Despite the existence of various molecular marker systems there are still limitations in distinguishing between closely related species based on molecular divergence, especially when hybridization events have occurred in the past. The characterisation of plant small nucleolar RNA (snoRNA) genes and their organisation into multigene clusters provides a potential nuclear marker system which could help in resolving the phylogenetic history of plants and might be applicable in DNA barcoding. Using closely and distantly related Senecio species, I investigated a combination of fragment length and sequence variation of snoRNA genes/snoRNA gene clusters to assess the utility of this marker system for barcoding and resolving species relationships. SnoRNA gene and gene cluster sequences identified in Arabidopsis thaliana were used to find homologues in other species and subsequently used for the design of universal primers. Most of the universal primer pairs designed were successful in amplifying snoRNA fragments in most Senecio species and fragment length variation between and within species could be detected. Furthermore, the combination of some fragment length datasets produced by different primer pairs enabled the separation of species and the detection of reticulate evolution indicating a high potential of snoRNA gene/gene cluster fragment length polymorphisms (SRFLPs) for phylogenetic reconstructions in Senecio and other plant genera. Most of the examined gene clusters showed a similar gene order in Senecio and Arabidopsis. However, the majority of these clusters appeared to exhibit more copies in Senecio, some of which were distinguishable by a combined sequencing/fragment profiling approach, and shown to be putative single copy regions with the potential to be used as co-dominant markers. However, a high number of paralogues and possible differences in copy number between species excludes these regions from being used in DNA barcoding. This is because specific primers would have to be developed for specific copies which would preclude development of a universal application for barcoding. None of the regions showed enough sequence variation to delimit distinctly closely related Senecio species and were therefore also considered to be unsuitable for DNA barcoding. Although most snoRNA genes and gene clusters might be inapplicable for DNA barcoding, they are likely to be valuable for phylogenetic studies of species groups, genera and families. On this scale, specific primers might act universally and the number of paralogous copies is likely to be equal across the species group of interest.

L'environnement des organismes vivants est par définition fluctuant, toutes variations aléatoires du milieu de vie constituent un stress pour les cellules. Au fil de l'évolution, une forte pression de sélection a façonné le fonctionnement cellulaire jusqu'aux réponses complexes élaborées par les organismes vivants. Mes travaux s'inscrivent autour des mécanismes moléculaires de la réponse au stress et plus particulièrement les stress génotoxiques. La première partie de l'étude décrit finement la réplication de l'ADN en condition de stress réplicatif. Ainsi, nous avons montré que les pools de dNTPs sont limitants pour la progression des fourches de réplication en phase S normale et en stress, et que leurs niveaux conditionnent le programme temporel de réplication. De plus, nous avons mis en évidence un mécanisme d'adaptation au stress réplicatif et aux dommages constitutifs dans des mutants caractérisés par de l'instabilité génétique (CIN) via l'activation du checkpoint de dommage conduisant à l'expansion des pools de dNTPs. Pour finir, nous montrons que l'augmentation des niveaux de dNTPs facilite la réplication en présence de lésions de l'ADN, d'une manière indépendante des ADN polymérases translésionnelles. Le second projet apporte de nouveaux éléments sur le rôle de Crt10 in vivo, préalablement identifié comme un régulateur transcriptionnel des gènes de la Ribonucléotide Réductase (RNR). Nos données indiquent que les mutants crt10Δ ont des niveaux de dNTP similaires à ceux des cellules sauvages, et que cette mutation a un très faible impact sur l'expression des gènes RNR, malgré un phénotype de vitesse de progression des fourches accrue. Nous montrons que le mutant crt10Δ est caractérisé par un défaut d'entrée en phase S et d'initiation des origines de réplication. L'origine de ce défaut pourrait résider dans les fonctions de Crt10 impliquant la régulation de la biosynthèse des ribosomes au sein du complexe Rtt101-Mms1. Le troisième projet identifie MRX (Mre11-Rad50-Xrs2) comme un acteur de la voie de terminaison des ARN non codants. MRX s'associe à des loci recrutant également le complexe de terminaison Nrd1-Nab3-Sen1 à l'échelle du génome entier. L'inactivation de RAD50 se traduit par une perte d'efficacité de terminaison et l'accumulation de transcrits bicistroniques, ainsi qu'une dérégulation du niveau d'ARNs non codants instables (CUT) et de leurs gènes associés. Tout comme Sen1, MRX pourrait intervenir dans la résolution des collisions entre les machineries de transcription et de réplication. / A fluctuating environment is a powerful mean of selection