The silica-based cell walls of diatoms are outstanding examples of nature’s capability to synthesize complex porous structures with genetically controlled patterns from the nanometer scale to the range of hundreds of micrometers. Formation of the cell wall building blocks (valves and girdle bands) occurs in membrane-bound compartments, termed silica deposition vesicles (SDVs), which are unique organelles in silica-forming protists. Isolation of the SDVs has not yet been achieved, which has severely hampered the efforts to understand the mechanisms of biological silica morphogenesis. The present thesis aimed to address this shortcoming. The foundation was the development of an improved cell cycle synchronization and a fluorescence labeling method for the model diatom Thalassiosira pseudonana that enabled rapid identification of valve SDVs in a cell lysate. Correlative fluorescence and electron microscopy allowed visualizing the development of valve silica with unprecedented spatio-temporal resolution. Elemental analysis and demineralization of immature valves provided the first direct chemical evidence that silica morphogenesis is an interplay of inorganic and organic molecules inside the valve SDVs. Cryo TEM imaging of valve SDVs indicated the formation of organic patterns that precede silica depostion. From these observations, an organic biomolecule dependent, liquid-liquid phase separation based model for pore formation in the diatom T. pseudonana was proposed. The second part of this thesis was focused on the enrichment of valve SDVs from T. pseudonana and the subsequent proteomics based identification of more than 40 potential valve SDV proteins. Among these, three diatom-specific proteins contained conserved protein protein interaction domains (ankyrin-repeats) and were surprisingly predicted to be located in the cytoplasm. The fluorescent tagging of the three proteins (termed dANK1-3) confirmed their association with the valve SDVs. When the respective dank genes were knocked out by CRISPR/Cas9, the valves displayed permanent anomalies in the quantity and the pattern of ~22 nm sized pores. Double knockout mutants lacking both dank1 and dank3 were almost completely devoid of pores. The analysis of valve morphogenesis in the single and double knockout mutants revealed phenotypic changes that were consistent with the liquid-liquid phase separation based model for pore pattern formation in diatom biosilica. The work of this thesis has provided for the first time direct access to valve SDVs, which has opened entirely new possibilities for studying the composition, properties, and working mechanism of an organelle that forms a complex shaped mineral.

Pathological angiogenesis, or new blood vessel formation, is involved in many pathologies, including cancer and serious eye diseases. While traditional anti-angiogenic therapies target vascular endothelial growth factor receptors to reduce or inhibit new vessel formation, this approach has several downsides, including unpleasant side effects and low efficacy over time. Therefore, identifying new targets to treat pathological angiogenesis is still needed. CMG2, one of the two identified anthrax toxin receptors, has been proposed as an alternative target to treat pathological angiogenesis. CMG2’s role as a cell surface receptor that mediates anthrax toxin internalization is very well documented. One physiological function for CMG2, not related to anthrax intoxication, is suggested by the observation that loss-of-function mutations in CMG2 cause hyaline fibromatosis syndrome (HFS), a genetic disease that results in accumulations of extra-cellular matrix (ECM) protein in different parts of the body. While the complete molecular mechanism for CMG2’s role in regulating angiogenesis has not been determined, this dissertation addresses multiple ways CMG2 regulates pathological angiogenesis. We have discovered that CMG2 plays a role in mediating ECM homeostasis via endocytosis of ECM proteins and protein fragments as a way to generate angiogenic signals from the cell. We have also demonstrated that a fragment from Col IV, S16, is endocytosed into the cells by interacting with CMG2, and S16 treatment to endothelial cells leads to a significant reduction in cell migration. Also, an endothelial cell migration assay with CMG2 knockout cells results in abolished directional migration, indicating that CMG2 is required for endothelial cell chemotaxis. Notably, we have identified that bFGF, VEGF, and PDGF are involved in CMG2 mediated chemotaxis but not insulin and sphingosine-1-phosphate (S1P). While recent literature reports show that CMG2 works closely with RhoA GTPase, which is commonly known to regulate cell migration, we have also observed that inhibition of RhoA also reduced cell chemotaxis towards VEGF but not S1P. These results could be leveraged to develop new classes of therapeutic molecules to treat pathological angiogenesis induced by multiple various growth factors via targeting CMG2.

