Le virus de la grippe est un virus à ARN négatif appartenant à la famille des Orthomyxoviridae qui se compose de 7 membres dont les virus influenza A, B, C et D. Le génome viral comprend 7 à 8 particules ribonucléoprotéiques (RNP) au sein desquelles l’ARN viral (ARNv) est recouvert de multiples copies de nucléoprotéines (NP) et est associé à l’ARN polymérase virale via ses extrémités 3’ et 5’. Au cours de ce travail de thèse, nous nous sommes tout d’abord focalisés sur l’étude biochimique de NP A et NP B et avons pu mettre en évidence des comportements différents en ce qui concerne leurs propriétés d’oligomérisation en présence ou en absence d’ARN et en fonction de la concentration en sel. Pour la première fois nous avons pu observer une structure similaire aux RNP mais reconstituée uniquement à partir de NP A et d’un ARN de 12 nucléotides. Nous avons pu formuler l’hypothèse que 12 nucléotides de l’ARN serait fixés à la NP avec une forte affinité tandis que le reste de l’ARN fixerait la NP avec une affinité beaucoup plus faible. En parallèle nous avons résolu la structure cristallographique de la nucléoprotéine de la grippe de type D et réaliser la caractérisation de son interaction avec l’importine-α7 humaine. Enfin nous avons étudié la fixation de l’ARN sur NP D et mis en évidence l’importance de l’extrémité C-terminale dans le processus de fixation à l’ARN. Ces informations ont permis de formuler de nouvelles hypothèses quant au fonctionnement du virus de la grippe et permettre d’inscrire ce projet de thèse dans une dynamique globale de lutte contre ce virus. / Influenza virus is a negative RNA virus belongs to the Orthomyxoviridae family which consists of 7 members including influenza viruses A, B, C and D. The viral genome comprises 7 to 8 ribonucleoprotein particles (RNP) in which the viral RNA (vRNA) is coated with multiple copies of nucleoproteins (NP) and is associated with the viral RNA polymerase by its 3 'and 5' ends. In this thesis, we first focused on the biochemical study of NP A and NP B and we demonstrate that there are different behaviors with regard to their oligomerization properties in the presence or absence of RNA and as a function of the salt concentration. For the first time we were able to observe a structure very similar to RNP which was reconstituted only from NP A and a 12 nucleotide RNA. Thus, we formulate the hypothesis that 12 nucleotides of the RNA would bind NP with a very strong affinity while the rest of the RNA would bind NP with a lower affinity. In parallel, we solved the crystallographic structure of the nucleoprotein of influenza D virus and we characterized its interaction with human importin-α7. Finally, we studied the binding of RNA on NP D and we demonstrated the importance of the C-terminal end in the RNA binding process. This thesis project made it possible to formulate new hypotheses concerning the functioning of the influenza virus and to include this thesis project in a global dynamic of combating the influenza virus.

