Abstract Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyzes the hydroxylation of lysine residues in collagens and other proteins. It occurs as a post-translational event. The hydroxylysine residues participate in the formation of covalent cross-links to stabilize the collagenous structure in tissues. The hydroxylysine residues can be glycosylated to galactosyl- or glucosylgalactosylhydroxylysine residues. Novel human lysyl hydroxylases, 2a, 2b and 3 isoforms, were characterized in this study. Lysyl hydroxylases 2a and 2b are alternatively spliced forms of lysyl hydroxylase 2. Lysyl hydroxylase 2b contains an additional exon of 63 nucleotides. The polypeptide size of lysyl hydroxylase 2a is 737 amino acids, lysyl hydroxylase 2b is 758 amino acids and lysyl hydroxylase 3 is 738 amino acids. The putative signal peptide is 25 amino acids in lysyl hydroxylases 2a and 2band 24 amino acids in lysyl hydroxylase 3. Lysyl hydroxylases 2aand 2b contain 7 possible N-glycosylation sites and lysyl hydroxylase3 contains 2 sites. Tissue distribution of novel isoforms were studied on Northern blots. The expression of lysyl hydroxylases 2a, 2b and 3 differ from the expression of previously characterized lysyl hydroxylase 1. Lysyl hydroxylase 1 is expressed constitutively in all tissues whereas the expression of novel isoforms is more strictly regulated. Lysyl hydroxylase 2 is highly expressed in heart, placenta, liver and pancreas. Lysyl hydroxylase 2b expression is highest in heart and skeletal muscle and lysyl hydroxylase 3 expression is highest in heart, placenta and pancreas. Brain, lung and kidney contain the lowest amounts of these isoforms. Novel isoforms were expressed as recombinant proteins in baculovirus expression system in vitro. All these novel isoforms were able to hydroxylate lysine residues in short collagenous peptides. A more detailed kinetic analysis was performed on lysyl hydroxylase 2a and 2b in order to find out if they differed from each other. The binding affinity of ascorbate and peptide substrate is different in lysyl hydroxylase 2a from 2b. Chromosomal assignments were carried out on human lysyl hydroxylases 2 and 3. Lysyl hydroxylase 2 was localized to chromosome 3q23–q24 and lysyl hydroxylase 3 to chromosome 7q36.

Abstract Lysyl hydroxylase (E.C. 1.14.11.4, protocollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyses the formation of hydroxylysine in collagens and other proteins with collagen-like sequences. The hydroxylysine residues participate in the formation of collagen crosslinks and serve as attachment sites for carbohydrate units. The importance of lysine hydroxylation is demonstrated by the critical manifestations found in patients with the type VI variant of the Ehlers-Danlos syndrome, which is caused by a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate. The binding site for the C-5 carboxyl group of 2-oxoglutarate is characterized here by site-directed mutagenesis. Two conserved and one non-conserved amino acid residues at the possible binding site in human lysyl hydroxylase 1 were converted individually to alanine or lysine and the mutant polypeptides were expressed in insect cells. Mutation of arginine-700 to alanine inactivated the enzyme completely, whereas mutation of the other two residues had only a minor effect. In addition, the Km of the arginine-700 to lysine mutant polypeptide for 2-oxoglutarate was increased 10-fold. The results thus indicate that this conserved arginine is the residue that binds the C-5 carboxyl group of 2-oxoglutarate in lysyl hydroxylases. A novel human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3, was identified, cloned and characterized here. The overall amino acid sequence identity between the novel human lysyl hydroxylase isoenzyme and the other human lysyl hydroxylase isoenzymes is about 60%. The highest expression levels of the mRNA for lysyl hydroxylase 3 among the tissues studied were found in the placenta, pancreas and heart. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to function as a lysyl hydroxylase in vitro hydroxylation experiments using short synthetic peptides as substrates. No differences in catalytic properties were found between the recombinant lysyl hydroxylases 3 and 1. The structure of the human gene for lysyl hydroxylase 3 was determined in the last part of this work. The gene is shown to be only 11.6 kb in size and to contain 19 exons. Transcription was found to be initiated at multiple sites, and the introns contained 15 full-length Alu retroposons or partial Alu fragments of more than 100 bp. The present characterization of the exon-intron organization of the gene will provide a basis for further studies to determine whether there is any genetic disease that is attributable to mutations in this gene.

