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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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Variation of abnormalities of foramen obturatum and retropubic space and its relation to complications of tape surgery
Hubka, Petr ; Mašata, Jaromír (advisor) ; Smetana, Karel (referee) ; Krofta, Ladislav (referee)
Introduction: The knowledge of anatomy is crucial for introduction of new surgical methods. It is also of especial use while dealing with surgical complications during surgeries with limited surgical field, where the way of approach limits the management of complications. It was assumed that common anatomical variations would influence different efficacy of surgeries and would explain potential complications. Methods: During anatomical dissections fifty female cadavers were dissected and tension-free vaginal tapes TVT-S H, TVT-S U, TVT Abbrevo and Ajust were studied. A novel descriptive system for localisation of the tape was created. During the dissection the tape was located and its localisation and fixation was described. Results: Common anatomical variation in the sample was corona mortis with frequency of 72 %. Preperitoneal fatty plug, which is recognized by some authors as the first stage of obturator hernia, was found in 40 % in obturator canal. The proper fixation of TVT-S H was achieved in 53.6 %. In 10.5 % urinary bladder was injured. In case of TVT-S U the proper fixation occurred in 63.8 %. In two cases the inserter was nearby corona mortis. Within the group of TVTO Abbrevo the tape was fixated properly into the obturator complex (consists of the obturator membrane and obturator muscles) in...
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Glycobiology of the head and neck cancer
Szabo, Pavol ; Smetana, Karel (advisor) ; Říhová, Blanka (referee) ; Brábek, Jan (referee)
Povrch buněk je bohatě pokryt oligosacharidy, které jsou v plazmalemě ukotvené pomocí proteinů a lipidů. Oligosacharidy zprostředkují vzájemnou vazbu mezi buňkami nebo vazbu buněk k složkám extracelulární matrix. Galektiny jsou živočišné lektinů které mají afinitu k oligosacharidům obsahujícím β-galaktózu. Jsou to multifaktoriální proteiny, které se účastňují řady reakcí v organizmu, jako jsou mezibuněčné interakce, interakce buněk s mezibuněčnou hmotou, proliferace i apoptóza a sestřih pre-mRNA. Proteiny po translaci procházejí různými strukturálnimy úpravami, které mají vliv na jejich funkci. Galektin-3 je možný prognostický ukazatel u nádorů vycházejících z vrstevnatých dlaždicových epitelů je fosforylován na N-konci. Prokázali jsme, že tato posttranslační modifikace nemá vliv na jeho vazebnou reaktivitu. Jiný endogenní lektin, galektin-1 je charakteristickou molekulou nádorového stromatu a granulační tkáně hojícího se poranění. Zjistili jsme, že galektin-1 indukuje na TGF-β nezávislý in vitro přechod normálních fibroblastů na myofibroblasty včetně produkce sítě extracelulární matrix bohaté fibronektinem a galektinem-1. Tento poznatek je využitelný v terapii hojení ran a v tkáňovém inženýrství. Dnes je jasné, že nádorové stroma ovlivňuje i biologické vlastnosti nádoru (lokální agresivita,...
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Interaction of bone and vascular cells with materials constructed for tissue replacement and drug delivery system
Vandrovcová, Marta ; Bačáková, Lucie (advisor) ; Smetana, Karel (referee) ; Jirka, Ivan (referee)
Interactions between biomaterials and cells play an important role in tissue engineering. It has been repeatedly shown that the cell behaviour strongly depends on physical and chemical properties of the material surface. In our study we focused on materials used for the construction of bone implants and replacements to support cell adhesion, growth and osteogenic diferenciation and to lead to an integration between an implant and a bone tissue. First we tested an influence of different micrpattern of fullerens' C60 and composites' C60/Ti films to adhesion of bone cells MG 63, their initial spreading, growth, viability and formation of cytosceletal protein actin. Some of these films were additionally iradiated with Au+ ions, which led to the conversion of some C60 molcules into amorphous carbon (a-C). We confirmed that pattern influenced the distribution of cells without decrease viability. Thus, the production of patterns could be used to direct cell adhesion. In the second study, we also observed the influence of the thickness of coated layer. We found that more selective cell growth can be achieved by depositing of thicker fullerens' film. Our study with osteoblast-like MG 63 cells cultured on PLGA films revealed, that surface coated with chosen components of extracellular matrix (colagen I and...
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Role of Smarca5 (Snf2h) during transcription of transfected DNA template.
Zikmund, Tomáš ; Stopka, Tomáš (advisor) ; Smetana, Karel (referee)
Cellular and tissue characteristics are results of dynamic regulation of gene expression. DNA wrapped into proteins, referred to as chromatin, requires involvement of mechanisms guiding accessibility of specific sequences. In higher organisms, chromatin remodeling proteins are indispensable in regulating chromatin structure including ISWI ATPase SMARCA5. SMARCA5 is involved in almost any transaction on DNA including transcription, however precise in vivo role of SMARCA5 in these processes remains unknown. To advance understanding of specific role of SMARCA5 in the development of chromatin structure during transcription we devised cellular model in which SMARAC5 level is manipulated while chromatin structure development and transcriptional response are monitored. Our data indicate that the transfected DNA template that is transcribed is enriched with histone H3 and its specific methylation of Histone H3 lysine (K) 4, a mark of active chromatin structure. Overexpression of SMARCA5 results within the reporter gene coding sequence in ~2,5-3 fold increase of both H3 occupancy an its modification H3K4Me3. Increased DNA template commitment into chromatinization is associated with repression of reporter gene expression. These results are supported by studies indicating dynamic development of nucleosomal...
