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The role and function of stromal enzymes in keratoconus pathogenesis
Ďuďáková, Ľubica ; Jirsová, Kateřina (advisor) ; Svozílková, Petra (referee) ; Ardan, Taras (referee)
Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...
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Cell and Molecular Characterization of Failed Human Corneal Grafts. The Role of Matrix Metalloproteinases in Recurrent Corneal Melting.
Brejchová, Kristýna ; Jirsová, Kateřina (advisor) ; Smetana, Karel (referee) ; Heissigerová, Jarmila (referee)
The aim of this work was to investigate the contribution of matrix metalloproteinases (MMPs) to recurrent corneal melting. Twenty three melted corneas from seven patients were separated into three groups: a) patients with primary Sjögren's syndrome, b) those with rheumatoid arthritis and c) those with other corneal melting underlying pathologies. Eleven cadaverous corneas served as controls. The presence of MMP-1, -2, -3, -7, -8, -9, and -13 was detected using indirect enzyme immunohistochemistry. The active forms of MMP-2 and -9 and MMP- 3 and -7 were examined by gelatin and casein zymography, respectively. The concentrations of active MMP-1 and -3 were measured using activity assays. Increased immunostaining intensity for MMP-1, -2, -3, -7, -8 and -9 was shown in the corneal epithelium and the stroma of almost all melted corneas from all three groups compared to the negative or slightly positive staining of the controls. In the endothelium, immunostaining for MMP-2 and MMP-9 was increased in most specimens of groups II and III and group I, respectively. A markedly higher level of active MMP-2 was detected in six, and active MMP-9 in all, pathologic specimens compared to the controls. In contrast to the completely negative controls, the proenzymes of MMP-3 and -7 were detected in almost all melted...
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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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Use of corneal endothelium and amniotic membrane for transplantation purposes.
Šmeringaiová, Ingrida ; Jirsová, Kateřina (advisor) ; Netuková, Magdaléna (referee) ; Čejková, Jitka (referee)
Part I: Endothelial cells form the posterior layer of the cornea and are important for maintaining its transparency. Dysfunctional endothelium can only be restored by transplantation. The global shortage of donor corneas requires the search for alternative treatments. The preparation of the graft by tissue engineering methods is complicated by low proliferative capacity of endothelium. To date, no endothelium-specific marker has been defined and the existence of endothelial stem cells has not been confirmed yet. We have prepared a protocol for culturing endothelial cells from research-grade tissue - corneoscleral rims obtained after transplantation or corneas excluded from the transplant process. We monitored localization of selected proteins, including stem cell markers, in native tissue and in primary cell cultures. We prepared up to 6.4 cm2 of endothelium from one cornea/rim, which had cellular features comparable to the native endothelium. This approach can increase the amount of endothelium for research or transplantation purposes. Using indirect immunohistochemistry, we showed that none of the previously proposed endothelial molecular markers is specific for these cells. We detected the expression of stem cell markers throughout the endothelial layer. In the porcine cornea model, we monitored...
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The culture of limbal and mesenchymal cells on various feeders for their use in ophthalmology.
Trošan, Peter ; Jirsová, Kateřina (advisor) ; Heissigerová, Jarmila (referee) ; Netuková, Magdaléna (referee)
P.Trošan Ph.D. Thesis Abstract Limbal stem cell deficiency (LSCD) is a disease characterized by the deficiency of stem cells in the limbus, which are responsible for the homeostasis and renewal of the corneal epithelium. This disorder results in corneal neovascularization, chronical inflammation and opacification, which may lead to loss of vision. The most successful treatment is the transplantation of limbal tissue or cultured limbal epithelial cells (LECs) onto the damaged ocular surface. The human amniotic membrane (HAM) is used as the feeder of the LECs culture, as well as for the LSCD treatment. HAM is also widely used in clinical practice, particularly for the treatment of chronic wounds. This dissertation is particularly concerned on cell therapy for LSCD, on preparation of cells suitable for grafting onto the ocular surface, on the improvement of the LECs culture conditions, and on the preparation of appropriate carrier for the transfer of cells onto the damaged cornea. During my work I have used a wide spectrum of methods, e.g. cell cultures (LECs, mesenchymal stem, amniotic epithelial, conjunctival epithelial, goblet and 3T3 cells), immunohisto- and immunocytochemistry, microscopy, proliferation and colony forming assays, reverse transcription and quantitative real-time PCRs and statistical...
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Nutrition for oncological diseases
Strejčková, Vendula ; Jirsová, Kateřina (advisor) ; Aschermannová, Alexandra (referee)
Sufficient and quality nutrition is an important support for mastering serious oncological treatment and debilitating illness. Educating the public about the risk of oncological disease related malnutrition and the importance of a balanced diet, incidence of new malignanccies increases every year. 94 000 people as the newly diagnosis were in 2015. Nearly 542 000 people lived with cancer diagnosis in 2015. Not only among patient it is widespread idea that malnutrition is something that can not concern us because we are doing good. Many patients do not have insight into their nutritional status. They do not think they are at risk of malnutrition. But if someone gets cancer, sooner or later, the symptoms of the disease will appear, and along with demanding treatment that brings a lot of side effects, and worsing of oral intake, and weight loss is coming. The theoretical part describes the causes of the cancer disease, its influence on the organism, the most common types of anticancer treatment together with the side effects and the possibilities of their management. The second half of the theoretical work deals with the evaluation of nutritional risk in oncological patients, nutritional recommendations and in case of insufficient nutrition for any reason, as well as nutritional support. Objective...
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The role and function of stromal enzymes in keratoconus pathogenesis
Ďuďáková, Ľubica ; Jirsová, Kateřina (advisor) ; Svozílková, Petra (referee) ; Ardan, Taras (referee)
Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...
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