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Viral vectors based on tobacco mosaic virus for the expression of recombinant proteins in plants
Kroupa, Daniel ; Moravec, Tomáš (advisor) ; Cvrčková, Fatima (referee)
(eng): Over the past few decades, plant viruses and vectors derived from them have become not only an integral part of the work in laboratories, but also began to be used commercially for the production of vaccines, drugs, enzymes and other proteins / peptides. One of the most commonly used viruses for the expression of heterologous proteins is tobacco mosaic virus. In this work, I would like to focus on plant viruses and vectors derived therefrom. Compare it with other expression systems used in plants and describe how their benefits could be further used and combined with modern methods of cloning. Key words: viral vector, expression, Tobacco mosaic virus, plant expression systems, Golden gate cloning
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Identification and characterization of proteins interacting with plant formins
Houšková, Anežka ; Cvrčková, Fatima (advisor) ; Abrhámová, Kateřina (referee)
Formins are evolutionarily conserved proteins participating in actin and microtubule organisation, affecting thus also intracellular transport, cell growth, morphogenesis and cell polarity. All formins contain FH2 domain, known to dimerize and act as a nucleator of actin. Angiosperms have two formin clades, Class I and Class II, which are distinguished by domain organisation. Based on knowledge from animal models and protein sequence homology, two groups of candidate membrane-associated formin interactors have been proposed in Arabidopsis (Cvrčková, 2013). First group of candidates consists of FYVE domain-containing proteins FAB1A (At4g33240) and FAB1B (At3g14270), the other contains proteins with BAR and SH3 domains AtSH3P1 (At1g31440), AtSH3P2 (At4g346600) and AtSH3P3 (At4g18060). Yeast two hybrid assay was used to examine protein interactions of selected proteins from both candidate groups (FAB1A, SH3P2 and SH3P3) with FH2 domains representing both plant formin clades. The same experimental setup was also used to test dimerization among FH2 domains of plant formins. Translational fusions of FH2 domains from Class I formins AtFH1 (At3g25500), AtFH5 (At5g54650) and Class II representatives AtFH13 (At5g58160) and AtFH14 (At1g31810) with the GAL4 activation domain have been co-expressed in yeast with GAL4...
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New breeding techniques in crop plants
Brousková, Mariana ; Opatrný, Zdeněk (advisor) ; Cvrčková, Fatima (referee)
Nowadays when human population and demand for quantity and quality of food constantly grow, there is effort to improve technologies, which are used to functioning currently agriculture. Currently spectrum of crop plants, which are available for people, is one of main basis of agriculture. This spectrum is enriched of new cultivars or new crop by classical breeding for centuries. From the end of 20th century spectrum is further expanded on gentically modified plants. However, there were distrustfulness of human society and therefore genetically modified plants were legislatively restricted about their use and cultivation. Inter alia, for these reasons effective and socially acceptable alternatives of standart GM techniques were searched. Set of methods, which was named "New breeding techniques"- NBTs, provides such benefits. This work summarizes NBTs and explains basic principles of their use.
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Plant synapses: more than a metaphor?
Pavlovic, Jakub ; Cvrčková, Fatima (advisor) ; Sikorová, Lenka (referee)
Synapses are generally defined as places where cell-to-cell signal transmission especially between neurons can occur, involving transmission of signal by local recycling of exo- and endocytosis vesicles. Possible existence of synaptic signal transduction in plants is still subject of discussion. However, existence of plants homologues of proteins related to those found in synapse support this theory. Cell-to-cell contacts similar to electric, immunological and chemical synapses were described in plants. Here I describe some proteins involved in function or synapse development whose plant homologs are known, or were discovered by myself. Protein RHD3 was described as ortholog of mammalian atlastin. Mutation in atlastin cause hereditary spastic paraplegia where morphology of long axons is changed. Similar change of cell morphology is found also in rhd3 mutans, whose root-hairs have altered phenotype. These root-hairs are shorter and posses wavy appearance. Further I succeeded in identification of possible homolog of two other proteins. It is homolog of Dlg-MAGUK, which is involved in proper function and synapse development. Second whose possible homolog I found is RIMs- BP 2. RIMs-BP 2 forms bifunctional connections between fusion apparatus of synaptic vesicle and Ca2+ channels. Last protein was...
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Pharmacologically active compounds in hops
Pýchová, Kateřina ; Cvrčková, Fatima (advisor) ; Mašková, Petra (referee)
The hop plant (Humulus lupulus) is used not only in brewing but also in phytopharmacology and phytotherapy. Among the isolated or chemically produced substances used for healing are xanthohumol, 8-prenylnaringenine and α and β-bitter acids. Nowadays, a lot of attention is paid to their effect on humans - especially to their anticancer activity. But there is a lack of information about the importance of these substances for the hop plant itself. Most of the plant secondary metabolites originally serve for protection of the plant against pathogens and herbivores. The selected metabolites are not an exception. The purpose of this work was to summarize the published data about the biological importance of selected hop secondary metabolites and the effects they have on other organisms than plants, with an emphasis on humans.
