Search

The cellular environment modifies cellular phenotypes, in particular, the stress response phenotype, which easily exhibits high phenotypic heterogeneity due to the common characteristics of its regulatory networks. The aim of this work is to quantify and interpret the impact of collagen type I, a major component of the cellular environment, on the phenotypic heterogeneity of the cellular response. Our approach combines in an original way the monitoring of the response of a single cell and the mathematical modeling of the network. After a detailed statistical description of the phenotypic heterogeneity of the cellular response, the mathematical modeling explains how the observed changes can be explained by an induced increase in the average expression of a central protein of the regulatory network. The predictions of the data-driven model are fully consistent with the biochemical measurements performed. The framework presented here is also a new general methodology to study phenotypic heterogeneity, although we focus here on the response to proteotoxic stress in HeLa cells.

A common signature of cell adaptation to stress is the improved resistance upon priming by prior stress exposure. In the context of hyperthermia, priming or preconditioning with sublethal heat shock can be a useful tool to confer thermotolerance and competitive advantage to cells. In the present study, we develop a data-driven modeling framework that is simple and generic enough to capture a broad set of adaptation behaviors to heat stress at both molecular and cellular levels. The model recovers the main features of thermotolerance and clarifies the tradeoff principles which maximize the thermotolerance effect. It therefore provides an effective predictive tool to design preconditioning and fractionation hyperthermia protocols for therapeutic purpose.

We developed an experimental methodology to monitor response dynamics of single human cells to hydrogen peroxide. Our approach is based on fluidic control of both magnitude and time-evolution of the external perturbation, and on high-throughput imaging of intracellular fluorescent redox reporters. We applied step stimulus to MCF7 cells with hydrogen peroxide concentration in the range of 10 to 500μM. First, our data highlights dynamic adaptation of Reactive Oxygen Species (ROS) scavenging system at several time scales. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) level is rapidly restored within 10 min after a transient decrease, while glutathione (GSH) redox potential is slowly driven back toward pre-stimulus level (within one hour). Extra-cellular glucose is necessary for adaptation of both NADPH level and GSH system. Second, our results also reveal large cell-to-cell variability in the dynamic response to external ROS. Our experimental approach is readily usable to monitor other cellular redox systems such as thioredoxins. As response-heterogeneity can lead to fractional killing, we finally discuss how our methodology can be an opportunity to link dynamics of ROS scavenging and cellular fate such as cell death.

Cell-to-cell variability in stress response is a bottleneck for the construction of accurate and predictive models that could guide clinical diagnosis and treatment of diseases as for instance cancers. Indeed such phenotypic heterogeneity can lead to fractional killing and persistence of a subpopulation of cells resistant to a given treatment. The heat shock response network plays a major role in protecting the proteome against several types of injuries. We combine high-throughput measurements and mathematical modeling to unveil the molecular origin of the phenotypic variability in the heat shock response network. Although the mean response coincides with known biochemical measurements, we found a surprisingly broad diversity in single cell dynamics with a continuum of response amplitudes and temporal shapes for several stimuli strengths. We theoretically predict that the broad phenotypic heterogeneity is due to network ultrasensitivity together with variations in the expression level of chaperons controlled by heat shock factor 1. We experimentally confirm this prediction by mapping the response amplitude to concentrations chaperons and heat shock factor 1 expression level.

In this chapter, we will discuss singlet oxygen direct production schemes that imply only natural transition of molecular oxygen that we name direct photoactivation. Our purpose will be to highlight that despite its low efficiency compared to nondirect production methods (ca. 104–105 times less efficient that photosensitized production), direct photoactivation can be seen as a powerful tool to study the chemistry and biology of singlet oxygen. First, a brief history of the discovery of the molecular oxygen electronic transitions under consideration in this chapter is presented. We will briefly review pioneering studies that have allowed a better understanding of peculiar spectroscopic properties of O2. Secondly, we discuss recent works that focus on singlet oxygen direct photoactivation in liquid solvents at room temperature and atmospheric pressure. These studies have led to estimation of absorption cross sections of molecular oxygen bands in homogeneous liquids as well as in heterogeneous solutions. Finally, we will discuss singlet oxygen direct photoactivation in biological systems as an alternative to traditional photodynamic therapy. We highlight potential outcomes of this production scheme for both therapeutic use and to study cellular oxidative stress response.

