Introdução: O carcinoma de células renais (CCR) representa uma causa crescente de mortalidade por câncer. Apesar da sua curabilidade em estádios iniciais, agentes citotóxicos e imunomodulatórios têm homogeneamente alcançado nenhum ou pouco benefício no tratamento do CCR avançado. Um melhor entendimento da biologia tumoral pode levar ao desenvolvimento de terapias mais eficientes e dirigidas aos mecanismos moleculares de manutenção do tumor. Sinalização aumentada através do receptor do fator de crescimento epitelial (EGFR), sistema de quinases proteicas ativadas por mitógenos (MAPK) e via da fosfatidilinositol 3-quinase (PI3K) assim como estimulação do complexo de início de tradução (CIT) foram caracterizados em linhagens celulares e amostras tumorais de CCR. Nós investigamos o efeito de um inibidor de EGFR (gefitinibe), de um inibidor de MAPK (UO126) e de um inibidor do CIT (rapamicina) nos intermediários de sinalização celular e nos elementos do CIT assim como no crescimento in vitro de linhagens celulares de CCR. Métodos: Linhagens celulares de CCR (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN e KRCY) foram mantidas em condições ideais para a cultura de células de mamíferos e expostas a drogas e/ou inibidores nas concentrações e por períodos de tempo variáveis. A fosforilação de intermediários da sinalização celular foi determinada utilizando-se western blots. Nível de mRNA para genes de interesse foram determinados por qRT-PCR. Crescimento celular foi avaliado por método colorimétrico em diferentes concentrações de gefitinibe, UO126 e rapamicina, isoladamente ou em combinação. Resultados: UO126 levou a defosforilação não apenas de intermediários de MAPK como também de substratos do alvo da rapamicina em mamíferos (mTOR) revelando sinalização cruzada entre essas vias. Nós identificamos ainda que ERK5 é a quinase potencialmente responsável por esta sinalização cruzada. No entanto, tratamento com UO126 não afetou o nível de mRNA para o substrato de mTOR 4EBP1. Gefitinibe foi capaz de bloquear a sinalização iniciada por EGF na via de PI3K em todas as linhagens wt-PTEN e de bloquear a sinalização através de ERK1/2 e ERK5 ao menos parcialmente em todas as linhagens celulares. Rapamicina mostrou-se um potente inibidor do crescimento na maioria das linhagens celulares de CCR e tal efeito foi freqüentemente amplificado com a combinação com UO126 ou gefitinibe.Conclusão: EGFR, MAPK e CIT são alvos promissores no tratamento do CCR / Background: Renal cell carcinoma (RCC) is an increasing cause of cancer mortality. Despite its curability in early stages, conventional cytotoxic and immunomodulatory agents have been homogeneous in providing minimal or no benefit in the treatment of advanced RCC. Better understanding of tumor cell biology may lead to development of more efficient targeted therapies. Signaling intensification through epithelial growth factor receptor (EGFR), mitogenactivated protein kinase (MAPK) pathway, Phosphatidylinositol 3-kinase (PI3K) pathway and overactivation of the translation initiation complex (TIC) has been previously characterized in RCC cell lines and tumor samples. We investigated the effect of an EGFR inhibitor (gefitinib) as well as a MAPK inhibitor (UO126) and a TIC inhibitor (rapamycin) in the intermediates of cell signaling, in the elements of TIC and in the in vitro growth of RCC cell lines. Methods: RCC cell lines (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN and KRCY) were maintained on standard mammalian cells culture conditions and exposed to drugs and/or inhibitors in variable concentrations and for variable periods of time as required in each experiment. Phosphorylation status of signaling intermediates were determined using western blots. Levels of mRNA for genes of interest were determined by qRT-PCR. Cell growth was assessed by colorimetric method in control conditions or in different concentrations of gefitinib, UO126 or rapamycin, alone or in combination. Results: UO126 caused dephosphorylation not only of MAPK intermediates but also of mammalian target of rapamycin (mTOR) substrates revealing crosstalk between these pathways. We also identified ERK5 as a kinase potentially responsible for such cross talk. However, treatment with UO126 did not affect mRNA levels of the downstream target of mTOR 4EBP1. Gefitinib was able to block EGF signaling through PI3K in all wt- PTEN cell lines and the signaling through ERK1/2 and ERK5 at least partially in all cell lines. Rapamycin was found to be a potent growth inhibitor in most RCC cell lines and such effect was often increased by its combination with UO126 or gefitinib. Conclusion: EGFR, MAPK and CIT are promising targets for the treatment of RCC

