Bei konventionellen Mikroarray-Experimenten zur Genexpressionsanalyse wird fluoreszenz- oder radioaktiv-markierte cDNA oder RNA mit immobilisierten Proben hybridisiert. Für ein gut detektierbares und auswertbares Ergebnis werden jedoch pro Array mindestens 15 - 20 &#181;g Hybridisierungstarget benötigt. Dazu müssen entweder 15 - 20 &#181;g RNA direkt durch Reverse Transkription in markierte cDNA umgeschrieben werden oder bei Vorhandensein von weniger Startmaterial die RNA amplifiziert werden (Standard- Affymetrix-Protokolle, Klur et al. 2004). Oft sind damit zeit- und kostenintensive Probenpräparationen verbunden und das Ergebnis ist nicht immer reproduzierbar. Obwohl es inzwischen einige Protokolle gibt, die dieses Problem zu lösen versuchen (Zhang et al. 2001, Iscove et al. 2002, McClintick et al. 2003, Stirewalt et al. 2004), eine optimale, leicht handbare und reproduzierbare Methode gibt es weiterhin nicht, weshalb in dieser Arbeit ein weiterer Lösungsansatz gesucht wurde.<br> In der vorgestellten Arbeit werden zwei einfache Methoden beschrieben, mit denen Gene aus geringen RNA-Mengen nachgewiesen werden können: erstens die On Chip- RT-PCR mit cDNA als Matrize und zweitens diese Methode als One-Step-Reaktion mit RNA als Matrize.<br><br> Beide Methoden beruhen auf dem Prinzip der PCR an immobilisierten Primern auf einer Chipoberfläche. Diese Möglichkeit der exponentiellen Amplifikation ist reproduzierbar und sensitiv.<br><br> In Experimenten zur Etablierung des On-Chip-PCR-Systems wurden für die Immobilisierung der Primer verschiedene Kopplungsmethoden verwendet. Die affine Kopplung über Biotin- Streptavidin erwies sich als geeignet. Die On-Chip-Reaktion an kovalent gebundenen Primern wurde für amino-modifizierte Primer auf Epoxy-Oberflächen sowie für EDC-Kopplung auf silanisierten Oberflächen gezeigt. Für die letztgenannte Methode wurde die On-Chip-PCR optimiert, dass Spottingkonzentrationen der Primer von 5 - 10&#181;M schon ausreichend sind. Der Einsatz von fluoreszenz-markierten Primern während der PCR ermöglicht eine unmittelbare Auswertung nach der Synthese ohne zusätzliche Detektionsschritte. In dieser Arbeit konnte außerdem mit der vorgestellten Methode der simultane Nachweis zweier Gene gezeigt werden. Die Methode kann noch als Multiplex-Analyse ausgebaut werden, um so mehrere Gene in gleichzeitig einem Ansatz nachweisen zu können.<br><br> Die Ergebnisse der Versuche mit Matrizen aus unterschiedlichen Zelltypen deuten darauf hin, dass die On-Chip-RT-PCR eine weitere optimale Methode für den Nachweis von gering exprimierten Genen bietet. / The detection of low quantities of mRNA is often difficult and methods like microarray analysis require large amount of starting material or have to be amplified before application. The reverse transcription polymerase chain reaction (RT-PCR) is often the chosen method to detect specific RNA sequences on account of its high sensitivity. The solid phase amplification technology by On-Chip-PCR provides a combination of amplification of rare target material and its on chip detection in one step.<br><br> In this report a novel application for the On-Chip-PCR technology is described. It was suitable to identify mRNA sequences and genes, respectively. For this approach we amplified cDNA sequences using immobilized specific primers and fluorescent labeled primers. They were used for genes coding subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm) and Atpase (Atp2a2). The cDNA templates were synthesized before the On-Chip application by Reverse Transcription from mRNA. For this application only at most 500 pg of total-RNA preparation was sufficient for detectable results and no pre-amplification steps were needed.<br><br> Furthermore the handling of RT-PCR could be minimized by using a One-step- RT-PCR protocol. This method used immobilized primers on glassy supports detecting specific mRNA sequences from 5 pg or less of total RNA preparations.<br> Moreover to the detection of low quantities of RNA preparation, low abundant genes could be detected by this method.

