Thesis (D. Min.)--McCormick Theological Seminary, 1997. / Includes bibliographical references (leaves 45-47).

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Aurora A is an important mitotic regulator that has been found to be up-regulated in a variety oftumours provoking a great deal of attention and the development of a number of small moleculeAurora kinase inhibitors. Most of these inhibitors though have predominantly targeted Aurora B,meaning that our understanding of the role of the kinase activity of Aurora A is comparatively lesswell developed.MLN8054 however, is a small molecule inhibitor that has been reported in vitro to have a highdegree of specificity towards Aurora A activity. In this thesis, I show in vivo that MLN8054 can beused to specifically inhibit Aurora A activity, and exploit this quality to probe the role of Aurora Aactivity in human cells. I was consequently able to show that Aurora A activity not only has a clearrole in spindle formation, where it is required for the determination of K-fibre length and in thedegree of centrosome separation, but also in the regulation of microtubule organisation. Despite thespindle deformities seen after inhibiting Aurora A activity, the majority of HeLa and DLD-1 cellswere still able to form bipolar spindles capable of attaching to kinetochores. These spindlestructures did not however, assert normal levels of force through the kinetochores, and cells wereconsequently unable to efficiently align their chromosomes, causing significant delays to mitoticprogression. Cells were still able to divide in the absence of Aurora A activity, although thedetection of segregation defects and aneuploid progeny indicates a role for Aurora A activity in thefaithful segregation of the genetic material. Importantly however, Aurora A activity was not foundto have a prominent role in the spindle assembly checkpoint.Increasing the potency of Aurora A inhibition by using a drug-resistant cell line confirmed theobservations made in HeLa and DLD-1 cells, emphasising that although Aurora A activity isrequired for spindle assembly, cells can still activate the spindle checkpoint and divide in itsabsence. I therefore propose that Aurora A activity is required for the formation of normal spindlestructures capable of efficiently aligning and evenly dividing chromosomes during cell division.These roles were attributed in part to the kinase activity of Aurora A in the regulation of TACC3and chTOG localisation on the spindle and centrosomes.Interestingly however, Aurora A activity did not appear to be required for spindle assembly in nontransformedcells, which were able to more efficiently align their chromosomes and dividefollowing Aurora A inhibition than the cancer cell lines. Furthermore, the non-transformed cellsaccumulated with 2N DNA after longer-term Aurora A inhibition, as opposed to the cancer celllines, which exhibited profound aneuploidy following the equivalent treatment. This finding isencouraging, as consistent with recently published reports, it indicates that Aurora A inhibitionmay be successfully used in order to specifically target cancer cells.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Ciência da Computação. / Made available in DSpace on 2012-10-19T14:03:11Z (GMT). No. of bitstreams: 1 197383.pdf: 854414 bytes, checksum: e009a842e9e0148f1e1a79a8c829056d (MD5)

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The kinetics of protein phosphorylation and dephosphorylation are tightly controlled by specific kinases and phosphatases; disturbances are often disease-causing. Phosphorylation kinetics are normally monitored using radioactive isotopes of phosphorus, or by using stop-flow techniques. Approaches using mass spectrometry are severely limited by the lack of a stable isotope of phosphorus (other than 31P). The principal aim of this study is to develop a new method to incorporate 18O label into phosphorylation sites of phosphoproteins with a view of applying this method to enhance the detection of phosphorylation by mass spectrometry and to analyze the phosphorylation kinetics of proteins. Aurora-A kinase was selected to explore the possibility of using 18O-labelling to monitor phosphorylation kinetics. The kinase is well characterized, phosphorylated both in human cells and when expressed in recombinant form in E. coli and it contributes to development of some cancers when deregulated. Applying different mass spectrometric approaches resulted in the identification of 19 phosphorylation sites of Aurora-A including five new sites. Using H3P18O4 as a label donor to incorporate 18O into Aurora-A phosphorylation sites showed partial and inconsistent label incorporation. Alternatively, H218O was used to investigate the possibility of label incorporation. Preliminary results, however, showed high complex data which hampered precise identification of phosphopeptides and their labelling state. The labelling experiment was then redesigned in which induction took place in label free medium to allow the light version of the kinase to accumulate, before chasing with 18O label. This design successfully introduced fully labelled P18O3 into Aurora-A phosphorylation sites. LC ESI Q-ToF analysis of 18O labelled Aurora-A sample isolated according to this protocol identified 30 phosphopeptides showing label incorporation, which is double the number of phosphopeptides identified by MASCOT using the same MS analysis. The method was also used to investigate phosphorylation kinetics of Aurora-A. The results suggested differential regulation of phosphorylation sites of Aurora-A as some sites showed early phosphorylation while others were phosphorylated at later stages. Overall, a new approach was developed for enhanced detection of phosphorylation sites and analysis of phosphorylation kinetics.

