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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. / 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.

Dentale Implantate sind heutzutage ein fester Bestandteil der modernen Zahnheilkunde. Durch die steigende Anzahl gesetzter Implantate steigt auch die absolute Zahl an Misserfolgen. Der Hauptgrund für den Verlust von Implantaten ist die Periimplantitis. Sie stellt eine Entzündung des Weich- und Hartgewebes um dentale Implantate dar, welche im ultimativen Stadium den Verlust der Integrität des Implantats zur Folge hat. Das Ziel dieser Arbeit soll es sein, einen Überblick über die Genexpression des entzündeten periimplantären Gewebe zu erlangen um so die Periimplantitis auf mikrobiologischer Ebene zu charakterisieren. Um das zu erreichen wurde umgebendes Gewebe von Implantaten mit Periimplantitis und von Implantaten ohne Periimplantitis von menschlichen Patienten gewonnen. Für die Ermittlung der Ergebnisse wurde die Microarray-Technik angewandt (Affymetrix Gene- Chip R Human Gene 1.0 ST Array) und um diese Ergebnisse zu validieren wurde die qPCR-Technik verwendet (real-time-PCR). Die Ergebnisse zeigen, dass klassische Entzündungsmarker wie IL-1, -8 und -6 sind in ihrer Expression stark erhöht. Die erhöhte Expression von CD24 zusammen mit weiteren Cluster of Differentiation und das Expressionsmuster der Interleukine zeigen, dass das unspezifische Immunsystem eine Hauptrolle bei der Entzündung um Implantate spielt. Makrophagen und Monozyten sind sehr wahrscheinlich die am stärksten vertretene Spezies von Abwehrzellen im von Periimplantitis betroffenen Gewebe. Marker für den Abbau von Gewebe, die MMPs-1, -12 und -13 sind in ihrer Expression erhöht, ihre Inhibitoren, die TIMPs sind in ihrer Expression erniedrigt. MMP-12 ist die Makrophagen spezifische Elastase und somit ein weiterer Indikator für die tragende Rolle von Makrophagen bei der Periimplantitis. Kollagen-3 ist das einzige Kollagen, welches den Ergebnissen dieser Arbeit zufolge in seiner Expression erhöht ist. Kollagen-3 findet sich neben Haut- und Muskelzellen in der Wand von Blutgefäßen. Transkriptions- und Wachstumsfaktoren wie RUNX2, SOX2 und FGF18 sind den Ergebnissen dieser Arbeit nach im entzündeten periimplantären Gewebe in ihrer Expression erniedrigt. Fazit: Anhand der Ergebnisse dieser Arbeit lässt sich die Periimplantitis als ein gut durchblutetes, granulationsgewebe-ähnliches-Gewebe mit einer herabgesetzten Kompetenz zur Regeneration charakterisieren. Das unspezifische Immunsystem ist Hauptakteur bei der Periimplantitis und die hauptsächlich anwesenden Immunzellen sind Makrophagen und Monocyten. Die Gewebehomöostase ist in Richtung Gewebeabbau verschoben.

Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...

Most viruses can infect only a reduced range of organisms and an effective replication is possible only in selected hosts. These hosts are called permissive for the virus. Molecular principles of a nonpermissiveness and viral mechanisms of overcoming replication obstacles are still not clearly elucidated. This thesis discusses the molecular causes of the cellular nonpermissiveness against a model retrovirus - Rous sarcoma virus. The research is conducted on duck cells which are semipermis- sive to the subgroup C of Rous sarcoma virus. The virus can enter those cells, but it is not able to produce enough infectious viral progeny. Two blocks of the viral replication cycle in the duck cells are described in the thesis. The first one is the probably not optimal cellular receptor recognition. The second one is in the late phase of the replication cycle when the viral proteins are synthesized. The amount of the envelope glyco- protein coding mRNA is reduced due to the altered splicing ratios, and the virions produced from the duck cells are less infectious. This block is recessive and can be partially omitted by cell fusions with permissive chicken cells; therefore, the block is not caused by specific restriction fac- tors in sensu stricto. Additionally, the influence of mutations in duck adapted Rous...

