificacions post-traduccionals. Tanmateix, molts detalls de la
regulació de Cdk1 romanen desconeguts, com és el cas de com les activitats CDK de
fase G1 o de fase M són inhibides en resposta a determinats estressos cel·lulars.
Quan la progressió del cicle cel·lular és amenaçada per la presència d’estressos
genotòxics tals com l’estrès replicatiu o la presència de dany al DNA, un mecanisme de
vigilància, l’anomenat checkpoint de la fase S, s’activa per tal de protegir la integritat
del genoma. Al llevat de gemmació, el checkpoint de la fase S és mediat per la quinasa
Mec1 (ATR/ATM a humans) i la seva quinasa efectora Rad53 (Chk2 a humans).
Per explorar si la quinasa efectora Rad53 regula Cdk1 en resposta a estrès
genotòxic, hem explorat dues qüestions principals: (1) la fosforilació de Cdk1 per
Rad53 i (2) la regulació per Rad53 de proteïnes associades a Cdk1.
Pel que fa a la primera qüestió, aprofitant un assaig quinasa in vitro amb Rad53,
mostrem que Cdk1 és fosforilat directament per Rad53. Hem identificat
proteòmicament dos llocs de Cdk1 (Ser46, Ser258) fosforilats per Rad53 in vitro. Les
cèl·lules dirigides per l’al·lel no-fosforilable (Cdk1-2A) mostren un fenotip wee,
compatible amb una activitat CDK incrementada/desregulada. Les cèl·lules dirigides
per l’al·lel fosfomimètic (Cdk1-2E) són allargades i més grans que les cèl·lules
silvestres, compatible amb una activitat CDK reduïda. A més, assignem i quantifiquem
les diferents formes fosforilades de Cdk1 in vivo mitjançant electroforesi Phos-tag.
Respecte la segona qüestió, hem identificat proteòmicament proteïnes associades
a Cdk1 en presència d’estrès replicatiu en forma dependent de Rad53. Hem estudiat en
detall el producte del gen de funció desconeguda YPL014W, que bategem Cip1 (per
Cdk1 Interacting Protein 1), que obté la puntuació més alta en l’anàlisi. Les nostres
dades mostren que Cip1 és una proteïna regulada al llarg del cicle cel·lular. A més,
l’abundància de Cip1 s’incrementa en forma dependent de Rad53 en presència d’estrès
replicatiu. La sobre-expressió de Cip1 bloqueja les cèl·lules a fase G1 i estabilitza el
CKI de fase S Sic1 in vivo. A més, Cip1 interacciona específicament amb el complex de
fase G1 Cln2-Cdk1, però no amb el complex de fase S Clb5-Cdk1 or el de fase M
Clb2-Cdk1. Cip1 inhibeix l’activitat Cln2-CDK tant in vivo com in vitro. Els nostres
resultats suggereixen que Cip1 pot ser un nou CKI de l’activitat CDK de fase G1.Cyclin dependent kinases are drive cell division cycle progression in eukaryotic cells. In the model eukaryotic organism Saccharomyces cerevisiae (budding yeast) a single Cyclin Dependent Kinase, Cdk1, is essential and sufficient to drive the cell cycle. Alternately bound to G1, S and G2/M phase cyclins, Cdk1 regulates cell cycle transcriptional programs, chromosome replication and segregation, spindle dynamics, polarized cell growth, morphogenesis, etc. Misregulated CDK activity induces unscheduled proliferation as well as genomic instability. Given its essential function in cell cycle progression, Cdk1 is tightly regulated by binding partners (cyclins, Cks1 and Cyclin dependent Kinase Inhibitors -CKIs) and post-translational modifications. However, many details on Cdk1 regulation remain unknown, such as how G1 or mitotic CDK activities are inhibited in response to challenging conditions. When the cell cycle progression is challenged by genotoxic stress such as DNA replication stress or DNA damage, a surveillance mechanism, the S phase checkpoint is activated to protect the integrity of the genome. In the budding yeast the S phase checkpoint is mediated by the Mec1 kinase (ATR/ATM in humans) and its downstream effector kinase Rad53 (Chk2 in humans). To explore whether the effector kinase Rad53 regulates Cdk1 in response to genotoxic stress, we have been exploring two main avenues: (1) Cdk1 phosphorylation by the S phase checkpoint effector kinase Rad53 and (2) Rad53 dependent regulation of Cdk1 associated factors. With respect to the first question, taking advantage of a Rad53 in vitro kinase assay, we show that recombinant Cdk1 is directly phosphorylated by Rad53. We also proteomically identified two sites of Cdk1 (Ser46, Ser258) phosphorylated by Rad53 in vitro. Cells carrying the non-phosphorylatable Cdk1 allele (Cdk1-2A) display a wee phenotype, compatible with increased/unrestrained CDK activity. Cells carrying the phosphomimetic Cdk1 allele (Cdk1-2E) are elongated and larger in size than wild type cells. Moreover, we also assign and quantify the different phosphorylation forms of Cdk1 in vivo using Phos-tag electrophoresis technology. With respect to the second question, we have proteomically identified proteins associated with Cdk1 in the presence of replication stress in a Rad53 dependent manner. The product of the unknown function gene YPL014W, which we name Cip1 (for Cdk1 Interacting Protein 1), with the highest score, is further studied. Our data shows that Cip1 is a cell cycle regulated protein. In addition, the abundance of Cip1 increases in a Rad53 dependent manner upon DNA replication stress. Overexpression of Cip1 blocks cells in G1 and stabilizes the S-phase-Cdk1 inhibitor Sic1 in vivo. Moreover, Cip1 specifically interacts with G1 phase Cln2-Cdk1 but not with S phase Clb5-Cdk1 or M phase Clb2-Cdk1. Cip1 inhibits Cln2-CDK activity both in vivo and in vitro. Our finding suggests that Cip1 may be a novel CKI of G1 phase CDK activity.
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Chen, Jian 1969. "Regulation of CAK activity of Cdk7 in Drosophila melanogaster." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82842.
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Cdk7 (Cyclin-dependent kinase 7) is conserved from yeast to human and involved in multiple functions. Cdk7 acts as a CAK (Cdk activating kinase) in a trimeric complex with Cyclin H and Mat1. The CAK activity is required for the full activation of the Cdks that directly regulate the cell cycle transitions. In addition, Cdk7 is the kinase subunit of TFIIH, the general transcription/DNA repair factor IIH. TFIIH is required for the general transcription of messenger RNAs by RNA polymerase II and for the transcription-coupled nucleotide excision repair functions. As in other systems, Drosophila Cdk7 has multiple functions. In order to understand how different functions of Cdk7 are regulated, I performed genetic screens to identify the regulators or downstream factors of multiple functions of Cdk7. Several candidate dominant suppressors and enhancers were identified in these screens. One strong suppressor of cdk7, xpd, encodes another subunit of TFIIH. The genetic suppression by xpd attracted me to further characterize the biological significance of this interaction. I showed that Xpd does have a novel function in regulating CAK activity of cdk7 , it down-regulates mitotic CAK activity. Furthermore, I found that Xpd protein levels are cell cycle dependent, being down-regulated at the beginning of the mitosis. Based on these data, I propose a model that mitotic down-regulation of Xpd results in increased CAK activity, positively regulating mitotic progression. Simultaneously, this down-regulation can be expected to contribute to the mechanisms of mitotic silencing of basal transcription.
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Woodbury, Erika L. "Regulation of spindle stability by Cdk1 and the APC." Diss., Search in ProQuest Dissertations & Theses. UC Only, 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:3261256.
