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Lipid emulsions in lonf-term parenteral nutition
Ševela, Stanislav ; Novák, František (advisor) ; Cahová, Monika (referee) ; Lahoda Brodská, Helena (referee)
The combination of intestinal failure and parenteral nutrition is a condition associated with mild chronic inflammation and organ injury, the most significant of which is hepatic injury. This crossover-controlled trial investigated the effects of different intravenous lipid emulsions (IVLE) enriched with increased doses of omega-3 polyunsaturated fatty acids (n-3 PUFA) from fish oil (FO) in patients on home parenteral nutrition (HPNP) for chronic intestinal failure. Twelve patients were consecutively given three different IVLEs, with an FO emulsion added after each IVLE. The study also included twelve paired healthy controls for comparison. The aim of this study was to investigate how long-term administration of these IVLEs affected primarily the erythrocyte and plasma fatty acid (FA) spectrum, as well as the inflammatory response, antioxidant status, markers of liver function and bile acid spectrum in HPNP, and how these changes differed from healthy subjects. We demonstrated successful incorporation of n-3 PUFA into erythrocyte phospholipids and corresponding changes in the plasma FA spectrum, characterized in particular by significant increases in n-3 versus n-6 PUFA, with a decrease in the n-6/n-3 PUFA ratio. We also found a more prolonged effect on these changes with high dose FO...
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Application of Selected Methods for Oxidative Stress Analysis
Lízalová, Martina ; Tremlová, Bohuslava (referee) ; Beklová, Miroslava (referee) ; Vávrová, Milada (referee) ; Lojek, Antonín (advisor)
Chronic pancreatitis (CP) is a heterogeneous disease defined as chronic inflammatory changes of the pancreatic tissue caused by variety of aetiologies. Oxidative stress accompanying the inflammatory processes has been suggested as an important factor contributing to CP development. The aim of this study was to determine levels of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), together with nitrites, the total antioxidant capacity, cytokines, biochemical and haematological parameters in the plasma of patients with CP and control subjects. Levels of MDA and 4-HNE were analyzed using high-performance liquid chromatography. The total antioxidant capacity of plasma against peroxyl radicals was evaluated using chemiluminescence determination. Nitrites were determined using Griess reaction. Cytokines - TNF-alfa; TNF RI; PDGF-AB; TGF-beta, together with myeloperoxidase and hyaluronan were determined using ELISA Kits. Biochemical and haematological parameters were measured by standard methods.
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Use of stress factors for the production of lipid substances by yeasts of the genus Metschnikowia
Tručková, Marie ; Márová, Ivana (referee) ; Němcová, Andrea (advisor)
This Master´s thesis consists of two parts. The theoretical part is focused on the influence of various stress factors in the cultivation process, such as osmotic stress, oxidative stress, dehydration, cultivation time, pH or temperature on yeasts of the Metschnikowia genus. The practical part deals with osmotic and oxidative stress. Oxidative stress was performed under two different conditions (various cultivation time and temperature). The properties of yeast production were monitored mainly by gas chromatography and marginally also by flow cytometry. The analysis was performed for Metschnikowia pulcherrima, Metschnikowia andauensis, Metschnikowia chrysoperlae, Metschnikowia sinensis, Metschnikowia zizyphicola and Metschnikowia shanxiensis strains. The results showed that both oxidative stress and osmotic stress induce better lipids production. The amount of lipids, especially the fatty acid composition, also varied depending on the strains studied and the culture conditions used. The production of unsaturated fatty acids was further demonstrated in this work. The most suitable medium for the production of lipids and unsaturated fatty acids was the salt medium. It is clear that yeasts of the genus Metschnikowia are highly adaptable yeasts. Therefore, they might be potentially auspicious biotechnology producers.
