Les mécanismes régissant le trafic et la localisation des ARN à l'intérieur du noyau sont encore mal compris. La protéine PHAX (Phosphorylated adaptator for RNA export) est un facteur nécessaire à l'export nucléaire des précurseurs des snARN, ainsi qu'à l'adressage des précurseur des snoRNA coiffés vers les corps de Cajal (Ohno et al., 2000 ; Boulon et al., 2004 ). De manière remarquable, la liaison de PHAX à ces ARN est strictement dépendante de leur taille, et celle-ci est perdue au-delà d'une certaine longueur d'ARN (Ohno et al., 2002). Cependant, in vitro, PHAX est capable de s'associer aux ARN, mais il le fait sans spécificité de séquence ni de taille. L'étude des mécanismes impliqués dans la distinction des petits ARN non-codants des grands ARN comme les ARN messagers, est malgré tout essentielle car elle dirige ces ARN vers des voies de maturation différentes. Lors de ma thèse j'ai mis en place un projet qui aborde deux questions : (i) déterminer comment le facteur PHAX sélectionne les petits ARN, (ii) caractériser l'ensemble des ARN liés à PHAX dans le but de découvrir de nouveaux petits ARN non-codants. J'ai observé que PHAX forme un complexe avec le CBC, comme attendu, et avec une protéine non caractérisée à l'époque, et que nous avons nommée PBP1 (PHAX binding protein 1). J'ai pu déterminer que ces quatre protéines, CBC20-CBC80-PHAX-PBP1 forment un complexe, que j'ai appelé complexe CBCAP. In vivo, ce complexe est bien associé avec les précurseurs de snARN et snoARN. De façon très intéressante, le complexe CBCAP est aussi associé à de nombreux ARNs messagers de petite taille (inferieurs à 1500pb). De manière intéressante, les ARN liés par le CBCAP sont également parmi les plus abondants dans la cellule. L'expression de ces ARN est de plus affectée suite à la déplétion des protéines PHAX ou PBP1, et, de manière surprenante, ces protéines ont souvent des effets opposés sur la stabilité de ces ARN. PHAX est préférentiellement requis pour la production des snRNA, tandis que PBP1 est plutôt nécessaire à la production des snoRNA coiffés et des ARN messagers. Le complexe CBCAP pourrait donc sélectionner les ARN selon leur taille et il distinguerait plusieurs types d'ARN (codants ou non-codants; exportés ou retenus), af in qu'ils soient orientés vers la voie de maturation adéquate. De manière remarquable, la production de PBP1 est elle-même contrôlée par des signaux de croissance cellulaire. Ainsi, PBP1 permettrait de réguler l'expression des ARN les plus abondants dans la cellule, et jouerait ainsi un rôle clef dans le contrôle de la croissance cellulaire. Au cours de ma thèse, j'ai aussi observé par analyse tilling array, la co-précipitation avec PBP1 et de PHAX avec des petits ARN non annotés. J'initie la caractérisation de ces nouveaux ARN. / RNA localisation determines the fate of RNA. However inherent transport mechanisms still remain unclear. The protein PHAX is required for snRNA and snoRNA maturation as it both exports precursors of snRNA to the cytoplasm via the recruitment of the CRM1 export machinery, and transports precursors of capped snoRNA to the cajal bodies. Interestingly, PHAX associates with and transports mainly small RNA through a mechanism that is not specific of the RNA sequence. To determine how PHAX recognises specifically small RNA, we characterised the PHAX native complex. We purified a complex that we called CBCAP, which contains the CBC (cap binding complex), the protein that we called PBP1 (PHAX binding protein 1) and the transporter PHAX. As expected, this complex is associated with precursors of snRNA and snoRNA. Interestingly, we also co-purified with CBCAP small coding RNA (under 1500bp). Furthermore, expression of these RNA is affected by PBP1 and PHA X depletion. Surprisingly, these two proteins have an opposite effect on RNA stability. PHAX is required for snRNA expression, whereas PBP1 seems to control capped snoRNA and small mRNA expression. The complex CBCAP might thus be involved in the distinction of RNA size and seems to discriminate coding from non-coding RNA in order to address them to the appropriate processing pathway.

