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Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii
Žídek, Radim
Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...
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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
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Sexual dimorphism of rat gut microbiota composition and intestinal immunity
Kalousová, Pavla ; Kovařík, Miroslav (advisor) ; Jílek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Pavla Kalousová Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Title: Sexual dimorphism of rat gut microbiota composition and intestinal immunity Background and Aim: Many factors can influence the composition of gut microbiota and the immune system. It is well-known that one of those factors is sex. This sexual dimorphism can lead to a specifically adjusted treatment of diseases for different gender and nutritional interventions. This study focuses on analysing sexual differences in gut microbiota and intestinal immunity in adult rats. Methods: Caecal content from 12-week-old female and male Wistar rats were collected and analysed by DNA-sequencing technique to characterize microbiota composition. ELISA test was performed to quantify the concentration of IgA in faeces and gut wash. Flow cytometry determined the concentration of IgA-coating bacteria in faecal samples. Results: Metagenomic analysis revealed that female gender presents 1 phylum, 4 families, 13 genera, and 13 species which are not present in male rats. Only one male-specific colonization was observed at the species level. Quantitative analysis showed a higher proportion of Firmicutes phylum in males which was associated with...
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Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii
Žídek, Radim
Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...
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Sexual dimorphism of rat gut microbiota composition and intestinal immunity
Kalousová, Pavla ; Kovařík, Miroslav (advisor) ; Jílek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Pavla Kalousová Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Title: Sexual dimorphism of rat gut microbiota composition and intestinal immunity Background and Aim: Many factors can influence the composition of gut microbiota and the immune system. It is well-known that one of those factors is sex. This sexual dimorphism can lead to a specifically adjusted treatment of diseases for different gender and nutritional interventions. This study focuses on analysing sexual differences in gut microbiota and intestinal immunity in adult rats. Methods: Caecal content from 12-week-old female and male Wistar rats were collected and analysed by DNA-sequencing technique to characterize microbiota composition. ELISA test was performed to quantify the concentration of IgA in faeces and gut wash. Flow cytometry determined the concentration of IgA-coating bacteria in faecal samples. Results: Metagenomic analysis revealed that female gender presents 1 phylum, 4 families, 13 genera, and 13 species which are not present in male rats. Only one male-specific colonization was observed at the species level. Quantitative analysis showed a higher proportion of Firmicutes phylum in males which was associated with...
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Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii
Žídek, Radim ; Kozmik, Zbyněk (advisor) ; Janečková, Lucie (referee) ; Macůrková, Marie (referee)
Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...
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Bioaktivní látky ve vodním prostředí a jejich vliv na ryby - zaměření na cytochromy P450 v rybách
SAKALLI, Sidika
The term "biologically active compounds" covers a wide range of substances originating from natural or synthetic origins. These compounds can enter the aquatic environment through wastewater treatment plants, manufactural discharge or they are naturally present in the aquatic plants or microorganisms. Their adverse effects on fish has been widely studied and accepted. This thesis is focussed on the adverse effect of several bioactive compounds (i.e. pharmaceuticals, phytochemicals, or complex mixture of pollutants) on fish using different experimental design as in vitro, in vivo and in situ. In the first part of this thesis, in vitro effects of pharmaceuticals and phytochemicals or their combinations on fish CYP system were investigated. Moreover, effects of standard carrier solvents used in enzyme activity assays were also investigated. An important finding in this study was the lack of effects of either dexamethasone, quercetin, or indole-3-carbinol on EROD activity; however, when these agents were combined, EROD activity was strongly inhibited. This demonstrates that combination of compounds might exert different effects than single compounds, and the effects of mixture compounds cannot be predicted from the effects of individual compounds. In the second part of the thesis, the effects of chronic exposures of rainbow trout to dexamethasone and clotrimazole were investigated. The results regarding dexamethasone showed similarities with in vitro studies, and both in vitro and in vivo exposure of dexamethasone did not alter the CYP enzyme activities. On the other hand, in vivo exposure of clotrimazole yielded conflicting results with findings from the in vitro studies. Clotrimazole induced EROD activity in chronically exposed rainbow trout, and BFCOD activity showed biphasic pattern in which it was inhibited at environmentally relevant concentrations and induced at high concentrations. Thus, the observed effects suggest that clotrimazole could negatively affect fish CYPs at environmentally relevant concentrations. However, in the environment, the effects of clotrimazole and dexamethasone on fish CYPs system might be modified because of unknown compounds in these mixtures. Therefore, further investigations were done to identify the effects of mixture compounds using an in situ model. The last part of the dissertation addresses the effects of cocktail PPCPs on common carp under natural conditions. In situ studies provide valuable information on both hepatic and intestinal CYP activities. Both EROD and BFCOD activities were affected by the PPCPs that are present in the exposed fish. Moreover, changes in intestinal CYP activities suggest that fish can ingest some of these contaminants through their feed. Therefore, the intestines might be responsible for elimination of some of these pollutants and act as the first barrier of pollutant entry in fish. Despite the extensive studies concerning aquatic pollution, further studies are necessary. Development of new pharmaceuticals, their occurrence in the aquatic environment, and their effects on non-target organisms should be continuously monitored.
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Study of interactions of antiviral drugs with intestinal drug efflux ABC transporters
Huličiak, Martin ; Červený, Lukáš (advisor) ; Vokřál, Ivan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martin Huličiak Supervisor: PharmDr. Lukáš Červený, Ph.D Title of diploma thesis: Study of interactions of antiviral drugs with intestinal drug efflux ABC transporters P-gp, MRP2 and BCRP are efflux transporters, members of the family of ATP binding cassette (ABC) transporters. These transporters are located on the apical membrane of the intestinal epithelium, where they may limit absorption of orally administered drugs. Study of drug interactions with/on intestinal efflux transporters is necessary to provide safe and effective treatment. The Caco-2 cell line is FDA recommended in vitro model of intestinal barrier and it is used for bidirectional testing of substrates and inhibitors of ABC transporters in preclinical research. However, this methodology has several shortcomings, so the need of introduction of new experimental models is increasing and the ex vivo method based on human or rat intestine is a promising option. Precision-cut intestinal slices (PCIS) represent a mini-model of the organ and contain all types of cells of the tissue. We used both in vitro model using Caco-2 cell monolayers for drug transport study and in our lab established ex vivo method of PCIS for accumulation study...
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In vitro cultivation of the trematode species Trichobilharzia regenti
Vrbová, Kristýna ; Kašný, Martin (advisor) ; Skelly, Patrick (referee)
The class Trematoda includes many pathogenic representatives. Main subject of this thesis, avian schistosome Trichobilharzia regenti, is a close relative to the important human pathogen Schistosoma mansoni (family Schistosomatidae). In vitro cultivation of trematodes enables closer understanding of their biology and parasite- host interactions; however, no trematode species has been successfully kept in vitro from the egg stage to the adults producing eggs. Many studies are focused on the problematic of S. mansoni cultivation, but data concerning T. regenti cultivation remain scarce. Only the ability of T. regenti cercariae to transform into schistosomula in vitro was documented, with following survival in a culture medium for a few days. Comparison of eight transformation methods was performed with T. regenti cercariae. Based on the number of tailless cercarial bodies obtained, five transformation methods were selected for further evaluation of the early schistosomula characteristics (glycocalyx shedding, penetration glands emptying and survival in vitro). It was observed that the largest quantity of cercarial bodies can be obtained by using a syringe needle or the BeadBeater cell disrupter. The largest quantity of schistosomula meeting the criteria of early schistosomulum was recorded after...
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