|
Introduction of Western blotting method for detection of AMPK cascade activation in LS174T cell line
Dubecká, Michaela ; Pávek, Petr (advisor) ; Nachtigal, Petr (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Michaela Dubecká Supervisor: Doc. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Introduction of Western blotting method for detection of AMPK cascade activation in LS174T cell line LKB1/AMPK is the main cellular energetic pathway, which acts as the sensor of various extra- and intracellular signals. In response to the signals, it regulates energy metabolism and the maintenance of cellular homeostasis, so its major role lies in the survival, growth and development of the whole organism. This pathway is of significant importance because it has the potential to suppress tumoral progress in the cell. Metformin, the widely used drug in the treatment of type 2 diabetes, exerts a significant antitumour activity. However, the direct mechanism of metformin's action is still unknown. Metformin induces cellular stress similar to the metabolic stress via inhibiting mitochondrial respiratory chain complex I, which results in an increase of AMP levels in plasma. AMP then binds to the AMPKγ subunit, so metformin mediates the activation of AMPK. AMPK is suddenly phosphorylated on Thr172 of the AMPKα kinase domain via LKB1, which mediates the downregulation of many downstream kinases. The...
|
|
Soluble form of the scavenger receptor for hemoglobin (sCD163) in pregnancies complicated by preterm prelabor rupture of membranes
Kacerovský, Marian ; Tošner, Jindřich (advisor) ; Calda, Pavel (referee) ; Procházka, Martin (referee)
Preterm prelabor rupture of membranes (PPROM) is responsible for approximately 30% of all preterm deliveries. Histological chorioamnionitis (HCA) has been found in 50-80% of PPROM cases and is associated with higher rates of adverse maternal and neonatal outcome. The scavenger receptor for hemoglobin (CD163) is a transmembrane glycoprotein expressed almost exclusively on monocytes and macrophages. Its main function is the binding of hemoglobin-haptoglobin complexes. CD163 also serves as a surface receptor that recognizes intact bacteria and triggers cytokine production function. Moreover, it participates in the late down-regulatory phase of both acute and chronic inflammation. The soluble form of CD163 (sCD163) most likely represents the extracellular domain of CD163, which can be shed from the surface and released into the body fluid. The main aim of this thesis was to investigate sCD163 in pregnancy complicated by PPROM and relationships with HCA and funisitis. The first specific aim was to determine amniotic fluid sCD163 levels in uncomplicated pregnancies. Amniotic fluid samples were taken from 31 women who underwent amniocentesis for genetic testing in the second trimester, as well as from 32 women at term, 21 of whom had and 11 of whom did not have uterine contractions. The sCD163 levels in...
|
|
Adsorption of metal ions on humic acids
Pokorná, Markéta ; Mgr. Martina Havelcová, Ph.D (referee) ; Klučáková, Martina (advisor)
The Bachelor´s thesis deals with humic acids and their ability to bind metal ions on their surface. Humic acids are generally aromatic polycyclic compounds, which contain phenolic and carboxylic functional groups on their sidechains. Because of their functional groups, they are able to form stable complex compounds. In this thesis, the adsorption of lead ions was studied by electroanalytical method called flow-through coulometry.
|
|
Effect of intravenously applicated lactoferine on early faze; of myocardial ischemia II. Biochemical examinations
Písaříková, Veronika ; Semecký, Vladimír (advisor) ; Mladěnka, Přemysl (referee)
Lactoferrin (Lf) is an 80-kDa iron-binding glykoprotein. Lf is present physiologically in exocrine secretions, eg. tears, saliva, milk, sinovial fluid, seminal fluid and in the secondary granules of neutophils. The precise function of Lf in organism is considered to be very complex and it is still a hot subject of scientific disputation. Lf was documented to act as antimicrobial, antiinflamatory and antitumoral agent. Another of its properties is ability to inhibit hydroxyl radical formation via Fenton reaction. Afinity of Lf to iron is about 260times higher than that of transferrin. Isoproterenol (ISO) has been used as a model compound to induce infarct-like lesions in the rat and various other animal species. Administration of necrotic dose of ISO caused ischaemia followed by damage of the myocardium. Ischaemia alters iron homeostasis and redox-active free iron, which catalyses ROS-generation. The only possibility for myocardial tissue recovery in acute moycardial infarction represents the reperfusion of ischemic myocardium. But the whole process is associated with damage of myocardium due to burst of hydroxyl radical catalyzed by free iron (Fenton reaction). In our experiment we studied the effects of Lf in a catecholamine model of myocardial injury. We had four experimental groups divided according to...
|
|
Adenylate cyclase toxin of Bordetella pertussis, its conformation and ion balance in host cell.
