Drug transport across the intestinal epithelium is roughly predicted from permeability values obtained from Caco-2 cell monolayers. This thesis examines the important role of pH and extracellular additives for increasing the reliability and predictivity of the in vitro screening system, Caco-2. It was shown that the passive transport of ionizable compounds may be biased by a false efflux or uptake component, when applying a physiological pH-gradient across the membrane. pH also affected the amount of compound available at the transporter-binding site. Therefore, pH dependence should be considered in studies of such compounds and of drug-drug interactions involving efflux transporters. It was also shown that proton-dependent apical uptake or basolateral efflux should be studied both with and without a pH gradient over the whole monolayers. The two extracellular additives, bovine serum albumin (BSA) and the solubilizing agent, Cremophor® EL, also influenced Caco-2 permeabilities. BSA applied to the receiver side increases, and to the donor side decreases drug permeation according to the drug’s protein binding capacity. Thus, the absorptive transport for both passive and active compounds is favoured, giving a physiologically sound improvement of the Caco-2 cell model. Inclusion of BSA increased both the predictivity and quality of permeability studies, particularly of highly lipophilic, BCS class II compounds. Passive and active transport processes could also be distinguished after accounting for unbound concentrations. The overall effect of Cremophor® EL on the permeability to a drug was compound-specific and probably dependent on micellar incorporation. Cremophor® EL can therefore not be recommended. Neither pH nor BSA affect the functionality of transporters such as P-glycoprotein. However, efflux ratios of ionizable or protein bound drugs are altered in the presence of a pH-gradient or BSA, indicating that an experimental system without protein or pH gradient can over- or underestimate active and passive efflux in drug transport.

L’étude de la réponse membranaire suite au stress éthanolique chez Saccharomyces cerevisiae vise à comprendre les mécanismes impliqués dans la survie des eucaryotes aux concentrations en éthanol élevées. La membrane cellulaire, par sa position entre l’environnement intra et extracellulaire, est la première cible des perturbations éthanoliques. Les expérimentations effectuées dans cette étude visent à caractériser le maintien de l’intégrité fonctionnelle en relation avec la composition en stérols membranaires chez la levure S. cerevisiae soumise à différents types de perturbations éthanoliques (augmentation de la concentrations en éthanol dans le milieu de croissance, chocs éthanoliques d’amplitude croissante et suivi au cours du temps ; choc à 20% pendant 15 minutes puis suivi au cours du temps après retour dans un milieu sans éthanol (« pulse » 20% d’éthanol)). Les résultats obtenus démontrent l’importance de l’ergostérol dans le maintien de l’intégrité membranaire et supportent également l’hypothèse du rôle « vecteur » de l’éthanol vis à vis d’une oxydation, dont l’efficacité serait dépendante de la nature des stérols présents au niveau membranaire. Les premiers résultats analysant la cinétique de transcription de gènes impliqués dans la réponse au stress oxydatif suggèrent une formation plus importante de formes réactives de l’oxygène (ROS), induite par le choc éthanol chez un mutant Δerg6, affecté dans la voie de biosynthèse de l’ergostérol. / The study of membrane response induced by ethanol stress in Saccharomyces cerevisiae aims to understand mechanisms involved in the survival of eukaryotic cells submitted to high ethanol concentrations. The cell membrane by its position between the intra- and extracellular environment is the first target of ethanolic perturbations. Experiments performed in this study aimed to characterize the maintain of the functional integrity of the membrane in relation to the sterol composition in the yeast S. cerevisiae submitted to different types of ethanolic disturbances: increasing concentrations of ethanol in the growth medium; ethanolic shocks of increasing magnitude; shock 20% ethanol for 15 minutes and then return in a medium without ethanol ("pulse" 20% ethanol)). Our results demonstrate the importance of ergosterol in maintaining membrane integrity and also support the hypothesis of the vector role of ethanol in cell oxidation, whose effectiveness is dependent on the nature of sterols at the membrane level. In addition, our results analyzing the kinetics of transcription of genes involved in oxidative stress response suggest an increased formation of reactive oxygen species (ROS) induced by ethanol in the Δerg6 mutant, affected in the biosynthetic pathway of ergosterol.

