Grammaire analytique et grammaire philosophique

Dans un premier temps, je voudrais développer la thèse selon laquelle les mathèmes de Lacan peuvent être considérés comme la grammaire de l'inconscient structuré comme un langage¹. Cela Lacan ne l'a jamais dit, mais je considère que cette hypothèse éclaire considérablement le statut des mathèmes et, plus généralement, le jeu de langage de la psychanalyse. Dans un second temps, je comparerai cette grammaire de Lacan à la grammaire philosophique de Wittgenstein. J'attends de cette comparaison un surcroît de lumière quant à la compréhension de la relation entre philosophie et psychanalyse.

Introduction to fluorescence probing of biological membranes.

Fluorescence is one of the most powerful and commonly used tools in biophysical studies of biomembrane structure and dynamics that can be applied on different levels, from lipid monolayers and bilayers to living cells, tissues, and whole animals. Successful application of this method relies on proper design of fluorescence probes with optimized photophysical properties. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Being smaller than the membrane width they can sense the gradients of these parameters across the membrane. We present examples of novel dyes that achieve increased spatial resolution and information content of the probe responses. In this respect, multiparametric environment-sensitive probes feature considerable promise.

Tuning excited-state proton transfer dynamics of a 3-hydroxychromone dye in supramolecular complexes via host-guest steric compatibility.

The photophysics of 2-(2'-benzofuryl)-3-hydroxychromone (BFHC) is remarkably modulated in its complexes with macrocyclic hosts such as ß-cyclodextrin (ß-CD), hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and methyl-ß-cyclodextrin (M-ß-CD). BFHC exhibits dual emission bands, attributable to excited normal (N*) and tautomer (T*) forms, where the latter originates from the former through an excited-state intramolecular proton transfer (ESIPT) reaction. Fluorescence lifetimes of the tautomer, along with the intensity ratio (IT*/IN*) of the dual emission bands, and the fluorescence quantum yield (Φ) of the dye, increase significantly in the order ß-CD < HP-ß-CD < M-ß-CD to indicate increasing hydrophobicity of the dye environment in the host CD cavity. In accordance with this increasing hydrophobicity of the dye environment, the ESIPT dynamics of BFHC becomes increasingly fast in the host cavity in the order ß-CD < HP-ß-CD < M-ß-CD. Binding constant data and molecular modeling studies indicate that the increasing order of the faster ESIPT dynamics originates from an increasingly tight host-guest spatial fit, which causes increasingly strong dehydration of the BFHC dye. Steric compatibility in size and shape between the host cavity and the guest, which modulates the tightness of the host-guest spatial fit and hence the extent of hydration, is a key factor for tuning the proton transfer dynamics since water molecules perturb the ESIPT reaction and quench the fluorescence of BFHC.

Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

Sentinel lymph nodes fluorescence detection and imaging using Patent Blue V bound to human serum albumin.

Patent Blue V (PBV), a dye used clinically for sentinel lymph node detection, was mixed with human serum albumin (HSA). After binding to HSA, the fluorescence quantum yield increased from 5 × 10(-4) to 1.7 × 10(-2), which was enough to allow fluorescence detection and imaging of its distribution. A detection threshold, evaluated in scattering test objects, lower than 2.5 nmol × L(-1) was obtained, using a single-probe setup with a 5-mW incident light power. The detection sensitivity using a fluorescence imaging device was in the µmol × L(-1) range, with a noncooled CCD camera. Preclinical evaluation was performed on a rat model and permitted to observe inflamed nodes on all animals.

Ab initio study of the solvent H-bonding effect on ESIPT reaction and electronic transitions of 3-hydroxychromone derivatives.

The electronic transitions occurring in 4-(N,N-dimethylamino)-3-hydroxyflavone (DMAF) and 2-furanyl-3-hydroxychromone (FHC) were investigated using the TDDFT method in aprotic and protic solvents. The solvent effect was incorporated into the calculations via the PCM formalism. The H-bonding between solute and protic solvent was taken into account by considering a molecular complex between these molecules. To examine the effect of the H-bond on the ESIPT reaction, the absorption and emission wavelengths as well as the energies of the different states that intervene during these electronic transitions were calculated in acetonitrile, ethanol and methanol. The calculated positions of the absorption and emission wavelengths in various solvents were in excellent agreement with the experimental spectra, validating our approach. We found that in DMAF, the hydrogen bonding with protic solvents makes the ESIPT reaction energetically unfavourable, which explains the absence of the ESIPT tautomer emission in protic solvents. In contrast, the excited tautomer state of FHC remains energetically favourable in both aprotic and protic solvents. Comparing our calculations with the previously reported time-resolved fluorescence data, the ESIPT reaction of DMAF in aprotic solvents is reversible because the emitting states are energetically close, whereas in FHC, ESIPT is irreversible because the tautomer state is below the corresponding normal state. Therefore, the ESIPT reaction in DMAF is controlled by the relative energies of the excited states (thermodynamic control), while in FHC the ESIPT is controlled probably by the energetic barrier (kinetic control).