Motlová, Lucia ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Adenylate cyclase (CyaA, ACT) toxin is one of the major virulence factors of Bordetella pertussis. Although CyaA binds to many types of membranes, it is assumed that the integrin CD11b/CD18 is its receptor which is expressed on the surface of myeloid cells. CyaA belongs to the family of RTX toxin-hemolysins. CyaA acts on the host cells by two independent activities. One of them is the conversion of ATP to cyclic AMP, which is catalyzed by adenylate cyclase (AC) domain after its translocation into the cytosol of the host cell, which leads to the entry of calcium cations into the host cell. Translocation is probably initiated by interaction of CyaA monomer with the target membrane. The second activity is the formation of CyaA channel selective for cations, which probably causes colloid osmotic lysis of target cells. The channel forming activity is provided by RTX hemolysin domain which most probably forms oligomers, although it was found that CyaA as a monomer causes leakage of potassium cations from the host cell. It is also not clear whether the oligomerization of CyaA would occur in solution, or after interaction with the host membrane. The aim of this study was to examine the flow of sodium ions on the membrane of murine macrophages J774A.1, which express integrin CD11b/CD18 on their surface....
|
|
High-strength macrodefect-free composites for refractory applications
Ryšková, Martina ; Másilko, Jiří (referee) ; Šoukal, František (advisor)
The subject of the bachelor thesis is the study of heat-resistant macrodefect-free (MDF) cements based on calcium-aluminate cement and organic polymer polyvinylalcohol. Macrodefect-free cements are particularly important in excellent mechanical parameters, which include tensile strength in bending. These materials are characterized by their low porosity and the absence of microcracks. The main role of the MDF cements takes binding and non-flush interaction with polymer cement, which affect the chemical and physical-mechanical properties. The main problem in applying the MDF cements is the low water resistance. To prepare macrodefect-free samples were used calcium-aluminate cement (SECAR 51, SECAR 71) and polymer polyvinyl Gohsenol GH 17S. They were observed changes in mechanical properties depend on the type of curing samples were cured at laboratory temperature and 70°C. Samples annealed at 1000 ° C and then tested for tensile strength in bending before and after annealing, compared to mechanical properties. Thermal stress was tested by doping a microscope TGA-DTA-EGA and TMA analysis.
|
|
Panels of steroid receptor reporter cell lines for compound profiling and development of selective ligands for estrogen receptor alpha and beta
Sedlák, David ; Bartůněk, Petr (advisor) ; Anzenbacher, Pavel (referee) ; Kasal, Alexander (referee)
of the Ph.D. thesis Steroid hormone receptors represent a major target in the drug discovery. As ligand inducible transcription factors, their activity can be modulated by small lipophilic molecules. The first part of this work describes a preparation of two panels of potent, selective and robust luciferase reporter cell lines on the unified cellular background in U2OS osteosarcoma cell line. This system consists of two panels of stable luciferase reporter cell lines for estrogen receptor α (ERα), estrogen receptor β (ERβ), androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and progesterone receptor (PR). The first panel of reporter cell lines relies on the expression of the chimeric steroid receptors created by the replacement of the N-terminal part of the steroid receptor molecule by Gal4 DNA binding domain (Gal4 DBD) binding to 9 copies of Gal4 upstream activation sequences (Gal4 UAS) in the promoter of the pGL4 luciferase reporter vector. In the second panel of reporter cell lines the activation of either synthetic promoter containing multiple hormone response elements or viral promoter derived from MMTV LTR is mediated by full- length exogenously expressed steroid receptors. We have extensively validated both panels using 28 well established ligands, carefully...
|
|
Characterization and function of Factor C from the tick \kur{Ixodes ricinus}
HARTMANN, David
Factor C is a multi-domain serine protease which recognizes Gram-negative bacteria via binding to lipopolysaccharides and triggers hemolymph clotting cascade in the horseshoe crab. A closely related molecule was also found to be present in the genome of the tick Ixodes scapularis. In this work, the full sequence of Factor C ortholog from Ixodes ricinus (IrFC) was determined. IrFC is mainly expressed in tick hemocytes and the heavy chain of the activated molecules is present in tick hemolymph as confirmed by Western blotting with antibodies raised against recombinant fragments of IrFC. The function of the IrFC in tick innate immunity was assessed using its silencing by RNA interference.
|
guest ::