Les membranes cellulaires jouent un rôle important dans l’absorption des médicaments et la distribution de ceux-ci dans le corps humain. Elles contiennent différents transporteurs membranaires qui sont responsables des profils pharmacocinétiques, d’innocuité et d’efficacité des xénobiotiques. Lors du développement d’un médicament, il s’avère donc indispensable, de prédire l’interaction des nouveaux composés avec les transporteurs présents dans l’organisme. Le but du projet de recherche est de créer un nouvel outil pour étudier le comportement de la glycoprotéine-P (P-gp), un transporteur membranaire responsable du rejet de nombreux composés, sur différents médicaments. Pour cela, un modèle non cellulaire est développé en utilisant des protéoliposomes : des liposomes dans lesquels des transporteurs sont incorporés. La méthodologie consiste tout d’abord à produire, extraire et purifier la protéine d’intérêt à partir de deux systèmes d’expression : MDCK-MDR1 (cellules de chien transfectées avec le gène humain MDR1) et Pichia pastoris (levures) fin de déterminer les avantages et les limites de ces deux types cellulaires. Différentes méthodes de reconstitution dans des protéoliposomes ont ensuite été testées avec la P-gp obtenue. Puis, l’activité ATPasique de la P-gp reconstituée a été évaluée en présence de différents substrats. Les protocoles de culture cellulaire, d’extraction et de purification des deux systèmes d’expression ont été implémentés avec succès au sein du laboratoire. Les résultats montrent que les rendements obtenus sont supérieurs avec les levures qu’avec les cellules de mammifère. En outre, Pichia pastoris offre les avantages d’être facile et rapide à cultiver et peu couteux. Les premiers résultats d’activité ATPasique obtenus avec la P-gp reconstituée en protéoliposomes étaient prometteurs mais n’ont pas été reproduits en raison de la dégradation de la protéine membranaire. Les prochaines études du projet porteront sur un autre transporteur membranaire de la famille ABC, BCRP, une protéine de plus petite taille qui devrait montrer une plus grande stabilité pour mener à bien les tests. / Cellular membranes play an important role in the absorption and distribution of drugs in the human body. They contain different membrane transporters, which are responsible for the pharmacokinetic properties of drugs, as well as the safety and efficiency of their diffusion. When developing a new drug, it is thus of utmost importance to study the way that it will interact with the transporters present within the body. The aim of this study was to evaluate a new tool for measuring permeability in order to understand the function and mecanisms of P-glycoprotein (P-gp). P-gp is a transporter that is responsible for the rejection of many different compounds found in various drugs. This study thus seeks to use proteoliposomes to develop non-cellular models of membrane permeability including efflux and uptake transporters. This novel model of permeability will be utilized to study the underlying mechanisms of membrane permeability to xenobiotics. The human P-gp was produced, extracted and purified using two different expression systems: MDCK-MDR1 cells (Madin-Darby canine kidney cells transfected with the human MDR1 gene) and Pichia pastoris. Both expression systems were studied in order to compare the strengths and weaknesses of each system. We then tested different methods of reconstituting the P-gp into protéoliposomes. Finally, we measured the level of ATPase activity using different substrates. The protocols of cell culture, extraction and purification of both expression systems were accomplished in a laboratory during this study. These results demonstrated that expressing P-gp using yeast was more effective than that of mammalian cells. Furthermore, working with Pichia pastoris offers multiple advantages: expressing P-gp was easier, faster and cheaper than working with mammalian cells. The first measurements of ATPase activity using reconstituted P-gp proteoliposomes were promising, however they proved difficult to reproduce due to the possible degradation of the membrane protein.Further studies in this project will look to evaluate another ABC membrane transporter, BCRP. This smaller protein should prove to be more stable than P-gp, facilitating experimentation.

