Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Etela Kouklíková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines Cyclin-dependent kinase inhibitors (CDKi) are considered as a suitable treatment especially in patients with wrong prognosis or advanced stage of cancer. It has only recently been discovered that CDKi are able to influence the activity of some enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. AKR and SDR enzymes belong to a group of carbonyl reducing enzymes that are involved in the metabolism of endobiotics and xenobiotics. An important group of drugs that are metabolized by these enzymes to less efficient compounds are anthracyclines. The aim of this diploma thesis was to find out whether purvalanol A, roscovitin, dinaciclib, AZD5438 and R547 can affect the expression of the most important anthracycline reductases (AKR1A1, AKR1B10, AKR1C3, AKR7A2 and CBR1) in human HepG2 and HL-60 cell lines. Expression of anthracycline reductases in cells exposed to CDKi was evaluated at mRNA level by RT-qPCR and at protein level by Western blotting. The...

Submitted by THIAGO DE SOUZA CANDIDO null (thiago.s.candido@gmail.com) on 2016-09-30T14:48:19Z No. of bitstreams: 1 Tese Thiago de Souza Candido.pdf: 8667516 bytes, checksum: a5ceff1a03a2f39c134a0633e677f4df (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-10-04T14:33:04Z (GMT) No. of bitstreams: 1 candido_ts_dr_araiq.pdf: 8667516 bytes, checksum: a5ceff1a03a2f39c134a0633e677f4df (MD5) / Made available in DSpace on 2016-10-04T14:33:04Z (GMT). No. of bitstreams: 1 candido_ts_dr_araiq.pdf: 8667516 bytes, checksum: a5ceff1a03a2f39c134a0633e677f4df (MD5) Previous issue date: 2016-09-15 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fungo Neurospora crassa tem sido amplamente usado como um organismo modelo para os aspectos fundamentais da biologia dos eucariotos. Neste trabalho, foi investigado o papel funcional de uma ciclina de N. crassa (PCL-1), codificada pela ORF NCU08772 e ortóloga a Pcl10 de Saccharomyces cerevisiae. Na levedura, a proteína Pcl10, em conjunto com a proteína quinase dependente de ciclina Pho85, fosforila a enzima glicogênio sintase, a enzima regulatória da síntese de glicogênio. A fosforilação resulta na inativação da enzima e, portanto, em diminuição do acúmulo de glicogênio. A linhagem pcl-1 de N. crassa apresentou um atraso na germinação dos conídios e um retardo na progressão do ciclo celular quando comparado com a linhagem selvagem, sugerindo que esta ciclina pode regular o desenvolvimento e a divisão celular. Além disto, a linhagem nocauteada acumulou níveis mais elevados de glicogênio que a linhagem selvagem indicando o papel na regulação do metabolismo deste carboidrato. A fosforilação da enzima glicogênio sintase de N. crassa (GSN) foi analisada na linhagem nocaute através de análises de atividade enzimática, e os resultados mostraram que a GSN apresentou baixo índice de fosforilação, portanto alta atividade durante o crescimento, um resultado que pode explicar o alto acúmulo de glicogênio observado. Este resultado foi confirmado por análise de 2D-PAGE seguida por western blot, utilizando anticorpos anti-GSN. GSN apresentou na linhagem mutante isoformas menos fosforiladas que a enzima presente na linhagem selvagem. As proteínas recombinantes PHO85, PCL-1 foram utilizadas em ensaios de fosforilação in vitro da enzima GSN e os resultados mostraram que a enzima GSN foi fosforilada pelo complexo PHO85/PCL-1 no sítio putativo Ser636. O papel da ciclina na resposta ao cálcio foi investigado, e os resultados mostraram que a linhagem pcl-1 mostrou uma resistência ao estresse provocado por altas concentrações de cálcio quando comparada com a linhagem selvagem. Análises de expressão gênica por RT-qPCR foram realizadas e a linhagem pcl-1 mostrou estar envolvida na regulação de genes do metabolismo do cálcio. Os resultados indicam que a proteína PCL1 de N. crassa pode ser uma ciclina multifuncional e pode estar envolvida na regulação de vários processos celulares essenciais dependendo do ambiente. / The fungus Neurospora crassa has been widely used a model organism for the fundamental aspects of eukaryotes biology. This work investigated the functional role of a N. crassa cyclin (PCL-1), encoded by the ORF NCU08772, and orthologous to the Saccharomyces cerevisiae Pcl10 cyclin. In yeast, Pcl10 protein, together with the Pho85 cyclin-dependent protein kinase, phosphorylates the glycogen synthase enzyme, the regulatory enzyme in glycogen synthesis. Phosphorylation results in enzyme inactivation and therefore in decreased glycogen accumulation. The N. crassa pcl-1 strain showed a delay in conidia germination and in the progression of the cell cycle compared to the wild-type strain, suggesting that the cyclin may regulate development and cell division. Furthermore, the mutant strain accumulated higher glycogen levels than wild-type strain indicating its role in the regulation of the