Macromolecular interactions play very important roles in regulation of all levels of biological processes. Aberrant macromolecular interactions often result in diseases. By applying a combination of spectroscopy, calorimetry, computation and other techniques, the protein-protein interactions in the system of the Shaw2 Kv channel and the protein-RNA interactions in West Nile virus RNA-cellular protein TIAR complex were explored. In the former system, the results shed light on the local structures of the key channel components and their potential interaction mediated by butanol, a general anesthetic. In the later studies, the binding modes of TIAR RRM2 to oligoU RNAs and West Nile virus RNAs were investigated. These findings provided insights into the basis of the specific cellular protein–viral RNA interaction and preliminary data for the development of strategies on how to interfere with virus replication

Motiviert durch die in der Literatur bisher unvollständige Beschreibung der Relaxation hochgeladener Ionen vor Festkörperoberflächen, besonders in Bezug auf den Eintrag potenzieller Energie in Oberflächen und der Aufstellung einer vollständigen Energiebilanz, werden in dieser Arbeit komplementäre Studien präsentiert, die sowohl die Ermittlung des Anteils der deponierten potenziellen Energie als auch die Ermittlung der emittierten potenziellen Energie ermöglichen. Zum Einen wird zur Bestimmung des eingetragenen Anteils der potenziellen Energie eine kalorimetrische Messanordnung verwendet, zum Anderen gelingt die Bestimmung der emittierten potenziellen Energie mittels doppeldifferenzieller Elektronenspektroskopie. Für vertiefende Studien werden Materialien unterschiedlicher elektronischer Strukturen (Cu, n-Si, p-Si und SiO2 ) verwendet. Im Falle der Kalorimetrie wird festgestellt, dass die eingetragene potenzielle Energie linear mit der inneren potenziellen Energie der Ionen wächst. Dabei bleibt das Verhältnis zwischen der eingetragenen potenziellen Energie und der inneren potenziellen Energie nahezu konstant bei etwa (80 ± 10) %. Der Vergleich von Cu, n-Si und p-Si zeigt im Rahmen der Messfehler keine signifikanten Unterschiede in diesem Verhältnis. Es liegen jedoch deutlich unter jenem von SiO2. Die Elektronenspektroskopie liefert ein dazu komplementäres Ergebnis. Für Cu und Si konnte ebenfalls eine lineare Abhängigkeit zwischen emittierter Energie und innerer potenzieller Energie festgestellt werden. Das Verhältnis wurde hierfür bis zum Ladungszustand bis Ar7+ zu etwa (10 ± 5) % unabhängig vom Ladungszustand bestimmt. Im Gegensatz dazu liefert SiO2 eine nahezu verschwindende Elektronenausbeute. Für Ar8+ und Ar9+ steigt die Elektronenausbeute wegen der Beiträge der LMM-Augerelektronen für alle untersuchten Materialien leicht an. Der Anteil der emittierten Energie eines Ar9+ -Ions wird für Cu und Si zu etwa 20 % und für SiO2 zu etwa 10 % angegeben. Diese Ergebnisse sind in guter Übereinstimmung mit den Kalorimetrieexperimenten und erfüllen die Energiebilanz. Zusätzlich werden die experimentellen Ergebnisse mit einer Computersimulation modelliert, welche auf dem erweiterten dynamischen klassischen Barrierenmodell basiert. Aus diesen Rechnungen kann zudem jener Anteil der deponierten potenziellen Energie erhalten werden, welcher durch Bildladungsbeschleunigung vor der Oberfläche in kinetische Energie umgewandelt wurde. / Motivated by the incomplete scientific description of the relaxation of highly charged ions in front of solid surfaces and their energy balance, this thesis describes an advanced complementary study of determining deposited fractions and re-emitted fractions of the potential energy of highly charged ions. On one side, a calorimetric measurement setup is used to determine the retained potential energy and on the other side, energy resolved electron spectroscopy is used for measuring the re-emitted energy due to secondary electron emission. In order to study the mechanism of energy retention in detail, materials with different electronic structures are investigated: Cu, n-Si, p-Si and SiO2 . In the case of calorimetry, a linear relationship between the deposited potential energy and the inner potential energy of the ions was determined. The total potential energy which stays in the solid remains almost constant at about (80 ± 10) %. Comparing the results of the Cu, n-Si and p-Si targets, no significant difference could be shown. Therefore we conclude that the difference in energy deposition between copper, n-doped Si and p-doped Si is below 10 %, which is significantly lower than using SiO2 targets. For this purpose, electron spectroscopy provides a complementary result. For Cu and Si surfaces, an almost linear increase of the re-emitted energy with increasing potential energy of the ion up to Ar7+ was also observed. The ratio of the re-emitted energy is about (10 ± 5) % of the total potential energy of the incoming ion, almost independent of the ion charge state. In contrast, an almost vanishing electron emission was observed for SiO2 and for charge states below q=7. For Ar8+ and Ar9+, the electron emission increased due to the contribution of the projectile LMM Auger electrons and the re-emitted energy amounts up to 20 % for Cu and Si and around 10 % for SiO2 .These results are in good agreement with the calorimetric values. In addition, the experimental results are compared with computer simulations based on the extended dynamical over-the-barrier model. From these calculations, the ratio of deposited potential energy that is transformed into kinetic energy before deposition due to the image charge acceleration can be maintained.

