Spatial and temporal dynamics of polymer chains play critical roles in their rheological properties, which have a significant influence on polymer processing and fabrication of polymer-based (nano) materials. Many theoretical and experimental studies have aimed at understanding polymer dynamics at the molecular level that give rise to its bulk phase properties. While much progress has been made in the field over the past ~60 years, many aspects of polymers are still not understood, especially in complicated systems such as entangled fluids and polymers of different topologies. In addition, the physical properties of biological macromolecules, i.e. DNA, are expected to affect the spatial organization of chromosome in a cell, which has the potential impact on a broad epigenetics research. Here, we propose new methods for simultaneous visualization of diffusive motion and conformational dynamics of individual polymer chains, two most important factors that characterize polymer dynamics, based on a new single-molecule tracking technique, cumulative-area (CA) tracking method. We demonstrate the applicability of the CA tracking to the quantitative characterization of the motion and relaxation of individual topological polymer molecules under entangled conditions, which is possible only by using the newly-developed CA tracking, using fluorescently-labeled linear and cyclic dsDNA as model systems. We further extend the technique to multi-color CA tracking that allows for the direct visualization and characterization of motion and conformation of interacting molecules. We also develop a new imaging method based on recently developed 3D super-resolution fluorescence microscopy technique, which allows direct visualization of nanoscale motion and conformation of the single molecules that is not possible by any other methods. Using these techniques, we investigate spatial and temporal dynamics of polymers at the single-molecule level, with special emphasis on the effect of topological forms of the molecules and the confined geometry on their spatiotemporal dynamics. Our results demonstrate that the new methods developed in this thesis provide an experimental platform to address key questions in the entangled topological polymer dynamics. The research will provide a platform for developing new polymer-based materials and open the possibility of studying spatial organization of DNA in a confined geometry from physics point of view.

This thesis focuses on autofluorescence of flavins in gram-negative bacteria Cupriavidus necator H16 and its mutant strain PHB-4. The main methods used were fluorescence microscopy and flow cytometry. To confirm the presence of flavins, excitation and emission spectra of the bacterial suspension were measured, which were compared with flavin standards. In the part of testing cells without stress response, the autofluorescence of bacteria in PBS buffer and cell suspensions stained with fluorescence probe BODIPY 493/503 was measured. The ratio of short fluorescence lifetime to long autofluorescence lifetime, and its dependence on fluorescence probe was compared with previous conditions. Autofluorescence of the supernatant was measured; it was found that the relative amplitude of long lifetime was multiple times higher than in the cell. In the part devoted to the stress response, this thesis was focused on the amount of dissolved oxygen in the production medium and the effect on bacterial autofluorescence. Then differently concentrated hydrogen peroxide was used, the best results were obtained from the concentration of 100 mM in media. For comparison a combination of hydrogen peroxide with ferro-ammonium sulphate was used, but there was no big difference. Sodium azide and antimycin A were selected as substances that directly influence on bacterial respiratory chain. Both compounds affected change in the ratio of the relative amplitudes, but the distribution of these lifetimes and the autofluorescence change over time was affected only by sodium azide.

Práca je zameraná na navrhnutie vhodnej metódy analýzy smerovosti neuritov. Využité boli snímky neurónov z fluorescenčnej mikroskopie. Pred samotnou segmentáciou bolo potrebné snímky predspracovať, pričom sa postupne využila úprava kontrastu, ostrenie a adaptívna filtrácia pomocou Weinerovského filtru. Jednotlivé návrhy metód segmentácie pozostávali z prostého prahovania, narastaním oblastí a využitím morfologických operácií. Následná analýza smerovosti využívala smer gradientov v obraze. Navrhnutá metóda bola využitá aj ako klasifikátor, ktorý dokázal rozdeliť jednotlivé snímky do skupín podľa smeru rastu.