carbohydrate metabolism. The phosphorylation rate of the N. crassa glycogen synthase (GSN) was analyzed in the mutant strain by enzymatic activity assays, and the results showed that GSN was less phosphorylated during growth; therefore, high activity, and this result may explain the high glycogen accumulation observed in the mutant strain. This result was confirmed by 2D-PAGE gels followed by western blot using anti-GSN antibodies. The GSN isoforms presented in the mutant strain were less phosphorylated than the enzyme present in the wild-type strain. The recombinant proteins PHO85 and PCL-1 were used in in vitro phosphorylation assays of GSN enzyme, and the results showed that the enzyme was phosphorylated by the PHO85/PCL-1 complex at the putative site Ser636. The role of the cyclin in the response to calcium was investigated, and the results showed that the pcl-1 strain is more resistant than the wild-type strain to the stress caused by high calcium concentration. Gene expression analysis by RTqPCR was performed to analyze genes involved in calcium metabolism, and the pcl-1 strain showed to regulate the expression of some calcium metabolism genes. The results indicate that the N. crassa PCL1 cyclin may be multifunctional and may be involved in the regulation of several cellular processes depending on the environment. / FAPESP: 2012/08652-4

Made available in DSpace on 2015-04-20T12:31:17Z (GMT). No. of bitstreams: 1 rodolfo.pdf: 816515 bytes, checksum: e22fa8b8a75851c23c61ffb380635b34 (MD5) Previous issue date: 2012-12-13 / The goiters stand out among the hyperplastic thyroid pathologies by high prevalence in the Amazon region. Manaus, in recent decades, receives patients with goiter from the states of Pará to Acre in search of treatment of their pathologies. This work evaluated the immunoexpression of cyclin D1 in a qualitative way in surgical specimens with histopathologic diagnosis of goiter patients undergoing thyroidectomy in Manaus / AM. Forty-one selected patients with a histopathologic diagnosis of goiter, and from the paraffin blocks of histopathology, immunohistochemistry analysis submitted to the expression of cyclin D1. Data were analyzed according to the association between the expression of cyclin D1 and the diagnosis of goiter adopting the index of statistical significance (p <0.05) and the odds ratio (OR). Of the 41 patients, 36 (87.8%) were women, with a mean age of 48 years. Twenty (48.8%) patients were positive for the expression of cyclin D1 and the observed correlation between histopathology and the results were positive for cyclin D1 (Pearson correlation = 26.1%) for goiters. The chance of a female patient have resulted positive for cyclin D1 is 4.5 times higher than in male patients (OR = 4.5). But even if the absolute majority of cases positive for cyclin D1 is female, a statistical correlation was not significant for the genre - Fisher exact test (p-value = 0.16). Commonly regarded as a marker of cell differentiation in malignant thyroid lesions, where may perform a function the cell multiplication, this study showed that cyclin D1 may be present also in benign hyperplastic lesions, such as goiters on Amazon. / Os bócios destacam-se entre as patologias hiperplásicas tireoidianas pela elevada prevalência na região Amazônica. Manaus, nas últimas décadas, recebe pacientes portadores de bócio oriundos dos estados do Pará até o Acre em busca do tratamento de suas patologias. Este trabalho avaliou a imunoexpressão de ciclina D1 de forma qualitativa em peças cirúrgicas com o diagnóstico histopatológico de bócio de pacientes submetidos à tireoidectomia na cidade de Manaus/AM. Selecionados quarenta e um pacientes com diagnóstico histopatológico de bócio, e, a partir dos blocos de parafina da histopatologia, submetidos a análise imunohistoquímica da expressão da ciclina D1. Os dados foram analisados segundo a associação entre a expressão da ciclina D1 e o diagnóstico de bócio adotando-se o índice de significância estatística (p<0,05) e o odds ratio (OR). Dos 41 pacientes, 36 (87,8%) eram mulheres, com média de idade de 48 anos. Vinte (48,8%) pacientes apresentaram positividade para a expressão de ciclina D1 e a correlação observada entre o diagnóstico histopatológico e os resultados para ciclina D1 foi positiva (Correlação de Pearson = 26,1%) para bócios. A chance de um paciente feminino ter resultado positivo para ciclina D1 é 4,5 vezes maior do que em pacientes masculinos (OR = 4,5). Mas, mesmo que a maioria absoluta dos casos positivos para ciclina D1 seja do gênero feminino, a correlação estatística não se mostrou significante para o gênero - teste Exato de Fisher (p-valor = 0,16). Comumente considerada como marcador de diferenciação celular nas lesões tireoidianas malignas, onde pode exercer uma função de multiplicação celular, este estudo mostrou que a ciclina D1 pode estar presente, também, em lesões hiperplásicas benignas, como os bócios no Amazonas.

