|
|
Preparation of X. tropicalis recombinant growth factors and their characterization in testicular tissue culture.
Borecká, Marianna ; Krylov, Vladimír (advisor) ; Drobná Krejčí, Eliška (referee)
In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells
|
|
|
WASH complex and its interactome in human pathology
Pácalt, Ondřej ; Libusová, Lenka (advisor) ; Drobná Krejčí, Eliška (referee)
Efficient transport of cargo to its correct destination is required for the proper functioning of eukaryotic cells. Vesicular trafficking is one of the important means of intracellular transport. Impairment of this process often leads to serious pathologies. Sorting and recycling is the crucial part of vesicular trafficking as it enhances its efficiency. The WASH complex has a key role in the regulation of branched actin patches formation. If this occurs on the membrane of endosomes, then it affects sorting, recycling and cargo trafficking. Mutations in the WASH complex or its interacting partners cause diseases such as hereditary spastic paraplegia, Parkinson disease or light intellectual disability. Despite certain advance in the understanding of above-mentioned pathologies, mechanism of the pathogenesis is still elusive. Research in this field can reveal basic molecular mechanisms responsible for the complexity of cargo sorting, recycling and trafficking and thus provide better opportunities for treatment of affected individuals.
|
|
|
Preparation of Xenopus tropicalis transgenic testicular stem cell culture.
Vegrichtová, Markéta ; Tlapáková, Tereza (advisor) ; Drobná Krejčí, Eliška (referee)
Testicular stem cells (TSCs) are relatively accessible potential source of pluripotent cells, which are particularly important for their application in regenerative medicine. Xenopus tropicalis is a useful model organism to study the migration and differentiation potential of stem cells. This amphibian is characteristic by outer fecundation and embryonic development of a great amount of embryos after fertilization. Oocytes and embryos are large enough (about 1 mm) to be suitable for micromanipulation micromanipulations. Laboratory of Developmental Biology, Faculty of Science, Charles University in Prague succeeded in the establishment of a mixed cell culture of TSCs growing on feeder layer of pre- Sertoli cells. This culture was derived from the testes of juvenile Xenopus tropicalis male. In the study of their differentiation potential it was found, that leukemia inhibitory factor (LIF) is the decisive factor allowing rapid proliferation of stem cells and their forming into characteristic colonies. This protein is produced by both types of cells which are present in the culture. The mouse LIF has the same positive effect on the proliferative potential of stem cells, which points at the evolutionary conservation of metabolic pathways associated with the maintenance of the stemness. RT-PCR analysis...
|
|
|
WASH complex and its interactome in human pathology
Pácalt, Ondřej ; Libusová, Lenka (advisor) ; Drobná Krejčí, Eliška (referee)
Efficient transport of cargo to its correct destination is required for the proper functioning of eukaryotic cells. Vesicular trafficking is one of the important means of intracellular transport. Impairment of this process often leads to serious pathologies. Sorting and recycling is the crucial part of vesicular trafficking as it enhances its efficiency. The WASH complex has a key role in the regulation of branched actin patches formation. If this occurs on the membrane of endosomes, then it affects sorting, recycling and cargo trafficking. Mutations in the WASH complex or its interacting partners cause diseases such as hereditary spastic paraplegia, Parkinson disease or light intellectual disability. Despite certain advance in the understanding of above-mentioned pathologies, mechanism of the pathogenesis is still elusive. Research in this field can reveal basic molecular mechanisms responsible for the complexity of cargo sorting, recycling and trafficking and thus provide better opportunities for treatment of affected individuals.
|
|
|
Preparation of Xenopus tropicalis transgenic testicular stem cell culture.
Vegrichtová, Markéta ; Tlapáková, Tereza (advisor) ; Drobná Krejčí, Eliška (referee)
Testicular stem cells (TSCs) are relatively accessible potential source of pluripotent cells, which are particularly important for their application in regenerative medicine. Xenopus tropicalis is a useful model organism to study the migration and differentiation potential of stem cells. This amphibian is characteristic by outer fecundation and embryonic development of a great amount of embryos after fertilization. Oocytes and embryos are large enough (about 1 mm) to be suitable for micromanipulation micromanipulations. Laboratory of Developmental Biology, Faculty of Science, Charles University in Prague succeeded in the establishment of a mixed cell culture of TSCs growing on feeder layer of pre- Sertoli cells. This culture was derived from the testes of juvenile Xenopus tropicalis male. In the study of their differentiation potential it was found, that leukemia inhibitory factor (LIF) is the decisive factor allowing rapid proliferation of stem cells and their forming into characteristic colonies. This protein is produced by both types of cells which are present in the culture. The mouse LIF has the same positive effect on the proliferative potential of stem cells, which points at the evolutionary conservation of metabolic pathways associated with the maintenance of the stemness. RT-PCR analysis...
|
|
|
Comparison of migration and morphogenesis of neural crest cells in Ray-finned fishes: towards identification of developmental sources of craniofacial diversity
Štundl, Jan ; Černý, Robert (advisor) ; Drobná Krejčí, Eliška (referee)
Extensively migrating population of neural crest cells, which contributes to many tissues and builds up most of craniofacial vertebrate structures, has a crucial role in embryonic development of vertebrate body. The migratory pathways of neural crest cells are thought to be very conserved throughout the vertebrates and cranial neural crest migration is defined by progression of three migratory streams: trigeminal, hyoid and a common branchial stream. In this diploma thesis, migration of cranial neural crest was analysed using embryos of the Senegal bichir (Polypterus senegalus) and of sterlet (Acipenser ruthenus), which represent two basal-most lineages of extant ray-finned fishes. A combination of several techniques was used in both species in order to study cranial neural crest cells from their sites of origin to post- migratory stages and the pattern of migration was compared and discussed in revealed embryonic context. In the Senegal bichir the hyoid neural crest stream was shown to migrate first and it is also the most abundant; this heterochrony shift is apparently related to formation of external gills, which in bichir are situated on the hyoid arch only. In sterlet, neural crest cells migrate in a classic pattern of three progressive streams but their dynamics and patterning is influenced by...
|
|
|
Preparation of X. tropicalis recombinant growth factors and their characterization in testicular tissue culture.
Borecká, Marianna ; Krylov, Vladimír (advisor) ; Drobná Krejčí, Eliška (referee)
In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells
|
guest ::
