This master thesis deals with the study of thermodynamic and structural aspects of the interactions of humic acids with organic ions. Methylene blue (dye) was used as a model organic ion. The interactions were studied by isothermal titration calorimetry, dissolution calorimetry and by diffusion in diffusion cells.

Over the last years, varieties of technologies for biofilm analysis were developed and established. They work on different principles and deliver information about biofilms on different information levels. In this work, chip-calorimetry was applied as an analytical tool that measures heat produced from biofilms. Any change of metabolism in biofilms is reflected by a changed heat flow. The heat, which is the integral of the heat flow vs. time, is quantitatively related to the growth stoichiometry of the biofilm, as described by the Hess’ Law. The heat flow is related to the growth kinetics with the reaction heat as proportionality factor. The results from the calorimetric measurement thus, deliver general information about growth stoichiometry and kinetics. The other interpretation of calorimetric results bases on the assumed proportionality between heat flow and oxygen consumption rate (- 460 kJ/mol ). This ratio is called oxycaloric equivalent. Because in case of aerobic growth the majority of oxygen is consumed in catabolic processes during the electron transport phosphorylation, calorimetry is assumed to provide information about the catabolic side of the metabolism. The newly developed chip-calorimeter applied in this work is much more suitable for biofilm studies compared to conventional microcalorimeters due to the flow-through design of the calorimetric chamber. The measurement of undisturbed growing biofilms and the comparison with conventional biofilm analysis tools (i.e. plate counts, confocal laser scanning microscopy (CLSM), and the determination of intermediates’ concentrations (e.g. ATP)) demonstrate the proper functionality of the calorimetric method and the related cultivation procedure by delivering measurement results in the range of literature values. However, when the biofilms were challenged with antimicrobial agents i.e. antibiotics, bacteriophage, and predatory bacteria, the calorimetric results surprisingly deviated from the reference analyses. By combining the results of the calorimetric and reference analyses, additional information about the antimicrobial effects on biofilms can be acquired. Combination of heat measurement and plate counts, which is one of the most conventional approaches, demonstrated that antimicrobials (especially the bactericidal acting kanamycin) could cause the loss of culturability while the cells were still metabolically active. The measurement of ATP content resulted in values out of the typical range, which indicated that antimicrobial treatments disturbed the cellular ATP regulation and the ATP concentration was no longer linearly correlated to the cell number. ATP measurements are therefore not suitable for antimicrobial susceptibility testing. The comparison of heat profiles with the biovolume determined by quantification of microscopic images shows an elevated cell specific heat production rate after the introduction of some antimicrobials (antibiotics and bacteriophage). In case of antibiotics, this can be explained as a consequence of the bacterial defense mechanisms. Most of the described defense mechanisms against antibiotics need biological energy and therefore drive the electron transport phosphorylation (ETP). In case of biofilm treatments with bacteriophage, the trigger of increasing ETP might be the synthesis of phage proteins, hull material, and genetic information molecules. In aerobic conditions, oxygen is used as terminal electron acceptor. Elevated ETP leads therefore to an increase in oxygen consumption, which correlates to the heat production using oxycaloric equivalent as a factor. These correlations explain the increase of cell specific heat productions as biofilms were challenged by antibiotics and bacteriophage. However, also a decrease of specific heat production was observed (in case of predatory bacteria). Here, the predatory bacteria activity caused various damages in host cells, including the interruption of ETP. With these experiments, chip-calorimetry was demonstrated as a promising complementary tool in biofilm research, which provides deeper insights about metabolic activity and alterations. It benefits from the noninvasive handling and the online, real-time measurement that allow the method to be applied for monitoring purposes. Furthermore, its miniaturized dimension allows easy integration in more complex analytic systems and also reduces experiment costs with minimal media/chemical consumption. This thesis also demonstrates the potential development of chip-calorimetry to be more suitable for routine analyses. The use of superparamagnetic beads as matrix to grow biofilms allows regulated transfer of biofilm samples into and from the measurement chamber. This was an initial step towards automation and higher-throughput analysis. One further outcome of the thesis is based on the highly interesting fact about the elevated heat production rate of the host cells induced by the phage infection observed in the chip- calorimetric experiments. The volume specific detection limit of the chip-calorimeter is lower compared to a commercial microcalorimeter. Thus, the infection effect of phages was additionally measured in microcalorimeter to get better quantitative information about the thermal effect of the infection. The results showed that the immediate heat increase after the addition of phage into the solution of the host cells appeared to be quantitatively related to the infection factor, MOI (Multiplicity of Infection). Unfortunately, microcalorimetric measurements in closed ampoules are often subjected to the oxygen limitation. Thus, this problem of microcalorimetric measurement has been addressed. The combination of experimental results and mathematical modeling showed that the rate of metabolism in the static ampoules is defined by the diffusion rate of oxygen into media. This factor has to be considered while designing biological experiments in closed calorimetric measuring chambers and interpreting the calorimetric results for their biological meaning. Some possible solutions to overcome the oxygen bioavailability problem are e.g. to design the experiments with low biomass, or by using media with elevated density to float the biomass at the interface to air and thus to reduce the diffusion path.

