Cinétique enzymatique | EPFL Graph Search

La cinétique enzymatique a pour objet d'identifier et de décrire les mécanismes des réactions biochimiques, catalysées par les enzymes (réaction enzymatique), en étudiant leur vitesse c'est-à-dire leur évolution en fonction du temps. En partant des enzymes isolées et en allant vers les systèmes métaboliques organisés et intégrés, la cinétique enzymatique permet de décrire quantitativement les propriétés catalytiques des enzymes et les mécanismes mis en place pour leur régulation. Les enzymes jouent un rôle central dans la régulation des processus biologiques. Elles sont généralement constituées de molécules protéiques issues de la traduction du génome, à l'exception des ribozymes constitués d'ARN. Les enzymes interviennent en diminuant la barrière énergétique entre les réactants permettant d'accélérer les réactions des milliers de fois plus qu'en absence de catalyse. L'activité catalytique des enzymes est hautement spécifique, c'est-à-dire qu'une enzyme donnée, parmi les milliers

Hydrophilic Nanogels

Catherine Ann Schütz

Hydrophilic nanocarriers, formed by electrostatic interaction between their oppositely charged components, are recognized as nanomaterials with an enormous potential as vectors in biomedical and pharmaceutical applications. However, comprehensive information about their fate in various biological environments is still lacking. In general, due to their size, nanoparticles are able to enter and transport molecules into cells via pathways forbidden for bigger structures of even identical chemical nature. Research under defined conditions aiming at a better understanding of nanoparticle-cell interactions is crucial for the development of safe and successful nanocarriers for biomedical and pharmaceutical applications. Nanogels produced by electrostatic interaction of two types of the polysaccharide chitosan and pentasodium triphosphate were the subject of the research of this thesis. Additional surface coating with sodium alginate conferred to the nanogels a negatively charged surface enabling sufficient stability under physiological and in vivo relevant conditions. Of particular interest was the comparison of the two chitosan types, the first one obtained from usual animal sources while the second one became newly available from fungal sources. Prerequisite for the comparison in terms of nanogel formation, stability, interaction with cells and potential applications was the detailed analysis and characterization of the chitosan samples. The identification of almost identical degree of deacetylation, very similar molar mass distribution and solution behavior, but a somewhat higher molar mass of the animal chitosan as the only difference, allowed for a direct comparison. The animal and fungal derived chitosan samples yielded similar nanogels in relation to formation, size distributions and zeta potential. The nanogels were spherical with a hydrodynamic diameter in the range of 450 nm and a zeta potential of approx. -65 mV. The physical characteristics of the nanogels were clearly influenced by the ionic strength and temperature of the suspension. High deformability of the nanogel network was demonstrated by filtration through pores much narrower than the gel diameter. Temperature cycles in the range of 4°C to 37°C proved reversibility of size and zeta potential. The nanogels were stable in both simulated in vivo environment and in conditioned cell culture media. Exposure of seven human cancerous and non-cancerous cell lines to the nanogels, to identify the influence on the cell metabolic activity, revealed cell-type, nanogel dosage and exposure time dependence. However, nanogels prepared from animal and fungal chitosan showed similar behavior in vitro in all cell lines tested. The results obtained so far confirm fungal derived chitosan as an alternative to the animal derived chitosan. Nanogels carrying model protein antigen cargoes were taken up by porcine monocyte-derived dendritic cells, and kinetic analysis using an auto-quenched protein revealed a time-dependent endocytic processing of the nanogel cargo. These in vitro analyses were elaborated with an in vivo murine immunization experiment. Nanogels carrying ovalbumin (OVA) induced a dose-dependent antibody response over 6 weeks post-vaccination. An effective memory response was induced in nanogel-OVA immunized mice following a 0.1 µg OVA boost injection. Nanogels were also prepared carrying recombinant vaccine antigen against Neospora caninum. These induced a protective immune response following both intranasal and intraperitoneal vaccination in mice challenged with the tachyzoite form of the parasite. Overall, the results obtained in the framework of this research extend the knowledge on chitosan-based nanogels intended as nanocarriers for biomedical/pharmaceutical applications and contribute to further development of effective nanogel-based delivery systems.