The structure of the excited $2^{3}$S and $2^{3}$P triplet states of $^{3}$He and $^{4}$He in an applied magnetic field B is studied using different approximations of the atomic Hamiltonian. All optical transitions (line positions and intensities) of the 1083 nm $2^{3}$S-$2^{3}$P transition are computed as a function of B. The effect of metastability exchange collisions between atoms in the ground state and in the $2^{3}$S metastable state is studied, and rate equations are derived, for the populations these states in the general case of an isotopic mixture in an arbitrary field B. It is shown that the usual spin-temperature description remains valid. A simple optical pumping model based on these rate equations is used to study the B-dependence of the population couplings which result from the exchange collisions. Simple spectroscopy measurements are performed using a single-frequency laser diode on the 1083 nm transition. The accuracy of frequency scans and of measurements of transition intensities is studied. Systematic experimental verifications are made for B=0 to 1.5 T. Optical pumping effects resulting from hyperfine decoupling in high field are observed to be in good agreement with the predictions of the simple model. Based on adequately chosen absorption measurements at 1083 nm, a general optical method to measure the nuclear polarisation of the atoms in the ground state in an arbitrary field is described. It is demonstrated at $B\sim$0.1 T, a field for which the usual optical methods could not operate.

We report on optical pumping experiments in helium-3 using a new broad-band, all-fiber laser at 1083 nm. The kinetics of polarization build-up are compared to the numerical results of an appropriate model. Effects of laser power and bandwidth are discussed.

Optical pumping of helium makes use of the 2 3S–2 3P transition at 1083 nm. We report on a study of this transition in magnetic fields up to 1.5 T. Based on these results, an optical method to measure nuclear polarisation in arbitrary field has been developed. Preliminary results on optical pumping at 0.1 T are presented.

Singlet oxygen is produced through the O2 [3∑g–] → O2 [1Δg] transition of molecular oxygen upon direct 1270 nm laser irradiation in water. The non water-soluble 1,3-diphenylisobenzofuran (DPIBF) is encapsulated in lipid nanocapsules (LNCs) in D2O and used to monitor the formation of singlet oxygen in these microheterogeneous systems. A two-pseudophase kinetic model for 1O2 distribution is applied to LNCs dispersions in D2O. From a simple analytical expression of the trap disappearance rate, both the σ1270 absorption cross section of the O2 [3∑g–] → O2 [1Δg] transition and the reactivity of singlet oxygen toward DPIBF can be evaluated simultaneously and independently in LNCs dispersions in D2O, and σ1270 can be extrapolated to pure deuterated water. The water-soluble chemical trap tetrapotassium rubrene-2,3,8,9-tetracarboxylate (RTC) is used to monitor the formation of singlet oxygen in water and D2O, and σ1270 is also determined in these solvents. Absorption spectrum for the transition O2 [3∑g–] → O2 [1Δg...

Hypericin (Hy), a naturally occurring photosensitizer (PS), is extracted from Hypericum perforatum plants, commonly known as St. John's wort. The discovery of the in vitro and in vivo photodynamic activities of hypericin as a photosensitizer generated great interest, mainly to induce a very potent antitumoral effect. However, this compound belongs to the family of naphthodianthrones which are known to be poorly soluble in physiological solutions and produce non-fluorescent aggregates (A. Wirz et al., Pharmazie, 2002, 57, 543; A. Kubin et al., Pharmazie, 2008, 63, 263). These phenomena can reduce its efficiency as a photosensitizer for the clinical application. In the present contribution, we have prepared, characterized, and studied the photochemical properties of Hy-loaded lipid nanocapsule (LNC) formulations. The amount of singlet oxygen (1O2) generated was measured by the use of p-nitroso-dimethylaniline (RNO) as a selective scavenger under visible light irradiation. Our results showed that Hy-loaded LNCs suppressed aggregation of Hy in aqueous media, increased its apparent solubility, and enhanced the production of singlet oxygen in comparison with free drug. Indeed, encapsulation of Hy in LNCs led to an increase of 1O2 quantum yield to 0.29-0.44, as compared to 0.02 reported for free Hy in water. Additionally, we studied the photodynamic activity of Hy-loaded LNCs on human cervical carcinoma (HeLa) and Human Embryonic Kidney (HEK) cells. The cell viability decreased radically to 10-20% at 1 μM, reflecting Hy-loaded LNC25 phototoxicity.

Singlet oxygen ((1)O(2)) is an electronic state of molecular oxygen which plays a major role in many chemical and biological photo-oxidation processes. It has a high chemical reactivity which is commonly harnessed for therapeutic issues. Indeed, (1)O(2) is believed to be the major cytotoxic agent in photodynamic therapy. In this treatment of cancer, (1)O(2) is created, among other reactive species, by an indirect transfer of energy from light to molecular oxygen via excitation of a photosensitizer (PS). This PS is believed to be necessary to obtain an efficient (1)O(2) production. In this paper, we demonstrate that production of (1)O(2) is achieved in living cells from PS-free 1270 nm laser excitation of molecular oxygen. The quantity of (1)O(2) produced in this way is sufficient to induce an oxidative stress leading to cell death. Other effects such as thermal stress are discriminated and we conclude that cell death is only due to (1)O(2) creation. This new simplified scheme of (1)O(2) activation can be seen as a breakthrough for phototherapies of malignant diseases and/or as a noninvasive possibility to generate reactive oxygen species in a tightly controlled manner.