Listeriosis is a potentially lethal disease caused by the Gram-positive facultative intracellular pathogen Listeria monocytogenes (L.m.). L.m. is found ubiquitously in the environment and infects humans via ingestion of contaminated food. Contaminated products are usually derived from ruminants and involve dairy products and different kinds of processed meat. Listeriosis is a potential lifethreatening disease with a total mortality rate of 20-30 %. The development of listeriosis may lead to meningitis and septicemia or other invasive diseases. Pregnant women are of increased risk of developing listeriosis and a materno-fetal infection commonly lead to spontaneous abortion or still-birth. Regulation of gene expression, and specifically virulence gene expression, is essential for pathogenic bacteria to be equipped for handling counteractions from the host as well as thriving in the often hostile environment. In pathogenic Listeria, virulence gene expression is under the control of the global virulence gene regulator PrfA. The expression of prfA is highly regulated at the transcriptional, post-transcriptional and post- translational level. We have identified a novel type of post-transcriptional regulation of prfA-mRNA by a trans-acting riboswitch element (SreA). By binding to the leader region of prfA-mRNA, SreA negatively regulates the expression of prfA. To our knowledge, this is the first description of a cis-acting riboswitch capable of functioning as a small RNA in trans, regulating targets on distant sites. To date, there have been around 100 sRNAs identified in Listeria monocytogenes, but experimental data is still limited. We have characterized a blood induced sRNA, Rli38, which is important for full virulence during oral infection of mice. Our data suggest that Rli38 regulates the expression of at least two proteins; OppD (Oligopeptide transport protein) and IsdG (heme degrading monooxygenase). Both of these proteins have been implicated in the infectious cycle of L.m. We speculate that the virulence phenotype of an ∆rli38 mutant is possibly mediated through the effect of these proteins. L.m. is a complex pathogen, able to infect and replicate in a variety of organs and cause several distinctive forms of disease. These qualities of L.m. generate difficulties in simulating human listeriosis in animal models, as entailed by the multitude of models used in the field. In this work, we have evaluated the use of an alternative animal model in studying listeriosis. Our results describe the differentiated virulence potential of wildtype bacteria and a ∆prfA mutant strain in the chicken embryo by live/death screening and organ colonization. Large differences in mean time to death were found between wild-type and the ∆prfA strain and ∆prfA cells displayed a considerable defect in colonization of the embryonal liver. The results presented in this thesis show that the chicken embryo infection model is a valuable and convenient tool in studying end-outcome and organ colonization of Listeria monocytogenes. Taken together, this thesis describes the characterization of two previously unknown sRNAs in the human pathogen Listeria monocytogenes and the use of an alternative infection model for simulating listeriosis.

: Filamentous hemagglutinin (FHA) is adhesive protein molecule that is secreted by Gram- negative bacterium Bordetella pertusis, the causative agent of whooping cough (pertussis). The C-terminal segment of FHA plays a crucial role in host-pathogen interaction, however, the structural features are still unknown. Here, we identified the C-terminal residue of FHA and processed form of FHA (FHA*) as alanine residues in position 2304 and 2228, respectively. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy demonstrated that the C-terminal segment of FHA(FHA 1995-2228) is characterized by alpha-helical contribution without any compact protein fold. Moreover, suppression of transcription of small regulatory RNA pairing to the 5'-end of fhaB transcript resulted in two- fold increase of FHA production. These data suggested that the C-terminal segment of FHA appear to be an unstructured protein and FHA secretion is negatively regulated by small regulatory RNA. (In Czech) Keywords: Bordetella pertussis, filamentous hemagglutinin, small RNA

Respiratory syncytial virus (RSV) is the leading cause of respiratory illness in children worldwide. RSV has a negative sense RNA genome, which is the template for viral mRNA transcription and genome replication, and encodes a polymerase to carry out viral RNA synthesis. The promoters for RSV transcription and genome replication are found in a 44-nucleotide (nt), 3´-extragenic region called the leader (Le). Replication is initiated opposite the first nt of the Le, and transcription of the first gene begins at position +45, at a gene start (GS) sequence. However, transcription is also dependent on sequence within Le1-12. Interestingly, Le nucleotides 3-12 bear strong similarity to a GS signal. We hypothesized that this GS-like sequence is the recruitment site for transcribing polymerase. To test this hypothesis, we examined RNA synthesis events at the Le promoter. We identified a previously undescribed RNA initiation site at Le position +3 (Le+3) that was used frequently during RSV infection. Initiation at Le+3 led to the production of a small ~25 nt RNA. Le+3 initiation was shown to occur independently of replication initiation at +1, indicating it is a bona fide initiation site. Mutation of Le1-12 to increase similarity to a GS resulted in elongation of Le+3 RNA and a decrease in transcription initiation at the GS, demonstrating that the Le initiation sequence alters polymerase processivity and impacts downstream transcription events. Preliminary experiments to determine the function of the small RNA showed that it increased levels of viral RNA replication, suggesting it may be involved in influencing a switch from transcription to replication. These studies suggest a model for RSV transcription initiation, whereby the transcribing polymerase enters at the 3´–end of the genome, initiates RNA synthesis from Le+3 and generates a small RNA, and is then positioned to initiate transcription at the first GS. The small RNA that is generated may act as a feedback molecule to promote RNA replication. These findings provide a greater understanding of polymerase behavior at the promoter and may inform rational drug and vaccine design.

