Photoaffinity labeling of the T cell receptor on cloned cytotoxic T lymphocytes by covalent photoreactive ligand.

The interaction of the T cell antigen receptor with a photoreactive antigenic peptide derivative bound covalently to the H-2Kd (Kd) molecule was studied by photoaffinity labeling on cloned, CD8 positive cytotoxic T lymphocytes. The Kd-restricted Plasmodium berghei circumsporozoite peptide 253-260 (YIPS-AEKI) was conjugated with iodo-4-azidosalicylic acid at the N terminus and with 4-azidobenzoic acid at the T cell receptor residue Lys-259. Cell-associated or soluble Kd molecules were photoaffinity-labeled with the peptide derivative by selective photoactivation of the N-terminal photoreactive group. Incubation of cell-associated or soluble covalent Kd-peptide derivative complexes (ligands) with cytotoxic T lymphocytes that recognized this peptide derivative and activation of the orthogonal photoreactive group resulted in specific photoaffinity labeling of the T cell receptor. The labeling was inhibitable by an anti-Kd antibody and was absent on Kd-restricted cytotoxic T lymphocytes of different specificity. The binding of the soluble ligand reached a maximum after 2-4 min at 37 degrees C, after 30 min at 18 degrees C, and after 3 h at 4 degrees C. In contrast, binding of the cell-associated ligand reached a transient maxima after 50 and 110 min at 37 and 18 degrees C, respectively. The degree of binding at 37 degrees C was approximately 30% lower than that at 18 degrees C. No binding took place at 4 degrees C. Inhibition studies with antibodies and drugs indicated that the binding of the cell-associated, but not the soluble ligand, was highly dependent on T cell-target cell conjugate formation, whereas the binding of the soluble ligand was greatly dependent on CD8.

Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin.

Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Synthesis and anticancer activity of long-chain isonicotinic ester ligand-containing arene ruthenium complexes and nanoparticles

Arene ruthenium complexes containing long-chain N-ligands L1 = NC5H4-4-COO-C6H4-4-O-(CH2)9-CH3 or L2 = NC5H4-4-COO-(CH2)10-O-C6H4-4-COO-C6H4-4-C6H4-4-CN derived from isonicotinic acid, of the type [(arene)Ru(L)Cl2] (arene = C6H6, L = L1: 1; arene = p-MeC6H4Pr i , L = L1: 2; arene = C6Me6, L = L1: 3; arene = C6H6, L = L2: 4; arene = p-MeC6H4Pr i , L = L2: 5; arene = C6Me6, L = L2: 6) have been synthesized from the corresponding [(arene)RuCl2]2 precursor with the long-chain N-ligand L in dichloromethane. Ruthenium nanoparticles stabilized by L1 have been prepared by the solvent-free reduction of 1 with hydrogen or by reducing [(arene)Ru(H2O)3]SO4 in ethanol in the presence of L1 with hydrogen. These complexes and nanoparticles show a high anticancer activity towards human ovarian cell lines, the highest cytotoxicity being obtained for complex 2 (IC50 = 2 μM for A2780 and 7 μM for A2780cisR)

Tandem use of solid-phase extraction and dispersive liquid-liquid microextraction for the determination of mononitrotoluenes in aquatic environment.

Solid-phase extraction (SPE) in tandem with dispersive liquid-liquid microextraction (DLLME) has been developed for the determination of mononitrotoluenes (MNTs) in several aquatic samples using gas chromatography-flame ionization (GC-FID) detection system. In the hyphenated SPE-DLLME, initially MNTs were extracted from a large volume of aqueous samples (100 mL) into a 500-mg octadecyl silane (C(18) ) sorbent. After the elution of analytes from the sorbent with acetonitrile, the obtained solution was put under the DLLME procedure, so that the extra preconcentration factors could be achieved. The parameters influencing the extraction efficiency such as breakthrough volume, type and volume of the elution solvent (disperser solvent) and extracting solvent, as well as the salt addition, were studied and optimized. The calibration curves were linear in the range of 0.5-500 μg/L and the limit of detection for all analytes was found to be 0.2 μg/L. The relative standard deviations (for 0.75 μg/L of MNTs) without internal standard varied from 2.0 to 6.4% (n=5). The relative recoveries of the well, river and sea water samples, spiked at the concentration level of 0.75 μg/L of the analytes, were in the range of 85-118%.

