The thesis comprehensively investigates 2 pcs of ferrous and 14 pcs of bronze Celtic artefacts from a region of Zdejciny by Beroun. The study by means of ore microscopy, x-ray fluorescent microspectrometry, chemical microanalysis, Raman microspectrometry and x-ray powder diffractive analysis has showed that the bronze artefacts are made mainly of bronze. The phases present in the studied items correspond to fields and + of the Cu-Sn phase diagram. Content of Sn in bronze ranges between 4 and 33 wt.%. For the bronze selected items there was also Vickers microhardness measured. There were two artefacts where areas with a significant lead enrichment were found. Corrosive products were also deeply analysed and identified.

This thesis concerns my theoretical calculations and simulations in comparison with experimental measurements acquired by means of surface science techniques on bare surfaces and molecules adsorbed on surfaces. In the beginning of the thesis I briefly describe the density functional theory (DFT) method, which is used for calculations of geometric and electronic structure of surfaces and absorbed molecules. It is followed by a quick overview of the scanning probe microscopy (SPM) and X-ray spectroscopy techniques that provided experimental context for my calculations. In the later part of my thesis I introduce publications on which I participated. In these works, direct results of my DFT calculations or simulations based on DFT outputs helped to reveal geometric and electronic structure of acetophenone adsorbed on Si(111) surface, on-surface oligomerized organic molecules and boron atoms used for substitational doping of graphene. The simulations also enable us to probe a laser initiated CO hydrogenation in real time. A big part of my work was the development of some simulation methods for SPM imaging of molecules on surfaces with flexible tip apexes. These simulation methods helped to create a comprehensive overview of SPM techniques performed with flexible tip apexes. 1

Introduction: Relation of diabetes mellitus (DM) to the diabetic keratopathy and various stages of corneal nerve fiber damage has been well accepted. A possible association between changes in the cornea of diabetic patients and diabetic retinopathy (DR), DM duration, and age at the time of DM diagnosis were evaluated. Neuropathies are among the most common long-term complications of diabetes mellitus. Good glycemic control is essential in prevention of this complication. DM patients with similar mean glucose levels or glycated hemoglobin (HbA1c) levels often exhibit differences in evaluation of diabetic complications. One reason for these differences may be the differences in glucose variability. DM patients with similar mean glucose levels or HbA1c levels often exhibit differences in glucose variability Hypothesis: Diabetes mellitus damages the subbasal nerve fibers of the corneal and affects the density of epithelial, endothelial and stromal cells. Corneal changes in patients with DM are dependent on the degree of diabetic retinopathy (DR), age at diagnosis, duration of DM, and compensation parameters. Purpose: To compare changes in cell density in individual layers of cornea and status of subbasal nerve fibers in patients with type 1 DM (DM 1) and in healthy subjects. To evaluate the dependence...

In the past decades, numerous single-molecule techniques have been developed to investigate individual bio-molecules and cellular machines. While a lot is known about the structure, localization, and interaction partners of such molecules, much less is known about their mechanical properties. To investigate the weak, non-covalent interactions that give rise to the mechanics of and between proteins, an instrument capable of resolving sub-nanometer displacements and piconewton forces is necessary. One of the most prominent biophysical tool with such capabilities is an optical tweezers. Optical tweezers is a non-invasive all-optical technique in which typically a dielectric microsphere is held by a tightly focused laser beam. This microsphere acts like a microscopic, three-dimensional spring and is used as a handle to study the biological molecule of interest. By interferometric detection methods, the resolution of optical tweezers can be in the picometer range on millisecond time scales. However, on a time scale of seconds—at which many biological reactions take place—instrumental noise such as thermal drift often limits the resolution to a few nanometers. Such a resolution is insufficient to resolve, for example, the ångstrom-level, stepwise translocation of DNA-binding enzymes corresponding to distances between single basepairs of their substrate. To reduce drift and noise, differential measurements, feedback-based drift stabilization techniques, and ‘levitated’ experiments have been developed. Such methods have the drawback of complicated and expensive experimental equipment often coupled to a reduced throughput of experiments due to a complex and serial assembly of the molecular components of the experiments. We developed a high-resolution optical tweezers apparatus capable of resolving distances on the ångstrom-level over a time range of milliseconds to 10s of seconds in surface-coupled assays. Surface-coupled assays allow for a higher throughput because the molecular components are assembled in a parallel fashion on many probes. The high resolution was a collective result of a number of simple, easy-to-implement, and cost-efficient noise reduction solutions. In particular, we reduced thermal drift by implementing a temperature feedback system with millikelvin precision—a convenient solution for biological experiments since it minimizes drift in addition to enabling the control and stabilization of the experiment’s temperature. Furthermore, we found that expanding the laser beam to a size smaller than the objective’s exit pupil optimized the amount of laser power utilized in generating the trapping forces. With lower powers, biological samples are less susceptible to photo-damage or, vice versa, with the same laser power, higher trapping forces can be achieved. With motorized and automated procedures, our instrument is optimized for high-resolution, high-throughput surface-coupled experiments probing the mechanics of individual biomolecules. In the future, the combination of this setup with single-molecule fluorescence, super-resolution microscopy or torque detection will open up new possibilities for investigating the nanomechanics of biomolecules.

Mit zunehmendem Alter zeigt das Verbundverhalten von Multifilamentgarnen aus alkaliresistentem Glas (AR-Glas) oder Kohlenstoff in Abhängigkeit von der Zusammensetzung der zementgebundenen Matrix eine sehr unterschiedliche Entwicklung. Während bei AR-Glas teilweise drastische Verluste des Leistungsvermögens zu verzeichnen sind, treten diese bei Kohlefasern nicht auf. Zur Untersuchung der Phänomene wurden beidseitige Garnauszugversuche durchgeführt und die Interphase zwischen Filamenten und Matrix im Rasterelektronenmikroskop (ESEM) untersucht. Die unterschiedlichen mechanischen Eigenschaften stehen in Zusammenhang mit verschieden ausgeprägten Mikrostrukturen der Interphasen. Welche Ursachen die unterschiedliche morphologische Entwicklung der Interphasen hat, ist Gegenstand aktueller Arbeiten.

Both testate amoebae and fungi are common inhabitants of coniferous litter. Their interactions in this environment were rarely studied, although they reach high biodiversity and can play a significant role in nutrient cycling in this environment. In this study, a cultivation of litter needles in the damp chambers was used to investigate interactions between fungi and testate amoebae. Observation of spruce litter needles in environmental scanning electron microscope was used to better characterize testate amoebae communities directly on the needles. Additionally, two experiments changing the biotic conditions in the microcosm were used to follow a principle of the interactions. Three species of testate amoebae from litter needles were able to colonize the filter paper on the bottom of the damp chambers. Occurrence of Phryganella acropodia and Assulina muscorum on the filter paper was significantly fuelled by the presence of mycelium. Assulina muscorum was associated with the fungal spores and Arcella discoides was attracted by sporulating colonies of Cladosporium spp. in the damp chambers. By contrast, no association of putatively mycophagous Phryganella acropodia with fungal spores was observed. Arcella discoides was attracted both by live and death mycelium in additional experiment. Interestingly, the...

Pavel Jůda - Abstract The cell nucleus is a complex cellular organelle. The nucleus and nuclear processes are organized into functionally and morphologically separated nuclear subcompartments. This thesis is particularly concerned with the three following nuclear subcompartments: sites of DNA replication, Polycomb bodies and nuclear inclusions constituted of inosine monophosphate dehydrogenase 2 (IMPDH2). First, we examined the relationship between MCM proteins and DNA replication. Using immunofluorescent labeling of cells extracted prior fixation and applying cross-correlation function analysis, we showed that MCM proteins are present at the sites of active DNA synthesis. Our results contributed to the solving of the first part of so-called MCM paradox. Second, we studied the structural basis of the Polycomb bodies. Based on fluorescence microscopy studies, Polycomb bodies have been considered to be the nuclear subcompartments formed by the accumulation of Polycomb proteins in the interchromatin compartment. In our work, using correlative light electron microscopy and experimental changes in macromolecular crowding, we clearly showed that a Polycomb body is a chromosomal domain formed by an accumulation of heterochromatin structures, rather than a typical nucleoplasmic body. Third, we were interested in...

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The methods of analysis with a focused ion beam, commonly termed as nuclear microscopy, include quantitative physical processes like PIXE and RBS. The element concentrations in a sample can be quantitatively mapped with a sub-micron spatial resolution and a sub-ppm sensitivity. Its fully quantitative and non-destructive nature makes it particularly suitable for analysing biological samples. The applications in biomedical research are manifold. The iron overload hypothesis in Parkinson\\\''s disease is investigated by a differential analysis of human substantia nigra. The trace element content is quantified in neuromelanin, in microglia cells, and in extraneuronal environment. A comparison of six Parkinsonian cases with six control cases revealed no significant elevation in iron level bound to neuromelanin. In fact, a decrease in the Fe/S ratio of Parkinsonian neuromelanin was measured, suggesting a modification in its iron binding properties. Drosophila melanogaster, or the fruit fly, is a widely used model organism in neurobiological experiments. The electrolyte elements are quantified in various organs associated with the olfactory signalling, namely the brain, the antenna and its sensilla hairs, the mouth parts, and the compound eye. The determination of spatially resolved element concentrations is useful in preparing the organ specific Ringer\\\''s solution, an artificial lymph that is used in disruptive neurobiological experiments. The role of trace elements in the progression of atherosclerosis is examined in a pilot study. A differential quantification of the element content in an induced murine atherosclerotic lesion reveals elevated S and Ca levels in the artery wall adjacent to the lesion and an increase in iron in the lesion. The 3D quantitative distribution of elements is reconstructed by means of stacking the 2D quantitative maps of consecutive sections of an artery. The feasibility of generating a quantitative elemental rodent brain atlas by Large Area Mapping is investigated by measuring at high beam currents. A whole coronal section of the rat brain was measured in segments in 14 h. Individual quantitative maps of the segments are pieced together to reconstruct a high-definition element distribution map of the whole section with a subcellular spatial resolution. The use of immunohistochemical staining enhanced with single elements helps in determining the cell specific element content. Its concurrent use with Large Area Mapping can give cellular element distribution maps.

New concepts in assembly technology boost our daily life in an unknown way. High end semiconductor industry today deals with functional structures down to a few nanometers. ITRS roadmap predicts an ongoing decrease of the “DRAM half pitch” over the next decade. Packaging of course is not intended to realize pitches at the nanometer scale, but has to face the challenges of integrating such semiconductor devices with smallest pitch and high pin counts into systems. Advanced techniques of nondestructive evaluation (NDE) with resolutions in volume better than 1 micrometer vixen size are urgently needed for the safety and reliability of electronic systems, especially those that are used in long living applications. The development speed of integrated circuits is still very high and is not expected to decrease in the next future. The integration density of microelectronic devices is increasing, the dimensions become smaller and the number of I/O's is getting higher. The development of new types of packages must be done with respect to reliability issues. Potential damage sources must be identified and finally avoided in the new packages. In power electronics production the condition monitoring receives a lot of interest to avoid electrical shortcuts, dead solder joints and interface crac king. It is also desired to detect and characterize very small defects like transportation phenomenon or Kirkendall voids. For this purpose, imaging technologies with resolutions in the sub-micron range are required.