Introdução: A ciclina D1 constitui um importante regulador do ciclo celular e pode funcionar como co-regulador de transcrição. A superexpressão da ciclina D1 tem sido associada ao desenvolvimento e progressão do câncer. A degradação irregular da ciclina D1 pode ser responsável pelos seus níveis elevados em algumas neoplasias malignas. A ciclina D1, além disso, modula indiretamente a estrutura da cromatina e transcrição de genes envolvidos na proliferação e diferenciação. Mutações, amplificação e superexpressão da ciclina D1, que alteram a progressão do ciclo celular, são observadas frequentemente em várias apresentações de neoplasias malignas, incluindo o carcinoma epidermóide de laringe, e as elucidações inferidas destas observações podem trazer melhor entendimento à oncogênese. Objetivo: O objetivo deste estudo foi comparar a expressão imunoistoquímica de ciclina D1 e a ocorrência de metástase linfática em estádios III e IV de carcinoma epidermóide de laringe. Métodos: Estudo retrospectivo, coorte longitudinal, por avaliação imunoistoquímica e quantificação digital da imunoexpressão nuclear e citoplasmática de ciclina D1 em espécimes de tumor preservados em parafina oriundos de pacientes consecutivos submetidos à cirurgia oncológica radical, entre 1999 e 2004. A sobrevida global dos pacientes foi avaliada, bem como a idade, sexo, tabagismo, estado de comprometimento linfático, grau de diferenciação e estadiamento (pTNM). A análise estatística teve como significância valores de p< 0.05. A curva de sobrevida foi elaborada utilizando o método de Kaplan-Meier. Resultados: Houve imunomarcação citoplasmática em 566 (1,2%) células, imunomarcação nuclear em 13788 (29,6%) células, a relação do IPc e IPn foi de 0,007 (0,7%), ausência de imunomarcação celular foi observada em 32210 (69,1%) células, perfazendo um total de 46554 (100%) células investigadas. Entre os 28 (59,5%) casos que não apresentaram metástase linfática, o IPn foi de 26,8 (9,7 - 46,9) e o IPc foi de 0,1 (0 0,3); naqueles 19 (40,4%) em que foi observada metástase linfática, o IPn foi de 26,7 (16,7 39,0) e o IPc foi de 0,3 (0 - 1,0). Conclusões: Não houve associação estatisticamente significante entre expressão nuclear e citoplasmática de ciclina D1, em carcinoma epidermóide primário de laringe, e ocorrência de metástase linfática cervical, graus de diferenciação histológica, bem como recidiva loco-regional e metástase hematogênica. O presente estudo não subsidia a superexpressão de ciclina D1 como fator limitante de sobrevida global / Background: Cyclin D1 is an important regulator of cell cycle progression and can function as a transcriptional co-regulator. The overexpression of cyclin D1 has been linked to the development and progression of cancer. Abnormal cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. Recent findings have identified novel mechanisms involved in the regulation of cyclin D1 stability. Cyclin D1 belongs to the family of cyclin proteins which function as the regulatory subunits of cyclin/cyclin dependent kinase (Cdk) holoenzymes that regulate entry into and progression through the cell cycle. Cyclin D1 expression is induced upon stimulation by growth factors (e.g. EGF, IGF-I/II), aminoacids, hormones, and oncogenes such as Ras, Src, ErbB2, and SV40 T antigen. Cdk4 and Cdk6 can partner with cyclin D1 in early to mid-G1 phase to phosphorylate and inactivate the cell cycle inhibitory function of the retinoblastoma protein (pRB) in cooperation with cyclin E/Cdk2. Cyclin D1 is also known to modulate local chromatin structure and transcription of genes involved in proliferation and differentiation through CDK-independent association with histone acetylases (e.g. CBP, P/CAF) and deacetylases. Mutations, amplification or overexpression of cyclin D1, which alters cell cycle progression, are observed frequently in a variety of tumors, including laryngeal squamous cell carcinoma, and may contribute to oncogenesis. Methods: This was a retrospective study by immunohistochemical determination of cyclin D1 in fixated and paraffin-embedded tumour especimens from 47 consecutive patients with squamous cell carcinoma in larynx treated by curative oncological surgery from 1999 to 2004. Survival of patients was related to age, gender, nodal status and stage at termination of treatment. Significant differences were considered for p<0.05. Results: Cytoplasmic immunostain was observed in 566 (1,2%) cell, nuclear immunostain in 13788 (29,6%) cell, relationship between PIc and PIn was 0,007 (0,7%), absent cell immunostain was observed in 32210 (69,1%), and total 46554 (100%). Among 28 (59,5%) cases with no lymph node metastasis, PIn was 26,8 (9,7 - 46,9) and PIc was 0,1 (0 0,3); those 19 (40,4%) with lymph node metastasis, had a PIn of 26,7 (16,7 39,0) and PIc of 0,3 (0 -1,0). Conclusions: According to these results, it has been concluded that cyclin D1 showed nuclear and cytoplasmic expression in larynx squamous cell carcinoma; however, tumor cyclin D1 expression was not significantly associated with lymph node metastasis when quantified by quantitative or semiquantitative methods. Besides, cyclin D1 expression showed no influence in overall survival