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Zhu, Rui, and 朱睿. "Liver-intestine cadherin (CDH17) in hepatocellular carcinoma: molecular analysis and clinicalimplications." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43703793.
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Rafael, Tiago João Reuter Hirst de Sousa. "Proliferação de mastócitos em felinos. Imunomarcação para CD117 e MMP-9." Master's thesis, Universidade Técnica de Lisboa. Faculdade de Medicina Veterinária, 2011. http://hdl.handle.net/10400.5/3607.
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Dissertação de Mestrado Integrado em Medicina VeterináriaExistem, no gato, várias formas de proliferação anormal de mastócitos na pele e noutros tecidos. Embora em alguns casos se trate indubitavelmente de neoplasias, outros há que podem corresponder a reacções de hipersensibilidade. Opostamente ao cão, não há um sistema de classificação nos felinos que permita prever a evolução clínica, embora, normalmente, se tratem de processos benignos. No presente estudo, procurou-se caracterizar vários casos de mastocitoma cutâneo, extracutâneo, mastocitose cutânea e sistémica, para alguns parâmetros epidemiológicos, clínicos e histiopatológicos, comparando estes dados com o que foi observado anteriormente noutros estudos. Recorreu-se a métodos de imunohistoquímica para a proteína de membrana ckit (CD117) e para a metaloproteinase MMP-9 de modo a definir a sua utilidade como indicadores de prognóstico, à semelhança do que foi feito para o cão. Verificaram-se poucas diferenças entre esta amostra e estudos recentes e concluiu-se que o padrão de expressão da marcação para c-kit pode realmente ser indicativo do comportamento biológico destes tumores. Quanto à marcação para a MMP-9 não foi possível tirar idênticas conclusões.
ABSTRACT - FELINE MAST CELL PROLIFERATION. CD117 AND MMP-9 IMMUNOSTAINING - In the cat there are different forms of abnormal mast cell proliferation in the skin and other tissues. Even though some may undoubtedly be viewed as neoplasia, there are others that might as well be considered a mere hypersensitivity reaction. Contrarily to the dog, there is no classification system that helps to predict the outcome of these lesions, even thought they are normally benign. In the present study, some epidemiologic, clinical and histopathologic parameters were characterized for cases of mast cell tumors of the skin, non cutaneous mast cell tumors, systemic mastocytosis and cutaneous mastocytosis, comparing this data to what has been observed in previous studies. Immunohistochemistry methods were also used to identify the membrane protein c-kit and MMP-9 in order to estimate their usefulness as prognostic tools in the same way they are used in dogs. Few differences were seen between this data and recent studies. It was concluded that the expression pattern of c-kit may indeed be linked to the biologic behavior of these tumors. As for the immunostaining of MMP-9, it was not possible to achieve such conclusions.
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Heusse, Emmanuelle. "Expression de TAL1 et de CD117 dans les leucémies aigües myéloi͏̈des." Paris 5, 1999. http://www.theses.fr/1999PA05P166.
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Link, Patrick. "THE ROLE OF THE MECHANICAL ENVIRONMENT ON CD117+ ENDOTHELIAL CELL ANGIOGENESIS." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5970.
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Angiogenesis is a complex process coordinating cell migration, proliferation, and lumen formation. Changes to the microenvironment regulate angiogenesis through mechanotransduction and cytokine signals. In pulmonary hypertension, something in the process becomes abnormal, resulting in changes to the microenvironment and the formation of a glomerulus of dysfunctional capillaries, called a plexiform lesion. Endothelial cells, expressing CD117 (CD117+ EC clones) increase in the plexiform lesions of pulmonary hypertension, independent of pro-angiogenic VEGF signaling. We hypothesize that the mechanical environment and the macromolecular composition of the extracellular matrix, both, contribute to the aberrant angiogenesis. When we changed the mechanical environment, we changed the angiogenic potential and cellular phenotype of CD117+ Endothelial cell clones. Turbulent flow, pathologic substrate stiffness, and pathologic stretch increased Endothelial-to-mesenchymal markers, such as acta2, cnn1, snail, and slug in CD117+ EC clones while CD117- ECs showed minimal change. We perturbed the mechanical environment of CD117+ EC clones and identified changes in Bone Morphogenic Protein-2, an often overlooked pro-angiogenic cytokine. We coupled changes in the mechanical environment to Rho GTPase intracellular signaling, to predict how changes to the mechanotransduction would affect angiogenesis through a computational model. In our model of angiogenesis, we found vessel synchronicity to depend on both which cell undergoes mitosis, and also at which phase of GTPase cycling the cell undergoes mitosis. We believe changes to the GTPase cycling may be the mechanism linking mechanotransduction to the abnormal vessels found in pulmonary hypertension. We are the first group to look at the role of the ECM composition, independent of stiffness. Our results show diseased ECM composition alone leads to phenotypic changes indicative of PH progression. In conclusion, these results provide a possible cytokine implicated in the mechanotransduction of PH, established a computational model of angiogenesis which provides a mechanotransduction mechanism of disease progression, and established that the ECM composition alone is capable of phenotypic changes leading to disease progression.
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Soni, Deena. "Studies on regulation of mitotic transition by cyclin B1/CDK1." Connect to text online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1099070698.
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Thesis (Ph. D.)--Case Western Reserve University, 2005.[School of Medicine] Department of Environmental Health Sciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.
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Lamas, Cíntia Betite. "Clonagem, expressão e purificação da quinase dependente de ciclina 10 (CDK10) humana." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-02022015-112005/.
Full textAbstract:
Quinases dependentes de ciclinas (CDKs) compreendem uma família de proteínas que podem ser subdivididas em dois grupos funcionais majoritários baseados na sua função no ciclo celular e/ou controle transcricional. Já foram identificadas mais de 30 CDKs humanas. A CDK10 é uma proteína quinase dependente de ciclina pertencente ao grupo de quinases relacionadas à Cdc2. CDK10 é essencial na fase G2/M do ciclo celular, possivelmente no progresso dessa fase, monitorando a replicação completa do DNA e permitindo que as células passem desse ponto de restrição. Essa proteína é um importante determinante de resistência à terapia endócrina para câncer de mama. Portanto, é um alvo potencial para o desenvolvimento de inibidores, uma vez que está presente em células cancerosas. O estudo da estrutura e função da CDK10 deverá ser realizado após sua clonagem, expressão e purificação. O cDNA da CDK10 foi amplificado por reação em cadeia da polimerase (PCR), e posteriormente, o produto foi aplicado em gel de agarose para análise e purificação. O vetor de clonagem recombinante foi obtido, o qual foi clonado em células competentes e sequenciado. A obtenção do plasmídeo recombinante para expressão deu-se pela inserção do DNA nos vetores de expressão pET28a(+) e pET23a(+) (Novagen). A proteína foi expressa em células competentes e analisada por eletroforese em gel de poliacrilamida. Em seguida, a proteína obtida foi purificada em coluna de afinidade por níquel e em coluna de afinidade por ATP. Com a otimização dos resultados obtidos, será possível, futuramente, caracterizar bioquimicamente a CDK10 e elucidar suas estruturas secundária e terciária. O estudo da CDK10 irá contribuir para o entendimento da sua relação estrutura-função e sua relação com oncogenes e supressores de tumor, e o estudo estrutural para o desenvolvimento de inibidores químicos de baixo peso molecular que possa inibir especificamente a CDK10.Cyclin-dependent kinases (CDKs) comprise a family of proteins that can be subdivided into two major groups based on their functional role in cell cycle and / or transcriptional control. Over 30 human CDKs have been identifies. CDK10 is a cyclin-dependent kinase protein that belongs to the cdc2-related kinases group. CDK10 is essential in phase G2 / M of the cell cycle, possibly in the progress of this phase, monitoring the complete DNA replication and allowing the cells to pass through this restriction point. This protein is an important determinant of resistance to endocrine therapy for breast cancer. Therefore, it is a potential target for the development of inhibitors, since it is present in cancerous cells. The study of the structure and function of CDK10 must be performed after its cloning, expression and purification. cDNA of CDK10 was amplified by polymerase chain reaction (PCR) and then the product was applied into agarose gel for analysis and purification The cloning vector was obtained, which was cloned into competent cells and sequenced. The obtaining of the recombinant plasmid for expression was due to the insertion of DNA into expression vectors pET28a(+) and pET23a(+) (Novagen). The protein was expressed in competent cells and analyzed by electrophoresis on polyacrylamide gel. Then, the obtained protein was purified by nickel affinity column and ATP affinity column. With the optimization of the results obtained, it will be possible, in the future, to biochemically characterize CDK10 and elucidate its secondary and tertiary structures. The study of CDK10 will contribute to the understanding of its structure-function relationship and its relationship with oncogenes and tumor suppressors, and the structural study for the development of low molecular weight chemical inhibitors that can specifically inhibit CDK10. The structural studies will contribute to the development of chemical inhibitors of low molecular weight that may this and other CDKs.
