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Effect of a new FLT3 inhibitor on induction cell death in leukemia cell lines
Hotáryová, Andrea ; Čečková, Martina (advisor) ; Novotná, Eva (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Andrea Hotáryová Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Effect of a new FLT3 inhibitor on induction of cell death in leukemia cell lines Approximately 30% of patients with acute myeloid leukemia have a Flt3 kinase mutation, which is clearly a negative prognostic factor of the disease. Therefore, the development of new Flt3 inhibitors represents a major advance in the therapy of AML in these patients. Although standard treatment with chemotherapeutics (cytarabine and anthracycline) and allogeneic stem cell transplantation, which have been used in AML therapy for the past 40 years, lead to complete remission in many cases, they do not reduce the frequency of relapse and bring many risks associated with the effect on healthy cells of the body. The purpose of developing targeted therapy, including Flt3 kinase inhibitors, is to prevent this adverse effect of chemotherapeutics. The aim of the research carried out as part of this diploma thesis was to determine the effect of the newly synthesized Flt3 inhibitor K1872 on the proliferation of leukemic cells with wild type Flt3 kinase (THP-1) and cells with the most frequently occurring Flt3-ITD mutation (MOLM- 13 and...
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Functional assessment of Bcl-2 family proteins in mitochondrial metabolism and beyond
Sovilj, Dana ; Anděra, Ladislav (advisor) ; Mráček, Tomáš (referee) ; Živný, Jan (referee)
(CZ) Od jejich prvotní identifikace v háďátku C. elegans a také v lidských buňkách před více než 30 lety jsou proteiny z rodiny Bcl-2 spojovány s indukcí, regulací a potlačením mitochondriální apoptotické signalizace, ale také se mohou uplatňovat při modulaci neapoptotických signálních dráh. V této studii jsme si stanovili za hlavní cíl rozšířit stávající znalosti o neapoptotických rolích hlavních proapoptotických proteinů z Bcl-2 rodiny, BAX a BAK, zejména pak na jejich roli v buněčném metabolismu. Pomocí genové editace využitím CRISPR/Cas9 jsme eliminovali expresi těchto proteinů v lidských nádorových buňkách různého tkáňového původu a v těchto Bax/Bak- deficitních buňkách jsme primárně analyzovali mitochondriální respiraci a buněčnou glykolýzu. Zatímco eliminace exprese Bax a Bak neměla žádný patrný vliv na glykolýzu ve všech testovaných buněčných liniích, v závislosti na buněčném typu modulovala mitochondriální respiraci. Eliminace exprese Bax a Bak v buňkách rakoviny tlustého střeva HCT-116 neměla vliv na mitochondriální respiraci, ale zjevně ovlivnila mitochondriální respiraci v Bax/Bak-deficitních buňkách odvozených od glioblastomu (U87) a lymfomů (HBL-2, UPF1H, UPF1G). Bax/Bak -/- buňky U87 významně upregulovaly mitochondriální respiraci a akcelerovaly svou proliferaci a také nádorový růst...
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Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2
Kalábová, Dana ; Obšilová, Veronika (advisor) ; Pavlíček, Jiří (referee) ; Žáková, Lenka (referee)
Molecular mechanism of 14-3-3 protein dependent regulation of caspase-2 Abstract Caspase-2 is a protease standing apically in the cascade of reactions leading to apoptosis. Properly functional apoptosis eliminates damaged cells, autoreactive lymphocytes or redundant groups of cells in ontogeny. The process of caspase-2 activation must be precisely regulated. One of the described ways of caspase-2 regulation causing its inhibition is posttranslational modification phosphorylation with subsequent binding of the regulatory scaffold protein 14-3-3. The aim of this dissertation is to explain the molecular mechanism of this regulation. To understand the interaction between the proteins, it was necessary to first identify the phosphorylation sites in the caspase-2 molecule recognized by the 14-3-3 protein and then describe the detailed structure of the binding complex. The structure was characterized by a number of biochemical and biophysical methods, such as analytical ultracentrifugation, native electrophoresis in TBE buffer, polarization-fluorescence assay, hydrogen/deuterium exchange coupled to mass spectrometry, or crystallization; and the results led to stimulating conclusions. Activation of caspase-2 begins with its binding to adaptor proteins, cleavage and dimerization of the catalytic subunits. The...
