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Chemical enucleation in mammalian oocytes
Černá, Martina ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The common techniques of enucleation are inexact and time-consuming. Nuclear DNA staining with Hoechst is frequently used, whereas the staining agent is toxic for the oocyte and has negative impact on its further development. Chemical enucleation is a method which facilitates the production of cytoplasts in higher amount and shorter time. Chemical enucleation has a potentional use in the field of biotechnology, especially in nuclear transfer methods (therapeutical, reproductive cloning). The chemical enucleation was successfully used on several model animals: mouse, sheep, cattle. The technique is beeing continually improved on pigs, regarding their similarity to human where it should be consequently applied. It was discovered that same chemicals have different effect on oocytes of each species and therefore it is necessary to examine each species separately. In our study we use porcine oocytes for chemical enucleation. We determined demekolcine in concentration 0,4 Ug/ml and acting time 30 minutes as the most suitable for chemical enucleation. Furthermore we used cytochalasin B in concetration 7,5 Ug/ml and acting time 10 minutes. The protrusion rate correlates with quality of oocytes. In other experiment we focused on distribution of mitochondria after fusion of normal and enucleation oocyte. We found...
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Effect of the sperm ubiquitination in the early embryonic development in pig
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The intracellular sperm injection (ICSI) technique is a very effective tool for the fertilization research. In the newly established laboratory at the Faculty of Science of the Charles University it was necessary to introduce this method and define the early developmental potential of fertilized oocytes. After fertilization oocytes were incubated to the blastocyst stage with a success comparable with other laboratories (17%). The ubiquitin-proteasome system which plays a major role in a protein degradation within cells is involved in a regulatory mechanism of sperm maturation. It is also responsible for a penetration of a vitelline membrane. In these processes ubiquitin residues are localized extracellulary. High level of sperm ubiquitination correlates with their low quality. Hypotetically it can be expected that the ubiqutination of impaired sperm cells can be used as a negative marker for their recognition and degradation by 26S proteasome complex localized. Experiments in this diploma thesis were designed based on the hypothesis that the executive part of the selective mechanism is the 26S proteasome. Therefore the effect of MG132 peptide inhibition of the 20S proteasome on the pronuclei formation and subsequent early embryonic development after ICSI was studied. Inhibition of 20S proteasome...
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Expressing Causation in the Czech and Spanish Language
Petr, Jaroslav ; Čermák, Petr (advisor) ; Zavadil, Bohumil (referee)
Expressing Causation in Czech and Spanish Language The subject of this thesis, as is evident from the title, consists in expression of causation in Spanish and its equivalents in the Czech language. We dealt with the issue of the causation in both formal and practical perspective. As a basic material, we have used not only Spanish, but also Czech philological publications. We based particularly on the article written by Petr Čermák and Pavel Štichauer called Španělské a italské faktitivní konstrukce hacer/fare + sloveso a jejich české ekvivalenty, as well as from selected chapters of Gramática descriptiva de la lengua española written by different authors. The work is divided into a theoretical part (Chapters 1-4) and a practical part (5-6). In introduction, we set ourselves the goals, we have outlined the research methodology using a parallel corpus InterCorp, which we described in detail. At the theoretical level, we discussed the question of the translation direction and its effect on our research. Furthermore, we defined the terms of causation, causative verb and factitiveness, factitive verb, to be able to continue working with it. We have commented the form of the causative verbs in Czech and their most distinctive features, including a test for distinguishing causative verbs from non-causative verbs....
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Recognition of expressed double-stranded RNAs in mammalian cells
Vaškovičová, Michaela ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
Long double-stranded RNA (dsRNA) is a unique structure formed during viral replication or transcription of repetitive elements. Mammalian cells evolved several mechanisms how to respond to dsRNA. dsRNA can be engaged in one of three pathways: interferon response, RNA editing, and RNA interference (RNAi). RNAi is evolutionary conserved effect of dsRNA, which results in sequence-specific messenger RNA degradation. However, in mammals, RNAi is functional only in mouse oocytes, which express truncated version of Dicer (DicerO ). In somatic cells, dsRNA triggers sequence-independent interferon pathway. The main aim of this Master's thesis was to examine how specific double-stranded RNA-binding proteins (DRBPs) influence distribution of long dsRNA into RNAi and sequence-independent pathways. We used a luciferase-based reporter RNAi assay to monitor sequence-specific and sequence-independent effects of dsRNA co-expressed with selected DRBPs. Our results suggest that none of the tested DRBPs is sufficient to stimulate RNAi in somatic cells. Interestingly, the overexpression of either TARBP2 or PACT suppressed RNAi in cells expressing DicerO . Moreover, microRNA pathway, which employs the same protein factors as RNAi, is not inhibited by TARBP2 or PACT. Therefore, we propose that DRBPs overexpression...