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Zhu, Rui. "Liver-intestine cadherin (CDH17) in hepatocellular carcinoma molecular analysis and clinical implications /." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43703793.
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Y, Hu Nie Kdam Siriwan Grisurapong. "Cultural factors in male adolescent alcohol use among ede ethnic minority in central highland Vietnam /." Abstract, 2008. http://mulinet3.li.mahidol.ac.th/thesis/2551/cd417/4938061.pdf.
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TAGLIALATELA, ANGELO. "CDK12 IS A NOVEL ONCOGENE WITH CLINICAL AND PATHOGENETIC RELEVANCE IN BREAST CANCER." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/219124.
Full textAbstract:
Breast cancer heterogeneity demands new reliable prognostic markers and therapeutic targets for the personalized management of patients. Over recent years, knowledge on the involvement of different types of kinases in cancer has guided the design of a variety of kinase inhibitors as novel molecularly targeted anti-cancer agents.
In a previous high-throughput screening performed by in situ hybridization (ISH) and immunohistochemistry (IHC) on breast cancer tissue microarrays (TMAs), the cyclin-dependent kinase 12 (CDK12) was found to be frequently overexpressed in breast cancer and its overexpression correlated with clinical/pathological parameters of aggressive disease. CDK12 was therefore proposed to be a novel prognostic biomarker in breast cancer.
In the present thesis, we extend these preliminary studies and demonstrate, by IHC analysis on TMAs comprising large cohorts of breast cancer patients, that CDK12 overexpression is significantly associated with clinical/pathological parameters of poor prognosis (high tumor grade, high Ki67 proliferative index, positive ERBB2 status) and with a higher risk of disease recurrence and death. We also show, either in human breast tumors or in breast cancer cell lines, that CDK12 overexpression is due to the amplification of the CDK12 gene.
Through the use of amenable cell models in diverse in vitro and in vivo assays, we provide evidences that CDK12 is a bona fide oncogene in breast cancer. By genome-wide expression analysis, we show that alterations in CDK12 expression are associated with changes in the transcription and splicing of genes involved in cancer-relevant cellular processes, such as DNA damage response, cell cycle and epithelial-to-mesenchymal transition.
In conclusion, we have established that CDK12 is a novel prognostic biomarker in breast cancer and represents a potential novel molecular target for therapeutic intervention in breast cancer.
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Ren, Ping. "Control of cytokinesis by the mitotic cyclin dependent kinase M-Cdk1." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/458616.
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La citocinesi és el darrer procés regulat del cicle de divisió cel·lular mitòtica eucariota.
Les cèl·lules entren en citocinesi un cop la segregació dels cromosomes s’ha
completat satisfactòriament. Durant la citocinesi les cèl·lules es separen físicament,
donant lloc a dues cèl·lules filles. Els defectes en el control de la citocinesi donen lloc
a aneuploïdies i inestabilitat genòmica. Un controlador clau de la citocinesi és
l’activitat Quinasa Dependent de Ciclina (M-Cdk1). El nostre projecte deriva de la
correlació observada entre l’entrada en citocinesi i l’eliminació de l’activitat M-Cdk1.
Complementària a aquesta observació, l’expressió d’un al·lel hiper-estable de la
ciclina mitòtica Clb2 bloca la citocinesi. Per tant, la mateixa activitat que empeny les
cèl·lules a entrar en mitosi i que hi promou l’anafase, impedeix que la citocinesi pugui
iniciar-se prematurament. Aquestes observacions suggereixen que una o més
proteïnes, essencials per disparar la citocinesi, són inhibides per fosforilació per MCdk1.
La identitat d’aquest/s substrat/s crític/s és desconeguda. Per tant, l’objectiu
d’aquesta tesi ha estat el d’avançar en la comprensió de com l’activitat M-Cdk1 evita
la citocinesi prematura durant l’anafase, a l’organisme eucariota model
Saccharomyces cerevisiae. El treball realitzat revela que, en presència dominant
d’activitat M-Cdk1 elevada, la Xarxa de Sortida de Mitosi (Mitotic Exit Network,
MEN) està, contra tot pronòstic, parcialment activada en presència d’activitat M-Cdk1
elevada i dominant, i és la responsable de l’alliberament de la fosfatasa Cdc14 al
citoplasma en aquestes condicions.Cytokinesis is the final regulated process in the eukaryotic mitotic cell division cycle. Cells enter cytokinesis once that chromosome segregation is satisfactorily completed. During cytokinesis cells physically separate, giving place to two daughter cells. Defects in the control of cytokinesis result in aneuploidies and genomic instability. A key controller of cytokinesis is the mitotic Cdk1 (M-Cdk1) activity. Our project derives from the observed correlation between the onset of cytokinesis and the termination of M-Cdk1 activity. Complementary to such observation, expression of a hyperstable allele of the mitotic cyclin Clb2, blocks cytokinesis. Thus, the very same activity that pushes cells into mitosis and promotes mitotic entry and anaphase, blocks the occurrence of premature cytokinesis. These observations suggest that one or more proteins, essential to trigger cytokinesis, are inhibited by M-Cdk1 phosphorylation. The identity of such critical M-Cdk1 substrate/s is unknown. Therefore, the goal of this thesis is to gain insight on how M-Cdk1 prevents premature cytokinesis during anaphase in the model eukaryotic organism Saccharomyces cerevisiae. The thesis work reveals that the Mitotic Exit Network is unexpectedly partially activated in the presence of prevailing high M-Cdk1 activity, and drives the release of the Cdc14 phosphatase to the cytoplasm under such conditions.
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Soni, Deena V. "Studies on the regulation of mitotic transition by cyclin B1/Cdk1." Case Western Reserve University School of Graduate Studies / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=case1099070698.
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Martin, Ina Verena. "Analysis of the structure and function of the S.pombe DNA ligase I protein Cdc17." Thesis, University of Edinburgh, 2002. http://hdl.handle.net/1842/11101.
