This thesis deals with preparation of geopolymeric binders for inhibition of toxic elements, especially arsenic. It studies influence of content progress of different phases in prepared binders on leachability of As during the setting. The aim of this study is to determine the type of inhibition or the cause of leachability of As in prepared binders respectively. Fly ash from high-temperature coal combustion, slag, cement kiln dust and sodium hydroxide solution were used for experimental works. FT-IR, TG-DTA, XRD analyses after different hydration times were used. The leachates were analyzed by ICP-MS. Calorimetric measurements of tested mixtures fresh pastes were implemented to learn about setting process. Hardened binder specimens were tested for bending and compressive strength.

The theoretical part of this thesis deals with cellulolytic enzymes, their microbial producers, the possibilities of using such enzymes in the industry and how can be enzymes - not only cellulolytic - immobilized. Experimental part examines the preparations created by immobilizing various amounts of the commercially used cellulolytic complex Cellulast 1.5L onto various synthetic carriers made of polyethylene terephthalate - commercially used Sorsilen, PET carrier and glutaraldehyde-treated PET carrier. Enzyme activity of these preparations was determined by Somogyi - Nelson method by spectrophotometry. For the highest activity immobilized preparation was determined the temperature- and the pH-optimum. The difference in effects change between the free and immobilized enzyme by measuring viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.

In this thesis the use of waste materials for the microbial production of important metabolites is reported. The first part is focused on the use of waste paper (a lignocellulosic material) as a non-traditional source for the production of bioethanol. The second part is focused on the immobilization of cellulolytic enzymes, which are used for the hydrolysis of lignocellulosic materials. First, the waste paper (cardboard) was pre-treated using a blender and a vibratory mill. The pre-treated cardboard was used for the production of ethanol by the method of simultaneous saccharification and fermentation. This method was optimized with free cells of Saccharomyces cerevisiae. Then strains suitable for the immobilization were selected. Strains of S. cerevisiae and Pichia kudriavzevii were immobilized by encapsulation into the polyvinyl alcohol carrier and tested again for the ethanol production by simultaneous saccharification and fermentation. In the second part of the work a carrier from waste polyethylene terephthalate bottles was prepared and used for the immobilization of the cellulolytic complex. The basic characteristics were determined, such as optimal pH and optimal temperature, storage, operational and thermal stability, enzyme kinetics and the mode of action of the enzyme. Compared to two other commercial carriers this carrier showed to be suitable for the immobilization of the cellulolytic complex.

11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate Mgr. Jana Kubačková Supervisor Assoc. Prof. Jarmila Zbytovská, Mgr., Dr. rer. nat. Co-supervisor PharmDr. Ondřej Holas, PhD. Title of Doctoral Thesis Preparation of pharmaceutical formulations based on polymeric and lipid carriers Nanomedicine allows application of nanoscaled drug delivery carriers to achieve a therapy that can be tailored in terms of e.g. controlled release, site-specific delivery and protection of an active substance. From multiple nanoplatforms available for drug delivery, advantage was taken of biocompatible and biodegradable polymers and lipids to enable targeted intracellular delivery, delivery of a poorly water-soluble drug and delivery of a sensitive macromolecule. In the study with biodegradable polymeric nanomaterial we worked with experimental poly(lactic-co-glycolic acid) (PLGA) polymers. The formulations were optimised for targeting to phagocytic macrophages - of size up to 300 nm and negative surface charge. For this purpose, two linear and one branched PLGA were screened in combination with one of four surfactants in low concentrations (0.1-1%). These PLGA polymers were formulated into nanoparticles and loaded with a hydrophilic fluorescent dye Rhodamine B...

