The topic of this thesis is pre-welding process specification for heat reisisting T24 steel with TIG method. This is concerned welding waterwalls during construction electrical steam boilers. The main advantage T24 steel, except high heat resistance, is T24 doesn´t need to pre-heating neither post-welding heat treatment. Two tubes from T24 steel was flash welding and evaluating weld metal with several tests in this experiment. ALSTOM Power, s.r.o is submitter of this project.

The mesenchymal stromal cell (MSC) is of great interest for its immunomodulatory and regenerative properties. However, to research and use these MSCs it is essential to identify and characterize them as such. They need to fulfill the MSCs' minimal criteria which assess the differentiation potential, cell surface markers, and adherence. In this study, cells donated from human bone marrow were identified as MSC according to the minimal criteria. Methods used were flow cytometry, immunofluorescent staining, and ELISA. Furthermore, the population was cultured in three different media (DMEM-LG with either 10% FBS, 2% FBS, or 10% FBS supplemented with 10% conditioned media from human urinary bladder carcinoma cells (T24)) for 21 days whereupon tested for the mesenchymal characteristics, cells were counted and size measured at every passage. All cultures maintained their mesenchymal character, however, cells grown in 2% FBS became a considerably more heterogenous population regarding cell size and granularity, perhaps because of senescence. Additionally, these cells somewhat decreased in proliferation and resulted in 1 x 106 cells after 21 days, however, this was not a significant decrease when compared to the 10% FBS culture which had 2.16 x 106 cells after 21 days (p=0.061). On the contrary, the culture supplemented with T24 conditioned media resulted in a significantly higher cell count with 4.75 x 106 cells (p=0.008). Further studies could investigate which components in the conditioned media contributed to the proliferation. Moreover, the cell population in this study could not be characterized as MSC with certainty as additional cell surface markers should be tested.

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As Photodynamic Therapy (PDT) becomes increasingly popular as a treatment modality for some solid tumours, the need for a better understanding of the mechanism(s) of action and resistance are paramount. To this end we have generated Photofrin® PDT-induced resistant variants to numerous cell lines including the colon cancer cell line HT29. There is significant evidence indicating that stress proteins play an important role in determining the outcome of PDT on a cell. In this thesis the roles of the mitochondrial Heat Shock Protein 60 (Hsp60) as well as the endoplasmic Glucose Related Protein 78 (GRP78) were examined in the HT29 cells and their Photofrin induced resistant variant HT29-P14. The expression and role of these two stress proteins were also examined in T24 Bladder carcinoma cells and their GRP 78 stable-overexpressing clones Hsp60 protein was expressed at slightly higher basal levels in the resistant HT29-P14 cells relative to the parental HT29 cells. After incubation alone or PDT action, a temporal and dose dependent induction of Hsp60 was observed and this too was found to be significantly greater in the resistant cells. In the T24 model, no Hsp60 induction was observed following drug incubation or PDT. GRP78 protein levels were increased by PDT action but not by Photofrin® incubation alone in all cell lines tested. In the T24 model, GRP78 transfection resulted in a stable 2-fold increase in protein levels and a 10-20-fold increase in cell survival after PDT at the highest dose tested. A temporal and dose dependent response was noted in all cells and induction of GRP78 protein was lower in the stable overexpresser such that all cell lines had similar post induction levels. In the HT29 and HT29-P14 resistant cells, GRP78 protein levels were similar at basal level, and, both cell lines exhibited the same temporal and dose dependent increases in expression post PDT. Finally, broad scale expression profiling using a "stress" microarray in the HT29 and HT29-P14 resistant variants revealed a very similar expression profile for the 168 of the 169 stress proteins tested with the exception of the small Heat Shock Protein 27 (Hsp27). As confirmed by northern and western blot analysis, Hsp27 is over 20 fold greater at the transcriptional level and 10-15 fold greater at the translational level in the HT29-P14 resistant variant. These findings implicate Hsp27, Hsp60 and GRP78 as possible mediators of cellular sensitivity to Photofrin-mediated PDT. Specifically, Hsp27 appears to play a role in the increased resistance of our induced resistant HT29-P14 cells. / Thesis / Master of Science (MS)

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