We report on the development of a tunable Raman fiber ring laser especially designed for the investigation of the 3Σ(-)(g) →1 Δg transition of molecular oxygen. Singlet oxygen (1Δg) is a reactive species of importance in the fields of biology, photochemistry, and phototherapy. Tunability of the Raman fiber ring laser is achieved without the use of an intracavity tunable bandpass filter and the laser thus achieves a slope efficiency only obtained up to now in Perot-Fabry cavities. A measurement of the action spectrum of a singlet oxygen trap is made in air-saturated ethanol and acetone to demonstrate the practical application of the tunable Raman fiber ring laser for the investigation of the 3Σ(-)(g) →1 Δg transition of molecular oxygen.

Large amounts of hyperpolarized 3He gas with high nuclear polarization rates are required for use in neutron spin filters or nuclear magnetic resonance imaging of human lung. Very high efficiency can be obtained by metastability exchange optical pumping using multimode lasers to excite the 23S-23P transition at 1083 nm. Broadband Ytterbium-doped tunable fiber lasers have been designed for that particular application. Different options for the architecture of the fiber oscillator are presented and compared. Emphasis is given to a linear cavity configuration that includes a high reflectivity fiber mirror and a low reflectivity tunable fiber Bragg grating. Optical measurements are performed to finely characterize the spectral behavior of the lasers. Atomic response is also quantitatively probed to assess the optimal design of the oscillator for optical pumping. Multimode operation matching the 2 GHz Doppler-broadened helium resonance line and tunability over more than 200 GHz are demonstrated. Boosting the output of this fiber laser with a Yb-doped fiber power amplifier, all-fiber devices are built to provide robust, high power turn-key sources at 1083 nm for improved production of laser polarized 3He., Comment: Submitted July 2003, published online December 2003 (http://www.springerlink.com). V3 symbols corrected in Figs 10-11; minor final changes included

Metastability exchange optical pumping of helium-3 usually operates at low pressures. This is not well suited for the production of a highly polarised and dense gas of helium-3 required in particular for lung MRI applications. We show that an improvement results from performing the optical pumping at higher pressures in the presence of a sufficiently high magnetic field.We first point out the importance of correlations between atomic velocity and nuclear orientation for excited atoms in the plasma which are coupled to the 1083 nm optical pumping light. We have studied the effect of these correlations on optical pumping efficiency through systematic experimental measurements in conjunction with a detailed model.We then focus on non standard conditions for optical pumping in high magnetic field and high pressure. Here we performed a detailed spectroscopic study of Zeeman effects on the 1083 nm transition of helium. It gave a preciseidentification of positions and intensities of all transitions, and in turn allowed us to we develop a new optical measurement method of nuclear polarisation in arbitrary field. We have demonstrated that the presence ofintense 1083 nm light significantly increases the population of helium metastable molecules in the plasma, in particular at high pressure., Un inconvénient important du pompage optique indirect de l'hélium-3 traditionnel est qu'il opère à des pressions faibles qui ne sont en particulier pas favorables aux applications médicales d'IRM nécessitant la production d'hélium-3 gazeux très polarisé et dense : une voie prometteuse est d'utiliser un fort champ magnétique quipermet d'opérer plus efficacement à plus forte pression.Dans une première partie nous faisons apparaître l'importance des corrélations entre vitesse et orientation nucléaire pour les atomes excités présents dans le plasma et soumis à la lumière de pompage optique à 1083 nm. Nous avons étudié leur effet sur l'efficacité du pompage optique tant au moyen d'un modèle détaillé que par des mesures expérimentales systématiques. Nous montrons la grande importance des caractéristiques spectrales fines des lasers à 1083 nm.Dans la deuxième partie, nous nous intéressons plus spécifiquement à des conditions non standard de pompage optique, en présence d'un fort champ magnétique et à forte pression. Nous avons réalisé une étude spectroscopique détaillée de l'effet Zeeman de la transition à 1083 nm de l'hélium qui permet d'identifier précisément les positions de ces transitions et leurs intensités, ce qui nous a permis de mettre au point une technique originale de mesureoptique de la polarisation nucléaire en champ quelconque. Nous avons démontré que la présence de lumière à 1083 nm augmente significativement la population de molécules métastables au sein d'un plasma d'hélium, en particulier à forte pression.

