La demande énergétique mondiale toujours en croissance, combinée aux préoccupations environnementales, constitue le moteur qui anime la recherche de nouvelles énergies. La fusion thermonucléaire est une des technologies qui offrent la possibilité d'une production d'énergie abondante, sûre et non polluante ; cependant son apprivoisement n'est pas encore au point ! La présence d'impuretés dans les plasmas est un des obstacles majeurs à vaincre pour atteindre les conditions nécessaires pour que la fusion thermonucléaire devienne suffisamment efficace. Leur densité dans le plasma dépend des matériaux de revêtement des parois et du contrôle des conditions aux limites, conditions qui définissent leur processus de génération et leur diffusion dans la région centrale. Les pertes énergétiques ont été considérablement réduites par l'utilisation de revêtements à basse charge nucléaire (Z), principalement ceux à base de carbone (comme le graphite). Ainsi, les chercheurs se sont intéressés aux effets des impuretés hydrocarbonées se dégageant des parois des réacteurs, à savoir leur structure et les voies de leur fragmentation, en particulier, les caractéristiques de leurs processus collisionnels avec les autres constituants du plasma. L'ion moléculaire est un composant essentiel de nombreux plasmas astrophysiques où l'ionisation et la dissociation se font principalement avec des électrons ; sa concentration est élevée dans l'ionosphère, région de l'atmosphère située à une altitude comprise entre 90 et 250 Km. Le rôle important joué par cet ion dans la modélisation ionosphérique nous a mené à réaliser une série de mesures des sections efficaces de ses différents processus collisionnels par impact d'électrons. La majorité des travaux expérimentaux antérieurs ne concernait que la molécule neutre . Ce travail de thèse a porté sur l'étude expérimentale de l'ionisation simple, de l'excitation dissociative et de l'ionisation dissociative par impact d'électrons des ions hydrocarbonés polyatomiques et et de l'ion diatomique . Nous avons utilisé la technique des faisceaux croisés et animés pour mesurer les sections efficaces absolues de ces processus. Nous avons d'abord mesuré les distributions d'énergie cinétique des produits de ces processus dans le référentiel du laboratoire, en fonction du champ magnétique d'analyse, pour différentes valeurs d'énergie d'électrons. Ensuite, nous avons mesuré les sections efficaces apparentes pour les mêmes processus en fonction de l'énergie des électrons. Concernant les processus dissociatifs, la mesure des distributions d'énergie cinétique des fragments dans le référentiel du laboratoire a montré que ces fragments sont, généralement, distribués dans des gammes angulaires et énergétiques larges et que leurs divergences angulaires, dans la plupart des cas, excèdent l'ouverture de la fente d'analyse et en conséquence, leur transmission n'est que partielle. Nous avons alors calculé leurs coefficients de transmission en fonction de l'énergie des électrons, et nous avons corrigé les sections efficaces apparentes, pour chacun des processus dissociatifs. Nous avons ainsi pu obtenir les sections efficaces absolues de plusieurs voies dissociatives. Nous avons identifié leur seuil d'apparition en tenant compte des structures de l'ion moléculaire primaire et des ions produits. Nous avons déterminé aussi les distributions d'énergie cinétique des fragments produits dans le référentiel du centre de masse à partir des distributions énergétiques observées dans le référentiel du laboratoire, nous avons ainsi pu estimer l'énergie maximale libérée lors de chacun des processus dissociatifs. Les résultats ont montré que la section efficace de l'ionisation simple est faible en comparaison avec les sections efficaces d'excitation et d'ionisation dissociative. Celle-ci dépend du nombre, généralement réduit, des états liés stables de l'ion doublement chargé. Ils ont montré aussi que les courbes des sections efficaces des processus d'excitation dissociative et d'ionisation ont une allure très différente. Dans le premier cas, elle augmente abruptement au voisinage du seuil alors que dans le second cas, elle augmente progressivement avec l'énergie des électrons. Pour les processus de dissociation, nous avons besoin d'une analyse angulaire et d'une détection en coïncidence des fragments produits, permettant ainsi de déterminer la nature exacte du mécanisme et d'identifier avec plus de précision l'état ou les états moléculaires impliqués dans chacune des réactions. Les résultats obtenus sont concluants et ont permis d'améliorer l'étude théorique semi-classique menée par Probst et Märk (2004) concernant l'ionisation de . Ils ont montré aussi que les prédictions empiriques de Janev et Reiter (2002) relatives aux familles d'hydrocarbures ne sont pas encore au point. Elles sont en désaccord avec nos résultats, montrant ainsi qu'il y a grand besoin d'autres études expérimentales des processus collisionnels des hydrocarbures et d'autres ions moléculaires avant de pouvoir formuler une quelconque loi d'échelle ou une théorie capable de prédire le comportement de tels ions moléculaires.

