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Transgenic mouse: Experimental model for study of acute lymphoblastic leukemia.
Kozáková, Jana ; Semecký, Vladimír (advisor) ; Lenčo, Juraj (referee)
The TEL and AML1 genes occur in a markedly high numer of different aberrations in heamatological malignancies. We studied TEL/AML hybride gene in transgenic mice model, translocation t(12;21)(p13;q22). This hybride gene was searched by PCR methode in 76 individuals. After, they were examined for presence of mRNA of fusion gen TEL/AML1 using RQ-RT-PCR. Compared the blood cell analysis of transgenic and non-transgenic mice, we searched difference in the erytrocyte, lymphocyte, trombocyte count and lymphocyte ratio in peripheral blood. The TEL/AML1 positive Acute lymphoblastic leukemia is B cell precursor leukemia, therefor we used cytofloumetric analysis for study the ratio of the B lymphocytes of transgenic and non-transgenic mice.Then, we observed the leukemic cell marker of pre-B lymphocytes (B220+ /CD43+ ). Presence of the pre-B lymphocytes in peripheral blood show incomplete differenciation and proliferation of B lymphocytes, cosequently leucemogenesis.
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Identification of purified biotransformation enzymes by means of mass spectrometry
Křížová, Andrea ; Lenčo, Juraj (advisor) ; Řehulka, Pavel (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Andrea Křížová Supervisor: PharmDr. Juraj Lenčo, Ph.D Title of diploma thesis: Identification of purified biotransformation enzymes by means of mass spektrometry Biotransformation enzymes are involved in the metabolism of a wide range of both endogenous compounds and xenobiotics. Regarding the metabolism of xenobiotics, many of those enzymes have not been characterized at all or only marginally. To get better knowledge about how the biotransformation enzymes affect compounds with significant impact on the organism as a whole, they need to be purified, identified and subsequently thoroughly characterized. The classical electrophoresis is still irreplaceable for protein purification and characterization. Moreover, combination of electrophoresis with mass spectrometry (MS) represents an inseparable tandem for protein identification. However, to obtain unambiguous outputs from mass-spectrometry based protein identification, it is essential to avoid unwanted contamination of the sample during sample preparation. Wearing disposable protective equipments, using MS grade chemicals, solutions filtering are examples of rules for sample preparation for MS analysis that were established at the...
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Optimization of mobile phase composition for analysis of peptides in mixed-mode chromatography
Tužil, Ondřej ; Lenčo, Juraj (advisor) ; Řehulka, Pavel (referee)
Faculty of Pharmacy in Hradec Králové, Charles University Department of Analytical Chemistry Candidate: Ondřej Tužil Supervisor: PharmDr. Juraj Lenčo Ph.D. Title: Optimization of mobile phase composition for analysis of peptides in mixed-mode chromatography In bottom-up proteomic analyzes performed by liquid chromatography with mass detection, the mobile phase is acidified with weak acids, which do not suppress the intensity of the analytes in the detection to the same extent as commonly used acids for separations without mass detection. Such a mobile phase additive is formic acid, which is most commonly used at a concentration of 0.1 % (v / v). This work was focused on finding the lowest possible concentration of formic acid, which in combination with the multimodal stationary phase of the ACQUITY UPLC CSH C18 column from Waters would still ensure efficient separation of peptides, while increasing the response in the mass spectrometer. First, separations of peptide standards were performed by liquid chromatography with UV detection with decreasing amounts of formic acid in the mobile phase. It has been found that significantly lower concentrations of formic acid can be used for satisfactory peptide separation parameters. The three lowest concentration levels were used for mass detection analyzes,...
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Top-down, middle-up, and bottom-up RPLC analyses of biopharmaceuticals using a single column
Hývnar, Ondřej ; Lenčo, Juraj (advisor) ; Chocholouš, Petr (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Ondřej Hývnar Supervisor: PharmDr. Juraj Lenčo, Ph.D. Title of diploma thesis: Top-down, middle-up and bottom-up RPLC analyses of biopharmaceuticals using a single column Nowadays, different columns are needed for top-down, middle-up and bottom-up analyses of biopharmaceuticals using liquid chromatography. The aim of this thesis was to determine whether it is possible to use for all these types analyses a new column BioResolve RP mAb Polyphenyl of the company Waters, intended mainly for top-down and middle-up analyses. The biopharmaceuticals trastuzumab, bevacizumab, panitumumab and aflibercept were analyzed as intact for top-down analysis, reduced, cleaved by IdeS protease, cleaved by IdeS protease and reduced for middle-up analysis and cleaved by endopeptidase Lys-C and trypsin for bottom-up analysis. Top-down and middle-up analyses were performed using liquid chromatography with UV detection. We verified the ability of the column to analyze biopharmaceuticals prepared this way in our conditions. We found that although the column can handle intact biopharmaceuticals, it gives better results in middle-up analyses. Bottom-up analyzes were performed using liquid chromatography coupled with...
