Le Lupus Erythémateux Systémique est une pathologie inflammatoire chronique. L’étiologie de cette maladie auto-immune est encore méconnue bien que certains facteurs génétiques et environnementaux aggravants aient été mis en évidence. Les traitements proposés aux patients ont pour but de réduire les symptômes et aucun remède curatif n’a encore été mis au point. Nous avons observé de forts taux de sCD95L dans le sérum de patients atteints de LES comparé à celui de sujets sains. Nos données indiquent que ce facteur soluble agit comme une cytokine pro-inflammatoire et promeut la transmigration des lymphocytes T CD4+ Th17 dans les organes, au détriment des lymphocytes T régulateurs (Treg). L’accumulation de ces cellules Th17 est responsable du maintien d’une réponse inflammatoire chronique chez les patients lupiques. Nous mettons en évidence qu’il existe une interaction directe entre le récepteur CD95 et la Phospholipase Cγ1, par l’identification du Calcium Inducing Domain impliqué dans ce recrutement. L’identification du couple CD95/CD95L comme acteur aggravant le LES et la mise en évidences des mécanismes cellulaires et moléculaires sous-jacents nous ont conduit à l’élaboration de stratégies thérapeutiques innovantes. En collaboration avec les chimistes et modélisateurs de notre Unité, nous avons généré une petite librairie d’inhibiteurs, composée de peptides et peptidomimétiques sélectifs. Parmi ces composés, le TAT-CID est une protéine piège comprenant la zone d’interaction de CD95 (domaine CID des aa 175 à 210) à la PLCγ1. L’injection de ce peptide dans un modèle de souris lupiques restaure la fonction biologique rénale de ces souris et diminue la production d’auto-anticorps (anti-DNA) et de complexes immuns, marqueurs biologiques associés à la progression de la pathologie. En parallèle, le criblage d’une librairie chimique commerciale constituée de médicaments approuvés par la FDA et l’EMA a permis d’identifier un inhibiteur efficace de notre interaction. In vivo, ce composé est capable de réduire drastiquement les signes cliniques de la pathologie lupique. Ces nouvelles données enrichissent notre compréhension du processus mis en place par le système CD95/CD95L dans l’aggravation du LES, et nous permettent de proposer des outils thérapeutiques interessants. Ces molécules pourraient représenter de nouvelles options thérapeutiques originales et attrayantes pour prévenir l’inflammation dans les pathologies inflammatoires chroniques. / Systemic Lupus Erythematosus (SLE) is a chronic inflammatory disease. The etiology of this autoimmune disease is still unknown although some aggravating genetic and environmental factors have been identified. The treatments used for patients are intended to reduce the symptoms and no curative one has been developed yet. We observed high levels of sCD95L in the serum of patients with SLE compared to healthy subjects. Our data indicate that this soluble factor acts as a pro-inflammatory cytokine and promotes the transmigration of CD4 + Th17 T lymphocytes to the detriment of regulatory T lymphocytes (Treg) in the enflammed organs of patients. The accumulation of these Th17 cells is responsible for maintaining a chronic inflammatory response in lupus patients. We show that there is a direct interaction between the CD95 receptor and the phospholipase Cγ1, by the identification of the Calcium Inducing Domain involved in this recruitment. The identification of the CD95/CD95L couple as an aggravating factor in SLE context and the underlying cellular and molecular mechanisms led us to the development of innovative therapeutic strategies. In collaboration with the chemists and modellers of our research Unit, we have generated a small library of inhibitors, composed of selective peptides and peptidomimetics. Among these compounds, TAT-CID is a decoy peptide comprising the interaction zone of CD95 (CID domain 175 to 210 aa) with PLCγ1. Repeated treatments of lupus-prone mice with this peptide restore the biological function of these mice and decrease the production of autoantibodies (anti-DNA) and immune complexes, biological markers associated with the progression of the pathology. In parallel, the screening of a commercial chemical library consisting of FDA and EMA-approved drugs allowed us to identify an effective inhibitor of our targeted interaction. In vivo, this compound is able to drastically reduce the clinical signs of lupus pathology. These new data enrich our understanding of the process implemented by the CD95/CD95L system in the aggravation of SLE, and allow us to propose interesting therapeutic tools. These molecules could represent novel and attractive therapeutic options for preventing inflammation in chronic inflammatory pathologies.

