Les cocktails enzymatiques tels que Rovabio® sont utilisés en nutrition animale comme complément alimentaire pour aider les animaux à mieux assimiler les fibres présentes dans leur ration alimentaire composée principalement de blé en Europe. Le mécanisme de déconstruction enzymatique du son de blé, partie du grain majoritairement composée de fibres, considérées comme difficilement hydrolysables et donc assimilables reste encore incompris, c’est pourquoi ces travaux de thèse s’appuient sur l’utilisation d’un bioréacteur instrumenté combinant des analyses physiques in-situ et biochimiques ex-situ afin d’avoir un point de vue global de ce phénomène. Cette approche multi-échelle est originale car rarement considérée en nutrition animale où les études in-vivo sont privilégiées. Cos travaux ont ainsi permis de mettre en évidence que l’action de Rovabio® se caractérise par une première phase de fragmentation notamment des grosses particules concomitante avec une forte solubilisation. La déconstruction du son de blé se poursuit ensuite par une fragmentation mais cette fois sans aucune solubilisation de polysaccharides. L’ajout d’une xylanase seule, en tant qu’enzyme la plus active du cocktail, solubilise la même quantité d’arabinoxylane mais ne permet pas une fragmentation importante des particules, contrairement au Rovabio®. Ces résultats confirment donc l’importance de la richesse et de la diversité d’un cocktail enzymatique pour déconstruire efficacement des structures aussi complexe que le son de blé. Cependant, en dépit de cela, seulement 37%w/w de matière sèche est solubilisée, même en excès de Rovabio®. Cette incapacité du cocktail enzymatique à dégrader complètement ces fibres semblerait provenir d’une inaccessibilité des enzymes à leur substrat. Nous avons ainsi montré que le rendement d’hydrolyse enzymatique est amélioré en augmentant la surface spécifique des particules (traitement mécanique) et/ ou en désorganisant l’architecture de la structure des fibres par l’ajout d’un complexe enzymatique particulièrement riche en pectinases. Néanmoins, si ces deux voies améliorent les performances du cocktail, elles ne permettent toujours pas une hydrolyse totale du son de blé. Finalement ce travail souligne l’intérêt d’enzymes ou de protéines actives capables d’attaquer les structures minoritaires du réseau lignocellulosique assurant sa résistance et sa cohésion, ce qui permet ainsi aux enzymes d’avoir un meilleur accès à leurs substrats. / Enzyme cocktails, such as Rovabio®, which is rich of hydrolytic enzymes are used as feed additives to favor degradation of non-starch polysaccharides present in wheat, a major feed in poultry industry. The deconstruction mechanism of wheat bran, part of the seed mainly composed of fiber, is still fairly unclear. This PhD aims to highlight these mechanisms using a multi-instrumented bioreactor that allowed to combine in-situ physical and ex-situ biochemical analyses. This multiscale approach stands as an alternative and original approach which is rarely considered in animal nutrition. This work highlights that Rovabio® action occurred in two concurrent process, namely fragmentation and solubilization phenomena which take place within the first 2 h after addition of the enzyme cocktail. It is then followed by a particle fragmentation which was not accompanied by any sugars solubilization. Thus, in spite of the abundant and very active hydrolytic enzyme activities in Rovabio®, the deconstruction of destarched wheat bran was however limited to 37% of w/w. At variance to Rovabio®, xylanase added alone was capable of solubilization activity (same final release of xylose and arabinose) but the fragmentation was much weaker by only disorganizing the fibrous network and hence led to particle disaggregation. Altogether, these results confirmed the importance of the enzyme mixtures which act in a synergistic manner to readily solubilize wheat bran. Our results also indicated that the limitation of Rovabio® action upon wheat bran degradation may come from physical inaccessibility of the substrate as it could be partially overcome by enhancing the substrate specific surface by a mechanical treatment and/or due to some missing or limiting enzyme activity as shown by a slight increase in solubilization following addition of some pectinases cocktails that are poorly represented in Rovabio®. Nevertheless, these complementary actions were still insufficient for complete hydrolysis of wheat bran. To conclude, this work draws attention to plant cell wall-deconstructing enzymes or active proteins which are able to attack the biomass minor structures and disorganize its network in order to increase substrate accessibility to enzymes that cleave backbone structures.