Membrane fusion is essential for survival in eukaryotic cells. Many physiological processes such as endocytosis and exocytosis are mediated by membrane fusion, which is driven by highly specialized and conserved family of proteins. Neuronal soluble Nethylmaleimide- sensitive factor attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane during neurotransmitter release; however, the mechanism for SNARE-mediated membrane fusion remains to be established. In the current work, we aimed at investigating this mechanism using atomic force microscope (AFM) spectroscopy. We established an AFM lipid bilayer system, which proved effective in detecting fusion of bilayers and measuring compression forces required to generate fusion. It also revealed that SNARE-mediated membrane fusion proceeds through an intermediate hemifused state. Using this system, we revealed the energy landscape for membrane fusion using a dynamic force approach. We carried out compression force measurements at different compression rates and a significant reduction in the force was observed when SNAREs were present in the bilayers. The results also indicated that a single energy barrier governed membrane fusion in our experimental system. The energy barrier is characterized by its width and height, which determine the slope of the activation potential. With SNAREs in the opposing (trans) bilayers, the width of the barrier increased > 2 fold, which is interpreted as an increase in the compressibility of the membranes and subsequently a greater ease in their deformation and fusion under compression. Moreover, specific perturbations to the SNARE interaction interfered with the observed facilitation of membrane fusion, which indicated the involvement of SNAREs in the observed fusion facilitation and increase in the fusion rate. Furthermore, dissociation kinetics analysis of the SNARE interaction revealed a strong binding force during trans SNARE-complex formation, and a correlation between the strength of the SNARE interaction and the degree of fusion facilitation was established. In conclusion, the present findings provide support for a mechanism for SNAREmediated membrane fusion, where trans-interaction between SNAREs provides close apposition of the membranes and reduces fusion energy requirements by locally destabilizing the bilayers, in which the SNAREs are anchored, through pulling on or tilting of their transmembrane segments.

Though mass spectrometry has earned a central role in the field of proteomics due to its versatility in a wide range of experiments, challenges and complications are still encountered when using mass spectrometry to characterize protein structures, post-translational modifications (PTMs), and abundances. In this dissertation, analytical methods utilizing mass spectrometry have been developed to address challenges associated with both qualitative and quantitative protein characterization. The effectiveness of using multiple pepsin-like proteases, both separately and in mixtures, combined with online proteolysis using a special triaxial probe has been demonstrated on an amyloid beta peptide related to the onset of Alzheimer’s disease. These findings have broad implications in protein structural characterization studies using hydrogen-deuterium exchange mass spectrometry. A wider range of proteases (Lys-C, Glu-C, and trypsin) and multiple fragmentation methods (collisionally activated dissociation, electron transfer dissociation, and decision tree) have been utilized in the discovery-based PTM characterization of extracellular cellulosome proteins of the bioenergy-relevent organism Clostridium thermocellum, resulting in the identification of 85 previously unknown modification sites in 28 cellulosome proteins. These modifications may contribute to the structure and/or function of the cellulosome protein complex. By using peptide internal standards and a triple quadrupole mass spectrometer operating in selected reaction monitoring mode, a method has been developed for the absolute quantitation of the Clostridium thermocellum cellulosome protein machine in samples ranging in complexity from purified cellulosome samples to whole cell lysates as an alternative to a previously-developed enzyme-linked immunosorbent assay method of cellulosome quantitation. The precision of the cellulosome mass concentration in technical replicates is better than 5% relative standard deviation for all samples, indicating high precision of cellulosome mass concentration for this method. Though methods and results presented in this dissertation have implications in the study of Alzheimer’s disease and bioenergy research, more broadly this dissertation focuses on development of methods to contend with some of the more complex challenges associated with protein characterization currently presented to the field of proteomics.