for living organisms, which evolved complex signaling networks to integrate these variations and direct swift and efficient cellular responses. The aim of my work is the identification and characterization of molecular mechanisms involved in the tolerance of replicative stress and DNA damage. First, we show that changes in dNTP pools affect several aspects of replication dynamics in budding yeast. dNTP levels are limiting for normal S-phase progression and determine the temporal program of replication during a replicative stress. Interestingly, we also observed that chromosomal instability (CIN) mutants display expanded dNTP pools due to the constitutive activation of the DNA damage checkpoint. Since increased dNTP levels promote forks progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replicative stress by upregulating dNTP pools. Secondly, we bring new lights on the role of Crt10 in vivo, which has been initially identified as a negative regulator of Ribonucleotide Reductase (RNR) genes expression. Deletion of CRT10 neither leads to expanded dNTP pools, nor to a massive deregulation of RNR genes, although crt10Δ cells exhibit faster fork progression. The crt10Δ mutant accumulates at the G1/S transition and exhibits a strong defect of origin firing that could account for its replication phenotype. Moreover, we observed a global decrease in ribosome biogenesis in crt10Δ. The physical interaction of Crt10 with several members of the ribosome biogenesis pathway and its role in the Rtt101-Mms1 complex suggest that Crt10 may regulate ribosome levels in vivo. At last, we identified MRX (Mre11-Rad50-Xrs2) as a bona fide member of the transcription termination of non-coding RNA (ncRNA). ChIP-seq reveals that MRX localized at the same loci than the Nrd1-Nab3-Sen1 complex in vegetative growth. rad50Δ cells exhibit transcriptional read-through and upregulation of unstable cryptic transcripts (CUTs) leading to a misregulation of their associated gene. Finally, MRX seems to be involved in the resolution of branched structures emanating from collision between transcription and replication machineries, as it is the case for Sen1.

Les ARN non-codant (ARNnc) assurent différentes fonctions vitales permettant aux bactéries de s'adapter rapidement aux conditions changeantes de leur environnement L'analyse de données transcriptomiques de la souche Pseudomonas brassicacearum NFM421 en réponse à différents stress en utilisant des puces à ADN qui contiennent aussi bien les régions codantes que les régions non codantes a révélé la modulation de deux ARNnc potentiels en réponse à des métaux lourds (Cd et U), dénommés IrsZ et IrsY. De plus, le génome de P. brassicacearum a été entièrement séquencé et une centaine d'ARNnc potentiels a été identifié par l'utilisation d'outils bioinformatiques de prédiction d'ARNnc. Ce travail vise à caractériser les deux ARNnc potentiels, IrsZ et IrsY, et à déterminer leur fonction chez P. brassicacearum. L'analyse bioinformatique de leur séquence ne révèle pas d'homologue dans la base de données des ARNnc. Nous avons validé expérimentalement par différentes approches techniques l'expression des deux ARNnc candidats dans différentes conditions de cultures et sous différents stress. Ceci a conduit notamment à révéler la modulation par le fer de l'expression des deux ARNnc IrsZ et IrsY. Leur expression est fortement activée par de fortes concentrations en fer. Cependant, en réponse à un stress oxydant causé par le peroxyde d'hydrogène, l'expression des deux ARNnc est réprimée. Cette répression est exacerbée chez les bactéries surexprimant oxyR. Nos travaux semblent indiquer que IrsZ et IrsY agissent comme des senseurs du statut intracellulaire du fer. / Regulatory non-coding RNAs (ncRNA), act as regulators of translation and message stability. They modulate a wide range of physiological responses to environmental stimuli. Due to their biological interest, different bioinformatics tools and experimental approaches have been developed for detecting new ncRNA. Transcriptome analysis of the plan root-associated bacterium Pseudomonas brassicacearum NFM421 strain in response to various stresses, using microarrays containing coding as well as non-coding DNA fragments, revealed the modulation of two potentials ncRNA in response to heavy metals (Cd and U), named IrsZ and IrsY. Furthermore, P. brassicacearum genome was completely sequenced and hundreds of potentials ncRNA have been predicted by using computational tools. This work aims at characterizing the two potentials ncRNA, IrsZ and IrsY, and to determine their function in P. brassicacearum. No homologous was found in the ncRNA database. We validated the expression of the two potential ncRNA by different experimental approaches in different culture conditions and under different stresses. This led to reveal that both IrsZ and IrsY are modulated by iron. Their expression is strongly activated by high concentrations of iron. However, the expression of both Irs ncRNA is suppressed under oxidative stress generated by hydrogen peroxide. This repression is exacerbated in P. brassicacearum overexpressing oxyR. Our work suggests that IrsZ and IrsY act as sensors of intracellular iron status.