O EPP-AF® é um extrato padronizado de própolis quimicamente caracterizado e com eficácia e segurança pré-clínica estabelecidas. O objetivo principal deste trabalho foi realizar um ensaio clínico de segurança para avaliar a influência do EPP-AF® na atividade da P-gp e das principais isoformas CYP, através de um teste in vivo tipo coquetel de fármacos marcadores administrados em doses subterapêuticas. Foram investigados 16 voluntários adultos sadios antes e após a exposição a 375 mg de EPP-AF® por via oral durante 15 dias. As amostras seriadas de sangue foram colhidas até 12 h após a administração do coquetel contendo midazolam (0,2 mg), cafeína (10 mg), omeprazol (2 mg), metoprolol (10 mg), losartana (2 mg) e fexofenadina (10 mg). Foram desenvolvidos e validados três métodos analíticos empregando LC-MS/MS para quantificar as concentrações plasmáticas de fexofenadina, losartana, E-3174 (método 1), omeprazol, 5-OH-omeprazol, midazolam, metoprolol, ?-OHmetoprolol (método 2) e cafeína (método 3). Os métodos não apresentaram efeito matriz ou efeito residual e mostraram-se lineares para os analitos nos intervalos de 0,05-20 ng/mL (fexofenadina); 0,03 - 5 ng/mL (losartana e E31-74); 0,1 - 50 ng/mL (omeprazol), 0,3 - 50 ng/mL (5-OH-omeprazol), 0,01 - 10 ng/mL (midazolam), 0,05 - 50 ng/mL (metoprolol e ?- OH-metoprolol) e 5 - 1000 ng/mL (cafeína). Os parâmetros farmacocinéticos dos compostos foram calculados com base nas curvas de concentração plasmática versus tempo (AUC) empregando o programa Phoenix® WinNonlin®. Os valores das razões das AUC0-t e Cmax após e antes da exposição ao EPP-AF®, apresentados como média geométrica (IC90%) foram de 0,74 (0,62 - 0,89) e 0,90 (0,76 - 1,07) para fexofenadina; 0,88 (0,80 - 0,97) e 0,86 (0,76 - 0,98) para losartana; 0,96 (0,83 - 1,11) e 0,91 (0,79 - 1,04) para E-3174; 1,18 (0,91 - 1,54) e 1,21 (0,87- 1,70) para omeprazol; 1,12 (0,95 - 1,31) e 1,22 (0,95 - 1,67) para 5-OHomeprazol; 1,14 (1,03 - 1,28) e 1,21 (1,00 - 1,46) para o midazolam; 1,04 (0,92 - 1,18) e 0,94 (0,80 - 1,12) para o metoprolol; 1,05 (0,99 - 1,12) e 0,99 (0,88 - 1,12) para ?-OH-metoprolol; 0,97 (0,77 - 1,21) e 0,87 (0,69 - 1,11) para a cafeína. Quando observadas as razões metabólicas das AUC0-t E3174/losartana, 5-OH-omeprazol/omeprazol e ?-OHmetoprolol/ metoprolol encontramos, respectivamente, 1,11 (0,98 - 1,25); 0,94 (0,81 - 1,10) e 1,01 (0,88 - 1,16), indicando que, com exceção do CYP2D6, a administração de EPP-AF® nas condições estudadas apresenta potencial para inibição das isoformas CYP2C19 e CYP3A4 e indução das enzimas CYP1A2, CYP2C9 e do transportador de efluxo P-gp, embora as suas magnitudes encontram-se abaixo dos limites definidos pelos órgãos reguladores e portanto não apresentam relevância clínica / EPP-AF® is a standardized extract of propolis chemically characterized and with established pre-clinical efficacy and safety. The main objective of this work was to perform a clinical trial to evaluate the effect of EPP-AF® on P-gp and the major CYP isoforms activity, through an in vivo assay using the cocktail approach with sub-therapeutic doses. Sixteen healthy adult volunteers were investigated before and after exposure to orally administered 375 mg/day of EPP-AF® for 15 days. Serum blood samples were collected up to 12 h after the administration of midazolam (0.2 mg), caffeine (10 mg), omeprazole (2 mg), metoprolol (10 mg), losartan (2 mg) and fexofenadine (10 mg). Three analytical methods were developed and validated applying LC-MS/MS to quantify plasma concentrations of fexofenadine, losartan, E-3174 (method 1), omeprazole, 5-OH-omeprazole, midazolam, metoprolol, ?-OH-metoprolol (method 2), and caffeine (Method 3). Neither matrix effect nor carryover effect were observed. The methods were linear for the analytes in the ranges of 0.05 - 20 ng/mL (fexofenadine); 0.03 - 5 ng/ml (losartan and E-3174); 0.1 - 50 ng/mL (omeprazole), 0.3 - 50 ng/mL (5-OH-omeprazole), 0.01 - 10 ng/mL (midazolam), 0.05 - 50 ng/mL (metoprolol and ?-OH-metoprolol) and 5 - 1000 ng/mL (caffeine). The pharmacokinetic parameters of the compounds were calculated based on plasma concentration versus time (AUC) curves applying Phoenix® WinNonlin® software. AUC0-t and Cmax ratios after and before the EPPAF ® exposure, presented as geometric mean (CI 90%) were 0.74 (0.62 - 0.89) and 0.90 (0.76 - 1.07) for fexofenadine, 0.88 (0.80 - 0.97) and 0.86 (0.76 - 0.98) for losartan, 0.96 (0.83 - 1.11) and 0.91 (0.79 - 1.04) for E-3174, 1.18 (0.91 - 1.54) and 1.21 (0.87 - 1.70) for omeprazole; 1.12 (0.95 - 1.31) and 1.22 (0.95 - 1.67) for 5-OH-omeprazole, 1.14 (1.03 - 1.28) and 1.21 (1.00 - 1.46) for midazolam, 1.04 (0.92 - 1.18) and 0.94 (0.80 - 1.12) for metoprolol, 1.05 (0.99 - 1.12) and 0.99 (0.88 - 1.12) for ?