Berlin, Freie Universiẗat, Diss., 2005. / Dateiformat: zip, Dateien im PDF-Format.

Berlin, Freie Universiẗat, Diss., 2004. / Dateiformat: zip, Dateien im PDF-Format.

Universiẗat, Diss., 2006--Würzburg.

Die STATs (signal transducers and activators of transcription) sind eine Familie latent zytoplasmatischer Transkriptionsfaktoren, die Signale von der Zellmembran in den Zellkern weiterleiten. Ein Mitglied der Proteinfamilie, STAT3, ist aufgrund übermäßiger Tyrosinkinase-Aktivität in einer breiten Vielzahl von Krebszelllinien und menschlichen Tumoren konstitutiv-aktiv. Um kleine organische Moleküle zu identifizieren, die die Funktion der SH2-Domäne von STAT3 blockieren und dadurch die Aktivität und die Dimerisierung des Proteins inhibieren, wurde ein Hochdurchsatz-Verfahren entwickelt, welches auf Fluoreszenzpolarisation beruht. Das Prinzip dieses Verfahrens war die Bindung eines Fluorescein-markierten Phosphotyrosin-Peptids, welches von gp130, einer Untereinheit des Interleukin-6-Rezeptors, abgeleitet war, an nicht phosphoryliertes STAT3-Protein. Der Kd Wert dieser Bindung betrug 150 nM und der Assay war stabil im Hinblick auf die Salzkonzentration, der Konzentration an Dimethylsulfoxid und der Zeit. Der Assay wurde auf ein 384-Lochplattenformat angepasst und wies einen Z’-Wert von 0,87 auf. Das Fluorscein-markierte Phosphotyrosin-Peptid band spezifisch an die SH2-Domäne von STAT3 und die Bindung konnte durch Phosphotyrosin-Peptide unterschiedlich stark inhibiert werden. Die Hochdurchsatz-Analyse mehrerer Substanzbibliotheken führte schließlich zur Identifikation eines spezifischen STAT3-Inhibitors, Stattic (STAT three inhibitory compound). Stattic ist das erste nicht-peptidische kleine Molekül, welches selektiv die Funktion der STAT3-SH2-Domäne beeinträchtigte. Dabei spielte der Aktivierungszustand von STAT3 in vitro keine Rolle. Die gleichzeitige Inkubation mit Stattic führte im Fluoreszenzpolarisations-Assay zur Inhibition der Bindung des Fluorescein-markierten Phosphotyrosin-Peptids an die SH2-Domäne von STAT3. Diese antagonistische Reaktion stellte sich als stark temperaturabhängig heraus und hatte in vitro bei der physiologisch relevanten Temperatur von 37°C nach 60 Minuten einen IC50 Wert von 5,1 µM. Zusammen mit einer Abhängigkeit von der Zeit wiesen die Ergebnisse auf eine irreversibel ablaufende Reaktion unter Knüpfung einer kovalenten Bindung zwischen Stattic und STAT3 hin. Die Inhibition war spezifisch gegenüber der Bindung verschiedener Fluorescein-markierten Phosphotyrosin-Peptide an die jeweiligen Proteine STAT1, STAT5b und Lck und Stattic hatte ebenfalls nur einen sehr geringen Effekt auf die Proteindimerisierung von c-Myc/Max und Jun/Jun. Die genauere Betrachtung der Kinetik der antagonistischen Reaktion zeigte eine signifikante Verlangsamung der Reaktionsgeschwindigkeit beim Vergleich zwischen STAT3 und STAT1 bzw. STAT3 und STAT5b. Die Inhibierung der Bindung des entsprechenden Fluorescein-markierten Phosphotyrosin-Peptids an das Protein Lck durch Stattic war hingegen nicht zeitabhängig. Diese Versuche zeigten eine deutliche Präferenz der Bindung von Stattic an das Protein STAT3. Die Verdrängung des Fluorescein-markierten Phosphoytrosin-Peptids von der STAT3-SH2-Domäne durch Stattic verlief kompetitiv zur Inhibition mit einem Phophotyrosin-Peptid, welches an die SH2-Domäne von STAT3 bindet. In Verbindung mit den vorherigen Experimenten wies dies auf eine kovalente Bindung von Stattic innerhalb des STAT3-Proteins