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Targeted editing of the Arabidopsis CYP98A3 gene using the CRISPR/Cas9 method
Šamaj, Matej
Lignin is an abundant biopolymer, which fulfils several important functions including facilitation of polar water transport and mechanical strengthening of plant body. It is required for proper growth and development of plants but also for plant defence responses. Complex lignin biosynthesis involves the activity of numerous enzymes catalysing diverse chemical reactions that are stepwise generating various precursors for lignin formation. One such enzyme of lignin biosynthetic pathway, P‐COUMAROYL SHIKIMATE 3′‐HYDROXYLASE (C3′H), is encoded by the gene CYP98A3/ C3′H and convert p-coumaroyl shikimate to caffeoyl shikimate. CRISPR/Cas systems adapted from prokaryotes, where they provide adaptive immunity, have very quickly evolved to unique and powerful tool for precise gene editing. The CRISPR/Cas9 technology allows to specifically target desired sites within the genomes to subsequently perform very precise mutagenesis of selected gene(s). The purpose of this thesis was to employ the CRISPR/Cas9 method to perform targeted editing of the CYP98A3/C3′H gene in transformed Arabidopsis thaliana plants. Selected transformed plants were verified to confirm the presence of CRISPR/Cas9-mediated gene editing. The verified T2 generation putative mutant plant shows altered phenotypes of leaves, leaf rosettes, stems and inflorescences accompanied by lower deposition of lignin as revealed by histochemical staining by basic fuchsin and microscopic observations.
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Volume-regulated anion channels in astrocytes- in vitro and in situ analysis
Harantová, Lenka ; Anděrová, Miroslava (advisor) ; Vargová, Lýdia (referee)
Astrocytes need to preserve constant volume in the face of osmolarity perturbations to function properly. To regain their original volume after hyposmotically induced swelling, they extrude intracellular electrolytes and organic osmolytes, such as inorganic ions, excitative amino acids or polyols, accompanied by osmotically driven water. This process is termed regulatory volume decrease and is ensured by various ion channels and transporters. Recently, much attention has been focused on the ubiquitous volume-regulated anion channels activated by cell swelling. VRACs are moderately outwardly rectifying with intermediary conductance, permeable to inorganic anions and organic osmolytes and sensitive to broad-spectrum anion channels blockers. Using patch-clamp technique we aimed to characterize VRACs in cultured cortical astrocytes isolated from neonatal Wistar rats and to elucidate the effect of intracellular Na+ on VRAC activity. In addition, we also intended to characterize these channels in situ in brain slices of 10 - 12 days old rats, focusing mainly on hippocampal astrocytes. To induce astrocytic swelling, we exposed astrocytes to hypotonic solution (250 mOsm). In agreement with previous findings, we showed that cultured cortical astrocytes activate VRAC currents upon exposure to hypotonic stress, which...
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Histological and histochemical methods used in study of development of internal male sex organs
VÁVROVÁ, Martina
Presented thesis devotes to comparative study of usability of fixation agents and colouring methods for the study of histology and histochemistry of Culex pipiens s.l. mosquito males. The study was carried out in males of Culex pipiens quinquefasciatus Say, which is a significant transmitter of filariasis. Histological and histochemistry studies of the structure of the whole abdomens or prepared testicles were carried out on the material fixed by fixation agents Carnoy and Bouin of Dubosque-Brasil modification. Fixed tissue was cast in paraplast or resin. Histological sections were done by means of automatic microtome Leica and half-thin sections by means of ultra microtone Reichert. Section preparations were coloured by Mayer or Harris haematoxylin or by colouring method according to Mallory. Semi-thin sections from materials cast in resin were coloured by toluidine blue. Fixation agent Bouin, Dubosque-Brasil modification proved as more convenient than Carnoy, either for the preparation of paraffin sections or for fabrication of half-thin sections from tissues cast in resin. The next advantage of this fixation is that fixed tissue can be stored for unlimited period of time without over fixation of the tissue as it is in the case of fixation by Carnoy. For general histological preparations Mallory colouring is apparently the most convenient from the used methods; it provides better orientation in coloured tissue thanks to the whole spectrum of colours-pink to red colouring of cell nuclei, blue colouring of tissue and secretions. In case of colouring of male genital organs all used colourings (Mallory, Harris and Mayer haematoxylin) seem to be comparable. Casting in resin with connection to colouring by toluidine blue (also histochemistry proof for carbohydrates) has shown to be suitable for visualisation of finer structures, in male genital organs particular developmental phases of male sex cells were distinguished.
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