Rozprawy doktorskie na temat „Motility”

Les kinésines sont des protéines moteur liées au cytosquelette de microtubules. Elles convertissent l’énergie provenant de l’hydrolyse de l’ATP en un travail mécanique. Leur fonction typique est de se déplacer le long du microtubule pour véhiculer des charges. La plupart des kinésines sont des dimères. Elles comprennent un domaine moteur, qui porte à la fois les sites de liaison du nucléotide et du microtubule, un domaine intermédiaire de dimérisation et une partie dite « queue » qui confère la spécificité des charges à transporter. Mon objectif est d’établir le mécanisme moléculaire à la base de la motilité, avec un intérêt particulier pour la détermination des variations structurales du domaine moteur de la kinésine le long de son cycle mécano-chimique. Au cours de ma thèse, mon objet d’étude principal a été la kinésine-1 humaine, encore appelée kinésine conventionnelle.J’ai étudié plus particulièrement deux aspects du cycle mécano-chimique de la kinésine-1, en combinant des approches de biologie structurale et l’étude de mutants. Les deux aspects concernent l’étude de la fixation de la kinésine-ADP au microtubule, conduisant à l’éjection du nucléotide et à une liaison forte de la kinésine au microtubule. Dans un premier temps, j’ai déterminé la structure du domaine moteur de la kinésine-1, dépourvue de nucléotide, et sous forme d’un complexe avec la tubuline. La tubuline est la protéine constitutive des microtubules. Cette structure était la donnée principale qui nous manquait dans le cycle structural de la kinésine. En comparant cette structure avec celle de la kinésine dans un état ATP, on peut rendre compte des changements de conformation de la kinésine selon le mouvement de trois sous-domaines du domaine moteur. Cette analyse explique notamment le lien entre la fixation de l’ATP et l’ouverture d’une poche hydrophobe distante de 28 Å du site du nucléotide. Cette cavité va accommoder le premier résidu du neck linker, conduisant à la stabilisation de ce peptide situé en partie C-terminale du domaine moteur. En s’ordonnant, le neck linker va faire avancer la charge ainsi que l’autre domaine moteur de la kinésine dimérique. Il lie ainsi la fixation de l’ATP au mouvement. L’étude de l’effet de mutations du neck linker montre aussi comment, réciproquement, le neck linker bloque la kinésine dans la conformation active pour l’hydrolyse de l’ATP. Ceci diminue la probabilité que l’ATP soit hydrolysé avant que l’étape mécanique se soit produite; cet aspect est essentiel pour rendre compte de la processivité de la kinésine-1.Ces données structurales suggèrent également comment la fixation de la kinésine-ADP au microtubule accélère l’éjection de l’ADP. Pour étudier cet aspect plus en détail, j’ai étudié l’effet de mutations sur la vitesse de largage de l’ADP. L’idée était de mimer à l’aide de mutations la fixation au microtubule. J’ai identifié ainsi deux séries de mutants qui présentent une vitesse accélérée de largage spontané de l’ADP, ce qui suggère deux voies pour interférer avec la fixation du nucléotide. J’ai ensuite déterminé la structure de deux de ces mutants dépourvus de nucléotide, ainsi que celle de la kinésine de départ également dans une forme apo, obtenue par digestion de l’ADP. En absence de microtubule, la kinésine dépourvue de nucléotide adopte une conformation soit à l’image de celle de la kinésine-ADP, ou proche de celle de la kinésine-apo liée à la tubuline. Dans un contexte naturel, seule la deuxième conformation est compatible avec la fixation au microtubule. L’ensemble de ces résultats suggère que le microtubule accélère l’éjection du nucléotide par un double mécanisme : en interférant avec la liaison du magnésium et en déstabilisant le motif P-loop de liaison du nucléotide
Kinesins are a family of microtubule-interacting motor proteins that convert the chemical energy from ATP hydrolysis into mechanical work. Many kinesins are motile, walking along microtubules to fulfill different functions. Most kinesins are dimers, the monomer comprising a motor domain, a dimerizing stalk domain, and a tail domain. The motor domain contains both the nucleotide-binding site and the microtubule-binding site. I am interested in the molecular mechanism of kinesin's motility. In particular I want to establish the structural variations of the kinesin motor domain along with the mechanochemical cycle of this motor protein. During my thesis, I have focused my work on the human kinesin-1, also named conventional kinesin, which is the best characterized kinesin.I have studied two aspects of the kinesin mechanochemical cycle, by combining structural and mutational approaches. Both aspects rely on the binding of ADP-kinesin to a microtubule, which leads to the release of the nucleotide and to a tight kinesin-microtubule association. First I determined the crystal structure of nucleotide-free kinesin-1 motor domain in complex with a tubulin heterodimer, which is the building block of microtubule. This structure represented the main missing piece of the structural cycle of kinesin. Three subdomains in the kinesin motor domain can be identified through the comparison of my structure with ATP-analog kinesin-1-tubulin structure. The relative movements of these subdomains explain how ATP binding to apo-kinesin bound to microtubule triggers the opening of a hydrophobic cavity, 28 Å distant from the nucleotide-binding site. This cavity accommodates the first residue of the “neck linker”, a short peptide that is C-terminal to the motor domain, allowing the neck linker to dock on the motor domain. The docking of the neck linker is proposed to trigger the mechanical step, i.e. the displacement of the cargo and the stepping of the dimeric kinesin. By studying mutants of the neck linker, I have shown that, reciprocally, this peptide locks kinesin in the ATP state, which is also the conformation efficient for ATP hydrolysis. Doing so, it prevents the motor domain from switching back to the apo-state. It prevents also an untimely hydrolysis of ATP, before the mechanical step has occurred. These features are required for movement and processivity.Second, these structural data also suggest how the binding of ADP-kinesin to tubulin enhances nucleotide release from kinesin. To further study this step of the kinesin cycle, I studied the effect of kinesin-1 mutations. These mutations were designed in isolated kinesin to mimic the state when kinesin is bound to a microtubule. I identified two groups of mutations leading to a high spontaneous ADP dissociation rate, suggesting that there are two ways to interfere with ADP binding. Then I determined the crystal structures of the apo form of two mutants as well as that of the nucleotide-depleted wild type kinesin. It showed that apo-kinesin adopts either and ADP-like conformation or a tubulin-bound apo-like one. In the natural context, the second one is stabilized upon microtubule binding. Overall, the mutational and structural data suggest that microtubules accelerate ADP dissociation in kinesin by two main paths, by interfering with magnesium binding and by destabilizing the nucleotide-binding P-loop motif

Introduction: The reasons for the rapid resolution of diabetes (DM) following bariatric surgery in a significant proportion of patients with morbid obesity remain unclear. This thesis investigates the putative role of changes in gastrointestinal (GI) motility and GI hormones as well as the possible significance of alterations in energy expenditure that occur as a consequence of weight loss. Methodology: My preliminary studies involved a systematic review of GI motility in obesity, and retrospective studies measuring GI motility with alternative methods including capsule endoscopy and hydrogen breath test. Subsequent to this I measured changes in GI motility in two very different patient cohorts; one following bariatric surgery for morbid obesity and the other a group of patients with proven gastroparesis treated with gastric neuromodulation (GNM). Parallel to the above I conducted studies of indirect calorimetry in these patients in an attempt to determine if changes in energy expenditure which occur as a consequence of weight loss were significant. Results: In our prospective study temporary GNM significantly improved gastric emptying and nutritional intake. There was conclusive evidence to causally relate alterations in GI motility and Glucagon like peptide -1 (GLP-1) with weight loss and resolution of DM following bariatric surgery. An interesting "spin off" result of my studies was validation of capsule endoscopy (CE) as a means of assessing GI motility. My results obtained from measure if indirect calorimetrty clearly show that standard equations tend to over estimate the energy requirements of this group. The implications of this are discussed. Conclusions: 1. Fast pouch emptying; an early and exaggerated GLP-1 response contributes in resolution of type 2 diabetes following RYGB. 2. GNM is an effective treatment for gastroparesis. 3. Capsule endoscopy may be used to assess GI motility. 4. Prediction equations over estimate energy requirements in morbidly obese patients.

