Universiẗat, Diss., 2003--Düsseldorf.

In contrast to mammals, some fish and amphibians have retained the ability to regenerate complex body structures or organs, such as the limb, the tail, the eye lens or even parts of the heart. One major difference in the response to injury is the appearance of a mesenchymal growth zone or blastema in these regenerative species instead of the scarring seen in mammals. This blastema is thought to largely derive from the dedifferentiation of various functional cell types, such as skeletal muscle, skin and cartilage. In the case of multinucleated skeletal muscle fibres, cell cycle re-entry into S-phase as well as fragmentation into mononucleated progenitors is observed both in vitro and in vivo. In order to identify molecules that initiate dedifferentiation of cells at the wound site in amphibians we have established a cellular assay with a cultured newt myogenic cell line. Using this assay we have found a serum activity that stimulates cell cycle re-entry in differentiated multinucleated newt myotubes. The activity is present in serum of all mammalian species tested so far and, interestingly, thrombin proteolysis amplifies the activity from both serum and plasma. We think this serum factor provides a link between wounding and regeneration and its identification will be a key step in understanding the remarkable differences in wound healing between mammals and amphibians. In the course of this PhD thesis we have characterized the serum factor as a thermo-labile, pH- and proteinase K-sensitive, high molecular weight protein that is resistant to denaturing conditions such as SDS, urea or organic solvents. Surprisingly, under denaturing conditions the activity behaves as a low molecular weight protein that displays charge heterogeneity on isoelectric focusing. Using these characteristics of the serum factor we have performed a systematic investigation of commonly used protein chromatography modes and separation techniques to develop a successful purification procedure. After four column chromatography steps -- cation exchange, hydrophobic interaction, heparin affinity and size exclusion chromatography under denaturing conditions -- we have achieved a 2,000-fold purification starting from a commercially available Crude Bovine Thrombin preparation. This represents about 40,000-fold purification over bovine serum. Silver stained gels of the most purified fractions revealed ten major protein bands. In order to finally identify the cell cycle re-entry factor, we are currently analyzing the purification by quantitative mass spectrometry by correlating the abundance of tryptic peptides with activity in sequential fractions across a chromatography run.

Urodele amphibians have the remarkable ability to re-grow lost body parts. This regenerative response after injury in urodeles involves dedifferentiation of fully differentiated cells into proliferative cells. One well-studied example of this is the dedifferentiation of multinucleated muscle cells into mononucleate cells resembling their precursors, the myoblasts. To form these mononucleate cells the differentiated myotubes in vivo must re-enter and complete the cell cycle; they again proliferate and produce progeny. A key question is what factors induce the myotubes to re-enter the cell cycle and proliferate. Early events of cell cycle re-entry can be studied in the A1 cell line, a myogenic cell line isolated from the Notophthalmus viridescens hindlimb, which traverses cell cycle until G2 in response to serum. In particular, it was found that thrombin cleavage induces a factor in serum of all animals tested so far to promote S phase re-entry in A1 myotubes. We have used this S phase re-entry of the A1 cell line to purify the serum activity and developed a 5-step purification protocol that enriches the activity almost 2 000 fold over the starting material, or 40 000 fold over serum. To conveniently produce and test potential candidates for their ability to induce S phase re-entry in A1 myotubes, we also developed an overexpression- and purification system for emerging candidates. Candidates were then tested for this activity with or without prior incubation with thrombin. We identified Bmp proteins as the first pure molecules that were found in fractions across the purification of the activity and that could also induce cell cycle re-entry in a dose-dependent manner when recombinantly added to the A1 myotubes. Furthermore, this response could be blocked in a dose-dependent manner by the known bmp-inhibitor noggin. Finally, we showed that inhibition of Bmp signaling in vivo causes defects in axolotl tail regeneration.

Sterine sind essentielle Komponenten der Zellmembran, deren Konzentration und Lokalisierung genau kontrolliert wird. Die Hefe Saccharomyces cerevisiae ist ein fakultativ anaerober Organismus, der in Abwesenheit von Sauerstoff auxotroph für Sterine wird. Die Proteine Aus1p und Pdr11p gehören zur Familie der ABC Proteine und spielen eine wichtige Rolle in diesem Prozess, da die gleichzeitige Deletion beider Protein die Aufnahme von Sterinen unter anaeroben Wachstumsbedingungen blockiert.In dieser Arbeit wurde das Gen AUS1 in voller Länge kloniert. Methoden für die Extraktion und Reinigung dieses Transporters wurden entwickelt, damit dieser detailliert charakterisiert werden kann. Mit Hilfe von Detergenzien wurde das Protein löslich gemacht und zeigte ATP-Bindung und -Hydrolyse. Die ATP-Hydrolyse konnte durch die Mutation eines konservierten Lysins zu Methionin im Walker A Motif verhindert. Genauso konnte die ATP-Hydrolyse auch durch klassische Inhibitoren von ABC Transportern inhibiert werden. Nach der Rekonstitution von Aus1p in Proteoliposomen wurde die ATPase Aktivität spezifisch durch Phosphatidylserin in einer stereoselektiven Weise stimuliert.Zusätzlich konnte gezeigt werden, dass Änderungen im zellulären PS Spiegel die Aus1p-abhängige Aufnahme von Sterin an die Membran beeinflussen. Diese Ergebnisse schlagen eine für die Aktivität des Transporters wichtige, direkte Interaktion zwischen Aus1p und PS vor.Da es sich bei der Aufnahme von Sterin um einen komplexen Prozess handelt, könnten Komponenten exisitieren, die mit Aus1p interagieren. Der Hefestamm, der die Immunpräzipitation von Aus1p mit seinem Interaktionspartner ermöglicht, wurde erzeugt und der Einfluß von Mannoproteinen auf Sterinaufnahme wurde getestet. Außerdem wurde eine Methode entwickelt, mit der Aus1p in Giant Unilamellar Vesicles rekonstituiert werden kann. Mit diesen Liposomen kann das Verhalten und die Aktivität von Aus1p in Membranen mit einer komplexen Lipidzusammensetzung untersucht werden. / Sterols are essential components of cellular membranes and their concentration and localization are tightly controlled. Saccharomyces cerevisiae is a facultative anaerobic organism which becomes auxotrophic for sterols in the absence of oxygen. However, the precise mechanism of sterol uptake remains to be revealed. Two proteins belonging to ABC protein family, Aus1p and Pdr11p were proposed to play a critical role in this process as simultaneous deletion of both of them blocks sterol uptake under anaerobiosis. In the present work, the full length AUS1 gene was cloned. An extraction and purification procedures were then developed to allow for detailed characterization of the transporter. The detergent solubilized protein was shown to bind and hydrolyse ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1p was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. Furthermore, it was demonstrated that Aus1p-dependent sterol uptake, but not Aus1p expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1p and PS which is critical for the activity of the transporter. Because of the complexity of sterol incorporation process efforts were made to identify additional components of the sterol uptake machinery that interact with Aus1p protein. The yeast strain allowing for immunopercipitation of Aus1p with its interaction partners was generated and previously proposed influence of mannoproteins on the sterol uptake was tested. Additionally, method was developed to reconstitute Aus1p protein into Giant Unilamellar Vesicles. These liposomes can be used further for testing of the behaviour and activity of Aus1p in the membranes with complex lipid composition.

Durch die Kombination von SKY-, Array-CGH-, und Expressionsnanalysen wurden ausgewählte MM-Zelllinien auf Aberrationen hin untersucht und diese genauer analysiert. 32 Myelom-Patienten wurden mittels Array-CGH-Analyse untersucht und aufgrund ihrer Aberrationen und der klinischen Daten durch eine anschließende Clusteranalyse in 4 Subgruppen unterteilt.

Durch die Kombination von SKY-, Array-CGH-, und Expressionsnanalysen wurden ausgewählte MM-Zelllinien auf Aberrationen hin untersucht und diese genauer analysiert. 32 Myelom-Patienten wurden mittels Array-CGH-Analyse untersucht und aufgrund ihrer Aberrationen und der klinischen Daten durch eine anschließende Clusteranalyse in 4 Subgruppen unterteilt.

Urodele amphibians have the remarkable ability to re-grow lost body parts. This regenerative response after injury in urodeles involves dedifferentiation of fully differentiated cells into proliferative cells. One well-studied example of this is the dedifferentiation of multinucleated muscle cells into mononucleate cells resembling their precursors, the myoblasts. To form these mononucleate cells the differentiated myotubes in vivo must re-enter and complete the cell cycle; they again proliferate and produce progeny. A key question is what factors induce the myotubes to re-enter the cell cycle and proliferate. Early events of cell cycle re-entry can be studied in the A1 cell line, a myogenic cell line isolated from the Notophthalmus viridescens hindlimb, which traverses cell cycle until G2 in response to serum. In particular, it was found that thrombin cleavage induces a factor in serum of all animals tested so far to promote S phase re-entry in A1 myotubes. We have used this S phase re-entry of the A1 cell line to purify the serum activity and developed a 5-step purification protocol that enriches the activity almost 2 000 fold over the starting material, or 40 000 fold over serum. To conveniently produce and test potential candidates for their ability to induce S phase re-entry in A1 myotubes, we also developed an overexpression- and purification system for emerging candidates. Candidates were then tested for this activity with or without prior incubation with thrombin. We identified Bmp proteins as the first pure molecules that were found in fractions across the purification of the activity and that could also induce cell cycle re-entry in a dose-dependent manner when recombinantly added to the A1 myotubes. Furthermore, this response could be blocked in a dose-dependent manner by the known bmp-inhibitor noggin. Finally, we showed that inhibition of Bmp signaling in vivo causes defects in axolotl tail regeneration.

In contrast to mammals, some fish and amphibians have retained the ability to regenerate complex body structures or organs, such as the limb, the tail, the eye lens or even parts of the heart. One major difference in the response to injury is the appearance of a mesenchymal growth zone or blastema in these regenerative species instead of the scarring seen in mammals. This blastema is thought to largely derive from the dedifferentiation of various functional cell types, such as skeletal muscle, skin and cartilage. In the case of multinucleated skeletal muscle fibres, cell cycle re-entry into S-phase as well as fragmentation into mononucleated progenitors is observed both in vitro and in vivo. In order to identify molecules that initiate dedifferentiation of cells at the wound site in amphibians we have established a cellular assay with a cultured newt myogenic cell line. Using this assay we have found a serum activity that stimulates cell cycle re-entry in differentiated multinucleated newt myotubes. The activity is present in serum of all mammalian species tested so far and, interestingly, thrombin proteolysis amplifies the activity from both serum and plasma. We think this serum factor provides a link between wounding and regeneration and its identification will be a key step in understanding the remarkable differences in wound healing between mammals and amphibians. In the course of this PhD thesis we have characterized the serum factor as a thermo-labile, pH- and proteinase K-sensitive, high molecular weight protein that is resistant to denaturing conditions such as SDS, urea or organic solvents. Surprisingly, under denaturing conditions the activity behaves as a low molecular weight protein that displays charge heterogeneity on isoelectric focusing. Using these characteristics of the serum factor we have performed a systematic investigation of commonly used protein chromatography modes and separation techniques to develop a successful purification procedure. After four column chromatography steps -- cation exchange, hydrophobic interaction, heparin affinity and size exclusion chromatography under denaturing conditions -- we have achieved a 2,000-fold purification starting from a commercially available Crude Bovine Thrombin preparation. This represents about 40,000-fold purification over bovine serum. Silver stained gels of the most purified fractions revealed ten major protein bands. In order to finally identify the cell cycle re-entry factor, we are currently analyzing the purification by quantitative mass spectrometry by correlating the abundance of tryptic peptides with activity in sequential fractions across a chromatography run.

No description available.