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The role of ATM in breast cancer
Soukupová, Jana ; Pohlreich, Petr (advisor) ; Souček, Pavel (referee) ; Foretová, Lenka (referee)
Incidence of breast cancer is continuously increasing in the Czech Republic. Tumor development is a result of gene alterations'accumulation, particulary associated with genes involved in regulation of cell growth and division. Hereditary carcinomas account for approximately 5-10% of all breast tumors and in 60-80% cases are caused by a germline mutation in the major predisposition genes BRCA1 and BRCA2. Nevertheless, other genes, mostly of lower penetrance, may play a role in breast pathogenesis such as the ATM tumor-suppressor gene. ATM is the apex of the repair pathway of DSB. This protein kinase activates through phosphorylation of its substrates cell cycle checkpoints, which leads either to the delay of the cell cycle progression until DSB are repaired or to the promotion of apoptosis. To sum up, the ATM gene seems to have a role in breast cancer development in a minority of the high-risk families in our population which is significantly lower compared to BRCA1/2 and it also seems to be involved in pathogenesis of sporadic breast cancer. Despite the ATM gene's length, we do not perform the preventive screening of this gene in breast cancer high-risk families. Nevertheless, we offer the molecular diagnostics of ATM to ataxia telangiectasia patients.
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Importance of Transport Proteins and Biotransformation Enzymes for Defending Role of the Placenta
Vacková, Zuzana ; Štaud, František (advisor) ; Trejtnar, František (referee) ; Mičuda, Stanislav (referee)
Placenta is a unique organ which ensures a number of vital functions necessary for normal course of pregnancy and development of a new individual. In addition to its main function of oxygen supply and nutrient and waste product exchange, placenta also serves as an endocrine, metabolic and protective organ. Placenta is considered to be one of the physiological barriers of the organism which regulates transport of both endogenous and exogenous compounds between two compartments - maternal and fetal blood circulations. Up to recently, the placental barrier was supposed to be formed only by cellular layers which separate maternal and fetal blood - syncytiotrophoblast and fetal capillary endothelium. However, it has been demonstrated that the activity of placental efflux transport proteins and metabolic enzymes contributes considerably to the protective function of placental barrier. Efflux transporters are membrane proteins which actively (along with consumption of ATP) "pump" a diversity of substrates out of the cell. It has been shown that the kinetics of transport of various substances across the placenta is affected predominantly by two transporters: P-glycoprotein (P- gp) and breast cancer resistance protein (BCRP). Compared to these transporters, placental biotransformation enzymes are considered...
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Antibodies against sand flies saliva in domestic animals from endemic areas of visceral leishmaniasis
Košťálová, Tatiana ; Volf, Petr (advisor) ; Kopecký, Jan (referee)
The main aim of this thesis was to test sera of domestic animals from endemic areas of visceral leishmaniasis in north and northwest Ethiopia for antibodies against presumed vector P. orientalis salivary glands and to clarify behavior of the sand fly and the role of domestic animals in transmission of visceral leishmaniasis. Specific IgG antibodies against P. orientalis saliva were tested in dogs, cattle, goats, sheep and donkeys. Above the cut-off value there was 76 % analyzed dogs, 15 % cattle, 26 % goats, 60 % sheep and 45 % donkeys. Dogs, goats and donkeys revealed significantly higher anti-P. orientalis antibodies compared to control animals. In the case of cattle, goats and donkeys cut-off levels were very high and is questionable if observed results are relevant. Canine sera were screened also for anti-P. orientalis IgG2 and 88% of Ethiopean sera revealed significantly higher IgG2 than control sera. The next aim was to detect if canine sera from Ethiopia with anti-P. orientalis antibodies react with other bloodsucking insect saliva by using immunoblotting method. In saliva of sand fly Sergentomyia schwetzi, which is abundant in Ethiopia, canine sera reacted mostly with 36, 37 and 51 kDa proteins. Mosquito Culex quinquefasciatus is cosmopolitan species and canine sera most intensely...
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The Role of Gut Microbiota and Lipopolysachaide Content of the Diet in the Development, Maturation and Function of the Immune System
Hrnčíř, Tomáš ; Tlaskalová - Hogenová, Helena (advisor) ; Prokešová, Ludmila (referee) ; Macela, Aleš (referee)
Mammals are essentially born germ-free but the epithelial surfaces are promptly colonized by astounding numbers of bacteria soon after birth. The most extensive microbial community is harboured by the distal intestine. The gut microbiota outnumbers ~10 times the total number of our somatic and germ cells. The hostmicrobiota relationship has evolved to become mutually beneficial. Studies in germfree mice have shown that gut microbiota is essential for the proper development of the immune system. The pivotal role of the innate immune system in the complex and dynamic host-microbiota interactions has become increasingly evident. The principal aims of the present study were: firstly, to determine whether LPS-rich sterile diet can promote maturation of the immune system in germ-free mice, secondly, to elucidate whether gut microbiota and LPS-rich sterile diet influence the LPS susceptibility, and finally, to investigate a role of the adaptive immunity in endotoxin shock. Our data clearly show that both live gut microbiota and LPS-rich sterile diet increase susceptibility to endotoxin shock. Further, we demonstrate that immunodeficient SCID mice, which lack mature B and T cells, are more sensitive to endotoxin shock than immunocompetent Balb/c mice. In addition, we show that not only live gut microbiota but also...
