Le système nerveux est un réseau complexe qui détecte et analyse les informations. Ces informations sont transmises entre cellules grâce à des connections synaptiques et des jonctions communicantes. Ce réseau n’est pas statique et évolue au cours du développement, de l’apprentissage mais aussi durant le processus de vieillissement – naturels ou pathologiques. Comprendre le système nerveux et son fonctionnement requiert une analyse des connections synaptiques in vivo chez un animal model tel que Caenorhabditis elegans. Cependant les techniques actuellement disponibles pour C. elegans sont laborieuses, ne dépendent pas forcément d’une interaction trans-synaptique ou fixent la synapse. Par conséquent, ces approches ne permettent pas de réaliser des études de populations et dynamiques des modifications synaptiques. Dans ce manuscrit, je décris tout d’abord une nouvelle technique pour visualiser les synapses in vivo chez le vers C. elegans. Cette technique appelé iBLINC (in vivo Biotin Labeling of INtercellular Contacts) qui consiste en la biotinylation d’un peptide par une ligase d’Escherichia coli, BirA. Ces deux molécules sont fusionnées à des protéines trans-membranaires qui forment un complexe à la synapse. La biotinylation est détectée grâce à une streptavidin monomérique taguée avec un fluorophore qui est secrétée dans l’espace extracellulaire. J’ai démontré que cette technique est directionnelle et dynamique. En utilisant iBLINC pour visualiser des synapses faisant partie du circuit sensoriel de C. elegans, une évolution du nombre et de la taille des synapses a pu être observée avec l’âge. Il semblerait que ce changement soit dépendant du segment de la zone synaptique observée. Ces résultats ont été corroborés par l’observation d’une diminution du nombre de vésicules pendant le vieillissement grâce à un marqueur pré-synaptique des synapses GABAergique de la jonction neuromusculaire. Pour conclure, ce manuscrit décrit une nouvelle technique permettant d’observer les synapses chez le vers vivant et démontre une évolution naturelle du nombre de synapses et du nombre de vésicules pré-synaptiques avec l’âge. / The nervous system is a complex network that senses and processes information and is essential for the survival of both vertebrates and invertebrates as it is involved in behavior responses. Information within the network is transmitted through specialized cell-cell contacts, including synaptic connections. Importantly, the network is not static and is believed to change during development and learning, as well as during pathological or normal age-related decline. Studying the nervous system in vivo requires the use of animal models such as Caenorhabditis elegans. Understanding of behavior and development requires visualization and analysis of synaptic connectivity. However, existing methods are laborious and may not depend on trans-synaptic interactions, or otherwise ‘trap’ the synapses by fixing the connections, thus precluding dynamic studies. In order to study synaptic modifications, we developed a new transgenic approach for in vivo labeling of specific connections in C. elegans, called iBLINC (in vivo Biotin Labeling of INtercellular Contacts). iBLINC involves the biotinylation of an acceptor peptide (AP) by the Escherichia coli biotin ligase BirA. Both are fused to two interacting post- and pre-synaptic proteins, respectively. The biotinylated acceptor peptide fusion is detected by a monomer streptavidin fused to a fluorescent protein that is transgenically expressed and secreted into the extracellular space. The method is directional, bright and dynamic. Moreover it correlates well with electron micrograph reconstruction. Using this new technique to observe synapses, which are part of the thermosensory circuitry of C. elegans, during aging, we could conclude that the connection pattern varies with age and within a population. Changes of the number and size of the synapses were observed during aging. Some segments of the synaptic region seem to be more affected than others by the aging process. Those results were corroborated by using a GABAergic pre-synaptic marker which allowed us to visualize a decline of the vesicle number with aging. In summary, in this thesis I explained a new in vivo trans-synaptic method to visualize synapses in C. elegans. Then I demonstrated that a natural decline in the number of synapses as well as the number of vesicles occurs during aging.

