Tumble dryers are becoming more and more common in ordinary households as a complement to the washing machine. Tumble dryers offer a fast drying cycle independent on weather conditions and require small space. They do, however, considering the large number of units use a large amount of electricity. The main objective in this thesis is to identify possibilities in order to reach a reduced electricity use for domestic tumble-drying of clothes. This involves an investigation of the condensing tumble dryer in order to point out possible energy efficiency improvements. The purpose of the energy label, which indicates the energy efficiency of the tumble dryer, is also studied, whether it matches the actual laundry habits. Finally, suggestions for technical development of the tumble dryer are made in line with today’s consumer behaviour. The performance of the condensing tumble dryer has been studied using a design of experiments to create a statistical model in Paper I. This model was used to find the best settings for the power supply to the heater, the internal airflow and the external airflow in order to reach a high specific moisture extraction rate (SMER) and a low leakage ratio. A low external airflow and high power supply to the heater gives the highest SMER. To reach the lowest values for the leakage ratio, a low internal airflow should be applied together with a high external airflow. The use of a statistical model gave valuable information of the performance of the existing tumble dryer. For further improving the energy efficiency of the dryer, the amount of leakage and its location was investigated in Paper IV. By studying energy and mass balances from experiments, pressure measurements and modelling, the effects of leakage on the process were evaluated. As the location of the leakage is so important for the energy efficiency, the worst-case scenario where leakage is located between the heater and the drum is used as a start point in the study. It was determined that there is a large leakage of air between the heater and the drum leading to a significant loss in energy recovery. The drying loads used by consumers are getting smaller, often less than 3 kg dry load, while the maximum capacity of the dryers are increasing, up to 7 or 8 kg. In Paper II, tests were made with different loads in order to investigate if the energy label serves its purpose as today’s standard is set at the dryers’ maximum capacity. The results from this study show that the energy efficiency when drying a small load is significantly lower than for a large load. In order to encourage a production of tumble dryers with higher energy efficiency for small loads, where the dryer is most frequently used, the standards for the energy label should be revised. Today, manufacturers do not gain any benefits by improving the performance for partial loads. A mathematical model over a venting tumble dryer was established in Paper III with the aim of testing different control strategies in order to improve the energy efficiency of the tumble dryer for partial loads. The ideas behind the different strategies were to minimize the heat losses during the drying process and to increase the residence time for the air in the drum and thereby increase the moisture content of the air leaving the drum. Using such a control strategy it is possible to reach an improvement of SMER by approximately 4% when drying small loads. In order to reach larger improvements, however, a more extensive product development will be necessary. Finally, the results in this thesis points at the necessity of including not only the technical development of the tumble dryer, but also the policy tools involved and the consumers’ habits in order to reach a reduced electricity use for drying clothes in households.

Nitrones are alternatives to azides in rapid strain-promoted 1,3-dipolar cycloadditions with cyclooctynes. To evaluate the differences between nitrones and azides we have performed kinetic studies of strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions of biarylazacyclooctynone (BARAC) with various acyclic and cyclic nitrones. The reactions were conducted under pseudo first-order reaction conditions using UV-visible spectroscopy. The reactivity of the acyclic nitrones was evaluated by varying the stereoelectronic and steric character of substituents at both the α-aryl and nitrogen positions. Cyclic nitrone reactivity was assessed according to the size of the ring and additional steric and strain effects. The obtained second-order rate constants for reactions of BARAC with cyclic nitrones were found to be greater than those for acyclic nitrones. However, all nitrones employed in the kinetic studies herein displayed significantly greater reactivity than azides in the analogous cycloadditions with BARAC. It is of particular note that the five-membered cyclic nitrones showed exceptional reactivity and, if used as rapid alternatives to azides in reactions with BARAC, can increase the reaction rates by up to 50 fold. An attempt to synthesize an allylated BARAC analogue is also described; the rearrangement reaction leading to the unexpected products is reported. The reaction rate for the novel rearrangement under both neutral and acidic conditions was obtained and plausible mechanisms for formation of products are proposed. Based on the results reported herein we anticipate that development of a labelling probe based on BARAC and a five-membered cyclic nitrone would allow for significant decrease of the concentrations of labelling reagents, thereby minimizing reaction time and reagent usage in life sciences applications. Nevertheless, a possible labelling decrease due to side reactions should be given consideration for prolonged labelling.

