Coordinated polarity and differentiation of cells in the plane of a tissue layer are essential to the development of multicellular organisms. Arabidopsis thaliana root hairs and trichomes provide model systems to study the pathways that control planar polarity and cell fate specification in plants. A concentration gradient of the plant hormone auxin provides an instructive cue that coordinates polar assembly of signalling complexes at plasma membranes of root epidermal cells; however, knowledge about additional players and cytoskeletal effectors driving cell polarization prior to hair emergence remains limited. On the other hand, epidermal cell fate specification is controlled by a well-characterized gene network of transcription factors that translate positional signals and cell-to-cell communication into tissue-wide patterning. Yet, new components are continuously found to interact with the patterning pathway, shedding light on its connections with diverse developmental processes. This thesis presents the SABRE (SAB) gene as a novel player in planar polarity establishment and root epidermal patterning. SAB is a large protein with sequence similarity to proteins present in all eukaryotes and affects planar polarity as well as orientation of cell divisions and cortical microtubules. Genetic interaction with the microtubule-associated protein gene CLASP further supports involvement of SAB in microtubule arrangement, suggesting a role for this gene in cytoskeletal organisation. Strikingly, SAB also interacts genetically with ACTIN7 (ACT7), and both ACT7 and its modulator ACTIN INTERACTING PROTEIN 1-2 (AIP1-2) contribute to planar polarity of root hair positioning. Cell-file specific expression of AIP1-2 depends on the epidermal-patterning regulator WEREWOLF (WER), revealing a connection between actin organization, planar polarity and cell fate specification. Consistent with this finding, SAB also functions in patterning of the root epidermis by stabilizing cell fate acquisition upstream of the core patterning pathway. These results unveil new roles for SAB in planar polarity and epidermal patterning and suggest that organization of the microtubule and the actin cytoskeleton are important to both planar polarity establishment and cell fate specification. / Samordning av polaritet och differentiering av celler inom ett vävnadslager är avgörande för utvecklingen av multicellulära organismer. Rothår och bladhår hos Arabidopsis thaliana utgör modellsystem för att studera signalvägar som kontrollerar planpolaritet och specifikation av cellers öde hos växter. En koncentrationsgradient av växthormonet auxin ger en instruktiv signal som koordinerar polär hopsättning av signalkomplex vid plasmamembranet i rotepidermisceller; dock är kunskapen om ytterligare aktörer och hur cytoskelettets aktörer påverkar cellpolaritet innan rothår bildas begränsad. Vad gäller differentieringen av epidermala cellers öde kontrolleras dessa genom ett väl karakteriserat nätverk av transkriptionsfaktorer som överför positionssignaler och cell-till-cell kommunikation till vävnadsomfattande mönsterbildning. Fortfarande hittas dock nya komponenter som interagerar med signalvägarna för mönsterbildning, vilket ger nya insikter om dess förbindelser med diverse utvecklingsprocesser. Denna avhandling presenterar genen SABRE (SAB) som en ny aktör i etableringen av planpolaritet och mönsterbildning av rotepidermis. SAB är ett stort protein som har sekvenslikhet med proteiner som finns i alla eukaryoter och det påverkar planpolaritet, orientering av celldelning och kortikala mikrotubler. Genetisk interaktion med genen för det mikrotubuli-associerade proteinet CLASP stärker ytterligare inblandningen av SAB i organiserandet av mikrotubler och antyder att denna gen har en roll i organiserandet av cytoskelettet. Slående är att SAB även interagerar genetiskt med ACTIN7 (ACT7) och att både ACT7 och dess modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2) bidrar till planpolaritet vid positionering av rothår. Cellfils-specifikt uttryck av AIP1-2 beror på den epidermala mönsterbildande genen WEREWOLF (WER), vilket påvisar ett samband mellan organisationen av aktin, planpolaritet och specifikationen av cellers öde. SAB fungerar även i mönsterbildning av rotens epidermis och stabiliserar förvärvet av cellöde uppströms av den centrala signalvägen för mönsterbildning. Dessa resultat visar på nya roller för SAB i planpolaritet och mönsterbildning av epidermis och indikerar att organiseringen av mikrotubler och aktin-cytoskelettet är viktiga både för etablerandet av planpolaritet och för specificeringen av cellers öde.

