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Current possibilities of laboratory diagnostics of pneumococcal infections
ČAPKOVÁ, Irena
Streptococcus pneumoniae (S. pneumoniae) can be common colonizing flora of human nasopharynx, but it also can be one of the main patogens causing invasive pneumococcal disease. It is diagnosed directly, using various methods, such as microscopy, cultivation, identification, or non-cultivation proof of antigen or deoxyribonucleic acid (DNA). First part of my thesis is dedicated to the description of the Streptococcus genus, including species S. pneumoniae. Morphology, physiology, antigenic structure, pathogenesis and pathogenicity of this bacterial race and species is described, as well as theoretical description of laboratory diagnostics methods. In methodics, the identification methods are described as they were used for diagnostics of S. pneumoniae in the Laboratory of medicinal microbiology, Department of bacteriology Nemocnice České Budějovice a.s. It also includes description of cultivation of biological samples, which was S. pneumoniae isolated from, and several identification tests which can differentiate S. pneumoniae from other viridans streptococci. Two basic, commonly used identification tests were used for diagnostics test of sensitivity to optochin and test of solubility in bile-sodium deoxycholate. Test of solubility using sodium deoxycholate is a basic test in diagnostics of S. pneumonia. Out of 127 species which were positive in the solubility test, 114 were also positively tested for sensitivity to optochin. Test of sensitivity to optochin had 89,9% accuracy. Four species primarily identified as viridans streptococci were tested using Matrix-Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI TOF MS). This specific, fast and accurate method cannot be fully used for identification of S. pneumoniae however, because its genotype is far too similar to the one of Streptococcus oralis/mitis. Two tests were used and described to prove the antigen S. pneumoniae imunochromatographic test and latex agglutination reaction. Out of 266 examined samples, antigen was found in fifteen cases in urine and cerebrospinal fluid using the imunochromatographic test, and in seven cases, the antigen was proved using the latex agglutination reaction. These two methods are highly specific and provide fast information about the presence of the antigen S. pneumoniae in the examined sample and subsequently about the possibility of pneumococcus infection. Another highly specific test used for diagnostics of severe pneumococcus diseases is DNA proof using PCR methods, which was successful in 5 cerebrospinal fluid samples, which were examined simultaneously in the Laboratory of molecular biology and genetics of Nemocnice České Budějovice a.s.
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Mantle cell lymphoma, gene expression in the disease pathogenesis
Břízová, Helena
At the molecular level mantle cell lymphoma (MCL) is characterized by a specific translocation t(11;14) leading to a transcriptional activation of the cyclin D1 gene. Cyclin D1 is a key regulator of the cell cycle and thus its overexpression leads to a proliferation advantage of the cells, which is important in the MCL pathogenesis. Moreover, proliferation activity is an important and the first biological prognostic factor for the MCL prognosis prediction. We developed an optimal approach to analyze quantitatively the cyclin D1 expression level in lymphoma specimens using real-time PCR. We detected the cyclin D1 overexpression in 97% MCL specimens. The developed method supported the MCL differential diagnosis with a high reliability, including a differential diagnosis of extranodal lymphomas. Moreover, the cyclin D1 mRNA expression level measurement also provided an approach to study a key molecule in the MCL pathogenesis at the molecular level. We demonstrated a direct relation between t(11;14), a cyclin D1 mRNA overexpression and a pathologic cyclin D1 protein synthesis in the MCL cells. The cyclin D1 mRNA level also correlated with the mRNA level of proliferation markers implying a quantitative cell cycle regulation by a controlled cyclin D1 level, which controls the proliferation degree, and...
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Significance of MLL gene aberrations in patients with acute myeloid leukemia
Šárová, Iveta
In acute myeloid leukemia (AML), predominantly in AML M5a, the most frequent recurrent aberration of chromosome 11 involves region 11q23. Molecular breakpoint studies of several translocations involving chromosomal band 11q23 led to the detection of a gene that was named MLL (myeloid/lymphoid leukemia). This gene is important for the proper HOX gene expression during ontogenesis and hematopoiesis. Chromosomal aberrations affecting the MLL gene occur in 5 - 10 % of AML cases and are very variable. Since that time, more than 70 different translocation partners of the MLL gene have been described. Aberrations of the MLL gene are associated with an aggresive type of the disease and its detection is needed for the treatment decision. Therefore, we investigated the occurrence of MLL abnormalities in bone marrow cells of the 66 newly diagnosed AML patients, using conventional cytogenetic and fluorescence in situ hybridization (FISH) analyses with a commercially available MLL Break Apart Rearrangement probe (Abbott VYSIS). Out of the 66 patients, we proved MLL abnormalities in 9 (13,6%): 5 (7,6%) showed translocation of MLL gene, in 3 (4,5%) we detected MLL gene amplification without any evidence of rearrangement and in 1 (1,5%) pacient only an extra copy of the MLL gene. The FISH results were verified by...