Biologiska läkemedel har under det senaste århundradet utökats, och under de senaste 25 åren så har proportionen av godkända biologiska läkemedel har ökat för behandlingen av sjukdomar, vaccin, och diagnostik. Det finns flera olika mekanismer för protein aggregering, och en av dessa är seeding, vilket innebär aggregering inducerat av tillsatta aggregat eller förekomsten av strukturförändringar i proteinet. I detta examensarbete har två terapeutiska proteiner, Somatropin och en monoklonal antikropp, studerats i form av aggregering. Denna studie har utförts genom att värma en del av proteinlösningen för att bilda aggregat och strukturförändrat protein, och sedan blanda detta med nativt protein till olika volymprocent. Dessa lösningar förvarades i olika temperaturer, 4°C, RT och 40°C för att undersöka temperaturberoendet. Med Dynamic Light Scattering (DLS) mättes storleksfördelningen och medelstorleken på proteinet, vilket visade att de seedade proverna ökade i medelstorlek med tiden. Detta indikerar att seedingen inducerade aggregering med tiden. / Biological pharmaceuticals have expanded their use over the last decade, and during the recent 25 years, the proportion of approved biologics has increased for the treatment of diseases, vaccines, and diagnostics. There are several aggregation mechanisms, and one is seeding, i.e., aggregation induced by pre-formed aggregates or the presence of conformational changed proteins. In this master thesis, two therapeutic proteins, Somatropin and one monoclonal antibody have been studied in terms of aggregation. The study has been performed by heating a part of a protein solution to induce aggregation and mixing this with native protein in different volume percentages. These were stored in different temperatures, 4°C, RT, and 40°C, to investigate the temperature dependence. With Dynamic light scattering (DLS), the size distribution and the average-sized particles were measured. This showed that there was a growth of average size in the seeded samples with time. This indicates that the seeding induced aggregation with time.

Efterfrågan på bättre och mer hållbart material ökar. Mer effektivt material kommer att behövas för att möta den ökande, globala efterfrågan. Hybrida organiska-oorganiska material är en typ av material som har varit av stort intresse nyligen, och kan beskrivas som en typ av material som består av både organiska och oorganiska komponenter. Denna avhandling har fokuserat på hybrida organiska-oorganiska material inspirerade av den klassiska perovskitstrukturen ABX3, där komponent A är en organisk katjon, komponent B är en divalent metalkatjon och komponent X är en anjon. Hybrida organiska-oorganiska material som är utgår från den klassiska perovskitstrukturen kan ha olika funktionella egenskaper och en bred variation av tänkbara applikationer. Några exempel på dessa egenskaper och möjliga applikationer inkluderar god fotokonduktivitet för solceller, utmärkt emissionsegenskaper för ljusdioder och justerbara dielektriska egenskaper för elektroniska växlar och sensorer.  De fysiska egenskaperna av det hybrida organiska-oorganiska materialet beror på kristallstrukturen av materialet, som i sig bestäms av valet av komponenter. På grund av de många möjligheter av organiska och oorganiska komponenter så finns det möjlighet att syntetisera helt nya hybrida organiska-oorganiska föreningar som kan ha nya eller förbättrade fysiska egenskaper.  