O uso de agentes químicos coadjuvantes da higienização bucal pode ser necessário para o controle da microbiota cariogênica de indivíduos com alto risco e atividade da doença cárie. Atualmente o agente antimicrobiano mais recomendado é o digluconato de clorexidina (CHX) devido ao seu amplo espectro de ação e efeito residual. Contudo, quando utilizado por longos períodos, este agente químico apresenta efeitos colaterais. Neste contexto, os polifenóis naturais, como a Epigalocatequina3galato (EGCG), derivada do chá verde, vêm sendo propostos como alternativa aos agentes antimicrobianos sintéticos. Entretanto, os polifenóis não apresentam estabilidade ao longo do tempo, podendo se oxidar rapidamente. Desta forma, o encapsulamento da EGCG em nanopartículas poderia aumentar a sua biodisponibilidade e estabilidade física e química, manter o efeito deste polifenol no tecido alvo e potencializar sua eficácia farmacológica. Assim, o presente estudo teve como objetivo desenvolver e caracterizar sistemas de encapsulamento de EGCG e avaliar, in vitro, sua atividade antimicrobiana frente a microorganismos cariogênicos. Inicialmente, foram preparadas nanopartículas poliméricas (NPP) e carreadores lipídicos nanoestruturados (CLN), que foram caracterizados e avaliados quanto à sua atividade antimicrobiana in vitro frente aos microorganismos Streptococcus mutans, Streptococcus sobrinus e Lactobacillus casei. Após a análise dos resultados microbiológicos, o CLN foi selecionado para o encapsulamento da EGCG (CLNEGCG), por apresentar atividade antimicrobiana frente à todos os microorganismos avaliados. O CLNEGCG foi preparado pelo método de emulsão e sonicação e caracterizado quanto ao diâmetro, índice de polidispersão (PdI), potencial zeta (PZ), eficiência de encapsulamento (EE), cristalinidade, capacidade de mucoadesão e morfologia. A atividade antimicrobiana in vitro da EGCG livre e encapsulada foi avaliada por meio da determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM). O diâmetro, PdI e o PZ dos CLN foram 228nm, 0,216 e 36,53mV, respectivamente, sendo que o encapsulamento da EGCG não alterou significativamente estes parâmetros. O CLN apresentou forma esférica, estabilidade por 330 dias e propriedade mucoadesiva devido a presença de quitosana na superfície do CLN. Além disso, a quitosana favoreceu o encapsulamento da EGCG obtendose uma EE de ~96%. As concentrações inibitórias e bactericidas mínimas do CLNEGCG (33,75 a 67,5 µg/mL) foram menores do que as verificadas para a EGCG livre (250 a 2.000 µg/mL), comprovando o aumento do potencial antimicrobiano com o encapsulamento da EGCG em nanocarreadores híbridos. De acordo com esses resultados, o CLNEGCG, desenvolvido no presente trabalho, constitui um sistema com potencial para o uso tópico bucal, pois além de ser estável e apresentar propriedade de mucoadesão e morfologia adequada, apresentaram alta atividade antimicrobiana frente aos principais microorganismos envolvidos no processo carioso. / The use of oral hygiene adjuvants may be necessary to control the cariogenic microbiota of individuals with high risk and caries disease activity. Currently the most recommended antimicrobial agent is chlorhexidine digluconate (CHX) because of its broad spectrum of action and residual effect. However, this chemical has side effects. In this context, as an Epigallocatechin3gallate (EGCG), a derivative of green tea, as an alternative to synthetic antimicrobial agents. However, there is no stability over time and can oxidize rapidly. Thus, the encapsulation of EGCG in nanoparticles can increase their bioavailability and physical and chemical stability, maintain the effect of this polyphenol on the target tissue and potentiate its pharmacological efficacy. Thus, the present study aimed to develop and characterize EGCG encapsulation systems and to evaluate, in vitro, its antimicrobial activity against cariogenic microorganisms. Initially, polymer nanoparticles (NPP) and nanostructured lipid carriers (CLN) were prepared and evaluated for their in vitro antimicrobial activity against Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei microorganisms. After the analysis of the microbiological results, the CLN was selected for the encapsulation of EGCG (CLNEGCG), due to its higher antimicrobial activity. The CLNEGCG was developed by emulsion and sonication method and was characterized in relation to diameter, polydispersity index (PdI), zeta potential (PZ), encapsulation efficiency (EE), crystallinity, mucoadhesion capacity and morphology. The in vitro antimicrobial activity of EGCG and its ability to evaluate minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The diameter, PdI and PZ of the CLNs were 228nm, 0.216 and 36.53mV, respectively, and the EGCG encapsulation did not significantly alter these parameters. The CLN showed a spherical structure, stability for 330 days and a mucoadhesive property due to a presence of chitosan on the CLN surface. In addition, a chitosan favored EGCG encapsulation resulting in an EE of ~ 96%. The minimum inhibitory and bactericidal concentrations of CLGEGCG (33.75 to 67.5 µg / mL) were lower than those for free EGCG (250 to 2,000 µg / mL), with increased antimicrobial potential with EGCG encapsulation in hybrid nanocarriers. According to the results, the CLNEGCG, developed in the present study, constitutes a system with potential for oral topical use, besides being stable and possessing mucoadhesion properties, presented high antimicrobial activity against the main microorganisms involved in the carious process.