Singlet oxygen plays a major role in many chemical and biological photo-oxidation processes. It has a high chemical reactivity, which is commonly harnessed for therapeutic issues. Indeed, singlet oxygen is recognized as the major cytotoxic agent in photodynamic therapy. In this treatment of cancer, singlet oxygen is created, among other reactive species, by an indirect transfer of energy from light to molecular oxygen via excitation of a photosensitizer. In this paper, we show that the conventional singlet oxygen production scheme can be simplified. Production of singlet oxygen is achieved in living cells from photosensitizer-free 1270 nm laser excitation of the electronic ground state of molecular oxygen. The quantity of singlet oxygen produced in this way is sufficient to induce an oxidative stress leading to cell death. Other effects such as thermal stress are discriminated, and we conclude that cell death is only due to singlet oxygen creation. This new simplified scheme of singlet oxygen activation can be seen as a breakthrough for phototherapies of malignant diseases and/or as a non-invasive possibility to generate reactive oxygen species in a tightly controlled manner.

Cell transfection aims at introducing foreign material inside a biological living cell. This intracellular drug transport process is at the heart of many diagnosis procedures and a key step in gene therapy. In view of these stakes, high performances are of importance, namely the success rate (positive and viable cells), the quality (uptake) and the throughput (treatment rate). Research has been conducted to understand how the spontaneous uptake of macromolecules (endocytosis) is limited while workarounds to bypass cell barriers for controlled intracellular drug release has received consistentpayoff. However, up-to-date techniques mimicking bioelements or introducing physical means suffer several drawbacks. Nevertheless, Gold nanoparticle (AuNP) mediated photoporation has stood out as a promising tool to permeabilize cellmembranes through laser induced Vapour NanoBubble (VNB) generation. Based on these VNBs, a novel microfluidic approach is presented in this work, offering high throughput prospects with no contact with AuNPs to retrieve AuNP-freetreated viable cells.We set up a new optofluidic experiment integrating the use of advected AuNPs to achieve VNB generation near suspended cells. This method allows high throughput, high efficiency and low cytotoxicity drug delivery. Based on hydrodynamic focusing, the approach allows the control and tuning of the cell-to-AuNP distance. With the optical setup used, throughput ranging from 10³ to 10⁴ cells /min can be achieved. Interestingly, it yields a better viability than the mixed AuNP-cell (80% vs. 40%). As a matter of fact, viability increases with distance whereas transfection rate decreases (30% vs 50%). Both a transfection and a rheological tool, our optofluidic device is a bridge toward triggered single cell transfection, circumventing barriers to the clinical deployment of emerging cellular therapies., La transfection de cellules vise à injecter du matériel génétique dans des cellules biologiques vivantes. L'incorporation intracellulaire de molécules exogènes est un processus nécessaire en diagnostique et une étape clef dans la thérapie génique. Ces enjeux nécessitent des performances en terme d'efficacité (cellules viables positives), de qualité (incorporation) et de rendement (cadence de traitement). Des recherches sur les mécanismes limitant l'incorporation naturelle de macromolécules (endocytose) sont menées et des techniques sont développées pour surmonter les barrières cellulaires et réaliser le transfert intracellulaire de manière contrôlée. Cependant, ces systèmes biologiques, chimiques ou physiques présentent certains inconvénients. La photoporation par intermédiaire de nanoparticules d'or (AuNP) réalise néanmoinsdes performances intéressantes en perméabilisant les membranes cellulaires grâce à des nano-bulles de vapeur (VNB) générées par laser. Une nouvelle approche est présentée ici de photoporation en environnement microfluidique dans le but d'améliorer le rendement et de garantir la séparation entre les cellules et les AuNP afin d'obtenir un échantillon sans éléments cytotoxiques.Nous avons développé un nouveau système opto-fluidique intégrant des AuNP en suspension pour générer des VNB au voisinage des cellules. Cette méthode permet un rendement et un taux de transfection élevés et réduit la cytotoxicité de la transfection. L'approche permet le contrôle de la distance entre cellules et AuNP. En l'état, le rendement du dispositif peut atteindre 10³ à 10⁴ cell. /min . Surtout, la séparation réalise une meilleure viabilité des cellules photoporées en comparaison avec la méthode sans séparation (80% contre 40%) mais réduit le taux de transfection (30% contre 50%) : l'augmentation de la distance améliore la viabilité. Outil rhéologique ou de transfection, ce dispositif est une étape supplémentaire vers la transfection efficace et bio-compatible de cellule unique, un atout majeur pour le développement clinique des nouvelles thérapies cellulaires.