Planar cell polarity (PCP) refers to the coordinated orientation, movement, or structure of cells within the plane of a tissue. Zebrafish PCP mutants such as the vangl2 mutant exhibit defects in convergent extension, neural tube morphogenesis, and ciliary positioning. Tmem14a is a putative tetraspanin protein that was identified as an potential interactor of Vangl2 in a membrane yeast-two hybrid screen. GFP-tagged versions of Tmem14a are localized to the trans-Golgi network in zebrafish neuroepithelial cells. Knockdown of Tmem14a activity results in convergent extension defects, an ectopic accumulation of cells in the neural tube, and disorganized cilia. The localization of GFP-tagged Tmem14a to the trans-Golgi network suggested that Tmem14a plays a role in the trafficking of core PCP components to the cell membrane. Indeed, the membrane localization of GFP-Vangl2 was disrupted in Tmem14a morphants. Thus, Tmem14a is an interactor of Vangl2 and a novel regulator of vertebrate planar cell polarity signaling.

Planar cell polarity (PCP) refers to the coordinated orientation, movement, or structure of cells within the plane of a tissue. Zebrafish PCP mutants such as the vangl2 mutant exhibit defects in convergent extension, neural tube morphogenesis, and ciliary positioning. Tmem14a is a putative tetraspanin protein that was identified as an potential interactor of Vangl2 in a membrane yeast-two hybrid screen. GFP-tagged versions of Tmem14a are localized to the trans-Golgi network in zebrafish neuroepithelial cells. Knockdown of Tmem14a activity results in convergent extension defects, an ectopic accumulation of cells in the neural tube, and disorganized cilia. The localization of GFP-tagged Tmem14a to the trans-Golgi network suggested that Tmem14a plays a role in the trafficking of core PCP components to the cell membrane. Indeed, the membrane localization of GFP-Vangl2 was disrupted in Tmem14a morphants. Thus, Tmem14a is an interactor of Vangl2 and a novel regulator of vertebrate planar cell polarity signaling.

The neural cell adhesion molecule L1 is a member of the immunoglobulin superfamily and it mediates many adhesive interactions during brain development. Mutations in the L1 gene are associated with a spectrum of X-linked neurological disorders known as CRASH or L1 syndrome. The objective of this thesis was to use the zebrafish model to investigate the molecular mechanisms of L1 functions and the pathological effects of its mutations. Zebrafish has two L1 homologs, L1.1 and L1.2. Inhibition of L1.1 expression by antisense morpholino oligonucleotides resulted in phenotypes that showed resemblances to L1 patients. However, knockdown of L1.2 expression did not result in notable neural defects. Furthermore, analysis of the expression pattern of L1.1 has led to the discovery of a novel soluble L1.1 isoform, L1.1s. L1.1s is an alternatively spliced form of L1.1, consisting of the first four Ig-like domains and thus a soluble secreted protein. L1.1 morphants exhibited disorganized brain structures with many having an enlarged fourth/hindbrain ventricle. Further characterization revealed aberrations in ventricular polarity, cell patterning and proliferation and helped differentiate the functions of L1.1 and L1.1s. While L1.1 plays a pivotal role in axonal outgrowth and guidance, L1.1s is crucial to brain ventricle formation. Significantly, L1.1s mRNA rescued many anomalies in the morphant brain, but not the trunk phenotypes. Receptor analysis confirmed that L1.1 undergoes heterophilic interactions with neuropilin-1a (Nrp1a). Peptide inhibition studies demonstrated further the involvement of L1.1s in neuroepithelial cell migration during ventricle formation. In the spinal cord, spinal primary motoneurons expressed exclusively the full-length L1.1, and abnormalities in axonal projections of morphants could be rescued only by L1.1 mRNA. Further studies showed that a novel interaction between the Ig3 domain of L1.1 and Unplugged, the zebrafish muscle specific kinase (MuSK), is crucial to motor axonal growth. Together, these results demonstrate that the different parts of L1.1 contribute to the diverse functions of L1.1 in neural development.

The seven-pass transmembrane atypical cadherin, Flamingo (also known as Starry Night) is evolutionally conserved in both structure and function in vertebrates and invertebrates. It plays important roles during the establishment of planar cell polarity (PCP) of epithelial tissues and during the development of axons and dendrites in both peripheral and central neurons. / This thesis looks at the role of Flamingo/Starry Night in axon growth and guidance in the embryonic abdominal peripheral nervous system (PNS) of Drosophila. It describes the expression pattern of Flamingo in the PNS and its environment. A combination of single cell labelling and immunohistochemical techniques was used to define the effect of mutations in flamingo as well as several genes coding for potential Flamingo interaction partners. Rescue- and over-/mis-expression experiments featuring targeted expression of either a wild type version or mutant versions of flamingo provide information on the cellular and molecular mechanisms by which Flamingo regulates sensory axon development. Loss of Flamingo function results in a highly penetrant axon stall phenotype. Both sensory and motor axons frequently halt their advance early along their normal trajectories. Flamingo appears to mediate an axon growth promoting signal upon contact of sensory growth cones with specific early intermediate targets. Expression of Flamingo in sensory neurons is sufficient to rescue the mutant sensory axon phenotype. This rescue is at least partially independent of most of the extracellular region of the Flamingo protein. While Flamingo was previously found to have homophilic adhesion properties in vitro and appears to function by a homophilic mechanism during the neurite development of several types of neurons, this study supports a heterophilic signalling mechanism by which Flamingo fulfils its role in abdominal sensory axon growth promotion.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (M.Communication Pathology)--Universiteit van Pretoria, 2002. / Summary in English and Afrikaans. Includes bibliographical references (leaves 142-159).