In dieser Dissertation wurden am Ferkelmodell (8-15 kg, drei Gruppen: „Kontrolle“ n=7, „Herz-Lungen-Maschine (HLM)“ n=10, „(-)-Epigallocatechin-3-Gallat (EGCG)“ n=6, die Kontrollgruppe wurde thorakotomiert, die HLM- und die EGCG-Gruppe wurden thorakotomiert und für 90 Minuten an eine HLM angeschlossen, die EGCG-Gruppe erhielt vor und nach der HLM-Zeit EGCG) drei Fragestellungen behandelt: Erstens wurde untersucht, ob die Verwendung einer HLM während eines kardiochirurgischen Eingriffes unter hypothermen Bedingungen mit nicht-pulsatilem Blutfluss und Kardioplegie die Niere schädigte. Dafür wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe mit Hämatoxylin-Eosin (HE) angefärbt und unterschiedliche Strukturen betrachtet, wobei histopathologische Veränderungen in der HLM-Gruppe auffielen. Paraklinisch fanden sich erhöhte nierenspezifische Blutwerte (Serumkreatinin und -harnstoff) in der HLM-Gruppe. Diese Ergebnisse waren hinweisend für eine funktionell relevante Schädigung der Niere durch die HLM. Unterstützend kam ein Absinken des Gesamteiweißes im Serum der HLM-Gruppe hinzu, was auf eine generelle Schädigung des Organismus durch die HLM hindeutete. Zweitens wurde betrachtet, ob die gesetzten Schäden die Merkmale eines Ischämie-Reperfusionsschadens aufwiesen. Hierzu wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe immunhistochemisch (Hypoxie-induzierter-Faktor-1-alpha-Tyramide- Signal-Amplification (HIF-1-alpha-TSA)-, Nitrotyrosin-3-Amino-9-Ethylcarbazol (Nitrotyrosin-AEC)- und Apoptose-induzierender-Faktor-Tyramide-Signal-Amplification (AIF-TSA)-Färbung) angefärbt. Dabei zeigte sich, dass sich die HLM-Gruppe in einer hypoxischen Situation befand (HIF-1-alpha Akkumulation in den Zellkernen), nitrosativem Stress ausgesetzt war (Nitrotyrosin in den Tubuli) und dass sie teilweise so stark geschädigt wurde, dass Apoptose induziert wurde (AIF in Zellkernen) – alle drei Färbungsergebnisse waren hinweisend für einen ischämischen Zustand, in dem sich die HLM-Gruppe befunden hat. Auch die Ergebnisse der durchgeführten renalen Reversed Phase High Performance Liquid Chromatography (RP-HPLC) deuteten auf ebendies hin. Unterstützend wirkten die Ergebnisse des arteriellen Laktats – die HLM-Gruppe zeigte eine Hyperlaktämie – und die Tatsache, dass einige der histologischen Merkmale für eine frühe Schockniere (welche ischämischen Ursprungs sein kann) in der HLM-Gruppe gefunden wurden. Dies alles zeigte, dass der HLM-assoziierte Nierenschaden vorrangig die Natur eines Ischämie-Reperfusionsschadens aufwies. Drittens wurde untersucht, ob EGCG diese HLM-assoziierte Schädigung abmildern konnte. Dafür wurden bei der EGCG-Gruppe alle oben genannten Untersuchungen durchgeführt. Die Ergebnisse zeigten, dass EGCG in der Dosierung 10 mg/kg eine renoprotektive Wirkung gegen die HLM-assoziierten Schäden hatte, und diese abmildern bzw. ihnen entgegenwirken konnte. Diese Ergebnisse sind für die pädiatrische Kardiochirurgie interessant, welche zum Beispiel bei der Korrektur angeborener Herzdefekte auf die Verwendung der HLM angewiesen ist. Komplikationen wie eine Nierenschädigung post operationem sind nicht selten und verkomplizieren den Verlauf. Die vorliegende Dissertation zeigt das renoprotektive Potential des in grünem Tee vorkommenden Katechins EGCG im Umfeld eines kardiochirurgischen Eingriffes mit Verwendung einer HLM. Die Wirksamkeit dieser Substanz ist wahrscheinlich darin begründet, dass sie mehr als ein Antioxidans ist. Neben seiner Radikalfänger- und Stickstoffmonoxidscavenger-Fähigkeiten ist EGCG außerdem antiapoptotisch wirksam. Derzeit wird die Kardiochirurgie mit Verwendung einer HLM in der Veterinärmedizin nur in wenigen Zentren angewendet. Es besteht für die Zukunft jedoch die Hoffnung, dass gerade für Kleintierbesitzer, die ihre Tiere als Familienmitglied betrachten, und auch für zoologische Einrichtungen bei der Diagnose eines Herzfehlers die Kardiochirurgie mit Verwendung einer HLM als Therapiemöglichkeit eine interessante und realistische Alternative zur bislang angewandten palliativen