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DNA ligases join breaks in double-stranded DNA and are therefore crucial enzymes in all aspects of DNA metabolism. Eukaryotic DNA ligase I homologues belong to the family of ATP-dependent ligases and join Okazaki fragments generated on the lagging strand during chromosomal DNA replication. DNA ligase I enzymes consist of C-terminal catalytic domains conserved across all ATP-dependent ligases, a middle conserved domain of unknown function and N-terminal extensions, which share a conserved PCNA binding motif, but otherwise show very limited sequence similarity. In this work structure-function analyses on the DNA ligase I homologue Cdc17 of Schizosaccharomyces pombe were performed. The presence of the middle conserved non-catalytic domain in addition to the catalytic domains was found to be essential for rescue of a cdc17 deletion strain. The N-terminal domain targets the enzyme to the nucleus and mitochondria and essential residues were identified which are required for targeting to both cellular compartments. Evidence suggests that mitochondrial Cdc17 function is required for survival of S. pombe. The PCNA binding motif is functional in vivo since its absence reduces cell viability. Despite these identified function overexpression of truncations lacking the N-terminal domain can complement a cdc17 deletion strain.
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Petretti, Clotilde. "Contrôle de la mitose par phosphorylation : une nouvelle fonction pour la protéine kinase CDK11." Rennes 1, 2006. http://www.theses.fr/2006REN1S137.
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La mitose est une étape fondamentale du cycle cellulaire qui aboutit à la répartition égale des chromosomes vers chacune des cellules filles. Les protéines kinases sont des enzymes-clé contrôlant plusieurs processus mitotiques. Les CDKs (Cyclin Dependent Kinases) sont une des grandes familles de protéines kinases dont certaines sont impliquées en mitose. Chez l’Homme, il en existe dix : CDK1 à CDK9 et CDK11. La sous-famille des protéines kinases CDK11 comprend deux isoformes : une forme courte CDK11p58 et une forme longue CDK11p110. Au cours de ce travail, J’ai étudié l’effet de la suppression de l’expression du gène CDK11 et démontré que la kinase était nécessaire à la progression de la mitose. J’ai également démontré que c’était l’isoforme courte CDK11p58 qui était la forme mitotique. La fonction de CDK11p58 est d’assurer la maturation du centrosome en contrôlant le recrutement des kinases mitotiques Aurora-A et Plk1
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Kroll, Sebastian Herbert Benjamin. "Towards the synthesis of selective CDK7 inhibitors as potential anti-cancer drugs." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/11657.
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Cyclin-dependent kinase 7 (CDK7) exhibits an interesting target for an anti-cancer therapy approach. CDK7’s triple role in phosphorylation (cell cycle, transcription, estrogen receptor (ER)) in cell regulation makes this kinase interesting. Phosphorylation of cell cycle CDK’s via its CAK-complex, of Ser-5 in RNA-PolII as part of the TFIIH-complex and phosphorylation of Ser-118 in ER all show the importance of this enzyme. Given that CDKs are over-expressed in many cancers, selective inhibition of CDK7 should result in cell cycle arrest and apoptosis predominately in tumour cells. Previously, BS-181 (Figure 0.1) has been reported as the first CDK7 selective inhibitor, which displayed a good in vitro and in vivo profile.1 Based on this initial lead compound, a library of rational-designed analogues was synthesised. Much of this library was based on a computer-aided-drug-design (CADD) approach by docking, which gave valuable insights in possible binding modes and helped to focus targeting the whole active site. [Molecular structure diagrams appear here. To view, please open pdf attachment] Figure 0.1: BS-181 and new analogues. Several of these novel inhibitors showed excellent selectivity versus CDK2 in particular, and potency against CDK7 in the 30 – 60 nM range for their IC50-values. Cellular assays confirmed the growth inhibitory properties of these new compounds, with GI50-values in the low μM range. This work also demonstrates what functional groups were tolerated in the 3-,5- and 7-position.
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Korsisaari, Nina. "Functional analysis of Cdk7-interacting proteins Mat1 and Hint in model organisms." Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/korsisaari/.
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Gamero, Angel Mauricio Castro. "Efeitos da Inibição Transcricional de Survivina e Cdk1 através do Ácido Tetra-O-Metil Nordihidroguaiarético em Células de Glioblastoma." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-29082013-140405/.
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O Glioblastoma é um dos tumores mais agressivos do sistema nervoso central e entre as diversas neoplasias possui um dos piores prognósticos. Mesmo com as novas estratégias de tratamento, a sobrevida de pacientes portadores de glioblastoma continua sendo muito baixa, sendo a temozolomida (TMZ) o agente mais comum usado no seu tratamento. O ácido tetra-o-metil nordihidroguaiarético (M4N), é um novo agente terapêutico que funciona como um repressor transcricional global de genes dependentes do fator de transcrição Sp1, tais como Survivina e Cdk1. No presente estudo, foram investigados os níveis de expressão do gene Survivina, suas variantes gênicas por splicing alternativo e Cdk1 em amostras tumorais e linhagens celulares de GBM. Adicionalmente, foram investigados os efeitos do M4N em combinação ou não com TMZ e/ou radiação em culturas primárias e linhagens celulares de GBM. Ensaios de qRT-PCR foram realizados para determiner a expressão de mRNA das variantes gênicas de Survivina e Cdk1. A proliferação celular foi analisada pelo ensaio XTT e os niveis de apoptose e variações do ciclo celular foram determinados por citometría de fluxo. Analises de combinação de drogas utilizando diferentes estratégias de administração (simultânea e seqüencial) foram realizados baseados no método de Chou-Talalay em linhagens celulares e culturas primárias de GBM. Para os ensaios de sobrevivência clonogênica, foram utilizadas as doses de 2, 4 e 6 Gy de radiação gamma. Todas as variantes por splicing alternativo de Survivina e o gene Cdk1 foram expressos em amostras (n=16) e linhagens celulares (n=6) de GBM, exceto a variante Survivina-2B que apenas foi expressa nas linhagens celulares de GBM. O tratamento com M4N diminuiu a expressão de Cdk1, Survivina e a variante Survivina-Ex3, enquanto que houve um aumento da expressão da variante Survivina-2B. O M4N diminuiu a proliferação celular de forma isolada e sinérgicamente quando combinada com TMZ. Além disso, o M4N aumentou os efeitos da radiação, principalmente quando associado com TMZ. O M4N causou morte celular apoptótica, diminuição do índice mitótico e parada do ciclo celular principalmente na fase x G2/M. Os resultados do presente estudo sugerem a potencial aplicação clínica de M4N em combinação com TMZ e radiação no tratamento do GBM.Glioblastoma (GBM), one of the most human malignant neoplasia, responds poorly to current treatment modalities, being temozolomide (TMZ) the most used drug in its treatment. TetraO-methyl Nordihydroguaiaretic Acid (M4N) is a global transcriptional repressor of genes dependents of Sp1 transcription factor, such as Survivin and Cdk1. In this study was evaluated the gene expression of Survivin, their spliced-variants and Cdk1 in GBM samples and cell lines. Moreover, it was investigated the effects of M4N combined or not with TMZ and/or radiation on primary cultures and cell lines of GBM. qRT-PCR assays were performed to determine the Survivin-spliced variants and Cdk1 gene mRNA expression in GBM tumor samples and cell lines. Cell proliferation was measured by XTT assay and cell cycle and apoptosis were determined by flow cytometry. Drug combination analyzes using different schedules of administration (simultaneous and sequential) were performed based in ChouTalalay method on GBM cell lines and primary cultures. For clonogenic survival, it was used the doses of 2, 4, and 6 Gy of gamma radiation. All Survivin-spliced variants and Cdk1 gene were expressed in GBM samples (n=16) and cell lines (n=6), except the Survivin-2B variant that was only expressed in GBM cell lines. M4N treatment down regulated the expression of Cdk1, Survivin and Survivin-Ex3 variant, while the Survivin-2B variant was up-regulated. M4N decreased the cell proliferation separately and synergistically with TMZ, moreover it enhanced the radiation effects, mainly when associated with TMZ. M4N also induced apoptotic cell death, decreased mitotic index and arrested the cell cycle mainly in G2/M phase. Our results suggest a potential clinical application of M4N in combination with TMZ and radiation in GB treatment.
