Etude spectroscopique de l'autofluorescence de cellules oesophagiennes saines et tumorales in vitro et développement d'un microspectroimageur confocal fibré destiné à l'exploration in vivo

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Alanine Screening Mutagenesis Establishes the Critical Inactivating Damage of Irradiated E. coli Lactose Repressor.

International audienceThe function of the E. coli lactose operon requires the binding of lactose repressor to operator DNA. We have previously shown that γ rradiation destabilizes the repressor-operator complex because the repressor loses its DNA-binding ability. It was suggested that the observed oxidation of the four tyrosines (Y7, Y12, Y17, Y47) and the concomitant structural changes of the irradiated DNA-binding domains (headpieces) could be responsible for the inactivation. To pinpoint the tyrosine whose oxidation has the strongest effect, four headpieces containing the product of tyrosine oxidation, 3,4-dihydroxyphenylalanine (DOPA), were simulated by molecular dynamics. We have observed that replacing Y47 by DOPA triggers the largest change of structure and stability of the headpiece and have concluded that Y47 oxidation is the greatest contributor to the decrease of repressor binding to DNA. To experimentally verify this conclusion, we applied the alanine screening mutagenesis approach. Tetrameric mutated repressors bearing an alanine instead of each one of the tyrosines were prepared and their binding to operator DNA was checked. Their binding ability is quite similar to that of the wild-type repressor, except for the Y47A mutant whose binding is strongly reduced. Circular dichroism determinations revealed small reductions of the proportion of α helices and of the melting temperature for Y7A, Y12A and Y17A headpieces, but much larger ones were revealed for Y47A headpiece. These results established the critical role of Y47 oxidation in modifying the structure and stability of the headpiece, and in reduction of the binding ability of the whole lactose repressor

Plasmon Waveguide Resonance: Principles, Applications and Historical Perspectives on Instrument Development

Plasmon waveguide resonance (PWR) is a variant of surface plasmon resonance (SPR) that was invented about two decades ago at the University of Arizona. In addition to the characterization of the kinetics and affinity of molecular interactions, PWR possesses several advantages relative to SPR, namely, the ability to monitor both mass and structural changes. PWR allows anisotropy information to be obtained and is ideal for the investigation of molecular interactions occurring in anisotropic-oriented thin films. In this review, we will revisit main PWR applications, aiming at characterizing molecular interactions occurring (1) at lipid membranes deposited in the sensor and (2) in chemically modified sensors. Among the most widely used applications is the investigation of G-protein coupled receptor (GPCR) ligand activation and the study of the lipid environment’s impact on this process. Pioneering PWR studies on GPCRs were carried out thanks to the strong and effective collaboration between two laboratories in the University of Arizona leaded by Dr. Gordon Tollin and Dr. Victor J. Hruby. This review provides an overview of the main applications of PWR and provides a historical perspective on the development of instruments since the first prototype and continuous technological improvements to ongoing and future developments, aiming at broadening the information obtained and expanding the application portfolio

Traumatismes de la dent permanente (complications et séquelles)

MONTROUGE-BUFR Odontol.PARIS5 (920492101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Journal of Drug Delivery Science and Technology

Trans-Resveratrol (RV) was encapsulated in multi-lamellar liposomes (MLLs) composed of a mixture of phosphatidylcholine (P100), Tween 80 (T80) and water. The P100-to-T80 ratio as well as their water content were chosen based on a previous study to promote transdermal transport of RV. The effect of RV on the size, elasticity, and charge of MLLs was evaluated as well as the effect of encapsulation on the apparent solubility of RV. The diffusion of RV and MLLs in artificial skin, namely Strat-M™, was monitored by confocal Raman imaging, and compared with that of a RV solution and empty MLLs, while the transdermal passage rate was measured by UV–vis spectrophotometry on both artificial and excised human skin. RV was found to remain localized in the outer layer of the skin with less than 3% passing through it over a 24-h period in both skin types. Encapsulation in MLLs drastically increased its transdermal passage: 73 ± 10% after 3 h of incubation on excised human skin, and 10% on Strat-M™ after 9 h. Whereas RV in its free form underwent cis isomerization, MLLs protected it up to 9h before undergoing chemical degradation after 24h

H/D Isotope Effects in Protein Thermal Denaturation: The Case of Bovine Serum Albumin

International audienceThe present work investigates the effects of HID isotopic sub stitution on the structural and thermodynamic stability of bovine serum albumin (BSA) in aqueous solution over the temperature range of 5-90 degrees C. Using far-ultraviolet circular dichroism, we have compared protein unfolding pathways in H(2)O and D(2)O. Our results show that BSA possesses similar conformations in H(2)O and D(2)O at temperatures below 50 degrees C but follows different unfolding pathways at higher temperatures. The presence of D(2)O retards the occurrence of irreversible thermal denaturation in BSA, as evidenced by a higher onset temperature of 58 degrees C, in contrast to 50 degrees C in H(2)O center dot D(2)O exhibits a protective effect on the domain structure during the early stages of domain denaturation. Following incubation at 90 degrees C over a period of minutes, D(2)O causes a rapid aggregation of BSA molecules. This behavior is not observed in H(2)O solutions. Meanwhile, H/D substitution does not influence the reversible structural transformation of the protein in a significant manner. Partly renatured BSA in H(2)O and D(2)O undergoes very similar reversible structural transformations during a second heating cycle

DUV Autofluorescence Microscopy for Cell Biology and Tissue Histology Biology of the Cell Biology of the Cell

International audienceAutofluorescence spectroscopy is a powerful tool for molecular histology and for following metabolic processes in biological samples as it does not require labelling. However, at the microscopic scale, it is mostly limited to visible and near infrared excitation of the samples. Several interesting and naturally occurring fluorophores can be excited in the UV and deep UV (DUV), but cannot be monitored in cellulo nor in vivo due to a lack of available microscopic instruments working in this wavelength range.To fulfil this need, we have developed a synchrotron-coupled DUV microspectrofluorimeter which is operational since 2010. An extended selection of endogenous autofluorescent probes that can be excited in DUV, including their spectral characteristics, is presented. The distribution of the probes in various biological samples, including cultured cells, soft tissues, bone sections and maize stems, is shown to illustrate the possibilities offered by this system.In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology

Synchrotron UV Fluorescence Microscopy Uncovers New Probes in Cells and Tissues

International audienceUse of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because it does not need external specific probes or labeling but instead allows use of the intrinsic fluorescence that exists for many biomolecules when excited in this wavelength range. Indeed, observation of label free biomolecules or active drugs ensures that the label will not modify the biolocalization or any of its properties. In the past, it has not been easy to accomplish DUV fluorescence imaging due to limited sources and to microscope optics. Two worlds were coexisting: the spectrofluorometric measurements with full spectrum information with DUV excitation, which lacked high-resolution localization, and the microscopic world with very good spatial resolution but poor spectral resolution for which the wavelength range was limited to 350 nm. To combine the advantages of both worlds, we have developed a DUV fluorescence microscope for cell biology coupled to a synchrotron beamline, providing fine tunable excitation from 180 to 600 nm and full spectrum acquired on each point of the image, to study DUV excited fluorescence emitted from nanovolumes directly inside live cells or tissue biopsies

Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase: ultrafast spectroscopy of a Trp→Phe mutant

International audienc