Das Glioblastom ist der häufigste hirneigene Tumor des Erwachsenen. Es ist hoch invasiv, stark proliferierend und mit einer schlechten Prognose assoziiert. Heutige Therapiean-sätze zielen, neben der möglichst vollständigen Resektion des Tumorgewebes, vor allem auf Apoptoseinduktion durch DNA-Schäden in Tumorzellen. Daher ist die Aufklärung der molekularen Grundlagen dieser Prozesse essentiell, um Verbesserungen bei den Behandlungsmöglichkeiten erzielen zu können. Der Proteasomenaktivator PA28γ wird im Hirngewebe stark exprimiert, über seine Funktion ist jedoch nur wenig bekannt. Er wurde als Interaktionspartner des Zellzyklus- und DNA-Schadensregulators Mad2b in einem Hefe Two-Hybrid Screen identifiziert. Im Rahmen dieser Arbeit wurde diese Wechselwirkung mittels eines GST-Pulldown Experimentes be-stätigt. Obwohl PA28γ in Verbindung mit der Zellproliferation gebracht wird, konnte in GBM-Zelllinien keine signifikante Änderung der Zellteilungsraten beobachtet werden. Allerdings unterstützte die vermehrte Expression von PA28γ die Apoptose. Um durch neue Interaktionspartner von PA28γ Hinweise auf dessen Funktion zu erhalten, wurde ein Hefe Two-Hybrid Screen durchgeführt: PA28gamma steuert den Abbau von p53 und verweist über die hier neu beschriebene Interaktion mit HIPK1 ebenfalls auf den programmierten Zelltod. Dieser pro-apoptotische Zusammen-hang wird unterstützt durch die Interaktion mit 1A6/DRIM-interacting protein. Die Inter-aktion der Sumo E2 Ligase Ubc9 mit PA28gamma war ein erster Hinweis für eine Sumoylierung des Proteasomenaktivators, die die PA28gamma Aktivität regulieren könnte. Gleichzeitig ist Ubc9, wie auch die E3-Ligase PIAS, im Zusammenhang mit Apoptose beschrieben worden. Diese Fragestellungen wurden in weiterführenden Arbeiten erforscht. Einen anderen Aspekt beleuchtet die Interaktion von PA28gammamit Catenin alpha. Durch diese Wechselwirkung könnte PA28gamma Einfluß auf Interzellulärkontakte nehmen. Gerade im Hin-blick auf das GBM, charakterisiert durch ausgeprägtes Migrations- und Invasionsverhal-ten, könnte die Regulation von Interzellulärkontakten von besonderer Bedeutung sein. Aufgrund der oben beschriebenen Eigenschaften von PA28gammasollte dieses Protein für eine Therapie mittels DNA-Schäden induzierter Apoptose erforscht werden. PA28gamma könnte bei diesen Vorgängen ein zentraler Faktor sein, dessen Manipulation die etablierten Therapieformen unterstützen und deren Wirkung verbessern. / Glioblastoma multiforme is the most prevalent brain tumor in adults, being highly invasive and proliferative. The prognosis is poor. Contemporary treatment options include complete tumor resection, followed by induction of DNA-damage by chemotherapeutics and gamma irradiation to induce apoptosis in the tumor cells. To optimize these treatment options, a full knowlegde of the involved molecular pathways is essential. The proteasome activator PA28gamma is highly expressed in brain tissue. However, little is known about its function. It was found in a yeast two-hybrid screen as an interaction partner of Mad2b, a regulator of cell cylce function and DNA-damage response. This thesis confirmed this interaction in a GST-pulldown assay. Although PA28gamma has been associated with cell proliferation, there was no significant effect on proliferation rates of GBM-cell lines detectable. However, PA28gamma was able to induce apoptosis in these cells. A yeast two-hybrid screen was performed using PA28gamma, to detect new, additional interaction partners. The aim was to get better knowlegde about the function of PA28gamma by elucidating its signaling network. Ubc9, a SUMO E2 conjugating enzyme, was detected. The interaction with Ubc9 was a first indication for a sumoylation of PA28gamma which might be involved in the regulation of PA28gamma protein activity. Ubc9 has been described in conjunction with regulation of apoptosis. It is known that PA28gamma regulates p53-degradation. In addition, the yeast two-hybrid screen revealed an interaction with the proteins HIPK1 and 1A6/DRIM-interacting protein. Together these data strongly suggest an involvement of PA28gamma in the regulation of apoptosis. Another new interaction partner of PA28gamma was Catenin alpha, which points to a putative role of PA28gamma in the regulation of cell-cell-contacts. Since Glioblastoma multiforme display strong invasive characteristics, the regulation of intercellular contacts could be of special importance for the treatment. The established therapeutic means target DNA-damage to induce apoptosis of the tumor cells. In view of the suggested role of PA28gamma in regulation of apoptosis and cellular contacts, PA28gamma is worth further research to support the standard-therapy by manipulation of the PA28gamma activity.

