Two-photon polymerization (2PP) has provided the field of cell biology with the opportunity to fabricate precisely designed microscaffolds for a wide range of studies, from mechanobiology to in... Show moreTwo-photon polymerization (2PP) has provided the field of cell biology with the opportunity to fabricate precisely designed microscaffolds for a wide range of studies, from mechanobiology to in vitro disease modelling. However, a multitude of commercial and in-house developed photosensitive materials employed in 2PP suffers from high auto-fluorescence in multiple regions of the spectrum. In the context of in vitro cell biological studies, this is a major problem since one of the main methods of characterization is fluorescence microscopy of immunostained cells. This undesired auto-fluorescence of microscaffolds affects the efficiency of such an analysis as it often overlaps with fluorescent signals of stained cells rendering them indistinguishable from the scaffolds. Here, we propose two effective solutions to suppress this auto-fluorescence and compare them to determine the superiority of one over the other: photo-bleaching with a powerful UV point source and auto-fluorescence quenching via Sudan Black B (SBB). The materials used in this study were all commercially available, namely IPL, IP-Dip, IP-S, and IP-PDMS. Bleaching was shown to be 61.7-92.5% effective in reducing auto-fluorescence depending on the material. On the other hand, SBB was shown to be 33-95.4% effective. The worst result in presence of SBB (33%) was in combination with IP-PDMS since the adsorption of the material on IP-PDMS was not sufficient to fully quench the auto-fluorescence. However, auto-fluorescence reduction was significantly enhanced when activating the IP-PDMS structures with oxygen plasma for 30 s. Moreover, we performed a cell culture assay using a human neuroblastoma cell line (SH-SY5Y) to prove the effectiveness of both methods in immunofluorescence characterization. SBB presented a lower performance in the study especially in presence of 2PP-fabricated microchannels and microcages, within which the differentiated SH-SY5Y cells migrated and extended their axon-like processes, since the SBB obstructed the fluorescence of the stained cells. Therefore, we concluded that photo-bleaching is the optimal way of auto-fluorescence suppression. In summary, this study provides a systematic comparison to answer one of the most pressing issues in the field of 2PP applied to cell biology and paves the way to a more efficient immunofluorescence characterization of cells cultured within engineered in vitro microenvironments. Show less
The studies presented in this thesis aimed to better understand specific C. difficile physiological processes: genome maintenance, DNA replication, and toxin regulation. As the available tools to... Show moreThe studies presented in this thesis aimed to better understand specific C. difficile physiological processes: genome maintenance, DNA replication, and toxin regulation. As the available tools to investigate C difficile were insufficient, a secondary goal was to develop novel methods for C. difficile studies. We developed a luciferase toolkit for use in C. difficile, not only for gene expression analysis, but also for protein-protein interaction studies in vivo, secretion of proteins or the analysis of complexes present at the cell surface. We investigated fluorescence microscopy for C. difficile, by comparing different fluorescent systems previously described, and introduced the use of the fluorophore HaloTag. Additionally, we analyzed C. difficile intrinsic fluorescence and report the potential limitations for live-cell microscopy. The development of the tools enabled the assessment of the localization of the TcdC C-terminus and the characterization of the HupA protein. Finally, we provide new insights into the chromosome remodelling and DNA replication by studying DnaA-dependent unwinding and characterizing the histone-like protein HupA. In the discussion, we present the implications of our findings and provide perspectives for further research on TcdC-mediated toxin regulation and chromosome dynamics, specifically during DNA replication. Show less
Taylor, R.; Lindorfer, M.; Oostindie, S.; Cook, E.; Zent, C.; Burack, R.; ... ; Parren, P. 2018
During developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome... Show moreDuring developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome condensation and segregation. Several unique protein families are involved in the control of this process, including that of the SsgA-like proteins (SALPs). While SsgA and SsgB are essential for sporulation-specific cell division in S. coelicolor, SsgC-G are responsible for correct DNA segregation/condensation, spore wall synthesis, autolytic spore separation, or exact septum localisation. The SALPs are a novel protein family that acts through timing and localisation of the activity of penicillin-binding proteins and autolysins, thus controlling important steps during the initiation and the completion of sporulation. The formation of septa is initiated by the formation of a ring of the tubulin-like protein FtsZ (the Z-ring), functioning as a scaffold for the construction of septa. Subsequently, other cell division proteins are recruited to the Z-ring, forming the divisome. In S. coelicolor, the cell division proteins FtsE and FtsX participate during autolytic spore separation, and most likely function by re-importing peptidoglycan subunits for recycling. The cytoskeletal protein MreB is involved in cell shape determination and chromosome segregation in many rod-shaped bacteria. In S. coelicolor, the actin-like proteins MreB and Mbl are not essential for vegetative growth but exert their function in the formation of environmentally stable spores, thereby primarily influencing the assembly of the spore wall. Show less
This thesis deals with various aspects of intracellular behavior of antitumor-active dinuclear platinum complexes, i.e. their anticancer activity, cellular distribution and nephrotoxicity. A major... Show moreThis thesis deals with various aspects of intracellular behavior of antitumor-active dinuclear platinum complexes, i.e. their anticancer activity, cellular distribution and nephrotoxicity. A major part of this thesis is focused on the investigation of cellular processing of dinuclear platinum anticancer drugs using fluorescence microscopy. Besides, design of new dinuclear platinum anticancer compounds is described, the relationships between the structure and nephrotoxicity of dinuclear complexes are discussed, and application of dinuclear platinum complexes in drug targeting is evaluated. Show less