Nuvarande hybrida organiska-oorganiska material som utgår från perovskitstrukturen använder huvudsakligen bly som divalent metalkatjon, och det beror på att den ger den bästa funktionella effekten. Blys toxicitet är dock en stor nackdel för nuvarande blybaserade hybrid oorganiska material. Möjligheten att ersätta bly med en annan divalent metall har undersökts under detta projekt. I denna avhandling så har den organiska katjonen cyclohexylammonium (CHA) varit i fokus som den organiska komponenten. Målet med detta examensarbete var att designa, syntetisera och karakterisera nytt hybrid organisk-oorganiskt material. De hybrida organiska-oorganiska föreningarna CHAZnBr3 och (CHA)2ZnBr4 syntetiserades för den första gången, så vitt författaren vet, och kommer vara i fokus i denna avhandling. De två nya hybrida organiska-oorganiska föreningarna blev strukturellt karakteriserade med X-ray Diffraction (XRD) och termiskt karakteriserade med Thermal Gravimetric Analysis (TGA) och Differential Scanning Calorimetry (DSC).  Den första föreningen, CHAZnBr3, kunde bestämmas att vara ortorombisk vid 298 K. Föreningen bestämdes vara termisk stabil upp till 490 K, och genomgår en fasövergång vid 445 K. Den andra föreningen, (CHA)2ZnBr4, kunde inte bestämmas strukturellt vid varken 100 K eller 298 K. Föreningen bestämdes vara termisk stabil upp till 490 K, och genomgår en fasövergång vid 230 K. Ytterligare karakterisering krävs för att bättre förstå egenskaperna hos dessa föreningar och deras möjliga användningsområden. / The demand for better and more sustainable material is increasing. More efficient materials will be needed to meet the growing global need. Hybrid organic-inorganic materials are one type of materials that have been of great interest recently, which can be described as a class of materials that mix organic and inorganic components. This thesis focused on hybrid organic-inorganic materials inspired by the classical perovskite crystal structure ABX3, where component A is an organic cation, component B is a divalent metal cation and component X is an anion. Hybrid organic-inorganic materials based on the classical perovskite structure may have various functional properties and may have a broad range of potential applications. Some examples of those properties as well as some and possible applications include good photoconductivity and power conversion efficiency for photovoltaic devices, excellent emission properties for light emitting diodes and tunable dielectric properties for electronic switches and sensors.  The physical properties of the hybrid organic-inorganic material are determined by the crystal structure of the material, which in turn will be decided by the choice of components. With the many possible choices for organic and inorganic components, there is an opportunity to synthesize completely new hybrid organic-inorganic compounds that may display new or superior physical properties. Current hybrid organic-inorganic materials based on the perovskite crystal structure mainly use lead as the divalent metal, since it currently gives the best performance. The toxicity of lead is a major drawback for current lead-based hybrid organic-inorganic materials. The possibility to replace lead with another divalent metal has been explored during this project. For this thesis, the organic cation cyclohexylammonium (CHA) has been of focus as the organic component. The aim of this thesis was to design, synthesize and characterize novel hybrid organic-inorganic compounds. The hybrid organic-inorganic compounds CHAZnBr3 and (CHA)2ZnBr4 were synthesized for the first time, to the best of our knowledge, and will be the focus of this thesis. The two new hybrid organic-inorganic compounds were structurally characterized by X-ray Diffraction (XRD) and thermally characterized by Thermal Gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC).  The first compound, CHAZnBr3, could be determined to be orthorhombic at 298 K. The compound was found to be thermally stable up 490 K, and to undergo a phase transition at 445 K.  The second compound, (CHA)2ZnBr4, could not be fully structurally solved at either 100 K or 298 K. The compound was found to be thermally stable up to 490 K, and to undergo a phase transition at 230 K.  Further characterization will be needed to better understand the properties of these two compounds and their possible applications.