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Phosphorescent transition metal complexes make up an important group of compounds that continues to attract intense research owing to their intrinsic bioimaging applications that arise from bright emissions, relatively long excited state lifetimes, and large stokes shifts. Now for biomaging assay a model organism is required which must meet certain criteria for practical applications. The organism needs to be small, with a high turn-over of progeny (high fecundity), a short lifecycle, and low maintenance and assay costs. Our model organism C. elegans met all the criteria. The ideal phosphor has low toxicity in the model organism. In this work the strongly phosphorescent platinum (II) pyrophosphito-complex was tested for biological applications as a potential in vivo hypoxia sensor. The suitability of the phosphor was derived from its water solubility, bright phosphorescence at room temperature, and long excited state lifetime (~ 10 µs). The applications branched off to include testing of C. elegans survival when treated with the phosphor, which included lifespan and fecundity assays, toxicity assays including the determination of the LC50, and recovery after paraquat poisoning. Quenching experiments were performed using some well knows oxygen derivatives, and the quenching mechanisms were derived from Stern-Volmer plots. Reaction stoichiometries were derived from Job plots, while percent scavenging (or antioxidant) activities were determined graphically. The high photochemical reactivity of the complex was clearly manifested in these reactions.

The microbes that inhabit the human body, our microbiota, greatly influence our physiology and propensity for disease. For instance, the gut microbiota metabolizes compounds from our diet to provide important nutrients. Similarly, the microbiota has the potential to impact drug response; directly by metabolizing drugs, or indirectly by providing metabolites to the host. The complexity of the mammalian microbiota, and the limited throughput of such models, prohibit a systematic interrogation of specific interactions between microbes and host drug response. Here, I use C. elegans and its bacterial diet as a suitable model with the scalability and genetic tractability to address these questions. In Chapter II, I describe host-bacteria-drug interactions involving the anti-pyrimidine drugs 5-FU and FUDR. In brief, we identified two main mechanisms by which bacteria affect the C. elegans response to anti-pyrimidines: (1) metabolic conversion into FUMP by uridine phospho-ribosyltransferase (upp) and (2) dietary supplementation of uracil. Chapter III will focus on a selective estrogen-receptor modulator, TAM, with no clear target in bacteria or C. elegans. I will describe my work characterizing a bacteria-dependent response to TAM involving fatty acid metabolism. Lastly, the Appendix will summarize my efforts to expand the sample space of tested host-microbe-drug interactions.

La maladie d’Alzheimer est une maladie neurodégénérative déterminée par deux caractéristiques : les plaques extracellulaires composées d’amyloïde-β et l’accumulation intracellulaire de tau hyperphosphorylée, appelée enchevêtrements neurofibrillaires. Malgré le nombre important d’études, la nature de la toxicité des espèces tau hyperphosphorylée et hypophosphorylée reste mal connue. Notre projet de recherche vise à caractériser quel état de phosphorylation de la tau contribue le plus à la toxicité neuronale ainsi que d’identifier les mécanismes sous-jacents. Pour répondre à ces objectifs, nous avons généré des modèles transgéniques de C. elegans exprimant soit une tau hyperphosphorylée humaine (12 glutamates pour mimer l’hyperphosphorylation de la tau trouvée chez des patients Alzheimer), une tau sauvage, ou une tau hypophosphorylée (12 alanines pour mimer l’hypophosphorylation), dans les motoneurones GABAergiques. Ensuite, pour caractériser nos modèles, nous avons mesuré leur comportement principalement avec des tests de locomotion en utilisant le logiciel WormLab. Nos résultats ont montré que la tau phosphorylée est l’espèce la plus toxique car la souche hyperP a montré une perturbation du système locomoteur se traduisant par une neurodégénérescence ainsi que des problèmes développementaux (longueur des vers). Puis nous avons testé certains médicaments utilisés dans des modèles de tauopathies, afin d’identifier des voies biologiques impliquées dans la toxicité de la tau hyperphosphorylée. Pour conclure, nos modèles vont être des outils utiles pour identifier des modificateurs génétiques et pharmacologiques dans la toxicité de la tau. / Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by two hallmarks: extracellular plaques composed of amyloid-β (Aβ) deposits and intraneuronal accumulation of hyper and abnormal phosphorylated tau, also called neurofibrillary tangles (NFT). Despite many decades of research, the nature hypophosphorylated or hyperphosphorylated Tau toxicity remains ill understood. Our research project aims to characterize which state of Tau phosphorylation contributes to neuronal toxicity and identify the underlying mechanisms. To assess these objectives, we generated transgenic C. elegans models expressing either a human hyperphosphorylated tau (incorporation of 12 glutamate residues to mimic Tau hyperphosphorylation found in AD’s patients) human wild type Tau, or a human hypophosphorylated tau (incorporation of 12 alanine residues to mimic Tau hypophosphorylation) in the GABAergic motoneurons. Then, to characterize our models, we measured their behavior mainly with locomotion’s test using WormLab software. Our results showed that hyperphosphorylation of tau is the most toxic species for our models because hyperP strain showed an impair in the locomotor system translating into neurodegeneration, as well as developmental problems such as worm length. Then we tested some drugs used in taupathies C. elegans models to see if we could identify some biological pathways implicated in the toxicity. To conclude, our models may be a useful tool to identify genetic and pharmacological modifiers of tau toxicity.

