Die monoklonale Proliferation maligner Plasmazellen im Knochenmark ist charakteristisch für das multiple Myelom (MM) und kann bei Erkrankten zu Störungen in der Hämatopoese sowie zu Knochenläsionen und Niereninsuffizienz führen. Die Weiterentwicklung und der Einsatz neuer Therapieoptionen konnten das Überleben von MM-Patienten zwar erheblich verbessern, jedoch gilt diese Krankheit weiterhin als unheilbar. Onkogene Mutationen und das Knochenmarkmikromilieu führen in MM-Zellen zur Entstehung eines onkogenen Signalnetzwerks, das das Wachstum und Überleben der Zellen aufrechterhält. Mutationen der GTPase RAS treten bei bis zu 50 % der MM-Patienten auf und tragen zum Überleben von MM-Zellen bei. Trotz der Häufigkeit und Bedeutsamkeit von onkogenem RAS, auch in anderen Tumorentitäten, ist die GTPase nach wie vor therapeutisch nicht angreifbar. Die GTPase RAL aus der Familie der RAS-GTPasen wird als Downstream-Effektor von RAS angesehen, der damit ebenfalls zur Aufrechterhaltung des Tumorzellüberlebens beitragen könnte. In einigen Tumorentitäten konnte bisher gezeigt werden, dass eine Überexpression von RAL in den Tumorzellen vorliegt und die Proliferation und Apoptose von Tumorzellen durch RAL beeinflusst wird. Daher stellte sich die Frage, ob RAL im MM ebenfalls das Überleben von Tumorzellen beeinflusst und ob eine direkte Verbindung zwischen onkogenem RAS und RAL besteht. In dieser Arbeit wurde die funktionelle Rolle von RAL sowie dessen Zusammenhang mit onkogenem RAS im MM untersucht. Hierbei konnte eine Überexpression von RAL in MM-Zellen im Vergleich zu MGUS oder normalen Plasmazellen beobachtet werden. In Knockdown-Analysen wurde gezeigt, dass RAL überlebensnotwendig für MM-Zellen ist. Dabei wurde in Western Blot-Analysen festgestellt, dass diese Überlebenseffekte unabhängig von MAPK/ERK-Signaling vermittelt werden. Es konnte teilweise jedoch eine Abhängigkeit von der AKT-Aktivität beobachtet werden. Da RAL-Knockdown Einfluss auf das Überleben von MM-Zellen hat, wurde eine pharmakologische Inhibition von RAL durch den Inhibitor RBC8 untersucht. RBC8 zeigte in höheren Dosen nur bei einem Teil der MM-Zelllinien eine Wirkung auf das Zellüberleben sowie auf die RAL-Aktivierung. Die Weiterentwicklung potenter RAL-Inhibitoren ist daher für eine klinische Translation einer RAL-Inhibition von großer Bedeutung. Zur Untersuchung des Zusammenhangs zwischen onkogenem RAS und der RAL-Aktivierung wurden RAL-Pulldown-Analysen nach Knockdown von onkogenem RAS durchgeführt. In diesen Experimenten wurde keine Abhängigkeit der RAL-Aktivierung von onkogenem RAS festgestellt. Darüber hinaus zeigten Genexpressionsanalysen nach RAS- bzw. RAL-Knockdown unterschiedliche Genexpressionsprofile. In Massenspektrometrie-Analysen wurden mögliche Effektoren, die mit RAL an der Beeinflussung des Zellüberlebens beteiligt sein könnten, untersucht. Hierbei wurden die Komponenten des Exozyst-Komplexes EXO84 und SEC5 als Interaktionspartner von RAL identifiziert. Nachdem gezeigt wurde, dass RAL ausschlaggebend für das Überleben von MM-Zellen ist, wurde eine Kombination von RAL-Knockdown mit klinisch relevanten Wirkstoffen analysiert. Diese zeigte bei der Kombination mit PI3K oder AKT-Inhibitoren verstärkte Effekte auf das Zellüberleben der MM-Zellen. Zusammenfassend wurde die Bedeutung von RAL für das Überleben von Tumorzellen im MM gezeigt und RAL als potentielles therapeutisches Target im MM beschrieben, welches unabhängig von onkogenem RAS reguliert wird. / Multiple myeloma (MM) is a hematologic neoplasia which is characterized by monoclonal proliferation of malignant plasma cells in the bone marrow leading to hematopoetic failure, bone lesions and renal failure. Although continuous development of existing therapeutics and new therapeutic options vastly improved MM patient survival, MM still remains an incurable disease. Oncogenic mutations and the bone marrow microenvironment contribute to a signaling network which sustains MM cell proliferation and survival. Within this network mutations of the RAS oncogene account for up to 50 % of MM patients. Despite its prevalence and importance not only in MM, RAS still remains undruggable. The GTPase-family Member RAL is considered as a RAS effector which might also influence maintainance of tumor cell survival. In several