-OH-metoprolol, 0.97 (0.77 - 1.21) and 0.87 (0.69 - 1.11) for caffeine. AUC0-t metabolic ratios of E3174/losartan, 5-OH-omeprazole/omeprazole and ?-OH-metoprolol/metoprolol we found to be, respectively, 1.11 (0.98 - 1.25), 0.94 (0.81 - 1.10 ) and 1.01 (0.88 - 1.16), indicating that, with the exception of CYP2D6, the administration of EPP-AF® under the conditions studied shows potential for CYP2C19 and CYP3A4 inhibition and CYP1A2, CYP2C9 and P-gp induction, although their magnitudes are below the limits defined by the regulatory agencies and therefore exhibit no clinical relevance

This dissertation deals with the profession of bartender between years 1958 and 1988 in socialist Czechoslovakia. It describes the development of mixology after the WW2 and difficulties it was facing in the socialist period up to late Eighties. Through interviews with contemporary witnesses, the author examines bad habits, shortcomings and abuses of the system behind the bar. Attention is paid to the availability and shortages of different kinds of alcohol and non-alcoholic drinks. The author also looks at bartenders as illicit moneychangers. The work analyses regulatory laws valid at this period and offers compact description of the most interesting and popular bars in Prague at that time.

It has been previously established that when engaged in a difficult attention intensive task, which involves repeating information while blocking out other information (the dichotic listening task), participants are often able to report hearing their own names in an unattended audio channel (Moray, 1959). This phenomenon, called the cocktail party effect is a result of words that are important to oneself having a lower threshold, resulting in less attention being necessary to process them (Treisman, 1960). The current studies examined the ability of a person who was engaged in an attention demanding task to hear and recall low-threshold words from a fictional story. These low-threshold words included a traditional alert word, "fire" and fictional character names from a popular franchise-Harry Potter. Further, the role of stimulus degradation was examined by including synthetic and accented speech in the task to determine how it would impact attention and performance. In Study 1 participants repeated passages from a novel that was largely unfamiliar to them, The Secret Garden while blocking out a passage from a much more familiar source, Harry Potter and the Deathly Hallows. Each unattended Harry Potter passage was edited so that it would include 4 names from the series, and the word "fire" twice. The type of speech present in the attended and unattended ears (Natural or Synthetic) was varied to examine the impact that processing a degraded speech would have on performance. The speech that the participant shadowed did not impact unattended recall, however it did impact shadowing accuracy. The speech type that was present in the unattended ear did impact the ability to recall low-threshold, Harry Potter information. When the unattended speech type was synthetic, significantly less Harry Potter information was recalled. Interestingly, while Harry Potter information was recalled by participants with both high and low Harry Potter experience, the traditional low-threshold word, "fire" was not noticed by participants. In order to determine if synthetic speech impeded the ability to report low-threshold Harry Potter names due to being degraded or simply being different than natural speech, Study 2 was designed. In Study 2 the attended (shadowed) speech was held constant as American Natural speech, and the unattended ear was manipulated. An accent which was different than the native accent of the participants was included as a mild form of degradation. There were four experimental stimuli which contained one of the following in the unattended ear: American Natural, British Natural, American Synthetic and British Synthetic. Overall, more unattended information was reported when the