Usant de certaines conditions, la croissance épitaxiale de GaN sur un large panel de substrats donne lieu à une assemblée de nanofils. Cette géométrie filaire peut permettre la croissance d'hétérostructures libres de tous défauts cristallins étendus, ce qui les rendent attractives pour créer des dispositifs de hautes performances. En premier lieu, mon travail de thèse a visé à clarifier le mécanisme de nucléation auto-organisé des nanofils de GaN sur substrat de silicium. Dans ce but, une étude approfondie de la couche tampon d'AlN, déposée préalablement à la nucléation des nanofils, a été réalisée, mettant en évidence une inattendue forte réactivité de l'Al avec le substrat. La nécessité de la polarité azote pour la croissance des nanofils de GaN a été mise en lumière, bien que des nanofils contenant dans leur cœur un domaine de polarité Ga ont également été observés. Dans ces nanofils, une paroi d'inversion de domaine est présente et a été démontrée être optiquement active, exhibant une photoluminescence à 3.45 eV. Ensuite des hétérostuctures filaires GaN/AlN ont été synthétisée pour des caractérisations structurales et optiques. Il a été montré que le mode de croissance de l'hétérostructure peut être changé en fonction du diamètre du nanofil. En dernier lieu, en prenant avantage de la géométrie cylindrique des nanofils, des mesures de diffusion de porteurs de charge ont été réalisées dans des nanofils de GaN et d'AlN. / Using specific conditions, GaN can be epitaxially grown on a large variety of substrates as a nanowire (NW) array. This geometry allows the subsequent growth of wire-like heterostructures likely free of extended defects, which makes them promising for increasing device controllability and performance. First, my PhD work has been devoted to the understanding of self-organized nucleation of GaN NWs on silicon substrates. For this purpose, a deep characterization of the growth mechanism of the AlN buffer deposited prior to NW nucleation has been done, emphasizing an unexpected large reactivity of Al with the substrate. The requirement of the N polarity to nucleate GaN NWs has been evidenced, although the possible existence of NWs hosting a Ga polar core has been observed as well. In these NWs, an inversion domain boundary is present and has been demonstrated to be optically active, having a photoluminescence signature at 3.45 eV. Next, GaN/AlN wire heterostructures have been grown for structural and optical characterization. It has been shown that by changing the wire diameter, different growth mode for the heterostructure could be reached.At last, thanks to the cylindrical geometry of NWs, the measurement of diffusion length for charge carriers in GaN and AlN NWs have been performed.

