Myoclonus-Dystonia (MD) is an inherited, rare, autosomal dominant movement disorder characterized by quick, involuntary muscle jerking or twitching (myoclonus) and involuntary muscle contractions that cause twisting and pulling movements, resulting in abnormal postures (dystonia). The first MD locus was mapped to 7q21-q31 and called DYT11; this locus corresponds to the SGCE gene. Our group previously identified a second MD locus (DYT15) which maps to a 3.18 Mb region on 18p11. Two patients were chosen to undergo next-generation sequencing, which identified 2,292 shared novel variants within the critical region. Analysis of these variants revealed a 3 bp duplication in a transcript referred to as CD108131, which is believed to be a long non-coding RNA. Characterization of this transcript determined that it is 863 bp in size, it is ubiquitously expressed, with high expression in the cerebellum, and it accounts for ~3% of MD cases.

A major virulence factor of the human malaria parasite Plasmodium falciparum is Plasmodium falciparum erythrocyte membrane protein 1(PfEMP-1). This protein is inserted into the erythrocyte membrane, giving cytoadherence properties. A family of genes called var, located sub-telomerically and in chromosome central clusters encode this protein. Var genes are expressed in a mutually exclusive manner, how this is controlled is unclear. A non-coding RNA (ncRNA) termed the GC-rich element (GRE) had been identified that is only located at the central clusters and is transcribed throughout the parasite lifecycle. A screen of the P. falciparum genome for novel ncRNAs identified ncRNAs from known classes. Novel transcripts were identified, but none in the proximity of var genes. We have investigated the role of the GRE in var gene regulation. A set of qRT-PCR primers have been designed and tested to follow var gene expression in the HB3 isolate, these are not cross-reactive with a published set for the 3D7 isolate. Alterations were made to the 3D7 set to remove cross-reactivity with HB3. Var gene expression was studied in 31 HB3 clones and progeny of the 3D7xHB3 genetic cross. Following var switching over five months in eleven HB3 clones showed that all of the clones ended up expressing var genes from the same central cluster on chromosome 4. GRE Transcription in these clones is linked to a specific class of var gene. Transcription from a single GRE locus occurs only when a var gene of the central UpsC class is expressed from the same cluster. Expression of other classes of var gene gives multiple transcripts from different GRE loci. Investigations into the in vitro binding properties of the GRE revealed an RNA:protein complex that can be resolved by electrophoresis. Proteomic analysis of the complex revealed predominantly ribosome proteins and translation factors.

La machinerie d'assemblage HSP90/R2TP est impliquée dans la biogenèse de complexes essentiels à l'expression génique et à la croissance cellulaire. Le complexe est constitué des protéines PIH1D1, RPAP3 et des AAA+ ATPases RVB1 et RVB2. Le R2TP, via son adaptateur NUFIP, permet l'assemblage de complexes ribonucléoprotéiques tels que les snoRNP à boîtes C/D, et le snRNP U4, tous deux impliqués respectivement dans la maturation des ARNr et ARNm. Le mode d'action du R2TP dans ces processus n'était pas bien compris. Pour étudier cela, une approche de protéomique, avec des tests d'interaction ARN/protéine et protéine/protéine, ainsi qu'une approche structurale, ont été utilisés. Un nouveau modèle a ainsi été établi. Le R2TP permettrait de former des pré-complexes d'assemblage contenant les protéines cœurs du complexe RNP avec des facteurs d'assemblage mais sans l'ARN. Les protéines RVB se détacheraient du R2TP pour rester associées à ce pré-complexe d'assemblage, par la suite, elles permettraient de le stabiliser tout en y incorporant de nouvelles protéines cœurs, et en relargant des facteurs d'assemblage ayant déjà accompli leur fonction dans le processus de biogenèse. Cette fonction de chaperon moléculaire des complexes en cours d'assemblage est très probablement régulée par hydrolyse de l'ATP par les ATPases RVB, et ceci sous le contrôle de co-facteurs, comme potentiellement la protéine BCD1. Dans le cas de l'assemblage des C/D snoRNP, il a été établi un modèle d'assemblage dans lequel le rôle des différents facteurs d'assemblage peut être prédit. ZNHIT3 aurait un rôle dans l'incorporation du snoARN naissant dans le pré-complexe, et NUFIP permettrait de garder la particule immature dans une conformation inactive afin de