Cellulose ethers and polyethylene glycols are used in drug formulations as water swelling or water soluble matrices. Polar lipids, for example monoglycerides, and surfactants can be used to solubilise hydrophobic or amphiphilic drugs and to formulate potential drug delivery vehicles such as emulsions, liposomes and cubic phases. In this thesis mixtures of these excipients are characterised in various environments, from dilute aqueous solutions to solid dispersions. Special focus has been on the understanding of the associating processes involved. Detailed understanding of the association of cellulose ethers, of varying hydrophobicity, and amphiphilic substances is presented. The hydrophobicity of the polymer was found to have an impact on the interaction scheme. The amphiphiles were found to bind at lower amphiphile concentrations to a more hydrophobic polymer thus influencing both micro- and macroscopic structure of the aggregates. The choice of counterion to the amphiphile has a small but significant effect on the interaction and the structure of the aggregates. Also amphiphilic drug molecules can interact with nonionic polymers in a similar way as surfactants in aqueous solution. Due to the higher cmc of the drug ibuprofen the interaction is largely influenced by the ionic strength of the solution. The type of amphiphile also influences the cooperativity of the amphiphile-polymer binding. In more concentrated systems liquid crystals are formed into which the polymer interact with the amphiphiles. Both cubic and sponge phases were found with relatively large polymers interacting with polar lipids. These phases were found to swell and shrink mainly controlled by the amount of polymer inside them. Also membrane interacting substances added to the sponge phase could influence the size of the water channels in the phase. In water free systems polymers and polar lipids were found to interact as well as forming solid dispersions. The behaviour of the phase separation between polymer and lipid depended on the concentration of the dispersed phase. The polar lipid was found to be distributed in the lamellar part of the semicrystalline polymer influencing the polymer folding.

Additives, incorporated in film coating formulations, and their process parameters are generally selected using a trial-and-error approach. However, coating problems and defects, especially those associated with aqueous coating systems, indicate the necessity of embracing a quality-by-design approach to identify the optimum coating parameters. In this study, the feasibility of using thermal and rheological measurements to help evaluate and design novel coating formulations has been investigated. Hydroxypropyl methylcellulose acetate succinate (HPMCAS), an enteric coating polymer, was used as the film forming polymer. Differential Scanning Calorimetry (DSC), Dynamic Mechanical Analysis (DMA), and Parallel Plate Shear Rheometery (PPSR) were used to evaluate the effect of different plasticisers on the performance of HPMCAS. The results illustrate that, for identical formulations, the DSC and DMA methods yielded up to 40% differences in glass transition temperature (Tg) values. Moreover, Tg measured using loss modulus signals were always 20-30 oC less than those measured using tan delta results in DMA testing. Absolute and relative Tg values can significantly vary depending on the geometry of the samples, clamp size, temperature ramping rate and the frequency of the oscillations. Complex viscosity data for different formulations demonstrated a variable shear thinning behaviour and a Tg independent ranking. It is, therefore, insufficient to rely purely on Tg values to determine the relative performance of additives. In addition, complex viscosity results, obtained using both the DMA and PPSR techniques at similar temperatures, are shown to be comparable. The results from both techniques were therefore used to produce continuous master curves for the HPMCAS formulations. Additionally, step strain tests showed that HPMCAS chains do not fully III disentangle after 105 seconds as predicted by the Maxwell model. Finally, in situ aqueous-based coating experiments proved that mixtures of triethyl acetyl citrate and acetylated monoglyceride (TEAC/AMG), even without cooling of the suspension, do not cause blocking of the spray nozzle whereas triethyl citrate (TEC) based formulae did. TEAC (alone or in a combination with AMG) exhibits superior wettability to HPMCAS than TEC/AMG formulations and can be used to enhance the efficiency and film quality of the dry coating process.

