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Introduction: At our post-genomic era the studies of epigenetic regulation constitutes one of the tools for understanding the function of genes. Epigenetic regulation can directly control the temporal and spatial gene activity or silencing. The molecular basis of these regulations are DNA bases modifications, chromatin remodeling and RNA interference. At the same time, these mechanisms have a special way of transferring genetic information to subsequent generations called epigenetic inheritance. It has been proven epigenetic deregulation of certain genes as cause for many disease. For this reason, the study of epigenome HLA genes seems particularly important because these genes play a fundamental role in regulating the immune system. Aims: The aim of this work is to create a description of epigenetic modifications within families. It is an analysis of histone modifications and DNA methylation in the promoter region of the gene HLA DQA1. The aim was also to compare the differences in epigenetic modifications between alleles and compared the differences in these modifications between generations. The results will be compared with the analysis of the level of expression of the gene HLA DQA1. Methods: From collected peripheral blood of donors were isolated DNA, RNA, and leukocytes. DNA was used for...

Το σύστημα του Συμπληρώματος απαρτίζεται από μια ομάδα πρωτεϊνών του πλάσματος και κυτταρικών υποδοχέων που παίζουν σημαντικό ρόλο στην άμυνα του ξενιστή μέσω της αλληλεπίδρασής τους τόσο με συστατικά της φυσικής, όσο και της προσαρμοστικής ανοσίας. Παράλληλα, μια ομάδα από ρυθμιστικές πρωτεΐνες του πλάσματος και της κυτταρικής μεμβράνης προστατεύει τα κύτταρα του ξενιστή από την αυτόλογη δράση του Συμπληρώματος. Η σύνθεση του τελικού λυτικού συμπλόκου MAC, που οδηγεί σε κυτταρόλυση, ρυθμίζεται από τις ρυθμιστικές πρωτεΐνες : CD59, vitronectin και clusterin. Μέχρι σήμερα οι ρυθμιστικές πρωτεΐνες της λυτικής οδού του Συμλπηρώματος έχουν ελάχιστα χαρακτηριστεί σε σπονδυλωτά πέραν των θηλαστικών. Οι οστεϊχθύες αποτελούν ένα σημαντικό μοντέλο, καθώς είναι οι μόνοι οργανισμοί που εμφανίζουν ένα πλήρως αναπτυγμένο και διευρυμένο σύστημα συμπληρώματος, μέσω διπλασιασμού πολλών γονιδίων του Συμπληρώματος. Προκειμένου να μελετηθεί η παρουσία και η φυλογενετική εξέλιξη του ρυθμιστικού μορίου της clusterin, το οποίο παρεμποδίζει την πρόσδεση του συμπλόκου C5b-7 στην μεμβράνη του κυττάρου-στόχου, με αποτέλεσμα την αναστολή της κυτταρόλυσης, έγινε κλωνοποίηση και χαρακτηρισμός του γονιδίου της clusterin στην ιριδίζουσα πέστροφα (Oncorhynchus mykiss irideus). Απομονώθηκαν δύο ισομορφές του γονιδίου της clusterin, clusterin-1 (TCLU-1) και clusterin-2 (TCLU-2) από cDNA βιβλιοθήκη ήπατος πέστροφας, με χρήση ειδικών ολιγονουκλεοτιδίων. Οι προκύπτουσες αμινοξικές αλληλουχίες των ισομορφών clusterin-1 και clusterin-2 παρουσιάζουν 89% ταυτοσημία μεταξύ τους, καθώς και 40 και 38% ταυτοσημία με την clusterin του ανθρώπου, αντίστοιχα. Η μοριακή δομή των συναγομένων πρωτεϊνών αντιστοιχεί στην α΄ αλυσίδα της clusterin και ομοιάζει με εκείνη των θηλαστικών. Γονιδιωματικοί κλώνοι απομονώθηκαν και για τις δύο ισομορφές και μελετήθηκε η οργάνωση εξωνίων-εσωνίων των αντίστοιχων γονιδίων. Η οργάνωση των γονιδίων clusterin-1 και clusterin-2, με τα μέχρι στιγμής δεδομένα, δείχνει αντιστοιχία εξωνίων - εσωνίων με το γονίδιο της clusterin του ανθρώπου, στην περιοχή ομολογίας τους. Ως προς το μέγεθος, τα αντίστοιχα εξώνια φαίνονται συντηρημένα, ενώ τα εσώνια ποικίλουν, εμφανίζοντας μικρότερο μέγεθος συγκριτικά με εκείνα της clusterin του ανθρώπου. Ανάλυση κατά Southern έδειξε ότι η clusterin απαντάται σε δύο τουλάχιστον αντίγραφα στο γονιδίωμα της πέστροφας. Τα γονίδια clusterin-1 και clusterin-2 εμφανίζουν διαφορική έκφραση στο επίπεδο του mRNA σε διάφορους ιστούς πέστροφας, όπως διαπιστώθηκε με RT-PCR. Παρατηρήθηκαν υψηλά επίπεδα mRNA του γονιδίου TCLU-1 στο ήπαρ και στο σπλήνα, ενώ η έκφραση του γονιδίου TCLU-2 παρατηρήθηκε αυξημένη στην καρδιά και στο έντερο, χαμηλή στο ήπαρ και στο σπλήνα δεν ανιχνεύτηκε. Τέλος, η φυλογενετική ανάλυση των πρωτεϊνών της clusterin από διάφορους οργανισμούς εμφάνισε την TCLU-1 να ομαδοποιείται με την TCLU-2, ενώ η όλη υποομάδα προηγείται φυλογενετικά των ορθόλογων πρωτεϊνών του pufferfish και του zebrafish και αυτές των ορθόλογων των άλλων οργανισμών που αναλύθηκαν. Συμπερασματικά, το γονίδιο της clusterin ανιχνεύεται στην ιριδίζουσα πέστροφα, σε δύο τουλάχιστον ισομορφές και παρουσιάζει ομολογία με την clusterin του ανθρώπου, τόσο στο επίπεδο οργάνωσης του γονιδίου, όσο και στο επίπεδο της αμινοξικής αλληλουχίας και της προκύπτουσας δομής. Η παρουσία ισομορφών από εναλλακτικό μάτισμα που ισχύει στην