No description available.

Cancer of the prostate (CaP) is the most common malignancy diagnosed in men in the Western society. During the last years, prostate specific antigen (PSA) has been used as a biomarker for CaP, although a high PSA value is not specific for CaP. Thus, there is an urgent need for new and improved diagnostic markers for CaP. In this thesis, the aim was to find a miRNA signature for diagnosis of CaP and to elucidate if differences in behavior between transition zone and peripheral zone tumors are reflected in miRNA expression. One of the major findings is anexpression signature based on nine miRNAs that with high accuracy (85%) could classify normal and malignant tissues from the transition zone of the prostate. The results furthermore show that the major differences in miRNA expression are found between normal and malignant tissues, rather than between the different zones. In addition, tumors arising in the peripheral zone have fewer changes in miRNA expression compared to tumors in the transition zone, indicating that the peripheral zone is more prone to tumor development compared to the transition zone of the prostate. A crucial step in pre-processing of expression data, in order to differentiate true biological changes, is the normalization step. Therefore, an additional aim of this thesis was to compare different normalization methods for qPCR array data in miRNA expression experiments. The results show that data-driven methods based on quantile normalization performs the best. The results also show that in smaller miRNA expression studies, only investigating a few miRNAs, RNU24 is the most suitable endogenous control gene for normalization. Taken together, the results in this thesis show the importance of miRNAs and the possibility of their future use as biomarkers in the field of prostate cancer.

Wie sich in den letzten Jahren gezeigt hat, ist Fettgewebe nicht nur ein inerter Fettspeicher, sondern produziert auch eine Vielzahl endokrin wirksamer Substanzen, die unter anderem auch an der Blutdruckregulation beteiligt sind. Da Adipositas ein wichtiger Risikofaktor für die Entwicklung der Hypertonie ist, sollte im Rahmen dieser Dissertation ein System zur quantitativen Untersuchung der Genexpression in Primärkulturen humaner Adipocyten entwickelt werden und dessen Funktionalität am Beispiel der hormonellen Regulation der Gene des Renin-Angiotensin-Systems demonstriert werden. Dies beinhaltete die Etablierung der Adipocytenisolierung und -kultivierung, eines Stimulationsassays, die Entwicklung einer der besonderen Größe und dem hohen Fettgehalt der Zellen angepaßten Zellzahl- und Vitalitätsbestimmungsmethode, die Untersuchung vier verschiedener RNA-Extraktionsmethoden auf ihre Eignung für Adipocyten und die Etablierung eines besonders sensitiven RT-PCR Systems zur Untersuchung der Genexpression mittels einer fluoreszenzmarkierten Sonde. Exemplarisch konnte anhand der Renin-Angiotensin-System-Gene die Funktionalität der Methoden demonstriert werden, indem nicht nur die Genexpression aller Komponenten des Renin-Angiotensin-Systems in humanen Adipocyten nachgewiesen wurden, sondern auch gezeigt werden konnte, dass Hydrocortison sowohl die Genexpression als auch die Dichte des Angiotensin II Typ 1-Rezeptors in der Adipocytenmembran stimuliert. Dieser Aspekt könnte möglicherweise nicht nur bei der besonderen Adipositasform des Cushing-Syndroms, sondern auch für die Entstehung der zentralen Adipositas von Bedeutung sein. / Adipose tissue has functions above-and-beyond storing fat. It also produces a variety of different endocrine substances, some of which influence blood pressure regulation. Obesity is a well known risk factor for the development of hypertension Thus, the genes regulating expression of vasoactive molecules in adipose tissue, possibly contributing to an increase in blood pressure are of great interest. The aim of this work was to develope a system for quantitative gene expression analysis in primary cultured human adipocytes and to demonstrate its utility for studying the hormonal regulation of genes encoding the renin-angiotensin-system. We established procedures for the isolation and culture of human adipocytes, as well as a stimulatory assay. We also developed methods for the determination of cell number and vitality. Above this, four RNA extraction protocols were evaluated regarding their suitability for adipocytes, and a very sensitive RT-PCR system for gene expression analysis using fluorescent labeled probes was established. As an example for the functionality of these methods we showed that all genes of the renin-angiotensin-system are expressed in human adipocytes. We also demonstrated that hydrocortisone stimulates the gene expression as well as the density of the angiotensin II receptor type 1 on cultured human adipocytes. This finding may be of interest for the development of the obesity phenotype found in cushing syndrome, but could also contribute to the development of central obesity.