The thesis describes the synthesis of chromatographic media using several different approaches, their characterizations and applications in liquid chromatography. The steps to achieve a separation column for a specific analyte are presented. The main focus of the study was the design of novel molecularly imprinted polymers. Attachment of monolithic polymeric substrates to the walls of fused silica capillaries was studied in Paper I. With a broad literature survey, a set of common methods were tested by four techniques and ranked by their ability to improve anchoring of polymers. The best procedure was thus used for all further studies. Synthesis of monoliths in capillary columns was studied in Paper II. With the goal of separating proteins without denaturation, various monoliths were polymerized in situ using a set of common monomers and cross-linkers mixed with poly(ethylene glycol) as porogen. The resulting network was expected to present “protein-friendly pores”. Chemometrics were used to find and describe a set of co-porogens added to the polymerization cocktails in order to get good porosity and flow-through properties. Assessment of the macroporous structure of a monolith was described in Paper III. An alternative method to mercury intrusion porosimetry was proposed. The capillaries were embedded in a stained resin and observed under transmission electron microscope. Images were then computed to determine the pore sizes. Synthesis of molecularly imprinted polymers grafted to a core mono-lith in a capillary was described in Paper IV. The resulting material, imprinted with local anaesthetics, was tested for its chromatographic performance. Similar imprinted polymers were characterized by microcalorimetry in Paper V. Finally, imprinted monoliths were also synthesized in a glass tube and further introduced in a NMR rotor to describe the interactions between stationary phase and template in Paper VI.

L’augmentation de la dépense énergétique (DE) par une augmentation de l'activité physique (AP) participe au maintien et à l’amélioration de la santé. La mesure à grande échelle de la DE totale (DET) en général et AP en particulier se heurte à des difficultés pratiques de recueil de données, de validité et aux coûts. Bien que dans la littérature de nombreux accéléromètres permettent d’estimer la DET, il y a encore des limites quant à la mesure de l’intensité de l’AP, élément qui influence l’état de la balance énergétique et le bénéfice pour la santé de l’AP. De plus, peu de comparaisons entre les différents accéléromètres sont disponibles. La présente étude avait pour but d’évaluer la capacité de deux accéléromètres (Actical et Sensewear Armband) pour estimer la DET en comparaison avec la technique de l’eau doublement marquée (EDM) ; d’évaluer la concordance entre les accéléromètres dans la mesure de la DE au repos (DER) en comparaison avec la technique de la calorimétrie indirecte (CI) et d’évaluer la DE liée à l’AP en comparaison avec la technique de la CI. Les résultats montrent qu’il y a une très bonne corrélation entre la CI et les accéléromètres dans la mesure de la DER(r > 0.80, p<0.001) et une bonne corrélation entre les accéléromètres et l’EDM dans la mesure de la DET(r>0.75, p<0.001). Pour la mesure de l’intensité de l’AP, l’ACT est plus précis (r=0.80, p<0.001)) que le SWA à la marche, le SWA est meilleur(r=0.80, p<0.001) au vélo. Cette étude permet d’affirmer que le SWA et l’ACT sont des alternatives valables pour mesurer la DE. / The increase in energy expenditure by the increase of physical activity contributes to maintaining and improving health. The large-scale measurement of total energy expenditure in general and physical activity in particular, is faced with difficulties of data collection, validity and cost. Although literature suggests that many accelerometers are used to estimate total energy expenditure, there are still limits as to the measurement of the intensity of physical activity, which is one of the factors that influences the energy balance and health benefits of physical activity. In addition, few comparisons between the different accelerometers are available. This study aimed to evaluate the ability of two accelerometers (Actical and SensWear Armband) to estimate total energy expenditure compared with the technique of doubly labelled water; to assess the correlation between the two accelerometers in the measurement of resting energy expenditure, compared with the technique of indirect calorimetry and finally to assess energy expenditure linked to physical (walking and cycling) at different intensities, in comparison with the indirect calorimetry technique. The results show a very good correlation between indirect calorimetry and the two accelerometers(r > 0.80, p<0.001) in the measurement of resting energy expenditure and a good correlation between accelerometers and the gold standard technique (doubly labeled water) in the measurement of total energy expenditure(r>0.75, p<0.001). To measure the intensity of physical activity, Actical is more accurate than SensWear Armband(r=0.80, p<0.001) at low intensity while the SensWear Armband is better at higher intensity (r=0.80, p<0.001). This study provides evidence that these two accelerometers are suitable alternatives for measuring energy expenditure.

