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Factors interacting with bacterial RNA polymerase
Sudzinová, Petra ; Krásný, Libor (advisor) ; Fišer, Radovan (referee)
The bacterial cell must be able to rapidly change its gene expression to survive unstable external conditions. Transcription is the key level that affects gene expression. The pivotal enzyme of transcription is RNA polymerase (RNAP). Activity of RNAP is tightly regulated by transcription factors (TFs). These factors affect RNAP in different ways. This work presents an overview of various proteins and others factors, description of their effects on transcription and also mechanisms of their actions. TFs could be divided according to various criteria. In this work, TFs are divided according to how they interact with RNAP: TFs interacting only with RNAP; TFs binding simultaneously DNA and RNAP; TFs interacting with RNA and RNAP. This work presents a comprehensive overview of various TFs that are involved in the bacterial cell's reprogramming of gene expression that is required to withstand the changes in the environment.
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The effect of 6S-like RNAs on physiological differentiation of Streptomyces coelicolor
Burýšková, Barbora ; Bobek, Jan (advisor) ; Branny, Pavel (referee)
The variety of bacteria and their genomes sometimes causes conservation of homologue molecules to be displayed not in sequence but in secondary and tertiary structures. In the case of the regulatory 6S RNA, sequence homologues have been found in over 100 bacterial species so far. However, none were found in the genus Streptomyces. The unique genome of these soil- dwelling bacteria, known for their capacity to produce antibiotics, has a high G/C content and diverges substantially from distantly related bacteria. Yet in the non-coding 6S RNA it is the secondary structure that is crucial for its function. The 6S RNAs trap sigma factors by mimicking target promoter sequences in order to help with switching sets of expressed genes during developmental transitions. 6S-like RNA genes in Streptomyces coelicolor have been computationally predicted by comparison of in silico modelled secondary structures of known 6S RNAs. The aim of this thesis was the verification of these 6S-like RNA predictions. The experimental approach was based on RNA co-immunoprecipitation (RNA CoIP), as well as RT- PCR from RNA samples. The outcomes of this project are the detection of six novel ncRNA transcripts with possible 6S-like RNA functions, which also served as the wet-lab verification of the in silico prediction technique...
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Rare cells in diagnostics and monitoring of gynecological diseases
Kiss, Imrich ; Bobek, Vladimír (advisor) ; Špaček, Jiří (referee) ; Klézl, Petr (referee)
In the last two decades there is an enormous effort to discover a non-invasive marker to diagnose, predict and monitor therapy effect of malignant diseases. Circulating tumor cells (CTC) have the ability to fulfil all these criteria. The introduction of the monothematic thesis deals with the problematics of CTC in general and its application in clinical medicine. It is followed by the author's first publication, which reviews the current status of CTC in gynecological malignancies. The next publication is an original article about CTC in patients with endometrial carcinoma. It reports a successful isolation of vital CTC in 75% of tested patients and identifies CTC as an individual marker of the disease without correlation to the stage, grade or lymph nodes involvement. The second part of this thesis deals with endometriosis, a benign but often recurrent disease worsening the life quality of women in reproductive age. The multicentre study presents a successful isolation of circulating endometrial cells (CEC) in patients with histologically proven endometriosis with various stages and symptoms. From the total of 423 samples 78.4% were CEC positive. Eleven patients were monitored during their menstrual cycle and CEC tested in different phases, being the early post-ovulatory period in which the...
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Osteogenesis and bone healing in congenital short femur
Frydrychová, Monika ; Dungl, Pavel (advisor) ; Havlas, Vojtěch (referee) ; Charvát, Jan (referee)
Introduction: Congenital short femur, or proximal femoral focal deficiency (PFFD), is a rare complex deformity of the lower extremity with femoral dominance. The clinical findings cover wide range of variety, from femoral absence till inconspicuous shortening of the femur. Aim of the study: 1. Molecular analysis of pseudoarthrosis tissue in congenital short femur with focusing on osteogenic and angiogenic gene expression in comparison with physiological bone. The differences in gene expression were expected. 2. Retrospective analysis of femoral healing after prolongation calculating the severity of affection, age, distance of elongation and complication. The extended healing according to severity type and age was expected compared to control group. Material and methods: The RNA from piece of one was isolated and transcription profile of possible 113 genes of osteogenesis and angiogenesis was detected by biochip technology (SuperArray Bioscience Corporation). 10 samples analyses were performed (7 of PFFD, 3 controls). The data of 57 PFFD patients indicated for elongation of the femur with the types Pappas III, IV, VII, VIII and IX and 12 patients in control group were evaluated retrospectively and statistically by GLS method. Results: The expected differences in gene expression in PFFD tissue...
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Factors affecting gene expression in Bacillus subtilis
Sudzinová, Petra ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Vohradský, Jiří (referee)
Bacterial DNA-dependent RNA polymerase (RNAP) is a key enzyme of bacterial transcription. Its activity must be tightly regulated. This could be done on the level of promoter DNA topology recognition, by changing the intracellular levels of metabolites, or by binding proteins, known as transcription factors. Even though the RNAP regulatory network has been intensively studied for decades, new regulators are still being described. The main focus of this Thesis is to characterize some of them: i) HelD, a novel RNAP interacting factor, with so far unknown protein 3D structure; ii) RNase J1, an enzyme with a unique mechanism of functioning; iii) Spx, a major regulator of gene expression in Bacillus subtilis, with still new roles to be defined and iv) the effect of the topological state of promoters on transcription. We identified HelD as an interacting protein of RNAP in Bacillus subtilis and described its biochemical properties. It stimulates transcription in an ATP-dependent manner, by enhancing recycling of RNAP molecules (Publication I). We published the first insight into the HelD structure by SAXS (small angle X-ray scattering) and deepened the understanding of HelD domain composition (Publication III). And finally, we were able to solve the cryo-EM structure of HelD:RNAP complexes from...