SHIP2 is a phosphatase that belongs to the family of the phosphoinositide 5-phosphatases. It is known to dephosphorylate PI(3,4,5)P3 to PI(3,4)P2 imparting a tight control of the PI 3-kinase pathway. Over the last decade, SHIP2 has been described as a tumor promotor or tumor suppressor in several cancer types such as glioblastoma, colorectal cancer or breast cancer cells. Several studies have proposed a role of SHIP2 in breast cancer cells, but its tumor promoting function was unclear at the beginning of this thesis especially in terms of its mode of regulation. In 2013, the INPPL1 gene that encodes SHIP2 has been found to be mutated in opsismodysplasia (OPS), a rare autosomal recessive disease characterized by delayed bone maturation but no molecular mechanism was provided to explain the mechanism. In this thesis, we first contributed to establish a negative regulation of SHIP2 on cell migration in 1321 N1 glioblastoma (GBM) cells. Our studies revealed a dephosphorylation activity of SHIP2 on PI(4,5)P2 at the plasma membrane to control cell migration. This study was done in collaboration with Dr. Elong Edimo in the lab. We have also shown that the regulation of cell motility cannot be generalized to all the GBM cells. In LN229 and U-251 GBM cells we observed a positive regulation of cell migration by SHIP2. We next took advantage of a unique model comparing fibroblasts derived from non-affected and OPS patients (in collaboration with Dr. Valérie Cormier-Daire). We have shown that the fibroblasts from the OPS patients are SHIP2 deficient and migrate slower as compared to fibroblasts from non-affected individuals. Finally, the major part of the thesis was the study of breast cancer cells: in the model MDA-MB-231 cells, we established a positive regulation of SHIP2 on cell migration. We extended this regulation on cell migration to different breast cancer cell models using a SHIP2 inhibitor AS1949490. We confirmed that this inhibitor blocks the phosphatase activity of SHIP2 and showed its selectivity towards SHIP2 in cell migration assay. In MDA-MB-231 cells we deciphered a second messenger role of PI(3,4)P2 to control cell migration. Our data in this model rely on the use of SHIP2 depleted cells obtained by lentiviral infection and shRNA. We confirmed the positive role of SHIP2 on cell migration in the model of rat chondrosarcoma SHIP2CRISPR cells (in collaboration with Dr. Pavel Krejci).A major goal of this thesis was achieved thanks to in-vivo studies: using MDA-MB-231 cells injected in SCID mice, we found a tumor promoting role of SHIP2 by determining the tumor weight. We also observed less lung metastasis of SHIP2 depleted injected cells as compared to control cells suggesting SHIP2 to be important for invasiveness of triple negative breast cancers. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cell migration is a prominent feature in cancer metastasis and the characterization of proteins related to migration is important in order to understand the behavior of tumor cells and their invasiveness during tumor progression. Among the proteins involved in cell migration, isoforms of non-muscle myosin type II play a pivotal role in several events related to cell migration, such as contractility, adhesion and cellular signaling. There are three isoforms of myosin type II that can be expressed in mammalian cells: myosin IIA (MIIA), myosin IIB (MIIB) and myosin IIC (MIIC). However, few studies have been conducted to characterize the expression and distribution patterns in cells of different types of tumors, including oral squamous cell carcinoma. The aim of the study was to analyze the expression and distribution of isoforms of myosin II (A, B and C) in surgical fragments of squamous cell carcinoma. Fragments of surgical specimen were collected from different regions of the tumor: a tumor-free zone, the center of the tumor and the invasion zone. These samples (n = 4) were fixed, crioprotected, cut on criostat, submitted to immunolocalization of MIIA, MIIB and MIIC and analyzed on confocal microscopy. The three isoforms of myosin II were expressed differently and showed distinct distribution in accordance with the region of the tumor sample. MIIA and MIIC were overexpressed at the center zone when compared with the free zone, whereas strong staining revealed MIIB at the zone invasion. Based on these observations, the isoforms of myosin IIC and IIA appeared to be more associated with tumor proliferation while MIIB appeared to be more involved in the invasive behavior of tumor, indicating that the isoforms can participate in different ways regulating the behavior and development of tumor type analyzed. / A migração celular é uma característica proeminente na metástase do câncer e a caracterização de proteínas relacionadas com a migração é importante para que se compreenda o comportamento de células tumorais e sua capacidade de invasão durante a progressão tumoral. Entre as proteínas envolvidas na migração celular, as isoformas de miosina não-muscular do tipo II desempenham um papel central em vários eventos relacionados com a migração de células, como a contratilidade, a adesão e a sinalização celular. Existem três isoformas de miosina do tipo II que podem ser expressas em células de mamíferos: miosina IIA (MIIA), miosina IIB (MIIB) e miosina IIC (MIIC). Entretanto, poucos estudos têm sido realizados para caracterizar a sua expressão e padrões de distribuição em células de diferentes tipos de tumores, incluindo carcinoma epidermóide. O objetivo do estudo foi analisar a expressão e distribuição de isoformas de miosina II (A, B e C) em fragmentos de peça cirúrgica de carcinoma epidermóide. Os fragmentos de peça cirúrgica foram coletados a partir de distintas regiões do tumor: a zona livre de tumor, o centro do tumor e a zona de invasão. Estas amostras (n = 4) foram fixadas, crioprotegidas, cortadas em criostato, submetidas à imunolocalização de MIIA, MIIB e MIIC e analisadas em microscopia confocal. As três isoformas de miosina II foram expressas diferentemente e apresentaram distribuição distinta de acordo com a região da amostra de tumor. MIIA e MIIC foram superexpressas na zona central quando comparadas com a zona livre, ao passo que MIIB revelou forte marcação para região de zona de invasão. Com base nas presentes observações, as isoformas de miosina IIA e IIC pareceram estar mais associadas ao evento de proliferação tumoral ao passo que MIIB pareceu mostrar-se mais envolvida com o comportamento invasivo do tumor, indicando que as isoformas podem participar de formas distintas na regulação do comportamento e do desenvolvimento do tipo de tumor analisado.