La division cellulaire asymétrique est un processus crucial dans le développement des organismes multicellulaires puisqu’elle permet la génération de la diversité cellulaire. Les cellules qui se divisent de façon asymétrique doivent tout d’abord se polariser et correctement orienter leur fuseau mitotique pour ségréger des déterminants cellulaires en deux entités distinctes. L’embryon du nématode C. elegans est un modèle robuste et largement utilisé pour étudier la division cellulaire asymétrique. Dans cet embryon, le point d'entrée du spermatozoïde détermine l'axe de polarité antéro-postérieur. Suite à la fécondation, le cortex embryonnaire est uniformément contractile et un complexe conservé formé des protéines PAR-3, PAR-6 et PKC-3 (nommé complexe PAR-3 ci-dessous) est localisé sur l'ensemble du cortex. La complétion de la méiose maternelle induit une relaxation corticale au postétieur et un flux cortical vers l’antérieur de l’embryon. Ces contractions corticales asymétriques mènent à la formation d'un domaine antérieur contenant le complexe PAR-3, tandis que le cortex postérieur, dont le complexe PAR-3 s’est délocalisé, est enrichi avec les protéines PAR-2 et PAR-1. Par conséquent, les domaines formés par les protéines PAR définissent un pôle antérieur et un pôle postérieur dans l'embryon suite au remodelage du cytosquelette. Les protéines PAR-4 et PAR-5 restent localisées de façon uniforme dans l'embryon. Curieusement, les protéines PAR exercent une régulation par rétroaction sur la contractilité corticale. Il a été montré qu’une des protéines PAR récemment identifiée, PAR-5, est orthologue à la protéine adaptatrice 14-3-3 et joue un rôle important dans la contractilité corticale. En dépit de son rôle central dans la contractilité corticale et le processus de polarisation cellulaire, le mécanisme par lequel PAR-5 régule la contractilité corticale n’est pas bien compris. Le but de ce projet est de mieux comprendre comment PAR-5 et ses interacteurs contrôlent la régulation des contractions corticales et, de ce fait, la polarité cellulaire. Dans un essai de capture de la protéine GST (GST pull-down), nous avons identifié plusieurs nouveaux interacteurs de PAR-5. Parmi ceux-ci, nous avons trouvé CAP-2 (protéine de coiffage de l'actine), qui a été identifiée dans des éxpériences de capture de 14-3-3 dans trois systèmes modèles différents. CAP-2 est un hétérodimère des protéines CAP, qui sont impliquées dans la régulation de l'actine. Nous avons trouvé que la déplétion des protéines CAP par interférence à l’ARN dans des vers de type sauvage mène à une augmentation létalité embryonnaire, ce qui suggère que ces protéines jouent un rôle important dans le développement embryonnaire. L'imagerie en temps réel d'embryons déplétés pour les protéines CAP montre qu’ils ont une diminution des contractions corticales avec un sillon de pseudoclivage mois stable, suggérant un défaut dans la régulation du cytosquelette d'actine-myosine. Ceci a également été confirmé par la diminution de la vitesse et du nombre de foci de NMY-2::GFP. En outre, ces embryons montrent une légère diminution de la taille du croissant cortical de PAR-2 lors de la phase d’établissement de la polarité. Les embryons déplétés en CAP-2 montrent également un retard dans la progression du cycle cellulaire, mais le lien entre ce phénotype et la régulation des contractions corticales reste à être précisé. La caractérisation des protéines CAP, des régulateurs du remodelage du cytosquelette, permettra d'améliorer notre compréhension des mécanismes qui sous-tendent l'établissement et le maintien de la polarité cellulaire, et donc la division cellulaire asymétrique. / Asymmetric cell division is a crucial step in organism development, as it allows the generation of cellular diversity. In order to achieve asymmetric division cells need to polarize their cell fate determinants and properly orient their mitotic spindle before division. The C. elegans embryo is a powerful and widely used model to study asymmetric cell division. In the embryo the sperm entry site determines the anterior-posterior axis of polarity. In the newly fertilized embryo, shortly after meiosis, the cortex is uniformly contractile and the conserved PAR-3/PAR-6/PKC-3 complex (hereafter referred to as the PAR-3 complex) is localised on the entire cortex. Entry of the sperm triggers posterior smoothening and anterior-directed cortical flows. Asymmetric cortical