Visual functions in the healthy and schizophrenic brain : from stimulus control to illusory perceptions using electrical neuroimaging

ABSTRACT (FRENCH)Ce travail de thèse basé sur le système visuel chez les sujets sains et chez les patients schizophrènes, s'articule autour de trois articles scientifiques publiés ou en cours de publication. Ces articles traitent des sujets suivants : le premier article présente une nouvelle méthode de traitement des composantes physiques des stimuli (luminance et fréquence spatiale). Le second article montre, à l'aide d'analyses de données EEG, un déficit de la voie magnocellulaire dans le traitement visuel des illusions chez les patients schizophrènes. Ceci est démontré par l'absence de modulation de la composante PI chez les patients schizophrènes contrairement aux sujets sains. Cette absence est induite par des stimuli de type illusion Kanizsa de différentes excentricités. Finalement, le troisième article, également à l'aide de méthodes de neuroimagerie électrique (EEG), montre que le traitement des contours illusoires se trouve dans le complexe latéro-occipital (LOC), à l'aide d'illusion « misaligned gratings ». De plus il révèle que les activités démontrées précédemment dans les aires visuelles primaires sont dues à des inférences « top- down ».Afin de permettre la compréhension de ces trois articles, l'introduction de ce manuscrit présente les concepts essentiels. De plus des méthodes d'analyses de temps-fréquence sont présentées. L'introduction est divisée en quatre parties : la première présente le système visuel depuis les cellules retino-corticales aux deux voix du traitement de l'information en passant par les régions composant le système visuel. La deuxième partie présente la schizophrénie par son diagnostic, ces déficits de bas niveau de traitement des stimuli visuel et ces déficits cognitifs. La troisième partie présente le traitement des contours illusoires et les trois modèles utilisés dans le dernier article. Finalement, les méthodes de traitement des données EEG seront explicitées, y compris les méthodes de temps-fréquences.Les résultats des trois articles sont présentés dans le chapitre éponyme (du même nom). De plus ce chapitre comprendra les résultats obtenus à l'aide des méthodes de temps-fréquenceFinalement, la discussion sera orientée selon trois axes : les méthodes de temps-fréquence ainsi qu'une proposition de traitement de ces données par une méthode statistique indépendante de la référence. La discussion du premier article en montrera la qualité du traitement de ces stimuli. La discussion des deux articles neurophysiologiques, proposera de nouvelles d'expériences afin d'affiner les résultats actuels sur les déficits des schizophrènes. Ceci pourrait permettre d'établir un marqueur biologique fiable de la schizophrénie.ABSTRACT (ENGLISH)This thesis focuses on the visual system in healthy subjects and schizophrenic patients. To address this research, advanced methods of analysis of electroencephalographic (EEG) data were used and developed. This manuscript is comprised of three scientific articles. The first article showed a novel method to control the physical features of visual stimuli (luminance and spatial frequencies). The second article showed, using electrical neuroimaging of EEG, a deficit in spatial processing associated with the dorsal pathway in chronic schizophrenic patients. This deficit was elicited by an absent modulation of the PI component in terms of response strength and topography as well as source estimations. This deficit was orthogonal to the preserved ability to process Kanizsa-type illusory contours. Finally, the third article resolved ongoing debates concerning the neural mechanism mediating illusory contour sensitivity by using electrical neuroimaging to show that the first differentiation of illusory contour presence vs. absence is localized within the lateral occipital complex. This effect was subsequent to modulations due to the orientation of misaligned grating stimuli. Collectively, these results support a model where effects in V1/V2 are mediated by "top-down" modulation from the LOC.To understand these three articles, the Introduction of this thesis presents the major concepts used in these articles. Additionally, a section is devoted to time-frequency analysis methods not presented in the articles themselves. The introduction is divided in four parts. The first part presents three aspects of the visual system: cellular, regional, and its functional interactions. The second part presents an overview of schizophrenia and its sensoiy-cognitive deficits. The third part presents an overview of illusory contour processing and the three models examined in the third article. Finally, advanced analysis methods for EEG are presented, including time- frequency methodology.The Introduction is followed by a synopsis of the main results in the articles as well as those obtained from the time-frequency analyses.Finally, the Discussion chapter is divided along three axes. The first axis discusses the time frequency analysis and proposes a novel statistical approach that is independent of the reference. The second axis contextualizes the first article and discusses the quality of the stimulus control and direction for further improvements. Finally, both neurophysiologic articles are contextualized by proposing future experiments and hypotheses that may serve to improve our understanding of schizophrenia on the one hand and visual functions more generally.

Spatially-resolved eigenmode decomposition of red blood cells membrane fluctuations questions the role of ATP in flickering.

