Title: Imaging of fluorescently labelled mitochondria using Biplane FPALM microscopy Author: Bc. Marek Dostál Department: Fyzikální ústav UK Supervisor: prof. RNDr. Jaromír Plášek, CSc. Consultants: RNDr. Petr Ježek, DrSc. Mgr. Hana Engstová, Ph.D. Abstract: In this thesis the results are presented of a search for suitable PCFP markers for a visualization inner and outer mitochondrial membranes by unique biplane FPALM microscope. We participated in debugging the measuring software for the purpose of 3D vizualization mitochondrial nets and determine their parameters. For this purpose were developed two methods. The first one can be applied to determine the parametrs of outer membranes and the second one is applicable to determine parametrs of inner membranes. On smaller statistical file we managed to confirm dependence quality of mitochondrial net on the cell culture conditions. Under stress conditions e.g. hypoxia the quality of mitochondrial net is changed. Keywords: mitochondria, Biplane FPALM, GFP, 3D visualization.

Despite the significant progress in medicine, infectious diseases are life-threatening thanks to an increasing number of multiresistant strains of microorganisms and late detection of pathological agents. An opportunistic fungus Aspergillus fumigatus cause respiratory system diseases called aspergillosis. The invasive pulmonary aspergillosis affects immunocompromised patients after inhalation of ubiquitous conidia of A. fumigatus and results in 450,000 deaths per year. The biofilm formation in the infected tissue protects A. fumigatus against antimicrobial drugs. Late therapy may not be effective. Infection of immunocompromised patients and biofilm formation is characteristic also for gram negative bacteria Pseudomonas aeruginosa, which is due to the production of many factors of virulence and multiresistance a dreaded opportunistic pathogen. Scanning electron microscopy (SEM) provides detail information about morphology of microorganisms with the resolution in range of tens of nanometers that allows to observe microorganisms in the infected tissue and its pathological changes. Mass spectrometry allows to detect infection and its course based on identification of characteristic microbial molecules. The aim of this study was to optimize sample preparation of tissues infected with A. fumigatus or P....

In this work phase transformations in metastable β (primarily Ti-15Mo) alloys were studied utilizing electrical resistance, dilatometry, transmission electron microscopy and X-ray and neutron diffraction. The materials Ti-15Mo, Ti-6.8Mo-4.5Fe-1.5Al (LCB), Ti-5Al-5V-5Mo-3Cr (Ti-5553), Ti-29Nb-1Fe-0.5Si (TNFS), Ti-15Mo-3Nb-3Al-0.2Si (Timetal 21S) and Ti-13Cr-1Fe-3Al (TCFA) (in wt. %) - were subjected to a solution treatment at a temperature above β transus and quenched into water. In this condition, the microstructure of the investigated materials consists of β matrix and ω particles. Samples quenched from important temperatures determined from in-situ electrical resistance and dilatometry measurements were studied by post-mortem TEM. In-situ X-ray and neutron diffraction provided direct observations of microstructure of Ti-15Mo alloy during linear heating and confirmed statements based on results of indirect methods, such as: the decrease of volume fraction of ω phase during heating at low temperatures (up to 250 ◦ C), complete dissolution of ω phase at 560 ◦ C and precipitation of α phase without ω particles serving as its direct precursors. X-ray diffraction experiment allowed to determine relative evolution of the size of ω particles while phase fraction evolution was derived from neutron diffraction. The...

Title: Raman microspectroscopy of living cells and biological tissues Author: Šárka Moudříková Department / Institute: Institute of Physics of Charles University Supervisor of the doctoral thesis: doc. RNDr. Peter Mojzeš, CSc., Institute of Physics of Charles University Abstract: Raman microscopy combines Raman spectroscopy with optical confocal microscopy and thus provides information on chemical composition of a sample with a µm3 resolution. In this thesis, Raman microscopy has been used to study microalgae-unicellular photosynthetic organisms that are greatly relevant for the Earth's environment as well as for biotechnological applications. Raman microscopy of photosynthetic organisms struggles with a highly intensive background of the spectra, which is formed by fluorescence of cellular photosynthetic apparatus. In this thesis, we have developed a fast and reliable photobleaching method that suppresses the unwanted background; this method has enabled us to study intracellular distribution of algal biomolecules such as proteins, starch, lipids and polyphosphate. We have investigated an evolution of these structures during a cell cycle of a model microalga Desmodesmus quadricauda. Next, we have developed a method for quantitative analysis of polyphosphate in a cellular culture of a microalga Chlorella...

Zuzana Kubínová: Study of Quantitative Parameters of Norway Spruce Needle Structure under the Effect of Elevated CO2 Concentration and Different Irradiance, Doctoral Thesis, Prague 2019 Abstract Atmospheric concentration of CO2 is increasing, while its influence on plants is still not fully elucidated. Norway spruce (Picea abies L. Karst.) is an abundant conifer tree in European temperate and boreal forests, which behave as carbon sink in the global carbon cycle. The physiological response to elevated CO2 concentration may be interconnected with changes in leaf anatomy and morphology. Needle structure is also determined by other factors in addition to CO2 concentration, irradiance being the most important one. Thus, effect of irradiance was also included in our studies. The effects of elevated CO2 concentration and irradiance on Norway spruce needle structure were studied using new applications of well-established quantitative methods and novel methods enabling effective and unbiased analysis of needle structural traits. The General Procrustes analysis showed to be effective for needle shape on cross section comparison and the disector method proved to be suitable for chloroplast number estimates. The influence of elevated CO2 concentration and different irradiance on needle structure was studied at two...

The microstructure, diffusion and phase transformations in aluminum-steel clad sheet were studied within the thesis by means of light optical microscopy, electron microscopy, resistometry and positron annihilation spectroscopy. Results of experimental methods were supplemented by finite element method employed for evaluation of electrical resistivity and simulation of diffusion between steel and aluminum. Several annealing experiments were carried out. The effective interdiffusion coefficient was evaluated by Boltzmann-Matano method from measured concentration profiles through the interface. Formation of an interfacial intermetallic phase was studied and orthorhombic phases Al13Fe4 and Al5Fe2 were identified present in the layer. Surprising results were obtained from the in-situ annealing in TEM (SEM) which go against the results found in a recent literature - the interfacial layer grows towards steel layer.

Natural killer (NK) cells represent indispensable part of the innate immunity as they are capable of promptly identifying virally infected or tumor cells and participating in the regulation of adaptive immune responses. These functions are ensured by the interplay between NK receptors, creating a complex regulatory system. Solving the receptors' structure may contribute to an overall understanding of NK cell biology. Presented thesis describes an elucidation of the structure of the inhibitory C-type lectin-like receptor (CTLR) Nkrp1b with an emphasis toward structural features (stalk, loop and oligomerization state) which might affect conformation or interactions of this receptor. The interaction of Nkrp1b with its ligand, Clr-b protein, is immunologically significant as it regulates NK cells' activity independently and monitors changes that are not visible to cytotoxic T lymphocytes. To study individual structural aspects of Nkrp1b, two protein variants were recombinantly prepared in bacterial expression system: entire ectodomain and ligand-binding domain lacking the stalk. Using a range of mass spectrometric techniques in combination with homology modeling and molecular dynamics, we proposed the Nkrp1b structure including its monomeric and dimeric arrangements. In addition, the oligomerization...

Magister Scientiae Dentium - MSc(Dent) / Die doel van hierdie in vitro studie was om mikrolekkasies te bepaal en te vergelyk in klas V kawiteite wat met verskillende amalgaambindingsisteme behandel is. Klas V kawiteitsvoorbereidings is vir elk van die drie sisteme in 16 kariesvrye menslike premolare voorberei sodat die okklusale rande in glasuur en die gingivale rande in dentien geëindig het. 'n Hoë koper sferiese amalgaam (Lojic) is onderskeidelik, volgens die vervaardigers se voorskrifte, met Amalgambond-Plus, Fuji-Plus en Optibond-Solo as bindmiddels in die kawiteite geplaas. Die tande is blootgestel aan 500 termiese siklusse tussen 5°C en 55°C in 'n basiese fuchsien oplossing as kleurstof, waarna hulle in hars ingebed, gesny en onder 'n stereomikroskoop by 100 x vergroting vir mikrolekkasie geëvalueer is. Diepte van kleurstofpenetrasie tussen die tandstruktuur en die vulsel is deur twee gekalibreerde operateurs vir elke groep bepaal. Statistiese analise van die data, deur middel van 'n Kruskal-Wallis analise (p < 0,05), het getoon dat Amalgambond-Plus betekenisvol meer mikrolekkasie toon as beide Optibond-Solo en Fuji-Plus. Optibond-Solo en Fuji-Plus het nie betekenisvol van mekaar verskil nie. Geen bindmiddel kon mikrolekkasie volledig elimineer nie. Die doel van die in vivo gedeelte van die studie was om mikrolekkasies te bepaal en te vergelyk in klas V kawiteite by vitale en non-vitale tande van 'n primaat wat met die Optibond-Solo amalgaambindingsisteem behandel was. Endodonsie was gedoen op sewe tande in die tweede kwadrant van 'n volwasse bobbejaan (papio Ursinus ursinus), ten einde non-vitale tande te bekom waarin die pulpale druk geëlimineer is. As vitale kontroles is dieselfde tande in die teenoorstaande kwadrant gebruik. Twaalf bukkale klas V kawiteite is in vitale-, sowel as non-vitale tande voorberei. 'n Hoë koper sferiese amalgaam (Lojic) is met Optibond-Solo as bindingsagens volgens die vervaardigers se voorskrifte in alle kawiteite geplaas. Na 90 dae is die tande geoës, vir 24 uur by 37°C in 'n basiese fuchsien oplossing geplaas, in hars ingebed, gesny en vir mikrolekkasie ondersoek onder 'n optiese mikroskoop by 100 x vergroting. Die gemiddelde lesings is vir elke groep verkry en met mekaar vergelyk. Die vitale groep het betekenisvol meer mikrolekkasie getoon as die non-vitale groep waar pulpale druk uitgeskakel was. Skandeer elektron mikroskopie (SEM) Ten einde die morfologie van die binding te illustreer, is een tand uit elke groep vir 48 uur in 10% HCI gedemineraliseer, waarna hulle vir 'n verdere 48 uur in 3% NaOCI gedeproteïniseer is. 'n Tweede groep IS vertikaal deur die kawiteite gefraktuur. Die voorbereide monsters is deur middel van 'n skandeer elektron mikroskoop (lO KV) by verskillende vergrotings ondersoek. Die vorming van 'n goed ontwikkelde hibriedlaag, met inkorporering van die bindingshars in die amalgaam, kon waargeneem word.

Common wheat and spelled wheat are one of the most important agricultural crops. Their use is mainly in the food industry and also in the feed industry. There is a strong interest in the differentiation of the common wheat from the spelled wheat in the last tens of years. This trend is especially in the food industry because of the adulteration of the spelled flour. This dissertation was focused on the differentiation of the common wheat from the spelled wheat by using the microscopic methods and on the detection of variability in the gene for gamma wheat gliadin by the molecular markers (RFLP method). Through the molecular analysis of the 15 tested varieties of the spelled wheat and one variety of the common wheat were obtained the results which make us able to detect the proportion of the spelled wheat in the common wheat.

Src kinase is a key element in many signaling pathways affecting cellular processes such as differentiation, proliferation, motility, or migration. Deregulation of its activity is associated with the promotion of cancer. Therefore, understanding its cellular function is vital. Src activity directly correlates with its structure; when Src is active, it adopts opened conformation, when inactive, it is in closed conformation stabilized by intramolecular interactions. Detection of the conformation can be used to analyze Src activity. In this thesis, conformation-sensitive FRET-based Src biosensor was improved using mNeonGreen as a new acceptor fluorophore in the existing design and the properties of the new biosensor were compared with the original Src biosensor. The new biosensor is able to detect changes in Src conformation and can be stably expressed in cells. Src activity in focal adhesion was analyzed and higher Src activity in these structures was confirmed. Although the new biosensor did not exhibit significantly better sensitivity to Src conformational changes, it still proved to be a useful tool to study Src activity, and mNeonGreens higher brightness makes it more suitable for microscopic experiments. Key words: Src, FRET, biosensor, live-cell imaging, mNeonGreen