Spectroscopie diélectrique hyperfréquence pour l'analyse cellulaire : pertinence pour l'évaluation de l'efficacité d'agents anti-cancéreux

Les techniques optiques dédiées à l'analyse de cellules entières classiquement utilisées dans les laboratoires de biologie cellulaire (microscopie, cytométrie en flux), nécessitent généralement un large éventail de fluorochromes et marqueurs spécifiques afin de cibler des composés ou des fonctions cellulaires. La grande puissance informative de ces techniques optiques conventionnelles exige une ou plusieurs étapes de marquage souvent précédées d'une fixation et d'une perméabilisation de l'échantillon cellulaire. Les marquages nécessaires requièrent donc une méthodologie lourde à mettre en œuvre, ils sont complexes, souvent coûteux et systématiquement chronophages. De plus, après avoir été marquées, les cellules sont la plupart du temps altérées et leur suivi au cours du temps est alors difficile. Dans ce contexte, la spectroscopie diélectrique hyperfréquence se positionne comme une méthode pertinente ne nécessitant aucune molécule exogène pour analyser les cellules, pouvant ainsi répondre au besoin de suivre des processus biologiques au cours du temps. En effet, cette méthode exploite l'interaction champ électromagnétique/matériel biologique avec l'extraction de la permittivité relative obtenue de l'échantillon biologique d'intérêt en fonction de la fréquence du champ appliqué. De plus, les mesures sont réalisées directement dans le milieu de culture des cellules permettant des analyses sur des temps longs sans altérer l'intégrité cellulaire. Le travail mené au cours de cette thèse se situe donc à l'interface entre trois vastes domaines scientifiques que sont la biologie cellulaire, l'électronique hyperfréquence et les micro-technologies. Ce travail a nécessité une succession d'étapes de mises au point, suivant un cheminement à complexité croissante, conduisant finalement à l'obtention de signatures hyperfréquences cellulaires spécifiques. Les résultats de suivi de la mort apoptotique de cellules de lymphome B (DOHH2) induite par des agents anti-cancéreux montrent que la technique permet de distinguer des évènements à des temps plus précoces qu'avec la cytométrie en flux. La spectroscopie diélectrique hyperfréquence présente donc des capacités de détection inédites par rapport aux techniques classiques de la biologie cellulaire. Actuellement utilisée sur des lignées de lymphome B, la technique offre de nombreuses applications potentielles sur cellules de patient et on peut imaginer son intérêt pour l'évaluation de l'efficacité de diverses drogues, avec l'objectif à plus long terme de mieux adapter les thérapies anti-cancéreuses aux patients.Optical techniques dedicated to the analysis of whole cells conventionally used in cell biology laboratories (microscopy, flow cytometry), generally require a variety of fluorochromes and specific markers to target cell compounds or functions. The high level of information provided by these conventional techniques requires one or more steps of labeling often preceded by fixation and permeabilization of the cell sample. Labeling therefore requires a heavy methodology, it is complex, often expensive and systematically time-consuming. In addition, after being labeled, the cells are usually altered and their monitoring over time is difficult. In this context, microwave dielectric spectroscopy is a relevant method that requires no exogenous molecule to analyze cells and so it can be used to monitor biological processes over time. Indeed, this method exploits the electromagnetic field / biological material interaction with the extraction of the relative permittivity obtained from the biological sample of interest versus the frequency of the applied field. Furthermore, the measurements are performed directly in the cell culture medium enabling cell analysis over long time without altering the cellular integrity. The work in this thesis is situated at the interface between three broad areas of science that are microwave electronics, cell biology and micro-technologies. This work involved a series of steps for the set-up with increasing complexity, ultimately leading to the production of specific microwave cell signatures. The results of monitoring of the apoptotic cell death of B-cell lymphoma (DOHH2) induced by anti-cancer agents show that the technique can distinguish events at earlier time compared to flow cytometry. Microwave dielectric spectroscopy therefore provides unprecedented detection capabilities compared to conventional techniques of cell biology. Currently used on B lymphoma lines, the technique has many potential applications and one can imagine to assess the effectiveness of various drugs on patient cells, with the aim to better adapt anticancer therapies to patients in the future

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Biological Cells Proliferation in Microwave Microsystems

International audienceThis paper presents the biological compatibility of a microwave analyzing microsystem of living cells through the indicator of cells proliferation. The cells under investigation correspond to adherent cells of Normal Rat Kidney (NRK). In a first time, both their adhesion and proliferation into the highfrequency-based micro-device have been successfully obtained. In a second step, microwave signals have been applied at different power levels. Experimental studies demonstrate that microwave power levels up to +8,6 dBm do not impact cells proliferation

