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Gene expression reporters in \kur{Drosophila melanogaster}.
ŠTROS, Jiří
Drosophila melanogaster is a widely used model organism in genetic research and in a number of other disciplines associated with medical and biotechnological issues. The first part of this thesis presents a review of some basic genetic tools and gene expression reporters used in D. melanogaster research with emphasis on the use of GFP reporter. The second part presents a description and results of my experimental work aimed at the reporter construct consisting of adenosine deaminase gene (ADGF-A) and GFP marker. When using gene expression reporters such as GFP, it is important to know whether the presence of reporter does not affect the studied process. The experiment described in this study tested whether the fusion protein consisting of GFP and adenosine deaminase is fully functional enzyme or whether the enzyme activity is influenced by the presence of fluorescent tag. Results of this work support the usefulness of using the fusion construct as a gene expression reporter.
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Fetal hemoglobin in myelodysplastic syndrome patients.
Staňková, Nora ; Beličková, Monika (advisor) ; Brdička, Radim (referee)
5 Abstract Aims Determination of gene expression of HBG1 gamma globin in myelodysplastic syndrome (MDS) patients in CD34+ pluripotent hæmatopoietic cells and connection of HBG1 gene expression with various subtypes of MDS. Furthermore, detection of single nucleotide polymorphisms rs 4671393 and rs 11886868 in these patients and in healthy Czech population donors and to determine whether a connection exists between the occurrence of the above polymorphisms and HBG1 gene over-expression, as demonstrated in some hæmatological disorders. Samples The source of genetic material to identify gene expression were 80 HBG1 RNA samples isolated from the pluripotent hematopoietic CD34+ cells of MDS patients. As a sample of healthy controls, 6 samples of commercially purchased CD 34+ cells from the Lonza com- pany were used. The source of genetic material for the detection of polymorphisms were 140 DNA samples isolated from purified granulocytes of MDS patients and 49 samples of DNA isolated from peripheral blood granulocytes from healthy Czech population donors. Methods Real-Time PCR was used to determine HBG1 gene expression and detection of single nu- cleotide polymorphisms. Taqman Gene Expression Assay was used to determine the level of expression and the results were evaluated using the comparative ΔΔCT method....
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Biosystematic studies in the family Cyperaceae
KOŠNAR, Jan
The thesis was focused on the microevolutionary mechanisms that contribute to morphological diversity in selected members of the sedge family (Cyperaceae). Natural hybridization, evidenced from both morphological characters and molecular markers, was revealed to be a potentially important source of diversification in the tropical spikerushes of Eleocharis subgenus Limnochloa. High levels of phenotypic plasticity of clonal growth but rare genetic (ecotypic) differentiation among contrasting morphotypes were found in the polymorphic species Carex nigra, which implied that taxonomic splitting of the species was unreasonable.
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The influence of individual genetic predisposition to the pharmacokinetics and pharmacodynamics of chosen opioids
Matoušková, Olga ; Perlík, František (advisor) ; Votava, Martin (referee) ; Mičuda, Stanislav (referee)
MUDr. Olga Matoušková - the dissertation theses The influence of individual genetic predisposition to the pharmacokinetics and pharmacodynamics of chosen opioids ABSTRACT Introduction: The aim of this thesis is to study the influence of polymorphism of CYP2D6 and MDR1 on the pharmacokinetics and pharmacodynamics of tramadol in healthy volunteers using measurement. A secondary objective is to evaluate these polymorphisms in relation to the analgesic efficacy and side effects of piritramide for acute postoperative pain. Materials and methods: In two prospective work studying the influence of genetic predisposition on the pharmacokinetic and pharmacodynamic parameters of tramadol, we included a total of 90 healthy volunteers. Clinical studies on opioid analgesia and influence of genetic predisposition to the pharmaco-therapeutic effects and side effects in patients with acute postoperative pain, we included a total of 161 patients with acute postoperative pain. Polymorphism genotyping CYP2D6 and MDR1 gene we performed PCR - RFLP analysis, to determine concentrations of tramadol and metabolite, we used gas and liquid chromatography and pharmacodynamic effects of opioids was evaluated by pupilometric measurement and visual analogue scale. Results and conclusion: Variability of the opioid effect is influenced by...
