|
|
Functional analysis of the ERK signaling pathway in epithelial cells
Galvánková, Kristína ; Vomastek, Tomáš (advisor) ; Rösel, Daniel (referee)
The MAPK/ERK pathway, which is evolutionarily conserved in eukaryotes is one of the most intensively studied signaling pathways and consists of a three-tier cascade of Raf- MEK-ERK protein kinases. A variety of extracellular signals are transduced from receptors to hundreds of substrates by a series of sequential phosphorylations leading from Raf to MEK to ERK. The ERK pathway regulates a plethora of cell- and extracellular signal- specific responses such as gene expression, proliferation, differentiation, migration, and apoptosis. The proper execution of these physiological processes requires a precise temporal and spatial regulation of the pathway and disruption of the regulatory mechanisms leads to pathological consequence such as tumor transformation. Specificity and regulation of signal transduction are provided in part by the presence of isoforms at each level of the ERK signaling pathway. The functional differences between the effector protein kinases ERK1 and ERK2 have been controversial for a long time, but it is still unclear how important they are in achieving an appropriate cellular response. In this work, we focused on the functional characterization of ERK1 and ERK2 isoforms in MDCK epithelial cells. Specifically, we examined the effects of ERK2 inactivation on cell morphology and...
|
|
|
Where transcription meets translation
Hegrová, Karolína ; Krásný, Libor (advisor) ; Mašek, Tomáš (referee)
Transcription and translation are key steps in gene expression. The RNA polymerase (RNAP) plays a major role in the transcription process, while the ribosome is involved in translation. In bacteria, these two processes are not separated. RNAP and the ribosome interact, and its called transcription- translation coupling. In this thesis, I discuss the mechanism of transcription and translation, with the main focus on transcription-translation interactions. I divide these interactions into indirect, which are caused by regulátory molecules, and direct, where the ribosome directly binds with RNAP. When physical binding occurs, either a tight junction between these molecules occurs or a bridge is formed by transcription factors. Then I describe regulatory function of this connection and explain the exceptions where transcription and translation don't link. In the last part of the thesis, I focus on elongation factor Tu (EF-Tu), its important role in metabolism, its interactions with MreB protein, and how this factor is used by some bacteriophages. Finally, I mention its possible role in transcription-translation interactions. Key words: transcription, translation, transcription-translation coupling, RNA polymerase, ribosome, transcription factors, EF-Tu
|
|
|
Molecular mechanisms in the development of the cochlea
Procházková, Šárka ; Pavlínková, Gabriela (advisor) ; Novák, Josef (referee)
Molecular mechanisms in the development of the cochlea The cochlea is the primary structure for the perception of sound. A spiraled cochlear duct contains the auditory organ, the organ of Corti, is innervated by sensory neurons of the spiral ganglion. The organ of Corti contains hair cells, the sensory receptors of the inner ear, that are responsible for the mechano-transduction of sound waves into electrical signals. The development of the cochlea is regulated by the cellular and molecular processes, including cochlear outgrowth, cellular differentiation, and patterning. The precise coordination of multiple processes during the transformation of a flat embryonic epithelium into the cellular mosaic of the adult structure requires a multitude of transcription factors and signaling pathways. These regulatory networks dictate cell fate and timing decisions during cochlear development. Interestingly, these mechanisms operate as well during sensory epithelial cell regeneration after damage and during stem cell directed differentiation, making developmental studies instrumental for improving therapies for hearing impairment.
|
|
|
Peak identification from ChIP-nexus data.
Drahoňovská, Lucie ; Převorovský, Martin (advisor) ; Gahurová, Lenka (referee)
Proteins play a very important role in live organisms. Their roles are for example structural, transport, regulatory or catalytic. What genes will be expressed, what proteins will be made and at what rate can have a strong impact on the function or even health of the organism. Gene expression is significantly regulated by transcription factors, whose activity may cause multiple diseases or disorders (Latchman 1997). Studying those factors and their function is therefore very important. Several methods were developed to this cause, ChIP-chip, ChIP-seq, ChIP-exo and ChIP-nexus. They enable us to study the binding sites of transcription factors and other DNA-binding proteins with various degrees of resolution. In this thesis I am going to describe the above mentioned methods and peak callers, softwares used for analysis of data obtained by those methods. I will also attempt to do peak calling of ChIP-nexus data of Cbf11 protein and compare the outcomes.
