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Physiological and pathological factors affecting absorption of drugs
Ryšánek, Pavel ; Šíma, Martin (advisor) ; Mičuda, Stanislav (referee) ; Dražanová, Eva (referee)
Absorption of drugs from the gastrointestinal tract after oral administration is a key pharmacokinetic process co-determining the subsequent pharmacodynamic response of the organism and therapeutic efficacy. This dissertation thesis is devoted to the study of factors that influence this parameter. Special emphasis is placed on the study of lymphatic absorption, i.e. the rate of absorption of the active substance via the intestinal lymphatic system. A number of in vivo studies have been carried out in laboratory rats. Pharmacokinetic studies have been performed by means of regular blood sampling from vascular catheters after oral administration of the drug. Lymphatic absorption was investigated in an anaesthetized mesenteric lymphatic duct cannulated rat model. Modern drugs were tested that were incorporated into innovative dosage forms by collaborating chemical and technological institutions. Abiraterone acetate, a lipophilic agent used in the therapy of prostate cancer, was well absorbed from the gastrointestinal tract after administration in the form of oil marbles. This technology also succeeded in reducing the otherwise very significant positive food-effect. Abiraterone acetate was not absorbed to any significant extent via the intestinal lymphatic system. In contrast, lymphatic absorption of...
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Plant virus-based biotechnology
Vaculík, Petr ; Čeřovská, Noemi (advisor) ; Ryšánek, Pavel (referee) ; Petrzik, Karel (referee)
The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...
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Production of heterologous proteins in plants - human papillomavirus (HPV 16) derived antigens
Folwarczna, Jitka ; Čeřovská, Noemi (advisor) ; Ryšánek, Pavel (referee) ; Kumar, Jiban (referee)
5 Abstract Even though prophylactic vaccine against human papillomavirus (HPV) is currently licensed, infections by the virus continue to be the major health problem mainly in developing countries. Considerable effort is being devoted to preparation of therapeutic vaccine and to decrease of the production costs of current vaccine. Viral proteins such as the E7 oncoprotein and the L2 capsid protein from HPV type 16 are promising targets for the development of the experimental anti-HPV vaccine. The aim of our work was optimization of expression of mutagenized E7 oncoprotein (E7ggg) fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein (CP) or Potato virus X (PVX) CP in viral vectors derived from these plant viruses. The impact of linkers connecting CP and E7ggg fusion partners on expression and stability of fusion proteins was examined. The fusion proteins were first expressed in Escherichia coli (E. coli) MC1061 to assess the characteristics of the recombinant protein prior to their transient expression in both non-transgenic or transgenic Nicotiana benthamiana (N. benthamiana). We have obtained the high level expression in E. coli, but most of the expressed proteins based on TMV CP remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular...
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Influence of nematophagous fungi on populations of important phytoparasitic nematodes, family Heteroderidae, in the Czech Republic
Hussain, Manzoor ; Ryšánek, Pavel (advisor) ; Vadlejch, Jaroslav (referee)
We report on studies aimed to evaluate the effects of five nematophagous fungi on population dynamics of northern root knot nematode, Meloidogyne hapla and sugar beet cyst nematode, Heterodera schachtii in laboratory and greenhouse trials. During our biocontrol studies, mainly five fungal species Arthrobotrys oligospora, Dactylella oviparasitica, Clonostachys rosea, Stropharia rugosoannulata, and Lecanicillium muscarium were isolated from eggmasses of nematodes during root and soil samples collected in Czech Republic and tested against Meloidogyne hapla in carrots while Heterodera schachtii in sugar beet, both in-vitro and in-vivo fashions. All fungi exhibited varied type of infection on nematodes but L. muscarium proved to be the most potential bioagent in controlling nematode population enormously. Lecanicillium muscarium not only halted nematode reproduction factor (Pf/Pi) but also escalated plant growth. Plant root systems treated with L. muscarium were observed colonized by fungi while seen under microscope which depicted that fungi provided a protecting shield or body guard against plant parasitic nematodes. Moreover, L. muscarium was further compared with commercially available nematicides and a fertilizer to justify its highly potential for diminishing nematode population. L. muscarium along with nematicides and fertilizer had dramatic effects in reduction of both nematodes (M. hapla, H. schachtii) population in soil but improvement of plant growth was only noticed in case of L. muscarium. The chemical nematicide, Dazomet had some negative effects which appeared in the form of chlorotic spots on leaves which further suggested that there were some the phytotoxic effects.