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

The aim of this thesis is to understand RNA-RNA interactions steering cellular functions, as in the case of this thesis the structure of mRNA(Sirt1) in complex with microRNA-34a (miR-34a). MiR-34a regulates the cancer protein p53 via Sirt1 modulation. This work will be the basis for future drug design and the understanding of misguided regulation in cancer. The miR-34a binds to the mRNA(Sirt1) 3’ untranslated region (3’-UTR) and will either inhibit the translation of the protein Sirtuin 1 by capturing its mRNA or by degrading it. p53, a key activator of miR-34a, prevents cancer development by inducing programmed cell death (apoptosis) on cells with DNA damage. In contrast, the protein Sirtuin 1 (Sirt1) has been shown to help cells with DNA damage to survive by down regulating the activity of protein p53 and will therefore increase the risk of cancer development. Studying the interaction between the mRNA(Sirt1) and miR-34a can present valuable information on the structure of the complex as well as the mode miR-34a uses to inhibit translation of mRNA(Sirt1) leading to down regulation of protein Sirtuin 1 and therefore prevent cancer development. For the elucidation of this question different biochemical and biophysical methods were applied, such as in vitro transcription, gel electrophoresis, RNA purification with gel, crush & soak and Cicular Dichroism (CD) melting studies. For this thesis work, the target sequence in mRNA(Sirt1) was optimized and purified so melting studies could be carried out. For future structural characterization using Nuclear Magnetic Resonance (NMR) studies with the miR-34a also produced in the lab. The mRNA(Sirt1) target sequence was produced and purified with the final yield of 0.02%. The results show that the sequence is highly ATP dependent and suggest the ratio between the nucleotides ATP/CTP to be 1:2. Low yield of purified mRNA(Sirt1) was received and still contained some impurities, which imply that another method than crush & soak should be used when purifying. The results, indicate that High-Preformance Liquid Chromatography (HPLC) might be a better solution for the pufication process. The melting profiles done on mRNA(Sirt1) show that the secondary structures decrease with an increase in temperature. Accroding to the results, the mRNA(Sirt1) sequence is folded in room temperature, though not very stable. The wavelength which provided the best resolution was at 268 nm and the melting point of mRNA(Sirt1) was determined to 44 °C. This thesis also contains an educational part, where an educational material was provided and testing was conducted for the subject Chemistry 2 for students age 18 and the material was evaluated with qualitative methods together with pedagogical methods. The study showed that the student can develope the different abilities stated in the curriculum with the material created. The results also showed that the students preferably choose cultural arguments when dicussing socio scientific question, rather than economical, democratic or utility arguments. / Syftet med studien är att förstå RNA-RNAinteraktioner som styr cellulära funktioner, i detta fall mRNA(Sirt1) i komplex med microRNA-34a (miR-34a). MiR-34a reglerar cancerproteinet p53 via modulation av Sirt1. Detta arbete kommer lägga grund för framtida läkemedelsdesign vid reglering av cancer. MiR-34a binder till den 3’ otranslerade regionen i mRNA(Sirt1) och hämmar antingen translationen av protein Sirtuin 1 (Sirt1) genom att fånga dess mRNA eller genom att försämra det. p53 förhindrar utvecklingen av cancer genom att framkalla programmerad cell död (apoptosis) av celler med skadat DNA. Det har visats att proteinet Sirtuin 1 hjälper celler med skadat DNA att överleva, genom att sänka aktiviteten av p53. På så vis ökar risken för utveckling av cancer. Genom att studera interaktionen mellan mRNA(Sirt1) och miR-34a kan värdefull information kring komplexets struktur fås. Samt hur miR-34a hämmar translationen av mRNA(Sirt1), vilket leder till minskad aktivitet av protein Sirt1. För att klarlägga denna fråga har olika biokemiska och biofysiska metoder använts, såsom in vitro transkription, gelelektrofores, RNA rening med gel och Circular Dichroism (CD). För detta arbete har målsekvensen i mRNA(Sirt1) optimerats och renats så CD smältstudier med kunde genomföras. Resultatet visar att mRNA(Sirt1) sekvensen renats med ett utbyte på 0.02 %. Sekvensen är beroende av ATP och förhållandet mellan ATP/CTP nukleotider bör vara 1:2. Resutatet visar på ett lågt utbyte som visar på att High-Performance Liquid Chromatography (HPLC) kan vara en bättre metod än Crush & soak för reningen av mRNA(Sirt1). Ur de smältprofiler som gjorts visade det sig att de sekundära strukturerna av mRNA(Sirt1) minskade med ökande temperatur. I enlighet med resultaten visar det att mRNA(Sirt1) är veckat i rumstemperatur men är inte stabil. Den bästa upplösningen erhölls vid 268 nm och mRNA(Sirt1) har en smältpunkt runt 44 °C. Detta arbete innehåller även ett utbildningskapitel, där ett utbildningsmaterial har skapats och testats på 18-åriga kemi 2 studenter i åldern 18 år. Materialet har utvärderats med hjälp av kvalitativa metoder tillsammans med pedagogiska metoder. Studien visade att de flesta förmågorna för kemi 2 kan utvecklas med hjälp av denna typ samhällsfrågor i det naturvetenskapliga klassrummet (SNI-fall) förutom förmågan att planera och genomföra experiment. Det argument som eleverna helst väljer att använda då de diskuterar det skapade SNI-fallet är Kulturargument och det minst använda är Demikratiargument.

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