La formation des métastases est un processus en plusieurs étapes à travers lequel les cellules néoplasiques se détachent de la tumeur primaire pour constituer des tumeurs secondaires à distance. Les capacités à migrer et à envahir, des cellules tumorales sont cruciales dans la cascade métastatique. Selon le type cellulaire et les stimuli présents dans le microenvironnement tumoral, les cellules peuvent se déplacer collectivement ou individuellement selon un programme de migration mésenchymateuse ou amiboïde. Différentes voies de signalisation sont liées à la régulation de la motilité cellulaire. Les GTPases Rho (Rac1, Cdc42 et RhoA) contrôlent la migration en régulant la dynamique du cytosquelette d’actine, la contraction acto-myosine et les microtubules. Rac1 régule la motilité mésenchymateuse en favorisant la formation des lamellipodes via un complexe multiprotéique, le « Wave Regulatory Complex (WRC) » et RhoA contrôle la motilité amiboïde en favorisant la contraction du cytosquelette d'acto-myosine. Les protéines Ral (RalA et RalB) appartenant à une autre famille de petites G, ont été récemment impliquées dans la régulation de la migration cellulaire. RalB, à travers le complexe « Exocyst » joue un rôle essentiel dans la motilité. Dans ce travail de thèse, nous avons étudié les mécanismes moléculaires par lesquels la voie RalB/Exocyste contrôle la motilité et l'invasion cellulaire. La première partie de ce travail démontre que l’Exocyste interagit avec SH3BP1, une protéine GAP (GTPase Activating Protein) (projet 1). Nous montrons que l’interaction entre SH3BP1 et Rac1 est nécessaire à l’activité de Rac1 au front de migration. Dans le projet 2, nous montrons que l’Exocyste interagit directement avec WRC, ce qui est un élément clé de la polymérisation de l'actine. Cette interaction est nécessaire à la localisation du complexe WRC au front de migration où il contrôle la formation de protrusions membranaires. Dans de nombreux carcinomes, la transition épithélio-mésenchymateuse (EMT) joue un rôle important dans la promotion de la migration, l’invasion et la formation des métastases. Le projet 3 a permis de mieux caractériser la plasticité de migration et l’invasion des cellules cancéreuses post-EMT et d’étudier la contribution de Ral dans l'invasion des cellules post-EMT. Nous montrons qu’après l’EMT les cellules envahissent la matrice individuellement ? en utilisant la contraction du cytosquelette d'acto-myosine. Nous montrons que RalB est nécessaire à l’invasion des cellules post-EMT, et à la contractilité cellulaire. Nous proposons que le rôle de RalB dans l'invasion passe par GEF-H1 qui est une protéine GEF (Guanine Nucleotide Exchange Factor) de Rho associée à l’Exocyste. Dans la dernière partie de ce manuscrit, nous présentons le logiciel « AVeMap » que nous avons développé afin d’automatiser la quantification des paramètres de la migration cellulaire.En résumé, dans ce travail de thèse nous montrons que la voie Ral/Exocyste est un organisateur moléculaire nécessaire à l’exécution à la fois de la motilité cellulaire contrôlée par Rac1 et à la motilité contrôlée par Rho
Metastasis is a multistep process by which cancer cells migrate away from the primary neoplastic mass to give rise to secondary tumors at distant sites. Thus, the acquisition of motility and invasive traits by tumor cells is a crucial step for metastasis to occur. Depending on the cell type and the environment, cells can move collectively keeping stable cell-cell contacts or as individual cells, which translocate by exploiting either mesenchymal or amoeboid motility programs.Different molecules and pathways have been linked to the regulation of cell motility. Rho small GTPases (Rac1, Cdc42 and RhoA) control cell migration through their actions on actin assembly, actomyosin contractility and microtubules. Rac1 drives mesenchymal-type motility by promoting lamellipodia formation via the Wave Regulator Complex (WRC). On the contrary, amoeboid motility is governed by RhoA which promotes cell movement via the generation of actomyosin contractile force. Another family of small GTPases, the Ral proteins, was recently involved in the regulation of cell migration. RalB, through the mobilization of its main effector the Exocyst complex, was shown to play an essential role in cell motility. In this work of thesis we investigated the molecular mechanisms through which RalB/Exocyst pathway controls cell motility and invasion.In the first part of this manuscript we show that Exocyst interacts with the RacGAP SH3BP1 (project 1). In mesenchymal moving cells Exocyst/SH3BP1 interaction is required to organize membrane protrusion formation by spatially regulating the activity of Rac1 at the cellular front. In addition, in project 2, we show that the Exocyst binds to the wave regulator complex (WRC), a key promoter of actin polymerization. We provide evidences for Exocyst to be involved in driving the WRC to the leading edge of motile cells, where it can stimulate actin polymerization and membrane protrusions. Reactivation of a developmental program termed epithelial-mesenchymal transition (EMT) was recently shown to promote motility, invasion and metastasis of neoplastic cells. Tumor cells undergoing EMT loose cell-cell contacts acquire a fibroblastoid phenotype and invade the surrounding tissues as individual cells. In project 3 we characterized the invasion plasticity of cancer cells after EMT and we investigated the molecular contribution of Ral to post-EMT invasion. We showed that upon EMT cells disseminate individually in a Rho-driven fashion exploiting the generation of actomyosin force to deform the extracellular matrix. We document that RalB silencing severely impairs actomyosin contractility and dissemination of post-EMT cells. We hypothesize that RalB regulates invasion by controlling the dynamics of the Rho pathway via the Exocyst-associated RhoGEF GEF-H1 in post-EMT cells. Finally, in the last part of this thesis manuscript, we present the PIV-based “AVeMap” software which has been developed to quantify in a fully automated way cell migration and its parameters (Project 4).Taken together the results presented in this thesis manuscript point out the Ral/Exocyst pathway as a key molecular organizer of the execution of both Rac1- and Rho-driven motility programs