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Identification of potential intraamniotic infection and inflammation biomarkers in amniotic fluid from preterm birth patients
Tambor, Vojtěch ; Lenčo, Juraj (advisor)
5 Summary In this work, we took advantage of modern proteomics in order to characterize intraamniotic infection and inflammation related changes in the proteome of amniotic fluid from preterm birth patients. Proteins with altered levels could subsequently serve as potential biomarkers for timely recognition of intraamniotic infection and inflammation. Due to the extremely high complexity and high dynamic range of amniotic fluid proteome, we first developed a technique - CysTRAQ - which enables proteome complexity reduction based on cysteinyl peptide capturing and features a multiplexed protein quantitation across four samples. The developed method was subsequently applied into the comparative proteomic analyses of amniotic fluid. Our study included patients with both principal phenotypes of spontaneous preterm birth - spontaneous preterm labour with intact membranes as well as preterm premature rupture of membranes - in order to provide a comprehensive insight into the proteomic background of the infectious and inflammatory processes occurring in amniotic fluid. By employing our CysTRAQ approach in combination with additional fractionation and separation techniques, we managed to describe a remarkable number of the amniotic fluid proteins. Owing to the quantitation feature of CysTRAQ, we were also able to...
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Analysis of intracellular pathogen Francisella tularensis expression upon cultivation under stressfull conditions and during the interaction with a host cell
Lenčo, Juraj ; Stulík, Jiří (advisor) ; Rychlík, Ivan (referee) ; Weiser, Jaroslav (referee)
Univerzita Karlova v Praze Lékařská fakulta v Hradci Králové Analýza exprese intracelulárního patogena Francisella tularensis kultivovaného za stresových podmínek a v průběhu interakce s hostitelskou buňkou Autoreferát dizertační práce Mgr. Juraj Lenčo Hradec Králové 2007 2 Dizertační práce byla vypracována v rámci doktorského studijního programu lékařská biologie na Ústavu lékařské biologie a genetiky Lékařské fakulty v Hradci Králové, Univerzity Karlovy v Praze a Ústavu molekulární patologie Fakulty vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno. Uchazeč: Mgr. Juraj Lenčo Ústav molekulární patologie, Fakulta vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno. Školitel: Doc. MUDr. Jiří Stulík, CSc. Ústav molekulární patologie, Fakulta vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno Školitel konzultant: Prof. MUDr. RNDr. Miroslav Červinka, CSc. Ústav lékařské biologie a genetiky, Lékařská fakulta v Hradci Králové, Univerzita Karlova v Praze Oponenti: RNDr. Ivan Rychlík, Ph.D. Oddělení bakteriologie Výzkumný ústav veterinárního lékařství Brno RNDr. Jaroslav Weiser, CSc. Mikrobiologický ústav Akademie věd České republiky Stanovisko k dizertační práci vypracovala oborová rada oboru lékařská biologie akreditovaného na Ústavu lékařské biologie a genetiky Lékařské...
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Use of proteomic analysis to identify potential biomarkers for cardiovascular disease
Fučíková, Alena ; Lenčo, Juraj (advisor) ; Hernychová, Lenka (referee) ; Pudil, Radek (referee)
Hypertrophic cardiomyopathy is one of the most common inherited diseases of the cardiovascular system. Although this disease is known for a long time, a suitable diagnostic procedure uncovering its early stages in patients with negative or unknown family history is still lacking. A development of the method for targeted proteomic analysis in combination with subsequent quantification of chosen hypothetical markers of hypertrophic cardiomyopathy was the main aim of this thesis. This method is able to detect very small amounts of chosen markers in the minimum amount of complex biological material. Moreover, in combination with properly used standards, targeted proteomic analysis enables quite precise quantification of many analytes in a relatively short time. Several previously described protein markers of hypertrophic cardiomyopathy were assayed and quantified using unique proteomic technique. Concurrently, a new potential protein marker - soluble fibronectin - was described. Protein concentrations were validated using enzymoimmunoanalytical method and obtained results were compared with targeted proteomic analysis findings. In the presented thesis, a new short method was developed for detection and quantification of potential markers of hypertrophic cardiomyopathy. The main objectives of this...
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Pros and cons of using elevated temperature for peptide separation using reversed phases
Šemlej, Tomáš ; Lenčo, Juraj (advisor) ; Řehulka, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Tomáš Šemlej Supervisor: PharmDr. Juraj Lenčo, Ph.D. Title of diploma thesis: Pros and cons of using elevated temperature for peptide separation using reversed phases Nowadays, powerful mass spectrometers are available for bottom-up LC-MS proteomic analyses. They allow to identify up to several thousand proteins in one bottom-up analysis. It has been recently demonstrated, however, that their capabilities cannot be fully exploited without significant improvements in peptide separation. One of the easiest, but often neglected, ways to enhance peptide separation efficiency is to increase column temperature. Elevated column temperature may contribute to more efficient separation especially by increasing low diffusivity of peptides. On the other hand, stability of both a stationary phase and peptides limits increasing column temperature to values affording maximum effect on separation efficiency. In this thesis, we explored the effect of increased column temperature on peptide separation efficiency using columns packed either with superficially porous particles or with fully porous particles. Elevated column temperature narrowed peaks for peptides eluted from fully porous particles whereas peak...
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