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Preparation and characterization of nanoliposomal carriers of hydrophobic cytostatics using nanofluidic mixing
Zelníčková, Jaroslava ; Mašek,, Josef (referee) ; Turánek, Jaroslav (advisor)
This diploma thesis is focused on preparation of liposome by relatively new method called nanofluidisation. This method allows the controlled preparation of small unilamellar liposomes in one step. In my thesis I was dealing with optimalization of liposomes preparation which carry hydrophobic cytostatics using this method. Cytotoxic effect of liposomes carrying hydrophobic cytostatics in vitro on cell lines A549 and MCF-7 was determined. In cytotoxicyty test I compared the effect of hydrophobic cytostatics (paclitaxel and derivates of vitamin E specifically alfa-Tos, alfa-TEA) that were incorporated into liposomes prepared via nanofluidisation method and lipid film hydration method. Moreover, a technology of lyophilisation in the presence of cryoprotectants for preparation of liposomes using the method of nanofluidisation was developed.
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Structural studies of selected protein complexes involved in signal transduction
Honzejková, Karolína ; Obšil, Tomáš (advisor) ; Bouřa, Evžen (referee) ; Pavlíček, Jiří (referee)
Protein-protein interactions are critical for most physiological and pathophysiological processes. Detailed characterization of these interactions is therefore essential not only to understand the nature of these events, but also to design strategies to target these interactions. This work focuses on the study of the structure and interactions of several proteins and their complexes. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the p38/JNK protein kinase pathways, thereby directing cells toward an inflammatory response or apoptosis. ASK1 interacts with thioredoxin (TRX), a small dithiol oxidoreductase, which inhibits ASK1, but the mechanism of this inhibition has not been clarified. CaMKK1 and CaMKK2 are Ca2+ /calmodulin (CaM)-dependent protein kinases that regulate cellular energy balance, memory, and inflammation, among others. Both are inhibited by 14-3-3 proteins, but despite their domain and sequence similarities, the extent of 14-3-3 protein- mediated inhibition is different. Estrogen receptor alpha (ERα) is a nuclear receptor involved in breast cancer. Tamoxifen, an ERα antagonist, is used to treat this disease, but resistance often develops. 14-3-3 proteins interact with ERα and inhibit its transcriptional activity,...
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Structural and functional characterization of the E3 ligase BIRC6
GRATZL, Sascha
The aim of the thesis was to characterize the ubiquitin ligase BIRC6 in several contexts. The goal was to expand the knowledge on the selectivity of E1-E2 recognition, to increase the known substrate range of BIRC6, and to elucidate the molecular interactions fulfilled by BIRC6 during autophagy via interaction with GABARAP. In addition, it was aimed to gain a better understanding on the general ubiquitination mechanism in E2/E3 hybrid enzymes, to identify the ubiquitination sites of known apoptotic and autophagic substrates and to enlighten substrate recognition by BIRC6.
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Pathofysiology of a tumour microenvironment of salivary glands cancer
Kuchař, Martin ; Skřivan, Jiří (advisor) ; Pokorný, Jaroslav (referee) ; Lohynská, Radka (referee)
Treatment options for salivary gland carcinomas (SGC), especially advanced ones, are limited. Immunotherapy, particularly therapy with immune checkpoint inhibitors (ICI), has brought significant progress and change in the treatment of malignant tumors. The effect and response to immunotherapy using ICI are largely driven by the characteristics of immune cells in the tumor tissue and, as it turns out, also in the peritumoral tissue. We conducted an immunohistochemical analysis of the expression of the immune checkpoint protein PD-1 and its ligand PD-L1 on the surface of tumor cells as well as tumor-infiltrating immune cells (TIIC) in samples of salivary carcinomas, separately in their centre and at their periphery. In addition to the above, an increasing amount of evidence suggests that resistance to ICI therapy is modulated by the interaction of the Fas receptor (CD95) and Fas ligand (FasL, CD178) between tumor cells and immune cells. We therefore decided to explore the expression and interaction of Fas-FasL between tumor cells and tumor-infiltrating immune cells in the centre of the tumor and in the peritumoral area of salivary carcinoma samples. Differential evaluation of the tumor centre and tumor periphery across various histological subtypes of SGC revealed the role of peripheral TIICs and...