unattended channel was Natural than Synthetic. This implies that synthetic speech does take more working memory processing power than even an accented natural speech. Further, it was found that experience with the Harry Potter franchise played a role in the ability to report unattended Harry Potter information. Those who had high levels of Harry Potter experience, particularly with audiobooks, were able to process and report Harry Potter information from the unattended stimulus when it was British Natural. While, those with low Harry Potter experience were not able to report unattended Harry Potter information from this slightly degraded stimulus. Therefore, it is believed that the previous audiobook experience of those in the high Harry Potter experience group acted as training and resulted in less working memory being necessary to encode the unattended Harry Potter information. A pilot study was designed in order to examine the impact of story familiarity in the attended and unattended channels of a dichotic listening task. In the pilot study, participants shadowed a Harry Potter passage (familiar) in one condition with a passage from The Secret Garden (unfamiliar) playing in the unattended ear. A second condition had participants shadowing The Secret Garden (unfamiliar) with a passage from Harry Potter (familiar) present in the unattended ear. There was no significant difference in the number of unattended names recalled. Those with low Harry Potter experience reported significantly less attended information when they shadowed Harry Potter than when they shadowed The Secret Garden. Further, there appeared to be a trend such that those with high Harry Potter experience were reporting more attended information when they shadowed Harry Potter than The Secret Garden. This implies that experience with a franchise and characters may make it easier to recall information about a passage, while lack of experience provides no assistance. Overall, the results of the studies indicate that we do treat fictional characters in a way similarly to ourselves. Names and information about fictional characters were able to break through into attention during a task that required a great deal of attention. The experience one had with the characters also served to assist the working memory in processing the information in degraded circumstances. These results have important implications for training, design of alerts, and the use of popular media in the classroom.

Human induced pluripotent stem cells (iPSCs) represent a scalable cell source for the generation of hematopoietic progenitor cells (iHPCs); however, a lack of efficient iHPC expansion in vitro currently limits translational applications. To address this translational bottleneck, we assessed a panel of stem cell agonist cocktails (SCACs), originally developed to enhance cord-blood derived HSPC (CB-HSPC) expansion, on iHPC expansion. Three SCACs and GAS6 (X2A, X2A+GAS6, SM6, or SMA) were supplemented during iHPC differentiation and subsequent expansion using the STEMdiff™ Hematopoietic Kit. This monolayer differentiation strategy yielded a population of CD34⁺CD43⁺ and CD45⁺CD34⁺ iHPC. SCAC supplementation during iHPC differentiation yielded up to 2.5-fold higher frequency of CD34⁺CD43⁺ hematopoietic progenitors and up to 2.9-fold higher frequency of CD45⁺CD34⁺CD45RA⁻CD90⁺ HSC-like cells compared to non-treated controls. Subsequent SCAC supplementation during 2 weeks of expansion culture also significantly increased iHPC expansion (X2A+GAS6: 3.8-fold, X2A: 3.5-fold, SM6: 2.8-fold, SMA: 2.0-fold). The expanded iHPCs retained high levels of CD34⁺CD43⁺ expression but we observed an increase in the expansion of HSC-like cell fraction. The collective expansion observed with the SCACs was 1.5- to 2.8-fold higher than UM171 treatment alone. Furthermore, all SCAC-supplemented iHPCs retained multilineage potency, producing erythroid and granulocyte-macrophage progenitors in CFU assays. However, prolonged expansion, beyond 7 days, reduced multilineage potential, indicating a limited expansion window. Although optimal timing and composition of SCAC supplementation remains to be refined, these results highlight that exploiting the additive and synergistic effects of multiple small molecules represents a promising approach for enhancing iHPC expansion yields and biomanufacturing.

