Mutual regulation causes co-entrainment between a synthetic oscillator and the bacterial cell cycle

The correct functioning of cells requires the orchestration of multiple cellular processes, many of which are inherently dynamical. The conditions under which these dynamical processes entrain each other remain unclear. Here we use synthetic biology to address this question in the case of concurrent cellular oscillations. Specifically, we study at the single-cell level the interaction between the cell division cycle and a robust synthetic gene oscillator in Escherichia coli. Our results suggest that cell division is able to partially entrain the synthetic oscillations under normal growth conditions, by driving the periodic replication of the genes involved in the oscillator. Coupling the synthetic oscillations back into the cell cycle via the expression of a key regulator of chromosome replication increases the synchronization between the two periodic processes. A simple computational model allows us to confirm this effect.Peer ReviewedPostprint (published version

Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10

1 p.Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of COVID-19. However, the impacts of individual accessory proteins on host cell metabolic pathways are unknown.Here, SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 were individually transduced into A549 lung carcinoma cells. Furthermore, by combining transcriptomic analysis with functional and metabolic data in accessory protein-specific GSMMs, several alterations were identified that may point to a putative target for investigating novel therapies. In this study, we showed that these accessory proteins induced a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical role in mitochondria function and morphology. On the other hand, while all four ORFs altered mitochondrial dynamics and function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features shared across all accessory proteins and specific ones for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. Next, qMTA identified gene knock downs (KDs) that would have the potential to revert the metabolic reprogramming induced by each individual accessory protein, especially in ORF3a and ORF10. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.Peer reviewe