Rinderpest virus (RPV) belongs to the order Mononegavirale which comprises non segmented negative sense RNA viruses including human pathogens such as Measles, Ebola and Marburg virus. RPV is the causative agent of Rinderpest disease in large ruminants, both domesticated and wild. The viral genome contains a non segmented negative sense RNA encapsidated by nucleocapsid protein (N-RNA). Viral transcription/replication is carried out by the virus encoded RNA dependent RNA polymerase represented by the large protein L and phosphoprotein P as (L-P) complex. Viral transcription begins at the 3’ end of the genome 3’le-N-P-M-F-H-N-tr-5’ with the synthesis of 55nt leader RNA followed by the synthesis of other viral mRNAs. A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end of the genome due to attenuation of polymerase transcription at each gene junction. The present study aims at functional characterization of Rinderpest virus transcription and the associated activities required for viral mRNA capping. In addition, an attempt has been made to understand the novel role of a host factor, Ebp1, playing a key role in virus multiplication in infected cells. The specific aims of the study are presented in detail below. 1. Development of in vitro transcription system for RPV mRNA synthesis and role of phosphorylation of P protein in transcription. The transition of viral polymerase from transcription to replication in infected cells has been a long standing puzzle in all paramyxoviruses. Earlier work carried out using RPV minigenome with a CAT reporter gene and studies with phosphorylation null mutant P, has revealed the importance of P phosphorylation for viral transcription in vivo. However, the contribution of other cellular factors in the viral transcription/replication switch could not be ruled out in these assays. In order to understand the specific role of P protein in transcription/replication, it was necessary to develop a cell free transcription system for viral mRNA synthesis. Hence, viral genomic RNA (N-RNA) was purified from RPV infected cells using CsCl density gradient centrifugation. The viral RNA polymerase consisting of L-P complex was separately expressed in insect cells and partially purified by glycerol gradient centrifugation. Glycerol gradient fraction containing the L-P complex was found to be active in viral transcription. Notably, the gradient of transcription of viral mRNA was observed in vitro with the partially purified recombinant L-P complex similar to in vivo. However, the recombinant polymerase complex failed to synthesis the 55nt leader RNA, in agreement with the recent finding in VSV that the transcriptase complex was unable to synthesize leader RNA and viral transcription is initiated at the N gene start site unlike the conventional 3’ entry mode. The newly developed in vitro reconstituted transcription system was used to analyze the effect of P phosphorylation on viral transcription. The results presented in chapter 2, indicate that phosphorylated P supports transcription whereas unphosphorylated P transdominantly inhibits the transcription in vitro suggesting the possible role of the status of P protein phosphorylation in determining transcription/replication switch. 2. Enzymatic activities associated with RPV L protein- role in viral mRNA capping. Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if RPV RNA polymerase possesses any of the capping and methylation activities, both virus as well as the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from infected cells. Using the purified virus and RNP complex, the first two activities required for mRNA capping vis-à-vis, RNA triphosphatase and guanylyltransferase were tested and the results are described in chapter 3 and 4. Purified virus as well as the RNP complex showed both RNA triphosphatase (RTPase) and Nucleotide triphosphatase activities. Neither purified N-RNA or recombinant P proteins show these activities suggesting that it is indeed mediated by viral L protein. By the metal dependency of the reaction and by the motif conservation with other reported RTPases, RPV L protein was assigned to the metal dependent RTPase tunnel family. Capping activity was also seen with the L protein present in RNP complex by its ability to form a covalent complex with GMP moiety of GTP. The specificity of the reaction with GTP, inhibition of Enzyme-GMP complex formation by the inorganic pyrophosphate and the susceptibility of Enzyme-GMP complex under acidic conditions clearly indicated that RPV L represents the viral guanylyl transferase. Further confirmation was obtained by the indirect capping assay in which Enzyme-GMP complex was formed when recombinant L protein was incubated with the cap labeled RNA due to the reversible nature of capping reaction. Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to RNA synthesis and modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Since domain II has already been assigned as the viral RNA dependent RNA polymerase, domain I and domain III were chosen for further characterization. Both domains were cloned, expressed and purified to homogeneity using recombinant baculovirus expression system. However, the recombinant domain III alone showed the NTPase activity where as neither domain I or III showed RTPase activity. This is expected since a part of the conserved RTPase motif was located in domain II in the multiple sequence alignment with other viral and yeast RTPases. In addition, the recombinant domain III also showed the characteristic enzyme-GMP complex formation but failed to be active in the indirect capping assay. Therefore, both domain II and domain III are likely to be involved in the co-transcriptional capping of viral mRNA. In support of this view, recent report in VSV suggests the presence of additional motif in domain II which is essential for viral mRNA capping. Preliminary evidence has been presented in the appendix section for the presence of N7 guanine methyl transferase activity with L protein although further experiments are needed to confirm this activity. 3. Role of host factor Ebp1 in negative sense RNA virus replication - a possible antagonist In recent years, many cellular factors such as actin, tubulin and profilin have been shown to be involved in viral transcription. Ebp1-ErbB3 binding protein was initially isolated as a cellular protein which binds to Influenza viral polymerase subunit PB1. Ebp1 selectively inhibits the influenza virus transcription in vitro whereas the cap binding and endonuclease activity of PB1 subunit of viral polymerase is unaffected. Till now there are no reports of the role of Ebp1 in non segmented negative sense RNA virus infection. The fifth chapter describes the role of Ebp1 in RPV infection and vice versa. RPV infection leads to down regulation of Ebp1 mRNA levels which in turn leads to decreased protein synthesis. Subsequently, it was found that Ebp1 interacts presumably with viral N protein, being a part of the viral RNP complex in both infected cells as well as in purified virion. Further, over expression of Ebp1 inhibits viral transcription and as a consequence the virus multiplication in vivo suggesting a mutual antagonism between virus and the host cell through Ebp1 protein.

