The grain-scale distribution and behaviour of melt and fluid in crystalline analogue systems

The production, segregation and migration of melt and aqueous fluids (henceforth called liquid) plays an important role for the transport of mass and energy within the mantle and the crust of the Earth. Many properties of large-scale liquid migration processes such as the permeability of a rock matrix or the initial segregation of newly formed liquid from the host-rock depends on the grain-scale distribution and behaviour of liquid. Although the general mechanisms of liquid distribution at the grain-scale are well understood, the influence of possibly important modifying processes such as static recrystallization, deformation, and chemical disequilibrium on the liquid distribution is not well constrained. For this thesis analogue experiments were used that allowed to investigate the interplay of these different mechanisms in-situ. In high-temperature environments where melts are produced, the grain-scale distribution in “equilibrium” is fully determined by the liquid fraction and the ratio between the solid-solid and the solid-liquid surface energy. The latter is commonly expressed as the dihedral or wetting angle between two grains and the liquid phase (Chapter 2). The interplay of this “equilibrium” liquid distribution with ongoing surface energy driven recrystallization is investigated in Chapter 4 and 5 with experiments using norcamphor plus ethanol liquid. Ethanol in contact with norcamphor forms a wetting angle of about 25°, which is similar to reported angles of rock-forming minerals in contact with silicate melt. The experiments in Chapter 4 show that previously reported disequilibrium features such as trapped liquid lenses, fully-wetted grain boundaries, and large liquid pockets can be explained by the interplay of the liquid with ongoing recrystallization. Closer inspection of dihedral angles in Chapter 5 reveals that the wetting angles are themselves modified by grain coarsening. Ongoing recrystallization constantly moves liquid-filled triple junctions, thereby altering the wetting angles dynamically as a function of the triple junction velocity. A polycrystalline aggregate will therefore always display a range of equilibrium and dynamic wetting angles at raised temperature, rather than a single wetting angle as previously thought. For the deformation experiments partially molten KNO3–LiNO3 experiments were used in addition to norcamphor–ethanol experiments (Chapter 6). Three deformation regimes were observed. At a high bulk liquid fraction >10 vol.% the aggregate deformed by compaction and granular flow. At a “moderate” liquid fraction, the aggregate deformed mainly by grain boundary sliding (GBS) that was localized into conjugate shear zones. At a low liquid fraction, the grains of the aggregate formed a supporting framework that deformed internally by crystal plastic deformation or diffusion creep. Liquid segregation was most efficient during framework deformation, while GBS lead to slow liquid segregation or even liquid dispersion in the deforming areas.

Systematic studies of the interaction between amyloid beta-protein and lipids

The amyloid peptide (Aß), a normal constituent of neuronal and non-neuronal cells, has been shown to be a major component of the extracellular plaque of Alzheimer’s disease (AD). The interaction of Aß peptides with the lipid matrix of neuronal cell membranes plays an important role in the pathogenesis of AD. In this study, we have developed peptide-tethered artificial lipid membranes by the Langmuir-Blodgett and Langmuir-Schaefer methods. Anti-Aß40-mAb labeled with a fluorophore was used to probe the Aß40 binding to the model membrane system. Systematic studies on the antibody or Aß-membrane interactions were carried out in our model systems by Surface Plasmon Field-Enhanced Fluorescence Spectroscopy (SPFS). Aß adsorption is critically determined by the lipid composition of the membranes. Aß specifically binds with membranes of sphingomyelin, and this preferential adsorption was markedly amplified by the addition of sterols (cholesterol or 25-OH-Chol). Fluorescence microscopy indicated that 25-OH-Chol could also form micro-domains with sphingomyelin as cholesterol does at the conditions used for the built-up of the model membranes. Our findings suggest that micro-domains composed of sphingomyelin and the sterols could be the binding sites of Aß and the role of sphingomyelin in AD should receive much more attention. The artificial membranes provide a novel platform for the study on AD, and SPFS is a potential tool for detecting Aß-membrane interaction. Numerous investigations indicate that the ability of Aß to form fibrils is considerably dependent upon the levels of ß-sheet structure adopted by Aß. Membrane-mediated conformational transition of Aß has been demonstrated. In this study, we focus on the interaction of Aß and the membranes composed of POPC/SM/25-OH-Chol (2:1:1). The artificial membrane system was established by the methods as described above. Immunoassy based on a pair of monoclonal antibodies (mAbs) against different epitopes was employed to detect the orientation of the Aß at the model membranes. Kinetics of antibody-Aß binding was determined by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The attempt has also been made to probe the change in the conformation of Aß using SPFS combined with immunoassay. Melatonin was employed to induce the conformational change of Aß. The orientation and the conformational change of Aß are evaluated by analysing kinetic/affinity parameters. This work provides novel insight into the investigation on the structure of Aß at the membrane surface.