Die Tumormikroumgebung (TME) spielt eine wichtige Rolle in Bezug auf das Ansprechen von Therapien, Tumorwachstum und die Bildung von Metastasen. In den letzten Jahren konnte belegt werden, dass Tyrosinkinaseinhibitoren (TKIs) Einfluss auf Zellen des TME haben und vor allem die dort vorherrschenden Zellen, Tumor-assoziierte Makrophagen (TAM), durch die TKIs moduliert werden können. Sie entsprechen meist dem M2-Phänotyp, sezernieren antiinflammatorische Zytokine und sind protumoral, indem sie u.a. die Metastasierung und das Tumorwachstum unterstützen. Zentrale Targets für die Reprogrammierung von Makrophagen stellen sowohl der NF-κB als auch die Inhibition des CSF1-Rezeptors dar. An diesen beiden Schlüsselstellen wirken u.a. TKIs. In den durchgeführten Versuchen wurden drei TKIs verwendet – Dasatinib, Src-Kinase-Inhibitor-I, Bosutinib – um die Ergebnisse von Vorarbeiten zu verifizieren und um zu untersuchen, ob ein Klasseneffekt in Bezug auf eine gesteigerte IL-12-Produktion vorliegen könnte. Ein wichtiger Ansatzpunkt in der Bekämpfung von Tumoren ist die Aktivierung von Immunzellen gegen Tumorzellen, in unserem Fall eine Modulation von TAM in Richtung M1-Makrophagen. Eine signifikante Änderung des Phänotyps konnte nicht festgestellt werden. Allerdings wurde eine gesteigerte IL-12-Produktion aller Makrophagensubtypen durch die Inkubation mit Dasatinib- bzw. Src-kinase-inhibitor-I oder Bosutinib gezeigt. IL-12 ist ein wichtiges proinflammatorisches Schlüsselzytokin des Immunsystems, indem es u.a. NK-Zellen und T-Zellen aktiviert. Die funktionellen Auswirkungen der verstärkten IL-12-Produktion in Hinblick auf NK-Zellen haben wir untersucht. Eine deutlich verstärkte Aktivierung anhand Aktvierungsmarker von NK-Zellen konnten wir nicht beweisen. Allerdings wurde eine erhöhte Zytotoxizität durch Ko-Kultivierung der NK-Zellen mit den unterschiedlichen Makrophagensubtypen und gleichzeitiger Inkubation mit Dasatinib demonstriert. Die erhöhte IL-12-Produktion von APCs sowie verringerte IL-10-Produktion und der Einfluss auf andere Immunzellen, hier am Beispiel der NK-Zellen, zeigen u.a. das therapeutische Potential der TKIs als antineoplastisch wirksame Substanz. Als alleinige Therapie ist deren Wirkungsbereich nach den hier vorliegenden Ergebnissen jedoch noch zu gering. In Kombination mit anderen Therapieoptionen stellen die TKIs allerdings ein mögliches Therapieregime dar. Der genaue Wirkmechanismus und die dadurch entstehenden Veränderungen sind noch genauer zu untersuchen. Ein weiteres Ziel ist in vitro etablierte Ergebnisse auch in die klinische Anwendung einfließen zu lassen. Der zweite Teil der Arbeit befasste sich mit einem proinflammatorischen Zytokin IL-32γ und dessen Wirkung auf Makrophagen. Wie bereits auch bei den TKIs wurde der Einfluss des Interleukins auf das Tumormikromilieu und die entsprechenden Auswirkungen untersucht. IL-32γ wirkt nicht nur selbst als proinflammatorisches Zytokin, sondern reguliert eine Vielzahl an weiteren Zytokinen. Der Einfluss von IL-32 auf das Tumormikromilieu und dessen Zellen stellt einen der zentralen Interessenspunkte dar. In unseren Versuchen konnte unter IL-32γ eine effektivere Antigenpräsentation der Makrophagen – gemessen an einer verstärkten Expression von CD80 und CD86 – gezeigt werden. Auf der anderen Seite wurde das antiinflammatorische Zytokin, IL-10, von IL-32γ-stimulierten Makrophagen ebenfalls verstärkt sezerniert. Eine Ko-Kultivierung von Makrophagen und NK-Zellen und gleichzeitige Inkubation mit IL-32γ führte bei NK-Zellen zu einer verstärkten Aktivierung sowie zu einer erhöhten Zytotoxizität. Die Auswirkungen auf NK-Zellen deuten eine antitumorale Wirkung von IL-32γ an. Das breite Wirkspektrum des Interleukins ist vielversprechend und könnte neue Therapiestrategien eröffnen, wofür allerdings weitere Versuche sowohl in vitro als auch in vivo notwendig sind, um das Interleukin und seine Wirkungen genauestens zu verstehen. / Tumor microenviroment (TME) plays an important role in therapies, tumor development and metastasis formation. In recent years it could be proven that tyrosinekinaseinhibitors (TKIs) have an influence on cells of the TME and that especially the main cells located there, tumor-associated macrophages (TAM), can be modulated by the TKIs. They usually are of the M2 phenotype, secrete anti-inflammatory cytokines and are protumoral in respect to fostering metastasis and tumor development. The NF-κB as well as the inhibition of the CSF1 receptor are central targets for the reprogramming of macrophages. TKIs, amonst others, impact on those two central points. Three TKIs were used in the experiments – Dasatinib, Src Kinase Inhibitor-I, Bosutinib – to verify the results of previous work and to examine whether a class effect can be measured in terms of increased IL-12 production. An important starting point in fighting tumors is the activation of immune cells against tumor cells, in our case a modulation of TAM in the direction of M1 macrophages. A significant change of the phenotype could not be determined. It could be shown, however, that the incubation with Dasatinib, Src-kinase-inhibitor I or Bosutinib resulted in increased IL-12 production of all macrophage subtypes. IL-12 is an important pro-inflammatory key cytokine of the immune system, activating NK cells and T cells, amongst others. We examined the functional impact of the increased IL-12 production in respect to NK cells. A significantly increased activation using activation markers of NK cells could not be proven. However, an increased cytotoxicity was demonstrated by co-culturing the NK cells with the different macrophage subtypes and simultaneous incubation with Dasatinib. The increased IL-12 production of APCs as well as the reduced IL-10 production and the influence on other immune cells, here using the example of NK cells, show, among other things, the therapeutic potential of TKIs as an antineoplastic substance. However, according to the results available here, its impact is still too small as an independent therapy. In combination with other therapy options, however, the TKIs represent a possible therapy regimen. The specific mechanism of action and the resulting changes remain to be further examined. Another goal is to incorporate the results established in vitro into clinical application. The second part of the thesis dealt with a pro-inflammatory cytokine IL-32γ and its effect on macrophages. As with the TKIs, the influence of interleukin on the tumor microenvironment and the corresponding effects were investigated. IL-32γ not only acts as a pro-inflammatory cytokine itself, but also regulates a large number of other cytokines. The influence of IL-32 on the tumor microenvironment as well as its cells is one of the main points of interest. Our experiments showed a more effective antigen presentation of the macrophages under IL-32γ – measured with an increased expression of CD80 and CD86. On the other hand, IL-32γ-stimulated macrophages also secreted more of the anti-inflammatory cytokine IL-10. The co-cultivation of macrophages and NK cells and simultaneous incubation with IL-32γ lead to an increased activation of NK cells as well as an increased cytotoxicity. The impact on NK cells indicate an anti-tumor effect of IL-32γ. The broad spectrum of action of interleukin is promising and could open up new therapeutic strategies, for which, however, further experiments in vitro and in vivo are required in order to understand the interleukin and its effects in enough detail.

No description available.

During the cell division dynamic processes take place, the origin of which are to find in the physical characteristics of cell components. The most important characteristics are the electrical charge and the energy of the moving base components in a viscous cytoplasm. During the emergence of the new hydrogen bonds takes place a competition of the complementary base components which are electrostatically attracted by the codogen matrix. Thus, the base components will be accelerated more and more in the course of replication, and the resulting binding energies become always larger in a monotonous sequence. We call this process “base rivalry”. It is shown that the strength of these new bindings depends on three factors: First it is dependent on the length of a monotonous sequence, second it is dependent on the viscosity of the cytoplasm, and third it is dependent on the replication speed. In the study in detail is stated, how it affects the effectiveness of the DNA repair mechanism, mutation susceptibility, and thus also affects the cancer susceptibility. This is a condition where the DNA repair mechanism fails: Because of the base rivalry, in a monotonous base sequence there is (for a short time) a high binding energy between the complementary bases from a critical sequence length upwards, and the effectiveness of the repair mechanism is strongly decreased. If a tautomeric base pair is behind the end of monotonous sequence, then an extension of the monotonous sequence is provoked so that, for example, the monotonous sequence CCCT irreparably changes itself into CCCC (see section 2.2). The author describes in detail how the base rivalry affects on the evolution and on the mutation of viruses. The probability for the emergence of an irreparable mutation (caused by base rivalry) will be calculated. The result is (for a large number of individuals) a mathematical connection between temperature and the length of monotonous DNA - sequences which are lengthened by base rivalry. In the study, there are preferentially used physical and statistical computations and therefore is to understand as theoretical work. For the examination of this theory, two different computations are necessary: 1. Statistical computation: It is safe to assume that an individual base component exists (for example, dGTP) having a very large fading time in the case of excitation (preferable, owing to rotation energy after it became lumpy). Such a base component is very rarely, so that it appears within a DNA-fragment either not or once at most. This is called the “elitist”. If it appears within the fragment, we can compute the probability for its appearance in a certain position during replication, namely in a monotonous sequence of this fragment. The calculation of the probability must be statistically, because the replication is a distribution on the codogen matrix. 2. Physical computation: If the elitist (accidentially) arrives at a monotonous sequence of the DNA-fragment, it will reach the end of this monotonous sequence because of its high base rivalry energy, and now we can the tunnel probability calculate for the conversion into the tautomeric form which leads to a mutated hydrogen bond at the end of monotonous sequence. This mutated hydrogen bond is irreparabel, if the fading time of the excited elitist higher is than the repair time of the DNA repair mechanism. Both probabilities have to be connected for the computation of the total probability of the irreparable mutation. The result of this connection is an interesting equation between temperature and monotonous sequence length which is irreparably lengthened, and this gives rise to the speculation that this theory as well as the resulting equation may have a certain importance for the theory of evolution, and may have an importance for the dangerous virus mutations. In the case of base rivalry within of a DNA - fragment, there is a connection between the base sequence (especially the monotonous sequence length) and the temperature at which irreparable mutations are be expected (equation [96a]. This work is a revision of the article 'Die Kalkulation irreparabler Mutationen” by the same author. Errors have been corrected in some chapters in the mathematical representation. Chapters 6 and 7 have been re-edited. Corrected excerpts from 'Tumour Physics' [25] and from 'Evolution Physics' [26] are used in this work. In the appendix [28] are supplementary remarks in order to understand the sections better. There is, too, a remark concerning the coherence between tumor development and cell - viscosity. This modified version differs from the previous one, since it (according to the latest considerations) is not realistic, the electrostatic attraction (between codogen basic components and the complementary basic components) attributable to the sum of all partial charges, but attributable to only one. The reason for this is that (in the course of attraction) not all part charges of the matrix and complements will be the same distance from each other, since they are located at different positions. This altered version has consequences in the sections 1, 2, 7, 8, and picture 7.

During the cell division dynamic processes take place, the origin of which are to find in the physical characteristics of cell components. The most important characteristics are the electrical charge and the energy of the moving base components in a viscous cytoplasm. During the emergence of the new hydrogen bonds takes place a competition of the complementary base components which are electrostatically attracted by the codogen matrix. Thus, the base components will be accelerated more and more in the course of replication, and the resulting binding energies become always larger in a monotonous sequence. We call this process “base rivalry”. It is shown that the strength of these new bindings depends on three factors: First it is dependent on the length of a monotonous sequence, second it is dependent on the viscosity of the cytoplasm, and third it is dependent on the replication speed. In the study in detail is stated, how it affects the effectiveness of the DNA repair mechanism, mutation susceptibility, and thus also affects the cancer susceptibility. This is a condition where the DNA repair mechanism fails: Because of the base rivalry, in a monotonous base sequence there is (for a short time) a high binding energy between the complementary bases from a critical sequence length upwards, and the effectiveness of the repair mechanism is strongly decreased. If a tautomeric base pair is behind the end of monotonous sequence, then an extension of the monotonous sequence is provoked so that, for example, the monotonous sequence CCCT irreparably changes itself into CCCC (see section 2.2). The author describes in detail how the base rivalry affects on the evolution and on the mutation of viruses. The probability for the emergence of an irreparable mutation (caused by base rivalry) will be calculated. The result is (for a large number of individuals) a mathematical connection between temperature and the length of monotonous DNA - sequences which are lengthened by base rivalry. In the study, there are preferentially used physical and statistical computations and therefore is to understand as theoretical work. For the examination of this theory, two different computations are necessary: 1. Statistical computation: It is safe to assume that an individual base component exists (for example, dGTP) having a very large fading time in the case of excitation (preferable, owing to rotation energy after it became lumpy). Such a base component is very rarely, so that it appears within a DNA-fragment either not or once at most. This is called the “elitist”. If it appears within the fragment, we can compute the probability for its appearance in a certain position during replication, namely in a monotonous sequence of this fragment. The calculation of the probability must be statistically, because the replication is a distribution on the codogen matrix. 2. Physical computation: If the elitist (accidentially) arrives at a monotonous sequence of the DNA-fragment, it will reach the end of this monotonous sequence because of its high base rivalry energy, and now we can the tunnel probability calculate for the conversion into the tautomeric form which leads to a mutated hydrogen Bond at the end of monotonous sequence. This mutated hydrogen bond is irreparabel, if the fading time of the excited elitist higher is than the repair time of the DNA repair mechanism. Both probabilities have to be connected for the computation of the total probability of the irreparable mutation. The result of this connection is an interesting equation between temperature and monotonous sequence length which is irreparably lengthened, and this gives rise to the speculation that this theory as well as the resulting equation may have a certain importance for the theory of evolution, and may have an importance for the dangerous virus mutations. If several base rivalries take place in a monotonous sequence of a DNA fragment over time and with decreasing cell temperature, an extension of the fragment and thus a DNA extension is provoked at each base rivalry (section 8.1). In the appendix [28] are supplementary remarks in order to understand the sections better. There is, too, a remark concerning the coherence between tumor development and cell - viscosity.

Bei einem als „Basenkonkurrenz“ bezeichneten Vorgang werden bei der Replikation irreparable Verlängerungen monotoner Sequenzen provoziert, die von der Zelltemperatur, der Zell - Viskosität und der monotonen Sequenzlänge abhängen. Dadurch können die im Laufe langer Evolutionsepochen entstehenden sehr langen monotonen Sequenzen und die Entstehung sehr langer DNAs erklärt werden. Vermutlich werden durch Basenkonkurrenz (bei Tautomerität oder zu kleiner Zell – Viskosität) auch die Entstehung von Tumoren und das Auftreten gefährlicher Virenmutationen provoziert.