clusterin του άνθρωπου δεν έγινε δυνατόν να πιστοποιηθεί. / The complement system consists of a group of plasma proteins and cell receptors that play a critical role in host defense, by interacting with components of both innate and adaptive immunity. Α group of plasma and cell membrane regulatory proteins protects the host cells from the autologous complement activation. The conformation of the lytic complex MAC, which results to the cytolysis, is regulated by the regulatory proteins: CD59, clusterin και vitronectin. Up to date, the regulatory proteins of MAC complex are not well characterized in low vertebrates. Bony fish represent an attractive model for studies on complement system, as they are, the only organisms that reveal a completely developed and extended system, via duplication and functional differentiation of many genes of the complement system. In order to elucidate the presence and the evolution of the clusterin regulatory molecule, which impedes the binding of C5b-7 complex to the membrane of the target cell, inhibiting cytolysis, we report here the cloning and characterization of the clusterin gene in rainbow trout (Oncorhynchus mykiss irideus). Two isoforms of clusterin gene, clusterin-1 (TCLU-1) and clusterin-2 (TCLU-2) were isolated from a trout liver cDNA library, by PCR using specific oligonoucleotides. The deduced amino-acid sequences of the isoforms TCLU-1 and TCLU-2 show 89% identity to each other, as well as 40 and 38% identity to human clusterin, respectively. The domain structure of the deduced proteins corresponds to the α΄ chain of clusterin and resembles to that of mammalian. Genomic clones were isolated for the two isoforms and intron-exon organization of corresponding genes was clarified. The organization of the clusterin genes TCLU-1 and TCLU-2, as the partial data have shown, is in line with the intron-exon organization of the human clusterin gene, at their homology region. Regarding the length, the corresponding exons seem to be conserved, but the introns in trout clusterin genes are shorter than human clusterin. Southern blot analysis has shown that clusterin can be found as two copies at least, in the trout genome. Also, the genes TCLU-1 and TCLU-2 reveal differential expression, at mRNA level, among several trout tissues, as it was found out by RT-PCR analysis. High levels of TCLU-1 mRNA are detected in liver and spleen. On the other hand, TCLU-2 is expressed mainly in heart and intestine, while is not detected any expression in spleen. Finally, the phylogenetic analysis of the clusterin proteins from various organisms, showed grouping of TCLU-1 and TCLU-2, while this subgroup precedes evolutionary the clusterin orthologs of pufferfish, zebrafish and the other examined organisms, afterwards. Conclusively, the clusterin gene in rainbow trout is detected as two isoforms and shows homology with human clusterin, not only at the level of gene organization, but also at the level of amino-acid sequence and domain architecture. The alternative splicing of human clusterin was not able to be certified in trout.

Artemisia annua L. is a well-known medicinal herb used in Traditional Chinese Medicine for many centuries and contains artemisinin, which exhibits antimalarial properties [1] and is a potential treatment for SARS-CoV-2 [2]. However, the neurological effect of A. annua and its compounds are poorly understood. We hypothesize that A. annua extracts and artemisinin will provide protection against oxidative stress in mouse neuronal Neuro2A cells. Superoxide dismutase 1 (SOD1) is an anti-oxidant enzyme that protects against oxidative damage [3]. Growth Arrest and DNA Damage-inducible 45 alpha (Gadd45a) is involved in DNA repair and is a common biomarker for DNA damage[4]. SOD1 and Gadd45a mRNA expression was measured through RT-qPCR and analyzed using the 2-∆∆Ct method. Cell viability was measured using NucBlue, Annexin V, and propidium iodide to differentiate between apoptosis and necrosis. Cell counts were quantified using ImageJ (NIH). Our results showed that lower concentrations of 100 µg/mL A. annua treatment significantly reduced apoptotic cell death during oxidative stress. We also saw a general trend of decreasing Gadd45a mRNA expression at lower concentrations of A. annua treatment and a significant decrease in SOD1 mRNA levels from 0 to 100 µg/mL. This study suggests that lower concentrations of A. annua treatment prior to oxidative stress can increase cell viability and could protect Neuro2A cells from oxidative stress. Further research is needed to elucidate the mechanism of A. annua’s and artemisinin’s medicinal properties and further test their potential neuroprotective effects.

No description available.