Le complexe Arp2/3, conservé sur le plan évolutif, joue un rôle central dans la nucléation d’actine branchée, qui entraîne la migration cellulaire, l’endocytose et d’autres processus cellulaire. Récemment, une petite protéine, Arpin, qui inhibe le complexe Arp2/3 au front du lamellipode a été découverte et caractérisée. Sur sa partie C-terminale, Arpin possède un motif acide (A), qui est homologue au motif A des différents NPF (Nucleation Promoting Factor). Il a été prédit qu’Arpin peut se lier à deux sites de liaison au complexe Arp2/3, similaire aux domaines VCA des NPF. Ici, nous utilisons la microscopie électronique de particules uniques pour obtenir une reconstruction 3D du complexe Arp2/3 lié à Arpin, à une résolution de 25 Å. Nous avons montré que la liaison d’Arpin induit la conformation ouverte, standard, du complexe Arp2/3. Nous avons confirmé qu’il y a deux sites de liaison sur le complexe Arp2/3 pour Arpin : un à l’arrière de la sous-unité Arp3, et le second localisé entre les sous-unités Arp2 et ARPC1. La distance entre le complexe Arp2/3 et Arpin (5nm) confirme qu’Arpin interagit avec son partenaire via sa queue acide C-terminale non structurée.Nous avons, ensuite, identifié Tankyrases1/2, comme un nouveau partenaire qui se lie à Arpin, par « pull-down ». De façon intéressante, les sites de liaisons d’Arpin aux Tankyrases et à Arp2/3 se chevauchent. Nous avons, par conséquent, démontré qu’il y a une compétition dose-dépendante entre le domaine ARC4 de Tankyrase1 et le complexe Arp2/3.Pour comprendre les principes de l’interaction entre Arpin et Tankyrases, nous avons créé un mutant d’Arpin (ArpinG218D) qui, in vitro, se lie toujours au complexe Arp2/3, mais plus aux Tankyrases. In vivo, ArpinG218D n’est pas capable d’inhiber le complexe Arp2/3, ce qui suggère que Tankyrase pourrait être nécessaire pour l’interaction entre Arpin et le complexe Arp2/3. Arpin est le facteur responsable du changement de direction des cellules migrantes. Nous avons donc analysé, la migration de cellules MCF10A exprimant soit la forme sauvage d’Arpin (ArpinWT) soit son mutant ArpinG218D en parallèle de la déplétion d’Arpin endogène. Les cellules exprimant ArpinG218D ont une persistance de migration supérieure, similaire à celles déplétées d’Arpin endogène. Nous avons, ainsi, fait l’hypothèse que le mutant ArpinG218D ne peut pas inactiver le complexe Arp2/3 car il n’est pas présent au niveau du lamellipode. Nous avons donc comparé la quantité de protéine d’ArpinWT et d’ArpinG218D dans la fraction membranaire de cellules migrantes. Une différence significative (44%) dans la quantité d’ArpinWT et d’ArpinG218D a confirmé notre hypothèse.Les Tankyrases sont des cibles thérapeutiques dans de nombreux cancers, mais il n’existe pas de modèle structural pour ces protéines grandes et flexibles. Dans ce travail, nous avons, pour la première fois, obtenu deux reconstructions 3D de Tankyrase1 et Tankyrase2 complètes liées à Arpin en utilisant la microscopie électronique de particules uniques. La résolution obtenue (27 Å) a été suffisante pour détecter un changement de conformation dramatique des domaines SAM et PARP de Tankyrase après fixation d’Arpin. Dans notre reconstruction, trois molécules d’Arpin se lient aux domaines ARC1, ARC4 et ARC5 de Tankyrase1. ARC5 a été montré pour être la partie le plus flexible de l’ensemble des domaines ARC.Grâce aux données que nous avons obtenues, nous avons suggéré un modèle de régulation de l’activité d’Arpin par les Tankyrases. Selon notre modèle, les Tankyrases se lient à Arpin dans le cytoplasme, changent sa conformation et amènent Arpin au niveau de la membrane dans le lamellipode. Traduisant les signaux extracellulaires, la GTPase Rac active Arpin, qui séquentiellement inactive le complexe Arp2/3, tandis que les Tankyrases sont libérées
The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. Recently, an inactivator of the Arp2/3 complex at the lamellipodium tip, a small protein, Arpin, was discovered and characterized. On its C-terminus, Arpin possesses an acidic (A) motif, which is homologous to the A-motif of various Nucleation Promoting Factors (NPFs). It was predicted that Arpin can bind at two binding sites to the Arp2/3 complex, similar to VCA domains of NPFs. Here, we used single particle electron microscopy to obtain a 3D reconstruction of the Arp2/3 complex bound to Arpin at a 25Å resolution. We showed that the binding of Arpin causes the standard open conformational of the Arp2/3 complex. We confirmed that there are two binding sites on the Arp2/3 complex for Arpin: one on the back of the Arp3 subunit, and the second is located between Arp2 and ARPC1 subunits. The distance between the Arp2/3 complex and Arpin (5 nm) supports the view that Arpin interacts with its partner via its unstructured C-terminal acidic tail.Next, using the pull-down assay, we identified the new Arpin binding partners, Tankyrases1/2. Interestingly, Tankyrases and the Arp2/3 complex possess overlapping amino acid sequences at Arpin binding sites. Hence, we demonstrated a competition between the ARC4 domain of Tankyrase1 and the Arp2/3 complex in a dose-dependent manner.To understand the principles of Tankyrases-Arpin interaction, we created a mutant Arpin (ArpinG218D) that lacks its ability to interact with Tankyrases, but not with the Arp2/3 complex in vitro. Interestingly, ArpinG218D was not able to inhibit the Arp2/3 complex in vivo, suggesting that Tankyrase may be necessary for Arpin-Arp2/3 complex interaction. Arpin is the turning factor of migrating cells, so we performed a migration analysis of MCF10-A cells expressing either wild type Arpin (ArpinWT) or mutant ArpinG218D in parallel with the depletion of endogenous Arpin. Cells expressing ArpinG218D had higher directional persistence, similar to the cells where the endogenous Arpin was knocked down. Thus, we suggested that mutant ArpinG218D cannot inactivate the Arp2/3 complex since it is not present at the lamellipodial tip. We compared the amount of protein for both ArpinWT and ArpinG218D in the membrane fraction of the migrating cells. A significant difference (44%) in the amount of ArpinWT and Arpin G218D was consistent with our hypothesis.Tankyrases are therapeutic targets in a variety of cancers, but currently there is no structural model available for these large and flexible proteins. In this work, we obtained for the first time two 3D reconstructions of full-length Tankyrase1 and Tankyrase1 bound to Arpin using single particle electron microscopy. The achieved resolution (27Å) was enough to detect a dramatic conformational change in Tankyrase SAM and PARP domains upon binding of Arpin molecules. In our reconstruction, three Arpins were bound to the ARC1, ARC4 and ARC5 domains of Tankyrase1. ARC5 was shown to be the most flexible part of the ARC cluster.Based on the obtained data, we suggested a model of regulation of the activity of Arpin by Tankyrases. According to our model, Tankyrases bind Arpin in the cytoplasm, change their conformational state and bring Arpin closer to the membrane in the lamellipodia. Deciphering the extracellular signals, Rac GTPase activates Arpin, which sequentially inactivates the Arp2/3 complex, while Tankyrases are released

The change from swarmer cell to non-motile reproductive cell was examined as a landmark event in differentiation of the purple non-sulphur bacterium, Rhodomicrobium vannielii, using synchronised swarmer cell populations. It was found that shed flagella from Rm. vannielii consisted of 3 proteins: flagellin, Mr 34k, hook protein, Mr 36k and a polypeptide of Mr 37k, possibly rod protein. No methyl-accepting chemotaxis proteins were detected in Rm. vannielii. Radioimmunoprecipitation was used to determine the period of flagellin synthesis during differentiation. Synthesis of flagellin was switched off in Rm. vannielii swarmer cells after 1-2 hours anaerobic incubation in the light. If swarmer cells were held anaerobically in the dark, flagellin synthesis continued for at least 6 hours. Thus, swarmer cells, which have a dispersal role in nature, detect whether environmental conditions are conducive to completion of the cell cycle, and regulate their gene expression accordingly. This contrasts with conclusions drawn from work with the non-photosynthetic aquatic bacterium, Caulobacter crescentus, whose cell cycle also includes a swarmer cell to non-motile reproductive cell transition. In order to study control of flagellin expression further, cloning of the gene(s) was attempted. Heterologous Southern hybridisations between restricted Rm. vannielii chromosomal DNA and the cloned 29k flagellin gene from C. crescentus showed that 55-60% homology existed between the two. This was judged to be too weak a signal to allow cloning of Rm. vannielii flagellin genes by hybridisation. Antibodies raised against Rm. vannielii flagellin and hook protein did cross react with C. crescentrus flagellins and hook protein. Screening a library of EcoRl digested Rm. vannielii chromosomal DNA in the vector λgt11 with anti-flagella antiserum indicated that Rm. vannielii flagellin and hook protein were not expressed in E. coli from DNA cut in such a way. Eleven Tn5-induced motility mutants of Rm. vannielii were isolated (3 of which were chemotaxis deficient), and these should enable the cloning of genes for motility from this organism in the future.

Listeria monocytogenes is a saprophytic food borne microorganism which is pathogenic to humans and animals. The pathogenicity and physiological features of L. monocytogenes have been studied for many years. Some characteristics of this microorganism have been described, such as its low temperature growth and adaptation, the expression of its flagellin gene, and the regulation of its virulence genes. Previous studies imply a possible reversed relationship that may exist between the regulation of the listerial flagellin gene, flaA and its virulence regulator gene, prfA.

Cell motility is required for many biological processes, including cancer metastasis. The molecular requirements for migration and, the morphology of migrating cells, can vary considerably depending on matrix geometry. Therefore, predicting the optimal migration strategy or the effect of experimental perturbation is difficult. This thesis presents a computational model of single cell motility that encompasses flexible cell morphology, actin polymerisation based protrusions, cell cortex asymmetry, plasma membrane blebbing, local cortex heterogeneity at the protein level, cell–extracellular matrix adhesion, and varying extracellular matrix geometries. This computational model is used to explore the theoretical requirements for rapid migration in different matrix geometries. The analysis reveals that confinement of the cell within the extracellular matrix brings profound changes in the relationship between cortical contractility and cell velocity. In confined environments with discontinuity, the relationship between adhesion and cell velocity is fundamentally altered: adhesion becomes dispensable for a large range of gap sizes in between the extracellular matrix filaments. The utility of the model is shown by predicting cancer cell behaviour in vivo, in terms of both cell velocity and the morphology of the motile cell. Furthermore, the model is challenged to predict the effects of selected biochemical perturbations that alter i) cortical contractility, ii) cell-ECM adhesion, and iii) signalling between the cell-ECM adhesion sites and intracellular regulators of cell motility machinery. Multiphoton intravital imaging is used to verify bleb driven migration of melanoma, breast cancer cells, and, surprisingly, endothelial cells at tumour margins. Intravital imaging of melanoma verified model predictions on cell velocity, cell morphology, nucleus behaviour, and effects of anti-invasive interventions. The model succesfully predicted melanoma velocities in vitro and in vivo. Moreover, it successfully predicted the effects of anti-invasive interventions, showing all perturbations will result in significant reduction in cell velocity in vitro, whereas only perturbation of cortical contractility will affect cell velocity in vivo. The model also successfully predicted the interactions of the cell nucleus with the cell cortex and the cell morphology upon intervensions. Overall, from measure ment of rather simple variables in vitro, the model has been able to predict the in vivo response of three very different putative anti-invasive interventions.

