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Ras-MAPK signalling regulates key cellular processes such as proliferation, differentiation and survival. Unsurprisingly, mutations in RAS genes are now recognized as potent oncogenic drivers. However, disruption of this pathway during development is associated with a family of disorders termed the Rasopathies. Shared clinical features include cutaneous, neurological and cardiac anomalies. At the outset of this study, the genetic etiology of three neurocutaneous disorders, microcephaly-capillary malformation syndrome (MIC-CAP), encephalocraniocutaneous lipomatosis (ECCL) and PHACE (Posterior fossa malformations, facial Hemangiomas, cerebral Arterial anomalies, Cardiovascular defects and Eye abnormalities) syndrome had not yet been established. This thesis identifies mutations in STAM-binding protein (STAMBP) in a cohort of individuals with MIC-CAP syndrome using whole-exome sequencing (WES). This gene encodes a deubiquitinating isopeptidase that regulates cell surface receptor-mediated endocytosis and sorting. Cell lines of individuals with MIC-CAP show reduced STAMBP expression, associated with accumulation of ubiquitinated protein aggregates, increased apoptosis and constitutive activation of the Ras-MAPK and PI3K-AKT pathways. WES also enabled the identification of post-zygotic mutations within the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1) in individuals with ECCL. Fibroblasts from affected individuals showed increased phosphorylation of the FGFRs consistent with receptor activation as well as insensitive signal transduction through the Ras-MAPK pathway. Neurocutaneous syndromes can feature striking vascular lesions such as the cerebral vasculopathy and large segmented facials hemangiomas seen in PHACE syndrome. The asymmetric and patchy vascular malformations coupled with a sporadic incidence and absence of familial recurrence suggested that PHACE might be caused by post-zygotic mutations. Interrogation of a discordant sib-pair using copy number analysis and WES did not identify causative mutations indicating the need for a comprehensive and targeted –omic approach to elucidate the molecular mechanism of this syndrome. Taken together, these findings expand the spectrum of the Rasopathies while providing novel pathomechanistic insights into the regulation of cellular proliferation and survival during development.

[Truncated abstract] Androgens inhibit the growth of human breast tumours and have been successfully used to treat breast cancer in women. Expression of the androgen receptor (AR), which mediates androgen action, is upregulated in breast cancer cells and the AR is the most frequently expressed steroid hormone receptor in breast tumours. AR levels and activity are modulated by the activity of other signalling pathways, however interactions between the AR and signalling pathways and the consequent alterations to the androgen responsiveness of breast cancer cells are largely uncharacterised. The extracellular signal regulated kinase (ERK1/2) pathway is hyperactivated in ~30% of breast tumours and these tumours are often associated with low oestrogen receptor-a (ERa) levels, reduced responsiveness to antioestrogen therapies and an overall poorer prognosis. In this thesis, the MCF-7 human breast cancer cell line which expresses ERa, progesterone receptor (PR) and the AR, was used to investigate ERK1/2-mediated regulation of the AR and the androgen responsiveness of cells. Inhibition of ERK1/2 signalling was achieved by treatment of cells with U0126, an inhibitor of MEK1/2, the upstream activator of ERK1/2. Hyperactivation of ERK1/2 signalling was achieved by stably transfecting cells with a plasmid encoding a constitutively active form of the MEK1 protein (¿MEK1), resulting in the isolation of two clonal cell populations stably expressing ¿MEK1, ¿C3 and ¿6B, and a monoclonal cell line stably expressing the empty vector, MT3-1. Steady state AR mRNA levels, quantitated using real-time RT-PCR, were increased following U0126 treatment of MCF-7, MT3-1 and ¿6B cells. Conversely, treatment of cells with 10-8M 5a-dihydrotestosterone (DHT) for up to 72 hours decreased AR mRNA levels, indicating that ERK1/2 hyperactivation did not alter the androgenresponsiveness of AR mRNA. '...' Overall levels of AR phosphorylation were enhanced in ¿6B cells in the absence and presence of ligand, indicating that ERK1/2 hyperactivation either directly or indirectly induced receptor phosphorylation. The AR is localised in the cytoplasm in the absence of ligand and was more rapidly translocated to the nucleus in the presence of DHT in ¿C3 cells, an effect that was abrogated in the presence of U0126, thereby indicating an ERK1/2-specific mechanism. AR transcriptional activity, measured using androgen responsive reporter plasmids was not significantly altered in ¿6B cells in either the absence or presence of DHT, although the trend towards enhanced AR activity may be confirmed in future studies using optimised reporter assays. Consistent with the cell cycle regulatory functions of ERK1/2 signalling, proliferation of ¿C3 cells and ¿6B cells was increased in comparison to that of MT3-1 and MCF-7 cells. Treatment of ¿C3 cells and MCF-7 cells with 10-10 – 10-8M DHT produced similar inhibition of proliferation (~40%) during 8 days of culture, with no evidence of cytotoxicity. The results obtained in this thesis demonstrate that while ERK1/2 signalling regulates AR phosphorylation, processing and intracellular localisation, ERK1/2 hyperactivation in breast cancer cells does not inhibit the anti-proliferative effects of androgens. These findings support the development of tissue-specific androgenic treatments for breast tumours including poor prognosis tumours exhibiting ERK1/2 hyperactivation.