Ph. D. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Hydrocarbon pollution is becoming a growing environmental concern in South Africa and globally. This inadvertently supports the need to identify enzymes for their targeted degradation. The search for novel biocatalysts such as monooxygenases, alcohol dehydrogenases and aldehyde dehydrogenases, have relied on conventional culture-based techniques but this allows sourcing of the biomolecules from only 1-10 % of the microbial population leaving the majority of the biomolecules unaccounted for in 90-99 % of the microbial community. The implementation of a metagenomics approach, a culture-independent technique, ensures that more or less than 100 % of the microbial community is assessed. This increases the chance of finding novel enzymes with superior physico-chemical and catalytic traits. Hydrocarbon polluted soils present a rich environment with an adapted microbial diversity. It was thus extrapolated that it could be a potential source of novel monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH) involved in hydrocarbon degradation pathways. Therefore, the aim of the study was to extract metagenomic DNA from hydrocarbon contaminated soils and construct a metagenomic fosmid library and screen the library for monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Accordingly, the fosmid library was constructed from metagenome of hydrocarbon-contaminated soil. Then the library was functionally screened using hexadecane, octadecene and cyclohexane as substrates and fifteen positive clones were selected. The fosmid constructs of the positive clones were sequenced using PacBio next generation sequencing platform. The sequences were de novo assembled and analysed using CLC Genomic Workbench. The open reading frames (ORF) of the contigs were identified by blasting the contigs against uniport database. Accordingly, four novel genes namely amo-vut1, aol-vut3, dhy-sc-vut5 and dhy-g-vut7 that showed close similarity with our target enzymes were further analysed in silico and codon-optimized as per Escherichia coli codon preference. The codon adjusted sequences were synthesised and cloned into pET30a(+) expression vector. However, it is worth noting that expression of amo-vut1 was not successful since it was later identified to be a multi-pass member protein, which made it insoluble despite the use of detergent to the effect. There is a need to meticulously genetically engineer amo-vut1 to remove the signal and other membrane-bound peptides while maintaining its activity. Yet the other three constructs were successfully transformed and expressed in E. coli BL21 (DE3). The enzymes were purified and characterized and cocktail for hydrolysis of hexanol was succesfully engineered based on AOL-VUT3, DHY-SC-VUT5 and DHY-G-VUT7. Therefore, novel enzymes were mined from metagenome of fossil-oil contaminated soil and effective hydrocarbon-degrading enzyme cocktails containing their combination were successfully engineered.

This thesis is a two-part project, where the first part is to find out what a design guide is,what it should contain and make a suggestion for one that fits Röshults design language.A design guide contains information that can help a designer follow a company’s designlanguage when developing a new product.The second part is to look at Röshults assortment for an opening for a new product anduse the guide suggestion to design it. / Detta examensarbete är ett tvådelat projekt. Där den första delen går ut på att ta reda påvad en designguide är, vad den ska innehålla och ge ett förslag på en som passarRöshults formspråk. En designguide innehåller information som kan hjälpa en designeratt följa ett företags formspråk när de utvecklar en ny produkt.Den andra delen är att titta på Röshults sortiment för en öppning för en ny produkt ochanvända guideförslaget för att designa den.

La biomasse lignocellulosique comprend les sous-produits agricoles et industriels pouvant être utilisés comme matière première dans des bioprocédés variés destinés à produire des molécules d'intérêt énergétique ou chimique. Ces ressources lignocellulosiques, peuvent notamment être fournies par l'industrie papetière qui est particulièrement adaptée pour les bio-raffineries modernes car elle est en capacité de produire en grande quantité un substrat ayant une faible teneur en lignine et sans composés inhibiteurs. La bagasse de canne à sucre est également un substrat prometteur par sa composition chimique simple et son abondance dans les pays tropicaux. Lors de l'utilisation de ces substrats, l'hydrolyse enzymatique constitue une étape cruciale permettant la transformation des fibres de cellulose en une source de carbone fermentescible. Si les aspects biochimiques de cette étape d'hydrolyse font l'objet de nombreuses recherches et de développements, les réactions sous haute teneur en matière sèche font apparaître des limitations physiques qui sont beaucoup moins étudiées et analysées mais constituent des verrous scientifiques et technologiques qui freinent actuellement l'utilisation de cette ressource abondante et durable. Ce travail s'inscrit dans ce contexte et propose l'étude de cette étape d'hydrolyse enzymatique de la lignocellulose en s'intéressant conjointement aux aspects biochimiques et physiques de façon à aller vers une compréhension et une maîtrise des transferts (de masse, de