Abstract This study utilized normal and defective epithelial cell cultures and epidermal skin samples to examine intra- and intercellular calcium signaling. The main interests of this thesis were Hailey-Hailey disease (HHD), Darier disease (DD), neurofibromatosis 1 (NF1) and transitional cell carcinoma (TCC). HHD and DD diseases are rare autosomal dominant skin disorders characterized by dissociation of epidermal keratinocytes (acantholysis) at the suprabasal layer of the epidermis. HHD and DD diseases are caused by mutations in the genes encoding the calcium pumps in the Golgi apparatus (hSPCA1) and endoplasmic reticulum (SERCA2b), respectively. Due to these mutations calcium uptake into the Golgi apparatus or ER is diminished, which is believed to cause abnormal cell junction protein processing and dissociation of keratinocytes. This study utilized electron probe microanalysis (EPMA) and demonstrated for the first time that lesional areas of HHD and DD and non-lesional areas of DD epidermis display abnormally low calcium content in the basal cell layer. Furthermore, ATP mediated calcium signaling was impaired in cultured HHD and DD keratinocytes and epidermal ATP receptor localization was disrupted. In conclusion, these results suggest that the low calcium content in the basal cell layer is the reason for suprabasal ruptures in HHD but not necessarily in DD lesions, and that abnormal ATP receptor localization contributes to the calcium signaling defects. NF1 deficient keratinocytes display abnormally low resting cytosolic calcium levels and it has been suggested that the calcium concentration in the lumen of the endoplasmic reticulum is decreased. This study demonstrated that NF1 keratinocytes rely mostly on ATP mediated calcium signaling while normal keratinocytes rely mostly on gap junctional intercellular communication (GJIC). Studies with TCC cells have demonstrated that gap junctions participate in intercellular calcium wave propagation. This thesis demonstrated that the ATP mediated pathway was also operational in calcium wave propagation in normal uroepithelial and TCC cell cultures. Furthermore, impaired calcium wave propagation in the TCC cell culture could be improved through PKC α/βI –isoenzyme inhibition with Gö6976. Gö6976 treatment increased connexin 26 clustering at plasma membrane but did not alter expression level of the protein. This thesis contains a wide repertoire of calcium detection techniques including a new cutting-edge technology for elemental calcium detection of epidermal samples. These techniques can be used for molecular specific analysis of calcium signaling in epithelial cells.

Objective: Primary transitional cell carcinoma (TCC) of the upper urinary tract represents 6–8% of all TCC cases. Nephroureterectomy with removal of a bladder cuff is the treatment of choice. The rates of TCC recurrence in the bladder after primary upper urinary tract surgery described in the literature range between 12.5 and 37.5%. In a retrospective analysis we examined the occurrence of TCC after nephroureterectomy for upper tract TCC in patients without a previous history of bladder TCC at the time of surgery. Methods: Between 1990 and 2002, 29 patients underwent primary nephroureterectomy for upper tract TCC. The mean age of the patients was 69.5 years. In 5 cases upper urinary tract tumors were multilocular, in the remaining cases unilocular in the renal pelvis (n = 12) or the ureter (n = 12). The follow-up was available for 29 patients with a mean follow-up of 3.37 (0.1–11.2) years. Results: 11/29 (37.9%) patients had TCC recurrence with 9/11 patients having bladder TCC diagnosed within 2.5 years (0.9–6.0) after nephroureterectomy. 13/29 patients are alive without TCC recurrence, 3/29 patients died due to systemic TCC progression and 5/29 died of unrelated causes without evidence of TCC recurrence. Conclusion: Our data indicate a high incidence of bladder TCC after nephroureterectomy for primary upper tract TCC of up to 6 years after primary surgery. Because of the high incidence of bladder TCC within the first 3 years of surgery, careful follow-up is needed over at least this period. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

<p><em>Background:</em> UC is the most common urogenital cancer, comprising up to 2% of all naturally occurring neoplasia in dogs and can be challenging to diagnose. With early diagnosis, the disease can be controlled in most dogs with a good quality of life. MiRNAs are small non-coding RNAs that function by post-transcriptional regulation of gene expression. Their abundant presence and stability in the body make them promising tools for disease diagnosis. </p> <p><em>Hypothesis:</em> A microRNA (miRNA) signature can be used to differentiate canine urothelial carcinoma (UC) from other lower urinary tract diseases.</p> <p><em>Literature review:</em> There is an overlap of miRNA expression changes between normal physiologic processes, non-infectious and non-inflammatory conditions, infectious and/or inflammatory conditions, and neoplasia. Additionally, the mechanism of action of these overlapping miRNAs varies depending on the disease process. There is a lack of standardization of miRNA evaluation and consistency within a single evaluation method. Herein we evaluate three papers on miRNA expression in canine UC and compared the reported expression profile to human UC literature and identified experimentally validated targets of the dysregulated miRNA. </p> <p><em>Methods and results:</em> <strong>(Aim 1)</strong> Using reverse transcriptase quantitative PCR (RT-qPCR), we assessed the effects of sample handling on miRNA expression in formalin-fixed Paraffin-embedded (FFPE) tissue and urine sediment. We showed that the time of tissue fixation in formalin does not alter the detection of miRNA expression, but the inclusion of the muscularis layer altered the miRNA expression profile in bladder tissue. Additionally, miRNAs in urine sediment were proven to be stable despite the storage temperature for up to two weeks. <strong>(Aim 2)</strong> Using Next Generation Sequencing (NGS) with validation of findings via RT-qPCR, we evaluated differential miRNA expression in bladder tissue collected from normal canine urothelium and the invasive type of UC (iUC) to elucidate the dysregulated pathways. We found that twenty-eight miRNAs were differentially expressed (DE). The DE miRNAs were most often associated with gene silencing by miRNA, miRNAs in cancer, and miRNAs involved in DNA damage responses. Proteins involved include HRAS, KRAS, ARAF, RAF1, MAPK1, MAP2K1, MAPK3, FGFR3, EGFR, HBEGF, RASSF1, E2F2, E2F3, ERBB2, SRC, MMP1, and UP3KA. <strong>(Aim 3)</strong> Using RT-qPCR, expression of miR-214, miR-181a, miR-361, and miR-145 were evaluated. We failed to reject the null hypothesis that the relative gene expression in all groups was the same for any miRNA, nor did we find any multivariate summary that could effectively differentiate UC from inflammatory and non-neoplastic transitional cells. </p> <p><em>Conclusions:</em>   The findings within this thesis highlight the need for standardized methods for miRNA evaluation, support the use of stored samples for miRNA expression analysis, and show the importance of isolating the tissue of interest in FFPE. We defined the miRNome of iUC and investigated numerous protein pathways affected by dysregulation of differentially expressed miRNA in urothelial carcinoma. While we failed to reject our null hypothesis that the miRNA signature we evaluated could be utilized as a diagnostic tool for canine urothelial carcinoma, we showed the promise of miRNA as diagnostic tools and highlight several novel pathways that miRNA regulation affects in this disease. </p>