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Recombinant expression of transcription factor TEAD1.
Řeháková, Jana ; Novák, Petr (advisor) ; Bláha, Jan (referee)
TEAD1 is one of the members of the TEAD transcription factor family. This transcription factor is very important, for example for development of organs. The structure of the entire TEAD1 transcription factor is not now yet known. Nevertheless, structure of two important domains are known. The TEA binding domain, which is important for the binding of the transcription factor TEAD1 to DNA, and the transactivation domain, to which coactivators bind. TEAD1 binds to DNA and upon coactivator bind it affects the transcription of various genes. Genes, that are affected by the transcription factor TEAD1 includes genes regulating proliferation, differentiation and apoptosis of cells. TEAD1 is also the target of the Hippo signalling pathway, which is active in adulthood and prevents abnormal growth of organs. Important for the activity of transcriptional factor TEAD1 are post-translation modifications, such as palmitoylation and phosphorylation. To discover the entire structure of the transcriptional factor TEAD1 and the way it interacts with DNA, the transcriptional factor TEAD1 was prepared recombinationally by expression in cells of Escherichia coli bacteria. Suitable conditions for production of the transcriptional factor TEAD1 were found and the cleavage of the histidine tag by thrombin was performed....
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The noncoding control region of human polyomaviruses
Pešek, David ; Saláková, Martina (advisor) ; Váňová, Jana (referee)
Genome of human polyomavirus consists of circular dsDNA around 5000 base and can be divided into three functional regions - the early viral gene region (EVGR), that encodes the regulatory T antigen and miRNAs, noncoding control region (NCCR) harboring the minimal cis- acting elements involved in viral replication and the late viral gene region (LVGR), that encodes the structural capsid proteins. Noncoding control region contains the origin of viral replication that overlaps the promoters that control expresion of early and late gene region. Noncoding control region sequences include a large number of various binding sites for cellular transcription factors involved in regulation expression from LVGR and EVGR. This thesis describes the organization of the most variable region of the PyV genome, NCCR, in chosen polyomaviruses SV40, BKPyV and JCPyV. This region often undergoes rearrangements, deletion and point mutations that affects exression of human polyomavirus. Key words: polyomavirus, noncoding control region, BKPyV virus, JCPyV virus, SV40, large T antigen, transcriptional factor
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Molecular mechanisms in transcriptional regulation of neurosensory development
Filová, Iva ; Pavlínková, Gabriela (advisor) ; Tureček, Rostislav (referee) ; Valihrach, Lukáš (referee)
The development, maturation, and viability of inner ear neurosensory cells depend on the spatiotemporal expression of multiple transcriptional factors. Based on three mouse models [Tg(Pax2-Isl1)], Sox2CKO, and Neurod1CKO, this thesis investigates the function of three transcriptional factors ISL1, SOX2 and NEUROD1 in the neurosensory development of the inner ear. The mouse mutant [Tg(Pax2-Isl1)] carries transgenic sequence containing Isl1 gene under Pax2 regulatory sequence in its genome. ISL1 ectopic expression driven by Pax2 regulatory sequence resulted in the enlarged cochleovestibular ganglion and accelerated neurite extension in [Tg(Pax2- Isl1)] embryos. In adult mutants, we detected an early onset of age-related hearing loss correlating with the worsening function of outer hair cells. These changes were associated with the loss of medial olivocochlear efferent neuron fibers innervating outer hair cells. For the first time, we showed that the age-related hearing loss (presbyacusis) might be caused by efferent innervation defects besides hair cell loss and spiral ganglion degeneration. In addition to presbyacusis, [Tg(Pax2-Isl1)] mice suffered from hyperactivity that was diminished by the administration of picrotoxin - channel blocker for GABA receptor chloride channels. This indicates that...
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Recombinant expression of transcription factor TEAD1.
Řeháková, Jana ; Novák, Petr (advisor) ; Bláha, Jan (referee)
TEAD1 is one of the members of the TEAD transcription factor family. This transcription factor is very important, for example for development of organs. The structure of the entire TEAD1 transcription factor is not now yet known. Nevertheless, structure of two important domains are known. The TEA binding domain, which is important for the binding of the transcription factor TEAD1 to DNA, and the transactivation domain, to which coactivators bind. TEAD1 binds to DNA and upon coactivator bind it affects the transcription of various genes. Genes, that are affected by the transcription factor TEAD1 includes genes regulating proliferation, differentiation and apoptosis of cells. TEAD1 is also the target of the Hippo signalling pathway, which is active in adulthood and prevents abnormal growth of organs. Important for the activity of transcriptional factor TEAD1 are post-translation modifications, such as palmitoylation and phosphorylation. To discover the entire structure of the transcriptional factor TEAD1 and the way it interacts with DNA, the transcriptional factor TEAD1 was prepared recombinationally by expression in cells of Escherichia coli bacteria. Suitable conditions for production of the transcriptional factor TEAD1 were found and the cleavage of the histidine tag by thrombin was performed....
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Role of Nkx2.5 on development and electrophysiology of the mouse heart
Hámor, Peter ; Sedmera, David (advisor) ; Neckář, Jan (referee)
Role of Nkx2.5 on development and electrophysiology of the mouse heart Prague 2015 Bc. Peter Hámor ABSTRACT The objective of this thesis is to investigate the role of Nkx2.5 gene dosage on electrophysiology of the mouse heart in prenatal stage of its development, in which the physiological functions of the heart fail to function properly. The main goal of this work is to search for differences in conduction of electric impulses through the embryonic mouse heart according to their genotype. Special method of capturing the conduction of electric impulse through myocardium was used for this purpose, called optical mapping. Thanks to this method I was able to construct images and videos capturing transition of the impulse with marked beginning of the activation and its direction in the heart. These outputs, or optical maps, help to define anomalies and defects compared with a normal functioning heart. The thesis focuses on the expression of the transcription factor Nkx2.5 and regulatory components related with the correct formation and physiology of the heart until 9.5 days post coitum. Individuals in this developmental stage were optically mapped and compared according to their genotypes - homozygous non-mutant, heterozygote and homozygous mutant mouse embryos exhibited some degree of similarity, while other...
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Functional role of ISLET1 in the neurosensory development of the inner ear.
Hampejsová, Zuzana ; Pavlínková, Gabriela (advisor) ; Peterka, Miroslav (referee)
Loss of hearing affects more than 10 % of the population, and one newborn in a thousand is born with defects of the inner ear. Transcriptional factors involved in the development of inner ear are important in our understanding of the causes of inner ear defects. ISLET1 is one of these factors. ISLET1 expression is detected in the sensory and neuronal cells of the inner ear. It participates in otocyst formation, and the specification and differentiation of cells of cochlea and vestibular system. The functional role of ISLET1 during inner ear development was investigated. Its role was studied by using Pax2-Isl1 transgenic mice that overexpress Islet1 under the control of the Pax2 promoter. Two transgenic lines were generated, Pax2-Isl1/300 and Pax2- Isl1/52. Two copies of the Pax2-Isl1 transgene were inserted to Pax2-Isl1/300 genome and one copy was inserted to the Pax2-Isl1/52 genome. Defects in sense of hearing were detected in both lines and circling behavior, a defect of balance, was detected in the Pax2-Isl1/300 transgenic mice. We observed high postnatal lethality in heterozygote transgenic mice. Pax2-Isl1/52 homozygote mutation is lethal at embryonic day 10 (E10,5). Pax2-Isl1/300 homozygote letality couldn't be detected because of the inability to breed heterozygote mutated mice of this line....
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