Yes / A key aim of Alzheimer disease research is to develop efficient therapies to prevent and/or delay the irreversible progression of cognitive impairments. Early deficits in long-term potentiation (LTP) are associated with the accumulation of amyloid beta in rodent models of the disease; however, less is known about how mGluR-mediated long-term depression (mGluR-LTD) is affected. In this study, we have found that mGluR-LTD is enhanced in the APPswe /PS1dE9 mouse at 7 but returns to wild-type levels at 13 months of age. This transient over-activation of mGluR signalling is coupled with impaired LTP and shifts the dynamic range of synapses towards depression. These alterations in synaptic plasticity are associated with an inability to utilize cues in a spatial learning task. The transient dysregulation of plasticity can be prevented by genetic deletion of the MAP kinase-activated protein kinase 2 (MK2), a substrate of p38 MAPK, demonstrating that manipulating the mGluR-p38 MAPK-MK2 cascade at 7 months can prevent the shift in synapse dynamic range. Our work reveals the MK2 cascade as a potential pharmacological target to correct the over-activation of mGluR signalling. / Wellcome Trust, Grant/Award Number: 200646/Z/16/Z

Sexual development begins with the process by which the bipotential gonads of the embryonic urogenital ridge develop into either testes or ovaries. In the mouse, sex determination occurs at around 11.5 dpc and depends on the presence or absence of the Y chromosome and the associated activity of the testis-determining gene, Sry, in supporting cell precursors. The mutually antagonistic male and female developmental pathways are regulated by many cellular and molecular processes, disruption of which can lead to disorders of sex development (DSDs). However, many of the molecular mechanisms regulating the differentiation of the two gonads are still unknown. The boygirl (byg) mutant was identified in an ENU-based forward genetic screen for embryos with gonadal abnormalities. On the C57BL/6J background, XY byg/byg homozygotes exhibited complete embryonic gonadal sex reversal. The defective gene in byg, Map3k4, is a component of the mitogen-activated protein (MAP) kinase signalling pathway and provides the first evidence for a function of this pathway in sex determination. This thesis describes experiments aimed at investigating the cellular and molecular basis of the sex reversal phenotype associated with the XY Map3k^4byg/byg mutant. Cellular characterisations revealed a defect in male-specific proliferation at 11.5 dpc, which was attributed to a defect in Sry up-regulation. Elucidation of the downstream kinases activated by MAP3K4 during sex determination was attempted, with particular focus on identifying a role for p38α MAP kinase (MAPK). Using a conditional knockout approach, the function of p38α in Steroidogenic factor-1 (Sf1)-positive somatic cells was assessed. However, specific inactivation in these cells did not affect gonad development. Conditional inactivation of Map3k4 itself in these Sf1¬-positive cells also did not disrupt gonad development, suggesting that this pathway is either initiated in a different cell lineage or at an earlier stage than deletion driven by Sf1-Cre can disrupt. Conditional inactivation of p38α in the Müllerian duct mesenchyme and ovarian granulosa cells using Amhr2-Cre did reveal a function for p38α in female fertility, but did not disrupt embryonic sexual development. Gene knockdown in organ culture was attempted to determine a role for multiple p38 MAPKs in all cell types of the gonad. Therefore, this thesis details further characterisations of a novel signalling pathway important for the expression of Sry, focussing on the role of the p38 MAPKs.