Neste estudo avaliou-se os efeitos do diabetes mellitus (DM) sobre dois sistemas: glândula parótida de ratos e células cultivadas in vitro. Foram avaliados respectivamente a composição da matriz extracelular e a migração de células expostas a elevada concentração de glicose. Na parótida observou-se aumento de colágenos III, IV e V, laminina e fibronectina, mediado por TGFb2. Em células isoladas observou-se que a glicose dificultou a polarização celular, reduziu a velocidade e direcionalidade de migração, reduziu a persistência e estabilidade das protrusões celulares e a maturação de adesões. Estas alterações estão relacionadas à ativação da GTPase Rac1, dependente de estresse oxidativo. Este estudo sugere, pela primeira vez, que: 1) a hipofunção salivar pode envolver um espessamento da lâmina basal de capilares e parênquima por mecanismos previamente observados em outros orgãos-alvo de complicações diabéticas e 2) que a glicose exerce um efeito direto sobre a migração celular, fator que pode contribuir para a cicatrização deficiente em indivíduos diabéticos. / In this study we evaluated the effects of DM on two different systems: the rat parotid gland and in vitro cultured cells. Extracellular matrix composition and the migratory behavior of cells exposed to a high glucose concentration (HG) were evaluated, respectively. In the parotid, DM led to an increase in collagens III, IV and V, laminin and fibronectin, through a TGFb2-dependent mechanism. In cultured cells, HG impaired cell polarization, reduced migration velocity and directionality, reduced the persistence and stability of protrusive cellular processes, as well as adhesion maturation. These effects were related to Rac1 GTPase activation, dependent on the oxidative stress promoted by HG. This study suggests, for the first time, that: 1) salivary hypofunction in DM might involve the thickening of capillary and parenchyma basal lamina, through mechanisms already described in other target organs for diabetic complications and 2) that glucose directly impairs cell migration, and this effect may contribute to the chronic wound healing observed in diabetic patients.

Os peptídeos INKKI e YPVQPFTE foram isolados a partir da hidrólise da b-caseína bovina e correspondem às seqüências 26-30 e 114-121 respectivamente. A atividade proliferativa foi avaliada em culturas primárias de linfócitos T. A atividade antitumoral in vitro foi realizada em culturas de B16F10. Foi utilizado grupo com 40 camundongos da linhagem C57BL/6J para avaliar a atividade antitumoral. Nossos resultados mostraram que o peptídeo INKKI apresentou resposta proliferativa semelhante ao mitógeno comercial PHA. O peptídeo YPVEPFTE mostrou ter ação proliferativa maior do que a apresentada pelo mitógeno comercial PHA. O peptídeo INKKI mostrou ação quimiotáxica. O tratamento in vitro mostrou que somente o pentapeptídeo INKKI induz seletiva atividade citotóxica para as células de melanoma. Os animais portadores de tumores dorsais apresentaram significativa inibição da capacidade de crescimento e a metastatização. Conclui-se que os peptídeos apresentam ação significativa tanto nos experimentos in vitro como in vivo sugerindo um possível papel fisiológico. / Peptides INKKI and YPVQPFTE were isolated from the bovine b-casein after hydrolysis corresponding to the 23-30 and 114-121 sequence, respectively. Evaluation of the proliferative activity in primary cultures of lymphocytes. The activity antitumor in vitro was accomplished culture of was studied. Groups with 40 C57BL/6J lines mice had been used to evaluate the antitumoral activity. Our results showed that peptide presented similar proliferative response to the PHA commercial mitogen. The peptide YPVEPFTE showed to have proliferative action larger than presented by the commercial mitogen. The peptide INKKI showed in the chemotactic action. The treatment in vitro had shows that the peptide INKKI induces selective citotoxicity. The bearing animals of dorsal tumors had presented significant inhibition of the capacity of growth and the spread of methastasis. Thus, the peptides casein present significant action in in vitro and in vivo experiments, suggesting a possible physiologic role.

