A healthy immune system's core challenge is to mount appropriate responses to an immense and unknown variety of antigenic stimuli. By unraveling the regulatory networks that drive and control immune-cell behaviors, we can begin to identify the means by which proper balance can be achieved and aberrant behaviors clinically corrected. Traditionally, major advances in our understanding of cellular immunological processes depended critically on both improved perturbation and enhanced observation methods. In my doctoral research, I have pursued both strategies to elucidate the differentiation and effector functions of adaptive immune Th17 cells. These cells exemplify the need for balance: while Th17 cells are needed to induce clearance of fungal infections and extracellular bacteria, irregular responses have been strongly implicated in autoimmunity. / Chemistry and Chemical Biology

Σκοπός της συγκεκριμένης ερευνητικής εργασίας ήταν η αποκάλυψη του ρόλου και της θέσης του PlCOUP-TF στο ρυθμιστικό γονιδιακό δίκτυο της νευρογένεσης του αχινού Paracentrotus lividus, αλλά και η διερεύνηση των επιδράσεων του συγκεκριμένου μεταγραφικού παράγοντα στα υπόλοιπα γονίδια που εμπλέκονται στην δημιουργία νευρώνων. Ο PlCOUP-TF είναι ένας μητρικός μεταγραφικός παράγοντας και ορφανός πυρηνικός υποδοχέας που φαίνεται να διαδραματίζει σημαντικό ρόλο στην δημιουργία νευρώνων σε μια ποικιλία οργανισμών. Τα υπό μελέτη γονίδια της νευρογένεσης είναι επίσης μεταγραφικοι παράγοντες με εξελικτικά συντηρημένες επικράτειες. Για τον προαναφερθέντα σκοπό, πραγματοποίηθηκαν πειράματα διπλής φθορίζουσας in situ υβριδοποίησης ώστε να αποκαλυφθούν οι περιοχές συνεντοπίσμου του PlCOUP-TF και των γονιδίων της νευρογένεσης σε ώριμα στάδια της ανάπτυξης. Επίσης, διεξήχθησαν πειράματα καταστολής της μητρικής έκφρασης του PlCOUP-TF με ενέσεις με ΜΑSO σε γονιμοποιημένα ωάρια και έλεγχος της έκφρασης των νευροειδικών γονιδίων στα ενεμένα και στα αντίστοιχα control έμβρυα. Ο έλεγχος της έκφρασης ήταν τόσο ποιοτικός (in situ χρωμογόνος υβριδοποίηση) όσο και ποσοτικός (Q-PCR). Με τα πειράματα αυτά, αποκαλύφθηκε ότι πιθανότατα ο συγκεκριμένος μεταγραφικός παράγοντας λειτουργεί ενεργοποιητικά για τα εν λόγω γονίδια. Συγκεκριμένα, φαίνεται η ύπαρξη κατασταλτικής δράσης του PlCOUP-TF πάνω σε καταστολέα των γονιδίων της νευρογένεσης (double negative gate) στην περιοχή του εμπρόσθιου νευροεξωδέρματος και σε συγκεκριμένες περιοχές της βλεφαριδωτής ζώνης. / Our aim was to identify the role of the orphan nuclear receptor PlCoup-TF in sea urchin embryonic neurogenesis and especially in the determination of the anterior neuroectoderm (ANE). To this end, we cloned a set of embryonic cDNAs encoding regulatory proteins expressed specifically in the ANE and we prepared antisense RNA probes for double fluorescence in situ hybridization for the following genes: PlCoup-TF, PlHbn, Plz81 and PlFoxG. We studied the spatial pattern of expression of ANE genes in conjunction with the expression pattern of PlCoup-TF in all embryonic stages of the sea urchin Paracentrotus lividus. A neurogenic territory specified within the animal pole of the embryo, is formed as a result of the interplay of the aforementioned regulatory factors that together constitute a sub-circuit within the embryonic gene regulatory network (GRN). We wanted to determine PlCoup-TF’s place within the GRN and specifically the ANE sub-circuit. Therefore, we knockdowned PlCoup-TF expression during embryogenesis by injecting specific morpholino antisense oligonucleotides (MASO) into sea urchin eggs and determine the expression pattern of the ANE specific genes by chromogenic in situ hybridization to resulting morphant embryos. The efficiency of the knockdown was measured by QPCR, where the amount of PlCoup-TF transcripts of morphants is compared to that of control embryos. Finally, we concluded that probably PlCoup-TF activate the ANE genes, by repressing repressor(s) of these genes (double negative gate) in ANE and in specific regions of CBE.

No description available.

