Shp-1 (Src homology 2 domain-containing phosphatase 1) est une tyrosine phosphatase retrouvée principalement chez les cellules hématopoïétiques, mais également chez les cellules épithéliales. Bien que Shp-1 soit reconnue comme étant un régulateur négatif de plusieurs voies de signalisation intracellulaire chez les cellules hématopoïétiques, son rôle dans les cellules épithéliales a été jusqu’ici très peu étudié. Afin de mieux comprendre son rôle dans les cellules épithéliales intestinales, nous avons généré un modèle murin de délétion conditionnelle de Shp-1 spécifiquement dans l’épithélium intestinal (Shp-1CEI-KO). De manière intéressante, dès l’âge de 6 semaines, les souris expérimentales présentent une intestinalomégalie associée à une légère augmentation de la prolifération cryptale. La taille des cellules épithéliales est également augmentée, suggérant de l’hypertrophie cellulaire chez les souris invalidées pour Shp-1. Parallèlement, la voie de signalisation PI3K/Akt/mTor est activée dans l’épithélium des souris mutantes. Nous avons également noté une production accrue de cellules caliciformes et de leurs précurseures, les cellules intermédiaires, en absence de Shp-1. Par contre, la maturation des cellules de Paneth semble grandement compromise vu la baisse importante d’expression du lysozyme et des RegIIIβ et RegIIIγ, de même que la faible densité de leurs granules de sécrétion. La comparaison du phénotype intestinal des souris Shp-1CEI-KO avec celui des souris PtenCEI-KO suggère que l’hyperactivation de la voie PI3K/Akt/mTor est responsable en partie des altérations phénotypiques observées chez la souris invalidée pour Shp-1. En conclusion, nos résultats montrent que la tyrosine phosphatase Shp-1 est un régulateur important de l’homéostasie de l’épithélium intestinal en contrôlant notamment la croissance cellulaire et la différenciation des cellules de la lignée sécrétrice.

No description available.

Mosaic, activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of class IA phosphatidylinositol 3-kinase (PI3K), are the cause of rare, developmental growth disorders collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). Given the pressing need for targeted therapy and evidence for tissue- and cell lineage-specific distribution of PIK3CA mutations in PROS, developmental models of this disease will be a key asset for preclinical drug testing and for a better understanding of PIK3CA activation in development. This PhD project addressed the lack of human, developmental PROS models by establishing isogenic series of human induced pluripotent stem cells (iPSCs) with endogenously expressed, activating PIK3CA mutations. This involved the optimisation of a CRISPR/Cas9 protocol for efficient knockin of different PIK3CA variants into human iPSCs. An isogenic iPSC series was established with cells expressing either wild-type PIK3CA or PIK3CA-H1047R, knocked into either one or both endogenous alleles. In parallel, mosaic patient- derived fibroblast cultures were reprogrammed to obtain isogenic wild-type and heterozygous iPSCs expressing PIK3CA-E418K. The models were used in comprehensive signalling studies, providing new insights into PI3K signalling in human iPSCs and how it is perturbed by genetic p110α activation. PIK3CA-E418K, a rare variant in both PROS and cancer, caused minimal pathway activation, in contrast to the highly recurrent variant PIK3CA-H1047R which induced strong PI3K signalling in both heterozygous and homozygous iPSCs according to a graded pattern. Studies of clinically relevant PI3K pathway inhibitors provided proof-of-concept that stem cell-based PROS models can be used for preclinical drug testing, and demonstrated that p110α is likely to be the main catalytic isoform mediating canonical PI3K signalling in human iPSCs. Differentiation assays revealed allele dose-dependent effects of PIK3CA-H1047R on stemness, with homozygous iPSCs exhibiting widespread transcriptome remodelling affect- ing genes implicated in cancer and development. Accordingly, these cells showed increased expression of pluripotency genes such as NANOG and NODAL, resulting in self-sustained "stemness" in embryoid body and teratoma assays. In comparison, heterozygous mutants behaved similar to wild-type controls under all differentiation paradigms. Furthermore, evidence was obtained that strong activation of PI3K signalling is fully compatible with definitive endoderm formation, arguing against cell-autonomous differentiation defects as the cause of endoderm sparing in PROS. In summary, these studies demonstrate the utility of human stem cell-based models of PROS for preclinical drug testing and for improved understanding of class IA PI3K signalling in human development. They are also likely to be useful in efforts to obtain a better understanding of PIK3CA-H1047R in human cancer.

