@phdthesis{Stroehle2020,
  author    = {Str{\"o}hle, Serge - Peer},
  title     = {Kultivierung von humanem Speicheldr{\"u}sengewebe in einer dreidimensionalen Polyurethanmatrix},
  doi       = {10.25972/OPUS-21688},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216887},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Bei Tumoren von Kopf und Hals kann prim{\"a}r oder adjuvant durch Bestrahlung therapiert werden. Die Folgen dieser Behandlung k{\"o}nnen Xerostomie, Karies, Infektionen, Dysphagie oder Mundgeruch sein. Diese Nebenwirkungen vermindern die Lebensqualit{\"a}t des Patienten. Unterschiedliche Behandlungsans{\"a}tze haben aufgrund von therapiebedingten Einschr{\"a}nkungen nicht den Weg in den klinischen Alltag gefunden. Eine Alternative zu den vorhandenen Behandlungsans{\"a}tzen kann das Tissue Engineering sein. Das Ziel einer Normalisierung der Speichelproduktion nach Behandlung soll durch eine implantierbare, k{\"u}nstliche Speicheldr{\"u}se erreicht werden. Kann humanes natives Speicheldr{\"u}sengewebe der Parotis auf gradientenfreiem dreidimensional aufgebauten Polyurethan wachsen und seine Funktionalit{\"a}t beibehalten? Humane Parotiszellen wurden von 20 Patienten im Alter von 42 - 90 Jahren durch Operation entnommenen und in Polystyrol-Zellkulturflaschen mit dem N{\"a}hrmedium BEGM herangez{\"u}chtet. Es erfolgte eine 2D-Zellverteilung der reinen Parotiskultur. Zur Kontrolle der Vitalit{\"a}t zwischen den Passagen wurde eine Trypan-Blau F{\"a}rbung verwendet. Als Tr{\"a}germaterial der Zellen wurde eine biokompatible, abbaubare Matrix aus ε-Polycaprolacton verarbeitet. Die {\"U}bertragung der humanen Parotiszellen wurde mit einer Kleberproteinl{\"o}sung, bestehend aus den Hauptbestandteilen Aprotinin, Fibrinogen und der Thrombinl{\"o}sung durchgef{\"u}hrt. 7,14 und 21 Tage nach Aufbringung wurde der {\"U}berstand der zeitgleich entnommenen Konstrukte zur {\"U}berpr{\"u}fung des α-Amylase konserviert. Zus{\"a}tzlich wurden an den 3 Untersuchungstagen Konstrukte f{\"u}r die Anfertigung von histologischen Schnitten, quantitativer PCR, indirekter Immunfluoreszenz und zur Elektronenmikroskopie entnommen. Zur {\"U}berpr{\"u}fung der Funktionalit{\"a}t der angez{\"u}chteten Speicheldr{\"u}senzellen wurde das Enzym α-Amylase und das Wasserkanalprotein Aquaporin 5 herangezogen. Bei der Kultivierung der humanen Speicheldr{\"u}senzellen konnte durch den Vitalit{\"a}tstest Trypan-Blau F{\"a}rbung in Kombination mit einer Neubauerz{\"a}hlkammer eine konstant hohe Anzahl an vitalen Zellen bis zur 4. Passage nachgewiesen werden. Durch die Lebend/Tot F{\"a}rbung auf FDA/EB Basis der Konstrukte {\"u}ber die Untersuchungszeit von 14 Tagen konnte keine Vermehrung von avitalen Zellen mikroskopisch festgestellt werden. Die statistische Auswertung mittels Boxplots des ELISA berechnete f{\"u}r den ersten Untersuchungstag einen Median auf niedrigem Niveau von 4,4 U/l und sank im weiteren Zeitverlauf am Untersuchungstag 21 auf die niedrigsten Median von 2,2 U/l ab. α-Amylase konnte an allen 3 Tagen mittels quantitativer PCR und indirekter Immunfluoreszenz belegt werden. Aquaporin 5 als Funktionsnachweis war in der vorliegenden Studie nicht signifikant durch quantitative PCR beweisbar. Die Rasterelektronenmikroskopie bildete adh{\"a}rente Zellen in kugeliger Form aus den besiedelten Matrices nach 7 Tagen Kultivierung ab. Durch die Transmissionselektronenmikroskopie konnten Zellen, die Zellforts{\"a}tze ausgebildet hatten nach 14 Tagen beobachtet werden. Der Versuch, histologische Schnitte auf Grundlage der Paraffineinbettung oder Kryo-Konservierung zu erzeugen, musste frustran abgeschlossen werden. Eine Kultivierung von Speicheldr{\"u}senzellen auf einer Matrix aus ε-Polycaprolacton ohne Gradienten ist eingeschr{\"a}nkt umsetzbar. Die Studie konnte zeigen, dass das Wachstum der Zellen auf konstant niedrigem Niveau {\"u}ber den Untersuchungszeitraum von 21 Tagen lag. Der Funktionsnachweis von α-Amylase auf absinkendem niedrigem Niveau sowie fehlender Best{\"a}tigung von Aquaporin 5 kann als station{\"a}re Phase des Wachstums interpretiert werden. Zur Verbesserung der Zellentwicklung sollte die besiedelte Matrix zu einem 3D-Zellwachstum anregen. Bei sequenziell entstehender Polarit{\"a}t der Zellen k{\"a}me es zu einer Verbesserung der Vitalit{\"a}t sowie der vermehrten Ausbildung von α-Amylase und Aquaporin 5. Dies k{\"o}nnte in einer Kombination der Zellkultur aus Parotiszellen mit Kokulturen aus humanen Myoepithelzellen und Parenchymzellen erreicht werden. Sehr gute Ergebnisse des Zellwachstums und der Zellfunktion konnten aktuell in anderen Studien auf der Tr{\"a}gersubstanz Matrigel oder durch Rebesiedelung von dezellularisierten Organen beobachtet werden.},
  subject      = {Ohrspeicheldr{\"u}se},
  language  = {de}
}
@article{Scheer1982,
  author    = {Scheer, Ulrich},
  title     = {A novel type of chromatin organization in lampbrush chromosomes of Pleurodeles waltlii: visualization of clusters of tandemly repeated, very short transcriptional units},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-41087},
  year      = {1982},
  abstract  = {A novel chromatin configuration is described in lampbrush chromosomes of Pleurodeles waltlii oocytes which is different from transcriptionally inactive chromatin as weil as from the various forms of transcribed chromatin hitherto described. This novel type of chromatin is not arranged in Christmas tree-Iike configurations of densely packed lateral ribonucleoprotein (RNP) fibriIs but is characterized by a periodic alternating pattern of thick and thin regions which occur in clusters 01 some 10,000 repeats. Each thickened unit with an average length of 45 nm contains two c10sely spaced particles, the putative RNA polymerases, and each thickened unit is separated from the next one by a beaded chromatin spacer with a length of about 80 nm. This chromatin spacer contains on average two particles of approximately 14 nm in diameter, assumed to be nucleosomes. The thickened regions are interpreted to represent short transcriptional units containing approximately 130 base pairs of DNA which are separated from each other by nontranscribed spacers of 240-400 base pairs of DNA. The possibility is discussed that these transcriptional units represent 5S rRNA or tRNA genes.},
  language  = {en}
}
@article{Scheer1972,
  author    = {Scheer, Ulrich},
  title     = {The ultrastructure of the nuclear envelope of amphibian ooctyes: IV. On the chemical nature of the nuclear pore complex material},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-39500},
  year      = {1972},
  abstract  = {In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from mature Xenopus laevis oocytes were manually fractioned into nucleo· plasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60- 90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9\% of the total nuclear RNA of the mature Xenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41 X 10- 16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270 X 10- 15 g/fJ-3) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules. Additionally, the results of the chemical analyses as well as of the 3H-actinomycin D autoradiography and of the nucleoprotein staining method of Bernhard (1969) speak against the occurence of considerable amounts of DNA in the nuclear pore complex structures.},
  language  = {en}
}
@article{FrankeKartenbeckKrienetal.1972,
  author    = {Franke, Werner W. and Kartenbeck, J{\"u}rgen and Krien, S. and VanderWoude, W. J. and Scheer, Ulrich and Morr{\´e}, D. J.},
  title     = {Inter- and intracisternal elements of the Golgi apparatus: A system of membrane-to-membrane cross-links},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-39514},
  year      = {1972},
  abstract  = {Electron opaque cross-bridge structures span the inter- and intracisternal spaces and provide membrane-to-membrane connections between adjacent cisternae of dictyosomes of pollen tubes of Clivia and Lilium. Additionally, the classic intercisternal rods, characteristic of intercisternal regions near the maturing face of dictyosomes, are connected with the adjacent membranes through similar cross-bridge elements. We suggest that these structural links are responsible for maintaining the flattened appearance of the central parts of Golgi apparatus cisternae as well as for the coherence of cisternae within the stack. Observations on other plant (e.g. microsporocytes of Canna) and animal cells (e.g. rodent liver and hepatoma cells, newt spermatocytes) show that such an array of membrane cross-links is a universal feature of Golgi apparatus architecture. The cross-bridges appear as part of the complex "zone of exclusion" which surrounds dictyosomes, entire Golgi apparatus and Golgi apparatus equivalents in a variety of cell types.},
  language  = {en}
}
@phdthesis{Elsner2022,
  author    = {Elsner, Clara Dorothea},
  title     = {Ultrastructural analysis of biogenesis and release of endothelial extracellular vesicles},
  doi       = {10.25972/OPUS-28852},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-288526},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Extracellular vesicle (EV)-mediated intercellular communication through exosomes, microvesicles (MVs) and apoptotic bodies has been shown to be implicated in various physiological as well as pathological processes such as the development and progression of atherosclerosis. While the cellular machinery controlling EV formation and composition has been studied extensively, little is known about the underlying morphological processes. This study focuses on a detailed ultrastructural analysis of the different steps of EV formation and release in Myocardial Endothelial (MyEnd) and Aortic Endothelial (AoEnd) cells cultured under serum starvation and inflammatory stimulation with TNF-α. Detailed morphological analyses were conducted applying and comparing different high- resolution light and electron microscopic methods. In this study, we could depict all steps of MV biogenesis named in literature. However, during the study of exosome biogenesis, we discovered a yet undescribed process: Instead of a direct fusion with the plasma membrane, multivesicular bodies were incorporated into a new distinct cellular compartment bound by fenestrated endothelium first. This may present a novel step in exosome biogenesis and warrants further study. Regarding the conditions of cell cultivation, we observed that the commonly used serum starvation causes MyEnd cells, but not AoEnd cells, to enter apoptosis after 48 hours. When preparing functional EV studies, we therefore recommend assessing the morphological condition of the serum-starved cells at different cultivation points first. When evaluating MV production, a statistical analysis showed that the more time AoEnd cells spent in cultivation under serum starvation, the higher the percentage of MV producing cells. However, additional TNF-α stimulation induced a significantly higher MV production than serum starvation alone. Lastly, our results show that TNF-α stimulation of AoEnd cells in vitro leads to the upregulation of CD44, an adhesion molecule critical in the early stages of atherosclerosis. CD44 was then depicted on the surface of generated MVs and exosomes. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells. This could be a novel mechanism by which MVs contribute to the development and progression of atherosclerotic disease and should be clarified by further studies.},
  subject      = {Vesikel},
  language  = {en}
}