Red blood cells (RBCs) present unique reversible shape deformability, essential for both function and survival, resulting notably in cell membrane fluctuations (CMF). These CMF have been subject of many studies in order to obtain a better understanding of these remarkable biomechanical membrane properties altered in some pathological states including blood diseases. In particular the discussion over the thermal or metabolic origin of the CMF has led in the past to a large number of investigations and modeling. However, the origin of the CMF is still debated. In this article, we present an analysis of the CMF of RBCs by combining digital holographic microscopy (DHM) with an orthogonal subspace decomposition of the imaging data. These subspace components can be reliably identified and quantified as the eigenmode basis of CMF that minimizes the deformation energy of the RBC structure. By fitting the observed fluctuation modes with a theoretical dynamic model, we find that the CMF are mainly governed by the bending elasticity of the membrane and that shear and tension elasticities have only a marginal influence on the membrane fluctations of the discocyte RBC. Further, our experiments show that the role of ATP as a driving force of CMF is questionable. ATP, however, seems to be required to maintain the unique biomechanical properties of the RBC membrane that lead to thermally excited CMF.

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block

Topographic organization of V1 projections through the corpus callosum in humans.

The visual cortex in each hemisphere is linked to the opposite hemisphere by axonal projections that pass through the splenium of the corpus callosum. Visual-callosal connections in humans and macaques are found along the V1/V2 border where the vertical meridian is represented. Here we identify the topography of V1 vertical midline projections through the splenium within six human subjects with normal vision using diffusion-weighted MR imaging and probabilistic diffusion tractography. Tractography seed points within the splenium were classified according to their estimated connectivity profiles to topographic subregions of V1, as defined by functional retinotopic mapping. First, we report a ventral-dorsal mapping within the splenium with fibers from ventral V1 (representing the upper visual field) projecting to the inferior-anterior corner of the splenium and fibers from dorsal V1 (representing the lower visual field) projecting to the superior-posterior end. Second, we also report an eccentricity gradient of projections from foveal-to-peripheral V1 subregions running in the anterior-superior to posterior-inferior direction, orthogonal to the dorsal-ventral mapping. These results confirm and add to a previous diffusion MRI study (Dougherty et al., 2005) which identified a dorsal/ventral mapping of human splenial fibers. These findings yield a more detailed view of the structural organization of the splenium than previously reported and offer new opportunities to study structural plasticity in the visual system.

Determination of oseltamivir and oseltamivir carboxylate in human plasma by liquid chromatography-tandem mass spectrometry

Introduction: Oseltamivir phosphate (OP), the prodrug of oseltamivir carboxylate (OC; active metabolite), is marketed since 10 years for the treatment of seasonal influenza flu. It has recently received renewed attention because of the threat of avian flu H5N1 in 2006-7 and the 2009-10 A/H1N1 pandemic. However, relatively few studies have been published on OP and OC clinical pharmacokinetics. The disposition of OC and the dosage adaptation of OP in specific populations, such as young children or patients undergoing extrarenal epuration, have also received poor attention. An analytical method was thus developed to assess OP and OC plasma concentrations in patients receiving OP and presenting with comorbidities or requiring intensive care. Methods: A high performance liquid chromatography coupled to tandem mass spectrometry method (HPLC-MS/MS) requiring 100-µL aliquot of plasma for quantification within 6 min of OP and OC was developed. A combination of protein precipitation with acetonitrile, followed by dilution of supernant in suitable buffered solvent was used as an extraction procedure. After reverse phase chromatographic separation, quantification was performed by electro-spray ionization-triple quadrupole mass spectrometry. Deuterated isotopic compounds of OP and OC were used as internal standards. Results: The method is sensitive (lower limit of quantification: 5 ng/mL for OP and OC), accurate (intra-/inter-assay bias for OP and OC: 8.5%/5.5% and 3.7/0.7%, respectively) and precise (intra-/inter-assay CV%: 5.2%/6.5% and 6.3%/9.2%, respectively) over the clinically relevant concentration range (upper limits of quantification 5000 ng/mL). Of importance, OP, as in other previous reports, was found not to be stable ex vivo in plasma on standard anticoagulants (i.e. EDTA, heparin or citrate). This poor stability of OP has been prevented by collecting blood samples on commercial fluoride/oxalate tubes. Conclusions: This new simple, rapid and robust HPLC-MS/MS assay for quantification of OP and OC plasma concentrations offers an efficient tool for concentration monitoring of OC. Its exposure can probably be controlled with sufficient accuracy by thorough dosage adjustment according to patient characteristics (e.g. renal clearance). The usefulness of systematic therapeutic drug monitoring in patients appears therefore questionable. However, pharmacokinetic studies are still needed to extend knowledge to particular subgroups of patients or dosage regimens.