Insulin signalling is an essential process in humans by which the level of plasma glucose is maintained within the physiologically healthy range. Insulin activates the phosphoinositide 3 kinase (PI3K) signalling pathway that generates the phospholipid messenger PtdIns(3,4,5)P3, which in turn enhances the activity of two important proteins, AKT and Rac1. This then leads to increase the presence of the glucose transporter 4 (GLUT4) at the plasma membrane that enhances the intake of glucose, particularly in skeletal muscle cells and adipocytes. Insulin signalling also triggers interconversion of several other phosphoinositides (PIs) which play pivotal roles in different steps of glucose regulation. PtdIns5P is an important PI that is robustly increased after insulin treatment in the skeletal muscle cell line, L6 myotubes. Many of PtdIns5P`s functions are not fully understood. To gain more knowledge of the role of PtdIns5P in insulin signalling in muscle cells, the PtdIns5P kinase phosphatidylinositol-5-phosphate 4-kinase a (PIP4K2a) was over-expressed in L6 myotubes as a way of removing PtdIns5P, and the consequences in insulin signalling were studied. Although PtdIns5P is converted by PIP4K2a to PtdIns(4,5)P2 which is a precursor of the potent PI PtdIns(3,4,5)P3, previous studies revealed that the increase in PtdIns(3,4,5)P3 induced by insulin in control cells is diminished in cells overexpressing PIP4K2a, for unknown reasons. Additionally, although the phosphorylation of the serine/threonine protein kinase AKT was not affected in these L6 cells, glucose uptake was attenuated. The current study investigates the possible causes of attenuating glucose uptake in PIP4K overexpressing myotubes by examining the small GTPase Rac1 which plays an important role in the cytoskeleton re-arrangement that is necessary for GLUT4 translocation. Furthermore, the possible roles of PI phosphatases that may cause the disturbance on the levels of PIs in response to insulin were evaluated. Additionally, the potential role of PtdIns5P in Rac1 activation in L6 myotubes was further investigated by delivering synthetic PtdIns5P using a carrier-based delivery approach. The results showed that the attenuation of glucose uptake documented in previous studies occurred as a result of a defect in the process of translocating GLUT4 from intracellular storage to the plasma membrane. Rac1 activity was significantly reduced in cells expressing PIP4K2a. Quantifying the level of PIs suggested that PIP4K2a expression increases the removal of PtdIns(3,4,5)P3 by the PI 5-phosphatase, SKIP. Silencing the expression of SKIP by siRNA restored the level of PtdIns(3,4,5)P3 but Rac1 activity and the attenuation GLUT4 translocation were not rescued possibly as a result of removing PtdIns5P by PIP4K2a. On the other hand, exogenous delivery of PtdIns5P in L6 myotubes activates both Rac1 and GLUT4 translocation in the absence of insulin. However, activating GLUT4 translocation by the exogenous PtdIns5P requires PI3K activity since redistribution of GLUT4 to the plasma membrane is inhibited by the PI3K inhibitor, wortmannin. Removing PtdIns5P reduces Rac1 activity and stimulates SKIP that inhibits PtdIns(3,4,5)P3 increase which attenuates GLUT4 translocation and hence glucose uptake. These results emphasise the critical role played by PtdIns5P which seems to serve as a regulator of insulin signalling, both directly and/or by regulating other enzymes involved in the metabolism of PIs.

