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Advective transport between atmospheric layers
Zajíček, Radek ; Šácha, Petr (advisor) ; Karami, Khalil (referee)
The transport in the middle atmosphere is controlled by the global-scale Brewer-Dobson circulation. Advective transport is an important component of the Brewer-Dobson circulation. Using the transformed Eulerian mean framework this thesis focuses in detail on advective transport in the middle atmosphere based on data from the ERA5 reanalysis and chemistry-climate models within the Coupled Model Intercomparison Project Phase 6 (CMIP6) and the Chemistry-Climate Model Initiative (CCMI-1). Part of the thesis concerns the derivation of a new method for analytical decomposition of transport changes into partial kinematic factors such as accelerating residual circulation, changes in the vertical structure of the middle atmosphere and others.
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Synthesis of peptides as potential ligands of aspartate protease HIV and MAY1 and confirmation of their inhibitory activity
Klikarová, Ivana ; Šácha, Pavel (advisor) ; Žáková, Lenka (referee)
The lentivirus known as the human immunodeficiency virus (HIV) is transmitted through blood and body fluids, causing the destruction of CD4 lymphocytes and leading to opportunistic infections that define acquired immunodeficiency syndrome (AIDS). The urgent need for new antiretroviral drugs stems from concerns about the long-term toxicity of existing drugs, HIV-1 variants resistant to treatment, and frequent changes in patient treatment. Drug development is focused on inhibitors of two viral enzymes, reverse transcriptase and protease. Antiretroviral therapy uses protease inhibitors in combination with nucleoside analogs to effectively suppress viral replication, prolonging the lives of HIV-infected patients and reducing morbidity. Cryptococcus neoformans and cryptococcus gattii infections primarily affect the immunocompromised population and have high morbidity and mortality rates. Resistance to commonly used antifungals has been emerging, making it more difficult to treat these infections. Protease inhibitor components used in antiretroviral therapies have shown some clinical efficacy in these opportunistic infections, particularly in Major Aspartyl peptidase 1, an aspartate protease belonging to the same family of proteases as the HIV protease. To search for low molecular weight peptide ligands,...
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Adaptation of enzymatic assays for high-throughput screening with use of ECHO-MS
Kráľová, Zuzana ; Mertlíková Kaiserová, Helena (advisor) ; Šácha, Pavel (referee)
High throughput screening for potential inhibitors of enzyme purine nucleoside phosphorylase (PNP) was developed during the work on this thesis. Products of PNP-catalysed reaction, hypoxanthine and ribose-1-phosphate, were quantified using mass spectrometry. Advantages and disadvantages of an Echo® MS device were explored and compared to the previously used radioenzymatic method. The emphasis was put on assay robustness and automatization of the protocol. Acoustic droplet ejection technique (ADE) and contactless dispensing of chemicals were employed, using Echo® 550 and Certus Flex® devices. These allowed to reduce reaction volume from original 20 µl to 5 µl and perform the whole reaction in a 384-well plate, decreasing the costs and increasing the throughput. Using a developed Echo® MS method, the time of analysis was shortened from 8 minutes per sample for radiometric analysis to 0,2 minute per sample. Furthermore, enzyme assay parameters, such as PNP concentration, composition of reaction mixture, incubation time and reaction termination were optimized, following the rules of HTS. The method developed in this work was employed for PNP inhibitors screening and their IC50 determination. Moreover, the HTS method enabled screening of the whole library of IOCB in-house synthesized compounds (~ 8000)....
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Type of the scheme and tuning dependence of the parameterized orographic gravity wave drag distribution in global climate models.
Hájková, Dominika ; Šácha, Petr (advisor) ; Gisinger, Sonja (referee)
Orographic gravity waves (OGWs) are ubiquitous in the atmosphere and they have an important influence on the dynamics and energy transport especially in the middle- atmosphere. As such, they have to be included in global climate models. Current global models have a low resolution and for that reason OGW effects have to be parameterized. This thesis focuses on the most important output of the OGW parameterizations - the resulting OGW drag. This drag is transported upwards from the surface by the wave and is distributed in the atmosphere. Parameterization schemes differ in many aspects such as the topography description, inclusion of non-linear effects, tuning of the free parameters and others. We have reviewed and described 7 different parameterizations, which are used in 9 different CMIP6 models. After comparing drag data from each of the models we find unexpectedly great differences in the vertical distribution of the drag as well as the magnitude. Focusing on 4 hotspots around the globe, we proposed hypotheses based on the knowledge of the parameterization schemes that can partially explain the inter-model differences. The thesis can pave the way for a more systematic research of the OGW parameterizations in the future, with an ultimate goal of lowering the amount of uncertainty of the future climate...