[Truncated abstract] Reactive oxygen species (ROS) have been implicated to play a major role in many pathological conditions including heart attack and stroke. Their ability to modulate the extracellular signal-regulated protein kinase (ERK) and c-Jun Nterminal kinase (JNK) signalling pathways, thereby influencing cellular response has been well-documented. Recent studies implicate a central role for mitochondria in ERK and JNK activation by ROS although the mechanisms remained unresolved. Using Jurkat T-lymphocyte as a cell model, this study demonstrated increased mitochondrial ROS production as a result of decreased mitochondrial complex activities mediated by hydrogen peroxide treatment. This is the first study to show that mitochondria are not essential for activating ERKs, however damaged mitochondria producing ROS can be expected to cause sustained ERK activation . . . This study revealed that JNK and its upstream kinases MKK4, MKK7 and ASK1 are associated with the mitochondria. Furthermore, findings from this study imply that JNK resides in the mitochondrial matrix. This study is the first to demonstrate that mitochondrial JNK can be activated in a cell-free environment by signals originating from the mitochondria. Experimental work using isolated mitochondria demonstrated that mitochondrial JNK can be activated by ROS generated from the mitochondria themselves. Flavin-containing proteins appear to be the main sources of mitochondrial-ROS which signal through redoxsensitive proteins to activate mitochondrial JNK.

[Truncated abstract] The enlargement of the heart, also known as myocardial hypertrophy, is thought to be a compensatory process that maintains the mechanical function of the heart in response to stress factors such as pressure or volume overload. Although this process is initially compensatory, it frequently results in heart failure and death. Cardiac hypertrophy is a complex process involving changes in the individual cardiac muscle cells, cardiac myocytes. As well as the morphological changes that result from hypertrophy, there are molecular changes within each cell that regulate the hypertrophic process. These molecular changes involve many different pathways within the cardiac myocytes and remain poorly understood . . . Both STAT3α and β overexpression resulted in the upregulation of the VEGF, MnSOD and SOCS-3 genes. This indicates that in the heart, STAT3β is able to activate the gene expression of these genes in a similar manner to STAT3α. However, STAT3α or β activation alone is not enough to induce cardiac hypertrophy. In conclusion, the results presented in this thesis determined a novel role for ERK in the induction of cell death in the heart and revealed many changes in cardiac gene expression following ERK activation. These genes may be the mediators of ERK responses and their identification provides valuable information and direction for further research in this area. One consequence of ERK activation was the negative regulation of the STAT3 pathway. Further investigation revealed for the first time that the STAT3 proteins themselves may not be involved in the induction of cardiac hypertrophy and that STAT3β, initially thought to be a transcriptional repressor, can induce the expression of genes that are known to be activated by STAT3α in the heart. Therefore, these results help to better understand the roles of these two signalling pathways in the heart.

