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Application for the Data Processing in the Area of Evolutionary Biology
Radakovič, Lukáš ; Burgetová, Ivana (referee) ; Očenášek, Pavel (advisor)
This Bachelor’s thesis describes the design and implementation of the application that has the task to verify the accuracy of the algorithm. Purpose of the algorithm is to analyze mechanisms used in the creation of the phylogenetic tree. The application allows users to specify different parameters of phylogenetic tree, its generation and subsequent analysis using an algorithm. Results of the analysis are written to the output file, giving the user the option of setting file path. Studied algorithm correctly estimates the participation of specific mechanism in the tree formation. Estimates of the absolute and relative share of changes in chromosome number and genome size are less than accurate results.
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Molecular aspects of mammalian oocyte and embryo quality
Chyská, Anna ; Vaškovičová, Michaela (advisor) ; Jansová, Denisa (referee)
According to statistics in the Czech Republic, up to 20 % of couples experience infertility, which is still rising. One of the infertility treatment options is to undergo one of the assisted reproduction methods. In vitro fertilization (IVF) is among the most commonly used methods. In order to increase the success rate of fertilization, it is preferable to have a quality oocyte with specific characteristics. Such an oocyte is then a prerequisite for a good quality embryo, a correct course of pregnancy, and proper fetal development. Meiosis, a key step in oocyte formation, is regulated by complex signalling pathways. If these pathways are dysfunctional, the chances of the oocyte developing into a competent embryo are reduced. For example, errors in segregation during meiosis lead to aneuploidy, which is a major cause of miscarriages and birth defects. Therefore, understanding the signaling pathways can help identify the origin of the errors that result in aneuploidy and thus contribute to improving women's reproductive health. Key words: aneuploidy, spindle, in vitro fertilization, spindle assembly checkpoint, oocyte quality, chromosome segregation
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Optimization of digital polymerase chain reaction for application in non-invasive prenatal diagnostics
Šenkyřík, Pavel ; Korabečná, Marie (advisor) ; Vodička, Radek (referee)
Digital PCR (Polymerase Chain Reaction) is a method that enables absolute quantification of DNA sequences and therefore finds application in many diagnostic disciplines. We focused on the diagnosis of trisomy 21, which manifests itself as Down syndrome. Information about the fetal genome can be obtained from free fetal DNA that is released into the mother's bloodstream from the placenta during pregnancy. In the first trimester, fetal DNA constitutes approximately 5-10% of free DNA and is therefore a suitable target for non-invasive prenatal testing (NIPT). However, we need a technique that is able to distinguish this relatively small fraction of free fetal DNA and differentiate it from free maternal DNA. Precisely digital PCR provides various ways to achieve this goal thanks to its wide multiplexing possibilities. We focused on optimizing the multiplex reaction, which in one fluorescence channel distinguishes the number of copies of the reference chromosome 18 and the observed chromosome 21. The resulting determination of this ratio provides us with information regarding the balance between the chromosomes and can thus confirm or refute the presence of a trisomic fetal fraction. We tested the usability of the proposed method for clinical application by examining DNA samples isolated from 25 plasma...
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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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Molecular genetic characterization of the rare tumours of the urogenital tract.
Šteiner, Petr ; Vaněček, Tomáš (advisor) ; Španielová, Hana (referee)
The aim of this study was molecular characterization of four types of renal tumours (papillary renal cell carcinoma [PRCC], tubulocystic renal carcinoma [TCRC], pseudorossette forming renal carcinoma [PRRC] and unclassified renal carcinomas [URC]) and two types of rare tumours of the testes (Adult type of granulosa cell tumours [ATGCTs] and Incompletely differentiated sex cord stromal tumours [ISCSTs]). In case of TCRC the activity of signalling pathways involved in angiogenesis was studied. The aim was to determine the suitability of antiangiogenic agents for treatment of TCRC. Next, the methylation profile of 24 tumor suppressor genes was studied in TCRC and PRCC in order to analyze their similarity. Eventual differences could be helpful tool in differential diagnostics. Also, spectrum of chromosomal aberrations was analyzed by array-CGH in one case of PRRC and two cases of URC. Any unique aberration found would be useful in differential diagnostics of these tumors. Last, but not least, the specificity of mutation c.402C>G of FOXL2 gene for ovarian ATGCTs was verified by studying its occurrence in testicular ATGCTs and ISCSTs. Analysis of mRNA levels did not reveal any enhanced activity of the studied signalling pathways. Cluster analysis of methylation profiles showed close relationship between PRCC a...
