Myofibrilläre Myopathien sind Erkrankungen der Muskulatur mit teilweiser Beteiligung der Herzmuskulatur, die oft einen frühzeitigen Tod zur Folge haben. Dabei macht sich das Leitsymptom einer Myofibrillären Myopathie, eine progressive Muskelschwäche, typischerweise in der vierten Lebensdekade bemerkbar.<br /> Die Myofibrilläre Myopathie, die durch einen spontanen Basenaustausch von Prolin gegen Leucin an Position 209 im BAG3-Gen verursacht und als P209L-BAG3opathie bezeichnet wird, ist hingegen durch einen frühen Ausbruch und einen sehr progressiven Krankheitsverlauf, unter anderem mit einer selten vorkommenden schweren restriktiven Kardiomyopathie, charakterisiert. Das Cochaperon BAG3, welches in Zellen der quergestreiften Muskulatur an die Z-Scheiben lokalisiert, trägt als Bestandteil des CASA-Komplexes dazu bei, dass verbrauchte Z-Scheibenproteine dem Lysosom zugeführt und dort degradiert werden.<br /> In elektronenmikroskopischen Aufnahmen betroffener Muskeln kann eine Disintegration von Z-Scheiben und eine Aggregatbildung durch Akkumulation fragmentierter Filamente, Mitochondrien und Vesikel beobachtet werden.<br /> Warum es zu den diesen pathologischen Veränderungen und der Bildung von Aggregaten kommt und wie diese mechanistisch zu erklären sind, ist bis heute nicht verstanden. Deshalb sind die Aufklärung der Mechanismen, die dieser Krankheit zu Grunde liegen, und die darauf basierende Erforschung möglicher Behandlungsmethoden von großem Interesse.<br /> In dieser Arbeit wurde daher die Generierung und Charakterisierung unterschiedlicher transgener BAG3^P209L-eGFP-mES-Zelllinien und Mausmodellen etabliert. Dazu erfolgte die Herstellung unterschiedlicher BAG3^P209L-Konstrukte, die zur Visualisierung der Lokalisation des Proteins an einen eGFP-Reporter gekoppelt (BAG3^P209L-eGFP) und untersucht wurde. Hierbei war die Expression einerseits unter Kontrolle des Kardiomyozyten-spezifischen αMHC-Promotors, was eine Untersuchung des Einflusses der BAG3^P209L-Mutation auf die Herzmuskulatur und die daraus resultierenden restriktive Kardiomyopathie erlaubt. Andererseits sollte ein weiteres transgenes Mausmodell mit einer induzierbaren ubiquitären BAG3^P209L-eGFP-Expression (β-Aktin-Promotor) generiert werden, um die Expression des mutierten Cochaperons auch in der Skelettmuskulatur und in anderen Organen untersuchen zu können. Auch den Beginn der Krankheit und die Progression der Myofibrillären Myopathie verfolgen zu können, war hierbei von großem Nutzen. Bei diesem Modell kann die BAG3^P209L-eGFP-Expression in vitro durch Applikation einer Cre-Rekombinase oder in vivo durch Einkreuzen eines transgenen Cre-Männchens induziert werden.<br /> Während die Expression von BAG3^P209L-eGFP in den transgenen Tieren heterogen und nicht hoch genug war, um eine Beeinträchtigung der Entwicklung bzw. veränderte Herzfunktion herbei zu führen, konnten der humanen Pathologie entsprechend in über der Hälfte der BAG3^P209L-eGFP-exprimierenden Kardiomyozyten ein Rückgang der Z-Scheibenstruktur und eine BAG3^P209L-eGFP- und α-Aktinin-Aggregatbildung detektiert werden. Die degenerativen Muskel-veränderungen und die Bildung der BAG3^P209L-eGFP-Aggregate stand dabei in Zusammenhang mit einer deutlich verringerten Mobilität des BAG3^P209L-eGFP-Proteins. Diese Kardiomyozyten zeigten ebenso eine Dislokalisation der Z-Scheibenproteine Myopodin und Filamin C, Interaktionspartner von BAG3 und Substrat im CASA-Mechanismus, was auf eine Beeinträchtigung des CASA-Mechanismus hinweist. Ebenso konnte Desmin, das in der quergestreiften Muskulatur vorherrschende Intermediärfilament, in den Aggregaten nachgewiesen werden, was zeigte, dass die Expression von BAG3^P209L-eGFP in Kardiomyozyten zu einer sekundären Desminopathie führte. Im Einklang mit der beobachteten Auflösung des Zytoskeletts stand dabei die schwächere Titinexpression in den Bereichen, in denen BAG3^P209L-eGFP-Aggregaten vorlagen.<br /> Durch Verwendung des CAG-Promotors in einem weiteren transgenen Mausmodell konnte eine konditionale BAG3^P209L-eGFP-Expression erreicht werden. Dies bietet den Vorteil den Beginn der Erkrankung zu evaluieren und erlaubt die Untersuchung der BAG3^P209L-eGFP-Expression insbesondere in der Skelettmuskulatur und anderen Organen.<br /> Erste Untersuchungen von Organen eines embryonalen Entwicklungsstadiums zeigten eine ubiquitäre Expression des Proteins, die besonders in Zellen der quergestreiften Muskulatur sehr stark war....