This diploma thesis describes marking of goods and services which help raise competitive producers on the market and also help to consumers in decision of purchasing products. In this time there are on the Czech market many labels describing the quality of goods. There is the special focus on the eco-labelling, which is label for positive effect on the environment. In this thesis there is described the history of voluntary approaches in chosen countries. My work analyses labels and their functions on the Czech market. There are also views of consumers on marking goods and services. At the conclusion I investigate producers' options on labels of goods.

Heparin glycosaminoglycans mediate a range of biological events, including anticoagulation as well as a diversity of cell proliferation and differentiation processes. Heparin saccharides have been shown to act as inhibitors against angiogenesis and metastasis of tumour cells. This thesis describes work developing chemistry towards varying length oligosaccharide sequences with potential to offer variable sulfation patterns. The main synthetic components to this work were contribution to developing scalable syntheses of an orthogonally protected L-Iduronic acid unit and a differentially protected D-glucosamine unit. The synthetic work also evaluated a recently reported diazo transfer reagent, which allowed for earlier placement of azide protection over that of previously developed routes within the group. This provided a cheaper, more atom efficient route towards protected D-glucosamine building blocks. Glycosylation of the developed D-GlcN donor units with the L-Ido acceptor allowed the production of key disaccharides which facilitated an efficient iterative glycosylation strategy towards longer oligosaccharides, ultimately providing a differentially protected pentasaccharide. The project evaluated methods towards generating various dimeric heparin type systems through forming new O4 ether linkages between GlcN residues across various short linker fragments. The most successful of these dimerisations used a methallyl dichloride core which allowed for further derivatisation towards dihydroxylated species, the analysis of which highlighted some interesting proton NMR data. The final aspect of this project began development of chemistry towards non-reducing end-labelled oligosaccharide sequences by implementation of a masked aldehyde unit on the C4 hydroxyl of GlcN synthesised from the allylated GlcN precursor via dihydroxylation chemistry. Incorporation of this moiety (protected as a 1,2-dibenzyl glycol) within both a trisaccharide and a pentasaccharide was achieved. Further development of this chemistry should allow for late step oxidative cleavage to reveal the reactive aldehyde, potentially allowing for attachment of various amine functionalised fluorophores via reductive amination. Radiolabelling of such a species should also be possible through sodium borotritide reduction for example.

Cette thèse vise à développer de nouvelles méthodes efficaces pour incorporer des isotopes de l’hydrogène dans les molécules organiques complexes, après une introduction portant sur les applications et la synthèse des molécules marquées par le deutérium et tritium. Les méthodes permettant le marquage, par échange isotopique direct, d’hétérocycles azotés par des isotopes de l’hydrogène restent perfectibles, voire inexistantes dans certains cas, malgré la récurrence de ce type de sous-structures dans les molécules d’intérêt pharmacologique. Pour cette raison, la majeure partie de ce travail a consisté au développement de nouvelles méthodes d’incorporation d’atomes de deutérium et de tritium sur des hétérocycles azotés catalysées par des nanoparticules métalliques. Dans un premier chapitre, la mise au point, le champ d’application d’une méthode de marquage mettant en jeu l’utilisation de nanocatalyseurs de ruthénium seront discutés. Dans ce cadre, des calculs théoriques ont permis de rationaliser les regiosélectivités obtenues expérimentalement et d’identifier notamment des intermédiaires clefs inédits. D’un point de vue applicatif, cette méthode a permis de synthétiser des étalons internes deutérés pour la quantification LC-MS mais aussi des molécules complexes tritiées ayant des activités spécifiques élevées en une étape de synthèse. Dans un autre chapitre, la synthèse et la réactivité de nouveaux nanocatalyseurs de nickel permettant de réaliser des échanges isotopiques sélectifs seront discutés. / This PhD thesis deals with the development of new efficient methods for the incorporation of hydrogen isotopes into organic molecules, which represents a serious issue especially