La diffusion des entérobactéries multirésistantes aux antibiotiques (MDR) à l’échelle mondiale constitue une menace de santé publique majeure. Résistantes à au moins trois classes d’antibiotiques, les entérobactéries MDR entrainent des infections échappant aux traitements de première intention. La première partie de ce travail s’intéresse à l’épidémiologie moléculaire des souches d’entérobactéries MDR isolées dans les infections et les colonisations des patients hospitalisés en Languedoc-Roussillon, en France, et dans un pays où cette épidémiologie est encore peu connue, l’Algérie. Nous avons montré, dans notre région et au niveau national, que la résistance aux carbapénèmes était essentiellement liée à des modifications de la perméabilité membranaire (87,4% des entérobactéries résistantes, au niveau national). Dans la deuxième partie de ce travail, nous avons étudié les modulations de la perméabilité membranaire et de l’efflux chez Escherichia coli ST131, l’exemple-type d’un clone MDR. Nous avons montré que ce clone mondial présentait une remarquable adaptabilité à la pression antibiotique. Cette adaptabilité avait un impact significatif sur la virulence et le fitness de E. coli. Les capacités de formation de biofilm et la virulence chez Caenorhabditis elegans étaient augmentées chez les souches de phénotypes « efflux ». Inversement, les souches de phénotypes « imperméabilité » présentaient un faible potentiel de virulence, associé à une diminution significative de la formation de biofilm et de la mobilité par swimming. / The spread of multidrug-resistant (MDR) Enterobacteriaceae is a major public health threat worldwide. Resistant to at least three classes of antibiotics, MDR Enterobacteriaceae cause infections for which first-line treatments are inefficient. The first part of this work focused on the molecular epidemiology of MDR Enterobacteriaceae strains isolated in infections and colonizations of patients hospitalized in Languedoc-Roussillon, in France and in Algeria, a country where few data are currently available. We showed in our region and nationally, that resistance to carbapenems was mainly due to changes in membrane permeability (87.4% of resistant Enterobacteriaceae, nationally).In the second part of this work, we studied the modulation of membrane efflux and permeability in the quintessential example of an international MDR high-risk clone, Escherichia coli ST131. We showed that this global clone had a remarkable adaptability to antibiotic pressure. This adaptability had a significant impact on the virulence and the fitness of E. coli. The biofilm formation and virulence capacities in Caenorhabditis elegans model were increased in strains overexpressing an efflux system. Conversely, the strains with altered porins expression had a low potential virulence, associated with a significant reduction in biofilm formation and swimming mobility.

Salinity continues to be one of the world’s most serious environmental problems in agriculture. The increasing world population and urbanization are forcing farmers to utilize marginal lands as well as poor quality water. One of the strategies in dealing with salinity is growing salt tolerant plants and there has been increased need to understand the effects of salinity on crops. Owing to its high nutritive value and wide adaptability to diverse environments, amaranth is considered a promising crop for marginal lands and semiarid regions. The objective of the study was to investigate the response of amaranth to salinity stress and evaluate stress amelioration by calcium and seed priming. Salinity tolerance during germination and early seedling growth was examined for six genotypes of amaranth (Amaranthus species) at different salt concentrations ranging from 0 to 200 mM NaCl or Na2SO4. Enhancement of germination was observed at 25 mM, while increasing salt concentrations reduced the germination percentage as well as germination rate. A.tricolor and Accession ’83 were able to germinate in 200 mM NaCl while there was no germination at 200 mM Na2SO4 in all the genotypes. Overall, Accession ’83 was the most resistant and A. hybridus the most sensitive genotype, particularly at high salt concentrations. Inhibition of germination was greater in Na2SO4 than in NaCl salinity treatments. Amaranth was more salt tolerant at germination than at seedling growth. Seedling