O melanoma cutâneo é a neoplasia de pele de maior mortalidade. A imprevisibilidade de sua evolução é uma de suas características principais, o tratamento do tumor primário é, atualmente, de pouca morbidade e, na doença disseminada, as opções terapêuticas são pouco eficazes. É fundamental a pesquisa de marcadores tumorais que permitam a previsão da evolução, melhor compreensão da patogênese do melanoma e possibilitem a descoberta de alvos moleculares. Nesse contexto, estudos genéticos mostraram a importância da regulação do ciclo celular, especialmente a passagem da fase G1-S. Importantes fatores envolvidos compõem a cascata da proteína Rb (p16, ciclina D1, CDK4 e pRb) e da proteína p53 (p53 e p21). Objetivo: verificar a frequência da expressão de p16, ciclina D1,CDK4, pRb, p53 e p21 em melanomas cutâneos de cabeça, pescoço e tronco e sua relação com prognóstico. Métodos: Estudo retrospectivo envolvendo 46 pacientes (sendo 67,3% homens, idade média 57,7 ± 15,8 anos) com melanoma cutâneo de cabeça, pescoço e tronco que foram tratados pela mesma equipe com seguimento mínimo de dois anos. Foram estudados fatores clínicos (topografia do tumor primário, tempo de seguimento, ocorrência de metástases e óbito relacionado), histopatológicos (tipo histológico, índice de Clark, índice de Breslow) e análise imuno-histoquímica pela técnica de micro-array para as proteínas reguladoras do ciclo celular p16, ciclina D1, CDK4, pRb, p53 e p21. Resultados: Houve proporção igualitária entre as topografias (23 casos em tronco, 23 em cabeça e pescoço). Treze pacientes com Clark I (29,5%), cinco com II (11,3%), 16 com III (36,5%), 10 com IV (22,7%) e nenhum com Clark V. A média das medidas de Breslow foi 0,96 (DP=1,01). O seguimento médio foi de 77 meses (DP=47). Oito dos 46 pacientes (17,3%) tiveram evolução desfavorável, com seis óbitos relacionados. A idade foi mais elevada no grupo com evolução desfavorável (p=0,04). Houve expressão de p16 em 80%, ciclina D1 em 58,9%, CDK4 em 43,5%, pRb em 58,5%, p53 em 53,6% e p21 em 52,3% dos melanomas. Em análise univariada, a expressão do p21 foi relacionada com evolução desfavorável (p=0,04), o que não foi observado com a expressão dos outros marcadores (p>0,05). Conclusão: A expressão da proteína p21 nos melanomas cutâneos de cabeça, pescoço e tronco foi relacionada com evolução desfavorável, o que não ocorreu com outros fatores envolvidos na regulação do ciclo celular / Melanoma is the most lethal skin cancer. The outcome of melanoma is not predictable in most cases. Although the treatment for the primary tumor is well tolerated, there are no effective therapeutic options in disseminated disease. Efforts are being made in the search for tumoral markers that may predict outcome, increase the comprehension of melanoma pathogenesis, and may also help the search for molecular targets. In this issue, genetic studies concerning the regulation of cell cycle, including the G1-S checkpoint, are important. The retinoblastoma protein (pRb) pathway (p16, cyclin D1, CDK4 and pRb) and the p53 pathway (p53 and p21) are part of this regulation. Objectives: to verify the expression of p16, cyclin D1, CDK4, pRb, p53 and p21 in head, neck and trunk melanomas, and its correlation with prognosis. Method: Retrospective study approved by institution ethics committee. Fourtysix head, neck and trunk melanoma patients (67.3% men, mean age 57.7±15.8) treated by a single surgeon with minimum 2-years follow-up were enrolled. Clinical factors (primary tumor location, follow-up period, metastasis or related deaths), pathologic (histological subtype, Clark and Breslow index) and microarray immunohystochemical analysis of the cell cycle proteins p16, cyclin D1, CDK4, pRb, p53 and p21. Results: Location of the primary tumor was equal for head/neck and trunk (50% each). Thirteen patients were classified as Clark I (29.5%), five as Clark II (11.3%), 16 as Clark III (36.5%), 10 as IV (22.7%), none as Clark V. Mean Breslow measure was 0.96±1.01. Mean follow-up was 77±47 months. Eight patients (17.3%) had bad outcome, with six related deaths. Patients with worse outcome had a higher mean age at diagnosis (p=0,04). Expression of p16 was positive in 80%. Cyclin D1 was positive in 58.9%. CDK4 was positive in 43.5%. pRb was positive in 58.5%. p53 was positive in 53.6%. p21 was positive in 52.3%. Univariated analysis showed that p21 expression was related to worse outcome (p=0,04), while the other markers were not (p>0,05). Conclusion: p21 expression in head, neck and trunk melanomas was related to worse outcome. Expression of the other cell cycle regulators proteins was not