Angsterkrankungen sind häufige Krankheitsbilder mit bislang nicht vollständig geklärter multifaktorieller Ätiologie. Neben Umwelt- und psychosozialen Faktoren zeigen Studien eine signifikante familiäre Häufung und lassen eine genetische Komponente mit einer Heritabilität in einem Bereich von 30-60 % vermuten. Da hierbei am ehesten von einem komplexen Zusammenspiel verschiedenster Gene mit unterschiedlicher Relevanz auszugehen ist, stellen miRNAs eine bedeutende Größe dar, da sie es vermögen auf transkriptioneller Ebene Einfluss auf die Regulierung einer Vielzahl von Genen zu nehmen. Verschiedene Aspekte liefern Hinweise darauf, dass eine Neurotransmitterdysregulation eine wichtige Komponente in der Pathogenese von Angsterkrankungen einnimmt – insbesondere veränderte noradrenerge Signalwege sind hierbei entscheidend beteiligt. Dies macht den Noradrenalin-Transporter bzw. SLC6A2 zu einem interessanten Kandidatengen, und stellt die Bezugsgröße der angestellten Untersuchungen in dieser Arbeit dar. miRNAs, welche die SLC6A2-Expression modulieren, können somit Einfluss auf zentrale Verarbeitungswege von Angst nehmen. Im ersten Teil der vorliegenden Arbeit wurden potentielle miRNA-Regulatoren von SLC6A2 in silico ermittelt und in einem weiteren Schritt in vitro überprüft. Zehn der miRNAs (hsa-miR-378g, hsa-miR-330-5p, hsa-miR-4781-5p, hsa-miR664b-3p, hsa-miR-4715-3p, hsa-miR-579-3p, hsa-miR-3921, hsa-miR-3622b-5p, hsa-miR-4773, hsa-miR-532-3p) zeigten hierbei eine relevante Abnahme der Luciferase-Aktivität als Hinweis auf ihre funktionelle Relevanz und stellen damit die Basis der nachfolgenden Untersuchungen dar. Im zweiten Teil der Arbeit wurden Einzelbasenpolymorphismen im Bereich der zuvor ermittelten miRNA-Gene sowie eines SNP innerhalb der 3’-UTR von SLC6A2 mittels Fall-Kontroll-Studie in einer Population von Patienten mit Panikstörung und entsprechenden Kontrollen untersucht. Eine nominelle Assoziation ließ sich für das (minor) T-Allel von rs2910931 (stromaufwärts von MIR579) (p-allel = 0,004) sowie das (major) A-Allel von rs2582372 (p-allel = 0,023) feststellen. In Einklang hiermit ließ sich weiterhin für rs2910931 eine signifikante Assoziation zwischen der Anzahl der (minor) T-Allele und dem ASI-Wert (β = 0,371, p = 0,029, 95 %-CI 0,039-0,702) sowie dem ACQ-Wert (β = 0,012, p = 0,041, 95 %-CI 0,000-0,023) ermitteln. Somit zeigt sich eine Einflussnahme der genetischen Variante um MIR579 auf die Feinmodulation der Noradrenalin-Homöostase als möglichem ätiopathogenetischen Faktor von Angsterkrankungen. / Anxiety disorders are common conditions with a multifactorial etiology that has not yet been fully understood. In addition to environmental and psychosocial factors, studies show a significant familial clustering and suggest a genetic component with a heritability in the range of 30-60%. Since a complex interaction of various genes with different relevance can be assumed, miRNAs are an important factor, since they are able to influence the regulation of a large number of genes at the transcriptional level. Various aspects provide evidence that neurotransmitter dysregulation is an important component in the pathogenesis of anxiety disorders - in particular, altered noradrenergic signaling pathways are crucially involved. This makes the norepinephrine transporter, or SLC6A2, an interesting candidate gene, and represents the reference parameter for the studies conducted in this work. miRNAs that modulate SLC6A2 expression can thus influence central processing pathways of anxiety. In the first part of the present work, potential miRNA regulators of SLC6A2 were identified in silico and, in a further step, tested in vitro. Ten of the miRNAs (hsa-miR-378g, hsa-miR-330-5p, hsa-miR-4781-5p, hsa-miR664b-3p, hsa-miR-4715-3p, hsa-miR-579-3p, hsa-miR-3921, hsa-miR-3622b-5p, hsa-miR-4773, hsa-miR-532-3p) here showed a relevant decrease in luciferase activity as an indication of their functional relevance and thus form the basis of subsequent studies. In the second part of the work, single base polymorphisms in the region of the previously identified miRNA genes as well as a SNP within the 3'-UTR of SLC6A2 were investigated by case-control study in a population of patients with panic disorder and corresponding controls. A nominal association could be detected for the (minor) T allele of rs2910931 (upstream of MIR579) (pallel = 0.004) as well as the (major) A allele of rs2582372 (pallel = 0.023). Consistent with this, a significant association between the number of (minor) T alleles and the ASI value (β = 0.371, p = 0.029, 95%-CI 0.039-0.702) as well as the ACQ value (β = 0.012, p = 0.041, 95%-CI 0.000-0.023) could further be determined for rs2910931. Thus, an influence of the genetic variant around MIR579 on the fine modulation of noradrenaline homeostasis as a possible etiopathogenetic factor of anxiety disorders is revealed.

MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs. Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations. Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages. Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases. / Ph. D.

Fibromyalgia syndrome (FMS) is a largely heterogeneous chronic pain syndrome of unclear pathophysiology, which lacks objective diagnostics and specific treatment. An immune-related shift towards a pro-inflammatory profile is discussed at a systemic level. Small fiber pathology (SFP) and local participation of non-neuronal skin cells like keratinocytes in cutaneous nociception are potential peripheral contributors. Small RNAs, particularly microRNAs (miRs) and newly described tRNA fragments (tRFs) act as posttranscriptional key regulators of gene expression and may modulate systemic and peripheral cell pathways. On cellular level, the exact mechanisms of keratinocyte-intraepidermal nerve fiber (IENF) interaction in the skin are insufficiently understood. Via small RNA sequencing and quantitative real-time PCR, we investigated miR and tRF signatures in whole blood cells and skin biopsy-derived keratinocytes of female FMS patients versus healthy controls. We applied gene target prediction analysis to uncover underlying cellular pathways affected by dysregulated small RNAs. Altered FMS small RNAs from blood were compared with their expression in disease controls, i.e. Parkinson`s patients and patients with major depression and chronic pain. Association of SFP with small RNAs was investigated via correlation with clinical parameter. To explore keratinocyte-nerve fiber interactions with high relevance for SFP and cutaneous nociception, we adapted a super-resolution array tomography (srAT) approach and expansion microscopy (ExM) for human skin samples. Further, we created a fully human 2D co-culture model of primary keratinocytes and induced pluripotent stem cell derived sensory neurons. Blood miR deregulation indicated systemic modulation of immune processes exerted by CholinomiRs and by miRs targeting the FoxO signaling pathway. Short sized tRFs were associated with mRNA metabolism and splicing. This supports the hypothesis of an inflammatory/autoimmunity component in FMS. Expression of blood small RNAs in FMS were discriminative against disease controls, highlighting their potential as objective biomarker. Blood small RNAs were predominantly upregulated and correlations between miR and clinical parameter reflected rather pain in general than SFP. In FMS keratinocytes, a downregulation of miRs and tRFs was evident. Pathways for adenosine monophosphate-activated protein kinase (AMPK), adherens junction, and focal adhesion were predicted to be affected by miRs, while tRFs may influence proliferation, migration, and cell growth. Similar to blood miRs, altered miRs in keratinocytes correlated mostly with widespread pain and pain severity parameter. TRFs were partially associated with more severe IENF loss. Small RNAs in FMS keratinocytes may modulate pathways that define how keratinocytes interact with each other and with IENF. These interactions include nerve fiber ensheathment, a conserved epithelial mechanism, which we visualize in human epidermis and a fully human co-culture model. Additionally, we revealed plaques of connexin 43, a pore forming protein involved in intercellular communication, at keratinocyte- nerve fiber contact sites. Objective quantification of these morphological findings in FMS and other diseases with SFP may inherit diagnostic value similar to IENF density. We provide evidence for distinct miR and tRF signatures in FMS with implications for systemic immune regulation and local cell-cell interaction pathways. In the periphery we explored novel keratinocyte-nerve fiber interactions relevant for SFP and cutaneous nociception. / Das Fibromyalgie Syndrom (FMS) umfasst ein sehr heterogenes chronisches Schmerzsyndrom mit ungeklärter Pathophysiologie, ohne objektive Diagnostik und