Protection of human foetus - summary The thesis gives a detailed analysis of the extent and standard of protection of the human foetus in the Czech legal system, concentrating on the aspects of criminal law. In the introductory part it presents a brief chronological survey of the synopses of the most significant instruments which constitute the subject of the thesis. The legal institutions concerned are - in the minimum necessary extent - put in the context of several key international agreements, with the emphasis laid on the provisions of the Convention on Biomedicine whose concept significantly influenced that of the Czech medical law. Also the role of civil law and its connection - both as regards the terminology and content - with the other relevant provisions of the legal system are briefly mentioned in the third chapter. However, the thesis focuses mainly on criminal law and therefore its greatest part is concerned with the means by which criminal code guarantees the protection of human foetus. Not only does it give a survey of selected criminal offences and their matters of issue, but it also underlines their connection with the provisions of the first part of the law in question as far as the interpretation and application are concerned. A brief separate chapter deals with the connection of...

Protection of a human foetus The aim of this thesis is to deal with the protection of the human foetus in the scope of the Czech legal environment as well as in the relation with the international law. The work provides the insight into the specific law areas as constitutional law, criminal law and also private law and deals with the specific rules contended in the single act as the Interruption of the pregnancy act and the new Specific medical services act. The substantial part of the legal discussion is supported by examination of the decisions of the courts both international and national which reflect the protection of human foetus. The thesis focuses on the protection of human foetus in its complexity and shall provide the comparative material which compares the protection of the human foetus within the specific law disciplines. The analysis of single legal problems and issues of the protection of the human foetus is not exhaustive and focuses on the protection and state of the human foetus in specific statutes. The protection of the human foetus represents quite discussable issue which provides a large number of questions to be answered and leads to the discussions which are reflected in this work. The key issue is researched under the actual conditions of the legal system and also with the...

The thesis is focuses on possibilities of harvesting drone brood, it‘s conservation and processing – especially in home conditions. It deals with some nutritive aspects of samples of dry basis of the drone brood which was gained by various methods of dry-ing. Primarily by it‘s fat content, of some mineral substances, micronutrients and heavy metals. Regarding the mentioned samples, furthermore there are monitored the diffe-rences in the microbial profile of the observed samples and differences, which happen after some time of the storing. With respect to the samples of dry basis and the final product in the form of a nutrition ball, there was performed a sensory evaluation and a questionnaire survey.

The work is divided into two chapters. In the first one the World's and Czech poets (chosen on the basis of Orten's list of reading) are analyzed. Those authors could be understood as impersonators or predecessors of the existential tendencies and it's provable Orten knew their works well (i.a. Rilke, Rimbaud, Akhmatova, Mácha, Weiner, Halas, Holan, Zahradníček, Bonn and Daniel). The second and more voluminous chapter consists of the motives' and topics' analysis of Orten's literary work, focused mainly on the poetry and less on the other literary genres - notes, letters, prose. The most of his poems and notes is based on the factual life experience and their ethos is founded on both existential and Christian moral philosophy. There's also an influence of some other intellectual tendencies and artistic styles like folk literature, romanticism, impressionism, less intensively expressionism, naturalism, psychoanalysis, surrealism and naturism. To get the coherent interpretation of Orten's work it's necessary to reflect (beside the existential motives) the pathos of the intimate traumatic relationship to the father, the girl(friend)s and the unborn child as well as the deeply Christian understanding of reality (connected with the biblical /The Old Testament/ dialogical relation to the God, the...