Einleitung: Kurzkettige Fettsäuren (SCFA) stellen das hauptsächliche Energiesubstrat für Wiederkäuer dar. In Anbetracht des - bedingt durch höhere Milch-, Mast und Reproduktionsleistung - steigenden Energiebedarfs von Hauswiederkäuern wie Milchkuh und Mastbulle ist es von zentraler Bedeutung, die Mechanismen zur Resorption dieser Energielieferanten bzw. Ansatzpunkte für die Beeinflussung dieser Transportprozesse genau zu kennen. Dieses Wissen kann möglicherweise dabei helfen, zukünftig die Energieaufnahme der Tiere zu unterstützen bzw. sogar effizienter zu gestalten. Ziele der Untersuchungen: Deshalb war es Ziel der vorliegenden Arbeit, die Mechanismen zur Resorption von SCFA zu charakterisieren, wobei der Schwerpunkt auf den Transport aus den Pansenepithelzellen ins Blut gelegt wurde, da hierzu im Gegensatz zu ihrer Aufnahme aus dem Pansenlumen in die Epithelzellen noch sehr wenig bekannt war. In einem zweiten Schritt sollte untersucht werden, inwiefern die nachgewiesenen Mechanismen einer Regulation unterliegen und über welche Signalwege diese vermittelt werden könnte. Materialien und Methoden: Zur Charakterisierung der beteiligten Resorptionsmechanismen wurden Epithelstücke aus dem ventralen Pansensack von Schafen in Ussing-Kammern eingespannt und mit Hilfe radioaktiv markierten Azetats, Butyrats und L-Laktats der Transport dieser Substrate unter verschiedenen Bedingungen sowie verschiedenen Hemmstoffeinflüssen untersucht. Zur Charakterisierung regulativer Einflüsse wurden die Epithelstücke über sechs bzw. 24 Stunden mit Butyrat inkubiert und anschließend RNA bzw. Totalprotein extrahiert. Hiermit konnten Veränderungen in mRNA- und Proteinexpression mittels quantitativer Echtzeit-PCR bzw. Western Blot nachgewiesen werden. Ergebnisse: Die Untersuchungen der vorliegenden Arbeit konnten zeigen, dass der Transport von SCFA über die basolaterale Membran des Pansenepithels hauptsächlich proteinvermittelt erfolgt. Eine signifikante Beteiligung lipophiler Diffusion, d.h. ein passiver Transport, kann weitgehend ausgeschlossen werden. Der aktive Transport wies eine bikarbonatabhängige und eine bikarbonatunabhängige Komponente auf. Der Einsatz von Hemmstoffen verschiedener Transportproteine ergab deutliche Hinweise darauf, dass der Monocarboxylattransporter (MCT) 1 eine Rolle beim bikarbonatgekoppelten Transport von Azetat bzw. allgemein unmetabolisierten SCFA spielt. Diese Hinweise wurden untersetzt durch die Beobachtung, dass MCT 1, aber auch der apikal bzw. intrazellulär lokalisierte MCT 4 durch langfristige Inkubation des Epithels mit Butyrat sowohl auf mRNA- als auch auf Proteinebene signifikant erhöht exprimiert wurden, was als Anpassungsreaktion an eine Substratakkumulation interpretiert werden kann. Außerdem wurde auch die mRNA-Expression des Putativen Anionentransporters (PAT) 1 durch Inkubation mit Butyrat erhöht, was für eine Beteiligung auch dieses Transportproteins am SCFA-Transport über das Pansenepithel spricht. Allerdings ist im Gegensatz zu MCT 1 die Lokalisation des PAT 1 in der basolateralen Membran noch fraglich. Die Expressionssteigerung von Zielgenen des Nukleären Faktors ĸB und des Peroxisomenproliferator-aktivierten Rezeptors α sowie des Hypoxie-induzierbaren Faktors selbst deuten weiterhin darauf hin, dass die Steigerung der Transportkapazitäten von MCT 1 und 4 und auch PAT 1 über diese Signalwege vermittelt wird. Schlussfolgerungen: Zusammenfassend konnte in dieser Arbeit erstmals der Transport von SCFA über die basolaterale Membran des Pansenepithels näher charakterisiert werden, sodass es nun möglich ist, zusammen mit den bereits vorliegenden Befunden für die apikale Membran ein komplettes Modell dafür zu erstellen. Auch wurden Erkenntnisse zu regulativen Einflüssen auf diesen Transport gewonnen, die es zukünftig ermöglichen könnten, die Resorption der SCFA aus dem Pansen nutritiv oder eventuell pharmakologisch zu beeinflussen.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für Wiederkäuer 3 2.2 Metabolismus von SCFA im Pansenepithel 4 2.2.1 Aufrechterhaltung des Konzentrationsgradienten vom Pansenlumen ins Epithel 4 2.2.2 Produktion von HCO3- aus CO2 durch die Carboanhydrase 5 2.2.3 Bereitstellung von Energie für die Epithelzellen 5 2.2.4 Bereitstellung von wasserlöslichen, glukosesparenden Energiesubstraten für die periphere Zirkulation 5 2.2.5 Verhinderung möglicher Schädigungen durch Butyrat 6 2.3 Transportmechanismen für kurzkettige Fettsäuren 7 2.3.1 Para- versus transzelluläre Resorption 7 2.3.2 Transzelluläre Resorption mittels lipophiler Diffusion 7 2.3.3 Proteinvermittelte SCFA-Permeation 9 2.3.4 Permeation von SCFA aus dem Epithel ins Blut 11 2.4 Beeinflussung der SCFA-Resorption auf Genexpressionsebene 17 2.4.1 Beeinflussung der Genexpression durch Butyrat 17 2.4.2 Beeinflussung der Genexpression durch Hypoxie 20 2.4.3 Mechanismen für die Regulation der Genexpression durch Butyrat (-Metaboliten) und Hypoxie 21 2.5 Fragestellungen dieser Arbeit 26 3 Ergebnisse 28 3.1 Publikation 1 28 3.2 Publikation 