gezielt wirkende Behandlungsmöglichkeiten. Auf systemischer Ebene wird eine entzündungsfördernde Verschiebung von Immunprozessen diskutiert. In der Peripherie stellen die Kleinfaserpathologie (SFP) und Beteiligungen nicht-neuronaler Hautzellen, beispielsweise Keratinozyten, an kutaner Nozizeption potenziell beitragende Faktoren dar. Kleine RNAs, vor allem microRNAs (miRs) und die kürzlich beschriebenen tRNA Fragmente (tRFs) agieren als posttranskriptionelle Schlüsselregulatoren der Genexpression und könnten daher systemische und periphere Zellprozesse modulieren. Die genauen zellulären Mechanismen bei der Interaktion von Keratinozyten mit intraepidermalen Nervenfasern (IENF) in der Haut sind nur unzureichend verstanden. Mittels Sequenzierung von kleinen RNAs und quantitativer Real-Time PCR untersuchten wir miR und tRF Signaturen in Vollblutzellen und in durch Hautbiopsie gewonnene Keratinozyten von FMS Patientinnen im Vergleich zu gesunden weiblichen Kontrollen. Um zugrundeliegende Zellprozesswege aufzudecken, die von der Deregulierung kleiner RNAs betroffen sind, verwendeten wir Vorhersageprogramme für regulierte Gene. In FMS verändert vorliegende kleine RNAs im Blut verglichen wir mit ihrer Expression in Krankheitskontrollen, d.h. Parkinson Patientinnen und Patientinnen mit schwerer Depression und chronischem Schmerz. Die Beziehung zwischen SFP und kleinen RNAs wurde mittels der Korrelation mit klinischen Parametern untersucht. Zur Erforschung von Keratinozyten-Nervenfaser Interaktionen, mit großer Relevanz für SFP und kutane Nozizeption, adaptierten wir eine superauflösende Array-Tomographie (srAT) Methodik und Expansionsmikroskopie (ExM) für humane Hautproben. Außerdem entwickelten wir ein rein humanes 2D Ko-Kultur Zellmodell, bestehend aus primären Keratinozyten und sensiblen Neuronen, die aus induzierten pluripotenten Stammzellen generiert wurden. MiR Deregulierungen in Blut wiesen auf systemische Modulierung von Immunprozessen hin, ausgeübt durch CholinomiRs und miRs, die auf den FoxO Signalweg einwirken. Die tRFs mit kurzer Fragmentlänge waren mit mRNA Metabolismus und Splicing verknüpft. Diese Ergebnisse unterbauen die Hypothese einer entzündungsfördernden/autoimmunen Komponente in FMS. Die Expression kleiner RNAs aus FMS Blut war unterschiedlich zu Krankheitskontrollen, was ihr Potenzial als objektive Biomarker hervorhebt. Kleine RNAs im Blut waren überwiegend erhöht exprimiert und Korrelation zwischen miRs und klinischen Parametern spiegelten eher Schmerzen im Allgemeinen wider als SFP. In Keratinozyten von FMS Patientinnen war eine Herunterregulierung von miRs und tRFs ersichtlich. Der Signalweg der Adenosinmonophosphat aktivierten Proteinkinase (AMPK), sowie Adherens Junction und Fokale Adhäsion waren prognostiziere Prozesse unter Einfluss von miRs. Ähnlich wie bei den Blut miRs, korrelierten veränderte miRs in Keratinozyten vor allem mit der Verbreitung des Schmerzes über den Körper und der Schmerzintensität. TRFs waren teilweise mit einem höheren Verlust an IENF verknüpft. Kleine RNAs in Keratinozyten von FMS Patientinnen könnten jene Prozesse modulieren, die festlegen, wie Keratinozyten miteinander und mit IENF interagieren. Diese Interaktionen beinhalten den konservierten Mechanismus der Nervenfaserumhüllung, den wir in humaner Epidermis und einem komplett humanen Ko-Kultur Modell auflösen konnten. Zusätzlich zeigten wir Anhäufungen von Connexin 43, einem an interzellulärer Kommunikation beteiligten porenformenden Protein, an Keratinozyten-Nervenfaser Kontaktstellen. Eine objektive Quantifizierung dieser morphologischen Befunde in FMS und weiteren Erkrankungen mit SFP könnte einen diagnostischen Wert vergleichbar mit dem der IENF Dichte innehaben. Wir liefern Belege für klare miR und tRF Signaturen in FMS mit Bedeutung für systemische Immunregulation und lokale Zell-Zell Interaktionsprozesse. In der Peripherie erkundeten wir neueartige Keratinozyten-Nervenfaser Interaktionen relevant für SFP und kutane Nozizeption.

