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Das Ribonukleoprotein, Telomerase wird vor allem für die Aufrechterhaltung der Telomerlänge benötigt und ist normalerweise nur in Keimbahnzellen, Stammzellen und anderen Zellen mit erhöhter Regenerationsfähigkeit aktiv. Die Aktivierung der Telomerase ist darüber hinaus ein wichtiger Faktor während der Krebsentstehung. Fast das komplette Spektrum humaner Tumore zeichnet sich durch hohe Telomerase-Aktivität aus. Vor allem maligne Tumore besitzen eine sehr aktive Telomerase, unlimitiertes Wachstum und Immortalität ermöglicht. Die Aktivität der Telomerase wird vor allem über die Expression der katalytischen Untereinheit hTERT reguliert, die unter der strikten Kontrolle verschiedener Tumorsuppressorgene liegt. Zu den wichtigsten Regulatoren der hTERT-Expression gehört auch der bekannte Tumorsuppressor p53. Über die Rolle des p53-Familienmitglieds p73 in der Regulation der Telomerase-Aktivität war bisher nur wenig bekannt. Im Rahmen dieser Arbeit konnte ein regulatorischer Einfluss von p73 nachgewiesen werden. Dabei wurden deutliche Unterschiede in der Funktion der N-terminalen Isoformen TAp73 und DeltaNp73 beobachtet. TAp73 erwies sich sowohl nach Überexpression als auch nach Induktion des endogenen TAp73 als ein effizienter Repressor der hTERT-Expression. Im Gegensatz dazu konnte durch die Hemmung des endogenen TAp73 mittels RNAi die Expression von hTERT in verschiedenen Zelllinen induziert werden. Zusätzlich zu der Funktion als Tumorsuppressor scheint p73 auch in verschiedene Differenzierungsprozesse involviert zu sein. Die Expression von p73 korreliert zwar mit der Hemmung der Telomerase-Aktivität während der myeloischen Differenzierung von HL60-Zellen, hat hier aber keine Bedeutung für die Repression von hTERT. Die N-terminal verkürzte Isoform DeltaNp73 wirkt im Gegensatz zu TAp73 als effizienter Aktivator der hTERT-Expression. DeltaNp73 induziert die hTERT-Expression einerseits über seine dominant-negative Funktion auf die pro-apoptotischen p53-Familienmitglieder und andererseits über die Hemmung repressiver RB-E2F-Komplexe. Im Rahmen dieser Studie erwies sich p73 somit als ein wichtiger Regulator der Telomerase Aktivität, wobei sich eine duale Rolle als negativer (TAp73) und auch als positiver (DeltaNp73) Regulator der Telomerase Aktivität herausstellte.
Alveolar echinococcosis, which is caused by the metacestode stage of the small fox tapeworm Echinococcus multilocularis, is a severe zoonotic disease with limited treatment options. For a better understanding of cestode biology the genome of E. multilocularis, together with other cestode genomes, was sequenced previously. While a few studies were undertaken to explore the E. multilocularis transcriptome, a comprehensive exploration of global transcription profiles throughout life cycle stages is lacking. This work represents the so far most comprehensive analysis of the E. multilocularis transcriptome. Using RNA-Seq information from different life cycle stages and experimental conditions in three biological replicates, transcriptional differences were qualitatively and quantitatively explored. The analyzed datasets are based on samples of metacestodes cultivated under aerobic and anaerobic conditions as well as metacestodes obtained directly from infected jirds. Other samples are stem cell cultures at three different time points of development as well as non-activated and activated protoscoleces, the larval stage that can develop into adult worms. In addition, two datasets of metacestodes under experimental conditions suitable for the detection of genes that are expressed in stem cells, the so-called germinative cells, and one dataset from a siRNA experiment were analyzed. Analysis of these datasets led to expression profiles for all annotated genes, including genes that are expressed in the tegument of metacestodes and play a role in host-parasite interactions and modulation of the host's immune response. Gene expression profiles provide also further information about genes that might be responsible for the infiltrative growth of the parasite in the liver.
Furthermore, germinative cell-specific genes were identified. Germinative cells are the only proliferating cells in E. multilocularis and therefore of utmost importance for the development and growth of the parasite. Using a combination of germinative cell depletion and enrichment methods, genes with specific expression in germinative cells were identified. As expected, many of these genes are involved in translation, cell cycle regulation or DNA replication and repair. Also identified were transcription factors, many of which are involved in cell fate commitment. As an example, the gene encoding the telomerase reverse transcriptase (TERT) was studied further. Expression of E. multilocularis tert in germinative cells was confirmed experimentally. Cell culture experiments indicate that TERT is required for proliferation and development of the parasite, which makes TERT a potentially interesting drug target for chemotherapy of alveolar echinococcosis.
Germinative cell specific genes in E. multilocularis also include genes of densoviral origin. More than 20 individual densovirus loci with information for non-structural and structural densovirus proteins were identified in the E. multilocularis genome. Densoviral elements were also detected in many other cestode genomes. Genomic integration of these elements suggests that densovirus-based vectors might be suitable tools for genetic manipulation of tapeworms. Interestingly, only three of more than 20 densovirus loci in the E. multilocularis genome are expressed. Since the canonical piRNA pathway is lacking in cestodes, this raises the question about potential silencing mechanisms. Exploration of RNA-Seq information indicated natural antisense transcripts as a potential gene regulation mechanism in E. multilocularis. Preliminary experiments further suggest DNA-methylation, which was previously shown to occur in platyhelminthes, as an interesting avenue to explore in future.
The transcriptome datasets also contain information about genes that are expressed in differentiated cells, for example the serotonin transporter gene that is expressed in nerve cells. Cell culture experiments indicate that serotonin and serotonin transport play an important role in E. multilocularis proliferation, development and survival.
Overall, this work provides a comprehensive transcription data atlas throughout the E. multilocularis life cycle. Identification of germinative cell-specific genes and genes important for host-parasite interactions will greatly facilitate future research. A global overview of gene expression profiles will also aide in the detection of suitable drug targets and the development of new chemotherapeutics against alveolar echinococcosis.