faciliter l'obtention de la structure active du snoRNP grâce aux RVB. Avec les travaux sur la biogenèse de la particule U4, il a été mis en évidence l'existence d'un pré-complexe cytoplasmique contenant PRP31/NUFIP/R2TP/complexe SMN qui serait important pour l'assemblage de la snRNP U4 avec non seulement les protéines Sm, mais aussi la protéine PRP31. / The HSP90/R2TP machinery is involved in the biogenesis of essential complex for gene expression and cell growth. The complex consists of proteins PIH1D1, RPAP3 and the AAA+ ATPases RVB1 and RVB2. The R2TP, via its NUFIP adaptator, allows assembly of ribonucleoprotein complexes like box C/D snoRNP, and the U4 snRNP, both involved in the maturation of mRNA and rRNA respectively. The mode of action of R2TP in these processes is not well understood. In this study, a proteomic approach, with tests of interaction RNA/protein and protein/protein and a structural approach, were used. A new model has been established. The R2TP would form an assembly pre-complex containing RNP core proteins with assembly factors but not RNA. RVB proteins detach from R2TP to remain associated with the assembly pre-complex, and then, would stabilize it while incorporating new core proteins. They would also release assembly factors that already have accomplished their function in the biogenesis process. This function of molecular chaperone complex during assembly is most likely regulated by ATP hydrolysis by the RVB ATPases, and this under the control of co-factors as potentially BCD1 protein. In the case of the assembly of box C/D snoRNP, it was established an assembly model in which the roles of the various assembly factors can be predicted. ZNHIT3 has a role in the incorporation of the nascent snoRNA in the pre-complex and NUFIP would keep the immature particle into an inactive conformation to facilitate the formation of the active structure of the snoRNP through RVB. With the study of the biogenesis of the U4 particle, it was revealed the existence of a cytoplasmic pre-complex containing PRP31/NUFIP/R2TP/SMN complex that would be important for the assembly of the U4 snRNP with not only Sm protein but also the PRP31 protein.

A visao sobre o dogma central da biologia molecular passou por aperfeicoamentos na virada deste seculo. Muito se deve ao interesse por pesquisas feitas para compreensao do que ate entao eram regioes do genoma conhecidas como DNA Lixo. Neste contexto, projetos de transcriptoma, avancos em tecnologias de sequenciamento, bem como analises em bioinformatica, contribuiram para elucidar o que estava sendo transcrito. Tais regioes foram denominadas como RNAs nao-codificadores ou non-coding RNA (ncRNA) que eram transcritas, mas nao traduzidas em proteinas. Apesar da quantidade de metodos para o estudo in silico dos ncRNAs, existem lacunas a serem preenchidas nas pesquisas desta molecula, tais como: metodos de anotacao em geral, caracterizacao de novas classes e mecanismos alternativos de busca por similaridade de sequencia primaria. Alem disso, nao se havia uma ferramenta que reunisse num unico local as informacoes dos bancos de dados publicos de ncRNA disponiveis. Neste trabalho, buscou-se preencher tais lacunas, contribuindo para o desenvolvimento de metodos computacionais nas pesquisas em ncRNAs. Foram utilizados os genomas de Hymenoptera e Diptera como sistema biologico para aplicar e testar os metodos desenvolvidos. / The classical vision of the central dogma of molecular biology was not changes dramatic until the end of the 20th century. At this time the scientific communities were interesting to understand what have in the regions of the genome known as \"Junk DNA\". Transcriptome projects together with sequencing Technologies anda bioinformatics analysis help to elucidate that this transcripts were regions that do not coding proteins and maybe has function. These transcripts are called non-coding RNA (ncRNA). Although there are a lot of computational approaches to the in silico research of ncRNA, there is a gap of research about this molecule such: approaches to the general annotation of ncRNA; identification of new classes of ncRNA; and alternatives search mechanisms of ncRNA. Besides that, there are not any central repository of public non-coding RNA databases that could help search for the information about it. In this report, we fill this gap. We tried to contributing to the development of computational methods in research on ncRNAs. We also used the Hymenoptera and Diptera genomes as a biological system to apply and test our developed approaches.