Nowadays, polymer chemists undertake considerable efforts to achieve the preparation of new macromolecules and a perfect control over the macromolecular engineering, i.e. the mass parameters but also over the chain and end-group compositions, topology, etc… is definitively expected. In addition, more complex architectures, such as brush (co)polymers, jellyfish-like topologies…, are required to improve or drastically modify the physicochemical properties of the materials. As a direct consequence of the development of such complex molecular objects, sophisticated techniques are required for the in-depth characterization of the macromolecules, since the exact compositions and structures should be fully and unambiguously identified. Given the fact that the usual characterization tools such as Nuclear Magnetic Resonance (NMR) and Gel Permeation Chromatography (GPC) are extensively used, their abilities have been intensively developed to account for the increasing complexity and diversity of the targeted molecules. Nevertheless, all the usual techniques are averaging methodologies since they only provide pieces of information about the polymer mixture instead of affording data on the individual macromolecules. Since few decades, mass spectrometry (MS) has become as used as NMR and GPC for polymer characterization. In the context of large molecules analysis, MS undoubtedly underwent an impressive craze with the development of two modern ionization procedures, namely Electrospray Ionization (ESI) and Matrix-assisted Laser Desorption/Ionization (MALDI). Those ionization procedures permit the vaporization of macromolecules allowing the intact polymers to be analyzed without a too extensive level of degradation. ESI and MALDI are often considered as soft ionization methods since they offer the possibility to observe ions corresponding to the intact molecules. After their production in the ion source, ions corresponding to the polymer molecules can be mass analyzed by the mass spectrometer and important parameters such as the molecular weight distribution (Mn and Mw), polydispersity index (PDI), the nature of the monomer units and the end-groups can be derived from the measure of the mass-to-charge ratios of the produced ions. In the first part of the present thesis, we studied the MS behavior of different classes of polymers when submitted to ESI and MALDI ionizations. The investigations were devoted to the validation of MS as a truly reliable methodology for fragile polymers such as aliphatic polyesters for instance. In this context, a preliminary MS investigation on semi-telechelic polyethers revealed the importance of the source parameters for the characterization of polymers presenting fragile moieties. We also demonstrated the huge importance of the matrix molecule selection for the MALDI analyses of polymers. In particular, we introduced a new matrix for the MALDI measurements of electroconjugated polymers such as polythiophenes. After the study of the influence of the source parameters on the MS data, a complete study by single stage MS and double stage MS (MS/MS) on newly synthesized polylactides (PLA) was performed. The PLA samples were prepared following original procedures using carbene as catalyst. Finally, to achieve the MS study of PLA ions, we used ion mobility-mass spectrometry (IM-MS) experiments to obtained information on the tridimensional structure of the gas phase PLA ions. In particular, we put a special emphasis on the influence of the charge and size of the polymer chains on their gas-phase conformations. The conclusions derived from the MS/MS and IM-MS results were fully supported by theoretical calculations. In the second part of the thesis, the acquired MS experience was applied to the fine characterization of macromolecules presenting complex architectures obtained by two different polymerization procedures: (i) cobalt-mediated radical polymerization of inter alia acrylonitrile and vinyl acetate and (ii) ring-opening polymerization (ROP) of lactones using non-organometallic catalysts. In particular, mass spectrometry was used to tune the experimental conditions for the ROP of â-lactones using different phosphazenes as catalysts. As an ultimate conclusion, this work points to the very efficient synergy between polymer synthesis, mass spectrometry and theoretical calculations. We believe that this thesis paves the way for innumerable possibilities in the future.