contractions result in the formation of an anterior domain containing the PAR-3 complex, while the posterior-pole cortex, depleted of the PAR-3 complex, is enriched in PAR-2 and PAR-1 proteins. Therefore PAR domains define an anterior and a posterior pole of the embryo in response to cytoskeleton remodelling. The PAR-4 and PAR-5 proteins remain localized uniformly throughout the embryo. Intriguingly, the PAR proteins exert a feedback regulation on cortical contractility. PAR-5, one of the lately identified PAR proteins, was shown to be an ortholog of the adaptor protein 14-3-3 and to play an important role in cortical contractility. Despite its central role in cortical contractility and henceforth the polarization process, little is known on how PAR-5 regulates cortical contractility. The aim of this project is to better understand the regulation of cortical contractions via the PAR-5 protein and its interactors, and how they control cell polarity. In a GST pull down assay we identified several new interactors of PAR-5. Among these we found CAP-2 (actin capping protein), which was also pulled down with 14-3-3 in three different model systems. CAP-2 has been implicated in actin regulation. Interestingly we found that depletion of CAP proteins by RNA interference in wild type worms results in increased embryonic lethality, suggesting an important role in embryonic development. Live imaging of embryos depleted of CAP proteins shows that these embryos have decreased cortical contractions with a less stable pseudo cleavage furrow, indicating a defect in the regulation of the actin-myosin cytoskeleton. This was further confirmed by the decreased velocity and the number of NMY-2::GFP foci in CAP depleted embryos. Furthermore, these embryos show mild decrease in PAR-2 domain size during the polarity establishment phase. cap-2(RNAi) embryos also show a delay in cell cycle progression, however the role of the cell cycle delay in the regulation of cortical contractions has to be determined. The characterization of CAP proteins, which are cytoskeleton-remodeling regulators, will improve our understanding of the mechanisms underling the establishment and maintenance of cell polarity, and thereby asymmetric cell division.

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) protects against redox stress by providing reducing equivalents to antioxidants such as glutathione and thioredoxin. NADPH levels decline with aging in several tissues, but whether this is a major driving force for the aging process has not been well established. Global or neural overexpression of several cytoplasmic enzymes that synthesize NADPH have been shown to extend lifespan in model organisms such as Drosophila suggesting a positive relationship between cytoplasmic NADPH levels and longevity. Mitochondrial NADPH plays an important role in the protection against redox stress and cell death and mitochondrial NADPH-utilizing thioredoxin reductase 2 levels correlate with species longevity in cells from rodents and primates. Mitochondrial NADPH shuttles allow for some NADPH flux between the cytoplasm and mitochondria. Since a decline of nicotinamide adenine dinucleotide (NAD + ) is linked with aging and because NADP + is exclusively synthesized from NAD + by cytoplasmic and mitochondrial NAD + kinases, a decline in the cytoplasmic or mitochondrial NADPH pool may also contribute to the aging process. Therefore pro-longevity therapies should aim to maintain the levels of both NAD + and NADPH in aging tissues.

In meiner Dissertation klärte ich wesentliche Fragen zu 7-Transmembrandomänen-unabhängigen Funktionen von Adhäsions-G-protein-gekoppelten Rezeptoren auf. Diese einzigartigen Zelloberflächenmoleküle sind essentiell für die Physiologie des Menschen, jedoch auf molekularer Ebene erst in Grundzügen verstanden. Unter anderem haben sie als G-protein-gekoppelte Rezeptoren die Fähigkeit, Signale aus der zellulären Umgebung mittels ihrer 7-Transmembrandomäne in die Zelle weiterzuleiten. Jedoch sind sie auch im Stande unabhängig von ihrer 7-Transmembrandomäne agieren. In meiner Arbeit zeigte ich wie solche Funktionen in vivo implementiert werden können und präsentierte weiterhin, dass die Rezeptoren in diesem Kontext entgegen ihrer 'Natur' im Stande sind, Signale auf benachbarten Zellen auszulösen. Meine Arbeit bildet somit die Grundlage zur weiteren Erforschung der komplexen Signalmechanismen von Adhäsions-GPCRs.