Aurora is a light phenomenon in the night sky that fascinates many people. This project has aimed to develop a detector that can send alerts to users and tell whether or not there is an aurora event. Many want to see the aurora, but not as many want to stay up all night to have the chance to see them. By using a multi-layer interference filter in front of a photodiode on a single Complementary metal-oxide-semiconductor, CMOS, available on the market, I have managed to produce a detector that gives promising results regarding the ability to at least distinguish intense aurora events.

Les sarcomes sont des cancers rares touchant toutes les zones du corps humain, caractérisés par une grande diversité de nature, de comportement clinique et de réponse aux thérapies existantes, certains étant de bon pronostic, d’autres très difficilement curables.Leur traitement de référence est la chirurgie ; la radiothérapie et les protocoles de chimiothérapie n’ayant que peu évolué lors des 30 dernières années.Récemment des caractéristiques génétiques leur étant propres ont été découvertes, prédictives de leur agressivité et contre lesquelles il est possible de diriger des drogues spécifiques pouvant améliorer le pronostic et diminuer les effets secondaires des thérapies conventionnelles.C’est sur l’inhibition d’Aurora Kinase A et B et CHK1 que s’est focalisé ce travail avec le test des effets de deux nouvelles drogues sur 9 types de cellules cancéreuses sarcomateuses avec des résultats très prometteurs, qu’il conviendra de conforter par d’autres expériences, notamment sur l’animal. / Sarcomas are rare cancers, which may arise in all parts of human body. They are characterized by great diversity in their nature, clinical behavior and response to existing therapeutics. Some are of good prognosis and others hard to cure.Their treatment essentially relies on surgery and radiotherapy or chemotherapy haven’t know major breakthrough over the last 3 decades.Recently new genetics abnormalities linked to sarcomas have been discovered. Their analysis can predict their aggressiveness and it is now possible to develop targeted therapies against them. This could help improving cancer prognosis and/or limiting conventional drugs adverse effects.Our work focused on Aurora Kinase A and B and CHK1 inhibition, testing the effects of 2 new drugs on 9 types of sarcoma cells with promising results, which we will confort by other experiments, including on the animal.

Introdução: Tumores adrenocorticais (TAC) são raros, correspondendo somente a 0,2% de todas as neoplasias pediátricas, sendo que a maioria dos casos são diagnosticados no Brasil e estão associados com a mutação TP53 p.R337H. A cirurgia é o único tratamento efetivo conhecido para os TAC, sendo os tumores em estadios avançados frequentemente fatais. A família das Aurora-quinases é formada por três membros (Aurora-A, -B e -C) os quais atuam em diversas fases do ciclo celular, como alinhamento dos cromossomos, formação do fuso mitótico e citocinese. Diferentes trabalhos mostraram a expressão alterada de membros desta família em vários tipos de tumores e a inibição da atividade destas proteínas tem sido considerada uma potencial abordagem para o tratamento do câncer. Objetivo: A partir da análise da expressão dos genes Aurora-A e Aurora-B em amostras de TAC pediátrico, foram investigados os efeitos do AMG 900, um pan-inibidor de aurora quinases, na proliferação, apoptose, síntese hormonal e perfil transcricional da linhagem H295A. Além disso, foram avaliados os efeitos do AMG 900 combinado