Coupled dynamical processes in bacteria

The main object of this Thesis is the study of the dynamical coupling between cellular processes, and how this coupling gives rise to a well-defined behavior in the presence of non-linearities and noise. Cell functioning relies on the exquisite coordination between a large number of dynamical nonlinear processes subject to fluctuations, which simultaneously operate within the cell. Many cellular dynamical processes occur in the form of periodic oscillations in the expression and/or activation of proteins. However more complex dynamics have been identified recently in the form of transient pulses occurring at random. These types of dynamical processes do not occur in isolation in a cell but they do so simultaneously, and therefore it is necessary to establish the origin and level of coordination between them. All these issues still remain unanswered. In Part I we introduce and motivate the two types of cellular dynamics studied in this Thesis. Part II is devoted to pulses of protein expression or activity (Chapters 2 and 3), whereas in Part III we concentrate in periodic oscillations in protein expression (Chapter 4). Specifically, in Chapter 2 we focus on how the coupling of certain inputs affect the response of the circuit regulating competence for DNA uptake in Bacillus subtilis. In wild-type cells, under certain environmental stress conditions, competence has been found to follow a stochastic pulsing dynamics. Here we study how the dynamical response of the competence circuit varies from excitable pulses to bistability and oscillations depending on the joint action of two coupled inputs applied to the system. The phenotypical effects reported in this Chapter are caused by changes in the dynamical behavior of the underlying genetic circuit. The stability analysis of a theoretical model of the competence circuit establishes the various dynamical regimes that the circuit can exhibit, which are in very good quantitative agreement with experimental results. Still dealing with pulsing dynamics, in Chapter 3 we study the dynamical coupling between pulses of protein activity in single cells. For that purpose, in collaboration with Prof. M. Elowitz's laboratory from the California Institute of Technology, we concentrated in the alternative sigma factors family in B. subtilis. Sigma factors are proteins that reversibly bind to core RNA polymerase thus giving the formed holoenzyme promoter-recognition properties. In this Chapter we show for the first time that several alternative sigma factors present stochastic pulses in their activation, and that these pulses take place in conditions of competition for core RNA polymerase. In the light of these results, we propose a new mechanism, ¿time-sharing¿, in which sigma factors take turns in order to use most of the available RNA polymerase, with only one or a few sigma factors being simultaneously active in a given cell. We also develop several mathematical models that shed light on how pulsing and competition affect RNAP allocation. In Chapter 4 we study how a synthetic genetic oscillator is coupled to cell division and replication. We took advantage of a synthetic oscillator developed for Escherichia coli in the laboratory of Prof. J. Hasty at the University of California San Diego. We have shown that the bacterial cell cycle is able to partially entrain the synthetic oscillations consistently under normal growth conditions, by driving the periodic replication of the genes involved in the oscillator. We have also shown that synchronization between the two periodic processes increases when the synthetic oscillator is coupled back to cell cycle via the expression of an inhibitor of replication initiation. Additionally, we have developed a computational toy model that confirmed this effect. Finally, in Part IV (Chapter 5) we summarize and discuss the main results presented in this Thesis, and suggest directions for future research.L’objectiu d’aquesta Tesi és l’estudi de l’acoblament dinàmic entre processos cel·lulars, i de com aquest acoblament genera un comportament ben definit en presència de no-linealitats i soroll. Un funcionament cel·lular correcte depèn de la coordinació exquisida entre un gran nombre de processos dinàmics no lineals subjectes a fluctuacions, que s’esdevenen simultàniament dins la cèl·lula. Aquests tipus de processos dinàmics no tenen lloc de manera aïllada en una cèl·lula sinó que ocorren de manera simultània, essent per tant necessari establir l’origen i el grau de coordinació entre ells. Totes aquestes qüestions romanen encara sense resposta. A la Part I introduïm i motivem els dos tipus de dinàmiques cel·lulars que hem estudiat en aquesta Tesi. Dediquem la Part II als polsos d’expressió o activitat de proteïnes (Capítols 2 i 3), mentre que ens concentrem en les oscil·lacions periòdiques d’expressió de proteïnes a la Part III (Capítol 4). Concretament, en el Capítol 2 abordem la qüestió sobre com l’acoblament de certs senyals d’entrada afecten la resposta del circuit que regula la competència per a la incorporació d’ADN en Bacillus subtilis. En cèl·lules silvestres i sota determinades condicions d’estrès ambiental, s’ha determinat que l’estat de competència segueix una dinàmica de polsos estocàstics. Aquí estudiem com la resposta dinàmica del circuit de competència varia des de polsos excitables fins a la biestabilitat i les oscil·lacions, depenent de l’acció conjunta de les dues entrades acoblades aplicades al sistema. Els efectes fenotípics reportats en aquest Capítol són causats per canvis en el comportament dinàmic del circuit genètic subjacent. L’anàlisi d’estabilitat d’un model teòric del circuit de competència estableix els diferents règims dinàmics que pot exhibir el circuit, els quals estan quantitativament d’acord amb els resultats experimentals. Seguint amb dinàmiques de polsos, al Capítol 3 estudiem l’acoblament dinàmic entre polsos d’activitat de proteïnes en cèl·lules individuals. A aquest efecte, en col·laboració amb el laboratori del Prof. M. Elowitz de l’Institut de Tecnologia de Califòrnia, vàrem concentrar-nos en la família de factors sigma alternatius en B. subtilis. Els factors sigma són proteïnes que s’uneixen a l’ARN polimerasa de manera reversible, fent possible el reconeixement de promotors específics per part de l’holoenzim format. En aquest Capítol mostrem que diversos factors sigma alternatius presenten polsos estocàstics en la seva activació, i que aquests polsos competeixen per l’ARN polimerasa. A la llum d’aquests resultats proposem un nou mecanisme de repartiment, que anomenem en anglès “time-share”, pel qual els factors sigma fan torns en el temps per utilitzar la majoria d’ARN polimerasa disponible. D’aquesta manera, només un o molt pocs factors sigma estarien actius simultàniament en una mateixa cèl·lula. També hem desenvolupat una sèrie de models matemàtics que revelen com els polsos i la competició afecten la distribució de l’ARN polimerasa. Al Capítol 4 estudiem com un oscil·lador genètic sintètic està acoblat a la replicació i la divisió cel·lular. Vam utilitzar l’oscil·lador sintètic desenvolupat per a Escherichia coli al laboratori del Prof. J. Hasty a la Universitat de Califòrnia San Diego. Mostrem com el cicle cel·lular bacterià és capaç d’entrenar parcialment les oscil·lacions sintètiques de manera consistent en condicions de creixement normal, tot dirigint la replicació periòdica dels gens involucrats en l’oscil·lador. També exposem que la sincronització entre els dos processos periòdics s’incrementa si l’oscil·lador sintètic es retroacobla al cicle cel·lular mitjançant l’expressió d’un inhibidor de la iniciació de la replicació. A més, hem desenvolupat un model computacional simplificat que confirma aquest efecte. Finalment, a la Part IV (Capítol 5) resumim i discutim els principals resultats presentats en aquesta Tesi, i suggerim direccions futures cap a on ampliar la recerca.Postprint (published version