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Study of Activity of Enzyma and Low Molecular Weight Antioxidant Systems
Macuchová, Simona ; Kráčmar, Stanislav (referee) ; Čertík, Milan (referee) ; Márová, Ivana (advisor)
Oxidative processes play important role in cell physiology and pathology as well. Balance of these processes is supplied by cooperating antioxidative systems; function of antioxidant defense systems depens on high levels of antioxidants in organism. Presented work is focused on developement and optimization of methods for analysis of important enzyme and non-enzyme antioxidants as well as total antioxidant capacity of selected types of biological material. Extractions and analyses of vitamin E, carotenoids, superoxide dismutase, catalase, peroxidase and lipoxygenase in barley and malt were optimized. RP-HPLC and HPLC/ESI-MS were used for analysis of vitamin E, phenolic and carotenoid content, spectrophotometry was used for enzymes activity analysis. A new methods for catalase and lipoxygenase activities were developed and compared with direct UV methods. Superoxide dismutase activity was determined by commercial diagnostic kit. A colorimetric method was used for peroxidase activity determination. Some kinetic parameters of enzymes were provided too. Optimized methods were used in the analyses of antioxidants in plant material - in barley and malt - in sets of samples of 6 varieties cultivated in four different locations for two years. Content of individual antioxidants differed depending on the variety, but usually were not found significant differences in the levels, depending on growing location. Perhaps climatic conditions have the greatest influence on levels of low molecular weight and enzymatic antioxidants at the specific location; oxidation processes are influenced both the quantity of moisture, both by sunlight, which induces oxidative processes in cultivated plants. The activity of antioxidants in barley caryopses is rapidly increasing during the malting process; an elevated temperature and moistness first induces activation the enzyme systems including antioxidant. In caryopsis is metabolic activity increased during which we can expect an increased production of radicals; for this purpose can antioxidant systems be activated that protect cells from damage by oxidative stress. In the second part of work optimized methods were applied in two clinical trials focused on study of the influence of exogenous antioxidants intake on metabolic and antioxidant status in human organism. In the first clinical study influence of food supplement containing polyunsaturated fatty acids and vitamin E on metabolism of hyperlipidaemics was evaluated. After 3-month supplemenation a lipid profile was improved and serum antioxidant levels increased. The second experiment was focused on enzyme and non-enzyme antioxidant levels in healthy subjects after temporarily intake of specific foods rich in antioxidants. After two-month intake plasma phenolic substances were slightly increased. Total antioxidant capacity and activities of enzyme antioxidants were not affected. Results of both clinical exeriments showed that supplying of antioxidants in natural form or in the form of food supplements does not markedly affect metabolism of healthy subjects, while in patients with chronic diseases antioxidant supplementation can positively influence metabolic status. Results of this work showed that optimized methods are suitable for analyses of antioxidant status parameters and also for monitoring of exogenous antioxidant intake.
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Measurement of antioxidant markers at children with malignant tumours
Hořavová, Lenka ; Masařík, Michal (referee) ; Provazník, Ivo (advisor)
Tumour disease belongs to one of the most serious health problems of our time. Finding of new indicators to recognise the beginning of the illness is the key role for today‘s research. Oxidative stress is known to be related to the disharmony between the prooxidant concentration and antioxidant mechanisms. Moreover, it is shown that these changes are very important for patients. Further it appears that these changes are very significant with path physiologically critically ill patients. Direct measuring of the reactive oxygen radicals or oxidative stress’ markers is still very hard in clinical medicine. The oxidative stress’ level is watched as the change of the antioxidant activity or alternatively as the antioxidant status. Work is focused on the spectrophotometric fixing of the antioxidant activity and markers signalising the damage of organism. The automated photometric analyzer is going to be used to gain the experimental data. Data will be processed statically.
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Comparison of sequence variations in genes of biotransfromation enzymes in some carcinoma
Turková, Lucie ; Tavandzis, Spiros (referee) ; Bóday, Arpád (advisor)
Xenobiotic biotransformation process and its capacity is crucial for xenobiotic chemicals elimination that may cause damage toward cell structures. The effectiveness of the enzymes included in this process depends on the gene variants that encodes them. The aim of this work was to compare certain polymorphisms of selected genes between cases and control groups. Studied polymorphisms were null genotypes of the glutathione S-transferase gene M1 and T1 and the insertion of TA dinucleotide in the promotor region of UDP-glucuronosyl transferase 1A1. The number of cases group was six included patients with colorectal, lung, prostate, breast, pancreatic and head and neck cancer. Total number of analysed individuals was 1 118 for cancer cases and 470 for healthy controls. The control group was divided into two groups, the first one was called general and the second one was called special included healthy individuals with no cancer history in their closest family members. Gilbert syndrome (GS) is caused by homozygous insertion of the TA dinucleotide in the TATA box of the gene UGT1A1 and it causes elevated bilirubin levels. Bilirubin is a potent antioxidant in human body, so the aim was to attest its protective effect toward cancer. We expected lower frequency of GS as a protective factor in the cases groups compared with controls. This hypothesis was confirmed in the breast cancer group (GS frequency 10,0 %) and pancreatic cancer group (GS frequency 11,1 %). In the general and special control groups the frequency of GS was 16,0 % and 15,4 % respectively. Although the other case groups show lower frequency of GS, the results weren´t statistically significant. Null GSTM1 genotype was observed with 50,4 % frequency in the general control groups and with 55,3 % frequency in the special control group. Neither the one of the cases groups hasn´t showed significantly lower percentage of null genotype. Despite expectation we observed statistically significant lower frequency of null genotype in the group of lung and pancreatic cancer group (37,4 % and 39,3 % respectively). According to this study, we can say that the lack of glutathione S-transferase M1 activity is not a risk factor for cancer development. Null genotype of GSTT1 wasn´t identified in both control groups at all. In case groups of breast and prostate cancer, there was only one individual carrying the null GSTT1 genotype. Statistically significant higher frequency of this polymorphism was observed in patients with colorectal cancer (9,7 %), lung cancer (17,2 %), pancreatic cancer (3,0 %) and head and neck cancer (15,9 %). In these groups the lack of glutathion S-transferase T1 activity might be considered as risk factor for cancer development. Nevertheless, for further verification it needs to take more investigation in this field, especially enlarge the number of patient in the case groups of head and neck, lung and pancreatic cancer.
