Plant cells are mostly immobile, therefore it is crucial for them to distinguish a direction of the signals coming into the cell and on the other hand they have to precisely target their own signals. To achieve this communication, plant cells use endomembrane system and secretory vesicles, which are recruited to the specific membrane domains. This ability is important for the plant defense against pathogenic microorganisms and it even forms a part of the innate plant immunity. Two complexes, the exocyst and SNARE, play a prominent role in the process of polarized secretion. In this work, we focused on a possible interaction between these two complexes in preinvasive defense and particularly, we studied the exocyst subunit EXO70B2 and SNARE protein SYP121. We obtained double mutant plants of EXO70B2 and SYP121 by utilizing the reverse genetics approach. These mutant plants did not show any obvious phenotype under standard conditions in comparison with Wt plants. However, we observed marked defects of secretory pathway in double mutant exo70B2/syp121 after infection by pathogenic fungi Blumeria graminis f. sp. hordei. Using histochemical staining, we described problems with the deposition of defensive papilla and secretion of haustorial encasement. We prove that these defects are not connected with...

Trichomes are fine epidermal outgrowths covering aerial organs of most land plants. Although unicellular trichomes of Arabidopsis thaliana have long been used as a model system in plant cell and developmental biology, surprisingly little is known about the processes involved in cell wall biogenesis during the last stage of trichome maturation. A role of EXO70H4, a putative subunit of the vesicle tethering complex exocyst, in trichome maturation has recently been identified in our laboratory. Image analysis, histochemical detection and FT-IR spectroscopy methods were used in this study to analyze cell wall defects of the exo70H4 LOF mutant, revealing the mutation causes altered deposition of pectins and possibly also lignins and hemicelluloses. Transgenic lines with EXO70 paralogues driven by the EXO70H4 promoter were prepared and their analysis revealed that the closest paralogue EXO70H3, unlike EXO70A1 and EXO70B1, can complement the exo70H4 mutation. Based on the results, questions concerning trichome cell wall composition, the role of EXO70H4 in trichome maturation and functions of the plant exocyst complex are discussed. Keywords: Arabidopsis, trichome, cell wall, secretory pathway, exocyst complex, EXO70H4, FT-IR spectroscopy

of the thesis The presented work explores the function and regulation of intracellular signaling that utilizes phospholipase D (PLD) and phosphatidic acid (PA), especially in the context of cellular morphogenesis of plants. PLDs cleave membrane phospholipids to phosphatidic acid, which has important biophysical and signaling role in many contexts, such as stress response, regulation of cytoskeletal dynamics and vesicular transport. Vesicular transport is essential in focused tip growth of plant pollen tubes and root hairs. Part of the work deals with NADPH oxidases, that are an emerging counterpart of PLD/PA signaling. Tobacco pollen tubes served as the main experimental model, as it enables assessing of changes in secretory pathway after pharmacological or genetic treatments. A technique utilizing antisense oligonucleotides was used for selective knock-down of PLD isoforms, NADPH oxidase and newly studied family of lipid phosphate phosphatases (LPPs) in pollen tubes. This enabled to assess functions of individual isoforms. For studying of selected gene families, various bioinformatic tool were utilized, such as dendrogram construction, analysis of available expression data and creating of virtual proteome. These tools together enabled to select potentially important genes for further experimental...

During the course of evolution the early land plants gained extensive innovations that can be seen in modern day plants. The polar growth is an ancient feature of eukaryotic cells and is one of preadaptations that helped plants in successful colonization of land. The polar growth in plants regulates not only the direction of cell expansion and structural properties of cell wall but especially also the orientation of cell division, and is governed by various factors, including the exocyst complex. The exocyst is a well conserved vesicle tethering multi-subunit complex involved in tethering of secretory vesicles to the target membrane. The essential role of the exocyst complex in regulation of various cellular processes in Angiosperms is now well documented. Here I present results of a doctoral project that contributed to phylogenetic analyses of the land plant exocyst complex and especially to uncovering functions of three moss exocyst subunits, namely EXO70 (isoform PpEXO70.3d), SEC6 and SEC3 (isoforms PpSEC3A and PpSEC3B) in the model organism Physcomitrella patens. Various knock-out (KO) mutants in several moss exocyst subunits (Ppexo70.3d, Ppsec6, Ppsec3a and Ppsec3b) show pleiotropic defects directly or indirectly linked to the cell polarity regulation. Cell elongation and differentiation,...