The propulsion mechanics driving the movement of living cells constitutes one of the most incredible engineering works of nature. Active cell motility via the controlled movement of a flagellum beating is among the phylogentically oldest forms of motility, and has been retained in higher level organisms for spermatozoa transport. Despite this ubiquity and importance, the details of how each structural component within the flagellum is orchestrated to generate bending waves, or even the elastic material response from the sperm flagellum, is far from fully understood. By using microbiomechanical modelling and simulation, we develop bio-inspired mathematical models to allow the exploration of sperm motility and the material response of the sperm flagellum. We successfully construct a simple biomathematical model for the human sperm movement by taking into account the sperm cell and its interaction with surrounding fluid, through resistive-force theory, in addition to the geometrically non-linear response of the flagellum elastic structure. When the surrounding fluid is viscous enough, the model predicts that the sperm flagellum may buckle, leading to profound changes in both the waveforms and the swimming cell trajectories. Furthermore, we show that the tapering of the ultrastructural components found in mammalian spermatozoa is essential for sperm migration in high viscosity medium. By reinforcing the flagellum in regions where high tension is expected this flagellar accessory complex is able to prevent tension-driven elastic instabilities that compromise the spermatozoa progressive motility. We equally construct a mathematical model to describe the structural effect of passive link proteins found in flagellar axonemes, providing, for the first time, an explicit mathematical demonstration of the counterbend phenomenon as a generic property of the axoneme, or any cross-linked filament bundle. Furthermore, we analyse the differences between the elastic cross-link shear and pure material shear resistance. We show that pure material shearing effects from Cosserat rod theory or, equivalently, Timoshenko beam theory or are fundamentally different from elastic cross-link induced shear found in filament bundles, such as the axoneme. Finally, we demonstrate that mechanics and modelling can be utilised to evaluate bulk material properties, such as bending stiffness, shear modulus and interfilament sliding resistance from flagellar axonemes its constituent elements, such as microtubules.

A thesis submitted in fulfilment of the requirements for the degree of Masters in Molecular Genetics and Biomedicine
Motile ciliary dysfunctions cause specific Ciliopathies that affect mainly the respiratory tract, fertilization and left-right body establishment. The embryonic organ where left-right decisions are first taken is called the organizer, a ciliated organ where a leftward cilia driven fluid-flow is generated. The organizer is named node in the mouse and Kupffer’s vesicle (KV) in zebrafish. The correct left-right axis formation is highly dependent on signaling pathways downstream of such directional fluid-flow. Motile cilia need to be coordinated and Ciliary Beat Frequency (CBF) is characteristic of different types of cilia depending on their function. Using zebrafish as a model, our group has been studying cilia length regulation and motility in wild-type and deltaD-/- mutant embryos. Recently, we showed that Notch signalling was directly involved in the control of cilia length in the KV cells given that the deltaD-/- mutant present shorter KV cilia. The goal of this project was to characterize the CBF of deltaD-/- KV cilia vs. wild-type cilia and reveal how potential differences in CBF impact on KV fluid flow, using spectral analysis associated with highspeed videomicroscopy. By decomposing and comparing the obtained CBF with Fast Fourier Transform, we identified two major populations of motile cilia in wild-type as well as in deltaD-/- mutant embryos. However, we found the CBF populations had differential relative contributions and different distributions between wild-type and mutant embryos. Furthermore, by measuring the velocity of native particles we studied the KV fluid-flow and concluded that the dispersion of the flow velocity was much wider in the deltaD-/- mutants. On the other hand, based on a gene expression study of motility genes downstream of DeltaD, we concluded that motility related genes (dnah7, rsph3 and foxj1a) were deregulated in the mutants. During this project we generated data that led to new hypotheses that will allow us to test the causality between the described correlations.

We ask the question "what will a realistic nanobot look like?". The answer is something like a bacterium (such as e. coli) or a sperm. Both of these have a propulsion mechanism (a flagellum), a capsule containing a chemical payload and a system of sensors to detect food or the target for the payload. It is be soft and wet, just like biology, and to exemplify this we have built a series of biomimetic devices. Our progress in the development of responsive polymer-based molecular devices is be discussed with examples of vesicles of controlled size, synthetic muscles & flagella, and microparticles fitted with a jetpack. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/35017

The effect of nitric oxide (NO) upon gallbladder motility as examined in a group of volunteers after fatty meal during infusion with NO donors (glyceryltrinitrate and sodium nitroprusside), normal saline and hydralazine as a hypotensive control agent. Postprandial gallbladder emptying was significantly reduced during infusion with the NO donors. This inhibitory effect was independent of hypotension and CCK release. This inhibitory effect of NO donors was also observed on isometric contraction of isolated gallbladder muscle strips. The effect of NO on the SO was examined by infusion of glyceryltrinitrate to the ampulla during SO manometry. Basal tone and phasic activity were both suppressed. This finding may have therapeutic application for stone extraction during endoscopic retrograde cholangiopancreatography. Symptoms were assessed in 100 patients before and six months after laparoscopic cholecystectomy (LC) with standard questionnaire. 13% of patients had persistent biliary symptoms and abdominal bloating, constipation and consumption of antidepressant were fund to be significantly more prevalent in these subjects compared to patients who had successful operations. SO dysfunction is a cause of post-cholecystectomy pain. We hypothesised that LC could destroy cholecysto-sphincteric nerves leading to SO dysfunction. SO manometry was performed in a group of volunteers before and six months after LC. Following LC, the SO was not inhibited by CCK. This could lead to relative post-prandial biliary obstruction and result in post-cholecystectomy pain in susceptible individuals and to dilatation of the common bile duct. Several issues and concepts arose from the work of this thesis. The mechanism underlying the early release of CCK needs further investigation. The clinical relevance of the effects of NO upon biliary tract motility remains to be explored. It is hoped that future research in this area will help to clarify these issues.

Mammalian spermatozoa acquire the capacity for motility during passage through the epididymis. This study on rat spermatozoa shows that pH, cAMP and protein kinase C (PKC) all play an important role in the initiation of motility. pH has the most critical role and until the initial pH change in spermatozoa has occurred between the caput and caudal epididymal regions of the rat, second messengers are not effective in stimulating motility, but they are involved once such pH change has occurred. The spermatozoa of Fucus serratus differ from mammalian spermatozoa in that they are released into the sea prior to fertilisation and the motility of these spermatozoa is initiated upon their release into sea water. The ionic composition of sea water plays an important role in this activation and it is evident that the presence of Na+ is vital for the Initiation of motility. This study shows that a Na+/H+ exchanger, a N+-dependent bicarbonate/chloride exchanger and a Na+/K+ pump, which regulate the concentration of Na+, are present in Fucus serratus and integrated activity of these exchangers/pumps causes an increase in intracellular pH (pHi). An elevation in pHi correlates to an increase in motility, mediated through the activation of the dynein ATPase of the flagella. Motility and respiration of these spermatozoa are closely linked, probably because the ATP produced by respiration is used primarily by the dynein ATPase. Second messengers have also been Implicated in the initiation/regulation of motility and respiration. Indirect evidence shows cAMP and PKC are present and regulate motility, possibly through the phosphorylation and thereby activation of key regulatory proteins, such as the Na+/H+ exchanger. A rise in intracellular Ca2+ is also associated with the activation of Fucus serratus spermatozoa but the exact mechanism by which such a rise regulates motility remains unclear.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that causes motor, visual, and sensory symptoms. Patients also experience constipation, which is not yet understood, but could involve dysfunction of the enteric nervous system (ENS). Autoimmune targeting of the ENS occurs in other autoimmune diseases that exhibit gastrointestinal (GI) symptoms, and similar mechanisms could lead to GI dysfunction in MS. Here, we characterize GI dysmotility in the experimental autoimmune encephalomyelitis (EAE) model of MS and test whether autoantibodies targeting the ENS are present in the serum of MS patients. Male SJL or B6 mice were induced with EAE by immunization against PLP139-151, MOG35-55, or mouse spinal cord homogenate, and monitored daily for somatic motor symptoms. EAE mice developed GI symptoms consistent with those observed in MS. In vivo motility analysis demonstrated slower whole GI transit, and decreased colonic propulsive motility. EAE mice had faster rates of gastric emptying, with no changes in small intestinal motility. Consistent with these results, ex vivo evaluation of isolated colons demonstrated that EAE mice have slower colonic migrating myoelectric complexes and slow wave contractions. Immunohistochemistry of EAE colons exhibited a significant reduction in GFAP area of ENS ganglia, with no changes in HuD, S100, or neuron numbers. To test whether antibodies in MS bind to ENS structures, we collected serum samples from MS patients with constipation and without constipation, and healthy control patients without constipation. Immunoreactivity was tested using indirect immunofluorescence by applying serum samples to guinea pig ENS tissue. MS serum exhibited significantly higher immunoreactivity against guinea pig ENS than control patients, which was particularly evident in MS patients who did not experience constipation. There was no significant difference in immunoreactivity between MS patients with and without constipation. Targets of human MS and mouse EAE serum include enteric glia and neurons. Taken together, these data validate EAE as a model for constipation in MS, and support the concept that this symptom involves changes within the neuromuscular system of the colon. EAE mice develop symptoms consistent with constipation that affects functional ENS networks and may result in structural or phenotypic changes at the cellular level. Serum immunoreactivity suggests that autoantibodies could play a role in the development of constipation in MS by targeting the ENS itself.