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Bernis, Cyril. "Caractérisation de nouveaux mécanismes de régulation de la kinase mitotique Cdk1-cycline B." Montpellier 2, 2007. http://www.theses.fr/2007MON20246.
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Le but de ce travail a été de caractériser de nouvelles voies de régulation de la kinase mitotique Cdk1 associée à la protéine régulatrice, Cycline B. La cycline B se lie à Cdk1 et l'active, ceci est nécessaire au bon déroulement de la mitose. Malgré cela, la kinase est maintenue inactive avant la mitose suite à la phosphorylation par les kinases Myt1 et Wee1 sur la thréonine 14 et la tyrosine 15 de Cdk1. Ce sont les phosphatases de la famille de Cdc25 qui permettent de déphosphoryler ces sites et d'activer Cdk1-cycline B lors de la transition G2/M. Ce complexe Cdk1-cycline B est inactivé lors de la sortie de mitose quand la cycline B est ubiquitiné (par le complexe E3-ubiquitine ligase APC (Anaphase Promoting Complex)) et dégradée par le protéasome. Nos travaux ont montré que Pin1 (une peptidyl-prolyl isomérase) empêche la protéolyse de l'inhibiteur mitotique Emi1 (Early Mitotic Inhibitor) en phase G2. En effet le rôle d'Emi1est de permettre l'accumulation de la cycline B, en bloquant l'APC avant l'entée en mitose. Par la suite nous avons montré qu'Emi1 était également stabilisé par Pin1 au cours de la méiose dans les ovocytes de xénope. D'autre part, une autre étude a permis de montrer que la protéine kinase MPS1 (requise au départ lors du point de contrôle du fuseau mitotique) était impliquée dans la transition G2/M, probablement en régulant négativement l'activité de Cdc25. Enfin, nous avons montré que l'isoforme Cdc25C, outre ses fonctions lors de la transition G2/M, est requise pour le maintient de l'activité de Cdk1-cycline B lors de la métaphase de deuxième division de méiose dans les ovocytes de xénope. De façon surprenante, lors de ce travail nous avons mis en évidence que la kinase Wee1, qui est montrée comme inactive en phase M, possède malgré tout une activité de phosphorylation non négligeable à ce niveau, sur Cdk1
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El, Dika Mohammed. "Régulation de la phase M du cycle cellulaire par CDK1, PP2A et CDC6." Thesis, Rennes 1, 2013. http://www.theses.fr/2013REN1S068.
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L'objectif de cette thèse est de mieux comprendre la régulation de la phase M du cycle cellulaire. Nos expériences ont été effectuées dans des extraits acellulaires d’embryons de Xenopus laevis. Tout d'abord, nous montrons que le moment de l'entrée en phase M est précisément déterminé par un équilibre entre l'activité de la protéine kinase CDK1 et l’activité d’une protéine phosphatase sensible à l'acide okadaïque, PP2A. Nous montrons également le rôle de la protéine CDC6 dans la régulation de l'entrée dans la première phase M embryonnaire. En effet, CDC6 inhibe CDK1 et à travers cette action régule la dynamique de cette kinase lors de l'entrée et de la progression en phase M. Ces résultats mettent en évidence un nouveau contrôle qui précise le moment du clivage embryonnaire. Ce contrôle joue un rôle clé dans la coordination entre les mécanismes de régulation du cycle cellulaire et le programme de développement de l'embryonThe aim of this thesis is to understand better the regulation of the M-phase of the cell cycle. Experiments were done in cell-free extracts of Xenopus laevis one-cell embryos. Firstly, we show that the timing of the M-phase entry is precisely determined by a balance between the activity of CDK1 kinase and okadaic acid sensitive phosphatase, mainly PP2A. Secondly, we show the role of CDC6 protein in regulation of the entry into the first embryonic M-phase. CDC6 inhibits CDK1 and through this action regulates the dynamic of this kinase upon M-phase entry and during M-phase progression. This mechanism discovered during my PhD allows controlling precisely the timing of embryonic cleavage. This control plays a key role in coordinating the cell cycle regulating machinery and the development program of the embryo
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Ji, Jun-Yuan. "Functions of Cdk1-cyclin B in regulating the early embryonic mitoses in Drosophila /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/5124.
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Šupák, Marek. "Příprava myších monoklonálních protilátek proti cyklin-dependentní kináze 13." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2019. http://www.nusl.cz/ntk/nusl-401868.
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The aim of this master‘s thesis is to prepare a monoclonal antibody against cyclin-dependent kinase 13 (CDK13). The theoretical part focuses on antigen-antibody binding, which is essential for the use of monoclonal antibodies in the determination of CDK13 as well as the transcription that this kinase affects. This section is also devoted to Western blot and ELISA methods for detection of newly generated antibodies. Furthermore, the antibodies and the antigen definition are stated, which are later on discussed. The practical part is devoted to the preparation of antigen - its isolation and purification on a peristaltic pump. It also addresses immunization, its course, and the amount of antigen used to immunize mice. After immunization, the work focuses on fusion of sp-2 cells and splenocytes, which were first removed from the immunized mouse and purified. After the fusion alone, selection of hybridomas on HAT selection medium is mentioned, followed by detection first by ELISA and later by Western blotting. The resulting hybridomas with positive ELISA response are frozen for further testing at the Veterinary Research Institute in Brno, where the entire practical part of this thesis was carried out. These frozen hybridomas are further tested by immunoprecipitation to conclude this thesis.
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CHIAVETTA, Roberta. "Study of the combined effects of CDK1 inhibitors and senolytic drugs for the clearance of aneuploid-senescent cells." Doctoral thesis, Università degli Studi di Palermo, 2022. http://hdl.handle.net/10447/533837.
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Despite the progresses in discovering new therapeutic drugs and treatments, cancer is still one of the main causes of death. The biggest part of available treatments, moreover, is not always effective against tumour spread and it also has negative effects on the healthy tissues of the individual. For this reason, it is extremely relevant to find new strategies to avoid side effects during the anti-cancer therapies. Aneuploidy, an aberrant number of chromosomes in the cell, is a typical condition of cancer cells caused mainly by segregation errors and chromosomal instability (CIN). CIN is a process by which higher rate of chromosome segregation defects occurs by different mechanisms (chromosome mis-alignments, spindle alterations, mitotic defects cytokinesis failure…) resulting in aneuploidy that, by inducing proteotoxic stress, energy stress and DNA damages, affects proliferation of normal cells. On the other hand, CIN and aneuploidy allow cancer cells to escape pathways leading to cell death (apoptosis), cell cycle arrest and cellular senescence. The reactivation of pathways leading to apoptosis or cellular senescence is a powerful strategy to halt proliferation of cancer cells. In particular, cellular senescence, an irreversible cell cycle arrest, presents itself as an effective mean to stop proliferation of cancer cells that could be then killed specifically, for example by senolytic drugs. Generally, cellular senescence originates from a G1 arrest of the cell cycle caused by DNA damage or other cellular stresses/alterations (such as defects in chromosome segregations).