Sensing micelle hydration by proton-transfer dynamics of a 3-hydroxychromone dye: role of the surfactant headgroup and chain length.

The dynamics of the excited-state intramolecular proton-transfer (ESIPT) reaction of 2-(2'-furyl)-3-hydroxychromone (FHC) was studied in micelles by time-resolved fluorescence. The proton-transfer dynamics of FHC was found to be sensitive to the hydration and charge of the micelles, demonstrated through a decrease of the ESIPT rate constant (k(PT)) in the sequence cationic → nonionic → anionic micelles. A remarkably slow ESIPT with a time constant (τ(PT)) of ~100 ps was observed in the anionic sodium dodecyl sulfate and sodium tetradecyl sulfate micelles, whereas it was quite fast (τ(PT) ≈ 15 ps) in the cationic cetyltrimethylammonium bromide and tetradecyltrimethylammonium bromide micelles. In the nonionic micelles of Brij-78, Brij-58, Tween-80, and Tween-20, ESIPT occurred with time constants (τ(PT) ≈ 35-65 ps) intermediate between those of the cationic and anionic micelles. The slower ESIPT dynamics in the anionic micelles than the cationic micelles is attributed to a relatively stronger hydration of the negatively charged headgroups of the former than the positively charged headgroups of the latter, which significantly weakens the intramolecular hydrogen bond of FHC in the Stern layer of the anionic micelles compared to the latter. In addition, electrostatic attraction between the positively charged -N(CH(3))(3)(+) headgroups and the negatively charged 4-carbonyl moiety of FHC effectively screens the intramolecular hydrogen bond from the perturbation of water molecules in the micelle-water interface of the cationic micelles, whereas in the anionic micelles, this screening of the intramolecular hydrogen bond is much less efficient due to an electrostatic repulsion between its negatively charged -OSO(3)(-) headgroups and the 4-carbonyl moiety. As for the nonionic micelles, a moderate level of hydration, and the absence of any charged headgroups, causes an ESIPT dynamics faster than that of the anionic but slower than that of the cationic micelles. Furthermore, the ESIPT rate decreased with a decrease of the hydrophobic chain length of the surfactants due to the stronger hydration of the micelles of shorter chain surfactants than those of longer chain surfactants, arising from a less compact packing of the former surfactants compared to the latter surfactants.

Lactose-ornithine bolaamphiphiles for efficient gene delivery in vitro.

The development of new nonviral vectors characterized by high transfection efficiency and low cytotoxicity remains an important challenge in the field of gene delivery. Unsymmetrical bolaamphiphiles (bolas) appear as new emerging candidates for this application. In this work, new unsymmetrical bolas, bearing neutral lactonic acid and cationic ornithine residues at the two ends of a hydrophobic spacer, were synthesized and their properties were compared to analogues bearing a gluconic acid residue. The new bolas showed DNA binding and condensation at higher N/P ratios than their gluconic analogues, probably due to their larger neutral head group. Whereas the size of the complexes of the new bolas with DNA (bolaplexes) increased with N/P, as a result of charge neutralization, their formulations with DOPE at high N/P were of small size (ca. 200 nm). These DOPE formulations showed high transfection efficiency in different cell lines (HeLa, COS-7 and HepG2), close to that of jetPEI. Their cytotoxicity was relatively low, which allowed repetitive transfection in vitro. Fluorescence imaging showed that the bolaplexes bind rapidly to cell surface and internalize mainly through endocytosis. This work suggests a new type of efficient nonviral vectors based on bolaamphiphiles.

Specific implications of the HIV-1 nucleocapsid zinc fingers in the annealing of the primer binding site complementary sequences during the obligatory plus strand transfer.

Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (-) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(-)PBS annealing mechanism by activating a loop-loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (-)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(-)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can 'freeze' PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.

New unsymmetrical bolaamphiphiles: synthesis, assembly with DNA, and application for gene delivery.