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.

Hintergrund: Der Insulin-like growth factor receptor (IGF1R) spielt eine zentrale Rolle bei Wachstumsprozessen. Heterozygote IGF1R-Mutationen führen durch eine partielle IGF1-Resistenz zu Kleinwuchs. Methoden: Auxologische und endokrinologische Daten des Patienten wurden erhoben. Anhand von Fibroblasten wurde die IGF1R-Deletion charakterisiert und die Auswirkungen auf die mRNA- und Protein-Expression sowie die Signaltransduktion untersucht. Ergebnisse: Der Junge, der eine heterozygote Exon 6 Deletion im IGF1R – durch Alu-Rekombination verursacht – und eine heterozygote SHOX-Variante (p.Met240Ile) in seinem Genom vereint, kam ‚appropriate for gestational age‘ zur Welt, entwickelte aber postnatal eine Wachstumsretardierung. Die Endokrinologischen Daten waren unauffällig. Der Patient zeigt keine Stigmata, die bei anderen IGF1- oder SHOX-Mutationsträgern beschrieben wurden. Durch Nonsense-Mediated mRNA Decay kommt es zu einer Dosisreduktion der IGF1-Rezeptoren und einer entsprechenden verminderten Aktivierung der Rezeptoren, nicht aber des Signalwegs. Zusammenfassung: Der Patient trägt eine bisher unbeschrieben heterozygote IGF1R-Deletion, die zu Kleinwuchs führt. Ursächlich dafür ist eine durch die Mutation verursachte Dosisreduktion der IGF1-Rezeptoren.

BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...

