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Streptomycetes surface growth and differentiation on inert microbeads- morphology and proteome study
Tesařová, Eva ; Weiser, Jaroslav (advisor) ; Lichá, Irena (referee)
Streptomyces, filamentous Gram-positive bacteria are producers of more than 70% of antibiotics used in human therapy and agriculture. They are remarkable because of their complex life cycle (morphological differentiation) which leads to a formation of dormant spores able to survive unfavorable living conditions and allowing long-term survival of the organism. Soil represents their mostly natural living environment. In laboratory conditions they are cultivated in liquid media or on agar. We have developed in our laboratory two phase cultivation system which allows quantitative and reproducible preparation of samples for proteomic, transcriptomic and metabolomic analyses of Streptomycetes differentiation. The system is composed of inert micro- beads submerged in liquid medium. We used two types of micro-beads in our studies, glass and zirconia/silica beads. We followed the surface growth and differentiation of Streptomycetes on both types of beads using optical and electron microscopy (SEM) techniques. We observed major growth and higher antibiotic production on glass beads. Another difference we observed was in size and shape of colonies. In further research, using comparative proteomics, we attempted to identify proteins which might be responsible for recognition and adhesion of Streptomycetes to...
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Analýza regulace komplexů cytoplazmatických poly(A) polymeráz
Novák, Jakub ; Staněk, David (advisor) ; Hrossová, Dominika (referee)
The regulation of gene expression is achieved at many levels. Chromatin-based gene regulation has been the central focus of many decades of research; however, posttranscriptional control mechanisms are emerging as a fundamental complement to direct protein synthesis. This thesis is focused on a specific mechanism of posttranscriptional control - the translational regulation of mRNAs in the cell cytoplasm. This control is a consequence of the balance between translational repression and activation and hinges on the selective recognition of regulated mRNAs by RNA-binding proteins and their ability to recruit RNA modifying proteins. In this thesis, Caenorhabditis elegans germline was used to study translational control of the germ cell-enriched gene, gld-2. Mutants of known RNA-binding proteins of the PUF and CPB protein families were analyzed by performing Western blots, using anti-GLD-2 antibodies. Yeast 3-Hybrid system was used to identify the cis-regulatory sites in the gld-2 mRNA conferring translational regulation by members of PUF and CPB protein families. Potential autoregulatory loop of gld-2 gene expression was also investigated. This thesis shows that FBF proteins positively regulate expression of gld-2 and bind to a conserved sequence in the 3'UTR of its mRNA. Mutations of gld-2 negatively affect...
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Contribution to study of rivalry between definite article and demonstrative adjective
Berthótyová, Júlia ; Štichauer, Jaroslav (referee) ; Tláskal, Jaromír (advisor)
This theoretical thesis deals with the relationship bertween two kinds of determiners in French: the definite article and the demonstrative adjective. As they have the same distribution, we can call this relationship the rivalry. The present work tries to promote the comprehension of this specific subject by assembling all available theories and other contributions. Chapters are ranged according to their degree of generatization: we start by quite general approaches and progress to more and more specific ones. The first chapter presents the general ground of our theme: noun determination. In the chapter two, we analyze some French grammar books in order to find some reference about the rivalry. The chapter three exposes selected theories that are related to the problem of the rivalry between the definite article and demonstrative adjective. The chapter four presents three classifications of use of demonstrative adjective. Even if this part of work seems not to be in close relation to the main subject, we found in these classifications many observations of the rivalry and that's why we devoted whole chapter to a brief synthesis of classifications. The chapter five gives a summary of the research carried out by Hélne Manuélian. In contrast to above mentioned works, the research of Manuélian is based on a...
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Position control of cross-table dedicated manufacturing machine
Plocek, Jaroslav ; Vetiška, Jan (referee) ; Houška, Pavel (advisor)
The bachelor thesis treats the drive solution of the two axis cross-table of a single-purpose manufacturing machine. The first chapter analyzes the concept of drives and compares different variants using either a direct linear drive or a ball screw with a rotary servo drive. The thesis further deals with sizing the motor and the power unit assembly and the description of the whole measuring system. The structure of cascading position controller, its description and the calculation of current and speed control loops is covered in the next part. The last section discusses practical settings of all control parameters.
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Functional studies of Ser/Thr protein kinases and phosphatases of Pseudomonas aeruginosa
Goldová, Jana
Reversible protein phosphorylation is considered the universal language for intracellular communication in all living organisms. This process, catalysed by protein kinases and phosphatases, enables the translation of extracellular signals into cellular responses and also allows for adaptation to a constantly changing environment. In recent years, a number of bacterial eukaryotic-type Ser/Thr protein kinases and phosphatases have been identified. However, their precise functions and substrates are not yet well defined. The genome of opportunistic human pathogen Pseudomonas aeruginosa contains at least five genes encoding putative eukaryotic-type Ser/Thr protein kinases and phosphatases. In the first part of this study, we have attempted to establish the role of Ser/Thr protein kinase PpkA and phosphatase PppA, which belong to type VI secretion system H1-T6SS. Double mutant strain ∆pppA-ppkA was prepared in P. aeruginosa PAO1 background. Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pigment pyocyanin. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the...
