National Repository of Grey Literature 24 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Structural and functional study of viral RNA polymerases
Dubánková, Anna ; Bouřa, Evžen (advisor) ; Bařinka, Cyril (referee) ; Plevka, Pavel (referee)
Viral RNA-dependent RNA polymerases (RdRps) are enzymes essential for viral multiplication. The general function of RdRp is universal for all RNA viruses: to recognise viral RNA, bind it and synthesize the complementary RNA strand. This series of steps is absolutely crucial for viral infection. It is important to mention that the non-infected cell is incapable of replicating any RNA. The host cell thus does not naturally express any RdRps. I chose RdRps for my research because these enzymes are key to viral replication and thus an excellent target for antivirals. This study characterises polymerases from ​Picornaviridae and Flaviviridae families, in depth. Picornaviral replication takes place in viral-induced membrane structures called Replication Organelles (ROs), where the polymerase is localised to the membrane. In this study, we investigated the recruitment of picornaviral polymerase membrane. Subsequently, we focused on the activation of picornaviral RdRp induced by the insertion of the very first residue into the protein core. Next, we focused on the flaviviral RdRps specifically from yellow fever virus (YFV) and Zika virus (ZIKV). This study reports the first structure of a full length YFV polymerase and a model of ZIKV polymerase in complex with RNA. The model of ZIKV RdRp in complex with...
Protein engineering as a tool for the production of antibody derivatives
Šulc, Josef ; Bařinka, Cyril (advisor) ; Mikulecký, Pavel (referee)
This thesis deals with production and properties of disulfide-stabilized single-chain variable fragments of the 5D3 antibody (dsscFv), which specifically recognizes and binds to glutamate carboxypeptidase II (GCPII), an antigen closely related to the prostate carcinoma processes and other tumor diseases. Small antibody fragments are in current focus of development of diagnostic and therapeutic reagents. However, compromised stability of antibody derivatives often results in low production yield or loss of function. Introduction of structural changes by protein engineering is often used to solve the issue. The aim of the study was based on enhancement of protein stability by the introduction of interdomain disulfide bond into the structure of single-chain variable fragment. The effect of modification was evaluated by estimation of production yield and affinity of studied protein. The aforementioned antibody derivative was produced using an Escherichia coli expression system, using specific signal sequences leading the production to the bacterial periplasm. The attempted stabilization was carried out by introducing mutations at LV-G44 and HV-G100 positions, replacing glycines with cysteines. The binding affinity of the derivative for human GCPII was determined using ELISA. This thesis also shows a solved 3D...
Analysis of Histone Deacetylase 6/Kinesin Interactions
Nedvědová, Jana ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Intracellular transport is provided by two major types of molecular motors kinesins and cytoplasmic dynein. Kinesin-1 is a molecular motor that transports molecules and organelles along microtubule tracks anterogradely. Specific protein-protein interactions are required to activate kinesin-1 as the free kinesin exist in an autoinhibited state. The activation of kinesin-1 induces its conformational change, enables microtubule binding and ATP hydrolysis necessary for the directional cargo transport. HDAC6 is a multifunctional protein composed of several domains. It plays an important role in many microtubule dependent processes as HDAC6 is a major tubulin deacetylase. It has been shown that HDAC6 manipulation (inhibition/genetic ablation) affects transport along microtubules but the exact mechanisms are unknown. The effect can be caused either by deacetylation microtubules or direct interaction with molecular motors. This thesis is focused on characterization of interactions between kinesin-1 and HDAC6 that have not been described so far. To this end, we expressed and purified various constructs of kinesin-1 and HDAC6 and tested their interactions by microscale thermophoresis (MST) and hydrogen deuterium exchange (HDX) to determine affinity and interaction sites, respectively. MST data revealed that...
Deciphering the biological role of Ddi1-like protein family
Sivá, Monika ; Grantz Šašková, Klára (advisor) ; Bařinka, Cyril (referee) ; Stopka, Pavel (referee)
Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...
Generation and Characterization of Glutamate Carboxypeptidase II (GCPII)-Deficient Mice
Vorlová, Barbora ; Šácha, Pavel (advisor) ; Eckschlager, Tomáš (referee) ; Bařinka, Cyril (referee)
Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein, which consists of short intracellular and transmembrane domains, and a large extracellular domain possessing carboxypeptidase activity. In the human body, GCPII fulfils a neuromodulatory function in the brain and facilitates folate absorption in the small intestine. In addition to the brain and small intestine, high level of GCPII is also present in the prostate and kidney. However, GCPII function in these tissues has not been determined yet. To study the role of GCPII in detail, several research groups attempted to inactivate GCPII encoding gene Folh1 in mice. Surprisingly, the experiments led to rather conflicting results ranging from embryonic lethality to generation of viable GCPII-deficient mice without any obvious phenotype. This dissertation project aimed to dissect the discrepancy using alternative strategy for gene modification. For this purpose, we designed TALENs that specifically targeted exon 11 of Folh1 gene and manipulated mouse zygotes of C57BL/6NCrl genetic background. We analysed all genetically modified mice of F0 generation for presence of TALEN-mediated mutations and established 5 different GCPII-mutant mouse colonies from founder mice that altogether carried 2 frame-shift mutations and 3 small in-frame...
Proteolytic systems of the blood fluke (Schistosoma mansoni).