An der Interpretation extrazellulärer Signale beteiligte Regulationsnetzwerke sind von zentraler Bedeutung für alle Organismen. Extrazelluläre Signale werden gewöhnlich durch enzymatische Kaskaden innerhalb weniger Minuten in den Zellkern weitergeleitet, wo sie langsame Änderungen der Genexpression bewirken und so das Schicksal der Zelle beeinflussen. Im ersten Teil der Arbeit wird durch mathematische Modellierung untersucht, wie die MAPK Kaskade Signale von der Zellmembran in den Kern weiterleitet. Es wurden Netzwerkeigenschaften herausgearbeitet, die verhindern, dass die MAPK Kaskade fälschlicherweise durch genetische Mutationen aktiviert wird. Desweiteren wurde eine versteckte positive Rückkopplungsschleife identifiziert, welche die Aktivierung der MAPK Kaskade oberhalb eines gewissen Schwellwert-Stimulus verstärkt. Der zweite Teil der Arbeit konzentriert sich darauf, wie Änderungen der Genexpression auf langsamer Zeitskala in das Signalnetzwerk rückkoppeln. Eine systematische Genexpressionsdaten-Analyse ergab, dass transkriptionelle Rückkopplung in Eukaryoten generell über Induktion kurzlebiger Signalinhibitoren geschieht. Dynamische Modellierung und experimentelle Validierung von Modellvorhersagen ergab, dass das Inhibitorprotein SnoN als zentraler negativer Feedback Regulator im TGFbeta Signalweg fungiert. Der dritte Teil der Arbeit untersucht, wie die Genexpressionsmaschinerie intrazelluläre Signale interpretiert (“dekodiert“). Eine experimentelle und theoretische Analyse der cyanobakteriellen Eisenstress-Antwort ergab, dass IsrR, eine kleine regulatorische RNA, die Genexpression auf ausreichend starke und lange Stimulation beschränkt. Des Weiteren wurde ein “Reverse Engineering“-Algorithmus auf Hochdurchsatz-RNAi-Daten angewendet, um die Topologie eines krebsrelevanten Transkriptionsfaktornetzwerks abzuleiten. Zusammenfassend wurde in dieser Dissertation gezeigt, wie mathematische Modellierung die experimentelle Analyse biologischer Systeme unterstützen kann. / Intracellular regulatory networks involved in sensing extracellular cues are crucial to all living organisms. Extracellular signals are rapidly transmitted from the cell membrane to the nucleus by activation of enzymatic cascades which ultimately elicit slow changes in gene expression, and thereby affect the cell fate. In the first part of this thesis, the Ras-MAPK cascade transducing signals from the cell membrane to the nucleus is analyzed using mathematical modeling. Model analysis reveals network properties which prevent the MAPK cascade from being inappropriately activated by mutations. Moreover, the simulations unveil a hidden positive feedback loop which ensures strong amplification of MAPK signalling once extracellular stimulation exceeds a certain threshold. The second part of the thesis focuses on how slow gene expression responses feed back into the upstream signalling network. A systematic analysis of gene expression data gathered in mammalian cells demonstrates that such transcriptional feedback generally involves induction of highly unstable signalling inhibitors, thereby establishing negative feedback regulation. Dynamic data-based modelling identifies the SnoN oncoprotein as the central negative feedback regulator in the TGFbeta signalling pathway, and corresponding model predictions are verified experimentally in SnoN-depleted cells. The third part of the thesis focuses on how intracellular signals are decoded by the downstream gene expression machinery. A combined experimental and theoretical analysis of the cyanobacterial iron stress response reveals that small non-coding RNAs allow cells to selectively respond to sufficiently strong and sustained stimuli. Finally, a reverse engineering approach is applied to derive the topology of a complex mammalian transcription factor network from high-throughput knock-down data. In conclusion, this thesis demonstrates how mathematical modelling can support experimental analysis of biological systems.