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Identification and molecular characterization of the putative immunophilins (IMMs) in the oilseed rape pathogens Leptosphaeria maculans, Leptosphaeria biglobosa, and Plasmodiophora brassicae
Sandhu, Khushwant Singh ; Ryšánek, Pavel (advisor) ; Radovan, Radovan (referee)
Oilseed rape is largely infected by several phytopathogens and two most economical important diseases are blackleg caused by fungus species complex Leptosphaeria maculans and L. biglobosa and clubroot caused by protist P. brassicae. The sequenced genomes of these phytopathogens provide opportunity to uncover various aspects related to disease infection, host pathogen interactions, plant disease resistance, and evolution of pathogens. Considering these we focused on one of the most conserved family called immunophilins (IMMs) in these genomes. IMMs are comprised of three structurally unrelated sub-families including cyclophilins (CYPs), FK506-binding proteins (FKBPs), and parvulin-like proteins (PARs). We identified putative members of IMMs in each phytopathogen using bioinformatics approaches. We further characterized the IMMs based on domain architecture, subcellular localization, exon-intron organization, transcriptomic expression patterns, gene ontology terms, conserved motifs presents and evolutionary analyses. IMMs are performing several vital roles in plants, animals and fungi. However, in phytopathogens their roles are not well established except for cyclophilin that implicates in pathogenicity in some phytopathogens. Therefore, we exploited the role of cyclophilin in L. maculans and L. biglobosa using expression profiles and in P. brassicae using Magnaporthe oryzae cyclophilin deletion mutant. Overall, we concluded that the cyclophilin acts as a virulence determinant in our studied phytopathogens. However, delineating the precise role of other IMMs would also be imperative. Taken together, our findings for the first time shed light on the highly conserved IMM family in the oilseed rape pathogens.
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Diagnotics of the important apple viruses
Winkowska, Lucie ; Ryšánek, Pavel (advisor) ; Petr, Petr (referee)
Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) are economically the most important viruses in pome fruit trees, which are distributed worldwide and can caused significantly yield reduction. The major control strategies (namely pathogen detection, exclusion by crop certification or quarantine, control in infected orchards by eradication from infected cultivars and rootstocks, etc.) rely heavily on accurate and sensitive detection methods and on perfect knowledge of pathogens.
In the doctoral thesis the diagnostic method quantitative RT-PCR (qRT-PCR) was optimized for detection and quantification of four studied viruses. The results suggested that qRT-PCR method was the most reliable technique in comparison with conventional diagnostic methods DAS/I-ELISA and RT-PCR.
In our study the concentration of ASGV, ASPV and ACLSV, measured by qRT-PCR, were stable during vegetation and in different plant tissue. Only the concentration of ApMV changed during vegetation in leaves and inner bark. This result indicates that changes of virus concentration observed by DAS/I-ELISA and RT-PCR in plant tissues are caused by other way (inhibitors, plant senescence, lower sensitivity, ect.) than by changes of virus concentrations in plant.
Under the monitoring (at all 351 trees were tested) it was showed, that studied viruses were more spread in orchards and gardens then in wild apple trees.
Selected virus isolates from wild apple trees and apples from orchards and gardens were sequenced and molecular variability was studied also with already published isolates. However individual isolates of studied viruses were similar. The variability associated with geographic origin or with type of planting has not been confirmed.
