National Repository of Grey Literature 27 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Novel therapeutic targeting of the quorum sensing transcriptional regulation in multi-antibiotic resistant bacterium Staphylococcus aureus
Maďarová, Anna ; Maloy Řezáčová, Pavlína (advisor) ; Cherepashuk, Ivan (referee)
The threat of antibiotic resistance is deemed as a ticking time-bomb, yet in spite of this, the number of newly discovered antibiotics continues to steeply decline. Each year, infections caused by multi-drug resistant bacteria kill more than 4.95 million people, which demonstrates that in order to tackle this crisis, a novel approach is desperately required. The solution could be found in targeting bacterial transcriptional regulators that are responsible for the control of various virulence factors and can be highly species-specific. This also applies to the most widespread multi-drug resistant bacterium Staphylococcus aureus, whose virulence is intricately linked to the transcriptional regulation of the quorum sensing system. The aim of this bachelor thesis is to summarize current knowledge about clinically relevant transcriptional regulators that are associated with this system (AgrA, SarA, SarR, MgrA, Rot), with focus on their 3D structures, mechanisms of action, and inhibition. Key words: antibiotic resistance, transcriptional regulation, quorum sensing, Staphylococcus aureus
Design, preparation and structural studies of biologically relevant protein variants of cancer-related Carbonic Anhydrase IX
Fejfarová, Adéla ; Maloy Řezáčová, Pavlína (advisor) ; Novotný, Marian (referee)
3 Abstract Carbonic anhydrase IX (CA IX) represents an attractive target for the development of anticancer drugs as it is overexpressed in various types of solid tumors. By its catalytic activity, CA IX assists the cancer cells to maintain the optimal intracellular pH and to acidify the extracellular milieu promoting tumor development. There are twelve enzymatically active carbonic anhydrases (CAs) present in human body, all sharing a high sequence identity and a typical β-sheet structural fold of the well-studied catalytic domain. Although, the activity of CAs is efficiently inhibited by sulfonamide-containing compounds, the design of inhibitor selective to the cancer-related CA IX has been hampered by the high sequence conservation. CA IX has several unique features compared to other members of the family, which investigation may help in the development of selective drug compounds. Namely, it is a type I transmembrane dimeric protein with unique N-terminal proteoglycan-like (PG) domain, extracellular catalytic domain, and short cytoplasmic C-terminal segment. The above denoted traits make CA IX subject of structure-based drug design efforts. However, the expression and purification in high yield as well as crystallization experiments has been challenging. Therefore, protein variant bearing six amino acid...
Structure-assisted design of inhibitors targeting medicinally relevant enzymes
Djukic, Stefan ; Maloy Řezáčová, Pavlína (advisor) ; Kutá-Smatanová, Ivana (referee) ; Kolenko, Petr (referee)
Structure-assisted drug discovery is a powerful approach that utilizes detailed knowledge on 3D structure to design and optimize new inhibitors targeting medically relevant enzymes. X-ray crystallography is a widely used structural biology technique since it provides detailed snapshot of protein-inhibitor complex, which is used to analyze protein- inhibitor interactions. PNP plays an important role in salvage pathway of purine metabolism, it is a target in treatment of T-cell malignancies and/or parasitic infections. Our effort focused on human and M. tuberculosis PNP, and our aim was to develop new inhibitors with high selectivity and specificity. Our inhibitors are acyclic nucleoside phosphates with 9- deazahypoxanthine nucleobase that contain three moieties binding to all three regions of the active site: purine, phenyl and phosphonate moieties. The best inhibitors have IC50 values as low as 19 nM (human) and 4 nM (M. tuberculosis). The presence of short substituents at central phenyl moiety, such as methoxy and bromide group, decreases inhibitor's affinity towards human PNP, but does not affect affinity towards mycobacterial PNP. At the same time, bulky substituents, such as fluorinated phenyl ring, decrease inhibitor's affinity towards human PNP but increase affinity towards mycobacterial...