medikamentösen Therapie darstellen kann. / In this dissertation a piglet model (8-15 kg, three groups: “control” n=7, “extracorporeal circulation (EC)” n=10, “EGCG” n=6, the control-group was thoracotomized, the EC- and the EGCG-group were thoracotomized and underwent cardiopulmonary bypass (CPB) for 90 minutes, and the EGCG-group received EGCG before and after the CPB) is presented. Three questions were raised and answered: Firstly, it was investigated if the use of a CPB during cardiac surgery with hypothermia, non-pulsatile blood flow and cardioplegia caused damage to the kidney. In order to answer this question, paraffin slices of the kidney of the control- and the EC-group were stained with hematoxylin-eosin (HE), and different structures were evaluated – this staining showed histopathological changes in the EC-group. Paraclinical, the EC-group showed elevated kidney-specific blood parameters (serumcreatinine and -urea). These findings indicated a functionally relevant impairment of the kidney caused by the CPB. Supporting this, the EC-group also showed a decline of the total amount of proteins in the serum, which was suggestive of a generalized injury of the body by the CPB. Secondly, it was investigated whether the injury of the kidney might have been caused by an ischemia/reperfusion injury. Therefore, paraffin slices of the kidney of the control- and the EC-group were immunhistochemically stained (hypoxia-induced-factor-1-alpha-tyramidesignal-amplification (HIF-1-alpha-TSA)-, nitrotyrosine-3-amino-9-ethylcarbazole (nitrotyrosine-AEC)- and apoptosis-inducing-factor-tyramide-signal-amplification (AIF-TSA)-staining). These stainings revealed, that the EC-group had suffered from a hypoxemic situation (accumulation of HIF-1-alpha in the nuclei), from nitrosative stress (presence of nitrotyrosine in the tubuli), and that the kidney was partly damaged to the point of an induction of apoptosis (presence of AIF in the nuclei) – all three of these findings indicated, that the kidneys of the EC-group were put into an ischemic situation. The findings of the renal reversed phase high performance liquid chromatography (RP-HPLC) indicated the same thing. This was also supported by the blood parameter of lactate – the EC-group showed a hyperlactemia – and by some histological findings in the EC-group, which were characteristical for an early shock-kidney (which may be caused by ischemia). Taken together, these findings showed that the CPB-associated kidney injury was primarily caused by an ischemia/reperfusion injury. Thirdly, it was investigated, whether EGCG might attenuate the CPB-associated kidney injury. For that purpose, all of the investigation methods mentioned above were carried out with the samples of the EGCG-group. The findings showed that EGCG (dose: 10 mg/kg) had a protective effect on the kidney, protecting it against the damage caused by the CPB, and was able to partly attenuate this damage and partly even fully counteract it. These findings are of interest for pediatric cardiac surgery, which for example for the correction of innate heart defects depends on the use of CPB. Complications – like acute renal injury post operationem – occur frequently and complicate the recovery. This dissertation demonstrates the renoprotective potential of the natural compound EGCG in the setting of cardiac surgery with the use of CPB. The reason for the effectiveness of EGCG in this situation probably is that EGCG is more than an antioxidant. EGCG not only works as a radical- and nitric-oxide-scavenger, but also is antiapoptotic. In veterinary medicine cardiac surgery with CPB is done by few centers only. However for the future there is hope that people – especially pet owners who view their companion animals as family members, and zoos – become more and more willing to and interested in having an animal diagnosed with a heart defect treated with cardiac surgery including the use of an CPB, instead of – like its usually done nowadays – only giving palliative medication to the animal.