The Use of Thromboelastography in Evaluation of Coagulation in Females with Physiological or Pathological Pregnancy MUDr. Ferdinand Polák Abstract Introduction: The target of this study was to compare thromboelastography coagulation parameters in the following three groups: a) healthy pregnant women, b) healthy non-pregnant women and c) pregnant women with pathological pregnancy and also to compare it to reference limits for the common population. If appropriate, we would propose recommendations for new reference ranges for pregnant women in their third trimester. Materials and methods: Prospective observational study, comparing, by using thromboelastography, the blood samples of 60 healthy women in their third trimester of pregnancy (group GRAV) to the samples of the control group of 43 healthy non-pregnant fertile women (group NON-GRAV) and to the samples of 50 women with pathological pregnancy (preeclampsia, fetal death) in their third trimester (group PATOL). Selective percentiles were used to determine new reference limits. Results and conclusions: We found statistically significant differences between groups GRAV and NON-GRAV. Therefore, we established, based on our results, new thromboelastography reference limits for pregnant women. Coagulation changes during pathological pregnancy are less...

Criminal Aspects of Artificial Abortion This diploma thesis deals with the issue of artificial abortion, especially its criminal aspects. Legal aspects are not the most important aspects of artificial abortion. Social, ethical or ideological aspects are of the same importance but this diploma thesis cannot analyse all of them. The main issue with artificial abortion is whether it is possible to force a pregnant woman to carry a child and give birth to a child when she cannot or does not want to do so, and whether a state can prosecute a pregnant woman or anyone else for interrupting her pregnancy. The thesis is divided into an introduction, seven chapters and conclusion. After a short introduction, the author of this thesis briefly defines the concept of artificial abortion. The following chapter focuses on two ideological groups - pro choice and pro life. The first group prefers mother's right to self-determination. The second group regards a human foetus as a person and considers killing the human foetus as a murder. The third chapter is dedicated to historical development of artificial abortion. The author regards historical development as an important part of this thesis, especially for understanding the current legislation. The fourth chapter analyzes the current legislation, the Abortion Act...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Barbora Gorová Supervisor: Assoc Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of thesis: Iron-chelating properties of fruit extracts of various elderberries Inorganic iron is the major food source of iron in humans. It plays role in many biochemical reactions. Thus, iron metabolism disorders can lead to different diseases associated with lack of iron or iron overload. One of the possible treatment modalities of the latter represents the administration of iron chelators. Elderberry, Sambucus nigra L. (Adoxaceae), has been used in traditional medicine. The fruits of elderberry are a rich source of cyanidin-based anthocyanins as the main component. There are important differences, both in chemical and physical properties between several cultivars of elderberry. Anthocyanins might interact with metals in the gastrointestinal tract by formation of chelates. However, data on metal interactions with anthocyanins are sparse. The main aim of this study was to perform the analysis of interaction of iron with elderberry fruit extracts as a rich and cheap source of anthocyanins with cyanidin as the aglycon. In this in vitro study ten elderberry fruit exctracts were tested for iron chelating activities...

The Use of Thromboelastography in Evaluation of Coagulation in Females with Physiological or Pathological Pregnancy MUDr. Ferdinand Polák Abstract Introduction: The target of this study was to compare thromboelastography coagulation parameters in the following three groups: a) healthy pregnant women, b) healthy non-pregnant women and c) pregnant women with pathological pregnancy and also to compare it to reference limits for the common population. If appropriate, we would propose recommendations for new reference ranges for pregnant women in their third trimester. Materials and methods: Prospective observational study, comparing, by using thromboelastography, the blood samples of 60 healthy women in their third trimester of pregnancy (group GRAV) to the samples of the control group of 43 healthy non-pregnant fertile women (group NON-GRAV) and to the samples of 50 women with pathological pregnancy (preeclampsia, fetal death) in their third trimester (group PATOL). Selective percentiles were used to determine new reference limits. Results and conclusions: We found statistically significant differences between groups GRAV and NON-GRAV. Therefore, we established, based on our results, new thromboelastography reference limits for pregnant women. Coagulation changes during pathological pregnancy are less...