|
| |
|
|
STUDY OF POTENTIAL PHARMACOLOGICAL PROTECTION OF CARDIAC CELLS AGAINST OXIDATIVE STRESS AND ANTRACYCLINE ANTICANCER DRUGS
Jansová, Hana ; Šimůnek, Tomáš (advisor) ; Hrdina, Radomír (referee) ; Jun, Daniel (referee)
CHARLES UNIVERSITY IN PRAGUE FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ DEPARTMENT OF BIOCHEMICAL SCIENCES Candidate: Mgr. Hana Jansová Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of Doctoral Thesis: STUDY OF POTENTIAL PHARMACOLOGICAL PROTECTION OF CARDIAC CELLS AGAINST OXIDATIVE STRESS AND ANTRACYCLINE ANTICANCER DRUGS Development of cardiovascular disorders is associated with various risk factors and oxidative stress plays an important role in many of them. Iron-catalysed production of highly toxic and reactive hydroxyl radicals may contribute to oxidative stress. Chelation of free iron seems to be a promising strategy to prevent the propagation of oxidative stress. However, the use of classic iron chelators in pathological conditions without iron overload is associated with the risk of toxicity due to the iron depletion. Hence, this study deals with cardioprotective properties of iron chelators as well as prochelators derived from them. We focused on prochelators with almost no affinity for iron ions until they are activated under disease-specific oxidative stress conditions. For a long time, it has been assumed that oxidative stress is also the main denominator in an anthracycline-induced cardiotoxicity. However, the previous studies suggested alternative mechanism(s). Therefore in the...
|
|
|
Ovlivnění buněčné direrenciace u nádorových buněk analýzou exprese proteinu Serca3 jako markeru diferenciace
Šerá, Pavlína ; Pávek, Petr (advisor) ; Šimůnek, Tomáš (referee)
In the present work we investigated the process of cellular differentiation induced by histone deacetylase inhibitors in cell lines derived from various types of non- small cell lung cancer (NSCLC). Our objective was to study the cross-talk between the process of cellular differentiation and expression of Sarco-Endoplasmic Reticulum Calcium ATPases (SERCA proteins), enzymes that transport calcium from cytosol to the endoplasmic reticulum. Various lung cancer cell lines were grown in vitro, subjected to treatments by various short chain fatty acid-derived histone desacetylase inhibitors, and SERCA expression was determined in a semi-quantitative Western blot format using the IID8 and PLIM430 anti-SERCA monoclonal antibodies that recognise SERCA2 and SERCA3, respectively. Our experiments show that the expression of SERCA proteins and cellular differentiation are interconnected in all lung studied cancer cell lines (A549, Calu-3, ChaGoK-1, NCI-H292 and NCI-H460). In particular, the induction of the expression of SERCA3 protein was observed after the induction of cellular differentiation by histone deacetylase inhibitors such as sodium butyrate, valerate and phenylbutyrate. Induction of SERCA3 expression was specific, because SERCA2 levels were not modified by the treatments. Taken together, our...
|
|
|
Study of the protective properties of the series of novel aroylhydrazone iron chelators against the oxidative stress-induced cardiomyocyte injury
Pravdíková, Kateřina ; Šimůnek, Tomáš (advisor) ; Lenčová, Olga (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Pravdíková Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Study of the protective properties of the series of novel aroylhydrazone iron chelators against the oxidative stress-induced cardiomyocyte injury. Oxidative stress is an imbalance between oxidants and antioxidants in favor of oxidants. In this process occurs the formation of reactive oxygen species (ROS) via the Haber-Weiss reaction, where a redox-active iron participates as the catalyst. ROS play an important role in the pathogenesis of many diseases, including cardiovascular diseases. After clarifying the role of iron in these processes, attention has focused on iron chelators. They may remove free iron ions, which thus cannot catalyse radical reactions, and prevent further development of oxidative damage to the myocardium. The aim of this work was to study the cardioprotective effects of newly synthesized aroylhydrazone derivatives of iron chelator SIH against the toxic effects of hydrogen peroxide in vitro. Their own toxicities were also examined. H9c2 cell line derived from rat embryonic cardiac myoblasts was used for evaluation of the protective and toxic effects of chelators. The evaluation...