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Human F1Fo-ATPsynthase deficiency
Suldovská, Sabina ; Tesařová, Markéta (advisor) ; Černá, Leona (referee)
F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...
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Diagnostic morphological features of PDGFRA-mutated gastrointestinal
Daum, Ondřej ; Hes, Ondřej (advisor) ; Mandys, Václav (referee) ; Ehrmann, Jiří (referee) ; Zámečník, Josef (referee)
Daum O., Grossmann P., Vanecek T., Sima R., Mukensnabl P., Michal M. (2006): Diagnostic morphological features of PDGFRA-mutated gastrointestinal stromal tumors: Molecular genetic and histological analysis of 60 cases of gastric GISTs. Ann. Diagn. Pathol. In Press Summary In this study, 60 gastrointestinal stromal tumors (GISTs) of the stomach were analyzed to elucidate the possible relation of their morphology to mutation status of KIT and PDGFRA genes. The patients included 27 men and 33 women with a mean age of 63,8 years (range 12 to 92). Only one tumor occurred before the age of 21 years. KIT mutations were detected in 31 cases (51,7%), PDGFRA mutations in 22 cases (36,7%), and seven cases (11,7%) were KIT and PDGFRA wild type. When the mutation status was correlated with histological features of the tumors, epithelioid or mixed epithelioid/spindle cell pattern and mast cell infiltration were found as the most reliable signs of PDGFRA mutation. Neoplastic rhabdoid cells and multinucleated giant cells, also previously reported as features of PDGFRA mutated GISTs, seemed to be less specific but still helpful markers in our study. Finally, tumor infiltrating lymphocytes and myxoid stroma do not seem to be valuable histological signs. Daum O., Klecka J., Ferda J., Treska V., Vanecek T., Sima R., Mukensnabl...
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Laboratory diagnosis of mycobacteria, with a focus on the bacteriological proof
VELKOVÁ, Martina
Laboratory methods play an important role in disease diagnostic. Although there are currently available fast molecular genetic methods and methods based on cell response, basic diagnostic method of direct evidence is still microscopy and cultivation. Microscopy (staining by Ziehl-Neelsen and fluorescence microscopy) is the primary method for most of the specimens, especially for sputum. The sensitivity of this method is not very high because for the detection of a positive finding in 1 mm3 is needed at least 105 microbes. However, the method is important because it can fastly proof extensive clinical and epidemiological serious diseases. The ?Golden standard? is today the cultivation on solid egg media. Nowadays it is filled in automatic detection system, which accelerates the time of detection. The thesis was carried out in the Laboratory of Medical Microbiology, Bacteriology department, Hospital Ceske Budejovice in the period from 1.1.2013 till 6.6.2013, and included 400 sputa, which were processed and examined. The purpose was to compare the effectiveness of decontamination methods with HCl and with NALC, and to monitor the recovery and detection for the automatic detection system and conventional cultivation. In the automatic system using the BACTECTM MGITTM 960 Non-Radiometric fluorescent technology is cultivation in liquid Middlebrook 7H9 medium supplemented with antimicrobials (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, azlocilin). The classical culture used solid egg culture medium: Lowenstein-Jensen and culture medium by Ogawa. The individual results then show that the method of HCl had the overall contamination of 1 % of the samples. For each methods in 5 % BACTECTM MGITTM 960 and 4 % in the conventional cultivation. In comparison the method NALC, had the overall contamination of 21 % of the samples. For the individual methods in 26 % of BACTECTM MGITTM 960 and 50 % in the conventional cultivation. From these results it is obvious that the method with the NALC has lower efficiency, the proportion of contamination compared to the method with HCL increased by 20 %. Decontamination with the NALC method recommended by the manufacturer for BACTECTM MGITTM 960 was found to be unsatisfactory and was canceled. Comparing the recovery and the detection of the strains isolated in individual methods showed that the BACTECTM MGITTM 960 exhibits greater sensitivity than conventional cultivation, since the total of 17 strains isolated BACTECTM MGITTM 960 captured 15 against 10 strains isolated in conventional cultivation. There is also a significantly shorter time to detect positive samples. The average detection time for BACTECTM MGITTM 960 was 16.2 days, while a conventional culturing was 31 days. It was confirmed that the BACTECTM MGITTM 960 system achieves better results, but optimization is achieved by combining the two methods used. There is an apparent decrease in detection of mycobacteria from the processed statistical data of the strains isolated during the period 2010 - 2012, but a substantial reduction does not occur. Noticeable is only the decline of the isolated stains of Mycobacterium bovis BCG, which can be explained by the fact that the vaccination against tuberculosis is since 2011 no longer carried out across the board, but according to the new legislation in force only in high-risk groups. An interesting fact is that every year the highest laboratory detection is found in the age group of over 60 years.