Dissociation dynamics of the ionized molecules HCl and the deuterated system DCl has been studied in gas-phase using synchrotron based photoelectron spectroscopy (PES). The inner-valence "(4σ)-1" photoionization band for DCl and HCl was recorded using maximum resolution in order to probe an interference pattern between a dissociative and a bound electronic state. For HCl+, we clearly observed distorted Fano-type peaks even for modest resolution, whereas for DCl+, the pattern was hardly discernible. The observation in HCl+ has been explained by a coupling between two adiabatic electronic states, where the bound state was populated through non-adiabatic curve-crossing. The nuclear motion of HCl+ is too fast for the Born-Oppenheimer approximation to be fully valid in this case. Whereas for DCl+, with larger reduced mass and therefore slower nuclear motion, the non-adiabatic coupling is less pronounced, and the vibrational progression vanishes. A comparative study between PES and threshold photoelectron spectra (TPES) of the inner-valence bands of HCl and DCl has been performed, showing differences in intensities and shapes of the vibrational bands. These differences were attributed to the fact that the sudden approximation, which can be assumed to be valid for PES, is violated in the case of TPES. A resonant Auger electron spectroscopy study of HCl and DCl has been performed, which shows an interference pattern between atomic and molecular Auger- and photoelectron channels. The atomic features are associated with ultra-fast dissociation of the molecules, on the same time scale as the Auger decay. The observation shows that the excited molecular system has to be regarded as a superposition of fragmented and molecular states. A study of the X-state of HCl+, populated via a core-excited state, shows a selective population of the final state. The explanation was shown to be that the magnetic orientation of the core-hole is transferred to the final state of the molecule. A setup for data acquisition of Photo-Electron Photo-Ion Photo-Ion COincidence (PEPIPICO) measurements using a Time-Of-Flight (TOF) spectrometer has been developed. A Time-to-Digital Converter (TDC) card has been linked together with the data treatment program Igor as a user interface. Furthermore, the PEPIPICO spectrometer has been characterized to provide a solid basis for the analysis of experimental data.

The work in this thesis has demonstrated the advantages and limitations of using MS based technologies in protein and peptide structural studies. Tandem MS, specifically electron capture dissociation (ECD) have shown the ability to provide structural insights in molecules containing the slightest of all modifications (D-AA substitution). Additionally, it can be concluded that charge localization in molecular ions is best identified with ECD and to a lesser degree using CAD. Fragment ion abundances are a quantifiable tool providing chiral recognition (RChiral). An analytical model demonstrating the detection and quantification of D-AAs within proteins and peptides has been achieved. ECD has demonstrated the ability to quantify stereoisomeric mixtures to as little as 1%. Chirality elucidation on a nano LC-MS/MS time scale has been shown. The structures of various stereoisomers of the mini protein Trp Cage were explored, each providing unique ECD fragment ion abundances suggestive of gas phase structural differences. The uniqueness of these abundances combined with MDS data have been used in proposing a new mechanism in c and z fragment ion formation in ECD. This mechanism suggests initial electron capture on a backbone amide involved in (neutral) hydrogen bonding. The wealth of solution phase (circular dichroism), transitition phase (charge state distribution, CSD) and gas phase (ECD) data for Trp Cage suggest that at low charge states (2+) the molecule has a high degree of structural similarity in solution- and gas- phases. Furthermore, quantitative information from CSD studies is garnered when using a “native” deuteriated form as part of the stereoisomeric mixture. It has also been shown that the stability of the reduced species after electron capture is indicative of the recombination energy release, which in turn is linked to the coulombic repulsion- a structural constraint that can be used for approximation of the inter-charge distance for various stereoisomers.

The main aim of this thesis has been to investigate trauma and dissociation among Swedish adolescents and to evaluate the psychometric properties such as reliability and various kinds of validity of three screening instruments for assessment of dissociation and other symptoms of post traumatic stress. The three instruments in question have been Dis-Q-Sweden, A-DES and TSCC, the symptoms measured by these instruments are neither easy to capture nor easy for the adolescent to talk about. Therefore these self report scales are essential. A second aim has been to compare the results with results from other countries and to develop preliminary Swedish norms for the clinician to use. Age and gender differences have been looked upon as well as assessed symptoms connected to known experienced trauma/sexual and/or physical abuse and self-reported trauma in normal and clinical populations. The populations, in this thesis have been children and adolescents age 10 -19 years old from the general population; the clinical groups have had the same age range. All children and adolescents in the clinical groups have been sexually and/or physically abused. Participants have answered the questionnaires Dis-Q-Sweden, A-DES and/or TSCC and their answers have been statistically analysed. All three instruments have been shown to have good reliability, such as internal consistency and test-retest. Validity has been established through factor analyses, concurrent, and criterion related validity. Clinical groups with known experienced trauma/sexual abuse and/or physical abuse gave significantly higher scores on all the instruments compared to normative groups. Also self-reported trauma in a normative group gave significantly higher scores even if the significances are not as high as between the normative and clinical groups. Girls scored significantly higher than boys in both the clinical and normative groups. Girls in the age range 14-15 years old gave the significantly highest scores on both Dis-Q-Sweden and ADES. Swedish adolescents gave lower mean scores on all three instruments than have been reported from other studies in other countries. The scores from the clinical groups gave about the same mean as have been reported elsewhere. The conclusion from this thesis is that all the three questionnaires Dis-Q-Sweden, A-DES and TSCC have shown satisfactory psychometrics properties and can very well be used by Swedish clinicians in Child and Adolescents Psychiatry.