Introdução: O carcinoma de células renais (CCR) representa uma causa crescente de mortalidade por câncer. Apesar da sua curabilidade em estádios iniciais, agentes citotóxicos e imunomodulatórios têm homogeneamente alcançado nenhum ou pouco benefício no tratamento do CCR avançado. Um melhor entendimento da biologia tumoral pode levar ao desenvolvimento de terapias mais eficientes e dirigidas aos mecanismos moleculares de manutenção do tumor. Sinalização aumentada através do receptor do fator de crescimento epitelial (EGFR), sistema de quinases proteicas ativadas por mitógenos (MAPK) e via da fosfatidilinositol 3-quinase (PI3K) assim como estimulação do complexo de início de tradução (CIT) foram caracterizados em linhagens celulares e amostras tumorais de CCR. Nós investigamos o efeito de um inibidor de EGFR (gefitinibe), de um inibidor de MAPK (UO126) e de um inibidor do CIT (rapamicina) nos intermediários de sinalização celular e nos elementos do CIT assim como no crescimento in vitro de linhagens celulares de CCR. Métodos: Linhagens celulares de CCR (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN e KRCY) foram mantidas em condições ideais para a cultura de células de mamíferos e expostas a drogas e/ou inibidores nas concentrações e por períodos de tempo variáveis. A fosforilação de intermediários da sinalização celular foi determinada utilizando-se western blots. Nível de mRNA para genes de interesse foram determinados por qRT-PCR. Crescimento celular foi avaliado por método colorimétrico em diferentes concentrações de gefitinibe, UO126 e rapamicina, isoladamente ou em combinação. Resultados: UO126 levou a defosforilação não apenas de intermediários de MAPK como também de substratos do alvo da rapamicina em mamíferos (mTOR) revelando sinalização cruzada entre essas vias. Nós identificamos ainda que ERK5 é a quinase potencialmente responsável por esta sinalização cruzada. No entanto, tratamento com UO126 não afetou o nível de mRNA para o substrato de mTOR 4EBP1. Gefitinibe foi capaz de bloquear a sinalização iniciada por EGF na via de PI3K em todas as linhagens wt-PTEN e de bloquear a sinalização através de ERK1/2 e ERK5 ao menos parcialmente em todas as linhagens celulares. Rapamicina mostrou-se um potente inibidor do crescimento na maioria das linhagens celulares de CCR e tal efeito foi freqüentemente amplificado com a combinação com UO126 ou gefitinibe.Conclusão: EGFR, MAPK e CIT são alvos promissores no tratamento do CCR / Background: Renal cell carcinoma (RCC) is an increasing cause of cancer mortality. Despite its curability in early stages, conventional cytotoxic and immunomodulatory agents have been homogeneous in providing minimal or no benefit in the treatment of advanced RCC. Better understanding of tumor cell biology may lead to development of more efficient targeted therapies. Signaling intensification through epithelial growth factor receptor (EGFR), mitogenactivated protein kinase (MAPK) pathway, Phosphatidylinositol 3-kinase (PI3K) pathway and overactivation of the translation initiation complex (TIC) has been previously characterized in RCC cell lines and tumor samples. We investigated the effect of an EGFR inhibitor (gefitinib) as well as a MAPK inhibitor (UO126) and a TIC inhibitor (rapamycin) in the intermediates of cell signaling, in the elements of TIC and in the in vitro growth of RCC cell lines. Methods: RCC cell lines (PRC3, WT8, SKRC-02, SKRC-17, SKRC-39, SKRC-45, ACHN and KRCY) were maintained on standard mammalian cells culture conditions and exposed to drugs and/or inhibitors in variable concentrations and for variable periods of time as required in each experiment. Phosphorylation status of signaling intermediates were determined using western blots. Levels of mRNA for genes of interest were determined by qRT-PCR. Cell growth was assessed by colorimetric method in control conditions or in different concentrations of gefitinib, UO126 or rapamycin, alone or in combination. Results: UO126 caused dephosphorylation not only of MAPK intermediates but also of mammalian target of rapamycin (mTOR) substrates revealing crosstalk between these pathways. We also identified ERK5 as a kinase potentially responsible for such cross talk. However, treatment with UO126 did not affect mRNA levels of the downstream target of mTOR 4EBP1. Gefitinib was able to block EGF signaling through PI3K in all wt- PTEN cell lines and the signaling through ERK1/2 and ERK5 at least partially in all cell lines. Rapamycin was found to be a potent growth inhibitor in most RCC cell lines and such effect was often increased by its combination with UO126 or gefitinib. Conclusion: EGFR, MAPK and CIT are promising targets for the treatment of RCC

La première partie de mon travail de thèse a été de définir le rôle joué par le récepteur nucléaire Rev-erb alpha dans les mécanismes de synchronisation par la nourriture d'une horloge circadienne putative, non encore localisée, appelée " horloge alimentaire ". La seconde partie de mon travail a consisté à étudier la participation de Rev-erb alpha dans les régulations des métabolismes glucidique et lipidique. L'ensemble de nos données indique que le répresseur transcriptionnel Rev-erb alpha joue un rôle charnière dans les fonctions circadiennes ainsi que dans le métabolisme. En effet, d'un point de vue circadien, l'absence de Rev-erb alpha altère la synchronisation à l'heure des repas - démontré par une réduction des sorties comportementales et physiologiques de l'horloge alimentaire, ainsi que par l'absence d'ajustement du rythme de la protéine d'horloge PER2 dans l'oscillateur cérébelleux. Sur le plan métabolique, la délétion de ce gène modifie notamment le métabolisme des lipides - démontré par une accumulation excessive de tissu adipeux, une utilisation préférentielle des acides gras, ainsi qu'une perte de contrôle de l'expression de la Lipoprotéine lipase.