The introductory part of this thesis is focused on the theoretical analysis of solved problems as examining the toxicity of naphthoquinones plumbagine and juglone, especially with regard to their allelopatic action. The next section is focuses on the plant stress, caused by the action of stress factors leading to their death. There is an experimental protocol and the possibilities of determination the effect of naphthoquinones on the plant model. Experimental section discusses changing growth parameters of the plant samples in different concentrations of the naphthoquinone. There is examined cell viability and changes in the synthesis of the secondary metabolites. Data obtained by using spectrophotometric and microscopic analysis are evaluated with STATISTICA software and statistical significance are plotted.

This work deals with the study of fluorescence lifetime and spectral changes of nanoparticles in cell biology. It describes the principle of fluorescence, fluorescence microscopy and laser confocal microscope Leica TCS SP8. The classic FLIM method, the Lambda Square mapping and the division of nanoparticles are introduced there. In the practical part, the created program for the evaluation of fluorescence lifetime and spectral changes is described. The program can show two-dimensional lambda maps, the fluorescence lifetime and spectral shift in the space area. In the final part of the thesis, an experiment with rhodamine nanoparticles is carried out and it is evaluated using the created program and then discussed.

This thesis focuses on fluorescent analysis of viability and PHA content in bacterial cultures, the main methods of investigation were flow cytometry and fluorescent microscopy. In order to determine viability of C. necator H16, several viability probes were tested, nevertheless, only BacLightTM kit and propidium iodide can be used to estimate portion of viable and live bacterial cell in samples. Further, Acridine orange was used to monitor physiological state of bacterial culture and two hydrophobic probes, Nile Red and BODIPY 493/503, were used to investigate PHA content in bacterial cells. Application of BODIPY 493/503 seems to be promising since this probe does not require permeabilization of bacteria cells and it can be used along with propidium iodide. Furthermore, several fluorophores were tested in the microscopic part. In was found that concentrations used in cytometric analyses were too high for microscopic use. Emission from the SYTO9 fluorophore is seen mainly in the green channel but because of the high concentration some emission was visible in the red channel. Cells stained with BODIPY 493/503 had very high fluorescence intensities when the stain concentration was 10 . At the same time, negative amplitudes of fluorescence were measured in both strains of C. necator, but in case of C. necator H16 that amplitude was much more pronounced. In this strain surprising high concentration of BODIPY stain was observed on the surface of PHB granules. Anisotropy of the fluorophore was nearing 0 which is very surprising.

This diploma thesis deals with the possibilities of multiparametric fluorescence spectroscopy, since the main objective of this experiment was to evaluate the possibilities of multiparametric measurements in the fluorescence spectroscopy laboratory. A suitable fluorescence probe was proposed for this type of experiment that shows high sensitivity for pH changes in the environment, SNARF-4F AM, based on a literature research. The fluorophore was dissolved in solutions of different pH and this system was examined using a time-resolved spectrofluorimeter. The method named TRES (time-resolved emission spectra) was used to obtain the emission spectra of the probe and to find the emission maximum. Fluorescence intensity decay measurements as a function of wavelengths allowed to create deconvolution of the emission spectra, which provided information about the fluorescent lifetime and the relative representation of the states of probes in the solution. Later, the probe was dissolved in solutions of different density and pH - this system served for anisotropic measurements, during which the individual correlation-rotational times of the fluorophore were obtained. The obtained results were then used as the basis for multiparametric analysis, which was performed by using a fluorescence correlation microscope and a spectrograph. This combination allows to measure the necessary fluorescence parameters in one step. A standard operating procedure was created for the spectrograph’s control. On the basis of the obtained information the suitability, accuracy and sensitivity of the multiparametric analysis were qualified.

En biologie développementale, l'étude d'organismes modèles vise à comprendreles mécanismes génétiques responsables de la morphogenèse chez le vivant. Lamicroscopie confocale à fluorescence permet aujourd'hui d'observer in vivo àl'échelle de la cellule et avec une haute fréquence temporelle le développementd'organismes. Les séquences d'images 3D+t ainsi obtenues nécessitent d'avoirdes outils de traitement d'images adaptés.Dans cette thèse, nous construisons des outils dédiés à l'étude dudéveloppement de deux organismes, l'embryon de l'ascidie Phallusiamammillata et le bouton floral d'Arabidopsis thaliana.Nous développons d'abord une méthode de comparaison de segmentationsadaptée aux images de tissus épithéliaux d'organismes en développement.Nous nous appuyons sur cet outil pour valider notre seconde contribution quiporte sur la mise en place d'un outil de détection et de reconstruction demembranes cellulaires conçu afin de procéder à la segmentation de cellulesdans les images d'ascidies et d'arabidopsis.Nous utilisons ensuite l'outil de segmentation de membranes précédemmentintroduit pour construire une stratégie de recalage spatial inter-individusappliquée aux embryons d'ascidies. Enfin, nous élaborons une stratégie derecalage spatio-temporel inter-individus appliquée à des séquences d'images 3Dde méristèmes floraux / In developmental biology, the study of model organisms aims for theunderstanding of genetic mechanisms responsible of morphogenesis. Today,fluorescent confocal microscopy is a means for in vivo imaging of developingorganisms at cell level with a high spatio-temporal resolution. To handle such3D+t image sequences, adapted computer-assisted methods are highlydesirable.In this thesis, we build dedicated tools for the study of two developingorganisms, the ascidian Phallusia mammillata's embryo and the Arabidopsisthaliana's floral meristem.We first develop a method for segmentation comparison adapted to developingorganism epithelial tissue images. This tool is then used to validate our secondcontribution that is about the development of a cell membranes detection andreconstruction tool for cell shape segmentation process applied to ascidian andarabidopsis images.We then use the previously introduced membrane detection tool to build aninter-individual spatial registration strategy applied to ascidian embryo images.Finally, we develop an inter-individual spatio-temporal registration strategyapplied to 3D image sequences of arabidopsis floral meristems

Vesicles are a type of biological or biomimetic particle consisting of one or more often spherical bilayers made up of amphipathic molecules, creating a closed system. They can function as an encapsulating device, holding hydrophilic molecules on the inside of the bilayer membrane(s) or hydrophobic molecules in the non-polar interstitial space in the middle of the bilayers. Because of this capacity to carry molecules, vesicles are a premier system for drug delivery and even theranostics in vivo. A peptide-based approach to release of encapsulated molecules has previously been developed but since drug delivery vesicles are in the size range of nanometers, the mechanisms have not been visualized. This project aims to produce giant unilamellar vesicles as a model system used to visualize membrane interactions vital to the understanding and further development of smaller vesicle-based systems for drug delivery. Giant unilamellar vesicles were produced successfully and a preparation protocol was established. Additionally, some membrane interactions were investigated using fluorescence microscopy.

The nonapoptotic form of regulated cell death known as ferroptosis is an attractive target for combating numerous diseases. Ferroptosis is an iron-dependent death of cells by lipid peroxidation. Pharmacological inhibition of anti-ferroptotic pathways is a promising therapeutic avenue for treatment of cancer, and death by ferroptosis has been implicated in numerous neurodegenerative and ischemia-reperfusion-driven diseases. Therefore, demystifying the dynamics of lipid peroxidation in this cell death process opens a window to understanding disease processes and how to treat them. This dissertation makes use of ferroptosis-modulating compounds as chemical probes to elucidate the roles of different subcellular membranes in ferroptotic lipid peroxidation. Chapters two and three explore the structure-activity-distribution relationship of fatty acids and the ferroptosis inducer FINO2, respectively, and together demonstrate the endoplasmic reticulum as a driver of lipid peroxidation in ferroptosis. Chapter two makes use of stimulated Raman scattering imaging, while chapter three uses confocal fluorescence imaging. Chapter four shifts gears to focus on development of FINO2 as a drug lead, performing structure activity relationship analysis to increase the potency and pharmacological properties of the analogs. Altogether, this work answers questions about how cells die by ferroptosis, and provides footwork for how we can better modulate ferroptosis against cancer and other illnesses.