|
|
|
Molecular mechanism of animal cells adaptation on hyperosmotic induced stress
Novotná, Jana ; Vávra, Jiří (advisor) ; Převorovský, Martin (referee)
Various types of cells animal tissues consist of need to adapt to intracellular as well as extracellular osmotic changes in order to maintain homeostasis. Hypertonicity (increased osmolarity) is one of the factors activating complex cellular reactions. In order to manage such stress, a cell needs to incorporate regulation pathways that can either regulate ion transporters through WNK kinases or activate gene expression of transporters of compatible osmolytes through the transcription factor TonEBP/NFAT5. Physiologically exposed to hypertonic conditions are cells in renal medulla, cartilage, inner ear tissues, in specialised neurons or buccal epithelium. This work includes basic and current knowledge about the adaption of cells to increased osmolarity of outer environment. Key words: Hyperosmolarity, osmotic stress, TonEBP/NFAT5, WNK kinases, NF-κB, HSP70
|
|
|
The role of innate lymphoid cells in influenza virus infection
Mouyabi, Flaviancia ; Hrdý, Jiří (advisor) ; Kössl, Jan (referee)
Innate lymphoid cells (ILCs) are recently discovered group of innate immune cells. They do not have antigen-specific receptors but they can be activated by cytokines similarly to T lymphocytes. ILCs have a crucial role in the regulation of inflammation, tissue repair, containment of commensals, anti-infection immunity and regulation of tissue homeostasis. The presence of mouse and human ILCs can be detected in the lung during and after influenza virus infection when ILCs contribute to the restoration of damaged lung parenchyma. ILCs directly or indirectly provide protection against viral infections by secretion of various cytokines and co-operation with other cells (e.g. T cells, macrophages). Overall, lung ILCs are important in immune responses and tissue homeostasis, but further studies on this topic are needed to fully understand their role. The aim of this thesis was to specifically characterize these cells, focus on their function in the lung, and describe their role in the course of influenza virus infection.
|
|
|
Study of the mammalian oncogenic transcription factors in the yeast model
Novák, Josef ; Zámostná, Blanka (advisor) ; Šťovíček, Vratislav (referee)
Yeast serves as a useful tool for studying cellular processes and therefore a large amount of techniques and protocols has been developed. There are special methods for studying the transcriptional factors in yeast, such as modified yeast two-hybrid screens, yeast one-hybrid screens and systems studying ability of transcription factors to transactivate a reporter gene. Oncogenes from AP-1 complex, Myc and Myb protein families are described in this work. Using a yeast model the structural-functional properties of proteins can be easily studied and in some cases even their ability of oncogenic transformation can be predicted (FASAY or ability of c-Myc to transactivate a reporter gene). However, results from yeast models must be confirmed in mammalian cells. 1
|
|
|
Potential of genetic engineering for breeding plants tolerant to abiotic stresses: cold resistance in rice
Lotová, Gabriela ; Lipavská, Helena (advisor) ; Janská, Anna (referee)
With increasing population and climate change, there has been an increase in efforts to breed more efficient crops. Genetic engineering has opened unprecedented breeding possibilities in developing plants with desired traits. Transgenic crops with better qualities, including resistance to adverse environmental conditions, can contribute to solving problems of hunger and malnutrition in developing countries. Although society perceives genetically modified crops rather negatively, these crops are widely used as feed for livestock and outside Europe also for human nutrition. Because of the complexity of resistance to abiotic stress, the utility of genetic manipulations for the breeding of resistant plants was previously not anticipated. However, it turned out that modification of the stress signalling cascade or transcription factors can lead to success. This thesis summarizes the possibilities of genetic modification of crops, which may result in better tolerance to cold, and is mainly focused on rice. Part of the work deals with transduction of cold signal, whose modification can also result in increased tolerance to cold. Another part deals with transcription factors that activate expression of low temperature- resistant genes. The greatest attention is paid to CBF/DREB transcription factors that...
|
|
|
Utilization of protein radical foootprinting for stuctural biology
Polák, Marek ; Novák, Petr (advisor) ; Junková, Petra (referee)
(In English) The reaction of highly reactive oxygen radicals with protein solvent-accessible residues can be utilized to map protein landscape. Fast photochemical oxidation of proteins (FPOP) is an MS- based technique, which utilizes highly reactive radical species to oxidize proteins and map protein surface or its interactions with their interaction partners. In this work, FPOP was employed to study protein-DNA interactions. First, a full-length of FOXO4-DBD was successfully expressed and purified. The ability of the protein to bind its DNA-response element was verified by electrophoretic and MS-based techniques, respectively. Optimal experimental conditions were achieved to oxidize the protein itself and in the presence of DNA, respectively. Oxidized samples were analyzed by bottom-up and top-down approach. In the bottom-up experiment, modification of individual residues was precisely located and quantified. Different extend of modification was observed for protein alone and in complex with DNA. To avoid experimental artifacts analyzing multiply oxidized protein, standard bottom up approach was replaced by a progressive top-down technology. Only a singly oxidized protein ion was isolated, and further fragmented by collision-induced dissociation (CID) and electron-capture dissociation (ECD),...
|
|
|
Chromatin immunoprecipitation of selected transcription factors
Smetanová, Jitka ; Vališ, Karel (advisor) ; Převorovský, Martin (referee)
The TEAD family of transcription factors regulates expression of genes affecting cell proliferation, differentiation and apoptosis. The activity of a particular transcription factor called TEAD1 is regulated by the Hippo signalling pathway. The Hippo pathway has been implicated to play a role in cancer suppression, however its precise mechanism remains unclear. MYC and GLUT1, genes which are coding two key regulators of glycolysis, were recently described as potential targets of the Hippo signalling pathway in human leukemia cells. In this diploma thesis, I tried to confirm the proposed interaction of the transcription factor TEAD1 with regulatory sequences of MYC and GLUT1 genes using chromatin immunoprecipitation (ChIP) analysis in human leukemic cells. However, I failed to successfully isolate TEAD1 complexes using ChIP. So, I discuss in my diploma thesis also possible reasons for this outcome, including biological and methodological issues. (In Czech) Key words: Transcriptional regulation, TEAD transcription factors, chromatin immunoprecipitation, leukemia
|
guest ::