Les ARNnc sont impliqués dans la régulation de l’expression de gènes chez les Procaryotes, les Archées et les Eucaryotes. Cette régulation peut être effectuée au niveau transcriptionnel ou post-transcriptionnel. Elle fait parfois intervenir des modifications des histones comme la méthylation ou l’acétylation. J’ai étudié le gène TIR1 dont l’expression est fortement réduite lorsqu’un ARNnc codant antisens nommé TIR1axut est stabilisé. J’ai montré que cette régulation est dépendante de l’histone déacétylase Hda1. De plus, j’ai montré que l’acétylation de H3K14 et H3K18 ne sont pas directement impliquées dans la régulation de TIR1 mais qu’un résidu polaire est nécessaire pour la répression de TIR1 en présence de l’ARNnc antisens. En outre, j’ai mis en évidence que la répression de TIR1 par son XUT est en parti post-transcriptionnel, mais ne fait pas varier la stabilité de l’ARNm. Finalement, j’ai tenté en vain de comprendre le ciblage de l’activité histone déacétylase de Hda1 le long de TIR1 en cherchant la présence d’hybride ARN/ADN grâce à un anticorps reconnaissant ce type de structure. / NcRNAs are involved in gene regulation in Prokaryotes, Eukaryotes and Archaea. This regulation could be transcriptional or post-transcriptional. Histone modifications could be involved such as methylation or acetylation. I studied TIR1 gene whose expression is highly reduced when an antisense ncRNA called TIR1axut is stabilized. I showed that this regulation is Hda1-dependant. In addition to that, I showed that H3K14ac and H3K18ac are not directly responsible for TIR1 repression but a polar residue is required for a proper silencing of TIR1 in a XUT depending manner. Moreover, I showed that TIR1 repression is due to a post-transcriptional effect but does not affect mRNA stability. Finally, I tried in vain to understand Hda1 targeting on TIR1 searching for RNA/DNA hybrids using an antibody that recognizes such structures.

Non-coding RNAs (ncRNAs) have important biological functions such as regulation of gene expression and disease causality. These ncRNAs exert their functions by interacting with other molecules, such as messenger RNAs (mRNAs). Thus RNA-RNA interaction studies are important for understanding the gene regulation mechanism and for curing ncRNAs related diseases. This thesis contributes to RNA-RNA interaction prediction problem, construction of a comprehensive human micro RNAs (miRNAs)-related database and data mining on high throughput RNA-RNA interaction data. / On RNA-RNA interaction prediction problem, a novel energy model is proposed and a GA based algorithm is developed, namely RIPGA. The experiments results show that the novel energy model outperforms the state of the art model, which is called Turner energy model, RIPGA with novel energy model also outperforms two state of the art programs, which are called inRNAs and RactIP. / On construction of a comprehensive human miRNA-related database, data are collected and cleansed from three state of the art databases related to human miRNAs, which are called miR-TarBase, miRBase and HMDD v2.0. A network is constructed from these data to present the complete relationships of the miRNAs because the relationships are only partial covered by the existing databases. A website and database are setup for data query, visualization and analysis functions to complement the existing databases. / On data mining on high-throughput RNA-RNA interaction data, four characteristics of RNA-RNA interaction are identified from the high-throughput data. We believe these characteristics are potential explanations of the high degree of connectivity of some miRNAs. These characteristics are also important scientific knowledge for future research on RNA-RNA interaction and control for biomedical applications. / 非編碼核糖核酸(ncRNAs)有重要的生物功能，例如：基因表現的調控和疾病的因果關係。這些ncRNAs透過與其他分子的互相作用來發揮作用，例如：信使核糖核酸(mRNAs)。因此，核糖核酸互相作用的研究對理解基因表現的調控和治愈與ncRNAs有關的疾病十分重要。本論文集中解決核糖核酸互相作用的預測問題、建設人類微核糖核酸(miRNAs)綜合數據庫和高通量核糖核酸互相作用數據的數據挖掘。 / 針對核糖核酸互相作用的預測問題，我們提出了一個新的能量模型和開發了一個基於遺傳算法的算法，即RIPGA。實驗數據顯示新的能量模型比最先進的模型﹐即特納能量模型做得更好。而RIPGA亦比最先進的inRNAs和RacIP做得更好。 / 針對建設人類微核糖核酸綜合數據庫，我們收集及潔淨來自三個最先進的數據庫的數據，即miRTarBase，miRBase和HMDDv2.0。我們由這些數據建設了一個網絡來表達完整的miRNAs關係，因為現存的數據庫只覆蓋了部份的關係。我們亦設置了網站和數據庫，並提供數據查詢、可視化及分析的功能，以補足現存的數據庫。 / 針對高通量核糖核酸互相作用數據的數據挖掘，我們從高通量核糖核酸互相作用數據中認出四個核糖核酸互相作用的特點。我們相信這些特點是部份高連通miRNAs的可能的解釋。這些特點對核糖核酸互相作用及控制的未來研究和生物醫學應用亦是重要的科學知識。 / Cheung, Kwan Yau. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 149-154). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Sous conditions de stress métabolique ou environnemental, l’activation instantanée de voies moléculaires puissantes permet aux cellules de prévenir la formation et l’accumulation d’agrégats protéiques toxiques. HSF1 (Heat Shock Factor 1) est le facteur de transcription majeur capable d’orchestrer cette réponse cellulaire au stress et cela via l’activation de protéines au rôle protecteur nommées chaperonnes. Cependant, il est aujourd’hui évident que les fonctions initialement attribuées au facteur HSF1 s’étendent bien au-delà de l’activation de la transcription de chaperonnes. En effet, il a été démontré que HSF1 joue un rôle essentiel dans l’activation et le remodelage de régions répétées appartenant à l’hétérochromatine péricentromérique sous stress thermique et plus récemment qu’HSF1 contribuerait significativement au processus de tumorigenèse dans différents types de cancers. Dans cette étude, nous avons identifié pour la première fois les régions subtélomériques comme étant une nouvelle cible génomique d’HSF1 sous conditions de stress thermique. Nous avons démontré, que la liaison directe et spécifique d’HSF1 avec plusieurs de ces régions sous stress thermique est à l’origine d’une surexpression de longs ARNs non codants issus des télomères, aussi connus sous le nom de TERRA. De façon intéressante nous avons trouvé que cette transcription était corrélée à un enrichissement de la marque épigénétique répressive H3K9me3 au niveau télomérique. De plus, nos données ont permis de démontrer que l’intégrité de la chromatine télomérique était significativement atteinte sous conditions de stress thermique. Nous observons à la fois, une dissociation partielle de la protéine TRF2 (Telomeric repeat-binding factor 2) et une accumulation de dommages à l’ADN détectés grâce au marqueur moléculaire H2AX-P, au niveau des télomères. Finalement, nos résultats ont également permis de souligner un rôle d’HSF1 dans le maintien de cette intégrité télomérique. L’ensemble de ce travail établit un premier lien entre la voie cellulaire puissante de réponse au stress, son acteur majeur HSF1 et les régions de l’hétérochromatine télomérique, dans des lignées de cellules humaines cancéreuses. Ces données fournissent des indications précieuses sur une voie de maintien de télomères sous stress et nous permettant de proposer un modèle dans lequel cette nouvelle fonction d’HSF1 aux télomères pourrait être étroitement liée à l’expression des ARNs non codants télomériques. Sur la base de nos données ainsi que sur les multiples publications démontrant l’implication d’HSF1 dans la tumorigenèse, la définition exacte du rôle d’HSF1 au niveau de l’intégrité des télomères dans un contexte pathologique comme le cancer apparait aujourd’hui comme un défi prometteur. / In response to metabolic or environmental stress, cells rapidly activate powerful defense mechanisms to prevent the formation and accumulation of toxic protein aggregates. The main orchestrator of this cellular response is HSF1 (Heat Shock Factor 1), a transcription factor involved in the up-regulation of protein-coding genes with protective roles. However, it is now becoming clear, that HSF1 function extends beyond what was previously predicted and that HSF1 can contribute to pericentromeric heterochromatin remodeling and activation as well as to efficiently support malignancy. In this study, we identify subtelomeric DNA as a new genomic target of HSF1 upon heat shock (HS). We show that HSF1 binding to subtelomeric regions plays an essential role in the upregulation of TERRA lncRNAs transcription and in the accumulation of repressive H3K9me3 histone mark at telomeres upon HS. Additionally, we demonstrate that HS significantly affects telomere capping and telomere integrity. We bring evidence of a partial TRF2 telomeric-binding factor dissociation and we reveal an accumulation of DNA damage at telomeres using the DNA damage marker H2A.X-P. In line with this, we bring solid evidences that under heat shock, HSF1 contributes to preserve telomere integrity by significantly limiting telomeric DNA damage accumulation. Altogether, our findings therefore reveal a new direct and essential function of HSF1 in transcription activation of TERRA and in telomere protection upon stress in human cancer cell lines. This work provides new insights into how telomeres are preserved under stressful heat shock conditions and allow us to propose a model where HSF1 may exert its protective function at telomeres via the expression of TERRA ncRNAs. Based on our results and given the important role of HSF1 in tumor development, defining the role of HSF1 with regard to telomere stability in tumor development already emerges as a promising challenge.