Characterization of cellulose by its molecular weight distribution is afforded after polymeranalogeous dissolution. Additionally, a molecular dispersion of the polymer is a prerequisite. Common processes are dissolution of cellulose in dimethylacetamide-lithiumchloride, nitration and carbanilation. Degradation of the polysaccharide chains can occur in each of the mentioned processes. It is shown that degradation in pyridine occurs via beta-elimination at carbonyl groups along the cellulose chains. Carbanilierung in DMSO is much more pronounced. It comprises oxidation along the Pfitzner-Moffatt-mechanism and subsequent beta-elimination at the thus formed carbonyl-groups. This was elucidated with model systems and by investigation of the carbanilation in different media. Carbonyl groups of cellulose are masked through reaction with phenylisocyanate. This was shown with model. Therefore, the determination of carbonyl groups in cellulose-tricarbanilates is not possible. The separation of low-molecular weight byproducts was optimised. The influence of pretreatment and preactivation of cellulose-samples on the completeness of the conversion was studied. A standard protocol for the carbanilation of cellulose is provided. / Cellulose wird u.a. durch ihre Molmassenverteilung charakterisiert, deren Ermittlung ein polymeranaloges Verfahren zur molekulardispersen Auflösung des Polymers erfordert. Hierfür sind die Direktlösung, die Nitrierung und die Carbanilierung in Gebrauch. Bei allen Prozessen kann es zum Abbau der Polysaccharidketten kommen, wobei diesen Prozessen wenig Beachtung geschenkt wurde. In der Arbeit wird gezeigt, daß der Abbau bei der Carbanilierung in Pyridin durch Beta-Eliminierung an vorhandenen Carbonylgruppen erfolgt. Die Carbanilierung in DMSO fällt stets stärker aus als bei Einsatz von Pyridin und umfasst die Prozesse Oxidation nach dem Pfitzner-Moffatt-Mechanismus und anschließende Beta-Eliminierung an den neu gebildeten Carbonylgruppen. Dies wird durch Untersuchungen an Modellsystemen und am Polymer herausgearbeitet. Carbonylgruppen an Cellulose werden durch die Umsetzung mit Phenylisocyanat maskiert, was an Modellverbindungen gezeigt wurde (Bildung von Endioldicarbanilaten und carbanilierten Halbacetalen). Ihre Bestimmung in Cellulosecarbanilaten ist daher nicht möglich. Die Abtrennung von niedermolekularen Nebenprodukten der Umsetzung wurde optimiert. Der Einfluss der Vorbehandlung und Voraktivierung von Celluloseproben auf die Vollständigkeit der Umsetzung wurde eingehend untersucht. Es wird ein Standardverfahren zur Carbanilierung von Cellulose angegeben.

The chondroitin sulphate proteoglycan (CSPG) versican forms complexes with hyaluronan (HA), which are essential in a range of functions including cellular proliferation and migration. Four isoforms of versican result from alternative splicing. Furthermore, biological roles have been identified for the proteolytic cleavage product of versican which contains the N-terminal G1 hyaluronan binding domain. All of these versican forms have different tightly regulated tissue expression profiles. Consequently, impaired regulation is associated with a number of disease pathologies. For example the largest variants (V0/V1) have been shown to be negative indicators of disease outcome in a number of malignant cancers and are a marker of disease progression in atherosclerosis. Interestingly, the smaller versican isoform V3 which lacks CS chains has been demonstrated to have the potential to reverse disease associated phenotypes. The motivation for carrying out the work in this thesis was to try and gain a better understanding of how versican functions on a molecular scale. In this regard, the first aim was to investigate the structure of the hyaluronan binding region of versican using a construct called VG1. The structure of VG1 was analysed in the presence and absence of hyaluronan oligomers. This revealed an insight into the multi-modular structure of the versican hyaluronan binding region and demonstrated that on binding to HA, VG1 under goes a conformational change. Furthermore, the interaction between VG1 and longer lengths of hyaluronan (pHA) was investigated. This demonstrated that when VG1 binds to pHA it is does so with positive cooperativity, packing very close to neighbouring VG1 molecules along a chain of HA. One consequence of this interaction was to reorganise pHA into a helical conformation, an organisation that was confirmed by a number of solution phase techniques. The effect of this reorganisation of pHA by VG1 on HA/CD44 interactions was also assessed. Previously the interaction between CD44 (a cell surface hyaluronan receptor) and long chains of HA (>30 kDa) was shown to be irreversible; however we demonstrate that VG1 can reverse this. Furthermore, a TSG-6 enhanced CD44/interaction was also completely reversed by the addition of VG1. This provides an indication that a functional hierarchy of hyaluronan binding proteins may exist which could have important implications in understanding the function of hyaluronan complexes. Currently, we do not know whether intact versican molecules could interact with HA in the same way as VG1. However, preliminary data suggests that the CS-containing variants (i.e. V0, V1 and V2) would not, whereas V3 and versican fragments could. This work provides an exciting mechanistic insight into the function of versican variants and their breakdown products.