2 41 4 Diskussion 54 4.1 Transport von SCFA über die basolaterale Membran des Pansenepithels 54 4.1.1 Transport mittels lipophiler Diffusion 57 4.1.2 SCFA werden bevorzugt über die basolaterale Membran transportiert 58 4.1.3 SCFA(-Metaboliten) werden bikarbonatabhängig über die basolaterale Membran transportiert 59 4.1.4 SCFA(-Metaboliten) werden durch einen Anionenaustauschmechanismus ins Blut ausgeschleust 61 4.1.5 Azetat wird durch einen pHMB- und CHC-sensitiven Mechanismus transportiert 63 4.2 Der Transport von SCFA über das Pansenepithel unterliegt regulativen Einflüssen 68 4.2.1 Einfluss von Butyrat(-Metaboliten) auf die Expression von potentiellen SCFA Transportern 68 4.2.2 Mechanismen für die Regulation der Expression durch Butyrat(-Metaboliten) 72 4.3 Theoretisches Modell des SCFA-Transports und dessen Regulation auf Genexpressionsebene auf Grundlage der Ergebnisse der vorliegenden Arbeit 74 5 Zusammenfassung 76 6 Summary 78 7 Literaturverzeichnis 80 Danksagung 98 / Introduction: The main energy source for ruminants are short chain fatty acids (SCFA). Considering the ever increasing energy requirements of cattle due to increasing milk yield and meat production, it is crucial to identify the mechanisms for the resorption of these energy sources as well as possibilities to influence these transport mechanisms. This knowledge could help support the animals’ energy uptake or even making it more efficient. Aim: Thus, the aim of the present study was to characterise mechanisms for the resorption of SCFA focusing on their transport from the epithelial cells into the blood. In particular, since – compared to the research findings on the uptake of SCFA from ruminal lumen into the cells – so far only very little was known regarding this side of the epithelium. In a second step, the study aimed to elucidate whether the mechanisms observed are subject to regulatory processes and which signalling pathways are involved. Materials and methods: To characterise the transport mechanisms involved, epithelial pieces from the ventral sac of ovine rumen were mounted in Ussing chambers. Using radioactively labelled acetate, butyrate and L-lactate, the transport of these substrates was investigated under different conditions and by applying different inhibitors for potential SCFA transport proteins. To characterise regulatory influences, epithelial pieces were incubated with butyrate for six and 24 hours, respectively. Subsequently, total RNA and protein were extracted to detect changes in mRNA and protein expression using quantitative real time PCR and western blot, respectively. Results: The present study could show that transport of SCFA across the basolateral membrane of rumen epithelium is mainly realised by protein-mediated mechanisms. A significant participation of lipophilic diffusion, i.e. a passive transport, can almost entirely be excluded. The active transport could be divided into a bicarbonate-dependent and a bicarbonate-independent part. The experiments with inhibitors of different transport proteins showed clear evidence of an involvement of monocarboxylate transporter (MCT) 1 in the bicarbonate-dependent transport of acetate and non-metabolised SCFA in general. This evidence was supported by the finding that the expression of MCT 1 but also of the apically and intracellularly localised MCT 4 was increased significantly on both mRNA- and protein-level after long-term incubation of the epithelium with butyrate. This can be interpreted as an adaptation to a substrate accumulation. Additionally, butyrate incubation led to an increased mRNA expression of putative anion transporter (PAT) 1, which makes an involvement of this transport protein in SCFA transport across ruminal epithelium likely as well. However, in contrast to MCT 1 the localisation of PAT 1 in the basolateral membrane is still questionable. The increased expression of target genes of nuclear factor ĸB and peroxisome-proliferator activated receptor α as well as of hypoxia inducible factor strongly point to an involvement of these pathways in the increased expression of MCT 1 and 4 as well as PAT 1. Conclusions: In summary, this study could characterise the transport of SCFA across the basolateral membrane of ruminal epithelium in detail for the first time. This enables us to draw a complete model of ruminal SCFA transport. Also, evidence for regulatory influence on this transport processes was found, perhaps making it possible to influence resorption of SCFA from rumen by nutritive or pharmacological means in the future.