Cellular model | EPFL Graph Search

A cellular model is a mathematical model of aspects of a biological cell, for the purposes of in silico research. Developing such models has been a task of systems biology and mathematical biology. It involves developing efficient algorithms, data structures, visualization and communication tools to orchestrate the integration of large quantities of biological data with the goal of computer modeling. It involves the use of computer simulations of cellular subsystems, such as the networks of metabolites and enzymes which comprise metabolism, signal transduction pathways and gene regulatory networks. Overview The eukaryotic cell cycle is very complex and is one of the most studied topics, since its misregulation leads to cancers. It is possibly a good example of a mathematical model as it deals with simple calculus but gives valid results. Two research groups have produced several models of the cell cycle simulating several organisms. They have recently produced a generic euk

Reversible attachment of Cell-Penetrating Peptides for the efficient delivery of α-synuclein to HeLa cells and primary cortical neurons

Carole Desobry

The discovery of alpha-synuclein (a-syn) as the main component of Lewy Bodies (LBs), the pathological hallmarks of Parkinson's disease (PD), and the identification of mutations in the gene coding for a-syn in familial form of PD have reinforced the central role of a-syn in the etiology of PD. Nevertheless, the molecular mechanisms by which a-syn is involved in neurodegeneration are still not fully understood. Many studies have raised the potential role of post-translational modifications (PTMs) in regulating a-syn physiological properties but also its pathogenicity in PD development. However, a-syn can be modified simultaneously by several PTMs making it extremely challenging to understand which of those PTMs is responsible in regulating the cellular properties of a-syn. Therefore, our laboratory developed a strategy to study the role of individual PTMs using semi-synthesis, enabling the generation of site-specifically modified a-syn. However, in the absence of an efficient system to internalize these semi-synthetic proteins in cells, those proteins have been limited to biophysical and structural studies. Therefore, we proposed to use cell penetrating peptides (CPPs) to deliver site-specifically modified a-syn inside cells. In a first step, we compared CPPs to deliver a-syn inside the cells. Strikingly, only N-terminal CPP fusions were efficient in delivering it to primary cortical neurons. In order to explain the differences, we performed biophysical assays and established that C-terminal CPPs formed a hairpin with the C-terminus of a-syn, preventing penetration of the plasma membrane. N-terminal fusions displayed a more transient interaction that allowed uptake. In addition, we showed that the CPPs were enhancing the aggregation of a-syn. Among the CPPs tested, TAT fused at the N-terminus was one of the most efficient. Therefore, we evaluated its capacity to deliver a-syn in different cellular models, its subcellular localization and its stability in the cells. We started by evaluating the uptake and molecular properties of TAT- a-syn in HeLa cells and in primary cortical neurons. We found that it was rapidly and efficiently internalized and distributed in the cytosol as punctuated structures. Once internalized, TAT-a-syn was cleared from the cells through autophagy and the proteasome without promoting any cellular toxicity. Finally, TAT-a-syn phosphorylated on Serine 129 or fibrillar TAT-a-syn were also successfully delivered to cells thereby showing that our tool could be used to deliver modified a-syn in cells. However, our data suggested that TAT could influence the properties of a-syn. Thus, we aimed at improving our delivery tool by developing two reversible approaches to relieve a-syn from the influence of TAT after its uptake: 1) a photocleavable linker to release a-syn from TAT upon UV irradiation or 2) a disulfide bond which can be cleaved once in the cell. Both approaches were successful in delivering a-syn to neurons. While the photocleavable version did not allow full release, TAT-S-S-a-syn was efficiently reduced and efficient in delivering pS129 a-syn. In summary, our study allowed developing a versatile tool capable of carrying a-syn inside the cells and allowing its study in the absence of constraints originating from the presence of the CPP sequence in fusion with a-syn. We believe that this tool will be useful for the study of a-syn's PTMs, interactions and cross-talk between modifications.