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Amaryllidaceae alkaloids of haemanthamine structural type and their semisynthetic derivatives as potential drugs in the treatment of Alzheimer's disease.
Peřinová, Rozálie ; Cahlíková, Lucie (advisor) ; Lapčík, Oldřich (referee) ; Mučaji, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy and Pharmaceutical Botany Candidate: Ing. Rozálie Peřinová Supervisor: prof. Ing. Lucie Cahlíková, Ph.D. Title of doctoral thesis: Amaryllidaceae alkaloids of haemanthamine structural type and their semisynthetic derivatives as potential drugs in the treatment of Alzheimer's disease. To deepen the knowledge about the Amaryllidaceae alkaloid haemanthamine, which was isolated at our workplace as part of previous phytochemical studies, derivatives of this alkaloid were synthesized. First, series of aliphatic (3-12) and aromatic ester derivatives (13-66) were prepared, and then, to compare the structure-activity relationship, ether derivatives (67-80) were prepared from the most active substituents. All synthesized compounds were identified using the following structural analysis methods: NMR, HPLC/MS, and HRMS, including testing physical properties such as optical rotatability. After structure confirmation, all derivatives were subjected to screening studies for their inhibitory potential against hAChE and hBuChE. The selected derivatives were tested for their inhibitory potential against another enzyme, GSK-3β, which plays a significant role in the pathogenesis of AD. In cooperation with the Faculty of Medicine in...
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The effect of synthetic modified mRNAs induced proliferation on pancreatic beta cells
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a chronic disease caused by the loss of pancreatic beta cells due to autoimmune destruction or increased apoptosis. Beta-cell deficiency results in reduced insulin production, which plays an important role in glucose metabolism. The number of beta-cells in the body is one of the main factors that influence the development of this chronic disease. Therefore, it is necessary to find a way by which the number of beta-cells of the organism can be increased and thus the insulin production can be restored in a natural way without any need for the use of insulin infusions. However, the ability of beta-cells to divide decreases with age and is virtually nil in adulthood. The study of the cell cycle, especially the early and late cyclins and cyclin-dependent kinases, which act as cell cycle regulators, thus appears to be a promising way to restore natural insulin-producing tissues. In order to increase the number of beta cells entering the cell cycle, we focused on studying the effect of in vitro transcribed (IVT) mRNAs, encoding cyclins type D and cyclin dependent kinases 4 and 6 on stimulating cell division of isolated beta-cells. We found that transfection IVT mRNAs for type D cyclins in combination with cyclin-dependent kinases 4 and 6 significantly increased the proliferation of beta-cells...
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The role of a specific miRNAs in the regulation of apoptosis during physiological and pathophysiological processes in the CNS
Kaslová, Tereza ; Romanyuk, Natalyia (advisor) ; Klassen, Ruslan (referee)
MicroRNAs are small non-coding RNAs of 20 to 24 nucleotides in size that are able to post- transcriptionally regulate gene expression by binding to mRNA. This paper focuses on how these microRNAs are generated and how they are able to regulate at the level of proteins involved in programmed cell death - apoptosis. By what mechanisms apoptosis occurs, what proteins are involved and what changes the cell undergoes are further discussed in this thesis. The precise influence of this post-transcriptional regulation is presented by using selected microRNAs that influence apoptosis during the development of the central nervous system, as well as during and as a consequence of the neurodegenerative diseases and damage that can affect it. Finally, it will also introduce the use of microRNAs as potential biomarkers, due to changes in their levels associated with various diseases, and as direct therapeutic targets. Keywords Apoptosis, microRNA, cell death, central nervous system, neurodegenerative diseases, gene expression regulation
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