| |
|
The study of photodynamic phenomenon on melanom cell lines
KOLÁČKOVÁ, Zdeňka
Photodynamic therapy (PDT) is a medical method combining the use of photodynamic active substance and light in presence of oxygen. It extends the treatment possibilities of tumor and non-tumor disorders. It complements surgical treatment, radiotherapy, chemotherapy and immunotherapy. An increased accumulation of photosensitive substance in pathological focus is the base of PDT. Subsequent irradiation by light of suitable wavelenght evokes photodynamic reactions leading to formation of reactive oxygen species and to biological answer leading to tumor cells damage. The final effect depends on sensitizer type, its concentration in target tissue and on used source of radiation. Aim of the thesis is to prove photodynamic properties of newly developed photodynamic active substance phtalocyanin CIAIPcS2 and possibilities of its usage to induce photodynamic phenomenon in melanom cells. Owing to absorption of light in sensitizer the formation of excited states happens, and then the excited form of sensitizer reacts directly with substrate. During this reaction free radicals of substrate form or transfer of energy from sentitizer to oxygen and formation of highly reactive singlet oxygen happen. Free radicals, especially radicals of lipid components of cell membranes, are the major cause of tumor destruction. Fruitfulness of PDT depends not only on type of sensitizer and level of oxygen in tumor cell but also on used light source. Luminiscence diodes (LEDs) were used as the source of light. Formation of ROS after PDT was detected with the help of fluorescent molecular probe CM-H2DCFDA on spectrofluorimeter Synergy HT and on fluorescent microscope Olympus IX 81 with image analysis. According to our results the production of ROS depends on concentration of sensitizer CIAIPcS2 and the radiation dose. We proved that the sensitizer CIAIPcS2 is suitable photodynamic active substance and evokes photodynamic phenomenon in tumor cells.
|
|
Dendritic cells in health and disease
Horváth, Rudolf ; Špíšek, Radek (advisor) ; Krejsek, Jan (referee) ; Stříž, Ilja (referee)
During the past decades several spectacular finding s have been made in the field of immunology. Elucidating the functions of the antigen presenting cells (APCs) belong to the most important. Dendritic cells (DCs) represent a specific group of APCs with a unique ability to initiate primary immune responses. Despite the fact that, in vivo, they are very rare and difficult to isolate, DCs came very fast into the focus of scientific interests. Development of novel laboratory techniques facilitated a robust expansion of their research. With time it has been proven that DCs play a pivotal role in initiation, maintenance and control of the immune responses. The extraordinary features of DCs were soon investigated in human clinical trials, where DCs have been particularly used as vectors for vaccination protocols, especially in the treatment of tumors. However, DCs capability to polarize the outcome of immune response and the potential to induce or suppress immunity under specific circumstances led to the idea that they might be also used in the treatment of autoimmune and allergic diseases or in transplantation medicine as well. There is a need to stress that most of the knowledge has been obtained from the in vitro generated DCs, but advanced technological methods bring us the opportunity to study DCs directly...
|
|
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...
|
|
The role of dendritic cells in various pathological states
Sochorová, Klára ; Bartůňková, Jiřina (advisor) ; Tučková, Ludmila (referee) ; Stříž, Ilja (referee)
Dendritic cells (DCs) represent one of the most important components of the immune system. DCs are the most effective antigen presenting cells with a unique ability to stimulate naive T cells. They ensure the crosstalk between innate and adaptive immunity. They participate in anti-infectious and anti-tumor immune reaction as well as in the induction of tolerance. It is clear, that the defect in DC can be fatal for the organism. In our work we studied the biology of DCs and the disturbances of DCs function in pathological states. We analyzed DCs in patients with Bruton's tyrosin kinase deficiency, we compared the effect of vitamin D analogs, calcitriol and paricalcitol, on DCs and we set up the protocol of DC generation for the immunotherapy of ovarian cancer. In the study concerning btk deficiency and dendritic cell function we found profound impairment of IL-6 and TNF production in response to the stimulation by Toll-like receptor ligand 8. In the second part of our work we compared the effect of calcitriol and paricalcitol on DCs. Both drugs inhibited DCs maturation and decreased their ability to induce proliferation of antigen specific T cells. In the third part of our work we set up the protocol for the DCs, which are able to induce tumor specific immune responses. We optimized the form of tumor antigen...
|
|
Plant alkaloids and their effects on enzymes metabolizing xenobiotics
Višněvská, Kateřina ; Stiborová, Marie (advisor) ; Černá, Věra (referee)
Sanguinarine and chelerythrine are quaternary benzo[c]phenanthridine alkaloids. The first step in sanguinarine metabolism is its reduction to dihydrosanguinarin. Antimicrobial and anti-inflammatory activities of these alkaloids are used in dentistry and as feed additives. Sanguinarine and chelerythrine induce apoptosis of cells. Fluorescence of these alkaloids and intercalation into DNA could be utilized to use the alkaloids as supravital DNA probe. Negative effect of sanguinarine and chelerythrine is their genotoxicity. Cytochrome P450 and peroxidase oxidize ellipticine to detoxication and activation metabolites. Ellipticine is a potent antineoplastic agent exhibiting the multimodal mechanism of its action. Ellipticine intercalates into DNA and inhibits topoisomerase II. Covalent DNA aducts are mediated by CYP or peroxidase oxidation of ellipticine. The anti-tumor activity of ellipticine and its derivatives is caused by a combination mechanism of cell cycle arrest and induction of the apoptotic pathway. Pharmacological efficiencies and geneotoxic side effects of ellipticine is dependent on levels and activities of cytochrome P450 or peroxidase in target tissues. Aristolactams are the major metabolites of biotransformation of aristolochic acid. Nitroreduction is the crucial step in formation of an...