for drug discovery and drug development processes. After giving an introduction about hydrogen isotopes and their applications in organic molecules, the course will proceed to an overview of different chemical transformations for establishing deuterium or tritium labels on molecular frameworks. The possibilities to label N-heterocycles by hydrogen isotopes through hydrogen isotope exchange (HIE) are still very restricted and even impossible for some representatives despite the strong recurrence of these substructures in numerous biologically active molecules. For this reason, the emphasis of the practical part will lie on the development of new methods for the incorporation of deuterium and tritium on N-heterocycles through metal nanoparticle catalysis. In the first chapter, HIE through ruthenium nanocatalysts will be optimized and the application range will be demonstrated. In this context, DFT-based calculations allowed to explain experimental regioselectivities and to identify new keyintermediates. In terms of application, it was shown that the ruthenium-catalyzed method is useful for the synthesis of deuterium labelled internal standards for LC-MS quantifications and for the tritiation of complex molecules displaying satisfying specific activities. In the next chapter, the synthesis of new nickel nanoparticles and their potential to catalyze selective HIE on N-heterocyclic derivatives will be discussed.

Les Métallo Protéases Matricielles (MMP) en tant qu'endopeptidases à zinc ont une large gamme de fonctions biologiques allant du remodelage tissulaire à la modulation de la réponse cellulaire. Une modification de leur activité protéolytique est souvent associée à de nombreux désordres biologiques. In vivo, ces protéases sont soumises à de nombreuses modifications post-traductionnelles. Elles sont sécrétées sous formes latentes à l'extérieur des cellules pour être ensuite transformées en forme fonctionnelles. Ces dernières sont ensuite inhibées par des inhibiteurs endogènes. En raison de leur sécrétion dans l’espace extra cellulaire, les MMP sous formes actives ont longtemps été considérées comme de simples ciseaux moléculaires capable de dégrader uniquement la matrice extracellulaire. Cependant, le remodelage tissulaire ne constitue pas la fonction unique et encore moins la fonction principale de ces enzymes. Elles peuvent en effet cliver une grande variété de substrats non matriciels et à ce titre sont impliquées dans la progression tumorale, l'immunité et l'inflammation. Pour ajouter une complexité supplémentaire à la biologie des MMP, il a été récemment montré que certaines MMP ont une localisation intracellulaire associée à des fonctions non protéolytiques. Ces observations, mais aussi celles montrant que ces protease participent à la progression de la maladie alors que d'autres ont une fonction protectrice, soulignent la nécessité de mieux documenter leur activation spatiale et temporelle dans divers contextes biologiques.Le profilage protéique basé sur l'activité vise à analyser l'état fonctionnel des protéines dans des échantillons biologiques complexes. À cette fin, des sondes basées sur l'activité (ABP), qui réagissent avec les enzymes en s’appuyant sur leur mécanisme catalytique, ont été développées pour la détection d’enzymes sous formes actives, notamment dans le cas des protéases à sérine et à cystéine. Une sonde basée sur l’activité (ABP) est classiquement composée : i) d’un groupement réactif conduisant à la modification covalente de résidus au sein du site actif de l’enzyme, ii) d’un motif de liaison imposant la sélectivité au groupement réactif et iii) d’un groupement rapporteur permettant la détection des enzymes ciblées. Cette approche ne s’applique toutefois pas aux MMP, pour lesquelles il n’existe pas de résidus nucléophiles conservés au sein du site actif. À cet égard, tous les ABP ciblant les MMP comportent un groupement photo activable qui, sous irradiation UV, favorise la formation du complexe covalent. De telles sondes photo sensibles ont permis de détecter les MMP sous leurs formes actives dans des tissus et des fluides, mais pas chez les animaux vivants au sein desquels l’étape de photo-activation ne peut être réalisé.Dans ce contexte, en nous appuyant sur un contexte structural favorable et en exploitant la chimie de l'acyl imidazole (LDAI) dirigée par un ligand, nous avons identifié une nouvelle série de sondes capables de modifier de manière covalente les MMP sans recourir à la photo-activation. Nous avons ainsi validé la capacité de ces sondes à marquer de manière sélective et efficace la MMP12 humaine in vitro et dans des protéomes complexes. Dans ce dernier