Carrapatos são artrópodes hematófagos de distribuição cosmopolita que parasitam vertebrados e transmitem uma grande variedade de agentes infecciosos para o homem e animais domésticos. Cobaias, diferentemente de cães e camundongos, são capazes de desenvolver resistência a carrapatos Rhipicephalus sanguineus após sucessivas infestações. Ao comparar o tipo de resposta imune desenvolvida por cobaias e cães frente a carrapatos observou-se que cobaias re-infestadas desenvolvem uma pequena reação de hipersensibilidade imediata e uma forte reação de hipersensibilidade tardia à inoculação cutânea com antígenos de carrapatos. Já em cães e camundongos, é notada somente uma forte reação de hipersensibilidade imediata. Também foi verificado que células dos linfonodos de cobaias infestadas três vezes com carrapatos (resistentes) proliferam intensamente na presença de saliva de carrapatos, diferentemente do que ocorre com células de cães e camundongos re-infestados (suscetíveis) que não proliferam. Esses achados sugerem o envolvimento de um padrão Th1 de resposta imune na aquisição de resistência, no entanto essa hipótese ainda não foi confirmada. Assim sendo, no atual trabalho procurou-se verificar a expressão de mRNA de citocinas na pele e linfonodos de cobaias infestadas e re-infestadas com carrapatos. Para tal foram delineados primers e padronizadas reações de PCR para detectar a expressão de mensagem das citocinas IL-12p40, IFN-gama e TNF-alfa, pertencentes a um padrão Th1, e IL-4, IL-5, IL-10 e TGF-beta, pertencentes a um padrão Th2 de resposta imune. Os resultados obtidos demonstraram que cobaias sucessivamente infestadas apresentaram um aumento significativo na intensidade de mensagem para IL-12p40 nos linfonodos, tanto comparado com animais uma vez infestados (aumento de 2,6 vezes), quanto comparado com os controles (aumento de 13 vezes). Embora a análise estatística não tenha apontado uma diferença significativa houve elevação consistente na intensidade de mRNA para IFN-gama nos linfonodos de cobaias re-infestadas comparadas às infestadas apenas uma vez (aumento de 2,4 vezes). Também foi observado um aumento significativo na intensidade da mensagem para IL-5 nos linfonodos de cobaias infestadas uma vez quando comparadas aos controles (aumento de 5 vezes). Não foi detectada expressão de mensagem para IL-4 e IL-10 nas amostras analisadas. Já a expressão de mensagem para TGF-beta foi observada em todos os animais (experimentais ou controles), sugerindo que essa citocina possa ter uma expressão constitutiva em cobaias. Tomados em conjunto, os resultados sugerem o envolvimento predominante de um perfil de citocinas de padrão Th1 na aquisição de resistência em cobaias a carrapatos. Nossos resultados poderão auxiliar o desenvolvimento de novas abordagens para o controle de carrapatos, como, por exemplo, sugerir adjuvantes mais adequados a serem utilizados em vacinas anti-carrapatos. O conhecimento gerado não se restringe à indução de proteção contra carrapatos como também a possibilidade de aumentar a resistência de hospedeiros a patógenos transmitidos por carrapatos que poderiam ser controlados por uma resposta tipo Th1. / Ticks are hematophagous arthropods of cosmopolitan distribution and are significant vectors of several diseases for humans and animals. Guinea pigs, unlike dogs and mice, develop resistance to Rhipicephalus sanguineus ticks after successive infestations. When the immune reaction between tick-infested guinea pigs and dogs/mice are compared, guinea pigs develop both immediate and strong delayed type hypersensitivity reactions while dogs and mice develop only a strong immediate reaction. Additionally was shown that lymph node cells from tick-infested guinea pigs (resistant hosts) proliferate intensely when cultured with tick saliva, differently to what is observed with cells from tick-infested dogs and mice (susceptible hosts). These findings propose the contribution of a Th1 cytokine pattern on the acquired immune response to ticks; however this hypothesis still has to be tested. This being so, in this study we investigated the expression profile of genes coding for selected cytokines on R. sanguineus infested guinea-pigs. Messenger RNA for IL-12-p40, IFN-gamma and TNF-alfa (Th1 cytokine pattern) and IL-4, IL-5, IL-10 and TGF-beta (Th1 cytokine pattern) was measured in skin and lymph nodes biopsies from tick-infested guinea pigs. Our results demonstrated that repeatedly tick-infested guinea pigs presented a significant increase on the intensity of message for IL-12p40 in the lymph nodes compared to both, one time tick-infested guinea pigs (raise of 2.6 times) or controls (raise of 13 times). Although the statistical analysis did not point out differences, there was a consistent increase on the mRNA intensity for IFN-gamma on the lymph nodes from re-infested guinea pigs compared to one time tick-infested animals (raise of 2.4 times). In addition, a significant enhance on the intensity of message for IL-5 on the lymph nodes from one time tick-infested guinea pigs compared to the controls (raise of 5 times). No message for IL-4 and IL-10 was detected on the analyzed tissues. In contrast, TGF-beta was detected on tissues collected from all animals (experimental or controls), suggesting a spontaneous production of this cytokine in guinea pigs. Taken together, these data suggest that a T helper 1-type pattern of cytokine production might be associated with the resistance expressed by guinea-pigs to ticks. Moreover, our results can provide new approaches to control ticks, i.e. suggest adjuvants to be added to anti-tick vaccines that preferably induce a Th1-type of response.