INTRODUÇÃO: Os astrocitomas difusamente infiltrativos são os tumores mais freqüentes de Sistema Nervoso Central (SNC) com uma taxa de 5-7 novos casos por 100.000 pessoas ano. São tumores altamente invasivos e estão associados com alterações de alguns genes como EGF (fator de crescimento epidérmico) e o EGFR (receptor do fator de crescimento epidérmico), que podem criar um aumento da atividade mitogênica, acarretando aumento de proliferação e maturação celular, apoptose, angiogênese e metástase. O nível de expressão destes genes pode ser influenciado por alterações genéticas, como a presença de polimorfismos. Uma mudança única de base (SNP) pode alterar a expressão gênica e, sendo assim, estar associada ao aumento do risco de desenvolver astrocitomas. Nesse trabalho, foram analisados 2 SNPs na região não traduzida (c.-191C>A e c.-216G>T) e um SNP no exon 16 (c.2073A>T) do gene EGFR, e um outro SNP na região não traduzida no gene EGF (c.61A>G). Os SNPs foram associados a expressão gênica do EGFR e a sobrevida dos pacientes. MÈTODOS: Foi realizado um estudo caso-controle com 193 casos de astrocitomas difusamente infiltrativos e 200 controles por amplificação por PCR seguido de digestão enzimática. Os produtos digeridos das amostras foram analisados por eletroforese em gel de agarose e poliacrilamida e corados com brometo de etídeo. A expressão gênica foi realizada após extração de RNA do tecido tumoral seguida de transcrição reversa e PCR em tempo real. Testes de qui-quadrado, odds ratio (OR), intervalo de confiança 95% (IC95%), t de Student e curvas de Kaplan-Meier foram realizados para análises estatística. RESULTADOS: A análise das freqüências dos genótipos dos polimorfismos mostrou uma diferença na distribuição entre casos e controles para o polimorfismo c.2073A>T. Pacientes com o genótipo TT apresentou um menor risco para astrocitoma quando comparados com o genótipo AA (OR=0,51, IC95%=0,29-0,99). Nenhuma correlação foi encontrada para os outros polimorfismos analisados. Também não foi encontrada correlação entre os genótipos dos polimorfismos e os níveis de expressão de EGFR e a sobrevida dos pacientes. CONCLUSÃO: Nosso trabalho mostrou haver um possível fator de proteção quando o paciente é portador do genótipo TT, o que pode levar a uma diminuição do risco de desenvolver o tumor. Pacientes com genótipo TT do polimorfismo c.2073A>T do gene EGFR apresentam um menor risco para astrocitomas difusamente infiltrativos do que os com o genótipo AA. / INTRODUCTION: Diffusely infiltrative astrocytomas are the most frequent tumors of the Central Nervous System (CNS) with a rate of 5-7 new cases in 100,000 individuals per year. They are highly invasive, and they are associated to alterations in some genes as EGF (epidermal growth factor) and EGFR (epidermal growth factor receptor), which may increase mitogenic activity, leading to increase of proliferation, cellular maturation, apoptosis, angiogenesis, and metastasis. Genetic alterations, as presence of polymorphisms of single nucleotide change (SNP) could influence their expression level, and thus could be associated to increased risk in developing astrocytomas. In the present study, two SNP of non-coding region (c.-191C>A and c.-216G>T) and one SNP in exon 16 (c.2073A>T) of EGFR, and another SNP of non-coding region of EGF (c.61A>G) were analyzed. The SNPs were associated to EGFR expression level and to survival time. METHOD: a case-control study of 193 of diffusely infiltrative astrocytomas and 200 controls was carried out, with PCR amplification and enzymatic digestion, which products were analyzed in agarose gel or polyacrylamide gel electrophoresis stained by ethidium bromide. EGFR expression level was studied by real time PCR after RNA extraction followed by reverse transcription of tumor tissues compared to epileptic non-neoplastic brain tissues. Stastistical analysis were performed by chi-square, odds ratio (OR), 95% confidence interval (95% CI), Student-t test and Kaplan Meier. RESULTS: The polymorphic genotype frequency was