No description available.

Scaffolding proteins belonging to the Membrane Associated GUanylate Kinase (MAGUK) superfamily function as adaptors linking cytoplasmic and cell surface proteins to the cytoskeleton to regulate cell-cell adhesion, cell-cell communication and signal transduction. We have identified a novel Drosophila MAGUK member, Varicose (Vari), the homologue of vertebrate scaffolding protein PALS2. Similar to its vertebrate counterpart, Varicose localizes to pleated Septate Junctions (pSJs) of all embryonic, ectodermally derived epithelia and peripheral glia. In vari mutants, essential SJ proteins NeurexinIV and FasciclinIII are mislocalized basally and the cells develop a leaky paracellular seal. Localization of SJ protein Discs Large is not affected, indicating Vari is not involved in cell polarization. In addition, vari mutants display irregular tracheal tube diameters and have reduced lumenal protein accumulation suggesting involvement in tracheal morphogenesis. We found that Vari is distributed in the cytoplasm of optic lobe neuroepithelium and is required for proper ommatidial patterning. As well, Vari is expressed in a subset of neuroblasts and differentiated neurons of the nervous system. We also present a novel MAGUK function in wing hair alignment during adult morphogenesis. We conclude that Varicose is involved in scaffold assembly at the SJ and has a role in patterning adult epithelia and in nervous system development. / Thesis / Doctor of Philosophy (PhD)

The ability of tissues and cells to move and rearrange is central to a broad range of diverse biological processes such as tissue remodeling and rearrangement in embryogenesis, cell migration in wound healing, or cancer progression. These processes are linked to a solidlike to fluid-like transition, also known as unjamming transition, a not rigorously defined framework that describes switching between a stable, resting state and an active, moving state. Various mechanisms, that is, proliferation and motility, are critical drivers for the (un) jamming transition on the cellular scale. However, beyond the scope of these fundamental mechanisms of cells, a unifying understanding remains to be established. During embryogenesis, the proliferation rate of cells is high, and the number density is continuously increasing, which indicates number-density-driven jamming. In contrast, cells have to unjam in tissues that are already densely packed during tumor progression, pointing toward a shape-driven unjamming transition. Here, we review recent investigations of jamming transitions during embryogenesis and cancer progression and pursue the question of how they might be interlinked. We discuss the role of density and shape during the jamming transition and the different biological factors driving it.