cas, jusqu’à 50ng de hMMP12 correspondant à 0,05% du protéome total peuvent être détectés. Nous avons également déterminé l'identité de l’unique résidu modifié de façon covalente au sein du site actif de la hMMP-12 et vérifié que cette modification avait peu d'impact sur l’activité protéolytique de cette dernière. Nous avons démontré que cette approche permettait de détecter des MMP endogènes. Enfin, nous avons étendu cette stratégie de marquage à un panel plus large de MMP.En développant la première stratégie de marquage des formes actives de MMP «sans photo-activation», il semble maintenant possible d’envisager la détection de ces enzymes à la fois dans les protéomes complexes et in vivo. / Matrix MetalloProteases (MMPs) as zinc endopeptidases have a wide range of biological functions, and changes in their proteolytic activity underlie many biological disorders. Since their proteolytic activity has to be tightly controlled to prevent tissue destruction, theses proteases are subjected to numerous posttranslational modifications in vivo. They are secreted under latent forms outside of the cells, and are subsequently processed into their functional form that can be further inhibited by endogenous inhibitors. Due to their delineated area of activation, MMP active forms have long been considered for their unique ability to degrade extracellular substrates. However, turnover and breakdown of the extracellular matrix are neither the sole nor the main function of MMPs. These enzymes can indeed process a wide variety of non-matrix substrates and are involved in the regulation of multiple aspects of tumor progression, immunity and inflammation. To add further complexity to MMPs biology, some members within the family were recently reported to have intracellular localization associated to non-proteolytic functions. These observations but also those evidencing that some MMPs participate in disease progression while others have a protective function, stress the need to better document their spatial and temporal activation in various biological contexts.Activity-based protein profiling (ABPP) aims to analyze the functional state of proteins within complex biological samples. To this purpose, activity-based probes (ABPs) that react with enzymes in a mechanism-based manner have been successfully developed for the profiling of several enzymes including serine and cysteine proteases. A typical Activity-Based probe (ABP) is composed of i) a reactive warhead, which reacts in a covalent manner with enzyme active site residues, ii) a targeting moiety that imposes selectivity upon the reactive group and iii) a detectable group for subsequent analyses. This approach is not applicable to MMPs, which lack a targetable nucleophile involved in the catalysis. In this respect, all ABPs directed to MMPs are affinity-based probes (AfBPs) containing within their structure a photo cross-linking group that promotes the formation of a covalent complex upon UV-irradiation. Such photoactivatable probes have been successfully developed for the detection of MMPs under their active forms in fluids and tissue extracts, but not in living animals where the photo-activation step is not feasible.By relying on a favorable structural context and by exploiting the ligand-directed acyl imidazole (LDAI) chemistry, we have identified a novel series of AfBPs capable of covalently modifying matrix metalloproteases without making use of photo-activation. These active-site-directed probes whose structure was derived from that of a MMP12 selective inhibitor harbored a reactive acyl imidazole in their P3' position. They demonstrated their labelling specificity in vitro by covalently modifying a single Lysine residue within the MMP-12 S3' region. We also showed that these probes only targeted functional states of hMMP-12 and spared forms whose active site was occluded either by a synthetic or a natural inhibitor. We have validated the ability of these chemical probes to efficiently label human MMP12 in complex proteomes. In this case, down to 50 ng of hMMP12 corresponding to 0.05% of the whole proteome can be labelled and detected by in-gel fluorescence analysis. We demonstrated that this approach also allowed detecting endogenous MMPs secreted by stimulated-macrophages. In addition, by modifying the nature of the targeting moiety, we have extended this affinity-labeling approach to six other MMPs.By developing the first “photo activation-free” strategy to covalently modify active forms of MMPs, the unresolved proteomic profiling of native MMPs should be now accessible both in complex proteomes and in preclinical model in which MMPs are potential relevant targets.

