No description available.

Proteins play a major role in living systems and present a wide spectrum of functionalities. Many different types of proteins are involved into biological processes, such as the catalysis of biochemical reactions, cellular membrane transport, immune system response and DNA replication. However, some proteins and peptides might become harmful to living organisms; for example, their abnormal aggregation causes neurodegenerative disorders including Alzheimer disease (AD). One of the causes of AD is the presence of amyloid beta peptides Aβ(1-42), Aβ(1-40), which self-assemble into insoluble fibrils and plaques, which surround neuronal cells impeding synapsis. The number of AD patients is increasing, but a cure has not been founded yet. Therefore, it is crucial to investigate the mechanisms underlying amyloid aggregation and screening for compounds able to prevent this irreversible process. Microfluidics permits characterising the physicochemical properties of proteins, investigate their aggregation and study their interactions with other molecules. Chemical kinetics allows studying the microscopic events occurring during protein self-assembly. The combination of these two techniques provides a powerful tool for the identification of compounds inhibiting the aggregation process. In this thesis by using microfluidics, chemical kinetics and other biophysical assays, I have investigated the proteins isoelectric point (pI) and the inhibition of aberrant Aβ(1-42) self-assembly process. Firstly, I describe the development of a microfluidic platform allowing for the measurement of the protein pI, in a gradient-free manner. This approach overcomes a fundamental limitation of convectional techniques that is the achievement of a stable and well-controlled pH gradient. Secondly, I investigate the inhibiting effect of llama nanobodies on Aβ(1-42) aggregation. The findings from this study show that nanobodies target monomeric species with high affinity whereas interactions with fibril surfaces are weak. Finally, I discuss the use of other compounds inhibiting specific nucleation stages. These include the chaperones clusterin and brichos, as well as soot and pure carbon nanoparticles. Importantly, the addition of both chaperones to Aβ(1-42) solutions has an additive inhibitory effect on aggregation. My findings will improve the characterization of the physicochemical properties of proteins as well as providing promising candidates for the inhibition of specific stages of amyloid beta aggregation opening the way to possible cures for AD disease.

La forte progression démographique mondiale induit une demande d’énergietoujours en hausse. Ceci se traduit par un fort développement de nouvelles énergiesrenouvelables qui nécessitent, de par leur nature intermittente, des dispositifs de stockagede l’énergie. Parmi eux les supercondensateurs permettent un stockage électrostatique decharges (supercondensateurs à base de carbones activés), mais certains systèmes, ditspseudocapacitifs, font en outre intervenir des réactions redox rapides de surface.L’association des deux systèmes permettent d’accéder à des propriétés intéressantes, enparticulier pour le système MnO2/carbone activé. Cependant, les oxydes de manganèse sontd’excellents matériaux pseudocapacitifs mais assez peu conducteurs électroniques.L’objectif de ce travail est d’améliorer cette conductivité en les associant avec des oxydes decobalt conducteurs. Pour cela, une approche « architecturale » de synthèse de matériaux aété choisie. En partant d’oxydes de Mn et de Co lamellaires, ceux-ci sont exfoliés pourobtenir des nanofeuillets de nature différente. S’ensuit une étape de réempilement pouraboutir à un matériau lamellaire alterné. L’analyse structurale et morphologique desmatériaux prouve que des nanocomposites très finement divisés sont obtenus. Lespropriétés électrochimiques obtenues pour ces nanocomposites s’avèrent meilleures quecelles des matériaux initiaux, tant en densité d’énergie qu’en puissance. Cette stratégieoriginale est prometteuse et ouvre la voie à des réempilements de différente nature,notamment le graphène. / The ever increasing demand of renewable energies imposes, due to theirintermittent nature, the development of performant energy storage devices. Supercapacitorsare reliable devices that offer a high power density and numerous investigations are focusingon increasing their energy densities. In particular, asymmetric "metal oxides / activatedcarbons" supercapacitors are possible candidates. The MnO2/carbon system is the mostinvestigated system, due to its capability to work in aqueous medium at potentials up to 2 V,as well as to the low cost and environmental friendliness of manganese. Nevertheless, thissystem suffers from the poor electronic conductivity of manganese. This work reports anoriginal strategy for novel electrode materials involving exfoliation and restacking processesof lamellar “building blocks”: lamellar manganese oxides for their pseudocapacitiveproperties and lamellar cobalt oxyhydroxides for their high electronic conductivity. Thematerial engineering strategy focuses on the exfoliation of the lamellar materials intooligolamellae. The obtained suspensions are then restacked through various strategies andnew well defined mixed oxides are obtained. After structural and morphologicalcharacterization, it is clear that these nanocomposites present an intimate mix of the twoinitial phases. The electrochemical responses are hereby enhanced, proving the intertwinedrelationship between structure, morphology and properties. Furthermore, this architecturalapproach of building novel electrode materials is original and efficient and can easily betransposed to other “building blocks”, including graphene.