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Relationship between Large-scale Chromatin Organization and Nucleolus in Human Cells
Kalmárová, Markéta ; Raška, Ivan (advisor) ; Nedvídek, Josef (referee) ; Smetana, Karel (referee)
In human cells ribosomal genes are organized as clusters called Nucleolus Organizer Regions (NORs) that are situated on the short arms of acrocentric chromosomes. It was found that essential components of the RNA polymerase I transcription machinery, including Upstream Binding Factor (UBF), can be detected on some NORs, termed "competent" NORs, during mitosis. The competent NORs are believed to be transcriptionally active during interphase. But since individual NORs cannot be observed in the cell nucleus, their interphase status remained unclear. To address this problem, we detected the competent NORs by two commonly used methods, UBF immunofluorescence and silver staining, and combined them with FISH for visualization of rDNA and/or specific chromosomes. We found that the numbers of competent NORs on specific chromosomes were largely conserved in the subsequent cell cycles, with certain NOR-bearing homologues displaying a very stable pattern of competence. Importantly, those and only those NORs, which were loaded with UBF, incorporated bromo-uridine in metaphase after stimulation with roscovitine and in telophase, suggesting that competent and only competent NORs contain ribosomal genes transcriptionally active during interphase. Applying premature chromosome condensation with calyculin A, we visualized...
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Glycobiology of squamous epithelium - in vitro and in vivo
Dvořánková, Barbora ; Smetana, Karel (advisor) ; Mandys, Václav (referee) ; Ulbrich, Karel (referee)
The skin is the largest organ of human body and its severe damage can cause even death of a patient. As the allografting of skin can not create permanent closure of wounds, the treatment (especially in cases of burn injuries) is rather demanding and prolonged. In my work I focused on determination of glycobiologic characteristcs of squamous cell epithelium, especially the skin, under normal and pathological conditions. Those results which we have obtained could be used for development of new methods of keratinocyte cultivation for clinical purposes. Epidermis was the very first human tissue that was prepared in vitro and returned back to the patient however the results of these transplantations were not satisfactory. (...) A new method of cultivation and transfer of keratinocytes on polymer support was developed. This way of cultivation of epidermal grafts exhibits some advantages compared with confluent sheets attached to textile. Namely the shortening of the cultivation period and the deletion of enzymatic detachment of the cells, that negatively influence their viability, are the main benefits. First of all the hydrogel support protects the cells from infection and desiccation after its application to the wound bed. So the optimal microclimate for the attachment and spreading of transplanted cells is...
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Expression of CD44 molecules in urothelial bladder cancer
Kuncová, Jitka ; Mandys, Václav (advisor) ; Smetana, Karel (referee) ; Hes, Ondřej (referee) ; Záťura, František (referee)
The main aim of presented work was to contribute to clarify the significance of changes in expression of adhesion molecules and CD44 in the malignant behavior of urothelial carcinomas of the urinary bladder. The individual stages to meet this goal were: 2.1 Detailed determination of expression of CD44s and CD44v6 protein in urothelial carcinomas of the urinary bladder in relation to the degree of tumor differentiation. 2.2 Determine the relationship of CD44 protein expression changes depending on the characteristics histomorphologic urothelial carcinoma of the urinary bladder, and other indicators of biological behavior of tumor (proliferative activity, p53 expression). 2.3 Evaluation of expression of CD44s and CD44v6 protein in two phenotypically distinct cell lines, urothelial carcinomas (HT1197 and 5637) and determine relationships CD44 expression on morphological and growth characteristics of tumor cells of these lines.
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The Use of Multichannel Flow Cytometry in Biomedicine and Experimental Biology
Kideryová, Linda ; Smetana, Karel (advisor) ; Elleder, Milan (referee) ; Klíma, Jiří (referee)
Flow-cytometry is a process on which large numbers of single cells are quantitatively and qualitatively analyzed. This method gives information about size, granularity surface or intracellular markers of every single cell in suspension. In modern biology is worthy to perform quick, objective multiparametric analyses of cell phenotype. This project was focused on cells, which analyses are complicated by extreme rareness or lack of clearly identifying specific markers. Analysis of stromal cells of the investigated tumors (histiocytoma and tumor fibroblasts originating in squamous epithelium: basalioma (BCCF) and spinalioma (SCCF) elucidated alteration of gene expression induced by tumor cells. Tumor-derived stromal fibroblasts acquire distinct properties to shape a microenvironment conducive to altering the phenotypic characteristics of normal epithelial cells in vitro. Reproducible, quick and highly sensitive method of detection extremely rare non-haematopoetic cells (EPC, CEC) was established. Numbers of CFU-En correlate neither with circulating endothelial progenitors nor with matured endothelial cells detected by flowcytometry. These colonies are formed in cooperation of CD14 + and CD4+ cells. Numbers of endothelial progenitors and matured endothelial cells are closely related with vessel endothelium...
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