Les petites molécules bioactives sont des acteurs clés dans la recherche biomédicale et les études de chimie biologiques, pour leur application potentielle comme sondes moléculaires pour enquêter les systèmes biologiques, et la possibilité de développer de nouveaux traitements puissants et sélectifs. Dans ce travail, nous avons exploré l'utilisation de plusieurs classes de petites molécules capables d'interférer avec des cibles biologiquement significatives, comme les acides nucléiques et les lysosomes, et déclencher des réponses biologiques. A partir de l'étoposide, l'un des agents anti-néoplasiques le plus employé en clinique, nous avons développé une nouvelle classe de petites molécules actives capables de cibler différemment les deux isoformes humaines de la topoisomérase II, TOP2A et TOP2B, qui jouent des rôles différents dans l'inhibition de la croissance des cellules cancéreuses, et le développement de tumeurs malignes secondaires. Dans un autre projet, nous avons utilisé une stratégie d'étiquetage « click in situ » pour enquêter sur la localisation subcellulaire d'un nouvel inhibiteur de la réaction d'échange nucléotidique des protéines Arf, un outil potentiel pour l'étude du trafic cellulaire et la signalisation liés a ces protéines. Enfin, nous avons étudié l'origine de l'efficacité d'un nouveau agent de ciblage du compartiment lysosomal: l'artesumycin, un hybride moléculaire des petites molécules bioactives la marmycine A et l'artésunate, qui possède une activité antiproliferative accrue en comparaison avec les deux produits naturels utilisés indépendamment. Nos résultats fournissent une précieuse contribution dans ces domaines de recherche. / Bioactive small molecules are key players in biomedical research and chemical biology studies given their potential application as molecular probes to investigate biological system, together with the possibility to develop new potent and selective therapeutics. In this work, we explored the use of several classes of small molecules able to interfere with therapeutically relevant targets, from nucleic acids to lysosomes, and evaluate ensuing biological responses. Starting from etoposide, one of the most clinically employed anti-neoplastic agents, we developed a new class of active small molecules able to differentially target the two human topoisomerase II isoforms, TOP2A and TOP2B, which have been shown to play different roles in inhibiting cancer cell growth and initiating secondary malignancies. In a separate project, we aimed to use an in situ click labelling strategies to investigate the subcellular localization of a new inhibitor of the nucleotide exchange reaction of Arf proteins, a potential tool for the study of Arf-related cellular trafficking and signalling. Finally, we studied the origins of the synergy displayed by the new lysosome targeting agent artesumycin, a molecular hybrid of the bioactive small molecules marmycin A and artesunate, which has been shown to possess enhanced antiproliferative activity in comparison to the two natural product used independently. Our results provide valuable contributions for future advances in these research areas.

IntroductionApproximately 5-10% of the general population report an antibiotic allergy. It has been reported that labeling of medical records with antibiotic hypersensitivity are often incorrect. As a result, antibiotic treatment choice will be increasingly difficult resulting in prolonged hospital visit, increased use of broad-spectrum antibiotics, increased frequency of side effects and the development of antibiotic resistance.AimThe primary aim was to investigate to what extent medical records were labelled with antibiotic allergy and whether these labels were adequately documented. The secondary aim was to investigate the difference in the impact of the label on the doctors’ choice of antibiotics depending on whether the doctor worked at a clinic of infectious diseases or not.MethodsA retrospective cohort study based on medical records labeled with antibiotic allergy in patients admitted to the Clinic of Infectious Diseases and the Emergency Ward at the Clinic of Medicine between 1st of January to 30th of June 2018.ResultsOf the total 1720 patients there were 132 (7,7%) patients marked with antibiotic allergy. Of these, only 21 patients (15.8%) were correctly labelled. There was no significant difference in the impact of the label on the choice of prescription between the two wards.ConclusionA substantial number of medical journals have a label for antibiotic allergy and the quality of the label is often poor with only 21 (15.8%) correct documented labels. We argue the need of education on antibiotic allergy and how to label and medical records.