emergence, survival and growth were reduced by salinity and at much lower concentrations than at seed germination. Differences in salt tolerance were noted among the genotypes. Salinity stress was initiated at different growth stages (cotyledon stage, 2-leaf stage and 4-leaf stage) in order to determine whether tolerance of amaranth differs with the stage of development. The treatment either continued until termination of the experiment or for 14 days at each stage. Amaranth plants were less sensitive to salinity when the stress was initiated at the 4-leaf stage. Lower salt concentrations had less detrimental effects than higher concentrations when applied at the cotyledon stage. Application of low salt concentration at cotyledon stage for 14 days did not have any effect on plant growth. The results indicate that it is feasible to use saline water for growing amaranth with minimum yield losses if salt concentration, duration of exposure and time of salinization are carefully managed. Differences in salinity tolerance among amaranth genotypes were analyzed in terms of plant survival, growth, gas exchange, water use and leaf anatomical changes. A. hypochondriacus and A. cruentus showed greater tolerance to salinity since they survived in 200 mM NaCl treatment and the reduction in growth at 50 and 100 mM was lower than that of A. tricolor and Accession ’83. A. hypochondriacus and A. cruentus were more efficient water users and partitioned photosynthates towards shoot growth as opposed to the other two genotypes. Photosynthetic rate, stomatal conductance, stomatal density and apertures were reduced by salinity but were higher in A. tricolor than in A. cruentus. Salinity resulted in A. cruentus developing thicker leaves compared to A. tricolor. Productivity on saline soils can be increased by growing genotypes more tolerant to salinity. The interactive effect of salinity and water stress on amaranth plant growth was evaluated. It was found that the reduction in shoot growth was greater in plants submitted to water stress than in those submitted to salt or salt + water stress. Water use efficiency was increased while leaf water and osmotic potentials were reduced by the salinity stress treatments. In drying soil plants previously salinized had a greater degree of osmotic adjustment, so that plants were able to continue growth for a longer period compared to water stressed plants. The effect of calcium in ameliorating salt stress was investigated. Supplementary calcium, either as CaSO4 or CaCl2 ameliorated the negative effects of salinity on growth, gas exchange, membrane permeability and mineral uptake. In a separate experiment it was shown that it is feasible to mitigate the adverse effects of salinity on amaranth seed germination, seedling survival and growth by seed priming and that the positive effect of priming persisted to vegetative growth stage. Priming with CaSO4 + NaCl showed a greater positive response than priming with the individual salts. / Thesis (PhD (Plant Production andSoil Science))--University of Pretoria, 2007. / Plant Production and Soil Science / unrestricted

Organelles are crucial compartments in living cells for carrying out biological events, and cells normally employ compartmentalization to spatially manage their cellular material transport, signaling, and metabolic processes. Engineering biomimetic nanoreactors to replicate biological processes has attracted a lot of interest in recent years. pH-responsive and photo-crosslinked polymersomes, for example, as synthetic vesicles, have tuable membrane permeability and mechanical stability, and may be utilized to build artificial organelles by encapsulating bioactive molecules in their cavity. Most existing reports of stimuli-responsive polymersomes for enzymatic cascade reactions are based on a simple mix of two types of polymersomes loaded with different enzymes, whereas cells process multi-enzyme catalytic systems in which intracellular biological reactions are carried out by combining two or more enzymes in the same organelle. In fact, the most of sophisticated biological functions and features of cells are based on self-organization, the coordination and connection between their cell organelles determines their key functions. Therefore, spatially ordered and controllable self-assembly of polymersomes to construct clusters