<p>A C. elegans model of nicotine use and aversion resistance following chronic low-dose nicotine pretreatment. Model was then applied to various receptor knockouts  (acr-5, acr-15, acr-16, dop-1, and dop-2) to determine the role of these receptors in aversion resistance. </p>

Development of a single-cell embryo into an adult multi-cellular organism features the establishment of upto three anatomical body axes - anteroposterior, dorsoventral and left-right. It has been observed in many organisms that these body axes can consistently orient relative with respect to the geometric features of the embryo in many organisms. One such example is observed in the model organism Caenorhabditis elegans (C. elegans), where the Anteroposterior (AP) axis coincides with the geometric long axis of the ellipsoidal embryo -- the shape being imposed by the surrounding eggshell. In C. elegans, the Anteroposterior axis is established at the one-cell stage via its polarization by PAR polarity proteins. This cell polarization proceeds via a self-organized mechanochemical feedback between the PAR proteins and mechanical flows in the actomyosin cortex, resulting in the formation of two mutually exclusive domains of Anterior PAR and Posterior PAR proteins on the cortex denoting the future anterior and posterior end of the embryo -- and thus establishing the Anteroposterior axis. The initial orientation of the Anteroposterior axis is determined by the site of sperm entry at fertilization. However, the nascent Anteroposterior axis that forms after fertilization is observed to actively re-orient -- indicated by the movement of the PAR domains and concurrent migration (here termed posteriorisation) of the sperm-donated male pronucleus -- such that it aligns with the long axis of the ellipsoidal embryo, if it is not already aligned. In effect, the site of sperm entry only determines which half of the embryo becomes the posterior half of the embryo. This phenomenon of active re-orientation of the Anteroposterior axis, that ensures that the Anteroposterior axis aligns with the long axis of the embryo, is termed Anteroposterior axis alignment. The work described in this thesis investigates the mechanism of this Anteroposterior axis alignment in the C. elegans embryo. Anterior-directed flows in the actomyosin cortex observed during Anteroposterior axis establishment have also been found to be essential for Anteroposterior axis alignment. In this thesis, two possible mechanisms of Anteroposterior axis alignment are considered, both of which are consequences of these cortical flows. Cortical flows at the embryo surface can drive flows in the bulk cytoplasm in the embryo, generating cytoplasmic flows which point towards the sperm-donated male pronucleus as it posteriorises. Previous studies have proposed that these cytoplasmic flows could push onto the male pronucleus, and due to the ellipsoidal geometry of the embryo, drive it towards the closest tip of the embryo. This proposed mechanism is referred to as the cytoplasmic flow-dependent mechanism in this thesis. Another mechanism proposed in this thesis postulates that the reorientation of the Anteroposterior axis occurs via the repositioning of the pseudocleavage furrow. The pseudocleavage furrow is a contractile ring-like structure that forms at the boundary of the two PAR domains during Anteroposterior axis establishment. The pseudocleavage furrow forms as a result of compressive alignment of actin filaments in the actomyosin cortex due to cortical flows. In cases where the Anteroposterior axis is not aligned with the long axis of the embryo, the pseudocleavage furrow is not perpendicular to the long axis of the embryo. In such cases, active anisotropic stresses generated in the actomyosin cortex could force the rotation of the pseudocleavage furrow akin to an elastic rubber-band on an ellipsoid, and cause the Anteroposterior axis to re-orient towards the long axis of the embryo. This proposed mechanism is referred to as the pseudocleavage furrow-dependent mechanism in this thesis. This thesis investigates the role played by the two mechanisms in Anteroposterior axis alignment. This is accomplished in the following way: a theoretical model of the Anteroposterior axis alignment is introduced, consisting of a description of the actomyosin cortex as an active nematic fluid present on the 2D surface of a fixed ellipsoid representing the embryo. This description of the cortex incorporates both the cytoplasmic flow-dependent