Une surprolifération cellulaire associée à de l’aneuploïdie est un marqueur couramment retrouvé dans les cancers et une faible instabilité génétique peut-être un élément aggravant (sinon déclencheur) de la tumorigénèse. Récemment, il a été montré sur un modèle de cellules cancéreuses en culture qu’une forte aneuploïdie compromet la prolifération cellulaire en entraînant la mort de ces dernières. Au cours de ma thèse, nous avons souhaité tester si cette hypothèse se vérifiait in vivo en utilisant comme modèle, les tumeurs du système nerveux central de la larve de D. melanogaster. Nous avons fait le choix d’utiliser des mutants pour des gènes impliqués dans la formation du fuseau mitotique et la ségrégation des chromosomes (Sas-4 ou AurA) afin d’induire ces tumeurs. Pour générer l’aneuploïdie, nous avons choisi d’associer les mutations sas-4 ou aurA avec des mutations pour des gènes essentiels du SAC, Mad2 ou BubR1ken. Nous avons ensuite analysé par immunofluorescence et microscopie l’effet de la perte du SAC sur la prolifération des Nb. Pour sas-4, la perte du SAC cause l’apparition d’une forte aneuploïdie et une baisse du nombre de Nb associée à une forte réduction de taille des cerveaux. Cela compromet totalement la capacité des cerveaux mutants à induire des tumeurs lorsqu’on les injecte dans l’abdomen de mouches adultes saines. Dans le cas d’aurA, ni hausse de l’aneuploïdie dans le tissu ni baisse de la prolifération des Nbs n’ont été observés. Par ailleurs, la même forte proportion de mouches injectées avec des cerveaux aurA ou aurA mad2 développant une tumeur a été constaté. Afin de mieux comprendre pourquoi le mutant aurA ne réagit pas comme le mutant sas-4 à la déplétion du SAC, nous avons entrepris une analyse détaillée des mutants aurA et aurA mad2. Nous avons d’abord observé que, malgré la perte du SAC, 1) il existe toujours un délai en mitose dans aurA mad2 et 2) il existe un délai entre la satisfaction du SAC et l’entrée en anaphase dans aurA. Comme l’entrée en anaphase est dépendante de la dégradation de la CycB et de la Sécurine via l’APC/C, nous avons analysé le comportement de la CycB (couplé à une étiquette GFP) par vidéo-microscopie en temps réel et observé un défaut de la régulation de la dégradation de cette dernière dans le mutant aurA ainsi que dans le double mutant aurA mad2. Ces observations nous ont permis de proposer un nouveau rôle pour la kinase AurA dans la régulation de la dégradation de la CycB en fin de mitose. / Cellular overproliferation associated with aneuploidy is a common hallmark of cancers. Low genetic instability may be a contributing factor of tumorigenesis. Recently, it was shown on a cellular cancer model in culture that strong aneuploidy compromises cell proliferation by causing cell death. During my thesis, we have test if this hypothesis was verified in vivo by using as a model, the tumours of the larval central nervous system of D. melanogaster. We decided to use mutants involved in mitotic spindle formation and chromosome segregation (Sas-4 or AurA) to induce these tumours. To generate aneuploidy, we chose to associate these mutations with mutations in genes essential for the SAC, Mad2 or BubR1ken. We then analysed the effect of the SAC depletion on the Nb proliferation. For sas-4, loss of the SAC leads to high aneuploidy and a decrease in Nb number associated with brain size reduction. It completely undermines the ability of mutant brain to induce tumors when injected into the abdomen of healthy adult flies. In the case of aurA, nor increase of aneuploidy in tissue or decrease in nb proliferation have been observed. Moreover, the same proportion of flies injected with aurA or aurA mad2 brains developed tumours. To better understand why the aurA mutant not react as the sas-4 mutant to the SAC depletion, we undertook a detailed analysis of aurA and aurA mad2 mutants. We first observed that despite the SAC depletion, 1) there is always a delay in mitosis in aurA mad2 and 2) there is a delay between SAC satisfaction and anaphase onset in aurA. Since anaphase onset is dependent of the CycB and Securine degradation via the APC / C, we analysed the behaviour of the CycB (coupled with a GFP tag) by real-time videomicroscopy and observed a defect in the regulation of CycB degradation in aurA and in the double aurA mad2 mutant. These observations lead us to propose a new role for AurA kinase in regulating the degradation of CycB at the end of mitosis.

