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Cell wall integrity signalling pathway and yeast colony morphology
Reslová, Gabriela ; Schierová, Michaela (advisor) ; Seydlová, Gabriela (referee)
In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...
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Methodology of transgenic mouse preparation.
Reslová, Gabriela ; Komrsková, Kateřina (advisor) ; Pěknicová, Jana (referee)
The thesis deals with methods used for preparation of transgenic mice and with comparison of them in terms of efficiency and suitability of applications for research purposes. Technologies for the transfer of gene constructs involve microinjection of DNA into the pronukleus of fertilized oocyte, which seems to be the most reliable one. Another possibility is associated with the use of sperm, which carry the incorporated gene construct into the oocyte during in vitro fertilization, or with the use of retroviral vectors by which the oocytes are transfected. Somatic cell nuclear transfer into enucleated oocyte, the use of embryonic stem cells which are incorporated into strange embryo or transgenesis by liposomes are techniques far less explored and not so often used. The next section of this thesis introduces to the reader individual steps which are necessery for successful transgenesis. It is focused on ovarian stimulation which is necessery to obtain sufficient amount of oocytes, as well as on stimulation of recipient mother in embryo transfer as well as on capacitation of sperm required for in vitro fertilization. The thesis also deals with backward analysis confirming positivity of transgene expression by genotyping of pups and by crossbreeding of transgene positive and compares advantages and...
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Cell wall integrity signalling pathway and yeast colony morphology
Reslová, Gabriela ; Schierová, Michaela (advisor) ; Seydlová, Gabriela (referee)
In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...
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Methodology of transgenic mouse preparation.
Reslová, Gabriela ; Komrsková, Kateřina (advisor) ; Pěknicová, Jana (referee)
The thesis deals with methods used for preparation of transgenic mice and with comparison of them in terms of efficiency and suitability of applications for research purposes. Technologies for the transfer of gene constructs involve microinjection of DNA into the pronukleus of fertilized oocyte, which seems to be the most reliable one. Another possibility is associated with the use of sperm, which carry the incorporated gene construct into the oocyte during in vitro fertilization, or with the use of retroviral vectors by which the oocytes are transfected. Somatic cell nuclear transfer into enucleated oocyte, the use of embryonic stem cells which are incorporated into strange embryo or transgenesis by liposomes are techniques far less explored and not so often used. The next section of this thesis introduces to the reader individual steps which are necessery for successful transgenesis. It is focused on ovarian stimulation which is necessery to obtain sufficient amount of oocytes, as well as on stimulation of recipient mother in embryo transfer as well as on capacitation of sperm required for in vitro fertilization. The thesis also deals with backward analysis confirming positivity of transgene expression by genotyping of pups and by crossbreeding of transgene positive and compares advantages and...
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