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Characterization of the ABC-F protein Sco0636 in Streptomyces coelicolor
Pinďáková, Nikola ; Balíková Novotná, Gabriela (advisor) ; Mikušová, Gabriela (referee)
The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...
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3D scene reconstruction using Clifford algebras
Hrubý, Jan ; Návrat, Aleš (referee) ; Hrdina, Jaroslav (advisor)
Tato diplomová práce má za cíl seznámit čtenáře se stále ještě relativně novou a neznámou oblastí matematiky, s geometrickou algebrou. Nejdříve jsou uvedeny základní definice a poté jsou studovány vlastnosti obecné geometrické algebry. Další velká část textu se věnuje Konformní geometrické algebře, která je v současnosti jedna z nejvíce zkoumaných a aplikovaných geometrických algeber. Jsou popsány její algebraické a geometrické vlastnosti, konkrétně schopnost reprezentovat určité geometrické objekty jako vektory. Taktéž umožňuje počítat jejich průniky a konformní transformace. Další část textu je zaměřena na aplikace Konformní geometrické algebry, nejdříve k popisu kinematiky robotické ruky a poté v binokulárním viděni.
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Inhibitors of the eukaryotic translation initiation factor eIF4E1
Pospíšilová, Klára ; Pospíšek, Martin (advisor) ; Roithová, Adriana (referee)
Protein synthesis is one of the most important processes that take place in a cell. Thus there are various mechanisms in a cell that regulate it. If that regulation fails it may lead to serious pathologies. An example of this is an abnormal increase in the production of eukaryotic translation initiation factor 4E1 which occurs in some types of cancer including head and neck squamous cell carcinoma, colorectal cancer, cervical cancer or lung cancer. Enhanced availability of factor 4E1 enables transformed cells to undertake a more intense translation. The expression of individual proteins is not increased to the same extent though. The enhancement in the level of factor 4E1 has a more significant effect on oncogenic proteins. Malignant transformation caused by an incorrect regulation of factor 4E1 can be precluded by an application of compounds that impair the activity of factor 4E1. A summarization of 4E1 inhibitors is the subject of this work.
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Visualization of RNA and its translation in mammalian oocyte
Bašus, Kryštof ; Tětková, Anna (advisor) ; Macůrková, Marie (referee)
Translation is one of the fundamental biological processes. It is assumed that the locali- zation of mRNA and its translation in a certain time and at specific locations plays a role in many cellular processes. The mammalian oocyte becomes transcriptionally inactive when it reaches its full size and utilizes only RNA that has been synthesized and stored in the early stages of development. Thus the regulation of protein synthesis at the translational level is cri- tical for the correct completion of meiotic maturation of oocytes and early development of embryos. To monitor cellular physiology, it is necessary to be able to visualize and monitor spe- cific molecules and processes at single cell level. This is enabled by the development of light microscopy and fluorescence probes that specifically bind to certain organelles, cellular struc- tures, proteins or other molecules. In this thesis I describe selected methods of visualization of RNA, global translation as well as translation of specific transcripts, and proteins. The methods that this thesis describes are RNA FISH, visualization of translation using methionin analogs, FUNCAT, SUnSET, FlAsH, ReAsH, TRICK, SINAPS, FUNCAT-PLA, PURO-PLA. Key words: Translation, RNA, Protein, Visualization, Oocyte
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Eukaryotic translation initiation factor 3 and its role in plant translation regulation
Raabe, Karel ; Michailidis, Christos (advisor) ; Retzer, Katarzyna (referee)
After transcription, mRNA translation is another highly regulated process in gene expression. In plants, translation regulation plays an important role during progamic phase, fertilization and seed development, where synthesized transcripts are stored and selectively translated later in development. Translation regulation is also broadly used in stress responses as a fast and flexible tool to change gene expression; therefore, it plays an essential role in the survival strategy of sessile organisms like plants. Both regulation of the global translational rate as well as selective regulation of specific transcripts modulate the final gene expression response. Most of the regulatory mechanisms are concentrated in the stage of initiation, which is facilitated by several translation initiation factors. Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these factors, consisting of 12 conserved subunits. Its key function in the initiation is to scaffold the formation of the translation initiation complex and in the scanning mechanism accuracy. In past decades, additional eIF3 functions were discovered acting upon the whole translation cycle, including its importance in global and specific translation regulation. The aim of this work is to review eIF3 functions and to...