The role of the NG2 proteoglycan in migration and characterization of the interaction with the intracellular binding partner, Syntenin

NG2 is a transmembrane proteoglycan with two N-terminal LNS domains and a C-terminal PDZ-binding motif. It is expressed in the developing and adult CNS by oligodendroglial precursor cells and subpopulations of perisynaptic glia and elsewhere by many immature cell types. In order to elucidate the functions of the protein and the heterogenous cell population which expresses it, we undertook to identify and characterise interaction partners of the molecule. The presence of the C-terminal PDZ recognition site in NG2 suggested PDZ-domain proteins as intracellular binding partners. In this work, interaction between the PDZ protein Syntenin and NG2 has been characterised. Syntenin is known to be involved in plasma membrane dynamics, metastasis and adhesion. Syntenin may thus link NG2 to the cytoskeleton, mediating migration of developing oligodendrocytes to axonal tracts prior to myelination, as well as process movement of NG2+ perisynaptic glia. NG2 is involved in cell spreading and polyclonal antibodies against NG2 inhibit the migration of immature glia and cell lines expressing the molecule. In this work we have characterised the segments of the extracellular portion of NG2 that are involved in migration. We found that the extracellular region immediately preceding the transmembrane segment is most important for cell motility. As part of this thesis, biochemical approaches to identify a trans-binding ligand interacting with the extracellular part of NG2 was also explored.

Fluidodinamica biomedica computazionale del flusso sanguigno in presenza di affezioni strumentali e/o patologiche

La tesi di Dottorato studia il flusso sanguigno tramite un codice agli elementi finiti (COMSOL Multiphysics). Nell’arteria è presente un catetere Doppler (in posizione concentrica o decentrata rispetto all’asse di simmetria) o di stenosi di varia forma ed estensione. Le arterie sono solidi cilindrici rigidi, elastici o iperelastici. Le arterie hanno diametri di 6 mm, 5 mm, 4 mm e 2 mm. Il flusso ematico è in regime laminare stazionario e transitorio, ed il sangue è un fluido non-Newtoniano di Casson, modificato secondo la formulazione di Gonzales & Moraga. Le analisi numeriche sono realizzate in domini tridimensionali e bidimensionali, in quest’ultimo caso analizzando l’interazione fluido-strutturale. Nei casi tridimensionali, le arterie (simulazioni fluidodinamiche) sono infinitamente rigide: ricavato il campo di pressione si procede quindi all’analisi strutturale, per determinare le variazioni di sezione e la permanenza del disturbo sul flusso. La portata sanguigna è determinata nei casi tridimensionali con catetere individuando tre valori (massimo, minimo e medio); mentre per i casi 2D e tridimensionali con arterie stenotiche la legge di pressione riproduce l’impulso ematico. La mesh è triangolare (2D) o tetraedrica (3D), infittita alla parete ed a valle dell’ostacolo, per catturare le ricircolazioni. Alla tesi sono allegate due appendici, che studiano con codici CFD la trasmissione del calore in microcanali e l’ evaporazione di gocce d’acqua in sistemi non confinati. La fluidodinamica nei microcanali è analoga all’emodinamica nei capillari. Il metodo Euleriano-Lagrangiano (simulazioni dell’evaporazione) schematizza la natura mista del sangue. La parte inerente ai microcanali analizza il transitorio a seguito dell’applicazione di un flusso termico variabile nel tempo, variando velocità in ingresso e dimensioni del microcanale. L’indagine sull’evaporazione di gocce è un’analisi parametrica in 3D, che esamina il peso del singolo parametro (temperatura esterna, diametro iniziale, umidità relativa, velocità iniziale, coefficiente di diffusione) per individuare quello che influenza maggiormente il fenomeno.

From lipid bilayers to synaptic vesicles: atomic force microscopy on lipid-based systems

The aim of this thesis was to apply the techniques of the atomic force microscope (AFM) to biological samples, namely lipid-based systems. To this end several systems with biological relevance based on self-assembly, such as a solid-supported membrane (SSM) based sensor for transport proteins, a bilayer of the natural lipid extract from an archaebacterium, and synaptic vesicles, were investigated by the AFM. For the characterization of transport proteins with SSM-sensors proteoliposomes are adsorbed that contain the analyte (transport protein). However the forces governing bilayer-bilayer interactions in solution should be repulsive under physiological conditions. I investigated the nature of the interaction forces with AFM force spectroscopy by mimicking the adsorbing proteoliposome with a cantilever tip, which was functionalized with charged alkane thiols. The nature of the interaction is indeed repulsive, but the lipid layers assemble in stacks on the SSM, which expose their unfavourable edges to the medium. I propose a model by which the proteoliposomes interact with these edges and fuse with the bilayer stacks, so forming a uniform layer on the SSM. Furthermore I characterized freestanding bilayers from a synthetic phospholipid with a phase transition at 41°C and from a natural lipid extract of the archaebacterium Methanococcus jannaschii. The synthetic lipid is in the gel-phase at room temperature and changes to the fluid phase when heated to 50°C. The bilayer of the lipid extract shows no phase transition when heated from room temperature to the growth temperature (~ 50°C) of the archeon. Synaptic vesicles are the containers of neurotransmitter in nerve cells and the synapsins are a family of extrinsic membrane proteins, that are associated with them, and believed to control the synaptic vesicle cycle. I used AFM imaging and force spectroscopy together with dynamic light scattering to investigate the influence of synapsin I on synaptic vesicles. To this end I used native, untreated synaptic vesicles and compared them to synapsin-depleted synaptic vesicles. Synapsin-depleted vesicles were larger in size and showed a higher tendency to aggregate compared to native vesicles, although their mechanical properties were alike. I also measured the aggregation kinetics of synaptic vesicles induced by synapsin I and found that the addition of synapsin I promotes a rapid aggregation of synaptic vesicles. The data indicate that synapsin I affects the stability and the aggregation state of synaptic vesicles, and confirm the physiological role of synapsins in the assembly and regulation of synaptic vesicle pools within nerve cells.

