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Testicular Degeneration of Transgenic Porcine Model of Huntington's Disease
Skřivánková, Monika ; Motlík, Jan (advisor) ; Roth, Jan (referee) ; Petr, Jaroslav (referee)
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by an extended (≥36) CAG repeat in the huntingtin gene. Its hallmark is brain athrophy, but huntingtin is widely deposited in all tissues of the body, most notably in the brain and testes. Its pathogenic effect is conditioned by the formation of cytotoxic forms of aggregates and fragments, which occur in both brain and peripheral tissues. Testicular atrophy has been demonstrated in postmortem samples from human patients with Huntington's disease and in transgenic mouse models. We investigated reproductive decline in a large animal model of Huntington's disease. A transgenic (tgHD) minipig model was created by inserting a lentiviral vector into the genome of a pig. Vector contained a truncated form of the N terminal part of huntingtin gene. Boars of this transgenic line showed a reduced ability to produce offspring from 13 months of age. We confirmed apoptosis of seminiferous epithelial cells and Sertoli cells, and a production of morphologically damaged spermatozoa, which were unable to efficiently fertilize the oocyte under in vitro conditions. We found a reduction of mitochondrial metabolism parameters in the sperm of tgHD boars. These changes were not dependent on the age of the boars., It is directly related to the...
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RNA interference in mouse oocytes and somatic cells
Táborská, Eliška ; Svoboda, Petr (advisor) ; O´Connell, Mary Anne (referee) ; Petr, Jaroslav (referee)
RNA interference (RNAi) is a pathway, which employs Dicer to process long double stranded RNAs (dsRNA) from endogenous or exogenous sources into short interfering RNAs (siRNA). siRNAs are loaded onto Argonaute proteins to mediate sequence-specific post-transcriptional RNA targeting resulting in regulation of protein-coding genes and retrotransposons or antiviral immune response. Another small RNA pathway - PIWI-associated RNA (piRNA) pathway is suppressing retrotransposons in the germline. In mice, canonical RNAi pathway activity is negligible in somatic cells where a full-length Dicer produces gene-regulatory microRNAs (miRNA) but RNAi is highly active in oocytes, which express a truncated oocyte-specific Dicer isoform (DicerO ). DicerO lacks an N-terminal DExD helicase domain and has higher cleavage activity of long dsRNAs. Deletion of oocyte specific DicerO promoter leads to transcriptome aberrations, which include upregulation of putative RNAi targets and MT retrotransposons and, consequently, to meiotic spindle defects and female sterility. In contrast, the piRNA pathway is non-essential in mouse oocytes, potentially because of overlapping functions of RNAi. The PhD thesis aims to understand biological significance of mammalian endogenous RNAi and to explore consequences of re-activated RNAi...
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Bisphenol S influence on selected markers of meiotic maturation of porcine oocytes
Černíková, Terezie ; Petr, Jaroslav (advisor) ; Krylov, Vladimír (referee)
Bisphenol A is a widely used chemical in the manufacture of plastics. The presence of BPA in the environment adversely affects human health due to contamination of air, drinking water and food. Growing concerns about the effects of BPA have led to its regulation in production and development of alternative chemicals to BPA, such as bisphenol S (BPS). However, the effects of BPS were not properly tested before its introduction to production and the effects on human reproduction are still unknown. For this reason, it is desirable to test the effect of BPS on mammalian oocyte development. This study hypothesizes that BPS exposure causes inhibition of meiotic maturation of porcine oocytes in vitro. This study aims to investigate the potency of BPS at low concentrations corresponding to normal human exposures to selected porcine oocyte proteins. The results of this study demonstrate the negative effect of BPS on the progression of meiotic maturation and reaching the mature oocyte stage. In addition, the results show an increase in the formation of defective meiotic spindles and a disruption of mitochondrial integrity after exposure to BPS concentrations. However, the effect of BPS on double-strand breaks was not demonstrated in this study, in contrast to the case of BPA. Taken together, the results show...
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The development of tourism potential in Ghana using social media as a marketing tool
Petr, Jaroslav
The aim of my bachelor thesis is to analyze the development of tourism using social media as a marketing tool. The first part, the literature review, deals with the terms of world tourism, tourism in Africa, tourism in Ghana and types of social media. The second practical part deals with the current situation in which social media help in the development of tourism and which are most suitable for presentation. The practical part is based on the research of states, more specifically cities, which increased the share of tourism thanks to marketing on social networks. The practical part also includes a research questionnaire on local tourism in Ghana. The conclusion and recommendations that Ghana or the agency that represents them on social networks should be implemented based on research and analysis.
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Mechanism and risk factors for the development of monozygotic twins in mammals
Žabková, Světlana ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
Monozygotic twins result from the splitting of one embryo in early embryonic development. The developmental stage, in which the splitting occurs, is the main factor determining the degree of sharing fetal sacs. The etiology and mechanism of monozygotic twinning are still unknown. The human and two species of armadillo, Dasypus novemcinctus and Dasypus hybridus are the only mammals that regularly produce monozygotic multiple pregnancies. The spontaneous occurrence of monozygotic twinning is 0,45 % of all births. The monozygotic twin pregnancies have been reported to occur at a higher rate following the increasing interest of assisted reproduction technologies. In certain consideration, we understand them as a side effect of infertility treatment because monozygotic twins result in a higher rate of prenatal mortality, premature birth and congenital anomalies than singleton pregnancies. This bachelor thesis aims to summarize knowledge about the formation of monozygotic twins in mammals. Also, it tries to discuss potential mechanisms and risk factors which could influence their formation. Monozygotic twins are an important model for explaining the genetic predisposition of some diseases. The thesis also introduces the ways of experimental production of monozygotic twins. Keywords: monozygotic twins,...