In cell culture, entry into mitosis of many adherent mammalian cells is accompanied by substantial changes in cellular architecture. Flat, spread-out interphase cells detach from the extracellular matrix and become more spherical. These changes in cell shape are mediated by rearrangements in the actin cytoskeleton, a dynamic network of actin filaments that are organized by actin-binding proteins. Filamin A (FLNa) is a 280 kD actin-binding protein that crosslinks actin filaments into parallel bundles or three-dimensional orthogonal networks. We previously identified FLNa as an in vitro substrate of cyclin-dependent kinase 1 (Cdk1), a kinase that regulates entry into mitosis, and hypothesized that Cdk1 phosphorylation of FLNa regulates mitotic actin remodelling. Using mass spectrometry and a p-FLNa antibody, we show that FLNa is phosphorylated in vivo in HeLa cells on multiple Cdk1 sites, including serines 1084, 1459 and 1533. All three sites match the phosphorylation consensus sequence of Cdk1. We further show that p-FLNa is almost fully dephosphorylated by anaphase, consistent with it being a cell cycle-regulated substrate. Using a phospho-specific antibody, we find that p-FLNa has decreased cortical actin localization compared to total FLNa in mitotic cells. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine and expressed this FLNa mutant (FLNa-S1084A, S1459A, S1533A, referred to as “FLNa-AAA GFP”) in FLNa-deficient human M2 melanoma cells. FLNa-AAA GFP-expressing cells have enhanced FLNa-AAA GFP localization at sites of contact between daughter cells and this correlates with defects in cell division and impaired cell migration. Therefore, mitotic delocalization of cortical FLNa is critical for successful cell division and interphase cell behaviour.

Until now, the role of cyclin dependent kinase 5 (CDK5) in cardiac pathophysiology has not been explored. While CDK5 has been well studied in the neuroscience/Alzheimer’s field as a cyclin-independent kinase, there is currently no investigation into the cardiac-specific role of CDK5. Recently, it was established that inhibition of CDK5 in stem cell derived cardiomyocytes from individuals with Timothy Syndrome (TS) rescued the delayed inactivation phenotype; TS is a fatal genetic long QT syndrome (LQTS) caused by delayed inactivation of the L-type voltage gated Ca2+channel CaV1.2. While it is evident that CDK5 plays an important role in regulating CaV1.2 function, its role in cardiac tissue remains to be elucidated. To determine whether CDK5 is essential for cardiac function, two separate mouse models were established—a cardiac-deficient Cdk5 mouse model (Cdk5 flox x αMHC-MerCreMer+) and a Cdk5 activation mouse model via overexpression of Cdk5’s known activator, p35 (Cdk5r1/p35 OE x αMHC-MerCreMer+). Immediately after spatiotemporal induction of deficiency/activation of Cdk5 in adult mice, echocardiography, histology and proteomic analysis were performed to examine effects on cardiac structure and function. Analysis of cardiac function and morphology in Cdk5 deficient mice revealed severe systolic dysfunction and a dilated cardiomyopathy-like phenotype. These results were further validated by a pathway analysis of quantified global proteome changes. Conversely, mice with an activation of Cdk5 displayed only minor changes in cardiac function with a modest reduction in fractional shortening and ejection fraction. Notably, these mice did not have any significant changes in cardiac chamber morphology, nor any significant changes to their global proteome. Interestingly, however, phosphoproteomic analysis revealed over 3,000 differentially phosphorylated proteins. Pathway and gene ontology analysis of proteome changes revealed significant hits related to cell adhesion. Evidence for the extensively studied role of CDK5 in the brain has demonstrated a critical role for CDK5 kinase activity in the regulation of cell adhesion. Alterations in cell adhesion are observed in a number of cardiac pathologies including heart failure and dilated cardiomyopathy; it is therefore plausible that CDK5 potentially regulates cardiac function via cell adhesion mechanisms. A comparison of the phospho-proteome acutely after Cdk5 depletion vs the phospho-proteome acutely after Cdk5 activation, allowed for the identification of a novel cardiac-specific Cdk5 substrate, beta taxilin (Txlnb). Validation of this potential phospho-substrate with an in situ proximity ligation assay demonstrated the co-localization of