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The role of caudal organ in chemical communication in field mice of the genus Apodemus
Obstová, Lucie ; Stopka, Pavel (advisor) ; Petrák, Jiří (referee)
This thesis deals with the chemical communication of wood mouse of genus Apodemus with a focus on the special sebaceous gland on the ventral side of the tail - the caudal organ. Role of the caudal organ in chemical communication is unclear. To illustrate the function of the sebaceous glands, I decided to use metods of 454 pyrosequencing, proteomic analysis and qPCR. I created a database of transcriptionally active genes of caudal organ using 454 pyrosequencing. In proteomic analysis, I focused on proteins with a molecular weight 14-18kDa to verify the presence of the lipocalins, proteins involved in chemical communication. I compared the relative mRNA abundance of selected genes between individuals of the opposite sex using qPCR. In my research, I found that the caudal organ is active organ and caudal organ could participate in chemical communication. I detected a different relative mRNA abundance of genes in males and females at the mRNA level, which could play a role in differences in development of caudal organ between individuals of the opposite sex. Finally, I suggested possible ways of using the caudal organ in the chemical communication in wood mouse genus Apodemus from the obtained results. Key words: caudal organ, Apodemus, chemical communication, FABP5, Scd1
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Cytochrome P-450: Study of structure and interactions using chemical modification, photo-initiated cross-linking and mass spectrometry
Ječmen, Tomáš ; Šulc, Miroslav (advisor) ; Petrák, Jiří (referee) ; Šebela, Marek (referee)
ABSTRACT Mixed function oxygenase system participates in biosynthesis of endogenous and metabolism of exogenous substances (e.g. drugs or chemical procarcinogens) in an organism. Substrates are biotransformed by terminal oxygenases - cytochromes P450 (P450). Catalytic properties of certain P450s (e.g. studied isoform 2B4) are altered in the presence of a redox partner - cytochrome b5 (cyb5). Both cytochromes are anchored by hydrophobic domains in a lipid membrane of endoplasmic reticulum whereas their catalytic domains are exposed to cytosol. Two zero-length cross-linking approaches were employed to extend present knowledge of P450 2B4 and cyb5 protein structure and protein-protein interactions: (1) interlinking of carboxylate and primary amine groups of amino acids by water soluble 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and (2) photo-initiated cross-linking by photo-labile methionine analog (pMet), which links to any amino acid after activation by UV-irradiation, either in hydrophilic or hydrophobic environment. pMet was incorporated to methionine site(s) of cyb5 during recombinant expression in E. coli, which was carried out in limit medium supplemented with amino acid analog. Optimization of experimental conditions led to ~20-30% substitution of the natural amino acid. Covalent...
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Analysis of bone marrow plasma in order to identify new biomarkers in childhood acute lymphoblastic leukemia
Kováč, Martin ; Hrušák, Ondřej (advisor) ; Schwarz, Jiří (referee) ; Petrák, Jiří (referee)
Leukemia, a malignant disorder of lymphoid or myeloid progenitor cells, is the most frequent malignant disease of childhood. The most common subtype is acute lymphoblastic leukemia (ALL). In acute leukemia, several clinical symptoms may be caused by soluble proteins secreted by AL cells into the bone marrow (BM) microenvironment. We aimed at identifying proteins in BM plasma of children with ALL, which may be responsible for ALL aggressiveness or for microenvironment-mediated survival of ALL cells. LBMp (leukemic bone marrow plasma) at the diagnosis of ALL was compared to CBMp (control bone marrow plasma) and CPBp (control peripheral blood plasma) using cytokine antibody array and two-dimensional electrophoresis (2-D PAGE). Cytokine antibody array for 79 proteins identified 23 proteins expressed differentially with a statistical significance of p<0.05; of those, 2 proteins (TIMP-1 and LIF) withstood Bonferroni correction for multiple comparisons (p<0.00064). On the other hand little difference was observed between CBMp and CPBp. Using 2-D PAGE, we were able to detect 397 protein spots per gel, among which 16 showed statistically significant differences in protein levels, when comparing LBMp and CBMp by MALDI-TOF analysis. The highest difference has been found for haptoglobin in LBMp and retinol binding...
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"The cancer cell proteome and its changes after anti-cancer drug treatment".
Tylečková, Jiřina ; Kovářová, Hana (advisor) ; Strnad, Miroslav (referee) ; Petrák, Jiří (referee)
Cancers represent a group of unprecedented heterogeneous diseases and currently available anti-cancer therapies provide highly variable efficacy with unsatisfactory cure rates. A wide range of proteomic technologies are being used in quest for newer approaches which could significantly contribute to the discovery and development of selective and specific cancer biomarkers for monitoring the disease state and anti-cancer therapy success. Taking into consideration the above aspects, this research was undertaken to study cancer cell proteomes and their changes after anti-cancer treatment with specific focus on: (a) response to conventional anthracycline/anthracenedione drugs with respect to their different clinical efficacy and (b) identification of novel targets for therapy in cancer cells resistant to biological drugs such as inhibitors of (b1) cyclin-dependent kinases and (b2) Aurora kinases. This study identified several interesting key aspects related to the effects of daunorubicin, doxorubicin and mitoxantrone. With the main focus on early time intervals when the influence of apoptosis is minimised, changes common for all three drugs belonging mainly to metabolic and cellular processes were observed. More importantly, significant changes in proteins involved in the generation of precursor...
