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Molecular biology investigation of somatostatin receptors in diagnostics of pituitary tumors
Drastíková, Monika ; Beránek, Martin (advisor) ; Fryšák, Zdeněk (referee) ; Patočka, Jiří (referee)
Molecular biology investigation of somatostatin receptors in diagnostics of pituitary tumors The overwhelming majority of pituitary tumors are benign, with 25-30% of them representing clinically non-functioning pituitary adenomas (NFA). The treatment of choice for NFA is transsphenoidal neurosurgery. However, the procedure is often not completely successful and frequently tumor remnants remain, resulting in regrow over the long-term follow-up period. This fact has led to the development of new therapy strategies using pharmacological treatment: somatostatin analogs (SA) and estrogen receptor modulators. SA are effectively used in the treatment of acromegaly, neuroendocrine tumors and Cushing's disease. In contrast, pharmacological treatment of NFA has for the most part been unsuccessful. Low SA effectiveness could be associated with the variable expression of target receptors on the adenomas. The aim of the study was to determine the somatostatin (SSTR subtypes 1-5) and estrogen receptor 1 (ER1) expression profile. To obtain more complex receptor profile expression in the pituitary, we also investigated dopamin receptor 2 (D2R). Methods The group of patients was made up of 105 men and 101 women (20-87 years old; median 61). 144 samples were NFA, 44 growth hormone secreting (GHomas), 9 corticotrophin...
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Molecular mechanisms of Francisella tularensis pathogenesis
Fabriková, Daniela ; Beránek, Martin (advisor) ; Krejsek, Jan (referee)
Title of Ph.D. thesis: Molecular mechanisms of Francisella tularensis pathogenesis Key words: Francisella tularensis, pattern recognition receptors, inhibition, TRAF6 and TRAF3 complexes Annotation Francisella tularensis is a highly infectious intracellular pathogen and the causative agent of the disease called tularemia. An important aspect of Francisella tularensis virulence represents the capacity to subvert the host immune response by inhibiting or disrupting of the innate immune cell functions. The initial stage of infection is characterized by the massive bacterial replication without apparent inflammatory response, which is crucial for the development of effective host defense against invading pathogen. The aim of this Ph.D. thesis was to describe the early pattern recognition receptors (PRR) signaling response to Francisella tularensis subsp. holarctica LVS (F. tularensis LVS) in primary bone marrow- derived macrophages. The obtained data show the capacity of F. tularensis LVS to simultaneously activate and suppress Toll-like receptors, RIG-I-like receptors, and cytosolic DNA sensors signaling pathways. F. tularensis LVS modulates these PRR pathways by the suppression of K63-linked polyubiquitination events and by the inhibition of the assembly of TRAF6 and TRAF3 signaling complexes. The use of the...
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Contribution of Next Generation Sequencing for Laboratory Diagnostics
Votýpka, Pavel ; Beránek, Martin (advisor) ; Macháček, Miloslav (referee)
5 ABSTRACT Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavel Votýpka Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Consultant: Mgr. Nikola Ptáková Title of diploma thesis: Contribution of Next Generation Sequencing for Laboratory Diagnostics The endeavor to sequence the whole human genome lead not only to the knowledge acquisition regarding the human genetic information but as well to the development of new sequencing methods and technologies. In order to keep up with progress in genetic field in many clinical and research laboratories the new massive parallel sequencing equipment is being utilized. On the market are currently established four leading platforms - Illumina, Solid, Ion Torrent and 454 Life Technologies. The process of sequencing analysis can be summarized into three main steps - the sequencing library preparation, sequencing itself, variant calling and data analysis. Each part of the sequencing analysis exhibits certain specifics, we need to count with and as well its pitfalls, we need to avoid or to minimalize their impact on the analysis final result. Recently new methods termed sequencing of the 3rd generation are being developed, enabling sequence of a single DNA molecule to be determined without previous...
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Thiopurine methyltransferase - clinical importance of genotyping and phenotyping
Černá, Blanka ; Beránek, Martin (advisor) ; Šimůnek, Tomáš (referee)
Charles University in Prague Faculty of pharmacy in Hradec Králové Department of Biochemical science Candidate: Blanka Černá Supervisor: Doc. PharmDr. Martin Beránek, PhD. Title of Diploma Thesis: Thiopurine S-methyltransferase - clinical importace of genotyping and phenotyping. Thiopurine S-methyltransferase catalyzes S-methylation thiopurine's drugs such as 6-mercaptopurine and thioguanine. TPMT genetic polymorphisms represent an important role in clinical pharmacogenetics. The differences in TPMT activity result from mutations in gene for TPMT. The polymorphisms are important factor in efficacy of treatment by thiopurine drugs. Patients inheriting low activity of enzyme TPMT have mutated allels, patients inheriting high activity of TPMT are usualy wild types. TPMT gen was genotypized by method real-time PCR in volunteers (n=55) with autoimmune diseases. The average of patient's age was 16,7 years. From blood collected into EDTA DNA was isolated by using QIAmp Mini Kit (Quiagen, Germany) and it was used for genotyping of TPMT. Genotyping was carried out by real-time PCR in LightCycler (Roche, Germany). TPMT was phenotypized in Hradec Králové in Medical Faculty of Charles University in Department of Pharmacology. The lysate of suspension of erythrocyte was used for phenotyping (The blood was...