com diferentes quimioterápicos. Metodologia: Os níveis de expressão dos genes Aurora-A e Aurora-B foram analisados em 60 crianças com TAC através das técnicas de RT-qPCR e imuno-histoquímica. A proliferação celular foi avaliada por coloração com Giemsa e a apoptose foi realizada por citometria de fluxo. A análise de combinação de drogas foi feita com base no método de Chou-Talalay e o ensaio de microarray foi realizado utilizando a plataforma da Agilent. Resultados: A expressão dos genes Aurora-A e Aurora-B foi associada com estadios avançados da doença e a expressão do Aurora-A foi associada com a presença da mutação TP53 p.R337H. O tratamento com o AMG900 causou a inibição da proliferação, aumento da apoptose e sensibilizou as células para os inibidores de topoisomerase II (doxorrubicina e etoposídeo). Adicionalmente, o AMG 900 levou à redução da síntese de hormônios bem como modulou a expressão de genes envolvidos com esta atividade. A inibição das aurora-quinases alterou a expressão de genes associados com a regulação da fase G1 do ciclo celular e afetou a expressão de genes da via de sinalização Notch. Conclusão: A inibição das aurora-quinases pelo AMG 900 pode ser uma alternativa para o tratamento dos tumores adrenocorticais. / Introduction: Pediatric adrenocortical tumors (ACT) are rare malignancies representing only 0.2 % of all pediatric cancers. Most cases are diagnosed in Brazil and are associated with TP53 p.R337H mutation. Surgery is the only effective treatment known for the ACT however this approach has a small impact on survival in advanced disease. The Aurora kinase family is comprised of three members (Aurora-A, -B and -C) which act at different phases of the cell cycle, such as chromosomes alignment, mitotic spindle formation and cytokinesis. Several studies have demonstrated altered expression of members of this family in various types of tumors and the functional inhibition of the aurora kinases have been considered as a potential approach to cancer treatment. Aim: On the basis of analysis of Aurora-A and Aurora-B gene expression in the samples from pediatric ACT, we investigated the effects of AMG 900, a pan-aurora kinase inhibitor, on proliferation, apoptosis rate, hormone synthesis and transcriptional profile of H295A cell line. Furthermore, we evaluated the effects of AMG 900 combined with different chemotherapeutic agents. Methods: The mRNA expression levels of Aurora-A and Aurora-B genes were analyzed in 60 children with ACT by RT-qPCR and immunohistochemistry. Cell proliferation was assessed by Giemsa staining and apoptosis was performed by flow cytometry. Drug combination analysis was made on the basis of Chou- Talalay method. Microarray experiments were carried out using the Agilent human microarray. Results: Aurora-A and Aurora-B overexpression was associated with advanced disease. Patients carrying the TP53 p.R337H mutation presented significantly higher expression values of Aurora-A. Treatment with AMG900 caused inhibition of proliferation, increased apoptosis and sensitized the cells to topoisomerase II inhibitors (doxorubicin and etoposide). Additionally, the AMG 900 led to decreased synthesis of hormones and modulated the expression of genes involved in this activity. Finally, Aurora kinases inhibition altered the expression of genes associated with G1 cell cycle phase regulation and affected the Notch signaling pathway target genes. Conclusion: These data suggest that Aurora kinase inhibition by AMG900 may be a new therapeutic approach to adrenocortical carcinoma treatment.