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Plasmodiophora brassicae on winter rape
Řičařová, Veronika ; Ryšánek, Pavel (advisor) ; Jaroslav, Jaroslav (referee)
Winter oilseed rape (Brassica napus) is an important crop in the Czech Republic. Clubroot disease caused by the pathogen Plasmodiophora brassicae Wor. is a serious and still-growing problem for oilseed rape growers. Research on P. brassicae in the Czech Republic is therefore important for the development of effective strategies to manage clubroot under Czech environmental conditions.
One of the aims of this study was monitoring of this pathogen. The disease was previously widespread in commercial vegetable production and in hobby gardens. Since 2010, oilseed rape clubroot started to spread across the whole country, whereas it had previously only been observed in the northeast. Clubroot occurrence was monitored for five years by the Union of Oilseed Growers and Processors on the basis of disease symptoms present on oilseed rape fields. The presence of P. brassicae and clubroot symptoms were reported in all regions of the Czech Republic, except the Ústecký Region, and in 31 out of 76 districts. At present, at least 130 fields are known to be infested by the pathogen, but this number is very likely underestimated. Some soil samples were also tested by conventional PCR (polymerase chain reaction) to evaluate the possibility of their usage. All 14 suspected samples tested positive by PCR.
The next aim was to evaluate the pathotype composition of P. brassicae populations from the Czech Republic, according to the three evaluation systems, and to determine soil inoculum loads for representative fields via traditional end-point PCR as well as quantitative PCR analysis. There were considerable differences between the populations of P. brassicae, and the number of pathotypes varied depending on the evaluation system and the threshold used to distinguish susceptible vs. resistant plant reactions. This is the first study comparing the effect of different thresholds. Using an index of disease (ID) of 25 % to distinguish susceptible vs. resistants reactions, there was a total of five pathotypes identified based on the differentials of Williams, five with the system of Somé et al., and 10 with the European Clubroot Differential (ECD) set. However, based on a threshold of 50%, there were five pathotypes according to the evaluation system by Williams, four based on the differentials of Somé et al. and 8 with the ECD set. Changing of the thresholds led to the reclassification of some pathotypes. Pathotypes 7 by Williams was the most frequent in both thresholds. High amounts of pathogen DNA were found in many of the field soils analysed by quantitative PCR.
Experiments with P. brassicae-resistant cultivars of winter oilseed rape were conducted in an infested field and greenhouse. In the greenhouse, six resistant cultivars were grown in infested soil collected from various fields in the Czech Republic and assessed for index of disease (ID %). The best results bring cultivar Mentor (2+- 0.7 %) closely followed by cultivar SY Alister (5+-1.1 %), the highest ID had cultivar CHW 241 (30+-3.8%). In the field experiment, seven resistant cultivars were grown, and disease development was monitored monthly. The lowest index of disease brought cultivar Andromeda (3+- 0.8 %) and PT 235 (4+-1.5 %), the highest ID has cultivar CWH 241(46 +- 6.5 %) in the first season and in the second season any cultivar achieved 25 % ID. Yields were measured at the end of the cropping season. The highest yield was achieved by cultivar SY Alister (6.1 t/ha) in the first season and cultivar PT 242 (5.03 t/ha) in the second season. The inoculum level was measured across the field by (qPCR), and a map of the infestation was created. The highest spore concentration was found on the field entrance. Collectively, the information obtained on the effectiveness of host resistance and pathogenic diversity of P. brassicae populations from the Czech Republic may help to more effectively manage clubroot in this country.
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Bionomics and physiology of stored product mite Tyrophagus putrescentiae (Acari: Acaridida)
Rybanská, Dagmar ; Ryšánek, Pavel (advisor) ; Vladimír, Vladimír (referee)
The infestation of foodstuffs by mites is connected to health risks and economic losses. Tyrophagus putrescentiae (Schrank, 1781) is a cosmopolitan generalist feeder that prefers foodstuffs of high-fat and high-protein content. The stored product mite T. putrescentiae is an emerging and predominant pest of dry dog food. It is an emerging source of allergens in stored products and homes. The results of this study have important implications for predictive models of stored-product mite populations in dry dog food. High-fat and -protein food accelerated T. putrescentiae population growth compared with the high-carbohydrate diet.