Candida glabrata est une levure pathogène opportuniste, apparaissant aujourd’hui comme la deuxième cause de candidémie en Europe et en Amérique du Nord. Cette levure présente de nombreuses particularités génomiques telles que la présence de nouveaux domaines structuraux au sein d’ARN non-codants ubiquitaires. Le premier aspect de cette thèse a consisté en l’étude de la sous-unité ARN atypique de la Ribonucléase P nucléaire de C. glabrata. Cet ARN contient trois grand domaines additionnels octroyant au transcrit une taille trois fois plus élevée que la moyenne des sous-unités ARN des RNase P eucaryotiques. Les expériences réalisées ont permis une meilleure compréhension du rôle de ces domaines additionnels et ont démontré la présence inédite de la protéine Rcl1 au sein du complexe de la RNase P. Dans un second temps ce travail de thèse a aussi contribué à l’amélioration des outils d’édition du génome de C. glabrata existants. De nouvelles cassettes intégratives de faible taille et positivement sélectionnables ont été mises au point. Ces éléments présentent toutes les caractéristiques permettant leur utilisation dans la modification du génome de souches sauvages et d’isolats cliniques de C. glabrata. / Candida glabrata is an opportunistic pathogenic yeast, and is today the second causative agent of candidemia in Europe and North America. This yeast has many genomic peculiarities such as the presence of new structural domains within ubiquitous non-coding RNAs. The first aspect of this thesis was the study of the atypical RNA subunit of the nuclear Ribonuclease P of C. glabrata. This RNA contains three large additional domains giving the transcript an overall size more than three times larger than the average eukaryotic RNase P RNA subunits. The experiments performed led to a better understanding of the role of these additional domains and demonstrated for the first time the presence of the Rcl1 protein within the RNase P complex. Secondly, this thesis work also contributed to the improvement of existing genome editing tools in C. glabrata. New small and positively selectable integrative cassettes have been developed. These elements exhibited all the required characteristics for their use in wild-type strains and clinical isolates of C. glabrata.

Orientador: Cesar Martins / Resumo: Os cromossomos B ocorrem em cerca de 2.828 espécies de diferentes táxons, sendo basicamente heterocromáticos e compostos de DNAs repetitivos. Recentemente, análises genômicas em larga escala estão sendo utilizadas para elucidar questões acerca dos cromossomos supranumerários. Os peixes ciclídeos recebem grande interesse científico, uma vez que muitas espécies passaram por um rápido e extenso processo de radiação adaptativa. Em algumas espécies do grupo, como Astatotilapia latifasciata, foi descrita a presença de cromossomos B. Neste trabalho foi caracterizado o perfil de expressão proteico em tecidos específicos na A. latifasciata e realizada análise funcional da presença do cromossomo B nesta espécie de teleósteo, elucidando a influência que este pode acarretar em vias metabólicas específicas. Além disso, esses dados foram integrados com os resultados de RNA-Seq dessa espécie, e construídas sub redes de co-expressão e interação proteína-proteína. Também foi calculada a entropia de Shannon, a qual não apresentou diversidade na expressão dos transcritos em cada biblioteca comparada. Além disso, foi analisada a expressão diferencial de RNAm em cada tecido em relação a presença do cromossomo B e ao sexo. Dentre os transcritos diferencialmente expressos, a análise de enriquecimento funcional apresentou processos relacionados ao ciclo celular, resposta imune e resposta ao estresse. Na maioria dos casos analisados entre a expressão de proteínas, os transcritos up regulated e as sub... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The B chromosomes occur in about 2,828 species of different taxa, being heterochromatic and composed of repetitive DNA.Recently, large-scale genomic analyzes are being used to elucidate questions about supernumerary chromosomes.Cichlid fish are of great scientific interest, since many species have gone through a rapid and extensive process of adaptive radiation.In some species of the group, such as Astatotilapia latifasciata, the presence of B chromosomes was described.In this work, we characterize the profile of protein expression in specific tissues in A. latifasciata and performed a functional analysis of the presence of the B chromosome in this species of teleost, elucidating the influence that it can cause in specific metabolic pathways.In addition, we integrate these data with the RNA-Seq results of this species, and construct sub-networks of co-expression and protein-protein interaction.The Shannon entropy was also calculated, which did not show diversity in the expression of the transcripts in each library. In addition, differential expression analysis was performed on each tissue separately and the relationship between the presence of B chromosome and sex chromosome was analyzed. Among the differentially expressed transcripts, functional enrichment analysis presented processes related to the cell cycle, immune response and stress response. In relation to abundance of proteins, the up regulated transcripts and the sub-networks we identified genes like the Aurora kinas... (Complete abstract click electronic access below) / Doutor

Análises de dados gerados em larga escala revelaram que os transcriptomas são mais extensos e complexos do que inferido previamente. Hoje é evidente que a maioria dos genomas de eucariotos são quase inteiramente transcritos e sob regulação atrelada a estágios de desenvolvimento. O controle da expressão gênica envolve RNAs não codificadores (ncRNAs) regulatórios, inclusive em processos pós-transcricionais. A regulação de expressão gênica em nível pós-transcricional é crucial em diferentes organismos, mas é particularmente central nos tripanossomatídeos. Os parasitos do gênero Leishmania (Ordem Kinetoplastidae, família Trypanosomatidae) provocam doenças infecto-parasitárias conhecidas como leishmanioses e em seu ciclo de vida apresenta-se sob três formas principais de desenvolvimento: promastigotas procíclicos, promastigotas metacíclicos e os amastigotas. Este trabalho teve como objetivo identificar e caracterizar computacionalmente ncRNAs putativos de Leishmania em diferentes estágios de desenvolvimento. A associação de abordagens em larga escala e ferramentas de bioinformática possibilitaram a identificação de 11.376 ncRNAs putativos em L. braziliensis e, em um estudo preliminar, de 37 ncRNAs putativos em L. donovani. Adicionalmente, o transcriptoma de L. braziliensis foi analisado comparativamente entre os três estágios de desenvolvimento do parasito. Em L. donovani, dos 37 ncRNAs putativos identificados, 34 estavam em UTRs (Untranslated Regions) e 3 em regiões intergênicas. Preditores de características específicas de ncRNAs foram utilizados e 32 ncRNAs putativos tiveram pelo menos uma predição positiva. Todos os candidatos estão conservados inteira ou parcialmente em pelo menos 3 espécies de Leishmania. Cinco ncRNAs de L. donovani foram confirmados por experimentos de Northern blotting. A análise do transcriptoma de L. braziliensis revelou uma diferença de expressão gênica entre os estágios de desenvolvimento que variou entre 52% e 71%, dependendo dos estágios comparados. Também foram definidos os limites das 5UTRs e 3UTRs de 81% e 38% das CDSs anotadas, respectivamente. Propusemos uma metodologia para identificação de ncRNAs putativos utilizando dados de sequenciamento de RNA-total. Essa metodologia identificou 11.376 ncRNAs putativos em L. braziliensis, sendo que todos os candidatos foram analisados por programas preditores de características específicas de ncRNAs e apresentaram pelo menos uma predição positiva, além de não possuírem semelhança com domínios proteicos conhecidos. A análise de conservação demonstrou que de 27% a 41% dos ncRNAs putativos identificados são conservados em outras espécies de Leishmania. Foram encontrados de 27% a 38% de ncRNAs putativos com regulação atrelada ao estágio de desenvolvimento, dependendo dos estágios comparados. Assim, além da identificação e descrição de ncRNAs em Leishmania foram encontrados candidatos com regulação atrelada ao desenvolvimento e padrões foram descortinados com o processo de análise proposto e executado. Portanto, esse trabalho contribui significativamente para ampliar a compreensão dos processos de regulação de expressão gênica em Leishmania e oferecerá à comunidade um conjunto grande e importante de informações sobre a organização genética do parasito, diferenças genéticas e regulatórias ao longo do desenvolvimento, além de informações do transcriptoma de forma global. / High-throughput data analyses indicated that transcriptomes are more extensive and complex than previously supposed. Currently, it is evident that most eukaryotic genomes are almost entirely transcribed and under regulation tied to developmental stages. Control of gene expression involves regulatory non-coding RNAs (ncRNAs), including post-transcriptional processes. Post-transcriptional regulation is particularly relevant for the regulation of gene expression in trypanosomatids, as compared to other organisms. Parasites of the genus Leishmania (Order Kinetoplastidae, family Trypanosomatidae) causes infectious-parasitic diseases known as leishmaniasis, and their life cycle comprises three development stages: procyclic promastigotes, metacyclic promastigotes and amastigotes. This study aimed to computationally identify and characterize Leishmania putative ncRNAs at different stages of development. Large-scale approaches combined with bioinformatics tools allowed the identification of 11,376 putative ncRNAs in L. braziliensis and, in a preliminary study, of 37 putative ncRNAs in L. donovani. In addition, the L. braziliensis the complete transcriptome was analyzed comparatively between the parasite development stages. In L. donovani, of the 37 putative ncRNAs identified, 34 were in UTRs (Untranslated Regions) and 3 in intergenic regions. Predictors of ncRNAs specific characteristics were used and 32 putative ncRNAs had at least one positive prediction. All candidates are conserved, partially or entirely, in at least three Leishmania species. Five L. donovani ncRNAs were confirmed by Northern blotting experiments. Analysis of the L. braziliensis transcriptome revealed differences in gene expression levels between developmental stages, ranging from 52% to 71%, depending on the compared stages. The boundaries of the 5\'UTRs and 3\'UTRs were also defined for 81% and 38% of the annotated CDSs, respectively. We developed a methodology for the identification of putative ncRNAs using total RNA sequencing data. This methodology allowed the identification of 11,376 putative ncRNAs in L. braziliensis, and all candidates were analyzed by predictive programs of ncRNAs specific characteristics and presented at least one positive prediction, in addition to bearing no similarity to known protein domains. The analysis showed that from 27% to 41% of the putative ncRNAs identified are conserved in other Leishmania species. We found 27% to 38% of putative ncRNAs with regulation associated to the developmental stage, depending on the compared stages. Consequently, besides identification and characterization of ncRNAs in Leishmania, candidates with developmental-related regulation were found and patterns were uncovered with the proposed and implemented analysis. Thus, this work contributes significantly to improve understanding of gene expression regulation processes in Leishmania and will offer to the community important information about the parasite genetics and regulatory differences along the development, besides of L. braziliensis transcriptome information.

L’ARN non-codant 7SK forme la charpente d’un complexe, 7SK snRNP, qui régule l’activité du facteur d’élongation de la transcription P-TEFb, intervenant dans la levée des pauses transcriptionelles chez les métazoaires. Le 7SK snRNP comprend les protéines LARP7, essentielle pour la stabilité de l’ARN 7SK et MePCE, participant à sa coiffe. Dans le cadre d’une investigation du rapport entre structure et fonction de l’ARN7SK, le projet était de comprendre commen la protéine LARP7 reconnait et assemble l’ARN dans le 7SK snRNP. La protéine LARP7, membre d’une famille reliée à laprotéine La, est spécifique de 7SK. Les éléments responsables de l’interaction ont été analysés par des méthodes biochimiques dans des complexes reconstitués à partir d’ARN synthétique et de protéines recombinantes. Le module La, dans la région N-terminale, reconnaît et lie les trois uridines à l’extrémité 3’ de l’ARN et, additionellement,une séquence conservée au pied de la tigeboucle en 3’, induisant une conformation fermée de l’ARN. L’autre extrémité de la protéine comprend un domain RRM de reconnaissance de l’ARN, qui se lie à la boucle apicale de la tige-boucle 3’. La protéine LARP7 reconnaît également une région conservée au centre de l’ARN. Dans l’ensemble, LARP7 utiliserait ses domaines terminaux et central pour envelopper l’ARN et le stabiliser. Au cours de ces travaux, une interaction directe du domaine C-terminal avec la tige-boucle 5’ a également été mise en évidence. Celle-ci comprend le site de liaison à la HEXIM, la protéine qui déclenche l’interaction avec P-TEFb et un rôle fonctionnel de LARP7 est envisagé. / The non-coding RNA 7SK is the scaffold for the 7SK snRNP complex that regulates PTEFb, the positive transcription elongation factor, which relieves transcription pauses in metazoans. The 7SK snRNP comprises the proteins LARP7, essential for 7SK stability and MePCE, involved in capping. In the frame of an investigation of how the structure of the7SK RNA sustains its function, the project was to understand how is the RNA recognized and assembled in the 7SK snRNPby the associated protein LARP7. LARP7, a La-related protein is specific for 7SK. The elements responsible for the interaction were investigated by biochemical approaches in vitro with complexes reconstituted from purified recombinant proteins and transcribed RNA. The La-module of LARP7 recognizes and binds the triplet of uridines at the 3’-end of the 7SK RNA and additionally binds to a conserved region at the foot of the 3’-hairpin.This may stabilize a closed conformation of the 7SK. On the other end of the LARP7molecule, the C-terminal domain comprising a RRM (RNA Recognition Motif) binds to the apical loop of the 3’hairpin. Further investigations showed that a conserved region in the core of the RNA is also involved. On the whole, this strongly suggests thatLARP7 wraps around 7SK using its N terminal, C-terminal and linker domains to ensure the RNA stabilization into a functional core. In the course of the investigation, was revealed a direct interaction of the C-terminal domain of LARP7 with the 5’-hairpin of the RNA, which is responsible for 7SK function as it contains the binding site of HEXIM, the protein which bridges 7SK and P-TEFb. A possible functional role of LARP7 is envisioned.

