In animals, some of the most critical regulators of gene expression are nuclear hormone receptors (NRs) and their coregulators, specifically the Mediator complex. Of particular interest are the NRs implicated in metabolic and developmental regulation and in carcinogenesis: thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). In this work, I venture to elucidate some aspects of gene expression regulation by these NRs: the degree of evolutionary conservation of signalling based on NRs and their coregulators; the mechanisms of negative regulation by NRs; and possible implications of these findings for clinical medicine. State-of-the-art bioinformatical, genome editing and microscopic techniques are applied at three levels of animal evolution to study NRs and Mediator. Reverse genomics in human patients suffering from the syndrome of resistance to thyroid hormones β are used to infer the structure and function of TRβ subdomains. Alignments, binding studies and in vivo experiments in Trichoplax adhaerens allow identification of a close orthologue of human RXR at the basis of metazoan evolution. Employing database queries, genome editing and microscopy, we describe a correct orthologue of the Mediator subunit 28 in Caenorhabditis elegans, indicating a complete homology of the Mediator complex...

Sandra Pavlíčková Physiological effects mediated by the brain angiotensin receptors Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Pharmacy Department of Biological and Medical Science Supervisor: Doc.MUDr. Josef Herink, DrSc. Renin-angiotensin system (RAS) is one of the oldest and the most important hormonal systems. "Classic" angiotensin system regulates the blood pressure and homeostasis of water and electrolytes. The main bioactive peptide of this system is angiotensin II (Ang II), which, according to recent research, does not affect only one organ. Installation of the so-called "tissue" RAS, which in many cases complements "systemic" Ang II system, changes the view on RAS. It was established, that RAS components are located on other non- typical places, which cannot be incorporated to the known endocrinal function of this system. Especially, discovery of angiotensin components and receptors in brain led to formation of new hypothesis and functional concepts about local effects of RAS, based on local synthesis of Ang II and angiotensin IV (Ang IV). Current studies found that physiological effects of the central angiotensin receptors can play an important role in neuroprotection and brain perfusion, can affect stress and behavioral disorders and influence...

4 Abstract Senescence is a specific cell state distinquished by cessation of cell division and proliferation and changes in gene expression. Normal cells enter senescence after distinct number of cell divisions or in case of an unrepairable damage. Senescence in cancer cells can be induced by subliminal stress as sublethal treatment with certain drugs. Senescent cancer cells persist in the tissue and may secrete a number of factors and nutrients affecting surrounding cells. Senescence can thus change the response of cancer cells to various apoptogens during cancer therapy. In this study, we focused on the elucidation of presumed differences between normal proliferating and senescent cancer cells in their response to selected apoptogens. Implementing bromodeoxyuridine (BrdU)-mediated replication stress in cancer cells derived from pancreatic (PANC-1) or mesothelioma (H28) tumors, we efficiently forced these cells to acquire senescent phenotype. We document that these senescent cells gain higher resistance to combined TRAIL and homoharringtonine (HHT) treatment and enhance sensitivity to other apoptogens such as FasL, camptothecin and mVES. These cells also showed increased expression of anti-apoptotic protein c-FLIP in senescent cells and changes in the expression of some Bcl-2 family proteins....

Purinergic receptors are membrane ion channels that are activated by extracellular ATP. In vertebrates, seven genes encode subunits of P2X receptors. The subunits, designated P2X1-7, are 40 - 50% identical in amino acid sequences. P2X receptors are composed of three subunits and are found as homo- and heterotrimers in tissues of vertebrates. P2X receptors have a wide distribution in the organism, functional receptors are found in neurons, glial cells, muscle cells and also in nonexcitable tissues as epithelial, endothelial, and in hemopoietic tissue. Purinergic signalling plays an important role in pain transmission, at CNS injury and immune processes. P2X receptor subunit consists of two transmembrane domains, extracellular domain and intracellular N-and C-termini. Each transmembrane domain contains two amino acids conserved across all P2X subunits. In the first transmembrane domain receptor P2X2 are that Gly30 and Tyr43. In previous experiments performed on P2X2 receptor, electrophysiological measurements demonstrated that substitution of conserved Tyr43 in the first transmembrane domain with alanine prolongs the deactivation time of ion channel after agonist wash out. This work is focused on clarifying the role of conserved tyrosine in the process of opening and closing of ion channel of P2X...

This diploma thesis focuses on study of the expression of purinergic receptors P2X and P2Y in the cells of neonatal adenohypofysis. Purinergic receptors are activated by binding of extracellular ATP and their activation leads to an increase in intracellular concentrations of calcium ions. The aim of this thesis was to determine by microfluorimetry method whether neonatal pituitary cells contain purinergic receptors. In addition, the influence of various agonists and antagonists of purinergic receptors on intracellular concentrations of ions Ca2+ in neonatal pituitary cells was monitored. The influence of extracellular ATP on secretion of a luteinizing hormone was observed by radioimmunoassay and it was compared with the effect of a hormone GnRH. It was found that neonatal pituitary cells are less sensitive to extracellular ATP than adult pituitary cells, but the relative sensitivity to other agonists of purinergic receptors is similar to the one of adult pituitary cells. In the mixed population of neonatal pituitary cells there were identified ATP- and 2 MeSATP- sensitive purinergic receptors P2X, as well as ADP-sensitive receptors P2Y. Further, the BzATP-sensitive receptors P2X7, PPADS-sensitive receptors P2X2 and BDBD-sensitive receptors P2X4 were identified. Finally, it was proved that...