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Evaluation of antibiotic-induced mitochondrial superoxide production in adherent cells
Ingrová, Kateřina ; Chmelíková, Larisa (referee) ; Zumberg, Inna (advisor)
The theoretical part of this bachelor´s thesis contains a description of the effect of reactive oxygen species on oxidative stress by mitochondria and the consequences of antibiotics use in cell line culturing. The cell line studied in this bachelor´s thesis is the mesenchymal stem cells (MSCs). The practical part describes the procedure of the experiment including cell culturing, passaging and cell labeling. The proposed experiment was repeated with sufficient number of repetitions. Finally, confocal microscopy images were processed in the MATLAB programming environment.
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Influence of selected parameters on biotechnological production of polyhydroxyalkanoates
Eremka, Libor ; Pekař, Miloslav (referee) ; Obruča, Stanislav (advisor)
The aim of this work is to study microbial production of polyhydroxyalkanoates (PHA). Theoretical part is focused on production of PHA using microoganisms and transgenic plants. Bacterial strain Cupriavidus necator H16 was used for laboratory production of PHA. Various waste oils were used as sole carbon and energy source. Salt of propionic adic and 1-propanol were used as intermediate for 3-hydroxyvalerate monomer (3HV) unit. Incorporation of 3HV to polymer can improve material features of PHA. The major part of experimental work was focused to study influence of aeration (concentration of dissolved oxygen) to bacterial growth, selected metabolic pathways and formation of PHA. Furthermore, influence of aeration to monomer composition of polymer was evaluated. According to experimental conclusion of this work it was approved dependance between aeration and monomer composition of PHA. Moreover, it was approved that higher concentration of oxygen supports bacterial growth and influences PHA content in cells. In addition, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH inhibits TCA cycle and enhances accumulation of PHA in cells. For this reason, specific enzymatic aktivity of several selected intracelular enzymes were measured, including those enzymes which can generate NADPH.
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Pathophysiological and genetic factors affecting serum uric acid level.
Hasíková, Lenka ; Závada, Jakub (advisor) ; Hrnčíř, Zbyněk (referee) ; Horák, Pavel (referee)
Introduction: Serum uric acid level (SUA) depends on the balance between its production and excretion. SUA is associated with several transmembrane proteins responsible for reabsorption (mainly URAT1 and GLUT9) and secretion (ABCG2) on the apical and basolateral membranes of the proximal tubules in the kidney, and in the case of ABCG2, it also correlates with its significant excretion through the gastrointestinal tract. Gout is a metabolic disease caused by the deposition of urate crystals in the joints and tissues. Chronic hyperuricemia is a primary risk factor for the development of gout; however, gout patients usually have a lower SUA during an acute gout attack than in the intercritical periods. The exact mechanism of this phenomenon is unknown. It has been speculated that the systemic inflammatory response can explain this discrepancy. The aim of the study is to determine whether treatment with specific inhibitors of the proinflammatory cytokine TNF (TNFi) affects SUA in patients with systemic rheumatic disease (SRD), and whether changes in SUA correlate with changes in selected proinflammatory cytokines or with the biomarker of oxidative stress, allantoin. Another aim is to determine the frequency and effect of allelic variants in the ABCG2 urate transporter gene in patients with primary...
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Heme sensor proteins as potential biomarkers of cellular and oxidative stress processes induced by ionizing radiation
Vávra, Jakub ; Martínková, Markéta (advisor) ; Souček, Pavel (referee) ; Tichý, Aleš (referee)
[IN CZECH] Ionizing radiation is a potential inducer of the oxidative stress processes in cells. As a result, reactive radicals are formed in the intracellular space modifying the essential biomolecules. Ionizing radiation has either direct (radiation sickness) or indirect (malignant processes) effect on the organism. Therefore, a fast determination of the dose is required when suspected irradiation of the organism occurs. However, a method routinely applicable, fast enough and at the same time suitable for dose estimation based on biomarkers has not been developed so far. The aim of this thesis is to describe new properties of the selected heme sensor proteins and discuss their potential importance for the cellular adaptation to oxidative stress conditions. Specifically, the thesis is focused on two eukaryotic proteins, heme regulated inhibitor (HRI) and transcription factor p53. The study of functional regulation as well as the conformational changes of these proteins induced by heme is greatly emphasized. Besides, the optimization of the key experimental methods was conducted. Specifically, Phos-tag electrophoresis was applied for the kinetics study of HRI wild type and its Gly202Ser mutant form, which is a characteristics of lung cancer development. Unsurprisingly, for both HRI forms studied,...
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