Selon la tradition, Samuel de Champlain aurait retenu le site de Québec pour ses qualités défensives. Mais se pourrait-il qu'à défaut de comprendre Champlain, la tradition ait préféré questionner la nature au lieu de questionner le fondateur? Cette question constitue notre point de départ. Notre projet consiste à comprendre Québec à l'aune d'une morphogenèse traduisant une mobilisation des acteurs par les formes sensibles des paysages, préalablement investies de valeurs. La fondation de Québec aurait été le fait d'un investissement de représentations symboliques auxquelles le promontoire aurait fait écho. C'est que depuis la Renaissance, la localisation de certains acteurs serait influencée par une coïncidence entre des formes symboliques (idéaux, modèles...) et les images mentales que peuvent suggérer les formes des paysages. Dans ce processus, les artistes auraient la capacité de nous révéler ces coïncidences. Dans cette optique, quatre acteurs auraient lourdement infléchie la morphogenèse de Québec. Champlain, malgré ses prétentions, aurait finalement été contraint d'occuper une position attribuée, son «choix» pour Québec relevant notamment d'une coïncidence entre l'intuition qu'il pouvait avoir de la géopolitique amérindienne et des formes paysagères pouvant suggérer les discontinuités découlant de cette géopolitique. Dans le regard de Montmagny, le promontoire de Québec aurait fait écho à la posture que s'attribuait alors le sujet baroque. Le sujet aurait cherché à exprimer son unicité en s'associant avec les saillances du paysage. Mais au même moment, d'autres acteurs baroques — les communautés religieuses — auraient elles aussi été mobilisées par le promontoire de Québec. Une mimesis d'appropriation aurait été amplifiée par un paysage pouvant suggérer le rang social de ces acteurs. De cette rivalité devait émerger l'organisation axiale de Québec. Cette axialité a eu et a encore, une forte incidence sur le devenir de Québec. L'organisation du domaine bâti de Québec — des faubourgs enserrant un centre — aurait évoqué l'individualisme romantique, incitant les bourgeoisies à vouloir occuper le bourg fortifié. Modernes, elles voulaient détruire les murs de Québec. Or, Lord Dufferin les forçait à conserver ces derniers. La morphogenèse de Québec prenait une nouvelle tangente sous l'influence d'une confusion entre un idéal individualiste, l'organisation du paysage de Québec et une position centrale qui changeait de valeur. Les bourgeoisies se aisaient prendre dans un «piège paysager». Depuis lors, la morphogenèse de Québec ne ferait que s'inscrire à l'intérieur du lourd héritage de la «vieille capitale», au détriment de quartiers entiers. Aujourd'hui, nous pouvons constater que certains espaces, sous l'influence d'idéaux spécifiques, sont l'objets d'investissements de valeurs. Le bourg fortifié, dans la foulée de son classement, est l'objet d'une valorisation par des acteurs inscrits dans des trajectoires longues. Il est de ce fait de plus en plus un espace vide. Aussi, sous l'influence des discours environnementaux, le bassin de Québec serait en train d'émerger comme espace attractif, et ce, en concurrence avec les investissements récents dans le nouveau centre-ville. / According to tradition, the site for the construction of Québec was chosen by Samuel de Champlain for its defensive properties. Yet, while having no insight of the founder’s intentions, could it be that the above tradition preferred to investigate nature instead of investigating Champlain’s motives? This question constitutes the starting point of this research endeavor. It aims at understanding Québec from the very start of its morphogenesis resulting from a succession of actors influenced by the perceived landscape forms pre-invested with human values. Hence, the founding of Québec would have been the result of representation inputs of a symbolic nature to which echoed its promontary, considering that since Renaissance the location of certain actors could have been determined through the coincidence between symbolic forms ( ideals, models...) and the mental images as generated by the forms of the landscape. In this process, artists tend to unveil these coincidences. In that perspective, four actors have strongly inflected the morphogenesis of Québec. Champlain, notwithstanding his claims, would have been constrained to occupy a designated position, his «choice» for Québec resulting from a coincidence between his possible intuition of the ameridian geopolitics and the landscape forms suggesting the potential discontinuities created by this geopolitics. As seen by Montmagny, the Québec promontory evoqued the posture to which the Baroque individual pretended: his tendency to express his wholesomeness by his association with the stricking features of the landscape. But at the same time, other Baroque actors — the religious communities — invested the Québec promontory: an appropriative mimesis would have been amplified by a landscape able to suggest the social position of these actors. From this rivalry resulted the axial organisation of Québec which had and still has a strong incidence upon the spatial evolution of the agglomeration. The organization of the built form of Québec — faubourgs enclosing a centre — would have evoqued the romantic individualism, encouraging the (modern) bourgeoisies to occupy the fortified bourg; they wanted to destroy its walls, while Lord Dufferin insisted in conserving them. From then on, the morphogenesis of Québec took a new direction under the influence of a confusion between the individualistic ideal, the structured landscape of Québec and a central position undergoing a value change. Hence, the bourgeoisies were caught into a «landscape trap». Since then, the morphogenesis of Québec is still submitted to the constraining legacy of the «old Québec», creating prejudice to surrounding neighbourhoods. As a result, one can ascertain that today some portions of the agglomeration, under the influence of specific ideals, are subjected to new value investments. The fortified bourg, following its designation as a Heritage precinct (UNESCO), is subjected to a valorization process by actors set in long term trajectories; hence, it more and more becomes an “empty place”. Moreover, influenced by the environmental discourse, the walled City and its harbour seem to emerge as a space of attraction, this being in competition with the recent investments in the new CBD.