NMR studies on supramolecular aggregates in solution and the solid state

The aim of this work presented here is the characterization of structure and dynamics of different types of supramolecular systems by advanced NMR spectroscopy. One of the characteristic features of NMR spectroscopy is based on its high selectivity. Thus, it is desirable to exploit this technique for studying structure and dynamics of large supramolecular systems without isotopic enrichment. The observed resonance frequencies are not only isotope specific but also influenced by local fields, in particular by the distribution of electron density around the investigated nucleus. Barbituric acid are well known for forming strongly hydrogen-bonded complexes with variety of adenine derivatives. The prototropic tautomerism of this material facilitates an adjustment to complementary bases containing a DDA(A = hydrogen bond acceptor site, D = hydrogen bond donor site) or ADA sequences, thereby yielding strongly hydrogen-bonded complexes. In this contribution solid-state structures of the enolizable chromophor "1-n-butyl-5-(4-nitrophenyl)-barbituric acid" that features adjustable hydrogen-bonding properties and the molecular assemblies with three different strength of bases (Proton sponge, adenine mimetic 2,6-diaminopyridine (DAP) and 2,6-diacetamidopyridine (DAC)) are studied. Diffusion NMR spectroscopy gives information over such interactions and has become the method of choice for measuring the diffusion coefficient, thereby reflecting the effective size and shape of a molecular species. In this work the investigation of supramolecular aggregates in solution state by means of DOSY NMR techniques are performed. The underlying principles of DOSY NMR experiment are discussed briefly and more importantly two applications demonstrating the potential of this method are focused on. Calix[n]arenes have gained a rather prominent position, both as host materials and as platforms to design specific receptors. In this respect, several different capsular contents of tetra urea calix[4]arenes (benzene, benzene-d6, 1-fluorobenzene, 1-fluorobenzene-d5, 1,4-difluorobenzene, and cobaltocenium) are studied by solid state NMR spectroscopy. In the solid state, the study of the interaction between tetra urea calix[4]arenes and guest is simplified by the fact that the guests molecule remains complexed and positioned within the cavity, thus allowing a more direct investigation of the host-guest interactions.

Dynamics, rheology and critical properties of colloidal fluid mixtures

Liquids under the influence of external fields exhibit a wide range of intriguing phenomena that can be markedly different from the behaviour of a quiescent system. This work considers two different systems — a glassforming Yukawa system and a colloid-polymer mixture — by Molecular Dynamics (MD) computer simulations coupled to dissipative particle dynamics. The former consists of a 50-50 binary mixture of differently-sized, like-charged colloids interacting via a screened Coulomb (Yukawa) potential. Near the glass transition the influence of an external shear field is studied. In particular, the transition from elastic response to plastic flow is of interest. At first, this model is characterised in equilibrium. Upon decreasing temperature it exhibits the typical dynamics of glassforming liquids, i.e. the structural relaxation time τα grows strongly in a rather small temperature range. This is discussed with respect to the mode-coupling theory of the glass transition (MCT). For the simulation of bulk systems under shear, Lees-Edwards boundary conditions are applied. At constant shear rates γ˙ ≫ 1/τα the relevant time scale is given by 1/γ˙ and the system shows shear thinning behaviour. In order to understand the pronounced differences between a quiescent system and a system under shear, the response to a suddenly commencing or terminating shear flow is studied. After the switch-on of the shear field the shear stress shows an overshoot, marking the transition from elastic to plastic deformation, which is connected to a super-diffusive increase of the mean squared displacement. Since the average static structure only depends on the value of the shear stress, it does not discriminate between those two regimes. The distribution of local stresses, in contrast, becomes broader as soon as the system starts flowing. After a switch-off of the shear field, these additional fluctuations are responsible for the fast decay of stresses, which occurs on a time scale 1/γ˙ . The stress decay after a switch-off in the elastic regime, on the other hand, happens on the much larger time scale of structural relaxation τα. While stresses decrease to zero after a switch-off for temperatures above the glass transition, they decay to a finite value for lower temperatures. The obtained results are important for advancing new theoretical approaches in the framework of mode-coupling theory. Furthermore, they suggest new experimental investigations on colloidal systems. The colloid-polymer mixture is studied in the context of the behaviour near the critical point of phase separation. For the MD simulations a new effective model with soft interaction potentials is introduced and its phase diagram is presented. Here, mainly the equilibrium properties of this model are characterised. While the self-diffusion constants of colloids and polymers do not change strongly when the critical point is approached, critical slowing down of interdiffusion is observed. The order parameter fluctuations can be determined through the long-wavelength limit of static structure factors. For this strongly asymmetric mixture it is shown how the relevant structure factor can be extracted by a diagonalisation of a matrix that contains the partial static structure factors. By presenting first results of this model under shear it is demonstrated that it is suitable for non-equilibrium simulations as well.

