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Study of the microRNAs in patients with head and neck squamous cell carcinoma
Hudcová, Kristýna ; Šulc, Miroslav (referee)
Searching for markers of disease is at the forefront of scientific interest. Biomarker of a specific disease should help in disease management - whether for diagnosing a disease or predicting its progress. We picked microRNAs - short RNAs that regulate mRNA translation, due to their stability and function in the regulation of many cellular processes. Therefore they can be used as a biomarker for many diseases. In our study we focused on neck squamous cell carcinoma (HNSCC), since this type of cancer is widespread and clinically important markers are missing. It is in our interest to seek new kinds of markers, suitable for early diagnosis of the disease and the most accurate assessment of prognosis. We selected 3 candidate miRNA (miR-29c-3p, miR-200b-5p, miR-375-3p) and used qRT-PCR method to profile their expression in tissues of patients with HNSCC. MiRNA expressions in 42 tumor samples were compared with the miRNA expression in adjacent tissue of the tumor. Adjacent tissue surrounds the tumor and has interesting properties, as it looks like a histological normal tissue, but on closer inspection it is not. It is affected by tumor tissue and may contribute to tumor progression. We managed to distinguish between these two tissues using miR-29c-3p and miR-375-3p. Additional analyzes were focused on...
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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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The role of ultraviolet light in communication of birds
Šulc, Michal ; Sedláček, Ondřej (advisor) ; Albrecht, Tomáš (referee)
Recently, it has been found that the visual system of birds and humans differ remarkably, especially in the ability to perceive ultraviolet radiation. This component of the spectrum significantly affects various types of the birds' behaviour. UV reflection may be an indicator of the qualities of males, i.e. females often prefer individuals with expressive UV ornamentation. Likewise, parents can distribute the delivered food unevenly among their nestlings, depending on the nestlings' reflection of ultraviolet radiation. Recent studies also showed that the hosts of brood parasites may use UV spectrum as a key for recognizing the parasitic eggs from their own. The aim of my bachelor thesis is to review the current knowledge of the influence of UV radiation on sexual selection of birds, the interaction between parents and offspring and interactions between brood parasites and their hosts.
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Recombinant expression of RhoA protein with affinity tag.
Filipová, Barbora ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
The rate of intestinal infections associated with antibiotics has been rising steeply. Virulent toxins of C. difficile, Yersinia and many other bacteria target the RhoA protein. The protein is a GTPase involved in signalling pathways, its major function being the regulation of the actin cytoskeleton. The above mentioned toxins render this protein non- functional, which may lead up to a fatal destruction of the cell. The present diagnostic methods of these infections are insufficient. The use of protein chips and mass spectrometry to detect any RhoA protein modifications seems to provide a feasible method of determining these intestinal bacterial diseases. Therefore the purpose of this study is to use recombinant methods to prepare the desired RhoA protein with a bound affinity probe which will serve to its immobilisation on a biochip. The thus prepared protein will be subsequently used to diagnose the previously described diseases. In Czech.
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Study of structure and interaction of human lymphocyte receptors
Bláha, Jan ; Vaněk, Ondřej (advisor) ; Šulc, Miroslav (referee) ; Obšil, Tomáš (referee)
Natural killer (NK) cells are an essential part of immune system, providing self-surveillance of virally infected, stress transformed or cancerous cells. NKR-P1 receptors and their ligands from clec2 gene family represent an alternate missing-self recognition system of NK cells based on interaction of highly related C-type lectin-like receptors. Human NKR-P1 has been described more than twenty years ago but still remains the sole human orthologue of this receptor family, particularly numerous in rodents. On binding to its cognate ligand LLT1, NKR-P1 can relay inhibitory or co-stimulatory signals. Although being interesting targets for their potential role in tumor immune evasion and autoimmunity, nature of their interaction is still unclear. To elucidate the architecture of their interaction, we developed a generally applicable method for recombinant expression of human NKR-P1 and LLT1 and their homologues based on transfection of HEK293S GnTI- cells. Further, we described a stabilizing mutation His176Cys, that enables for expression of highly stable and soluble LLT1. Finally, we have crystallized LLT1 and human NKR-P1 in different glycosylation states both as individuals and in complex. While both structures of LLT1 and NKR-P1 follow the classical C-type lectin-like superfamily fold, contrary to...
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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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Evolution and function of polymorphism in warningly coloured prey
Fárová, Monika ; Exnerová, Alice (advisor) ; Šulc, Michal (referee)
Polymorphism can be expected in warningly colored prey if the prey is protected from predation by nothing else but its coloration. On the contrary, in defended prey, polymorphism was only until recently considered a controversial phenomenon due to its longer and costly avoidance learning. Individual moprhs can vary in different components of warning coloration: color, pattern, melanization degree, and internal and external contrast of colour patterns. This makes it difficult for predators to learn and remember warning signals of defended prey and avoid it in the future. Predator selection pressures and mechanisms leading to polymorphism differ between defended and undefended prey. For undefended prey, it is a negative frequency-depended selection that supports rare morphs or the multiple models hypothesis for one mimetic species. For defended prey, the polymorphism can occur temporarily (i. e., be unstable) and it also can be allowed by spatial heterogeneity of morphs or, as for undefended prey, one species can mimic multiple models. Quasi-Batesian mimesis could also contribute to the existence of the polymorphism, due to mechanisms similar to those in unprotected prey. Apart from selection by predators, there are other factors, that contribute to the existence of polymorhism in prey warning...
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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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Preparation of DNA-binding domain of Forkhead transcription factor FOXO3
Dolejš, Vojtěch ; Šulc, Miroslav (advisor) ; Ptáček, Jakub (referee)
This bachelor thesis is part of a project aiming for the development of low molecular compounds which would be capable to inhibit the interaction between human transcription factor FOXO3 and DNA. Main goal of this thesis is preparation of 15 N-labelled DNA-binding domain of FOXO3 protein (FOXO3-DBD) and verification of its native structure using 1 H- 15 N HSQC NMR experiment. FOXO transcription factors are important and evolutionary conserved regulatory proteins, which are involved in many crucial cellular processes. The activity of FOXO proteins is regulated by posttranslational modifications, out of which the most important are phosphorylation, acetylation and ubiquitination. Forkhead transcription factors participate in a variety of different cellular functions, although its expression is limited to specific tissues. They contain approximately 100 amino acids long DNA-binding domain composed of several parts. Among its main functions belong the regulation of cell cycle and apoptosis, proliferation and cell differentiation, metabolism control and stress-response regulation. Some types of tumor cells have developed resistance against chemotherapy by increasing activity of FOXO3 transcription factors. For this reason, it is necessary to look for means to specifically suppress the function of this...
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