For everyone hospitalization represents a change accompanied by various restrictions. Anyway, this cannot mean a change in rights. Each patient is unique {--} they have specific needs, unchangeable principles, a value system of their own. A patient can be defined as a person under medical care, as an ill person undergoing treatment. The term {\clq}qpatient is derived from the Latin word {\clq}qpatiens`` {--} patient, suffering. The patients' rights are laid down in the Code of Patients' Rights which became effective in the Czech Republic in 1992. It is the knowledge of rights that is important before all for the strengthening of an equal relationship between the doctor and the patient The risks which represent a threat during the hospitalization can be lowered by consistent observation of the patients' rights as well as the patient's satisfaction with the care provided can be heightened. The basic individual rights of the patients include the right to silence to be preserved about the facts that the health worker became familiar with in connection with medical care or treatment, the right to being informed about their state of health, the right to gaining necessary information in order to be able to make a decision before a new diagnostic or therapeutical procedure is started up. The other rights include refusing a treatment and being informed about the consequences of the decision, the right to privacy during medical care or treatment and to a considerate, sensitive and worthy approach during the treatment. The thesis comprises the present state of problems in question, the aims of the thesis, determination of hypotheses, methodology, the results of the research, discussion, conclusion, the list of sources used, key words and supplements. The present state portrays the history of ethical codes, legal standards and the problems of medical law, the patients' rights, individual patients' rights, accreditation standards of the patients' rights. The aim of the thesis is to find out whether the patients are made acquainted with their rights after being taken to hospital, whether they are informed about the character of the disease, about suggested treatment and possible treatment alternatives. To map out whether there is the need of education being taken into account with each patient and whether the nurses keep notes in the documentation and find out the nurses' knowledge in the sphere of the patient's immobility. here have 7 hypotheses and 5 statistical hypotheses been specified in the thesis. Quantitative research approach was applied for the research. Questioning was used as a method, a questionnaire as a technique. The questionnaire for the patients was anonymous and included 26 questions. It focused on making the patient acquainted with their rights, on awareness of the state of health, the character of the disease and the suggested treatment, on the education of the patient by the nurse and on respecting the patient's intimacy. It included closed and semi-closed questions. At the end of the questionnaire the patients had a chance to express their possible comments, suggestions, impulses concerning the problems in question. The questionnaire for the nurses was anonymous and included 32 questions. It had its focus on observing the patients' rights, the informed patients' consent, their education, on the problems of restricting the patient on the bed and on respecting the patient's intimacy. The questionnaire included closed and semi-closed questions. The representative sample was made up of 218 patients and 205 nurses. The results were compiled in tabular and chart form, and subsequently processed by means of the SPSS statistical programme. The discussion includes a summary of the research knowledge in comparison with the sources used.

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Michaela Šitnerová Title of Thesis: Protein macromolecules immobilization onto polymer carriers Enzymes are unique biocatalysts because of their properties. They are highly specific, selective and functional even under mild reaction conditions. The method of immobilization is used to increase their operational stability, activity and possible reuse. This process allows the wide use of enzymes in industry, for example in the food industry, analytical chemistry, chemical synthesis and in the pharmaceutical industry. The aim of my thesis was immobilized enzyme acetylcholinesterase (AChE) on the surface pellets of microcrystalline cellulose (MCC). Used method was simple sorption, immobilization using glutaraldehyde, and TEMPO oxidation using MCC. Well known Ellman's method served to measure the activity of AChE. The absorbance of the solution with the immobilized AChE was measured spectrophotometrically at 412 nm.

- 5 - Abstract Hydrogen/deuterium exchange coupled to mass spectrometry (HX-MS) utilizes the spontaneous exchange of protein backbone amide hydrogens for deuterium atoms from solution to gain information about changes in protein structure. To localize these changes to specific areas of the protein, enzymatic digestion by aspartate proteases is used. The proteases' ability to produce small overlapping peptides and to provide full sequence coverage of the studied protein is essential for pinpointing the protein regions of interest. In this study recombinant proteases nepenthesin I (Nepenthes gracilis) and rhizopuspepsin (Rhizopus chinensis) were prepared and compared to commercially available proteases porcine pepsin A and aspergillopepsin (Aspergillus saitoi). The comparison was performed using various activity assays, where the effects of pH, temperature and denaturing and reducing agents on the activity of the proteases were studied. All four proteases were also immobilized on a polymeric resin POROS and their activity in an online HX-MS digestion setup was tested using myoglobin as a model substrate.

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Helena Badalcová Title of Thesis: Immobilization of protein macromolecules onto polymer carriers: An overview Since the 70s, the immobilised enzymes have been getting the attention of not only scientific and laboratory workers, but also industrial companies. Enzymes are unique biocatalysts, which are distinguished by their specificity, environment-friendliness and the ability to react under mild conditions can be easily subject of denaturation or inhibition. With regard to the usually high cost of purchase, the use of these enzymes could often be disadvantageous. Immobilization techniques offer an efficient solution to this problem and greatly simplify the use of enzymes in industry and research. Compared to the free forms, immobilized enzymes show greater activity, stability and allow repeated use as well as easier separation from products. This thesis contains an overview of the basic methods of immobilization - physical absorption and covalent bonds to the carrier, entrapment, encapsulation and carrier- free techniques using cross-linking. Finally, we outline possible biomedical applications as well as the use of immobilised enzymes in biosensors.

Baker´s yeast (Saccharomyces cerevisiae) were magnetically modified by three different methods, namely, surface modification by magnetic fluid, entrapment of cells into alginate and covalent immobilization on particles of magnetic chitosan. The ability of H2O2 decomposition was tested for all types of modification. It is apparent that the most amount of hydrogen peroxid was degraded by magnetic fluid - modified cells (84-95%), while the efficiency of cell which were modified by other methods was much lower (40-60%). Thanks to immobilization on particles of magnetic chitosan, we made completely new type of magnetic material, which was tested for adsorption of Crystal violet and Safranin O. It was founded that magnetic chitosan adsorbs no dyes, so all adsorption belongs to immobilized yeast. The maximum adsorption capacities were determined using Langmuire isotherm at 69,4 mg/g for Crystal violet and 99,0 mg/g for Safranin O.