Fescue toxicosis is problematic for ruminant livestock, causing weight loss and low productivity when fed endophyte-infected forages. Complete underlying mechanisms of toxicosis are unknown therefore; the objective of the study was to determine if ruminally dosed ergot alkaloids impact rumen motility. Cannulated steers were pair-fed a forage diet and ruminally dosed with endophyte-free (E-) or endophyte-infected (E+) tall fescue seed. An 8-h period of rumen motility collection began 4-h after feeding by monitoring pressure change via a wireless telemetry and transducer system. In experiment 1, steers were paired by weight and assigned to E- or E+ treatment. Overall, E+ steers had more frequent contractions. On d 7 - 9, both treatments had lower frequencies and E- steers had greater amplitude of contractions, which corresponded with decreased DM intake. In experiment 2 steers remained in pair, but switched treatment. During the 57 d E+ steers received titrated levels of ergovaline + ergovalinine. There was no difference between treatments for frequency or amplitude of contractions, but increasing dosage, decreased frequency (d 1 - 44) and amplitude, coinciding with lower DM intakes. Alteration in rumen motility associated with changes in intake may be responsible for the decreased productivity in ruminants consuming E+ forages.

This thesis describes investigations into mechanisms responsible for cancer cell motility in vivo. Chapters 1 and 2 provide a review of current literature in this field and also describe the techniques used to generate the following the results. Chapter 3 describes a candidate-based approach to investigate whether ROCK1 might be regulated by phosphorylation. Mutagenesis of ROCK1 was carried out at 3 chosen sites (T233 T380 T398) in the activation loop and the hydrophobic domain and the phenotypes of the mutants were analysed. Chapter 4 describes a parallel approach finding phosphorylation sites in ROCK1 by mass spectrometry. From these results T518 was chosen for further investigation and its possible function is investigated. Chapter 5 describes an siRNA screen designed to identify novel regulators of the cortical acto-myosin cytoskeleton. The read-out for this was based on the disruption of rounded blebbing morphology of A375 cells cultured on 3D gel matrices. The rounded morphology is similar to that observed in amoeboid cancer cell motility in vivo, therefore we hypothesised that genes required for contracted, rounded morphology might also be required for motility. Results identified PDK1 amongst other genes as a potential regulator of contractile forces in A375 cells and the role of PDK1 was investigated further. It was found that PDK1 was required both in vitro and in vivo for amoeboid cell motility. Chapters 6,7 and 8 detail the investigations into the mechanism of how PDK1 regulates the cytoskeleton and amoeboid cell motility. It was shown that PDK1 was responsible for the localisation of ROCK1 but not ROCK2 at the plasma membrane. This regulation was achieved by the direct binding of ROCK1 to PDK1. It was further found that PDK1 was able to compete with and prevent RhoE, a negative regulator of ROCK1, from binding. Chapter 9 investigates the relationship between cell morphology, motility and pigment production. It was found that it was possible to image melanin containing vesicles using multiphoton excitation, and using this technique, the motile behaviour of pigmented melanoma cells was observed in vivo. It was found that motile invasive cells tended to contain less melanin than non-motile cells suggesting that they were less well differentiated. This chapter details investigations into what differences in signalling could be responsible for a switch to a de-differentiated, more invasive/metastatic phenotype. The final chapter discusses the findings contained within this thesis and the possible implications.

La motilité des cellules est un phénomène fondamental en biologie souvent étudié sur des surfaces planes, conditions peu physiologiques. Nous avons analysé la migration cellulaire dans une matrice cellulaire 3D contenant de la fibronectine fluorescente. Nous démontrons que les cellules y sont confinées, et déforment leur environnement de manière cyclique avec une période de ~14 min avec deux centres de contractions à l’avant et à l’arrière de la cellule qui contractent avec un déphasage de ~3.5 min. Une perturbation de ces cycles entraîne une réduction de la motilité. Par l’utilisation d’inhibiteurs spécifiques, nous avons identifié l’acto-myosine comme étant l’acteur principal de ce phénomène. En imposant des contractions-relaxations locales par ablations laser, nous avons déclenché la motilité cellulaire ce qui confirme notre hypothèse. L’ensemble de cette étude met en évidence un nouveau mécanisme fondamental de dynamique cellulaire impliqué dans le mouvement des cellules
Cell motility is an important process in Biology. It is mainly studied on 2D planar surfaces, whereas cells experience a confining 3D environment in vivo. We prepared a 3D Cell Derived Matrix (CDM) labeled with fluorescently labeled fibronectin, and strikingly cells managed to deform the matrix with specific patterns : contractions occur cyclically with two contraction centers at the front and at the back of the cell, with a period of ~14 min and a phase shift of ~3.5 min. These cycles enable cells to optimally migrate through the CDM, as perturbation of cycles led to reduced motility. Acto-myosin was established to be the driving actor of these cycles, by using specific inhibitors. We were able to trigger cell motility externally with local laser ablations, which supports this framework of two alternating contractions involved in motion. Altogether, this study reveals a new mechanism of dynamic cellular behaviour linked to cell motility

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Physics, 2005.
Includes bibliographical references (p. 61-64).
This thesis contains work on three separate topics: the spontaneous motility of functionalized particles, the designability of RNA secondary structures, and the statistical mechanics of homopolymer RNAs. For the work on spontaneous motility, we were motivated by in vitro experiments investigating the symmetry-breaking motility of functionalized spherical beads to develop a general theory for the dynamics of a rigid object propelled by an active process at its surface. Starting from a phenomenological expansion for the microscopic dynamics, we derive equations governing the macroscopic velocities of the object near an instability towards spontaneous motion. These equations respect symmetries in the object's shape, with implications for the phase behavior and singularities encountered at a continuous transition between stationary and moving states. Analysis of the velocity fluctuations of such an object reveals that these fluctuations differ qualitatively from those of a passive object. For the work on designability, we investigated RNA folding within a toy model in which RNA bases come in two types and complementary base pairing is favored. Following a geometric formulation of biopolymer folding proposed in the literature, we represent RNA sequences and structures by points in a high-dimensional "contact space." Designability is probed by investigating the distribution of sequence and structure points within this space. We find that one-dimensional projections of the sequence point distribution approach normality with increasing RNA length N.
(cont.) Numerical comparison of the structure point distribution with a Gaussian approximation generated by principal component analysis reveals discrepancies. The third and final project concerns the statistical mechanics of homopolymer RNAs. We compute the asymptotics of the partition function Zn and characterize the crossover length scale governing its approach to its leading asymptotic behavior. Consideration of restricted partition functions in which one or more base pairs are enforced leads to an interesting connection with ideal Gaussian polymers. We introduce the notion of gapped secondary structures and analyze the partition function Z?,) for RNAs of length n with gap at p. Another length scale emerges whose scaling agrees with that of the crossover scale found earlier.
by Allen Lee.
S.M.

Gastrointestinal motility investigations involve the study of the motions of the bowel. Their goal is to understand the nature of the physical interactions that take place between the gut wall and its contents in both health and disease. The aim of this project was to develop a computerised system for the measurement, inspection, analysis and collation of prolonged, ambulatory manometric records from within the human small bowel. This was planned to evolve from existing analogue techniques using a specially commissioned digital data-logger. This thesis describes the testing of the data-logger by comparison with an accepted standard technique, investigating its long-term stability and the effect of different sampling rates. Then the development of a means of detecting intestinal contractions with minimal artefact is described. When validated against 6 experienced human observers the program had a sensitivity of between 84-95 % and a positive predictability of 98% in a noise free signal falling to 37% in a signal containing many movement artifacts and few contractions. The inter- and intra-observers response was found to be variable displaying a high degree of subjectivity in their assessment with respect to the computer. Further methods for the classification of intestinal activity in the temporal domain such as contraction frequency and the occurrence of clusters of contractions are described. A new parameter, Psw is proposed which provides an indication of the relative inter-contractile separations. The effect of meals and disease on Psw, contraction and cluster incidence is investigated and all three parameters are found to be markedly affected by feeding. A program for the identification of contractile propagation across adjacent sites is described, with reference to both a computer model and data from healthy subjects. A cross-correlation method is devised for the accurate assessment of propagation velocities. Finally, the form of database currently used for the clinical reporting of routine small bowel motility investigations is described with suggestions for improvements and additions to this database which are planned.