The polymeric gel-forming mucins provide the structural framework of saliva and the mucus barriers that cover the mucosal surfaces of the alimentary canal. Dietary compounds may influence the barrier properties of these protective layers. The effects of green tea polyphenols, which have many health benefits but have low bioavailability and contribute to the astringency of green tea, on the structural properties of the mucins in the alimentary canal are investigated here. Using well characterised, highly purified salivary mucins MUC5B and MUC7, and porcine gastric mucins, the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) on mucins were studied here. Using rate-zonal centrifugation coupled to agarose gel electrophoresis, atomic force microscopy and particle tracking microrheology, EGCG, at concentrations found in a cup of green tea, caused increased aggregation of MUC5B in human whole saliva, and increased aggregation and viscosity of purified MUC5B. It was revealed using recombinant proteins of the N- and C-terminal regions of MUC5B that EGCG had these effects by aggregating the terminal globular protein domains of MUC5B. In contrast, MUC5B trypsin-resistant high molecular weight glycopeptides were not aggregated by EGCG, demonstrating that the oligosaccharide-rich, highly-glycosylated regions of mucins are not involved in the EGCG-induced aggregation of mucins. EGCG also caused the majority of MUC7 in human whole saliva to aggregate, and purified MUC7 also showed substantial aggregation in the presence of EGCG.Porcine gastric mucins were also used in order to model human gastric mucins. First, the identity of the porcine gastric mucins was explored using tandem mass spectrometry and immunohistochemistry. This revealed that Muc5ac was expressed by the surface epithelium and was the prominent mucin in porcine gastric mucus. Muc6 was expressed by gastric submucosal glands, but was not a major component of the secreted mucus barrier. Porcine Muc5ac and Muc6 were shown to be aggregated by EGCG. These data demonstrate that mucins from both saliva and the stomach are substantially altered by EGCG. This may contribute to the astringency and low bioavailability of EGCG. In contrast, the green tea polyphenol epicatechin (EC) did not cause aggregation of salivary mucins or porcine gastric mucins, suggesting that the galloyl ring of EGCG (which is absent in EC) is important for its aggregation of mucins, and that EC has different mechanisms of astringency. The structure of the mucins in the alimentary canal was studied using Raman spectroscopy, Raman optical activity (ROA) and Tip-enhanced Raman spectroscopy (TERS). The secondary structure of the oligosaccharide-rich regions of mucins was shown to be largely disordered, with some contribution of poly-proline II helix. The N- and C-terminal regions of MUC5B were largely β-sheet in structure, with some disordered structure also present in the C-terminal region. Raman spectroscopy could reliably distinguish between MUC5B glycoforms, demonstrating the sensitivity of this technique to mucin glycosylation and secondary structure. The first TERS spectra along the length of a MUC5B chain are reported, and suggest that patterns may exist in the glycosylation of MUC5B. Therefore, Raman spectroscopies are novel tools that shed new light on mucin structure and in future may be useful for studying the changes to mucin structure during interactions, such as those with polyphenols.

