It is the purpose of this thesis to implement primordial germ cell (PGC) transplantation, one of the new biotechnological reproductive methods, and for this to explain the details that we have to know about embryo development and PGC migration in pikeperch. We provide several specific useful methods such as GFP labelling and blastodisc surgery which are required for efficiency assessment of the transplantation technique. The main results of the publications in the thesis could be informative and useful for generation of germline chimera by using pikeperch. We described pikeperch embryo development to first feeding at 15°C in detail and demonstrated effects of temperature on the rate of embryogenesis to determine temperature limits for slowing development with minimum negative effects on growth and survival rate. We also developed a technique to soften the pikeperch chorion by enzyme in order to remove it by forceps for in depth observation. Additional groups of eggs were fertilised and incubated at different temperatures to document embryo developmental stages, developmental rate, and survival. The optimum fertilisation and incubation temperature was 15°C, with the highest fertilisation, survival, and hatching rates. Embryo development was drastically slowed down at 10 °C, with 45% of fertilised embryos surviving to hatching. Development was accelerated at 20 °C, with a 56% survival rate of fertilised embryos. After the series of experiments to characterize the embryo development of pikeperch, it could be a valuable model percid for research in which flexible incubation temperatures is required. We described the important early embryonic events, namely, yolk syncytial layer (YSL) formation and midblastula transition (MBT) during the blastula stage in pikeperch embryos. The chorion was removed as we described in the first study. The YSL was formed after the breakdown of marginal cells during the 512- to 1k-cell stage. Cell division analysis by 4'-6-diaminido-2-phenylindole (DAPI) staining revealed that transition from synchronous to asynchronous division occurred after 1k-cell stage. Our results indicate that MBT starts after this stage. Next, we performed blastodisc isolation assay to find the competent stage for embryonic manipulation. Embryos were manipulated by using a microneedle every hour from the 512-cell to the sphere stage, and then developmental rates were evaluated at the hatching stage. The highest survival rate was obtained when we performed this manipulation at the 1k-cell stage. These results clearly showed that the MBT is the best stage for transplantation of PGCs or any cells in pikeperch. We described PGC migration and performed blastomere transplantation in pikeperch. PGCs were visualised by injection of synthesised green fluorescent protein (GFP) within the 3'untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to the location at the gonadal ridge. We conducted blastomere transplantation at the blastula stage. Donor embryos were labelled with GFP-nos3 3'UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve blastomere transplantation chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that blastomere transplantation can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.

My thesis "The Analysis of the Genetic Source of the Prestice Black-Pie Pig" deals with the evaluation of the population of the Prestice Black-Pie pigs from the point of view of their numbers, the structure of their population and their reproductive qualities, using the data of the Association of Pig Breeders in Bohemia and Moravia. First the carcass value and meat quality of this original Czech meat bacon breed were evaluated and then a slaughter dissection and laboratory analyses of the meat from the analyzed group of pigs were done. The numbers of PBP pigs have increased significantly especially since 2012 and so has the number of the farms; however in many of them there are fewer than 10 PBP pigs. This is why the PBP pig breeding is rather fragmented and it is difficult to sustain sufficient genetic diversity of the closed population. This is also connected with unequal numbers of the 10 genealogical lines. The Akoga and Amperor lines were the most numerous. Since 2015 the numbers of pigs from different lines have been more balanced. In 2016 two sows from the renewed Apolon line, which had been born from a cryopreserved seed, were put in the farms. The influence of the line was evidential in the statistics: in reproduction as well in productive qualities. Therefore, to sustain sufficient genetic diversity in a small population we need to keep in the stock the maximum number of equally represented genealogical lines. A detailed analysis of reproductive indicators of the years 2013 - 2016 found 9.4 piglets born alive and 8.7 weaned piglets, the farrowing interval of 181 days, the average values of relevant indicators did not meet the requirements of the breeding objective; only one farm met them. Statistically speaking, there were significant differences between the individual farms. In bigger farms there were higher reproductive qualities. Between 1998 and 2016 the average daily gain in weight was 530 g for gilts, 578 g for boars, the muscle fibre ratio for gilts was 58.5 % and for boars 59.3 %, the back fat thickness was the same for both, 1.1 cm. The requirements of the breeding objective were met, except for the lower weight gain for gilts. The average daily weight gain between the years 2009 and 2013 for 577 gilts was 524 g, the average muscle fibre ratio was 59.1 % and the average back fat thickness was 1.03 cm; in all indicators there were statistically evidential differences between individual farms. In bigger farms the muscle fibre share was higher by 0.9 %. In a group of PBP pigs I studied the influence of slaughter weight and gender on carcass value. The statistics clearly showed that the bigger the slaughter weight was, the higher the back fat thickness was, and also the higher the weight of meat parts was, but the share of meat parts was reduced, and the area of MLLT increased, too. The slaughter weight had no influence on the muscle fibre share. The statistics also showed that with the increase in slaughter weight there was an increase in drip loss and the meat was tenderer. There was also a statistically inconclusive increase in intramuscular fat. The statistics also showed that the barrows in the analyzed group had a lot higher average daily gain, a statistically inconclusive higher back fat thickness, lower share of muscle fibre and a statistically conclusive lower share of ham and higher share of intramuscular fat. In the meat of the analyzed group of pigs I studied the content of fatty acids. There was a higher share of undersaturated fatty acids and a high share of n-6 PUFA:n-3 PUFA, which is not considered satisfactory from the nutritional viewpoint.