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für Wiederkäuer 3 2.2 Metabolismus von SCFA im Pansenepithel 4 2.2.1 Aufrechterhaltung des Konzentrationsgradienten vom Pansenlumen ins Epithel 4 2.2.2 Produktion von HCO3- aus CO2 durch die Carboanhydrase 5 2.2.3 Bereitstellung von Energie für die Epithelzellen 5 2.2.4 Bereitstellung von wasserlöslichen, glukosesparenden Energiesubstraten für die periphere Zirkulation 5 2.2.5 Verhinderung möglicher Schädigungen durch Butyrat 6 2.3 Transportmechanismen für kurzkettige Fettsäuren 7 2.3.1 Para- versus transzelluläre Resorption 7 2.3.2 Transzelluläre Resorption mittels lipophiler Diffusion 7 2.3.3 Proteinvermittelte SCFA-Permeation 9 2.3.4 Permeation von SCFA aus dem Epithel ins Blut 11 2.4 Beeinflussung der SCFA-Resorption auf Genexpressionsebene 17 2.4.1 Beeinflussung der Genexpression durch Butyrat 17 2.4.2 Beeinflussung der Genexpression durch Hypoxie 20 2.4.3 Mechanismen für die Regulation der Genexpression durch Butyrat (-Metaboliten) und Hypoxie 21 2.5 Fragestellungen dieser Arbeit 26 3 Ergebnisse 28 3.1 Publikation 1 28 3.2 Publikation 2 41 4 Diskussion 54 4.1 Transport von SCFA über die basolaterale Membran des Pansenepithels 54 4.1.1 Transport mittels lipophiler Diffusion 57 4.1.2 SCFA werden bevorzugt über die basolaterale Membran transportiert 58 4.1.3 SCFA(-Metaboliten) werden bikarbonatabhängig über die basolaterale Membran transportiert 59 4.1.4 SCFA(-Metaboliten) werden durch einen Anionenaustauschmechanismus ins Blut ausgeschleust 61 4.1.5 Azetat wird durch einen pHMB- und CHC-sensitiven Mechanismus transportiert 63 4.2 Der Transport von SCFA über das Pansenepithel unterliegt regulativen Einflüssen 68 4.2.1 Einfluss von Butyrat(-Metaboliten) auf die Expression von potentiellen SCFA Transportern 68 4.2.2 Mechanismen für die Regulation der Expression durch Butyrat(-Metaboliten) 72 4.3 Theoretisches Modell des SCFA-Transports und dessen Regulation auf Genexpressionsebene auf Grundlage der Ergebnisse der vorliegenden Arbeit 74 5 Zusammenfassung 76 6 Summary 78 7 Literaturverzeichnis 80 Danksagung 98

Com o início da fortificação de farinhas com ácido fólico (AF) no Brasil, a partir 2004, a população passou a estar exposta de modo compulsório a maiores quantidades desta vitamina. Sabe-se que o AF na sua forma sintética pode não ser completamente convertido para formas metabolicamente ativas, levando ao aparecimento de uma fração não metabolizada no organismo. Este fato é mais preocupante nos indivíduos que além da fortificação, fazem uso terapêutico dessa vitamina, como em pacientes com anemias hemolíticas (esferocitose hereditária (EH), β-talassemia heterozigótica (β-TH), entre outras). O objetivo desse estudo foi avaliar se as concentrações séricas de AF não metabolizado (UMFA) afetam a metilação global do DNA; a expressão de RNAm de genes da DHFR, MTHFR, interferon-γ, TNF-α e interleucina (IL)-8; e a citotoxicidade de células NK. Foram incluídos 27 pacientes com EH, 50 indivíduos com β-talassemia heterozigótica (β-TH) e 136 indivíduos saudáveis. Outros 30 indivíduos saudáveis foram incluídos num estudo de intervenção com 5mg/dia de AF durante 90 dias. Foram realizadas as análises de: ácido fólico sérico, eritrocitário e UMFA, vitamina B12, homocisteína total; expressões de RNAm dos genes de MTHFR, DHFR, IFN-γ, TNF-α e IL-8; citotoxicidade das células NK; metilação global do DNA e citocinas séricas IL6, IL-8, IL10, IFN-γ e TNF-α. Resultados: Os pacientes EH apresentaram maiores concentrações de AF (sérico, eritrocitário e UMFA) que seus controles, sendo que 55,5% (método microbiológico) e 74,1% (método LC-MS/MS) apresentaram concentrações suprafisiológicas da vitamina, e 74,1% apresentaram concentrações aumentadas de UMFA. No grupo β-TH foi observado maiores concentrações de folato eritrocitário comparado ao controle e 22% dos indivíduos tinham concentrações suprafisiológicas de AF. No estudo de intervenção, após 45 e 90 dias de uso de AF as concentrações suprafisiológicas estavam presentes em 93,3% dos indivíduos e 100% deles apresentavam concentrações aumentadas de UMFA. Não foram observadas diferenças nas taxas de metilação global do DNA entre os grupos EH e β-TH quando comparados aos seus controles e não foi verificada correlação entre metilação global e concentrações de UMFA. Tanto EH quanto β-TH apresentaram maior expressão de RNAm de IL-8, quando comparados aos seus controles. No grupo de intervenção houve maior expressão de IL-8 após 45 dias de uso de AF quando comparado ao período pré-intervenção. Foi mostrada uma correlação inversa entre as concentrações de folato eritrocitário com o número de células NK no grupo EH e com a capacidade citotóxica das células NK no grupo total (EH + controle). No grupo intervenção foi observado menor número e menor capacidade citotóxica das células NK após 90 dias de uso de AF. Conclusões: O uso de AF 5mg/dia foi associado com aumento expressivo das concentrações de folato sérico, eritrocitário, UMFA, na expressão de RNAm de genes da citocina inflamatória IL-8 e redução do número e da citotoxicidade das células NK. Dessa forma, altas doses de AF podem resultar em alterações de componentes do sistema imune podendo prejudicar os mecanismos de vigilância celular das células NK. Os nossos achados sugerem que é importante o acompanhamento terapêutico dos pacientes que fazem uso de AF, especialmente aqueles indivíduos que fazem uso crônico desta vitamina por longo tempo, como os pacientes