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Characterization of the Caenorhabditis elegans pop-1 gene
Jakšová, Soňa ; Vacík, Tomáš (advisor) ; Macůrková, Marie (referee)
The TCF/LEF transcriptional factors regulate the target genes of the Wnt signalling pathway - one of the key signalling mechanisms involved in development of multicellular organisms. The TCF/LEF genes produce a number of various protein isoforms, which consequently leads to a great functional diversity of the TCF/LEF proteins. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes, whose isoforms have not been studied yet. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels during seven developmental stages of C. elegans. Further, we also determined the expression profile of two important partners of pop-1, the bar-1 and sys-1 genes, whose protein products function as transcriptional co-activators. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1
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Relation of tumor genotype and phenotype to diagnosis, prognosis and prediction of colorectal cancer
Pitule, Pavel ; Králíčková, Milena (advisor) ; Vožeh, František (referee) ; Slabý, Ondřej (referee)
Colorectal cancer is one of the most common type of malignity. Despite of the existence of numerous studies focused on this carcinoma, there are still many unknown features regarding its diagnosis, treatment or prognosis. In the thesis we focused on the identification of novel prognostics markers that could be useful for the stratification of patients based on the disease outcomes. In the first study we immunohistochemically assessed expression of two proteins associated with cancer stem cells in the samples of primary colorectal cancer and matched liver metastasis. Goal of the study was to evaluate relation among expression of CD44 and CD133 and overall survival and disease free interval in our set of patients. We observed that increased ratio of CD133 positive compared to CD133 negative tumor glands resulted in longer disease free interval, finding which is opposite to the general view on the CD133 role in the cancer development. Our hypothesis is that we analyzed confined group of patients and followed a bit different goal, where we measured ratio between positive and negative glands in the view-field and not the intensity of staining as the previous studies did. Our second study was focused on the transcriptional analysis of the selected set of twelve genes using frozen samples from colorectal...
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The role adipose tissue in development of insulin resistance and other metabolic disorders in patients with pheochromocytoma
Klímová, Judita ; Petrák, Ondřej (advisor) ; Kříž, Jan (referee) ; Mlíková Seidlerová, Jitka (referee)
Pheochromocytoma and functional paraganglioma (PPGL) are rare neuroendocrine tumors characterized by catecholamines overproduction, which give a rise to disorders of glucose, lipid, and energy metabolism. The role of adipose tissue in these processes remains unclear. Our aim was to determine the gene expression profile in subcutaneous and visceral adipose tissue of patients with PPGL focusing on endocrine functions of adipose tissue, occurrence of brown (BAT) and beige adipose tissue (BeAT), all in connection with other measured metabolic and energy parameters and levels of circulating adipokines. We demonstrate signs of UCP1-dependent norepinephrine induced thermogenesis connected with overexpression of DIO2 in retroperitoneal VAT of PPGL and higher expression of key transcriptional factors of brown/beige adipogenesis, namely PPARGC1α, CEBPB and PRDM16. However, classic murine BAT or BeAT gene signature in VAT of PPGL was not detected. In subcutaneous adipose tissue (SAT) of PPGL we found signs of possible BeAT transformation, however without simultaneously undergoing UCP1-dependent thermogenesis. We also demonstrate that patients with PPGL have higher serum levels of FGF21 compared to healthy controls and these levels do not differ from obese patients. Furthermore, successful tumor removal...
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The use of "omics" methods in molecular-epidemiologic study in newborns from different localities of the Czech Republic
Hoňková, Kateřina ; Rössner, Pavel (advisor) ; Gábelová, Alena (referee) ; Bláha, Luděk (referee)
The "omics" is a concept of biological disciplines that globally characterizes and quantifies biomolecules involved in the key functions of an organism. The "omics" methods are used e.g. in molecular epidemiology, where they help to evaluate potential biomarkers that identify the impact of environmental factors for human health. In this thesis, the "omics" methods were applied in samples collected from newborns born in localities of the Czech Republic mostly differing by pollution levels from industrial sources. The principal aim was to determine whether environmental changes during prenatal development can affect gene expression and its regulation in newborns. The thesis further aimed to evaluate the level of air pollution at the time of biological samples collection. Using the whole genome approach, differentially expressed genes (DEGs) in newborns from districts Karvina and Ceske Budejovice (CB) were identified. In a pilot study of a small group of newborns from districts Most and CB, differentially methylated CpG sites in DNA were assessed. These sites attenuate gene activity and could be responsible for long-term changes at the genetic level. Finally, the aim was to find differentially expressed small non-coding RNA (DE miRNA) in newborns from Most and CB. Samples of umbilical cord blood from...
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Gene expression differences during regeneration in model organisms
Netušil, Jiří ; Šindelka, Radek (advisor) ; Hason, Martina (referee)
With new progressive methods allowing us to study natural regeneration in model organisms, we have an opportunity to gain important insights into the very essence of this process. These insights might help us radically improve the current state of therapeutic approaches based on tissue replacement. Many different animal models display an incredible ability to restore various body parts, allowing them to escape predators and avoid premature death. While invertebrate models give us a chance to investigate the fundamental elements of regeneration, vertebrates represent systems often more resembling human biology. This thesis outlines the variability of regeneration in frequently studied model organisms with a special emphasis on the impact of gene expression. Keywords: regeneration, model organism, gene expression, injury
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