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Ein Schlüsselereignis, welches dem prognosebestimmenden Organversagen bei systemi-schen Entzündungsprozessen und Sepsis vorangeht, ist die Entwicklung einer mikrovas-kulären endothelialen Schrankenstörung. Das vaskuläre endotheliale (VE-) Cadherin als mechanischer Stabilisator der Endothelbarriere spielt dabei eine wichtige Rolle. In der Inflammation werden Spaltprodukte von VE-Cadherin (sVE-Cadherin) gebildet. Ge-genstand der vorliegenden Arbeit war die Untersuchung der Hypothese ob diese Spalt-produkte selbst an der Störung der endothelialen Barrierefunktion beteiligt sind. Es wurde hierfür humanes sVE-Cadherin bestehend aus den extrazellulären Domänen EC1-5 (sVE-CadherinEC1-5) generiert. In Messungen des transendothelialen elektrischen Widerstands (TER), mit Immunfluoreszenzfärbungen und Western Blot Analysen wird gezeigt, dass sVE-Cadherin dosisabhängig die Barriere Integrität in primären humanen dermalen Endothelzellen stört. Dies führt zu einer Reduktion von VE-Cadherin und den assoziierten Proteinen α-, γ- und δ-Catenin und ZO-1, die nach der Applikation von sVE-Cadherin an den Zellgrenzen reduziert sind. Die Interaktion zwischen VE-PTP und VE-Cadherin wird durch sVE-CadherinEC1-5 reduziert. Durch pharmakologische Hem-mung der Phosphataseaktivität von VE-PTP mittels AKB9778 wird der durch sVE-CadherinEC1-5-induzierte Verlust der Endothelbarriere aufgehoben. Dagegen zeigt die direkte Aktivierung von Tie-2 mittels Angiopoetin-1 keinen protektiven Effekt auf die durch sVE-CadherinEC1-5 gestörte Endothelbarriere. Weitere Analysen zeigen eine erhöh-te Expression von GEF-H1 durch sVE-CadherinEC1-5. Diese ist ebenfalls durch AKB9778 hemmbar. Zusätzlich zu diesen Untersuchungen wurden die Konstrukte EC1-4 und EC3-5 in ver-schiedene Vektoren kloniert, um zu bestimmen, ob die extrazelluläre Domäne 5 von VE-Cadherin die dominante Rolle bei den sVE-Cadherin-vermittelten Effekten spielt. Zusammenfassend zeigen diese Untersuchungen zum ersten Mal, dass sVE-CadherinEC1-5 unabhängig von proinflammatorischen Auslösern über die Aktivierung des VE-PTP/RhoA-Signalweges den Zusammenbruch der Endothelbarriere mitversursacht. Dies stellt einen neuen pathophysiologischer Mechanismus dar, der zum Gesamtverständnis der entzündungsinduzierten Barriereveränderungen des Endothels beiträgt. / A key prognostic event preceding organ failure in sepsis and systemic inflammatory pro-cesses is dysfunction of the microvascular endothelial barrier. The transmembrane pro-tein vascular endothelial (VE-) cadherin is an important prerequisite to stabilize endothe-lial barrier. VE-cadherin is cleaved under inflammatory conditions which results in the release of soluble VE-cadherin (sVE-cadherin). The main hypothesis of this thesis is to investigate whether sVE-cadherin itself directly disrupts the endothelial barrier in the absence of proinflammatory stimuli. Human sVE-cadherin consisting of extracellular domains EC1-5 (sVE-cadherinEC1-5) was generated and applied onto primary human dermal endothelial cells (HDMECs) for structural and functional analysis. Measurements of transendothelial electrical resistance (TER) and 4 kDa FITC-dectran flux revealed that sVE-cadherinEC1-5 dose-dependently disrupts endothelial barrier integrity. This was confirmed by immunostaining and im-munoblotting analysis which showed that sVE-cadherinEC1-5 treatment reduced overall levels of VE-cadherin and the associated proteins α-, γ- and δ-catenin and ZO-1 as well as their distribution at the cell border of HDMECs. sVE-cadherinEC1-5 treatment reduced the interaction between the phosphatase VE-PTP and VE-cadherin. Accordingly, phar-macological inhibition of VE-PTP using AKB9778 reversed sVE-cadherinEC1-5-induced endothelial barrier loss. Further analysis showed that the increased expression of GEF-H1 by sVE-cadherinEC1-5 is also attenuated by AKB9778. In addition to these studies, the constructs EC1-4 and EC3-5 were cloned into different vectors to determine wheth-er the extracellular domain 5 of VE-cadherin plays the dominant role in sVE-cadherin-mediated effects. In summary, these studies show for the first time that sVE-cadherinEC1-5 actively con-tributes to breakdown of the endothelial barrier independently of proinflammatory stim-uli via activation of the VE-PTP/RhoA signaling pathway. This represents a new patho-physiological mechanism that adds to the understanding of inflammation-induced endo-thelial barrier changes.