Infectious diseases caused by pathogenic microorganisms are one of the largest socioeconomic burdens today. Although infectious diseases have been studied for decades, in numerous cases, the precise mechanisms involved in the multifaceted interaction between pathogen and host continue to be elusive. Thus, it still remains a challenge for researchers worldwide to develop novel strategies to investigate the molecular context of infectious diseases in order to devise preventive or at least anti-infective measures. One of the major drawbacks in trying to obtain in-depth knowledge of how bacterial pathogens elicit disease is the lack of suitable infection models to authentically mimic the disease progression in humans. Numerous studies rely on animal models to emulate the complex temporal interactions between host and pathogen occurring in humans. While they have greatly contributed to shed light on these interactions, they require high maintenance costs, are afflicted with ethical drawbacks, and are not always predictive for the infection outcome in human patients. Alternatively, in-vitro two-dimensional (2D) cell culture systems have served for decades as representatives of human host environments to study infectious diseases. These cell line-based models have been essential in uncovering virulence-determining factors of diverse pathogens as well as host defense mechanisms upon infection. However, they lack the morphological and cellular complexity of intact human tissues, limiting the insights than can be gained from studying host-pathogen interactions in these systems. The focus of this thesis was to establish and innovate intestinal human cell culture models to obtain in-vitro reconstructed three-dimensional (3D) tissue that can faithfully mimic pathogenesis-determining processes of the zoonotic bacterium Campylobacter jejuni (C. jejuni). Generally employed for reconstructive medicine, the field of tissue engineering provides excellent tools to generate organ-specific cell culture models in vitro, realistically recapitulating the distinctive architecture of human tissues. The models employed in this thesis are based on decellularized extracellular matrix (ECM) scaffolds of porcine intestinal origin. Reseeded with intestinal human cells, application of dynamic culture conditions promoted the formation of a highly polarized mucosal epithelium maintained by functional tight and adherens junctions. While most other in-vitro infection systems are limited to a flat monolayer, the tissue models developed in this thesis can display the characteristic 3D villi and crypt structure of human small intestine. First, experimental conditions were established for infection of a previously developed, statically cultivated intestinal tissue model with C. jejuni. This included successful isolation of bacterial colony forming units (CFUs), measurement of epithelial barrier function, as well as immunohistochemical and histological staining techniques. In this way, it became possible to follow the number of viable bacteria during the infection process as well as their translocation over the polarized epithelium of the tissue model. Upon infection with C. jejuni, disruption of tight and adherens junctions could be observed via confocal microscopy and permeability measurements of the epithelial barrier. Moreover, C. jejuni wildtype-specific colonization and barrier disruption became apparent in addition to niche-dependent bacterial localization within the 3D microarchitecture of the tissue model. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D host environment deviated from those obtained with conventional in-vitro 2D monolayers but mimicked observations made in vivo. Furthermore, a genome-wide screen of a C. jejuni mutant library revealed significant differences for bacterial factors required or dispensable for interactions with unpolarized host cells or the highly prismatic epithelium provided by the intestinal tissue model. Elucidating the role of several previously uncharacterized factors specifically important for efficient colonization of a 3D human environment, promises to be an intriguing task for future research. At the frontline of the defense against invading pathogens is the protective, viscoelastic mucus layer overlying mucosal surfaces along the human gastrointestinal tract (GIT). The development of a mucus-producing 3D tissue model in this thesis was a vital step towards gaining a deeper understanding of the interdependency between bacterial pathogens and host-site specific mucins. The presence of a mucus layer conferred C. jejuni wildtype-specific protection against epithelial barrier disruption by the pathogen and prevented a high bacterial burden during the course of infection. Moreover, results obtained in this thesis provide evidence in vitro that the characteristic corkscrew morphology