Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Ivana Turňová Supervisor of Diploma Thesis: Mgr. Monika Kuchařová, Ph.D. Consultant: prof. MUDr. Zdeněk Zadák, CSc. Title of Diploma Thesis: Determination of fatty acids in human tissues The fatty acids are non-negligible component of lipids as one of the basic nutrients. This thesis in its theoretical part presents above all the group of polyunsaturated fatty acids (PUFA), which are important structural units of the cell membranes, they are also the precursors to several significant biologically active substances. In the human organism PUFA participate in many physiological and pathological processes this way, where they cause the large spectrum of actions. Onwards the thesis describes the gas chromatography method that is used in chemical analysis of lipids for the fatty acid determination as the gold standard. Experimentally, the fatty acid representation was determined in the blood, muscle, heart, liver and kidney among 26 cadavers divided into two groups according to the presence/absence of inflammatory process at the time of death. The data obtained were statistically analysed. The results were descriptively evaluated and there were indicated possible explanations of differences...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Author of Diploma Thesis: Bc. Barbora Kuříková Supervisor of Diploma Thesis: Mgr. Monika Kuchařová, Ph.D. Consultant: prof. MUDr. Zdeněk Zadák, CSc. Title of Diploma Thesis: Determination of the fatty acids spectrum in patients undergoing treatment with taxanes The diploma thesis deals with the determination of fatty acids spectrum in erythrocytes in patients treated with taxanes. The theoretical part describes general features of fatty acids, their synthesis and degradation. Then it is focused on the topic of breast cancer, taxane treatment and negative side effects associated with taxane treatment, especially polyneuropathy. The gas chromatography, which is commonly used in practice, is also described in this part. The experimental part is divided into three parts. The first part describes working process, chromatographic analysis and evaluation of this analysis. In the second part there is comparing of spectrum of fatty acids of patients without and with polyneroupathy. And in the last part there is a comparing of spectrum of fatty acids of patients before taxane treatment, closely after treatment and about month after the treatment. Results of the experimental part are evaluated at the end...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Alena Čunátová Supervisor of Diploma Thesis: Mgr. Monika Kuchařová, Ph.D. Title of Diploma Thesis: The fluctuation of fatty acids levels in the tissues of suddenly deceased persons and comparison with values in pathological states Fatty acids and their metabolites are significantly involved in many physiological and pathological processes. This thesis monitors the levels of selected fatty acids in human tissues and the effect of long-term disease on their stores. In addition to the general characteristics, the theoretical part focuses on the intake of polyunsaturated fatty acids and their importance in the human body. It also deals with the effects of eicosanoids and other fatty acid metabolites. Methods used in fatty acid bioanalysis are also described. In the experimental part, the proportion of fatty acids in seven tissue types was compared between two groups of donors. One of them included 8 relatively young, healthy, suddenly deceased individuals. The second group included 12 elderly polymorbid patients. The obtained tissue samples were adjusted using extraction and derivatization techniques. Gas chromatography with flame ionization detection was used for analysis. The measured...

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Background: Endothelial dysfunction is a major factor in the development of atherosclerosis, thrombosis and heart disease. Evidence suggests dietary fat composition may modify cardiovascular risk, as well as surrogate markers of cardiovascular risk such as blood pressure, arterial stiffness and endothelium-dependent vasodilation. Aim: To investigate the impact of dietary fat composition on endothelial function and associated markers of vascular health. Methods: The effects of oils rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were separately investigated in a parallel-design, placebo-controlled randomised controlled trial (n=48, 6 weeks, 2.9 g/d), carried out in free-living healthy young men. Following a 2 week run-in period taking placebo capsules (olive oil), participants underwent baseline measurements of finger capillary density, endothelial progenitor cell numbers (EPC), platelet-monocyte aggregate numbers (PMA), ambulatory blood pressure (ABP), pulse wave analysis (PWA), digital volume pulse analysis (DVP), and gave blood samples for plasma lipid, glucose, insulin, nitric oxide metabolites (NOx) and isoprostanes. The same measurements were made at the study endpoint, 6 weeks. An in vitro investigation of the effects of physiologically-relevant fatty acid profiles on microvascular endothelial cell nitric oxide and prostacyclin production was also performed. Results: Neither EPA nor DHA supplementation influenced EPCs, capillary density, PMA, ABP, PWA, DVP or plasma cholesterol, triacylglycerol, glucose, insulin, NOx or isoprostanes compared to placebo. However, ambulatory night-time heart rate was increased following EPA supplementation compared to DHA. Furthermore, both EPA and DHA decreased plasma non-esterified fatty acids (NEFA) compared to placebo. The in vitro investigations suggested that the composition of circulating NEFA may differentially affect endothelial function in the microvasculature. Conclusion: Dietary EPA and DHA at relatively high doses do not improve a number of novel markers of vascular function, including microvascular function and a marker of endothelial repair in young healthy men. EPA and DHA have differing effects on heart rate during sleep, suggesting that further research is required into the possible adverse effects of higher doses of individual marine fatty acids in at-risk individuals. Further work is required to elucidate the role of physiological fatty acid profiles on endothelial function.