The success in gene therapy relies strongly on new efficient gene delivery vectors. Nonviral vectors based on lipids and polymers constitute an important alternative to the viral vectors. However, the key problem with these vectors is the poor structural control of their DNA complexes. In the present work, following new design we synthesized unsymmetrical bolaamphiphiles, molecules bearing neutral sugar (gluconic acid) and dicationic ornithine head groups connected by different long hydrophobic spacers. Within this design, a positively charged headgroup is expected to bind DNA, the hydrophobic spacer is to drive the formation of a monolayer membrane shell around DNA, while the neutral group is to be exposed outside of the complex. Our fluorescence and gel electrophoresis data showed that self-assembly of bolas and their interaction with DNA depend strongly on the bola structure. The size of bola/DNA complexes (bolaplexes) estimated from dynamic light scattering data was ∼100 nm at low N/P (cationic nitrogen/DNA phosphate molar ratio), while at higher N/Ps it was significantly larger due to neutralization of their surface charge. Atomic force microscopy studies revealed nanostructural rod-shaped or spherical morphology of the bolaplexes. Transfection efficiency of the bolaplexes in vitro was significant when either DOPE or chloroquine were used as helping agents, suggesting that the key barrier for their internalization is the endosomal escape. Finally, all bolas showed low cytotoxicity (cell viability >80%). The present results show that bolas are prospective candidates for construction of nonviral gene delivery vectors. We believe that further optimization of polar head groups and a hydrophobic spacer in the bolas will lead to vectors with controlled small size and high transfection efficiency.

Extracellular haemoglobin, oxidative stress and quality of red blood cells relative to perioperative blood salvage.

BACKGROUND: The quality of blood-pack units in perioperative blood salvage was studied with respect to the presence of extracellular haemoglobin (Hb) and in terms of oxidative stress and the mechanical properties of red blood cells (RBC). METHODS: The study was performed on blood-pack units and patients' blood after retransfusion. Results were compared to those obtained in patients prior to autotransfusion. Free Hb, non-protein thiols (as antioxidant marker) and markers of oxidative stress (conjugated dienes, thiobarbituric reactive substances and protein carbonyl content) were measured. The mechanical properties of RBC were evaluated by their osmotic fragility, membrane fluidity by measuring the fluorescence anisotropy of an extrinsic probe and permeability by measurement of extracellular K(+) and lactate dehydrogenase. RESULTS: Despite washing, extracellular Hb concentrations remained high (up to 0.7 g/L in blood-pack units) and was associated with a decrease of haptoglobin in patients, despite a concomitant inflammatory syndrome. In blood-pack units, we observed a decrease in antioxidant markers along with an increase in oxidative stress markers in association with an alteration of RBC membrane properties. CONCLUSIONS: Haemolysis must be limited during perioperative blood salvage in order to prevent exposure to oxidative stress and improve the efficiency of autotransfusion.

Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: effects of cholesterol depletion and apoptosis.

Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.

Unusually slow proton transfer dynamics of a 3-hydroxychromone dye in protic solvents.

The photophysics of a 3-hydroxychromone dye, 2-(2-furyl)-3-hydroxychromone (FHC) was explored in different types of protic solvents by steady-state and time-resolved fluorescence spectroscopy. FHC exhibits a dual emission, attributable to the excited normal (N*) and tautomer (T*) forms resulting from an excited state intramolecular proton transfer (ESIPT) reaction (N*-->T*). The ESIPT rate decreases with an increase in the hydrogen-bond donating ability of protic solvents. The proton-transfer dynamics is found to be unusually slow ( approximately 10(3) times slower than 3-hydroxyflavone) in strong hydrogen-bond donating solvents like methanol, trifluoroethanol or formamide, where it occurs on a time scale of approximately 100-260 picoseconds. This slow dynamics is likely to be related to the intermolecular solvent-solute H-bonding interactions that compete with the intramolecular H-bond in FHC required for the ESIPT reaction. These data provide a physical background for recent applications of this dye as a fluorescence probe of the microenvironment in biomolecules.

Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes.

We review the main trends in the development of fluorescence probes to obtain information about the structure, dynamics, and interactions in biomembranes. These probes are efficient for studying the microscopic analogs of viscosity, polarity, and hydration, as well as the molecular order, environment relaxation, and electrostatic potentials at the sites of their location. Progress is being made in increasing the information content and spatial resolution of the probe responses. Multichannel environment-sensitive probes that can distinguish between different membrane physicochemical properties through multiple spectroscopic parameters show considerable promise.