mechanism and the pseudocleavage furrow-dependent mechanism. RNAi experiments in the C. elegans embryo that remove the pseudocleavage furrow, in conjuction with numerical simulations using the theoretical model, show that the pseudocleavage furrow-dependent mechanism is the predominant mechanism that drives Anteroposterior axis alignment, while cytoplasmic flow-dependent mechanism plays only a minor role. RNAi experiments that modify the geometry of the C. elegans embryo -- specifically, generate rounder embyros -- show that embryo geometry can influence the rate of re-orientation of the Anteroposterior axis during Anteroposterior axis alignment -- with slower Anteroposterior axis alignment in rounder embryos. Such an relation between embryo geometry and Anteroposterior axis alignment is found to be consistent with pseudocleavage furrow-dependent mechanism, both via predictions made using the theoretical model and using a simplified effective model of a contractile ring (or elastic rubber-band) on a fixed ellipsoid. Altogether, the work presented in this thesis shows Anteroposterior axis alignment observed in the C. elegans embryo is driven primarily by the anisotropic stresses in the actomyosin cortex that generate the pseudocleavage furrow. The work here also shows that the Anteroposterior axis alignment process is sensitive to the geometry of the embryo. In effect, active mechanical flows in the actomyosin cortex translate the ellipsoidal geometry of the embryo into a robust orientation of the Anteroposterior axis of the C. elegans embryo. Mechanical flows such as these are not exclusive to C. elegans, nor are specific orientations of the body axes with respect to the embryo geometry. The results in this thesis thus point towards a possibly general role of the interactions between mechanical flows and embryo geometry to properly orient the body axes of the developing embryos of many multi-cellular organisms.:Contents Abbreviations iii Abstract iv 1 Introduction 1 1.1 Cytoskeleton 3 1.1.1 Main constituents of the cytoskeleton 3 1.1.2 Actomyosin cortex 7 1.2 Hydrodynamic theory of active fluids 8 1.2.1 Conservation Laws 9 1.2.2 Continuously broken symmetries 11 1.2.3 Irreversible thermodynamics of active fluids 13 1.2.4 Constitutive equations of active nematic fluids 19 1.3 C. elegans as a model organism 21 1.3.1 Early embryogenesis in C. elegans 22 1.4 AP axis establishment in C. elegans 24 1.4.1 PAR polarity system . 24 1.4.2 Mechanism of AP axis establishment 26 1.4.3 AP axis alignment 27 1.5 Overview 29 2 A theoretical model for AP axis alignment 30 2.1 A model of AP axis establishment in C. elegans 30 2.1.1 Turing-like system for PAR polarity system 31 2.1.2 Active isotropic description of actomyosin cortex 33 2.1.3 Guiding cues for AP axis establishment 34 2.1.4 Full model of AP axis establishment in [1] 35 2.2 A model of pseudocleavage furrow formation in C. elegans 36 2.2.1 Dynamics of Actin alignment 37 2.2.2 Active stress generated by alignment of actin filaments 38 2.3 A model of AP axis alignment in C. elegans 39 2.3.1 A thin film active nematic description of the cortex 40 2.3.2 Description of the Cytoplasm and Male pronucleus 46 2.3.3 Numerical simulations of the theoretical model 48 3 Materials and Methods 52 3.1 Culture conditions, strains and worm handling 52 3.2 Genetic perturbations by RNAi 53 3.3 Time-lapse microscopy 53 3.4 Image analysis 54 3.4.1 Pre-processing 54 3.4.2 Tracking posteriorisation of the male pronucleus 56 3.4.3 Measuring cortical flows 66 3.4.4 Measuring cytoplasmic flows 67 3.5 Data analysis 67 4 Experimental investigation of AP axis alignment 71 4.1 Characterising AP axis alignment in unperturbed embryos 71 4.2 Cortical flows are required for AP axis alignment 76 4.3 Role of Pseudocleavage furrow in AP axis alignment 83 4.3.1 Removing Pseudocleavage furrow via RNAi 83 4.3.2 Comparing numerical simulations to experimental results 88 4.4 Role of embryo geometry in AP axis alignment 99 4.4.1 Rounder embryos show slower AP axis alignment 99 4.4.2 Relation between embryo geometry and AP axis alignment 108 4.5 Additional experiments 118 4.5.1 Exploring relation between embryo geometry and AP axis alignment in ima-3 RNAi embryos 118 4.5.2 Are pseudocleavage furrow-dependent and cytoplasmic flow-dependent mechanisms sufficient to explain AP axis alignment? 121 4.5.3 Role of microtubules in AP axis alignment 127 5 Conclusions and Outlook 134 Appendix 139 Bibliography 142 List of publications 156 Acknowledgements 157