com anemias hemolíticas, mulheres que desejam engravidar e gestantes. / With the beginning of the fortification of flour with folic acid (FA) in Brazil, since 2004, the population has been exposed compulsorily to larger amounts of this vitamin. It is known that FA in its synthetic form can not be completely converted to metabolically active forms, leading to the appearance of an unmetabolized fraction in the body. This fact is more worrying in subjects beyond fortification, make therapeutic use of this vitamin, such as patients with hemolytic anemia (hereditary spherocytosis (HS), β-thalassemia heterozygotic (β-TH), among others). The aim of this study was to evaluate if serum concentrations of unmetabolized FA (UMFA) affect the global DNA methylation; mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin (IL)-8 genes; and on cytotoxicity of NK cells. It was included 27 patients EH, 50 subjects β-TH and 136 healthy subjects. Another 30 healthy subjects were included in an intervention study with 5 mg/FA daily during 90 days. Analyzes performed were: serum and erythrocyte folate, and UMFA, vitamin B12, tHcy; mRNA expression of MTHFR, DHFR, IFN-γ, TNF-α and IL-8 genes; cytotoxicity of NK cells; global DNA methylation and serum cytokines IL6, IL-8, IL10, IFN-γ and TNF-α. Results: EH patients presented higher FA concentrations (serum, erythrocytes and UMFA) than controls ones, and 55.5% (microbiologic method) and 74.1% (LC-MS/MS method) showed supraphysiologic concentrations of vitamin, and 74.1% presented increased concentrations of UMFA. In β-TH group, it was observed higher erythrocyte folate concentrations compared with the control and 22% of subjects had supraphysiological concentrations of FA. In the intervention study, after 45 and 90 days of FA use, supraphysiological concentrations were present in 93.3% of subjects and 100% of them showed increased concentrations of UMFA. It was not observed differences in global methylation of DNA between EH and β-TH groups when compared to their controls and it was not observed significant correlation between global DNA methylation and UMFA levels. Both EH and β-TH showed higher mRNA expression of IL-8 gene, when compared to controls. In the intervention group, there was higher mRNA expression of IL-8 gene after 45 days of FA use when compared to pre-intervention period. It was demonstrated an inverse correlation between erythrocyte folate levels and the number of NK cells in EH group; and cytotoxic capacity of NK cells on total group (EH + control). In intervention group, it was observed fewer number and lower cytotoxic capacity of NK cells after 90 days of AF use. Conclusions: The use of AF 5mg daily was associated with a significant increase in serum and erythrocytes folate levels, accompanied by increase in UMFA levels, an increase in mRNA expression of IL-8 gene, and reduction of the number and the cytotoxicity capacity of NK cells. Thus, high doses of AF can result in modifications of the immune system components, which may damage the cell surveillance mechanisms of NK cells. Our findings suggest that is important the therapeutic follow up of patients that are using AF, especially those subjects with chronic use of this vitamin, such as patients with hemolytic anemia, women who wish to become pregnant and pregnant women.

Com o início da fortificação de farinhas com ácido fólico (AF) no Brasil, a partir 2004, a população passou a estar exposta de modo compulsório a maiores quantidades desta vitamina. Sabe-se que o AF na sua forma sintética pode não ser completamente convertido para formas metabolicamente ativas, levando ao aparecimento de uma fração não metabolizada no organismo. Este fato é mais preocupante nos indivíduos que além da fortificação, fazem uso terapêutico dessa vitamina, como em pacientes com anemias hemolíticas (esferocitose hereditária (EH), β-talassemia heterozigótica (β-TH), entre outras). O objetivo desse estudo foi avaliar se as concentrações séricas de AF não metabolizado (UMFA) afetam a metilação global do DNA; a expressão de RNAm de genes da DHFR, MTHFR, interferon-γ, TNF-α e interleucina (IL)-8; e a citotoxicidade de células NK. Foram incluídos 27 pacientes com EH, 50 indivíduos com β-talassemia heterozigótica (β-TH) e 136 indivíduos saudáveis. Outros 30 indivíduos saudáveis foram incluídos num estudo de intervenção com 5mg/dia de AF durante 90 dias. Foram realizadas as análises de: ácido fólico sérico, eritrocitário e UMFA, vitamina B12, homocisteína total; expressões de RNAm dos genes de MTHFR, DHFR, IFN-γ, TNF-α e IL-8; citotoxicidade das células NK; metilação global do DNA e citocinas séricas IL6, IL-8, IL10, IFN-γ e TNF-α. Resultados: Os pacientes EH apresentaram maiores concentrações de AF (sérico, eritrocitário e UMFA) que seus controles, sendo que 55,5% (método microbiológico) e 74,1% (método LC-MS/MS) apresentaram concentrações suprafisiológicas da vitamina, e 74,1% apresentaram concentrações aumentadas de UMFA. No grupo β-TH foi observado maiores