The major sorting station of biosynthetic material destined for the cell surface or secretion is the trans Golgi Network, TGN. This organelle sorts proteins and lipids into vesicular transport carriers that are targeted via different pathways to distinct membrane compartments of the cell. The molecular principles that operate in cargo sorting at the TGN are still not very well understood. Especially, we know very little about the sorting of lipids. It was postulated that a sorting mechanism based on clustering of lipid rafts, dynamic membrane domains enriched in sphingolipids and sterols, could be an important part of the picture. My thesis study dealt with the elucidation of the molecular sorting principles at the TGN and their exploitation for cell surface polarity in the yeast Saccharomyces cerevisiae. To this end, we conducted a genome wide screen that identified yeast mutants defective in cell surface delivery of the model cargo protein FusMid-GFP. The most striking result of this screen was that mutant strains with defects in ergosterol (the major yeast sterol) and sphingolipid biosynthesis lost sorting competence. To elucidate a direct role for sphingolipids and ergosterol in cargo sorting and secretion we sought to characterize the lipid composition of secretory vesicles. Hence, we established a vesicle purification protocol based on an immunoisolation strategy. Additionally, in collaboration with the group of A. Shevchenko, we developed a mass spectrometry methodology that allows the comprehensive and quantitative lipid analysis of subcellular organelles. Preliminary results corroborate our genetic evidence. The data show that the vesicles are enriched in sphingolipids and decreased in phosphatidylcholine indicating a role for raft clustering in cargo sorting at the TGN. The studies of cell polarity during yeast mating also unraveled a role for raft clustering. We could identify that the lipid bilayer at the tip of the mating projection was more ordered than at the plasma membrane enclosing the cell body and that this was dependent on sphingolipid synthesis. The results of my thesis suggest that in the yeast Saccharomyces cerevisiae fundamental cell biological processes such as cargo sorting and vesicle formation at the TGN as well as cell surface polarity during mating employ raft clustering mechanisms.

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Thesis deals with relationship between histomorphological and molecular-genetic findings of selected salivary gland tumors. Author, as a molecular-cytogeneticist mainly focused on detection of tumor-specific translocations of the salivary gland tumors which can serve as differential diagnostic markers. The thesis is composed as a commented files of authors own publications, and it is divided into four parts. First part deepens the knowledge of salivary adenoid cystic carcinoma. It was proved, that t(6;9)(q22-23;p23-24) resulting in fusion of transcription factors MYB-NFIB, or more rarely t(8;9) resulting in MYBL1-NFIB fusion represent robust differential diagnostic marker of adenoid cystic carcinoma. Further it was proved, that the 1p36 deletion can serve as an unfavorable prognostic indicator of adenoid cystic carcinoma, as the patients with 1p36 deletion had significantly lower survival. Second part summarizes new developments about mammary analogue secretory carcinoma (MASC), which was described by our group as a new salivary tumor entity characterized by translocation t(12;15)(p13;q25) resulting in ETV6-NTRK3 fusion. Another novel observation is a discovery of ETV6-RET fusion in a subset of MASC cases. Further, the first two MASCs of nasal mucosa origin have been described. Third part consists...