Cdk5-Txlnb in wildtype mouse cardiac tissue sections. When looking at co-localization in Cdk5 deficient tissue sections, no signals were observed. Lastly, our lab obtained donor cardiac tissue samples from individuals who passed away due to either heart failure or non-cardiac causes (serving as control cardiac tissue). Analysis of cardiac tissue samples revealed a significant increase in both CDK5 and p35 expression in heart failure samples. Dysregulation of phosphorylation has been implicated in cardiac dysfunction, with known contribution to contractile failure and a number of cardiac pathologies including cardiomyopathies. These findings further support a role for CDK5 in cardiac function. In conclusion, it appears that CDK5 is imperative for the maintenance of healthy cardiac function. Cardiac-specific homozygous and heterozygous Cdk5 deficiency revealed severe systolic dysfunction along with a dilated cardiomyopathy-like phenotype. While the effects of Cdk5 activation in the heart need to be further investigated, initial findings report significant downstream effects on the phosphorylation of a number of proteins, including Txlnb. Moreover, Txlnb was identified as a potential novel cardiac-specific substrate of Cdk5. The importance of identifying a role for CDK5 in the heart extends beyond this study. CDK inhibitors have been at the forefront of drug development for cancer therapeutics and immunotherapy. While modulation of CDK5 activity may be beneficial in one physiological system, it may prove deleterious in another. It is therefore imperative that the full range of molecular and physiological roles of each CDK be fully elucidated prior to therapeutic application. Furthermore, outcomes from this study have the potential to be translational for drug discovery and the development of new therapeutic avenues for heart disease.

Les protéines se liant à l’ARN de la famille IMP sont les protéines oncofoetales conservées, qui régulent le transport, la stabilité et la traduction de plusieurs ARNm cibles. Les IMPs sont impliqués dans la tumorigenèse et dans le développement embryonnaire par le contrôle de la prolifération cellulaire, la différenciation, la migration, la polarisation et d`autres processus cellulaires. IMP-3 est difficilement détectable dans des tissus adultes normaux, mais il est surexprimé dans les nombreux cancers, où il a été caractérisé comme un marqueur d’agressivité et de la croissance tumorale rapide, ainsi que d’un pronostic défavorable pour les patients. Dans notre étude, nous avons utilisé une lignée cellulaire RD de rhabdomyosarcome (RMS), où IMPs étaient initialement décrits comme des protéines régulatrices de l`ARNm de IGF-2. Nous avons essayé d'élucider le mécanisme par lequel IMP3 régule l’expression des cyclines D1 et D3, contribuant ainsi à la compréhension des processus oncogéniques dans les RMS et autres cancers.Nous avons montré que IMP3 régule l'expression des cyclines D1 et D3 d'une manière significative in vivo. Nous avons également démontré, qu'en absence de IMP3, les ARNm des cyclines sont exportés vers le cytoplasme et s’associent avec les polyribosomes, mais ne sont pas traduits. En outre, l'inhibition d`IMP3 n'a pas d'influence sur la stabilité des ARNm des cyclines. Nous démontrons que dans des cellules cancéreuses humaines, IMP3 interagit avec plusieurs protéines se liant à l'ARN, et que nombre de ces protéines a un effet sul l’expression des cyclines, ce que suggère l'existence d'un complexe régulateur multiprotéique sur les 3'UTR des cyclines D1 et D3. Nos résultat montrent que l'inhibition de deux protéines clés de RNA-induced silencing complex (RISC) (AGO2 et GW182/TNRC6), rétablit les niveaux d'expression des cyclines D1 et D3, qui ont été considérablement diminués en l’absence d’IMP3 ou de ses partenaires protéiques ILF3/NF90 et PTBP1. Nous concluons que les complexes d`IMP3 et RISC peuvent concourir pour la régulation des ARNm des cyclines. Nous avons également identifié les miARNs qui peuvent être impliqués dans ce processus, ainsi que les domaines fonctionnellement importants dans les 3 'UTR des cyclines, où se passe la competition entre les complexes d’IMP-3 et RISC. Nos résultats sont compatibles avec l'existence de IMP3 - contenant complexe multiprotéique, qui est associé à 3'UTRs des cyclines et régule leur traduction en les protégeant contre la répression traductionnelle par miRISC. / RNA-binding proteins of the IMP family (IGF2 mRNA-binding proteins 1-3) are conserved oncofetal