Erosion Based Visibility Preprocessing

This paper presents a novel method for computing visibility in 2.5D environments based on a novel theoretical result: the visibility from a region can be conservatively estimated by computing the visibility from a point using appropriately "shrunk" occluders and occludees. We show how approximate, yet conservative, shrunk objects can be efficiently computed in an urban environment. The technique provides a tighter potentially visible set (PVS) compared to the original method in which only occluders are shrunk. Finally, theoretical implications of the shrinking theorem are discussed, opening new research directions

Microwave dielectric spectroscopy of cell membrane permeabilization with saponin on human B lymphoma cells

International audienceThis paper aims to contribute to the demonstration of the microwave dielectric spectroscopy relevance for biological applications. We demonstrate indeed that microwave dielectric spectroscopy may significantly identify the membrane permeabilization (and its consequence on cells) of cells induced by chemo-treatment. Standard deviations on both real and imaginary parts (resp.) of the permittivity between several experiments are below 0.08 and 0.06 (resp.), whereas the contrasts on these two parameters reach 1.92 and 1.30 (resp.) at 10 GHz. The contrasts to the standard deviation ratio are then above 22, which permits to convince the biologists that the technique can significantly and efficiently detect the viability state of a cells population in their traditional culture medium. Moreover, these results permits to predict that such a technique can evaluate the viability of a cells population with few percents of accuracy

Label-free discrimination of human lymphoma cell sub-populations with microwave dielectric spectroscopy

International audienceThis paper deals with the label-free microwave die-lectric characterization of B lymphoma cell lines, the OCI-LY7, OCI-LY10 and OCI-LY18 ones, and demonstrates that the technique is able to discriminate these three sub-population cell lines with their different dielectric properties. Proof is given that such a differentiation is not due to a variation of cell size, viability or cells concentration, but to internal molecular differences. This constitutes a major result as it demonstrates for the first time that microwave dielectric spectroscopy is sufficiently sensitive using di-electric properties variations to distinguish very similar biological cell lines of the same phenotype, B lymphoma cell lines in our case, whereas such a detection is traditionally performed with marker-based and costly equipment or using heavy genetic methods

Why using High Frequency Dielectric Spectroscopy for biological analytics?

International audienceHeterogeneity of cancer patients increases difficulties to find the best treatment for each patient. To adapt the treatment, the solution would be to detect the patient response in culture dish, instead to wait several months the result of the treatment measured by the disease regression. This would allow testing several treatments with different doses for the best response. Classical analytical techniques such as flow cytometry, confocal microscopy, immunohistochemistry, western blot, gene expression profiling, proteomics or metabolomics are not usually used for this kind of tests because of the heaviness of their implementation. High Frequency Dielectric Spectroscopy (HFDS) could be the solution while this approach compared to others presents several advantages: miniaturizing, processing speed, small sample, no invasive preparations such as fixation, no labeling and possible real time measurement. This opens the door to the development of HFDS for non-invasive biological analysis

Microwave dielectric spectroscopy for biological cells suspensions analysis and proliferation evaluation

International audienceThis paper presents the capabilities of microwave dielectric spectroscopy for biological cells suspensions analysis. The developed biosensor integrates both microfluidic channel for biological samples manipulation together with a microwave circuit for dielectric spectroscopy. Experimental investigation validates the success and accuracy of the microwave dielectric spectroscopy technique. This technique is applied with various cells concentrations in suspension in their culture medium. Results demonstrate that both real and imaginary parts of the contrast of the dielectric permittivity are proportional to the cells concentration with a correlation coefficient (R-square) greater than 0.99. The technique is consequently suitable for proliferation evaluation of cells. More widely, this work contributes to settle down the capabilities of microwave sensing for biochemical and bio-cellular analysis applications

Sub-microliter microwave dielectric spectroscopy for identification and quantification of carbohydrates in aqueous solution

International audienceThis paper presents the technique and associated miniature RF biosensor dedicated to the microwave dielectric spectroscopy of carbohydrates in aqueous solution. Small amounts of liquid in the range of microliter are required. This sensor includes an area for electromagnetic field analysis that operates in the near field, while a microfluidic channel allows precisely bringing and locating the liquid solution to be studied on the sensing zone. Different carbohydrates were measured from 40 MHz to 40 GHz with relevant concentrations for biologists and chemists. Derived from the extracted complex relative permittivity of the examined liquids, this paper shows that each biomolecules type exhibits a specific dielectric signature, which allows their identification and quantification