Introduction: This thesis investigates the effect of isolated epigallocatechin-3-gallate on weight reduction in the adult population. An overview of Czech and foreign literature was provided on topics of obesity, the possibility of using green tea in its treatment and the positive effects of green tea on human health, followed by a systematic review and meta-analysis. Methodology: Using a three-phase systematic search strategy, published studies were searched in the Scopus database using predefined keywords. The aim of the search was to find randomized placebo-controlled studies lasting at least 8 weeks, which examined the effect of isolated EGCG in the absence of other active substances on the change of body parameters. The extracted data from the primary studies were then evaluated using meta-analysis and forest plot. As a result, a standardized mean difference with a 95 % confidence interval was calculated using a random effect model. Results: Six1 studies met the inclusion criteria, one2 of which was excluded due to insufficient evaluation in the critical appraisal of methodological quality. The remaining 5 studies provided a total sample of 325 participants who were diagnosed with a change in BMI induced by EGCG consumption. The result was a standardized average difference of -0.01, 95 % CI:...

L’altération de la barrière hématoencéphalique (BHE) par les cellules tumorales et les cellules immunes circulantes peut mener à la neuroinflammation. Les cellules leucémiques promyélocytaires HL-60 sont un excellent modèle pour étudier et comprendre les mécanismes de signalisation moléculaires qui caractérisent le développement tumoral et métastatique. La cancérogenèse peut s’accompagner de modulations de l’expression de biomarqueurs tels que la cyclooxygénase-2 et la métalloprotéase-9. Les recherches décrites dans ce mémoire relatent l’analyse des biomarqueurs inflammatoires et invasifs régulés lors de la différenciation induite par le PMA des cellules HL-60 en macrophages. Le statut de différenciation cellulaire pourrait avoir un impact sur les gènes cibles de la voie NF-κB. Nous émettons l’hypothèse que le PMA active la voie NF-κB et que cette signalisation peut être renversée par l’(-)-épigallocatéchine-gallate (EGCG). En effet, une régulation à la hausse de l’expression de plusieurs gènes combinée à la diminution de l’expression d’IκB mettent en évidence l’implication de la voie NF-κB dans l’activation des mécanismes pro-inflammatoires et pro-invasifs. Les mêmes observations sont faites dans les cellules différenciées appelées «macrophages-like». L’EGCG, un polyphénol dérivé du thé vert, a un potentiel chimiopréventif. Il est capable d’inhiber la signalisation moléculaire passant par la voie NF-κB dans les cellules HL-60 traitées simultanément par l’EGCG et le PMA, mais pas dans les cellules «macrophages-like». Cette différence peut s’expliquer par une modulation de l’expression du récepteur de surface cellulaire de l’EGCG, le récepteur à la laminine de 67 kDa, et de son précurseur de 37 kDa. Collectivement, nos résultats montrent que le statut de différenciation des cellules promyélocytaires HL-60 concorde avec l’activation des mécanismes favorisant le développement d’un cancer et des métastases. Cet effet peut être prévenu par l’utilisation d’agents naturels tel l’EGCG. Le ciblage de biomarqueurs liés au statut de différenciation des cellules tumorales impliquées dans la perturbation de la barrière hématoencéphalique qui cause la neuroinflammation permettrait l’avancement des connaissances dans la prévention de la cancérogenèse. / Blood-brain barrier (BBB) disruption by circulating tumor and immune cells leads to secondary inflammatory infections. Promyelocytic HL-60 cells represent an excellent model to study and to get a better understanding of the molecular signaling mechanisms involved in carcinogenesis and metastasis. The research described in this thesis shows the analysis of several inflammatory and invasive biomarkers regulated during PMA-induced differentiation of promyelocytic HL-60 cells into macrophages. Carcinogenesis involves some modifications in the expression of biomarkers such as cyclooxygenase-2 and matrix metalloprotease-9. The differentiation status could have an impact on the NF-κB signaling pathway that regulates the target genes, given that these target genes expression varies during cell differentiation. We hypothesize that the activation of the NF-κB pathway by PMA can be reverse by (-)-epigallocatechin-gallate (EGCG). Indeed, the up-regulation of downstream genes combined with the down-regulation of IκB expression showed the significant implication of the NF-κB signaling pathway to activate pro-inflammatory and pro-invasive mechanisms linked to carcinogenesis. The same evidence exhibits in the differentiated cells called «macrophages-like». Moreover, the green tea polyphenol, EGCG, shows chemopreventive property since it better inhibited NF-κB signaling in cells treated simultaneously with EGCG and PMA compared to the «macrophages-like». This difference could be due, in part, to the down-regulation of the 67 kDa laminin receptor, known to be the non-integrin membrane receptor for EGCG. All together, our results suggest that the differentiation status of promyelocytic cells is linked to the activation of mechanisms involved in carcinogenesis and metastasis. These phenomena can be prevented by using natural agents such as EGCG. Targeting the specific biomarkers linked to the differentiation status of tumor cells and involved in the disruption of the BBB may help reduce secondary neuroinflammation and enable the advancement of knowledge towards carcinogenesis prevention.

L’altération de la barrière hématoencéphalique (BHE) par les cellules tumorales et les cellules immunes circulantes peut mener à la neuroinflammation. Les cellules leucémiques promyélocytaires HL-60 sont un excellent modèle pour étudier et comprendre les mécanismes de signalisation moléculaires qui caractérisent le développement tumoral et métastatique. La cancérogenèse peut s’accompagner de modulations de l’expression de biomarqueurs tels que la cyclooxygénase-2 et la métalloprotéase-9. Les recherches décrites dans ce mémoire relatent l’analyse des biomarqueurs inflammatoires et invasifs régulés lors de la différenciation induite par le PMA des cellules HL-60 en macrophages. Le statut de différenciation cellulaire pourrait avoir un impact sur les gènes cibles de la voie NF-κB. Nous émettons l’hypothèse que le PMA active la voie NF-κB et que cette signalisation peut être renversée par l’(-)-épigallocatéchine-gallate (EGCG). En effet, une régulation à la hausse de l’expression de plusieurs gènes combinée à la diminution de l’expression d’IκB mettent en évidence l’implication de la voie NF-κB dans l’activation des mécanismes pro-inflammatoires et pro-invasifs. Les mêmes observations sont faites dans les cellules différenciées appelées «macrophages-like». L’EGCG, un polyphénol dérivé du thé vert, a un potentiel chimiopréventif. Il est capable d’inhiber la signalisation moléculaire passant par la voie NF-κB dans les cellules HL-60 traitées simultanément par l’EGCG et le PMA, mais pas dans les cellules «macrophages-like». Cette différence peut s’expliquer par une modulation de l’expression du récepteur de surface cellulaire de l’EGCG, le récepteur à la laminine de 67 kDa, et de son précurseur de 37 kDa. Collectivement, nos résultats montrent que le statut de différenciation des cellules promyélocytaires HL-60 concorde avec l’activation des mécanismes favorisant le développement d’un cancer et des métastases. Cet effet peut être prévenu par l’utilisation d’agents naturels tel l’EGCG. Le ciblage de biomarqueurs liés au statut de différenciation des cellules tumorales impliquées dans la perturbation de la barrière hématoencéphalique qui cause la neuroinflammation permettrait l’avancement des connaissances dans la prévention de la cancérogenèse. / Blood-brain barrier (BBB) disruption by circulating tumor and immune cells leads to secondary inflammatory infections. Promyelocytic HL-60 cells represent an excellent model to study and to get a better understanding of the molecular signaling mechanisms involved in carcinogenesis and metastasis. The research described in this thesis shows the analysis of several inflammatory and invasive biomarkers regulated during PMA-induced differentiation of promyelocytic HL-60 cells into macrophages. Carcinogenesis involves some modifications in the expression of biomarkers such as cyclooxygenase-2 and matrix metalloprotease-9. The differentiation status could have an impact on the NF-κB signaling pathway that regulates the target genes, given that these target genes expression varies during cell differentiation. We hypothesize that the activation of the NF-κB pathway by PMA can be reverse by (-)-epigallocatechin-gallate (EGCG). Indeed, the up-regulation of downstream genes combined with the down-regulation of IκB expression showed the significant implication of the NF-κB signaling pathway to activate pro-inflammatory and pro-invasive mechanisms linked to carcinogenesis. The same evidence exhibits in the differentiated cells called «macrophages-like». Moreover, the green tea polyphenol, EGCG, shows chemopreventive property since it better inhibited NF-κB signaling in cells treated simultaneously with EGCG and PMA compared to the «macrophages-like». This difference could be due, in part, to the down-regulation of the 67 kDa laminin receptor, known to be the non-integrin membrane receptor for EGCG. All together, our results suggest that the differentiation status of promyelocytic cells is linked to the activation of mechanisms involved in carcinogenesis and metastasis. These phenomena can be prevented by using natural agents such as EGCG. Targeting the specific biomarkers linked to the differentiation status of tumor cells and involved in the disruption of the BBB may help reduce secondary neuroinflammation and enable the advancement of knowledge towards carcinogenesis prevention.

In dieser Dissertation wurden am Ferkelmodell (8-15 kg, drei Gruppen: „Kontrolle“ n=7, „Herz-Lungen-Maschine (HLM)“ n=10, „(-)-Epigallocatechin-3-Gallat (EGCG)“ n=6, die Kontrollgruppe wurde thorakotomiert, die HLM- und die EGCG-Gruppe wurden thorakotomiert und für 90 Minuten an eine HLM angeschlossen, die EGCG-Gruppe erhielt vor und nach der HLM-Zeit EGCG) drei Fragestellungen behandelt: Erstens wurde untersucht, ob die Verwendung einer HLM während eines kardiochirurgischen Eingriffes unter hypothermen Bedingungen mit nicht-pulsatilem Blutfluss und Kardioplegie die Niere schädigte. Dafür wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe mit Hämatoxylin-Eosin (HE) angefärbt und unterschiedliche Strukturen betrachtet, wobei histopathologische Veränderungen in der HLM-Gruppe auffielen. Paraklinisch fanden sich erhöhte nierenspezifische Blutwerte (Serumkreatinin und -harnstoff) in der HLM-Gruppe. Diese Ergebnisse waren hinweisend für eine funktionell relevante Schädigung der Niere durch die HLM. Unterstützend kam ein Absinken des Gesamteiweißes im Serum der HLM-Gruppe hinzu, was auf eine generelle Schädigung des Organismus durch die HLM hindeutete. Zweitens wurde betrachtet, ob die gesetzten Schäden die Merkmale eines Ischämie-Reperfusionsschadens aufwiesen. Hierzu wurden Paraffinschnitte der Niere aus der Kontroll- und der HLM-Gruppe immunhistochemisch (Hypoxie-induzierter-Faktor-1-alpha-Tyramide- Signal-Amplification (HIF-1-alpha-TSA)-, Nitrotyrosin-3-Amino-9-Ethylcarbazol (Nitrotyrosin-AEC)- und Apoptose-induzierender-Faktor-Tyramide-Signal-Amplification (AIF-TSA)-Färbung) angefärbt. Dabei zeigte sich, dass sich die HLM-Gruppe in einer hypoxischen Situation befand (HIF-1-alpha Akkumulation in den Zellkernen), nitrosativem Stress ausgesetzt war (Nitrotyrosin in den Tubuli) und dass sie teilweise so stark geschädigt wurde, dass Apoptose induziert wurde (AIF in Zellkernen) – alle drei Färbungsergebnisse waren hinweisend für einen ischämischen Zustand, in dem sich die HLM-Gruppe befunden hat. Auch die Ergebnisse der durchgeführten renalen Reversed Phase High Performance Liquid Chromatography (RP-HPLC) deuteten auf ebendies hin. Unterstützend wirkten die Ergebnisse des arteriellen Laktats – die HLM-Gruppe zeigte eine Hyperlaktämie – und die Tatsache, dass einige der histologischen Merkmale für eine frühe Schockniere (welche ischämischen Ursprungs sein kann) in der HLM-Gruppe gefunden wurden. Dies alles zeigte, dass der HLM-assoziierte Nierenschaden vorrangig die Natur eines Ischämie-Reperfusionsschadens aufwies. Drittens wurde untersucht, ob EGCG diese HLM-assoziierte Schädigung abmildern konnte. Dafür wurden bei der EGCG-Gruppe alle oben genannten Untersuchungen durchgeführt. Die Ergebnisse zeigten, dass EGCG in der Dosierung 10 mg/kg eine renoprotektive Wirkung gegen die HLM-assoziierten Schäden hatte, und diese abmildern bzw. ihnen entgegenwirken konnte. Diese Ergebnisse sind für die pädiatrische Kardiochirurgie interessant, welche zum Beispiel bei der Korrektur angeborener Herzdefekte auf die Verwendung der HLM angewiesen ist. Komplikationen wie eine Nierenschädigung post operationem sind nicht selten und verkomplizieren den Verlauf. Die vorliegende Dissertation zeigt das renoprotektive Potential des in grünem Tee vorkommenden Katechins EGCG im Umfeld eines kardiochirurgischen Eingriffes mit Verwendung einer HLM. Die Wirksamkeit dieser Substanz ist wahrscheinlich darin begründet, dass sie mehr als ein Antioxidans ist. Neben seiner Radikalfänger- und Stickstoffmonoxidscavenger-Fähigkeiten ist EGCG außerdem antiapoptotisch wirksam. Derzeit wird die Kardiochirurgie mit Verwendung einer HLM in der Veterinärmedizin nur in wenigen Zentren angewendet. Es besteht für die Zukunft jedoch die Hoffnung, dass gerade für Kleintierbesitzer, die ihre Tiere als Familienmitglied betrachten, und auch für zoologische Einrichtungen bei der Diagnose eines Herzfehlers die Kardiochirurgie mit Verwendung einer HLM als Therapiemöglichkeit eine interessante und realistische Alternative zur bislang angewandten palliativen medikamentösen Therapie darstellen kann. / In this dissertation a piglet model (8-15 kg, three groups: “control” n=7, “extracorporeal circulation (EC)” n=10, “EGCG” n=6, the control-group was thoracotomized, the EC- and the EGCG-group were thoracotomized and underwent cardiopulmonary bypass (CPB) for 90 minutes, and the EGCG-group received EGCG before and after the CPB) is presented. Three questions were raised and answered: Firstly, it was investigated if the use of a CPB during cardiac surgery with hypothermia, non-pulsatile blood flow and cardioplegia caused damage to the kidney. In order to answer this question, paraffin slices of the kidney of the control- and the EC-group were stained with hematoxylin-eosin (HE), and different structures were evaluated – this staining showed histopathological changes in the EC-group. Paraclinical, the EC-group showed elevated kidney-specific blood parameters (serumcreatinine and -urea). These findings indicated a functionally relevant impairment of the kidney caused by the CPB. Supporting this, the EC-group also showed a decline of the total amount of proteins in the serum, which was suggestive of a generalized injury of the body by the CPB. Secondly, it was investigated whether the injury of the kidney might have been caused by an ischemia/reperfusion injury. Therefore, paraffin slices of the kidney of the control- and the EC-group were immunhistochemically stained (hypoxia-induced-factor-1-alpha-tyramidesignal-amplification (HIF-1-alpha-TSA)-, nitrotyrosine-3-amino-9-ethylcarbazole (nitrotyrosine-AEC)- and apoptosis-inducing-factor-tyramide-signal-amplification (AIF-TSA)-staining). These stainings revealed, that the EC-group had suffered from a hypoxemic situation (accumulation of HIF-1-alpha in the nuclei), from nitrosative stress (presence of nitrotyrosine in the tubuli), and that the kidney was partly damaged to the point of an induction of apoptosis (presence of AIF in the nuclei) – all three of these findings indicated, that the kidneys of the EC-group were put into an ischemic situation. The findings of the renal reversed phase high performance liquid chromatography (RP-HPLC) indicated the same thing. This was also supported by the blood parameter of lactate – the EC-group showed a hyperlactemia – and by some histological findings in the EC-group, which were characteristical for an early shock-kidney (which may be caused by ischemia). Taken together, these findings showed that the CPB-associated kidney injury was primarily caused by an ischemia/reperfusion injury. Thirdly, it was investigated, whether EGCG might attenuate the CPB-associated kidney injury. For that purpose, all of the investigation methods mentioned above were carried out with the samples of the EGCG-group. The findings showed that EGCG (dose: 10 mg/kg) had a protective effect on the kidney, protecting it against the damage caused by the CPB, and was able to partly attenuate this damage and partly even fully counteract it. These findings are of interest for pediatric cardiac surgery, which for example for the correction of innate heart defects depends on the use of CPB. Complications – like acute renal injury post operationem – occur frequently and complicate the recovery. This dissertation demonstrates the renoprotective potential of the natural compound EGCG in the setting of cardiac surgery with the use of CPB. The reason for the effectiveness of EGCG in this situation probably is that EGCG is more than an antioxidant. EGCG not only works as a radical- and nitric-oxide-scavenger, but also is antiapoptotic. In veterinary medicine cardiac surgery with CPB is done by few centers only. However for the future there is hope that people – especially pet owners who view their companion animals as family members, and zoos – become more and more willing to and interested in having an animal diagnosed with a heart defect treated with cardiac surgery including the use of an CPB, instead of – like its usually done nowadays – only giving palliative medication to the animal.