As alterações genéticas mais frequentes em tumores de pulmão são mutações pontuais que ativam o oncogene KRAS. Apesar destas mutações estarem ligadas à oncogênese de forma causal, diferentes abordagens para inibir as proteínas RAS diretamente fracassaram na clínica. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis, será necessário identificar as vias sinalizadoras ativadas pela proteína KRAS, que são críticas para a oncogênese. O objetivo deste projeto foi identificar novos alvos terapêuticos na oncogênese pulmonar induzida pela KRAS. Este projeto se baseou na seguinte hipótese: (1) a KRAS oncogênica leva à ativação das quinases mitóticas Aurora A e/ou B e (2) que as quinases Aurora A e/ou B são alvos terapêuticos relevantes no câncer de pulmão induzido pelo oncogene KRAS. Esta hipótese foi formulada com base em estudos anteriores mostrando que a quinase Aurora A fosforila diretamente componentes das vias efetoras de RAS, e que a Aurora A e Aurora B cooperam com a RAS oncogênica na transformação maligna. Para testar esta hipótese, nós inicialmente determinamos se a forma oncogênica da KRAS induz a expressão das quinases Aurora A e B. Para tanto, nós usamos 3 modelos celulares: (1) uma linhagem primária epitelial pulmonar imortalizada e seu par isogênico transformado pela KRAS oncogênica; (2) células tumorais pulmonares H1703 manipuladas geneticamente para expressar a forma oncogênica da KRAS de forma induzível; e (3) células de adenocarcinoma pulmonar portadoras de mutações oncogênicas em KRAS H358 e A549 manipuladas geneticamente para expressar short hairpin RNAs (shRNAs) para KRAS de forma induzível. Em todos os casos, a expressão da forma oncogênica da KRAS se correlacionou positivamente com a expressão de Aurora A e B. Para validar as quinases Aurora A e B como alvos relevantes do ponto de vista terapêutico, nós usamos, nas células mencionadas acima, abordagens genéticas ou farmacológicas para inibir a expressão ou atividade das quinases Aurora A e B. Nas células A549 e H358, portadoras da forma oncogênica da KRAS, a inibição da expressão das quinases Aurora A ou B por interferência de RNA de forma induzível, bem como o tratamento com um inibidor dual destas quinases, reduziu o crescimento, viabilidade e tumorigenicidade celulares in vitro. Mais importante do que isso, no modelo celular primário isogênico, bem como na linhagem H1703 com expressão induzível de KRAS oncogênica, a inibição farmacológica dual das quinases Aurora A e B levou a uma redução no crescimento, viabilidade e tumorigenicidade celulares de forma dependente da presença da KRAS oncogênica, sugerindo que a inibição das quinases Aurora A e B afeta especificamente células transformadas pela KRAS. Em conclusão, nossos resultados apoiam a nossa hipótese de que as quinases Aurora são alvos da KRAS oncogênica no pulmão, e sugerem a inibição das quinases Aurora como uma nova abordagem para a terapia do câncer de pulmão induzido pela forma oncogênica da KRAS. / The most frequent genetic change found in lung tumors are activating point mutations in the KRAS gene, which have been causally linked to the oncogenic process. Unfortunately, different approaches to target RAS proteins for therapy have been unsuccessful. Therefore, in order to select better targets for lung cancer therapy, key cancer-relevant KRAS downstream pathways will need to be identified. The overall objective of this study was to identify novel therapeutic targets in KRAS-mediated lung cancer. This project was based on the following hypothesis: (1) KRAS activates mitotic kinases Aurora A and/or B; and (2) Aurora A and/or B are relevant therapeutic targets in KRAS-induced lung cancer. This hypothesis was formulated on the basis of published studies showing that Aurora A directly phosphorylates RAS effector pathway components, and Aurora A and B both cooperate with oncogenic RAS to promote malignant transformation. In order to test this hypothesis, we first determined whether oncogenic KRAS induces Aurora kinase expression. For that purpose, we used three different cell-based models: (1) an immortalized primary lung epithelial cell line and its isogenic KRAS-transformed counterpart, (2) H1703 lung cancer cell line engineered to express oncogenic KRAS inducibly, and (3) KRAS positive lung cancer cell lines H358 and A549 stably expressing inducible shRNAs targeting KRAS. In all cases, KRAS expression positively correlated with Aurora A and Aurora B expression. In order to validate Aurora A and/or B as therapeutically relevant KRAS targets in lung cancer, we used genetic and/or pharmacological approaches in the abovementioned cells to inactivate Aurora A or B. In KRAS positive H358 and A549 cell lines, inducible shRNA-mediated knockdown of Aurora A or B, as well as treatment with a dual Aurora A and B inhibitor, decreased growth, viability and tumorigenicity in vitro. More importantly, in the primary isogenic model and in the H1703 KRAS-inducible cell line, dual pharmacological inhibiton of Aurora A and B reduced growth, viability and tumorigenicity in an oncogenic KRAS-dependent manner. This suggests that Aurora kinase inhibition therapy can specifically target KRAS transformed cells. In conclusion, our results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy.