Syftet med arbetet var att utveckla en metod för att rena fram cytokrom c-Id1 ur periplasma från Ideonella dechloratans. Inledningsvis undersöktes proteinets stabilitet vid låga pH. Flera olika kromatografiska metoder provades under arbetets gång. Till de inledande experimenten användes flow-through fraktioner från rening av annat protein som startmaterial. Senare försök gjordes på periplasma från I. dechloratans. Stabilitetsundersökningen gjordes genom att proteinlösningens spektrum registrerades. Lösningen reducerades sedan med ditionit och ett nytt spektrum registrerades. Ett differensspektrum beräknades och absorbansmaximum vid 550 nm användes som mått på mängden cytokrom c-Id1. Undersökningen visade att proteinet är stabilt vid pH ner till 3.0. Första försöken med respektive kromatografisk metod, förutom gelfiltrering, gick ut på att undersöka om proteinet kunde adsorberas till kolonnen eller inte. När proteinet adsorberats till kolonnen ändrades målet med försöken till att försöka eluera det så rent som möjligt. Den slutliga reningsmetoden innehåller tre kromatografiska steg, varav ett behöver optimeras ytterligare för att ge rent protein. Första steget i reningen är katjonbyteskromatografi. Det andra stegetär hydrofob interaktionskromatografi och det sista steget är anjonbyteskromatografi. Efter reningen fanns små mängder föroreningar kvar. Två oönskade proteiner, båda i liten mängd fanns kvar i provet. För att få proteinet helt rent krävs ytterligare optimering av reningsmetoden. Störst potential till förbättring finns troligen i steget med anjonbytaren. Misstankar om att proteinet lätt dimeriserar eller reagerar på annat vis under lagring har funnits under hela arbetet. Resultat från bland annat gelfiltrering har indikerat att så är fallet. Arbetet har inte gettsvar på om cytokrom c-Id1 förändras med tiden eller inte. Bestämning av totala mängden protein samt mängden cytokrom c-Id1 efter reningen gjordes. Dessa analyser visar att 69 μg protein, varav 36 μg cytokrom c-Id1 återstod efter rening från en odling på fyra liter. Halten cytokrom c-Id1 var alltså 52 %. / The aim of this study was to develop a method to purify cytochrome c-Id1 from periplasmic extract of Ideonella dechloratans. First the stability of the protein at low pH was investigated. Several different chromatographic methods were tested during the study. In the first experiments the starting material was flow through fractions from purification of another protein. Later experiments were performed with periplasmic extract from I. dechloratans. The stability determination was made by recording a spectrum from the protein solution. Proteins were then reduced, by dithionite, and a new spectrum was recorded. A difference spectrum was calculated and the absorbance at 550 nm was used as a measure of the amount of cytochrome c-Id1. The investigation showed that the protein is stable at pH 3.0 and higher. The first experiments with each chromatographic method, except gel filtration, were made in order to see whether the protein could be adsorbed to the column or not. When the protein had been adsorbed to the column the target of the experiments changed. The target was then to elude the protein as pure as possible. The resulting purification method contains three chromatographic steps. One of these needs to be further optimized in order to give pure protein. The first step is cation exchange chromatography. The second step is hydrophobic interaction chromatography and the last step is anion exchange chromatography. After purification with this method, small amounts of contaminants were still present. Two unwanted proteins are present, in small amounts. In order to get the protein completely pure further optimization of the method is required. The biggest optimization potential is probably in the anion exchange chromatography step. During this work there have been some indications that the protein easily dimerizes or reacts in some other way upon storage. Results from for example gel filtration have indicated that so is the case. This work has not given an answer to whether cytochrome c-Id1 changes upon storage or not. The total amount of protein as well as the amount of cytochrome c-Id1 after the purification was determined. The analyses showed that the total amount of protein was 69 μg and the amount of cytochrome c-Id1 was 36 μg from four liters of culture. This means that the cytochrome c-Id1 content afterthe purification was 52 %.

The transition from pluripotency to differentiation is a key event in the life of all complex multicellular organisms. In the development of the mouse, the pluripotent epiblast undergoes gastrulation and gives rise to three multipotent germ layers, which will in turn form the tissues of the adult body. The events leading up to gastrulation have been extensively studied in vivo in developing embryos, and modelled in vitro making use of embryonic stem (ES) cells. Bone morphogenic protein (BMP) signalling plays a key role in these processes. BMP can in fact maintain ES cells in a self-renewing state by inhibiting their differentiation into neural ectoderm, whilst at the same time being required for the specification of mesoderm in the developing embryo (Winnier et al. 1995, Ying et al. 2003a). A key intracellular target of BMP is the transcriptional regulator Id1, which can recapitulate the effects of BMP in the preservation of ES cell pluripotency and in the inhibition of neural specification from pluripotent cells (Ying et al. 2003a). This thesis will focus on understanding the roles of this molecule in the early decisions affecting the transition from pluripotency to differentiation. In particular, I aim to study the expression pattern of Id1 in cultures of pluripotent cells, and to clarify which extracellular and intracellular molecules regulate the expression of the factor; I aim to understand how forced Id1 expression inhibits the differentiation of pluripotent cells, and whether Id1 may play a similar role in the regulation of the asynchronous exit from pluripotency observed in differentiating wild-type cells; finally, I aim to characterise the expression pattern of Id1 in the early stages of post-implantation development at the single-cell resolution, and to understand how the expression of the molecule correlates with the previously characterised expression patterns of key signalling molecules and transcription factors. The generation of a reporter ES cell line expressing the yellow fluorescent protein Venus fused to the C-terminus of Id1 allowed me to assess the expression of the factor in culture on a single-cell basis, making use of immunofluorescence and flow cytometry. I observed that expression of Id1 is reliant on active BMP signalling and low Activin/Nodal signalling, and I characterised the combinatory effects of the two pathways on Id1 expression. Furthermore, I demonstrated that high Nanog expression is incompatible with high Id1 expression in ES cell cultured in the presence of LIF and serum, which raises the possibility that Nanog may be affecting the expression of Id1 in vivo, both in pre-implantation and in post-implantation embryos. I generated ES cell lines overexpressing Id1 and observed that the factor inhibits differentiation of pluripotent cells into neural ectoderm by delaying their exit from a post-implantation epiblast-like pluripotent state, and ultimately favouring mesodermal specification. This suggests that Id1 is acting at a specific stage of differentiation and that the differentiation process itself is following a similar developmental pathway to what is observed in the peri-gastrulation stage embryo. I performed single-cell transcriptional analysis on differentiating wild-type ES cells and observed that Id1 is not expressed at an appropriate point in time to affect the asynchronous the exit from pluripotency observed in neural adherent monolayer differentiation, which suggests that other factors must be responsible for this phenomenon. Finally, I addressed the expression pattern of Id1 protein in the embryonic tissue of gastrulating mouse embryos by imaging chimaeric embryos generated using the Id1- Venus reporter ES cells. I observed that Id1 is expressed in the proximal regions of streak stage embryos; in the epiblast and migrating mesendoderm of bud stage embryos; in cardiac, lateral and allantoic mesoderm and in foregut endoderm in headfold stage embryos. These expression patterns fit with the reported expression of BMP molecules at these stages of development, and suggest that Id1 expression is primarily dependent on BMP expression in early post-implantation embryos. However, I also observed Id1 expression in a ring of cells surrounding the node in headfold stage embryos, a previously uncharacterised expression pattern not directly attributable to BMP expression. This raises the intriguing question of what is regulating Id1 expression and what roles Id1 may be playing in this key embryonic structure.

Für die Progression des Wachstums maligner Tumoren und ihre Metastasierung ist die Angiogenese, die Bildung neuer Blutgefäße aus bereits existierenden, eine essentielle Voraussetzung. In dieser Arbeit konnte gezeigt werden, dass die proangiogenen Transkriptionsfaktoren c-Jun, Id1 und Id3 in ihrer Stabilität gegenüber dem Ubiquitin/26S-Proteasom-System durch das COP9-Signalosom (CSN) kontrolliert werden. Dieses bildet einen multimeren Proteinkomplex, der deutliche Homologien mit dem Lid-Subkomplex des 26S-Proteasoms aufweist. Sowohl c-Jun als auch Id3 binden an die Untereinheit CSN5. Id3 interagiert zusätzlich mit CSN7. Rekombinantes c-Jun, ein bekanntes Substrat der CSN-assoziierten Kinasen CK2 und PKD, wird durch Curcumin, einen Hemmstoff dieser Kinasen, deutlich destabilisiert. Daneben induziert Curcumin hochmolekulare Formen von c-Jun, bei denen es sich höchstwahrscheinlich um Ubiquitin-Konjugate handelt. Ferner beschleunigt Curcumin, ebenso wie die CK2- und PKD-Inhibitoren Emdodin, DRB und Resveratrol, in HeLa-Zellen