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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.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and occurs in ~1 of every 700 live births. Individuals with DS present craniofacial abnormalities, specifically an undersized, dysmorphic mandible which may lead to difficulty with eating, breathing, and speech. Using the Ts65Dn DS mouse model, which mirrors these phenotypes and contains three copies of ~50% Hsa21 homologues, our lab has traced the mandibular deficit to a neural crest cell (NCC) deficiency in the first pharyngeal arch (PA1 or mandibular precursor) at embryonic day 9.5 (E9.5). At E9.5, the PA1 is reduced in size and contains fewer cells due to fewer NCC populating the PA1 from the neural tube (NT) as well as reduced cellular proliferation in the PA1. We hypothesize that both the deficits in NCC migration and proliferation may cause the reduction in size of the PA1. To identify potential genetic mechanisms responsible for trisomic PA1 deficits, we generated RNA-sequence (RNA-seq) data from euploid and trisomic E9.25 NT and E9.5 PA1 (time points occurring before and after observed deficits) using a next-generation sequencing platform. Analysis of RNA-seq data revealed differential trisomic expression of 53 genes from E9.25 NT and 364 genes from E9.5 PA1, five of which are present in three copies in Ts65Dn. We also further analyzed the data to find that fewer alternative splicing events occur in trisomic tissues compared to euploid tissues and in PA1 tissue compared to NT tissue. In a subsequent study, to test gene-specific treatments to rescue PA1 deficits, we targeted Dyrk1A, an overexpressed DS candidate gene implicated in many DS phenotypes and predicted to cause the NCC and PA1 deficiencies. We hypothesize that treatment of pregnant Ts65Dn mothers with Epigallocatechin gallate (EGCG), a known Dyrk1A inhibitor, will correct NCC deficits and rescue the undersized PA1 in trisomic E9.5 embryos. To test our hypothesis, we treated pregnant Ts65Dn mothers with EGCG from either gestational day 7 (G7) to G8 or G0 to G9.5. Our study found an increase in PA1 volume and NCC number in trisomic E9.5 embryos after treatment on G7 and G8, but observed no significant improvements in NCC deficits following G0-G9.5 treatment. We also observed a developmental delay of embryos from trisomic mothers treated with EGCG from G0-G9.5. Together, these data show that timing and sufficient dosage of EGCG treatment is most effective during the developmental window the few days before NCC deficits arise, during G7 and G8, and may be ineffective or harmful when administered at earlier developmental time points. Together, the findings of both studies offer a better understanding of potential mechanisms altered by trisomy as well as preclinical evidence for EGCG as a potential prenatal therapy for craniofacial disorders linked to DS.

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:...

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Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is the result of trisomy of human chromosome 21 (Hsa 21) and occurs in approximately 1/700 live births. Mouse models of DS have been crucial in understanding the gene-phenotype relationships that underlie many DS anomalies. The Ts65Dn mouse model, trisomic for half of the Hsa 21 orthologs replicates many DS phenotypes including craniofacial alterations such as a small, dysmorphic mandible, midface, and maxilla. Other mouse models, such as the Ts1Rhr which contains a triplication of 33 Hsa 21 orthologs, have been used to better understand the genes responsible for craniofacial alterations. Our laboratory has demonstrated that the postnatal mandibular phenotype found in Ts65Dn mice can be traced back to an original neural crest cell (NCc) deficit in the developing first pharyngeal arch (PA1) at embryonic day 9.5 (E9.5). Furthermore, evidence suggested that both a proliferation deficit in the PA1 and a migration deficit in the NCC from the neural tube (NT) could be the mechanism behind this deficit. However, the molecular mechanisms behind these deficits remain to be elucidated. Due to the involvement of the Hsa 21 genes DYRK1A and RCAN1 in regulation of signaling pathways including NFATc (NFAT2), a transcription factor known to influence cellular proliferation and, later, bone development, we hypothesized that dysregulation of these genes could underlie the cellular deficit in the PA1. Furthermore, we hypothesized that targeting Dyrk1a by decreasing activity or available protein could ameliorate the established deficits. Through the use of RNA isolation techniques and cell culture systems of cell from the PA1 and NT of E9.5 Ts65Dn, Ts1Rhr, and control embryos, we established that trisomic genes Dyrk1a and Rcan1 ara dysregulated in both structures and that these two genes may interact. Furthermore, we established that a proliferation deficit in the Ts65Dn PA1 and a migration deficit in the Ts65Dn PA1 and NT exists at E9.5 and can be rescued to euploid levels in vitro with the addition of the Dyrk1a inhibitor, EGCG, a green tea polyphenol. We also confirmed that harmine, a more highly studied and specific Dyrk1a inhibitor, is capable of similar effects on proliferation of PA1 cell from E9.5 Ts65Dn embryos. Furthermore, when Ts65Dn pregnant mothers were treated with EGCG in vivo, the cellular deficit found in the developing E9.5 embryonic PA1 was rescued to near euploid volume and NCC number. Treatment with EGCG did not adversely impact litter size or embryonic development. Interestingly, euploid embryonic volume increased with EGCG treatment. Expression analysis of the E9.5 PA1 of EGCG treated Ts65Dn and control embryos revealed dysregulation of several genes involved in craniofacial and developmental pathways including Dyrk1a, Rcan1, Ets2 and members of the sonic hedgehog pathways. Our novel results provide a foundation for better understanding the molecular mechanisms of craniofacial development and may provide evidence-based therapeutic options to improve the quality of life for individuals with DS.

