Drabbová, A.: Title of Diploma Thesis: Centaurea cyanus L. (Asteraceae) alkaloids and their biological activity related to Alzheimer's disease. Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové 2020. Ethyl acetate and chloroform alkaloids extracts were prepared from Centaurea cyanus L. seeds. A novel alkaloid AD-1 (Adrabbin) was isolated from the ethyl acetate extract by common chromatografic methods (column chromatography, preparative TLC). Its structure was elucidated by mass spektrometry, NMR analysis and determination of optical rotation. The alkaloid AD-1 (Adrabbin) possesses a unique structure which consists of a cyclic indole moiety. Also LC-MS analysis of fractions obtained by flash chromatography was performed. In those fractions were detected molecular ions related to compounds previously isolated from other Centaurea species. Alkaloid AD-1 (Adrabbin) was tested on ability to inhibit human cholinesterases, prolyl oligopeptidase and gylcogen synthase kinase 3β. The compound was considered against human cholinesterases inactive (IC50 values > 100 µM). Interestibgly, the alkaloid inhibited prolyl oligopeptidase the same intensity as a standard berberine (AD-1: IC50 143,0 ± 6,0 µM; berberin: IC50 142,0 ± 21,0 µM). The novel compound...

This thesis focuses on the importance of intermediate products of biosynthetic and catabolic pathway of cholesterol. The aim of the first part of the thesis is mainly to investigate, whether statins (HMG- CoA reductase inhibitors) possess antitumor properties and to compare the differences in antitumor potential of individual statins. The other part of the thesis aims at the utilization of 7α-hydroxycholest-4-en-3-one (C4), a promising marker of cholesterol 7α-monooxygenase (CYP7A1) activity and bile acid malabsorption. We demonstrated antitumor effect of statins on an experimental model of pancreatic cancer. Individual statins, however, differed significantly in their efficacy, depending on their physico-chemical properties. Our data suggests, that the most likely (but not the only) mechanism of antitumor effect of statins is decreased prenylation of signaling proteins, especially Ras protooncogene. We set up a reliable method for measurement of C4, which facilitated our research in CYP7A1 regulation. We demonstrated, that promoter polymorphism -203A>C might affect CYP7A1 activity, that diurnal variability of CYP7A1 activity might be triggered by insulin, and that insulin resistance in patients with non-alcoholic fatty liver disease impedes the feedback regulation of CYP7A1, which may lead to disease...

Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Pharmaceutical Botany Candidate: Simona Víchová Supervisor: RNDr. Jaroslav Jenčo, Ph.D. Title of dissertation thesis: The use of separation techniques in phytochemical analysis The alkaloid extract obtained from stems with immature capsules from Papaver rhoeas L. was subjected to a phytochemical study. The summary extract was separated into individual fractions by Flash chromatography. After performing biological tests for inhibitory activities of acetylcholinesterase and butyrylcholinesterase, the fraction PPR-11 and PPR-12 was selected. The fraction showed the highest activity against butyrylcholinesterase (95.55 ± 0.96 and 97.03 ± 0.79% inhibition of butyrylcholinesterase at 50 /g / ml). The combined PPR 11-12 fractions were purified by preparative chromatography to individual subfractions, which were purified by preparative thin layer chromatography. A monocomponent crystalline substance of alkaloid origin was obtained from the subfraction named PPR 11-12Fr # 8-A. Based on instrumental analysis, the analyte was identified as an alkaloid of the aporphine type (+)-caaverine. The inhibitory activities against acetylcholinesterase and butyrylcholinesterase for (+)-caaverine, were determined and compared with...

V. Hojgrová: Alkaloids of Vinca minor L. and their biological activity VIII. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany. Number of pages 69. This diploma thesis deals with the isolation of alkaloids from Vinca minor L. from the family Apocynaceae. Separation of alkaloids from the selected fraction (VM 215-258) or from their subfractions (VM 34-41, VM 86, VM 87-113) was performed by preparative TLC. Two pure alkaloids were isolated from the subfraction (VH 34-41). The first VH-1 alkaloid not yet isolated and the second VH 2 alkaloid: (-)-raucubainin. Alkaloids were identified by EI-MS, LC-MS, NMR and optical rotation and were compared with data in the literature. Isolated alkaloids were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and prolyloligopeptidase (POP) inhibitory activity and for cytotoxicity. Both substances did not show significant cholinesterase inhibitory activity IC50 against AChE after measurement, only (-)-raucubainin showed a slight activity against BuChE (IC50 = 94 ± 7 μM), VH-1 was found to be inactive (IC50 > 100 μM). POP inhibitory activity has so far only been tested for (-)-raucubainin; was found to be inactive (IC50 > 1000 µM). The results of the cytotoxic activity of the alkaloids...

The intestinal microbiota is composed of up to 100 trillion microorganisms of which bacteria are overwhelming majority. The microbiota affects the development of the immune system, defence against pathogens, host nutrition, vitamin synthesis or fat storage and its composition is changing throughout life. Some studies point to an association between microbiota composition and the development of inflammatory bowel disease. One of the treatment options is anti-TNFα antibodies therapy, which uptake or antagonize the TNFα cytokine that otherwise mediates inflammation in the intestinal mucosa. The aim of the thesis was to examine how this treatment affects the composition of the intestinal bacteriome in paediatric patients with Crohn's disease, and to find specific bacterial taxa, whose abundance changes during the treatment. By inclusion of patients with juvenile idiopathic arthritis, also treated with anti-TNFα, the study aims to discern specific effects of therapeutically induced intestinal restitution (observable in patients with Crohn's disease) from general effects of anti-TNFα therapy. Stool samples from healthy children were used to determine "healthy" bacteriome. The composition of the bacteriome was studied by profiling the variable region of the V4 gene of 16S rDNA from patients stool samples...

Adamcová, M.: Study of biological activity of alkaloids isolated from Argemone grandiflora (Papaveraceae) I. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2015. The aim of this study was isolation of substances from total diethyl ether alkaloid extract of Argemone grandiflora Sweet, their identification and assessment of their inhibition activity towards acetylcholinesterase, butyrylcholinesterase and prolyl oligopeptidase. Using common chromatografic methods, four alkaloids were isolated, that was identified as (+)-laudanosine, protopine, (-)-argemonine a (-)-platycerine. These substances was tested for their inhibition activity IC50: (+)-laudanosine (IC50 AChE = 617,00 ± 46,55 μM, IC50 BuChE = 644,77 ± 55,52 μM, IC50 POP = not mesured yet); protopine (IC50 AChE = 229,98 ± 21,02 μM, IC50 BuChE = 208,87 ± 17,67 μM, IC50 POP ˃ 1000 μM); (-)-argemonine (IC50 AChE = 4677,75 ± 1241,08 μM, IC50 BuChE = 885,45 ± 119,50 μM, IC50 POP = 337 ± 83,1 μM); (-)-platycerine (IC50 AChE = 223,65 ± 19,61 μM, IC50 BuChE = 1651,25 ± 327,7 μM, IC50 POP = 687 ± 74 μM). In comparison with the standards galanthamine (IC50 AChE = 1,710 ± 0,065 μM, IC50 BuChE = 42,30 ± 1,30 μM) and huperzine A (IC50 AChE = 0,033 ± 0,001...

Malý, L.: Study of biological activity of alkaloids isolated from Fumaria officinalis L. (Fumariaceae) II. Diploma Thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2014, 49 pp. Obtained diethylether extract of Fumaria officinalis L. was separated to fractions in column chromatography with petrol, chloroform and ethanol. Preparative TLC and crystalisation led to isolation of five alkaloids from fraction. Alkaloids were identified by GC-MS and NMR specters, optical rotation and melting point as protopine, cryptopine, (-)-fumaricine, (+)-fumariline and (+)-parfumidine. Isolated alkaloids were tested for their inhibition activity towards acetyl- and butyrylcholinesterase and towards prolyloligopeptidase. Activities were compared with standards. Natural inhibitor galanthamine showed IC50 AChE 1.710 ± 0.065 µM, IC50 BuChE 42.30 ± 1.30 µM. Best inhibition activity showed protopine (IC50 AChE 345.4 ± 24 µM, IC50 BuChE 239.6 ± 22.3 µM) and cryptopine (IC50 AChE 477.71 ± 47.33 µM, IC50 BuChE 270.82 ± 39.12 µM). The highest prolyloligopeptidase inhibition activity showed (+)-parfumidine with IC50 POP 99.2 µM, which was more active than used natural inhibitor baicaline (IC50 POP 605.9 ± 0.021 µM). Synthetic POP...

This thesis describes the life of a family with a dying child. The objective of the thesis is to describe the experience of its members after the diagnosis has been decided, during treatment and after the child's decease. It discusses the phases of going through the disease and death of one's own child for the parents and other close persons and how such a critical event in a family's life changes its values and attitudes. The theoretical part of this thesis deals with experiencing of all stages of the child's disease by their closest persons and with the psychology of the ill child. In this context, it briefly describes the history and idea of care of the incurably ill and the possibility of family support provided by non- governmental organizations. The practical part of the thesis contains the findings of the qualitative research conducted using the phenomenology approach on how the parents supported the child in the terminal stage of their disease, what they went through during this difficult time of life and how they remember that time after certain time has elapsed. Respondents include the parents or closest persons of children aged over one year that deceased as a result of a malignant disease.

Kostelník, J.: Study of biological activity of alkaloids isolated from Fumaria officinalis L. (Fumariaceae) I. Diploma thesis, Charles University in Prague,Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2014, 63 p. The aim of this study was to isolate alkaloids from joined fraction no. 55-67 (A2) obtained from the total alkaloid fraction of extract of Fumaria officinalis L. (Fumariaceae) plant. Using chromatography methods three alkaloids were isolated and then identified by structural analysis (GC-MS, NMR). Three alkaloids were isolated by using common chromagografic methods and then identified by structural analyses optical rotation and melting point as (-)-O- methylfumarophycine, (-)-sinactine a (-)-stylopine. Inhibitory activity of isolated alkaloids was assessed against human erythrocyte acetylcholinesterase, human butyrylcholineesterase and prolyl oligopeptidase. The results were expressed as IC50 values ((-)-stylopine: IC50 AChE and IC50 BuChE > 1000 μM, IC50 POP > 1000 mM; (-)-O-methylfumarophycine: IC50 AChE = 963.10 ± 135.98 µM, IC50 BuChE = 1771.0 ± 380.94 µM, IC50 POP - unmeasured; (-)-sinactine IC50 AChE = 632.0 ± 68.12 µM, IC50 BuChE = 8154.3 ± 981.42 µM, IC50 POP = IC50 POP = 52.9 ± 1.8 µM). None of alkaloids isolated showed...

Stable porcine ES cell lines have not been succesfully established yet. Ability to selfrenew or to differentiate has been limited in different porcine ES-like cell lines so far. PiPSCs represent an alternative to pESCs. PiPSCs can be generated by reprogramming of somatic cells by introduction of several transcription factors on viral vectors and were established by several groups. However, the majority of piPS cell lines depend on transgene expression because of incomplete reprogramming and weak activation of endogenous pluripotency genes. Transgene expression can infuence differentiation potential of piPSCs. Therefore, we have used integrative and reexcisable PiggyBac transposons to generate viral free piPSCs. At the same time, small molecules (low-molecular inhibitors) with potential to increase reprogramming efficiency and to activate endogenous pluripotency genes were used in the reprogramming media. This strategy has a potential for generation of naive piPSCs. Successful excision of transgenes would generate transgene-free piPSCs with uncompromised differentiation potential. Pig (Sus Scrofa) is at the same time an important animal model in preclinical stage research of the diseases. Somatic cells used for generation of piPSCs were isolated from pigs carrying mutated huntingtin. Integration of the...