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Recombinant preparation of DNA binding domain of transcription factor TEAD4
Zákopčaník, Marek ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
6 Abstract Transcription factors play a key role in the management of cell growth and differ- entiation and their deregulation is associated with many cancers. TEAD proteins utilise highly conserved DNA binding domain to recognise specific DNA sequences. This domain could facilitate new drug design and development. The goal of this master thesis includes recombinant preparation of DNA binding domain of transcriptional factor TEAD4 extended by a part of an unstruc- tured variable sequence, which connects this domain with transactivation domain. Purification steps include affinity chromatography followed by size exclusion chro- matography. The characterization of produced protein was performed by mass spectrometry and finally, native gel electrophoresis was used to prove the ability of the produced protein to bind DNA. During purification steps, a fragmentation from C-terminus was observed. Based on analysis of the mass spectra, three most represented forms of produced protein were described all of which were fragmented. The most abundant form (55%) consisted of amino acids 30-131 from TEAD4 protein. Second most abun- dant form (18%) consisted of amino acids 30-144 and the third form consisted of amino acids 30-81. Native gel electrophoresis verified the ability to bind DNA, the efficiency was however lower...
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Spirituality of advent and christmas in Bohuslav Reynek's work
Šulc, Martin ; Sládek, Karel (advisor) ; Šmejdová, Barbora (referee)
The bachelor thesis deals with exploring the spiritual reflection of advent and the Christmas period in the poetic literary work, but also in the life of the poet and painter Bohuslav Reynek. The aim of the thesis is to gain a deeper understanding of the issue and to penetrate the spirituality of advent and Christmas through the person and poetic work of Bohuslav Reynek, based on the Roman Catholic Christian pre-understanding. The main tool is the available literature dealing with the issue.
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The novel combinations of experimental approaches: mass spectrometry (MS) and photo-induced surface labelling, electron release (PIER), or cross-linking (PIXL)
Tuzhilkin, Roman ; Šulc, Miroslav (advisor) ; Kavan, Daniel (referee)
Countless electron transport/transfer (ET) processes occur in living organisms every day. Therefore, their study is a crucial field of modern structural and functional proteomics. In many cases model proteins like azurin from P. aeruginosa are utilised in experiments. This blue copper protein is favoured due to a characteristic absorbance maximum at 630 nm in Cu(II) redox state of the central Cu atom. During its oxidation to Cu(I) state the A630 value decreases allowing UV-Vis detection of ET reaction progress. We have introduced a structural photoinducible analogue of canonical amino acid Met - L-2-amino-5,5-azihexanoic acid (photo-Met) - into azurin structure to study oligomerization in solution via photo-induced cross-linking (PIXL). Using previously optimised protocols for recombinant expression in E. coli B834 we have inserted photo-Met into azurin moieties: wild type azurin and Az2W mutant where two adjacent W residues with confirmed role in electron hopping across protein-protein interface are present. The incorporation percentage of photo-Met in analysed samples was determined after SDS-PAGE and in-gel protease digestion via MALDI-TOF MS. PIXL was employed to study azurin-azurin interaction and oligomerization under different total concentrations of protein (in range of 15-300 µM). The...
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Význam sanitace v potravinářském průmyslu
Šulc, Marek
This Bachelor thesis was elaborated in the years 2011-2013 at the Institute of Post-Harvest Technology of Horticultural Products on the Faculty of Horticulture, Mendel University in Brno. The work is aimed on the importance of sanitation and hygiene in food production. This work presents cleaning technology, resources of contamination and categorization of performed sanitation. Following is an overview of foodborne illness in food and basic provisions of food law.
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The preparation and characterisation of analogues of insulin and IGF-2 selective for both isoform of insulin receptor and IGF-1 receptor
Mlčochová, Květoslava ; Žáková, Lenka (advisor) ; Obšil, Tomáš (referee) ; Šulc, Miroslav (referee)
Insulin and insulin-like growth factor 1 (IGF-1) and 2 (IGF-2) are related protein hormones with different but overlapping biological functions. All the hormones interact with a receptor within the insulin-IGF system (insulin receptor A and B, IGF-1 receptor), however with different affinity. The different interaction with individual receptors is just one of the main tools for regulation of the system that is essential for the proper functioning of the organism. Although the residues directly interacting with receptors are mainly located in A and B domains, the C and D domains probably play a role in receptor specificity. Here, we firstly focused on the impact of D domains of IGF-1 and 2 (D1 and D2 domains) and C domain of IGF- 2 (C2 domain). To probe the impact of C and D domains, we prepared insulin analogues containing a part of or an entire domain following a pattern seen in IGFs. The receptor-binding affinities of these analogues and their receptor autophosphorylation potentials were characterised. Our results revealed that the initial part of D1 domain has a detrimental effect on IR affinity that is only slightly enhanced by the rest of the D1 domain. D2 domain has rather neutral effect on IR affinity. We further showed that the addition of amino acids derived from the C2 domain to the...