Keloid disease (KD) is a benign fibroproliferative dermal disease of unknown aetiopathogenesis that occurs in genetically susceptible individuals. KD shows high heterogeneity within the lesion, harbouring different immune cell profiles, which are poorly characterised in KD at different lesional sites. Although, it has long been appreciated that chronic inflammation and dermal fibrosis is associated with other fibrotic diseases (e.g. scleroderma), this link has not, yet, been established in KD through direct evidence. Additionally, the limited availability of a simple KD animal model has hindered our understanding of the underlying pathogenesis of KD. Therefore, the main objectives were a) to identify and profile different immune cells at defined KD lesional and histological sites, b) to further characterize the potential contribution of viral particles in KD by investigating the gene and protein expression profile of toll like receptors that recognise viral particles in KD, and c) to develop an optimized long-term serum-free organ culture (OC) model for KD research as a tool for probing novel hypotheses in KD pathobiology deduced from a) and b) and to also validate the reliability and instructiveness of this novel ex vivo KD model with conventional (e.g. dexamethasone) and potential future anti-KD compounds [(-)-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) knock-down by siRNA]. To achieve above objectives, different cellular and molecular techniques were applied. Immune profiling of KD (chapter 2) at defined lesional and histological sites generated the first comprehensive analysis of KD-associated inflammatory cell infiltrates. This work demonstrated for the first time the presence of specific type of chronic inflammation in KD that resembles the formation of tertiary lymphoid tissues (TLTs) (in 14.7%, out of 68 KD cases). Although, these TLTs are not strictly linked to defined lesional sites within the KD, they are similar in structure to mucosa-associated lymphoid tissue (MALT). Therefore, we named this phenomenon as keloid-associated lymphoid tissue (KALT). Immunophenotyping of KD lesional sites also showed a predominance of T-cells, B-cells, M2 macrophages and OX40L+ degranulated mast cells in intralesional and perilesional sites of KD compared to normal skin and normal scar tissue. In the epidermis, Langerhans cells showed no changes, whereas the intra-epidermal T-cells were significantly increased in both the intralesional and perilesional sites of KD with an increased CD4:CD8 ratio. Intra-epidermal B-cells were only rarely found in KD. Interestingly, there was no significant statistical difference between intralesional and perilesional sites of KD immunophenotyping. These abnormal immune profiles suggest the persistence of non-resolving inflammation presence towards unknown stimuli, which require further investigation. The chronic inflammation could be followed by a reparative phase in a repetitive manner leading to KD formation. Evaluation of toll-like receptor (TLR) gene and protein expression in KD showed a significant increase in the expression of intra-epidermal TLR-6, -7 and dermal TLR-8. Since these TLRs are typically up regulated during anti-viral responses, these results further support the hypothesis that certain viruses or yet unidentified ligand may play a role in KD pathogenesis (chapter 3). A successful long-term, serum-free keloid OC model was established using a 4 mm sized punch biopsy embedded in collage matrix as air liquid interface in supplemented William’s E medium for up to 6 weeks (Chapter 4).

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.

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