Thermal Casimir effect at fluid interfaces: Fluctuation-induced forces between anisotropic colloids

We study the effective interaction between two ellipsoidal particles at the interface of two fluid phases which are mediated by thermal fluctuations of the interface. Within a coarse-grained picture, the properties of fluid interfaces are very well described by an effective capillary wave Hamiltonian which governs both the equilibrium interface configuration and the thermal fluctuations (capillary waves) around this equilibrium (or mean-field) position. As postulated by the Goldstone theorem the capillary waves are long-range correlated. The interface breaks the continuous translational symmetry of the system, and in the limit of vanishing external fields - like gravity - it has to be accompanied by easily excitable long wavelength (Goldstone) modes – precisely the capillary waves. In this system the restriction of the long-ranged interface fluctuations by particles gives rise to fluctuation-induced forces which are equivalent to interactions of Casimir type and which are anisotropic in the interface plane. Since the position and the orientation of the colloids with respect to the interface normal may also fluctuate, this system is an example for the Casimir effect with fluctuating boundary conditions. In the approach taken here, the Casimir interaction is rewritten as the interaction between fluctuating multipole moments of an auxiliary charge density-like field defined on the area enclosed by the contact lines. These fluctuations are coupled to fluctuations of multipole moments of the contact line position (due to the possible position and orientational fluctuations of the colloids). We obtain explicit expressions for the behavior of the Casimir interaction at large distances for arbitrary ellipsoid aspect ratios. If colloid fluctuations are suppressed, the Casimir interaction at large distances is isotropic, attractive and long ranged (double-logarithmic in the distance). If, however, colloid fluctuations are included, the Casimir interaction at large distances changes to a power law in the inverse distance and becomes anisotropic. The leading power is 4 if only vertical fluctuations of the colloid center are allowed, and it becomes 8 if also orientational fluctuations are included.

Interplay of pi–pi stacking and hydrogen bonding in conjugated supramolecular systems studied by solid-state NMR

In this work supramolecular organic systems based on rigid pi-conjugated building blocks and flexible side chains were studied via solid-state NMR spectroscopy. Specifically, these studies focussed on phenylene ethynylene based macrocycles, polymer systems including polythiophenes, and rod-coil copolymers of oligo(p-benzamide) and poly(ethylene glycol). All systems were studied in terms of the local order and mobility. The central topic of this dissertation was to elucidate the role of the flexible side chains in interplay of different non-covalent interactions, like pi-pi-stacking and hydrogen bonding.Combining the results of this work, it can be concluded that the ratio of the rigid block and the attached alkyl side chains can be crucial for the design of an ordered pi-conjugated supramolecular system. Through alkyl side chains, it is also possible to introduce liquid-crystalline phases in the system, which can foster the local order of the system. Moreover in the studied system longer, unbranched alkyl side chains are better suited to stabilize the corresponding aggregation than shorter, branched ones.The combination of non-covalent interactions such as pi-pi-stacking and hydrogen bonding play an important role for structure formation. However, the effect of pi-pi-stacking interaction is much weaker than the effect of hydrogen bonding and is only observed in systems with a suitable local order. Hence, they are often not strong enough to control the local order. In contrast, hydrogen bonds predominantly influence the structural organization and packing. In comparison the size of the alkyl side chains is only of minor importance. The suppression of certain hydrogen bonds can lead to completely different structures and can induce a specific aggregation behavior. Thus, for the design of a supramolecular ordered system the presence of hydrogen bonding efficiently stabilizes the corresponding structure, but the ratio of hydrogen bond forming groups should be kept low to be able to influence the structure selectively.

Molecular interaction of the human metalloprotease meprin beta with the amyloid precursor protein, ADAM10, and ADAM17 based on proteomics

