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Molecular biological evidence of Pneumocystis jirovecii (carinii) with application of the real-time PCR
Roučková, Vítězslava ; Fendrich, Zdeněk (advisor) ; Vopršalová, Marie (referee)
Pneumocystis carinii is an opportunistic pathogen, which often causes fatal pneumonia in patients under immunosuppressed or immune deficient conditions due to AIDS, cancer chemotherapy, or immunosuppressive therapy for organ transplantation. Different techniqes of microscopy and a nested polymerase chain reaction (PCR) are widely used for detection of this opportunistic fungus. But these methods are less sensitive and time-consuming. We focused our attention on the level of specific DNA by a quantitative PCR technique. This procedure has the advantage of greater precision and more objectivity. In this report we describe a real-time PCR assay suitable for use with the LightCycler system. We were successful in implementation and optimalization of quantitative real-time PCR for Pneumocystis carinii. We have reached sensitivity 1000 copies of DNA Pneumocystis carinii /ml. We have set a limit of detection to be 50 copies of DNA Pneumocystis carinii /ml. Real-time PCR procedure was optimized and validated in laboratory of molecular biology of Department of clinicall biochemistry and diagnostics of Fakultni Nemocnice in Hradec Kralové. A standard routine was established to be utilized in general practice
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Diagnostic imaging methods in senology, gynecology and obstetrics
PILAŘOVÁ, Martina
This thesis describes depicting methods in the senology, gynaecology and obstetrics. "In the present times keep using a new method of depositing diagnostics . The development of these methods goes ahead very fast. The breakthrough was the discovery of the diagnostic imaging W. C. Röntgen, who discovered beams X in the mid-20th century. It is the discovery that started the era of the evolution of modern diagnostics. The theoretical part deals with the anatomy of the breast and female genital organs. Further on, there is simply described the principle and the utilization of imaging techniques in the gynaecology, obstetrics and senology. In the senology part there is also the mention of the breast cancer. The Breast cancer is one of the most common cancers affecting women. It is a hormone-dependent tumour, therefore, the origin often is involved in hormonal stimulation. On the occurrence of the breast cancer is also involved in genetic factors. The most common risk factors include former the breast cancer, obesity, and exposure to ionizing radiation. The basic symptoms are as changing breast shape, size, pain, edema, ulceration. Highly effective prevention is self-examination of the breast, another preventive measure is the mammography screening. The Gynecologic section contains a list of the most common tests and a brief description. The final theoretical part is obstetrics, where I focused on a main topic- magnetic resonance imaging. This method is very useful but unfortunately , nowadays, in this field is still very rare. Harmfulness of this method is unproven, but is not recommended for use during the first trimester of pregnancy. The use of magnetic resonance imaging has its specifics and display area. It is used for example in the diagnosis of certain intracranial lesions, such as the absence of corpus calosum, intracerebral tumours, the detection of small lesions in the spine, unclear pathological structures and possibly congenital infection of the foetus. Its perspective is probably the possibility of evaluation of metabolic events during the development. The aim of my work is to analyse the radiological examination methods in senology, gynaecology and obstetrics. Another objective is to determine the number of specialized centres of prenatal diagnosis in the country. The hypothesis of the thesis were formulated as follows: Number of radiological examination methods increases. The practical part provides information relating primarily to the total number of radiological examination methods in Gynecology at the Hospital Budějovice (s.r.o) among 2010 and 2012. These investigations are primarily related to gynaecological diagnosis. All data were obtained from the hospital computer system. In this work was compared data of patients: ? Due to the age of the patients ? Due to the most common diagnosis ? Due to the total number of examinations in the years 2010-2012 Furthermore, the data from the internet portal of the Institute of Health Information and Statistics of the country ( the Czech Republic) were used. As a curiosity, I used data from the first ultrasound in pregnancy and newborn mortality. While this is not the subject of my work, but the issue took me personally therefore I gave her a portion of my work. The results of this work demonstrate achievement of the objectives that have been identified in this work. We managed to confirm the hypothesis that the use of radiodiagnostic imaging is rising every year. The awareness of people about the possibilities of testing facilities is on the increase and the facilities of department radiological imaging methods are gradually improving.
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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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Molecular aspects of interspecific hybridization of sturgeons related to polyploidy and in situ conservation
HAVELKA, Miloš
Sturgeons (Chondrostei: Acipenseriformes) display markedly disjunct distributions with a wide occurrence in the northern hemisphere. Their unique benthic specializations, conserved morphology, evolutionary age, the variation in their basic diadromous life history, and the large public interest due to their near extinction or critically endangered status make sturgeons and paddlefishes one of the most interesting group of vertebrates. In addition to that, ploidy diversity of Acipenseriformes possessing three ploidy groups having ~ 120 chromosomes, ~ 240 ? 270 chromosomes and ~ 360 chromosomes provides unique model for investigation of evolutionary processes which were going through the genome duplication events. Sturgeons are also notoriously known for their strong propensity to interspecific and intergeneric hybridization which can result in hybrids with various ploidy levels. All these facts make sturgeon genetics and cytogenetics a thriving but also complicated area for research. In the present work, the role of genome duplication and functional reduction evens in evolution of sturgeon species as well as sturgeons? ploidy levels and ploidy relationships among Acipenseriformes were investigated using molecular markers. In addition to that, clarification of origin of abnormal ploidy levels and observation of segregation pattern of microsatellite alleles in the course of hybridization of polyploid sturgeon species were included into this study. With regard to the all considerations and observations provided by this study we concluded that evolution of sturgeon species is still widely dynamic and ongoing process which might goes through the allopolyplodization as well as autopolyplidization events.