|
|
|
Thiopurine methyltransferase - clinical importance of genotyping and phenotyping
Černá, Blanka ; Beránek, Martin (advisor) ; Šimůnek, Tomáš (referee)
Charles University in Prague Faculty of pharmacy in Hradec Králové Department of Biochemical science Candidate: Blanka Černá Supervisor: Doc. PharmDr. Martin Beránek, PhD. Title of Diploma Thesis: Thiopurine S-methyltransferase - clinical importace of genotyping and phenotyping. Thiopurine S-methyltransferase catalyzes S-methylation thiopurine's drugs such as 6-mercaptopurine and thioguanine. TPMT genetic polymorphisms represent an important role in clinical pharmacogenetics. The differences in TPMT activity result from mutations in gene for TPMT. The polymorphisms are important factor in efficacy of treatment by thiopurine drugs. Patients inheriting low activity of enzyme TPMT have mutated allels, patients inheriting high activity of TPMT are usualy wild types. TPMT gen was genotypized by method real-time PCR in volunteers (n=55) with autoimmune diseases. The average of patient's age was 16,7 years. From blood collected into EDTA DNA was isolated by using QIAmp Mini Kit (Quiagen, Germany) and it was used for genotyping of TPMT. Genotyping was carried out by real-time PCR in LightCycler (Roche, Germany). TPMT was phenotypized in Hradec Králové in Medical Faculty of Charles University in Department of Pharmacology. The lysate of suspension of erythrocyte was used for phenotyping (The blood was...
|
|
|
Development of methods for in vitro testing of potential drugs against Alzheimer disease
Hrabinová, Martina ; Šimůnek, Tomáš (advisor) ; Soukup, Ondřej (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Healthcare bioanalytics - Specialist in Laboratory Methods Department of Biochemical Sciences Candidate: Bc. Martina Hrabinová Supervisor: Doc. PharmDr. Tomáš Šimůnek Ph.D. Supervisor specialist: Pplk. Doc. PharmDr. Daniel Jun Ph.D. Title of diploma thesis: Development of method for in vitro testing of potential drugs Alzheimer disease Alzheimer's disease (AD) is a neurodegenerative disease with increasing incidence. Although many decades have passed since the disease was discovered, there is no causal therapy yet. Currently available therapy consists in treatment with central acetylcholinesterase inhibitors and memantine, improving the patient's quality of life. The aim of this study was to develop a simple colorimetric method for the determination of prolyl oligopeptidase (POP) and beta secretase (BACE) activity, important enzymes associated with AD pathogenesis, and to use this method for screening of potential AD drugs and to determine their antioxidant and antiradical activity. Results showed that tested substances were weak inhibitors of POP compared to standard inhibitor Z- Gly-Pro- prolinal. Standard cholinesterase inhibitors used in AD therapy showed no ability to inhibit POP. The method for determination of BACE activity...
|
|
|
Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer
Macháček, Miloslav ; Šimůnek, Tomáš (advisor) ; Řezáčová, Martina (referee) ; Mosinger, Jiří (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of biochemical sciences Candidate Mgr. Miloslav Macháček Supervisor doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of Doctoral Thesis Study of novel phthalocyanine and azaphthalocyanine photosensitizers for the photodynamic therapy of cancer. Photodynamic therapy (PDT) of cancer is non-invasive treatment modality for solid tumour treatment using three basic components - molecular oxygen, light and photosensitizer (PS). These elements are essentially non-toxic on their own, but in the combination they induce reactive oxygen species (ROS; singlet oxygen mainly) production, causing damage to cellular components and subsequent cell death. Type of cell demise is dependent mainly on the type of PS, length of irradiation and subcellular localization of the drug. Singlet oxygen is highly reactive and is capable of limited diffusion in biological environment. Apart from direct cytotoxic effect, vascular shutdown (oxygen and nutrition deprivation) and activation of immune system are involved in tumour eradication. Combination of effective compound with delivery system, conjugation with targeting substances or synthesis of highly effective non-aggregating water soluble compounds are the main pathways in design of modern PSs. In our...