Vor dem Hintergrund der Zunahme von Übergewicht, Adipositas und daraus resultierender gesundheitlicher Gefährdungen wie Herz-Kreislauferkrankungen und Stoffwechselerkrankungen, zum Beispiel durch mangelndes Ernährungswissen, wird seit längerem über eine einfache erweiterte Lebensmittelkennzeichnung diskutiert. Bisher sind erweiterte Kennzeichnungsmodelle auf Lebensmittelverpackungen zu finden. Eingangs wird das Modell „Kennzeichnung unter Verwendung der GDA“ dargestellt, welches der Verband der europäischen Lebensmittelindustrie (CIAA) für eine vereinfachte Nährwertkennzeichnung für verpackte Lebensmittel entwickelt hat. Die erweiterten Nährwertangaben werden mit dem Modell „1 plus 4“ als Schwerpunkt des nationalen Aktionsplans in Deutschland zur Prävention von Fehlernährung, Bewegungsmangel, Übergewicht und damit zusammenhängende Krankheiten als „Leitfaden für erweiterte Nährwertinformationen auf Lebensmittelverpackungen“ durch das Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV) veröffentlicht. Das Modell „Ampelsystem“ wurde von der britischen Lebensmittelbehörde Food Standard Agency (FSA) entwickelt, mit dem Ziel, dem Verbraucher verständliche Informationen zu geben. Ein positiver Nebeneffekt der Ampel ist es, die Lebensmittelhersteller zu motivieren, die Inhaltsstoffe und die Zusammensetzung ihrer Produkte zu prüfen. Die Verkaufszahlen britischer Unternehmen zeigen, dass die Verbraucher ihren Lebensmitteleinkauf auf das System ausrichten. Abschließend wird das schwedische „Keyhole-Modell“ dargestellt. In diesem werden die Lebensmittel für den Verbraucher anhand der Kategorien „gesund“ oder „weniger gesund“ hervorgehoben. Die zentrale staatliche Behörde für Ernährung und Trinkwasser (NFA) führte das „grüne Schlüsselloch“ ein. Das „Keyhole-Modell“ soll zur gesunden Ernährung beitragen. Zusammenfassen lässt sich konstatieren, dass keines der dargestellten Modelle mehrheitlich akzeptiert wird. Zwar verfolgen alle Modelle das gleiche Ziel, die Sensibilisierung der Verbraucher als Hilfestellung für die Auswahl gesunder Lebensmittel, aber die Ansätze der Modelle sind verschiedenartig. Die Entscheidung für ein europäisches einheitliches Modell steht noch aus. / Regarding the increase of overweight, obesity and resultant health hazards such as cardiovascular diseases and metabolic diseases, for example by lack of nutrition knowledge, simple advanced food labelling has been discussed for some time. So far, advanced identification models can be found on food packaging. At first, the model identification using the GDA is shown, which has been developed by the Association of European food industry (CIAA) for a simplified nutrition labelling for packaged foods. The expanded nutrition information are published with "one plus four" model as the focus of the national plan of action in Germany on the prevention of malnutrition, physical inactivity, obesity and related diseases as a "Guide for expanded nutrition information on food packaging" by the Federal Ministry of Food, Agriculture and Consumer Protection (BMELV). The "traffic light system“ model has been developed by the British Food Administration Food Standard Agency (FSA) to give consumers clear information. A positive side effect of the traffic light system is to motivate food manufacturers to check the ingredients and the composition of their products. The sales of British companies show that consumers adjust their food shopping to the system. Finally, the Swedish "keyhole" model is shown. In this, the food is categorised for the consumer as "healthy" or "less healthy". The central government agency for food and drinking water (NFA) introduced the "green keyhole”. The "keyhole” model is to contribute to a healthy nutrition. Summarizing it can be stated that none of the models presented is accepted by majority. Although all the models follow the same objective, the promotion of consumer awareness as an aid for the selection of healthy food, but the approaches of the different models are different. The decision for a European standard model is still pending.

Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.:Abstract Table of contents 1 Introduction 1.1 The transforming principle 1.2 Chromatin 1.2.1 Nucleosomes 1.2.2 The 30 nm fibre: a mirage? 1.2.3 Histone code 1.3 Histone variant H2A.Z 1.3.1 H2A.Z and transcription 1.4 Single molecule studies of chromatin 1.4.1 Chromatin under tension 1.4.2 Open nucleosome 1.4.3 Twisted chromatin 1.5 Single molecule techniques 1.5.1 Atomic force microscopy 1.5.2 Foerster resonance energy transfer 1.5.3 Magnetic tweezers 1.5.4 Worm-like chain model 2 Aims of the project 3 Cut and paste method for internal DNA labelling 3.1 Introduction 3.2 Experimental design 3.3 Results 3.3.1 Sequence design and cloning 3.3.2 Labelling and religation efficiency 3.3.3 Structural modifications 3.3.4 Labelling of multiple loci 3.3.5 Opposite-strand labelling 3.4 Discussion 4 Reconstituting chromatin 4.1 Long array of NPSs 4.1.1 Polymer physics applied to molecular cloning 4.1.2 Preventing homologous recombination 4.2 Expression and purification of histone proteins 4.2.1 Protein expression 4.2.2 Inclusions bodies 4.2.3 Histone purification 4.2.4 Octamer reconstitution and isolation 4.2.5 H2AvD 4.3 Reconstitution of nucleosomal arrays and biochemical analysis 4.3.1 Reconstitution procedure 4.3.2 Biochemical analysis 4.4 Tweezers construct with nucleosomes 5 Eviction of nucleosomes 5.1 Nucleosome eviction 5.1.1 A two-stage process 5.1.2 Chromatin fibres 5.1.3 Reassembly of nucleosomes 5.1.4 Distinct populations within nucleosome eviction events 5.1.5 Nicked and supercoilable nucleosomal arrays 5.2 Eviction of H2AvD-nucleosomes 5.2.1 H2AvD-nucleosomes bind less inner turn DNA 5.2.2 H2AvD-nucleosomes evict at lower forces 5.2.3 Likelihood of nucleosome reassembly 5.2.4 Gradual weakening of nucleosomes 5.2.5 Analysis software NucleoStep 5.3 Towards a rotor-bead assay on chromatin 5.4 Discussion 5.4.1 Nucleosome eviction in two stages 5.4.2 The fate of dimers in single molecule experiments 5.4.3 Structural origin and biological relevance of the mechanical properties of H2AvD-nucleosomal core particles 5.4.4 Monolithic or dynamic nucleosomes 6 Conclusions Bibliography Appendix 6.1 Internal labelling Procedure 6.1.1 Cloning 6.1.2 Nicking & cutting 6.1.3 The replace reaction 6.1.4 Purification 6.1.5 Ligation (optional) 6.1.6 Opposite strand labelling 6.1.7 Assessing the results of the labelling reaction 6.2 Chromatin reconstitution 6.2.1 Long array of NPSs 6.2.2 Expression and purification of histone proteins 6.2.3 Reconstitution of nucleosomal arrays and biochemical characterization 6.2.4 Simple Phenol:chloroform isolation of DNA 6.3 Magnetic tweezers experiments 6.3.1 Flow cell assembly 6.3.2 Functionalization of flow cells 6.3.3 Magnetic tweezers and rotor bead measurements 6.3.4 Force calibration List of Figures List of Tables List of publications Acknowledgements Declaration of originality