La migration cellulaire possède deux volets antagonistes ; nécessaire à plusieurs processus physiologiques tels que la réponse immunitaire, elle peut également induire la mort d’un organisme en permettant les cellules cancéreuses d’envahir des organes sains. In vivo, la migration s’effectue dans des milieux complexes et confinés qui imposent une forte déformabilité aux cellules migratoires. Récemment, divers études ont montré que le noyau impose la limite de la déformabilité cellulaire lors de la migration en 3D (Wolf et al. JCB, 2013; Harada et al. JCB, 2013). Il a, en effet, été montré que la migration cellulaire peut être augmentée en diminuant la rigidité nucléaire (Wolf et al. JCB, 2013). Cependant, il existe une limite de rigidité nucléaire en dessous de laquelle la migration cellulaire peut être inhibée via l’inhibition de la survie cellulaire (Harada et al. JCB, 2013). Les cellules cancéreuses qui migrent à des vitesses relativement faibles (µm/heure) et ont des noyaux rigides surmontent la limite imposée par la déformation nucléaire en dégradant et élargissant le milieu extracellulaire. Les cellules immunitaires telles que les neutrophiles qui migrent rapidement (10 µm/mn) et ont des noyaux mous sont connus pour mourir aux sites d’infections. Les cellules dendritiques, de la famille des cellules immunitaires, ont une fonction de présentation d’antigènes qui requiert à la fois une grande capacité migratoire et de survie. Elles représentent donc un modèle cellulaire intéressant pour l’étude de la déformation nucléaire chez les cellules qui migrent rapidement et survivent longtemps. Durant mon doctorat, j’ai étudié le mécanisme grâce auquel les cellules dendritiques déforment leurs noyaux afin de migrer de manière efficace en milieux confinés tout en préservant un haut taux de survie. J’ai utilisé un système expérimental nouveau et original consistant en des microcannaux avec des constrictions (Heuzé et al. MMB, 2011). Ces canaux, combinés à des manipulations génétiques et de la video microscopie nous ont permis de montré que les cellules dendritiques possèdent un mécanisme spécifique, indépendant de celui utilisé pour leur migration, leur permettant de déformer leurs noyaux tout en migrant dans des milieux hautement confinés. Ce mécanisme est basé sur la génération d’un réseau d’actin, autour du noyau, nucléé par Arp2/3 et indépendant du moteur Myosin II. Ce réseau d’actine co-localise avec des sites de rupture de la Lamin A/C. De plus, réduire la quantité de Lamin A/C dans les cellules dendritiques inhibe la formation de ce réseau d’actin perinucléaire. Basés sur ces résultats, nous avons proposé un nouveau mécanisme de déformation du noyau lors de la migration en milieux confinés basé sur Arp2/3 qui, en nucléant un réseau d’actine autour du noyau permet de casser la lamin A/C diminuant ainsi la tension de surface nucléaire et permettant le passage noyau. / Cell migration has two opposite faces; necessary for many physiological processes such as immune response, it can also lead to the organism death by allowing metastatic cells to invade new organs. In vivo migration often occurs in complex 3D environments which impose high cellular deformability. Recently, cellular deformability during 3D migration has been shown to be limited by the nucleus (Wolf et al. JCB, 2013). For instance, cell migration can be increased by decreasing nuclear stiffness. However, below a given nuclear stiffness 3D cell migration can be reduced as a result of impaired cell survival (Harada et al. JCB, 2014). Cancer cells which display slow migration and have rather stiff nuclei have been shown to overcome the physical limits of 3D migration through adhesion combined to matrix degradation or high actomyosin contraction (Wolf et al. JCB, 2013). Immune cells such as neutrophils which are fast moving cells with soft nuclei have been reported to die at sites of infection. Interestingly, dendritic cells function as antigen presenting cells requires high migratory ability as well as high survival. They thus constitute an interesting model for studying nuclear deformation in fast moving and long lived cells. During my PhD, I studied the mechanism by which dendritic cells deform their nuclei to achieve proper migration in highly confining space while preserving a high survival rate. I used an original micro fabricated experimental set up (Heuzé et al. MMB, 2011) consisting of microchannels with constrictions to mimic cellular transmigration. Those channels combined with genetic manipulation and live cell imaging followed by image processing were used to assess the mechanism dendritic cells use to deform their nucleus, which we found to be specific and not required for cell motility per se. I showed that dendritic cells overcome the physical limitation imposed by nuclear deformation through small gaps by nucleating an Arp2/3 based actin network around the nucleus. Surprisingly, the formation of this actin network is independent of myosin II based contraction. This actin accumulation around the nucleus co-localized with sites of nuclear Lamin A/C breakage. Moreover, Lamin A/C depletion in dendritic cells leads to the disappearance of this actin ring and the release of the need for Arp2/3 for nuclear deformation. We thus propose a new mechanism of nuclear squeezing through narrow gaps based on an Arp2/3 nucleated actin meshwork which, by transiently breaking the Lamin A/C network, releases the nuclear surface tension and allows nuclear thus cell passage through micrometric constrictions. Lamin A/C repolymerization around the nucleus at the exit of constrictions would then restore nuclear stiffness, allowing cell survival. Interestingly, this actin accumulation around the nucleus was also observed in vivo in migrating macrophages but not in HL-60 derived neutrophils. Taken together, our data suggest that the Arp2/3 based nuclear squeezing mechanism would be a general feature of highly migratory cells which need to survive long enough to accomplish their functions.