Difficulties in controlling and forecasting avocado fruit ripening and the highly perishable nature of the crop once harvested, are the major causes of concern for avocado traders. In particular, the simultaneous presence of many suppliers may account for increased fruit variability during ripening. Avocado is a climacteric fruit with consistent ethylene production after harvest which is also related to high perishability. However, the mechanisms regulating ethylene biosynthesis and mesocarp softening are not completely understood. In order to study such effects, avocado fruit from different growing areas and harvested at various maturity stages, were investigated and the biochemical and physiological changes during ripening at both 18 and 23°C were studied. Mesocarp softening and fatty acid content discriminated fruit maturity and growing area, respectively, whereas C7 sugars (D-mannoheptulose and perseitol) discriminated length of fruit shelf life. For the first time, oleic acid content presents in the oil mesocarp was found to depend on fruit sources making of this a suitable indicator of avocado fruit growing area. In contrast, sugar content declined along fruit maturity and ripening. In particular the mannoheptulose presents in avocado mesocarp might be use to estimate avocado fruit shelf life. Indeed, fruit harvested late in season were found to have a lower C7 content than earlier harvest fruit and a faster softening, regardless fruit source. However, sugars content changed between growing area, thus a general C7 threshold defining fruit storability seems to be not definable. Furthermore, other possible indicators of fruit maturity and/or ripening stage have been searched in the cell wall constituents of avocado mesocarp. Thus, the structural carbohydrates profile of avocado mesocarp investigated with a new immunological method changed during ripening and harvest time (early and late season), suggesting a possible effect of cell wall composition on fruit ripening regulation. Also, the possible use of ethylene application in reducing the high heterogeneity noted on imported fruit from South Africa was also evaluated through different consignments. Results showed ethylene efficacy changed depending on harvest time and fruit dimension with less efficacy of the treatment on fruit harvested at the end of the season and characterised by smaller size.One of the most commercialized avocado cultivars, Hass, is peculiar in that its skin colour changes from green to deep purple as ripening progresses. The most common ripening indicator of avocado fruit is the mesocarp firmness and the destructive nature of this evaluation increases losses in the avocado industry. The availability of a non-destructive indicator of fruit ripening represents an important advantage for avocado consumers and importers. Thus, the possible relationship between mesocarp softening, skin colour were objectively evaluated (C*, L*, and H°), and the main pigment, cyanidin 3-O-glucoside, was investigated. Cyanidin 3-Oglucoside was confirmed to be the main anthocyanin present in avocado cv. Hass peel, regardless of preharvest factors. However, differences in its content were noted between shelf life temperatures. A higher relationship between hue angle and firmness was detected in late harvest fruit, whereas no correlation was found between anthocyanin content and firmness. Avocado skin is also involved in defence mechanisms due to the presence of antifungal and phenolic compounds. These phenolic compounds represent a natural protection against pathogenic infections and seem to be down regulated during ripening. The main phenolics were identified and quantified, using a new analytical method which was validated and optimised. Epicatechin, chlorogenic acid and procyanidin B2 were found to be present in the skin tissue and quantified using this assay and found to vary during shelf life and seasons. Although phenolics were present in minor amounts, in avocado pulp they are involved in mesocarp discoloration incidence, and therefore with fruit postharvest quality. Due to a lack of information, a new straightforward method for the identification and quantification of the main phenolics present in avocado mesocarp was developed. Finally, a commercial trial was undertaken to ensure that the results obtained in the laboratory can be reproduced in the market place. In conclusion, postharvest markers can define avocado fruit maturity and growing area and give guidelines in the control of avocado shelf life. Moreover, new methods for the investigation of the phenolic profiles (peel and mesocarp) and the characterisation of cell wall structures can be further tools in the management of avocado fruit postharvest quality.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Medical Sciences

Three studies were designed to evaluate the promoting effect of cyclopropenoid fatty acids (CPFA) on avian myeloblastosis virus (AMV) infection in White Leghorn chickens. In the first study effects on growth parameters, blood serum and erythrocyte lipid composition of White Leghorn chickens were investigated at 100 ppm and 300 ppm dietary CPFA. In comparison to controls, chicks fed dietary CPFA had reduced body weights and food intake. At both levels, CPFA increased the saturated fatty acids and decreased monoenes in blood serum and erythrocyte triacylglycerols and phospholipids. In a second study, the effects of high dietary (300 ppm) CPFA on AMV infectivity were investigated. Chicks were inoculated with 0.1 ml AMV at 7 days of age. A diet containing CPFA was fed before and after inoculation. CPFA/AMV treated chicks showed a marked reduction in body weight gain and food consumption in comparison to controls. CPFA appeared to promote AMV infection along with unexpected violent hemorrhagic manifestations. These hemorrhagic manifestations appeared to cause death before AMV induced myeloblast proliferation in the circulating blood. In comparison to controls, CPFA altered the phospholipid composition of isolated myeloblasts and the cholesterol/phospholipid ratio of isolated myelobasts, myeloblast plasma membrane, and virus particles. Analysis of the acyl group composition of choline and ethanolamine phospholipids from CPFA treated myeloblasts and myeloblast plasma membrane revealed an increase in saturated fatty acid and a decrease in monoenes. In both, CPFA appeared to affect ethanolamine more than choline phospholipids. In contrast, the alterations observed in CPFA treated virus particle choline and ethanolamine fatty acyl chains were reversed from those found in the myeloblast and myeloblast plasma membrane. In addition, ATPase specific activity was altered in relation to the CPFA induced lipid changes in the myeloblast plasma membrane and virus particle. It was shown that as saturation increased enzyme activity increased. As a third investigation, the effects of low dietary (75 ppm and 150 ppm) CPFA on AMV infection was conducted in an attempt to eliminate the hemorrhagic manifestations. A difference that was observed between this investigation and the 300 ppm CPFA/AMV study was less severe hemorrhagic responses. Histological examination of CPFA treated liver and spleen by light microscopy revealed heavy infiltration of mitotic myeloblasts into each tissue. Analysis of the number of virus particle/ml by reverse transcriptase revealed 300 ppm dietary CPFA stimulated the synthesis and release of virus particles. This was in contrast with 150 ppm dietary CPFA which retarded the synthesis and release of virus particles. / Graduation date: 1982