to simulate complex intracellular biological functions has attracted widespread attention. Here, a simple one-step copper-free click strategy is present to crosslink nanoscale pH-responsive and photo-crosslinked polymersomes (less than 100 nm) to micron-level clusters (more than 90% in 0.5-2 µm range). Various influencing factors in the clustering process and subsequent purification methods were studied to obtain optimal clustered polymeric vesicles. Even if co-clustering the separately loaded polymeric vesicles with different enzymes (glucose oxidase and myoglobin), the overall permeability of the clusters can still be regulated through tuning the pH values on demand. Compared with the conventional enzyme cascade reaction through simple blending polymersomes, the rate of enzymatic cascade reaction increased significantly due to the interconnected complex microstructure established. The connection of catalytic nano-compartments into clusters confining different enzymes of a cascade reaction provide an excellent platform for the development of artificial systems mimicking natural organelles or cells. Although pH-responsive polymersomes present a good membrane permeability in response to alternate pH values and good stability in swelling/shrinking behavior owing to the photo-crosslinked membrane, they are still insufficient to simulate more complex biological activity. The intrinsic pH values for molecules transport are always acidic, whereas the majority of cellular action occurs at physiological pH levels. Due to the closed membrane, the enzyme reaction cannot be carried out efficiently under simulated physiological conditions (pH 7.4). To generate a permeable membrane at a physiological pH value, a new stimulus element must be introduced into existing polymersomes. To self-assemble pH- and light-responsive as well as photo-crosslinked polymersomes, a single azobenzene unit is used as a junction molecule between the hydrophilic and hydrophobic segments of block copolymer. To compare light utilization, block copolymers based on donor-acceptor-substituted azobenzene junction and ether-substituted azobenzene junction were prepared. Besides, the photo-isomerization of novel macroinitiators, block copolymers and polymersomes was also studied to get responsive wavelength ranges of light. The dye release experiments proven the hydrophobic dye on the membrane of polymersomes can release from the membrane under light irradiation. Despite the fact that blue light (400-500 nm) has a higher release efficiency than UV light (365 nm) and ether-substituted azobenzene polymersomes have a slightly higher release efficiency than donor-acceptor-substituted azobenzene polymersomes, the mechanism is still unknown due to the different power of light sources. Furthermore, based on the results of light-driven enzyme reaction, more experiments are required to confirm the light-induced membrane permeability, such as photo-oxidation of substrates and photo-induced deactivation of enzyme. But in general, photo-induced membrane disorder does squeeze the tiny cargo out of the membrane. The single azobenzene unit as the linkage between hydrophilic and hydrophobic block induced membrane pertubation proposes a novel concept in which a trace of azobenzene unit can affect cargo mobility on the membrane of polymersomes and even propagate the fluidity of water molecules to the entire membrane, thereby resulting in membrane permeability. This approach offers a unique framework for the development of biomimetic behaviors under physiological simulated conditions.:Part I Fundamentals 1 Theoretical Background 1.1 Polymersomes 1.1.1 Polymersomes Formation 1.1.2 Self-Assembly Principles of Amphiphilic Block Copolymers (BCPs) 1.1.3 Preparation Methods of Polymersomes 1.1.4 Cargo Loading in Polymersomes 1.2 Clustering Methods of Synthetic Vesicle 1.2.1 Host-Guest Interaction 1.2.2 DNA Hybridization 1.2.3 Copper-Free Click Chemistry 1.3 Stimuli-Responsive Polymersomes with Controllable Membrane Permeability 1.3.1 pH-Responsive Polymersomes 1.3.2 Light-Responsive Polymersomes 2 Motivation and Aim Part II Experiments 3 Materials and Methods 3.1 Materials 3.2 Analytical Methods 4 Clustered pH-Responsive Polymersomes for Enzymatic Cascade Reaction 4.1 Synthetic Methods and Characterization of Block Copolymer (BCP) for Self- Assembly of Polymersomes 4.1.1 