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Molecular genetic approaches used in preimplantation genetic analysis of human embryos in assisted reproduction
Limbergová, Jana ; Machoň, Ondřej (advisor) ; Šimoník, Ondřej (referee)
Preimplantation genetic testing is one of the main clinical procedures that take place in assisted reproduction centres around the world. It is sought after by couples for many different reasons. The causes of problems may relate to hereditary diseases that potential parents do not wish to pass on to their offspring, or may include the risk of transmission of chromosomal alterations. In addition, infertility may be caused by an increased risk of embryo aneuploidy, which correlates with mother's increased age and other factors that this bachelor thesis discusses. The bachelor thesis also focuses on up-to-date approaches to cytogenetic examinations for reproductive genetics and discusses recent clinical molecular methods that are used to improve the diagnosis and therapy of infertile couples. These are, for example, microarray methods, quantitative real-time PCR or next-generation sequencing and methods of whole-genome amplification. This work also summarizes methods that are gradually being abandoned, e.g. fluorescent in situ hybridization, and compares their advantages and disadvantages. Key words: preimplantation genetic testing, aneuploidy, structural rearrangements, monogenic diseases, biopsy, fluorescent in situ hybridization, microarray methods, comparative genomic hybridization, quantitative...
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Splice variants of the gene coding for GCPII and their role in cancer development
Jindrová, Helena ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
Alternative splicing is a mechanism of generating distinct proteins that are encoded by the same gene. These proteins differ in amino acid sequence, overall structure and function. Splicing dysregulations have been shown to be implicated in several pathologic processes including cancer. For example, non-physiological splicing of osteopontin was proved to play a key role in cell progression of breast cancer. Glutamate carboxypeptidase II (also called prostate specific membrane antigen, PSMA) is present in both normal prostate and prostate cancer. Several splice variants of PSMA have been described and it has been suggested that the overexpression of some of them could be involved in the progression of prostate cancer. Nevertheless, more detailed investigation of each of the PSMA splice variant in terms of their occurrence in prostate cancer cells remains to be performed. This thesis focuses on the exploration of the expression of PSMA splice variants with deleted exons 6 and 18 in samples of a cell line derived from human prostate cancer, benign prostate hyperplasia and prostate cancer. For this purpose, RT-PCR was utilized to determine the ratio of deletions of exons 6 and 18 in cDNA of the prostate specific membrane antigen. Furthermore, the ratio of deletions of exon 6 and 18 was determined in...
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Identification of membrane transporters involved in carbon flux in arbuscular mycorrhizal symbiosis
Konečný, Jan ; Kohout, Petr (referee)
Arbuscular mycorrhizal symbiosis is one of the oldest and still most common interactions. This relationship between small soil fungi and plant roots, especially herbs, is the subject of research with, among other things, a promising use in sustainable agriculture. However, intensive research is also taking place at the cellular and molecular level, as the exact mechanisms of functioning of this symbiosis are still shrouded in mystery. Understanding these processes is desirable not only in the boom of genetic engineering. A commonly used model organism for the research of plant symbioses is a smaller legume of the genus medick - Medicago truncatula. Using a microarray for all known M. truncatula genes and by comparing mycorrhizal and non-mycorrhizal plants, the author identified several genes that could participate in symbiosis and he wrote a thesis from these results. In a follow-up experiment with an intervention in plant physiology, the authors of the article (Konečný et al., 2019) point to the probable involvement of some previously unreported genes in symbiosis, but also to different modes of regulation in genes whose probable symbiotic function is already published. This rigorous thesis focuses on the introduction of the first-author publication, a closer explanation of the experiment, which is not...