<p>The intricate interplay of biomolecules acting together, rather than alone, provides insight into the most basic of cellular functions, such as cell signaling, metabolism, defense, and, ultimately, the creation of life. Inherent in each of these processes is an evolutionary tendency towards increased efficiency by means of biolgocial synergy-- the ability of individual elements of a system to produce a combined effect that is different and often greater than the sum of the effects of the parts. Modern biochemists are challenged to find model systems to characterize biological synergy.</p><p>We discuss the multicomponent, enzyme complex the cellulosome as a model system of biological synergy. Native cellulosomes comprise numerous carbohydrate-active binding proteins and enzymes designed for the efficient degradation of plant cell wall matrix polysaccharides, namely cellulose. Cellulosomes are modular enzyme complexes, comparable to enzyme "legos" that may be readily constructed into multiple geometries by synthetic design. Cellulosomal enzymes provide means to measure protein efficiency with altered complex geometry through assay of enzymatic activity as a function of geometry.</p><p>Cellulosomes are known to be highly efficient at cellulose depolymerization, and current debates on the molecular origins of this efficiency suggest two related effects provide this efficiency: i) substrate targeting, which argues that the localization of the enzyme complex at the interface of insoluble cell wall polysaccharides facilitates substrate depolymerization; and ii) proximity effects, which describe the implicit benefit for co-localizing multiple enzymes with divergent substrate preferences on the activity of the whole complex.</p><p>Substrate targeting can be traced to the activity of a single protein, the cellulosomal scaffoldin cellulose binding module CBM3a that is thought to uniquely bind highly crystalline, insoluble cellulose. We introduce methods to develop a molecular understanding of the substrate preferences for CBM3a on soluble and insoluble cellulosic substrates. Using pivaloylysis of cellulose triacetate, we obtain multiple soluble cello-oligosaccharides with increasing degree of glucose polymerization (DP) from glucose (DP1) to cellodecaose (DP10) in high yield. Using calorimetry and centrifugal titrations, cello-oligosacharides were shown to not bind Clostridial cellulolyticum CMB3a. We developed AFM cantilever functionalization protocols to immobilize CBM3a and then probe the interfacial binding between CBM3a and a cellulose nanocrystal thin film using force spectroscopy. Specific binding at the interface was demonstrated in reference to a control protein that does not bind cellulose. The results indicate that i) CBM3a specifically binds nanocrystalline cellulose and ii) specific interfacial binding may be probed by force spectroscopy with the proper introduction of controls and blocking agents.</p><p>The question of enzyme proximity effects in the cellulosome must be answered by assaying the activity of cellulosomal cellulases in response to cellulosome geometry. The kinetic characterization of cellulases requires robust and reproducible assays to quantify functional cellulase content of from recombinant enzyme preparations. To facilitate the real-time routine assay of cellulase activity, we developed a custom synthesis of a fluorogenic cellulase substrate based on the cellohexaoside of Driguez and co-workers (vide infra). Two routes to synthesize a key thiophenyl glycoside building block were presented, with the more concise route providing the disaccharide in four steps from a commercial starting material. The disaccharide building blocks were coupled by chemical activation to yield the fully protected cellohexaoside over additional six steps. Future work will include the elaboration of this compound into an underivatized FRET-paired hexasaccharide and its subsequent use in cellulase activity assays.</p><p>This dissertation also covers an experimental system for the evaluation of methonium desolvation thermodynamics. Methonium (-N+Me3, Am) is an organic cation widely distributed in biological systems. The appearance of methonium in biological transmitters and receptors seems at odds with the large unfavorable desolvation free energy reported for tetramethylammonium (TMA+), a frequently utilized surrogate of methonium. We report an experimental system that facilitates incremental internalization of methonium within the molecular cavity of cucurbit[7]uril (CB[7]).</p><p>Using a combination of experimental and computational studies we show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by a remarkably small desolvation enthalpy of just 0.5±0.3 kcal*mol-1, a value significantly less endothermic than those values suggested from gas-phase model studies (+49.3 kcal*mol-1). More surprisingly, the incremental withdrawal of methonium surface from water produces a non- monotonic response in desolvation enthalpy. A partially desolvated state exists, in which a portion of the methonium group remains exposed to solvent. This structure incurs an increased enthalpic penalty of ~3 kcal*mol-1 compared to other solvation states. We attribute this observation to the pre- encapsulation de-wetting of the methonium surface. Together, our results offer a rationale for the wide biological distribution of methonium and suggest limitations to computational estimates of binding affinities based on simple parameterization of solvent-accessible surface area.</p> / Dissertation

This work presents an in-depth study of the crystal structures and hydrogen sorption potential of the Li - N - H and Li - Na - N - H systems. The structures of the materials have been studied using X-ray and neutron diffraction, Raman spectroscopy and inelastic neutron scattering. The behavior of the materials during heating was studied using variable temperature X-ray diffraction, intelligent gravimetric analysis in conjunction with neutron diffraction, intelligent gravimetric analysis combined with mass spectrometry and differential scanning calorimetry. The role of cation disorder in the Li - N - H (D) system has been explored, indicating that crystallographic ordering of the Li⁺ ions within lithium amide and lithium imide significantly affects the hydrogen sorption properties of the materials. Order-disorder transitions were observed both during hydrogen desorption from ordered LiNH₂ and during deuterium adsorption on ordered Li₂ND. Such transitions were not observed in disordered samples of the materials. The intrinsic disorder and the stoichiometry of Li - N - H(D) materials was shown to depend strongly on the techniques used during their synthesis. Studies regarding the synthesis, crystal chemistry and decomposition properties of the mixed Li / Na amides are presented. Two distinct mixed Li / Na amides of formulae Li₃Na(NH₂)₄ and LiNa₂(NH₂)₃ were observed in the LiNH₂ / NaNH₂ phase space. Na was also seen to be soluble in LiNH₂, forming sodium-doped LiNH₂ . Li₃Na(NH₂)₄ and Na-doped LiNH₂ were found to exhibit significant cation non-stoichiometry, whereas LiNa₂(NH₂)₄ was shown to exist as a line phase material. Thermogravimetric and calorimetric studies of the mixed Li / Na amides suggested that these materials decompose primarily with loss of H₂.