Characterization of cellulose by its molecular weight distribution is afforded after polymeranalogeous dissolution. Additionally, a molecular dispersion of the polymer is a prerequisite. Common processes are dissolution of cellulose in dimethylacetamide-lithiumchloride, nitration and carbanilation. Degradation of the polysaccharide chains can occur in each of the mentioned processes. It is shown that degradation in pyridine occurs via beta-elimination at carbonyl groups along the cellulose chains. Carbanilierung in DMSO is much more pronounced. It comprises oxidation along the Pfitzner-Moffatt-mechanism and subsequent beta-elimination at the thus formed carbonyl-groups. This was elucidated with model systems and by investigation of the carbanilation in different media. Carbonyl groups of cellulose are masked through reaction with phenylisocyanate. This was shown with model. Therefore, the determination of carbonyl groups in cellulose-tricarbanilates is not possible. The separation of low-molecular weight byproducts was optimised. The influence of pretreatment and preactivation of cellulose-samples on the completeness of the conversion was studied. A standard protocol for the carbanilation of cellulose is provided. / Cellulose wird u.a. durch ihre Molmassenverteilung charakterisiert, deren Ermittlung ein polymeranaloges Verfahren zur molekulardispersen Auflösung des Polymers erfordert. Hierfür sind die Direktlösung, die Nitrierung und die Carbanilierung in Gebrauch. Bei allen Prozessen kann es zum Abbau der Polysaccharidketten kommen, wobei diesen Prozessen wenig Beachtung geschenkt wurde. In der Arbeit wird gezeigt, daß der Abbau bei der Carbanilierung in Pyridin durch Beta-Eliminierung an vorhandenen Carbonylgruppen erfolgt. Die Carbanilierung in DMSO fällt stets stärker aus als bei Einsatz von Pyridin und umfasst die Prozesse Oxidation nach dem Pfitzner-Moffatt-Mechanismus und anschließende Beta-Eliminierung an den neu gebildeten Carbonylgruppen. Dies wird durch Untersuchungen an Modellsystemen und am Polymer herausgearbeitet. Carbonylgruppen an Cellulose werden durch die Umsetzung mit Phenylisocyanat maskiert, was an Modellverbindungen gezeigt wurde (Bildung von Endioldicarbanilaten und carbanilierten Halbacetalen). Ihre Bestimmung in Cellulosecarbanilaten ist daher nicht möglich. Die Abtrennung von niedermolekularen Nebenprodukten der Umsetzung wurde optimiert. Der Einfluss der Vorbehandlung und Voraktivierung von Celluloseproben auf die Vollständigkeit der Umsetzung wurde eingehend untersucht. Es wird ein Standardverfahren zur Carbanilierung von Cellulose angegeben.

This diploma thesis investigated thermal stability and the degradation of hyaluronan (HA) in HA with a molecular mass of 90–130 kDa and in HA with a molecular mass of 1 500–1 750 kDa. The following methods were used for the research: rheology, SEC-MALLS, TGA and DSC. Low-molecular HA was subject to time dependency of degradation investigation, where it was dried at a temperature of 90 °C for a period of 30 minutes and 60 minutes prior to the preparation of the solutions itself. High-molecular HA was investigated not only from the point of view of time but from the point of view of temperature dependency of degradation as well. In the case of investigating the time dependency of degradation, high-molecular HA was dried at a temperature of 75 °C at a time range from 15 minutes to 120 minutes prior to the preparation of the solutions. During the preparation of the solutions for discovering the temperature dependency of degradation, the high-molecular HA was then dried for a period of 30 minutes at a temperature range from 60 °C to 90 °C. For low-molecular HA, thermal stability was proven. Therefore, there is no decrease in the molecular mass and the solutions did not demonstrate a significant decrease of viscosity. For high-molecular HA, thermal stability was not proven. Degradation due to the temperature of drying as well as the time of drying occurred, which was demonstrated by a significant decrease in molecular mass and viscosity of the solutions. While in the case of using a drying temperature of 60 °C, a decrease in the molecular mass occurred by approximately 5 %, the molecular mass decreased by approximately 20 % at a drying temperature of 90 °C compared to undried HA. Due to this reason, high-molecular HA was also further investigated by means of the TGA method, where the decrease of humidity of HA samples in relation to the drying temperature was observed. The DSC method was also used. The objective of the DSC method was to find out temperatures, at which evaporation of humidity contained in an HA sample in relation to its form (undried HA, dried HA and lyophilized HA) occurs. This method further finds out the heat necessary to evaporate humidity from an HA sample. To conclude this research, the results obtained for high-molecular HA were compared with the results of other drying processes – lyophilized proved to be a very gentle drying method because a decrease in the molecular mass for lyophilized HA compared with undried HA almost did not occur.