Die Zinkendopeptidasen Meprin α und β sind Schlüsselkomponenten in patho(physiologischen) Prozessen wie Entzündung, Kollagenassemblierung und Angiogenese. Nach ihrer Entdeckung in murinen Bürstensaummembranen und humanen Darmepithelien, wurden weitere Expressionsorte identifiziert, z.B. Leukozyten, Krebszellen und die humane Haut. Tiermodelle, Zellkulturen und biochemische Analysen weisen auf Funktionen der Meprine in der Epithelialdifferenzierung, Zellmigration, Matrixmodellierung, Angiogenese, Bindegewebsausbildung und immunologische Prozesse hin. Dennoch sind ihre physiologischen Substrate weitgehend noch unbekannt. Massenspektrometrisch basierte Proteomics-Analysen enthüllten eine einzigartige Spaltspezifität für saure Aminosäurereste in der P1´ Position und identifizierten neue biologische Substratkandidaten. Unter den 269 extrazellulären Proteinen, die in einem Substratscreen identifiziert wurden, stellten sich das amyloid precursor protein (APP) and ADAM10 (a disintegrin and metalloprotease 10) als sehr vielversprechende Kandidaten heraus. Mehrere Schnittstellen innerhalb des APP Proteins, hervorgerufen durch verschiedenen Proteasen, haben unterschiedlichen Auswirkungen zur Folge. Die β-Sekretase BACE (β-site APP cleaving enzyme) prozessiert APP an einer Schnittstelle, welche als initialer Schritt in der Entwicklung der Alzheimer Erkrankung gilt. Toxische Aβ (Amyloid β)-Peptide werden in den extrazellulären Raum freigesetzt und aggregieren dort zu senilen Plaques. Membran verankertes Meprin β hat eine β-Sekretase Aktivität, die in einem Zellkultur-basierten System bestätigt werden konnte. Die proteolytische Effizienz von Meprin β wurde in FRET (Fluorescence Resonance Energy Transfer)-Analysen bestimmt und war um den Faktor 104 höher als die von BACE1. Weiterhin konnte gezeigt werden, dass Meprin β die ersten zwei Aminosäuren prozessiert und somit aminoterminal einen Glutamatrest freisetzt, welcher nachfolgend durch die Glutaminylzyklase in ein Pyroglutamat zykliert werden kann. Trunkierte Aβ-Peptide werden nur in Alzheimer Patienten generiert. Aufgrund einer erhöhten Hydrophobie weisen diese Peptide eine höhere Tendenz zur Aggregation auf und somit eine erhöhte Toxizität. Bis heute wurde keine Protease identifiziert, welche diese Schnittstelle prozessiert. Die Bildung der Meprin vermittelten N-terminalen APP Fragmenten wurde in vitro und in vivo detektiert. Diese N-APP Peptide hatten keine cytotoxischen Auswirkungen auf murine und humane Gehirnzellen, obwohl zuvor N-APP als Ligand für den death receptor (DR) 6 identifiziert wurde, der für axonale Degenerationsprozesse verantwortlich ist. rnIm nicht-amyloidogenen Weg prozessiert ADAM10 APP und entlässt die Ektodomäne von der Zellmembran. Wir konnten das ADAM10 Propeptid als Substrat von Meprin β identifizieren und in FRET Analysen, in vitro und in vivo zeigen, dass die Meprin vermittelte Prozessierung zu einer erhöhten ADAM10 Aktivität führt. Darüber hinaus wurde ADAM10 als Sheddase für Meprin β identifiziert. Shedding konnte durch Phorbol 12-myristate 13-acetate (PMA) oder durch das Ionophor A23187 hervorgerufen werden, sowie durch ADAM10 Inhibitoren blockiert werden. rnDiese Arbeit konnte somit ein komplexes proteolytisches Netwerk innerhalb der Neurophysiologie aufdecken, welches für die Entwicklung der Alzheimer Demenz wichtig sein kann.rn

Aeroelastic stability of structures: flutter analysis using Computational Fluid Dynamics

Thanks to the increasing slenderness and lightness allowed by new construction techniques and materials, the effects of wind on structures became in the last decades a research field of great importance in Civil Engineering. Thanks to the advances in computers power, the numerical simulation of wind tunnel tests has became a valid complementary activity and an attractive alternative for the future. Due to its flexibility, during the last years, the computational approach gained importance with respect to the traditional experimental investigation. However, still today, the computational approach to fluid-structure interaction problems is not as widely adopted as it could be expected. The main reason for this lies in the difficulties encountered in the numerical simulation of the turbulent, unsteady flow conditions generally encountered around bluff bodies. This thesis aims at providing a guide to the numerical simulation of bridge deck aerodynamic and aeroelastic behaviour describing in detail the simulation strategies and setting guidelines useful for the interpretation of the results.