Mechanistic and structural studies of the cGAS-STING signalling pathway
Vavřina, Zdeněk ; Maloy Řezáčová, Pavlína (advisor) ; Hudeček, Jiří (referee) ; Kolenko, Petr (referee)
The cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) signalling pathway plays a crucial role in the innate immune system. It is activated by pathogen double-stranded DNA (dsDNA) or cyclic dinucleotides, which are secondary messengers of bacteria. This activation leads to the expression of type I interferons and proinflammatory cytokines. The present dissertation examines the interaction between cGAS and its substrates and the relation between the STING protein and its agonists from a mechanistic and structural point of view. The enzyme cGAS is a metazoan intracellular sensor of dsDNA. Upon its binding to DNA, it synthesizes the cyclic dinucleotide 2′,3′-cGAMP, which activates the adaptor protein STING. Besides 2′,3′-cGAMP, STING can also be activated by 3′,3′-cyclic dinucleotides that serve as secondary messengers in bacteria. We investigated various dinucleotide cyclases to better understand their substrate specificity and utilized them for the preparation of novel cyclic dinucleotides activating STING. As the most appropriate for the preparation of 2′,3′-cyclic dinucleotides, we identified mouse cGAS. Additionally, we utilized the enzymes DncV from Vibrio cholerae and DisA from Bacillus thuringiensis for the synthesis of 3′,3′-cyclic dinucleotides. These enzymes exhibit...
Preparation and characterization of human cellular cofactors of retroviral integration.
Čermáková, Kateřina ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee)
Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular binding partner of Human Immunodeficiency Virus type 1 (HIV-1) integrase. It is a human nuclear protein, which has been implicated in transcriptional regulation and cell survival. The role of LEDGF/p75 in HIV integration is well characterized, the HIV integrase binding domain (IBD) was identified and structural studies, which provide detail information about this interaction, were done. However, very little is known about its physiological function. As a transcriptional co-activator, LEDGF/p75 is implicated not only in HIV replication, but also in human cancer and autoimmunity. Key feature for both, the viral and cellular role of this protein, is its ability to act as a molecular adaptor tethering proteins to the chromatin fiber. Recently, PogZ (Pogo transposable element derived protein with zinc finger domain) was identified and validated as a new cellular interaction partner of LEDGF/p75. It was shown, that their interaction is mediated by IBD of LEDGF/p75 and the C-terminal domain of PogZ. To gain more insight in this interaction, we have initiated structural studies of their complex. Structural information is crucial for understanding the LEDGF/p75 biological role and might help in design of inhibitors selectively blocking...
Regulation of purine nucleotide metabolism as a pharmacological target
Brinsa, Vítězslav ; Maloy Řezáčová, Pavlína (advisor) ; Hlouchová, Klára (referee)
Purine nucleotides are essential basic building blocks for DNA and RNA synthesis. They can also serve as energy storage and transfer unit and play an important role in cell signalling and regulation of variety of biochemical processes. It is crucial for the cells to maintain a sufficient supply of purine nucleotides in order to secure its survival and cell division. Level of purine nucleotide pool in the human body is regulated via purine nucleotide metabolism, which consists of three coordinated processes: de novo synthesis pathway, salvage pathway and degradation pathway of purine nucleotides. Regulation of those three pathways is under control of various mechanisms including regulation on the level of enzyme expression, allosteric regulation of enzyme activity or forming a multienzime complexes, i. e. purinosomes in the de novo synthesis pathway. Phosphoribosyl pyrophosphate synthetase I (PRS-I) and cytosolic purine 5'-nucleotidase (cN-II) play an important role in purine nucleotide metabolism. These enzymes contribute significantly to the purine nucleotide pool regulation by means of their allostericaly regulated activity. Malfunctions of their catalytic activity are connected with various pathologies such as gout, hyperuricosuria, neurological dysfunctions and acute lymphoblastic leukaemia...
