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Stimulation of pancreatic β-cell proliferation by synthetic mRNA
Volejníková, Anna ; Hodek, Petr (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a severe metabolic disease associated either with loss or functional impairment of insulin-producing β-cells. A major goal in diabetes research is to develop effective ways of pancreatic β-cells restoration. One of the possible approaches is the stimulation of β-cell proliferation. Transcription factor c-Myc is one of the promising targets for stimulation of β-cell proliferation. However, the adenovirus-mediated long-term overexpression of this oncoprotein is associated with risk of cellular dedifferentiation, apoptosis and development of cancer. Therefore, the applicability of an alternative approach for the c-Myc ectopic expression, based on the in-vitro transcribed synthetic mRNA, was verified in this thesis. Use of in vitro transcribed c-Myc-mRNA allows achieving short-term overexpression of transcription factor c-Myc in host cells. High level of c-Myc expression by the β-cells, was detected following the transfection with c-Myc-mRNA. However, within the 48 hours the expression level of c-Myc protein declined to the basic level. The physiological degradation of c-Myc-mRNA by the host cells is a key factor that reduces the risk of cancer development. Transfection of rat islet cells with c-Myc-mRNA resulted in a significantly increased β-cell proliferation. Under the...
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Stimulation of pancreatic β-cell proliferation by synthetic mRNA
Volejníková, Anna ; Hodek, Petr (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a severe metabolic disease associated either with loss or functional impairment of insulin-producing β-cells. A major goal in diabetes research is to develop effective ways of pancreatic β-cells restoration. One of the possible approaches is the stimulation of β-cell proliferation. Transcription factor c-Myc is one of the promising targets for stimulation of β-cell proliferation. However, the adenovirus-mediated long-term overexpression of this oncoprotein is associated with risk of cellular dedifferentiation, apoptosis and development of cancer. Therefore, the applicability of an alternative approach for the c-Myc ectopic expression, based on the in-vitro transcribed synthetic mRNA, was verified in this thesis. Use of in vitro transcribed c-Myc-mRNA allows achieving short-term overexpression of transcription factor c-Myc in host cells. High level of c-Myc expression by the β-cells, was detected following the transfection with c-Myc-mRNA. However, within the 48 hours the expression level of c-Myc protein declined to the basic level. The physiological degradation of c-Myc-mRNA by the host cells is a key factor that reduces the risk of cancer development. Transfection of rat islet cells with c-Myc-mRNA resulted in a significantly increased β-cell proliferation. Under the...
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Development of a model system to study adhesion of Burkholderia cenocepacia to epithelial lung cells of patients with cystic fibrosis
Volejníková, Anna ; Bořek Dohalská, Lucie (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF) is a common autosomal recessive disorder that primarily affects epithelial cells in respiratory system. As a consequence of lung damage the patients are chronically colonized by a number of pathogenic microorganisms such as Pseudomonas aeruginosa or Burkholderia cepacia komplex. Due to the seriousness of these bacterial infection and its primary resistance to commonly used antibiotics the suitable therapeutic alternatives are being sought. The passive immunization with yolk antibodies seems to be a suitable alternative. This thesis studied the bacterial adhesion of Burkholderia cepacia, the opportunistic human pathogen, to lung epithelial cells of healthy individuals and patients with CF, respectively. The aim of this study was to develop a model system suitable for this research. Two types of immortalised cell lines have been used for this purpose. CuFi-1 is the cell line derived from patient with CF caused by ∆F508 mutation , NuLi-1 is a healthy epithelial cell line. The strains of B. cenocepacia (ST28 a ST32) were fluorescently labeled with fluorescent cell linker PKH26. Furthermore, the way of fluorescent labeling of cell lines CuFi-1 and NuLi-1 using PKH67 dye was optimized. Using this model system, the adhesion of bacteria to cell line CuFi-1 was up to three times higher...
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