Aims: The overall aim of this thesis was to elucidate psychological processes and development, maintenance or recovery related to fibromyalgia based on in depth interviews. In a next step women with fibromyalgia, women without long-lasting pain and women with long-lasting pain were compared using psychometric instruments selected or developed based on qualitative results. Methods: Twenty-one women with fibromyalgia and 8 women recovered were interviewed. Their narrations were analysed using Grounded Theory. Tentative theory was built. The I Myself Scale (IMS) was constructed to mirror self-regulation prior to onset of symptoms and complemented with an instrument on current self-regulation: Structural Analysis of Social Behaviour (SASB) and SF-36 mirroring health related quality of life, regarding the two pain groups. The groups were compared using analysis of variance, principal components analysis paired with discriminant analysis and profile analysis. Results: Analyses of the interviews resulted in core concepts of an “unprotected self” (current fibromyalgia) or a “strong but not enough to be weak” self (recovery). Data patterns indicated that the women as children were unprotected in relation to stimuli and affects. Relationships with the parents were characterised by strain and low levels of support. The recovery group had as children simultaneously been able to develop obvious competence and capability to receive help. Psychological vulnerability was in adult life compensated for through pronounced helpfulness and dissociation/repression including intense activity. An increase in mental load such as localised pain or psychosocial crisis preceded onset of fibromyalgia accompanied by impaired cognitive functioning. The state of fibromyalgia meant maintained high levels of mental load such as difficulties of the selfstructures, impaired cognitive functioning and somatic symptoms. The recovery group experienced substantial social support and often used mastering strategies to ease symptoms. A decrease in strain as improved life conditions and cease of overexertion preceded recovery. Health was thereafter maintained through careful management as seeking low levels of strain and pacing of activity. Recovery ‘on parole’ also meant personal growth and use of efficacious defences. Psychometrical testing confirmed qualitative data patterns of self-regulation connected to fibromyalgia. Impaired selfreference/ understanding of health needs and others not being asked for help and advice was reported before onset of symptoms. Dissociation or repression including intense activity and self-loading were also employed. SASB and SF-36 indicated that women with fibromyalgia experienced higher levels of mental “load” than the other pain group. Conclusion: Qualitative data indicated that life prior to onset of fibromyalgia and current fibromyalgia held qualities of impaired self-regulation in relation to mental and physical load. The state of recovery relied on improved self-regulation allowed by conditions of life. Quantitative data patterns confirmed qualitative results on impaired self-protection before onset of fibromyalgia and a specifically high level of mental load during the state of fibromyalgia. Psychological disregulation is discussed and hypothesised to cause but also later in the process parallel alterations in somatic homeostatic functions. Recovery could mean that biological regulation regarding strain is replaced with more of “psycho-social” regulation as careful pacing of work. Implications for treatment are suggested.

No description available.

The paradigm of drug discovery have been to find the strongest possible binder to the target by high-throughput screening (HTS) but high affinity interactions are related to low kinetic off rates and thus result in severe side-effects and non-approved drugs. Lead molecules working in a transient manner (KD > µM) will allow for rapid off rates and possibly less side-effects. In this study the peak profile method applied to weak affinity chromatography (WAC) was evaluated as a simple way to provide the kinetics of the interaction and thereby allowing for high-throughput determinations. In the peak profile formula all band-broadening effects except the stationary mass transfer is subtracted which simplifies the calculations for the kinetics of the interaction tremendously. The technique was evaluated by screening of 3 different benzamidines at 3 linear flow-rates using zonal chromatography and human α-thrombin as immobilized target protein. The kinetics of the interaction could unfortunately not be determined. This was possibly due to the flow-rates not being high enough as indicated by a low critical ratio (η < 1). Higher flow-rates would increase the contribution to band-broadening due to kinetic effects but would also require more precise estimation of peak variance.