The main aim of this thesis was to study the genetic variation of several performance traits under pond management conditions and to estimate future perspective of sustainable selective breeding program in common carp. In the present study, Amur mirror carp (AM) and Hungarian synthetic mirror carp (HSM) were used. Most of the studied performance traits showed sufficient genetic variation (h2 = 0.12 1.0), suggesting a good potential for genetic improvement of the traits through selective breeding. It was shown that selection of common carp under pond management conditions should be optimally applied after the second overwintering. Winter survival is often a bottleneck for common carp production. As a result, the genetic background of winter survival and traits that might be correlated to the survival was studied. Main focus was given to the muscle fat and body weight and their dynamics through winter period and successive third growing period. It was found that selection for i) lower fat content before and after winter, ii) lower decrease in muscle fat content and/or body weight during winter, iii) higher condition factor may lead to better survival during both winter and third growing period and growth of fish during the third growing season. Unfortunately, direct selection for slaughter yields is impossible on live breeding candidates. Therefore, morphological predictors that can be measured in vivo are considered as an interesting alternative. As a result, external and internal measures were combined on 1553 fish by linear regression to predict log-log residuals (Logr) of slaughter yields. It was found that the accuracy of the prediction of slaughter yields may be solid. From the genetic point of view, model-predicted (h2 = 0.48 0.63) and even individual predictors (h2 = 0.34 0.72) of slaughter yields were highly heritable and favourably genetically correlated to the Logr yields. Hence, selection on trait predictors has an interesting perspective for genetic improvement of slaughter yields in common carp. Furthermore, genetic parameters of fillet fatty acid content and performance traits in market size common carp cultured under semi-intensive pond conditions were estimated. For flesh FA composition analysis 158 individuals were processed. Heritability estimates of total muscle fat, some FA groups and most performance traits were moderate to high (0.23 0.62). Interestingly, genetic correlations showed that genetic improvement of growth via selective breeding under Central European pond conditions without changing the production technology would very likely negatively affect carp flesh quality with respect to FA composition. Finally, restriction-site-associated DNA sequencing (Rad-seq) was used to identify and genotype single nucleotide polymorphism (SNP) markers for subsequent parentage assignment, construction of a medium density genetic map (12,311 SNPs), and testing of efficiency of marker-assisted (MAS) and genomic selection (GS) for growth and Koi herpes virus (KHV) resistance. No genome-wide significant QTL was identified for growth. However, genomic prediction of its breeding value may outperform the traditional pedigree-based prediction, resulting in an 18% improvement in prediction accuracy. On the other hand, genome-wide significant QTL affecting resistance to KHV was identified on linkage group (LG) 44 explaining approximately 7% of the additive genetic variation. The presented results add further evidence supporting the application of selective breeding in common carp cultured even under traditional pond management conditions. However, it is evident that the rearing technology will need suitable modifications. In addition, real economic impact of selective breeding on carp culture should be verified by calculation of realized heritabilities, real genetic gains and the total fish biomass yield of genetically improved stocks from a pond area unit.

RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...

This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.

This master´s thesis discusses cytotoxicity of selected naphthoquinones on prostatic cell cultures. The introductory part is dedicated to general characteristic of naphthoquinones with focus on their cytotoxicity, testing of cytotoxicity and mechanisms of cytotoxicity. This part is followed by chapters about cytotoxicity, characteristics and biological activities of selected naphthoquinones; plumbagin and naphthazarin. The last part of this thesis’ theoretical section speaks about fluorescence microscopy and its use in research of naphthoquinones cytotoxicity. The practical part is dedicated to evaluation of cytotoxical tests’ results and to analysation of pictures of cells obtained by fluorescence microscope. At the end of thesis, all finding are summarized and put in the context.

This master's thesis deals with the geographic information systems   with an emphasis on so - called vector analysis . The aim of the thesis is to create a library that implements the internal representation   of geographic data and performs analytical operation over them .  The thesis describes the logical structure of the application and the hierarchy of abstract classes . In the form of pseudo -code is shown a set of individual computing operations . Work is strongly based on geographic system GRASS. This GIS is connectable through  import and export vector data in shapefile format. Concept of   storing layers is designed for ease of data manipulation and  their portability . The main idea behind the application is to split the library into individual modules . These modules are  independent , but also can benefit from each other's functions . In conclusion are demonstrated implemented vector analysis and  representation of their calculation .

This master’s thesis deals with methods for detection of myocardial ischemia in the ECG signal. There is explained the principle of spreading of electrical activity through the heart muscle and its manifestations on the ECG. There are also mentioned the causes of myocardial ischemia and various methods of its detection in the ECG signal. In great detail there is explained the process of implementation of the two selected detection methods of myocardial ischemia in MATLAB. These methods are tested on the data from The PTB Diagnostic ECG Database. Finally, there is the presentation of detection results on used data and overall assessment of created algorithms.

Contrary to the classical mouse inbred strains with unnatural genetic variability, wild-derived strains offer a more suitable model for evolutionary immunology. Toll-like receptors (TLRs) belong to initial detectors of invading pathogens. Although TLRs recognise conserved structures they were shown to be polymorphic. This polymorphism is associated with various diseases. In my thesis, I describe variability of Tlr1, 2 and 6 in 24 inbred strains derived from two subspecies of house mouse (Mus m. musculus and M. m. domesticus). These Tlrs exhibit different levels in variability among the strains. In Tlr1 the polymorphic sites are spread along the whole exodomain. Tlr6 is quite conserved (a lower amount of substitutions located far from the binding region and with minor modifications in the amino acid residue properties). Tlr2, on the contrary, contains some substitutions with substantial alternations of residue properties that are located within or nearby the binding region and the subspecies differ at these sites. All alleles of M. m. domesticus and M. m. musculus, except for Tlr1 PWD, Tlr2 STAIL, are phylogenetically separated. The strains and the subspecies vary in the production of IL-1β, IL-12 a NO after stimulation by TLR1, 2 and 6 ligands. This trend is, however, presumably influenced by the effect of...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavla Martinková Supervisor: PharmDr. Anna Jirkovská, PhD. Title of diploma thesis: Antiproliferative activity of novel dexrazoxane analogues and their effect on antitumor effectiveness of anthracyclines Athracycline antibiotics (such as daunorubicin, doxorubicin or epirubicin) belong to the most common terapeutics of both solid tumors and hematological malignities. Unfortunately the serious and life-threatening adverse effect cardiotoxicity compromises their clinical usefulness. The only approved protection against anthracycline cardiotoxicity so far is dexrazoxane. Despite the outstanding cardioprotective ability, dexrazoxane use is very limited mainly due to its possible side effects. So we were directed towards synthesis of dexrazoxane analogues with better pharmacological properties. The aim of this diploma thesis was to assess the antiproliferative activity of novel analogues of both dexrazoxane (MK-15 and ES-5) and ADR-925 (JR-159 and KH- TA4) and their influence on the antiproliferative effectiveness of anthracyclines. Moreover, we aimed to study their chelating properties and their inhibition of the topoisomerase II in solution. We tested the antiproliferative activity of...