Cell migration is critical in generating complex animal forms during development; misregulation of migration contributes to pathological conditions such as cancer metastasis. Thanks to its easily traceable cell lineages in a transparent body and a compact genome accessible to a wealth of genetic manipulations, the use of the nematode C. elegans as a model system has greatly advanced our understanding of mechanisms governing cell migration conserved through higher organisms. Among several migration processes in C. elegans, sex myoblast (SM) migration is an attractive system that has a simple and well-defined migratory route along the ventral side from the posterior to the precise center of the gonad. A multitude of guidance mechanisms control SM migration, many of which are likely to be conserved in other migratory processes. Similar to vertebrate systems, C. elegans uses Rho family small GTPases to regulate the engine of cell motility, the actin cytoskeleton, in response to guidance cues. The differential utilizations of Rho GTPases in distinct processes in vivo remain a central question in the study of Rho GTPases. I investigated how Rho GTPases regulate different aspects of SM migration, and found that Cdc-42/CDC42 functions in the anteroposterior migration, whereas MIG-2/RhoG and CED-10/Rac1 control ventral restriction independently of FGF and SLIT/Robo signaling. The relative difficulty in perturbing SM migration using constitutively active Rho GTPases compared to other migration processes illustrates the robustness of the mechanisms that control SM migration. On a technical aspect, I established a nematode larval cell culture system that allows access to postembryonic cells. Compared to the flourishing genetic researches in C. elegans, there are few studies of molecules that also extend to the subcellular level in postembryonic development, mainly due to the lack of a larval cell culture system. I developed a novel method combining SDS-DTT presensitization of larval cuticles and subsequent pronase E digestion. My method efficiently isolates both low- and high-abundance cell types from all larval stages. This technical advance will not only facilitate studies such as regulation of actin dynamics with high-resolution microscopy, but is beginning to be used by researchers to tackle cell-type specific questions through profiling methods as gene expression analysis. / Ph. D.

Yes / Abstract Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial: GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments. Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity.

Titre de l'écran-titre (visionné le 30 octobre 2023) / Le processus complexe de division des cellules eucaryotes repose en grande partie sur l'organisation et la régulation méticuleuses de divers composants cellulaires, tels que les microtubules et le cortex d'actine. La coordination entre ces deux structures est essentielle au cours de la division cellulaire car un défaut de cette dernière peut conduire à une aneuploïdie. Le rôle des microtubules est central dans la division cellulaire, en particulier lors de la mitose. Lorsque les cellules entrent en mitose, elles subissent un processus de remodelage rapide et dynamique, au cours duquel les microtubules de l'interphase se désassemblent et se réassemblent, à partir des centrosomes, en microtubules plus courts et plus dynamiques. Lorsque la rupture de l'enveloppe nucléaire (NEBD) se produit, ces microtubules s'étendent et atteignent les chromosomes, formant un fuseau bipolaire au centre duquel les chromosomes seront alignés. Une fois les chromosomes alignés correctement, le point de contrôle d'assemblage du fuseau est satisfait et les cellules entrent en anaphase, au cours de laquelle les chromosomes se séparent. L'importance du cytosquelette d'actine au cours de la mitose est largement reconnue. Lors de l'entrée en mitose, les cellules perdent leurs adhésions focales, leurs extrémités se rétractent, elles s'arrondissent et le fuseau mitotique se forme. La mise en place de cette réorganisation cellulaire nécessite l'activation du complexe Cyclin B/CDK1 puis sa relocalisation vers le noyau. La géométrie de l'adhésion cellulaire peut influencer la position du fuseau mitotique et la mémoire des événements cellulaires entre générations, soulignant l'importance du cytosquelette d'actine dans le processus d'entrée en mitose. Alors que les changements dans la dynamique des microtubules et du cytosquelette d'actine lors de l'entrée en mitose semblent indépendant l'un de l'autre, des observations récentes suggèrent qu'il y aurait des interactions entre le fuseau mitotique et le cortex d'actine par l'intermédiaire des GTPases (Matthews et al., 2012). Les GTPAses de la famille RHO sont activées par les facteurs d'échange nucléotidiques (GEFs) en réponse à une pléthore de stimuli extracellulaires, régulant plusieurs réponses cellulaires telles que