Very few vertebrates survive extended periods of time without oxygen. Entry into metabolic depression is central to surviving anoxia, which is supported by overall suppression of protein synthesis, yet requires increased expression of specific proteins. Studying the rapid and complex regulation of gene expression associated with survival of anoxia may uncover new mechanisms of cellular biology and transform our understanding of cells, as well as inform prevention and treatment of heart attack and stroke in humans. Small non-coding RNAs (sncRNAs) have emerged as regulators of gene expression that can be rapidly employed, can target individual genes or suites of genes, and are highly conserved across species. There are diverse types of sncRNAs, some coopted from degradation of longer RNAs in the cell. The sncRNA revolution has yielded a large body of literature revealing the roles of sncRNAs in a myriad of biological processes, from development to regulation of the cell cycle and apoptosis, to responding to stress, including freezing, dehydration, ischemia, and anoxia. Given the regulatory complexity required to survive anoxia, examining sncRNAs in the context of extreme anoxia tolerance has the potential to expand our understanding of the role that sncRNAs may play in basic cell biology, as well as in response to stresses such as anoxia. A comparative model including anoxia-tolerant and anoxia-sensitive phenotypes allows us to better identify sncRNAs that likely play a critical role in anoxia tolerance. Embryos of A. limnaeus are the most anoxia tolerant vertebrate known and are comprised of a range of anoxia-tolerance phenotypes. These characteristics create a unique opportunity for comparative study of the role of sncRNAs in anoxia tolerance in phenotypes with a common genomic background. The overall goals of this project were to: (1) describe the sncRNA transcriptome and changes in its expression in response to anoxia in the embryos of A. limnaeus and in other anoxia-tolerant vertebrates, and (2) to identify specific sncRNAs of interest based on these sequencing projects and to follow-up on their biogenesis, localization, and function in A. limnaeus embryos and a continuous cell line derived from A. limnaeus embryos. Chapter 2 focuses on the identity and expression of sncRNAs in embryos of A. limnaeus in 4 embryonic stages that differ in their anoxia tolerance and physiology. Chapter 3 explores sncRNA expression in brain tissue (the most oxygen-sensitive organ) in other anoxia-tolerant vertebrates: the crucian carp, western painted turtle, leopard frog, and epaulette shark. This allows us to assess the similarities and differences in sncRNA biology in species that evolved anoxia independently, and put the findings from A. limnaeus in an evolutionary context. Chapter 4 describes the establishment of the AL4 anoxia-tolerant cell line derived from A. limnaeus embryos, which allows for more detailed study of particular sncRNAs of interest in Chapter 5. Using whole embryos of A. limnaeus and the AL4 cell line, Chapter 5 describes the expression, localization, and possible biogenesis and mechanism of action of mitochondria-derived sncRNAs, known as mitosRNAs. Chapter 6 summarizes the findings and discusses potential future directions. The work in this dissertation represents the first global survey of sncRNA expression in anoxia tolerant vertebrates. While many interesting patterns of expression were identified, the most interesting discovery is the expression of sncRNAs that are generated in the mitochondria, but have the potential to function in other compartments of the cell. This discovery could transform the way we view the role of the mitochondria in regulating gene expression in eukaryotic cells.