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Procalcitonin as an early marker of bacterial infection
Mikulová, Lenka ; Beránek, Martin (advisor) ; Drastíková, Monika (referee)
Introduction: Procalcitonin (PCT) is a 13 kD protein with a chain of 116 amino acids. It consists of parafollicularcells of the thyroid gland. Inflammatory procalcitonin is made up of 114 amino acids. Procalcitonin from plasma in the course of inflammatory process, does not consistof C-cells of the thyroid gland but it is generatedin the liver, and, to a lesser extent, in the lungs, kidneys, testes, prostate and small intestine. Objectives: 1. To compare the levels and the dynamics of C-reactive protein (CRP) and procalcitonin in the inflammatory process. in three patients with different diagnoses. To compare the number of examined PCT according to indicated VFN (General University Hospital) departments in the years 2009, 2012 and 2015. 3. To study the dynamics of the number of examined PCT in the years 2001- 2016. Methods: The levels of CRP and PCT were measured in the ÚLBLD laboratory of the General University Hospital in Prague in the period from May 20to June 17, 2016. The CRP was measured by immunoturbidimetricanalyzerUnicelDxC 880i. The PCT was measured by theCobas analyser using the electrochemiluminescence technology. In order to evaluate the data, graphs were processed with levels of bothanalytesfor 3 patients. Further, data needed for the evaluation of the above mentioned objectives no. 2...
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Genetic markers for monitoring post-transplant chimerism
Řehounková, Michaela ; Beránek, Martin (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Řehounková Supervisor: doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Genetic markers for monitoring post-transplant chimerism The aims of the thesis: Data processing of patients, who underwent allogenic hematopoietic stem cell transplantation in a period from 2010 to 2014 in University Hospital Hradec Kralove and whose state of chimerism was monitored at the Section of Molecular Biology at the Institute of Clinical Biochemistry and Diagnostics. Consequently, analysis of the possible relationship between selected clinical parameters and used genetic markers for chimerism quantification was carried out after the processing of acquired data. Finally, the possible influence of treatment success and mortality by chosen clinical parameters was evaluated. Methods: Analysis of short tandem repeat loci, which uses genetic variability between donor and recipient of transplanted graft, was employed for quantification of post- transplant chimerism. DNA of donor and recipient was isolated by QIAmp DNA Blood Mini Kit (QIAGEN, Germany), amplified by AmpFlSTR Identifier Kit (Applied Biosystems, USA) and separated by capillary electrophoresis (analyzer ABI 3130-4, Applied...
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Genetic polymorphism in the HFE gene in the Czech population
Červinková, Barbora ; Beránek, Martin (advisor) ; Šimůnek, Tomáš (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Barbora Červinková Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Title of diploma work: Genetic polymorphism of the HFE gene in Czech population Outline: The main objective of the thesis was to map the incidence of the three mutations (C282Y, H63D, S65C) in the HFE gene in the Czech population. Consequently, the obtained results were compared with the reported occurrence in the world. Methods: The work involved the isolation of DNA from buccal swabs obtained from 167 donors (65 men, 102 women; average age for men was 31, for women 28). Isolated DNA strings were amplified by PCR methods using 5'-CAG ATC CTC ATC TCA CTG-3' and 5'-CTG GAT AAC CTT GGC TGT ACC CCC-3' primers for C282Y mutation, 5'-GCC ACA TCT GGC TTG AAA TT-3' and 5'-ACA TGG TTA AGG CCT GTT GC GCC ACA- 3' primers for H63D and S65C mutations. DNA samples were treated with restriction enzymes Rsa I, Bcl I and Hinf I, for C282Y, H63D and S65C mutations, respectively. Finally, the restriction fragments were separated by gel electrophoresis (2 % agarose gel). Results: C282Y mutation was present in 0 (0 %) and 19 (11,38 %) samples as homozygote or heterozygote, respectively. Moreover, C282Y mutation was present in 3 (1,80 %)...
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