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Cellular Reprogramming as a Tool for Harvesting Patient-specific Stem Cells
Pisal, Rishikaysh ; Mokrý, Jaroslav (advisor) ; Hampl, Aleš (referee) ; Jendelová, Pavla (referee)
Cellular reprogramming as a tool for harvesting patient specific stem cells In the year 2006, Dr. Yamanaka surprised the entire field of medicine, by reporting a technique of inducing pluripotency in somatic cells. In his article, he had displayed that fibroblasts could be reprogrammed to pluripotent stem cell state, by ectopic expression of four transcription factors namely OCT4, SOX2, c-MYC and KLF4. His discovery made a paradigm shift in the field of reprogramming because previous methods of reprogramming were dependent on use of human oocytes and this raised ethical concerns. Moreover, his technique of cellular reprograming broadened the spectrum of application of somatic cells in regenerative medicine. Objectives of my research were focused on; development of an optimised protocol for detection of mycoplasma that commonly infects animal tissue culture; detailed characterization of reprogrammed clones; targeted differentiation of iPSC towards myogenic lineage, and construction of an expression vector, optimised for miRNA expression. For detecting mycoplasma infection, we adapted the protocol of Uphoff et al. (2002). By skipping the DNA extraction step (reported in the original protocol) and instead directly using cell culture supernatant and a robust polymerase enzyme for performing PCR, we...
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Manipulating the mammalian oocyte and embryo - Biological and epigenetic aspects
Fulková, Helena ; Hozák, Pavel (advisor) ; Hampl, Aleš (referee) ; Motlík, Jan (referee)
CONCLUSIONS . By antibodies against ďfferent covalent histone modifications and 5-methylcytosine, we have partialty characterised the epigenetic changes taking place during the oocyte mauration and in early mammalian embryogenesis in the mouse and pig, respectively. o We have also characterised thc epigenetic repogramming activities of cytoplasts derived from oocytes at different stages of maturation after somatic cell nuclear transfer. . We have evaluated the epigenetic effec$ of selected procedures that are currently used for embryo production. . Finally, we have developed a new cryopreservation scheme for oocyte nuclear material storage. orrr research is engaged in the development ofnew bíotechniques as well as elucidating and characterising the epigenetic pÍocesses that take place during normal and abnormal embryogenesis. Abnormal embryonic development is for example often observed in somatic cell nuclear transfer embryos. These techniques can also be potentially used not only in human medicine but also for valuable livestock and endangered species preservation Oy e.g. interspecies nuclear transfer). Especially in human meďcine, attention to the ethical issues associated with these techniques must be paid. It is also clear tbat many biological problems still do exist and these should not be...
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Utilization of biocompatible hydrogels based on 2-hydroxyethylmethacrylate to bridge the spinal cord lesion
Lesný, Petr ; Syková, Eva (advisor) ; Rokyta, Richard (referee) ; Hampl, Aleš (referee)
Utilization of biocompatible hydrogels based on 2-hydroxyethylmethacrylate to bridge the spinal cord lesion MUDr. Petr Lesný Biocompatible hydrogels based on copolymers of 2-hydroxyethylmethacrylate are suitable for implantation into a central nervous tissue lesion. Our prepared hydrogels were macroporous with predominantly communicating pores, and their mechanical properties were similar to those of spinal cord tissue. After we confirmed their biocompatibility by the growth of mesenchymal stem cells and determined the hydrogel diffusion properties, we implanted the prepared hydrogels into acute spinal cord injury in laboratory rats and studied the process of spinal cord tissue regeneration within the implants. When the hydrogels were implanted in subacute phase of spinal cord injury, we observed favorable impact on the spinal cord tissue regeneration and reduction of pseudocyst formation. Using histological and immunohistochemical methods, we observed the ingrowth of central nervous tissue elements (connective tissue, vessels, neurofilaments, Schwann cells, astrocyte processes) into the hydrogels implanted into the spinal cord hemisections. We have found that copolymerization with a positively charged ammonium salt increases the adhesion of the cells to the hydrogels in vitro and the ingrowth of...
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The application of functional tests to measure DNA repair capacity in molecular epidemiological studies
Slyšková, Jana ; Vodička, Pavel (advisor) ; Hampl, Aleš (referee) ; Kment, Milan (referee)
DNA repair is a vital process of a living organism. Inherited or acquired defects in DNA repair systems and cellular surveillance mechanisms are expected to be important, if not crucial factors in the development of human cancers. DNA repair is a multigene and multifactorial process which is most comprehensively characterized by the phenotypic evaluation of DNA repair capacity (DRC). DRC represents a complex marker with high informative value, as it comprises all genetic, epigenetic and non-genetic factors, by which it is modulated. Accordingly, DRC reflects the actual capability of the cell, tissue or organism to protect its DNA integrity. The present PhD study was focused on investigating DRC, which specifically involves base and nucleotide excision repair pathways, in human populations with different characteristics. The main aim was to answer substantial questions on the possible use of DRC as biomarkers in epidemiological studies. The study was in fact designed to understand the extent of physiological variability of DRC in a population, its modulation by genetic and non-genetic factors, tentative adaptability to high genotoxic stress and, finally, its involvement in cancer aetiology. In order to explore these issues, DRC, in respect to genetic and environmental variability, was investigated...