Diese Arbeit untersucht den Einsatz von keramischen Materialien in der Trinkwasseraufbereitung mittels Filtration und fokussiert dabei die Entfernung von Viren. Herkömmliche, auf Kieselgur basierende Tiefenfilter (Filterkerzen) mit Porengrößen im unteren Mikrometerbereich, werden hinsichtlich ihres Rückhaltevermögens gegenüber Kolloiden (Viren sowie Polystyrolpartikel) untersucht, um deren Einsatzfähigkeit in der Entfernung von Mikroorganismen im Allgemeinen abschätzen zu können. Ferner wird gezeigt, wie durch ein einfaches Verfahren solche Filter modifiziert werden können, um auch kleinste Viren mit ca. 30 nm Durchmessern aus dem Rohwasser zu entfernen. Die Zugabe von MgO während der Granulierungsstufe im Herstellungsprozess der Filterkerzen bewirkt eine erhebliche Verbesserung des Virenrückhalts bis zu über 99.9999%. Die experimentellen Ergebnisse wurden dabei mit theoretischen Modellen verglichen, um Aussagen über die Mechanismen der Virenentfernung treffen zu können.

Protein therapeutics offer numerous advantages over small molecule drugs and are rapidly becoming one of the most prominent classes of therapeutics. Unfortunately, they are delivered almost exclusively by injection due to biological obstacles preventing high bioavailability via the oral route. In this work, numerous approaches to overcoming these barriers are explored. PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) (P(IA-co-NVP)) hydrogels were synthesized, and the effects of monomer ratios, crosslinking density, microparticle size, protein size, and loading conditions were systematically evaluated using in vitro tests. P(IA-co-NVP) hydrogels demonstrated up to 69% greater equilibrium swelling at neutral conditions than previously-studied poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels and a 10-fold improvement in time-sensitive swelling experiments. Furthermore, P(IA-co-NVP) hydrogel microparticles demonstrated up to a 2.7-fold improvement in delivery of salmon calcitonin (sCT) compared to methacrylic acid-based systems, with a formulation comprised of a 1:2 ratio of itaconic acid to N-vinylpyrrolidone demonstrating the greatest delivery capability. Vast improvement in delivery capability was achieved using reduced ionic strength conditions during drug loading. Use of a 1.50 mM PBS buffer during loading yielded an 83-fold improvement in delivery of sCT compared to a standard 150 mM buffer. With this improvement, a daily dose of sCT could be provided using P(IA-co-NVP) microparticles in one standard-sized gel capsule. P(IA-co-NVP) was also tested with larger proteins urokinase and Rituxan. Crosslinking density provided a facile method for tuning hydrogels to accommodate a wide range of protein sizes. The effects of protein PEGylation were also explored. PEGylated sCT displayed lower release from P(IA-co-NVP) microparticles, but displayed increased apparent permeability across a Caco-2 monolayer by two orders of magnitude. Therefore, PEG-containing systems could yield high bioavailability of orally delivered proteins. Finally, a modified SELEX protocol for cellular selection of transcellular transport-initiating aptamers was developed and used to identify aptamer sequences showing enhanced intestinal perfusion. Over three selection cycles, the selected aptamer library showed significant increases in absorption, and from an initial library of 1.1 trillion sequences, 5-10 sequences were selected that demonstrated up to 10-fold amplification compared to the naïve library. These sequences could provide a means of overcoming the significant final barrier of intestinal absorption. / text

The dependencies of electrophoretic mobility on pH were measured for a set of 14 markers used for isoelectric focusing that were developed by the group of Šlais and that are based on substitutions on the nitrophenol core, and for a kit consisting of 5 pI markers developed by Shimura, which have an oligopeptide structure. The dissociation constants and limiting electrophoretic mobilities of these compounds were obtained from the dependencies with the use of the program AnglerFish. The isoelectric point values of the compounds were consequently calculated using the obtained data. A comparison of the obtained pI values with the values that have been declared in literature, albeit gained by different analytical methods, has been made. Key Words capillary zone electrophoresis, isoelectric focusing, pI markers, isoelectric point, thermodynamic dissociation constant, limiting ionic mobility

The aim of the diploma thesis is to characterize the adsorption properties of probiotic bacteria. Characterization was performed using of designed and optimized spectrophotometric method of determination of adhesion of probiotic bacteria to mucin as the main viscoelastic component of mucus. In order to provide a more detailed description of the adhesion interactions involved in the adhesion of probiotic bacteria to mucin, the viability of selected probiotic bacterial strains Lactobacillus rhamnosus CCM 1825, Lactobacillus plantarum CCM 7039, Lactobacillus delbrueckii subsp. bulgaricus CCM 7190, Lactobacillus acidophilus CCM 4833, Lactobacillus casei CCM 4798, Bifidobacterium breve CCM 7825, Bacillus coagulans CCM 2658 and a potential probiotic strain of Lactobacillus zeae CCM 7069. Moreover, bacterial adhesion to carbohydrates was also determined for all the bacteria tested. The last proposed and optimized technique was dynamic and electrophoretic light scattering characterizing surface properties such as -potential, average size distribution and isoelectric point. From this method, information was obtained on aggregation of bacterial cells and on electrostatic interactions. The combination of these methods was used as a complex tool to characterize adhesion of the tested bacterial cultures as a very specific, sensitive and key parameter of a successful probiotic microorganism that is influence by the multiple effects.