Synthesis of Poly(Ethylene Glycol) (PEG) Macroinitiator 4.1.2 Synthesis of Photo-Crosslinker 4.1.3 Synthesis of BCP with Different Terminal Groups 4.1.4 Synthesis of Bis-BCN Poly(ethylene glycol) Crosslinker (BisBCN-PEG) 4.2 Formation of Empty and Loaded Psomes-N3 4.2.1 Formation and Photo-Crosslinking of Empty-Psomes-N3 4.2.2 Preparation of Cy5 Labeled BSA (BSA-Cy5) 4.2.3 Preparation of RhB Labeled Myo (Myo-RhB) 4.2.4 Preparation of Cy5 Labeled GOx (GOx-Cy5) 4.2.5 Formation and Photo-Crosslinking of Loaded Psomes-N3 4.3 Preparation and Purification of Clustered Empty-Psomes-N3 II 4.3.1 Preparation of Clustered Empty-Psomes-N3 at Different Conditions 4.3.2 Optimized Preparation of Clustered Empty-Psomes-N3 4.3.3 Purification Method of Clustered Empty-Psomes-N3 4.3.4 DLS Measurement of the Empty-Psomes-N3 in the Supernatant 4.3.5 Quantification of Removed Psomes-N3 after Centrifugal Purification 4.4 Preparation and Purification of Clustered Enzyme-Psomes-N3: Enzymatic Cascade Reaction 4.4.1 Preparation of Clustered GOx or Myo Loaded Psomes-N3 (GOx-Psomes-N3 or Myo-Psomes-N3) 4.4.2 Enzyme Activity of Myo Samples 4.4.3 Enzyme Activity of GOx Samples 4.5 Preparation and Purification of Co-Clustered Enzyme-Psomes-N3: Enzymatic Cascade Reaction 4.5.1 Preparation of Co-Clustered Myo/GOx-Psomes-N3 4.5.2 Enzyme Activity of Co-Clustered Myo/GOx-Psomes-N3 Samples 5 Light-Driven Enzyme Reaction Based on pH-Responsive Polymersomes 5.1 Synthetic Methods and Characterization of Block Copolymers with Single Azobenzene Unit 5.1.1 Synthesis of Block Copolymer with Donor-Acceptor-Substituted Azobenzene Linkage between Hydrophilic and Hydrophobic Segments (BCP-DA-Azo) 5.1.2 Synthesis of Block Copolymer with Ether Substituted Azobenzene Linkage between Hydrophilic and Hydrophobic Segments (BCP-Azo) 5.2 Photo-Isomerization of Macroinitiator and Block Copolymer with Azobenzene Linkage 5.2.1 Photo-Isomerization of PEG-DA-Azo Macroinitiator Based on Blue Light Irradiation or UV Irradiation 5.2.2 Photo-Isomerization of PEG-Azo Macroinitiator Based on Blue Light Irradiation or UV Irradiation 5.2.3 Photo-Isomerization of BCP-DA-Azo (-) Based on Blue Light Irradiation or UV Irradiation 5.2.4 Photo-Isomerization of BCP-Azo (-) Based on Blue Light Irradiation or UV Irradiation 5.3 Formation and Characterization of Polymersomes with Azobenzene 5.3.1 Self-Assembly of Polymersomes with Azobenzene III 5.3.2 Photo-Isomerization of Psomes-DA-Azo (-) Based on Blue Light Irradiation or UV Irradiation 5.3.3 Photo-Isomerization of Psomes-Azo (-) Based on Blue Light Irradiation or UV Irradiation 5.3.4 Photo-Crosslinking of Polymersomes with Azobenzene 5.3.5 DLS Measurement of Photo-Crosslinked Polymersomes with Azobenzene through pH Titration 5.3.6 Photo-Stability of Polymersomes with Azobenzene 5.3.7 In-Situ Loaded Nile Red in Non-Photo-Crosslinked Polymersomes with Azobenzene (NR-Psomes-DA-Azo (+) or NR-Psomes-Azo (+)) 5.3.8 In-Situ Loaded Myo in Photo-Crosslinked Polymersomes with Azobenzene (Myo-Psomes-DA-Azo (+) or Myo-Psomes-Azo (+)) 5.4 Light-Induced Dye Release from Polymersomes with Azobenzene 5.4.1 Fluorescence Photobleaching of Nile Red under Blue Light or UV Irradiation 5.4.2 Nile Red Release under Blue Light or UV Irradiation 5.5 Light-Driven Enzyme Reaction Based on Polymersomes with Azobenzene Part III Results and Discussions 6 Clustered pH-Responsive Polymersomes for Enzymatic Cascade Reaction 6.1 Aim and Strategy 6.2 Photo-Crosslinked and pH-Responsive Polymersomes 6.2.1 Synthesis and Characterization of Block Copolymers (BCPs) 6.2.2 Formation and Characterization of Polymersomes 6.3 Preparation and Purification of Clustered Empty-Psomes-N3 6.3.1 Key Parameters of Clustering Process 6.3.2 Purification Methods of Clustered Empty-Psomes-N3 6.4 Preparation and Purification of Clustered Empty-Psomes-N3 and Enzyme-Psomes- N3 90 6.4.1 Formation and Characterization of Enzyme in-Situ Loaded Psomes-N3 (Enzyme- Psomes-N3) 6.4.2 Enzyme Location in Polymersomes 6.4.3 Deeper Characterization of Clustered Empty-Psomes-N3 and Clustered Enzyme- Psomes-N3 6.5 Clustered Enzyme-Psomes-N3 for Enzymatic Cascade Reaction 6.5.1 Influence of Enzyme Activity on Clustering Condition IV 6.5.2 Mixed Enzyme-Psomes-N3 for Enzymatic Cascade Reaction 6.5.3 Co-Clustered Enzyme-Psomes-N3 for Enzymatic Cascade Reaction 6.6 Summary 7 Light-Driven Enzyme Reaction