L’autisme est un syndrome neuro-développemental hétérogène à l’étiologie génétique complexe. Afin d’identifier les dérèglements initiaux responsables de ce mal-développement cérébral, des travaux antérieurs au sein de notre équipe se sont basés sur des cellules représentatives des stades précoces de l’ontogenèse : les cellules souches olfactives. Le gène MOCOS, codant pour la sulfurase du cofacteur à molybdène, a été trouvée sous-exprimé chez la majorité des patients autistes comparés à des sujets contrôles de même âge et du même sexe.Nous avons ensuite postulé que la dissection minutieuse des mécanismes moléculaires pouvant rendre compte de cette dérégulation aiderait à trouver des mécanismes sous-jacents contribuant aux troubles du spectre autistique (TSA). Ceci a conduit à l'identification de COSMOC, un long ARN non codant généré à partir d'une transcription divergente dans la région promotrice de MOCOS, dont l'expression est diminuée chez 10 des 11 patients autistes de notre cohorte. A l’aide de diverses techniques de biologie moléculaire, nous avons montré que la déplétion de COSMOC induit : (1) une sous-expression de MOCOS, (2) une déstabilisation de l'organisation de la chromatine, ce qui suggère une fonction de régulateur transcriptionnel, et (3) une altération du métabolisme des lipides et de l’homéostasie redox de la cellule, deux voies dérégulés dans les TSA. Par ailleurs, COSMOC régule de l’expression de la PTBP2 (polypirimidine track biding protein 2), un facteur d’épissage contrôlant l’expression de nombreuses protéines synaptiques. En conclusion, la dérégulation de COSMOC pourrait expliquer certains des dysfonctionnements observés dans les TSA. / Autism is a heterogeneous neuro-developmental syndrome with a complex genetic etiology. In order to unveil the initial disturbances responsible for this brain maldevelopment, previous works in our team relied on cells representative of the early stages of ontogenesis: olfactory stem cells. The MOCOS gene, coding for molybdenum cofactor sulfurase, was found under-expressed in most of autistic patients of our cohort when compared with age- and gender-matched control adults without any neuropsychiatric disorders. We postulated that the meticulous dissection of the molecular mechanisms involved this deregulation would help to unveil pathogenic mechanisms underlying autism spectrum disorders (ASD). This led to the identification of COSMOC, a long non-coding RNA, generated from a divergent transcription in the promoter region of MOCOS, whose expression is decreased in 10 out of 11 autistic patients in our cohort. Using various molecular biological techniques (interference RNA, DNA microarray, qPCR...), we showed that COSMOC depletion induces: (1) an under-expression of MOCOS, (2) a destabilization of chromatin organization, suggesting a transcriptional regulatory function, and (3) an alteration of cellular lipid metabolism and redox homeostasis, two deregulated pathways in ASD. In addition, COSMOC regulates the expression of PTBP2 (polypirimidine track biding protein 2), a splicing factor that controls the expression of many synaptic proteins, including PSD95. In conclusion, the deregulation of COSMOC may explain some of the dysfunctions observed in ASDs.

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis. The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds. To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations. In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future. / Koagulase-negative Staphylokokken besiedeln die menschliche und tierische Haut, sowie die Schleimhäute. Durch Läsionen oder das Einbringen von medizinischen Instrumenten wie Kathetern gelangen sie in tiefere Hautschichten oder die Blutbahn und können dort schwerwiegende Infektionen auslösen, vor Allem bei Risikopersonen. Besonders Staphylococcus epidermidis hat sich als Verursacher von nosokomialen Infektionen, aber auch als Pathogen in der Tierhaltung etabliert. Die Bakterien bilden bei der Besiedlung sogenannte Biofilme aus (d.h. eine Akkumulation der Keime, die von einer extrazellulären Matrix umgeben sind). Diese Matrix besteht neben Proteinen und eDNA hauptsächlich aus einem Polysaccharid, dem interzellulären Adhäsin PIA (engl.: polysaccharide intercellular adhesin). Dieses wird durch die Ica-Proteine synthetisiert, die im icaADBC-Operon (engl.: intercellular adhesin operon) kodiert sind. Das Operon hat große Bedeutung in