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Genetic, humoral and cellular factors of development of autoimmune diseases
Růžičková, Šárka ; Černá, Marie (advisor) ; Kalina, Tomáš (referee) ; Drda Morávková, Alena (referee)
Autoimmune diseases currently represent the most serious medical issues, mainly due to generally increasing number of patients with these diseases. Their pathogenesis is likely caused by hereditary factors, cellular and humoral interactions influenced by external environmental factors, whose knowledge is important both for diagnostics and therapy, and for theoretical immunology. The aim was to examine the correlation between genetic alterations and the production of autoantibodies, clinical manifestation of the disease; to identify joint autoantigens, further to demonstrate development of leukemic cells from originally autoreactive B cells, the role of B lymphocytes in the pathogenesis of the disease and finally to develop a method for detection of B cells recognizing a defined autoantigen. Several predisposing polymorphisms were revealed using genetic analysis however, they were not exclusively associated either to clinical forms of the disease or usable as prediction markers. In addition, the frequency of alleles IL-1RN * 2 and PD3.1 showed ethno- geographical differences and a critical role of size of sample cohorts in assessing of significance of particular polymorphism was demonstrated in GWA studies. The combination of examination of anti-CCP and IgM RF was found as the best and most...
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Importance of trombophilic mutations in clinical genetic.
Vavrušková, Klára ; Kuklík, Miloslav (advisor) ; Černá, Marie (referee)
Trombophilia means an increased disposition to creation of trombs. Health complication incurred as a consequence of hypercoagulation can be very serious. When a trombophilic mutation is found at a patient, it brings necassity of thrombosis - control in risk situations (e.g. pregnancy, operation) for the rest of the patient's life. There were filed 300 people (206 women and 94 men) with trombophilic mutations into my study of clinical signification of trombophilic mutations. These people were examinated in years 2008 - 2010. Most of positive medical findings - 266 people, were recorded in the area of MTHFR (C677T i A1298C) mutations. There were less findings in the field of FV Leiden and FII prothrombin mutations. Multipath trombophilic mutations were found at 99 patients. I accordance with foreign literature, our results advert to clinical consequences of trombophilic mutations like: repeated spontanious aborts, cerebrovascular akcident (CA), ischaemic heart disease (IHD), thrombosis, flebothrombosis, pulmonary embolism, varicose veins, aseptic necrosis of hip bone, arterial sclerosis and aortic stenosis. Mutations MTHFR C677T and MTHFR A1298C we found mainly at patients with CA, IM and IHD. Leiden mutation was most often found at patients with thrombosis, flebothrombosis and pulmonary embolism. We...
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Molecular aspects of genetic predisposition to type 2 diabetes mellitus and its monogenic forms
Pintérová, Daniela ; Černá, Marie (advisor) ; Bendlová, Běla (referee) ; Hubáček, Jaroslav (referee)
The aim of this work was to study some molecular aspects of genetic predisposition to T2DM and its monogenic forms. We searched for correlations of genotypes with many clinical and laboratory markers and tried to evaluate their significance. The consequences for possible changes in the treatment some particular patients were under discussion. Especially for those suffering from monogenic forms we were able to reach better compensation of DM, or at latest the same compensation for more acceptable therapy. We studied the associations of chosen candidate genes, PPAR, NF-B, its inhibitor IB, known polymorphisms and T2DM including diabetic complications. We used the association study approach to find out if there is any association between available genetic variants among our patients and T2DM with its phenotypic abnormalities (changed levels of total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides, further the presence of diabetic complications).
