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Coordination of growth and cell cycle progression in green algae
IVANOV, Ivan
Within the past century microalgae have gained importance both as model organisms in cell cycle research and as a biotechnological platform for the production of a variety of economically important compounds. This thesis examines the coordination of growth and cell cycle progression in green algae and attempts to explore the biotechnological relevance of some of the findings. Furthermore, the applicability of confocal Raman microscopy for both quantitative and qualitative analysis of storage biomolecules during the course of the cell cycle of Desmodesmus quadricauda is also investigated. Temperature and light shift experiments showed that there is no direct correlation between growth and cell cycle progression in D. quadricauda. Further analysis revealed that supraoptimal temperature has a profound effect on the cell cycle of Chlamydomonas reinhardtii causing a block in cell division, increase of cell size and over accumulation of starch. Starch production through supraoptimal temperature was successfully demonstrated in pilot scale experiments, however it was estimated that light availability within the culture poses a major limiting factor. Confocal Raman microscopy was successfully applied for the quantitative and qualitative analysis of storage biomolecules including starch, lipids, polyphosphates and guanine.
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Non-conventional bacterial signaling pathways
Krupička, Jiří ; Branny, Pavel (advisor) ; Beranová, Jana (referee)
Two component systems were traditionally considered as main phosphorylation systems of bacteria involved in cell signalling. Recently, attention focuses increasingly on bacterial eukaryote-like Ser/Thr protein kinases (eSTKs). These protein kinases are structurally similar to their eukaryotic counterparts. Some eSTKs possess additional domains such as extracellular PASTA domains that were discovered in a variety of gram-positive bacteria. It has been proved that these domains can act as sensors for unlinked peptidoglycan fragments. However, majority of environmental signal molecules still remains unknown. eSTKs phosphorylate a broad spectrum of substrates including proteins involved in various cell processes such as virulence, cell wall biosynthesis, cell division, and central and secondary metabolism. Cross talk between eSTKs and two component systems also occurs. In this thesis, the current knowledge about eSTKs and their significant substrates in different bacterial species is discussed.
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Live-cell tracking in time-lapse sequences
Zámečník, Tomáš ; Šorel, Michal (advisor) ; Křivánek, Jaroslav (referee)
Title: Live-cell tracking in time-lapse sequences Author: Tomáš Zámečník Department: Department of Software and Computer Science Education Supervisor: RNDr. Michal Šorel Ph.D., Oddělení zpracování obrazu ÚTIA AV ČR Abstract: This diploma thesis deals with methods of tracking particles in image sequences. It's goal is to design and implement a complete system for tracking of live cells, their motion and division. The thesis uses conclusions of published scientific papers, studies their application and analyzes possibilities for their mo- difications or improvement. As a result, there are two applications. First of them is a demonstrational pro- gram, provided as an attachment of this thesis. Second implementation is a mo- dule of commercial software NIS-Elements, by Laboratory Imaging, Ltd., which is used by both scientific and commercial institutions in the whole world. Keywords: cell tracking, particle tracking, cell division 1
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Spr0334, new protein of cell division in Streptococcus pneumoniae.
Štekerová, Nela ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...
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Regulation of penicillin-binding protein Pbp2a in Streptococcus penumoniae
Kubeša, Bohumil ; Doubravová, Linda (advisor) ; Pospíšil, Jiří (referee)
Regulation of penicillin-binding protein Pbp2a in Streptococcus pneumoniae Streptococcus pneumoniae is an extracellular human pathogen that encodes a unique eukaryotic-type Ser/Thr protein kinase StkP in its genome. This enzyme is involved in other cellular processes, such as cell division and cell wall synthesis, through phosphorylation with its substrates. A transmembrane protein MacP has been identified as a substrate of StkP. It is an activator of penicillin-binding protein PBP2a, which is involved in the synthesis of peptidoglycan with its transpeptidase and transglycosylase activities. We found that MacP is phosphorylated by the protein kinase StkP at positions T32 and T56. We confirmed that proteins MacP and PBP2a interact with each other and that phosphoablative and phosphomimetic mutations of the major phosphorylated residues of the MacP protein do not affect the interaction with PBP2a and do not fundamentally affect the function of MacP in vivo. Furthermore, we showed that the ∆macP mutation is synthethically lethal with the ∆pbp1a mutation, confirming that MacP is an activator of the PBP2a protein. MacP is located in the cell septum and interacts with a number of S. pneumoniae cell division proteins. Keywords: Streptococcus pneumoniae, cell division, MacP, PBP2a, phosphorylation
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Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae
Holečková, Nela
Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...
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Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae
Holečková, Nela
Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...
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Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae
Holečková, Nela ; Doubravová, Linda (advisor) ; Lichá, Irena (referee) ; Petříčková, Kateřina (referee)
Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...
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Identification of new substrates of Ser/Thr protein kinase StkP
Kleinová, Simona ; Ulrych, Aleš (advisor) ; Konopásek, Ivo (referee)
Streptococcus pneumoniae encodes single serine/threonine protein kinase StkP and its cognate protein phosphatase PhpP. This signalling couple phosphorylates/dephosphorylates many target proteins involved in various cellular processes. So far, only few ot them was characterized in detail. Global phosphoproteomic analysis in the ∆stkP mutant strain background resulted in the identification of protein Spr0175 as phosphorylated on threonine 7. The main aim of this work was to characterize this new substrate. The ∆spr0175 mutant strains were prepared in the wild type genetic background Rx and R6 and then monitored for their growth and cell morphology. Mutant strains exhibited morphological defects revealing potential involvement of Spr0175 in the process of cell division. In the wild type D39 the deletion was unsuccesful, which may entail possible essentiality of Spr0175 in D39 strain. The results obtained also confirmed that the Spr0175 is modified in in vitro and in vivo conditions at threonine 7. In vitro study also confirmed minor phosphorylation at T4 residue. By using co-immunoprecipitation assay we demonstrated that Spr0175 protein can form oligomeric structures. Another aim of this work was cellular localization of Spr0175. By using fluorescent microscopy we showed that GFP-Spr0175 fusion...
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Control of cell division of Streptococcus pneumoniae by unique signaling pathway
Kubincová, Hana ; Branny, Pavel (advisor) ; Fišer, Radovan (referee)
Genome of S. pneumoniae contains only one copy of the gene coding eukaryotic type protein kinase StkP and corresponding phosphatase PhpP. These two enzymes form a functional signaling pair regulating cell division, which could be used in the future for the design of new bacteriostatic compounds. Not only kinase and phosphatase are important components of the system, but also other members of this pathway - specific substrates of these enzymes. The identification of the Ser/Thr phosphoproteom with a focus on the membrane fraction provided information not only about already known substrates such as LocZ, Jag and DivIVA but also about an unknown protein P15 with a molecular weight about 15 kDa. In this thesis the protein was identified as rhodanase (spr0595) by MS MALDI TOF. However, its subsequent deletion did not confirm it as a StkP/PhpP substrate. Therefore we investigated another substrate, protein FtsA, which has already been identified as a substrate of this kinase in a previous study (Beilharz et al., 2012). FtsA is an essential cell division protein that anchors FtsZ filaments into the membrane. Phosphorylation of this protein was detected on the Thr residue at position 404. Using phosphoablative substitution we found out, that Thr404 is indeed phosphorylated by protein kinase StkP, however, FtsA...
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