|
|
Role of chemopreventive compounds and food additives in the process of metabolism and activation of carcinogens by cytochromes P450
Fousová, Petra ; Hodek, Petr (advisor) ; Hansíková, Hana (referee)
The origin of malignant tumors have different internal and external factors and also have been shown that, for majority of exogenous chemical carcinogens, the genotoxicity depends on metabolic activation through enzymes. Members of the subfamilies CYP1A are involved in activation of precarcinogens, for example the heterocyclic amine PhIP. One of the main approaches to achieve a reduction in a cancer risk is prevention. Recently, the consumption of dietary supplements containing various chemopreventive substances, such as flavonoids, has expanded. On the other hand, some negative effects of these compounds were also confirmed, especially their ability to induce cytochrome P450 and thus increase the risk of activating precarcinogens. In this study, the inductive effect of a single administration of chemopreventive compounds, namely the effect of -naphtoflavon to cytochrome P450, had been investigated. Furthermore, a sequential study was carried out. In this study rats were first given an inducer, the -naphtoflavon, and after a passed interval they were given the carcinogen PhIP. Finally, the effect of PhIP on the activity of cytochrome P450 has been studied in vitro. KEY WORDS: cytochrome P450, carcinogens, chemopreventive compounds
|
|
Carbohydrate dimers in tumor immunotherapy
Krupová, Monika ; Bezouška, Karel (advisor) ; Ryšlavá, Helena (referee)
Carbohydrate dimers in tumor immunotherapy Monika Krupová (Department of Biochemistry, Faculty of Science, Charles University in Prague) One possible approach to tumor immunotherapy is an activation of killer lymphocytes through specific ligands for their surface receptors. CD69 is a molecule greatly widespread among various cells of haematopoietic origin. Since the physiological ligand for this receptor is still unknown, ligand mimetics are used for modulation of its activity. The mimetics tested in this work are based on monomeric or oligomeric carbohydrated attached through two different chemical groups to the central linker of varying length, giving rise to thiourea and triazole series. The ability to precipitate soluble NKR-P1 and CD69 receptors was evaluated in precipitation assays and the optimal length of the linker for NKR-P1 receptor was found to be decyl. On the other hand, cross-linking of CD69 is not so dependent on the length of the linker. The aim of this work was to describe in vitro effect of the tested compounds on cellular signalization, natural killing of leukemic cell lines and activation-induced apoptosis. Compounds of triazole series containing two disaccharides (GalNAc β1→4 GlcNAc) linked by a linker were found to have the strongest effect on the production of...
|
|
Úloha Mst1 / FoxO dráhy při indukci apoptosy
Lettlová, Sandra ; Bezouška, Karel (advisor) ; Liberda, Jiří (referee)
Vitamin E analogue -tocopheryl succinate (-TOS) from the group of mitocans, the drugs targeting mitochondria, is a selective inducer of apoptosis in various cancer cell types, which involves the accumulation of reactive oxygen species (ROS). It was found that ROS generation causes p53-independent upregulation of the pro-apoptotic protein Noxa that induces apoptosis by displacement of the BH3-only protein Bak from its inactive complex with the anti-apoptotic protein Mcl-1 to form a pore in outer mitochondrial membrane. Current research has demonstrated that generation of ROS causes activation of Mst1, a component of the Hippo pathway that presents a universal size-control mechanism in all metazoans, whose deregulation is linked to tumorigenesis. Treatment of Jurkat cells with - TOS revealed that activated Mst1 kinase phosphorylates the Forkhead box O1 (FoxO1) transcription factor that then translocates to the nucleus and activates transcription of genes important for apoptosis induction, including NOXA. This explains the p53 independent apoptosis induction and presents Mst1-FoxO1-Noxa as a new pathway involved in the process. Current research has also documented that activated Mst1 kinase controls the expression of the c-MYC oncogene and its target genes, whose products are involved in glucose...
|
|
Monitoring of immune parameters during anti-tumor immunotherapy
Bílková, Pavla ; Palich Fučíková, Jitka (advisor) ; Fialová, Anna (referee)
Dendritic cells are the most effective antigen presenting cells in humans, they stimulate naive T lymphocytes and thus initiate specific immune response. The discovery of dendritic cells and understanding of their functions contributed to the idea of usingdendritic cells for the treatment of cancer. Anti tumor immunotherapy is a therapeutic strategy that aims to induce and maintain immune responses against tumor cells. Currently, immunotherapy based on dendritic cells has strong position among other anti cancer therapies and seems to be a promising therapeutic option for patients with tumors. In this work, I evaluated the effectiveness of treatment in patients with prostate cancer treated with immunotherapy based on dendritic cells. I focused on the detection of antigen specific T lymphocytes in peripheral blood against tumor antigens, PSA, NY ESO 1, MAGE A1 and MAGE A3. Using a 3 day standard protocol for the detection of antigen specific T cells using intracellular cytokine staining we were able to detect only a small percentage of this minor population. Only after extension of the protocol, we increased the sensitivity setting and we detected a significantly increased frequency of antigen specific T lymphocytes in the peripheral blood after one year DC vaccines application.
|
guest ::