EPFL2011

Inference methods for the study of interacting biological oscillators in single-cells

Colas Noé Droin

Biological oscillators are pervasive in biology, covering all aspects of life from enzyme kinetics reactions to population dynamics. Although their behaviour has been intensively studied in the last decades, the recent advances of high-throughput experimental technologies in the fields of omics and microscopy has called for the development of new analysis methods. Among the many types of models and quantitative analyses, parametric approaches are promising as they enable for a mechanistic or physical explanation of the phenomena under study. In particular, dynamical systems theory seems particularly adapted as the vast majority of oscillators can be modelled through differential equations. Dynamical systems parameters can also be easily opti-mized via maximum likelihood approaches. The validity of the inferred model can then be assessed from the quality of its predictions. We here present three different scientific questions regarding noisy biological oscillators, which are answered using maximum-likelihood inference approaches applied to parametric models. We first take interest in the characterisation of the influence of the cell-cycle over the circadi-an clock in individual mammalian cells. To this end, we develop a method combining a Hidden Markov Model with an Expectation-Maximization algorithm to infer their coupling from single-cell microscopy traces. We show that this coupling predicts multiple phase-locked states exhib-iting different degrees of robustness against molecular fluctuations inherent to cellular scale bio-logical oscillators. We then try to understand how the mammalian transcriptome behaves in the liver. Thence, we use single-cell RNA sequencing (scRNA-seq) along with mixed-models to investigate the interplay between gene regulation in space and time. Categorising mRNA expression profiles using mixed-effect models and smFISH validations, we find that many genes in the liver are both zonated and rhythmic, most of them showing multiplicative space-time effects. Finally, we look more closely at the cell-cycle, as it is one of the main drivers of gene expres-sion cell-to-cell heterogeneity in otherwise homogeneous cell populations. Here, we would like to understand if and how cell-cycle velocity changes depending on the phase of the cycling cells. To that end, we formulate the problem in terms of an autonomous dynamical system and use this to infer consistent dynamics for the cell-cycle from scRNA-seq data. Phase inference being paramount in all of these three studies, a short technical review on the topic is also provided at the end of this thesis, along with Julia scripts for the main inference methods presented. Various computational tools assisting the understanding of the scientific questions at stake are also presented, including a Python Dash web-app, a D3 widget and many Matplotlib animations and widgets.

EPFL2020

Model-based scale-up of a continuously operated consolidated bioprocess based on a microbial consortium for the production of ethanol

David Béat Dempfle

This work demonstrated that consolidated bioprocessing is a promising concept for conversion of lignocellulose to ethanol at industrial scale. CBP offers great cost saving potential, is feasible to be operated continuously and may be scaled up due to extensive knowledge of the process from a chemical engineering point of view.Cost savings of up to 27.5% of the total costs compared to conventional bioethanol production from lignocellulose, as stated by a techno-economic assessment, make continuous CBP the strongest lever to reduce processing costs of lignocellulosic ethanol. A cost sensitivity analysis identified scale and yield as the main cost-pushers from a process point of view, whereas the price level of the plant location has the highest impact on the investment conditions.To prove the feasibility of continuous CBP, and therefore the mentioned cost savings, experiments with a maximum titer of 3.258±0.007 g/L, a productivity of 0.025 g/(Lh) and constant enzyme production over 750h were conducted. Furthermore, the continuous experiments showed that experiments with identical volumetric oxygen transfer rate k_L a, but different oxygen fluxes per membrane area, showed titer differences of ca. 80% (1.83 g/L vs 3.26 g/L) in favor of setups with the large membrane surface. A rigorous process model was developed for continuously operated CBP. 9 species were considered (oxygen, glucose, total concentration Trichoderma reesei, secondary mycelia concentration of T. reesei, enzymes, cellulose, cellobiose, yeast density and ethanol). 8 of these 9 species needed to be modelled spatially resolved to account properly for mass transfer limitations. The fungal biofilm thickness d_f was found to be a critical parameter with an optimum for every membrane configuration. Smaller d_f reduced the fungal biofilm volume and thus, the enzyme production and larger d_f increased the diffusion path length causing shortage in nutrient supply as well as lower enzyme concentrations in the bulk. The enzyme synthesis rate of the secondary mycelia was fitted by reducing it by ca. 40% (0.67 FPU/(mLd) vs 1.152 (FPU/(mL*d) compared to batch models.With the process model as centerpiece, a scale-up framework consisting of the model and a set of non-dimensional parameters was developed to scale CBP from 2.7L laboratory scale to 130L pilot scale. The majority of the non-dimensional parameters could be kept constant during scale-up as requested by the similarity paradigm. Necessary changes were simulated with the model. Again, the fungal biofilm thickness d_f proved to be a relevant parameter for scale-up considerations. In general, the relative loss of biofilm volume is compensated by longer residence times since the residence time has by far the least impact on the process economics.Finally, popular rate-controlled separation techniques were investigated regarding their potential with CBP, since they offer the potential to reduce yeast inhibition, to avoid separation limitations by the azeotrope and they are not bound to the vapor-liquid-equilibrium of highly diluted ethanol-water mixtures. However, most of the mechanisms fail to handle the solids of the fermentation broth. Pervaporation, being the most promising concept for in-situ product removal, would be a cost-saving alternative to distillation for batch operation, but is limited by an unfavorable vapor-liquid equilibrium due to low bulk concentrations during continuous operation.

EPFL2022