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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...
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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...
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Study of Transport Mechanisms of Receptor-Specific Radiopharmaceuticals in the Kidney Cells.
Cihlo, Jan ; Lázníček, Milan (advisor) ; Šimůnek, Tomáš (referee) ; Komárek, Pavel (referee)
Renal accumulation of radiolabelled receptor specific substances can represent clinical problems for diagnosis and therapy of some typical malignancies due to radionephrotoxicity of these substances. The aim of this work was to design a suitable in vitro model for evaluating cumulative disposals of studied radiopharmaceuticals in the kidney and for finding possible mechanisms reducing the reabsorption of these substances by renal tubular cells. The suggested model designed for this purpose provides another opportunity for evaluation of their radionephrotoxical potential. The aforementioned in vitro method was standardised and used for the study of transport mechanism of some representatives of radiolabelled somatostatin analogues (111In/125I/90Y/177Lu-DOTA-Tyr3-octreotate, 111In/90Y/177Lu-DOTA-1-Nal3-octreotide), antibodies (111In/90Y/99mTc-AntiCD66) and albumin (FITC-albumin, 99mTc-albumin). All studied radioactive substances were radiolabelled with good practice and analyzed using HPLC and/or ITLC-SG methods. The radiolabelling technique of all studied specifically acting substances (radiolabelled peptides and antibodies) and also any concrete radionuclide showed no significant influence over the internalization into the renal OK cells. The difference of the internalized amount of FITC-albumin and...
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Vazebné vlastnosti enzymů purinnukleosidfosforylasa a thymidinfosforylasa
Strašilová, Kateřina ; Sochor, Jaroslav (advisor) ; Šimůnek, Tomáš (referee)
5. CONCLUSION In this study, the ligand binding properties of two enzymes - purine nucleoside phosphorylase and thymidine phosphorylase - were tested, exploiting a method of surface plasmon resonance in Biacore X instrument. PNP is a ubiquitous enzyme playing a key role in the purine salvage pathway and TP plays an important role in human organism too, especially in pathological processes like inflammation or cancer. Both of enzymes are potential tools for the enzymatic synthesis of nucleoside analogues, which may possibly be used as antiviral or anticancer agents and that are difficult to prepare by chemical synthesis, or are obtained in a low yields. Use of Biacore X instrument helps to reveal how the enzymes interact with their natural substrates or derivatives. This work was focused on searching for the most suitable conditions, at first for immobilizing procedure (coating the surface of the sensor chip with PNP and TP enzyme) and then for interaction analysis between the enzymes and their natural substrates. The present results can be used as starting point for additional investigations of interaction analyses between enzymes and semi-synthetic analogues or they can also contribute to optimize conditions of producing such compounds by biocatalysis. In particular, the use of enzymes as biocatalysts...
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Study of the novel phtalocyanines for the vascular-targeted photodynamic therapy of tumors
Brázdová, Petra ; Macháček, Miloslav (advisor) ; Šimůnek, Tomáš (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Petra Brázdová Supervisor: Mgr. Miloslav Macháček Title of diploma thesis: Study of the novel phthalocyanines for the vascular-targeted photodynamic therapy of tumors Cancer is one of the leading causes of death in developed countries, therefore a big effort is devoted on research of novel anticancer drugs. Photodynamic therapy (PDT) is currently a well-established method in this area, but targeting its effect on tumor vasculature has not been considered until recently. In comparison with PDT, vascular-targeted photodynamic therapy (VTP) takes the advantage of a much shorter interval between administration of the photosensitizer (PS) and irradiation (drug-light interval) and thus turning the effect of cytotoxic agents to tumor vasculature. It is intended to disrupt the oxygen and nutrients supply to malignant cells and thus cause their damage and death. Aim of this work is to evaluate the efficacy of newly synthesized PSs from the group of phthalocyanines and azaphthalocyanines using the VTP protocol under in vitro conditions. The PSs used in this study have been investigated recently with classical PDT protocol, in which they have demonstrated very promising activity against tumor cells...
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In vitro evaluation of photodynamic activity of tetrapyridoporphyrazine derivatives for treatment of solid tumours.
Jedličková, Adéla ; Macháček, Miloslav (advisor) ; Šimůnek, Tomáš (referee)
Charles University in Prague Faculty of Pharmacy in Hradci Králové Department of Biochemical Sciences Candidate: Bc. Adéla Jedličková Supervisor: Mgr. Miloslav Macháček Title of diploma thesis: In vitro evaluation of photodynamic activity of tetrapyridoporphyrazine derivatives for treatment of solid tumors Nowadays, cancerous diseases belongs among the main causes of death, incidence continues to rise. Therefore research in the area of tumor treatment significantly focuses on the development of new potential anticancer drugs and therapeutical approaches. Photodynamic therapy (PDT) belongs to clinically approved therapeutic methods for the treatment of the malignant and nonmalignant diseases. PDT is composed of three main components - photosensitiser (PS), light and oxygen, all of them are non-toxic on their own. However, their interaction leads to a generation of highly reactive molecules. This method is based on absorption of light with appropriate wavelenght by PS and subsequent energy transfer from photon to surrounding molecules, especially to oxygen. This transfer leads to a generation of cytotoxic reactive oxygen species (ROS) of which singlet oxygen plays a main role. ROS subsequently causes a damage of surrounding cell structures, which leads to irreversible damage of cell functions and...
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