O carcinoma epidermoide oral (CEO) possui alta incidência no Brasil, correspondendo a aproximadamente 95% das neoplasias malignas orais. A biologia molecular do carcinoma de cabeça e pescoço é complexa e se desenvolve a partir da disfunção de várias vias inter-relacionadas. A via das PI3K/mTOR é conhecida por regular várias funções celulares, como a regulação do ciclo celular, migração, angiogênese, morfologia e organização do citoesqueleto. O presente trabalho teve como objetivo avaliar o impacto do bloqueio da via PI3K e mTOR na sobrevida celular, na adesão e na morfologia de células endoteliais, relacionando-o com o processo angiogênico. Em cultura de células, observamos que as inibições farmacológicas de PI3K ou do mTOR diminuem a viabilidade celular do CEO. Houve redução da expressão de PARP, um marcador de apoptose e pS6, uma proteína dowstream na sinalização do mTOR, após o tratamento das células de CEO com os inibidores de mTOR. Além disso, demonstramos que o tratamento com os inibidores de mTOR diminuem a capacidade das células do CEO em formarem clones. Foi observado ainda que o cultivo de célula endoteliais da veia umbilical humana (HUVEC, do inglês Human Umbilical Vein Endothelial Cells) em meio condicionado (MC) proveniente de células de CEO, tratadas com inibidores de mTOR, resultaram em aumento da adesão celular e modificação na morfologia celular. Para investigar a influência dos fatores liberados pelas células tumorais na migração das células HUVECs, foi realizado ensaio de wound healing, em células HUVECs cultivadas em MC. Observamos maior migração das células HUVECs cultivadas em MC quando este era proveniente de células cancerosas tratadas com inibidor de mTOR. Ainda, investigamos a expressão da via VEGF nessa migração por meio do ensaio de ELISA. Observamos que no MC proveniente de células cancerosas tratadas com inibidor de mTOR havia maior presença de VEGF. Nossos dados sugerem que a via da PI3K/mTOR está envolvida na proliferação das células do CEO. Porém, a inibição de mTOR em células cancerosas pode liberar fatores, tais como VEGF, que influenciam na morfologia e migração de células HUVEC. / Oral squamous cell carcinoma (OSCC) has a high incidence in Brazil, corresponding to approximately 95% of oral malignancies. The molecular biology of head and neck carcinoma is complex and develops from the dysfunction of several interrelated pathways. The PI3K/mTOR pathway is known to regulate various cellular functions, such as cell cycle regulation, migration, angiogenesis, cytology, and cytoskeletal organization. The aim of the present study was to evaluate the impact of PI3K and mTOR pathway blockade on cell survival, endothelial cell adhesion and morphology, and to correlate it with the angiogenic process. In cell culture, we observed that pharmacological inhibitions of PI3K or mTOR decrease the OSCC\'s cellular viability. There was a reduction in the expression of PARP, a marker of apoptosis and pS6, a dowstream protein in mTOR signaling, after treatment of OSCC cells with mTOR inhibitors. In addition, we have demonstrated that treatment with mTOR inhibitors decreases the ability of the OSCC cells to form clones. It was further noted that human umbilical vein endothelial cell (HUVEC) culture in conditioned media (CM) from OSCC cells treated with mTOR inhibitors resulted in increased cell adhesion and modification in cell morphology. To investigate the influence of the factors released by the tumor cells in the migration of HUVEC cells, a wound healing assay was performed on HUVEC cells cultured in CM. We observed a greater migration of HUVECs cells cultured in CM when it came from cancer cells treated with mTOR inhibitor. Furthermore, we investigated the role of the VEGF pathway in this migration through the ELISA assay. We observed that in CM from cancer cells treated with mTOR inhibitor there was a greater presence of VEGF. Our data suggest that the PI3K/mTOR pathway is involved in the proliferation of OSCC cells. However, inhibition of mTOR in cancer cells may release factors, such as VEGF, which influence the morphology and migration of HUVEC cells.

Producing competent and fertilizable oocytes from in vitro grown primordial follicles could revolutionize female infertility treatment, particularly using fertility preservation approaches that use cryopreserved ovarian tissue. However, the protracted length of folliculogenesis in humans makes follicular culture complex, and the mechanisms controlling the tightly-regulated activation of primordial follicles remain largely unknown. The delicate balance between follicular recruitment and quiescence might be affected by preservation procedures, such as ovarian fragmentation or in vitro culture, that disrupt crucial pathways, such as the Hippo and PI3K/Akt/mTOR signaling pathways, that are involved in this process. When activated, these pathways induce massive recruitment of primordial follicles and accelerate follicular growth in vitro, with potential negative consequences on future oocyte developmental competence. Therefore, we hypothesized that the inhibition of the PI3K/Akt/mTOR pathway