hin. Eine abschließende Struktur-Wirkungs-Beziehung in vitro zeigte die Notwendigkeit sowohl von der Nitrogruppe als auch von der Doppelbindung der Vinylsulfongruppe in Stattic für die Bindung an STAT3 und untermauerte die These, dass Stattic kovalent innerhalb des STAT3-Proteins bindet. In zellbiologischen Systemen wurde die Wirksamkeit von Stattic anhand verschiedener molekularbiologischer Assays bestätigt. Stattic inhibierte selektiv die Tyrosinphosphorylierung von STAT3 in HepG2 Zellen, in NIH3T3/v-Src Zellen und in den Brustkrebszelllinien MDA-MB-231 und MDA-MB-435S. Aber auch bereits phosphorylierte STAT3-Proteine wurden durch Stattic in vitro an der Homodimerisierung gehindert, was in einer EMSA-Analyse gezeigt wurde. Somit inhibierte Stattic in vitro selektiv die Signalkette von STAT3 unabhängig von dessen Aktivierungszustand. Andere Signalketten oder die Funktion der in der Signalkette über STAT3 liegenden Tyrosinkinasen wurden in Zellen nicht beeinflusst. Im Folgenden konnte demonstriert werden, dass Stattic als direkter STAT3-Inhibitor dessen Lokalisierung in den Zellkern inhibierte, nicht jedoch die Lokalisierung des Gegenspielers STAT1. Weiterhin reduzierte der Einsatz von Stattic selektiv das von v-Src in NIH3T3 Zellen induzierte und von STAT3-abhängige Wachstum von Kolonien in Weichagar. Dass Stattic schließlich selektiv die Apoptoserate in Zellen mit konstitutiver STAT3-Aktivtät erhöhte, bestätigte die bisherigen Daten. Mit Stattic konnte daher ein neues biologisches Werkzeug generiert werden, um selektiv STAT3 in Zelllinien oder Tumoren in Tiermodellen auszuschalten, die eine konstitutive STAT3-Aktivität aufweisen. / Signal Transducers and Activators of Transcription (STATs) are a family of latent cytoplasmic transcription factors which signals from the cell membrane to the nucleus. One member of the protein family, STAT3, is constitutively activated by aberrant upstream tyrosine kinase activities in a broad spectrum of cancer cell lines and human tumors. A high-throughput assay based on fluorescence polarization was developed to identify small organic molecules blocking the function of the STAT3 SH2 domain and thereby inhibiting STAT3 activity and dimerization. The principle of the assay was the binding of a fluorescein-labeled phosphotyrosine-peptide derived from the interleukin-6 receptor subunit gp130 to unphosphorylated STAT3 with a Kd of 150 nM. The assay was stable with regard to salt concentration, dimethyl sulfoxide concentration, and time. It has been adapted to a 384-well format, with a Z’ value of 0.87. The fluorecein-labeled phosphotyrosine-peptide bound specifically to the STAT3 SH2 domain and this binding could be inhibited by different phosphotyrosine-peptides with varying activities. The high-throughput screening of a number of compound libraries finally lead to the identification of a specific STAT3 inhibitor, dubbed Stattic (STAT three inhibitory compound). Stattic is depicted as the first non-peptidic small molecule having an selective impact on the function of the STAT3 SH2 domain. Thereby the activation state of STAT3 was irrelevant in vitro. Simultaneous incubation with Stattic inhibited the binding of the fluorescein-labeled phosphotyrosine-peptide to the SH2 domain of STAT3 in the fluorescence polarization assay. This antagonistic reaction turned out to be strongly temperature-dependent and showed an IC50 of 5.1 µM in vitro at the physiological relevant temperature of 37°C after 60 minutes