Cette thèse introduit un modèle hydrodynamique minimal de polarisation, migration et déformation d'une cellule vivante confinée entre deux surfaces parallèles. Le cytoplasme cellulaire, décrit comme une goutte visqueuse, est entraîné par une force active contrôlée par un soluté diffusif. Une analyse de stabilité linéaire révèle que l'activité du soluté déstabilise d'abord un mode global de polarisation et de translation, induisant une motilité cellulaire par rupture spontanée de symétrie. Pour une activité plus grande, le système traverse une série de bifurcations de Hopf conduisant à des oscillations couplées de la forme de la goutte et de la concentration de soluté. Nous trouvons également des solutions non linéaires de type onde progressive associées à des formes polarisées ressemblant à des observations expérimentales. De plus, nous avons développé des simulations numériques de ce problème basées sur la méthode des éléments finis. L'étude numérique a mis en évidence la stabilité des solutions de type onde progressive, l’existence d’attracteurs oscillants et l’apparition d’une singularité du bord à temps fini. En intégrant des interactions mécaniques avec l'environnement extérieur, nous avons exploré la diffusion cellulaire en présence de parois et d'obstacles stationnaires, la migration à travers des micro-géométries imposées et les collisions cellule-cellule. Ces simulations ont capturé une gamme de motifs non triviaux résultant de la mémoire intrinsèque et de la déformabilité de la cellule. Globalement, notre étude fournit un paradigme mathématique de systèmes actifs déformables qui couplent l'hydrodynamique de Stokes à des transducteurs de force diffusifs
This thesis introduces a minimal hydrodynamic model of polarization, migration, and deformation of a biological cell confined between two parallel surfaces. Our model describes the cell cytoplasm as a viscous droplet that is driven by an active cytoskeleton force, itself controlled by a diffusive cytoplasmic solute. A linear stability analysis of this two-dimensional system reveals that solute activity first destabilizes a global polarization-translation mode, prompting cell motility through spontaneous-symmetry-breaking. At higher activity, the system crosses a series of Hopf bifurcations leading to coupled oscillations of droplet shape and solute concentration profiles. At the nonlinear level, we find traveling-wave solutions associated with unique polarized shapes that resemble experimental observations. In addition, we developed a numerical simulation of our moving-boundary problem based on the finite element method. The numerical study demonstrated the stability of our traveling-wave solutions, the existence of sustained oscillatory attractors, and the emergence of a finite-time pinch-off singularity. By incorporating mechanical interactions with the external environment, we explored cell scattering from stationary walls and obstacles, migration through imposed micro-geometries, and cell-cell collisions. These exercises capture a range of nontrivial patterns resulting from the intrinsic memory and deformability of the cell. Altogether, our work offers a mathematical paradigm of active deformable systems in which Stokes hydrodynamics are coupled to diffusive force-transducers

Thesis (M.S.) -- University of Maryland, College Park, 2008.
Thesis research directed by: Marine, Estuarine, Environmental Sciences Graduate Program. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

The work in this thesis set out to examine the importance of motility and chemotaxis to the behaviour of Rhizobium trifolii TA1 in soil. Mutants defective in chemotaxis or motility were therefore generated by Tn5- mutagenesis (Beringer et a1., 1978) of Rhizobium trifolii TA1. Attempts to produce totally non-chemotactic mutants of R. trifolii were unsuccessful, though several partially non-chemotactic mutants were isolated. These mutants lacked chemotaxis to a wide range of single carbon sources, but were responsive to yeast extract in every case. They were therefore considered unsuitable for investigating the role of chemotaxis in nodulation and migration of root nodule bacteria in soil, since the possibility remained that plant exudates or soil substrates could still elicit chemotactic responses. Non-motile mutants were obtained using transposon Tn-5 mutagenesis. These mutants were indistinguishable from the wild type in growth rates in both laboratory media and in the clover rhizosphere. They also produced as many nodules on clover plants (Trifolium subterraneum cv Mount Barker) as the wild type and were as effective as the parent at nitrogen fixation. When motile and non-motile (flagellate or nonflagellate) strains were inoculated simultaneously onto clover plants the non-motile strain was found in only about 15% of the resultant nodules and the motile strain in 85%. This implies that the non-motile mutants are at some disadvantage in competition with the motile parent. The vertical movement of root nodule bacteria was examined in vertical columns of steam-treated sand. The non-motile strain did not move away from the inoculation site whereas the motile one did. Other experiments to determine the nodulation pattern of motile and non-motile organisms, on the tap roots of clover in steamed soil, showed that the motile strain could produce nodules further down the clover tap root than the non-motile strains. Similar experiments examining the lateral movement of both motile and non-motile strains from a central inoculated clover plant again showed that the motile strain had a definite advantage over the non-motile strain in its ability to move through steamed soil to produce nodules on clover roots. When these experiments were carried out in untreated sand, quite different results were obtained. In experiments using soil columns to examine the vertical movement of root nodule bacteria, neither the motile or non-motile strains moved significantly from the inoculation point. Similarly, in experiments to examine lateral movement of root nodule bacteria, in non-treated soil, little movement of either motile or non-motile strain was observed. These experiments showed that in steam-treated soil, motile strains, but not non-motile strains, have the capacity to move considerable distances. However, this movement potential may not be achieved under natural conditions in soil.

This work explores bacterial motility from the mechanisms of propulsion of an individual cell to the complex behavior of collective motility. The shear modulus of bacterial flagella was measured by stretching isolated flagella with an optical trap and by measuring force extension curves of the stretched flagella shedding light onto the me-chanics involved in the motility of single micro-organisms. Experiments in concentrated suspensions of bacteria show collective behavior with large scale mixing on a time and length scale greater than can be understood from the standard model of "run and tumble" motility of a single organism are reported. To further understand the transition from individual to collective motility a novel form of motility where an individual bacterium can reverse direction without changing cell orientation is reported here. These experiments further the understanding of bacterial motility.