As food scares have hastened the growth of safety and quality standards around the world, certification schemes to assure various attributes of foods have proliferated in the global marketplace. High-value food commodities produced in the global south for export have been the subject of such schemes through third-party environmental certifications, providing regulatory and verification mechanisms welcomed by global buyers. As certification becomes more common, re-localization in the current global context can also mean the projection of place onto a food commodity to highlight its origin or attributes secured by transparent verification mechanisms. However, environmental food certification is often criticized for its inapplicability in the context of the global south, due to the extensive documentation requirements and high costs. The key question here is the process for small-scale producers in the global south to navigate increasing international regulation of food safety and quality. This dissertation examines (1) how the environmental standards (as defined by the global north) were translated in the rural global south through international certification schemes, and (2) what the implications are at the local level, especially where producers had not yet integrated into conventional global markets before the introduction of certification. The dissertation also analyzes the influence of such certification in determining the development trajectories of rural society in the global south. A case study is used to examine newly-introduced certified organic shrimp production in Ca Mau Province in Vietnam’s Mekong Delta. The selected shrimp production site is the first pilot organic shrimp project in Vietnam working with an international third-party certification scheme. It is located in rural Vietnam where, as in other parts of Southeast Asia, an accelerated process of agrarian transition is underway. Whereas elsewhere the trend with intensified regulation has been the consolidation of large-scale farms and the exclusion of small-scale farms from international agrofood markets, this case study demonstrates comparative advantages of small-scale farms over large-scale farms in producing sensitive high-value crops. This dissertation employs two main analytical approaches. The first approach is to examine the network of actors and the flow of information, payment and shrimp at the production level using environmental regulatory network (ERN). In contrast to chain analyses, which can be useful in identifying linear structure of supply chains for global commodities, ERN can capture the interrelatedeness of actors in the network built around environmental certification for agrofood products. The second analytical lens is that of agrarian transition. Countries experiencing agrarian transition at present are doing so in a very different international context from countries that accomplished their transitions in the past. Results of this research indicate that technical and financial constraints at the time of initial certification are not the primary obstacles to farmers getting certified, since the extensive farming method employed at the study site is organic by default. In spite of this, many farmers unofficially withdrew from the organic shrimp project by simply shifting their marketing channel back to a conventional one. Inefficient flows of information and payments, and a restrictive marketing channel within the environmental regulatory network that does not take into account local geographical conditions and farming practices, all contributed to limiting the farmers’ capacity and lowering their incentives to get involved in the network. The analysis also indicates that, by influencing those agrarian transition processes, food standards and certification based on values developed in the global north may modify, reshape and/or hold back agrarian transition processes in agricultural sectors of developing countries. The potential benefits of environmental certification are enhanced rural development, by generating opportunities for small-scale farmers to connect to global niche markets. The findings of this dissertation highlighted that such certification schemes or their environmental regulatory networks need to ensure information sharing and compensation for farmers. As an empirical finding, this dissertation also captures where ecological credibility and market logic meet: the success of this kind of certification depends on finding a balanced point where standards are ecologically (or ethically) credible to the level that does not attract too much criticism for being green washing, but not too unrealistic to become a disincentive for farmers to participate.

Complex multicellular organisms are composed of at least 200 cell types, which contain the same DNA "black box" of genetic information. It is the precise regime according to which they express their genes, exquisitely controlled by gene regulatory circuits, that defines their cellular identity, morphology and function. We have developed an in vivo biotinylation method that uses genetically encoded components in zebrafish, termed biotagging, for genome-wide regulatory analysis of defined embryonic cell populations. By labelling selected proteins in specific cell types, biotagging eliminates background inherent to analyses of complex embryonic environments via highly stringent biochemical procedures and targeting of specific interactions without the need for cell sorting. We utilised biotagging to characterise the in vivo translational landscape on polysomes as well as the transcriptional regulatory landscape in nuclei of migratory neural crest cells, which intermix with environing tissues during their migration. Our migratory neural crest translatome presented both known and novel players of the neural crest gene regulatory network. An in depth look into the active nuclear transcriptome uncovered a complex world of non-coding regulatory RNAs that potentially specify migratory neural crest identity and present evidence of active bidirectional transcription on regions of open chromatin that include putative cis-regulatory elements. Analysis of our transcribed cis-regulatory modules functionally links these elements to known genes that are key to migratory neural crest function and its derivatives. We also identified a novel cohort of circular RNAs enriched at regions of tandem duplicated genes. Last but not least, we recovered developmentally regulated long non-coding RNAs and transcribed transposable elements. To functionally dissect the biological roles of these factors, we have built two Ac/Ds-mediated in vivo toolkits for efficient screening of putative enhancers and for CRISPR/Cas9-based transcriptional modulation. Overall, our methods and findings present a comprehensive view of the active coding and non-coding landscapes of migratory neural crest on a genome-wide scale that refine the current regulatory architecture underlying neural crest identity.