Based on pH-Responsive Polymersomes 7.1 Aim and Strategy 7.2 Preparation and Characterization of Light-Responsive Polymersomes 7.2.1 Synthesis and Characterization of BCP with Different Types of Azobenzene Unit 7.2.2 Self-Assembly and Photo-Crosslinking of Light-Responsive Polymersomes 7.2.3 Characterization of Photo-Crosslinked Light-Responsive Polymersomes 7.3 Photo-Isomerization of Azobenzene Containing Polymeric Macromolecules and Vesicles 7.3.1 Photo-Isomerization of Azobenzene Containing PEG Macroinitiators 7.3.2 Photo-Isomerization of Azobenzene Containing BCPs and Polymersomes 7.4 Light-Driven Dye Release from Polymersomes with Azobenzene at Simulated Physiological Conditions 7.4.1 Characterization of In-Situ Nile Red Loaded Polymersomes 7.4.2 Light-Driven Dye Release from Polymersomes at Simulated Physiological Conditions 7.5 Light-Induced Enzyme Reaction in Polymersomes with Azobenzene at Simulated Physiological Conditions 7.5.1 Characterization of Polymersomes in-Situ Loaded Myoglobin 7.5.2 Light-Induced Enzyme Reaction in Polymersomes at Simulated Physiological Conditions 7.6 Summary 8 Conclusion and Outlook Reference List of Figures List of Tables List of Abbreviations and Symbols Appendix Acknowledgements Versicherung

No description available.

<p>Uvod: U pro&scaron;losti žučne kiseline su uglavnom razmatrane sa stanovi&scaron;ta njihove funkcije koju obavljaju u crevima jer posreduju u varenju masti i apsorpciji liposolubilnih vitamina. Nedavne studije potvrđuju da žučne kiseline ne igraju samo ulogu u varenju masti, nego se pona&scaron;aju i kao signalni molekuli koji stupaju u interakciju sa raznim receptorima uključujući nuklearne receptore i receptore vezane za G-proteine. Kao amfipatični molekuli one su sposobne da reaguju sa fosfolipidima ćelijskih membrana i da pobolj&scaron;avaju prolazak lekova kroz njih. Stoga se žučne kiseline razmatraju kao promoteri u bukalnim, okularnim i nazalnim farmaceutskim formulacijama. Cilj: Svrha ovog istraživanja je bila da se ispitaju žučne kiseline i njihovi okso derivati kao jedinjenja koja utiču na propustljivost ćelijskih membrana i prolazak lekova do ciljnih tkiva. Materijal i metod: Interakcije makrolidnih antibiotika i žučnih kiselina su ispitivane uz pomoć NMR difuzionih merenja i relaksacije paramagnetičnim jonima. Retencioni parametri odabranih žučnih kiselina su dobijeni kori&scaron;ćenjem hromatografije na normalnim fazama i evaluisani su primenom pet različitih softvera. In vivo ekaperimenti su sprovedeni na 126 eksperimentalnih životinja koje su bile podeljene u 21 grupu. Rezultati: Vezivanje žučnih kiselina za micele je indikovano razlikama u hemijskom pomeranju makrolida i pro&scaron;irenju signala kao posledica redukovane mobilnosti unutar micela. Dodatak micela žučnih kiselina povećava solubilizaciju makrolida za faktor približno 2-3. Sprovedena korelaciona analiza pokazala je značajnu zavisnost između faktora retencije i intestinalne apsorpcije, prodora u MDCK epitelne ćelije, permeabilnost kroz kožu, logBB i PPB%. Putem implementacije in vivo eksperimentalnog dela pokazano je da žučne kiseline utiču na prolazak makrolida u tkivo mozga, bubrega i jetre. Zaključak: Ispitivane žučne kiseline pokazuju dobre farmakokinetske karakteristike i olak&scaron;avaju prolazak makrolida kroz različite ćelijske membrane.</p> / <p>Introduction: In the past, bile acids were mostly considered to function in the intestine where they play a role in digestion of fats and mediate absorption of fat-soluble vitamins. Recent studies confirm that bile acids not only facilitate solubilization of fats but behave as signal molecules that interact with various receptors including nuclear receptors and G protein-coupled receptors. As amphipathic molecules they are able to interact with phospholipids of cells membranes and enhance drugs permeation. Thus, bile acids are considered as drug promoters in buccal, ocular, nasal, and transdermal dosage forms. Purpose: The purpose of this research was to investigate bile acids and its oxo derivatives as enhancers in drug permeability. Three research methods to evaluate the characteristics of bile acids and its properties were used. Material and method: The interaction between macrolide antibiotics and bile acids was investigated by NMR chemical-shift titration, self-diffusion measurements and paramagnetic relaxation