Les déplacements cellulaires, collectifs ou individuels, sont essentiels pour assurer des fonctions fondamentales de l'organisme (réponse immunitaire, morphogenèse), mais jouent également un rôle crucial dans le développement de certaines pathologies (invasion métastatique).Les processus cellulaires à l'origine du déplacement forment une activité complexe, auto-organisée et fortement multi-échelle en temps mais aussi en espace. Mettre en évidence des principes généraux de la migration est donc un enjeu majeur. Dans cette thèse, nous nous intéressons à la construction de modèles de migration individuelle qui prennent en compte ce caractère multi-échelle de manière minimale.Dans une première partie, nous nous intéressons à des modèles particulaires. Nous décrivons des processus intracellulaires clés de la migration de manière discrète au moyen de processus de population. Puis, par une renormalisation en grand nombre d'individus, taille infinitésimale et dynamique accélérée, nous obtenons des équations de dynamique continue et stochastique, permettant de faire le lien entre la dynamique intracellulaire et le déplacement macroscopique.Nous nous confrontons d'abord à la situation d'un leucocyte se déplaçant dans une artère, et développant des liaisons de différentes natures avec les molécules de la paroi, jusqu'à éventuellement s'arrêter. La dynamique de formation de liaisons est décrite par un processus stochastique de type Naissance et Mort avec Immigration. Ces liaisons correspondent à des forces de résistance au mouvement. Nous obtenons explicitement le temps d'arrêt moyen de la cellule.Puis, nous nous intéressons à la reptation cellulaire, qui se produit grâce à la formation d'excroissances au bord de la cellule, appelées protrusions, qui avancent sur le substrat et exercent des forces de traction. Nous modélisons cette dynamique au moyen d'un processus de population structurée par l'orientation de la protrusion. Le modèle continu limite obtenu peut être étudié pour la migration 1D, et donne lieu à une équation de Fokker-Planck sur la distribution de probabilité de la population de protrusion. L'étude d'une configuration stationnaire permet de mettre en avant une dichotomie entre un état non motile et un état de déplacement directionnel.Dans une seconde partie, nous construisons un modèle déterministe minimal de migration dans un domaine discoïdal non déformable. Nous nous basons sur l'idée selon laquelle les structures responsables de la migration renforcent la polarisation de la cellule, ce qui favorise en retour un déplacement directionnel. Cette boucle positive passe par le transport d'un marqueur moléculaire dont la répartition inhomogène caractérise un état polarisé.Le modèle comporte un problème de convection-diffusion sur la concentration en marqueur, où le champs d'advection correspond à la vitesse d'un fluide de Darcy modélisant le cytosquelette. Son caractère actif est porté par des termes de bord, ce qui fait l'originalité du modèle.Du point de vue analytique, le modèle 1D présente une dichotomie face à une masse critique. Dans les cas sous-critique et critique, il est possible de montrer l'existence globale de solutions faibles, ainsi que la convergence à taux explicite vers l'unique état stationnaire correspondant à un état non polarisé. Au delà de la masse critique et pour des masses intermédiaires, nous mettons en évidence deux états stationnaires supplémentaires correspondant à des profils polarisés. De plus, pour des conditions initiales assez asymétrique, nous démontrons l'apparition d'un blow-up en temps fini.Du point de vue numérique, des tests numériques en 2D sont effectués en volumes finis (Matlab) et éléments finis (FreeFem++). Ils permettent de mettre en évidence à nouveau des états motiles et non motiles. L'effet de perturbations stochastiques est étudié, permettant d'aborder des cas de réponse à des signaux extérieurs chimique (chimiotactisme) ou mécanique (obstacle). / Collective or individual cell displacements are essential in fundamental physiological processes (immune response, embryogenesis) as well as in pathological developments (tumor metastasis). The intracellular processes responsible for cell motion have a complex self-organized activity spanning different time and space scales. Highlighting general principles of migration is therefore a challenging task.In a first part, we build stochastic particular models of migration. To do so, we describe key intracellular processes as discrete in space by using stochastic population models. Then, by a renormalization in large population, infinitesimal size and accelerated dynamics, we obtain continuous stochastic equations for the dynamics of interest, allowing a relation between the intracellular dynamics and the macroscopic displacement.First, we study the case of a leukocyte carried by the blood flow and developing adhesive bonds with the artery wall, until an eventual stop. The binding dynamics is described by a stochastic Birth and Death with Immigration process. These bonds correspond to resistive forces to the motion. We obtain explicitly the mean stopping time of the cell.Then, we study the case of cell crawling, that happens by the formation of protrusions on the cell edge, that grow on the substrate and exert traction forces. We describe this dynamics by a structured population process, where the structure comes from the protrusions' orientations. The limiting continuous model can be analytically studied in the 1D migration case, and gives rise to a Fokker-Planck equation on the probability distribution for the protrusion density. For a stationary profile, we can show the existence of a dichotomy between a non motile state and a directional displacement state.In a second part, we build a deterministic minimal migration model in a discoïdal cell domain. We base our work on the idea such that the structures responsible for migration also reinforce cell polarisation, which favors in return a directional displacement. This positive feedback loop involves the convection of a molecular marker, whose inhomogeneous spatial repartition is characteristic of a polarised state.The model writes as a convection-diffusion problem for the marker's concentration, where the advection field is the velocity field of the Darcy fluid that describes the cytoskeleton. Its active character is carried by boundary terms, which makes the originality of the model.From the analytical point of vue, the 1D model shows a dichotomy depending on a critical mass for the marker. In the subcritical and critical cases, it is possible to show global existence of weak solutions, as well as a rate-explicit convergence of the solution towards the unique stationary profile, corresponding to a non-motile state. Above the critical mass, for intermediate values, we show the existence of two additional stationary solutions corresponding to polarised motile profiles. Moreover, for asymmetric enough initial profiles, we show the finite time apparition of a blowup.Studying a more complex model involving activation of the marker at the cell membrane permits to get rid of this singularity.From the numerical point of vue, numerical experiments are led in 2D either in finite volumes (Matlab) or finite elements (FreeFem++) discretizations. They allow to show both motile and non motile profiles. The effect of stochastic fluctuations in time and space are studied, leading to numerical simulations of cases of responses to an external signal, either chemical (chemotaxis) or mechanical (obstacles).