of C. jejuni indeed grants a distinct advantage in colonizing mucous surfaces. Overall, the results obtained within this thesis highlight the strength of the tissue models to combine crucial features of native human intestine into accessible in-vitro infection models. Translation of these systems into infection research demonstrated their ability to expose in-vivo like infection outcomes. While displaying complex organotypic architecture and highly prismatic cellular morphology, these tissue models still represent an imperfect reflection of human tissue. Future advancements towards inclusion of human primary and immune cells will strive for even more comprehensive model systems exhibiting intricate multicellular networks of in-vivo tissue. Nevertheless, the work presented in this thesis emphasizes the necessity to investigate host-pathogen interactions in infection models authentically mimicking the natural host environment, as they remain among the most vital parts in understanding and counteracting infectious diseases. / In der heutigen Zeit tragen insbesondere durch pathogene Mikroorganismen ausgelöste Infektionskrankheiten zur sozioökonomischen Belastung bei. Obwohl bereits jahrzehntelang an der Entstehung von Infektionskrankheiten geforscht wird, bleiben in zahlreichen Fällen die genauen Mechanismen, welche an den vielfältigen Interaktionen zwischen Pathogen und Wirt beteiligt sind, unbeschrieben. Gerade deshalb bleibt es für Wissenschaftler weltweit eine Herausforderung, neue Strategien zur Untersuchung des molekularen Kontexts von Infektionskrankheiten zu entwickeln, um präventive oder zumindest anti-infektive Maßnahmen ergreifen zu können. In den meisten Fällen ist jedoch das Fehlen geeigneter Infektionsmodelle, mit denen der Krankheitsverlauf im Menschen authentisch nachgestellt werden kann, eines der größten Hindernisse um detailliertes Wissen darüber gewinnen zu können wie bakterielle Pathogene die Krankheit auslösen. Zahlreiche Studien sind dabei auf Tiermodelle angewiesen, um die komplexen zeitlichen Abläufe zwischen Wirt und Pathogen im menschlichen Körper nachzuahmen. Während diese Modelle in hohem Maß dazu beigetragen haben, Aufschluss über diese Abläufe zu geben, sind sie doch sehr kostenintensiv, mit ethischen Bedenken behaftet und können nicht immer die Folgen einer Infektion im menschlichen Patienten vorhersagen. Seit Jahrzehnten werden daher alternativ in-vitro 2D Zellkultursysteme eingesetzt, um den Verlauf von Infektionskrankheiten zu erforschen, welche die Bedingungen im menschlichen Wirt wiederspiegeln sollen. Diese auf Zelllinien basierenden Modelle sind essentiell in der Entdeckung von Virulenzfaktoren diverser Pathogene, aber auch in der Aufklärung von wirtsspezifischen Abwehrmechanismen. Dennoch fehlt ihnen die morphologische und zelluläre Komplexität von intaktem menschlichen Gewebe. Dadurch sind die Erkenntnisse, die mit diesen Systemen über Infektionsverläufe gewonnen werden können, limitiert. Die vorgelegte Arbeit konzentriert sich auf die Etablierung und Weiterentwicklung intestinaler, humaner Zellkulturmodelle, um dreidimensionales Gewebe in vitro zu rekonstruieren mit dem Ziel, Pathogenese-beeinflussende Prozesse des zoonotischen Bakteriums C. jejuni nachzustellen. Das Fachgebiet der Gewebezüchtung wird üblicherweise für rekonstruktive Medizin eingesetzt und bietet exzellente Mittel zur in-vitro Herstellung organspezifischer Zellkulturmodelle, welche die unverkennbare Mikroarchitektur humanen Gewebes realistisch nachempfinden können. Die in dieser Arbeit verwendeten Modelle basieren auf einem extrazellulären Matrixgerüst, das aus der Dezellularisierung von Schweinedarm gewonnen wurde. Durch die Wiederbesiedelung mit human Kolonzellen und der Kultivierung unter dynamischen Bedingungen entwickelte sich ein hochpolarisiertes mucosales Epithel, das durch funktionale Zell-Zell-Kontakte (tight und adherens junctions) aufrechterhalten wird. Während andere in-vitro Infektionssysteme meist durch die Präsenz einer flachen Zellschicht limitiert werden, entwickelt das in dieser Arbeit eingeführte Gewebemodell die für den menschlichen Dünndarm charakteristische Architektur aus Villi und Krypten. Zunächst wurden experimentelle Bedingungen für die Infektion eines zuvor entwickelten, statisch kultivierten Dünndarmmodells mit C. jejuni etabliert. Dies beinhaltete die erfolgreiche Isolierung koloniebildender Einheiten, die Messung der epithelialen Barrierefunktion, sowie immunhistochemische und histologische Färbetechniken. Dadurch konnte die Anzahl der Bakterien sowie deren Translokalisierung über das polarisierte Epithel während des Infektionsprozesses nachvollzogen werden. Außerdem konnte die Beeinträchtigung von Zell-Zell-Kontakten durch konfokale Mikroskopie und Permeabilitätsmessungen der epithelialen Barriere beobachtet werden. Neben der Bestimmung der