different between case and controls for the polymorphism c.2073A>T. Patients with TT genotype presented lower risk to develop astrocytoma when compared to genotype AA (OR=0.51, CI95%=0.29- 0.99). No other correlation was observed for the remaining studied polymorphisms. There was neither correlation between the polymorphic genotypes and the EGFR expression levels nor with survival time. CONCLUSION: The present study showed a possible protection factor in developing astrocytomas for the patients harboring the genotype TT of c.2073A>T polymorphism of EFGR, thus the patients presenting TT genotype have lower risk to develop diffusely infiltrative astrocytoma than patients presenting the genotype AA.

Diese Dissertation betrachtet die Beziehung zwischen Parser und Grammatik bei Muttersprachlern (Native Speakers, NS) und Heritage- (Erb-) Sprechern (HS) des Griechischen, indem sie die Mechanismen untersucht, die einer pseudo-Lizenzierung bei Verletzungen der Kongruenz des grammatischen Geschlechts zugrunde liegen. Diese Verletzungen sind Fehler, die auftreten, wenn eine intervenierende Phrase (Attraktor) nicht mit den Genusmerkmalen des Kopfnomens übereinstimmt, ein Phänomen, das in der Literatur (Gender-)Agreement Attraktion, hier Attraktion von Genuskongruenz, genannt wird. Die Dissertation testet, ob eine solche Attraktion von Genuskongruenz im Griechischen vorhanden ist und ob ein- und zweisprachige Muttersprachler gleichermaßen anfällig für Fehler bei der Attraktion sind. Die Dissertation untersucht für die Gruppe der HS außerdem die Genuskongruenz beim Echtzeit-Sprachverstehen und -produzieren. In der Arbeit zeige ich, dass sowohl NS als auch HS anfällig für Attraktionsfehler bei der Genuskongruenz sind. Das zeigen die Reaktionszeitmuster und die Urteile. Gleichzeitig zeigten bei mündlichen Erzählungen beide Sprechergruppen die gleichen Übergeneralisierungsmuster für maskulines Genus bei belebten Nomen sowie bei mündlichen Erzählungen und beschleunigten Grammatikalitätsurteilen für Neutrum bei unbelebten Nomen. Zusammengenommen deuten diese Ergebnisse darauf hin, dass NS und HS anfällig für die Attraktion von Genuskongruenz sind und dass beide Gruppen ähnliche Hinweise zum Abruf des Genus verwenden und somit ähnliche Attraktionsmuster aufweisen. HS unterscheiden sich jedoch von NS in der Verarbeitung der Genuskongruenz an sich, insbesondere bei femininen Kopfnomen (markiertes Genus) in Objekt-Klitika, was darauf hindeutet, dass sowohl Markiertheit als auch Kongruenz an den Schnittstellen die Leistung von HS beeinflusst. Wenn Fehler auftreten, folgen beide Gruppen den gleichen Mustern der Übergeneralisierung. / This dissertation explores the relationship between the parser and the grammar in Native Speakers (NSs) and Heritage Speakers (HSs) of Greek by examining the mechanisms underpinning the illusory licensing of gender agreement violations: errors occurring when an intervening phrase (attractor) mismatches the gender cues of the head noun, a phenomenon which is usually called (gender) agreement attraction. In this work, I show that both NSs and HSs are prone to gender agreement attraction errors in the nominal domain of Greek, as their reaction time patterns and (speeded or scaled) judgements revealed. At the same time, both groups showed the same overgeneralization patterns of the masculine value in agreement errors with animate nouns in their oral narrations, and of the neuter value with inanimate nouns in their oral narrations and their online speeded judgements. Taken together, these results suggest that NSs and HSs are prone to gender agreement attraction in Greek and that both groups employ retrieval cues similarly showing similar attraction patterns. However, HSs differ from NSs in the processing of gender agreement per se, particularly with feminine head nouns (marked gender value) on object-clitics, suggesting that markedness as well as agreement at Interfaces influence HSs’ performance. Finally, when errors occur, both groups follow the same overegeneralization patterns.