might improve follicular growth by slowing down the activation process.In the first part of this thesis, we explored the potential benefit of inhibiting PI3K/Akt/mTOR signaling on the regulation of in vitro follicular activation and growth, as well as its impact on the Hippo pathway. The effect of everolimus (EVE), a specific mTORC1 inhibitor, was compared to the PI3K/Akt activators recently used to reinitiate the growth of residual follicles in the ovarian tissue of patients with premature ovarian insufficiency. We showed that short-term incubation of ovarian cortex with EVE partially delayed follicular recruitment while supporting follicle survival and steroidogenesis. However, morphological abnormalities were observed in all conditions, suggesting that EVE failed to protect follicles from accelerated in vitro growth-related defects.Our findings also provided evidence that ovarian fragmentation, which disrupts the Hippo pathway, contributes to the triggering of primordial follicle recruitment and early development of quiescent human follicles. Moreover, our data suggested that both PI3K/Akt and Hippo signaling could act synergistically to promote follicular activation and growth.In the second part of the project, we further investigated the integrity of EVE-treated follicles based on their ultrastructural and functional status. Our observations indicate that the integrity of oocyte and granulosa cells, as well as their physical contacts, were preserved in EVE and control conditions, although some in vitro grown follicles sustained cryopreservation- and culture- induced damage. We also found that short exposure to EVE allowed the maintenance of intra-follicular communication while preserving follicular developmental potential. Importantly, results obtained suggested that, at a similar developmental stage, cell coupling and oocyte growth may be improved in EVE-treated follicles.Altogether, these data provide better insight into the regulation of the follicular activation process and emphasize the importance of getting closer to physiological conditions to preserve follicle integrity. They also provide proof-of-concept evidence that reducing the initiation of growth is feasible, and suggest that mTORC1 inhibitors are a potentially useful pharmacological tool to regulate in vitro follicular growth. / La production d'ovocytes compétents et fécondables à partir de follicules primordiaux développés in vitro pourrait révolutionner les traitements liés à l'infertilité féminine, en particulier les approches de préservation de la fertilité à partir du tissu ovarien cryopréservé. Cependant, la longue durée de la folliculogenèse chez l'Homme rend la culture folliculaire complexe, et les mécanismes contrôlant l'activation des follicules primordiaux restent largement inconnus. L’équilibre fragile entre quiescence folliculaire et entrée en croissance pourrait être affecté par la fragmentation ovarienne ou la culture in vitro elle-même, qui perturbent deux voies de signalisation cruciales: les voies Hippo et PI3K/Akt/mTOR, respectivement. Lorsqu'elles sont activées, elles induisent un recrutement massif de follicules primordiaux et accélèrent la croissance folliculaire in vitro, avec des conséquences potentiellement néfastes sur la capacité future des ovocytes à devenir compétents. Par conséquent, nous avons émis l’hypothèse que l’inhibition de la voie PI3K/Akt/mTOR pourrait améliorer la croissance folliculaire via un ralentissement du processus d’activation.Dans la première partie de cette thèse, nous avons exploré le potentiel bénéfice d’une inhibition de la voie PI3K/Akt/mTOR sur la régulation de l'activation et de la croissance folliculaire in vitro, ainsi que son impact sur la voie Hippo. L’effet de l’évérolimus (EVE), un inhibiteur spécifique de mTORC1, a été comparé à ceux d’activateurs de PI3K/Akt, récemment utilisés afin d’initier la croissance des follicules résiduels au sein de tissus ovarien de patientes en insuffisance ovarienne précoce. Nous avons montré que l'exposition à court terme de cortex ovarien à l'EVE retardait partiellement le recrutement folliculaire tout en préservant la survie et la stéroidogenèse des follicules. Toutefois, des anomalies morphologiques ont été observées dans toutes les conditions, ce qui suggère que l’EVE ne préserve pas les follicules de défauts liés à une croissance accélérée.Nos résultats ont également prouvé que la fragmentation ovarienne, en perturbant la voie Hippo, contribue au recrutement et au développement précoce des follicules primordiaux. De plus, les données obtenues suggèrent que les voies PI3K/Akt/mTOR et Hippo pourraient agir de manière synergique pour promouvoir l'activation et la croissance folliculaire.Dans la deuxième partie du projet, nous avons étudié la qualité des follicules traités avec de l’EVE en se basant sur des critères ultrastructural et fonctionnel. Nos observations ont indiqué que l'intégrité des ovocytes et des cellules de la granulosa ainsi que leurs contacts physiques était préservée dans les conditions EVE et contrôle, bien que certains follicules en croissance présentent des signes de dommages