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Analysis of chromosomal aberrations in sperm by fluorescence in situ hybridization
Bendová, Petra ; Diblík, Jan (advisor) ; Novotná, Drahuše (referee)
The presented bachelor work is focused on the determination of frequency chromosomally abnormal sperm in the semen of healthy men (donors) with normal karyotype (46, XY). The important process, which plays an irreplaceable role in the development of numerical aberrations of chromosomes or structural abnormalities in the segregation of the gametes, is meiosis. Therefore, I devote much attention on meiosis in the theoretical part. The theoretical part is focused on the process of pre mature sperm (spermatogenesis), and the consequences of fertilize the oocyte by aneuploid sperm. In my work I present an overview of numerical abnormalities in autosomes and gonosomes and their frequency and distribution of gametes in healthy men. I also focused on the distribution and a brief description of structural aberrations affecting chromosomes and not least I paid attention on method of multicolor interphase fluorescence in situ hybridization, which in combination with sperm chromatin dekondenzation become irreplaceable and valuable research tool for rapid analysis of chromosomal abnormalities in large sperm samples. The experimental part of bachelor work deals with monitoring the frequency of selected numerical abnormalities in sperm samples of five donors aged 23 to 30 years with the use of I-FISH (fluorescence in situ...
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Giardia intestinalis karyotypes
Hudosová, Lenka ; Nohýnková, Eva (advisor) ; Král, Jiří (referee)
Giardia intestinalis is a parasitic protist that causes one of the most common diarrheal disease of parasite origin. The cell of Giardia contains two nuclei with unknown number of chromosomes until recently. Karyotype was determined five years ago using conventional cytogenetic method by Tůmová and collaborators. In my work, I assessed karyotype of four isolates, six lines and three clonal lines by the same method. It was confirmed, that two nuclei within one cell could differ in chromosome number, the differences found were 1, 2 or 6 chromosomes. Aneuploid number of chromosomes was found too. In case that both nuclei within single cell contained the same number of chromosomes, there were 10 chromosomes indentified in each nucleus. It was also revealed, that karyotype is not specific feature for different genetic groups (in this work assemblages A and E). Karyotype can be different even among lines and clonal populations derived from the same isolate. Changes in karyotype in the course of in vitro cultivation were detected within three populations. Results are discussed in relation to known facts.
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The increased diagnostic efficiency of QF-PCR for aneuploidy of amniotic fluid
Sedláková, Zdeňka ; Macek, Milan (advisor) ; Daňková, Pavlína (referee)
Quantitative fluorescence polymerase chain reaction (QF-PCR) is a molecular genetic method based on the amplification of microsatellites (Short tandem repeats, STR) and measurement of the peak heights of amplicons in the electropherogram. Currently, the QF-PCR deemed reliable, fast, and inexpensive method that is gradually replacing conventional cytogenetic analysis of aneuploidy (examination of long-term cultures of amniotic fluid). However, in certain cases it is impossible to determine the parental origin and meiotic aneuploidy by QF-PCR. The aim of this work was to verify the new dinucleotide STR markers on chromozomes 13, 16, 18, 21, and 22 and further increase the diagnostic efficiency of QF-PCR retaining other STR markers on chromozome 15, 16, 22 and to determine the population and the analytical characteristics of these markers. For all dinucleotide STR markers stutter occurred in high frequency and therefore there were found not to be suitable for routine diagnostics. STR markers for chromozomes 15, 16 and 22 were tested on 100 patients. We selected four informative markers for both chromozome 16 and 22, and three markers for chromozome 15. Thus, I expanded set of diagnostic STR markers in this thesis.
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Development of new molecular genetic aproaches in assisted reproductive technology
Limbergová, Jana ; Machoň, Ondřej (advisor) ; Šimoník, Ondřej (referee)
Preimplantation genetic testing is one of the major clinical procedures that takes place in centres of assisted reproduction all around the world. It is sought out by couples for many reasons discussed in this paper. The causes of couples' arrival to the centres might vary from inherited diseases that future parents do not wish to transfer to their offspring to the risk of transfer of chromosomal structural rearrangements. What's more, infertility might be caused by the greater risk of aneuploidy in embryos which correlates with higher maternal age and other factors. This paper also focuses on new approaches in cytogenetic examinations for reproductive medicine. Furthermore, it discusses clinical methods used to improve diagnosis and treatment of infertile couples and experimental methods that could become the base of new diagnostic tools. These are for example single nucleotide polymorphism methods, array-based methods, new generation sequencing and whole genome amplifications. It also looks back at methods that became old-fashioned, just like for example fluorescent in situ hybridisation, and states its pros and cons. Key words: preimplantation genetic testing, aneuploidy, structural rearrangements, monogenic diseases, biopsy
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Correlation of morphokinetic parameters of human preimplantation embryos with their chromosomal status
Kosařová, Zuzana ; Jelínková, Ladislava (advisor) ; Knytl, Martin (referee)
A significant amount of human preimplantation embryos is aneuploid. Preimplantation genetic testing of aneuploidies (PGT-A) enables us to examine the number of chromosomes in a few trophectoderm cells biopsied from developing embryos, and only euploid embryos are then recommended to be transferred. Biopsy of trophectoderm cells is an invasive method, PGT is quite expensive, the examination is not performed at all patients. Therefore, non-invasive methods for determining the highest quality embryos are searching. Non- invasive continual monitoring in a time-lapse system is able to observe the morphokinetic parameters of embryonic development, and can select the best developing embryos with a good prognosis for successful implantation. However, there is still the question of whether and how morphokinetic parameters and embryonic ploidy correlate. Most studies on this topic show that a combination of both of these approaches is the best way for selecting the highest quality embryo for transfer.
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