|
|
|
Assessment of the cytotoxic effects of selected transdermal permeation enhancers in fibroblasts
Tükörová, Katarína ; Šimůnek, Tomáš (advisor) ; Vávrová, Anna (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Katarína Tükörová Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Assessment of the cytotoxic effects of selected transdermal permeation enhancers in fibroblasts Transdermal permeation enhancers facilitate the process of drug penetration trough the skin barrier into the systemic circulation or into surrounding tissues. This approach has many advantages compared to traditional ways of drug administration, hence we observe intense research in this area in last decades. The mechanism of action of enhancers is not exactly elucidated yet. They reduce reversibly barrier properties of skin by nonspecific interactions with components of the stratum corneum (SC), i.e. proteins and lipids, or change the partitioning between the SC and the drug. The aim of our work was to determine the cytotoxic effects of selected transdermal permeation enhancers comparing IC50 values of new substances synthesized in our department with clinically used agents. The mouse embryonic fibroblasts 3T3 cell line was used in our assay. We used the MTT test to determine the viability of cells, only viable cells were able to reduce the yellow dye to blue formazan. Cells were treated with selected...
|
|
|
Study of the role of glutathione in the anthracycline-induced cardiomyocyte injury
Vávrová, Anna ; Šimůnek, Tomáš (advisor) ; Kvasničková, Eva (referee)
The clinical usefulness of anthracycline antineoplastic drugs has been limited by the risk of serious cardiotoxicity. Its precise mechanisms have not been fully elucidated, although the oxidative stress is widely believed to play a principal role. The anthracycline toxicity to heart may result from its high accumulation in myocardial tissue, richness on mitochondria producing reactive oxygen species and relatively poor antioxidant systems including superoxide dismutase, catalase and glutathione peroxidase. This study is focused on the role glutathione in daunorubicin (DAU)-induced cardiotoxicity both in vitro and in vivo. Total, reduced and oxidized glutathione (GSH/GSSG) have been determined by a spectrophotometric method using dithiobis(2-nitrobenzoic acid) in DAU (0.1 - 10 µM) - exposed H9c2 cardiomyoblast cell line and heart tissue from daunorubicin-treated rabbits (3mg/kg weekly, 10 weeks). In H9c2 cells, DAU induced significant toxicity (IC50=0.48 µM), levels of total glutathione were slightly but insignificantly decreased, the levels of oxidized glutathione were unafected. Neither buthionine sulfoximine (BSO, an inhibitor of glutathione synthesis) nor 2-oxo-4-thiazolidine-carboxylic acid (OTC, a glutathione synthetic precursor) had any significant effects of DAU cytotoxicity. This contrasted...
|
|
|
Gene expression profiling after experimental perinatal asphyxia and the effect of complement-derived anaphylatoxin C3a
Šourková, Hana ; Wsól, Vladimír (advisor) ; Šimůnek, Tomáš (referee)
Background: The complement system is involved in neuroprotection and brain repair after brain damage. To understand the molecular mechanisms of these processes, we performed gene expression profiling using quantitative real-time polymerase chain reaction (qPCR), which is the most accurate modern strategy for gene expression analysis. Project: Our project was directly aimed at expression profiling of selected genes potentially involved in loss and rescue of neural tissue during three weeks after hypoxic-ischemic brain injury, an experimental model of perinatal asphyxia. Recent experiments have shown that over-expression of C3a under the control of the GFAP promoter (C3a/GFAP) reduced hippocampal injury after left common carotid artery ligation in neonatal mice by 50%, compared to wild type mice. Here, we assessed how the local expression of C3a/GFAP transgene affects gene expression profiles. Gene expression was measured on samples from hippocampus ipsilateral and contralateral to the injury and ipsilateral part of cortex, taken at the time of injury, 6 and 24 hours; 3, 7 and 21 days after the injury. Results: Our data showed that the regulation of gene expression after hypoxic-ischemic injury differs in timing and intensity and may also be region dependent. The analysed genes belong to families...
|
guest ::