la prolifération, la différentiation et le mouvement. De nombreux événements de signalisation cellulaire sont contrôlés par les GTPAses de la famille RHO, notamment les changements dans la morphologie cellulaire, l'adhésion, la motilité, la progression du cycle cellulaire, la survie, l'apoptose et la cytokinèse. ARHGEF17, aussi appelé TEM4 (tumor endothelial marker 4), une protéineGEF de la famille RHO, qui est spécifique de RHOA/B/C, est impliquée dans la régulation du cytosquelette interphasique. Il a été démontré que TEM4 se lie directement à l'actine et aux filaments d'actine dynamiques nouvellement assemblés via son extrémité N-terminale, indépendamment de son activité RHOGEF. Dans les cellules en interphase, il est suspecté que TEM4 et RHOC suppriment la contractilité de la myosine et contrôlent le désassemblage des adhésions focales. TEM4 a aussi été identifiée comme une protéine essentielle à la mitose requise pour la localisation du fuseau monopolaire 1 (MPS1) au kinétochore. L'objectif de cette thèse est d'identifier les fonctions de TEM4 au cours du cycle cellulaire. Cette étude met en évidence le manque de spécificité de l'anticorps commercial le plus communément utilisé pour marquer TEM4 et démontre que cette protéine joue un rôle crucial dans l'entrée en mitose, l'alignement des chromosomes et la formation du fuseau mitotique. L'inhibition de l'expression de TEM4 empêche les cellules de s'arrondir et altère l'actine corticale dans les cellules mitotiques, entrainant une augmentation de RHOA actif, comme le montre la quantification de la RHOA-GTP au niveau du cortex mitotique, ce qui confirme sa fonction de régulateur de la contractilité cellulaire. Les phénotypes mitotiques observés en absence de TEM4 sont dus en partie à de faible niveaux de Cyclin B, expliquant le délai mitotique observé. L'expression exogène de Cyclin B rétablit la progression du cycle cellulaire, augmente l'index mitotique et résout l'excès de chromosomes retardés observé en absence de TEM4. Dans l'ensemble, cette étude apporte un nouvel éclairage sur l'importance de TEM4 dans le cycle cellulaire et contribue aux connaissances de son rôle dans les mécanismes cellulaires. / The intricate process of eukaryotic cell division relies heavily on the meticulous organization and regulation of various cellular components, such as the microtubules and the actin-based cortex. Proper coordination between these two structures is essential during cell division, as failure to do so can result in aneuploidy. The role of microtubules is central to cell division, particularly during mitosis. When cells enter mitosis, they undergo a rapid and dynamic remodeling process, during which the interphase microtubules disassemble and give rise to shorter, more dynamic microtubules emanating from centrosomes. Once Nuclear Envelope Breakdown (NEBD) occurs, these microtubules extend and reach the chromosomes, forming a bipolar spindle in the midzone of which captured chromosomes will align. Upon achieving proper orientation and satisfying the spindle assembly checkpoint, cells enter anaphase, during which the chromosomes separate. The importance of the actin cytoskeleton during the process of mitosis is widely recognized. Mitotic entry is characterized by the retraction of the cell margin, followed by the rounding up of the cell and the formation of the mitotic spindle. CDK1-Cyclin B activation and subsequent translocation into the nucleus are required for this process to occur. Upon activation of the complex and its translocation to the nucleus, there is a loss of cellular adhesion, followed by cell rounding up, culminating in mitotic entry. The geometry of cell adhesion can also impact the position of the mitotic spindle and the memory of cellular events between cell generations, highlighting the significance of the actin cytoskeleton to the mitotic entry process. While changes in the dynamics of the microtubule and actin cytoskeletons at the onset of mitosis appear to occur independently, recent evidence suggests that there may be crosstalk between the mitotic spindle and the actin cortex through GTPases (Matthews et al., 2012). RHO family GTPases are activated by guanine nucleotide-exchange factors (GEFs) in response to a plethora of extracellular stimuli, regulating several cellular responses, such as proliferation, differentiation, and movement. Among the numerous signaling events driven by RHO family GTPases are changes in cell morphology, adhesion, motility, cell cycle progression, survival, apoptosis, and cytokinesis. ARHGEF17, also called TEM4 (tumor endothelial marker 4), a RHO family GEF specific for RHOA/B/C, has been shown to regulate the interphase cytoskeleton. TEM4 has been shown to directly bind actin, specifically dynamic newly assembled actin filaments, via its N-terminus independently of its RHOGEF activity. In interphase cells, TEM4 and RHOC are proposed to suppress