Tato diplomová práce se zabývá popisem principů kultivace embryonálních fibroblastových buněk myší (3T3), lidských endoteliálních buněk odebraných z pupečníkové žily (HUVEC) a epiteliálních buněk vaječníku čínského křečka (CHO) v mikrofluidních systémech simulujících kapiláry. Byly provedeny literární rešerše v oblasti realizací experimentu “Scratch Wound Healing Assay” v mikrofluidních systémech s použitím fibroblastů a endotheliálních buněk. V práci jsou dále popsány principy konfokální a fluorescenční mikroskopie a metody zpracování obrazů pro sledování buněčné migrace. Experimentální nastavení pro mikrofluidní realizaci “Scratch Wound Healing Assay” s použitím trypsinu – EDTA pro vytvoření rýhy, a konfokálního mikroskopu Leica TCS SP8 X pro následující snímání pořízených dat bylo navrženo a otestováno s dostatečným počtem opakování. Vhodný algoritmus pro analýzu buněčné migrace byl napsán v programovacím prostředí Matlab. Závěrem této práce je diskuze získaných výsledků.

Single cell migration in artificial collagen gels as an in vitro model system in the context of cancer are studied. Cell and matrix mechanical properties are determined using atomic force microscopy and an advanced analysis method. Matrix pore-size is studied using a novel approach and analysis method. A novel, minimally invasive approach to determine the amount of displacement of the cell microenvironment due to force generation of single cells during migration in artificial 3D collagen gels is introduced. An automated analysis and user friendly software to analyze high-throughput cell invasion is introduced. These methods are used to study cell migration and mechanical properties of the breast cancer cell lines MDA-MB-231 and MCF-7 and the influence of cell nuclear elasticity is investigated. Using mouse embryonic fibroblasts, the role of focal adhesion kinase (FAK) during cell migration is studied using FAK deficient knock-out cell lines FAK-/- and control FAK+/+ as well as kinase-dead mutants FAKR454/R454 and control FAKWT/WT.:Abstract i Acknowledgements iii 1 Introduction 1 2 Background 5 2.1 Cancer — An ever-changing Disease 5 2.1.1 Carcinogenesis and Neoplasm 6 2.1.2 Hallmarks of Cancer 7 2.1.3 Metastasis— The malignant Progression of Cancer 7 2.1.4 Metastatic Cascade 9 2.2 The Cell— Where it begins 10 2.2.1 Actomyosin Complex 12 2.2.1.1 Actin Monomer 12 2.2.1.2 Polymerization 12 2.2.1.3 Structures 14 2.2.1.4 Actin Cortex 15 2.2.1.5 Filopodia 16 2.2.1.6 Lamellipodium 16 2.2.1.7 Invadopodium 17 2.2.1.8 Stress Fibers 17 2.2.1.9 Actin in Cancer and Metastasis 17 2.2.1.10 Myosin and Actin 18 2.2.2 Focal Adhesions 19 2.2.3 Microtubules 20 2.2.4 Intermediate Filaments 21 2.2.5 Cellular Stiffness 22 2.2.6 Nuclear Deformability 23 2.3 The Extracellular Matrix— Where it happens 24 2.3.1 Components and Structure 25 2.3.2 Collagen as a Model System 26 2.3.2.1 Collagen I Fibril Formation 27 2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28 2.4 Single Cell Migration— Why it spreads 29 3 Materials and Methods 31 3.1 Cell Culture 31 3.1.1 Cancer Cells 31 3.1.2 Mouse fibroblasts 32 3.1.3 Pharmacological treatment 34 3.2 Collagen matrices 34 3.3 Cell Elasticity 36 3.3.1 Atomic Force Microscopy 36 3.3.2 Preparation 37 3.3.3 Data Aquisition 38 3.3.4 Data Analysis 38 3.4 Matrix Stiffness 40 3.4.1 Preparation 40 3.4.2 Data Aquisition 41 3.4.3 Data Analysis 41 3.5 Invasion Assay 42 3.5.1 Preparation 42 3.5.2 Data aquisition 44 3.5.3 Data Analysis 44 3.6 Matrix Topology 48 3.6.1 Preparation 49 3.6.2 Data Acquisition 50 3.6.3 Data Analysis 51 3.6.3.1 Binarization 51 3.6.3.2 Pore-Size 53 3.6.3.3 Fiber Thickness 54 3.7 Fiber Displacement 55 3.7.1 Preparation 56 3.7.2 Data Aquisition 56 3.7.3 Data analysis 57 3.7.3.1 Fiber Displacement 59 3.7.3.2 Cell Segmentation 60 3.7.3.3 Shell Analysis 61 3.8 A toolset to understand Single Cell Migration and what influences it 62 4 Results 65 4.1 Cell Elasticity 65 4.1.1 Example Force-Distance Curves 66 4.1.2 Single Cell Elasticity 67 4.2 Matrix Stiffness 69 4.3 Invasion 71 4.4 Matrix Topology 75 4.5 Influence of Cell Nucleus on Cell Migration 79 4.5.1 Cellular Elasticity 79 4.5.2 Invasion 81 4.6 Fiber Displacement 89 4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93 4.7.1 FAK Knock-Out 93 4.7.2 Kinase-dead FAK Mutant 96 5 Discussion 103 References 107 / Die Einzelzellmigration in künstlichen Kollagennetzwerken als ein in vitro Modellsystem im