Studiens mål är att bidra till kunskapsutveckling om hur unga människor utvecklar handlingskompetens för hållbar utveckling. Med handlingskompetens för hållbar utveckling avses i studien vilja och förmåga att påverka livsstil och levnadsvillkor på ett sätt sominkluderar intergenerationellt och globalt ansvar. I avhandlingen introduceras begreppet avståndsmoral för att beskriva detta ansvar, som utsträcker sig i både tid och rum, till kommande generationer och till nu levande människor globalt. En utgångspunkt för studien är att hållbar utveckling innefattar idén om avståndsmoraliskt ansvar. Studiens huvudfråga är: Hur erfar avståndsmoraliskt aktivt handlande unga människor att de utvecklat handlingskompetens för hållbar utveckling? Studiens teorigrund är livsvärldsfenomenologisk. En upplevd verklighet undersöks i studien via berättelser. Genom ett strategiskt urval har tre intervjupersoner, som motiverar sina handlingar med avståndsmoraliska argument, valts att ingå i studien. Datainsamlingen har skett genom en kombination av öppna livsberättelseintervjuer ochhalvstrukturerade intervjuer. Analys och tolkning har metodologiskt stöd i berättelseforskningstraditionen och empirisk fenomenologisk forskning. Resultaten presenteras som tre citatrika levnadsberättelser om Karin, Carl och Matilda, tre unga vuxna med flera års engagemang i hållbarhetsfrågor. Syftet med levnadsberättelserna är att bidra till förståelse av det speciella i en enskild individs upplevelse av attutveckla handlingskompetens för hållbar utveckling. En integrerande analys redovisar mönster i form av likheter och skillnader mellan de tre individernas berättelser om att utveckla olika aspekter av handlingskompetens i avståndsmoraliska frågor. Sex gemensammakärnpoänger framstår som betydelsefulla: känslomässiga reaktioner, upplevd kompetens, kontrasterande perspektiv och normativ grund, handlingsimpregnering, tillit från och tillit till vuxna samt social gemenskap kontra utanförskap. Som motiv och drivkrafterför engagemanget framstår känslomässiga reaktioner som initierar önskan om förändring och vilja till handling, längtan efter meningsfullhet, önskan om att komma till sin rätt och längtan efter gemenskap. Resultaten diskuteras i förhållande till tidigare forskningoch en modell av en möjlig väg att utveckla aspekter av handlingskompetens för hållbar utveckling presenteras. Diskussionskapitlet utmynnar i fyra didaktiska utmaningar för enutbildning för hållbar utveckling. / The aim of the study at hand is to develop knowledge about the process of developing action competence for sustainable development. The overall research question explored is: How do young people experience that they have developed aspects of action competence forsustainable development? For the purposes of this study, action competence for sustainable development is defined as willingness and capability to act for changes in individual life style, as well as for structural changes of society, in a way that includes responsibility for present and future generations,globally. Life-world phenomenology provides the theoretical foundation of the study. Through purposive sampling, individuals have been found who, through different action strategies, engage in sustainability issues as for example climate change, bio-diversity and social justice. From a larger sample, three young adults have been selected for several life storyinterviews. Data has been analyzed and interpreted by use of narrative methodology. The result is presented as three stories, presented as thick descriptions, through whichthe winding paths towards aspects of action competence, as experienced, appear. This is followed by an integrative analysis presenting six themes that have emerged in theanalyses as relevant in the process of developing action competence for sustainable development: emotional reactions; perceived competence; contrasts and normative foundation;action permeation; trust and faith from adults and in adults; and social belonging in contrastto outsidership. Major motives and driving-forces for sustainability actions that emerge inthe stories are: emotional reactions initiating a desire for change and a desire to act; longingfor meaningfulness; a desire to feel comfortable with what you can contribute; and longing for belongingness. The findings are discussed in relationship to previous research and a modelof a possible way to develop aspects of action competence for sustainable development is introduced. This dissertation is part of a project supported by Formas, The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning.

- The aim of the thesis was to further process the selected buildings in the overall urban design animal farm. - Putting the individual buildings into a very rugged terrain and the subsequent harmony with the surrounding landscape - Create attractive objects for future visitor's animal farm - By ensuring the development of the territory