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Supply optimalization of assembly hall in a chosen company
Tuscherová, Helena ; Lukšů, Vladimír (advisor) ; Šafránek, Ondřej (referee)
The aim of the work is to make a proposal of a new process of material supply the hall and to customize the logistical process to the standard of the chosen company. Desription and valuation the actual estate of supply the assembly hall in a chosen company. Application fundamentals lean logistics in the assembly hall in the searched company. Interpretation of ascertained problems in the supply process of the assembly hall in a chosen company. Proposal of the corrective activities for eliminating detected problems.
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Prediction of chemosensitivity to 5-fluorouracil and its derivates in patients treated with chemotherapy for colorectal cancer
Zimovjanová, Martina ; Nečas, Emanuel (advisor) ; Cibula, David (referee) ; Vízek, Martin (referee)
Aim: 5-Fluorouracil (5-FU) is the most commonly used anticancer drug for colorectal cancer (CRC). Initially, we aimed to compare expression of 5-FU metabolic enzymes genes: thymidylate synthase (TS), thymidin phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in colorectal cancer and normal colon mucosa. We have tried to prove the correlation of the mRNA levels in fresh frozen tissues and protein expression using immunohistochemistry in paraffin-embedded colorectal cancer and adjacent normal tissues. Finally, we aimed to clarify the prognostic and predictive value of the expression of the 5-FU metabolic enzyme genes in patients with early colorectal cancer. (...) Results: We have found significantly higher DPD mRNA levels in normal colon tissue than in tumor tissue (p=0,03), but no significant differences in TS mRNA (p=0,34) and TP mRNA (p=1,0) levels in tumor and normal tissue. Immunohistochemically we have determined significantly higher TS expresion in tumor than in normal tissue (p=0,02) and significantly higher TP expression in stroma 9 cancer tissue than in normal tissue (p<0,001). No linear relationships were found between mRNA expression and protein TS and TP expression (p=0,28 resp. p=0,87). High TP mRNA expression associated with worse prognosis (p=0,05 HR 4,1), also high protein...
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The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor
Ulrych, Aleš ; Branny, Pavel (advisor) ; Pátek, Miroslav (referee) ; Španová, Alena (referee)
The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor WD-40 domains, also known as beta-transducin repeats, are highly conserved repeating amino acid units, which are found in a wide variety of eukaryotic proteins that have a range of different functions. In the late 1990s, the first WD-40 containing proteins were identified in prokaryotes, however the knowledge about their function is scarce. Streptomyces coelicolor is a gram-positive bacterium with complicated morphological and physiological differentiation in the course of its life cycle. The genome of Streptomyces coelicolor encodes 6 potential genes encoding proteins with WD-repeat motifs. To determine the function of two of these WD-40 genes (wdpB and wdpC), the deletion replacement mutants in both genes were prepared. Both mutants exhibited medium-dependent phenotypes, which are markedly evident on modified R3 plates. Phenotypic studies revealed that deletion of wdpB gene resulted in substantial reduction of aerial hyphae formation and reduced production of undecylprodigiosin. In addition, the hyphae of ΔwdpB mutant were unusually branched and showed the signs of precocious lysis. Delayed spore-containing hyphae were irregularly septated....
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Functional studies of Ser/Thr protein kinases and phosphatases of Pseudomonas aeruginosa
Goldová, Jana ; Branny, Pavel (advisor) ; Nešvera, Jan (referee) ; Španová, Alena (referee)
Reversible protein phosphorylation is considered the universal language for intracellular communication in all living organisms. This process, catalysed by protein kinases and phosphatases, enables the translation of extracellular signals into cellular responses and also allows for adaptation to a constantly changing environment. In recent years, a number of bacterial eukaryotic-type Ser/Thr protein kinases and phosphatases have been identified. However, their precise functions and substrates are not yet well defined. The genome of opportunistic human pathogen Pseudomonas aeruginosa contains at least five genes encoding putative eukaryotic-type Ser/Thr protein kinases and phosphatases. In the first part of this study, we have attempted to establish the role of Ser/Thr protein kinase PpkA and phosphatase PppA, which belong to type VI secretion system H1-T6SS. Double mutant strain ∆pppA-ppkA was prepared in P. aeruginosa PAO1 background. Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pigment pyocyanin. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the...
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Exocyst subunit AtSEC15b: its role in plant cell morphogenesis and characterization of its Rab interacting partner
Toupalová, Hana ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Organization of endomembrane compartments in all eukaryotic cells is dependent on continuous transport of membrane vesicles. Major part of the core regulators of intracellular membrane transport is represented by small GTPases from the Rab family. Rab GTPases cycle between the GTP-bound "active" and GDP-bound "inactive" forms. In their active form, they are able to interact with specific effectors and perform their functions. Exocyst is an octameric complex involved in regulation of secretion. It functions as an effector of Rab GTPases in yeast and mammals and tethers secretory vesicles to the plasma membrane prior to the actual membrane fusion. Using publicly available expression data, we have identified candidates from Rab GTPase family for the interaction with exocyst subunit AtSEC15b in plants and demonstrated that AtSEC15b specifically interacts with AtRABA4a GTPase. We also showed that, like in yeast and mammals, Arabidopsis Sec15b binds Rab GTPase also probably in GTP-dependent manner, implying that this interaction is well conserved throughout the eukaryotic kingdoms. We also successfully demonstrated the complementation of yeast thermo-sensitive mutant strain, sec15-1. Based on this observation we concluded that AtSEC15b is able to substitute the function of yeast SEC15 and restore the phenotype....
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