Mutual regulation causes co-entrainment between a synthetic oscillator and the bacterial cell cycle

The correct functioning of cells requires the orchestration of multiple cellular processes, many of which are inherently dynamical. The conditions under which these dynamical processes entrain each other remain unclear. Here we use synthetic biology to address this question in the case of concurrent cellular oscillations. Specifically, we study at the single-cell level the interaction between the cell division cycle and a robust synthetic gene oscillator in Escherichia coli. Our results suggest that cell division is able to partially entrain the synthetic oscillations under normal growth conditions, by driving the periodic replication of the genes involved in the oscillator. Coupling the synthetic oscillations back into the cell cycle via the expression of a key regulator of chromosome replication increases the synchronization between the two periodic processes. A simple computational model allows us to confirm this effect.Peer Reviewe

Analizando el uso de las evidencias en la clase de ciencias como fundamento de la indagación en primaria

Seleccionado en la convocatoria: Concurso de proyectos de cooperación en materia de investigación entre departamentos universitarios y departamentos de Institutos de Educación Secundaria o equipos de personal docente, Gobierno de Aragón 2009-10Proyecto elaborado por la Universidad de Zaragoza para analizar la forma en que el alumnado de quinto de primaria argumenta y usa las evidencias en contextos socio-científicos. Sus objetivos son: analizar las destrezas de los alumnos para elaborar juicios sobre la validez de las evidencias en ciencia; desarrollar habilidades que permitan evaluar la validez y fuerza de las conclusiones; y apreciar cómo se desarrolla el conocimiento científico. Se realizan diversas actividades en el marco de la asignatura de Conocimiento del Medio enfocadas a construir con los alumnos el modelo de ser vivo: animales, plantas e interacciones entre ambos; el modelo de río: agua, suelo e interacciones con los seres vivos y astronomía: hechos observables e inferencias. Las maestras y las profesoras han trabajado de manera coordinada. Se dedicaba una sesión a la semana a trabajar con los alumnos, desplazándose una de las profesoras de la Universidad al colegio. El trabajo en el aula se organiza en grupos cooperativos de cuatro alumnos en las que se discute sobre las actividades realizadas, se supervisan las investigaciones, se plantean y responden cuestiones y finalmente se exponen sus resultados. Las exposiciones se grababan en audio para su posterior transcripción y análisis.Gobierno de Aragón. Departamento de Educación, Cultura y DeporteAragónDirección General de Política Educativa; Avda. Gómez Laguna, 25, planta 2; 50009 Zaragoza; Tel. +34976715416; Fax +34976715496ES