induits par la cryopréservation et la culture. Nous avons également constaté qu'une courte exposition à l’EVE permettait de maintenir les communications intra-folliculaires tout en préservant le potentiel de développement des follicules. De façon importante, les résultats obtenus suggèrent qu’à un stade de développement similaire, le couplage cellulaire et la croissance des ovocytes pourraient être améliorés dans les follicules traités à l’EVE.En conclusion, ces données contribuent à une meilleure compréhension de la régulation de l'activation folliculaire in vitro, et soulignent l'importance de mimer les conditions physiologiques pour préserver l'intégrité des follicules. Elles apportent également la preuve qu’un ralentissement de l’initiation de la croissance est réalisable, et suggèrent que l’utilisation d’inhibiteurs de mTORC1 pourrait représenter un outil pharmacologique efficace pour réguler la croissance folliculaire in vitro. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

O carcinoma epidermoide oral (CEO) possui alta incidência no Brasil, correspondendo a aproximadamente 95% das neoplasias malignas orais. A biologia molecular do carcinoma de cabeça e pescoço é complexa e se desenvolve a partir da disfunção de várias vias inter-relacionadas. A via das PI3K/mTOR é conhecida por regular várias funções celulares, como a regulação do ciclo celular, migração, angiogênese, morfologia e organização do citoesqueleto. O presente trabalho teve como objetivo avaliar o impacto do bloqueio da via PI3K e mTOR na sobrevida celular, na adesão e na morfologia de células endoteliais, relacionando-o com o processo angiogênico. Em cultura de células, observamos que as inibições farmacológicas de PI3K ou do mTOR diminuem a viabilidade celular do CEO. Houve redução da expressão de PARP, um marcador de apoptose e pS6, uma proteína dowstream na sinalização do mTOR, após o tratamento das células de CEO com os inibidores de mTOR. Além disso, demonstramos que o tratamento com os inibidores de mTOR diminuem a capacidade das células do CEO em formarem clones. Foi observado ainda que o cultivo de célula endoteliais da veia umbilical humana (HUVEC, do inglês Human Umbilical Vein Endothelial Cells) em meio condicionado (MC) proveniente de células de CEO, tratadas com inibidores de mTOR, resultaram em aumento da adesão celular e modificação na morfologia celular. Para investigar a influência dos fatores liberados pelas células tumorais na migração das células HUVECs, foi realizado ensaio de wound healing, em células HUVECs cultivadas em MC. Observamos maior migração das células HUVECs cultivadas em MC quando este era proveniente de células cancerosas tratadas com inibidor de mTOR. Ainda, investigamos a expressão da via VEGF nessa migração por meio do ensaio de ELISA. Observamos que no MC proveniente de células cancerosas tratadas com inibidor de mTOR havia maior presença de VEGF. Nossos dados sugerem que a via da PI3K/mTOR está envolvida na proliferação das células do CEO. Porém, a inibição de mTOR em células cancerosas pode liberar fatores, tais como VEGF, que influenciam na morfologia e migração de células HUVEC. / Oral squamous cell carcinoma (OSCC) has a high incidence in Brazil, corresponding to approximately 95% of oral malignancies. The molecular biology of head and neck carcinoma is complex and develops from the dysfunction of several interrelated pathways. The PI3K/mTOR pathway is known to regulate various cellular functions, such as cell cycle regulation, migration, angiogenesis, cytology, and cytoskeletal organization. The aim of the present study was to evaluate the impact of PI3K and mTOR pathway blockade on cell survival, endothelial cell adhesion and morphology, and to correlate it with the angiogenic process. In cell culture, we observed that pharmacological inhibitions of PI3K or mTOR decrease the OSCC\'s cellular viability. There was a reduction in the expression of PARP, a marker of apoptosis and pS6, a dowstream protein in mTOR signaling, after treatment of OSCC cells with mTOR inhibitors. In addition, we have demonstrated that treatment with mTOR inhibitors decreases the ability of the OSCC cells to form clones. It was further noted that human umbilical vein endothelial cell (HUVEC) culture in conditioned media (CM) from OSCC cells treated with mTOR inhibitors resulted in increased cell adhesion and modification in cell morphology. To investigate the influence of the factors released by the tumor cells in the migration of HUVEC cells, a wound healing assay was performed on HUVEC cells cultured in CM. We observed a greater migration of HUVECs cells cultured in CM when it came from cancer cells treated with mTOR inhibitor. Furthermore, we investigated the role of the VEGF pathway in this migration through the ELISA assay. We observed that in CM from cancer cells treated with mTOR inhibitor there was a greater presence of VEGF. Our data suggest that the PI3K/mTOR pathway is involved in the proliferation of OSCC cells. However, inhibition of mTOR in cancer cells may release factors, such as VEGF, which influence the morphology and migration of HUVEC cells.