L'inférence des réseaux de régulation de gènes (RRG) à partir de données d'expression est un défi majeur en biologie. L’arrivée des technologies de mesure de transcriptomique à l’échelle de la cellule a suscité de nombreux espoirs, mais paradoxalement elles montrent une nouvelle complexité du problème d’inférence des RRG qui limite encore les approches existantes. Nous avons commencé par montrer, à partir de données d'expression en cellules uniques acquises sur un modèle aviaire de différenciation érythrocytaire, que les RRG sont des systèmes stochastiques à l'échelle de la cellule et qu'il y a une évolution dynamique de cette stochasticité au cours du processus de différenciation (Richard et al, PLOS Comp.Biol., 2016). C'est pourquoi nous avons développé par la suite un modèle de RRG mécaniste qui inclus cette stochasticité afin d'exploiter au maximum l'information des données expérimentales à l'échelle de la cellule (Herbach et al, BMC Sys.Biol., 2017). Ce modèle décrit les interactions entre gènes comme un couplage de processus de Markov déterministes par morceaux. En régime stationnaire une formule explicite de la distribution jointe est dérivée du modèle et peut servir à inférer des réseaux simples. Afin d'exploiter l'information dynamique et d'intégrer d'autres données expérimentales (protéomique, demi-vie des ARN), j’ai développé à partir du modèle précédent une approche itérative, intégrative et parallèle, baptisée WASABI qui est basé sur le concept de vague d'expression (Bonnaffoux et al, en révision, 2018). Cette approche originale a été validée sur des modèles in-silico de RRG, puis sur nos données in-vitro. Les RRG inférés affichent une structure de réseau originale au regard de la littérature, avec un rôle central du stimulus et une topologie très distribuée et limitée. Les résultats montrent que WASABI surmonte certaines limitations des approches existantes et sera certainement utile pour aider les biologistes dans l’analyse et l’intégration de leurs données. / Inference of gene regulatory networks from gene expression data has been a long-standing and notoriously difficult task in systems biology. Recently, single-cell transcriptomic data have been massively used for gene regulatory network inference, with both successes and limitations.In the present work we propose an iterative algorithm called WASABI, dedicated to inferring a causal dynamical network from timestamped single-cell data, which tackles some of the limitations associated with current approaches. We first introduce the concept of waves, which posits that the information provided by an external stimulus will affect genes one-byone through a cascade, like waves spreading through a network. This concept allows us to infer the network one gene at a time, after genes have been ordered regarding their time of regulation. We then demonstrate the ability of WASABI to correctly infer small networks, which have been simulated in-silico using a mechanistic model consisting of coupled piecewise-deterministic Markov processes for the proper description of gene expression at the single-cell level. We finally apply WASABI on in-vitro generated data on an avian model of erythroid differentiation. The structure of the resulting gene regulatory network sheds a fascinating new light on the molecular mechanisms controlling this process. In particular, we find no evidence for hub genes and a much more distributed network structure than expected. Interestingly, we find that a majority of genes are under the direct control of the differentiation-inducing stimulus. Together, these results demonstrate WASABI versatility and ability to tackle some general gene regulatory networks inference issues. It is our hope that WASABI will prove useful in helping biologists to fully exploit the power of time-stamped single-cell data.