EPFL2014

Fluorescent sensor proteins to assess the cellular uptake of therapeutic peptides and small molecules

Silvia Scarabelli

The determination of cell membrane permeability and of the absorption and clearance rates is an important step in the pharmacokinetic profiling of a new drug candidate. In a similar way, research in cell biology relies on the development of cell penetrating biochemical probes allowing to study and interfere with cellular functions and physiology. A variety of methods have been developed to estimate the ability of molecules to enter the intracellular space. They are based either on artificial membranes or on cellular models, with consequently different abilities to mimic the cellular permeation mechanisms. Howev-er, these techniques are often laborious, expensive and have to be combined to get a reliable characterization of the properties of the molecule of interest. This thesis introduces a new strategy to evaluate real-time absorption and clearance kinet-ics of different classes of molecules in living cells. The approach is based on the use of in-tracellular single-chain FRET-based biosensors with a versatile modular design. First, a proof-of-concept bioprobe for inhibitors of the enzyme human carbonic anhydrase II (HCAII) was developed and optimized for intracellular use: the cell-entry dynamics of a selection of well-known sulfonamide drugs was compared and monitored in real time. Then, we adapted the design of the sensory protein to generate a label-free biosensor for inhibitors of the p53-HDM2 interaction, a highly studied protein-protein interaction in cancer biology. The per-meability and the kinetics of cell entry and washout of various underivatized small molecule and peptidic inhibitors could be measured. This thesis represents a step towards the introduction of a general approach for a low-cost, rapid and minimally invasive evaluation of molecular permeabilities in the cell type of choice. Their modular design lends itself to be adapted to the detection of other families of molecules, provided that a binding protein for the molecule of interest is known.

EPFL2017

Integration of microfluidics and multimodal optics for translational bioanalytics

Daniel Migliozzi

Numerous analytical techniques for life sciences have been at the basis of the improvement of medical diagnostics and treatments. Current challenges in bioanalytics are related to the conception of methods enabling large amount of information to be extracted from biological samples, in order to be translated into concrete solutions for the patient. From this point of view, microfluidics constitutes a key element to automate, accelerate and standardize bioanalytical tests. At the same time, obtaining precise information from the analyte of interest is crucial to specifically address diagnoses and therapies. Since the high flexibility of optical readouts can give access to a multitude of diverse aspects of biological matter in multiplexed fashion, in this thesis we have chosen to integrate microfluidics and multimodal optics to address specific problems in translational bioanalytics: drug screening and tumor diagnostics. In the first part of the work, we approached the problem of drug screening on living organisms, and especially the possibility to provide as much information as possible on potential mechanisms of action and clinical targets. For this, we aimed at developing a method to study the model organism Caenorhabditis elegans, i.e. a small soil worm that is widely used by researchers to study physiopathology at whole-organism scale. In fact, while offering the systemic information that is missing with cellular models, this small organism is much easier to handle and grow in large amounts compared to rodents, for which the costs and the ethical concerns are substantial. Moreover, its large genetic homology with humans supports translational analysis and its genetic manipulation is well mastered to model human diseases. Thus, we developed a microfluidic array to select small separated populations of C. elegans specimens, and combined fluorescence and bright-field imaging along with high-throughput feature recognition and signal detection to identify the mode-of-action of an antibiotic. Also, we demonstrated real-time, very large field-of-view capability on multiplexed motility assays for the assessment of the dose-response relation of an anesthetic. Furthermore, to enable on-site antibiotic testing, we explored a method to empower low-magnification optical systems with dielectric micro- and macro-spheres for the detection of pathogens in liquid media. Such dielectric objects offer potential solutions to increase the resolution and the sensitivity of imaging systems in a cost-effective fashion. We first characterized the parameters that govern the resolution increase in the case of microsphere-assisted incoherent microscopy and demonstrated it by imaging silicon microstructures and fluorescent bacteria. Then, we developed a method to easily fabricate microfluidic chips that integrate custom designs and precise location of highly-diffractive macro-spheres, and used it for on-chip detection of bacteria in a flow. In the second part of the work, we aimed at developing a technique to assess the status of individual tumor and immune cells in the conventional tissue sections used in the clinics. The first step was to combine fluorescence-based image segmentation with automated microfluidics to quantify, at the single-cell level, the overexpression of two proteins used as biomarkers for breast cancer diagnostics. We first assessed our method on biological controls such as breast cancer cell lines, and then combined it with ...

EPFL2019