Le travail de thèse a porté essentiellement sur l'effet des conditions de prise en pression et en température dans une gamme (20-60°C/Patm-200 bar) d'un ciment pétrolier (Portland « classe G ») au très jeune âge (jusqu'à un jour) en condition saturée sur (i) la cinétique d'hydratation, (ii) les propriétés hydriques (perméabilité et porosité) et (iii) le développement des propriétés élastiques dynamiques obtenues par propagation d'ultrasons et quasi-statiques. Les propriétés quasi-statiques instantanées et différées ont été obtenues à partir d'essais mécaniques réalisés en compression simple à différents temps/degré d'hydratation.La partie I a permis de montrer que la pression appliquée pendant la prise a un effet négligeable sur la cinétique d'hydratation contrairement à l'effet accélérateur de la température de prise. Un modèle de cinétique d'hydratation tenant compte de l'effet accélérateur de la température a été mis en place afin de déterminer l'évolution des fractions volumiques des différentes phases en présence dans la pâte de ciment au cours de son hydratation. L'évolution de la phase (porosité capillaire + eau) obtenue par le modèle d'évolution des fractions volumiques est en bonne concordance avec l'évolution de la porosité connectée mesuré par porosimétrie dans la deuxième partie de travail de thèse. En partie II, concernant la caractérisation de la perméabilité, les valeurs obtenues ainsi que l'évolution des propriétés élastiques quasi-statiques obtenue expérimentalement ont permis de déterminer le temps de drainage pour chacune des conditions d'hydratation étudiées. Cette information permet de déterminer la nature drainée ou non drainée des essais mécaniques quasi statiques réalisés. En partie III, les essais mécaniques ont permis d'identifier un comportement granulaire faiblement cohésif au niveau du sommet du pic exothermique, puis un comportement solide cohésif avec progression des propriétés mécaniques au cours de l'hydratation à partir de la décroissance du flux thermique. Enfin, deux modèles par homogénéisation multi-échelles ont été proposés, la différence portant sur la schématisation de la pâte de ciment au très jeune âge. Les résultats ont été confrontés avec succès aux propriétés élastiques dynamiques et quasi-statiques drainées et non drainées obtenues expérimentalement / The thesis primarily concerned the effect of the set conditions in pressure and in temperature in a range (20-60°C/Patm-200 bar) of an oilwell cement (Portland cement class G) at very early age(up to one day) in saturated condition on (I) the hydration kinetics, (II) the hydrous properties (permeability and porosity) and (III) the development of the dynamic elastic properties obtained by ultrasonic propagation and quasi-static properties. The "instantaneous" and differed quasi-static properties were obtained by mechanical tests carried out in uniaxial compression with various times/degree of hydration

CD2AP is a member of the CD2AP/CIN85 family of adaptors and involved in several cellular processes, such as kidney podocyte development, actin mediated membrane trafficking and T cell activation. It contains three SH3 domains whose binding properties and interaction partners remain largely unexplored. The CD2AP SH3 interaction with the novel partner Rab5-activating GEF RIN3 was studied extensively by isothermal titration calorimetry (ITC), peptide scanning arrays, mutagenesis and X-ray crystallography. Mapping of the interaction regions showed that human RIN3 contains two binding sites for the CD2AP SH3 domains. From these studies, the CD2AP SH3 recognition motif P-x-P/A-x-x-R emerged. Two crystal structures (1.65 &Aring; and 1.2 &Aring;) of the SH3 1 and SH3-2 domains in complex with RIN3 epitopes 1 and 2 respectively revealed that these residues serve as anchoring points. With the aid of bioinformatics tools, this motif was used to conduct a peptide array-based screen for additional signalling partner candidates. One of the hits was the Arf-GAP ARAP1. ITC data indicate that the three SH3 domains differentially recognise three ARAP1 epitopes, with the first ARAP1 epitope binding to SH3-2 in the nanomolar range. A crystal structure (1.6 &Aring;) of the SH3-2 domain in complex with the first ARAP1 epitope implicates two additional anchoring residues that extend beyond the PPII helical region of the canonical motif. The CD2AP/ARAP1 interaction was confirmed in podocytes and cancer cells at the endogenous protein level. Even though RIN3 and ARAP1 are involved in membrane trafficking, a direct link to CD2AP had not been reported before. Other candidates from the peptide array analyses were also investigated by ITC. In conclusion, this study led to the elucidation of the CD2AP SH3-1 and SH3-2 domain binding signatures and the identification of putative novel binding partners for all three SH3 domains. Lastly, insight was gained into the binding preferences of CD2AP SH3-3.