Transient Receptor Potential channels (TRP) are a superfamily of cationic specific ionchannels that are regulated by various stimuli such as temperature, pH, mechanical stress, ligandsand ion concentration. The role of TRP channels in disease states such as autosomal dominantpolycystic kidney disease, cancer metastasis, and developmental defects lend credence to thebelief that they play an important part in epithelial morphogenesis events. The development ofsomites, neural tube closure and migration of neural crest cells to form things such as the faceand heart is a good developmental model for the aforementioned cellular processes. We haveshown that TRP channels can be found in the developing ectoderm, hindbrain, and heart and thatthe inhibition of TRP channels in a developing embryo results in phenotypes suggestingperturbation of cellular remodeling processes. This leads to the question of the specific role ofTRP channels in the epithelial mesenchymal transition and remodeling in developing chickembryos.

No description available.

No description available.

During animal development numerous organs with functions ranging from fluid transport to signal propagation develop into highly branched shapes and forms. To ensure organ function, the formation of their geometrical and topological as well as size-dependent properties is crucial. For example, organ geometry serves to maximize exchange area with its surroundings and organ topology controls the response to fluctuations and damage. Most importantly, organ size and proportion need to scale throughout animal growth to meet the demands of increasing body size. However, how organ geometry and topology are established and scaled in a self-organized manner, remains poorly understood. In this thesis, we present a novel theoretical framework to study the self-organized growth and scaling of branched organs. In this framework, we represent the organ outline by an infinitely thin interface and consider morphogen-controlled interface evolution in growing domains. We demonstrate that an instability in interface motion can lead to the self-organized formation of complex branched morphologies and show how the interplay between interface motion, morphogen dynamics, and domain growth controls the geometrical, topological, and size-dependent properties of the resulting structures. To understand the formation of branched structures from instabilities in morphogen-controlled interface growth, we first consider a range of different interface growth scenarios in non-growing domains. In a first approach, we present a stochastic lattice model with interface growth driven by a morphogen concentration gradient. We find a range of branched morphologies extending from self-similar fractal structures to almost circular structures with only a few branches depending on the morphogen gradient length scale. We present the Euler characteristic as an example of a topological invariant and employ it to introduce topological constraints into interface growth, leading to the formation of tree-like structures. In a second approach, we study a continuum model for morphogen-controlled interface growth. In this model, the interface has a constant tendency to grow and is inhibited by morphogen concentration. Additionally, we take into account a curvature dependency of interface growth, which leads to an effective stabilization of interface motion at small length scales. We identify branch distance and thickness as key morphological properties and discuss their regulation. We relate branch distance regulation to the interplay of destabilization from morphogen inhibition and stabilization from the curvature dependency of interface growth and explain branch thickness regulation in terms of mutual branch inhibition. By considering interface instability in different scenarios, we overall demonstrate the robustness of our approach. Finally, we apply our theoretical framework to study the branching morphogenesis of the planarian gut. The planarian gut is a highly branched organ that spans the entire organism and is responsible for the delivery of nutrients to the planarian body. Planarians undergo massive body size changes of more than one order of magnitude in organism length and thus constitute an ideal model organism to study the growth and scaling of branched organs. We reconsider our continuum model and include novel features needed to account for the organization of the planarian gut. We take into account external guiding cues that alter the orientation of branches and, most importantly, consider branching morphogenesis in a growing domain. We demonstrate that our model can account for the geometrical and topological properties of the gut and show that gut scaling can arise from to the interplay of branch growth and organism growth. Overall, we present a novel theoretical framework to study the growth and scaling of branched organs. In this framework, we demonstrate the self-organized formation of branched morphologies from instabilities in morphogen-controlled interface growth and show how the interplay of interface motion, morphogen dynamics, and system size determine geometry, topology, and size-dependent properties of the resulting structures.