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Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells
Ledvina, Vojtěch ; Klepárník, Karel
Caspases are proteases that play key role in the process of apoptosis, the programmed\ncell death. Among them, caspase-3 and -7 are main executioner caspases that cleave\nmany vital proteins during apoptosis and after their widespread activation, the process\ncannot be reversed. To analyze caspase-3/7 activation within single cells, a miniaturized\ndevice for parallel analysis of eight samples was developed. The assay is based on the\nmodified luciferin-firefly luciferase bioluminescence (BL) system. Individual\nsuspended cells were collected and transferred into detection microvials using a\nmicromanipulator. The bioluminescence was detected using a photon counting head\nwith cooled photcathode. The LOD suitable for detection of active caspase-3/7 in both\napoptotic and non-apoptotic cells was reached.
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Study of the composition and organization of cytochrome P450 system by covalent crosslinking
Koberová, Monika ; Hodek, Petr (advisor) ; Hansíková, Hana (referee)
The system of mixed function oxygenase (MFO system) participates in significant roles in the metabolism of endogenous compounds and xenobiotics. This system contains cytochrome P450, NADPH:cytochrome P450 reductase, and also there are assigned NADH: cytochrome b5 reductase and cytochrome b5. It was proved that cytochrome b5 can stimulate or inhibit cytochrome P450 (CYP)-dependent reactions and even change the ratio of resulting metabolites. The mechanism of cytochrome b5 action has not been fully elucidated yet. Elucidation of protein-protein interactions in MFO system and determination of topology of this system could explain the mechanism of cyt b5 action. The covalent cross- linking technique is suitable method for identifying protein-protein interactions within the membrane. Cytochrome b5 contains 3 methionines and in 2 cases the methionines are localized in a short hydrophobic C-terminal membrane anchor. Interactions with cytochrome P450 in the membrane environment can be identified by substitution of two methionine for photoactivatable analogue of methionine (photo-methionine) and subsequent photoactivation. This work is focused on expression and isolation of photo-cytochrome b5 (photo- cyt b5), cytochrome b5 analogue with incorporated photo-methionine. Conditions for photo-methionine...
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The effect of tick salivary proteins on innate immunity cells
PÁLENÍKOVÁ, Jana
Saliva of Ixodid ticks contains a whole array of pharmacologically active molecules with vasodilatory, antihemostatic, and immunomodulatory activities. This thesis focuses on two types of salivary proteins, serpins and cystatins, and their role in immunomodulation. These protease inhibitors are known to affect many biological functions. To better understand their role in tick saliva we examined their effect on dendritic cells and their ability to modulate the immune response after pathogen infection. As model pathogens, Borrelia spirochetes and tick-borne encephalitis virus were used.
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The endocrine function of adipose tissue and its importance for initiation and development of insulin resistance and diabetes
Haluzík, Michal ; Schreiber, Vratislav (advisor) ; Marešová, Dana (referee) ; Šeda, Ondřej (referee)
Endocrine production of adipose tissue is a very complex process affected by numerous endogenous and exogenous stimuli. Peroxisome proliferator-activated receptors-alpha (PPAR-) are important modulators of metabolic processes which can also affect endocrine function of adipose tissue. Recently, numerous novel factors produced by adipose tissue with important metabolic effects were identified. Some of them can directly bind PPAR receptors. One of the examples of these factors is fatty acid binding protein 4 (FABP4) which can directly bind PPAR receptors and indirectly modify its activation by changing availability of endogenous PPAR ligands -free fatty acids. We hypothesized that the mechanism of action of PPAR receptors to metabolic processes may partially lie in their complex interaction with adipose tissue-derived hormones. The unraveling of these interactions may have important consequences in finding approaches to treat patients with type 2 diabetes mellitus. (...) In summary, our data show an important role for the interplay of PPAR activation and endocrine function of adipose tissue in metabolic regulations which may have important clinical consequences.
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Role of Psb28 proteins in the biogenesis of the Photosystem II complex in the cyanobacterium Synechocystis sp. PCC 6803
BEČKOVÁ, Martina
The thesis focuses on the role of Psb28 proteins, namely the Psb28-1 and its homolog Psb28-2, in the biogenesis of the Photosystem II complex (PSII) in the cyanobacterium Synechocystis PCC 6803. The aims of this work were to localize the proteins within the cells, and to determine their function. A fraction of both Psb28 proteins was identified in the monomeric PSII core complexes but most proteins were found in the unassembled protein fraction associated with thylakoid membranes. Psb28-1 was mostly detected as a dimer while Psb28-2 as a monomer. Psb28-1 also differed from Psb28-2 by its higher affinity to the PSII core complex lacking CP43 antenna. Characterization of Psb28-less mutants suggested regulatory function of the proteins in PSII biogenesis in connection with chlorophyll biosynthetic pathway. Analysis of preparations isolated using FLAG-tagged versions of Psb28 proteins showed their association with Photosystem II - Photosystem I supercomplexes, especially under increased irradiance, and supported a role of Photosystem I in the PSII biogenesis.
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