myosin contractility and to control focal adhesion disassembly. TEM4 has also been identified as an essential mitotic protein required for the localization of Monopolar Spindle 1 (MPS1) at the kinetochore. This thesis aims to understand the functions of TEM4 during the cell cycle. This study highlights the lack of specificity of the most commonly used commercial antibody against TEM4 and provides evidence that TEM4 plays a crucial role in mitotic entry, chromosome alignment, and spindle formation. The downregulation of TEM4 prevents cells from rounding up and alters cortical actin in mitotic cells, leading to an increased activation of RHOA, as shown by quantification of RHOA-GTP at the mitotic cortex, confirming its function as a regulator of cellular contractility. The mitotic phenotypes observed in the absence of TEM4 are found to be partially due to low levels of Cyclin B, explaining the observed mitotic delay. The expression of exogenous Cyclin B restores cell cycle progression, increases the mitotic index, and rescues the excessively lagging chromosomes observed in the absence of TEM4. Overall, despite the limited literature available on TEM4, this study sheds new light on the importance of TEM4 in the cell cycle and contributes to our understanding of this protein's role in cellular processes.

Glaucoma is a degenerative disease that contributes greatly to vision loss and blindness worldwide. There are several treatments that focus on alleviating its symptoms, but few address the underlying pathophysiology, the blockage of aqueous outflow, and the onset of neuroretinal damage. Novel drugs under investigation have aimed to address this deficiency. Two of these Rho kinase inhibitors have been approved by national health agencies, and several are in clinical trials. This work investigates the promise of inhibitors of the Rho kinase signaling pathway to treat primary open-angle glaucoma.

Les bactériophages sont les virus spécifiques des bactéries. Ils sont considérés comme les entités biologiques les plus abondantes de la biosphère (1031 au total). Une grande partie des bactériophages sont dits tempérés de part leur propriété à intégrer leur génome dans celui de leur hôte et à s'y maintenir en état de réplication passive appelé lysogénie. Les gènes de prophages apportent de nouvelles propriétés à l'hôte via la conversion lysogénique. De nombreux prophages défectifs et fonctionnels sont maintenus dans les génomes bactériens. Nous avons émis l'hypothèse que des stratégies de maintien aient été sélectionnées pour maintenir cette source de gènes, même si elle est potentiellement dangereuse car les prophages peuvent être induits dans des conditions de stress.Nos résultats suggèrent que le maintien de la lysogénie d'une catégorie de prophages, qui présente une organisation génétique atypique du module de recombinaison spécifique de site, est sous le contrôle du facteur de terminaison de la transcription Rho. Pour ces prophages, qu'ils soient défectifs ou fonctionnels, leur induction par inactivation de Rho, fait intervenir une nouvelle voie d'induction lytique indépendante de la voie classique via la réponse SOS.Ces interactions hôtes-virus reflète la coévolution de ces microorganismes, qui permet l'acquisition de gènes via le transfert horizontal tout en contrôlant l'expression des gènes délétères. Ceci permet l'acquisition de nouvelles propriétés et l'adaptation de l'hôte à différentes conditions environnementales. / Bacteriophages are the most abundant biological entities in the biosphere. A majority of them are temperate phages that are able to integrate their genome into the host and replicate passively in a lysogenic state. Hosts frequently benefit from such massive gene acquisition through lysogenic conversion. As prophages may be beneficial to their hosts, we hypothesize that hosts adapted strategies for maintaining that gene source. Since prophages integrate into and excise from the host chromosome through site-specific recombination (SSR), we investigated whether regulation of SSR at the level of gene expression could be involved in the maintenance process. Our results suggest that lysogeny maintenance of a class of prophages, which all share a same unusual genetic organization, are controlled by the transcription termination factor Rho. Rho is not only involved in horizontally acquired gene silencing but also in prophage maintenance, which can be seen as an adaptation of the host to maintain prophage genes. For these prophages, whether defective or functional, their induction by the inactivation of Rho, involves a new pathway of lysogeny escape, which is independent of the classical pathway via the SOS response. This newly characterized interaction reflects the coevolution of host and viruses, which allows the acquisition of genes, and thus new properties, via horizontal transfer, while controlling the expression of deleterious genes.