Les fichiers accompagnant le document sont en format Microsoft Excel 2010. / Les modèles classiques de signalisation cellulaire eucaryotes sont généralement organisés en voies linéaires et hiérarchiques, impliquant un ensemble de facteurs restreint. Ces facteurs forment un circuit isolé qui transmet une information externe vers sa destination, d’où une réponse cellulaire sera alors engendrée. Or, ces modèles sont justement le fruit d’approches expérimentales réductionnistes qui ne permettent pas d’intégrer aisément la contribution de facteurs multiples, ni de faire une évaluation quantitative de l’apport des composantes du système. Le développement de techniques d’investigation plus holistiques, telles la génomique fonctionnelle et la protéomique, permettent d’examiner de manière systématique et quantitative l’apport d’ensembles larges de facteurs et de les mettre en relation avec d’autres systèmes cellulaires. Il y aurait donc lieu de réévaluer le modèle de voie de signalisation linéaire au profit d’un modèle de réseau de signalisation multiparamétrique, comportant plusieurs branches d’entrée et sortie de signal interagissant avec d’autres systèmes cellulaires. Cet ouvrage porte sur la voie RAS/MAPK, l’un des principaux axes de signalisation associé à la prolifération et la différenciation cellulaires. Le sujet y est d’abord abordé sous l’angle d’une perspective historique, en mettant l’emphase sur les contributions des études de génétique classique chez les organismes modèles D. melanogaster et C. elegans. Il fait ensuite état du développement du criblage par ARNi pan-génomique dans ces deux modèles en le comparant aux approches de criblage génétique classique. Le corps de l’ouvrage décrit ensuite les résultats expérimentaux d’une campagne de criblage par ARNi visant à dresser une carte globale des régulateurs de la voie chez la drosophile. Trois groupes de régulateurs identifiés dans ce crible ont été caractérisés de manière plus détaillée. Dans un premier article, nous démontrons que les composantes du complexe EJC ont un impact sur l’épissage de mapk; une découverte doublement intéressante puisque l’EJC était jusqu’alors associé qu’à la régulation post-épissage des ARNm. Une seconde publication fait état de l’ensemble des résultats du crible ARNi, mettant l’emphase sur un ensemble de facteurs d’épissage qui modulent également mapk. Nous y montrons que l’impact de ces facteurs sur l’épissage alternatif est différent de celui de l’EJC, suggérant ainsi deux modes de régulation distincts. Finalement, dans un troisième manuscrit, nous nous attardons au rôle d’Usp47, une déubiquitinase qui, contrairement aux autres facteurs identifiés dans le crible, régule l’expression de MAPK de manière post-traductionnelle. Nous y détaillons une stratégie de criblage d’interaction génétique par ARNi visant à identifier des facteurs reliés fonctionnellement à Usp47. Ce second crible a permis l’identification de trois facteurs reliés au « N-end rule », un mécanisme de dégradation des protéines caractérisé par la reconnaissance des résidus N-terminaux de protéines ou peptides. Il existait jusqu’alors très peu de données quant à la régulation de l’expression des composantes de la voie MAPK, ce qui rend la description d’un large réseau de régulateurs agissant sur l’expression de MAPK d’autant plus insoupçonnée. L’absence d’un réseau équivalent rattaché aux autres composantes de la voie laisse supposer que MAPK serait un noeud servant de point d’entrée à ce type de régulation dans le système RAS/MAPK. De plus, nos travaux témoignent de la capacité de la génomique fonctionnelle à mettre en relation différents systèmes cellulaires de manière plus globale et à quantifier les liens établis entre eux. / The classical model of eukaryotic cellular signalling generally involves hierarchically organized linear pathways involving a restricted set of elements. These generally function together as an insulated circuit, transmitting information from the outside to the intracellular compartment involved in eliciting a response. These models, often the fruit of reductionist experimental approaches, do not allow for the integration of multiple inputs nor for a gradation of responses. The recent emergence of more holistic investigation techniques has brought about the re-evaluation of these classical models in favor of multiparametric signalling networks. This thesis focuses on the RAS/MAPK pathway, one of the cell’s main proliferation and differentiation signalling conduits, beginning with a historical perspective covering the contributions of model organism genetics to the current pathway model. This provides context for the description of a whole-genome RNAi screen experiment that we carried out to obtain a global view of regulators in Drosophila. Three groups of factors emerging from this screen were then examined in more detail. A first article shows that the exon junction complex (EJC) plays a role in mapk alternative splicing, an observation that is unexpected given that this complex was not previously known to act on splicing. A second paper details the genome wide screening campaign and focuses on a large set of splicing factors that also regulate mapk, albeit in a distinct manner than the EJC’s. Finally, a manuscript in a third segment examines Usp47 function and finds it to control MAPK levels post-translationally. An RNAi-based genetic interaction screen is then used to identify factors functionally related to Usp47 capable of counteracting its impact on MAPK levels. Three such factors identified through this technique are linked to the N-end rule protein degradation pathway. Regulation of core pathway component expression is a poorly described process, which makes the identification of a large set of factors regulating MAPK expression all the more unusual. Moreover, the absence of such regulation linked to other pathway components suggests that MAPK may act as a node incorporating inputs of this type into RAS/MAPK signaling dynamics.