O problema de fatorar e decompor funções Booleanas é Σ-completo2 para funções gerais. Algoritmos eficientes e exatos podem ser criados para classes de funções existentes como funções read-once, disjoint-support decomposable e read-polarity-once. Uma forma fatorada é chamada de read-once (RO) se cada variável aparece uma única vez. Uma função Booleana é RO se existe uma forma fatorada RO que a representa. Por exemplo, a função representada por =12+134+135 é uma função RO, pois pode ser fatorada em =1(2+3(4+5)). Uma função Booleana f(X) pode ser decomposta usando funções mais simples g e h de forma que ()=ℎ((1),2) sendo X1, X2 ≠ ∅, e X1 ∪ X2 = X. Uma decomposição disjunta de suporte (disjoint-support decomposition – DSD) é um caso especial de decomposição funcional, onde o conjunto de entradas X1 e X2 não compartilham elementos, i.e., X1 ∩ X2 = ∅. Por exemplo, a função =12̅̅̅3+123̅̅̅ 4̅̅̅+12̅̅̅4 é DSD, pois existe uma decomposição tal que =1(2⊕(3+4)). Uma forma read-polarity-once (RPO) é uma forma fatorada onde cada polaridade (positiva ou negativa) de uma variável aparece no máximo uma vez. Uma função Booleana é RPO se existe uma forma fatorada RPO que a representa. Por exemplo, a função =1̅̅̅24+13+23 é RPO, pois pode ser fatorada em =(1̅̅̅4+3)(1+2). Esta tese apresenta quarto novos algoritmos para síntese de funções Booleanas. A primeira contribuição é um método de síntese para funções read-once baseado em uma estratégia de divisão-e-conquista. A segunda contribuição é um algoritmo top-down para síntese de funções DSD baseado em soma-de-produtos, produto-de-somas e soma-exclusiva-de-produtos. A terceira contribuição é um método bottom-up para síntese de funções DSD baseado em diferença Booleana e cofatores. A última contribuição é um novo método para síntese de funções RPO que é baseado na análise de transições positivas e negativas. / The problem of factoring and decomposing Boolean functions is Σ-complete2 for general functions. Efficient and exact algorithms can be created for an existing class of functions known as read-once, disjoint-support decomposable and read-polarity-once functions. A factored form is called read-once (RO) if each variable appears only once. A Boolean function is RO if it can be represented by an RO form. For example, the function represented by =12+134+135 is a RO function, since it can be factored into =1(2+3(4+5)). A Boolean function f(X) can be decomposed using simpler subfunctions g and h, such that ()=ℎ((1),2) being X1, X2 ≠ ∅, and X1 ∪ X2 = X. A disjoint-support decomposition (DSD) is a special case of functional decomposition, where the input sets X1 and X2 do not share any element, i.e., X1 ∩ X2 = ∅. Roughly speaking, DSD functions can be represented by a read-once expression where the exclusive-or operator (⊕) can also be used as base operation. For example, =1(2⊕(4+5)). A read-polarity-once (RPO) form is a factored form where each polarity (positive or negative) of a variable appears at most once. A Boolean function is RPO if it can be represented by an RPO factored form. For example the function =1̅̅̅24+13+23 is RPO, since it can factored into =(1̅̅̅4+3)(1+2). This dissertation presents four new algorithms for synthesis of Boolean functions. The first contribution is a synthesis method for read-once functions based on a divide-and-conquer strategy. The second and third contributions are two algorithms for synthesis of DSD functions: a top-down approach that checks if there is an OR, AND or XOR decomposition based on sum-of-products, product-of-sums and exclusive-sum-of-products inputs, respectively; and a method that runs in a bottom-up fashion and is based on Boolean difference and cofactor analysis. The last contribution is a new method to synthesize RPO functions which is based on the analysis of positive and negative transition sets. Results show the efficacy and efficiency of the four proposed methods.

Au cours de son développement depuis la cellule unique jusqu’à la forme adulte, l’embryon passe par de nombreuses étapes de morphogenèse. L'harmonie entre les cellules au cours de ces processus est assurée par l’intégration spatiale des signaux externes qui assurent la cohérence des polarités internes et externes des cellules. Ce travail de thèse se concentre sur la façon dont les cellules intègrent les informations spatiales dans la définition de leur polarité au cours de grandes transformations morphologiques comme la transition épithélium-mésenchyme et la dissémination des cellules tumorales. Nous avons utilisé la position du centrosome comme un indicateur de la polarité cellulaire interne en raison de son rôle actif dans l'organisation des microtubules et donc dans l'orientation du transport intra-cellulaire. La polarité corticale a été inférée à partir de la répartition spatiale des adhérences cellule-cellule (ACC) et cellule-matrice (ACM).Dans la première partie, nous avons étudié l'effet de l'amplification du nombre de centrosomes, une caractéristique fréquente dans les cellules tumorales, sur l’adhérence inter-cellulaire. L'amplification des centrosomes dans les cellules de la glande mammaire a conduit à la rupture des adhérences inter-cellulaires ainsi qu’à la genèse de protubérances cellulaire invasive. Cependant le matériel centrosomal étant plus développé, de nombreux microtubules supplémentaires émanait de ces clusters de centrosomes surnuméraires. L'utilisation de modèles cellulaires in vitro et de conditions de culture contrôlées ont révélées que la simple amplification des centrosomes est suffisante pour moduler le destin de cellules transformées et les rendre invasives. Cette étude a révélé que les mécanismes régissant l’orientation la polarité interne des cellules sont liés à l’arrangement spatial de la polarité corticale et que la diaphonie entre les deux perturbe la physiologie du