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Differences in histone acetylation in normoxia and hypoxia
Čepek, Pavel ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
Histones and their N and C terminal tails undergo different covalent modifications that regulate gene transcription. Among these histone modifications are methylation, ubiquitinilation, SUMOylation, ADP- ribosylation, phosphorylation, proline izomerization, deimination and acetylation. Histone acetylation is regulated by histonacetyltransferases (HATs) and histondeacetylases (HDACs). The balance between acetylation/deacetylation influences chromatin condensation and thus regulates gene transcription. Acetylation balance is disrupted in many human cancers and this fact can contribute to the development of malignant diseases. Histondeacetylase inhibitors (HDACi) can restore this acetylation imbalance. One of these HDACi is valproic acid (VPA) which has been used in treatment of epilepsy for decades. VPA shows antitumour effect in many studies. Decreased expression of n-myc oncoprotein, inhibition of tumour growth and angiogenesis are one of these anticancer effects observed in neuroblastoma cell lines after treatment with VPA. Despite the fact that exact mechanism of antitumour effect of VPA remains unclear, one of the most important mechanisms is hyperacetylation of histone H3 and H4. It is shown in this work that VPA increases acetylation of histones H3 and H4 in human neuroblastoma cell lines...
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Protein-nanodiamond conjugates: the preparation and characterization
Šlegerová, Jitka ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
Nanodiamonds represent a member of vast group of carbon nanomaterials. They are characterized by chemical stability, biocompatibility, low toxicity and possibility of surface chemical modification that enables further conjugation of biomolecules, namely proteins. The most important property of nanodiamonds is easily detectable fluorescence resistant towards photobleaching. This luminescence is produced by so called (N-V) centers that can be formed by irradiation of nanodiamonds using high energy beams. Fluorescence of nanodiamonds is useful for construction of fluorescent labels and probes, cellular targeting and internalization, controlled drug delivery as well as enzyme immobilization. Two main connection modes are suitable for biomolecules' attachment: non-covalent and covalent. The first one has been utilized for decoration of nanodiamonds by various proteins, e.g. lysozyme, cytochrome c, neurotoxin or antigen. The second possibility has been demonstrated also on various proteins and, furthermore, on glycoproteins, oligonucleotides, vitamins or growth factor. During the immobilization of biomolecule on the nanoparticle surface it is crucial to keep its function, for example the catalytic activity in enzymes. For such purposes, the oxime ligation (connection of aldehyde presented on biomolecule...
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Impact of the rRNA modifications on protein synthesis
Kročová, Eliška ; Pospíšek, Martin (advisor) ; Holá, Dana (referee)
A ribosome is a supramolecular structure, which mediates synthesis of all cellular proteins, and therefore is essential for cell life. The fact, that some nucleotides of ribosomal RNA are modified, is known for forty years. However only recently, successful deeper studies on how the individual modifications are synthesized and what is their effect on ribosome synthesis and function appear. Some particular nucleotide modifications are important for the ribosome formation (like m1 acp3 Ψ1191 SSU), some others influence proper function of the ribosome (e.g. Um2921, Gm2922, Ψ2923 LSU, m1 acp3 Ψ1191 SSU). Majority of modified nucleotides in eukaryotic rRNA is being recognized by small nucleolar RNA (snoRNA). Few nucleotides is, however, recognized and subsequently modified by specific proteins. These proteins also play crucial role in ribosome maturation. In thesis presented, current knowledge on the role of ribosomal RNA nucleotide modifications during their formation and maturation, and on their function is summarized and overviewed.
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Practical courses and modification of selected tasks from Biology olympiad
Filipová, Jana ; Janštová, Vanda (advisor) ; Hrozinka, Šimon (referee)
The aim of this thesis is to summarize the integration of hands-on activities into biology lessons and to compare them with the requirements in the high-school curriculum. Further it inquires the influence of hands-on activities on the knowledge of students and their interest in the subject, and it summarizes previous findings about this topic. Sources available show that hands-on activities have a positive impact and are benefitial to students. This thesis also gives some examples of chemical tests suitable to be carried out in lessons. These tests can serve as proofs of carbohydrates, lipides and proteins in plants.
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Posttranlational protein modifications in response to DNA damage
Kroupa, Michal ; Hodný, Zdeněk (advisor) ; Novotný, Marian (referee)
- 5 - Abstract Thousands of DNA lessions occur in each cell every day of which the most toxic are double-strand breaks (DSBs). Signaling of their presence and subsequent repair are mediated by so-called DNA-damage response mechanism (DDR), which involves accumulation of many effector proteins into DSBs sites. These molecular accumulation at DSBs are termed DNA damage foci. Depending on presence of sister chromatid, DSBs are repaired by two major mechanisms: by homologous recombination and by non-homologous end joining. Both pathways lead to activation of checkpoint kinases (Chk1 or Chk2) which iniciate checkpoints in cell cycle and allow repair of damaged DNA. Signaling of DNA damage and activation of these pathways are regulated by posttranslational protein modifications. These enzymatic reactions involve mainly phosphorylation, ubiquitination and sumoylation. Recently it was shown that ubiquitination of damaged chromatin is a prerequisite for sumoylation of tumor supressors BRCA1 and 53BP1. Failure in DNA damage recognizing mechanisms caused by disorders such as modifications or mutations of 53BP1 and BRCA1 genes can lead to subsequent disruption of genomic integrity and then a high risk for selection of cell clones with tumorigenic potencial. Current research is focused on regulation of posttranslational...