Infecções por Plasmodium sp. podem levar a um quadro respiratório grave, com complicações pulmonares denominadas lesão pulmonar aguda e síndrome do desconforto respiratório agudo (LPA/SDRA). Inflamação aguda, lesão do endotélio alveolar e do parênquima pulmonar, disfunção e aumento da permeabilidade da barreira alvéolo-capilar e, consequente, formação de edema, caracterizam esta síndrome. O modelo experimental, que utiliza o parasita murino Plasmodium berghei ANKA e camundongos da linhagem DBA/2, é empregado no estudo de mediadores imunológicos e fatores que propiciam o estabelecimento das lesões pulmonares associados à LPA/SDRA. Diversos estímulos podem atuar diretamente no aumento da permeabilidade endotelial por meio da desestabilização dos microtúbulos, rearranjo dos microfilamentos de actina e contração das células endoteliais, via sinalização de Rho-GTPases, causando disfunção da barreira endotelial. Desta forma, este trabalho tem como objetivo avaliar as alterações do citoesqueleto em células endoteliais primárias pulmonares de camundongos DBA/2 (CEPP-DBA/2), as vias de sinalização das principais Rho-GTPases e o estresse oxidativo, causados pela presença de eritrócitos parasitados com esquizontes de P. berghei ANKA (EP-PbA). As CEPP-DBA/2 foram estimuladas com TNF, VEGF ou IFNγ, em diferentes tempos de exposição, seguido da incubação com EP-PbA. Assim, foram realizados ensaios de imunofluorescência para análise do rearranjo de microfilamentos de actina e da desestabilização de microtúbulos. As vias de sinalização das Rho-GTPases foram avaliadas por Western blot, para as expressões proteicas de RhoA, Cdc42 e MLC. Além disso, ensaio fluorométrico foi realizado para detectar a produção de espécies reativas de oxigênio, resultantes do estímulo com eritrócitos parasitados. CEPP-DBA/2 estimuladas por EP-PbA, VEGF, TNF ou IFNγ, em associação ou não, apresentaram alterações morfológicas nos microfilamentos de actina e aumento dos espaços interendoteliais. Imagens de imunofluorescência também mostram desestabilização de microtúbulos e desfosforilação de FAK, causadas por EP-PbA. Os ensaios de permeabilidade validam que os eritrócitos parasitados com formas maduras de P. berghei induziram aumento da permeabilidade microvascular nas CEPP-DBA/2. Além disso, estas células, estimuladas com EP-PbA, demonstraram elevada produção de espécies reativas de oxigênio (EROs), o que pode estar contribuindo com o desenvolvimento de estresse oxidativo e com a injúria endotelial, assim como, com o aumento da permeabilidade vascular. O mais interessante é que estas alterações endoteliais podem estar relacionadas ao aumento da razão RhoA/Cdc42, da expressão proteica de MLC fosforilada e do sinal de ativação de RhoA. Em conjunto, estes resultados mostram envolvimento dos eritrócitos parasitados com esquizontes de Plasmodium berghei ANKA na desorganização do citoesqueleto e na disfunção da barreira alvéolo-capilar, via RhoA/Rho-kinase, o que pode estar contribuindo com a patogênese da LPA/SDRA associada à malária. / Infections by Plasmodium sp. can lead to a serious respiratory condition with pulmonary complications, named acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Acute inflammation, alveolar endothelium and lung parenchyma injuries, dysfunction and increased permeability of the pulmonary alveolar-capillary barrier and consequent formation of edema characterize this syndrome. Several stimuli can directly increase endothelial permeability through actin microfilaments rearrangement, via Rho- GTPases signaling, leading to endothelial barrier dysfunction. DBA/2 mice infected with Plasmodium berghei ANKA develop ALI/ARDS similar to that observed in humans. The purpose of this research was to assess cytoskeletal changes in DBA/2 mice primary microvascular lung endothelial cells (PMLEC), verify the signaling pathways of the Rho- GTPases and analyze the oxidative stress on these cells in the presence of P. berghei ANKA-infected red blood cells (PbA-iRBC). PMLEC were stimulated by TNF, VEGF or IFNγ followed by incubation with PbA-iRBC. Immunofluorescence assays were performed to analyze actin microfilaments rearrangement and microtubules destabilization. Western blot for RhoA, Cdc42 and MLC proteins were conducted to assess alterations in signaling pathways of Rho-GTPases. In addition, a fluorimetric assay was performed to detect the production of reactive oxygen species resulting from PbA-iRBC stimulus. P. berghei ANKA, VEGF, TNF and IFNγ stimuli, in association or not, caused morphological disturbances in actin microfilaments of PMLEC and an increase of intercellular spaces. Moreover, immunofluorescence images showed microtubules destabilization and FAK dephosphorylation in these cells, caused by PbA-iRBC. The permeability assay showed that PbA-iRBC induced an increase of microvascular permeability in PMLEC. In addition, PMLEC stimulated by PbA-iRBC, showed elevated production of ROS, which may be contributing to oxidative stress and increasing the damage of endothelial cells, as well as an increase of vascular permeability. Interestingly, these endothelial changes may be related to the increased RhoA/Cdc42 protein expressions ratio, augmented protein expression of phosphorylated MLC and RhoA activation signal. Taken together, these data demonstrate the involvement of P. berghei ANKA-infected red blood cells in cytoskeleton disorganization and alveolar-capillary barrier dysfunction, through of RhoA / Rho-kinase signaling pathway, which may contribute to ALI/ARDS pathogenesis.

Shigellen sind die Erreger der bakteriellen Ruhr beim Menschen, ihrem einzigen bisher bekannten Wirt. Ein wesentlicher Virulenzfaktor von Shigellen ist ihre Fähigkeit, in Epithelzellen des Intestinaltraktes einzudringen. Dabei induziert Shigella in der Wirtszelle Zytoskelettrearrangements, die zur Ausbildung einer blütenartigen Membranstruktur um das Bakterium herum führen, die schließlich über dem eindringenden Bakterium konfluiert und damit den Mikroorganismus internalisiert. Die Zytoskelettveränderungen sind essenziell für den Internalisierungsmechanismus und werden von der kleinen GTPase Rho gesteuert, wobei die Rho-Aktivität zeitlich und räumlich streng reguliert wird, um eine überschießende Bildung von F-Aktin auf Kosten des zellulären G-Aktin-pools zu verhindern. Myr5, ein atypisches Myosin der Klasse IX, ist das erste beschriebene Myosin mit einem Rho inaktivierenden GAP-Modul. Deshalb vermuteten wir, dass der Rho-Antagonist Myr5 während der Shigelleninvasion funktionell von Bedeutung sein könnte. Wir konnten zeigen, dass Myr5 bei der Shigelleninvasion in die zellulären Protrusionen rekrutiert wird. Dort kolokalisierte Myr5 mit F-Aktin und den Rho-Isoformen B und C, nicht jedoch mit RhoA. Die Rekrutierung von Myr5 in die Invasionszone erfolgte unabhängig von der Myosin-Kopf- und der GAP-Funktion. Die Resultate funktioneller quantitativer Untersuchungen zu einer möglichen Rolle während der bakteriellen Invasion sind kompatibel mit der Hypothese, dass sowohl die GAP-Funktion als auch die Myosin-Kopf-Funktion von Myr5 während unterschiedlicher Phasen der Shigelleninvasion von Bedeutung sind. / Shigella causes bacillary dysentery in humans, the only known host. A major feature of its pathogenic potential is the capacity to invade intestinal epithelial cells. Shigella entry into epithelial cells is considered a parasite induced internalization process requiring cytoskeletal rearrangements. Shigella induces a blossom-like membrane structure consisting of membrane sheaths that coalesce above and thus internalize the invasive microorganism. Cytoskeletal remodeling is an essential part of the entry process and is regulated by the small GTPase rho. Temporal and special regulation of rho activity is important to prevent excessive generation of F-actin in depense of the cellular G-actin pool. The class IX myosin myr5 is characterized by a GTPase activating protein (GAP)-module in the tail region. The GAP-module of myr5 is able to inactivate rho. We therefore hypothesized a potential role of myr5 in the regulation of rho activity during Shigella entry into epithelial cells. We could show that myr5 is recruited into bacterial entry spot. Myr5 colocalized with F-actin, rhoB and rhoC but not rhoA. Shigella-induced recruitment of myr5 did not require a functional myosin head or GAP-domain. The results of quantitative functional studies of a potential role of myr5 during bacterial entry suggest a dual role of the myosin head function and the GAP module of myr5 during different steps of the internalization process.

