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Contribution of Next Generation Sequencing for Laboratory Diagnostics
Votýpka, Pavel ; Beránek, Martin (advisor) ; Macháček, Miloslav (referee)
5 ABSTRACT Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavel Votýpka Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Consultant: Mgr. Nikola Ptáková Title of diploma thesis: Contribution of Next Generation Sequencing for Laboratory Diagnostics The endeavor to sequence the whole human genome lead not only to the knowledge acquisition regarding the human genetic information but as well to the development of new sequencing methods and technologies. In order to keep up with progress in genetic field in many clinical and research laboratories the new massive parallel sequencing equipment is being utilized. On the market are currently established four leading platforms - Illumina, Solid, Ion Torrent and 454 Life Technologies. The process of sequencing analysis can be summarized into three main steps - the sequencing library preparation, sequencing itself, variant calling and data analysis. Each part of the sequencing analysis exhibits certain specifics, we need to count with and as well its pitfalls, we need to avoid or to minimalize their impact on the analysis final result. Recently new methods termed sequencing of the 3rd generation are being developed, enabling sequence of a single DNA molecule to be determined without previous...
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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...
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In vitro effects of 3-hydroxytyrosol on renal hypoxia and inflammation
Kamasová, Terézia ; Boušová, Iva (advisor) ; Macháček, Miloslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Terézia Kamasová Supervisor: Asst. Prof. PharmDr. Iva Boušová, Ph.D. Prof. Alice Santos-Silva Maria João Valente, Ph.D. Title of diploma thesis: In vitro effects of 3-hydroxytyrosol on renal hypoxia and inflammation Chronic kidney disease (CKD) results from a group of heterogeneous disorders affecting the kidneys. The renal hypoxia and hypoxia-derived oxidative stress, renal fibrosis, and inflammation are highly prevailing conditions appearing in the diseased kidney, contributing to the progression of CKD. Phytochemicals are an essential part of contemporary therapeutic strategies for the treatment of various diseases. 3-Hydroxytyrosol (HT), a phenolic compound extracted from olives and olive-derived products (e.g. olive oil), is believed to carry a potent antioxidant, anti-inflammatory, antithrombotic, bactericidal and bacteriostatic activity. The aim of this work was to determine the preventive effect of HT in hypoxic renal cells and evaluate the effect of HT on hypoxia-related inflammation, fibrosis, and oxidative stress, in order to summarize the value of this phenolic compound as a promising novel remedy in the treatment of CKD. A cell line of human renal proximal tubular cells (HK-2) was...
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Biological evaluation of photodynamic activity of amphiphilic anionic photosensitizers
Halašková, Marie ; Macháček, Miloslav (advisor) ; Šimůnek, Tomáš (referee)
1 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Marie Halašková Supervisor: RNDr. Miloslav Macháček, Ph.D. Title of diploma thesis: Biological evaluation of photodynamic activity of amphiphilic anionic photosensitizers Tumor diseases are a global health problem and one of the most common causes of death in developed countries. Therefore, considerable effort is devoted to research and develop novel anticancer drugs. Common conventional therapies, such as chemotherapy, radiotherapy, surgery, or their combination, are often associated with severe side effects and high treatment costs. Photodynamic therapy (PDT) is therefore being increasingly recognized as an alternative treatment modality for the treatment of many malignant and non-malignant conditions. PDT is a clinically approved non-invasive treatment which consists of two relatively simple procedures: administration of a photosensitive drug and subsequent radiation of the tumor by light to activate the photosensitizer (PS). The activated photosensitizer reacts with molecular oxygen to form highly reactive oxygen species (ROS). This creates a state of oxidative stress in the tumor which leads to tumor cell death, tumor (micro)vasculature shutdown and induction of anti-tumor immune...
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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...
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In vitro study of newly synthesized potential cardioprotective drugs
Liptáková, Lucie ; Hašková, Pavlína (advisor) ; Macháček, Miloslav (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucie Liptáková Supervisor: RNDr. Pavlína Hašková, Ph.D. Title of master thesis: In vitro study of newly synthesized potential cardioprotective drugs Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are in an organism generated under normal or pathological conditions. There are antioxidant mechanisms, which protects the organism from their harmful effect. In case of imbalance between ROS/RNS production and antioxidant mechanisms, an oxidative stress is initiated. The oxidative stress is involved in the pathogenesis of many diseases, including cardiovascular desease. In consequence of higher presence of mitochondria and lower presence of antioxidants cardiomyocytes are more sensitive to the oxidative stress. Iron, by catalysing radical's reactions, significantly participates on formation and development of oxidative stress. Elimination of the free iron by iron chelators is one option how to prevent or moderate oxidative stress. The aim of this master theses was to study cardioprotective effect in presence of H2O2 and own toxicity of newly synthetized aroylhydrazone iron chelators (H21, H22, H23, H24, H25 and H26) on rat embryotic cardiomyoblasts H9c2. Protective and toxic...
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Study of cytoprotective properties of novel oxidative stress-activated aroylhydrazone pro-chelators of iron
Macháček, Miloslav ; Šimůnek, Tomáš (advisor) ; Lenčová, Olga (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Miloslav Macháček Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Study of cytoprotective properties of novel oxidative stress-activated aroylhydrazone prochelators of iron Oxidative stress plays an important role in many disorders including cardiovascular diseases. Fenton reaction catalysed by intracellular redox-active iron is an important source of highly reactive and toxic hydroxyl radicals. Hence, shielding of free iron ions seems to be an advantageous strategy for prevention of further propagation of oxidative stress and myocardial damage. Results from our laboratory as well as other groups have shown promising cardioprotective effect in vitro as well as in vivo experiments. On the other hand, excessive depletion of iron may result in toxicity to the cells. The aim of this project was therefore to examine new iron prochelators, which chelating activity develops only after activation by reactive oxygen species. Boronyl ester of salicylaldehyde isonicotinoyl hydrazone (BSIH) and its derivate BSIH-PD were examined in vitro for their potential protective properties against toxic reactive oxygen species (ROS). Furthermore, own toxicities of studied compounds...
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Effects of simple sugar consumption on cognitive functions in female rats
Podhorská, Gabriela ; Macháček, Miloslav (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Gabriela Podhorská Supervisor: Mgr. Miloslav Macháček, Ph.D. Title of diploma thesis: Effect of simple sugar consumption on cognitive functions in female rats The consumption of simple sugars, especially fructose, increased in the past few decades. Excessive sugar intake has been associated with the development of insulin resistance, hypertriglyceridemia and oxidative stress which lead to the impairment of cognitive functions. Nevertheless, it is still unclear whether these alterations in cognitive functions are exclusively caused by high caloric intake or if they are related to specific characteristics of fructose. Moreover, the mechanisms involved are not yet fully deciphered at the molecular level. Our research group supplemented Sprague-Dawley female rats with 10% w/v glucose in drinking water or with isocaloric fructose solution in a long-term study (7 months), simulating the chronic consumption of sugars in humans. Plasma parameters and expression of proteins, involved in metabolic pathways, were determined in the frontal cortex. Cognitive functions were evaluated through the novel object recognition (NOR) and Morris Water Maze (MWM) tests. Results, obtained in our study, showed a...
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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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