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Laboratory diagnosis of disorders of phagocytosis
POLÍVKOVÁ, Ivona
Phagocytosis is one of the oldest processes of absorbing particles like amoeba, which is one of the basic mechanisms of the immune system to defend the body against infections. Failure of these processes is clinically manifested like immune deficiency and it can cause a very serious complication which could leads to the death of the patient. Phagocytosis is performed by specialized cells (macrophages, dendritic cells, neutrophils and other phagocytic myeloid cells) which are able to absorb the target particles, especially microorganisms, dead cells and foreign objects. These processes are essential for stability of homeostasis. Absorption of micro-organisms leads to activation of adaptive immunity response, elimination of apoptotic and destroyed cells and starts the reparation processes of damaged tissue. Phagocytosis as a complex process can be divided into several phases: active movement of phagocytes to the inflammation zone, adherence, ingestion and intracellular degradation which leads to the killing of pathogens. There are two mechanisms of killing pathogens. First is independent on oxygen where antimicrobial substances are stored in azurophilic granules which could be released into phagolysosom. Second mechanism is oxygen-dependent called oxidation (respiratory) flare which leads to the formation of biologically active mediators where oxygen molecules have considerable oxidation potential. Defects in phagocytic system are mainly caused by low number or malfunction of neutrophils which leads to severe infections mainly caused by staphylococci, Enterobacteriaceae or fungi. Among malfunctioning of phagocytosis processes belongs LAD I and LAD II syndrome. The other possible disorder of phagocytic malfunction is defect in enzymes. The NADPH oxidase is necessary for bacterial lyses mechanism of phagocytes and lack of these enzyme caused serious inherit disorder called chronic granulomatous disease. Lack or completely missing of enzyme myeloperoxidase stored in the azurophilic granules of neutrophils and monocytes is more and more common autosomal recessive disorder of phagocytosis. In this diploma work I focused on detection of these rare and serious disorders defects using flow cytometry to detect respiratory burst activity of neutrophils in blood samples. This laboratory procedure is called a burst test. In this test we quantitatively evaluate the respiratory burst activity of granulocytes in heparinized blood samples using the flow cytometery. Principle of this method is an oxidation-reduction reaction of dihydrorhodamine 123 to green fluorescent rhodamine 123 using peroxide, hydroxyl radicals and superoxide anions which are activated by respiratory burst. This process is one of the significant characteristic of phagocytic cells which are characterized by multi-stage activation of NADPH oxidase. This oxidase catalyzes electron reduction of molecular oxygen to superoxide. This step plays a very important role in our immune system and allows to kill and degrade particles and bacteria in phagocyte cells. Since 2009 - 2012 I analyzed the results of 611 patients who were tested for respiratory burst of neutrophils on a request of their physician. Among all of these results we obtain only two positive results of reduction of stimulation index (SI) and significant decrease percentage of activated granulocytes. Both of these parameters: reduced percentage of (activated granulocytes stimulated by PMA and E. coli) and low stimulation index pointing to a potentially serious disorder of phagocytosis mechanism. In specific tests of these cases proved the enzyme defect in phagocytosis mechanism which is the most common disorder in this type of immunodeficiency. These results indicate that the major primary defects of phagocytosis are very rare and their detection is usually in childhood for suspicion of primary immunodeficiency. This test is very helpful for discovering a prime immunodeficiency.