tissu au point d’induire la formation de métastases tumorales.La deuxième partie de l'étude a porté sur l'exploration de la transition épithélium-mésenchyme (EMT). Nous avons étudié le rôle potentiel des mécanismes de régulation de la polarité pour diriger la précision des mouvements cellulaire au cours de l’EMT. Le remodelage des adhérences inter-cellulaires jouant un rôle central au cours de l’EMT, nous avons supposé qu'il était couplé à des changements de polarité interne. Nous avons suivi le positionnement du centrosome dans les cellules épithéliales et dans les cellules dans lesquelles l’EMT était induite par stimulation au TGFb. La libération des cellules mésenchymateuses de leur confinement nous a montré que la séparation des cellules après l’EMT était dépendante de l’inversion de polarité interne dans ces cellules. Ces résultats suggèrent que la dispersion des cellules observée pendant la formation du mésoderme au cours de la gastrulation impliquent un renversement actif et finement contrôlée du couplage entre l’axe de polarité interne et l’asymétrie des deux types d’adhérences cellulaires.Suite à l’étude de ces deux projets impliquant des dispersions cellulaires, nous avons développé un dispositif pour permettre le criblage de médicaments contre les dérèglements cellulaires impliqués dans la formation des métastases. Nous avons à nouveau utilisé un modèle simplifié de paires de cellules sur des micropattern pour détecter la capacité de dispersion des cellules suite à des stimulations externes comme celle induisant l’EMT. Le test, qui permet de mesurer le degré de séparation des cellules à l’aide d’une seule image, a été validé sur quatre lignées de cellules épithéliales différentes. Le dispositif final a été adapté à un format de plaque 96 puits en collaboration avec l’entreprise Cytoo afin de permettre des criblages à haut contenu. Ce kit a ensuite été validé en testant des médicaments connus contre l’EMT. / Development from single cell embryo to multicellular adult form of organism involves tremendous morphogenesis. The well defined and highly controlled morphonogenetic processes are crucial at every stage of development including gastrulation, organogensis, wound healing and tissue maintenance. The necessary harmony between cells for these processes is achieved by integration of internal and external polarity cues. This thesis work is focused on understanding how cells integrate polarity cues to drive morphogenetic event such as of Epithelial to mesenchymal transition (EMT) and cancer metastasis. We used centrosome position as an indicator of internal cell polarity due to its active role in organization of microtubules and orientation of internal traffic of endocytosed and secreted proteins; while cortical polarity was inferred by polarized distribution of cell-cell adhesions (CCA) and cell-matrix adhesions (CMA). In the first part, we studied effect of centrosome amplification, which is very common in human cancer; on CCA. Inducible centrosome amplification in mammary gland cells led to destabilization of CCA alongwith generation of invasive cell protrusions. Using a minimal model of tissue; confined on micropatterns, we demonstrated that cells with amplified centrosome correctly oriented their internal polarity axis like normal cells although increased centrosomal protein and peri-centriolar material emanated higher centrosomal microtubules. Use of in vitro models of cell lines and controlled culture conditions revealed that mere amplification of centrosome was sufficient to drive cell fate for cancer-like events in the absence of any additional external growth signals capable of affecting cortical polarity. This study revealed that internal polarity cues interact with cortical polarity signals and the crosstalk between the two governs the physiological state of the cell during transformation events like cancer metastasis. The second part of the study focused on exploring how internal polarity during EMT is modulated to drive precise spatial movements during development. Cell adhesion remodelling being central to EMT, we hypothesized that it was coupled to internal polarity changes. We monitored centrosome position in epithelial and in cells induced for EMT by TGFb and found that nucleus-centrosome axis was reversed. This phenomenon of polarity reversal strongly suggested that internal polarity cues and positioning of organelles is coupled to signals that polarize CCA and CMA distribution. A shift in the force balance between CCA and CMA was observed upon EMT and suggested that CMA forces dominated in mesenchymal cells and release of cells from confinement clearly revealed that ability of cell separation was dependent upon their internal polarity. These results demonstrated that scattering events observed during mesoderm formation during gastrulation or metastasis events in cancer involve active and tightly controlled reversal of internal polarity axis coupled to cortical polarity of cells. From the understanding of above two projects involving cancer-like scattering phenomenon, we developed a product to allow robust drug screening against cancer drugs. We once again used simplified two-cell model on micropattern geometries to develop an assay to detect scattering ability of cells after events like EMT. The assay was validated by EMT transformation of 4 different epithelial cells lines and detection of their scattering ability by single time point picture assay. We used internuclear distance between the cell-pair as the main parameter for scoring the scattering index of cells with possibility of automated image processing. The final product was manufactured in 96-well plate format by industrial collaborator Cytoo for high content screening. Preliminary validation using drugs against EMT constituted proof of principle for the product.