The cyclin-dependent kinase CDK1 is the most overexpressed kinase in malignant tumours compared to other CDKs. Its function is important for the correct cell cycle progression and its action is highly regulated, in order to preserve the right progression of mitosis. Once CDK1 is degraded, cell can successfully ultimate mitosis, proceeding from anaphase to telophase. Evidence showed how CDK1 and not its partner cyclin B1 is the main responsible of the right progression of mitosis1.
Aneuploidy has been suggested also as a trigger for cellular senescence and aging. Thus, I hypothesized that inducing a cell cycle arrest in the G2/M phase by inhibiting CDK1 could trigger mitotic errors leading to a subsequent G1 arrest and likely senescence of cancer cells. To this aim I have inhibited CDK1 by RNAi and the selective inhibitor RO-3306, an ATP-competitor which interferes with CDK1 activation, in cultured cancer cells and not transformed aneuploid human cells. Moreover, as a positive control of senescence, the flavonoid Curcumin was used due to its known senescence inducer activity in cancer cells. After senescence induction, cells have been treated with the senolytic drugs Fisetin and Quercetin in order to specifically eliminate senescent cells. Globally, my work shows that CDK1 depletion, by RNAi and pharmacological inhibition, leads to G2/M arrest followed by p21waf1/cip1 rise and triggers cellular senescence in cancer cells as shown by SA-βGal positive cells. Accordingly, even though with different mechanisms, Curcumin has induced cell cycle arrest in the G2/M phase, as well as senescence, in tumor cells. Interestingly, the percentage of senescent cells reduces following treatment with the drugs Quercetin and Fisetin, confirming the senolytic action of these compounds. Thus, combining CDK1 inhibition with senolytic drugs can be a powerful strategy for the clearance of aneuploid – senescent cancer cells.
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Dávila, André Vieira Peixoto. "Caracterização do gene CDK10 putativo e análise de sua possível atuação nos endociclos de Rhynchosciara americana." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-13092011-164136/.
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Rhynchosciara americana é estudada desde a década de 50 quando foi evidenciada a amplificação gênica em regiões de seus cromossomos politênicos. Os fenômenos de amplificação e a formação destes cromossomos gigantes se dão por meio da ocorrência de endociclos, regulados pela oscilação na atividade do complexo ciclinaE/CDK2. Nas glândulas salivares de nosso modelo, durante o desenvolvimento, há ocorrência de cromossomos politênicos. Foi seqüenciada uma mensagem de uma quinase dependente de ciclina (CDK10) que não apresenta qualquer ligação com os endociclos, descrita na literatura. Este transcrito teve sua sequencia revelada por experimentos de 5\'RACE. A sequência genômica foi parcialmente determinada. O perfil de expressão deste gene foi determinado nos ovários, glândulas salivares e corpo gorduroso. A presença da proteína CDK10 foi determinada por experimentos de western blot em glândulas salivares. A localização celular foi determinada nos ovários. Resultados sugerem a existência de duas isoformas deste gene nos tecidos analisados.Rhynchosciara americana is studied since the 50s decade when gene amplifications was discovered in some regions of its polytene chromosomes. The phenomena of amplification and the formation of these giant chromosomes happens through the endocycles, regulated by an activity oscillation of the complex CyclinE/CDK2. It is known that, in the salivary glands of our model, during it is development, there is the occurrence of these polytene chromosomes, formed since the beginning of the larval life. It was sequenced a message of CDK10, a cyclin depended kinase that shows no participation with the endocycles, as described at the literature. This transcript was fully sequenced by 5\'RACE experiments. Its genomic sequence was partially determined. The relative expression pattern was determined for ovaries, salivary glands and fat body. The CDK10 protein was observed by western blot experiments in the salivary glands. Its cellular location was determined at the ovaries. Results suggest the existence of two isoforms of the RaCDK10 gene at the tissues analyzed.
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Simonovic, Sinisa [Verfasser]. "The role of cyclin-dependent kinase 18 (CDK18) in clear cell renal cell carcinoma / Sinisa Simonovic." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2021. http://d-nb.info/1234984814/34.
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Bonnet, Christine. "Un motif sur la cycline B nécessaire à l'activation de CDK1 chez la levure ?" Paris 6, 2002. http://www.theses.fr/2002PA066509.
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Choi, Sung Hugh. "The Role of Dynamic Cdk1 Phosphorylation in Chromosome Segregation in Schizosaccharomyces pombe: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/453.
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The proper transmission of genetic materials into progeny cells is crucial for maintenance of genetic integrity in eukaryotes and fundamental for reproduction of organisms. To achieve this goal, chromosomes must be attached to microtubules emanating from opposite poles in a bi-oriented manner at metaphase, and then should be separated equally through proper spindle elongation in anaphase. Failure to do so leads to aneuploidy, which is often associated with cancer. Despite the presence of a safety device called the spindle assembly checkpoint (SAC) to monitor chromosome bi-orientation, mammalian cells frequently possess merotelic kinetochore orientation, in which a single kinetochore binds microtubules emanating from both poles. Merotelically attached kinetochores escape from the surveillance mechanism of the SAC and when cells proceed to anaphase cause lagging chromosomes, which are a leading cause of aneuploidy in mammalian tissue cultured cells. The fission yeast monopolin complex functions in prevention of mal-orientation of kinetochores including merotelic attachments during mitosis. Despite the known importance of Cdk1 activity during mitosis, it has been unclear how oscillations in Cdk1 activity drive the dramatic changes in chromosome behavior and spindle dynamics that occur at the metaphase/anaphase transition. In two separate studies, we show how dynamic Cdk1 phosphorylation regulates chromosome segregation. First, we demonstrate that sequential phosphorylation and dephosphorylation of monopolin by Cdk1 and Cdc14 phosphatase respectively helps ensure the orderly execution of two discrete steps in mitosis, namely sister kinetochore bi-orientation at metaphase and spindle elongation in anaphase. Second, we show that elevated Cdk1 activity is crucial for correction of merotelic kinetochores produced in monopolin and heterochromatin mutants.
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Souza, João Ricardo Malheiros de. "Injeção intracitoplasmática de espermatozoide: métodos de ativação oocitária e desestabilização da membrana espermática." Universidade Federal de Santa Maria, 2017. http://repositorio.ufsm.br/handle/1/11701.
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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPqDevelopment of bovine embryos produced by intracytoplasmic sperm injection (ICSI) is low compared to fertilized embryos. Deficient oocyte activation, inappropriate sperm capacitation, and lack of sperm decondensation are thought to be the main constrains affecting ICSI success in cattle. In the present study, activation compounds were tested to establish an effective protocol for activation of bovine oocytes, and then used for oocyte activation after ICSI. The activation approach consisted of exposing in vitro matured oocytes to Ionomycin (ION) following by a specific CDK1 inhibitor (RO-3306), a specific PKC activator (OAG) or both RO+OAG. In the first experiment, the rate of activation (pronuclear (PN) formation), cleavage, development to the blastocyst stage, and number of cells per blastocyst were evaluated after oocyte treatment. The PN rates were higher (P≤0.01) in the groups activated with ION+RO (48.5 %) and ION+RO+OAG (65.6 %) compared to ION (12.3 %) and ION+OAG (9.2 %). There was no significant effect between the RO concentrations tested (5, 7.5 and 10 μM) on oocyte activation. The PN rate was significantly higher (P≤0.01) when oocytes were exposed to RO for 240 min (84.6 %) compared to 60 (53.6 %) and 120 min (60.0 %). However, there was no difference between groups when treatment with RO started at 0, 30 or 60 min after on ION exposure. Cleavage rate was higher in ION+RO (70.2 %) and ION+RO+OAG (62.4 %) groups compared to ION (11.8 %) and ION+OAG (22.8 %). Blastocyst rate was also higher in the ION+RO+OAG (24.1 %) group, but not statistically different between ION+RO (19.7 %) and ION+OAG (9.5 %) groups. There was no development to the blastocyst stage after treatment with ION alone. The average cell number in blastocysts was not statistically different among treatments. In the second experiment, the effect of activation with ION+RO (10 μM for 240 min) was tested after ICSI using control (ICSI-Cont) or treated by electroporation (ICSI-El) sperm. Most oocytes presented a well-developed female PN (66.4%). Male PN formation was higher (P≤0.05) in the ICSI-El (33.3%) compared to the ICSI-Cont (9.4%) group. In conclusion, this study revealed that the specific inhibition of CDK1 after ION treatment is an effective approach to activate bovine oocytes. Male pronuclear formation after ICSI is increased by sperm electroporation, but is lower than female pronuclear formation. This indicates that deficient sperm decondensation and male PN formation rather than deficient oocyte activation is likely the main problem to develop an effective protocol for bovine ICSI.