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STR genotyping of Czech medieval population: archeologocal site in Mlekojedy (Litoměřice)
Brynychová, Veronika ; Hájek, Martin (advisor) ; Šimková, Halina (referee)
The aim of this diploma thesis was the initial genetic analysis of early mediaeval burial site from Mlekojedy polycultural locality (Litoměřice District, Czech Republic). Autosomal STR markers were chosen because of the following reasons. The high degree of polymorphism of these markers and the high extent of heterozygosity favor the use of STRs instead of mitochondrial DNA for the structural analysis of small populations. Usefulness of STR typing for validation purposes was demonstrated many times before. We used primers for miniSTRs to obtain the fullest results. Nuclear DNA was extracted from 35 % of bone samples and 91 % of teeth. We detected lower PCR amplification success rate of fragments longer than 150 bp and very high rate of allele drop-out which is sign of degraded DNA. Twelve reliable genotypes were determined for TH01 marker. Observed allele frequency and genetic diversity values were discussed in comparison with recent populations and other aDNA studies of burial sites. Keywords: ancient DNA, STR markers, miniSTR, early medieval burial site, Czech population
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Gene flow and its consequences for microevolution in Taraxacum (Asteraceae)
Marván, Richard ; Vančata, Václav (advisor) ; Brdička, Radim (referee) ; Lhota, Stanislav (referee)
Microsatellites(SIR) are very abundantand spread over the entire genome of all living organisms,so markers can be developedfor any genetic objective.Some SfRs exhibit very high levels of allelic variation.SIRs are generallyeasy to obtain and rapidlyassayed using polymerase chain reaction (PCR), requiringonly minute amountsof biologicalmaterial.Their distinctattributes made them attractiveand the popular tool of choice in a numberof applicationswithinthe area of biology' The core of this Íheslsconslsts of five chaptersrepresentingsťudieswhere we investigated autosomal or Y_linked SIRs Íocarry out our tasks. our objectives Were (i) to investigateallele frequencies for a set of autosomal SIRS, to estimatestandard population and forensic genetic parameters,and to perŤorman inter_populationcomparisonof allele frequencydata;(ii)to design a novel PCR-based Y chromosomeSIR multiplexassay for the analysisof highlydiverseloci, and to evaluateťhissysťemfor forensicand populationgenetic purposes;and (iii)to carryout the firststudy in a Pan paniscusgroup wherea combinationof behaviouraldata on male dominancerank and matingsuccess withgeneticdataonpaternityallowsthetestingof thepatternof relationshipsbetween thesevariables,and to evaluatepossib/econsequencesof the establishedgenetic relationshipsfor...
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Molecular- genetic analysis of the Thyroid carcinomas
Sýkorová, Vlasta ; Bendlová, Běla (advisor) ; Zamrazil, Václav (referee) ; Peterka, Miroslav (referee)
Introduction: Thyroid cancer represents more than 90% of endocrine tumors and its incidence, predominantly of papillary thyroid carcinoma (PTC), is still increasing in the Czech Republic. Several genetic changes are known, but thein impact to phenotype is still controversial. Aims: To study of the genetic causes (RET/PTC, BRAF and RAS alterations) and the role of RET polymorphisms in thyroid cancer (predominantly PTC), and to correlate genotype with phenotype. Subjects and Methods: Overall 234 PTC tissues, 8 poorly differentiated carcinomas, 3 anaplastic carcinomas, 23 medullary carcinomas, 6 follicular carcinomas and one follicular adenoma were analyzed. Samples of fresh frozen thyroid tissues, fine-needle aspiration biopsies and paraffin-embedded formalin-fixed tissue sections of patients with thyroid cancer and blood samples of healthy controls were used for analysis. The expression of RET/PTC rearrangements was detected on agarose gel. Five RET polymorphisms were analyzed using specific TaqMan probes. Detection of mutations in the BRAF gene and three RAS genes was performed by direct sequencing. Presence of alteration was correlated with clinicopathological parameters. Results: We found out that some RET polymophisms are associated with development of RET/PTC rearrangements in PTC and proved,...
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