enhancements. Retention parameters of selected bile acids are acquired by normal-phase thin layer chromatography and evaluated using five different softwares. In vivo experiments were conducted on 126 animals which were divided in 21 groups. Results: Binding bile acids to the micelles is indicated by differences in the chemical shift of the macrolides and line broadening as a consequence of reduced mobility in the micelle. Addition of bile micelles increases the solubility of macrolide antibiotics by a factor of approximately 2&ndash;3. Examined correlation analysis confirmed significant dependence between retention factor and intestinal absorption, MDCK epithelial cells, skin permeability, logBB and PPB%. Through the implementation of in vivo experiments it is shown that bile acids promote penetration of macrolides in brain tissue, kidney and liver. Conclusion: Investigated bile acids showed good pharmacokinetic properties and facilitate in macrolides permeation through various membranes.</p>

The field of direct inject mass spectrometry includes a massive host of ambient ionization techniques that are especially useful for forensic analysts. Whether the sample is trace amounts of drugs or explosives or bulk amounts of synthetic drugs from a clandestine laboratory, the analysis of forensic evidence requires minimal sample preparation, evidence preservation, and high sensitivity. Direct inject mass spectrometry techniques can rarely provide all of these. Direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry, however, is certainly capable of achieving these goals. As a multifaceted tool developed in the Verbeck laboratory, many forensic applications have since been investigated (trace drug and explosives analysis). Direct inject mass spectrometry can also be easily coupled to assays to obtain additional information about the analytes in question. By performing a parallel artificial membrane assay or a cell membrane stationary phase extraction prior to direct infusion of the sample, membrane permeability data and receptor activity data can be obtained in addition to the mass spectral data that was already being collected. This is particularly useful for characterizing illicit drugs and their analogues for a biologically relevant way to schedule new psychoactive substances.

<p>Transport into and across the cells of the human body is a prerequisite for the pharmacological action of drugs. Passive membrane permeability and active transport mechanisms are major determinants of the intestinal absorption of drugs, as well as of the distribution to target tissues and the subsequent metabolism and excretion from the body. In this thesis, the role of ATP-binding cassette (ABC) transporters and passive permeability on drug absorption and disposition was investigated. Particular emphasis was placed on defining the molecular properties important for these transport mechanisms. </p><p>The influence of different transport pathways on predictions of intestinal drug absorption was investigated using experimental models of different complexity. Experimental models that include the paracellular pathway gave improved predictions of intestinal drug absorption, especially for incompletely absorbed drugs. Further, the inhibition of the ABC transporters breast cancer resistance protein (BCRP/ABCG2) and multidrug-resistance associated protein 2 (MRP2/ABCC2) was experimentally investigated using structurally diverse datasets that were representative of orally administered drugs. A large number of previously unknown inhibitors were identified among registered drugs, but their clinical relevance for drug-drug interactions and drug-induced toxicity remains to be determined. The majority of the inhibitors affected all three major ABC transporters BCRP, MRP2 and P-glycoprotein (P gp/ABCB1), and these multi-specific inhibitors were found to be enriched in highly lipophilic weak bases. </p><p>To summarize, the present work has led to an increased knowledge of the molecular features of importance for ABC transporter inhibition and passive membrane permeability. Previously unknown ABC transporter inhibitors were identified and predictive computational models were developed for the different drug transport mechanisms. These could be valuable tools to assist in the prioritization of experimental efforts in early drug discovery.</p>