Der RAF-MEK-ERK-Signalweg steuert grundlegende, oftmals entgegengesetzte zelluläre Prozesse wie die Proliferation und Apoptose von Zellen. Die Dauer des vermittelten Signals wurde als entscheidener Faktor für die Steuerung dieser Prozesse identifiziert. Es ist jedoch nicht eindeutig geklärt, wie die verschiedenen früh und spät reagierenden Genexpressionsmodule kurze und lange Signale unterscheiden können und durch welche kinetischen Merkmale ihre Antwortzeit bestimmt wird. In der vorliegenden Arbeit wurden sowohl Proteinphosphorylierungsdaten als auch Genexpressionsdaten aus HEK293-Zellen gewonnen, die ein induzierbares Konstrukt des Proto-Onkogens RAF tragen. Hierbei wurde ein neues Genexpressionsmodul identifiziert, dass sich aus sofort induzierten aber spät antwortenden Genen zusammensetzt. Es unterscheidet sich in der Genexpressionsdynamik und Genfunktion von anderen Modulen, und wurde mit Hilfe mathematischer Modellierung experimenteller Daten identifiziert. Es wurde festgestellt, dass diese Gene aufgrund von langen Halbwertszeiten der vermitteltenden mRNA in der Lage sind spät auf das eingehende Signal zu reagieren und die Dauer des Signals in die Amplitude der Genantwort zu übersetzen. Trotz der langsamen Akkumulation und damit späten Antwortzeit, konnte aufgrund einer GC-reichen Promoterstruktur zunächst vermutet und mit Hilfe eines Markerverfahrens bestätigt werden, dass die Transkription dieser Gene instantan mit Beginn der ERK-Aktivierung startet. Eine vergleichende Analyse zeigte, dass das Prinzip der Signaldauer-Entschlüsselung in PC12-Zellen und MCF7-Zellen, zwei paradigmatischen Zellsystemen für die ERK-Signaldauer, konserviert ist. Insgesamt deuten die Ergebnisse der Untersuchung darauf hin, dass das neu identifizierte Genexpressionsmodul der Entschlüsselung der ERK-Signaldauer dient und das mRNA Halbwertszeiten sowohl hierfür, als auch für die zeitliche Abfolge der Genantwort eine entscheidende Rolle spielen. / The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals and what features govern their timing. Both protein phosphorylation and gene expression time course data was obtained from HEK293 cells carrying an inducible construct of the proto-oncogene RAF. A new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function was identified by mathematical modelling. It was found that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis showed that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, the findings of this study indicate that ILGs decode ERK signal duration and that both decoding capacity and gene expression timing are governed by mRNA half-life.

Made available in DSpace on 2016-04-29T13:18:02Z (GMT). No. of bitstreams: 1 Paulo A B Panetta.pdf: 2725022 bytes, checksum: 647e2f895cbc2bf81fd00346a8f0e9ab (MD5) Previous issue date: 2007-05-07 / Fifteen rats were randomized to one of the five following groups (with three rats in each group) to examine the effects of different durations of light ( delay signal ) upon the response acquisition and maintenance of lever press with delayed reinforcement, on the absence of shaping. Two phases construct the experiment, with fifteen experimental sessions and two sessions in extinction on each phase. For the rats randomized to Chain II Group, when Phase 1 was initiated, the duration of the light was initially the same of the delay, but was reduced gradually across phase until it was no longer present with the delay period. For rats randomized to Chain III Group, there was no delay signal initially, but its duration was increased across phase until it equalized the duration of the programmed delay (4 s). The duration of the delay signal was always the same of the delay for the rats employed on Chain I Group, while the was no delay signal for rats randomized to Tand Group. With the rats randomized to the Control Group there was no delay on reinforcement. Within subjects, response rates where higher as the duration of the delay signal increased, with the highest acceleration when its duration was minimal. Response rates where higher also to prior rates when the duration of the delay signal was reduced, with the highest acceleration when its duration was minimal. The findings are discussed in terms of the conditioned reinforcement of the delay signal by reduction the delay between its onset and the reinforcer / Quinze ratos foram distribuídos em cinco grupos com o objetivo de avaliar os efeitos de diferentes durações de luz, que eram apresentadas durante o período de atraso, sobre a aquisição e manutenção da resposta de pressão à barra com atraso de reforço, sem modelagem dessa resposta. O estudo foi dividido em duas fases com dezessete sessões cada (quinze sessões experimentais e duas em extinção). Na Fase 1, para os sujeitos do Grupo Encadeado III, a luz não estava presente nas sessões iniciais. Com o decorrer das sessões, a duração da luz foi aumentada gradualmente até que a sua duração cobrisse todo o período de atraso. Para os sujeitos do Grupo Encadeado II, duração da luz equivalia à totalidade do período de atraso nas sessões iniciais, sendo reduzida gradualmente a sua duração ao longo das sessões até não haver luz durante o período. Nas sessões dos sujeitos do Grupo Tandem não havia luz durante o período de atraso, enquanto nas sessões dos sujeitos do Grupo Encadeado I a duração luz sempre cobria a totalidade do período. Já nas sessões dos sujeitos do Grupo Controle não havia atraso de reforço. Decorrida a Fase 1, o período de atraso foi aumentado de 4 s para 8 s e iniciou-se a Fase 2, idêntica à anterior. Foi observada, em ambas as fases, um aumento na taxa de emissões e de reforços produzidos pelos sujeitos do Grupo Encadeado III na medida que a duração da luz era aumentada, com um aumento maior em ambas as fases quando a luz tinha sua duração mínima. Do mesmo modo, foi notado um pequeno aumento na taxa de emissões e de reforços produzidos pelos sujeitos do Grupo Encadeado II a partir da redução na duração da luz, com aumento maior quando a duração da luz era mínima. Os resultados do presente estudo foram discutidos a partir da noção de reforçador condicionado e da hipótese da redução do atraso proposta por Fantino (1969, 1977)