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Preparation of expression system of gamma-lactamase and expression testing
Magyerková, Monika ; Ingr, Marek (advisor) ; Šácha, Pavel (referee)
γ-lactamase is an enzyme clearing five-membered lactam cycles. Polyvinylpyrrolidone (PVP) is one of its potential substrates. Degradation of PVP by γ-lactamase is being studied due to its eventual use in waste-water purifying plants. The aim of the work was to prepare a synthetic gene from the bacterium Comamonas acidovorans and to clone it into the expression vector pET22b. PCA method was used for the synthesis of the γ-lactamase gene. 1725 bp long sequence of the γ-lactamase gene was split into two parts (synthons) which were synthesized individually. After the synthesis restriction cleavage and ligation to the vector pUC19 were performed. Competent cells E. coli, strain DH5α, were transformed by the obtained construct. After the sequence confirmation both synthons were cleaved by restriction endonucleases and connected by single-step ligation to the plasmid pET22b. Expression bacterial cells E. coli, strain BL21(DE3)RIL, were transformed by the recombinant plasmid containing the connected synthons and expression of the recombinant γ-lactamase was tested. Sequence of the clone producing a protein of the expected length was confirmed by sequencing analysis. The prepared plasmid will be used for the expression of recombinant γ- lactamase. (In English)
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Epigenetically based chemoresistance of cancer cells
Feriančiková, Barbara ; Eckschlager, Tomáš (advisor) ; Šácha, Pavel (referee)
Cancer, despite significant advances in diagnosis and treatment, is the second most common cause of death in economically advanced countries. The main reason for the failure of anticancer therapy is the development of chemoresistance, which can be either internal or acquired, and is primarily mediated by the activation of various key regulators (eg MDR, PI3K/Akt, etc.). Genetic and epigenetic mechanisms are involved in activating these pathwa- ys. Significant epigenetic mechanisms that can participate in chemoresistance include regula- tion of gene expression by microRNA (miRNA) and long noncoding RNA (lncRNA). Dere- gulated expression of these non-coding RNAs has been observed in many diseases and their involvement in the initiation and progression of malignant tumors has been demonstrated. In this study, we investigated the expression of long non-coding RNA MIAT in hypoxia (1% O2) in chemosensitive and chemoresistant neuroblastoma cell lines (NBL), as hypoxia is a significant negative prognostic factor of many tumors and is involved in chemoresistance. Relative expression of MIAT was influenced by the number of cultured cells, where expression was increased by culturing more cells. MIAT expression was also significantly increased after 6 hours of NBL culture UKF-NB-4 in hypoxic conditions, and...
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Nephropathy and tumour development caused by plant alkaloids aristolochic acid
Bárta, František ; Stiborová, Marie (advisor) ; Šácha, Pavel (referee)
Aristolochic acids (AA) are alkaloids contained in plant species of the family Aristolochiaceae. These plants are used since antiquity in traditional medicine to treatment of many varied diseases. There are known anti-inflammatory effects of these compounds, however these alkaloids exhibit mutagenic and carcinogenic properties. Despite of this fact, plant extracts AA are still used in traditional medicine, e.g. in China, India, Taiwan. Aristolochic acids are proven to be the cause of disease designated Aristolochic Acid Nephropathy (AAN, theretofore known as Chinese Herbs Nephropathy (CHN). This unusual nephropathy leads to a total renal failure. The late complication of this disease is the development of tumours in urothelial tissue of patients. AA can form persistent stable covalent DNA adducts. Formation of these DNA adducts lead to AT→TA transversion, the unique mutation in tumour suppressor gene p53 responsible for tumour formation. Balkan Endemic Nephropathy (BEN) is associated with AA, too. In this instance is supported also influence of another factors, e.g. mycotoxins (ochratoxin A). However, in all probability AA contribute to a development of this disease particularly. This hypothesis is supported by finding of AA-DNA adducts in tissues of patients suffering from AAN and BEN and that of...