L'adaptation d'une bactérie aux changements de son environnement est contrôlée par un réseau de régulation large et complexe, faisant intervenir de nombreux acteurs et modules différents. Dans ce travail, nous avons étudiés un module de régulation spécifique, contrôlant l'adaptation de la bactérie Escherichia coli à un changement de sources de carbone. Dans un milieu contenant du glucose et de l'acétate, la croissance est divisée en deux phases : les bactéries utilisent préférentiellement le glucose et commencent à métaboliser l'acétate qu'après l'épuisement du glucose. En effet, la présence du glucose réprime la transcription d'un gène nécessaire à la croissance sur acétate, le gène acs (codant pour l'acétyl-CoA synthétase). Le mécanisme régulateur fait intervenir le facteur de transcription Crp-AMPc et le système de transfert de phosphate (PTS), qui permet l'import du glucose. Plusieurs modèles décrivent en détail la cascade de réactions moléculaires à l'origine de cette « répression catabolique ». Cependant, certaines de nos observations expérimentales ne sont pas correctement prédites par les modèles actuels. Ces modèles doivent être révisés ou complétés. L'outil majeur que nous employons pour les expériences est la fusion transcriptionnelle : une région promotrice fusionnée en amont d'un gène rapporteur (GFP, luciferase). Avec ces constructions, nous mesurons la dynamique de l'expression génique dans différentes souches (mutants) et différentes conditions environnementales. Les observations à l'échelle de la population sont corroborées par des mesures similaires à l'échelle de la cellule unique. Nous utilisons cette même technologie pour construire de petits systèmes synthétiques qui sondent davantage le phénomène de répression catabolique. Nous avons ainsi créé un interrupteur génétique dont le fonctionnement est contrôlé par le flux glycolytique et nous avons construit un petit système de communication intercellulaire basé sur la molécule AMPc. Enfin, nous proposons une manière originale de mesurer l'état métabolique des cellules en utilisant la dépendance énergétique de la luciferase. / The adaptation of bacteria to changes in their environment is controlled by a large and complex regulatory network involving many different actors and modules. In this work, we have studied a specific module controlling the adaptation of Escherichia coli to a change in carbon sources. In a medium containing glucose and acetate, growth is divided into two phases : the bacteria preferentially use glucose and start to metabolize acetate only after glucose exhaustion. Indeed, the presence of glucose represses the transcription of a gene needed for growth on acetate : the acs gene (coding for acetyl-CoA synthetase). The regulatory mechanism involves the Crp-cAMP regulator and the phosphate transfer system (PTS), which is responsible for glucose import. Several models describe the cascade of molecular reactions responsible for this « catabolite repression ». However, our work shows that many of our experimental observations are incorrectly predicted by current models. These models have to be amended.We use transcriptional fusion, i.e., the fusion of a promoter region upstream of a reporter gene (GFP, luciferase), to measure the dynamics of gene expression in different genetic backgrounds and environmental conditions. Observations at the population level are corroborated by similar measurements at the single cell level. We use this same technology to construct small synthetic systems that probe further aspects of the phenomenon of catabolite repression. We have thus created a genetic toggle switch controlled by the glycolytic flux and we have built an inter-cellular communication system mediated by cAMP. Finally, we propose a novel way to measure the metabolic state of cells by using the energy dependence of the luciferase enzyme.

Em sistemas biológicos, o estudo da estabilidade das redes de regulação gênica é visto como uma contribuição importante que a Matemática pode proporcionar a pesquisas sobre câncer e outras doenças genéticas. Neste trabalho, utilizamos o conceito de ``canalização\'\' como sinônimo de estabilidade em uma rede biológica. Como as características de uma rede de regulação canalizada ainda são superficialmente compreendidas, estudamos esse conceito sob o ponto de vista computacional: propomos um modelo matemático simplificado para descrever o fenômeno e realizamos algumas análises sobre o mesmo. Mais especificamente, a estabilidade da maior bacia de atração das redes Booleanas - um clássico paradigma para a modelagem de redes de regulação - é analisada. Os resultados indicam que a estabilidade da maior bacia de atração está relacionada com dados biológicos sobre o crescimento de colônias de leveduras e que considerações sobre a interação entre as funções Booleanas e a topologia da rede devem ser realizadas conjuntamente na análise de redes estáveis. / In biological systems, the study of gene regulatory networks stability is seen as an important contribution that Mathematics can make to cancer research and that of other genetic diseases. In this work, we consider the concept of ``canalization\'\' as a consequence of stability in gene regulatory networks. The characteristics of canalized regulatory networks are superficially understood. Hence, we study the canalization concept under a computational framework: a simplified model is proposed to describe the phenomenon using Boolean Networks - a classical paradigm to modeling regulatory networks. Specifically, the stability of the largest basin of attraction in gene regulatory networks is analyzed. Our results indicate that the stability of the largest basin of attraction is related to biological data on growth of yeast colonies, and that thoughts about the interaction between Boolean functions and network topologies must be given in the analysis of stable networks.