Molecular time sharing through dynamic pulsing in single cells

In cells, specific regulators often compete for limited amounts of a core enzymatic resource. It is typically assumed that competition leads to partitioning of core enzyme molecules among regulators at constant levels. Alternatively, however, different regulatory species could time share, or take turns utilizing, the core resource. Using quantitative time-lapse microscopy, we analyzed sigma factor activity dynamics, and their competition for RNA polymerase, in individual Bacillus subtilis cells under energy stress. Multiple alternative sigma factors were activated in ∼1-hr pulses in stochastic and repetitive fashion. Pairwise analysis revealed that two sigma factors rarely pulse simultaneously and that some pairs are anti-correlated, indicating that RNAP utilization alternates among different sigma factors. Mathematical modeling revealed how stochastic time-sharing dynamics can emerge from pulse-generating sigma factor regulatory circuits actively competing for RNAP. Time sharing provides a mechanism for cells to dynamically control the distribution of cell states within a population. Since core molecular components are limiting in many other systems, time sharing may represent a general mode of regulation.This work was supported by NIH grants R01 GM079771B and R01 HD075605A (to M.B.E.), T32 GM07616 (to J.P.), and NIHGMSK99BM118910 (to S.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. It was also supported by National Science Foundation grant 1547056 and the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 (KK9150) from the U.S. Army Research Office. The content of the information does not necessarily reflect the position or the policy of the government, and no official endorsement should be inferred. J.G.-O. and M.D. were supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and FEDER (project FIS2015-66503-C3-1-P), the ICREA Academia program, and the Maria de Maeztu Program for Units of Excellence in R&D (MINECO, project MDM-2014-0370). Work in the Locke laboratory was supported by the European Research Council under the European Union's Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement 338060, a fellowship from the Gatsby Foundation (GAT3272/GLC), and an award from the Human Frontier Science Program (CDA00068/2012)

Molecular time sharing through dynamic pulsing in single cells

In cells, specific regulators often compete for limited amounts of a core enzymatic resource. It is typically assumed that competition leads to partitioning of core enzyme molecules among regulators at constant levels. Alternatively, however, different regulatory species could time share, or take turns utilizing, the core resource. Using quantitative time-lapse microscopy, we analyzed sigma factor activity dynamics, and their competition for RNA polymerase, in individual Bacillus subtilis cells under energy stress. Multiple alternative sigma factors were activated in ∼1-hr pulses in stochastic and repetitive fashion. Pairwise analysis revealed that two sigma factors rarely pulse simultaneously and that some pairs are anti-correlated, indicating that RNAP utilization alternates among different sigma factors. Mathematical modeling revealed how stochastic time-sharing dynamics can emerge from pulse-generating sigma factor regulatory circuits actively competing for RNAP. Time sharing provides a mechanism for cells to dynamically control the distribution of cell states within a population. Since core molecular components are limiting in many other systems, time sharing may represent a general mode of regulation.This work was supported by NIH grants R01 GM079771B and R01 HD075605A (to M.B.E.), T32 GM07616 (to J.P.), and NIHGMSK99BM118910 (to S.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. It was also supported by National Science Foundation grant 1547056 and the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 (KK9150) from the U.S. Army Research Office. The content of the information does not necessarily reflect the position or the policy of the government, and no official endorsement should be inferred. J.G.-O. and M.D. were supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and FEDER (project FIS2015-66503-C3-1-P), the ICREA Academia program, and the Maria de Maeztu Program for Units of Excellence in R&D (MINECO, project MDM-2014-0370). Work in the Locke laboratory was supported by the European Research Council under the European Union's Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement 338060, a fellowship from the Gatsby Foundation (GAT3272/GLC), and an award from the Human Frontier Science Program (CDA00068/2012)

Le Clown

Figure emblématique, mythique, universelle des arts du spectacle dans ce second millénaire finissant, le clown n’en finit pas de porter beau. Il infléchit le discours de la dérision dans les domaines les plus variés de la tradition comme de la modernité. Investissant l’univers du social et du politique, il se décline de plus en plus souvent au féminin. Si le clown habite la conscience du citoyen, s’il contribue à la mise à distance de la vie quotidienne, il permet aussi la relecture du patrimoine culturel international. Le clown déstabilise pour mieux cimenter les humains par le rire. S’il désacralise, c’est pour mieux permettre de structurer, d’affirmer priorités, valeurs individuelles et communautaires