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Recombinant preparation of DNA binding domain of transcription factor TEAD1
Kúdelová, Veronika ; Novák, Petr (advisor) ; Dračínská, Helena (referee)
TEAD proteins belong to a significant family of transcription factors that contribute to the regulation of organism growth and cell differentiation during its development by activating the expression of a wide variety of genes. This family shares two highly conserved sites, the TEA DNA binding domain, after which the proteins have been named, and the domain by which transcription factors bind other coactivators. Because TEAD proteins are not able to activate transcription themselves, they interact with a number of coactivators. These coactivators allow the transcription of the gene of interest to be regulated. Failure of TEAD protein activity regulation can lead to cancer. Therefore, TEAD family proteins nowadays play an important role in the development of new anticancer drugs. One way of inhibiting these proteins is to block the active site in their DNA binding domain, thus, to block their binding to DNA. This bachelor thesis deals with recombinant expression of said DNA binding domain of transcription factor TEAD1, which is extended by amino acids in unstructured regions. After finding suitable conditions of protein production, we proceeded to large volume production which was followed by purification and protein identity verification. Finally, the ability of the produced protein to interact...
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Structure and dynamics of mouse C-type lectin-like receptors.
Wallenfels, Lucie ; Novák, Petr (advisor) ; Obšilová, Veronika (referee) ; Šebela, Marek (referee)
Natural killer (NK) cells represent indispensable part of the innate immunity as they are capable of promptly identifying virally infected or tumor cells and participating in the regulation of adaptive immune responses. These functions are ensured by the interplay between NK receptors, creating a complex regulatory system. Solving the receptors' structure may contribute to an overall understanding of NK cell biology. Presented thesis describes an elucidation of the structure of the inhibitory C-type lectin-like receptor (CTLR) Nkrp1b with an emphasis toward structural features (stalk, loop and oligomerization state) which might affect conformation or interactions of this receptor. The interaction of Nkrp1b with its ligand, Clr-b protein, is immunologically significant as it regulates NK cells' activity independently and monitors changes that are not visible to cytotoxic T lymphocytes. To study individual structural aspects of Nkrp1b, two protein variants were recombinantly prepared in bacterial expression system: entire ectodomain and ligand-binding domain lacking the stalk. Using a range of mass spectrometric techniques in combination with homology modeling and molecular dynamics, we proposed the Nkrp1b structure including its monomeric and dimeric arrangements. In addition, the oligomerization...
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Multi-function Cultural Centre "Creative City" in Brno
Iermolovych, Ganna ; Novák, Petr (referee) ; Vojtová, Lea (advisor)
The master degree project presents design of conversion and extension of an old former prison situated between the streets Cejl, Soundni and Bratislavska for cultural, commercial and manufacturing purposes and forming multifunctional cultural center. The proposal consists of the conversion of two floors of existing building on the street Soudni, demolition of additional building on the street Bratislavska, while the additional building on street Cejl is belonging to other owner and will remain in existing status. Before the new building it is created a small square, that connects new and old building and the surroundings. Main goal is to create center for “creative” specialization, in which will be placed as working (studio, offices, conference hall) and performing spaces, but there also will be residential part (hostel and renting flats). Main purpose – create interesting space for collaboration of creative professions and to create the new cultural center.
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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...
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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 single stranded DNA by biophysical techniques
Svoboda, Jakub ; Schneider, Bohdan (advisor) ; Novák, Petr (referee)
DNA is the fundamental functional molecule of all domains of life. One of its characteristics is the ability to self-associate to form a double stranded helix. Nevertheless, even single stranded DNA can form non-canonical structures such as hairpins, triplexes or tetraplexes. One of the interesting single stranded sequences are REP elements. These sequences form a part of bacterial transposable elements, so-called insertion sequences, which can be found in a great number of copies in wide range of bacterial species. This thesis studies structure and properties of selected REP sequences. It presents the results of spectral measurements by technique of circular dichroism, melting curves from differential scanning calorimetry, and monocrystal diffraction data. Key words Single-stranded DNA, crystallography, circular dichroism, calorimetry, transpozone, REP elements, insertion sequences.
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Protein radical footprinting as a tool for protein therapeutcs validation.
Binar, Michal ; Novák, Petr (advisor) ; Ječmen, Tomáš (referee)
Mass spectrometry techniques are very important and useful tool in studying of protein structure. One of these techniques is the covalent labeling of proteins. The covalent labeling can be used for determination of the accessibility of protein surface and their dynamics in general. Although a lot of reagents have already been developer for this method, most of them are limited by their ability to selectively react with only some of amino acids, mostly lysine, arginine or tyrosine, and non-selective methods such as fast photochemical oxidation of proteins are very demanding and costly. Therefore, there is still a endeavor to develop new methods of covalent labeling. This new method could be trifluoromethylation, which is increasingly used in the modification of organic molecules, and whose potential we have decided to study. In this work we used a new group of formally electrophilic agents, which is formed on the basis of a cyclic hypervalent iodide nucleus. This group so-called Togni reagents was used as a tool for radical protein labeling. Since the structure was well characterized by X-ray, NMR and MS, the human carbonic anhydrase has been selected as a model protein. The modified protein was analyzed by a bottom up approach using a combination of liquid chromatography and high resolution tandem...
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The universal inclusivist soteriology (of Pauline type?) according to Pierre Teilhard de Chardin
Novák, Petr ; Houkal, Jan (advisor) ; Bouma, David (referee)
This bachelor thesis elaborates the topic of ontological and functional significance of the person of Jesus Christ, according to French philosopher and scientist PierreTeilhard de Chardin. At present time, his theological contribution is appreciated by Pope Benedict XVI and Francis. But many of his thoughts and genial writings were censored and forbidden in the preconciliar Catholic discourse during his lifetime. All his major works were published posthumously. However, he still remains a controversial author. The study aims to assess whether it could be considered the functional christology of Teilhard de Chardin as the universal inclusivist soteriology of Pauline type. The basis and frame of reference for coparison are proto-Pauline epistles of The New Testament (especially Romans, First Corinthians and Philippians). The inclusivist concept of salvation in the works of Teilhard de Chardin can be come the starting-point for ecumenical dialogue between the Christian denominations that share the Apostle Paul. Keywords christology, inclusivism, Pierre Teilhard de Chardin, Saint Paul, soteriology
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Study of molecular mechanisms of the signaling proteins regulation
Kylarová, Salome ; Obšilová, Veronika (advisor) ; Mikšík, Ivan (referee) ; Novák, Petr (referee)
EN The aim of this study was to investigate the regulatory mechanisms of two important signaling proteinkinases and promising therapeutic targets, ASK1 and CaMKK2. ASK1 kinase is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-JNK kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress. The function of ASK1 is associated with the activation of apoptosis and thus plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration or cardiovascular diseases. The natural inhibitor of ASK1 is a ubiquitous oxidoreductase, thioredoxin, which is probably bound to N-terminus of ASK1, thus preventing a homophilic interaction and subsequent ASK1 activation. It has been suggested, that upon oxidative stress and oxidation of thioredoxin active site, thioredoxin dissociates from ASK1, but the structural basis of this interaction remains unclear. Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of CaM kinase pathway that activates CaMKI, CaMKIV and AMPK involved in gene expression regulation or apoptosis activation. Function of this protein is often associated with neuropathology, carcinogenesis and obesity. CaM kinases are activated via binding Ca2+ sensor protein...
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