<p>The mechanical organization of biological tissue is crucial to the load-transmitting capacity of our bodies, and follows a hierarchical architecture that macroscopically results in organ’s formation and function. At the base of such a tightly regulated structure we find single cells, whose mechanical properties are decisive in shaping their interaction with the surrounding environment.<br /> Developing a fundamental understanding of this interplay over a wide range of length scales is essential to reach the deep working knowledge of the biomechanics of multicellular systems required for tissue engineering, surface design and nanomedicine. <br /> In this work, the mechanical properties of epithelial model systems were analyzed at different organizational scales (namely, from single cells to microtissue) by means of atomic force microscopy (AFM) micro- and nano-indentation experiments. Despite the intrinsic difficulty in characterizing soft and heterogeneous biological samples in terms of mechanical response, this technique still offers an exciting possibility to quantitatively probe their viscoelastic behavior. <br /> At first, a murine epidermal cell line completely devoid of keratin intermediate filaments (knock-out keratinocytes) was compared to its wild type counterpart in order to assess the role played by this cytoskeletal component in conferring mechanical stability to single cells. Then, cellular monolayers were analyzed, to validate the relevance of our findings also in a more physiological context. Despite its presence in organs such as skin and nails, which obviously serve a barrier function, the mechanical role of keratin in deeper tissue remained controversial for a long time. Reconstructed keratin polymer gels in fact display properties resemblant of viscoelastic solids; in vivo, the networks are formed of bundles that are relatively sparse and show lower connectivity than other cytoskeletal components. This fact, together with the low values of bending stiffness and extremely high extensibility reported for these filaments, would suggest that keratin networks confer resilience and elasticity to cells, rather than a scaffolding function against compressive stress. Our results though clearly pointed at a substantial softening of keratin-lacking cells, with elasticity moduli differences of 25% to 35% between wild type and knock-out according to the cellular region probed. The presented data represent the first proof of this effect on the single cell level. Validation of this result further came from the observation that the difference could be partially suppressed by reintroducing a single keratin protein in the mutant cells. <br /> In the second part of this work, a three-dimensional cell culture system mimicking the elementary unit of a human breast gland was analyzed in terms of its biomechanical and permeation properties. The cell line used for this purpose (MCF10A), when grown in an extracellular matrix-resembling environment, can develop into growth-arrested acinar structures which follow the same substantial maturation steps of a human breast gland; cells organize according to an apico-basal polarization scheme, secrete a dense matrix of cross-linked extracellular matrix proteins to surround them (the so called basement membrane) and finally develop a hollow lumen necessary, in vivo, for milk production and secretion. <br /> The centrality of breast gland tissue in a context of cancer research cannot be overstated: alveolar units are the hotspot for tumor formation, and as such have been the focus of much attention in the past years. Relatively little effort, though, has been dedicated to understanding the mechanical interplay of healthy breast gland microtissues with their surrounding environment, despite the fact that one of the hallmarks of cancer progression is a set of strong alterations in the mechanical phenotype of aberrant cells. <br /> Here, we offer an experimental analysis of the mechanical properties of healthy 3D acinar structures at different developmental stages, and briefly compare them with those of invasive microtissues. The application of different hyperelastic models to the interpretation of nanoindentation experiments is discussed, along with a tentative clarification of some of controversies arising during AFM data analysis. <br /> Additionally, a characterization of isolated basement membranes performed by means of atomic force microscopy imaging, scanning electron microscopy and superresolution light microscopy is reported; experimental evidence suggests that basement membranes act as fundamentally elastic materials whose thickness and structural stability change throughout the different developmental stages. To complement this biomechanical analysis, we investigated the acinar permeation properties; in short, data elucidate that the basement membrane acts as a passive diffusion barrier with a size-selectivity threshold for the retardation of macromolecular permeation of about 40 kDa and a pore size of at least 9 nm. At the same time, it offers a fundamental mechanical shielding function, reaching elastic modulus values of up to about 400 kPa in the fully matured state. Taken together, the presented data underline how intra- and extra-cellular polymer networks serve a crucial function in defining the mechanical properties of epithelial tissue.</p>...

In the past decades astrocytes have been demonstrated to play an important role in physiological brain processes but also neurodegenrative diseases. Their characteristic morphology allows them to closely contact neurons. ...

Massive stars have a strong influence on their environment by chemical enrichment of the surrounding interstellar medium due to strong stellar winds. The influence of mass loss and rotation on the main-sequence evolution ...

Natural products are most important in drug discovery and therapy, e.g. more than 80% of antibiotics are derived from microorganisms. Antimicrobial resistance is a global threat and thus an urgent need for new antimicrobial ...

TsdA enzymes are a phylogenetically widespread family of periplasmic <i>c</i>-type diheme cytochromes which catalyse both thiosulfate oxidation and tetrathionate reduction. The reaction directionality varies between enzymes ...

Combinations of different medicinal products are widely use in the medical practice and are in fact essential for the treatment of several diseases. The existing regulation for approval of medicinal products offers only a ...

The standard model of cosmology, founded on Einstein’s theory of gravity and on the Cosmological Principle (CP), has been understood as being successful in providing explanations for many observations so far. However, it ...

Metalloproteins are a class of proteins which contain metal ions in their active site. If these metal ions carry unpaired electrons, EPR spectroscopy can be used to identify and characterize these ions or even localize ...

Efficiently analyzing large amounts of high dimensional data derived from the simulation of industrial products is a challenge that is confronted in this thesis. For this purpose, simulations are considered as abstract ...