Analysis of serine racemase expression in the CNS of epileptic patients
Vorlová, Barbora ; Konvalinka, Jan (advisor) ; Maloy Řezáčová, Pavlína (referee)
Serine racemase is a pyridoxal-5'-phosphate dependent enzyme that converts L-serine to D-serine. D-serine is a recognized physiological co-agonist of N-methyl-D-aspartate type of glutamate receptors - key receptors that participate in the neurotransmission in the mammalian brain. Dysfunction of these receptors has been implicated in several neuropathologies, including schizophrenia, brain ischemia, neurodegenerative disorders and epilepsy. Serine racemase is thus a promising pharmaceutical target in these diseases. In this study, three anti-human serine racemase monoclonal antibodies were characterized and the best one was used for the Western blot detection of the enzyme in resected human epileptic tissues. For better interpretation of the results, accuracy of the tissue processing, the protein concentration determination and the Western blot quantification were verified. Finally, the activity of human serine racemase was determined with the L-serine-O-sulfate, the substrate with the highest-affinity to this enzyme. (Thesis in Czech)
Structural studies of transcription factors implicated in regulation of metabolism of pancreatic beta cells
Duchoslav, Vojtěch ; Maloy Řezáčová, Pavlína (advisor) ; Vaněk, Ondřej (referee)
Nkx6.1 is a homeodomain protein (37.8 kDa) and an important transcription factor, which regulates transcription of key genes in pancreatic ß-cells. Insufficient expression of this protein leads to reduced glucose uptake from blood as a consequence of suppressed transcription of the glucose transporter Glut2 and impaired glucose metabolism. Furthermore, the proliferation of pancreatic ß-cells is suppressed due to insufficient transcription of Cyclin D2, a protein regulating the mitosis. Moreover, the biosynthesis of insulin is impaired duet he diminished transcription of the genes coding for Ero1lb a Slc30a8, which as a consequence leads to reduced production of the mature insulin. Nkx6.1 could play a role in the pathogenesis of the type 2 diabetes , where ß-cells show diminished ability to compensate high demand for insulin. This malfunction is the cause of an insufficient ability to secrete insulin and death of pancreatic cells. Perhaps driven by misregulation of transcription of the genes that are involved in the mentioned processes. Nkx6.1 recognizes a strictly conserved 8-base pair DNA sequence (TTAATTAC). Its binding to DNA is regulated by an acidic domain at the C-terminus. Within the bachelor thesis, the resonances were assigned to the backbone atoms of the Nkx6.1 protein using nuclear...
Characterization of recombinant fragment of an antibody against CD3 marker.
Písačková, Jana ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee)
Monoclonal antibody MEM-57 recognizes CD3 antigen expressed on peripheral blood T-lymphocytes. CD3 surface glycoprotein complex associates with T-cell receptor and is responsible for the transduction of activation signal. Antibody MEM-57 has, therefore, a large diagnostic and therapeutic potential. It could be used in autoimmune diseases diagnostics, for classification of T-cell leukemias and, as an immunosuppressant, in transplantation. The most promising therapeutic use of MEM-57 antibody would be the construction of a "Bispecific T-cell Engager" (BiTE) antibody format with potential application in cancer therapy. In this format, single-chain variable fragment (scFv) of MEM-57 would be fused with an anti-tumor antigen scFv. The thesis is focused on biochemical and biophysical characterization of MEM-57 antibody scFv fragment. Recombinant antibody fragment scFv MEM-57, equipped with the pelB leader sequence, c-myc tag and His5 tag, was produced from a pET22b(+) vector into the periplasmic space of E. coli BL21 (DE3). Two-step purification protocol, employing nickel chelation affinity chromatography and ion-exchange chromatography, was developed to obtain high yield of pure protein. The antigen binding activity of scFv MEM-57 was confirmed by flow cytometry. Structural information on scFv MEM-57...

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