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Effect of endocrine disruptor bisphenol S on human health and reproduction
Procházková, Bára ; Petr, Jaroslav (advisor) ; Děd, Lukáš (referee)
Bisphenol S (BPS) is an environmental pollutant that has replaced bisphenol A (BPA) in plastic and paper products since 2011 as a safer "BPA-free" alternative. However, due to its rapid replacement, its potential adverse effects have not been investigated in detail, and due to its structural similarity to BPA, many studies suggest that it acts, like BPA, as an endocrine disruptor. Its use is wide and BPS is globally widespread, found in the environment and has been detected in both animal and human tissues. Nevertheless, an overall effect of BPS on an organism remains unclear. This work is focusing on gathering sources for its effect on reproductive ability and its association with metabolic disorders.
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The Generation of Transgenic Huntington's Disease Miniature Pig
Baxa, Monika ; Motlík, Jan (advisor) ; Procházka, Jan (referee) ; Petr, Jaroslav (referee)
Huntingtons's disease (HD) is devastating neurodegenerative disorder manifesting by motor disturbances, cognitive decline and personal changes. The huge effort to find a cure for HD has brought several promising therapeutic treatments on the scene. Each of the prospective approaches needs to be investigated for safety, tolerability and efficacy. Mouse and rat models were a lot helpful in examination of pathological mechanisms of HD, but they are not sufficient for completion of pre-clinical testing. Therefore, we aimed to generate transgenic HD minipig to overcome the gap between rodents and humans. Minipig transgenic for the first 548 aminoacids of human mutant huntingtin gene (TgHD) under the control of human HD promotor was manipulated by lentiviral transduction of porcine one-cell stage embryos. Currently, six generations of minipigs expressing single copy of N-truncated human mutant huntingtin protein (mtHtt) with a repetition of 124 glutamines are at disposal. The more the model simulates the disease symptoms the better it is for translational research as the efficacy of the cure can be finer evaluated. Hence, the second aim was to demonstrate HD-like phenotype in our model. Testicular degeneration that preceded the clinical symptoms onset was observed as a consequence of expression of mtHtt....
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Chemical communication of gametes
Otčenášková, Tereza ; Komrsková, Kateřina (advisor) ; Petr, Jaroslav (referee)
Fertilization is a multiple step process leading to fusion of female and male gametes resulting in a formation of a zygote. Besides direct gamete interaction via binding receptors localized on both oocyte and sperm surface, fertilization also involves communication based on chemical molecules triggering various signalling pathways. This work is aimed to characterize chemical communication of gametes of a model organism Mus musculus. For this purpose, modern proteomic and visualisation methods like nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS), selected reaction monitoring (SRM) and immunofluorescent microscopy were used. Lipocalins were identified as candidate proteins involved in communication including those from major urinary proteins (MUPs), LCN lipocalins and fatty acid binding proteins (FABPs). For the first time, we report their presence in the sperm acrosome. Based on lipocalins capacity to bind and transport other molecules, we propose that these proteins have a protective and/or signalling role for gametes. Furthermore, chemical communication between sperm and oocyte is based on chemotaxis which enables their interaction before their fusion. In this work, we detected that spermatozoa show chemotactic responses in the presence of L-glutamate. This amino acid naturally...
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Role and regulation of nuclear membrane during meiotic maturation of mammalian oocyte
Končická, Markéta ; Kubelka, Michal (advisor) ; Petr, Jaroslav (referee) ; Binarová, Pavla (referee)
Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...
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Creating a biosensor for miRNA effector complex formation using CRISPR nucleases
Petržílek, Jan ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
miRNAs are small regulatory RNAs, which function as post-transcriptional mRNA regulators. They direct ribonucleoprotein complexes to cognate mRNA to repress them by translational inhibition and degradation. miRNAs regulate thousands of mRNAs in mammals and have been recognized as regulatory factors in most cellular and developmental processes. Dysregulation of the miRNA pathway can lead to severe defects and diseases. Interestingly, a unique situation exists in mouse oocytes, where all the miRNA pathway components are present, yet the pathway is dispensable and nonfunctional, the molecular foundation of this phenomenon and its significance still remain unclear. In spite of the pronounced effects of the miRNA pathway in gene regulation in somatic cells, study strategies of the pathway bare limitations. Current methods for studying the activity of the miRNA pathway employ corelative studies (such as NGS) or reporter assays, which have relatively low throughput and are prone to artifacts. Here, I present design and development of a new strategy for directly monitor global miRNA pathway activity and integrity in near physiological conditions in living cells, which could also be employed in vivo for studies of mouse oocytes. The strategy is based on fluorescently tagged endogenous proteins of the...
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