klinischen Stämmen und wurde daher innerhalb der letzten beiden Jahrzehnte eingehend untersucht, auch im Hinblick auf seine Regulation. In der unmittelbaren Umgebung des icaADBC-Operons, stromabwärts des icaR Gens, das für den Repressor des ica-Operons (IcaR) kodiert, wurde ein großes Transkript identifiziert, von dem vermutet wird, dass es möglicherweise an der Regulation der Biofilmbildung beteiligt ist (Eckart, 2006). Ziel dieser Arbeit war es, dieses Transkript zu charakterisieren und seine Funktion in S. epidermidis aufzudecken. Die nicht-kodierende RNA, genannt IcaZ, hat eine Länge von ~400 nt und ist spezifisch für ica-positive S. epidermidis. Sie wird in der frühen bis mittleren exponentiellen Phase temperaturabhängig exprimiert. Stromaufwärts überlappt das icaZ-Gen und dessen Promotor mit der 3' UTR vom icaR-Gen. Stromabwärts wird das icaZ-Gen vom einem Transkriptionsterminator begrenzt, der auch für das tRNAThr-4-Gen benutzt wird, das auf dem gegenüberliegenden Strang in Richtung des icaZ-Gens lokalisiert ist. Die Deletion der RNA führte zu einem makroskopisch sichtbaren Biofilm-negativen Phänotyp mit deutlich verminderter PIA Bildung. Die Biofilmzusammensetzung wurde in vitro mittels eines klassischen Kristallviolett-Assays gemessen und die Biofilmbildung in vivo in Echtzeit mittels konfokaler Mikroskopie (CLSM) betrachtet. Dabei wurde mit einer peristaltischen Pumpe ein Mediumfluss appliziert. Die Mutante zeigte klare Defekte in der initialen Adhärenz und in der Zell-Zell Adhäsion. Sie bildete im Gegensatz zum Wildtyp keinen strukturierten Biofilm aus. Zur Komplementierung des Biofilms wurde die IcaZ von einem Plasmid exprimiert und die Biofilmzusammensetzung nach 18-20 Stunden Wachstum gemessen. Die Ergebnisse dieser Untersuchungen in den verschiedenen Mutanten waren nicht eindeutig. Um die Funktion von IcaZ aufzudecken, wurden Transkriptom- und Proteomvergleiche zwischen Wildtyp und Mutante gemacht. Diese lieferten einige Hinweise, aber da der metabolische Unterschied eines Biofilmbildners zu einem Nicht-Biofilmbildner zu groß war, wurde eine direktere Methode angewandt, die induzierte Expression (Pulsexpression). Zudem wurden potentielle Interaktionspartner der IcaZ mittels computer-basierter Bindungsvorhersagen analysiert. Die icaR mRNA kristallisierte sich dabei als Target heraus und die Interaktion zwischen IcaZ und icaR mRNA wurde mit Gelshift-Assays (EMSA) untersucht. Eine Bandenverschiebung wurde mit icaR 3' UTR und mit dem icaR-5'-3' UTR-Fusionsprodukt detektiert, wohingegen keine Interaktion zwischen IcaZ und icaA mRNA stattfand. Aufgrund dieser Assays wurde vermutet, dass IcaZ die Translation von icaR in S. epidermidis reguliert. In S. aureus fehlt die nicht-kodierende RNA IcaZ und für icaR mRNA wurde eine Autoregulation gezeigt, bei der die icaR 5' UTR mit der icaR 3' UTR intramolekular oder intermolekular durch Basenpaarung interagiert, wodurch die Shine-Dalgarno Sequenz blockiert wird und es aufgrund dessen zu einer Hemmung der Translation kommt. Die Umweltfaktoren, die dazu führen sind bisher unbekannt. Der Komplex wird durch eine Endoribonuklease, RNase III, abgebaut (Ruiz de los Mozos et al., 2013). In S. epidermidis wurde eine solche Interaktion theoretisch ausgeschlossen. Experimentelle Analysen dieser Arbeit haben gezeigt, dass diese Autoregulation in S. epidermidis nicht stattfinden kann und es wird angenommen, dass IcaZ diese Regulation übernimmt. Um die Interaktion zu visualisieren wurden GFP-Reporter Plasmide generiert, die aber für weitere Experimente noch zu verbessern sind. Zusammenfassend lässt sich sagen, dass IcaZ mit der icaR mRNA interagiert, was höchstwahrscheinlich zu einer Hemmung der Translation des Repressors IcaR führt und damit letztlich PIA-Synthese und Biofilmbildung positiv reguliert. Zusätzlich wurde gefunden, dass Ethanol die Expression der IcaZ-RNA induziert, während NaCl nur schwache Effekte zeigte und Glucose keinen Einfluss auf die Expression von icaZ hatte. Ethanol ist ein Bestandteil von Desinfektionsmitteln, die in Krankenhäusern verwendet werden und ist bekannt dafür Biofilmbildung auszulösen. Da die Bildung von Biofilmen auf medizinischen Geräten kritisch ist und diese die Behandlung von S. epidermidis Infektionen erschweren, tragen die Ergebnisse dieser Arbeit nicht nur zu einem besseren Verständnis des komplexen Netzwerks der Biofilmregulation bei, sondern haben möglicherweise auch praktischen Nutzen in der Zukunft.