Dual-fluorescence probe of environment basicity (hydrogen bond accepting ability) displaying no sensitivity to polarity.

3-Hydroxyquinolones (3HQs) are a new class of water soluble dual fluorescence probes that can monitor both polarity and basicity (H-bond accepting ability) parameters. Both parameters play an important role in proteins and lipid membranes. Nevertheless, no method exists actually to measure the basicity parameter separately from the polarity. To achieve this aim, we synthesized 2-benzofuryl-3-hydroxy-4(1H)-quinolone (3HQ-Bf) and characterized its photophysical properties by UV, steady-state and time-resolved fluorescence spectroscopy. Due to its extended conjugation and totally planar conformation, 3HQ-Bf is characterized by a high fluorescence quantum yield. In solution, this dye shows an excited state intramolecular proton transfer (ESIPT) reaction resulting in two tautomer bands in the emission spectra. The ESIPT reaction can be considered as irreversible and is governed by rate constants from 0.6 to 8 x 10(9) s(-1), depending on the solvent. The analysis of the spectral properties of 3HQ-Bf in a series of organic solvents revealed a marginal sensitivity to the solvent polarity, but an exquisite sensitivity to solvent basicity, as shown by the linear dependence of the logarithm of the emission bands intensity ratio, log(I(N*)/I(T*)), as well as the absorption or emission maxima wavenumbers as a function of the solvent basicity parameter. This probe may find useful applications through coupling to a protein ligand, for characterizing the H-bond acceptor ability at the ligand binding site as well as for studying the basicity changes of lipid membranes during their chemo- and thermotropic conversions.

Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone.

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.

Combining fluorescence lifetime and polarization microscopy to discriminate phase separated domains in giant unilamellar vesicles.

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42 mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.

Excited state proton transfer and solvent relaxation of a 3-hydroxyflavone probe in lipid bilayers.

The photophysics of a ratiometric fluorescent probe, N-[[4'- N, N-diethylamino-3-hydroxy-6-flavonyl]methyl]- N-methyl- N-(3-sulfopropyl)-1-dodecanaminium, inner salt (F2N12S), incorporated into phospholipid unilamellar vesicles is presented. The reconstructed time-resolved emission spectra (TRES) unravels a unique feature in the photophysics of this probe. TRES exhibit signatures of both an excited-state intramolecular proton transfer (ESIPT) and a dynamic Stokes shift associated with solvent relaxation in the lipid bilayer. The ESIPT is fast, being characterized by a risetime of approximately 30-40 ps that provides an equilibrium to be established between the excited normal (N*) and the ESIPT tautomer (T*) on a time scale of 100 ps. On the other hand, the solvent relaxation displays a bimodal decay kinetics with an average relaxation time of approximately 1 ns. The observed slow solvent relaxation dynamics likely embodies a response of nonspecific dipolar solvation coupled with formation of probe-water H-bonds as well as the relocation of the fluorophore in the lipid bilayer. Taking into account that ESIPT and solvent relaxation are governed by different physicochemical properties of the probe microenvironment, the present study provides a physical background for the multiparametric sensing of lipid bilayers using ESIPT based probes.

Liquid ordered and gel phases of lipid bilayers: fluorescent probes reveal close fluidity but different hydration.

Hydration and fluidity of lipid bilayers in different phase states were studied using fluorescent probes selectively located at the interface. The probe of hydration was a recently developed 3-hydroxyflavone derivative, which is highly sensitive to the environment, whereas the probe of fluidity was the diphenylhexatriene derivative, 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene. By variation of the cholesterol content and temperature in large unilamellar vesicles composed of sphingomyelin or dipalmitoylphosphatidlycholine, we generated different phases: gel, liquid ordered (raft), liquid crystalline, and liquid disordered (considered as liquid crystalline phase with cholesterol). For these four phases, the hydration increases in the following order: liquid ordered << gel approximately liquid disordered < liquid crystalline. The membrane fluidity shows a somewhat different trend, namely liquid ordered approximately gel < liquid disordered < liquid crystalline. Thus, gel and liquid ordered phases exhibit similar fluidity, whereas the last phase is significantly less hydrated. We expect that cholesterol due to its specific H-bonding interactions with lipids and its ability to fill the voids in lipid bilayers expels efficiently water molecules from the highly ordered gel phase to form the liquid ordered phase. In this study, the liquid ordered (raft) and gel phases are for the first time clearly distinguished by their strong difference in hydration.