Dynamic development of hydrofractures

Natürliche hydraulische Bruchbildung ist in allen Bereichen der Erdkruste ein wichtiger und stark verbreiteter Prozess. Sie beeinflusst die effektive Permeabilität und Fluidtransport auf mehreren Größenordnungen, indem sie hydraulische Konnektivität bewirkt. Der Prozess der Bruchbildung ist sowohl sehr dynamisch als auch hoch komplex. Die Dynamik stammt von der starken Wechselwirkung tektonischer und hydraulischer Prozesse, während sich die Komplexität aus der potentiellen Abhängigkeit der poroelastischen Eigenschaften von Fluiddruck und Bruchbildung ergibt. Die Bildung hydraulischer Brüche besteht aus drei Phasen: 1) Nukleation, 2) zeitabhängiges quasi-statisches Wachstum so lange der Fluiddruck die Zugfestigkeit des Gesteins übersteigt, und 3) in heterogenen Gesteinen der Einfluss von Lagen unterschiedlicher mechanischer oder sedimentärer Eigenschaften auf die Bruchausbreitung. Auch die mechanische Heterogenität, die durch präexistierende Brüche und Gesteinsdeformation erzeugt wird, hat großen Einfluß auf den Wachstumsverlauf. Die Richtung der Bruchausbreitung wird entweder durch die Verbindung von Diskontinuitäten mit geringer Zugfestigkeit im Bereich vor der Bruchfront bestimmt, oder die Bruchausbreitung kann enden, wenn der Bruch auf Diskontinuitäten mit hoher Festigkeit trifft. Durch diese Wechselwirkungen entsteht ein Kluftnetzwerk mit komplexer Geometrie, das die lokale Deformationsgeschichte und die Dynamik der unterliegenden physikalischen Prozesse reflektiert. rnrnNatürliche hydraulische Bruchbildung hat wesentliche Implikationen für akademische und kommerzielle Fragestellungen in verschiedenen Feldern der Geowissenschaften. Seit den 50er Jahren wird hydraulisches Fracturing eingesetzt, um die Permeabilität von Gas und Öllagerstätten zu erhöhen. Geländebeobachtungen, Isotopenstudien, Laborexperimente und numerische Analysen bestätigen die entscheidende Rolle des Fluiddruckgefälles in Verbindung mit poroelastischen Effekten für den lokalen Spannungszustand und für die Bedingungen, unter denen sich hydraulische Brüche bilden und ausbreiten. Die meisten numerischen hydromechanischen Modelle nehmen für die Kopplung zwischen Fluid und propagierenden Brüchen vordefinierte Bruchgeometrien mit konstantem Fluiddruck an, um das Problem rechnerisch eingrenzen zu können. Da natürliche Gesteine kaum so einfach strukturiert sind, sind diese Modelle generell nicht sonderlich effektiv in der Analyse dieses komplexen Prozesses. Insbesondere unterschätzen sie die Rückkopplung von poroelastischen Effekten und gekoppelte Fluid-Festgestein Prozesse, d.h. die Entwicklung des Porendrucks in Abhängigkeit vom Gesteinsversagen und umgekehrt.rnrnIn dieser Arbeit wird ein zweidimensionales gekoppeltes poro-elasto-plastisches Computer-Model für die qualitative und zum Teil auch quantitativ Analyse der Rolle lokalisierter oder homogen verteilter Fluiddrücke auf die dynamische Ausbreitung von hydraulischen Brüchen und die zeitgleiche Evolution der effektiven Permeabilität entwickelt. Das Programm ist rechnerisch effizient, indem es die Fluiddynamik mittels einer Druckdiffusions-Gleichung nach Darcy ohne redundante Komponenten beschreibt. Es berücksichtigt auch die Biot-Kompressibilität poröser Gesteine, die implementiert wurde um die Kontrollparameter in der Mechanik hydraulischer Bruchbildung in verschiedenen geologischen Szenarien mit homogenen und heterogenen Sedimentären Abfolgen zu bestimmen. Als Resultat ergibt sich, dass der Fluiddruck-Gradient in geschlossenen Systemen lokal zu Störungen des homogenen Spannungsfeldes führen. Abhängig von den Randbedingungen können sich diese Störungen eine Neuausrichtung der Bruchausbreitung zur Folge haben kann. Durch den Effekt auf den lokalen Spannungszustand können hohe Druckgradienten auch schichtparallele Bruchbildung oder Schlupf in nicht-entwässerten heterogenen Medien erzeugen. Ein Beispiel von besonderer Bedeutung ist die Evolution von Akkretionskeilen, wo die große Dynamik der tektonischen Aktivität zusammen mit extremen Porendrücken lokal starke Störungen des Spannungsfeldes erzeugt, die eine hoch-komplexe strukturelle Entwicklung inklusive vertikaler und horizontaler hydraulischer Bruch-Netzwerke bewirkt. Die Transport-Eigenschaften der Gesteine werden stark durch die Dynamik in der Entwicklung lokaler Permeabilitäten durch Dehnungsbrüche und Störungen bestimmt. Möglicherweise besteht ein enger Zusammenhang zwischen der Bildung von Grabenstrukturen und großmaßstäblicher Fluid-Migration. rnrnDie Konsistenz zwischen den Resultaten der Simulationen und vorhergehender experimenteller Untersuchungen deutet darauf hin, dass das beschriebene numerische Verfahren zur qualitativen Analyse hydraulischer Brüche gut geeignet ist. Das Schema hat auch Nachteile wenn es um die quantitative Analyse des Fluidflusses durch induzierte Bruchflächen in deformierten Gesteinen geht. Es empfiehlt sich zudem, das vorgestellte numerische Schema um die Kopplung mit thermo-chemischen Prozessen zu erweitern, um dynamische Probleme im Zusammenhang mit dem Wachstum von Kluftfüllungen in hydraulischen Brüchen zu untersuchen.