Nous présentons deux problèmes de biologie faisant appel à un traitement de données et des modèles issus de la physique statistique : la dynamique des populations en immunologie et la régulation génétique dans le développement embryonnaire. En immunologie, nous étudions le problème de la sélection somatique dans le système immunitaire adaptatif: la sélection cellulaire et la compétition qui s’y opèrent, constituant un système quasi Darwinien au sein de l’organisme. Dans un premier temps, nous considérons différentes hypothèses surla dynamique sélective : signaux déclenchant la division ou la mort cellulaire par liaison antigénique ou par cytokines, paramètres dynamiques de division, mort et fluctuations environnementales. Nous explorons leur influence sur la taille des clones dont la distribution à queue lourde a été observée à travers les espèces et les types de cellules. Deux familles de modèles émergent : un premier dans lequel le bruit est cohérent à l’échelle du clone et un second dans lequel le bruit varie de cellule à cellule. Nous montrons dans quelle mesure la distribution de taille de clones permet de déterminer le meilleur modèle et relions la forme de la distribution ainsi que l’exposant apparent de la loi de puissance aux paramètres biologiques. Dans un second temps, nous explorons les caractéristiques du réseau complexe et aléatoire formé par les clones et les antigènes : dimension, adjacence, dynamique. Nous nous intéressons à l’effet de la sélection dans le temps et à la vitesse d’évolution des clones.La deuxième partie de cette thèse est consacrée au développement embryonnaire. Dans l’embryon, il est essentiel pour le noyau de déterminer sa position avec une grande précision pour orienter la différentiation et construire un organisme structuré viable. Cette information positionnelle est acquise, transmise et conservée par la diffusion de protéines et l’activa- tion de circuits génétiques.Plus précisément, la formation de l’axe antéropostérieur chez la Drosophile est déterminée entre autres par l’activation du gène hunchback par la protéine Bicoid. Nous analysons des données issues d’expériences d’imagerie fluorescente dynamique dans les premiers cycles cellulaires de l’embryon. Nous construisons un modèle spécifique permettant d’analyser la fonction d’autocorrélation des traces temporelles de fluorescence qui prend en compte toutes les difficultés biologiques et expérimentales (bruit, calibration traces courtes, structure du gène artificiel) pour extraire les paramètre dynamiques d’activation de hunchback. Nous examinons différentes dynamiques potentielles (poisonnienne, markovienne ou non markovienne) et leur implication pour l’information dont la cellule dispose sur sa position ainsi que la précision de la lecture du gradient de Bicoid. / This work presents two problems of biology requiring data analysis and models from statistical mechanics: population dynamics in immunology and gene regulation in embryo development. In immunology I study the problem of somatic evolution in the adaptive immune system: selection of and competition among cells that form a close-to-Darwinian system within one individual. First, I consider different potential hypotheses for selective dynamics: division and death signals through antigen binding or cytokines, dynamical parameters for division, death and fluctuations of the environment. I explore their impact on clone sizes. Experimentally, these clone sizes show heavy tail distributions for different species and differentpools of cells. Two families of models emerge: models where noise is consistent at the level of the clone and models where it varies from cell to cell. I show how clone size distributions help discriminate between these models and relate the shape of the distribution and the exponent of the power law to biological parameters. Second, I explore the specifics of the complex stochastic network of clones and antigens: its dimensionality, connectivity and dynamics. I study the effect of selection at different time scales and the speed of evolution of the clones. The second part of this dissertation concerns embryo development. In the fly embryo, it is crucial that nuclei can evaluate their position within the organism accurately to determine cell fate and build a healthy organism. This positional information is obtained, transferred, and maintained through diffusion of proteins and activation of genetic networks. More specifically, the patterning of the antero-posterior axis in drosophila requires the hunchback gene, activated by the Bicoid protein. I analyze data from fluorescent live imaging in the early cell cycles of the embryo. I build a tailor-made model to analyze autocorrelation functions of fluorescence time traces overcoming all biological and experimental challenges (noise, calibration, short traces, transgene construct) to extract the parameters of hunchback activation. I examine several potential types of dynamics for gene switiching (Poisson, Markovian or non-Markovian) and predict their impact on positional information and the accuracy of bicoid gradient readout.

This thesis investigates how pairwise combinatorial gene and stimulus perturbation experiments are conducted and interpreted. In particular, I investigate gene perturbation in the form of knockout, which can be achieved in a pairwise manner by SGA or CRISPR/Cas9 methods. In the present literature, I distinguish two approaches to interpretation: the calculation of stimulus and gene interactions, and the identification of equality among phenotypes measured for distinct perturbation conditions. I describe how each approach has been applied to derive hypotheses about gene regulatory networks. I identify conflicts and uncertainties in the assumptions allowing these derivations, and explore theoretically and experimentally approaches to improve the interpretation of genetic interaction data. I apply the approaches to a well-studied gene regulatory branch of the DNA damage checkpoint (DDC) pathway of Saccharomyces cerevisiae, and confirm the known order of genes within this pathway. I also describe observations that seem inconsistent with this pathway structure. I explore this inconsistency experimentally and discover that high concentrations of the DNA alkylating drug methyl methanesulfonate cause a cell division arrest program distinct from a G1 or G2/M checkpoint or from DNA damage adaptation, that resembles an endocycle.