Ratiometric control for differentiation of cell populations endowed with synthetic toggle switches

We consider the problem of regulating by means of external control inputs the ratio of two cell populations. Specifically, we assume that these two cellular populations are composed of cells belonging to the same strain which embeds some bistable memory mechanism, e.g. a genetic toggle switch, allowing them to switch role from one population to another in response to some inputs. We present three control strategies to regulate the populations' ratio to arbitrary desired values which take also into account realistic physical and technological constraints occurring in experimental microfluidic platforms. The designed controllers are then validated in-silico using stochastic agent-based simulations.Comment: Accepted to CDC'201

Analysis and Control of Bacterial Populations in Synthetic Biology

Synthetic Biology is a new field of research that aims at engineering new functionalities in living beings. Analogously to electronic circuits, more advanced functionalities can be realised by putting together smaller functional modules that perform elementary tasks; however, the interaction of these basic pieces is somewhat complex and fragile. Therefore, to increase the robustness and reliability of the whole system, typical tools from Control Theory, such as feedback loops, can be employed. In the first part of this thesis we propose feedback control strategies to balance the gene expression of a bistable genetic circuit, known as genetic toggle switch, in an unstable region far away from its stable equilibria - a problem analogous to the stabilization of the inverted pendulum in mechanics. The effectiveness of the proposed control strategies is validated via realistic agent-based simulations of a bacterial population endowed with the genetic toggle switch. Later in the thesis we move towards the growth control of bacterial cells in bioreactors, introducing a novel open-source and versatile design of a turbidostat to host in vivo control experiments. In the last part, we want to control bioreactors to guarantee the coexistence of multiple species in the same environment. We analyse the dynamics of a simple one-chamber bioreactor, proposing control strategies to achieve the control goal. However, simple bioreactors have several drawback when the concentrations of multiple species are regulated at the same time; for these reason, we propose a novel layout for a bioreactor, with two growth chambers and a mixing one, to be used in multicellular in vivo control experiments

Combining calcium imaging with optogenetics in hippocampal primary cultures

Calcium imaging and optogenetics are two powerful tools in neuroscience research. Their combination can give great insights on the working of neural networks and circuits and can complement electrophysiological data. The present investigation shows that it is possible to analyse neuronal networks with optogenetic tools in vitro, where light is used to open and close ionic channels that have conduction properties identical to native channels. In this project we have opted for the optogenetic approach of Photoisomerizable Tethered Ligands (PTL). By tethering synthetic photoisomerizable compounds (PTLs, also called photoswitches) to engineered native proteins (channels or receptors), we gain the possibility of controlling them with light. In our case, these engineered proteins were ionotropic kainate-type glutamate receptors, called LiGluK2. In rat primary hippocampal cultures, we combined the optical stimulation of LiGluK2 with optical sensing by means of calcium imaging. Localized activation of LiGluK2 with confined illumination allowed us to control single neurons and analyse the effect of their activation on small neural networks. Different stimulation protocols were applied, and we found transfected neurons to have mainly three different kinds of responses in terms of calcium transients: single peaks, single prolonged calcium transients, multiple peaks. This activity affected surrounding neurons with variability, which can be ascribed to the physiological properties of the neurons involved and to the random connectivity of the analysed neural networks

Développement d'un système de différenciation modulable par la lumière pour la création et le contrôle de consortiums microbiens dans S. cerevisiae, sa caractérisation en cellule unique pour le développement de modèles prédictifs, et son utilisation pour l'expression hétérologue

Les consortiums microbiens artificiels cherchent à exploiter la division du travail pour optimiser des fonctions et possèdent un immense potentiel pour la bioproduction. Les approches de co-culture, le mode préférentiel pour générer des consortiums, restent limitées dans leur capacité à donner naissance à des consortiums stables ayant des compositions précisément ajustées. J'ai développé ici un système de différenciation artificielle dans la levure boulanger capable de générer à partir d'une seule souche des consortiums microbiens stables avec des fonctionnalités choisies et ayant une composition définie par l'utilisateur dans l'espace et dans le temps, grâce à une modification génétique pilotée par optogénétique. Grâce à une dynamique rapide, reproductible et ajustable par la lumière, mon système permet un contrôle dynamique de la composition des consortiums dans des cultures continues pendant de longues périodes. Je démontre également que notre système peut être étendu de manière simple pour donner naissance à des consortiums avec de multiples sous-populations. Cette stratégie de différenciation artificielle établit un nouveau paradigme pour la création de consortiums microbiens complexes qui sont simples à mettre en oeuvre, contrôlables avec précision et polyvalents à utiliser.En plus de cela, j'ai caractérisé le système au niveau de la cellule unique dans différents contextes en changeant la structure du bruit du facteur de transcription optogénétique qui induit la différenciation. J'ai découvert que le changement de la structure du bruit introduisait un couplage complexe entre les niveaux de la population de cellule et des cellules individuelles, qui ne peut être prédit par un simple modèle d'équations différentielles ordinaires. L'utilisation d'un modèle stochastique bien caractérisé a permis de rétablir la prévisibilité.Enfin, j'ai couplé le système de différenciation avec un system d'arrêt de croissance et de bioproduction de sorte que les cellules différenciées arrêtent de croître et commencent à produire une protéine d'intérêt. J'ai comparé l'efficacité de l'approche basée sur la différenciation avec des équivalents constitutifs et inductibles. J'ai constaté que la production n'était pas monotone par rapport à la fraction de différenciation mais qu'elle pouvait surpasser l'expression induite par un promoteur constitutif fort.Artificial microbial consortia seek to leverage division-of-labour to optimize function and possess immense potential for bioproduction. Co-culturing approaches, the preferred mode of generating a consortium, remain limited in their ability to give rise to stable consortia having finely tuned compositions. Here, I developed an artificial differentiation system in budding yeast capable of generating stable microbial consortia with custom functionalities from a single strain at user-defined composition in space and in time based on optogenetically-driven genetic rewiring. Owing to fast, reproducible, and light-tunable dynamics, my system enables dynamic control of consortia composition in continuous cultures for extended periods independently of the cell density. I further demonstrate that our system can be extended in a straightforward manner to give rise to consortia with multiple subpopulations. This artificial differentiation strategy establishes a novel paradigm for the creation of complex microbial consortia that are simple to implement, precisely controllable, and versatile to use.In addition to this, I characterized the system at the single cell level in different genetic contexts by changing the noise structure of the optogenetic transcription factor that drives differentiation. I found that changing the noise structure introduced complex coupling between the population and the single cell level, which cannot be predicted by a simple population model. A stochastic model of differentiation composed in a stochastic model of plasmid fluctuations not only restored predictability, but revealed mechanistic insights into the functioning of the system. The latter was exploited to demonstrate control of expression of a constitutively expressed gene (proxy for plasmid copy number).Lastly, I coupled the differentiation system with a growth arrest and production module such that differentiated cells stop growing and start producing a protein of interest. Growth arrest was effected via hijacking of the mating pheromone pathway and production was carried out by an orthogonal transcription factor. I developed a light inducible reference to assess the increase in production upon growth arrest. Comparing the efficiency of the differentiation-based approach with constitutive and inducible counterparts, I found that production was non-monotonic with respect to differentiation fraction and could outcompete constitutive expression. However, production did not increase upon growth arrest