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Dyr, Jan (advisor) ; Kodíček, Milan (referee) ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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Molecular mechanisms of Diamond-Blackfan anemia
Handrková, Helena ; Petrák, Jiří (advisor) ; Šebela, Marek (referee) ; Trka, Jan (referee)
Diamond-Blackfan anemia (DBA) is a rare congenital syndrome that presents with ane- mia and selective deficiency of erythroid precursors, while other blood lineages are usu- ally unaffected. Approximately half of the patients display additional somatic anoma- lies and growth retardation. The therapy is mostly symptomatic and is dominated by corticosteroids, other modalities include regular blood transfusions or hematopoietic stem cell transplantation. At the beginning of this work, only two DBA causal genes were known, RPS19 and RPS24, being mutated in approximately 1/4 of all DBA patients. The goals of this work were to study the consequences of the known DBA causal mutations on cellular level and to find novel DBA causal genes. To date, over a half of DBA patients have been reported to carry a mutation in one of nine known DBA causal genes, including RPS17, RPL11 and RPL5, that are reported in this dissertation. All confirmed DBA causal genes encode for ribosomal proteins (RPs) that were essential for ribosome assembly. We further hypothesized a non- ribosomal protein participating in this process might be involved in DBA pathogenesis, too. In one DBA patient, we identified a rare sequence variant in one such candidate, a protein arginine methyltransferase 3 (PRMT3). We reported that the patient PRMT3...
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Proteomics of biological fluids
Jarkovská, Karla ; Kovářová, Hana (advisor) ; Petrák, Jiří (referee) ; Ulčová-Gallová, Zdeňka (referee)
Reproductive diseases, mainly those resulting in the infertility affect the chances of human being to reproduce. On the contrary, the heart disease, cancer and degenerative diseases currently account for majority of deaths in the world. Usually, these lifestyle diseases need longer lifespan to become the cause of death. The proteins secreted by cells carry important information about the cell's well-being, as well as about the condition of the tissues formed by these cells. Once secreted, these proteins may also be transferred throughout the body by means of body fluids, many of which are easily accessible for further 'in-depth' studies. Cellular and secreted proteins are often a focus of studies using proteomic means and the revelation of protein alterations can lead us to new ideas about the molecular mechanisms of diseases as well as possible identification of proteins that may be used as new targets for pharmaceutical intervention or molecules that could be used for diagnostic or prognostic purposes. Taking into consideration the above aspects, this research was undertaken to find proteins that could: (a) characterise the human follicular fluid as microenvironment of the maturing oocyte, to increase understanding of reproductive processes to improve the techniques of assisted repro- duction;...
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Proteomic analysis of liver iron overload
Myslivcová, Denisa ; Petrák, Jiří (advisor) ; Kovářová, Hana (referee) ; Hrkal, Zbyněk (referee)
V našich experimentech jsme prokázali, že lidské jaterní buňky mění expresi proteinů v důsledku akutního toxického přetížení železem a následně v důsledku oxidativního stresu a že v játrech přetížených železem dochází ke změnám v močovinovém cyklu, metylačním cyklu, oxidaci mastných kyselin a metabolizmu cukrů. Dále jsme zjistili, že pro dědičnou hemochromatózu je typická odlišná jaterní exprese sarkozin dehydrogenázy, selenium binding proteinu 2, glutathion S-transferázy P1, karboxylesterázy 1 a thioredoxin-like proteinu 2. Identifikované proteiny v první a druhé studii přímo nebo nepřímo souvisejí s přetížením modelového organizmu železem. Jiné jsou spíše obecnou odpovědí buňky na stresové podmínky. Jsou to především PDI, enoláza, keratiny, HSP70, GST, GRP78/Bip a podjednotky proteazómu. Tyto proteiny patří mezi nejčastěji identifikované diferenciálně exprimované proteiny v proteomických studiích využívajících 2-DE (Petrak et al., 2008) a jejich expresní změny tedy nelze považovat za změny specifické pro metabolizmus železa či hemochromatózu. Výsledky naší studie nám poskytly nové informace nezbytné pro detailní molekulárních pochopení mechanizmů uplatňujících se v metabolizmu železa, které je podmínkou rozvoje nových postupů v prevenci i terapii onemocnění souvisejících s metabolizmem železa, především...
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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