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New Perspective on the Role of Gravity Waves in the Stratospheric Dynamics and Variability
Šácha, Petr ; Pišoft, Petr (advisor) ; Dameris, Martin (referee) ; Rieder, Harald (referee)
This thesis is concerned with the role of internal gravity waves (IGWs) in the stratospheric dynamics and variability demonstrating the effect of spatiotemporal distribution of their activity on the stratospheric dynamics and transport. The first part introduces a theoretical overview of the most recent as well as classical approaches used for description of the wave-mean interaction in the middle atmosphere. Methodology for an IGW analysis from the GPS radio occultation density data is described in the next chapter and the advantages of utilization of density data are listed. The third chapter presents results describing the peculiar dynamics and anomalous IGW activity in the Eastern Asia/Northern Pacific region. An important part is dedicated to a discussion of accuracy limits and usability of different IGW activity proxies. The possible impact of the localized IGW activity is investigated using a mechanistic middle and upper atmosphere model in the last chapter. Sensitivity simulations are used to demonstrate an important role of the spatial distribution of IGW activity for a formation of planetary waves and for the longitudinal variability of the Brewer-Dobson circulation. Implications for the middle atmospheric and climate change research are discussed along with consequences for parameterizations of...
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Structure and Function of Glutamate Carboxypeptidase II
Šácha, Pavel ; Konvalinka, Jan (advisor) ; Šedo, Aleksi (referee) ; Blahoš, Jaroslav (referee)
4 Závěr GCPII je důležitý protein, který hraje roli v mnoha fyziologických i patologických procesech. Proto bylo třeba získat větší množství enzymaticky aktivního proteinu pro jeho další biochemický výzkum. Heterologní expresí v hmyzích buňkách S2 bylo exprimováno a následně purifikováno dostatečné množství velmi čistého a aktivního enzymu. To umožnilo jeho biochemickou charakterizaci, krystalizaci a později vedlo i k vyřešení krystalové struktury. GCPII je aktivní v širokém rozmezí pH 6 - 8, s maximem kolem pH 7,5. Zjistili jsme, že kromě přirozeného substrátu Ac-Asp-Glu GCPII štěpí také acetylované dipeptidy Ac-Asp-Met, Ac-Glu-Met, Ac-Glu-Glu, Ac-Ala-Glu a Ac-Ala-Met. U těchto substrátů byly změřeny kinetické parametry štěpení. Nalezení dalších substrátů může vést k objevení dosud nepopsaných fyziologických rolí GCPII. Také byly porovnány hodnoty IC50 inhibitorů známých z literatury u námi připravené GCPII a GCPII izolované z potkaních mozků. IC50 bylo u všech méně specifických inhibitorů nižší u rekombinantní GCPII než u GCPII izolované z mozků. Rozdíly ve výsledcích vysvětlujeme vazbou méně specifických inhibitorů na jiné proteiny preparátů z mozku. Vůbec nepochybujeme o tom, že data získaná za použití čistého rekombinantního proteinu jsou přesnější než ta, ktará byla získána ze špatně definovaných...
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Application of the Nambu mechanics formalism in atmospheric dynamics
Procházková, Zuzana ; Šácha, Petr (advisor) ; Badin, Gualtiero (referee)
Nambu mechanics is a generalization of Hamiltonian mechanics that uses multiple conserved quantities as Hamiltonians. In this thesis, we review Nambu mechanics and its application on the equations of incompressible flow and shallow water equations. The Nambu form of the equations of incompressible flow is guessed based on its Hamiltonian form and derived conserved quantities. With the example of the shallow water equations a more general method of Nambu form derivation is illustrated. Based only on the knowledge of the Hamiltonian and the potential enstrophy moments conservation, the shallow water equations are written as a sum of the Nambu brackets and a Poisson bracket. For the classical potential enstrophy, the derived equations are up to constant factors equivalent to the known form of the shallow water equations. The notation by antisymmetric Nambu brackets is convenient for finding conservative schemes and the theory can be also used for example for the study of deviations of flow from stationary flow.
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