O desenvolvimento de embriões bovinos produzidos por injeção intracitoplasmática de espermatozóides (ICSI) é baixo em relação aos embriões fertilizados. Deficiência na ativação de oócitos, capacitação inadequada de espermatozóides e falta de descondensação de espermatozóides são os principais transtornos que afetam o sucesso de ICSI em bovinos. No presente trabalho, foram testados métodos de ativação com o intuito de estabelecer um protocolo efetivo para a ativação de oócitos bovinos após a ICSI. Para isso, um inibidor específico de CDK1 (RO-3306) e um ativador específico de PKC (OAG) foram utilizados após a incubação com Ionomicina (ION) para avaliar a retomada da meiose em oócitos bovinos. Além disso, a incubação em meio Fert durante 6 h e a eletroporação (El) de espermatozoides previamente a ICSI foram testadas para verificar o efeito sobre a descondensação do espermatozoide e formação do pró-núcleo (PN) masculino. Inicialmente oócitos bovinos foram incubados, com diferentes concentrações e períodos de exposição aos tratamentos. Foram avaliados conforme a taxa de ativação oocitária (formação de PN), clivagem, desenvolvimento a blastocisto e número de células nos embriões que se desenvolveram a blastocisto. As taxas de PN foram maiores (P≤0,01) nos grupos ativados com ION+RO (48,5 %) e ION+RO+OAG (65,6 %) comparado com ION (12,3 %) e ION+OAG (9,2 %). Não houve efeito significativo entre as concentrações 5,0, 7,5 e 10,0 μM de RO sobre a taxa de ativação. A taxa de ativação foi significativamente maior (P≤0,01) em oócitos tratados com RO por 240 min (84,6 %) comparado a 60 (53,6 %) e 120 min (60,0%). No entanto, não houve diferença significativa na taxa de ativação quando o tratamento com RO foi iniciado a 0, 30 ou 60 minutos após a incubação com ION. A taxa de clivagem foi inferior nos grupos ION (11,8 %) e ION+OAG (22,8 %) comparada aos grupos ION+RO (70,2 %) e ION+RO+OAG (62,4 %). A taxa de blastocistos também foi maior no grupo ION+RO+OAG (24,1 %), mas não houve diferença estatística entre os grupos ION+RO (19,7 %) e ION+OAG (9,5 %). Não houve desenvolvimento a blastocisto quando os oócitos foram tratados somente com ION. Não foi detectada diferença estatística entre os tratamentos sobre o número médio de células por blastocistos. No segundo experimento, espermatozoides não tratados (ICSI-Cont) ou tratados por electroporação (ICSI-El) foram microinjetados em oócitos, os quais foram ativados com ION+RO por 240 min e fixados cerca de 15 h após a injeção para determinar a taxa de formação de PNs masculino e feminino (2PN). A maioria dos oócitos apresentaram o PN feminino bem desenvolvido (66,4 %). A formação de 2PN (masculino e feminino) foi maior no grupo ICSI-El (33,3 %) comparado ao grupo ICSI-Cont (9,4 %). Em conclusão, esse estudo demonstrou que a inibição específica da CDK1 após o tratamento com ION promove a ativação de oócitos bovinos. O tratamento de espermatozoides com eletroporação melhora a formação do PN masculino após ICSI, mas a taxa é inferior a formação do PN feminino. Esses resultados indicam que a deficiente descondensação do espermatozoide é o principal limitante para estabelecer um protocolo eficaz para ICSI em bovinos.
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Piard, Juliette. "Déficience intellectuelle : identification de nouveaux gènes par une approche multicentrique." Thesis, Bourgogne Franche-Comté, 2018. http://www.theses.fr/2018UBFCE005.
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La déficience intellectuelle (DI) touche 1 à 3% de la population générale avec un excès de sujets de sexe masculin. Cette affection est caractérisée par une extrême hétérogénéité clinique et génétique rendant son élucidation complexe. La révolution technologique des outils permettant l’analyse du génome intervenue depuis les années 2000 avec l’analyse chromosomique sur microréseau et singulièrement depuis 2010 avec les applications du séquençage à haut débit a considérablement facilité l’identification de nouveaux gènes. Nous avons tiré avantage de ce phénomène pour identifier trois affections neurologiques à caractère familial Nous avons procédé selon une méthodologie structurée pour conduire, grâce à la mise en place d’un réseau de collaborations, à la découverte ou à la confirmation de l’existence de nouvelles formes de DI. 1.Séquençage de l'exome couplé à la recherche de variations du nombre de copies 2. Mise à jour d’une altération de séquence génique potentiellement causale retrouvée chez le cas index et chez les autres sujets atteints de la famille 3.Extension des résultats à d’autres familles par la constitution d’une cohorte de réplication 4.Élaboration d’une série d’études fonctionnelles venant conforter l’hypothèse de causalité par la création d’un modèle animal et/ou la réalisation d’études biochimiques spécifiquesL’application de cette méthodologie nous a permis de conduire à terme trois projets : L’individualisation d’une forme syndromique de DI récessive autosomique associée à une malformation du rachis cervical et liée aux mutations bi-alléliques de CDK10. La caractérisation d’une encéphalopathie récessive autosomique létale associée à une hypertonie sévère et à une arthrogrypose distale liée aux mutations bi-alléliques d’ATAD1. L’implication de FRMPD4 dans une nouvelle forme de DI non syndromique liée à l’XIntellectual disability (ID) impacts 1 to 3% of the general population with an excess of affected males. This condition is characterized by an extreme clinical and genetic heterogeneity making the deciphering of its causes more complex. The technological revolution that took place in the study of the genome over the last two decades has provided a useful tool for identification of new genetic entities. This is particularly true for chromosomal micro-array analysis since early 2000s and for next generation sequencing since 2011. We took advantage of this by identifying the molecular basis of three singular conditions. We applied a structured methodology and created a network of collaborations to define or confirm these new ID syndromes. 1. Whole exome sequencing alongside with array-CGH 2.Identification of a candidate gene sequence alteration in the index case and other affected patients of the family 3.Constitution and study of a replication cohort 4.Biochemical studies and/or animal models in order to support the assumption of causalityBased on this research strategy, we were able to complete the following projects : Discovery of a syndromic form of autosomal recessive ID associated with cervical spine defects due to bi-allelic CDK10 mutations. Identification of an ATAD1-related profound and lethal autosomal recessive encephalopathy with stiffness and distal arthrogryposis. Characterization of a FRMPD4-related X-linked non-syndromic ID
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Carrieri, Francesca Anna. "Molecular and biochemical characterisation of phosphorylation-dependent NICD1 turnover : novel roles for CDK1 and CDK2." Thesis, University of Dundee, 2018. https://discovery.dundee.ac.uk/en/studentTheses/ff090523-366c-4fd7-a4f7-964e5bc2b285.