Hintergrund: Der Insulin-like growth factor receptor (IGF1R) spielt eine zentrale Rolle bei Wachstumsprozessen. Heterozygote IGF1R-Mutationen führen durch eine partielle IGF1-Resistenz zu Kleinwuchs. Methoden: Auxologische und endokrinologische Daten des Patienten wurden erhoben. Anhand von Fibroblasten wurde die IGF1R-Deletion charakterisiert und die Auswirkungen auf die mRNA- und Protein-Expression sowie die Signaltransduktion untersucht. Ergebnisse: Der Junge, der eine heterozygote Exon 6 Deletion im IGF1R – durch Alu-Rekombination verursacht – und eine heterozygote SHOX-Variante (p.Met240Ile) in seinem Genom vereint, kam ‚appropriate for gestational age‘ zur Welt, entwickelte aber postnatal eine Wachstumsretardierung. Die Endokrinologischen Daten waren unauffällig. Der Patient zeigt keine Stigmata, die bei anderen IGF1- oder SHOX-Mutationsträgern beschrieben wurden. Durch Nonsense-Mediated mRNA Decay kommt es zu einer Dosisreduktion der IGF1-Rezeptoren und einer entsprechenden verminderten Aktivierung der Rezeptoren, nicht aber des Signalwegs. Zusammenfassung: Der Patient trägt eine bisher unbeschrieben heterozygote IGF1R-Deletion, die zu Kleinwuchs führt. Ursächlich dafür ist eine durch die Mutation verursachte Dosisreduktion der IGF1-Rezeptoren.:Inhaltsverzeichnis I Abkürzungsverzeichnis - 4 - 1 Bibliographische Beschreibung - 7 - 1.1 Referat - 7 - 2 Einleitung und Hintergrund - 9 - 2.1 Das menschliche Wachstum - 9 - 2.2 Das IGF-System als Regulator von Wachstum u. Entwicklung - 10 - 2.3 Der IGF1-Rezeptor - 11 - 2.4 Formen des Kleinwuchses - 12 - 2.5 IGF1R-Mutationen - 13 - 2.6 SHOX-Defizienz - 14 - 2.7 Der Nonsense-Mediated mRNA Decay - 15 - 2.8 Alu-Elemente - 16 - 2.9 Überleitung - 17 - 4 Originalpublikation - 18 - 5 Zusammenfassung der Arbeit - 31 - 5.1 Patientenbeschreibung - 32 - 5.2 Experimentelle Untersuchungen - 33 - 5.3 Interpretation - 35 - 5.4 Ausblick - 37 - 6 Literaturverzeichnis - 39 - III Curriculum vitae - 50 - IV Danksagung - 52 -

BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...

Das Enzym Glutaminsynthetase (GS) wird in Organen mit niedriger enzymatischer Aktivität in zumeist allen Zellen exprimiert. Auf der anderen Seite ist die Expression in Geweben mit hoher Aktivität auf spezialisierte Zellen beschränkt. So findet man in der Säugerleber Expression der GS nur in Hepatozyten, die in ein bis drei Zellreihen um die Zentralvenen lokalisiert sind. In der vorliegenden Arbeit wurde die Frage gestellt, ob der zwischen verschiedenen Spezies hoch konservierte 3’-Bereich der nicht-translatierten Region des GS-Gens an der Regulation der Expression und der Zonierung beteiligt ist. Hierzu wurden Reportergenstudien, transiente Transfektionen sowie Northern-Blot-Experimente unter Verwendung von primären Hepatozyten aus dem periportalen und perizentralen Bereich der Rattenleber durchgeführt. Die Ergebnisse der Arbeit lassen eine über das 3’-Ende vermittelte selektive Destabilisierung der GS-mRNA in periportalen (GS-negativen) Hepatozyten vermuten. Zudem zeigte sich, dass die Wechselwirkung des 3’-UTRs mit Bereichen des 5’-UTRs, bzw. dem GS-Promotor für die eigentliche Regulation verantwortlich ist. Es lässt sich vermuten, dass eine posttranskriptionale Regulation neben den in den letzten Jahren aufgeklärten Mechanismen der Regulation der Transkription mit zur Feinsteuerung der Expression der GS beiträgt.

Saccharomyces cerevisiae is an intron-poor organism with introns present in only 5% of its genes. The most prominent group of intron-containing genes are ribosomal protein (RP) genes. They are highly expressed and most of them are present as two paralogs. Parenteau et al. described the existence of intron- dependent intergenic regulatory circuits controlling expression ratios of RP paralogs. In this project, we did not confirm the regulation in 6 out of 7 tested regulatory circuits. We validated the regulation between RPL22 paralogs. We further showed that Rpl22 protein blocks the pre-mRNA splicing of both paralogs, with RPL22B paralog being more sensitive. Rpl22 protein binds to the stem-loop of RPL22B intron - disruption of the binding domain of Rpl22 proteins leads to loss of interaction. Moreover, the regulation seems to be working the same way in yeast Kluyveromyces lactis, which has only a single RPL22 copy. Overall, these results lead to better understanding of intergenic regulation, which adjusts the expression ratio between functionally different RPL22 paralogs. Key words introns, ribosomal protein genes, Rpl22, RPL22 paralogs, pre-mRNA splicing, Saccharomyces cerevisiae