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Sperm acrosomal reactien in selected species of mammals
Frolíková, Michaela ; Stopka, Pavel (advisor) ; Jonáková, Věra (referee) ; Petr, Jaroslav (referee)
Mammalian sperm must undergo the process of capacitation - series of physiological and biochemical modifications prior fertilization. In last stage of capacitation sperm undergoes acrosome reaction (AR). During AR the cell membrane of the sperm fuses with the outer acrosomal membrane and the contents of acrosomal vesicle are released into extracellular space. Sperm which did not undergo AR or sperm missing acrosome at all are unable to fertilize. AR results into dramatical changes in the sperm head. Most of the proteins present in plasmatic and outer acrosomal membrane are reorganized or lost. There are also significant changes in cytoskeletal and intraacrosomal proteins are released to extracellular space uncovering new surface domains. Some sperms undergo AR even without presence of inductor of AR during capacitation in vitro. This event is called spontaneous (accelerated) AR. The latest research indicates that spontaneous AR is natural part of the process of fertilization. Field mice (Apodemus) show high level of promiscuity leading to significant risk of sperm competition. Unique reproduction strategy where the sperms form so-called sperm trains was evolved in field mice. Spontaneous AR is probably enabling the dissociation of sperms from the sperm train. The spontaneous AR rate is dependent on...
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Retroviral infection of chicken testicular cells in a process of construction of transgenic poultry
Kalina, Jiří ; Škvor, Jiří (advisor) ; Petr, Jaroslav (referee) ; Jílek, František (referee)
Biotechnological research in chicken transgenesis is still lagging beyond the mammals mainly due to the specificities of avian reproductive system. This thesis is trying to offer functionally complex approach to the chciken transgenesis through one of the techniques. Common transfection techniques were applied on chicken blastodermal and testicular cells to affirm this approach. Our original techniqe of sterilization of chicken testes was improved and applied. Stained testicular cells of donor male were transplanted into sterilized acceptors testes and subseqent tubuli recolonization and sperm production were described. The retroviral- based vector was developed and transplanted testicular cells were successfuly infected. Carried marker transgene (Green fluorescence protein, GFP) was detected in DNA of produced sperms and no significant CpG methylation was detected when sreening infected cells. Through the flow cytometry, testicular cells used for transplantation were analyzed. All major spermatogonia-derived populations were described including the side population (SP), possible group of spermatogonial stem cells.
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Transgenic RNAi in mouse oocytes
Sarnová, Lenka ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
RNA interference (RNAi) is double-stranded RNA (dsRNA)-mediated mRNA degradation. RNAi has been widely used to investigate gene functions. Many methods to induce transient or stable RNAi have been developed. Transient RNAi can be induced by delivering of a long dsRNA or short interfering RNAs (siRNAs). Stable RNAi may be induced by introducing plasmids expressing a long or a short hairpin RNA. Both small and long RNAs have been used to induce transient RNAi in mouse oocytes. Nevertheless, only long hairpin-expressing system has been used to trigger stable RNAi in oocytes. Although, this system appeared to be highly efficient and specific, it has several disadvantages as complicated long inverted repeat cloning or limited possibility to test these vectors in the cell culture. Here, we constructed a short hairpin-expressing vector suitable for transgenic RNAi induction in mouse oocytes. The new vector, pTMP_ZP3_sh, was derived from a lentiviral short hairpin vector selected based on comparative study of different short hairpin-expressing plasmids. The pTMP_ZP3_sh vector was tested by targeting Moloney sarcoma oncogene (Mos) mRNA, which is a common model for RNAi in mouse oocytes. We designed several candidate short hairpin sequences and tested their efficacy. Subsequently, the most efficient one was selected...
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