Kolonisierungsrate von C. jejuni Isolaten und der dadurch hervorgerufenen spezifischen Zerstörung der epithelialen Barriere konnten die Bakterien auch innerhalb der 3D Mikroarchitektur des Gewebemodells lokalisiert werden. Außerdem konnte im Rahmen der 3D Gewebeumgebung beobachtet werden, dass Pathogenese-relevante Phänotypen von C. jejuni Mutantenstämmen im Vergleich zu konventionellen in-vitro 2D Zellschichten abwichen, diese aber dafür mit den in-vivo gemachten Beobachtungen übereinstimmten. Darüber hinaus wies die genomweite Suche einer C. jejuni Mutantenbibliothek signifikante Unterschiede zwischen bakteriellen Faktoren, die für die Interaktion mit nicht polarisierten Wirtszellen oder dem hochprismatischen Epithel des Gewebemodells bedeutsam oder entbehrlich waren, auf. Die Aufklärung der Funktion einiger bisher nicht charakterisierter Faktoren, die zu einer effizienten Kolonisierung menschlichen Gewebes beitragen, verspricht eine faszinierende Aufgabe für die zukünftige Forschung zu werden. Die vorderste Verteidigungslinie gegen eindringende Pathogene bildet die schützende, viskoelastische Mukusschicht, die mukosale Oberflächen entlang des menschlichen Gastrointestinaltrakts überzieht. Mit der Entwicklung eines mukusproduzierenden Gewebemodells in der hier vorgelegten Arbeit gelang ein entscheidender Schritt zur Erforschung der Wechselbeziehungen zwischen bakteriellen Pathogenen und wirtsspezifischen Muzinen. Während des Infektionsverlaufs wurde das unterliegende Epithel durch die Anwesenheit der Mukusschicht vor der Zerstörung durch die Mikroben geschützt und eine erhöhte bakterielle Belastung verhindert. Darüber hinaus liefern die Resultate dieser Arbeit einen in-vitro Nachweis für den bakteriellen Vorteil einer spiralförmigen Morphologie, um muköse Oberflächen zu besiedeln. Zusammenfassend unterstreicht diese Arbeit das Potential der hier entwickelten Gewebemodelle, entscheidende Eigenschaften des menschlichen Darms in einem leicht zugänglichen in-vitro Infektionsmodell zu vereinigen. Der Einsatz dieser Modelle im Rahmen der Infektionsforschung bewies deren Fähigkeit in-vivo beobachtete Infektionsverläufe widerzuspiegeln. Während diese Infektionsmodelle bereits organotypische Architektur und hochprismatische Zellmorphologie aufweisen, ist ihre Darstellung von menschlichem Gewebe noch nicht perfekt. Durch den Einsatz von humanen Primär- und Immunzellen wird es in Zukunft möglich sein, noch umfassendere Modellsysteme zu entwickeln, die komplexe multizelluläre Netzwerke von in-vivo Geweben aufweisen. Nichtsdestotrotz verdeutlicht die hier vorgelegte Arbeit wie wichtig es ist, die Interaktionen zwischen Wirt und Pathogen innerhalb von Infektionsmodellen zu erforschen, welche die natürliche Wirtsumgebung wiedergeben. Dies spielt eine entscheidende Rolle, um die Entstehung von Infektionskrankheiten nachvollziehen und ihnen entgegenwirken zu können.

Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of ArrF-encoding gene resulted in a 300old increase in the production of polyhydroxybutyrate (PHB), a polymer of industrial importance. This ∆arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. SDS-PAGE and MALDI-MS/MS revealed the overexpression of acetyl-CoA reductase, a phbBAC operon enzyme and the proteins that would alleviate the stress due to PHB accumulation in the ∆arrF mutant. Real-time RT-PCR revealed that phbR, phbB, phbA and phbC were upregulated in the mutant. Increased levels of activator PhbR in the mutant elevates the expression of phbB, phbA and phbC, resulting in the PHB overproduction. The proteins differentially expressed in the ∆arrF mutant were determined by gel-based proteomics and confirmed by real time RT-PCR. 6-phosphogluconolactonase that involve in the production of NADPH and acetyl-CoA, was upregulated, while the proteins involved in the TCA cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were overexpressed in the mutant. In addition, antioxidant proteins such as Fe-containing supeoxide dismutase (FeSOD), a putative oxidoreductase with unknown function, alkyl hydroperoxide reductase, flavorprotein WrbA and cysteine synthase were also upregulated, indicating that the PHB accumulation is highly stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase. Among the genes upregulated in the ∆arrF mutant, sodB gene coding for Fe-superoxide dismutase and phbF gene encoding PHB synthesis regulator appears to be negatively regulated by small RNA ArrF in an antisense mechanism. However, all the TCA cycle genes were downregulated in the ∆arrF mutant. In addition to the TCA cycles enzyme, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that several factors are responsible for the overproduction of PHB polymer in the ∆arrF mutant and one of which is the direct effect of small RNA ArrF on the expression of PhbF .

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