Terminaison de la transcription. Rho se fixe aux transcrits naissants au niveau d’un site Rut (Rhoutilization) libre à partir duquel il transloque le long de l’ARN (5’→3’) de façon ATP-dépendante pour rattraper le complexe d’élongation de la transcription et induire la dissociation de celui-ci. Il est généralement admis que les sites de fixation de Rho présentent une richesse en Cytosines et une pauvreté en Guanines, ainsi qu’une relative pauvreté en structures secondaires. Les études génomiques ou transcriptomiques n’ont pas dégagé d’éléments consensus ou de règles permettant de prédire les sites de terminaison Rho-dépendants. En combinant approches biochimiques et bioinformatiques, j’ai tenté de comprendre les mécanismes par lesquels Rho reconnait les transcrits.J’ai identifié un ensemble de déterminants de séquence qui, pris ensemble, possèdent un bon pouvoir prédictif et que j’ai utilisé pour construire le premier modèle computationnel capable de prédire la terminaison Rho-dépendante à l’échelle des génomes d’E. coli et Salmonella. J’ai caractérisé in vitro certains de ces terminateurs, en particulier dans les régions 5’UTR, avec l’espoir qu’ils soient impliqués dans des mécanismes de régulation conditionnelle. J’ai identifié des candidats dont l’activité pourrait être sous le contrôle de facteurs comme des petits ARN non codants (sRNA) ou latempérature. J’ai également développé une méthode fluorogénique pour détecter facilement la terminaison Rho-dépendante in vitro et ai commencé à adapter l’approche CLIP-seq à l’étude du transcriptome Rho-dépendant chez Salmonella. Collectivement, mes travaux offrent de nouveaux outils d’analyse et de prédiction de la terminaison Rho-dépendante, une meilleure cartographie des sites d’action de Rho chez E. coli et Salmonella, ainsi que de nouvelles pistes d’étude du rôle de Rhodans l’expression conditionnelle du génome. / Transcripts at a free Rut (Rho-utilization) site from which Rho moves along the RNA in an ATP dependentfashion to catch up with and dissociate the transcription elongation complex. It is generally believed that the Rut sites are, respectively, rich and poor in Cytosines and Guanines as well as relatively poor in secondary structures. Studies at the genomic or transcriptomic scale have notrevealed any stronger consensus features or rules for predicting potential Rho-dependent termination sites. By combining biochemical and bioinformatics approaches, I have explored the mechanisms by which Rho recognizes transcripts to induce transcription termination. I have identified a complex set of sequence determinants which, taken together, have good predictive power and which I used to build the first computational model able to predict Rho-dependent termination at the scale of Escherichiacoli and Salmonella genomes. I have characterized in vitro some of these terminators, particularly in 5'UTRs, with the hope that they will be involved in conditional regulatory mechanisms. I have identified several candidates whose activity may be under the control of factors such as small non-coding RNAs(sRNA) or temperature. I have also developed a fluorogenic method to easily detect Rho-dependent termination in vitro and have begun to adapt the CLIP-seq approach to the study of the Rhodependent transcriptome in Salmonella. Collectively, my work offers new tools for the analysis and prediction of Rho-dependent termination, a better mapping of the sites of probable Rho action in E.coli and Salmonella, as well as several lines of investigation of the role of Rho in the conditional expression of bacterial genomes.