Testing Spatial Autocorrelation in Weighted Networks : the Modes Permutation Test

In a weighted spatial network, as specified by an exchange matrix, the variances of the spatial values are inversely proportional to the size of the regions. Spatial values are no more exchangeable under independence, thus weakening the rationale for ordinary permutation and bootstrap tests of spatial autocorrelation. We propose an alternative permutation test for spatial autocorrelation, based upon exchangeable spatial modes, constructed as linear orthogonal combinations of spatial values. The coefficients obtain as eigenvectors of the standardised exchange matrix appearing in spectral clustering, and generalise to the weighted case the concept of spatial filtering for connectivity matrices. Also, two proposals aimed at transforming an acessibility matrix into a exchange matrix with with a priori fixed margins are presented. Two examples (inter-regional migratory flows and binary adjacency networks) illustrate the formalism, rooted in the theory of spectral decomposition for reversible Markov chains.

The brain uses single-trial multisensory memories to discriminate without awareness.

Multisensory experiences enhance perceptions and facilitate memory retrieval processes, even when only unisensory information is available for accessing such memories. Using fMRI, we identified human brain regions involved in discriminating visual stimuli according to past multisensory vs. unisensory experiences. Subjects performed a completely orthogonal task, discriminating repeated from initial image presentations intermixed within a continuous recognition task. Half of initial presentations were multisensory, and all repetitions were exclusively visual. Despite only single-trial exposures to initial image presentations, accuracy in indicating image repetitions was significantly improved by past auditory-visual multisensory experiences over images only encountered visually. Similarly, regions within the lateral-occipital complex-areas typically associated with visual object recognition processes-were more active to visual stimuli with multisensory than unisensory pasts. Additional differential responses were observed in the anterior cingulate and frontal cortices. Multisensory experiences are registered by the brain even when of no immediate behavioral relevance and can be used to categorize memories. These data reveal the functional efficacy of multisensory processing.

Fatal tolperisone poisoning: autopsy and toxicology findings in three suicide cases.

Tolperisone (Mydocalm) is a centrally acting muscle relaxant with few sedative side effects that is used for the treatment of chronic pain conditions. We describe three cases of suicidal tolperisone poisoning in three healthy young subjects in the years 2006, 2008 and 2009. In all cases, macroscopic and microscopic autopsy findings did not reveal the cause of death. Systematic toxicological analysis (STA) including immunological tests, screening for volatile substances and blood, urine and gastric content screening by GC-MS and HPLC-DAD demonstrated the presence of tolperisone in all cases. In addition to tolperisone, only the analgesics paracetamol (acetaminophen), ibuprofen and naproxen could be detected. The blood ethanol concentrations were all lower than 0.10 g/kg. Tolperisone was extracted by liquid-liquid extraction using n-chlorobutane as the extraction solvent. The quantification was performed by GC-NPD analysis of blood, urine and gastric content. Tolperisone concentrations of 7.0 mg/l, 14 mg/l and 19 mg/l were found in the blood of the deceased. In the absence of other autopsy findings, the deaths in these three cases were finally explained as a result of lethal tolperisone ingestion. To the best of our knowledge, these three cases are the first reported cases of suicidal tolperisone poisonings.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

Motifs de consultation en médecine du travail [Reasons for consultation in occupational medicine].

The inability to characterize more precisely the extent of occupational diseases limits the implementation of an effective preventive policy. Furthermore, not all work-related conditions are reported by the Swiss workers' compensation system. A seven-year (1986 to 1992) retrospective study of medical visits in an Institute of Occupational Health Sciences is presented. The objective of this study is to expand data on occupational diseases for clinical and public health intervention. 298 patients have been examined for a possible work-related condition. In 140 cases (47%), an occupational disease according to the Swiss Law was found. Respiratory tract was the main target of industrial pollutants. Respiratory irritation , solvent intoxications, contact dermatitis and asthma were the most frequent conditions seen. 97 workplace visits (32% of all medical visits) were necessary for diagnostic purposes. Painters (construction, cars) and other solvent exposed workers were at particular risk. Rare alpha-1-antitrypsin phenotypes were found several times in workers with respiratory diseases confirming the value of this test in occupational medicine. Despite many referral biases, direct clinical and public health applications of the data are possible. This study confirms the hypothesis that occupational respiratory diseases and intoxications are probably underreported in the workers' compensation statistics. Activities with an increased risk of work-related diseases have been identified so workplace intervention could be highly targeted. This study shows also that a more intense collaboration between primary care physicians, hospital services and occupational medical specialists is necessary to improve clinical and epidemiological surveillance of work-related health conditions.