Kontext von Krebs wurde studiert. Mechanische Eigenschaften von Zellen und der verwendeten Kollagennetzwerke wurden mithilfe der Atomic Force Microscopy (AFM) und weiterentwickelten Analysemethoden bestimmt. Die Porengröße der verwendeten Kollagennetzwerke wurde mit einer neuentwickelten Auswertemethode analysiert. Eine neuartige, minimal-invasive Methode zur Bestimmung der Verformung der Mikroumgebung von Zellen während der Migration verursacht durch Kräftegenerierung der Zelle wird beschrieben. Die Analyse des Invasions-Assays wurde automatisiert und eine nutzerfreundliche Software entwickelt, mit der große Datenmengen ausgewertet werden können. Diese Methoden wurden verwendet, um mechanische Eigenschaften und Migration der humanen Brustkrebszellinien MDA-MB-231 und MCF-7 zu studieren. Die Rolle der focal adhesion kinase (FAK) wurde mithilfe von embryonalen Maus-Fibroblasten studiert. Sowohl eine FAK knock-out Zellinie FAK-/- und Kontrolle FAK+/+, als auch eine kinase-dead Mutante FAKR454/R454 und Kontrolle FAKWT/WT wurden hinsichtlich ihrer Invasion und Verformung der Mikroumgebung analysiert.:Abstract i Acknowledgements iii 1 Introduction 1 2 Background 5 2.1 Cancer — An ever-changing Disease 5 2.1.1 Carcinogenesis and Neoplasm 6 2.1.2 Hallmarks of Cancer 7 2.1.3 Metastasis— The malignant Progression of Cancer 7 2.1.4 Metastatic Cascade 9 2.2 The Cell— Where it begins 10 2.2.1 Actomyosin Complex 12 2.2.1.1 Actin Monomer 12 2.2.1.2 Polymerization 12 2.2.1.3 Structures 14 2.2.1.4 Actin Cortex 15 2.2.1.5 Filopodia 16 2.2.1.6 Lamellipodium 16 2.2.1.7 Invadopodium 17 2.2.1.8 Stress Fibers 17 2.2.1.9 Actin in Cancer and Metastasis 17 2.2.1.10 Myosin and Actin 18 2.2.2 Focal Adhesions 19 2.2.3 Microtubules 20 2.2.4 Intermediate Filaments 21 2.2.5 Cellular Stiffness 22 2.2.6 Nuclear Deformability 23 2.3 The Extracellular Matrix— Where it happens 24 2.3.1 Components and Structure 25 2.3.2 Collagen as a Model System 26 2.3.2.1 Collagen I Fibril Formation 27 2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28 2.4 Single Cell Migration— Why it spreads 29 3 Materials and Methods 31 3.1 Cell Culture 31 3.1.1 Cancer Cells 31 3.1.2 Mouse fibroblasts 32 3.1.3 Pharmacological treatment 34 3.2 Collagen matrices 34 3.3 Cell Elasticity 36 3.3.1 Atomic Force Microscopy 36 3.3.2 Preparation 37 3.3.3 Data Aquisition 38 3.3.4 Data Analysis 38 3.4 Matrix Stiffness 40 3.4.1 Preparation 40 3.4.2 Data Aquisition 41 3.4.3 Data Analysis 41 3.5 Invasion Assay 42 3.5.1 Preparation 42 3.5.2 Data aquisition 44 3.5.3 Data Analysis 44 3.6 Matrix Topology 48 3.6.1 Preparation 49 3.6.2 Data Acquisition 50 3.6.3 Data Analysis 51 3.6.3.1 Binarization 51 3.6.3.2 Pore-Size 53 3.6.3.3 Fiber Thickness 54 3.7 Fiber Displacement 55 3.7.1 Preparation 56 3.7.2 Data Aquisition 56 3.7.3 Data analysis 57 3.7.3.1 Fiber Displacement 59 3.7.3.2 Cell Segmentation 60 3.7.3.3 Shell Analysis 61 3.8 A toolset to understand Single Cell Migration and what influences it 62 4 Results 65 4.1 Cell Elasticity 65 4.1.1 Example Force-Distance Curves 66 4.1.2 Single Cell Elasticity 67 4.2 Matrix Stiffness 69 4.3 Invasion 71 4.4 Matrix Topology 75 4.5 Influence of Cell Nucleus on Cell Migration 79 4.5.1 Cellular Elasticity 79 4.5.2 Invasion 81 4.6 Fiber Displacement 89 4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93 4.7.1 FAK Knock-Out 93 4.7.2 Kinase-dead FAK Mutant 96 5 Discussion 103 References 107

Organ formation is an important step during development of an organism that combines different scales from the molecular to the tissue level. Many organogenesis phenomena involve epithelial morphogenesis, where sheets of cells undergo rearrangements to form complex architectures – organ precursors, which subsequently develop into mature organs. Timely development of the characteristic architectures of the organ precursors is crucial for successful organogenesis and is determined by the choice of epithelial rearrangements that organise the constituent cells in space and time. However, for many organogenesis events the cellular dynamics underlying such epithelial rearrangements remain elusive. In the work presented here, I investigated the morphogenesis of the hemispherical retinal neuroepithelium (RNE), that serves as an organ precursor of the neural retina. Formation of RNE is an important event in vertebrates that shapes the optic cup and sets the stage for subsequent eye development. I investigated