tumor entities RAL is overexpressed in tumor cells and influences proliferation and apoptosis. Therefore, in MM RAL might also be controlled by oncogenic RAS and mediate cell survival of tumor cells. In this work, RAL’s functional role as well as the potential interconnection with oncogenic RAS was investigated. In MM cells RAL is ovexpressed compared to non-malignant MGUS or plasma cells. Knockdown analyses showed that RAL is essential for MM cell survival. These survival effects are transferred independently of MAPK/ERK signaling as shown by Western Blot analysis. However, to some extent RAL influenced MM cell survival dependently of AKT activity. Because RAL knockdown had a significant effect on MM cell survival a pharmacological inhibition was tested using the inhibitor RBC8. In a portion of MM cell lines RBC8 exerts effects on cell survival. But the effects of RBC8 on RAL activation were only visible at higher concentrations as shown by pulldown assays. Thus, subsequent development of potent RAL inhibitors is of major importance for clinical translation. To investigate whether RAL is directly activated by oncogenic RAS, RAL pulldown assays were performed after knockdown of oncogenic RAS. Strikingly, there was no direct connection between the presence of oncogenic RAS and RAL activation. Furthermore, gene expression profiles after RAS or RAL knockdown showed differing expression signatures. Potential effectors of RAL which might also influence MM cell survival were investigated in mass spectrometric analyses where the exocyst complex components EXO84 and SEC5 were identified as RAL interaction partners. Since RAL is of importance for MM cell survival, RAL knockdown was combined with clinically relevant agents. There was an enhanced induction of apoptosis upon combination of PI3K or AKT inhibitors with RAL knockdown. Taken together, the influence of RAL as a crucial mediator of MM cell survival was shown in this work. Therefore, RAL represents a potential therapeutic target which is regulated independently of oncogenic RAS.

Univ., Diss.--Freiburg (Breisgau), 2001.

A presente tese aborda o primeiro livro escrito em ídiche por Mêndele Môikher1 Sfórim, Dos Kleine Mêntshele (O Pequeno Homenzinho) através de várias leituras do mesmo, em mais de uma versão, às quais sucedeu-se uma traduçãocuidadosa para o português. Faz considerações e referências sobre vida e obra do autor, cognominado jocosamente, por Shólem Aleikhem, de o avô da moderna literatura ídiche. Ele foi também um dos precursores do ressurgimento da língua hebraica. Shólem Yákov Abramovitsh, seu verdadeiro nome, foi um dos importantes intelectuais conscientes do momento histórico e do processo linguístico de seu povo, no final do século XIX, no leste europeu, ao qual propunha auto respeito, profissionalização e não renegação do judaísmo. A escolha de seu pseudônimo, Mêndele, o vendedor de livros, serve de pretexto para a sua participação como personagem das histórias, dada a importância e atuação de tal profissional na sociedade retratada. O texto ídiche é permeado de frases e citações em hebraico que foram trazidas para o português no mesmo padrão de linguagem do texto original. / This dissertation studies Dos Kleine Mentshele The Little Man the first book ever written in yiddish by the writer Mendele Moikher Sforim through many readings of it in more that one version, followed by careful translation into Portuguese. It makes considerations and references about Mendeles life and work. He was nicknamed by Sholem Aleikhem the grandfather of the Modern Yiddish Literature. He was one of the precursors of the revival of the Hebrew language. Sholem Yakov Abramovitch, his real name, was one of the most important intellectuals aware of the historical moment and of the linguistic process of his people at the end of the 19th century in Eastern Europe, and suggested self respect, professionalization and no Jewish denial for the historical moment and the linguistic process. The choice of his pen name, Mendele, the book peddler, allows his participation as personage of his stories because the significance and acting od such professional in the described society. The Yiddish text is permeated by Hebrew sentences and quotations brought to Portuguese at the same linguistic level.

A presente tese aborda o primeiro livro escrito em ídiche por Mêndele Môikher1 Sfórim, Dos Kleine Mêntshele (O Pequeno Homenzinho) através de várias leituras do mesmo, em mais de uma versão, às quais sucedeu-se uma traduçãocuidadosa para o português. Faz considerações e referências sobre vida e obra do autor, cognominado jocosamente, por Shólem Aleikhem, de o avô da moderna literatura ídiche. Ele foi também um dos precursores do ressurgimento da língua hebraica. Shólem Yákov Abramovitsh, seu verdadeiro nome, foi um dos importantes intelectuais conscientes do momento histórico e do processo linguístico de seu povo, no final do século XIX, no leste europeu, ao qual propunha auto respeito, profissionalização e não renegação do judaísmo. A escolha de seu pseudônimo, Mêndele, o vendedor de livros, serve de pretexto para a sua participação como personagem das histórias, dada a importância e atuação de tal profissional na sociedade retratada. O texto ídiche é permeado de frases e citações em hebraico que foram trazidas para o português no mesmo padrão de linguagem do texto original. / This dissertation studies Dos Kleine Mentshele The Little Man the first book ever written in yiddish by the writer Mendele Moikher Sforim through many readings of it in more that one version, followed by careful translation into Portuguese. It makes considerations and references about Mendeles life and work. He was nicknamed by Sholem Aleikhem the grandfather of the Modern Yiddish Literature. He was one of the precursors of the revival of the Hebrew language. Sholem Yakov Abramovitch, his real name, was one of the most important intellectuals aware of the historical moment and of the linguistic process of his people at the end of the 19th century in Eastern Europe, and suggested self respect, professionalization and no Jewish denial for the historical moment and the linguistic process. The choice of his pen name, Mendele, the book peddler, allows his participation as personage of his stories because the significance and acting od such professional in the described society. The Yiddish text is permeated by Hebrew sentences and quotations brought to Portuguese at the same linguistic level.

Background: Small nucleolar RNAs (snoRNAs) are one of the most ancient families amongst non-protein-coding RNAs. They are ubiquitous in Archaea and Eukarya but absent in bacteria. Their main function is to target chemical modifications of ribosomal RNAs. They fall into two classes, box C/D snoRNAs and box H/ACA snoRNAs, which are clearly distinguished by conserved sequence motifs and the type of chemical modification that they govern. Similarly to microRNAs, snoRNAs appear in distinct families of homologs that affect homologous targets. In animals, snoRNAs and their evolution have been studied in much detail. In plants, however, their evolution has attracted comparably little attention. Results: In order to chart the phylogenetic distribution of individual snoRNA families in plants, we applied a sophisticated approach for identifying homologs of known plant snoRNAs across the plant kingdom. In response to the relatively fast evolution of snoRNAs, information on conserved sequence boxes, target sequences, and secondary structure is combined to identify additional snoRNAs. We identified 296 families of snoRNAs in 24 species and traced their evolution throughout the plant kingdom. Many of the plant snoRNA families comprise paralogs. We also found that targets are well-conserved for most snoRNA families. Conclusions: The sequence conservation of snoRNAs is sufficient to establish homologies between phyla. The degree of this conservation tapers off, however, between land plants and algae. Plant snoRNAs are frequently organized in highly conserved spatial clusters. As a resource for further investigations we provide carefully curated and annotated alignments for each snoRNA family under investigation.

Zugl.: München, Univ., Diss., 2006 / Includes bibliographical references (p. [700]-738) and index.

In this thesis we consider diverse aspects of existence and correctness of asymptotic solutions to elliptic differential and pseudodifferential equations. We begin our studies with the case of a general elliptic boundary value problem in partial derivatives. A small parameter enters the coefficients of the main equation as well as into the boundary conditions. Such equations have already been investigated satisfactory, but there still exist certain theoretical deficiencies. Our aim is to present the general theory of elliptic problems with a small parameter. For this purpose we examine in detail the case of a bounded domain with a smooth boundary. First of all, we construct formal solutions as power series in the small parameter. Then we examine their asymptotic properties. It suffices to carry out sharp two-sided emph{a priori} estimates for the operators of boundary value problems which are uniform in the small parameter. Such estimates failed to hold in functional spaces used in classical elliptic theory. To circumvent this limitation we exploit norms depending on the small parameter for the functions defined on a bounded domain. Similar norms are widely used in literature, but their properties have not been investigated extensively. Our theoretical investigation shows that the usual elliptic technique can be correctly carried out in these norms. The obtained results also allow one to extend the norms to compact manifolds with boundaries. We complete our investigation by formulating algebraic conditions on the operators and showing their equivalence to the existence of a priori estimates. In the second step, we extend the concept of ellipticity with a small parameter to more general classes of operators. Firstly, we want to compare the difference in asymptotic patterns between the obtained series and expansions for similar differential problems. Therefore we investigate the heat equation in a bounded domain with a small parameter near the time derivative. In this case the characteristics touch the boundary at a finite number of points. It is known that the solutions are not regular in a neighbourhood of such points in advance. We suppose moreover that the boundary at such points can be non-smooth but have cuspidal singularities. We find a formal asymptotic expansion and show that when a set of parameters comes through a threshold value, the expansions fail to be asymptotic. The last part of the work is devoted to general concept of ellipticity with a small parameter. Several theoretical extensions to pseudodifferential operators have already been suggested in previous studies. As a new contribution we involve the analysis on manifolds with edge singularities which allows us to consider wider classes of perturbed elliptic operators. We examine that introduced classes possess a priori estimates of elliptic type. As a further application we demonstrate how developed tools can be used to reduce singularly perturbed problems to regular ones. / In dieser Dissertation betrachten wir verschiedene Aspekte der Existenz und Korrektheit asymptotischer Lösungen für elliptische Differentialgleichungen und Pseudodifferentialgleichungen. Am Anfang betrachtet die Arbeit den Fall eines allgemeinen elliptischen Grenzwertproblems in partiellen Ableitungen. Hierbei hängen die Koeffizienten von einem kleinen Parameter ab. Solche Gleichungen wurden schon reichlich untersucht, aber es gibt immer noch theoretische Lücken. Unser Ziel ist eine allgemeine Theorie elliptischer Operatorklassen mit kleinen Parametern. Zu diesem Zweck untersuchen wir im Detail den Fall eines beschränkten Gebietes mit glattem Rand. Zuerst konstruieren wir formale Lösungen als Potenzreihe einer kleinen Variablen. Weiter untersuchen wir ihre asymptotischen Eigenschaften. Dazu reicht es aus, beidseitige A-Priori Abschätzungen für diejenigen Randwertproblemoperatoren zu bestimmen, die gleichmäßig stetig von den kleinen Parametern abhängen. Solche Abschätzungen gelten nicht in Funktionenräumen, die in der klassischen elliptischen Theorie benutzt werden. Um diese Beschränkungen zu überwinden, nutzen wir Normen abhängig vom kleinen Parameter. Änliche Normen finden sich oft in der Literatur, aber ihre Eigenschaften wurden unzureichend untersucht. Unsere theoretische Forschung zeigt, dass die gewöhnliche elliptische Methode korrekt durchgeführt werden kann. Die erhaltenen Abschätzungen erlauben das Fortsetzen der Normen auf kompakte Mannigfältigkeiten mit Rand. Unsere Forschung wird mit algebraischen Bedingungen für die Operatoren abgeschlossen. Wir zeigen, dass diese Bedingungen äquivalent zu der Existenz der A-Priori-Abschätzungen sind. Im zweiten Schritt erweitern wir das Konzept der Elliptizität mit kleinen Parametern zu allgemeineren Operatorklassen. Zuerst wollen wir den Unterschied in asymptotischen Mustern zwischen der erhaltenen Reihe und Lösungen ähnlicher Probleme untersuchen. Deshalb untersuchen wir die Wärmeleitungsgleichung in einem beschränkten Gebiet mit einem kleinen Parameter in der Zeitableitung. In diesem Fall tangiert der Rand die Charakteristik endlich oft. Es ist bekannt, dass die Lösungen unregulär im Allgemeinen in Umgebungen solcher Stellen sind. Wir nehmen an, dass der Rand an solchen Stellen nicht glätt sein kann und kaspydalische Singularitäten hat. Wir haben eine formale asymptotische Zerlegung gefunden und einen Schwellenwert gezeigt, sodass die asymptotische Eigenschaft der Reihe nicht mehr gilt, wenn der Randparameter diesen Schwellenwert übersteigt. Der letze Teil der Arbeit führt ein allgemeines Konzept der Elliptizit"at mit einem kleinen Parameter ein. Mehrere theoretische Erweiterungen auf Pseudodifferentialoperatoren wurden schon in früheren Studien vorgeschlagen. Als neuen Beitrag wenden wir die Analysis auf Manigfältigkeiten mit Kantensingularitäten an. Dies lässt es zu, allgemeinere gestörte Operatorklassen zu betrachten. Wir beobachten, dass die eingef"uhrten Klassen A-Priori-Abschätzungen elliptischer Gestalt haben. Als weitere Anwendung demonstrieren wir, wie die entwickelten Mittel zum Reduzieren singular gestörter Probleme zu regulären Fällen benutzt werden können.

The detection of non-protein-coding RNA (ncRNA) genes in bacteria and their diverse regulatory mode of action moved the experimental and bio-computational analysis of ncRNAs into the focus of attention. Regulatory ncRNA transcripts are not translated to proteins but function directly on the RNA level. These typically small RNAs have been found to be involved in diverse processes such as (post-)transcriptional regulation and modification, translation, protein translocation, protein degradation and sequestration. Bacterial ncRNAs either arise from independent primary transcripts or their mature sequence is generated via processing from a precursor. Besides these autonomous transcripts, RNA regulators (e.g. riboswitches and RNA thermometers) also form chimera with protein-coding sequences. These structured regulatory elements are encoded within the messenger RNA and directly regulate the expression of their “host” gene. The quality and completeness of genome annotation is essential for all subsequent analyses. In contrast to protein-coding genes ncRNAs lack clear statistical signals on the sequence level. Thus, sophisticated tools have been developed to automatically identify ncRNA genes. Unfortunately, these tools are not part of generic genome annotation pipelines and therefore computational searches for known ncRNA genes are the starting point of each study. Moreover, prokaryotic genome annotation lacks essential features of protein-coding genes. Many known ncRNAs regulate translation via base-pairing to the 5’ UTR (untranslated region) of mRNA transcripts. Eukaryotic 5’ UTRs have been routinely annotated by sequencing of ESTs (expressed sequence tags) for more than a decade. Only recently, experimental setups have been developed to systematically identify these elements on a genome-wide scale in prokaryotes. The first part of this thesis, describes three experimental surveys of exploratory field studies to analyze transcript organization in pathogenic bacteria. To identify ncRNAs in Pseudomonas aeruginosa we used a combination of an experimental RNomics approach and ncRNA prediction. Besides already known ncRNAs we identified and validated the expression of six novel RNA genes. Global detection of transcripts by next generation RNA sequencing techniques unraveled an unexpectedly complex transcript organization in many bacteria. These ultra high-throughput methods give us the appealing opportunity to analyze the complete RNA output of any species at once. The development of the differential RNA sequencing (dRNA-seq) approach enabled us to analyze the primary transcriptome of Helicobacter pylori and Xanthomonas campestris. For the first time we generated a comprehensive and precise transcription start site (TSS) map for both species and provide a general framework for the analysis of dRNA-seq data. Focusing on computer-aided analysis we developed new tools to annotate TSS, detect small protein-coding genes and to infer homology of newly detected transcripts. We discovered hundreds of TSS in intergenic regions, upstream of protein-coding genes, within operons and antisense to annotated genes. Analysis of 5’ UTRs (spanning from the TSS to the start codon of the adjacent protein-coding gene) revealed an unexpected size diversity ranging from zero to several hundred nucleotides. We identified and validated the expression of about 60 and about 20 ncRNA candidates in Helicobacter and Xanthomonas, respectively. Among these ncRNA candidates we found several small protein-coding genes that have previously evaded annotation in both species. We showed that the combination of dRNA-seq and computational analysis is a powerful method to examine prokaryotic transcriptomes. Experimental setups are time consuming and often combined with huge costs. Another limitation of experimental approaches is that genes which are expressed in specific developmental stages or stress conditions are likely to be missed. Bioinformatic tools build an alternative to overcome such restraints. General approaches usually depend on comparative genomic data and evolutionary signatures are used to analyze the (non-)coding potential of multiple sequence alignments. In the second part of my thesis we present our major update of the widely used ncRNA gene finder RNAz and introduce RNAcode, an efficient tool to asses local protein-coding potential of genomic regions. RNAz has been successfully used to identify structured RNA elements in all domains of life. However, our own experience and the user feedback not only demonstrated the applicability of the RNAz approach, but also helped us to identify limitations of the current implementation. Using a much larger training set and a new classification model we significantly improved the prediction accuracy of RNAz. During transcriptome analysis we repeatedly identified small protein-coding genes that have not been annotated so far. Only a few of those genes are known to date and standard proteincoding gene finding tools suffer from the lack of training data. To avoid an excess of false positive predictions, gene finding software is usually run with an arbitrary cutoff of 40-50 amino acids and therefore misses the small sized protein-coding genes. We have implemented RNAcode which is optimized for emerging applications not covered by standard protein-coding gene annotation software. In addition to complementing classical protein gene annotation, a major field of application of RNAcode is the functional classification of transcribed regions. RNA sequencing analyses are likely to falsely report transcript fragments (e.g. mRNA degradation products) as non-coding. Hence, an evaluation of the protein-coding potential of these fragments is an essential task. RNAcode reports local regions of high coding potential instead of complete protein-coding genes. A training on known protein-coding sequences is not necessary and RNAcode can therefore be applied to any species. We showed this with our analysis of the Escherichia coli genome where the current annotation could be accurately reproduced. We furthermore identified novel small protein-coding genes with RNAcode in this extensively studied genome. Using transcriptome and proteome data we found compelling evidence that several of the identified candidates are bona fide proteins. In summary, this thesis clearly demonstrates that bioinformatic methods are mandatory to analyze the huge amount of transcriptome data and to identify novel (non-)coding RNA genes. With the major update of RNAz and the implementation of RNAcode we contributed to complete the repertoire of gene finding software which will help to unearth hidden treasures of the RNA World.

Originally a Ph. D. Thesis--University of Essex, 1999. / Includes bibliographical references.

Originally a Ph. D. Thesis--University of Essex, 1999. / Includes bibliographical references.