Дисертації з теми "MRNA report"

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Os tripanossomatídeos são parasitas flagelados causadores de diversas doenças humanas e que apresentam algumas características moleculares bem distintas. Nestes organismos, o controle da sua expressão gênica é quase exclusivamente pós-transcricional e dados obtidos até o momento sugerem que a etapa de iniciação da tradução tem um papel importante neste controle. Durante a iniciação da tradução de eucariotos, o complexo trimérico eIF4F (formado pelas subunidades eIF4A, eIF4G e eIF4E) tem uma participação importante no reconhecimento do mRNA, facilitando o recrutamento ribossomal para iniciar a síntese proteica. Múltiplos homólogos das subunidades do complexo eIF4F foram identificados em tripanossomatídeos, mas não foi possível elucidar a função específica de cada um deles. Desta forma, este trabalho teve como objetivo o desenvolvimento de um ensaio in vivo para a avaliação do efeito da superexpressão desses homólogos de Trypanosoma brucei na tradução de um mRNA repórter, o qual codifica a proteína GFP (green fluorescent protein). Três linhagens repórter foram obtidas (4212, 4213 e 4235) e sete homólogos das subunidades do complexo eIF4F e um de PABP foram testados nestas linhagens. A análise da superexpressão foi feita através de ensaios de Western blot, seus perfis de crescimento foram avaliados por curvas de crescimento e a expressão de GFP foi avaliada através de citometria de fluxo. Os resultados apresentados mostram que as linhagens repórter 4213 e 4235 são viáveis para serem utilizadas na caracterização das proteínas envolvidas no controle da expressão gênica de tripanossomatídeos. Foi observado que as proteínas EIF4E1 e EIF4E2 provocam diminuição do crescimento, enquanto a superexpressão de EIF4E3, EIF4E4, EIF4E4W279A, EIF4G3, EIF4G4 e PABP1 não alteram o crescimento celular dos parasitas. Apesar da detecção de variações na expressão de GFP durante a superexpressão de alguns dos homólogos testados, não foi possível, contudo, confirmar que estas proteínas aumentam ou diminuem a tradução. Ensaios complementares ainda precisam ser feitos para confirmar a real função destes.
The trypanosomatids are flagellated parasites responsible for several human diseases and which display unique molecular characteristics. Control of gene expression in these organisms is almost exclusively post-transcriptional and the data generated so far suggest that the initiation stage of translation plays an important role in this control. During translation initiation in eukaryotes, the trimeric complex eIF4F (formed by the eIF4A eIF4G and eIF4E subunits) has a relevant role in mRNA recognition and facilitates ribosomal recruitment to start the protein synthesis. Multiples homologues for the eIF4F subunits have been identified in trypanosomes, but it has not been possible to elucidate their specific functions. Thus, this study aimed to develop an in vivo assay to evaluate the effect of the overexpression of these Trypanosoma brucei homologues during the translation of a reporter mRNA encoding for the green fluorescent protein (GFP). Three reporter strains were generated (4212, 4213 and 4235) in which seven homologues of eIF4F subunits and one of their PABP partner were overexpressed. Confirmation of overexpression was carried out by Western blot assays, growth profiles were evaluated by cell counting and GFP expression was assessed by flow cytometry. The results generated show that the reporter lines 4213 and 4235 are feasible for use in the characterization of proteins involved in the control of trypanosomatids gene expression. Overexpression of EIF4E1 and EIF4E2 was seen to induce a reduction in cell growth, while EIF4E3, EIF4E4, EIF4E4W279A, EIF4G3, EIF4G4 and PABP1 did not interfere with growh. Despite the detection of variations in GFP expression during overexpression of some of the homologues tested it was not possible to confirm if these proteins increase or decrease translation. Complementary assays still need to be done to confirm the true function of these factors.

Myotonic dystrophy (DM) is an inherited disease characterized by myotonia, insulin resistance, cardiomyopathy, and cognitive deficiencies. DM is a triplet repeat disorder, meaning that affected individuals carry anywhere between 50 and thousands of CTG/CCTG repeats in their genetic makeup. When transcribed into RNA, these repeats become “toxic” in the sense that they serve to bind and sequester important RNA binding proteins. One such family of proteins, the Muscleblind-like (MBNL) family, is important in the regulation of alternative mRNA splicing, and thus the sequestration of MBNL proteins leads to a number of mis-splicing events. Many of these events are directly correlated to DM symptoms. While there is no known cure for DM, the use of small molecules to treat symptoms is a well-characterized therapeutic tactic with immense promise. Pentamidine is a small molecule that was found to reverse mis-splicing in both DM cell and mouse models. Mechanistically, this molecule is particularly unique because unlike many small molecules, which physically displace MBNL from the toxic CUG RNA, pentamidine reduces CUG RNA levels, possibly through inhibition of CTG transcription. Chapter I summarizes alternative splicing mechanisms and regulation, defines MBNL protein structure and function, describes DM pathophysiology and molecular mechanism, and finally provides an overview of pentamidine characterization as a small molecule therapeutic. Chapter II reports the development of an in vitro T7 transcription assay, which allowed us to compare the relative efficacy by which pentamidine is able to inhibit the transcription of various repeat and non-repeat DNA sequences. This chapter further reports the characterization of a series of methylene linker analogues of pentamidine, which were also characterized through the T7 transcription assay. Chapter III details our thorough structure-activity relationship investigation of bisbenzamidine analogues of pentamidine, both in in vivo and in vitro models. Chapter IV describes our characterization of actinomycin D, a known transcription inhibitor and chemotherapeutic, within the DM disease framework. Chapter V summarizes these data, which ultimately serve as a proof of concept for the potential of CTG transcription inhibition in therapeutic contexts and broadly describe their application in other repeat diseases. This dissertation contains previously published and unpublished co-authored material.
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Friedreich ataxia (FRDA) is the most common inherited ataxia disorder, caused by a GAA repeat expansion mutation within the first intron of the FXN gene. The subsequent deficiency of frataxin protein leads to neurological disability, increased risk of diabetes mellitus, cardiomyopathy and premature death. The exact FRDA disease mechanism is not yet clear, despite some understanding of epigenetic, transcriptional and DNA repair system effects that lead to frataxin reduction. Previous studies have shown that mismatch repair (MMR) genes can affect other trinucleotide repeat disorders by destabilisation of the repeats. Furthermore, it has been proposed that frataxin deficiency might lead to cell malignancy by an as yet undefined mode of action. Therefore, the principle aim of this thesis was to use human and genetically altered mouse cells and tissues to understand the effects of MMR proteins on GAA repeat instability and FXN transcription, and also to identify potential changes in MMR transcription that might cause malignancy in FXN-defective human cells. Firstly, by using FXN and MMR genetically altered mice, MMR proteins were shown to be involved in both intergenerational and somatic GAA repeat instability, although their effects in the two systems were different. Thus, Msh2 or Msh3 were both found to protect against intergenerational transmission of GAA contractions, while loss of Msh2 or Msh3 reduced somatic GAA repeat expansions and increased levels of FXN transcription in brain and cerebellum tissues. Loss of Msh6 induced both intergenerational GAA repeat expansions and contractions, while the frequency of somatic GAA repeat expansions was reduced. Curiously, the level of FXN transcription was also reduced in Msh6-deficient brain and cerebellum tissues. On the other hand, Pms2 was found to protect against both intergenerational and somatic GAA repeat expansions, with loss of Pms2 causing increased GAA repeat expansions and decreased levels of FXN transcription in brain and cerebellum tissues. Finally, loss of Mlh1 led to a reduced frequency of both intergenerational and somatic GAA repeat expansions, but the level of FXN transcription was also reduced in brain and cerebellum tissues. Furthermore, upregulation of MMR mRNA expression was detected in human FRDA fibroblast cells, but downregulation was seen in FRDA cerebellum tissues, suggesting tissue-dependent control of FXN and MMR expression. In summary, these studies indicate that the MMR system can affect GAA repeat expansion instability and FXN transcription through different mechanisms of action. Furthermore, frataxin deficiency can also affect the levels of MMR mRNA expression in a tissue-dependent manner. These findings will assist future investigations aimed at identifying novel FRDA therapies.

Mutant mRNA and protein both contribute to the clinical manifestation of many repeat-associated neurodegenerative and neuromuscular disorders. The presence of nuclear RNA clusters is a feature shared amongst these diseases, such as C9ORF72/ALS and myotonic dystrophy 1/2 (DM1/2); however, this pathological hallmark has not been conclusively demonstrated in Huntington’s disease (HD) in vivo. Investigations into HD – caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene – have largely focused on toxic protein gain-of-function as a disease-causing feature, with fewer studies investigating the role of mutant HTT mRNA in pathology or pathogenesis. Here we report that in two HD mouse models, YAC128 and BACHD-97Q-ΔN17, mutant HTT mRNA is preferentially retained in the nucleus in vivo. Furthermore, we observed the early, widespread formation of large mutant HTT mRNA clusters (approximately 0.6 to 5 µm3 in size) present in over 50-75% of striatal and cortical neurons. Affected cells were limited to one cluster at most. Endogenous wild-type mouse Htt or human HTT mRNA containing 31 or fewer repeats did not form clusters. Additionally, the aberrantly spliced N-terminal exon 1-intron 1 RNA fragment, HTT1a, also formed clusters that fully co-localized with the mutant HTT mRNA clusters. These results suggest that multiple repeat-containing transcripts can coalesce to form a single cluster in a given cell. Treating YAC128 mice with antisense oligonucleotides efficiently silenced individual HTT mRNA foci but had limited impact on clusters. Our findings identify mutant HTT mRNA clustering as an early, robust molecular signature of HD, further supporting HD as a repeat expansion disease with suspected mRNA involvement.

Outbreaks of widely spread infectious diseases, such as Human Immunodefficiency Virus (HIV), Severe Acute Respiratory Syndrome (SARS) and Swineflu (H1N1) and hospital acquired infections, such as Meticillin-resistant Staphylococcus Aureus (MRSA) and Clostridium Diffcile, are serious health problems which have been tackled by the World Health Organization and international health protection agencies. Various statistical analyses have contributed a remarkable effect on providing scientific evidence on which to base political decisions and infection control strategies. In this project, we focused on two infectious diseases: HIV and MRSA and the research project is divided into two separate parts. One is the quantification of replication in HIV anonymous test reports and the other is the effect of patient movement between wards on the acquisition of MRSA. The first research project is concerned with the analysis of an anonymous HIV test dataset. The data is collected as a set of birthdays and it is possible that there is repeated sampling of the same person. The aim is to quantify the amount of replication in the HIV data using a maximum likelihood technique and then give the confidence intervals for the estimated amount of replication using the bootstrap method. The data were provided by the Public Health Laboratory Service (PHLS), Colindale, London in 1994, who were interested in a statistical method to estimate multiple counting that possibly existed in the database. The data consists of individual records of the number of AIDS cases diagnosed, with birthdates from 1901 to 1973. There were two datasets provided by the PHLS, one of which contained 1,134 records and was provided in 1991. The other dataset was provided in 1994 with the sample size 17,137. An estimate of the true number of distinct individuals as well as the percentage of replication was obtained by programming the maximum likelihood calculation in the languages R and C. This technique is based upon evaluation of the probability that two records with the same birthdate represent two separate individuals as opposed to the same person reported twice. The results for the 1991 dataset showed that there were five out of sixteen birth years (i.e. 31.25% of the observed records in the 1991 dataset) with replication in the true number of distinct individuals. In the results of the 1994 dataset, the majority of the birth years (57/73) recorded the correct number of distinct individuals in the observations. The 95% confidence intervals for the estimated amount of replication were calculated by applying a parametric bootstrap method. The results show that the birth years in the 1991 dataset with non-zero estimated amount of replication (the birth years of 1931, 1934, 1935, 1943 and 1944) have comparatively wide 95% bootstrap confidence intervals, which implies higher uncertainty of the true amount of replication. A similar conclusion was obtained from the results of 95% bootstrap con dence intervals for the 1994 dataset. Comparing the results within the same birth years recorded in the 1991 dataset and the 1994 dataset, the data indicate that the confidence intervals for the 1994 dataset are mainly narrower than the corresponding ones in the 1991 dataset. The conclusion of this study illustrates the drawback of recording the HIV patients only with date of birth, which has now been improved by combining with 'Soundex' codes for the surname and gender. The second part of the project aims to estimate the impact of patient movement within a hospital on the risk of MRSA acquisition by using data from the MRSA screening admission and discharge studies in Scotland which took place in two hospitals in 2010. The data consist of an admission-only database (7,181 patients), a discharge-only database (2,432 patients) and a combined admission-discharge cohort (2,792 patients). The third database has complete information on MRSA status on admission, on discharge, as well as data on the wards the patient was in while in hospital. In order to understand the effect of potential risk factors on MRSA acquisition, a multivariate logistic regression model was constructed to analyse the effects of the number of wards a patient was in on MRSA acquisition as well as other risk factors. Receiver Operating Characteristic (ROC) curves were plotted and the individual area under the curve (AUC) was also calculated for indicating the reliability and the accuracy of the prediction of the models. Furthermore, we modelled the dynamic patient movement and assessed the effect of being in a ward with MRSA by imputing the unknown date of transfer, simulating the missing length of stay (along with the simulation envelope). The timelines of MRSA infection and carriage pressure in each ward of the two hospitals were then mapped for all patients in the three databases, imputing where necessary. Patient movement was measured as a volume indicator in terms of the frequency of ward to ward transfer and as cohabiting in the same ward. By using logistic regression within a bootstrap simulation, we estimated the odds ratio of acquisition of MRSA association with being in a ward with MRSA present, which was given by averaging the estimated effects from the fitted models, and generating the 95% confidence intervals. The results indicate that the number of wards that patients had moved through and patients being in a ward with MRSA present do not affect the risk of acquiring MRSA significantly over and above the patient level risk factors such as age and the presence of open wounds or catheters. Some further work which can be done in an MRSA screening programme is suggested as an implementation study.

Background: The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. Results: Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMRA2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma.

Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\' 2′-O-Methyltransferase enzymecreates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48 h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

Parkinson's Disease (PD) is a late-onset neurodegenerative disease characterized by progressive motor dysfunction caused by a loss of dopaminergic neurons for which there is no known cure. Among the most common genetic causes of PD are mutations in the leucine-rich repeat kinase 2 gene (LRRK2), encoding a multi-domain protein with kinase activity. The LRRK2 G2019S mutation causes hyperactivity of the kinase domain and is the most frequent LRRK2 mutation in patients with familial PD, though its role in PD pathology remains unclear. Preliminary data from the lab of our collaborator, Dr. David Park, demonstrated through a genetic screen in Drosophila melanogaster that the deletion of rbp9 encoding an RNA-binding protein prevented pathology induced by PD-relevant mutations in the LRRK2 kinase domain. The neuronal homolog of RBP9 in humans is HuD, a member of the Hu family of RNA-binding proteins that regulates the expression of many transcripts involved in neuronal development, plasticity, and survival. In addition, HuD has been shown to modify the age-at-onset or risk of developing PD. Here, we studied the effect of LRRK2 on the post-transcriptional regulation of mRNAs bound by HuD in the context of PD. Our findings showed that HuD is a substrate for LRRK2 phosphorylation in vitro, and that LRRK2 G2019S hyperphosphorylates HuD. We demonstrated that LRRK2 kinase activity is required for the binding of several transcripts by HuD that encode PD-relevant proteins such as α-synuclein and neuronal survival factor BDNF. Our findings in human neuroblastoma cells indicated that LRRK2 regulates the protein levels of HuD mRNA targets α-synuclein and BDNF in a mechanism that can by modified by HuD. Finally, we showed that the combination of HuD knockout with LRRK2 G2019S expression in mice rescues aberrant expression of HuD targets in mice with only the LRRK2 G2019S mutation or the knockout of HuD alone. Together, our findings demonstrate that LRRK2 affects the post-transcriptional regulation of HuD-bound mRNAs, and suggest the use of HuD as a potential therapeutic target in patients with PD caused by the LRRK2 G2019S mutation.

Problemstellung: Die Tiergesundheit hat sich bei Milchkühen in den letzten Jahren weltweit negativ entwickelt. Wichtigster Ausdruck dafür ist die auf ca. 2,4 Jahre verkürzte Nutzungsdauer. Dabei spielt das Fettmobilisationssyndrom eine dominante Rolle. Das Fettgewebe ist nicht nur als reiner Energiespeicher, sondern als endokrines stoffwechselaktives Organ anzusehen. Untersuchungen an Menschen und Mäusen haben gezeigt, dass das Fettgewebe in Abhängigkeit von seiner Lokalisation im Körper unterschiedlich auf metabolische und hormonelle Stimuli reagiert. Es gibt Hinweise, dass auch für das Rind ähnliche Differenzen angenommen werden können. Zielstellung: Um die Eigenschaften des bovinen Fettgewebes und seine Rolle im Energiestoffwechsel besser charakterisieren zu können, war das Ziel der vorliegenden Untersuchung, die mRNA-Expressionen ausgewählter für den Fettstoffwechsel relevante Gene im bovinen Fettgewebe an verschiedenen Lokalisationen grundlegend in gesunden Rindern zu untersuchen. Material und Methoden: Die Probenentnahme erfolgte an 12 gesunden Schlachtkühen direkt nach der Tötung, die aufgrund Schwermelkbarkeit oder Unfruchtbarkeit geschlachtet wurden. Das Fettgewebe wurde aus dem Omentum majus, dem Depotfett der Niere, im kaudalen Beckendrittel (retroperitoneales Fett), dem Hüftbereich (subkutanes Fett) und dem Fett an der Herzbasis entnommen. Die Proben wurden in Flüssigstickstoff tiefgefroren, auf Trockeneis transportiert und bis zur Untersuchung bei -70°C gelagert. Die mRNA-Expression für die verschiedenen Gene (Hormonsensitive Lipase (HSL), Lipoproteinlipase (LPL), Fettsäuresynthase (FASN), Leptin, Adiponektin, Retinolbindungsprotein 4 (RBP4), Tumornekrosefaktor  (TNF) und Interleukin 6 (IL-6), Fettsäurebindungsproteine (FABP3, 4 und 5) und Glukosetransporter 4 (GLUT4)) , wurden mit einer quantitativen real time (RT)-PCR gemessen. Ergebnisse: Die mRNA-Expressionen der verschiedenen oben genannten Gene, ausgenommen IL-6 und FABP3, sind im bovinen Fettgewebe nachweisbar. Die mRNA-Expressionen unterschieden sich in den einzelnen Fettdepots nicht signifikant. Ausnahme hierbei bildete RBP4, dessen mRNA im pericardialen Fett signifikant höher exprimiert war als im subkutanen und omentalen Depot. Die mRNA-Expression des subkutanen, omentalen, perirenalen und pericardialen Fettdepots korrelierten signifikant positive untereinander. Schlussfolgerung: Die mRNA-Expressionen der in den Fettstoffwechsel involvierten und untersuchten Gene gesunder Rinder waren nachweisbar, unterschieden sich jedoch nicht signifikant von einander mit Ausnahme der RBP4 mRNA. Die positiven signifikanten Korrelationen zwischen dem subkutanen, omentalen, perirenalen und pericardialen Fettlokalisationen und gleichmäßigen Expressionen innerhalb der Gewebe deuten auf eine einheitliche Fettmetabolismus des gesamten Körpers. Verglichen mit Ergebnissen der Humanmedizin sind nur wenige Übereinstimmungen (HSL, LPL, GLUT4,TNF) zu eruieren. Weitere Studien mit gesunden Tieren im Vergleich zu erkrankten Rindern müssen klären, ob eine mögliche Verschiebung der mRNA-Konzentrationen auf das Fettmobilisationssyndrom hinweisen
Purpose: Over the last years, the situation of animal health concerning dairy cows has developed worldwide in an adverse way. Most important indicator is the shortened useful life of approx. 2.4 years. The fat mobilization syndrome plays a dominant role in this process. Apparently, fatty tissue does not only serve as a mere energy reservoir, but also as an endocrin organ with metabolic activity. Researches on humans and mice have shown fatty tissue to react on metabolic and hormonal stimuli in different ways, depending on its body localization. There are dues to anticipate, similar differences in cattle. Objectives: In order to better characterize the attributes of bovine fatty tissue and its purpose in metabolism, the present study aims examine basically the expression of mRNA in selected genes which are important for lipid metabolism in bovine fatty tissue of different localizations in healthy cattle. Methods and material: Samples where taken from twelve carcasses of healthy dairy cows slaughtered for reason of difficult milking or infertility directly after killing. Fatty tissue was taken from omentum major, kidney capsula, caudal pelvis area (retroperiteonal fat), hip area (subcutaneous fat), and cardiac base. It was instantly quick-freezed in liquid nitrogen, put on dry ice while transporting, and stored at -70°C until analysis. The expression of mRNA of different genes (hormone-sensitive lipase (HSL), lipoproteine lipase (LPL), fatty acid synthase (FASN), fatty acid binding proteine (FABP3,4 and 5), retinol binding proteine 4 (RBP4), adiponectine, glucose transporter 4 (GLUT4), leptin, interleukin-6 (IL-6), and tumor necrosis factor a (TNFα) was measured by means of a quantitative real-time (RT)-PCR. Results: The mRNA-expressions of all these different genes except IL-6 and FABP3 were detected in bovine fatty tissue. The differences of mRNA-expression between sample localization were not statistically significant. RBP4 was excepted, which mRNA showed a significantly higher expression in pericardial fat than in subcutaneous and omental fat, respectively. The correlation between mRNA-expressions of subcutaneous, omental, pericardial and perirenal fat was significant. Conclusions: The mRNA-expression of examined genes being involved in fatty tissue metabolism, were detected in healthy cattle, but were not significantly different, except RBP4. Significantly positive correlations between subcutaneous, omental, perirenal and pericardial localization and consistent expression indicate an integrative metabolism of the whole body. Compared to results of the human medicine only few analogies (HSL, LPL, GLUT4, TNF) were found. Further studies comparing healthy and diseased cattle will have to prove, if possible displacements of the mRNA-level can indicate the fat mobilization syndrome being present

À la suite de l’endosymbiose, le génome chloroplastique a rétréci et dépend maintenant du génome nucléaire pour son expression. Chez Chlamydomonas reinhardtii, les protéines Octotricopeptide repeat (OPR), codées dans le noyau, contrôlent l’expression d’ARNm chloroplastiques spécifiques. La répétition OPR est un motif dégénéré de 38 acides aminés, qui forme un tandem d’hélices α antiparallèles qui lient l’ARN. Une répétition OPR est prédite pour interagir avec un nucléotide spécifique grâce à des résidus variables à des positions précises. La succession de répétitions permet aux protéines OPR de se lier à une séquence donnée. En partant d’un « code OPR » théorique, j’ai cherché à étudier cette spécificité de reconnaissance. J’ai mute in vivo les cibles chloroplastiques de facteurs OPR pour empêcher l’interaction OPR/ARN, puis j’ai tenté de la restaurer en mutant les résidus conférant la spécificité dans les répétitions correspondantes. Étonnamment, les interactions OPR/ARN sont très résilientes, ce qui a complétement changé notre vision de ces interactions in vivo. Des études fonctionnelles complémentaires que j’ai réalisées sur les facteurs OPR MDB1 and MTHI1 ont révélé que l’expression des gènes chloroplastiques dépend probablement de systèmes de facteurs nucléaires. En coopérant ces facteurs auraient une affinité combinée plus forte et seraient ainsi plus résilients
Following endosymbiosis, the chloroplast genome shrunk and became reliant on the host genome for its expression. In Chlamydomonas reinhardtii, Octotricopeptide repeat proteins (OPR), encoded in the nucleus, control the expression of a specific organellar mRNA. The OPR repeat is a degenerate motif of 38 amino-acids, folding into a tandem of antiparallel α-helices which can bind to RNA. An individual OPR repeat is predicted to interact with one given nucleotide thanks to specificity-conferring residues at defined positions within the repeat. OPR proteins contain tracks of successive OPR motifs, thus they can bind to a specific RNA “target” sequence and act on it. I aimed to study this specificity, called the “OPR code”, starting with a draft code based on known OPR protein/mRNA couples. I mutated in vivo the chloroplast targets of some OPR factors to disrupt the OPR/RNA interaction, and then tried to restore it by mutating the specificity-conferring residues in the corresponding repeats. Surprisingly, OPR/RNA interactions seem very resilient, challenging our view of how the specificity is established in vivo. Complementary functional studies that I performed on the OPR factors MDB1 and MTHI1 revealed that chloroplast gene expression might rely on complex networks of nuclear factors. By cooperating those putative systems would be both more specific and more resilient

Los insecticidas piretroides presentan una alta gama de usos. Ciflutrin, un piretroide de Tipo II, es ampliamente utilizado en agricultura, en el control de plagas, para la salud humana y animal, siendo su principal órgano diana el sistema nervioso, específicamente las proteínas canales de voltaje de sodio. A pesar de ser considerados por causar baja toxicidad, estudios recientes han demostrado su efecto neurotóxico en mamíferos pequeños. Es por esos motivos, que este estudio tuvo como objetivos evaluar la distribución del piretroide ciflutrin sobre tres regiones cerebrales de ratas (hipocampo, cuerpo estriado e hipotálamo), y su efecto a dosis oral de 1, 5, 10 y 20 mg/kg p.c. por 6 días, sobre la expresión de genes relacionados a inflamación (NFκB, TNF-α e IL-6) y apoptosis (Bax, Bcl2, Casp-3), y sobre el estatus oxidativo en las mismas regiones cerebrales. Se pudo determinar que ciflutrin se distribuye ampliamente en las tres regiones estudiadas, siendo la mayor Cmax en hipotálamo, luego en cuerpo estriado e hipocampo. También en hipotálamo se encuentra la mayor razón AUCtejido/AUCplasma tras dosis oral de ciflutrin. Luego de administrar ciflutrin está fue eliminada (semivida de eliminación) de tejido nervioso en un rango entre 15-24 h. Asimismo, en hipocampo ciflutrin a dosis oral de 5, 10 y 20 mg/kg p.c. produjo un aumento significativo sobre la expresión de Bax y Casp-3, también hubo un incremento de NFκB sólo a dosis de 20 mg/kg; mientras que, a las dosis de 10 y 20 mg/kg pc se produjo un aumento deTNFα e IL6. En cuerpo estriado se produjo un aumento significativo sobre la expresión de Bax, Casp-3 e IL6. En Hipotálamo, ciflutrin sólo produjo un aumento significativo en genes relacionados a inflamación. Respecto de la actividad de las enzimas superóxido dismutasa (SOD) y glutatión reductasa (GR), se observó disminución de sus actividades a la dosis más alta de ciflutrin en hipocampo y cuerpo estriado. Nuestros resultados indican que ciflutrin produjo efectos citotóxicos sobre el sistema nervioso central de ratas que pueden contribuir al espectro general de neurotoxicidad causada por este piretroide, y que deben ser considerados como factores que contribuyen a la presentación de enfermedades neurodegenerativas.

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E‑binding protein (4E‑BP)‑1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator‑activated receptor (PPAR)γ expression. The small interfering RNA‑mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

Publicación a texto completo no autorizada por el autor
Logra aislar el mRNA que codifica la proteína PLA 2, utilizando una técnica simple. Esto es, amplificando el cDNA que codifica la PLA 2 D49 a través de RT-PCR. El análisis del cDNA codificador de PLA 2 muestra que esta tiene un marco de lectura abierto de 414 pares de bases que codifican un polipéptido de 138 aminoácidos, con un péptido señal de 16 residuos de aminoácidos, seguido de una secuencia polipeptídica de 122 aminoácidos que corresponden a la PLA 2. El análisis también muestra que la proteína está compuesta por 33 aminoácidos con carga: 11 aminoácidos ácidos, 22 aminoácidos básicos, 44 aminoácidos polares y 28 aminoácidos hidrofóbicos, con un punto isoeléctrico de 8.58 y una masa molecular de 14312.52 Da. El análisis de homología y árbol filogenético muestran que esta PLA 2 presenta un alto grado de homología y forma parte de las PLA 2 básicas N6F24D49. Además, se logró purificar dos isoformas de PLA 2 (fracciones V y VI) empleando cromatografía en HPLC de fase reversa con un alto grado de pureza, según lo revelado por la SDS-PAGE. Al realizar la repurificación de la fracción V, esta se muestra como un monómero, pues el espectro revela la presencia de una sola cadena polipeptídica con una masa molecular de 14238.71 Da. Según el análisis biológico, la PLA 2 V es capaz de elevar los niveles de CK plasmáticos cuando es inyectada por vía intramuscular y por vía intravenosa.
Tesis

O Diabetes Mellitus tipo 1 (DM1) resulta de um ataque autoimune contra as células pancreáticas, extinguindo a produção de insulina e levando à hiperglicemia. Evidências indicam uma associação entre o estresse oxidativo (que pode causar danos no DNA) e o DM1, sendo que apenas alguns trabalhos da literatura relataram a expressão de genes relacionados à respostas ao estresse oxidativo e reparo do DNA em DM1. Ainda, os microRNAs (reguladores pós-transcricionais da expressão gênica) estão envolvidos em vários processos biológicos e condições patológicas, mas informação sobre a expressão dos microRNAs em DM1 ainda é escassa. A fim de proporcionar um melhor entendimento sobre as vias de regulação de genes participantes de processos biológicos relevantes para o DM1, o presente estudo consistiu em analisar os perfis de expressão gênica (método de microarranjos) de microRNAs e de mRNAs (bem como de algumas proteínas) provenientes de células mononucleares do sangue periférico (PBMCs, do inglês peripheral blood mononuclear cells) de pacientes DM1 (n=19) em comparação com indivíduos sadios não diabéticos (n=11), dando maior enfoque a genes associados à resposta ao estresse oxidativo e reparo do DNA. Os resultados de expressão obtidos pelo método de microarranjos apontaram 44 microRNAs diferencialmente expressos (35 induzidos e nove reprimidos) nos pacientes DM1 e esses microRNAs apresentaram grande especificidade ao estratificar pacientes DM1 dos controles, incluindo hsa-miR-101, hsa-miR148a, hsa-miR-27b e hsa-miR-424, cujos dados de expressão foram confirmados por qRT-PCR. A análise funcional dos genes-alvo dos microRNAs, tanto dos induzidos quanto dos reprimidos, apontou 22 e 12 vias KEGG significativamente enriquecidas, respectivamente, incluindo vias relacionadas ao câncer. Com relação à análise de expressão de mRNAS, 277 genes diferencialmente expressos foram identificados nos pacientes DM1, sendo que 52% deles são potenciais alvos dos microRNAs diferencialmente expressos nos pacientes DM1. Dentre esses alvos foram encontrados genes candidatos ao desenvolvimento da doença, assim como genes implicados nos processos biológicos resposta ao estresse oxidativo e reparo do DNA, como UCP3, PTGS2, ATF3, FOSB, DUSP1 e TNFAIP3, cujos dados de expressão foram confirmados por qRT-PCR. Já a análise de grupos gênicos identificou 49 e 55 grupos gênicos significativamente expressos e enriquecidos em pacientes DM1, respectivamente, destacando-se vias relacionadas à sinalização apoptótica, resposta ao hidroperóxido, reparo do DNA por recombinação homóloga e resposta ao estresse do retículo endoplasmático. Quanto aos dados de expressão proteica (western blotting), PTGS2 e ATF3 não apresentaram níveis de expressão detectáveis em nenhum dos dois grupos estudados, enquanto que para DUSP1 não foi observada diferença estatisticamente significativa entre os grupos, apesar de os três genes se apresentarem induzidos em pacientes DM1. Os resultados do ensaio do gene repórter da luciferase demonstraram a ocorrência da interação entre hsa-miR-148a e DUSP1 em meio celular. Essa evidência aliada aos dados de western blotting, sugerem a possibilidade de hsa-miR-148a atuar na repressão traducional de DUSP1. Em conjunto, os resultados do presente estudo indicaram perfis distintos de expressão de microRNAs e mRNAs em PBMCs de pacientes DM1 comparados a indivíduos sadios, sendo que adicionalmente, dados inéditos relacionados à interação microRNAs-mRNAs em DM1 foram obtidos, principalmente associados à resposta ao estresse oxidativo e reparo do DNA, sugerindo um distúrbio na rede microRNA-alvo em pacientes DM1.
Type 1 Diabetes Mellitus (T1DM) results from an autoimmune attack against the pancreatic cells, ceasing insulin production, which causes hyperglycemia. Although associations between oxidative stress, which can cause DNA damage, and T1DM have been demonstrated, only a few studies have reported differential expression of genes associated with response to oxidative stress and DNA repair in T1DM patients. Moreover, microRNAs (post-transcriptional regulators of gene expression) are implicated in many biological processes and pathological conditions; however, only scarce information is available in the literature concerning the expression of microRNAs in T1DM. In order to better understand the regulatory pathways involved in biological processes that are relevant to T1DM, we aimed to investigate the microRNA and mRNA transcriptional expression profiles by microarray analysis (as well as expression of selected proteins) in peripheral blood mononuclear cells (PBMCs) from T1DM patients (n=19) compared with healthy non-diabetic individuals (n=11), emphasizing genes related to response to oxidative stress and DNA repair. Microarray expression results indicated 44 differentially expressed microRNAs (35 up- and nine down-regulated) in T1DM patients, with those microRNAs possessing a discriminatory power to clearly stratify the patients from the controls, including hsa-miR-101, hsa-miR148a, hsa-miR-27b, and hsa-miR-424, whose expression data were confirmed by qRT-PCR. Functional annotation analysis performed on the predicted targets of the differentially expressed microRNAs pointed 22 and 12 annotated KEGG pathways for the overexpressed and repressed microRNAs, respectively, many of them related to cancer. Regarding mRNA microarray results, we detected 277 differentially expressed genes in T1DM patients, with 52% of them being potential targets of the differentially expressed microRNAs in T1DM patients. Among these targets, we identified candidate genes for T1DM as well as genes involved in the biological processes response to oxidative stress and DNA repair, such as UCP3, PTGS2, ATF3, FOSB, DUSP1 and TNFAIP3, whose expression data were confirmed by qRT-PCR. Furthermore, out of the 49 and 55 significantly expressed/enriched gene sets in T1DM patients, respectively, five pathways related to apoptotic signaling, response to hydroperoxide, DNA repair via homologous recombination, and response to endoplasmic reticulum stress were of interest for the present work. Concerning protein expression results (western blotting), PTGS2 and ATF3 expression was not detected for either the patient or the control group, while significant difference in DUSP1 expression was not observed between the two groups, although the corresponding mRNAs of those genes were found induced. Regarding the luciferase assay, our results demonstrated that the interaction between hsa-miR-148a and DUSP1 occurs in the cellular milieu. Therefore, these findings together with those western blotting results suggest that hsa-miR-148a could play a role in DUSP1 translational repression. Altogether, our results indicate distinctive microRNA and mRNA expression profiles in PBMCs from T1DM patients relative to healthy non-diabetic individuals. Furthermore, we have provided novel data regarding microRNA-mRNA interactions in T1DM, in particular involving genes associated with response to oxidative stress and DNA repair, suggesting a perturbation in the microRNA-target network in T1DM patients.

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

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Determina las propiedades antitumorales e inmunomoduladoras del Lepidium meyenni (maca), que lo convierten en un excelente candidato para investigar su actividad hematopoyética. Se trataron ratones Balb/c a dosis de 200 mg/kg de extracto acuoso (EAc) de maca amarilla por vía oral durante 2 meses previo a la inmunosupresión (IS) con ciclofosfamida (CF), y se evaluó la producción de mRNA para las citoquinas hematopoyeticas: interleuquina 3 (IL-3), factor estimulador de colonias de granulocitos y monocitos (GM-CSF) e interleuquina 7 (IL-7) en el bazo y la médula ósea, tanto en los ratones tratados como en sus controles. Se realizaron cultivos de células mononucleares de médula ósea y se evaluó el efecto del EAc sobre la proliferación celular y producción de mRNA para las 3 citoquinas hematopoyéticas. La administración de EAc en ratones IS, incrementó (p<0.05) la producción de mRNA para las tres citoquinas en el bazo y de IL-7 en la médula ósea, dos días después de la IS; e IL-3 y GM-CSF en la médula ósea 5 días post-IS, al compararlos con los grupos no tratados con el extracto. En cultivos de médula ósea, el EAc en la dosis de 100 µg/ml, estimuló (p<0.05) la proliferación celular y la producción de mRNA para IL-7. Además, los ratones IS y tratados con EAc mostraron mayor recuento de células de médula ósea, sangre periférica, unidades formadoras de colonias endógenas en el bazo (CFU-S) y respuesta proliferativa a mitógenos de los linfocitos, cinco días después de la IS. El EAc de maca estimula la producción de mRNA para las tres citoquinas hematopoyéticas. La administración de EAc a ratones inmunocomprometidos puede revertir los efectos supresores de la ciclofosfamida.
Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica
Tesis

Transcription elongation by RNA polymerase II is regulated by an array of protein complexes. Among various elongation factors, Spt5 is conserved in the three kingdoms of life. I investigated functional interactions of its C-terminal repeats (CTR) domain with several elongation protein complexes in Saccharomyces cerevisiae. By using genetics and molecular biology methods, I established two major pathways in this thesis. The first describes how BUR kinase-mediated phosphorylation of CTR domain leads to co-transcriptional recruitment of the PAF complex to regulate histone modifications on active genes. The second describes how CTR phosphorylation facilitates recruitment of capping enzymes to enhance gene splicing. Finally, several Spt5-associated protein complexes were studied, and potential molecular mechanisms underlying these observations are proposed and discussed.

Das Neuroblastom ist der häufigste extrakraniell gelegene solide Tumor der pädiatrischen Onkologie. Der Verlauf der Erkrankung geht von spontaner Regression oder Differenzierung bis hin zu tödlich verlaufenden Erkrankungen. Die Mortalität von Patienten mit Tumoren in fortgeschrittenen Stadien ist immer noch sehr hoch. Die aggressivsten Tumoren sind die, die eine Amplifikation des Protoonkogens MYCN aufweisen. Eine Untergruppe dieser MYCN amplifizierten Tumoren weist eine Coamplifikation von DDX1 auf. Die Prognose dieser Patienten ist besser als die mit allein MYCN amplifizierten Tumoren, wenn auch immer noch schlechter als die von Patienten ohne MYCN Amplifikation. Das DDX1-Protein ist eine putative RNA-Helikase. Über seine genaue Funktion ist noch nicht viel bekannt. Ziel dieser Arbeit war es, potentielle Ziel-mRNAs von DDX1 zu identifizieren, um einen besseren Einblick in die Funktionen von DDX1 und mögliche Wege der Beeinflussung von Tumorverhalten und Prognose zu erhalten. Hierzu wurden eine DDX1 amplifizierte und eine nicht amplifizierte Zelllinie in Kultur genommen und eine Immunopräzipitation mit Zelllysaten der beiden Zelllinien durchgeführt – jeweils mit einem spezifischen Antikörper gegen DDX1 und einem unspezifischen Kontrollantikörper. Die Identifizierung der an DDX1 gebundenen mRNAs erfolgte mittels Microarray. Validiert wurden einige der im Microarray identifizierten RNAs mittels RT-PCR. CDK1, ATM und p18 ließen sich als spezifische Ziel-mRNAs von DDX1 identifizieren.

If has been proposed that a spontaneous deletion in the nicotinamide nucleotide transhydrogenase (Nnt) gene eliminating exons 7-11 in C57BL/6J (B6J) mice is associated with reduced glucose-stimulated insulin secretion in vitro, impaired glucose tolerance, higher epigonadal fat mass and altered susceptibility to diet induced obesity (DIO) of male B6J mice. A potential implication for NNT in human adipose tissue distribution has not been investigated so far. We therefore analyzed NNT mRNA expression in paired human samples of visceral (vis) and subcutaneous (sc) adipose tissue from 221 subjects with a wide range of BMI, insulin sensitivity and glucose tolerance. NNT mRNA expression is significantly higher in visceral fat of obese patients and correlates with body weight, BMI, % body fat, visceral and sc fat area, waist and hip circumference as well fasting plasma insulin. Multivariate linear regression analysis revealed visceral fat area, and % body fat, but not fasting plasma insulin and 2h OGTT glucose. In conclusion, our data suggest a functional relevance of NNT in the development of human obesity and visceral fat distribution.

The ability to reprogram adult somatic cells into induced pluripotent stem (iPS) cells could provide a valuable implement for in vitro disease modeling and drug discovery. More importantly, they may potentially serve as an unlimited source of cells for regenerative medicine. However, most of the iPS cells have been generated by retroviral vectors, and therefore they carry the risk of viral integration into the host genome. This problem prevents their use for clinical applications and regenerative medicine. mRNA-mediated delivery of reprogramming factors is an alternative approach for cellular reprogramming. mRNA-based reprogramming offers the advantage of being completely free of genomic integration and is therefore highly suitable for clinical translation. However, there are some limitations which must be overcome so that mRNA can be widely used for successful cellular reprogramming. In the current thesis, the attempt was to generate stable mRNA-iPS cells through overcoming those limitations. Several human donor cells were transfected with mRNA encoding reprogramming factors and the generation of two stable mRNA-iPS cell lines was shown. The resultant mRNA-iPS colonies were assessed for pluripotency markers. Their pluripotency features were evaluated by the viral-iPS cells produced by conventional retroviral vectors. It was noticed that the generation of mRNA-iPS cells was largely affected by the parental cells from which they were derived. However, characterization and evaluation of the generated mRNA-iPS cells proved their pluripotency states comparable to the viral-iPS cells. On the other hand, the aging hallmarks of the iPS cells were assessed in the second part of this thesis. The potential aging signatures of the iPS cells should be conducted before their use in clinical applications. Currently, there are controversial data regarding the ability of reprogramming to fully rejuvenate an aged somatic cell and reverse agerelated changes such as shortened telomeres, dysfunctional mitochondria and DNA damage. Moreover, mixed findings have been published regarding whether the iPS cells are fully rejuvenated or they might retain some of the aging hallmarks from the cells which they were derived. This thesis studied these controversies through the investigation of three hallmarks of aging including telomere length, mitochondrial alteration and DNA damage. Telomere elongation was indicated in the iPS cells. Furthermore, mitochondrial morphology and function were improved into more immature features in iPS cell lines than their corresponding fibroblasts. Moreover, the iPS cell lines were shown to have less amount of DNA damage compared to their parental fibroblasts. In summary, it can be concluded that generation of mRNA-iPS cells is largely affected by the primary donor cells from which they are derived. Furthermore, it seems that reprogramming enables reversion of aging signatures to a more youthful state.

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

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

A plethora of new functions of non-coding RNAs have been discovered in past few years. In fact, RNA is emerging as the central player in cellular regulation, taking on active roles in multiple regulatory layers from transcription, RNA maturation, and RNA modification to translational regulation. Nevertheless, very little is known about the evolution of this \Modern RNA World' and its components. In this contribution we attempt to provide at least a cursory overview of the diversity of non-coding RNAs and functional RNA motifs in non-translated regions of regular messenger RNAs (mRNAs) with an emphasis on evolutionary questions. This survey is complemented by an in-depth analysis of examples from different classes of RNAs focusing mostly on their evolution in the vertebrate lineage. We present a survey of Y RNA genes in vertebrates, studies of the molecular evolution of the U7 snRNA, the snoRNAs E1/U17, E2, and E3, the Y RNA family, the let-7 microRNA family, and the mRNA-like evf-1 gene. We furthermore discuss the statistical distribution of microRNAs in metazoans, which suggests an explosive increase in the microRNA repertoire in vertebrates. The analysis of the transcription of non-coding RNAs (ncRNAs) suggests that small RNAs in general are genetically mobile in the sense that their association with a hostgene (e.g. when transcribed from introns of a mRNA) can change on evolutionary time scales. The let-7 family demonstrates, that even the mode of transcription (as intron or as exon) can change among paralogous ncRNA.

Methicillin-resistenter Staphylococcus aureus (MRSA) ist im Rahmen des One-Health-Gedanken eine Gefahr für Mensch und Tier. Im Rahmen dieser Studie wurde eine Pferdeklinik auf ihre MRSA-Prävalenz in neu ankommenden Pferden und Umgebung getestet. Die beiden getrennten Abteilungen „Innere Medizin“ und „Chirurgie“ konnten dabei separat untersucht werden. Des Weiteren wurde ein Hygienekonzept entworfen, zu dem u.a. Aushänge zur Handhygiene, zusätzliche Händedesinfektionsmittelspender und Reinigungsanweisungen gehörten. Wir konnten zeigen, dass die Höhe der Prävalenz eingehender Pferde sich mit vorigen Untersuchungen deckte. Diese war jedoch in der chirurgischen Abteilung deutlich höher als in der für Innere Medizin. Die Maßnahmen führten zu einem teils signifikanten Rückgang der MRSA-Prävalenz in der Umgebung. Es kann schlussgefolgert werden, dass in Pferdekliniken, vergleichbar zur Humanmedizin, ein aktives Screening, in Verbindung mit einem adäquaten Hygienemanagement, effektive Mittel sind, um die MRSA-Last zu reduzieren.

Germ cells are a pool of cells that serve as a link between generations. These cells are separated from the somatic cells by specialized type of cytoplasm, called the germ plasm. Germ plasm contains, membraneless, electron dense subcellular structures, termed germplasm granules that contain numerous components of mRNA metabolism pathway. One of the most prominent protein families, commonly found in germplasm granules are DEAD-box helicases. While this protein family is currently heavily investigated, surprisingly little is known about their functions in germ plasm granules and the mechanisms of their association with the granules. This work identified novel biological and molecular roles of C. elegans’ LAF-1 in both somatic and germ cells. It reveals strong dependency of animal’s somatic, embryonic and post-embryonic development on LAF-1 activity, resulting in high penetrance developmental arrest phenotype. Moreover, this work documents requirement of LAF-1 for the fertility of the animal. Analysis of germ cells in the absence of LAF-1 activity reveals multilayered defects occurring at all stages of germ cell development and maturation. LAF-1 appears to be involved in the maintenance of proliferating potential of the germline stem cell pool and loss of LAF-1 significantly expands the region occupied by mitotic cells. Furthermore, loss of LAF-1 significantly affects expression of GLD-1, REC-8 and H3-S10P, implying that mitosis-to-meiosis boundary cannot be established correctly in the absence of LAF-1. This work solidifies previous conclusions that LAF-1 is a component of P granules, both in the adult germ cells and embryonic germ cell precursors and reveals that LAF-1 is required for correct assembly and dynamic behavior of P granules. Intrinsically disordered regions present in LAF-1 are indispensable for LAF-1’s association with P granules and its role in recruiting granule components. Lastly, LAF-1 associates with RNPs containing cytoplasmic polyA polymerases, indicating that LAF-1 might be involved in translational regulation. Altogether, the collected data describes biological functions of LAF-1 and elucidates the molecular mechanisms underlying these functions.

Einleitung: Kurzkettige Fettsäuren (SCFA) stellen das hauptsächliche Energiesubstrat für Wiederkäuer dar. In Anbetracht des - bedingt durch höhere Milch-, Mast und Reproduktionsleistung - steigenden Energiebedarfs von Hauswiederkäuern wie Milchkuh und Mastbulle ist es von zentraler Bedeutung, die Mechanismen zur Resorption dieser Energielieferanten bzw. Ansatzpunkte für die Beeinflussung dieser Transportprozesse genau zu kennen. Dieses Wissen kann möglicherweise dabei helfen, zukünftig die Energieaufnahme der Tiere zu unterstützen bzw. sogar effizienter zu gestalten. Ziele der Untersuchungen: Deshalb war es Ziel der vorliegenden Arbeit, die Mechanismen zur Resorption von SCFA zu charakterisieren, wobei der Schwerpunkt auf den Transport aus den Pansenepithelzellen ins Blut gelegt wurde, da hierzu im Gegensatz zu ihrer Aufnahme aus dem Pansenlumen in die Epithelzellen noch sehr wenig bekannt war. In einem zweiten Schritt sollte untersucht werden, inwiefern die nachgewiesenen Mechanismen einer Regulation unterliegen und über welche Signalwege diese vermittelt werden könnte. Materialien und Methoden: Zur Charakterisierung der beteiligten Resorptionsmechanismen wurden Epithelstücke aus dem ventralen Pansensack von Schafen in Ussing-Kammern eingespannt und mit Hilfe radioaktiv markierten Azetats, Butyrats und L-Laktats der Transport dieser Substrate unter verschiedenen Bedingungen sowie verschiedenen Hemmstoffeinflüssen untersucht. Zur Charakterisierung regulativer Einflüsse wurden die Epithelstücke über sechs bzw. 24 Stunden mit Butyrat inkubiert und anschließend RNA bzw. Totalprotein extrahiert. Hiermit konnten Veränderungen in mRNA- und Proteinexpression mittels quantitativer Echtzeit-PCR bzw. Western Blot nachgewiesen werden. Ergebnisse: Die Untersuchungen der vorliegenden Arbeit konnten zeigen, dass der Transport von SCFA über die basolaterale Membran des Pansenepithels hauptsächlich proteinvermittelt erfolgt. Eine signifikante Beteiligung lipophiler Diffusion, d.h. ein passiver Transport, kann weitgehend ausgeschlossen werden. Der aktive Transport wies eine bikarbonatabhängige und eine bikarbonatunabhängige Komponente auf. Der Einsatz von Hemmstoffen verschiedener Transportproteine ergab deutliche Hinweise darauf, dass der Monocarboxylattransporter (MCT) 1 eine Rolle beim bikarbonatgekoppelten Transport von Azetat bzw. allgemein unmetabolisierten SCFA spielt. Diese Hinweise wurden untersetzt durch die Beobachtung, dass MCT 1, aber auch der apikal bzw. intrazellulär lokalisierte MCT 4 durch langfristige Inkubation des Epithels mit Butyrat sowohl auf mRNA- als auch auf Proteinebene signifikant erhöht exprimiert wurden, was als Anpassungsreaktion an eine Substratakkumulation interpretiert werden kann. Außerdem wurde auch die mRNA-Expression des Putativen Anionentransporters (PAT) 1 durch Inkubation mit Butyrat erhöht, was für eine Beteiligung auch dieses Transportproteins am SCFA-Transport über das Pansenepithel spricht. Allerdings ist im Gegensatz zu MCT 1 die Lokalisation des PAT 1 in der basolateralen Membran noch fraglich. Die Expressionssteigerung von Zielgenen des Nukleären Faktors ĸB und des Peroxisomenproliferator-aktivierten Rezeptors α sowie des Hypoxie-induzierbaren Faktors selbst deuten weiterhin darauf hin, dass die Steigerung der Transportkapazitäten von MCT 1 und 4 und auch PAT 1 über diese Signalwege vermittelt wird. Schlussfolgerungen: Zusammenfassend konnte in dieser Arbeit erstmals der Transport von SCFA über die basolaterale Membran des Pansenepithels näher charakterisiert werden, sodass es nun möglich ist, zusammen mit den bereits vorliegenden Befunden für die apikale Membran ein komplettes Modell dafür zu erstellen. Auch wurden Erkenntnisse zu regulativen Einflüssen auf diesen Transport gewonnen, die es zukünftig ermöglichen könnten, die Resorption der SCFA aus dem Pansen nutritiv oder eventuell pharmakologisch zu beeinflussen.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für Wiederkäuer 3 2.2 Metabolismus von SCFA im Pansenepithel 4 2.2.1 Aufrechterhaltung des Konzentrationsgradienten vom Pansenlumen ins Epithel 4 2.2.2 Produktion von HCO3- aus CO2 durch die Carboanhydrase 5 2.2.3 Bereitstellung von Energie für die Epithelzellen 5 2.2.4 Bereitstellung von wasserlöslichen, glukosesparenden Energiesubstraten für die periphere Zirkulation 5 2.2.5 Verhinderung möglicher Schädigungen durch Butyrat 6 2.3 Transportmechanismen für kurzkettige Fettsäuren 7 2.3.1 Para- versus transzelluläre Resorption 7 2.3.2 Transzelluläre Resorption mittels lipophiler Diffusion 7 2.3.3 Proteinvermittelte SCFA-Permeation 9 2.3.4 Permeation von SCFA aus dem Epithel ins Blut 11 2.4 Beeinflussung der SCFA-Resorption auf Genexpressionsebene 17 2.4.1 Beeinflussung der Genexpression durch Butyrat 17 2.4.2 Beeinflussung der Genexpression durch Hypoxie 20 2.4.3 Mechanismen für die Regulation der Genexpression durch Butyrat (-Metaboliten) und Hypoxie 21 2.5 Fragestellungen dieser Arbeit 26 3 Ergebnisse 28 3.1 Publikation 1 28 3.2 Publikation 2 41 4 Diskussion 54 4.1 Transport von SCFA über die basolaterale Membran des Pansenepithels 54 4.1.1 Transport mittels lipophiler Diffusion 57 4.1.2 SCFA werden bevorzugt über die basolaterale Membran transportiert 58 4.1.3 SCFA(-Metaboliten) werden bikarbonatabhängig über die basolaterale Membran transportiert 59 4.1.4 SCFA(-Metaboliten) werden durch einen Anionenaustauschmechanismus ins Blut ausgeschleust 61 4.1.5 Azetat wird durch einen pHMB- und CHC-sensitiven Mechanismus transportiert 63 4.2 Der Transport von SCFA über das Pansenepithel unterliegt regulativen Einflüssen 68 4.2.1 Einfluss von Butyrat(-Metaboliten) auf die Expression von potentiellen SCFA Transportern 68 4.2.2 Mechanismen für die Regulation der Expression durch Butyrat(-Metaboliten) 72 4.3 Theoretisches Modell des SCFA-Transports und dessen Regulation auf Genexpressionsebene auf Grundlage der Ergebnisse der vorliegenden Arbeit 74 5 Zusammenfassung 76 6 Summary 78 7 Literaturverzeichnis 80 Danksagung 98
Introduction: The main energy source for ruminants are short chain fatty acids (SCFA). Considering the ever increasing energy requirements of cattle due to increasing milk yield and meat production, it is crucial to identify the mechanisms for the resorption of these energy sources as well as possibilities to influence these transport mechanisms. This knowledge could help support the animals’ energy uptake or even making it more efficient. Aim: Thus, the aim of the present study was to characterise mechanisms for the resorption of SCFA focusing on their transport from the epithelial cells into the blood. In particular, since – compared to the research findings on the uptake of SCFA from ruminal lumen into the cells – so far only very little was known regarding this side of the epithelium. In a second step, the study aimed to elucidate whether the mechanisms observed are subject to regulatory processes and which signalling pathways are involved. Materials and methods: To characterise the transport mechanisms involved, epithelial pieces from the ventral sac of ovine rumen were mounted in Ussing chambers. Using radioactively labelled acetate, butyrate and L-lactate, the transport of these substrates was investigated under different conditions and by applying different inhibitors for potential SCFA transport proteins. To characterise regulatory influences, epithelial pieces were incubated with butyrate for six and 24 hours, respectively. Subsequently, total RNA and protein were extracted to detect changes in mRNA and protein expression using quantitative real time PCR and western blot, respectively. Results: The present study could show that transport of SCFA across the basolateral membrane of rumen epithelium is mainly realised by protein-mediated mechanisms. A significant participation of lipophilic diffusion, i.e. a passive transport, can almost entirely be excluded. The active transport could be divided into a bicarbonate-dependent and a bicarbonate-independent part. The experiments with inhibitors of different transport proteins showed clear evidence of an involvement of monocarboxylate transporter (MCT) 1 in the bicarbonate-dependent transport of acetate and non-metabolised SCFA in general. This evidence was supported by the finding that the expression of MCT 1 but also of the apically and intracellularly localised MCT 4 was increased significantly on both mRNA- and protein-level after long-term incubation of the epithelium with butyrate. This can be interpreted as an adaptation to a substrate accumulation. Additionally, butyrate incubation led to an increased mRNA expression of putative anion transporter (PAT) 1, which makes an involvement of this transport protein in SCFA transport across ruminal epithelium likely as well. However, in contrast to MCT 1 the localisation of PAT 1 in the basolateral membrane is still questionable. The increased expression of target genes of nuclear factor ĸB and peroxisome-proliferator activated receptor α as well as of hypoxia inducible factor strongly point to an involvement of these pathways in the increased expression of MCT 1 and 4 as well as PAT 1. Conclusions: In summary, this study could characterise the transport of SCFA across the basolateral membrane of ruminal epithelium in detail for the first time. This enables us to draw a complete model of ruminal SCFA transport. Also, evidence for regulatory influence on this transport processes was found, perhaps making it possible to influence resorption of SCFA from rumen by nutritive or pharmacological means in the future.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für Wiederkäuer 3 2.2 Metabolismus von SCFA im Pansenepithel 4 2.2.1 Aufrechterhaltung des Konzentrationsgradienten vom Pansenlumen ins Epithel 4 2.2.2 Produktion von HCO3- aus CO2 durch die Carboanhydrase 5 2.2.3 Bereitstellung von Energie für die Epithelzellen 5 2.2.4 Bereitstellung von wasserlöslichen, glukosesparenden Energiesubstraten für die periphere Zirkulation 5 2.2.5 Verhinderung möglicher Schädigungen durch Butyrat 6 2.3 Transportmechanismen für kurzkettige Fettsäuren 7 2.3.1 Para- versus transzelluläre Resorption 7 2.3.2 Transzelluläre Resorption mittels lipophiler Diffusion 7 2.3.3 Proteinvermittelte SCFA-Permeation 9 2.3.4 Permeation von SCFA aus dem Epithel ins Blut 11 2.4 Beeinflussung der SCFA-Resorption auf Genexpressionsebene 17 2.4.1 Beeinflussung der Genexpression durch Butyrat 17 2.4.2 Beeinflussung der Genexpression durch Hypoxie 20 2.4.3 Mechanismen für die Regulation der Genexpression durch Butyrat (-Metaboliten) und Hypoxie 21 2.5 Fragestellungen dieser Arbeit 26 3 Ergebnisse 28 3.1 Publikation 1 28 3.2 Publikation 2 41 4 Diskussion 54 4.1 Transport von SCFA über die basolaterale Membran des Pansenepithels 54 4.1.1 Transport mittels lipophiler Diffusion 57 4.1.2 SCFA werden bevorzugt über die basolaterale Membran transportiert 58 4.1.3 SCFA(-Metaboliten) werden bikarbonatabhängig über die basolaterale Membran transportiert 59 4.1.4 SCFA(-Metaboliten) werden durch einen Anionenaustauschmechanismus ins Blut ausgeschleust 61 4.1.5 Azetat wird durch einen pHMB- und CHC-sensitiven Mechanismus transportiert 63 4.2 Der Transport von SCFA über das Pansenepithel unterliegt regulativen Einflüssen 68 4.2.1 Einfluss von Butyrat(-Metaboliten) auf die Expression von potentiellen SCFA Transportern 68 4.2.2 Mechanismen für die Regulation der Expression durch Butyrat(-Metaboliten) 72 4.3 Theoretisches Modell des SCFA-Transports und dessen Regulation auf Genexpressionsebene auf Grundlage der Ergebnisse der vorliegenden Arbeit 74 5 Zusammenfassung 76 6 Summary 78 7 Literaturverzeichnis 80 Danksagung 98

Systemische Infektionen mit S. aureus (MSSA und MRSA) und Infektionen des Gefäßzugangs bei HD-Patienten sind eine der wichtigsten Ursachen für Morbidität und Mortalität in dieser speziellen Population. Infektionsrisikos stellen die zunehmende Verwendung von Fremdkörpern, wie Katheter und Graft als Gefäßzugänge, sowie die intensivmedizinische Behandlung bei älteren und multimorbiden Patienten dar. Unter den bakteriell bedingten Infektionen bleiben Staphylokokken der am häufigsten nachgewiesene Stamm. Mit dem zunehmenden Gebrauch von Vancomycin zur Behandlung von MSSA-Infektionen hat das Vorkommen von MRSA zugenommen. Dies macht die Entwicklung von alternativen Antibiotikaregimen nötig, die eine Selektion von MRSA-Spezies verhindern. Unter dieser Überlegung wurde auf die Behandlung mit Vancomycin bei Zugangs-bezogenen Infektionen verzichtet. Es wurde im Jahr 2000 durch ein Standardregime bestehend aus Flucloxacillin und Rifampicin ersetzt. Mithilfe eines Screeningprogramms wurde nach MSSA- (n=88) und MRSA- (n=1) Kolonisationen gesucht. Dies gelang mit Hilfe von Querschnitts-Screenings und Indikations-Screeninguntersuchungen bei Aufnahme über den Zeitraum von 2000 bis 2010. Eine Besiedlung mit MRSA wurde bei nur einem Patienten während des 10-Jahres-Screenings registriert. Die gefundenen MSSA-Kolonisationen bei HD-Patienten beeinflussten die Morbidität und Mortalität nicht. Die Anzahl an HD-Patienten mit MSSA-Kolonisation nahm während des Beobachtungszeitraums von zehn Jahren ab Behandlungen mit dem Vancomycin-freien Regime waren generell erfolgreich und resultierten in einem Rückgang der klinischen und laborativen Infektionsmarker und/oder negativen Blutkulturen. Es konnte gezeigt werden, dass mit dem Gebrauch von vancomycinfreien Antibiotikaregimen ein erfolgreiches Management von Staphylokokkus-assoziierten Zugangsinfektionen bei HD-Patienten möglich ist.

Das Hygienemanagement in einer Pferdeklinik ist komplex. Zum einen erschweren bauliche Gegebenheiten wie rutschfeste Böden oder Holzwände sowie Einstreu und die hierdurch anfallende Staubbelastung die Reinigung und Desinfektion. Zum anderen bringen die Patienten ein vielfältiges, ständig wechselndes Keimspektrum in die Klinik ein. Nosokomiale Infektionen in Pferdekliniken haben in den letzten Jahren zugenommen weshalb Hygienemaßnahmen im Rahmen der Biosicherheit von zentraler Bedeutung sind. Im Rahmen dieser Doktorarbeit wurde das Hygienemanagement der Chirurgischen Tierklinik (inzwischen Teil der Klinik für Pferde) der Veterinärmedizinischen Fakultät in Leipzig analysiert. Ziel war es, zunächst das aktuelle Hygieneregime sowie die bakterielle Keimbelastung an ausgewählten Probenentnahmestellen zu erfassen und anschließend anhand der Ergebnisse Verbesserungsvorschläge zu erarbeiten. Zur Erhebung des Status Quo wurden Fragebögen zur allgemeinen sowie Raum-spezifischen Reinigung und Desinfektion angefertigt und zusammen mit dem verantwortlichen Klinikpersonal ausgefüllt. Einbezogen in die Auswertung des Hygienemanagements wurden auch bauliche Gegebenheiten sowie organisatorische Abläufe. Für die bakterielle Keimbelastung wurden Tupferproben auf MRSA, E. coli, coliforme Keime, ESBL-bildende Enterobacteriaceae, Salmonellen, Streptococcus (S.) equi subsp. equi, S. equi subsp. zooepidemicus, Rhodococcus hoagii und Acinetobacter baumannii untersucht. Jede Probenentnahmestelle wurde mindestens zweimal im Abstand von mindestens vier Wochen beprobt. In allen Proben sowie zusätzlich in Luftkeimsammelproben wurde die aerobe Lebendkeimzahl mittels Oberflächenspatelverfahren bestimmt. Darüber hinaus wurden die Patientenakten von Januar 2014 bis Juli 2016 auf Hinweise möglicher nosokomialer Infektionen ausgewertet. Die Keimbelastung in der Klinik variierte je nach Probenentnahmeort und war im OP-Bereich am niedrigsten und in den Stallungen am höchsten. ESBL-bildende E. coli und S. equi subsp. zooepidemicus wurden sporadisch nachgewiesen. Der Nachweis von MRSA gelang vom Bügel des Desinfektionsmittelspenders am Waschbecken, von einer Nasenbremse sowie aus einer Luftkeimsammelprobe, die in einer Stallgasse genommen wurde. Insgesamt ergab die Luftkeimsammlung eine Keimbelastung von bis zu 105 Kolonie-bildenden Einheiten (KbE)/m3 Luft im Stallbereich während im OP ein Keimgehalt von 102 KbE/m3 Luft messbar war. Hinsichtlich der Durchführung von Reinigung und Desinfektion wurden Schwachstellen durch die Erhebung des Status Quo aufgezeigt. Alle Ergebnisse wurden im Rahmen eines Vortrages in der Klinik vorgestellt und Verbesserungsvorschläge diskutiert. Ein Hygieneplan, der zu Beginn dieser Studie nicht vorlag, wurde eingeführt. Darüber hinaus erfolgte die Benennung einer Hygienebeauftragten. Die Auswertung der Patientendaten ergab, dass einige der vorangehend aufgeführten Keime, wie MRSA, S. equi subsp. zooepidemicus, S. equi subsp. Equi, und R. hoagii, im ausgewerteten Zeitraum durchaus eine Rolle bei Infektionen innerhalb der Klinik spielten. Allerdings gab es keinen Verdacht auf eine nosokomiale Infektion. In der internationalen Literatur sind wenige Studien zum Thema Hygienemanagement in Pferdekliniken zu finden. Die Keimbelastung in den Luftkeimsammelproben im Stallbereich war in der vorliegenden Studie fünf- bis ≥ zehnfach höher im Vergleich zu anderen Studien. Bei der Durchführung dieser Studie wurde deutlich, dass für ein gezieltes Hygienemanagement die Festlegung von Verantwortlichkeiten sowie das Vorliegen konkreter Handlungsanweisungen unabdingbar sind.:Inhalt 1 Einleitung 1 2 Literatur 2 2.1 Stallklima im Pferdestall 2 2.2 Bauliche Grundlagen einer idealen Pferdeklinik 3 2.3 Hygienemaßnahmen in der Pferdeklinik 4 2.3.1 Hygieneplan 5 2.3.2 Händehygiene 5 2.3.3 Reinigung 7 2.3.4 Desinfektion und Sanitation 8 2.3.5 Probennahmeverfahren für die mikrobiologische Untersuchung 12 2.3.5.1 Abklatschverfahren 12 2.3.5.2 Tupferabstrichverfahren 13 2.4 Beschreibung der ausgewählten Bakterienspezies 13 2.4.1 Methicillin-resistente Staphylococcus aureus (MRSA) 13 2.4.2 Streptokokken 14 2.4.2.1 Streptococcus equi subsp. equi 15 2.4.2.2 Streptococcus equi subsp. zooepidemicus 15 2.4.3 Escherichia coli und ESBL 16 2.4.4 Salmonellen 17 2.4.5 Acinetobacter baumannii 18 2.4.6 Rhodococcus hoagii (Rhodococcus equi/Prescotella equi) 19 3 Material und Methoden 20 3.1 Material 20 3.1.1 Bakterien 20 3.1.2 Nährmedien sowie deren Bestandteile 21 3.1.3 Biochemische Identifizierungssysteme 21 3.1.4 Chemikalien und Reagenzien 21 3.1.5 Geräte 22 3.1.6 Verbrauchsmaterialien 22 3.2 Methoden 23 3.2.1 Eigenschaften der verwendeten Nährmedien 23 3.2.2 Bestandsaufnahme in der Chirurgischen Tierklinik 27 3.2.2.1 Bauliche Gegebenheiten der Chirurgischen Tierklinik 27 3.2.2.2 Fragebogen über das Hygieneregime 28 3.2.2.3 Auswertung der Patientendaten 28 3.2.3 Probennahmen 29 3.2.3.1 Auswahl der Probenentnahmepunkte 29 3.2.3.2 Durchführung der Probennahmen 29 3.2.4 Probenbearbeitung 30 4 Ergebnisse 37 4.1 Bauliche Gegebenheiten der Chirurgischen Tierklinik 37 4.2 Hygienemaßnahmen in der Chirurgischen Tierklinik 40 4.2.1 Hygieneplan 40 4.2.2 Auswertung des Fragebogens 40 4.3 Auswertung der Patientendaten 42 4.4 Ergebnisse der Probennahmen 45 4.4.1 Instrumentenaufbereitung 46 4.4.2 Bildgebung 48 4.4.3 Klinikhalle und Mittelgang 49 4.4.4 Stall 55 4.4.5 OP-Saal 61 4.5 Hygieneplan 64 5 Diskussion 68 5.1 Bauliche Gegebenheiten 68 5.2 Hygienemaßnahmen 69 5.2.1 Hygieneplan und Desinfektionsmittel 69 5.2.2 Patientendaten 70 5.3 Probennahmen 71 5.3.1 Instrumentenaufbereitung 71 5.3.2 Bildgebung 71 5.3.3 Klinikhalle und Mittelgang 72 5.3.4 Stall 73 5.3.5 OP-Saal 74 5.3.6 Luftkeimsammelproben 75 6 Zusammenfassung 77 7 Summary 79 8 Literaturverzeichnis 81 9 Anhang 91 9.1 Fragebogen 91 9.2 Instrumentenaufbereitung 101 9.3 Klinikhalle und Mittelgang 102 9.4 Stall 105 9.5 OP-Saal 105 Tabellenverzeichnis 108 Abbildungsverzeichnis 109 Danksagung 110

RNA-ligand binding often depends crucially on the local RNA secondary structure at the binding site. We develop here a model that quantitatively predicts the effect of RNA secondary structure on effective RNA-ligand binding activities based on equilibrium thermodynamics and the explicit computations of partition functions for the RNA structures. A statistical test for the impact of a particular structural feature on the binding affinities follows directly from this approach. The formalism is extended to describing the effects of hybridizing small \modifier RNAs' to a target RNA molecule outside its ligand binding site. We illustrate the applicability of our approach by quantitatively describing the interaction of the mRNA stabilizing protein HuR with AU-rich elements [Meisner et al. (2004), Chem. Biochem. in press]. We discuss our model and recent experimental findings demonstrating the ffectivity of modifier RNAs in vitro in the context of the current research activities in the field of non-coding RNAs. We speculate that modifier RNAs might also exist in nature; if so, they present an additional regulatory layer for fine-tuning gene expression that could evolve rapidly, leaving no obvious traces in the genomic DNA sequences.