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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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IGF2 and preptin, biologically active peptides derived from pro-IGF2
Mrázková, Lucie ; Žáková, Lenka (advisor) ; Janovská, Petra (referee)
Pro-IGF2 is a 156 amino acid long protein and it is a prohormone from which two bio- logically active peptides are formed, IGF2 and preptin. IGF2 is an insulin-like growth factor 2 that, together with IGF1 and insulin, shares a similar 3-D structure and similar biological effects. It is a peptide whose sequence corre- sponds to amino acids 1-67 of pro-IGF2. It is produced mainly by the liver, but it is formed in most cells of our body. It is involved in the regulation of growth, differentiation, embryogenesis and in recent years the interest in IGF2 has been increasing mainly due to its function in the central nervous system. Preptin is a newly isolated 34 amino acid long peptide whose sequence corresponds to amino acids 69-102 (E-domain) of pro-IGF2. Preptin affects glucose metabolism as well as bone homeostasis. Disorders in glucose metabolism usually lead to diabetes mellitus, and disorders in bone homeostasis most often lead to osteoporosis. Preptin has been shown to increase the second phase of insulin secretion and to have a positive effect on the proliferation and differentiation of osteoblasts, bone cells promoting bone growth. Keywords: pro-IGF2, preptin, IGF2, E-domain, gene expression, imprinting IGF2
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The role of ERK1 and ERK2 protein kinases in the MAPK/ERK signaling
Galvánková, Kristína ; Vomastek, Tomáš (advisor) ; Dráber, Peter (referee)
The MAPK/ERK cascade is highly conserved signalling pathway regulating cellular processes which are necessary for cell life, such as proliferation, differentiation, apoptosis or cell migration. All these cellular responses are the result of the processing of extracellular signals through three-tier ERK cascade consisting of protein kinases Raf, MEK and ERK. The signal is transmitted by sequential phosphorylation where RAF phosphorylates MEK and MEK phosphorylates and activates ERK. Protein kinase ERK then phosphorylates and regulates a wide range of substrates at different locations in the cell. This affects the cellular response to the extracellular signal. Regulation of this pathway on every level is very important and is modulated by interaction partners and adaptor proteins. Deregulation of the pathway as well as mutations of individual protein kinases can lead to severe pathological consequences. At the level of ERK, there are two isoforms, ERK1 and ERK2, which are more than 80 % identical at the amino acid level. Their high sequence similarity has triggered the interest of many authors for more detailed examination of both isoforms in respect of their evolutionary conservation and whether they are functionally redundant or whether they have specific functions. The aim of this work is to...
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Role of matrix metalloproteinases in regulation of late embryonic healing process
Kikinderová, Paulína ; Šindelka, Radek (advisor) ; Procházka, Jan (referee)
Cutaneous wound healing could be distinguished into two main types: embryonic and adult. Embryonic healing in contrast to adult is faster, scar-less and consists of early, middle, and late phases. Actin ring is formed during the early phase and its cables pull the edges of the wound towards apposite sites during the following middle phase. De novo expression of healing specific genes is initiated also during middle phase. However, process of the wound healing continues under the closed wound in the late phase which has been poorly described. Adult wound healing is more complex, longer, and is divided into 4 phases: haemostasis, inflammation, proliferation, and remodelling phase. Adult wound healing might end with the scar. Pivotal role in the wound healing is given to matrix metalloproteinases (MMPs). These remodelling enzymes are important for releasing cytokines, inducing apoptosis, and degradation of extracellular matrix. Our laboratory performed temporal RNA-sequencing of the healing tissue using tailbud stage and swimming tadpole embryos. Results showed predominant expression of four mmps: mmp1.L, mmp7.S, mmp8.S, and mmp9.L. Injury or amputation caused the upregulation and their expression level peaked at 3-6 hours post injury which corresponds with late phase of healing. Thus, I focused on...
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Identification of non-coding RNAs of Clostridium beijerinckii NRRL B-598 using RNA-Seq data
Pomykalová, Barbora ; Sedlář, Karel (referee) ; Jurečková, Kateřina (advisor)
This bachelor thesis contains short introduction into bacterial small non-coding RNA problematic. It is oriented on their features and functions in organisms, especially in bacteria Clostridium beijerinckii NRRL B-598. Bachelor thesis also contains description of various laboratory methods for gene expression determination and suggests a detection method for small non-coding RNA in bacteria Clostridium beijerinckii NRRL B-598. Suggested method works with data, which were obtained by RNA-Seq technology. Within the framework of the bachelor thesis was suggested method implemented in programming and numeric computing platform MATLAB and its results were discussed.
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The Role of oncogenic microRNA - 155 and proto - oncogen MYB in chronic lymphocytic leukemia
Vargová, Karina
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B-CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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Laccase activity profiling in Trametes versicolor cultures degrading endocrine-disrupting compound Delor 103
Plačková, Martina
In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...
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The role of evolutionarily conserved proteins BIR-1/Survivin and SKP-1 in the regulation of gene expression
Kostrouch, David ; Kostrouch, Zdeněk (advisor) ; Dráber, Pavel (referee) ; Pacák, Karel (referee)
SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...
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