FGF signaling and cell state transitions during organogenesis

Organogenesis is a complex choreography of morphogenetic processes, patterns and dynamic shape changes as well as the specification of cell fates. Although several molecular actors and context-specific mechanisms have already been identified, our general understanding of the fundamental principles that govern the formation of organs is far from comprehensive. The application of the concept of ‘rebuild it to understand it’ from synthetic biology represents a promising alternative to the classical approach of ‘break it to understand it’ in order to distill biological understanding from complex developmental processes. According to this ‘rebuilding’ concept, in this study we sought to develop an experimental approach to induce the formation of organs from progenitor cells ‘on demand’ and to investigate the minimum requirements for such a process. The zebrafish lateral line chain cells are a powerful in vivo model for our study because they are a group of naïve multipotent progenitor cells that display mesenchyme-like features. In order to bring these cells to form organs, we used the well-known role of the FGF signaling pathway as a driver of organogenesis in the lateral line and developed an inducible and constitutively active form of the fibroblast growth factor receptor 1a (chemoFGFR). The cell-autonomous induction of this chemoFGFR in chain cells effectively triggered the formation of fully mature organs and thus enabled spatial and temporal control of the organogenesis process. Next, we asked what it takes to form an organ de novo. We used a combination of real-time microscopy, single cell tracking, polarity quantification, and mosaic analysis to study the cell behaviors that result from chemoFGFR induction. The picture that emerges from these analyses is that de novo organs form through a genetically encoded self-assembly process that is based on the pattern of chemoFGFR induction. In this scenario, cells expressing chemoFGFR aggregate into clusters and epithelialize as they sort out of non-expressing cells. We found that this sorting process occurs through cell rearrangement and slithering, which involves an extensive remodeling of the cell-cell contacts. Chain cells that do not express chemoFGFR can envelop these chemoFGFR expressing cell clusters and form a rim at the cluster periphery. This multi-stage process leads to the establishment of the inside-outside pattern of de novo organs, which is used as a blueprint for cell differentiation. In summary, in this study we provide insights into the mechanisms involved in the self-assembly of organs from a naïve population of progenitor cells

A Themed Issue in Honor of Professor Raphael Mechoulam: The Father of Cannabinoid and Endocannabinoid Research

During the last 60 years the relevance of cannabis (Cannabis sativa or Cannabis indica) ingredients, like the psychoactive Δ9-tetrahydrocannabinol (THC), cannabidiol, 120+ additional cannabinoids and 440+ non-cannabinoid compounds, for human health and disease has become apparent. Approximately 30 years after the elucidation of THC structure the molecular reasons for the biological activity of these plant extracts were made clearer by the discovery of endocannabinoids, that are endogenous lipids able to bind to the same receptors activated by THC. Besides endocannabinoids, that include several N-acylethanolamines and acylesters, a complex array of receptors, metabolic enzymes, transporters (transmembrane, intracellular and extracellular carriers) were also discovered, and altogether they form a so-called “endocannabinoid system” that has been shown to finely tune the manifold biological activities of these lipid signals. Both plant-derived cannabinoids and endocannabinoids were first discovered by the group led by Prof. Dr. Raphael Mechoulam, who has just celebrated his 90th birthday and clearly stood out as a giant of modern science. The many implications of his seminal work for chemistry, biochemistry, biology, pharmacology and medicine are described in this special issue by the scientists who reached during the last 20 years the highest recognition in the field of (endo)cannabinoid research, receiving the Mechoulam Award for their major contributions. I thank them for having accepted my invitation to be part of this honorary issue of Molecules, and Raphi for continuing to illuminate our field with his always inspiring investigations and new ideas

Ratiometric control for differentiation of cell populations endowed with synthetic toggle switches

We consider the problem of regulating by means of external control inputs the ratio of two cell populations. Specifically, we assume that these two cellular populations are composed of cells belonging to the same strain which embeds some bistable memory mechanism, e.g. a genetic toggle switch, allowing them to switch role from one population to another in response to some inputs. We present three control strategies to regulate the populations' ratio to arbitrary desired values which take also into account realistic physical and technological constraints occurring in experimental microfluidic platforms. The designed controllers are then validated in-silico using stochastic agent-based simulations

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin