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miRNA analysis of small extracellular vesicles isolated from plasma after diet intervention
Kratochvílová, Michala ; Koc, Michal (advisor) ; Šrámek, Jan (referee)
The aim of this thesis was to establish a protocol for the isolation of small extracellular vesicles (sEVs) from human plasma. Plasma samples were collected from 44 healthy women who underwent a 60-hour dietary intervention in the form of fasting as part of a clinical trial. These women were divided into two groups based on their BMI - lean and obese. Plasma was collected from each participant before and after the dietary intervention and sEVs were isolated using the polyethylene glycol precipitation method. Each sample was then characterized by the size of sEVs and their concentration. The amount of sEVs in plasma before the dietary intervention was the same for both groups and did not change with dietary intervention. However, the 60-hour fasting resulted in a change in sEVs size in the lean group. Another goal of this thesis was to analyze miRNA expression in sEVs. There were differences found in the expression of certain miRNA between the two groups. More specifically, differences were observed in the expression of hsa-miR-17-5p, hsa-miR-20a-5p and hsa-let-7b-5p. The 60-hour fasting period resulted in an increased expression of hsa-miR-5100 in the lean group and hsa- miR-27a-3p and hsa-miR-27b-3p in the obese group. However, these changes induced by the dietary intervention were not evaluated...
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Impact of weight loss in obese subjects on the sensitivity of adipose tissue cells in relation to stress of endoplasmatic reticulum.
Karlická, Michaela ; Rossmeislová, Lenka (advisor) ; Koc, Michal (referee)
Adipocytokines released by the adipose tissue play an important role in the regulation of immune and inflammatory responses. In case of obesity their production is badly regulated, which is one of the major factors contributing to the onset of a chronic low-grade overall inflammatory response. This response represents a risk factor for the progression of other diseases, such as atherosclerosis or type-2 diabetes. The main goal of this thesis consisted in comparing the secretion of selected adipocytokines (adiponectin, IL6 and MCP1), isolated from the adipose tissue prior to and after a dietary intervention, and this under basal conditions and during stimulated lipolysis. Based on the same profile, the secretion of the isoforms of adiponectin was compared subsequently. The concentration of adiponectin, IL6 and MCP1 was determined by the ELISA method, the Western Blot method was used to determine the distribution of the adiponectin isoforms. The thesis also concentrates on the gene expression of ATF3, ATF4 and HSPA5, factors engaged in the ER stress in the course of the differentiation of adipocytes. The changes in the gene expression were measured by the quantitative Real Time PCR method. To prove the correct process of the differentiation the genes aP2 and PPARg were used. Within the framework of...
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Molecular mechanisms of apoptosis induction in tumor cells
Koc, Michal ; Kovář, Jan (advisor) ; Červinka, Miroslav (referee) ; Haškovec, Cedrick (referee)
V. Závěr Tato práce se zaměřila na studium procesů uplatňujících se v indukci apoptosy různými faktory. V našem případě jsme indukovali buněčnou smrt u nádorových buněk deprivací železa, klinicky používanými taxany (paclitaxel, docetaxel) a nově syntetisovanými fotosensitivními látkami. Z presentovaných publikací jsme získali tyto závěry. (1) Studie týkající se indukce apoptosy deprivací železa prokázaly účast odlišných signálních drah vedoucí k indukci apoptosy u myších a lidských nádorových buněk. V obou případech byla použita B lymfomová buněčná linie citlivá k deprivaci železa. U myších buněk jsme popsali významnou část apoptotické signální dráhy u 38C13 buněk sensitivních k indukci apoptosy deprivací železa. Deprivace železa u těchto buněk vede k translokaci proapoptotického proteinu Bax z cytosolu na mitochondrie, která je následována zhroucením mitochondriálního membránového potenciálu v důsledku porušení integrity vnější mitochondriální membrány, uvolněním cytochromu c z mitochondrií, aktivací kaspasy 9 a následnou aktivací kaspasy 3. U lidských buněk jsme taktéž zaznamenali indukci apoptosy deprivací železa, avšak charakterem mechanismu odlišnou od apoptosy popsané u myších 38C13 buněk. V případě lidských Raji buněk sensitivních k indukci apoptosy deprivací železa docházelo pouze k aktivaci kaspasy...
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Molecular mechanisms of apoptosis induction in tumor cells
Koc, Michal
This work has focused on study of processes involved in apoptosis induction by various stimuli in cancer cells. In our case we induced programmed cell death in cancer cells by iron deprivation, commercially available taxanes docetaxel (Taxotere® ) and paclitaxel (Taxol® ) and application of novel porphyrins derivates P(-CD)l and P(-CD)2. From presented publications we can conclude: Studies dealing with iron deprivation clearly demonstrated participation different signal pathways which lead to apoptosis induction in mouse and human cancer cell lines. We used for study of apoptosis induction B lymphom cell line sensitive to iron deprivation in both mouse and human experiment model. In the case of sensitive mouse tumor cells (38C13), we have described a significant portion of the apoptotic pathway leading to apoptosis induction by iron deprivation. Iron deprivation in these cells leads to translocation of proapoptotic Bax protein from the cytosol to mitochondria which is succeeded by mitochondrial membrane potential disruption due to mitochondrial permeabilization, cytochrome c release from mitochondria, activation of caspase-9 and subsequent activation of caspase-3. In the case of sensitive human tumor cells (Raji) we detected as well apoptosis induction by iron deprivation, however, in character of...
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Molecular mechanisms of apoptosis induction in tumor cells
Koc, Michal ; Kovář, Jan (advisor) ; Červinka, Miroslav (referee) ; Haškovec, Cedrick (referee)
V. Závěr Tato práce se zaměřila na studium procesů uplatňujících se v indukci apoptosy různými faktory. V našem případě jsme indukovali buněčnou smrt u nádorových buněk deprivací železa, klinicky používanými taxany (paclitaxel, docetaxel) a nově syntetisovanými fotosensitivními látkami. Z presentovaných publikací jsme získali tyto závěry. (1) Studie týkající se indukce apoptosy deprivací železa prokázaly účast odlišných signálních drah vedoucí k indukci apoptosy u myších a lidských nádorových buněk. V obou případech byla použita B lymfomová buněčná linie citlivá k deprivaci železa. U myších buněk jsme popsali významnou část apoptotické signální dráhy u 38C13 buněk sensitivních k indukci apoptosy deprivací železa. Deprivace železa u těchto buněk vede k translokaci proapoptotického proteinu Bax z cytosolu na mitochondrie, která je následována zhroucením mitochondriálního membránového potenciálu v důsledku porušení integrity vnější mitochondriální membrány, uvolněním cytochromu c z mitochondrií, aktivací kaspasy 9 a následnou aktivací kaspasy 3. U lidských buněk jsme taktéž zaznamenali indukci apoptosy deprivací železa, avšak charakterem mechanismu odlišnou od apoptosy popsané u myších 38C13 buněk. V případě lidských Raji buněk sensitivních k indukci apoptosy deprivací železa docházelo pouze k aktivaci kaspasy...
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Epigenetics mechanisms
Šornová, Veronika ; Černá, Marie (advisor) ; Koc, Michal (referee)
Epigenetics is the study of heritable changes in gene activities that are not caused by changes in the DNA sequence. Epigenetic mechanisms can be employed at many levels, from transcription to translation. They include DNA methylation, histone modification, and with it connected chromatin modification, and RNA interference. The result is the change of chromatin conformation leading to decrease or increase of certain gene expression, X-chromosome inactivation or gene imprinting. Epigenetic regulation plays important role in etiopatogenesis of multifactorial diseases. Genetic predisposing factors (in autoimmune diseases there are genes of major histocompatibility complex) and environmental factors, which affect our genome just through epigenetic modifications, are involved in their manifestation. Key words: Epigenetic mechanisms, DNA methylation, histone modification, RNA interference, genomic imprinting, X-chromosome inactivation, multifactorial disease.
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Impact of weight loss in obese subjects on the sensitivity of adipose tissue cells in relation to stress of endoplasmatic reticulum.
Karlická, Michaela ; Rossmeislová, Lenka (advisor) ; Koc, Michal (referee)
Adipocytokines released by the adipose tissue play an important role in the regulation of immune and inflammatory responses. In case of obesity their production is badly regulated, which is one of the major factors contributing to the onset of a chronic low-grade overall inflammatory response. This response represents a risk factor for the progression of other diseases, such as atherosclerosis or type-2 diabetes. The main goal of this thesis consisted in comparing the secretion of selected adipocytokines (adiponectin, IL6 and MCP1), isolated from the adipose tissue prior to and after a dietary intervention, and this under basal conditions and during stimulated lipolysis. Based on the same profile, the secretion of the isoforms of adiponectin was compared subsequently. The concentration of adiponectin, IL6 and MCP1 was determined by the ELISA method, the Western Blot method was used to determine the distribution of the adiponectin isoforms. The thesis also concentrates on the gene expression of ATF3, ATF4 and HSPA5, factors engaged in the ER stress in the course of the differentiation of adipocytes. The changes in the gene expression were measured by the quantitative Real Time PCR method. To prove the correct process of the differentiation the genes aP2 and PPARg were used. Within the framework of...
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Molecular mechanisms of apoptosis induction in tumor cells
Koc, Michal
This work has focused on study of processes involved in apoptosis induction by various stimuli in cancer cells. In our case we induced programmed cell death in cancer cells by iron deprivation, commercially available taxanes docetaxel (Taxotere® ) and paclitaxel (Taxol® ) and application of novel porphyrins derivates P(-CD)l and P(-CD)2. From presented publications we can conclude: Studies dealing with iron deprivation clearly demonstrated participation different signal pathways which lead to apoptosis induction in mouse and human cancer cell lines. We used for study of apoptosis induction B lymphom cell line sensitive to iron deprivation in both mouse and human experiment model. In the case of sensitive mouse tumor cells (38C13), we have described a significant portion of the apoptotic pathway leading to apoptosis induction by iron deprivation. Iron deprivation in these cells leads to translocation of proapoptotic Bax protein from the cytosol to mitochondria which is succeeded by mitochondrial membrane potential disruption due to mitochondrial permeabilization, cytochrome c release from mitochondria, activation of caspase-9 and subsequent activation of caspase-3. In the case of sensitive human tumor cells (Raji) we detected as well apoptosis induction by iron deprivation, however, in character of...
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