The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.

L'équilibre arbre-herbe dans la savane tropicale est reconnu comme l'une des principales<p>énigmes de l'écologie des plantes. Les origines du difficile équilibre entre ces formes de vie<p>sur des dizaines de millions de kilomètres carrés à l'échelle mondiale sont en partie attribuées<p>aux perturbations fréquentes induites par les feux de végétation dont les effets varient dans<p>l'espace et dans le temps selon les conditions environnementales. Les résultats de recherche<p>dépendent de l’échelle et les conclusions tirées d’études locales sont rarement transposables à<p>d’autres échelles. La question du transfert d'échelle s’avère donc cruciale dans l'étude des<p>effets du feu, et nécessite une approche transdisciplinaire.<p>En raison de la variété des échelles couvertes, cette étude constitue une première dans la<p>confrontation de données sur l’historique des feux dérivé de l'imagerie satellitaire à des<p>données de terrain incluant des mesures détaillées sur la structure et la composition de<p>végétation, ainsi que des propriétés édaphiques et topographiques. Elle s’est focalisée sur la<p>composante ligneuse de par son caractère pérenne et son influence sur les processus<p>écologiques majeurs. Sur une zone de plus de 2000 km², le Parc National du W du Niger<p>(PNWN), où le feu est utilisé comme outil pour la gestion et la conservation des écosystèmes<p>semi-arides, une carte de l'historique des feux a été élaborée à partir d'images MODIS de 250<p>m de résolution spatiale et de résolution temporelle journalière couvrant une période de sept<p>années (2002-2009). Pour comprendre la variabilité, à la fois dans l'espace et le temps, de la<p>propagation du feu dans la végétation, nous avons étudié les caractéristiques de distribution<p>des feux en termes de régime du feu (i.e. période d'occurrence et fréquence) et de structure<p>spatiale (métriques paysagères). Les relations causales plausibles entre les régimes du feu, les<p>conditions édaphiques et topographiques à l'échelle régionale comme locale, et les<p>caractéristiques de la végétation ligneuse (composition et structure) ont été examinées à<p>travers des analyses multivariées et des modèles d'équations structurales. Nous avons aussi<p>examiné plus en détails les stratégies adaptatives mises en oeuvre par les ligneux, et les<p>interactions biologiques qui sous-tendent l'organisation spatiale des ligneux à travers une<p>approche des processus ponctuels.<p>Les résultats montrent que l'activité du feu dans le PNWN se caractérise par une hétérogénéité<p>spatio-temporelle induite principalement par les conditions édapho-topographiques via la<p>structure de la végétation ligneuse. Les feux précoces de gestion créent des pare-feux<p>efficaces, limitant une large extension des feux de saison tardifs. Cependant, ces feux tardifs<p>pourraient ne pas être aussi destructifs comme qu’on le suppose généralement. En effet,<p>l'adaptation des espèces aux différents régimes defeu correspond à des stratégies de croissance<p>contrastées. Dans le cas des feux tardifs, les surfaces terrières et hauteurs moyennes les plus<p>fortes sont rencontrées, permettant aux arbres de résister au feu. Quant aux zones non<p>affectées par les feux l'analyse "patron-processus" désigne clairement la facilitation entre<p>ligneux comme un processus fondamental de l'organisation spatiale périodique du couvert, une<p>structure émergente qui empêche le passage du feu. Bien qu’ils ne se substituent pas aux<p>études expérimentales, ces résultats basés sur une expérimentation naturelle à large échelle<p>apportent des informations nouvelles précieuses tant au niveau fondamental que pour la mise<p>en place d'une gestion raisonnée du PNWN.<p><p><p>The tree-grass equilibrium in tropical savanna is recognized as one of plant ecology's main<p>conundrums. The origins of the difficult balance between these life forms over tens of millions<p>of square kilometers worldwide are in part attributed to the frequent disturbances caused by<p>vegetation fires effects of which vary in space and time depending on local environmental<p>factors. Research results are scale-dependent and findings from local studies are rarely<p>transposable to higher levels of ecosystem organization. The question of scaling (scale<p>transfer) is therefore crucial in the study of fire effects, and requires a multidisciplinary<p>approach.<p>Because of the variety of scales covered, this study is a premiere in the confrontation of<p>satellite-imagery derived fire history data with detailed field data including measurements of<p>vegetation parameters (structure and composition), as well as soil and topographic properties.<p>The study focuses on the woody component, because of its perennial character and its<p>influence on major ecological processes. On an area of more than 2000 km², the W National<p>Park of Niger (WNPN) where fire is used as a tool for the management and conservation of<p>semi-arid ecosystems, a fire history map was elaborated from MODIS images with a 250 m<p>spatial resolution and a daily temporal resolution over a period of seven years (2002-2009). To<p>understand the variability, both in space and time, of fire propagation in vegetation, we studied<p>the fire distribution characteristics in terms of fire regime (i.e. timing and frequency) and<p>spatial structure (landscape metrics). Plausible causal relationships at regional and local scales<p>between fire regimes, edaphic and topographic conditions, and the woody vegetation<p>(composition and structure) characteristics were examined through multivariate analyses and<p>structural equations models. We also examined in detail the woody species adaptive strategies<p>as well biological interactions, which underlie their spatial organization, using point statistics.<p>Results show that the WNPN fire's activity is characterized by spatial and temporal<p>heterogeneity induced mainly by edaphic and topographic conditions via the structure of the<p>ligneous component. Prescribed early season fires create effective firewalls, limiting wide late<p>season fires. However, these late fires might not be as destructive as is commonly assumed.<p>Indeed, species adaptation to different fire regimes corresponds to contrasting growth<p>strategies. In the case of late fires, increased basal areas and mean tree heights were<p>encountered, enabling trees to resist fire and escape flames. As for the unburned areas, the<p>"pattern-process" analysis clearly indicates that facilitation between shrubs is a fundamental<p>process determining the woody cover periodic spatial organization, an emergent structure that<p>prevents fire spread.<p>Although they do not replace experimental studies, these results based on a large-scale natural<p>experiment provide valuable new information both on a fundamental level and for setting up<p>the rational management of the WNPN. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

「内発的地域イノベーション・エコシステム」とは、地域課題の革新的な解決を可能にする多様なプレイヤーによる機能的ネットワークであり、相互作用と共進化により持続する自律的システムである。人口減少を迎える小規模自治体において、いかにそうしたエコシステムを生み出し、地域公共財としての社会関係資本を蓄積しながら、しなやかに地域社会を維持・発展させていくことが可能なのかについて、国内の先進事例をもとに分析を行った。 / "Endogenous Regional Social Innovation Ecosystem" is that enable innovative solutions to regional challenges. It is a functional network of multi-sectoral players that is autonomous, sustained by interaction and co-evolution among the players. I analyzed based on advanced cases in Japan, about how it is possible for domestic small municipalities with declining populations to maintain and develop the local community in a flexible manner by creating such an ecosystem while accumulating social capital as local public goods. / 博士(ソーシャル・イノベーション) / Doctor of Philosophy in Social Innovation / 同志社大学 / Doshisha University