L'épissage alternatif des ARNs pré-messagers est le mécanisme majeur de diversification de l'information génétique chez les eucaryotes supérieurs. Près de 70% des gènes sont concernés par ce mécanisme. Au cours de la différenciation érythroïde, l'inclusion de l'exon 16 du pré-messager 4.1R représente un événement majeur pour la fonction érythrocytaire normale de la protéine 4.1R. Cet événement d'épissage est bloqué dans les cellules MEL surexprimant l'oncoprotéine Spi.1/PU.1. Mon travail de thèse avait pour but de comprendre ce mécanisme de blocage en étudiant le rôle de la voie de signalisation de la PI3K en relation avec la phosphorylation de Spi.1/PU.1 d'une part, et celle des facteurs d'épissage de la famille des protéines SR d'autre part. J'ai montré que l'inhibition de la PI3K active la production d'hémoglobine ainsi que l'épissage de l'exon 16 4.1R. Ensuite j'ai montré que la phosphorylation de Spi-1/PU.1 par la PI3K est nécessaire pour son activité d'inhibition de la différenciation et de l'épissage de l'exon 16 4.1R. Enfin, j'ai montré que le facteur d'épissage Srp40 de la famille des protéines SR est un activateur stade spécifique de l'inclusion de l'exon 16. La surexpression de Srp40 dans les cellules MEL active l'inclusion de l'exon 16 indépendamment de la différenciation.

<p>Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins by integrins and the following protein-protein interactions.</p><p>The part of the research that I did, focused on the activation of PI3K by integrins and the question whether Ras is included in that pathway. I also studied the conformation changes of the integrins and tried to identify factors which regulate these changes.</p><p>Known is that Ras can activate PI3K. But we wanted to know if this is a step in the activation of PI3K by integrins. So if this would be a fact then Ras must be activated by integrins.</p><p>To see if integrins could activate Ras I did a pull down assay. GTP loaded Ras was isolated through its affinity for Raf. Only when Ras is in its activated state then it is GTP loaded, otherwise it is GDP loaded. In the experiment we also compared the β1A and the β1B splice variants. As result we could see that both splice variants probably can activate Ras. By blotting with anti-PI3K antibody we looked if PI3K had bound to Ras but no clear result could be obtained.</p><p>Integrins presented on blood cells are mostly in the inactive state while adherent cells have integrins which are mostly in the active state. PI3K has been shown, for blood cells, to be involved in the conformation regulation of integrins. Possibly, there is a positive circle that for blood cells just has to be switched on. It could be that the integrins in adherent cells are active because the cells are adhesive. By being adhesive, PI3K is activated. PI3K may then activate the integrins, through which the integrins stay in the active state. This circle could be broken at two points: we could inhibit PI3K or we could make the cells un-adhesive. I analysed this in cell attachment assay and by binding of conformation-specific integrin antibodies in FACScan. From the results we could not find any evidence that the whole idea around the positive circle is correct. Surprisingly we saw that the integrin value at the surface decrease if you add PI3K inhibitor. This could be due to distribute recirculation of integrins from the cytoplasm to the cell surface.</p><p>β1- and β3-integrins are both widely spread, but no functional difference could be shown already. Previous results suggest that there is a difference between migrations of those two types. To ensure this suggestion I did a wound assay. Hereby I compared the migration of different cell types, with different integrins on their surface and on different ligands.</p>

<p>Integrins are a protein family of cell surface receptors, expressed in all cell types in the human body, except the red blood cells. Besides their importance in mediating physical connections with the surrounding environment, the integrin family members are also vital signalling mediators. They have no intrinsic kinase activity; instead the signals are transduced through conformational changes. </p><p>In this thesis, work is presented which is focused on molecular mechanisms of integrin signal transduction. The signal transduction was first studied from a structural point of view, determining the transmembrane domain borders of a few selected integrin family members and ruling out a signalling model involving a “piston-like” movement. </p><p>Then, downstream signalling events involved in the beta1 integrin-induced activation of Akt via the PI3kinase family were characterized. Our results identify a novel pathway for PI3K/Akt activation by beta1 integrins, which is independent of focal adhesion kinase (FAK), Src and EGF receptor. Furthermore, both beta1 integrins and EGF receptors induced phosphorylation of Akt at the regulatory sites Thr308 and Ser473, but only EGF receptor stimulation induced tyrosine phosphorylation of Akt.</p><p>Finally, signals from beta1 integrins underlying the morphologic changes during cell spreading were studied. A rapid integrin-induced cell spreading dependent on actin polymerisation was observed by using total internal reflection fluorescence (TIRF) microscopy. This integrin-induced actin polymerisation was shown to be dependent on PI3K p110alpha catalytic subunit and to involve the conserved Lys756 in the beta1-integrin membrane proximal part.</p>