chaleur) dans les réactions à forte concentration en substrat. La stratégie adoptée a consisté à réaliser et analyser des réactions d'hydrolyse sous différentes conditions opératoires en travaillant dans un premier temps sur des concentrations intermédiaires (suspension semi-diluée), c'est-à-dire en introduisant, mais de façon limitée, les complexités dues aux interactions entre particules/fibres de lignocellulose. Les résultats obtenus sont ensuite utilisés pour élaborer une stratégie adaptée aux fortes concentrations. Les aspects physiques analysés sont essentiellement le comportement rhéologique du milieu réactionnel ainsi que la morpho-granulométrie des objets en suspension. Différentes métrologies, tant in-situ que ex-situ, ont été mises en œuvre et apportent des résultats complémentaires. Les études ont été menées sur un substrat de référence, le papier Whatman, et deux substrats à vocation industrielle: la pâte à papier et la bagasse de canne à sucre. La stratégie d'étude porte sur les aspects suivants: (i) le suivi de l'évolution des comportements rhéologiques et des propriétés morphologiques des suspensions au cours de l'hydrolyse, (ii) l'étude des mécanismes d'hydrolyse lors de la dégradation des substrats, (iii) l'étude de l'impact de la composition et de la structure des substrats sur les cinétiques de solubilisation et d'hydrolyse, (iv) la quantification de la contribution des différentes activités enzymatiques, seules ou en mélange par une approche physique multi-échelle et (v) le contrôle et l'optimisation des conditions d'alimentation dans un procédé discontinu alimenté (fed-batch) afin d'atteindre des conditions de milieu concentré. Les chapitres 1 et 2 de ce document sont consacrés à une étude bibliographique du sujet et à la présentation des matériels et méthodes mis en œuvre. Le troisième chapitre présente les résultats obtenus et leur analyse. Il est constitué de trois sections: tout d'abord une étude des propriétés des différents enzymes ou cocktail d'enzymes utilisés, des substrats retenus et des suspensions avec, notamment, la détermination des régimes semi-dilués et concentrés. Ensuite sont présentées et analysées les hydrolyses effectuées en milieu semi-dilué. Les mécanismes d'hydrolyse (fragmentation, solubilisation, hydratation et séparation des agglomérats) sont étudiés pour diverses concentrations et divers enzymes/cocktails. Enfin les résultats en milieu concentré sont présentés dans une dernière section. / Lignocellulosic biomass consists of several agriculture and industrial by-products that can be used as raw material for several bioprocesses to obtain range of products. Among lignocellulosic sources, the pulp & paper industry is appropriated for modern bio-refining thank to pulp with low lignin content and free of inhibitory compounds. Besides, sugarcane bagasse is a very promising feedstock because of its simple chemical composition and its abundancy especially in tropical countries. In the bioconversion of lignocellulose, enzymatic hydrolysis is a crucial step that allows the transformation of cellulosic and hemicellulosic fibers into fermentable carbon sources. The lack of knowledge about physical limitations and hydrolysis mechanisms, especially at high dry matter content, stands as the main barrier which forbids the scale-up of bio-refinery processes. Thus, the efficient and sustainable use of lignocellulosic resources is currently a major challenge and need to be investigated. In this context, this PhD focused on the enzymatic hydrolysis of lignocellulose by both physical and biochemical approaches. The strategy consisted in carrying out and in analyzing the hydrolysis reactions under different operating conditions with semi-dilute suspensions. Then, obtained results were used to develop a hydrolysis strategy for concentrated suspensions. Different methodologies, in- and ex-situ analyses, were implemented and provided complementary results. From physical approach, analyses consisted in rheological behavior of suspensions as well as the morpho-granulometry of particles. The study was carried out on a reference substrate, Whatman paper, and on two industrial substrates, paper pulp and sugarcane bagasse. The strategy aimed to investigate different stakes: (i) evolution of rheological behaviors and the morphological properties of suspensions, (ii) hydrolysis mechanisms during the degradation of substrates, (iii) impact of substrate composition and structure on solubilization and hydrolysis kinetics, (iv) quantification of the contribution of single enzyme and enzyme mixture activities by multi-scale physical approaches and (v) control and optimization of feeding parameters for fed-batch process in order to access to concentrated suspension. Chapters 1 and 2 of this document are devoted to a research bibliographic and presentation of materials and methods. The third chapter presents obtained results and discussion in three sections. The first one is a study of the properties of different enzymes and substrates, in particular, the determination of semi-dilute and concentrated regime. Subsequently the enzymatic hydrolysis at semi-dilute regime is presented to highlight the hydrolysis mechanisms (fragmentation, solubilization, solvation and agglomerate separation) in relationship with enzyme mixtures and dosages. Finally, results in concentrated regime are discussed in the final section.