Acinetobacter baumannii i Ralstonia solanacearum són dues espècies de bacteris patògens filogenèticament no relacionats que, en els últims anys, han adquirit una gran rellevància a causa de l’impacte sanitari i agroalimentari que, respectivament, causen en tot el món. D’una banda, A. baumannii provoca infeccions nosocomials que, juntament amb l’augment de soques multiresistents, fan que algunes d’aquestes infeccions siguin pràcticament intractables. D’altra banda, R. solanacearum és l’agent causal del marciment bacterià, una malaltia letal que afecta a més de 200 espècies vegetals, disminuint la producció de nombrosos cultius d’interès per a la indústria agroalimentària. Una característica comuna de tots dos microorganismes és la seva capacitat de moure’s. A. baumannii pot desplaçar-se a través del moviment associat a pili de tipus IV, conegut com twitching; o mitjançant el moviment independent d’apèndixs, denominat surface-associated motility. En el cas de R. solanacearum, aquesta espècie exhibeix els moviments twitching i swimming, aquest últim associat a flagels. A l’ésser la motilitat bacteriana determinant per a la virulència de molts patògens, l’objectiu global de la present Tesi Doctoral és la identificació de gens implicats en la motilitat de tots dos microorganismes, permetent així identificar noves dianes terapèutiques. A la soca d’A. baumannii ATCC 17978, capaç de desplaçar-se exclusivament mitjançant surface-associated motility, es va avaluar la implicació en aquest procés de les proteïnes RecA (regulador positiu de sistema SOS) i A1S_2813 (anàloga al component quimiotàctic CheW). Els resultats obtinguts van mostrar que les dues proteïnes estan involucrades en la motilitat surface-associated motility, la resposta quimiotàctica i la virulència. A més, aquestes funcions són dutes a terme mitjançant la interacció específica entre les dues proteïnes. D’altra banda, a la soca de R. solanacearum GMI1000 es va avaluar la funció de les proteïnes PilI i ChpA, totes dues anàlogues als components del sistema quimiotàctic flagel·lar CheW i CheA, respectivament, i alhora homòlogues a components de sistema quimiotàctic associat a pili de tipus IV. Els resultats obtinguts van evidenciar la implicació de totes dues proteïnes a la motilitat twitching, la capacitat per transformar DNA de forma natural, de formar biofilm, d’adherir-se a les arrels de tomaquera i a la virulència. Paral·lelament, els estudis realitzats amb els mutants deficients dels gens que codifiquen les subunitats de pilina (PilA) i flagelina (FliC) van permetre determinar que, d’una banda, PilA està involucrat en la motilitat swimming, mentre que FliC participa en els processos de formació del biofilm i en l’adhesió a les arrels de tomaquera. Els resultats de la present Tesi Doctoral posen de manifest la importància de la motilitat bacteriana en el procés patogènic tant d’A. baumannii com de R. solanacearum. A més, la identificació de nous gens implicats en els sistemes descrits obre nous fronts de recerca per a la seva utilització com a dianes terapèutiques.
Acinetobacter baumannii y Ralstonia solanacearum son dos especies bacterianas patógenas, filogenéticamente no relacionadas, que en los últimos años han adquirido una gran relevancia debido al impacto sanitario y agroalimentario que, respectivamente, causan en todo el mundo. Por un lado, A. baumannii provoca infecciones nosocomiales que, junto al aumento de cepas multirresistentes, hacen que algunas de estas infecciones sean prácticamente intratables. Por otro lado, R. solanacearum es responsable de la marchitez bacteriana, una enfermedad letal que afecta a más de 200 especies vegetales, disminuyendo la producción de numerosos cultivos de interés para la industria agroalimentaria. Una característica común de ambos microorganismos es su capacidad de moverse. A. baumannii puede desplazarse, en función de la cepa, a través del movimiento asociado a los pili de tipo IV, conocido como twitching; o mediante un movimiento independiente de apéndices denominado surface-associated motility. En el caso de R. solanacearum, esta especie exhibe los movimientos twitching y swimming, este último asociado a flagelos. Al ser la motilidad bacteriana determinante para la virulencia de muchos patógenos, el objetivo global de la presente Tesis Doctoral es la identificación de genes implicados en la motilidad de ambos microorganismos, permitiendo así establecer nuevas dianas terapéuticas. En la cepa de A. baumannii ATCC 17978, capaz de desplazarse exclusivamente mediante surface-associated motility, se evaluó la implicación en este proceso de las proteínas RecA (regulador positivo del sistema SOS) y A1S_2813 (análoga al componente quimiotáctico CheW). Los estudios realizados mostraron que ambas proteínas están involucradas en el surface-associated motility, la respuesta quimiotáctica y la virulencia. Además, estas funciones son llevadas a cabo a través de la interacción específica entre ambas proteínas. Por otro lado, en R. solanacearum GMI1000 se evaluó la función de las proteínas PilI y ChpA, ambas análogas a los componentes del sistema quimiotáctico flagelar CheW y CheA, respectivamente, y a su vez homólogas a componentes del sistema quimiotáctico asociado a pili de tipo IV. Los resultados obtenidos determinaron la implicación de ambas proteínas en la motilidad twitching, la capacidad para incorporar DNA a través de la transformación natural, la formación de biofilm, la adherencia a raíces de tomatera y la virulencia. Paralelamente, los resultados obtenidos con los mutantes deficientes en los genes que codifican las subunidades de pilina (PilA) y flagelina (FliC) permitieron establecer que, PilA está involucrado en la motilidad swimming, mientras que FliC participa en la formación del biofilm y en la adhesión a raíces de tomatera. Los resultados de la presente Tesis Doctoral ponen de manifiesto la importancia de la motilidad bacteriana en el proceso patogénico tanto de A. baumannii como de R. solanacearum, abriendo nuevos frentes de investigación para su utilización como dianas terapéuticas.
Acinetobacter baumannii and Ralstonia solanacearum are two pathogenic bacterial species that, despite not being phylogenetically related, have acquired a great relevance in recent years, due to the sanitary and agri-food impact worldwide, respectively. On the one hand, A. baumannii causes nosocomial infections that, with an increasing number of multidrug resistant strains, some of these infections turn practically intractable. On the other hand, R. solanacearum is responsible of bacterial wilt, a devastating disease that affects more than 200 plant species, decreasing the production of many crops of interest for the agri-food industry. A common characteristic of both microorganisms is their ability to move. A. baumannii is able to move through type IV pili-associated motility called twitching, or by using an appendage-independent movement known as surface-associated motility. In the case of R. solanacearum, this species exhibits twitching and the flagella-associated motility called swimming. As bacterial motility is a determinant factor for the virulence of many pathogens, the main objective of this Doctoral Thesis is the identification of novel genes involved in the motility of both pathogens, which might allow the identification of therapeutic targets. In A. baumannii strain ATCC 17978, which is able to move exclusively through surface-associated motility, the involvement in motility of the RecA (positive regulator of the SOS system) and the A1S_2813 (analogous to the CheW chemotactic component) proteins was evaluated. Results obtained revealed that both proteins are involved in surface-associated motility, the chemotactic response and virulence. Furthermore, these functions are carried out through the specific interaction between both proteins. On the other hand, in R. solanacearum strain GMI1000, the function of the PilI and ChpA proteins, both analogous to the CheW and CheA components of the flagellar-chemotactic system, respectively, and homologous to components of the type IV pili-associated chemotactic system, was evaluated. Results obtained determined the involvement of both proteins in twitching motility, the ability to transform DNA naturally, the biofilm formation, the root attachment and the virulence. Simultaneously, analysis of the mutants deficient for the genes encoding the pilin (PilA) and flagellin (FliC) subunits showed that, while PilA is involved in swimming motility, FliC participates in the biofilm formation and tomato-root attachment processes. The results of this Doctoral Thesis demonstrate the importance of bacterial motility in the pathogenic process of both A. baumannii and R. solanacearum. Furthermore, the identification of new genes involved in the described systems opens up new research fronts for their usage as therapeutic targets.
Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologia

Tous les organismes, les animaux, les plantes et les microbes, sont composés de cellules polarisées, en affichant un positionnement asymétrique des organites sub-cellulaires ou des structures. Le contrôle de polarité a été étudié chez les eucaryotes pendant une longue période, et a été montré pour être impliqués dans de nombreux processus physiologiques, tels que l'embryogenèse, le cancer métastatique et les maladies dégénératives des neurones. Chez les procaryotes, des études de polarité ne sont apparues récemment avec le développement de la microscopie à fluorescence sensibles. Ces études ont révélé que les cellules procaryotes sont en fait très organisé et une masse croissante de la littérature a montré que les cellules bactériennes également utiliser des radeaux lipidiques, courbure membranaire, la paroi cellulaire et un cytosquelette complexe pour diriger le positionnement spécifique de structures subcellulaires.Petites GTPases de la superfamille Ras sont des éléments réglementaires polarisation répandue chez les eucaryotes. Malgré l'existence depuis longtemps de ces petites GTPases dans les génomes procaryotes, leur fonction a jamais été étudiée. Pendant ce travail de thèse, nous avons trouvé, pour la première fois, qu'une petite GTPase, MglA et de sa protéine apparentée Activation GTPase (GAP) MglB, directe une dynamique axe antéro-postérieur à la motilité directe en forme de tige deltaproteobacterium Myxococcus xanthus. Dans ce processus, MglA s'accumule dans son état lié au GTP au niveau du pôle leader de cellules, en activant les machineries motilité. Ce schéma de localisation est maintenue par MglB, qui localise le pôle opposé, le blocage de l'accumulation MglA à ce pôle à travers son activité GAP. Remarquablement, les deux protéines passer leur localisation synchrone, ce qui correspond à un changement dramatique dans la direction du mouvement cellulaire (inversion). Ce commutateur est réglementé par un système chimiosensoriels-like, Frz. Dans une deuxième partie de ce travail, nous avons identifié un régulateur de protéine de réponse, RomR qui est essentiel pour le regroupement polaire de MglA. Interdépendances complexes entre la localisation RomR, MglA et MglB indiquent que ces protéines pourraient constituer un complexe de polarité dynamique de trois protéines qui reçoit Frz de signalisation pour passer l'axe de polarité. En conclusion, les résultats de ce travail de thèse suggère que M. xanthus intégré un module de polarité eucaryotes-like (MglAB) dans un procaryote spécifique (Frz) réseau de signalisation pour réguler sa motilité. Une telle réglementation est distincte sous forme de petites protéines G des règlements, qui sont généralement couplés à la protéine G récepteurs couplés (GPCR) chez les eucaryotes. Enfin, ce travail ouvre la voie pour comprendre comment la réglementation seule la motilité cellulaire sont intégrés pour générer des comportements commandés multicellulaires donnant naissance à des structures primitives de développement, par exemple, la morphogenèse du corps fructifères. D'autre part, ce travail fournit également un exemple d'analyser les étapes évolutives donnant lieu à des réseaux de signalisation
All organisms, animals, plants and microbes, are composed of polarized cells, displaying asymmetric positioning of sub-cellular organelles or structures. Polarity control has been studied in eukaryotes for a long time, and has been shown to be involved in many physiological processes, such as embryogenesis, cancer metastasis and neuron degenerative diseases. In prokaryotes, polarity studies only emerged recently with the development of sensitive fluorescent microscopy. These studies revealed that prokaryotic cells are in fact highly organized and a growing body of literature has shown that bacterial cells also use lipid rafts, membrane curvature, the cell wall and a complex cytoskeleton to direct the specific positioning of subcellular structures.Small GTPases of the Ras superfamily are widespread polarization regulatory elements in eukaryotes. Despite the long known existence of such small GTPases in prokaryotic genomes, their function has never been studied. During this thesis work, we found, for the first time, that a small GTPase, MglA and its cognate GTPase Activating Protein (GAP) MglB, direct a dynamic anterior- posterior axis to direct motility of the rod-shaped deltaproteobacterium Myxococcus xanthus. In this process, MglA accumulates in its GTP-bound state at the leading cell pole, activating the motility machineries. This localization pattern is maintained by MglB, which localizes at the opposite pole, blocking MglA accumulation at this pole through its GAP activity. Remarkably, both proteins switch their localization synchronously, which correlates with a dramatic change in the direction of cell movement (reversal). This switch is regulated by a chemosensory-like system, Frz. In a second part of this work, we identified a response regulator protein, RomR which is essential for the polar clustering of MglA. Intricate localization interdependencies between Romr, MglA and MglB indicate that these proteins might constitute a dynamic three-protein polarity complex that receives Frz-signaling to switch the polarity axis. In conclusion, the results from this thesis work suggest that M. xanthus integrated a eukaryotic-like polarity module (MglAB) into a prokaryotic- specific (Frz) signaling network to regulate its motility. Such regulation is distinct form small G- protein regulations, which are generally coupled to G-protein coupled receptors (GPCRs) in eukaryotes. Finally, this work paves the way to understand how single cell motility regulations are integrated to generate ordered multicellular behaviors giving rise to primitive developmental structures, for example fruiting body morphogenesis. On the other hand, this work also provides an example to analyze the evolutionary steps giving rise to signaling networks