This master thesis deals with study of chitosan-lignohumate, chitosan-polystyrenesulfonate, chitosan-alginate and chitosan-carrageenan polyelectrolyte complexes. The work was motivated by research of finding suitable alternative substance for lignohumate. The molecular weights of substances were characterized using SEC-MALLS. A degree and a character of the interactions between polyelectrolyte were studied by isothermal titration calorimetry and dynamic light scattering method. The calorimetric experiments proved that decreasing concentration of samples causes decreasing of heat flow. The best calorimetric measurements were provided by adding chitosan into polymer solution. The interactions between chitosan and polyanions and influence of mixing order were proved also by measuring intensity of zeta potential, Z-average of particle size and turbidity. New chitosan-based materials have a big potential in agriculture and medicine.

This thesis, which is a continuation of the previous Bachelor thesis, is dedicated to the study of polyelectrolyte complexes between hyaluronan (HA) and bovine serum albumin (BSA). Interactions between HA and BSA and a formation of complexes were studied by SEC-MALLS method, where a molar mass, a root mean square (rms) radius, a hydronamic radius and an intrinsic viscosity of particles of the system were defined. Furthermore the interactions were studied by calorimetric measurement ITC, where thermodynamic character of complex formation was determined. Additionally, dynamic and electrophoretic light scattering methods (DLS and ELS) were applied, where hydrodynamic radius and a value of the zeta potential were defined. Also the effect of higher temperature on the character and complex formation was examined by SEC-MALLS and ITC measurements. The interactions were confirmed at any of the used media, but with different efficiency. It was also proved that complexes become smaller in their radii and viscosity with growing BSA concentration. Furthermore it was proved, that the high ionic strength hinders surface charges of HA and BSA molecules and minimizes their mutual interactions. At the higher ionic strength the radii of the complexes, their intrinsic viscosity and zeta potential are increased. The higher temperature has only minimal effect on the formation of the complexes.

Le virus de la stomatite vésiculaire (VSV) sert de modèle pour l'étude de la multiplication des virus (Mononegavirales) alors que la rage(RV) reste un sérieux problème de santé publique. Le génome de VSV et RV code notamment la nucléoprotéine (N) et la phosphoprotéine (P). N s'associe étroitement à l'ARN viral. Ce complexe N-ARN sert de matrice pour la réplication et la transcription virale. P est le cofacteur de la polymérase virale (L) et chaperonne N. En interagissant avec N-ARN (domaine C-terminal) et avec L (domaine N-terminal), P assure le lien physique entre l'ARN viral et L. La stœchiométrie de P, sa structure et son rôle exact pendant la transcription et la réplication restent incertains. Mon travail a consisté à une caractérisation biophysique et structurale de P et des complexes N-ARN-P pour mieux comprendre la dynamique du complexe de réplication de ces virus.<br />L'analyse biophysique montre que P RV & VSV existent sous forme de dimère allongé en solution. L'analyse bioinformatique a révélé une organisation modulaire, confirmé par des études biochimiques et biophysiques de mutants de P RV. La structure du domaine C-terminal de P VSV a été résolue par RMN et montre une homologie celle du C-ter de P RV. La caractérisation de l'interaction entre P et les anneaux N-ARN a révélé l'existence de deux types de complexes N-ARN-P (contenant un et 2 dimères de P par anneau). L'étude par ME des complexes nucléocapsides-P a permis de mettre en évidence un changement de conformation important.<br />Pour devenir accessible à L, l'ARN viral doit se dissocier localement de N. L'interaction N-ARN-P représente potentiellement une nouvelle cible pour le développement d'antiviraux.