proteins, regulating transport, stability and decay of multiple mRNAs. IMPs are involved in embryonic developement and tumorigenesis by controlling cell proliferation, differentation, migration, polarization and many other important aspects of cell function. IMP-3 is hardly detectable in normal adult tissues, but is overexpressed in many cancers, where it has been reported as a marker of tumor aggressiveness, rapid growth, and bad prognosis for patients. In our research we utilized a rhabdomyosarcoma (RMS) cell line RD, where IMPs were first described as IGF-2 mRNA regulating proteins. We aimed to elucidate the mechanism by which IMP3 regulates the expression of cyclins D1 and D3, thereby contributing to the understanding of oncogenic processes in RMS.In this study, we show that IMP3 regulates the expression of cyclin D1 and D3 in a significant manner in vivo. We also demonstrate that in the absence of IMP3, the mRNAs of the cyclins are exported to the cytoplasm and associated with polyribosomes, but not translated. IMP3 inhibition does not influence the stability of cyclin mRNAs. We demonstrate that in human cancer cells, IMP3 interacts with multiple RNA-binding proteins, and that a number of these IMP-3 partners impacts on the expression of cyclins D1 and D3. These observations suggest the existence of a regulatory IMP-3 containing RNP complex on the 3’UTR of mRNAs of cyclin D1 and D3. Our results show that an inhibition of two key proteins of RNA-induced silencing complex (RISC) (AGO2 and GW182/TNRC6) rescues the expression of cyclin D1 and D3 proteins, which is significantly decreased in the absence of IMP3 or its protein partners ILF3/NF90 and PTBP1. Therefore, IMP3 and RISC complexes can compete for cyclin mRNAs translational repression/activation. We also identified a number of miRNAs that can be involved in this process, and characterized functionally important regions within 3’ UTRs of the cyclins, where the competition between IMP-3 and RISC complexes takes place. Our results are consistent with the existence of IMP3 - containing multiprotein complex, which is associated with 3’UTRs of the cyclins and regulates their translation by protecting them from miRISC-dependent translational repression.

Cardiovascular diseases are the leading cause of death in humans throughout the world and “congenital heart defects” (CHDs) are the major cause of infant mortality and morbidity. GATA4 is one of the most critical and intensely studied cardiac transcription factor. It is important for proliferation of cardiomyocytes as well as their survival and adaptive response. The focus of the following thesis was to identify GATA4 mediators and cofactors in cardiac growth. The first part focused on cyclin D2 (CycD2), a growth inducible cell cycle protein. We identified Ccnd2 (gene encoding CycD2) as a direct transcriptional target of GATA4 in postnatal cardiomyocytes and Ccnd2 cardiomyocyte specific overexpression in Gata4 heterozygote mice was able to rescue their heart size and function. We further uncovered a novel regulatory loop between GATA4 and CycD2. CycD2 enhanced GATA4 activation of its target promoters. GATA4 was able to physically interact with CycD2 and its cyclin dependent kinase CDK4 suggesting that GATA4 recruits CycD2/CDK4 to its target promoters. Together, our data uncover a role of CycD2 in the developing and postnatal heart and provide novel insight for the potential of targeting the cell cycle in cardiac therapy. The second part of the project focused on KLF13, a cell specific cofactor of GATA4. KLF13 is a member of the Krϋppel-like transcription factors that are important regulators of cell proliferation and differentiation. Klf13 is highly enriched in the developing heart where it is found in both myocardial and endocardial cells. To determine its role in the mammalian heart, we deleted the Klf13 gene in transgenic mice. Klf13-/- mice were born at 50% reduced frequency and presented with variable cardiac phenotypes. Epithelial-mesenchymal transformation (EMT) was affected in these mice and reduced cell proliferation was evident in the AV cushion. These data uncover a role for a new class of transcription factors in heart formation and point to KLF13 as a regulator of endocardial cell proliferation and a potential CHD causing gene. Future discovery of more cardiac regulators and understanding the molecular basis of CHDs is essential for preventions of these defects and possible development of therapeutic approaches for myocardial repair.