concentrações de folato eritrocitário comparado ao controle e 22% dos indivíduos tinham concentrações suprafisiológicas de AF. No estudo de intervenção, após 45 e 90 dias de uso de AF as concentrações suprafisiológicas estavam presentes em 93,3% dos indivíduos e 100% deles apresentavam concentrações aumentadas de UMFA. Não foram observadas diferenças nas taxas de metilação global do DNA entre os grupos EH e β-TH quando comparados aos seus controles e não foi verificada correlação entre metilação global e concentrações de UMFA. Tanto EH quanto β-TH apresentaram maior expressão de RNAm de IL-8, quando comparados aos seus controles. No grupo de intervenção houve maior expressão de IL-8 após 45 dias de uso de AF quando comparado ao período pré-intervenção. Foi mostrada uma correlação inversa entre as concentrações de folato eritrocitário com o número de células NK no grupo EH e com a capacidade citotóxica das células NK no grupo total (EH + controle). No grupo intervenção foi observado menor número e menor capacidade citotóxica das células NK após 90 dias de uso de AF. Conclusões: O uso de AF 5mg/dia foi associado com aumento expressivo das concentrações de folato sérico, eritrocitário, UMFA, na expressão de RNAm de genes da citocina inflamatória IL-8 e redução do número e da citotoxicidade das células NK. Dessa forma, altas doses de AF podem resultar em alterações de componentes do sistema imune podendo prejudicar os mecanismos de vigilância celular das células NK. Os nossos achados sugerem que é importante o acompanhamento terapêutico dos pacientes que fazem uso de AF, especialmente aqueles indivíduos que fazem uso crônico desta vitamina por longo tempo, como os pacientes com anemias hemolíticas, mulheres que desejam engravidar e gestantes. / With the beginning of the fortification of flour with folic acid (FA) in Brazil, since 2004, the population has been exposed compulsorily to larger amounts of this vitamin. It is known that FA in its synthetic form can not be completely converted to metabolically active forms, leading to the appearance of an unmetabolized fraction in the body. This fact is more worrying in subjects beyond fortification, make therapeutic use of this vitamin, such as patients with hemolytic anemia (hereditary spherocytosis (HS), β-thalassemia heterozygotic (β-TH), among others). The aim of this study was to evaluate if serum concentrations of unmetabolized FA (UMFA) affect the global DNA methylation; mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin (IL)-8 genes; and on cytotoxicity of NK cells. It was included 27 patients EH, 50 subjects β-TH and 136 healthy subjects. Another 30 healthy subjects were included in an intervention study with 5 mg/FA daily during 90 days. Analyzes performed were: serum and erythrocyte folate, and UMFA, vitamin B12, tHcy; mRNA expression of MTHFR, DHFR, IFN-γ, TNF-α and IL-8 genes; cytotoxicity of NK cells; global DNA methylation and serum cytokines IL6, IL-8, IL10, IFN-γ and TNF-α. Results: EH patients presented higher FA concentrations (serum, erythrocytes and UMFA) than controls ones, and 55.5% (microbiologic method) and 74.1% (LC-MS/MS method) showed supraphysiologic concentrations of vitamin, and 74.1% presented increased concentrations of UMFA. In β-TH group, it was observed higher erythrocyte folate concentrations compared with the control and 22% of subjects had supraphysiological concentrations of FA. In the intervention study, after 45 and 90 days of FA use, supraphysiological concentrations were present in 93.3% of subjects and 100% of them showed increased concentrations of UMFA. It was not observed differences in global methylation of DNA between EH and β-TH groups when compared to their controls and it was not observed significant correlation between global DNA methylation and UMFA levels. Both EH and β-TH showed higher mRNA expression of IL-8 gene, when compared to controls. In the intervention group, there was higher mRNA expression of IL-8 gene after 45 days of FA use when compared to pre-intervention period. It was demonstrated an inverse correlation between erythrocyte folate levels and the number of NK cells in EH group; and cytotoxic capacity of NK cells on total group (EH + control). In intervention group, it was observed fewer number and lower cytotoxic capacity of NK cells after 90 days of AF use. Conclusions: The use of AF 5mg daily was associated with a significant increase in serum and erythrocytes folate levels, accompanied by increase in UMFA levels, an increase in mRNA expression of IL-8 gene, and reduction of the number and the cytotoxicity capacity of NK cells. Thus, high doses of AF can result in modifications of the immune system components, which may damage the cell surveillance mechanisms of NK cells. Our findings suggest that is important the therapeutic follow up of patients that are using AF, especially those subjects with chronic use of this vitamin, such as patients with hemolytic anemia, women who wish to become pregnant and pregnant women.