Diese Arbeit untersucht den Einsatz von keramischen Materialien in der Trinkwasseraufbereitung mittels Filtration und fokussiert dabei die Entfernung von Viren. Herkömmliche, auf Kieselgur basierende Tiefenfilter (Filterkerzen) mit Porengrößen im unteren Mikrometerbereich, werden hinsichtlich ihres Rückhaltevermögens gegenüber Kolloiden (Viren sowie Polystyrolpartikel) untersucht, um deren Einsatzfähigkeit in der Entfernung von Mikroorganismen im Allgemeinen abschätzen zu können. Ferner wird gezeigt, wie durch ein einfaches Verfahren solche Filter modifiziert werden können, um auch kleinste Viren mit ca. 30 nm Durchmessern aus dem Rohwasser zu entfernen. Die Zugabe von MgO während der Granulierungsstufe im Herstellungsprozess der Filterkerzen bewirkt eine erhebliche Verbesserung des Virenrückhalts bis zu über 99.9999%. Die experimentellen Ergebnisse wurden dabei mit theoretischen Modellen verglichen, um Aussagen über die Mechanismen der Virenentfernung treffen zu können.:Contents Chapter I Introduction 1 Chapter II Removal or inactivation of microorganisms, in particular viruses, for drinking water purposes with focus on small-scale, decentralised systems: A literature review 7 II. I Physical and chemical treatments 8 II. II Filtration processes 10 II. III Conclusions 15 Chapter III Mechanisms of adsorption in depth filtration 17 III.I Surface charge and the electrical double layer 18 III.II van der Waals interactions 22 III.III DLVO theory 23 III.IV Non-DLVO forces 25 III.V Extended DLVO Theory 27 Chapter IV Virus adsorption studies 29 IV.I A literature review 30 IV.I.I Virus concentration by adsorption-elution 33 IV.I.II Improved virus adsorption in filtration 35 IV.II The electrostatic enhanced adsorption approach 37 Chapter V Viruses 39 V.I Literature review 40 V.I.I Structure and morphology 40 V.I.II The viral life cycle 41 V.I.III Human pathogenic viruses in the aquatic environment 42 V.II Experimental 46 V.II.I The choice of viruses for adsorption studies 46 V.II.II Propagation and enumeration of the bacteriophages 48 V.II.III Characterisation of bacteriophages 51 V.III Results and discussion 54 V.III.I Production of high-titre and high-purity phage stocks 54 V.III.II Characteristics of bacteriophages 59 V.III.III Detection of a viral contaminant - the ‘Siphophage’ 64 Chapter VI The diatomaceous earth-based depth filter 69 VI.I Literature review 70 VI.I.I Diatomaceous earth 70 VI.I.II Retention of microorganisms in the DE-based depth filter 71 VI.II Experimental 73 VI.II.I Manufacturing the depth filter 73 VI.II.II Physical characterisation 74 VI.II.III Performing filter retention tests 75 VI.II.IV Latex retention test 76 VI.II.V Studying adsorption kinetics in a batch experiment 79 VI.II.VI Applying (X-)DLVO theory 80 VI.III Results and discussion 83 VI.III.I Characterisation of the depth filter 83 VI.III.II Latex removal in the depth filter 86 VI.III.III Filter performance on virus removal 94 VI.III.IV Batch-sorption experiments 99 VI.IV Summary and conclusions 102 Chapter VII The magnesium oxide modified depth filter 103 VII.I Experimental 104 VII.I.I Choice of the adsorbent material 104 VII.I.II Manufacturing the MgO-modified filter and characterisation methods 105 VII.II Results and discussion 106 VII.II.I The adsorbent: Magnesium oxide powder 106 VII.II.II Physical characterisation of modified depth filters 108 VII.II.III Virus removal in depth filters containing MgO 113 VII.II.IV Ageing behaviour of MgO modified filters 118 VII.II.V Discussion on the removal mechanisms 130 VII.III Summary and conclusions 134 Chapter VIII Summary, conclusions and outlook 135 VIII.I Summary and conclusions 136 VIII.II Outlook 137 Abbreviations, symbols and physical constants 139 Reference list 142