incubation. With regard to an time-dependency these results suggested an irreversible reaction with the formation of a covalent bond between Stattic and STAT3. The inhibitory reaction was specific over the binding of different fluorescein-labeled phosphotyrosin-peptides to the particular proteins STAT1, STAT5b and Lck, and Stattic also only marginally inhibited the protein dimerization of c-Myc/Max or Jun/Jun. A closer look on the kinetics of the reaction revealed a significant slowdown of the reaction speed comparing STAT3 to STAT1 or STAT3 to STAT5b. Stattic inhibited the binding of the corresponding fluorescein-labeled phosphotyrosine-peptides to Lck in a time-independent way altogether showing a clear preference of Stattic binding to STAT3. The displacement of the fluorescein-labeled phosphotyrosine-peptide from the STAT3 SH2 domain through Stattic was competitive to a phosphotyrosine-peptide binding to the SH2 domain of STAT3. With regard to other results, this result indicated Stattic covalently binding to STAT3. A structure-activity relationship in vitro showed the nitro moiety and the double bond within the vinylsulfone moiety of stattic being important for binding to STAT3. This confirmed the indication that Stattic covalently binds to STAT3 domain. The effectiveness of Stattic in cellular systems was proven by different molecular biological assays. Stattic selectively inhibited the tyrosine phosphorylation in HepG2 cells, in NIH3T3/v-src cells as well as in the breast cancer cell lines MDA-MB-231 and MDA-MB-435S. But also STAT3 proteins which already were phosphorylated could not dimerize after incubation with stattic in vitro which was shown with an EMSA analysis. Therby Stattic also inhibited STAT3 signaling in vitro regardless of STAT3 phosphorylation. Other signalling pathways or function of upstream tyrosinkinases in cells were not inhibited at the same time. It could be demonstrated that the direct STAT3 inhibitor Stattic specifically inhibited nuclear localization of STAT3, but not of its counterpart STAT1. Stattic reduced v-src induced STAT3 dependent colony growth of NIH3T3 cells in soft agar. The results were confirmed by Stattic selectively increasing the apoptotic rate in cell lines having constitutively active STAT3. In summary Stattic turned out to be a novel biological tool to selectively inhibit STAT3 in cell lines or tumor animal models which show constitutive active STAT3.

Zugl.: Berlin, Humboldt-Univ., Diss., 2007

Techn. Hochsch., Diss., 2004--Aachen.

Singapore är ett fascinerande land. På bara några få decennier har landet gått från att vara en liten instabil stadsstat utan större naturresurser till att bli ett ekonomiskt världscentrum. Mur hur fungerar egentligen Singapores politiska styrelseskick? Är landet verkligen en demokrati eller har det i själva verket diktatoriska drag? Denna uppsats syftar till att steg för steg undersöka landets politiska situation för att slutligen landa i en definition om vad Singapore egentligen står politiskt. Dem styrande hävdar att dem levererar en effektiv och okorrumperad regering till medborgarna medan vissa oppositionspolitiker menar att regeringen styr med auktoritära medel. Hur kan man definiera den politiska situationen i Singapore idag och framför allt vilken regimtyp kan landet klassas som? Detta arbete är en fallstudie av teorikonsumerande art där olika fakta kommer att analyseras och sedan sammanfattas i en slutlig analys där jag skall fastställa regimtypen.

A desnutrição modifica a resistência à infecção, alterando grande variedade de processos fisiológicos, incluindo a hematopoiese e a resposta imune. Os mecanismos exatos que comprometem o sistema imunológico em condições de desnutrição ainda não estão totalmente compreendidos, sendo que a desnutrição modifica a funcionalidade das células efetoras na resposta inflamatória/infecciosa, causando redução da síntese de citocinas pró-inflamatórias, além de alterações na hematopoiese. Os linfócitos têm papel-chave tanto na resposta imune inata quanto adaptativa. Nesse contexto, propusemo-nos a avaliar alguns aspectos da resposta linfoide esplênica em um modelo de desnutrição proteica. Camundongos BALB/c, machos, submetidos à desnutrição proteica, após perda de aproximadamene 20% do peso corpóreo, foram eutanasiados. Hemograma, mielograma, esplenograma e análise das concentrações séricas de proteína totais, albumina, pré-albumina e dosagem de imunoglobulinas G e M foram realizados. As células do timo e do baço foram coletadas e analisadas por citometria de fluxo - e foi realizado o ciclo celular. Para avaliação da linfoproliferação, as células esplênicas foram cultivadas, in vitro, e estimuladas com LPS ou ConA durante 72 horas. Foram quantificadas a capacidade de produção de INF-γ, IL-2 e IL-10 e a expressão de STAT- 1 e STAT-3. Os animais desnutridos apresentaram anemia, leucopenia e severa redução na celulariedade da medula óssea, com comprometimento no setor mieloide e diminuição da celulariedade do baço, com redução da população de linfócitos. Os animais desnutridos apresentaram alterações no desenvolvimento celular linfoide, alteração na capacidade de proliferação, menor produção de citocinas pró-inflamatórias, como IL-2, e, ao mesmo tempo, aumento da produção de IL-10. Os animais também apresentaram maior porcentagem de células CD3+ e CD4+ no baço em relação aos animais controle. Quando as células esplênicas foram estimuladas, apresentaram maior expressão de STAT-3 e menor expressão de STAT-1. Baseando-se nestes resultados, discutimos se deficiências nutricionais alteram a imunocompetência e aumentam o risco de infecções. / Malnutrition modifies the resistance against infection by changing a variety of physiological processes, including the hematopoiesis and the immune response. The exact mechanisms that compromise the immune system in conditions of malnutrition is not yet thoroughly understood, since malnutrition modifies the functionality of the effector cells in inflammatory / infectious response, causing the reduction of the synthesis of proinflammatory cytokines, besides causing alterations in hematopoiesis. The lymphocytes have a key role in both innate and adaptive immune responses, in this context, we proposed to evaluate some aspects of lymphoid spleen cells response in a model of protein malnutrition. Male mice BALB / c submitted to protein malnutrition after approximately 20% loss of body weight were euthanized. The complete blood count, bone marrow examination, spleen cells count and analysis of serum total protein, albumin, pre-albumin and immunoglobulin G and M were performed. The thymus and spleen cells were collected and analyzed by flow cytometry as well as the cell cycle performed. For evaluation of the lymphocyte proliferation the spleen cells were cultivated in vitro and stimulated with LPS or ConA for 72 hours and the ability to produce IFN-γ, IL-2 and IL-10 as well as the expression of STAT-1 and STAT -3, were quantified. The malnourished animals presented anemia, leukopenia and severe reduction in celulariedade bone marrow with myeloid commitment in the sector and decreased celulariedade spleen, with reduced lymphocyte population. The malnourished animals showed changes in lymphoid cell development, abnormal proliferation capacity, reduced production of proinflammatory cytokines such as IL-2, and at the same time, increased production of IL-10. The animals also had higher percentages of CD3 + and CD4 + spleen compared to control animals. When spleen cells were stimulated, showed higher expression of STAT-3 and lower expression of STAT-1. Based on these results, we discussed whether nutritional deficiencies alter immunocompetence and increase the risk of infections.

A desnutrição modifica a resistência à infecção, alterando grande variedade de processos fisiológicos, incluindo a hematopoiese e a resposta imune. Os mecanismos exatos que comprometem o sistema imunológico em condições de desnutrição ainda não estão totalmente compreendidos, sendo que a desnutrição modifica a funcionalidade das células efetoras na resposta inflamatória/infecciosa, causando redução da síntese de citocinas pró-inflamatórias, além de alterações na hematopoiese. Os linfócitos têm papel-chave tanto na resposta imune inata quanto adaptativa. Nesse contexto, propusemo-nos a avaliar alguns aspectos da resposta linfoide esplênica em um modelo de desnutrição proteica. Camundongos BALB/c, machos, submetidos à desnutrição proteica, após perda de aproximadamene 20% do peso corpóreo, foram eutanasiados. Hemograma, mielograma, esplenograma e análise das concentrações séricas de proteína totais, albumina, pré-albumina e dosagem de imunoglobulinas G e M foram realizados. As células do timo e do baço foram coletadas e analisadas por citometria de fluxo - e foi realizado o ciclo celular. Para avaliação da linfoproliferação, as células esplênicas foram cultivadas, in vitro, e estimuladas com LPS ou ConA durante 72 horas. Foram quantificadas a capacidade de produção de INF-γ, IL-2 e IL-10 e a expressão de STAT- 1 e STAT-3. Os animais desnutridos apresentaram anemia, leucopenia e severa redução na celulariedade da medula óssea, com comprometimento no setor mieloide e diminuição da celulariedade do baço, com redução da população de linfócitos. Os animais desnutridos apresentaram alterações no desenvolvimento celular linfoide, alteração na capacidade de proliferação, menor produção de citocinas pró-inflamatórias, como IL-2, e, ao mesmo tempo, aumento da produção de IL-10. Os animais também apresentaram maior porcentagem de células CD3+ e CD4+ no baço em relação aos animais controle. Quando as células esplênicas foram estimuladas, apresentaram maior expressão de STAT-3 e menor expressão de STAT-1. Baseando-se nestes resultados, discutimos se deficiências nutricionais alteram a imunocompetência e aumentam o risco de infecções. / Malnutrition modifies the resistance against infection by changing a variety of physiological processes, including the hematopoiesis and the immune response. The exact mechanisms that compromise the immune system in conditions of malnutrition is not yet thoroughly understood, since malnutrition modifies the functionality of the effector cells in inflammatory / infectious response, causing the reduction of the synthesis of proinflammatory cytokines, besides causing alterations in hematopoiesis. The lymphocytes have a key role in both innate and adaptive immune responses, in this context, we proposed to evaluate some aspects of lymphoid spleen cells response in a model of protein malnutrition. Male mice BALB / c submitted to protein malnutrition after approximately 20% loss of body weight were euthanized. The complete blood count, bone marrow examination, spleen cells count and analysis of serum total protein, albumin, pre-albumin and immunoglobulin G and M were performed. The thymus and spleen cells were collected and analyzed by flow cytometry as well as the cell cycle performed. For evaluation of the lymphocyte proliferation the spleen cells were cultivated in vitro and stimulated with LPS or ConA for 72 hours and the ability to produce IFN-γ, IL-2 and IL-10 as well as the expression of STAT-1 and STAT -3, were quantified. The malnourished animals presented anemia, leukopenia and severe reduction in celulariedade bone marrow with myeloid commitment in the sector and decreased celulariedade spleen, with reduced lymphocyte population. The malnourished animals showed changes in lymphoid cell development, abnormal proliferation capacity, reduced production of proinflammatory cytokines such as IL-2, and at the same time, increased production of IL-10. The animals also had higher percentages of CD3 + and CD4 + spleen compared to control animals. When spleen cells were stimulated, showed higher expression of STAT-3 and lower expression of STAT-1. Based on these results, we discussed whether nutritional deficiencies alter immunocompetence and increase the risk of infections.

Les cavéoles sont des invaginations en forme de coupelle à la membrane plasmique. Ces organelles multifonctionnelles jouent entre autres, un rôle clé dans la mécano-protection et la signalisation cellulaire. En effet, les cavéoles ont la faculté de s’aplanir en réponse à l’augmentation de la tension membranaire, afin de protéger la cellule des contraintes mécaniques. Les cavéoles jouant un rôle clé dans la signalisation cellulaire, nous avions émis l’hypothèse que le cycle mécano-dépendent de désassemblage/réassemblage des cavéoles constitue un interrupteur mécanique de certaines voies de signalisation. Ce projet consiste à élucider le mécanisme moléculaire responsable du contrôle de la voie de signalisation JAK-STAT par la mécanique des cavéoles. Dans ces travaux, nous avons pu démontré que la cavéoline-1 (Cav1), un constitutant essentiel des cavéoles est libérée et devient hautement mobile au niveau de la membrane plasmique. Considérant les propriétés de signalisation de Cav1, Nous avons testé l’effet du désassemblage des cavéoles sur la signalisation cellulaire. Un criblage à haut débit, nous a permis identifié la voie de signalisation JAK- STAT stimulée par l’IFN-α comme voie modèle pour cette étude. En effet, la transduction du signal JAK-STAT induit par l’IFN-α est modulée par la mécanique des cavéoles. Afin de disséquer le mécanisme moléculaire responsable du contrôle de la signalisation JAK-STAT par la mécanique des cavéoles, nous avons déterminé le rôle de Cav1 dans ce contrôle. Nous avons observé que Cav1 est un régulateur négatif de la phosphorylation de STAT3 dépendante de la kinase JAK1. De plus, nous avons démontré que Cav1 interagit avec JAK1 en fonction de la tension membranaire. Nous avons également démontré que cette interaction Cav1-JAK1 fait intervenir le « scaffolding domain » de Cav1 (CSD), et que celui-ci est responsable de l’abolition de l’activité kinase de JAK1. Par conséquent, l’interaction de Cav1 avec JAK1 empêche l’activation de STAT3 par la kinase JAK1. Ces résultats démontrent que les cavéoles sont des organelles de mécano-signalisation, qui, lors d’un stress mécanique, libèrent de la Cav1 non cavéolaire capable d’inactiver la kinase JAK1, empêchant ainsi, la transduction du signal JAK-STAT. / Caveolae are small cup-shaped plasma membrane invaginations. These multifunctional organelles play a key role in cell mechanoprotection and cell signaling. Indeed our laboratory reported that caveolae have the ability to flatten out upon membrane tension increase, protecting cells from mechanical strains. Since caveolae play a key role in cell signaling we hypothesized that the mechano-dependent cycle of caveolae disassembly/reassembly may constitute a mechanical switch for signaling pathways. In this project, we elucidated the molecular mechanism underlying the control of JAK-STAT signaling by caveolae mechanics. We showed that caveolin-1 (Cav1), an essential caveolar component is released and become highly mobile at the plasma membrane under mechanical stress. Considering that caveolae are important signaling hubs at the plasma membrane, we addressed the effects of the mechanical release of Cav1 on cell signaling. Using high throughput screening, we identified the JAK-STAT signaling pathway as a candidate. To further dissect the molecular mechanism underlying the control of JAK-STAT signaling by caveolae mechanics, we addressed the role of Cav1 in the control of JAK-STAT signaling stimulated by IFN-α. We found that Cav1 was a specific negative regulator of the JAK1 dependent STAT3 phosphorylation. Furthermore, the level of Cav1 interaction with JAK1 depended on mechanical stress. We could show that Cav1-JAK1 interaction was mediated by the caveolin scaffolding domain (CSD), abolishing JAK1 kinase activity, hence, interfering with STAT3 activation upon IFN-α stimulation. Altogether our results show that caveolae are mechanosignaling organelles that disassemble under mechanical stress, releasing non-caveolar Cav1, which binds to the JAK1 kinase and inhibits its catalytic activity, preventing thereby JAK-STAT signal transduction.