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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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Study of genetic factors modifying the risk of onset and progression of colorectal and pancreatic cancer
Mohelníková Duchoňová, Beatrice ; Souček, Pavel (advisor) ; Skálová, Lenka (referee) ; Macek, Milan (referee)
Introduction: The aim of this study was to evaluate the role of genetic and lifestyle factors in the risk of onset and progression of colorectal and pancreatic cancer. The first part deals with the etiological factors and the importance of polymorphisms in biotransformation enzymes and genetic alterations in the gene CHEK2 in the origin of these malignancies. In the second part, the ABC transporter genes were analyzed as potential prognostic and predictive markers of a treatment's outcome. Materials and methods: The polymorphisms and other genetic alterations were detected using real-time PCR, allelespecific PCR and PCR-RFLP methods in DNA which was extracted from the blood of patients. The frequency of polymorphisms was evaluated and their importance was assessed with regard to the available epidemiological data. Gene expressions were determined by qPCR in paired samples of tumor tissue and adjacent non-tumorous parenchyma. Results: A majority of the observed polymorphisms failed to show a relationship between their presence and the risk of any of these malignancies. CYP2A13 variant allele*7 coding inactive enzyme was found in 7 of 265 controls and in none of 235 pancreatic carcinoma patients. In contrast, GSTP1-codon 105 Val variant allele and GSTT1-null genotype were associated with an elevated...
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Prediction of the Secondary Structure of Proteins by Cellular Automaton
Šalanda, Vojtěch ; Vogel, Ivan (referee) ; Bendl, Jaroslav (advisor)
This thesis presents a new approach to the prediction of the secondary structure of proteins. It employs a new method based on cellular automata and its characteristic properties. The main objective is to increase speed of the prediction even at the cost of slight decrease of overall accuracy. Optimal parameters of cellular automata was found by genetic algorithm using suitable genetic operators. These parameters are incorporated into developed application for prediction. Finally, the results was compared with results of other tools for this purpose.
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Digital Filter Design on GPU
Vaverka, Filip ; Maršík, Lukáš (referee) ; Polok, Lukáš (advisor)
This thesis shows one of the approaches to the design of ditigal filters with infinite impulse response and specified order. The proposed solution is based on an evolutionary genetic algorithm and therefore allows for direct filter design from its specification. Its main contribution to this subject is that the implementation is parallel and it is acceleraded by GPU. The filters are designed in cascade representation. It also allows to specify both, the desired frequency and phase characteristics of filters.
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The Control of Chaos: Methods and Applications
Švihálková, Kateřina ; Dvořák, Jiří (referee) ; Matoušek, Radomil (advisor)
The diploma thesis is focused on the use of heuristic and metaheuristic methods to stabilization and controlling the selected systems distinguished by the deterministic chaos behavior. There are discussed parameterization of chosen optimization methods, which are the genetic algorithm, simulated annealing and pattern search. The thesis also introduced the suitable controlling methods and the definition of the objective function. In the theoretical part of the thesis there is a brief introduction to the deterministic chaos theory. The next chapters describes the most common and deployed methods in~the~control theory, especially OGY and Pyragas methods. The practical part of the thesis is divided into two chapters. The first one describes the~stabilization of the artifical chaotic systems with the time delayed Pyragas method - TDAS and its modification ETDAS. The second chapter shows the real chaotic system control. The Duffing oscillator system was chosen to serve this purpose.
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The influence of pH on iron chelation
Hrabalíková, Jitka ; Mladěnka, Přemysl (advisor) ; Hrdina, Radomír (referee)
v angličtině Iron is one of essential elements which are necessary for life of cells. Many important processes are connected with it and its insufficiency can lead to cell death. Iron excess can be dangerous, too; such situation can be triggered not only by acute intoxication, but it appears also chronically due to genetic abnormalities (hereditary hemochromatosis) or by frequent blood transfusions during treatment of thalassaemia, sideroblastic anemia or myelodysplastic syndrome. Iron chelators are used to cure these pathological states, but there have been currently analysed for the treatment of tumors or acute myocardial infarction as well. In pathogenesis of both of these disorders, iron and change of pH play significant roles. In this study, influence of pH on iron chelation abilities of various substances (deferoxamine, synthetic chelator SIH, flavonoids and synthetic coumarin 7,8-dihydroxy-4- methylcoumarin) was measured with the use of ferrozine as an indicator. The stability of complex chelator - iron in various pH was also tested. It was found that chelation activity of all tested substances and the stability of iron- chelator complex is increased with increasing pH. Strong chelators SIH and deferoxamine kept their effectiveness within pH from 7.5 to 5.5, whereas chelating activity of...
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