Motility of the small intestine is a result of complex neurochemical and hormonal interactions within the intestine. The net motility (contraction) of the intestine is a balance of the influences from the central nervous system, enteric nervous system and hormonal changes in the body. Recently, the discovery of several peptide neurotransmitters common to the brain and the intestine has stimulated new research into the influence of these novel neurotransmitter candidates on intestinal motility at the level of the enteric (intestinal) nervous system. The present studies examined the contractile actions of three families of peptides, the opioids, tachykinins and motilin. Each of these peptide groups has been localized in the intestine, and suggested to function in the control of intestinal motility. The peptides were administered by intraarterial injection to isolated segments of canine small intestine and the resulting contractile activity measured. The results of these experiments demonstrate that all of these peptides may elicit contractile activity of the intestine in very low doses. These actions were further examined, using pharmacological antagonists, to determine the mechanism of action and the receptor types involved in the contractile actions. The opioid peptide induced responses were found to be mediated by two receptor types, mu and delta, located on the enteric nerve and smooth muscle, respectively. Similarly, the tachykinin induced contractions were also found to be due to actions on two receptor types, SP-P and SP-K, located on the nerve and muscle layers, respectively. These data suggest that the opioids and tachykinins may have multiple functions in the intestine dependent on the site of action and the receptor type involved in the response. Administration of motilin induced long-lasting contractile patterns in the intestine. The results also suggest that the actions of motilin are mediated by intermediate neurons of the enteric plexes which synapse on terminal cholinergic motor neurons.

The TRPV4 cation channel is expressed in a broad range of tissues where participates in the generation of a Ca2+ signal. TRPV4 participation in osmo- and mechanotransduction contributes to important functions such as cellular and systemic volume homeostasis, among others. TRPV4 also responds to temperature and 4α-phorbol 12,13-didecanoate (4αPDD) thus showing multiple modes of activation. Besides, dozens of TRPV4 mutations have been linked to osteoarticular and neuromuscular diseases. However, little is known about the domains relevant for TRPV4 activation by different stimuli. This Thesis research aims to elucidate the participation of the N-terminal cytosolic tail in the gating of TRPV4 by physiological stimuli and the channel implication in cell motility. I provide evidences that activation of TRPV4 by hypotonic shocks and temperature requires PIP2 binding within the sequence 121KRWRK125 of the N-terminus tail and rearrangement of the cytosolic tails. My results also point to the participation of TRPV4 in cell migration by modulating the dynamics of trailing adhesions, a function that may require the interplay of TRPV4 with other cation channels present at the focal adhesion sites. Resumen El canal TRPV4 es un canal
El canal TRPV4 es un canal catiónico capaz de generar señales intracelulares de Ca2+ en diversos tejidos. La participación del canal TRPV4 en procesos de mecano-osmotransducción le implica en funciones tan importantes como la regulación del volumen celular y sistémico. El TRPV4 también se activa en respuesta a calor y al agonista sintético 4αPDD, lo que implica la presencia de varios modos de activación. Además, existen numerosas mutaciones en el TRPV4 que se han encontrado en pacientes que sufren de patología osteoarticular y neuromuscular. Sin embargo, aún se desconocen aspectos de su función relacionados con los mecanismos de activación. Mi trabajo de Tesis doctoral aborda el estudio de la región N-terminal del TRPV4, su participación en la activación del canal por estímulos fisiológicos y la relevancia del canal en proceso de migración celular. Esta Tesis doctoral proporciona evidencias de que el TRPV4 necesita unir PIP2 a través de la secuencia 121KRWRK125 de la cola N-terminal y que las colas se reorganicen para que el canal se abra en respuesta a estímulos osmóticos y de calor. Mis estudios también sugieren que el canal TRPV4 participa en la modulación de la adherencia de la cola durante el proceso de la migración celular, posiblemente interaccionando con otros canales presentes en las adhesiones focales.

Thesis (Ph. D.)--University of Oregon, 2006.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80 - 90). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph.D. )--University of Tennessee Health Science Center, 2007.
Title from title page screen (viewed on July 16, 2007). Research advisor: Lisa K. Jennings, Ph.D. Document formatted into pages (xiv, 150 p. : ill.). Vita. Abstract. Includes bibliographical references (p.130-150).

Cell motility has been found to play a role in many important body functions as well as the embryogenenis of mulitcellular organisms like vertebrates. From a physics point of view the interesting questions behind every motion are: Why is it moving? Where do the forces come from? Today we know that the motility of many cells is dependent on an active polymer network. Actin, one of the most abundant proteins in the body, is constantly polymerized, being moved around and depolymerized in motile cells. Until now, only forces outside the cell like traction forces could be measured. The direct measurement of the force generated by polymerizing actin filaments has only been measured by our lab and the lab of M. Radmacher. In these measurements fish keratocytes were used. Whereas I did these experiments, for the first time, on mammalian cells. To measure forward forces on neuronal growth cones I stabilized the SFM, as measurement times went up from minutes to hours. Furthermore measurements had to be performed at 37°C instead of room temerature, this induced drifts of the substrate. I incorporated an optical trap into the microscope to track the motion of the substrate. A feedback loop moved the SFM cantilever to minimize relative motion of substrate and cantilever. For keratocytes I directly measured the forces produced by actin polymerization and, to my knowledge for the first time, the forces associated with the retrograde actin flow using a SFM. The result was that both actin and myosin play important but different roles in motility. For actin it turned out that considering just the polymerization was not enough. Actin depolymerization and the resulting entropic forces are a completely new physical effect in actin based cell motility. With this new force in the force balance I can explain all effects observed in my experiments without introducing any new biochemical feedback loops. Finally I showed that neuronal growth cones are very soft and weak structures. They are at least one order of magnitude softer and weaker as for example fibroblasts or cells forming the blood vessel walls. As neurons are usually located in soft environments this does not impede their normal outgrowth. It could even serve as a safety mechanism that prevents cell from growing into wrong areas like breaching the blood-brain-barrier, on a physical level. For a neuron the wall of a blood vessel feels like a brick wall for us.