Fajtová, Pavla ; Horn, Martin (advisor) ; Bařinka, Cyril (referee) ; Sojka, Daniel (referee)
Schistosomiasis is a serious parasitic disease caused by blood flukes of the genus Schistosoma. It is a global health problem with more than 200 million people infected and 750 million people at risk. Current therapy relies on a single drug, praziquantel, for which there are concerns of emerging drug resistance. Proteases of schistosoma are promising target molecules for the development of new therapeutic strategies against schistosomiasis. This work focuses on the comprehensive characterization of proteolytic systems of Schistosoma mansoni and determination of their role in the interaction with the human host. First, the major proteolytic activities secreted by individual developmental stages of schistosoma that parasitize the human body were classified using functional proteomics. This analysis demonstrated their complex and specific distribution with predominant serine and cysteine proteases and metalloproteases. Second, tegumental and digestive proteases, namely prolyl oligopeptidase and cathepsins B, C and D, were identified by chemical genomics as suitable target molecules for therapeutic intervention. Prolyl oligopeptidase was biochemically characterized using a recombinant protein, its effective inhibitors were developed as templates for antischistosomal drugs, and a biological role of the...
Glutamate Carboxypeptidase II - Structural and Biochemical Characterization and Structure-Assisted Drug Design
Ptáček, Jakub ; Bařinka, Cyril (advisor) ; Obšil, Tomáš (referee) ; Brynda, Jiří (referee)
Glutamate carboxypeptidase II (GCPII) is a human membrane-bound metallopeptidase discovered more than 30 years ago. It has attracted attention of biomedical scientists thanks to its diverse tissue expression profile and different biological functions. GCPII is detected on the surface of astrocytes in both central and peripheral nervous systems where it is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. Glutamate, one of the hydrolytic products, is a potent excitatory neurotransmitter and its overproduction has been shown to be responsible for cell death in various neurological disorders by a so-called glutamate excitotoxicity mechanism. Together with the fact that NAAG acts neuroprotectively it has been postulated (and later confirmed) that GCPII inhibition has a therapeutic potential in such disorders. Prostate cancer (PCa) is the second most prevalent cancer in men and despite its slow progression it is prone to metastasize thus posing a life threat. GCPII has been found to be overexpressed in prostate tumor cells compared to the healthy tissue (therefore it is also termed prostate-specific membrane antigen - PSMA) thus representing an excellent biomarker of PCa validated by many publications and clinical studies....
Regulation of the activity of aspartic and serine proteases by selective natural inhibitors
Srp, Jaroslav ; Mareš, Michael (advisor) ; Novák, Petr (referee) ; Bařinka, Cyril (referee)
Proteases are involved in many physiological processes and their dysregulation is associated with various pathologies. Protease activity is effectively controlled by natural inhibitors. This PhD thesis is focused on the inhibitors of aspartic and serine proteases of animal and plant origin and provides the identification, biochemical characterization and structural description of their inhibition mechanisms. Plant Kunitz inhibitors are produced as defensive proteins, and they are able to block activities of a broad spectrum of proteases. In this thesis, the digestive proteolytic system of the Colorado potato beetle, a herbivore pest of potato plants, was described with the help of functional proteomics. It was shown that aspartic and serine proteases from this herbivore are effectively blocked by two potato Kunitz inhibitors (namely PCDI, PSPI). Using structural analysis, novel types of reactive centers were identified on PCDI and PSPI molecules for the inhibition of aspartic protease cathepsin D and the serine proteases trypsin and chymotrypsin. The analysis of the reactive center on a PCDI with the crystal structure of digestive cathepsin D from the Colorado potato beetle explained the mechanism of their interaction. Sphingolipids were identified as the first endogenous inhibitors of human...
Tubulin post-translational modifications
Kropáčková, Veronika ; Bařinka, Cyril (advisor) ; Dostál, Vojtěch (referee)
Microtubules composed of αβ-tubulin heterodimers are an integral part of the cellular cytoskeleton of eukaryotic organisms. They participate in the cellular transport, determine the distribution of membrane organelles and help defining cellular polarity. Microtubules are part of dynamic structures such as mitotic spindle, but they also form stable structures such as flagellar and ciliar axonemes. Microtubules have many distinct functions in cells and tissues and therefore must differ from each other in some way. Post-translational modificationsof αβ-tubulin in microtubules are the major source of their diversity and collectively define so-called tubulin code. Twelve posttranslational modifications of tubulin/microtubules have been identified so far. Unraveling the mechanisms involved in post-translational modifications of tubulin/microtubules, including the identification of responsible enzymes, is an important source of understanding of the effects of these modifications on biological functions. Key words: tubulin, post-translational modificaton, microtubules, acetylation, tyrosination, polyamination, polyglutamylation
Antibody derivatives for the detection of human glutamatecarboxypeptidase II
Bělousová, Nikola ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Prostate cancer is one of the most common human malignancies and, consequently it is critical to develop appropriate diagnostic and therapeutic tools. Glutamate carboxypeptidase II (GCPII) is currently being considered one of the most important prostate cancer markers due to its tissue- specific expression. Whereas in healthy prostatic tissue the expression levels of GCPII are low, the transformation into the tumor is associated with the substantial increase of GCPII expression, with the highest levels observed in androgen-independent metastatic tumors. GCPII is thus considered a promising marker for early phase as well as advanced metastatic stages of prostate cancer. Current research is focused on the development of highly sensitive and specific reagents that allow detection of small amounts of GCPII, for example in early stages of cancer. Antibody derivatives are promising molecules for this purpose because they have high affinity and specificity and minimum negative side effects. Protein engineering is a prefered approach for preparation of various antibody molecules that differ in size, binding properties, stability, solubility, and production means. Different types of derivatives are being developed for medical needs such as in vitro diagnosis, therapy, and in vivo imagingSmall molecular...

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