Les principaux substrats oxydés à l’exercice, soit les glucides, les lipides et les pro- téines ne contribuent pas tous au même niveau à la fourniture d’énergie lors de l’effort prolongé. De plus, le glucose peut provenir de différentes sources endogènes (muscle, foie) et exogènes. Plusieurs facteurs peuvent influencer leur contribution respective incluant : la masse musculaire impliquée et l’entraînement préalable, le sexe, l’état nutritionnel et les conditions environnementales. L’utilisation d’isotopes stables, tels que le carbone 13 (13C), combinée à la calorimétrie indirecte respiratoire corrigée pour l’excrétion d’urée dans l’urine et la sueur, permet de différencier les substrats endogènes et exogènes et d’évaluer la contribution de leur oxydation à la fourniture d’énergie. Ces méthodes d’investigation permettant d’apprécier la sélection des substrats lors de l’exercice prolongé avec ingestion de glucose ont permis d’effectuer les comparaisons qui ont fait l’objet des trois études de cette thèse. Dans la première étude, la sélection des substrats au cours d’un effort prolongé effectué avec les membres inférieurs ou les membres supérieurs a été comparée avec et sans ingestion de glucose. Une différence modeste fut observée entre la sélection des substrats selon le mode d’exercice avec l’ingestion d’eau, celle-ci favorisant légèrement l’oxydation des glucides lors de l’effort avec les membres supérieurs. La quantité de glucose exogène oxydée était plus faible lors de l’exercice avec les membres supérieurs qu’avec les membres supérieurs, mais sa contribution plus importante, conséquence d’une dépense énergétique plus faible. Dans la deuxième étude, on a comparé la sélection des substrats chez des sujets mas- culins et féminins et les effets d’une alimentation enrichie en glucides ou de l’ingestion de glucose, au cours d’un exercice prolongé d’une durée de deux heures. On reconnaît généralement que, pour une même puissance relative, les femmes utilisent moins de glucides et davantage de lipides que les hommes. Les effets séparés d’une alimentation riche en glucides ou de l’ingestion de glucose pendant l’exercice sur la sélection des substrats furent pourtant similaires chez les deux sexes. L’effet combiné des deux procédures de supplémentation est toutefois plus important chez la femme que chez l’homme, soutenant l’hypothèse qu’un léger déficit en glucides soit présent chez les femmes. Dans la troisième étude, l’oxydation des substrats et particulièrement celle d’amidon exogène au cours d’une marche prolongée à une faible puissance de travail a été décrite. Les individus qui pratiquent des activités physiques prolongées à des intensités faibles (< 40 %VO2max) sont encouragés à ingérer des glucides et de l’eau pendant l’effort, mais la contribution de leur oxydation à la fourniture d’énergie est relativement peu connue. Nous avons montré que, contrairement aux observations précédemment effectuées à jeun sans ingestion de glucides pendant l’effort, les glucides (incluant de source exogène) peuvent fournir une très grande partie de l’énergie lorsqu’ils sont ingérés à des intervalles réguliers au cours de l’exercice prolongé. Dans l’ensemble, les résultats des études expérimentales présentées dans cette thèse montrent que les glucides ingérés peuvent fournir une grande proportion de l’énergie pendant l’exercice prolongé. Toutefois, le mode d’exercice, le sexe et la puissance de travail mènent à des variations qui sont en grande partie liées à une dépense énergétique variable selon les conditions et les groupes d’individus ayant des caractéristiques différentes. / The main substrates oxidized during prolonged exercise, whether carbohydrates, fats or proteins, do not contribute equally to the energy supply. In addition, glucose can originate from different endogenous (muscle, liver) and exogenous sources. Many factors can affect their respective contribution including: active muscle mass, training, sex, nutritional state and environmental conditions. The use of stable isotopes, such as carbon 13 (13C), combined with indirect respiratory calorimetry corrected for urea excretion in urine and sweat, allows the differentiation of endogenous and exogenous substrates and the estimation of their contribution to the energy