La réparation de l’ADN par excision des nucléotides (NER) est un mécanisme capable de retirer une large variété de lésions causant une distorsion de la double hélice, comme celles causées par les rayons ultraviolets (UV). Comme toutes les voies de réparation de l’ADN, la NER contribue à la prévention de la carcinogénèse en prévenant la mutation de l’ADN. Lors de ce processus, il y a d’abord reconnaissance de la lésion par la protéine XPC/Rad4 (humain/levure) qui recrute ensuite TFIIH. Ce complexe déroule l’ADN par son activité hélicase et recrute l’endonucléase XPG/Rad2 ainsi que d’autres protéines nécessaires à l’excision de l’ADN. Lors de son arrivée au site de lésion, XPG/Rad2 déplace XPC/Rad4. TFIIH agit également lors de la transcription de l’ADN, entre autres par son activité hélicase. Outre cette similarité de la présence de TFIIH lors de la transcription et la réparation, il est possible de se demander en quoi les deux voies sont similaires. Nous nous sommes donc intéressés aux interactions impliquant TFIIH et la machinerie de réparation de l’ADN. Nous avons donc entrepris une caractérisation structurale et fonctionnelle de ces interactions. Nous avons découvert que Rad2 et Rad4 possèdent un motif d’interaction en nous basant sur d’autres interactions de la sous-unité Tfb1 de TFIIH. Par calorimétrie à titrage isotherme, nous avons observé que les segments de ces deux protéines contenant ce motif interagissent avec une grande affinité au domaine PH de Tfb1. Le site de liaison de ces segments sur Tfb1PH est très semblable au site de liaison du domaine de transactivation de p53 et au domaine carboxy-terminal de TFIIEα avec Tfb1PH, tel que démontré par résonance magnétique nucléaire (RMN). De plus, tous ces segments peuvent faire compétition les uns aux autres pour la liaison à Tfb1PH. Nous avons aussi démontré in vivo chez la levure qu’une délétion de Tfb1PH crée une sensibilité aux radiations UV. De plus, la délétion de multiples segments de Rad2 et Rad4, dont les segments d’interaction à Tfb1PH, est nécessaire pour voir une sensibilité aux rayons UV. Ainsi, de multiples interactions sont impliquées dans la liaison de Rad2 et Rad4 à TFIIH. Finalement, les structures des complexes Rad2-Tfb1PH et Rad4-Tfb1PH ont été résolues par RMN. Ces structures sont identiques entre elles et impliquent des résidus hydrophobes interagissant avec des cavités peu profondes de Tfb1PH. Ces structures sont très semblables à la structure de TFIIEα-p62PH. Ces découvertes fournissent ainsi un lien important entre la transcription et la réparation de l’ADN. De plus, elles permettent d’émettre un modèle du mécanisme de déplacement de XPC/Rad4 par XPG/Rad2 au site de dommage à l’ADN. Ces connaissances aident à mieux comprendre les mécanismes de maintient de la stabilité génomique et peuvent ainsi mener à développer de nouvelles thérapies contre le cancer. / The nucleotide excision repair pathway (NER) is a mechanism capable of removing a wide variety of helix-distorting lesions, such as those caused by ultraviolet irradiation (UV). As all DNA repair pathways, NER contributes to the prevention of carcinogenesis by preventing DNA mutation. During this process, the lesion is first recognized by the protein XPC/Rad4 (human/yeast), which then recruits TFIIH. This complex unwinds the DNA with its helicase activity and then recruits the endonuclease XPG/Rad2 and other proteins necessary for DNA excision. Upon arrival at the lesion site, XPG/Rad2 displaces XPC/Rad4. TFIIH also acts in DNA transcription, using its helicase activity. In addition to the similarity of the presence of TFIIH in transcription and DNA repair, it is possible to ask ourselves how the two pathways are similar. We were interested in the interactions involving TFIIH and the DNA repair machinery. We have therefore undertaken a structural and functional characterization of these interactions. We have found that Rad2 and Rad4 have a motif of interaction based on other interactions of the Tfb1 subunit of TFIIH. Using isothermal titration calorimetry, we found that segments of these two proteins containing this motif interact with high affinity to the PH domain of Tfb1. The binding site of these segments is very similar to Tfb1PH binding site of transactivation domain of p53 and the carboxyl-terminal domain of TFIIEα with Tfb1PH, as demonstrated by nuclear magnetic resonance (NMR). In addition, these segments can compete with each other for binding to Tfb1PH. We also demonstrated in vivo that deletion of Tfb1PH in yeast creates a sensitivity to UV irradiation. In addition, the deletion of multiple segments of Rad2 and Rad4, including segments of interaction Tfb1PH, is required to observe a sensitivity to UV. Thus, multiple interactions are involved in the binding of TFIIH to Rad2 and Rad4. Finally, the structures of the Rad2-Tfb1PH and Rad4-Tfb1PH complexes were solved by NMR. These structures are identical to each other and involve hydrophobic residues interacting with shallow grooves on Tfb1PH. These structures are very similar to the structure of TFIIEα-p62PH. These findings provide an important mechanistic link between transcription and DNA repair. In addition, they provide a model of the mechanism of the displacement of XPC/Rad4 by XPG/Rad2 at the damaged site. This knowledge helps to better understand the mechanisms of genomic stability and can lead to novel cancer therapies.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal