Human telomerase is a ribonucleoprotein complex that adds hexameric TTAGGG repeats to the ends of chromosomes in order to prevent their shortening. Telomerase activity has been evaluated for its diagnostic and prognostic value in cancer since it is observed in most malignancies but not in most normal somatic tissues. In this study telomerase activity was examined in tumor specimens obtained from cervix, endometrium and their non-cancerous regions by an improved telomeric repeat amplification protocol (TRAP) &ndash / silver staining assay. Appearance of characteristic TRAP leader with 6 base pair increments indicate a positive result and was observed in all cancerous and some of the non-cancerous tissues. Telomerase activities of carcinoma tissues and normal counterparts were compared by densitometric analysis after PCR. Significantly higher telomerase activity was observed in cervical carcinoma samples compared to normal adjacent tissue. No significant difference was observed between endometrium carcinomas and normal endometrial tissue in terms of telomerase activity. High telomerase activity in normal endometrium restricts the use of assay for detection of carcinogenesis. However, in cervical tissues an accurate quantification of telomerase activity by TRAP &ndash / silver stain assay may be valuable as a confirmatory assay.

Plattenepithelkarzinome der Kopf-Hals-Region sind durch einen aggressiven Krankheitsverlauf mit häufigem Auftreten von Lokalrezidiven und Zweitkarzinomen gekennzeichnet. Molekulare Veränderungen des Tumorrandes und der Tumor benachbarten Gewebe könnten in Ergänzung zur Histopathologie eine kurative chirurgische Tumorextirpation bestätigen. Wir untersuchten die Telomeraseaktivität in verschiedenen Entfernungen vom Tumorzentrum um ihre Verwendung als molekularen Marker zu prüfen. Wir untersuchten 80 kryokonservierte Gewebeschnitte von 40 Patienten mit einem Plattenepithelkarzinom im Kopf-Hals-Bereich, davon 33 karzinomfreie Tumorrandgewebe. 50% der 20 karzinomhaltigen Gewebe, 28 % der karzinomfreien Tumorrandgewebe und 35 % der tumorfernen Proben waren telomerasepositiv. Der Vergleich ergab keinen statistisch signifikanten Unterschied. Es konnte keine sichere Korrelation zwischen der Telomeraseaktivität in den untersuchten Geweben mit dem weiteren Krankheitsverlauf gefunden werden, allerdings zeigte die Telomerasepositivität in karzinomfreien Tumorrändern die Tendenz, mit einer besseren Prognose zu korrespondieren. Unsere Befunde sollten an einer größeren und homogeneren Patientengruppe überprüft werden. / Squamous cell carcinomas of the head-neck region are characterised by an aggressive course of disease and a strong tendency to loco-regional recurrence and second primary tumours. As a complement to histopathology, molecular changes of tumour marginal and tumor distant tissue may confirm curative surgical tumour extirpation. We tested telomerase activity at different distances to the tumour centre of squamous cell carcinomas to consider the use for molecular marker. We examined 80 cryo-conserved tissue samples from 40 patients with a squamous cell carcinoma of the head-neck region, among them 33 carcinoma-free margin tissues. 50 % of the carcinoma centres, 28 % of the carcinoma-free margin tissues and 35% of the tumour distant samples were telomerase-positive. The comparison showed no statistically significant difference. We have found no reliable correlation between telomerase activity in the carcinoma-free margin tissues and the further course of disease, but telomerase positivity in carcinoma-free tumour margins tended to correlate with a better prognosis. Confirmation of the results in a larger and more homogeneous patient group is needed.

Im Bereich der medizinischen Diagnostik spielen DNA-Chips eine immer wichtigere Rolle. Dabei werden Glas- oder Silikon-Oberflächen mit Tausenden von einzelsträngigen DNA-Fragmenten, sog. Sonden, bestückt, die mit den passenden DNA-Fragmenten in der zugefügten Patientenprobe verschmelzen. Die Auswertung solcher Messungen liefert die Diagnose für Krankheiten wie z.B. Krebs, Alzheimer oder für den Nachweis pathogener Erreger. Durch fortschreitende Miniaturisierung dieser Meßsysteme können bis zu 40.000 Genfragmente des Menschen in einer einzigen Messung analysiert werden. Neben den DNA-Fragmenten können Bio-Chips auch für andere biologische Komponenten wie Antikörper und Proteine eingesetzt werden, wobei bei letzteren neben der Bindung auch die Aktivität ein wichtiger Diagnoseparamter ist. <br /> <br /> Am Fraunhofer-Institut für medizinische Technik und am Lehrstuhl für Analytische Biochemie der Universität Potsdam wurden im Rahmen einer Doktorarbeit Methoden entwickelt, die es ermöglichen auf nukleinsäuremodifizierten Sensoroberflächen die Aktivität von Proteinen zu messen. Es wurden Nukleinsäuren auf Oberflächen optischer Sensoren verankert. Diese fungierten als Rezeptor für die Proteine sowie auch als Substrat für Restriktionsenzyme, die Nukleinsäuren schneiden und Polymerasen, die Nukleinsäuren synthetisieren und verlängern können.<br /> <br /> Seine Anwendung fand diese Messmethode in der Messung der Aktivität des Proteins Telomerase, das in 90% aller Tumore erhöhte Aktivität gegenüber gesunden Zellen aufweist. Die Vorteile dieses neuen Assays gegenüber älteren Methoden liegt im Verzicht auf radioaktiv-markierten Komponenten und einer deutlich verkürzten Analysezeit. Die Arbeit schliesst mit einem funktionsfähigen Nachweis der Telomeraseaktivität im Zellextrakt von gesunden und kranken Zellen. Der direkte Einfluß von Hemmstoffen auf die Aktivität konnte sichtbar gemacht werden, und steht daher bei der Entwicklung neuer Tumor-Diagnostika und Therapeutika zur Verfügung. / In the field of medical diagnostic the importance of DNA chips is growing continuously. On silica surfaces hundreds of single stranded DNA fragments are immobilised which finally detect the complementary sequences in samples of patients by hybridisation. These methods enable the detection of serious diseases as cancer, Alzheimer's disease or infection by pathogens. Biomolecules as nucleic acids, antibodies and proteins can be used as receptors on the solid surfaces whereas in case of proteins not only the binding but also the activity are of high interest for medical diagnosis.<br /> <br /> In this thesis a biosensoric approach has been developed to determine the activity of nucleic-acid modifying enzymes. Optical sensors as, e.g., the grating coupler, were used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of evanescent-field. Furthermore sensors based on total internal reflection fluorescence measured the activity of restriction enzymes and polymerases. The general approach included the immobilisation of oligonucleotides which acted as the receptor for the enzymes as well as the substrate for the enzymatic reaction. Enzymes as EcoRI and Klenow were used to establish a model system to measure the activity of DNA-modifying enzymes on optical surfaces. As most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe the new optical systems facilitated the analysis of the enzymatic activity by measuring the DNA-synthesis or restriction directly.<br /> <br /> These systems were finally used to detect the activity of the telomerase, an enzymatic marker for the cancerous development of cells. In 90% of cancer cells the activity of telomerase is higher than in normal cells. Additionally the increase of the telomerase activity in cells induced by carcinogenic substances was detected. Furthermore no purification steps of the samples were required as all measurements were performed with crude cell extract.<br /> <br /> Also the effect of inhibitors of the telomerase could be shown in real time measurements underlining the potential of this assay for further developments of new cancer therapeutics.

Giardia intestinalis is a protozoan causing diarrhea worldwide. Beside its medical importance, it is evolutionary distant protist with two nuclei within a cell adapted for parasitic life in the environment poor of oxygen. Its genome is small and compact in term of gene content and size. It is therefore an attractive model organism for studies of minimal requirements for cellular processes. Present work brings new partial information on different levels of chromosome integrity maintenance of this parasite. Our study presents characteristics of chromosome termini and their protection. We localized telomeres during all stages of the trophozoite cell cycle and determined the length of Giardia telomeres ranging from 0.5 to 2.5 kb, we proved an existence of an active telomerase enzyme synthesizing telomeric repeats in in this parasite, despite the fact that giardial telomerase is structurally divergent. Present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes. We described effects of commonly used drug for treatment of anaerobic infections, metronidazole, on DNA and cell cycle progression in susceptible and resistant cell lines. Incubation of cells with this drug causes phosphorylation of histone H2A in cell nuclei...