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Messenger RNA stability and microRNA activity in mouse oocytes
Flemr, Matyáš ; Svoboda, Petr (advisor) ; Motlík, Jan (referee) ; Hampl, Aleš (referee)
The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...
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New markers of colon cancer evolution in ulcerative colitis
Švec, Jiří ; Kment, Milan (advisor) ; Keil, Radan (referee) ; Hampl, Aleš (referee)
Background: Long-standing ulcerative colitis (UC) has an increased risk of evolving into colorectal cancer (CRC) and upregulated expression of cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), survivin, telomerase catalytic subunit (TERT), integrin-linked kinase (ILK) and transcription factors c- MYB and TCF-4, has been implicated in the development and progression of sporadic colorectal cancer. Nevertheless much less is known about their role in the process of UC-associated colon carcinogenesis. Methods: We analyzed the gene expression of these markers during the transition of colonic mucosa from chronic inflammation to epithelial neoplasia in biopsies of UC patients using quantitative real-time polymerase chain reaction and immunohistochemistry, and compared the expression profiles of this gene panel in samples of patients with CRC and in human tumor xenografts of SW620 malignant colonic cells. Additionally, we determined the expression of these genes in mouse models of sporadic and colitis-associated CRC in A/J and ICR mouse strains using quantitative RT-PCR and laser microdissection. Results: The transcript levels of survivin, c-MYB, COX-2, iNOS, and TCF-4 showed statistically significant increase during neoplastic transformation of UC-patient colonic mucosa, whereas TERT and ILK...
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Differentiation of Human Embryonic Stem Cells into Endothelial and Smooth Muscle Cells as a Model for Vascular Development
Obrtlíková, Petra ; Trněný, Marek (advisor) ; Mokrý, Jaroslav (referee) ; Hampl, Aleš (referee)
Aims of the study: We hypothesized that the optimal source of cell for vascular regeneration will be the progenitor cells derived from human embryonic stem cells (ESCs) which can differentiate both into endothelial cells (ECs) as well as vascular smooth muscle cells (SMCs). We propose to test if the population of human ESCs, H9 cell line, can serve this role. Material and methods: Human ESCs were cocultured with stromal cells S17, M2-10B4 or Wnt1 expressing M2-10B4 cell line to generate a CD34+ cell population. After that, CD34+ cells were sorted and cultured in media containing specific cytokines to generate ECs. To induce SMC differentiation from ECs, culture conditions were changed to media containing platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-b1). Phenotypic and functional characteristics of these populations were demonstrated by flow cytometry, immunohistochemistry, QRT- PCR, tube formation assay, and response to calcium signaling agonists. Results: CD34+ vascular progenitor cells derived from human ESCs give rise to ECs and SMCs. These two populations express cell specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when cocultured in Matrigel. Wnt1 overexpressing stromal cells...
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Molecular mechanisms regulating participation of neural crest on developmental processes and homeostasis in selected localizations
Krejčí, Eliška ; Grim, Miloš (advisor) ; Hampl, Aleš (referee) ; Jendelová, Pavla (referee)
This PhD thesis focuses on the study of neural crest (NC) properties and mechanisms regulating its development. It is based on four original papers, two of which are wholy concerning NC. In the first project we showed that transcription factor C-MYB is expressed in majority of cells in the developing chick embryo. Its expression level is elevated in neural folds and migrating NC cells. Other experiments showed that this elevated level of intracellular c-Myb influences induction and epithelial-mesenchymal transformation of NC cells, and that c-Myb lies within the BMP4 signalling cascade. In the second project we explored the spatio-temporal specification along the antero-posterior axis of the limb bud - a mechanism significantly regulating also NC development. We showed that deletion of regulatory element of Plzf transcription repressor gene caused decrease of Plzf expression in the limb buds followed by anterior expansion of its downstream genes (Hoxd10-13) expression domains only in the hind limb, with a resulting additional finger. The third paper studies developmental origin of medial border of mammalian scapula. We showed that its cells originate from somites and not in NC. In the last project we studied NC stem cells isolated from human adult hair follicles. These cells express NC stem cell...
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Primary Cilia and its Importance in Cell Response to Ionizing Radiation Exposure and Chemotherapy Drugs.
Filipová, Alžběta ; Řezáčová, Martina (advisor) ; Hampl, Aleš (referee) ; Hofer, Michal (referee)
Primary cilia and its importance in cell response to ionizing radiation exposure and chemotherapy drugs. Ionizing radiation, metabolic and genotoxic stress affect the cytoskeletal stability and morphology of cells by causing DNA damage in the form of single or double stranded breaks, the latter being the most critical. When DNA damage occurs the cell cycle becomes arrested and the repair machinery is engaged; however, if this damage is not repaired the cell enters programmed cell death by apoptosis. Primary cilia have been shown to act as physical- chemical sensors and their biological functions include the perception of the extracellular milieu, the regulation of organogenesis and cell polarity, as such, they are dynamically regulated during cell cycle progression. Further, the impairment or loss of primary cilia leads to the development of ciliopathies and other diseases including cardiovascular disorders, arthritis and, ultimately, cancer. Moreover, the presence and temporary formation of primary cilia is essential for the repair process of certain cell types. This work is focused on the evaluation of primary cilia changes caused by ionizing radiation, metabolic stress or cytostatic drugs (taxol and doxorubicin) in vitro. Our results showed a significantly higher number of ciliated C2C12 cells observed...
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