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In the developing vertebrate embryo, segmentation initiates very early and in a species-specific manner through the formation of somites, which later give rise to the vertebral column, most skeletal musculature and dermis. Somites are gradually and periodically pinched off from the anterior end of the presomitic mesoderm (PSM). The rhythmicity of somitogenesis is regulated by a molecular clock called the segmentation clock that drives the periodic expression of a number of "clock" genes in the PSM, with a periodicity that matches somite formation. Clock genes belong to the Notch, Wnt and FGF pathways, and Notch has been shown to be essential in mouse and chick for somite formation and for dynamic expression of clock genes. Since Notch signalling does not use second messengers, the pathway activity is exclusively driven by concentration of Notch intracellular domain (NICD). NICD itself is unstable and NICD production appears as pulsatile, spatiotemporal waves that traverse the rostrocaudal axis of the PSM in the manner of a clock gene. In a recent study from the Dale lab, using computational modelling together with experimental manipulation involving pharmacological inhibitors, it was shown that increasing the half-life of NICD and thus increasing its turnover time increases the period of the somitogenesis clock. However, that study did not determine the mechanism of action of the inhibitors used. NICD stability is a key requisite for its activity and to date the regulation of stability has been attributed to phosphorylation of the PEST domain by two kinases namely cyclin-dependent kinase-8 (CDK8) and GSK-3β, which then recruits the SCF Sel10/FBXW7 E3 ubiquitin ligase complex, that targets NICD for degradation by the proteasome. However, many of these studies have been conducted through overexpression assays in vitro and none of this regulation has been verified in the presomitic mesoderm. In this thesis, we demonstrated, for the first time, the interaction between NICD and FBXW7 at endogenous levels. We further identified phosphorylated residues within human NICD and we showed that a highly conserved site is crucial for NICD recognition by FBXW7. We also demonstrate for the first time that two new kinases can phosphorylate hNICD1 in the domain where this crucial residue lies Lastly, we demonstrate, using a variety of assays, that inhibition of these two kinases leads to increased levels of hNICD1 in vitro and mNICD1 in vivo and that this treatment also delays both the mouse somitogenesis clock and somite formation in the mouse PSM.
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Martens, Adam Arai. "Caracterização do gene CDK7 e análise de sua possível atuação nos endociclos de Rhynchosciara americana." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-26072012-142501/.
Full textAbstract:
CDKs são proteínas responsáveis pela ativação e progressão do ciclo celular e também apresentam função na ativação e alongamento na transcrição. Dentre elas, CDK7 atua em ambas as funções, fosforilando as CDKs do ciclo celular e fazendo parte do fator geral de transcrição TFIIH como subunidade catalítica. A partir de uma biblioteca de ESTs de glândula salivar, construído com mRNAs presentes durante o período em que ocorre o início do último ciclo replicativo da politenização e início da amplificação gênica, foram encontradas poucas mensagens relacionadas diretamente com o ciclo celular, correspondendo a 3.11% do ESTs. Dentre elas, foram encontradas as mensagens de Cdc2-like e Cdk7; portanto, foi realizada a caracterização do gene Cdk7 e a análise de sua atuação durante o desenvolvimento larval de Rhynchosciara americana. O gene Cdk7 apresenta 4 éxons, mais que em vertebrados. A sequência completa do mRNA foi obtida a partir de RACE, apresentando 1230 bases e uma ORF de 1020 bases. Perfis de expressão foram determinados por RT-PCR e Western blots. Modificações pós-traducionais foram analisadas por imunoblots em 2D. Seu perfil de expressão de mRNA e proteína apresentam variações durante o ciclo celular e entre os tecidos analisados; os imunoblots mostram a presença de uma fosforilação e possíveis modificações na cadeia lateral de alguns aminoácidos. O estudo de proteínas relacionadas ao ciclo celular nesse modelo é importante para um melhor entendimento dos ciclos celulares incomuns presentes em diferentes tecidos de insetos.CDKs are proteins responsible for activation and progression of cell cycle and also work on the activation and elongation of transcription. Among them, CDK7 acts in both functions, phosphorylating cell cycle CDKs and as a part of general transcription factor TFIIH as its catalytic subunit. From an EST library of salivary gland, constructed with mRNAs present during the period when the beginning of the last polyteny replicative cycle occurs, it was found only few messages directly related to cell cycle, corresponding to 3.11% of the ESTs. Among them, it was found Cdc2-like and Cdk7; therefore, it was performed the characterisation of Cdk7 gene and the analysis of its role during the larval development of Rhynchosciara americana. Cdk7 gene presents 4 exons, more than in vertebrates. Complete mRNA sequence was obtained via RACE, presenting 1230 bases and an 1020 bases ORF. Expression profiles were determined by RT-PCR and Western blots. Posttranslational modifications were analysed by 2D immunoblots. Its mRNA and protein expression profiles presented variations during cell cycle and between the studied tissues; immunoblots showed the presence of one phosphorylation and possible modifications on the side chain of some amino acids. The study of proteins related to cell cycle in this model is important for a better understanding of uncommon cell cycles in different insect tissues.
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Leclerc, Vincent. "Caracterisation fonctionnelle de deux couples cdk-cycline, cdk7-cych et cdk8-cycc, chez la drosophile." Nice, 1999. http://www.theses.fr/1999NICE5330.
Full textAbstract:
La famille des kinases dependant des cyclines, cdk, a ete isolee pour son role dans le controle du cycle cellulaire. Il apparait maintenant que des cdk jouent un role dans d'autres processus cellulaires. J'ai travaille a l'identification de la fonction de deux couples cdk-cyclines chez la drosophile. La cycline c est un membre distant de la famille des cyclines dont la fonction est longtemps restee enigmatique. J'ai identifie cdk8, le partenaire kinase de cycline c. Les deux proteines forment in vivo un complexe stable comprenant la grande sous-unite de l'arn polymerase ii (pol ii). Cdk8-cycline c est capable de phosphoryler in vitro le domaine c-terminal (ctd) de la pol ii. Le complexe cdk8-cycline c joue donc, chez les eucaryotes superieurs, un role similaire a celui du complexe homologue srb10-srb11 chez la levure dans le controle de l'activite de la pol ii. Cdk7 et ses deux partenaires cych et mat1 ont ete isoles initialement pour leur capacite a phosphoryler et activer les cdk (activite cak pour cdk-activating kinase). Ces trois partenaires se retrouvent egalement au sein du facteur general de transcription tfiih, ou ils ont une activite ctd kinase. Cependant, la fonction physiologique de cdk7 reste controversee. Afin d'analyser la fonction cdk7 in vivo j'ai isole les adnc codant pour la kinase cdk7 de drosophile et son partenaire cycline h. J'ai realise des mutations dominantes negatives de la kinase (cdk7 d n). Leur expression dans l'embryon precoce n'induit pas de perturbation du cycle cellulaire. En revanche, les proteines cdk7 d n induisent un retard du demarrage de la transcription zygotique. Ces resultats sont, d'une part, une confirmation in vivo du role de cdk7 dans le controle de la transcription. D'autre part, ils suggerent fortement que cdk7 ne soit pas responsable de l'activite cak au cours des cycles mitotiques precoces. Une autre kinase, homologue de la kinase cak1 de s. Cerevisiae, pourrait assumer cette fonction.
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Date, Dipali A. "Regulation and Post-translational modifications of Borealin." University of Toledo / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1279127511.
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