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Current possibilities of laboratory diagnosis of staphylococcal infections
CHRTOVÁ, Lucie
Laboratory diagnostics of Staphylococccal infections is based on direct evidence, like microscopy and cultivation, eventually on molecular genetics methods. The objective of this thesis is the presentation of nowadays possibilities in laboratory diagnostics of coagulase - negative staphylococcal infections and comparsion of two methods of identification used in the routine laboratory practice. First part of the thesis presents the genus Staphylococcus and the difference between it and the genuses Micrococcus and Peptococcus. The following part of this thesis shows the distribution of genus Staphylococcus in two main groups (Staphylococcus coagulase - positive and coagulase - negative), based on the ability to coagulate plasma. The description of these two main groups contains their morphological and cultivation features, antigen structure, virulence factors, pathogenesis and laboratory diagnostics. In the methodical part the pre-analytic and analytic phase is mentioned. The focus of the pre-analytic part the general priciples of collection and transport for microbiological analysis and the collection of material itself. The methodical part was performed in the České Budějovice Hospital - Laboratory of Medical Microbiology - Department of Bacteriology. This part of the thesis presents the differentiation of staphylococci by latex agglutination (PROLEX TM STAPH LATEX KIT) and then specific identification of 52 strains of coagulase-negative staphylococci isolated from central venous catheters , hemocultures and other clinical important materials is following. The specific identification of all the 52 strains of coagulase-negative staphylococci was performed by biochemical identification by STAPHYtest 16 (ErbaLachema) and parallel by mass spectrometry MALDI-TOF (system VITEK MSTM). The correct identification reached 96,2 % by the method of mass spectrometry MALDI-TOF and 67,3 % by biochemical identification STAPHYtest 16. The most frequent species isolated was Staphylococcus epidermidis (64 %), then Staphylococcus hominis ssp. hominis (10 %), Staphylococcus capitis (6 %), Staphylococcus warneri (6 %), Staphylococcus lugdunensis (4 %), Staphylococcus haemolyticus (4 %), Staphylococcus hominis (4 %), Staphylococcus caprae (2 %). The comparsion of both named methods shows the mass spectrometry more reliable, faster and simpler method, and more suitable for routine laboratory work.
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Evolution of sex-determining mechanisms and genomes in squamate reptiles (Reptilia: Squamata)
Pokorná, Martina ; Kratochvíl, Lukáš (advisor) ; Marec, František (referee) ; Vyskot, Boris (referee)
Evolution of sex determining mechanisms in squamate reptiles (Reptilia: Squamata) Martina Pokorná Ph.D. thesis Abstract This Ph.D. thesis is focused on the evolution of sex determining mechanisms and genomes in squamate reptiles. It is based on three published articles and two manuscripts. The evolution of sex determining mechanisms, sex chromosomes and genomes, and their organisation, was studied on a wide phylogenetic scale of the whole group of squamate reptiles and some lineages of other Sauropsids, as well as on the small phylogenetic range as a detailed comparative study inside individual lineages of squamates. This thesis is based upon the use of classical cytogenetic methods, methods of molecular cytogenetic (especially fluorescent in situ hybridisation) and the results were analysed using phylogenetic approaches. The results and outputs of this study represent an important contribution to the general knowledge of the principals of sex determination and the evolution of these phenomena not only in squamate reptiles but also in the whole group of amniotes. Using the results obtained during the work on this thesis we can conclude that sex chromosomes evolved in particular lineages of amniotes independently. This origin was in some cases followed by accumulation of microsatellite sequences on sex...
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A study of aneuploidy in gametes and embryos
Diblík, Jan ; Macek, Milan (advisor) ; Forejt, Julius (referee) ; Rubeš, Jiří (referee)
The thesis deals with improvement and clinical application of molecular cytogenetic methods for reproductive genetics. These methods include both clinical investigations used for improvement of diagnostic and therapeutic care for infertile couples and experimental methods that can become the basis of new diagnostic tools. The thesis concentrates on the study of aneuploidies, because they constitute a major complication of human reproduction especially by means of assisted reproductive technologies. Aims The main practical aim was the introduction of fluorescence in situ hybridization (FISH) for evaluation of chromosomes in sperm, polar bodies and blastomeres for prefertilisation and preimplantation diagnosis of aneuploidies. The main scientific objective was the study of chromosome localization in nuclei of blastomeres, that are removed from human embryos for preimplantation diagnosis. The aim of this study was to find, whether the localization of chromosomes in relation to the nuclear center and periphery is ruled by the same rules as in other cell types in later stages of development and whether the localization is influenced by aneuploidy. Next aim was to search for peripheral localization of chromosome X in embryos with more than one copies of the chromosome X. This could be a manifestation of X...
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