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Clinically relevant protein-protein interactions participating in process of bacterial pathogenesis B. pertusis
Málek, Albert ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
Whooping cought in human population was strongly suppressed during the 20th century. But in the past few years, the incidence of whooping cough began to rise. The origin of this disease is a pathogenic gram-negative bacteria Bordetella pertussis, which is becoming resistang to currecently used antibiotics or vaccination. B. pertussis attacks human respiratory system. One of it's virulent factors is adenylate cyclase toxin (ACT). It's secreted extracellularly from bacteria and binds to cytoplasmic membrane of host cells and translocates adenylatecyclase domain (dAC) to cytosol. This enzymatic domain is activated by non- covalent interaction with eukaryotic Calmodulin (CaM). After it's activation, dAC in high concentrations synthesizes cAMP, triggering host cell's apoptosis. We studied protein interaction of dAC with CaM by the PIXL method (Photo Induced Cross Linking) and mass spectrometry (MS). Mutant of dAC, with photo-methionine (pM), incorporated in position of Leucine 240 (dACL240pM) was expressed in transformed E.coli cells B834 in mineral medium containing pM. Expressed protein was isolated by affinite chromatography and characterized by MS (determined incorporation of pM was approximately 50 % in the final protein preparation). We performed a photochemical cross-linking with isolated...
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Body surfaces of reptiles as projection screens of evolution
Abramjan, Andran ; Frynta, Daniel (advisor) ; Rehák, Ivan (referee) ; Šulc, Michal (referee)
The PhD thesis deals mainly with the coloration (and also scalation) of selected reptile species and explores evolutionary or ecological contexts indicated by these traits. The main topics can be roughly divided into two groups: 'antipredation signals' and 'effects of parthenogenesis'. The dominant method used in most studies is visual modelling. The work consists of the following case studies. 1) Blue-tongue skinks use their conspicuous blue tongues to threaten potential predators. We found that the tongue has a relatively high UV reflectance, a typical feature of intraspecific communication in lizards. Using visual models, we investigated how the blue tongue is perceived by the conspecifics and predators (birds of prey). In both visual models, the UV-blue tongue appears more conspicuous against the natural background than a pink tongue. In addition, in the conspecifics model, its hue partially overlaps with hues of UV-blue spots, which are sexually selected traits in various species of lizards. Thus, the UV-blue tongue seems to contribute to the effectiveness of the deimatic display and its possible role in intraspecific communication cannot be ruled out either. 2) We detected UV reflectance in non-pigmented areas of the skin in the Leopard gecko (Eublepharis macularius). These form white patches...
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Analysis of posttranslational modifications of proteins by mass spectrometry
Musil, Dominik ; Šulc, Miroslav (advisor) ; Kukačka, Zdeněk (referee)
Viruses, etiological agents of many infectious diseases, are small noncellular particles that run their replication process solely inside host cells. It is generally assumed that the posttranslational modifications of viral capsid proteins are responsible for their infectivity (e.g. phosphorylation catalysed by kinases of host cells). The appropriate model for study of the viral phosphorylation "profile" relation with its infectivity is mouse polyomavirus of the Polyomaviridae family. By comparison of virions, as well as the major capsid protein VP1 of wild type mouse polyomavirus with viral mutant created by deletion of the part of the genome coding regulatory proteins of virus and produced in two different cell lines WOP and 3T6 was found by mass spectrometry a major phosphorylation in the three specific amino acids of VP1. These are considered important for proper morphogenesis of virions and their ability to infect host cells. The qualitative representation was not affected by the cell line selection. Furthermore, in case of VP1 "dimerization" detected on SDS-PAGE electrophoretogram the double phosphorylation of VP1 pThr63, pSer66 was confirmed in our experimental in vivo approach. Therefore, posttranslational modifications, specifically phosphorylation, could probably affect structural...
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Study of activation properties of insulin, IGF-1 and IGF-2 analogs towards the receptors for insulin and IGF-1
Chrudinová, Martina ; Selicharová, Irena (advisor) ; Šulc, Miroslav (referee) ; Skálová, Lenka (referee)
The insulin/insulin-like growth factor signaling axis is a complex system that is involved in the regulation of metabolism and body growth. It includes three related peptide hormones: insulin and two insulin-like growth factors (IGF-1 and IGF-2), and their receptors: two isoforms of insulin receptor (IR-A and IR-B), and IGF-1 receptor (IGF-1R). Whereas insulin is involved predominantly in regulation of the metabolism, IGFs participate mainly in the regulation of cell growth, proliferation and differentiation. Due to the similarities in the structures of both the hormones and the receptors, all the hormones can bind and activate all the receptors, although with different potencies. At which point and how are the "metabolic" and "mitogenic" functional outcomes of the system diversified, as well as the structural determinants responsible for the different affinities of the hormones to the individual receptors, is still not completely understood. Deregulation of the insulin/IGF system can result in many types of pathological states, mainly diabetes mellitus and cancer. Therefore, to deeply understand the functioning of the insulin/IGF system to the structural details is crucial for development of analogs with desirable properties, that are of great clinical interest. First of all, we developed a...
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