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Der Einzeller Eimeria tenella zählt zu den hochpathogenen Parasiten des Haushuhns und ist Verursacher der Blinddarm-Kokzidiose, eine Erkrankung, die zu hohen ökonomischen Belastungen in der Geflügelindustrie führt. Zur Zeit existiert noch kein Impfstoff, der auf Basis von einzelnen Antigenen wirksam ist. Insbesondere die Identifizierung und Charakterisierung von sekretorischen Proteinen, welche an der Parasit-Wirtszell-Interaktion beteiligt sind, könnte einen Beitrag zur Entwicklung einer Immunprophylaxe darstellen. Ziel dieser Arbeit war es, sekretorische E. tenella-Proteine zu identifizieren und ausgewählte cDNA-Sequenzen in DNA-Immunisierungsstudien zu überprüfen. Um ein DNA-Immunisierungsprotokoll für Hühner zu erarbeiten, wurden die cDNAs von drei schon bekannten E. tenella-Antigenen alleine oder in Fusion zur cDNA des stabilisierenden enhanced green fluorescence protein (EGFP) in zwei unterschiedliche DNA-Immunisierungsvektoren (pCDNA3, pVR1012) kloniert. Die Überprüfung der Serokonversion zeigte, dass nach DNA-Immunisierung von Hühnern mit beiden Vektoren bzw. mit und ohne EGFP-Fusion vergleichbare Immunantworten induziert wurden. D.h. mit dem etablierten DNA-Immunisierungsprotokoll konnte eine Expression heterologer (Eimerien) Gene im Huhn erzielt werden. Um neue sekretorische E. tenella-Proteine zu identifizieren wurden bioinformatische und experimentelle Methoden eingesetzt. Über die bioinformatischen Analysen wurden aus E. tenella-expressed sequence tag (EST)-Datenbanken 54 putativ sekretierte E. tenella-Sequenzen extrahiert für die aufgrund ihrer beschriebenen Funktion und Lokalisierung eine Beteiligung an der Wirts-Parasit-Interaktion abgeleitet wurde. Mittels funktioneller Komplementierung in Hefen konnten aus einer E. tenella-Sporozoiten-cDNA-Bank 25 sekretorische Sequenzen identifiziert werden. Die cDNA-Sequenzen von 13 neu identifizierten sekretorischen Proteinen wurden in DNA-Immunisierungsstudien überprüft. Die Ergebnisse zeigten, dass beide Methoden geeignet sind, um sekretorische Proteine von E. tenella zu identifizieren. Aufgrund der Erkenntnisse dieser Arbeit, sollten jedoch weitere verbesserte Immunisierungsprotokolle erarbeitet werden, um die immunprotektive Wirkung der isolierten Kandidaten-Antigene von E. tenella in Hühnern zu überprüfen. Wie die vorliegende Arbeit bestätigt, eignet sich die Methode der DNA-Immunisierung vermutlich als Grundlage solcher Verfahren, wobei zukünftig die Induktion der essentiellen intestinalen Immunantwort im Vordergrund stehen sollte. / The protozoan parasite Eimeria tenella is highly pathogenic in chickens and causes caecal coccidosis that contribute to high economic losses in poultry farming. At the moment no vaccine based on a single antigen is available. The identification and characterization of proteins that are involved in host parasite interaction could particularly contribute to the development of immunoprophylactic control strategies. In order to establish a DNA immunisation protocol, cDNA of three already known E. tenella antigens were subcloned alone or in fusion to the stabilising fusion partner enhanced green flurescence protein (EGFP) into two different DNA immunisation vectors (pCDNA3, pVR1012). Following DNA immunisation of chickens using both vectors with or without EGFP fusion, respectively, the induction of comparable immune responses were shown by serum conversion. This implies it was possible to achieve heterologous (Eimeria) gene expression in chickens with the established immunisation protocol. In order to identify new E. tenella secretory proteins bioinformatic and experimental approaches were used. Following bioinformatic analysis 54 putatively secretory E. tenella sequences were identified for which roles in host parasite interaction were surmised, due to their localisation and function. The identification of secreted proteins from a E. tenella sporozoite cDNA library using a functional complementation assay in yeast resulted in 25 unique sequences. The cDNA of 13 newly identified secretory proteins were tested in DNA immunisation studies. The results showed that both methods are capable of identifying secretory proteins of E. tenella. Owing to the results presented here, new immunisation protocols should be established to test the immune protective capacity of candidate antigens of E. tenella in chickens. The present study confirmed the method of DNA immunisation as a basic tool for such new protocols. In future, however, DNA immunisation protocols should focus on the induction of essential intestinal immune responses.

In this study, we examined the immunomodulatory effect of excretory/secretory products, crude adult extracts and crude larvae extracts from Hymenolepis diminuta on the intestinal epithelilal cell line from a rat. For determination of the immunomodulation effect of all H. diminuta extracts was used relative gene expression of TNFa, IL-17re and IL-33 from epithelial cells and it was tested using real-time PCR. Our result showed that excretory/secretory products had the strongest antiinflammatory effect on the epithelial cells. We assume that crude adult extracts play an important role in increase of gene expression of IL-33 and also in the immunomodulatory ability of H. diminuta in the host organism.