Extracellular glutamate-GABA balance in the developing neocortex

It has been shown in the study that glutamate transporters (EAAT) are capable to modulate GABA transports (GAT). Here we also report that DL-TBOA, a non-transportable glutamate uptake blocker, eliminates GAT-mediated GABA release, while D-aspartate, an EAAT substrate, does not block the latter. The strength or even the operating mode of GABA uptake/release could be influenced by the work of EAATs. Considering the interaction between EAATs and GATs we can conclude that ambient glutamate and GABA levels are mutually dependent. The EAAT-GAT crosstalk observed in this work is mediated by EAAT1 and GAT-2/3. Since both transporters are Na+ dependent and mainly glial, next we investigated the role of [Na+]i in astrocytic-mediated glutamate uptake. We tested whether [Na+]i changes affect paired-pulse plasticity of STCs recorded from cortical layer 2/3 astrocytes. We report that an elevation of [Na+]i induced either by using a high [Na+]i intrapipette solution or by application of GABA slows STCs kinetics and decrease paired-pulse facilitation (PPF) of STCs at short inter-stimulus intervals. Moreover, GAT inhibitors decrease PPF of STCs under control conditions, suggesting that endogenous GABA operating via GATs influences EAAT-mediated transport

Hydrodynamics and solid dosage form disintegration/dissolution : immediate release tablets and novel in situ polyelectrolyte gastroretentive drug delivery systems

Solid oral dosage form disintegration in the human stomach is a highly complex process dependent on physicochemical properties of the stomach contents as well as on physical variables such as hydrodynamics and mechanical stress. Understanding the role of hydrodynamics and forces in disintegration of oral solid dosage forms can help to improve in vitro disintegration testing and the predictive power of the in vitro test. The aim of this work was to obtain a deep understanding of the influence of changing hydrodynamic conditions on solid oral dosage form performance. Therefore, the hydrodynamic conditions and forces present in the compendial PhEur/USP disintegration test device were characterized using a computational fluid dynamics (CFD) approach. Furthermore, a modified device was developed and the hydrodynamic conditions present were simulated using CFD. This modified device was applied in two case studies comprising immediate release (IR) tablets and gastroretentive drug delivery systems (GRDDS). Due to the description of movement provided in the PhEur, the movement velocity of the basket-rack assembly follows a sinusoidal profile. Therefore, hydrodynamic conditions are changing continually throughout the movement cycle. CFD simulations revealed that the dosage form is exposed to a wide range of fluid velocities and shear forces during the test. The hydrodynamic conditions in the compendial device are highly variable and cannot be controlled. A new, modified disintegration test device based on computerized numerical control (CNC) technique was developed. The modified device can be moved in all three dimensions and radial movement is also possible. Simple and complex moving profiles can be developed and the influence of the hydrodynamic conditions on oral solid dosage form performance can be evaluated. Furthermore, a modified basket was designed that allows two-sided fluid flow. CFD simulations of the hydrodynamics and forces in the modified device revealed significant differences in the fluid flow field and forces when compared to the compendial device. Due to the CNC technique moving velocity and direction are arbitrary and hydrodynamics become controllable. The modified disintegration test device was utilized to examine the influence of moving velocity on disintegration times of IR tablets. Insights into the influence of moving speed, medium viscosity and basket design on disintegration times were obtained. An exponential relationship between moving velocity of the modified basket and disintegration times was established in simulated gastric fluid. The same relationship was found between the disintegration times and the CFD predicted average shear stress on the tablet surface. Furthermore, a GRDDS was developed based on the approach of an in situ polyelectrolyte complex (PEC). Different complexes composed of different grades of chitosan and carrageenan and different ratios of those were investigated for their swelling behavior, mechanical stability, and in vitro drug release. With an optimized formulation the influence of changing hydrodynamic conditions on the swelling behavior and the drug release profile was demonstrated using the modified disintegration test device. Both, swelling behavior and drug release, were largely dependent on the hydrodynamic conditions. Concluding, it has been shown within this thesis that the application of the modified disintegration test device allows for detailed insights into the influence of hydrodynamic conditions on solid oral dosage form disintegration and dissolution. By the application of appropriate test conditions, the predictive power of in vitro disintegration testing can be improved using the modified disintegration test device. Furthermore, CFD has proven a powerful tool to examine the hydrodynamics and forces in the compendial as well as in the modified disintegration test device. rn

Genexpressionsanalyse boviner mesenchymaler Stammzellen und deren in-vitro-differenzierten Folgelinien