yield. These methods, allowing the measurement of fuel selection during prolonged exercise with glucose ingestion, enabled the comparisons found in the three experimental studies part of this thesis. In the first study, fuel selection was compared during prolonged upper- and lower-body exercise with or without glucose ingestion. The difference in fuel selection between upper- and lower-body exercise when water was ingested was modest with a slightly higher reliance on carbohydrate oxidation during upper-body exercise. The amount of exogenous glucose oxidized was lower but its contribution to the energy yield was higher during upper-body exercise due to the lower energy expenditure. In the second study of the thesis, the effects on fuel selection in women and men of a diet rich in carbohydrate and glucose ingestion during prolonged exercise were compared. It is generally recognized that for a given relative workload, women rely less on carbohydrates and more on fat than men. The separate effects of a diet rich in carbohydrates or glucose ingestion during exercise on fuel selection were indeed similar in men and women. However, the combined effects of both procedures were larger in women than in men, supporting the suggestion that a small carbohydrate deficit could be present in women. In the third study, substrate oxidation and particularly that of exogenous starch during prolonged walking at a low workload was described. Subjects engaged in prolonged exercise at low workload (<40 %VO2max) are encouraged to ingest carbohydrate along with water before and during exercise but there is currently few data on the contribution to the energy yield of ingested carbohydrate in this situation. In contrast to what is observed in fasted subjects without carbohydrate ingestion during exercise, we have shown that carbohydrate (including exogenous starch) can supply a very large proportion of the energy yield when ingested before and at regular interval during prolonged exercise. Taken together, the results of the experimental studies show that ingested carbohydrates can supply a large fraction of the energy yield during prolonged exercise. However, the exercise mode, the sex and the workload sustained has shown some differences that are mostly due to variations in energy expenditure between experimental conditions or groups of individuals with different characteristics.

Thermal stability of theG37 tRNA methyltransferase proteins from Thermotoga maritima and Aquifex aeolicus have been compared using Differential Scanning Calorimetry. It was shown that the Thermotoga protein is remarkably stable and is denatured at temperatures in excess of 100 degrees Centigrade. The Aquifex aeolicus protein was less stable, denaturing broadly at temperatures between 55oC and 100oC. In contrast, the mesophilic E. coli protein was completely denatured at 55oC. Enzymatic activity of the proteins was measured at various temperatures. Both the Thermotoga and Aquifex enzymes are active at ambient temperatures, and display a significant decrease in activity when the temperature is raised above 50oC. This may relate to subtle changes in protein structure causing an effect on the tRNA based assay. Both enzymes contain inter subunit disulfide bonds which might contribute to thermal stability. Assays of the enzymes in the presence of high concentrations of Dithiothreitol (DTT) did not significantly reduce activity at higher temperatures, but did stimulate activity at lower temperatures. Site directed mutagenesis of non -conserved protein sequences within Thermotoga maritima were initiated in order to determine what structures might confer heat stability on the protein. Alanine mutagenesis of lysine residues 103,104 led to reduced catalytic activity, but did increased activity at higher temperatures. Aspartate is the most common residue at the relative position 166 in the variable loop of most TrmD genes. It has been shown that in E. coli this is essential for catalytic activity and possibly the residue which carries out N1 deprotonation on residue G37 in tRNA. In Thermotoga glutamate is present at this position. Alanine mutagenesis of this residue did not eliminate activity suggesting another nearby residue may function in this capacity in the Thermotoga TrmD protein.