den proteasomalen Abbau von c-Jun. Die c-Jun-abhängige Produktion von VEGF wird durch alle vier Kinase-Hemmstoffe signifikant reduziert. Verstärkt wird dieser Effekt noch durch den proteasomalen Inhibitor MG-132. Id3 wird nicht von den CSN-assoziierten Kinasen phosphoryliert. Allerdings hemmt es in einem Kinase-Assay die Phosphorylierung von c-Jun, ICSBP und CSN2. Curcumin und Emodin regen in HeLa-Zellen die Ubiquitinierung und den proteasomalen Abbau von Id3 an. Die Proteolyse von Id1 wird in HeLa-Zellen ebenfalls in Anwesenheit dieser beiden Hemmstoffe stimuliert. Mittels Kotransfektion von Id3 und His-markiertem Ubiquitin konnte eine verstärkte Ubiquitinierung von Id3 in Gegenwart von Curcumin direkt nachgewiesen werden. Außerdem wird Id3 durch die Überexpression von CSN2 stabilisiert. Auf diesen Daten basiert die Schlussfolgerung, dass die CSN-abhängige Phosphorylierung den Abbau von c-Jun und der beiden Id-Proteine über das Ubiquitin/26S-Proteasom-System inhibiert und dadurch ein interessantes neues Ziel einer antiangiogenen Tumortherapie repräsentiert. / Angiogenesis, the formation of new blood vessels from the existing vasculature, is a prerequisite for the progression of solid tumor growth and metastasis. In this study it is shown that the COP9 signalosome (CSN) regulates the stability of the angiogenic transcription factors c-Jun, Id1 and Id3 towards the ubiquitin/26S proteasome system. The COP9 signalosome constitutes a multimeric protein complex that shares sequence homology with the 26S proteasome lid complex. Both c-Jun and Id3 physically interact with the CSN subunit CSN5. In addition, Id3 can bind to CSN7. Recombinant c-Jun, a substrate of the CSN-associated kinases CK2 und PKD, is destabilized by curcumin, an inhibitor of these two kinases. Furthermore, curcumin induces high molecular weight c-Jun species, most likely ubiquitin conjugates. All tested inhibitors of the CK2 and PKD, emodin, DRB, resveratrol, as well as curcumin accelerate the degradation of c-Jun by the 26S proteasome in HeLa cells. The c-Jun-dependent expression of VEGF, the most potent angiogenic factor, is significantly reduced by the four kinase inhibitors. MG-132, an inhibitor of the 26S proteasome, also diminishes the production of VEGF. Id3 is not phosphorylated by the CSN-associated kinases. However, it inhibits c-Jun, ICSBP and CSN2 phosphorylation. Curcumin and emodin significantly induce ubiquitination and proteasome-dependent degradation of Id3 in HeLa cells. Proteasome-dependent degradation Id1 in HeLa cells is also stimulated by treatment with curcumin or emodin. Ubiquitination of Id3 is shown directly by cotransfection of HeLa cells with Id3 and His-tagged ubiquitin. Curcumin increases Id3-ubiquitin conjugate formation. In addition, overexpression of CSN2 leads to stabilization of Id3 protein. On the basis of these data it is concluded that CSN-mediated phosphorylation inhibits ubiquitination and proteasome-dependent degradation of c-Jun, Id1 and Id3. The COP9 signalosome thus represents an interesting new target for antiangiogenic tumor therapy.

Das Bone Morphogenetic Protein 2 (BMP2) gehört zur TGF-beta Superfamilie und findet seinen Fokus in der osteogenen Aktivierung und in der Anwendung bei der Frakturheilung. Es wird angenommen, dass weitere, bisher unbekannte Verbindungen existieren, die die BMP2-Signalübertragung und die osteogene Aktivität verbessern und somit zu einer verbesserten klinischen Wirksamkeit von BMP2 führen. Für den Stickstoffoxid (NO)-Signalweg ist bereits bekannt, dass im endothelialen Kontext eine Verbindung zum BMP2-Signalweg existiert. Ziel dieser Arbeit war es daher, eine Verbindung zwischen dem NO- und BMP2-Signalweg bezüglich der Regulierung des BMP2-abhängigen Signalwegs und der Osteoinduktion aufzuzeigen. Dies erfolgte durch Anwendung von Inhibitoren (LNAME, ODQ und LY83583) und Aktivatoren (L-Arginin, Deta NONOate, SNAP und YC-1) des NO-Signalwegs, in Kombination mit BMP2. Eine mögliche Verbindung zwischen dem BMP2- und NO-Signalweg, über eine Protein Kinase A (PKA) Brücke, wurde durch die Anwendung des PKA Inhibitors H89 untersucht. Zusammenfassend zeigen diese Ergebnisse, dass der NO-Stoffwechselweg den BMP2-vermittelten Signalweg und die osteoinduktive Aktivität modulieren kann, wobei PKA beide Signalwege im Rahmen der BMP Signalübertragung verbindet, jedoch nicht zu einer BMP2-vermittelten Osteoinduktion führt. / Bone Morphogenetic Protein 2 (BMP2) is a TGF-beta superfamily member, with a major focus on osteogenic activity and application in fracture healing. In order to improve efficiency of BMP2 in the clinic, it is assumed that additional, yet unknown compounds can improve BMP2 signalling and osteogenic activity. The Nitric Oxide (NO) pathway has previously shown to be connected with the BMP2 pathway in an endothelial context. Therefore, it was the aim of this study to unravel connections between the NO and BMP2 pathway in regulating BMP2 mediated signalling and osteoinduction. This was carried out through the application of inhibitors (LNAME, ODQ and LY83583) and activators (L-Arginine, Deta NONOate, SNAP and YC-1) of the NO pathway in combination with BMP2. A proposed connection between BMP2 and NO pathways via a Protein Kinase A (PKA) bridge was investigated by application of H89 inhibitor. In summary, these results show that the NO pathway can modulate BMP2 mediated signalling and osteoinductive activity. The PKA bridge connects NO and BMP2 only for the process of BMP2 signalling, but not for BMP2 mediated osteoinduction.