Mesenchymale Stamzellen (MSC) sind Vertreter der adulten Stammzellen. Sie bergen durch ihre große Plastizität ein immenses Potential für die klinische Nutzung in Form von Stammzelltherapien. Zellen dieses Typs kommen vornehmlich im Knochenmark der großen Röhrenknochen vor und können zu Knochen, Knorpel und Fettzellen differenzieren. MSC leisten einen wichtigen Beitrag im Rahmen regenerativer Prozesse, beispielsweise zur Heilung von Frakturen. Breite Studien demonstrieren bereits jetzt auch bei komplexeren Erkrankungen (z.B. Osteoporose) therapeutisch vielversprechende Einsatzmöglichkeiten. Oft kommen hierbei aus MSC gezielt differenzierte Folgelinien aus Zellkulturen zum Einsatz. Dies bedingt eine kontrollierte Steuerung der Differenzierungsprozesse in vitro. Der Differenzierung einer Stammzelle liegt eine komplexe Veränderung ihrer Genexpression zugrunde. Genexpressionsmuster zur Erhaltung und Proliferation der Stammzellen müssen durch solche, die der linienspezifischen Differenzierung dienen, ersetzt werden. Die mit der Differenzierung einhergehende, transkriptomische Neuausrichtung ist für das Verständnis der Prozesse grundlegend und wurde bislang nur unzureichend untersucht. Ziel der vorliegenden Arbeit ist eine transkriptomweite und vergleichende Genexpressionsanalyse Mesenchymaler Stammzellen und deren in vitro differenzierten Folgelinien mittels Plasmid - DNA Microarrays und Sequenziertechniken der nächsten Generation (RNA-Seq, Illumina Plattform). In dieser Arbeit diente das Hausrind (Bos taurus) als Modellorganismus, da es genetisch betrachtet eine hohe Ähnlichkeit zum Menschen aufweist und Knochenmark als Quelle von MSC gut verfügbar ist. Primärkulturen Mesenchymaler Stammzellen konnten aus dem Knochenmark von Rindern erfolgreich isoliert werden. Es wurden in vitro Zellkultur - Versuche durchgeführt, um die Zellen zu Osteoblasten, Chondrozyten und Adipozyten zu differenzieren. Zur Genexpressionsanalyse wurde RNA aus jungen MSC und einer MSC Langzeitkultur („alte MSC“), sowie aus den differenzierten Zelllinien isoliert und für nachfolgende Experimente wo nötig amplifiziert. Der Erfolg der Differenzierungen konnte anhand der Genexpression von spezifischen Markergenen und mittels histologischer Färbungen belegt werden. Hierbei zeigte sich die Differenzierung zu Osteoblasten und Adipozyten erfolgreich, während die Differenzierung zu Chondrozyten trotz diverser Modifikationen am Protokoll nicht erfolgreich durchgeführt werden konnte. Eine vergleichende Hybridisierung zur Bestimmung differentieller Genexpression (MSC vs. Differenzierung) mittels selbst hergestellter Plasmid - DNA Microarrays ergab für die Osteogenese mit Genen wie destrin und enpp1, für die undifferenzierten MSC mit dem Gen sema3c neue Kandidatengene, deren biologische Funktion aufzuklären in zukünftigen Experimenten vielversprechende Ergebnisse liefern sollte. Die Analyse der transkriptomweiten Genexpression mittels NGS lieferte einen noch umfangreicheren Einblick ins Differenzierungsgeschehen. Es zeigte sich eine hohe Ähnlichkeit im Expressionsprofil von jungen MSC und Adipozyten, sowie zwischen den Profilen der alten MSC (eine Langzeitkultur) und Osteoblasten. Die alten MSC wiesen deutliche Anzeichen für eine spontane Differenzierung in die osteogene Richtung auf. Durch Analyse der 100 am stärksten exprimierten Gene jeder Zelllinie ließen sich für junge MSC und Adipozyten besonders Gene der extrazellulären Matrix (z.B col1a1,6 ; fn1 uvm.) auffinden. Sowohl Osteoblasten, als auch die alten MSC exprimieren hingegen verstärkt Gene mit Bezug zur oxidativen Phosphorylierung, sowie ribosomale Proteine. Eine Betrachtung der differentiellen Genexpression (junge MSC vs. Differenzierung) mit anschließender Pathway Analyse und Genontologie Anreicherungsstatistik unterstützt diese Ergebnisse vor allem bei Osteoblasten, wo nun jedoch zusätzlich auch Gene zur Regulation der Knochenentwicklung und Mineralisierung in den Vordergrund treten. Für Adipozyten konnte mit Genen des „Jak-STAT signaling pathway“, der Fokalen Adhäsion, sowie Genen des „Cytokine-cytokine receptor interaction pathway“ sehr spannende Einsichten in die Biologie dieses Zelltyps erlangt werden, die sicher weiterer Untersuchungen bedürfen. In undifferenzierten MSC konnte durch differentielle Genexpressionsanalyse die Rolle des nicht kanonischen Teils des WNT Signalweges als für die Aufrechterhaltung des Stammzellstatus potentiell äußerst einflussreich ermittelt werden. Die hier diskutierten Ergebnisse zeigen beispielhaft, dass besonders mittels Genexpressionsanalyse im Hochdurchsatzverfahren wertvolle Einblicke in die komplexe Biologie der Stammzelldifferenzierung möglich sind. Als Grundlage für nachfolgende Arbeiten konnten interessante Gene ermittelt und Hypothesen zu deren Einfluss auf Stammzelleigenschaften und Differenzierungsprozesse aufgestellt werden. Um einen besseren Einblick in den Differenzierungsverlauf zu ermöglichen, könnten künftig NGS Analysen zu unterschiedlichen Differenzierungszeitpunkten durchgeführt werden. Zudem wären weitere Anstrengungen zur erfolgreichen Etablierung der chondrogenen Differenzierung zur vollständigen Analyse der Genexpression des trilinearen Differenzierungspotentials von MSC wünschenswert.