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Assessment of relative telomere length using qPCR: advantages and disadvantages of this method and shortcomings of commonly used reference markers
Krajzingrová, Tereza ; Velová, Hana (advisor) ; Králová, Tereza (referee)
Telomeres, the ends of eucaryotic chromosomes, consist of tandem repeated sequences, play important role in protection of chromosomal ends. In somatic cells telomeres are getting shorter with each cell division. Telomere length (TL) is directly related to cellular ageing (senescence). The rate of telomere shortening as well as ageing is individual and it differs among species and also among individuals. To the main factors, which can affect the rate of telomere shortening, belong stress, diseases, reproduction or also environmental conditions. Many methods could be used for TL determination. Each of them has its own limitation, which is good to know to be able to avoid the negative impacts on results. qPCR is currently the most used method. The most important factor, which could affect the results, is that qPCR measures besides proper telomeres also interstitial telomeric sequences. Furthermore, the choice of suitable reference marker followed by designing of highly specific primers is also crucial, if qPCR is used. Nevertheless, qPCR is suitable method especially because it offers fast and inexpensive relative TL measurement of large amount samples. My bachelor thesis is moreover a review of studies focused on telomere dynamics in birds and causes which influence different senescence measured by TL....
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Composition and activity of microbial communities in soil contaminated by heavy metals
Průchová, Pavla ; Kopecký, Jan (advisor) ; Zikánová, Blanka (referee)
The thesis focuses on studying changes of microbial communities living in the soil contaminated by heavy metals. Two sites with different degree of contamination were selected in the Příbram area. Respiration was measured in vitro in the soil samples supplemented with various carbon sources and different concentration of cadmium. The respiration showed that even at cadmium concentration of 1000 mg.kg-1 the community is viable and capable of utilization of substrates while increasing the respiration rate. Enviromental DNA from soil samples was isolated and 16S rRNA gene of actinobacteria was amplified. The terminal restriction fragment length polymorphism analysis showed a clear difference between the profiles of both sites. The shifts in the community profiles were observed also after the addition of substrates. The quantification of total bacteria and actinobacteria was performed by quantitative PCR based on amplification of part of the 16S rRNA gene. The more contaminated site contained slightly more bacteria, but almost twice the actinobacteria than the less contaminated one. The sequencing of amplicons of a part of 16S rRNA gene by Illumina showed an increase in proportion of actinobacteria and changes of their community structure in the more contaminated site. The conclusion was made that, high...
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Splice variants of the gene coding for GCPII and their role in cancer development
Jindrová, Helena ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
Alternative splicing is a mechanism of generating distinct proteins that are encoded by the same gene. These proteins differ in amino acid sequence, overall structure and function. Splicing dysregulations have been shown to be implicated in several pathologic processes including cancer. For example, non-physiological splicing of osteopontin was proved to play a key role in cell progression of breast cancer. Glutamate carboxypeptidase II (also called prostate specific membrane antigen, PSMA) is present in both normal prostate and prostate cancer. Several splice variants of PSMA have been described and it has been suggested that the overexpression of some of them could be involved in the progression of prostate cancer. Nevertheless, more detailed investigation of each of the PSMA splice variant in terms of their occurrence in prostate cancer cells remains to be performed. This thesis focuses on the exploration of the expression of PSMA splice variants with deleted exons 6 and 18 in samples of a cell line derived from human prostate cancer, benign prostate hyperplasia and prostate cancer. For this purpose, RT-PCR was utilized to determine the ratio of deletions of exons 6 and 18 in cDNA of the prostate specific membrane antigen. Furthermore, the ratio of deletions of exon 6 and 18 was determined in...
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The effect of IDGF2 protein on expression of selected genes in cell line Cl.8+
ROUHOVÁ, Lenka
The imaginal discs growth factors (IDGFs) are proteins able to induce proliferation of Cl.8+ cells proliferation in collaboration with insulin. They are also connected with response to starvation stress and innate immunity. A genome-wide analysis of Cl.8+ cells treated with IDGF2, which was performed in our lab, detected tens of genes transcriptionally induced by this protein. The aim of this work was to confirm this effect with some of these genes using quantitative PCR.
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