National Repository of Grey Literature 14 records found  1 - 10next  jump to record: Search took 0.01 seconds. 
The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan ; Honys, David (advisor) ; Paleček, Jan (referee) ; Smýkal, Petr (referee)
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
Enrichment techniques employed in phosphoproteomics
Fíla, Jan ; Honys, David (advisor) ; Zdráhal, Zbyněk (referee)
Reversible protein phosphorylation represents one of the most rapid and dynamic posttranslational modifications. Phosphorylation plays a key role in many cellular processes, e.g. cell division, signal transduction, cytoskeleton dynamics, translation regulation, protein targeting and metabolism. Unfortunately, the importance of phosphoproteins is often not mirrored in their abundance so enriching strategies are mostly inevitable. The enrichment can be performed either at the stage of phosphoproteins or at the stage of phosphopeptides. Alternatively, a combination of both strategies can be used. Most enriching strategies are based on chemical modifications, affinity of phosphates to positively-charged chromatography matrix or antibodies. Every method used has its advantages as well as disadvantages. Moreover, satisfactory coverage of phosphoproteome is very often reached by combination of several methods. In the past five years many protocols have been improved or alternatively new approaches have appeared. So in this bachelor thesis, the available enrichment strategies are reviewed. Key words: phosphoproteomics, phosphoprotein enrichment, phosphopeptide enrichment, IMAC, MOAC, titanium dioxide TiO2, aluminium hydroxide Al(OH)3, antibodies
Revealing phosphoproteins playing role in tobacco pollen activated in vitro
Fíla, Jan ; Honys, David (advisor) ; Fischer, Lukáš (referee)
5 Abstract Tobacco mature pollen rehydrates in vivo on a stigma tissue, and develops into the rapidly-growing pollen tube. This rehydration process is accompanied by the de-repression of stored mRNA transcripts, resulting in the synthesis of novel proteins. Furthermore, such metabolic switch is also likely to be regulated on the level of post-translational modifications of the already-present proteins, namely via phosphorylation, since it was shown to play a significant regulatory role in numerous cellular processes. Since only a minor part of proteins is phosphorylated in a cell at a time, the employment of various enrichment techniques is usually of key importance. In this diploma project, metal oxide/hydroxide affinity chromatography (MOAC) with aluminium hydroxide matrix was applied in order to enrich phosphoproteins from the mature pollen and the 30-minute in vitro activated pollen crude protein extracts. The enriched fraction was separated by both 2D-GE and gel-free liquid chromatography (LC) approaches with subsequent mass spectrometric analyses. Collectively, 139 phosphoprotein candidates were identified. Additionally, to broaden the number of phosphorylation sites identified, titanium dioxide phosphopeptide enrichment of trypsin-digested mature pollen crude extract was performed. Thanks to the...
The mechanisms of pollen incompatibility in the Brassicaceae family
Šesták, Petr ; Fíla, Jan (advisor) ; Vosolsobě, Stanislav (referee)
Sporophytic incompatibility (SI) represents one of the systems by which angiosperms prevent pollination by their own pollen or by the pollen from a genetically related plant. It is mostly studied in the Brassicaceae family, mainly due to its agricultural importance. Another reason is that the model plant Arabidopsis thaliana belongs to this family. In the last three decades, advances in molecular biological methods enabled the characterization of a large part of the signalling cascade that leads to the rejection of incompatible pollen. Then, the functions of various cellular components (e.g. cytoskeleton, exocyst or proteasome) involved in the incompatible response to pollination are studied mainly by live cell microscopy. Last but not least, the function of SI under various abiotic stresses was described to reveal their influence on SI mechanisms. The aim of this bachelor thesis is to summarize the new discoveries characterizing the molecular mechanisms of SI in the Brassicaceae family, to describe the processes leading to the germination of compatible pollen grain and to characterize the newly described proteins involved in cellular signalling leading to the rejection of incompatible pollen.
The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
Studying dimer formation and effectors of Arabidopsis thaliana nascent polypeptide-associated complex
Klodová, Božena ; Fíla, Jan (advisor) ; Robert Boisivon, Helene (referee)
The development of plant flowers represents a complex process controlled by numerous mechanisms. The creation of double homozygous mutant of both β subunits (sometimes also referred to as basic transcription factor 3) of nascent polypeptide associated complex in Arabidopsis thaliana (further referred to as nacβ1 nacβ2) caused quite a strong defective phenotype including abnormal number of flower organs, shorter siliques with a reduced seed set, and inferior pollen germination rate together with a lower ovule targeting efficiency. Previously, NAC complex was described to be formed as a heterodimer composed of an α- and β-subunit, which binds ribosome and acts as a chaperone in Saccharomyces cerevisiae. In plants, NACβ is connected to stress tolerance and to plant development as a transcription regulator. However, little is known of NAC heterodimer function in plants. In this thesis, yeast two hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC) assays were used to verify the NAC heterodimer formation in A. thaliana and to establish any potential interaction preferences between both NACβ paralogues and five NACα paralogues. To deepen the understanding about molecular mechanisms behind the nacβ1 nacβ2 phenotype, flower bud transcriptome of the nacβ1 nacβ2 double homozygous mutants...
The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...
The role of protein phosphorylation during progamic phase of tobacco male gametophyte development
Fíla, Jan ; Honys, David (advisor) ; Paleček, Jan (referee) ; Smýkal, Petr (referee)
v angličtině (English abstract) Tobacco male gametophyte has a strongly dehydrated cytoplasm and represents a metabolically inactive stage. Upon cytoplasm rehydration, pollen grain becomes metabolically active and after the activation is finished, the pollen tube growth through a selected pollen aperture starts. The rehydration together with metabolic activation are accompanied by the regulation of translation and post-translational modifications (mainly phosphorylation) of the existing proteins. In this Ph.D. thesis, there were identified phosphopeptides from tobacco (Nicotiana tabacum) mature pollen, pollen activated in vitro 5 min and pollen activated in vitro 30 min. The total proteins from the above male gametophyte stages were extracted. The protein extract was trypsinized and the acquired peptide mixture was enriched by MOAC (metal oxide/hydroxide affinity chromatography) with titanium dioxide matrix. The enriched fraction was subjected to liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Totally, there were identified 471 phosphopeptides, carrying 432 exactly localized phosphorylation sites. The acquired peptide identifications were mapped to 301 phosphoproteins that were placed into 13 functional categories, dominant of which were transcription, protein synthesis,...

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