Cardiotrophin-1 ist ein Zytokin der Familie Interleukin-6-Familie, zu der auch IL-11, CNTF, OSM und LIF gehören. Diese Substanzen wirken über die gemeinsame Rezeptoruntereinheit gp130. CT-1 induziert die Hypertrophie von Kardiomyozyten und inhibiert die Apoptose kardialer und Zellen. In verschiedenen Tiermodellen der Herzinsuffizienz konnte eine gesteigerte myokardiale CT-1 Expression beobachtet werden. Kardiomyopathien sind wiederum kardiale Erkrankungen, die mit einer Hypertrophie und Apoptose einhergehen und zu einer Herzinsuffizienz führen können. Man geht davon aus, dass 25-50 Prozent der familiär sind. Hierbei handelt es sich um eine monogenetische Erkrankung, die überwiegend autosomal-dominant vererbt werden. Daneben konnten aber auch modifizierende Polymorphismen in neurohumoralen Faktoren identifiziert werden. Basierend auf diesen Ergebnissen war das Ziel dieser Arbeit die Analyse der möglichen Beteiligung genetischer Varianten der kodierenden sowie der regulatorischen Region an der Pathogenese der Hypertrophen bzw. Dilatativen Kardiomyopathie. Zusätzlich sollte die mRNA-Expression von CT-1 in Myokardbiopsien von Patienten mit Herzinsuffizienz quantifiziert werden. Hierfür musste zunächst die Sequenzen der 5´-flankierenden Region identifiziert und bezüglich ihrer regulatorischen Eigenschaften analysiert werden. Es konnten 1,1 kb der 5´-flankierenden Region sequenziert werden. Die anschließende Luciferase-Reportergen-Analyse wies regulatorische Aktivitäten für den gesamten Bereich nach. Diese Region enthält zahlreiche cis-aktive DANN-Sequenzen aber keine TATA-Box. Für die Mutationssuche wurden 64 Patienten mit DCM, 53 Patienten mit HCM sowie 100 Kontrollpersonen mittels PCR-SSCP-Analyse untersucht. Es konnte eine kodierende Variante A92T bei jeweils einem DCM- bzw. HCM-Patienten identifiziert werden. Diese Substitution liegt in einem Bereich, der zwischen verschiedenen Spezies (Ratte, Maus, Mensch) konserviert ist. Diese Mutation könnte eine Veränderung der Sekundärstruktur bewirken und liegt in einem möglichen funktionellen Bereich. Die Promotorregion wies eine Basenpaarsubstitution bei -130 (G/T) sowie eine Deletion der Basen CTTT zwischen -992 und -995 auf. Der Polymorphismus an Position -130 fand sich tendenziell häufiger bei Patienten mit Dilatativer Kardiomyopathie. Die CTTT-Deletion konnte nur bei einer Patientin mit HCM nachgewiesen werden. Für die Quantifizierung der CT-1 mRNA wurden rechtsventrikuläre Endomyokardbiopsien von 6 Patienten mit eingeschränkter LVEF (CHI), 5 Patienten nach Herztransplantation (TX) sowie 3 Kontrollpatienten (KO) eingesetzt. Es konnte ein relativer Anstieg der CT-1 Expression um 82% bei den Patienten mit eingeschränkter LVEF festgestellt werden. Interessanterweise besteht eine enge Korrelation zur Schwere der eingeschränkten Herzfunktion sowie zur Zunahme der Hypertrophie. / Cardiotrophin-1 is a cytokine, which belongs to the interleukin-6 family, which includes IL-11, CNTF, OSM and LIF. These factors act via the receptor subunit gp130. CT-1 induces the hypertrophy of cardiomyocytes and inhibits the apoptosis of cardiac cells. Studies in animal models of congestive heart failure showed an enhanced expression of CT-1 in the myocardium. Cardiomyopathies are cardiac diesorders, which are charakterized by hypertrophy and apoptosis and which can terminate with congestive heart failure. About 25-50 percent of all cases are familial. It is a monogenetic mendelian disorder with an autosomal-dominant inheritance in most cases. Beside this, modifying polymorphisms in neurohunoral factors could be identified. Based on these facts, the aim of this study was to identify genetic variants within the coding and regulatory region of the CT-1 gene, which could influence the pathogenesis of hypertrophic or dilated cardiomyopathy. Additionally, the mRNA-expression of CT-1 in myocardial biopsies of heart failure patients should be quantified. First, it was necessary to sequence the 5´-untranslated region and to analyse its regulatory function. We could sequence 1.1 kb of the 5´-UTR. The luciferase reportergene assay showed a significant promoter activity for the whole region. The region contains various cis-active DNA sequences but no TATA-box.The TRANSFAC-analysis identified different binding sites for transcription factors but no TATA-box. The genetic material of 64 DCM and 53 HCM patients and 100 controls was screened for mutaions by using a PCR-based SSCP-analysis. A coding variant A92T could be identified for a patient with DCM and for an HCM patient. This mutation lies within a region which is conserved between different species (rat, mouse, human). This variant could disturb the secondary structure and lies in a probable functional region. Within the promoter we could identify a basepair substitution at position -130 (G/T) and a 4-basepair deletion between -992 and -995 (CTTTdel). The polymorphism at -130 showed a tendency for a higher occurrence in DCM patients. One HCM patient was heterozygous for the CTTT-deletion. To quantify the CT-1 mRNA we used endomyocardial biopsies of 6 patients with reduced LVEF (CHI), 5 patients after heart transplantation (TX) and 3 controls (KO). We performed a semiquantitative analysis by using HPLC and an external standard (PDH mRNA). We found an increased expression of CT-1 by 82% for patients with heart failure. Interestingly, we saw a tight correlation with to the reduction in LV function and to the degree of hypertrophy.