The results increase the medical importance of the mite allergens detected in the feces. The cocktail of allergens, i.e. feces covering chitin, mite allergens and bacterial toxins, represent high risks for the mammalian immune system. The association of the mite with Bacillus cereus is to the medical importance.
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Introducing targeted epigenetic changes in Solanum tuberosum and Nicotiana benthamiana by plant viruses
Dušek, Jakub ; Ryšánek, Pavel (advisor) ; Barbora, Barbora (referee)
In this thesis we have focused on the induction of targeted epigenetic changes using modified Potato Virus X vector (PVX, Potato virus X). The gene coding sequence phytoene desaturase (PDS) was isloated from Solanum tuberosum and Nicotiana benthamiana plants using PCR. Primers were specifically designed as universal for both plant species and their Tm temperatures was in the range of 5 °C. The primers also added flanking restriction sites XhoI and ClaI to simplify cloning of PDS PCR products into full length PVX viral vector in AS orientation. The PVX viral vector pGR106 was later introduced into the plants using Agrobacterium tumefaciens. Infection led to the degradation of the target transcript and to efficient gene silencing (PTGS posttranscriptional gene silencing). The process has been manifested by complete or partial fading of a green plant tissue phenotype..
Some of the plants were aslso inoculated in in vitro conditions, for which a novel technique has been implemented.
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Mutational analysis of Potato virus X (PVX) coat protein
Werschallová, Markéta ; Ryšánek, Pavel (advisor) ; Jan, Jan (referee)
The thesis deals with the mutational analysis of conserved amino acids of Potato virus X coat protein (PVX CP). The importance of selected amino acids for the spread of the virus in the plant should be determined. Nicotiana benthamiana was selected as an experimental plant. Mutations of the PVX CP were based on the comparison of PVX CP amino acid sequence with the sequence of Papaya mosaic virus coat protein (PapMV CP), the only representative of potexvirus, which includes PVX, with the described crystal structure of the CP. The importance of certain amino acids for interaction of coat protein subunits PapMV CP,CP and PapMV CP,RNA and thus for virus particles formation was experimentally determined. The available amino acid sequences of isolates and strains of PVX CP were obtained from the National Center for Biotechnology Information (NCBI) database, compared with each other and alsowith the sequence of PVX CP used in the Laboratory of Virology, Institute of Experimental Botany of the CAS. Codons encoding conserved phenylalanine and lysine at positions 33 and 118 in the PVX CP amino acid sequence were mutated using methods of molecular biology.
Five constructs of PVX CP mutants were prepared, Two deletion mutants of the PVX CP N-terminus which were created in the vector derived from PVX (pGR106) in which the cDNA sequence of PVX is integrated, the remaining 3 point mutants were prepared only as a product of SOE PCR. The reporter gene GFP for monitoring of infection and spread of mutants in plant tissue was cloned into pGR106 carrying the deletion mutants of the N-terminus of PVX CP (deletion of 2.-32. or 2.-33. amino acid). Both mutants are able to move only in a short distance from infected cells to adjacent cells within the inoculated N. benthamiana leaf. These two deletion mutants showed difference in the speed and in the extent of the GFP signal spread. Deletion mutant still possessing the codon for F33 showed faster onset of the GFP signal and was able to spread more rapidly to surrounding cells in comparison with deletion mutant, where the codon for F33 was removed. Other mutants carrying the point mutations were also prepared: the deletion mutant of the codon for F33 (deletion F33), the substitution mutant, in which the codon for phenylalanine at position 33 was replaced by the codon for alanine (F33A) and substitution mutant, where the codon for lysine at position 118 was replaced by the codon for alanine (K118A). Unfortunately, all three point mutants could not be cloned into the vector pGR106, therefore, the evaluation of their spread in N. benthamiana plants was not possible.
Based on obtained results it is possible to conclude that the amino acid F33 is important for the intercellular movement within the neighboring cells. To assess the importance of the amino acids F33 and K118 in the systemic infection, it would be necessary to evaluate also the point mutants deletion F33, F33A, and K118.
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