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Functional analysis of the ERK signaling pathway in epithelial cells
Galvánková, Kristína ; Vomastek, Tomáš (advisor) ; Rösel, Daniel (referee)
The MAPK/ERK pathway, which is evolutionarily conserved in eukaryotes is one of the most intensively studied signaling pathways and consists of a three-tier cascade of Raf- MEK-ERK protein kinases. A variety of extracellular signals are transduced from receptors to hundreds of substrates by a series of sequential phosphorylations leading from Raf to MEK to ERK. The ERK pathway regulates a plethora of cell- and extracellular signal- specific responses such as gene expression, proliferation, differentiation, migration, and apoptosis. The proper execution of these physiological processes requires a precise temporal and spatial regulation of the pathway and disruption of the regulatory mechanisms leads to pathological consequence such as tumor transformation. Specificity and regulation of signal transduction are provided in part by the presence of isoforms at each level of the ERK signaling pathway. The functional differences between the effector protein kinases ERK1 and ERK2 have been controversial for a long time, but it is still unclear how important they are in achieving an appropriate cellular response. In this work, we focused on the functional characterization of ERK1 and ERK2 isoforms in MDCK epithelial cells. Specifically, we examined the effects of ERK2 inactivation on cell morphology and...
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Role of bacteria and mucosal immune system and their interaction in the pathogenesis of inflammatory bowel disease
Du, Zhengyu ; Hudcovic, Tomáš (advisor) ; Prokešová, Ludmila (referee) ; Kamanová, Jana (referee)
Although the etiology and pathogenesis of inflammatory bowel disease (IBD) is not fully understood, it is generally accepted that the inflammation results from aberrant immune responses to antigens of gut microbiota in genetically susceptible individuals (Sartor et al., 2006). Alteration in intestinal microbiota has been found in IBD patients with increased abundance of certain bacteria and decreased abundance of others. Due to the complexity of the disease, multifaceted interactions between genetic factors, host immune response, gut microbiota and environment factors need to be taken into account. In this thesis, the pathogenesis of IBD was first reviewed in respect with the four factors mentioned above. Then we concentrated on the interaction between IBD-associated bacteria and mucosal immune system. We investigated the ability of mucosal-associated bacteria (MAB) from IBD patients to induce spontaneous colitis in germ-free (GF) mice and the impact of those bacteria on the development of dextran sulfate sodium (DSS)-colitis. Together with the analysis of the composition of gut microbiota of MAB colonized mice, we demonstrated the potential deleterious microbes were able to increase the susceptibility to DSS-colitis once they found a suitable niche. We revealed the mechanism of an E.coli strain...
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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee) ; Herichová, Iveta (referee)
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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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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Phenotypical characterization of the healthy human cornea and the alterations caused by posterior polymorphous corneal dystrophy
Reinštein Merjavá, Stanislava ; Jirsová, Kateřina (advisor) ; Martínek, Jindřich (referee) ; Čejková, Jitka (referee)
Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...
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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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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee) ; Herichová, Iveta (referee)
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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Phenotypical characterization of the healthy human cornea and the alterations caused by posterior polymorphous corneal dystrophy
Reinštein Merjavá, Stanislava ; Jirsová, Kateřina (advisor) ; Martínek, Jindřich (referee) ; Čejková, Jitka (referee)
Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...
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Development of tool for passive immunization of cystic fibrosis patients
Pacholíková, Lenka ; Hodek, Petr (advisor) ; Švédová, Martina (referee)
Cystic Fibrosis (CF) is one of the most common heriditary diseases. This congenital condition, caused by CFTR gene mutation, affects gastrointestinal and respiratory system especially. The affection of respiratory system is considered the most serious life-threatening symptom. Pacients suffer from reccurent infections proceeding to the development of chronic inflammation and progressive pulmonary tissue destruction. A typical specific microoganism colonizating pulmonary tissue of thouse suffering from CF is recognised as Pseudomonas aeruginosa. Pulmonary infections caused by this microorganism are the most often cause of death in patients suffering from CF. Antiobiotics are the first-line therapy of this condition currently. Nevertheless, the need to find alternatives occurs due to antibiotics resistance development. Passive immunization by specific hen-egg-yolk antibodies against P. aeruginosa is a possible alternative. An observation of IgY influence on a bacterial adhesion to pulmonary epithelial cells required an appropriate model. In this context an appropriate adhesion testing method based on P. aeruginosa and pulmonary epithelial cells visualisation was searched. At first bacterial cells labelling was tested by CellTracker, resazurin, FITC and consequently PKH 26. P. aeruginosa fluorescent...
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Role of bacteria and mucosal immune system and their interaction in the pathogenesis of inflammatory bowel disease
Du, Zhengyu ; Hudcovic, Tomáš (advisor) ; Prokešová, Ludmila (referee) ; Kamanová, Jana (referee)
Although the etiology and pathogenesis of inflammatory bowel disease (IBD) is not fully understood, it is generally accepted that the inflammation results from aberrant immune responses to antigens of gut microbiota in genetically susceptible individuals (Sartor et al., 2006). Alteration in intestinal microbiota has been found in IBD patients with increased abundance of certain bacteria and decreased abundance of others. Due to the complexity of the disease, multifaceted interactions between genetic factors, host immune response, gut microbiota and environment factors need to be taken into account. In this thesis, the pathogenesis of IBD was first reviewed in respect with the four factors mentioned above. Then we concentrated on the interaction between IBD-associated bacteria and mucosal immune system. We investigated the ability of mucosal-associated bacteria (MAB) from IBD patients to induce spontaneous colitis in germ-free (GF) mice and the impact of those bacteria on the development of dextran sulfate sodium (DSS)-colitis. Together with the analysis of the composition of gut microbiota of MAB colonized mice, we demonstrated the potential deleterious microbes were able to increase the susceptibility to DSS-colitis once they found a suitable niche. We revealed the mechanism of an E.coli strain...
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