The aim of this thesis was to study the changes that occur in cell line H9c2 after exposure to an oxygen level reduced to 2 % for 24 hours. We monitored changes in compartmentation of chosen members of β-adrenergic signaling system. We found an increase in expression of β1AR and β2AR. Only β2AR showed change in compartmentation after hypoxia, as they relocate from membrane rafts to non-rafts fractions of membrane. AC also showed an increase of expression and was located in membrane rafts. The next aim of this work was to monitore apoptotic markers to determine whether there are activated pro-apoptotic or anti-apoptotic signals under chosen conditions of hypoxia. There was an increase in expression of both pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. We compare ratios of Bcl-2 to Bax and we found that there is a bigger increase in protein Bax expression. Another apoptotic marker, caspase 3, was tested and we also found that there was an increase in expression of caspase 3 in cells after hypoxia. Furthermore, we studied possible activation of kinase signaling pathways that may contribute to protective effects of hypoxia. Expression of Akt and ERK kinases was increased after hypoxia, but we did not confirm activation by phosphorylation of these kinases. Levels of phosphorylated Akt...

β-Adrenergic signalization plays an important role in heart, regulating cardiac frequency and contractility. It is also involved in development of hypertension and heart hypertrophy. Spontaneous hypertensive rat strain is a common model for human essential hypertension, although the origin of blood pressure abnormalities in SHR remains unknown. Dysfunction in the regulation of fatty acid translocase Cd36 was suggested as a link to development of hypertension in SHR. Transgenic strain SHR-Tg19 (also known as SHR-Cd36) was obtained, harboring a wild type of FAT/Cd36. This thesis aimed to investigate key elements of β-adrenergic signaling in the heart of SHR-Tg19 compared to their SHR controls. Expression and distribution of β1- and β2-ARs were measured using radioligand binding and Western blot analysis along with expression of selected G proteins and expression and activity of adenylyl cyclase. Our experiments showed that there were no significant changes in the Gsα and Giα subunits expressions, along with the amount of β1-AR in both left and right ventricles, according to the Western immunoblotting, but radioligand binding showed an increase in the quantity of β-ARs, particularly β2 subtype. Alongside, an increased expression of β2- ARs was observed in the right ventricle. Different...

Lectin-saccharide interactions are involved in many biological processes essential for the survival and proper function of multicellular organisms. C-type lectin-like receptors, predominantly expressed by cells of the innate immune system, recognize saccharide structures on microbes and also aberrant glycosylation pattern of cancer cells. The NKR-P1 receptor family was among the first natural killer (NK) receptor families that were identified, however ligands for some of members remain still elusive. Recently, publications describing N-acetylglucosamine-terminated oligosaccharide structures as possible ligands for NKR-P1 receptor have been subjects for correction/retractions after investigation of the Ethical Committee of the Institute of Microbiology, ASCR, v. v. i. and Charles University in Prague. Re-evaluation of glycodendrimer effect, particularly effect of N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P), revealed mostly indirect role of NK cells on modulation of immune responses. Properly folded soluble recombinant rat NKR-P1A and mouse NKR-P1C lack binding activity to neoglycoproteins modified with GlcNAc-terminated structures. Moreover, new possible target cell populations (NKT cells and macrophages) for saccharide binding were identified.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lenka Linhartová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Molecular mechanisms of intractions - interactions of constitutive androstane receptor with selected stilbene compounds Constitutive androstane receptor (CAR), member of nuclear receptors family, is a major regulator of gene expression of phase I and II enzymes metabolizing endobiotics and xenobiotics. Changes in its activity can lead to pharmacokinetic drug interactions, ineffective treatment or higher toxicity of drugs simultaneously administered with CAR ligands. Recently another effects of this receptor, especially in homeostasis of bile acids, lipids and glucose have been discovered and CAR is now considered as a potential drug target for the treatment of metabolic diseases. Stilbenes represent a small group of plant polyphenols with typical 1,2-diphenylethylene nucleus. The most famous member is resveratrol, which has attracted great attention thanks to its antioxidant, anti-inflammatory, antiproliferative and cardioprotective effects. Others stilbene compounds such as pterostilben, piceatannol or pinosylvin have shown similar health beneficial effects as well. The aim of this diploma thesis was...

Morphine is a natural opioid which is used in medicine due to his potent analgesic and sedative effects. In the forefront of scientific interest is a chronic usage of opioids which can lead to a development of drug addiction. Morphine role in oxidative stress was described in last years. It was revealed its protective potencial by many studies. However, some studies described its pro-oxidative effect. The aim of this study was to determinate effect of chronic morphine on cell survival after oxidative stress caused by H202 analog - tBHP in the SH-SY5Y neuroblastoma cell line. The results verified morphine protective effect against oxidative stress. The highest protective effect of morphine was achieved in a concetration of 10 µM. It was desribed that morphine can induce activation of mu-opioid (MOR) and Toll-like 4 (TLR4) receptors signalling pathway on molecular level. The aim of this thesis was to evaluate the role of MOR a TLR4 in protective effect of morphine against oxidative stress by two methods. Firstly, it was used tests of oxidative stress on cell viability. The obtained results demonstrated majority role of TLR4 and minory role of MOR. Afterwards, we assesed changes in the expression of MOR a TLR4 after chronic morphine by SDS-PAGE electrophoresis. Results of these experiments did not...