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Characterization of eukaryotic translation initiation factor 3 subunits (eIF3) in A. thaliana male gametophyte
Linhart, Filip ; Hafidh, Said (advisor) ; Retzer, Katarzyna (referee)
From RNA-to-protein, translation initiation and protein synthesis is mediated by trans-acting factors that recognize mRNA features common to almost all eukaryotes. Eukaryotic translation initiation factor 3 complex (eIF3) is a highly conserved protein complex that recognizes 5'-CAP elements of the mRNA to initiate translation. eIF3 consists of nine subunits, three of them having two isoforms: eIF3A, eIF2B1, eIF3B2, eIF3C1, eIF3C2, eIF3D, eIF3E, eIF3F, eIF3G1, eIF3G2, eIF3H and eIF3K. This work deals with functional characterization, expression and subcellular localization of eIF3B1, eIF3B2 and eIF3E in Arabidopsis thaliana male gametophyte and interaction of eIF3E with the Constitutive photomorphogenesis 9 (COP9) complex as a regulatory complex of eIF3E post-translational control. Here we show that depletion of eif3b1 or eif3b2 is not gametophytic lethal and that the two protein might function redundantly, whereas, knockout of eIF3E causes male gametophyte lethality. Interestingly, eif3b1 show post-fertilization defects during embryogenesis, suggesting that its redundancy with eIF3B2 is restricted to the gametophyte. Gene expression studies revealed high expression of eIF3 subunits in actively dividing zones of leaf primordia, root meristem and root elongation zones as well as in the vegetative...
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Protein synthesis in cellular stress
Cienciala, Martin ; Pospíšek, Martin (advisor) ; Holá, Dana (referee)
Environmental stress is a daily bread for organisms across many different branches of life. Very complex response mechanisms have evolved to tackle such insults. Yeast Saccharomyces cerevisiae is adapted especially well for counteracting oxidative and osmotic stress. These unfavorable conditions usually lead to inhibition of protein synthesis. The GCN2 kinase is thought to be responsible for this phenomenon. General inhibition of protein synthesis is accompanied by an increase in expression of proteins engaging in stress response. Production of these proteins is often preceded by specialized regulatory processes, that operate on various stages of expression. This thesis will try to present the diversity and complexity of the individual regulatory layers.
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The role of stress granules and 4E-BP in heat-stressed cells of S. cerevisiae
Kolářová, Věra ; Hašek, Jiří (advisor) ; Zimmermannová, Olga (referee)
The cells are capable of very quick and specific reactions on stress conditions. Influence of translation, specifically initiation of translation by inhibition factors, is one of the main regulatory process. Two of eIF4E-binding proteins (4E-BP), Eap1p and Caf20p, are known as cap-dependent translation repressors in yeast Saccharomyces cerevisiae. We used in vivo fluorescent microscopy analysis to show different reaction of Caf20p and Eap1p to heat stress. Protein Caf20p does not react on heat shock and stays difused in cytoplasm. Contrary to Caf20p reaction, protein Eap1p accumulates in cytoplasm close to stress granules (SGs). This work shows that Eap1p is involved in stress granules assembly. In the absence of Eap1p, yeast cells react to the heat stress with small and less focused SGs. Dele- tion of CAF20 does not affect SG assembly. This points to specific function of SG in distribution of factors connected with stress reaction. Polysomal analysis shows that deletion of one of initiation translation repressors does not affect heat induced global repression of translation. In permisive condition deletion of EAP1 may cause defect in addition of 60S ribosomal subunits. Absence of protein Eap1p causes morphological defect. That point to a different reactions of Eap1p and Caf20p on heat stress and possible...
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