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The role of cell polarity signaling in the plasticity of cancer cell invasiveness
Gandalovičová, Aneta ; Brábek, Jan (advisor) ; Cvrčková, Fatima (referee)
Throughout the last few years cancer research has focused on studying the origin of secondary tumors, i.e. metastases, which are a direct outcome of the ability of cancer cells to disseminate from the primary tumor and invade the adjacent tissue. Generally, cancer cells migrate by two distinct mechanisms- amoeboid or mesenchymal. Whereas the mesenchymal migration mode can be described as "path generating", the amoeboid mode resembles a "path finding" way of migration. Both types of invasion are regulated by divergent signaling pathways that are closely related to cell polarity and cytoskeleton reorganization. Responsible for cell polarization are not only the polarity complexes Par, Scribble and Crumbs, but also phosphoinositides and Rho GTPases Rac, Rho and Cdc42, which, additionally, regulate the dynamics of the cytoskeleton. By a mutual interplay they regulate cell motility. It cannot come as a surprise that their deregulation commonly results in tumorigenesis. A more thorough comprehension of the signaling pathways leading to cancer cell invasiveness is a necessary step towards understanding the complex problem of metastasis. Key words: invasiveness, amoeboid, mesenchymal, cell polarity, motility, Rho GTPases, polarity complexes
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The architecture of regulatory network of metabolism
Geryk, Jan ; Flegr, Jaroslav (advisor) ; Cvrčková, Fatima (referee) ; Šafránek, David (referee)
The thesis focus on the modularity of metabolic network and foremost on the architecture of regulatory network representing direct regulatory interactions between metabolites and enzymes. I focus on the "modularity measure" in my first work. Modularity measure is quantitative measure of network modularity commonly used for module identification. It was showed that algorithms using this measure can produce modules that are composed of two clearly pronounced sub-modules. Maximum size of module for which there is a risk that is is composed of two sub-modules is called resolution limit of modularity measure. In my first work I generalize resolution limit of modularity measure. The generalized version provide insight to the origin of resolution limit in the null-model used by modularity measure. Moreover it is showed that the risk of omitting of sub-modular structures applies for bigger modules than mentioned in the original publication. The second work is focused on the question how does the modular structure of E. coli metabolic network change if we add regulatory interactions. I find that the modularity of modular core of network slightly increase after regulatory edges addition. The modularity increase is significant with respect to randomized ensemble of regulatory networks. Identified modules...
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Role of formins in the organization and dynamics of intracellular structures in Arabidopsis thaliana
Rosero Alpala, Elvia Amparo ; Cvrčková, Fatima (advisor) ; Baluška, František (referee) ; Malcová, Ivana (referee)
On the basis of detailed phenotypic examination of fh1 and fh2 mutants we observed that the main housekeeping Arabidopsis thaliana formin AtFH1 (At3g25500) and its closest relative, AtFH2 (At2g43800) are involved in both actin filaments and microtubule dynamics. fh1 mutants showed increased sensitivity to the actin polymerization inhibitor Latrunculin B (LatB). Formin mutants had cotyledon pavement cells which exhibited more pronounced lobes compared to the wild type, and alterations in vascular tissue patterning were found. The double fh1 fh2 homozygote was not obtained, suggesting that at least one functional formin gene is required for proper gametophyte development. Methods used to observe and quantify both architecture and dynamics of the cortical cytoskeleton from confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM) were standarized and allowed to find that mutants exhibited more abundant but less dynamic F- actin bundles and more dynamic microtubules than wild type seedlings, fh1 mutant phenotype observed in roots was further aggravated by a (heterozygous) fh2 mutation. The formin inhibitor SMIFH2 mimicked the alterations observed in fh1 mutants in plants, it has been the first report of this inhibitor in plants. Defects in membrane trafficking were...
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Transgenic crops and third world
Andrová, Julie ; Opatrný, Zdeněk (advisor) ; Cvrčková, Fatima (referee)
The issue of transgenic crops and the third world is mostly neglected in secondary education. In view of the fact that it is an interesting topic that gains greater importance worldwide, it would deserve the place in secondary education. It is not necessary to submit information about this topic only in the text form to students but teaching methods that force the students to gain information actively, can be used. Text for teachers was created in diploma thesis. The text could serve as a source of information for teachers in preparing for the lesson about this topic. Then test for students was created. The test consists of tasks that are inspired by the PISA. Students have to gain information actively from the texts in the tasks to solve the tasks correctly. The test consists of three thematic areas (transgenic crops, golden rice, vaccination and edible vaccines) and 16 questions (31 including the sub-questions). The test was verified on a sample of 82 grammar school students at the age of 16 to 19 years. The students were of the four classes in four grammar schools. The students recorded their answers in a data sheet. The data sheet included additional questions that sought the views and attitudes of students towards texts and questions in the test. The teachers completed the questionnaire. The...
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