Gifter och föroreningar förekommer sällan ensamma och studier om olika toxiska substanser genomförs ofta med endast en substans i taget. Det finns studier som har undersökt exempel på olika cocktails av substanser, men ibland är det svårt att identifiera alla källor som påverkar en organism. Vi har undersökt hur en kombination av ogräsmedlet Roundup och livsmedelsklassad polypropylenplast påverkar det sötvattenlevande kräftdjuret Daphnia magna. Vi fann att i högre doser orsakar Roundup 100% dödlighet innan könsmognad, men att det i små doser inte resulterar i lägre fitness än kontrollgruppen, utan till och med ökade den initiala överlevnaden när den tillsattes i plastbrukar. En cocktail av Roundup och plast orsakade en fördubblad utvecklingstid för äggen. Ägg från individer i plastburkar hade också en längre utvecklingstid men dessa individer hade även ett mycket högre antal ägg. Framtida studier bör undersöka avkommans fitness. Tidigt i studien upptäckte vi att det med stor sannolikhet fanns en tredje faktor som påverkade daphnierna negativt, som inte kunde förklaras av Roundup eller plastburkar, men som vi inte kunde identifiera. Därför vill vi också framhäva att experiment lätt kan påverkas av okända faktorer. / Toxins and pollutants rarely come alone and studies about various toxic substances are often performed with only one substance at a time. There are studies that have examined different cocktails of substances, but sometimes it can be difficult to identify all sources that affect an organism. We have investigated how a combination of the herbicide Roundup and Food Safe polypropylene plastic affect the freshwater crustacean Daphnia magna. We found that Roundup causes 100% mortality before reaching fertility in higher doses, but that in small doses does not result in lower fitness compared to the control group, but even increases the initial survival rate when Roundup is added to plastic jars. A cocktail of Roundup and plastic resulted in a doubled development time for the eggs. Eggs from individuals in plastic jars also had a longer development time but there was also a much higher number of eggs from these individuals. Futiure studies ought to investigate the fitness of the offspring. Early in the study we discovered that there was most likely a third factor influencing the daphnia negatively, that could not be explained by plastic jars or Roundup, but one that we could not identify. Therefore, we also wish to urge that experiments can easily be influenced by unknown factors.

Mon projet de doctorat a pour objet l'étude des facteurs pouvant influencer le métabolisme des médicaments et la variabilité interindividuelle dans la réponse aux médicaments. Mon projet cible plus précisément les Cytochromes P450 (CYP450), le système enzymatique majeur impliqué dans la biotransformation des médicaments. Mes travaux de recherche évaluent l'impact d’une condition pathologique, le diabète de type 2 (DT2), sur l'activité métabolique des CYP450s. Mes études comprennent un volet de métabolisme systémique chez le patient et un volet de métabolisme in vitro. Dans cette thèse, les résultats de mes recherches sont rapportés sous forme de présentation par articles. Le volet in vivo consistait en une étude de pharmacocinétique qui visait à évaluer l’impact du diabète sur l’activité métabolique de différentes isoformes des CYP450s en utilisant un cocktail de substrats-marqueurs. Des patients avec le DT2 et des sujets non diabétiques ont reçu une dose orale de notre cocktail VM/JT de substrats-marqueurs composé de caféine (CYP1A2), bupropion (CYP2B6), tolbutamide (CYP2C9), oméprazole (CYP2C19), dextrométhorphane (CYP2D6) et midazolam (CYP3A4/5) suivi d'une administration de chlorzoxazone (CYP2E1). Le protocole pour cette étude est détaillé dans l’article disponible à la section 2.1; manuscrit 1. Les concentrations plasmatiques et urinaires des médicaments marqueurs et de leurs métabolites spécifiques ont été quantifiées par LC-MS/MS suivant la méthode publiée dont l’article est disponible à l’annexe 1. Cette étude m’a permis de montrer que les patients avec le DT2 présenteraient une clairance systémique réduite via les isoformes CYP2B6, CYP2C19 et CYP3A. L’article présentant ces résultats se trouve à la section 2.1; manuscrit 2. Au cours de cette étude clinique, nous avons aussi évalué l’utilisation du 4-hydroxycholestérol comme biomarqueur endogène de l’activité du CYP3A dans une population avec le DT2 (objectif secondaire). Les conclusions démontrant la validité de ce biomarqueur sont disponibles dans l’article qui se trouve à la section 2.2; manuscrit 3. Le volet in vitro de mes travaux a permis d’évaluer au niveau du duodénum l’influence du DT2 sur l’expression de plusieurs CYP450s et transporteurs, ainsi que sur l’activité des CYP2B6, CYP2C9, CYP2J2 et CYP3A. Aucun impact significatif du DT2 n’a été mesuré sur l’expression d’ARNm des CYP450s et transporteurs testés exprimés dans des biopsies duodénales. Les niveaux d’activité mesurés à l’aide d’incubations avec des substrats-marqueurs des CYP450s dans des fractions S9 de biopsies duodénales étaient semblables chez des sujets avec le DT2 et des non diabétiques. L’article sur les résultats de ce volet in vitro est disponible à la section 2.3; manuscrit 4. Ces résultats suggèrent que les effets du diabète sur le métabolisme des substrat-marqueurs observés dans l’étude clinique peuvent s’expliquer par une modulation au niveau hépatique ou dans différentes sections de l’intestin. En accord avec ces résultats chez l’humain, notre groupe avait déjà rapporté que l’effet du diabète sur les CYP450s était isoforme et tissu spécifique chez la souris (annexe 2). L'objectif de ma thèse était de mieux comprendre les mécanismes sous-jacents à la variabilité dans la réponse aux médicaments observés chez les patients diabétiques, lesquels nécessitent fréquemment une polypharmacie. Les résultats de ces travaux permettront éventuellement d’optimiser la pharmacothérapie chez ces patients. / My PhD project evaluates factors that can influence drug metabolism and interindividual variability in drug response. More precisely, my thesis focuses on the major drug metabolizing enzymes, the cytochromes P450 (CYP450). My researches evaluated the impact of a pathological condition, namely type 2 diabetes (T2D), on CYP450 metabolic activities in two parts. First, the effect of diabetes on systemic metabolism was evaluated in patients. Then, in vitro experiments enabled us to measure the impact of T2D on organ-specific or metabolism. In this thesis, my research results are presented in 4 scientific papers. The in vivo part of my PhD research consisted of a pharmacokinetic study assessing metabolic activity of different isoforms of the CYP450s using a cocktail of probe drugs in T2D patients and non-diabetic subjects. All participants of both study groups received a dose of our oral VM/JT probe drugs cocktail consisting of caffeine (CYP1A2), bupropion (CYP2B6), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4/5) followed by a dose of chlorzoxazone (CYP2E1), alone. Study procedures are detailed in the protocol article (manuscrit 1) presented in section 2.1. Plasma and urine concentrations for all probe drugs and specific metabolites were quantified using a published LC-MS/MS method that is available in annexe 1. This study showed that patients with T2D exhibited reduced systemic clearances for the isoforms CYP2B6, CYP2C19 and CYP3A. Results of this pharmacokinetic research are presented in manuscrit 2 of section 2.1. As a secondary objective, this in vivo part of my PhD project enabled us to verify the validity of 4-hydroxycholesterol as an endogenous biomarker of CYP3A activity in a population with T2D. Conclusions showing its validity as an endogenous biomarker in such population are presented in section 2.2 (manuscrit 3). The in vitro part of my doctoral project evaluated in the intestines the influence of T2D on the mRNA expression of numerous CYP450 isozymes and drug transporters, as well as on metabolic activity of CYP2B6, CYP2C9, CYP2J2 and CYP3A. Using duodenal biopsies, no significant impact of T2D was detected on the mRNA expression levels of all tested CYP450s and transporters. Activity levels measured following incubations of probe-substrates in S9 fractions of duodenal biopsies obtained from patients with T2D and non-diabetic patients were similar. Results from this in vitro study are reported in section 2.3 (manuscrit 4) of this thesis. These results obtained in human subjects are in agreement with our previously published results showing isoform- and tissue-specific effects of T2D on CYP450s in mice (annexe 2). Overall, the central theme of this thesis is to better understand the underlying mechanisms of drug response variability observed in diabetic patients, whom often require polypharmacy, in order to eventually optimize drug therapy in those patients.