The Pentatricopeptide Repeat (PPR) gene family produces RNA-binding proteins that target organellar transcripts. The PPR family is expanded in land plants, with nearly 450 genes identified in Arabidopsis thaliana. In plants with a Cytoplasmic Male Sterility (CMS) phenotype, members of the PPR family can act as a RESTORER OF FERTILITY (Rf) and are part of a subset of genes called RESTORER OF FERTILITY-LIKE (RFL). Unlike other PPR transcripts, RFL transcripts are targets of both microRNA (miRNA) and trans-acting siRNA (tasiRNA) and produce secondary siRNA after initial miRNA- or tasiRNA-guided cleavage. We utilized the A. lyrata genome assembly and high-throughput sequencing of small RNA to examine the evolutionary dynamics of the PPR gene family and the pattern of small RNA targeting of RFL transcripts. We found an expanded set of 539 PPR genes in A. lyrata, 51 of which were in the RFL group, often in multiple collinear copies when compared to their A. thaliana orthologs. In-species RFL paralogs appear to be more related to one another than to their collinear orthologs, which is possible evidence of gene conversion or ectopic recombination. miRNA targeting of RFL transcripts is largely conserved with nearly two-thirds of all target sites maintained. TasiRNA targeting was less conserved with roughly one-third of comparable validated tasiRNA targets maintained in both species. However, when clusters of potential tasiRNA targets were considered, roughly two-thirds of target sites are conserved. Production of secondary siRNA from A. lyrata PPR transcripts is less well defined than in A. thaliana, with strong signals coming from phases that are not concordant with the miRNA- or tasiRNA-guided cleavage sites. / Graduation date: 2013

Immer mehr Berichte deuten darauf hin, dass nicht-kodierende RNAs an der Regulation des Immunsystems beteiligt sind. In dieser Arbeit wurden nicht-kodierende RNAs identifiziert, die durch zwei unterschiedliche immunologische Prozesse in zwei verschiedenen Zelltypen reguliert wurden. Zum einen wurde das Transkriptom von Multiplen Myelom-Zellen in Abhängigkeit von der Interleukin 6-Stimulation untersucht. Dabei wurden einige sehr lange, IL 6-regulierte macroRNAs identifiziert, die STAIRs (STAT3-induced RNAs). Bei den STAIRs handelt es sich wahrscheinlich um funktionelle, kontinuierliche, nicht-kodierende macroRNAs, die im Zellkern angereichert sind. Einige STAIRs dienen eventuell zusätzlich oder ausschließlich als Primärtranskript für gespleißte, lange ncRNAs (lncRNAs), die weitere Funktionen in der Zelle ausüben können. Die STAIRs weisen eine große Bandbreite an Gewebsspezifität auf und bei den Untersuchungen in dieser Arbeit zeigten sich Hinweise, dass sie sich für verschiedene Krebserkrankungen als Biomarker eignen könnten. Die zweite Transkriptomanalyse wurde bei der Aktivierung naiver T Zellen durchgeführt. Dabei offenbarte sich, dass die Zellen bei diesem Prozess einen dramatischen Wechsel ihres Transkriptionsprogrammes vollziehen und eine Vielzahl nicht Protein-kodierender Gene reguliert werden. Es wurde die Regulation von ncRNAs, die bisher noch nicht im Zusammenhang mit T Zellen beschrieben wurden, beobachtet und erneut unbekannte, differentiell exprimierte Bereiche identifiziert. Im Anschluss wurde STAIR18, eine nicht-kodierende RNA, die durch die beiden untersuchten Signalwege reguliert wird, eingehender untersucht. Es zeigte sich, dass STAIR18 im menschlichen Genom dupliziert ist und beide Loci die gespleißte, lange ncRNA152 in diversen Varianten transkribieren. ncRNA152 ist hauptsächlich im Zytoplasma lokalisiert und befindet sich dort anscheinend in perinukleären Aggregaten. Die verschiedenen ncRNA152-Isoformen scheinen unter-schiedliche Funktionen auszuführen. Einerseits ist eine Wirkung als competing endogenous RNA wahrscheinlich. Eine weitere Aufgabe der ncRNA152 scheint darin zu bestehen, das STAT3-Primärtranskript zu stabilisieren oder dessen Prozessierung zu fördern.

The studies in this thesis describe the application of genotyping and allele specific expression analysis to genetic studies. The role of the gene NPC1 in Triglyceride metabolism was explored in mouse models and in humans on the population level in study I. NPC1 was found to affect hepatic triglyceride metabolism, and to be relevant for controlling serum triglyceride levels in mice and potentially in humans. In study II the utility of the HapMap CEU samples was investigated for tagSNP selection in six European populations. The HapMap CEU was found to be representative for tagSNP selection in all populations while allele frequencies differed significantly in the sample from Kuusamo, Finland. In study III the power of Allele specific expression as a tool for the mapping of cis-regulatory variation was compared to standard eQTL analysis, ASE was found to be the more powerful type of analysis for a similar sample size. Finally ASE mapping was applied to regions reported to harbour long non-coding RNAs and associated SNPs were compared to published trait-associations. This revealed strong cis-regulatory SNPs of long non-coding RNAs with reported trait or disease associations.