RNE morphogenesis in the developing zebrafish embryo by visualising and investigating the cellular dynamics of the process in vivo. My findings show that the zebrafish RNE is shaped by the combined action of two different epithelial rearrangements – basal shrinkage of the neuroepithelial cells and involution of cells at the rim of the developing optic cup. The basal shrinkage of the neuroepithelial cells bends the neuroepithelial sheet and starts the process of invagination. However, my results show that the major player in RNE morphogenesis is rim involution. Rim involution translocates prospective RNE cells to their designated location in the invaginating layer and contributes to RNE invagination. My work unravelled the so far unknown mechanism of rim involution. I show that the rim cells involute by collective epithelial migration using directed membrane protrusions and dynamic cell-matrix contacts. If rim migration is perturbed, the prospective RNE cells cannot reach the invaginating layer. As a result, these migration-defective cells attain the RNE fate at an ectopic location and disrupt the tissue architecture. Therefore, rim migration coordinates the cellular location with the timing of RNE fate determination and orchestrates RNE morphogenesis in space and time. Overall, my work highlights how morphogenetic processes shape the organ precursor architecture and ensure timely organ formation. These findings provide important insights not only for eye development but also for epithelial morphogenesis and organogenesis in many other systems. / Für die Entwicklung eines Organismus ist die Bildung von Organen (Organogenese) von zentraler Bedeutung. Organogenese umfasst Prozesse auf allen Ebenen der Längenskala: von der molekularen Ebene, der Gewebeebene, bis hin zur Ebene des ganzen Organismus. Viele Phänomene der Organogenese beinhalten dabei Veränderungen von Epithelien, bei der sich Schichten von Zellen zu komplexen Strukturen - Organvorläufern - umwandeln. Diese entwickeln sich später zu vollständigen Organen. Die rechtzeitige Entwicklung der charakteristischen Architektur der Organvorläufer ist entscheidend für eine erfolgreiche Organogenese und wird durch die Wahl der epithelialen Umwandlungsprozessen bestimmt, welche die Zellen in Raum und Zeit koordinieren müssen. Für viele dieser Prozesse ist jedoch genau diese zugrundeliegende Zelldynamik unklar. In der hier vorgestellten Arbeit untersuchte ich die Bildung des hemisphärischen retinalen Neuropepithels (RNE). Das RNE ist der Organvorläufer der neuralen Retina, weshalb dessen korrekte Bildung die Voraussetzung für die korrekte Entwicklung der Augen ist. Ich untersuchte die RNE-Morphogenese in sich entwickelnden Zebrafisch-Embryos durch Visualisierung und Untersuchung der zellulären Dynamik der beteiligten Prozesse in vivo. Meine Ergebnisse zeigen, dass das RNE in Zebrafischen durch die kombinierte Umwandlung von zwei verschiedenen Epithelien geformt wird. Zum einen findet eine Verkleinerung des basalen Prozesses der neuroepithelialen Zellen statt, zum anderen die Involution von Randzellen. Die basale Verkleinerung der neuroepithelialen Zellen verbiegt die neuroepitheliale Schicht und führt zur Einstülpung des RNE. Meine Ergebnisse zeigten allerdings, dass Involution von Randzellen noch bedeutsamer für die RNE-Morphogenese ist. Die involution von Randzellen transportiert potenzielle RNE-Zellen in das Neuroepithel und trägt zur RNE-Einstülpung bei. Die Bedeutung meiner Arbeit liegt darin, den bisher unbekannten Mechanismus der Randzell-Involution entdeckt zu haben. Ich zeigte, dass die Randzellen sich aktiv durch kollektive epitheliale Migration bewegen indem sie gerichtete Membranforsätze und dynamische Zell zu Matrix Kontakte etablieren. Wird die Migration der Randzellen inhibiert, so führt dies dazu, dass diese Zellen die eingestülpte RNE Schicht nicht erreichen. Sie landen dann an den falschen Positionen, wo sie die Gewerbearchitektur stören können. Daher koordiniert die Randzellmigration die Position der Zellen und orchestriert die RNE-Morphogenese in Raum und Zeit. Insgesamt zeigt meine Arbeit, wie morphogenetische Prozesse die Organvorläuferarchitektur prägen und eine rechtzeitige Organbildung sicherstellen. Diese Erkenntnisse sind sowohl für das Verständnis der Augenentwicklung, als auch für das der epithelialen Morphogenese und Organogenese in anderen Systemen von großer Bedeutung.