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Pharmacological modifications of potential signal systems regulating metabolism of adipocytes and hepatocytes and their influence on obesity
Hodis, Jiří ; Farghali, Hassan (advisor) ; Kršiak, Miloslav (referee) ; Otová, Berta (referee)
v anglickém jazyce: Thesis abstract: Background and aims: Both obesity and metabolic syndrome form severe health problems in the whole world. Nevertheless the armament of pharmacotherapy for both diseases remains unsatisfactory. We aimed our work to main organs in risk of the mentioned diseases -liver and visceral fat using hepatocytes and visceral adipocytes as model. We detected 3 main metabolic and signalization activities- glycogenolysis, Nitric oxide (NO) production and transcription of inducible NO synthase (iNOS) in hepatocytes, lipolysis, NO production and iNOS transcription rate in adipocytes. We directed our interest to combination of peroxisome proliferation activator receptor γ (PPARγ) agonist, antagonist and β3 adrenergic agonist in the culture of epididymal rat adipocytes in the first part of our work. While in the second part we investigated the influence of β and α adrenergic mimetics, adrenergic blockers in the culture of rat high glycogen content hepatocytes. Methods: NO production was detected under the active agents treatments by detection of NO oxidative products NO2 and NO3 in media. Glycogenolysis was measured as free glucose rise released by hepatocytes into the media. NOS transcription level was extrapolated after comparative polymerase chain reaction with reverse...
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Effects of statins on Ras/Raf/ERK signaling in A431 cell line
Smutný, Tomáš ; Pávek, Petr (advisor) ; Trejtnar, František (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxikology Candidate: Tomáš Smutný Supervisor: Doc. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Effects of statins on Ras/Raf/ERK signaling in A431 cell line The ERK pathway is the signal transduction cascade, which plays an important role in different cellular functions e.g. cellular proliferation, differentiation, and survival. Its inappropriate activation is often presented in human cancers. The pathway consists of the many proteins, but the interest concentrates especially on members of the Ras subfamily (particularly H-Ras or K-Ras). Ras proteins are GTPases, which are significantly included in the oncogenesis of many human cancers. The key role in the Ras proteins function represents their posttranslation modifications with the lipid. The covalent binding of prenyl moiety to Ras protein facilitates its attachment to the plasmatic membrane, which allows its function. The prenyl groups are formed during the synthesis of the cholesterol as its precursors. The inhibition of the prenylation results in Ras proteins inactivation of the ERK signal transduction cascade and suppression of uncontrolled cell division where the activating mutations of Ras proteins are presented. Statins hypolipidemic...
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Gene silencing of ICAM-1 by lysine modified oligonucleotides
Kocourková, Aneta ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects on gene expression. In this study 2'-O-lysylaminohexyl group was linked to the uridine base, which replaces one, two or three thymine bases thus modifies the oligonucleotides. This exchange of bases was tested for improvement of silencing target protein expression. Effectivity of modifications in silencing target protein expression was examined with the alicaforsen sequence (DNA) and siRNA. Alicaforsen, currently in clinical trial 3, is a phosphorothioate targeting ICAM-1, which was the model used to evaluate the influence of modifications. The same target was chosen for siRNA to compare the efficiency of DNA and siRNA substances. For the first time, down-regulation of ICAM-1 was shown on the blood brain barrier cell line ECV304. Unmodified/modified antisense oligonucleotides and siRNA sequences were transfected into ECV304 cells with the help of a transfection agent lipofectamine 2000. After 24 hours of transfection cells were disrupted by a chemical lysis. Protein concentrations were determined by Bradford protein assay. ICAM-1 inhibition was assessed with western blot. The inhibitory effect of ICAM-1 was normalized to the corresponding actin and untreated cells. ICAM-1 protein levels were...
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Methylation profile in malignancy
Stojčeva, Nina ; Vodička, Pavel (advisor) ; Vondrejs, Vladimír (referee)
Epigenetic changes represent chemical modifications of the DNA molecule and histone proteins by which gene expression is altered. Among them, DNA methylation is a known mechanism of silencing of tumor-suppressor and DNA repair genes, with an important role in carcinogenesis. Many studies have been done in order to identify the methylation signatures of these genes in different types of cancer. In our study, we investigated the methylation status of promoter regions of eight mismatch repair genes (MLH1, MSH2, MSH3, MLH3, PMS1, PMS2, MSH6 and EXO1) in 45 sporadic colorectal cancer cases and 12 head and neck cancer patients. Two out of eight genes, MLH1 and MLH3, exhibited promoter methylation. The results from both groups of patients were concordant. We summarize that the methylation profiles of MLH1 and MLH3 promoters could be potential candidates for epigenetic biomarkers in colorectal cancer, and eventually in head and neck cancer. Further investigations, which would confirm this theory, should be carried out.
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Proteomic changes of malignant cells induced by short-term treatment with valproic acid.
Kostrouchová, Markéta ; Mandys, Václav (advisor)
Cancers are clonal disorders of multicellular organisms that arise by accumulation of genetic mutations and step-wise induction of epigenetic events that silence transcription of tumor suppressing genes and facilitate expression of tumor promoting genes. This allows cancer cells in combination with altered response of host organism to overcome the regulatory cascades that orchestrate proper cooperation of cells within the multicellular organism. Epigenetic regulation is executed on the level of DNA by methylation of CpG islands and on the level of post-translational modification of chromatin proteins, histones in the first place. Post-translational modifications of histones include histone phosphorylation, acetylation, methylation, biotinylation, poly(ADP ribosylation), ubiquitination and sumoylation. Histone acetylation is connected with transcription activation or "openness" of chromatin to regulation by transcriptional factors. Histones are acetylated by histone acetyltranferases (HATs). Histone acetylation is a dynamic process that is reversed by histone deacetylases (HDACs), enzymes that are able to remove the acetyl residue from the acetylated histones. A decrease in gene expression brought about by low acetylation of histones is part of the cancer specific transcription profile that is characterized...
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