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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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Intercellular interactions in skin tumors.
Kučera, Jan ; Smetana, Karel (advisor) ; Masařík, Michal (referee) ; Kovář, Marek (referee)
The dissertation is focused on the study of intercellular interactions in skin tumors. It is based on 5 original publications that cover several topics. We studied the origin of tumor-associated fibroblasts concerning the primary tumor population. We demonstrated using a mouse model that tumor-associated fibroblasts are produced from the host organism and thus did not arise from transformation directly from tumor cells. We also investigated the relationship between tumor-associated fibroblasts and keratinocytes. We have shown that tumor-associated melanoma fibroblasts affect keratinocytes which, under their influence, acquire the features typically observed in migrating cells and cells undergoing epithelial-mesenchymal transition. We studied the interactions between healthy fibroblasts and tumor cells. We have demonstrated that fibroblasts acquired from healthy skin from a patient suffering from melanoma are significantly different from control fibroblasts of healthy donors in the expression profile. Changes in distal fibroblasts support the view of melanoma as a systemic disease. We have further demonstrated that melanoma-associated fibroblasts do not carry a BRAF mutation, in contrast to BRAF positivity of melanoma cells. And therefore, they did not arise from the transition from melanoma. The...
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Physiological and pathophysiological role of GCPII in the body
Sedlák, František ; Konvalinka, Jan (advisor) ; Klener, Pavel (referee) ; Smetana, Karel (referee)
Glutamate carboxypeptidase II (GCPII) is a metalloprotease responsible for cleaving the neurotransmitter N-acetyl-aspartyl-glutamate in the central nervous system to N-acetyl aspartate and glutamate. At the same time, in the human small intestine, it facilitates folate absorption by cleaving γ-linked glutamate from folyl-poly-γ-glutamate. In humans, GCPII is also expressed in a number of other organs (e.g., kidney and prostate) and tumors, where its physiological function is unknown. In an attempt to characterize the physiological function of the enzyme, we first characterized the commercially available monoclonal antibodies against GCPII. Further, we developed a fully synthetic replacement based on a hydrophilic polymer with bound GCPII inhibitors. We evaluated the suitability of using a murine biomodel to study GCPII function in vivo. We found the difference in GCPII expression profile in mouse and human. We did not observe GCPII in either the mouse prostate or small intestine. To assess physiological and pathophysiological functions of the enzyme we analyzed a GCPII-deficient mouse model. Apart from the observation of enlarged seminal vesicles in older males, we did not detect any other obvious phenotype. Similarly, we confirmed that GCPII cannot cleave amyloid peptides (Aβ1-40 and Aβ1-42)....
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Role of fibroblasts in wound healing and cancer
Mateu Sanz, Rosana ; Smetana, Karel (advisor) ; Masařík, Michal (referee) ; Mokrý, Jaroslav (referee)
Fibroblasts are stromal cells ubiquitously present in the human body. They often appear in a quiescent state and can become activated in response to tissue remodeling signals. Activated fibroblasts acquire biosynthetic, pro-inflammatory and contractile properties, key functions for wound healing. In addition, the presence of permanently activated fibroblasts is one of the hallmarks of cancer. The purpose of this work is to investigate the differences between newborn and adult fibroblasts and keratinocytes in their implication in scarless wound healing, the origin of cancer associated fibroblasts (CAF)s and the influence of fibroblasts in melanoma invasion. Evidence suggests that wounds heal almost without scar in newborns. To understand the mechanisms that contribute to scarless wound healing we focused on the differences between newborn and adult fibroblasts and keratinocytes, which are cells present in human skin and participating in wound healing process. A comparison of the expression profile between newborn and adult fibroblasts showed differentially regulated genes related to the acute phase of the inflammatory response and ECM organization, traits involved in wound healing. We also found that newborn fibroblast showed higher differentiation potential, exhibited markers of pluripotency and...
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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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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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Healing wound as a model for the study of cell interactions
Gál, Peter ; Smetana, Karel (advisor) ; Motlík, Jan (referee) ; Brábek, Jan (referee)
Healing wound as a model for the study of cell interactions Abstract Galectins play an important role in the processes of cell proliferation, differentiation, migration and extracellular matrix formation. Furthermore, galectins are able to transfer cellular signals and to participate in cell interaction. It has been proven that galectins play an important role in the microenvironment formation of a tumor and/or healing wound. This study demonstrated significant role of galectins, in particular Galectin-1, in wound healing and cell interactions (endothelial cells, fibroblasts and keratinocytes) forming a part of the granulation tissue and tumor stroma. We have demonstrated that the extracellular matrix rich on Galectin-1 creates a suitable environment for the cultivation of keratinocytes. Galectin-1 also induces differentiation of fibroblasts into myofibroblasts. The knowledge of above mentioned processes is important to better understand the complexity of cancer biology and its parallel to wound healing. Key words: tissue repair, regeneration, galectin, tumor
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Structure-function organization of the cell nucleus.Microscopical analysis of nuclear subcompartments.
Jůda, Pavel ; Cmarko, Dušan (advisor) ; Mokrý, Jaroslav (referee) ; Kučera, Tomáš (referee) ; Smetana, Karel (referee)
Pavel Jůda - Abstract The cell nucleus is a complex cellular organelle. The nucleus and nuclear processes are organized into functionally and morphologically separated nuclear subcompartments. This thesis is particularly concerned with the three following nuclear subcompartments: sites of DNA replication, Polycomb bodies and nuclear inclusions constituted of inosine monophosphate dehydrogenase 2 (IMPDH2). First, we examined the relationship between MCM proteins and DNA replication. Using immunofluorescent labeling of cells extracted prior fixation and applying cross-correlation function analysis, we showed that MCM proteins are present at the sites of active DNA synthesis. Our results contributed to the solving of the first part of so-called MCM paradox. Second, we studied the structural basis of the Polycomb bodies. Based on fluorescence microscopy studies, Polycomb bodies have been considered to be the nuclear subcompartments formed by the accumulation of Polycomb proteins in the interchromatin compartment. In our work, using correlative light electron microscopy and experimental changes in macromolecular crowding, we clearly showed that a Polycomb body is a chromosomal domain formed by an accumulation of heterochromatin structures, rather than a typical nucleoplasmic body. Third, we were interested in...
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Glycobiology of the head and neck cancer
Valach, Jaroslav ; Smetana, Karel (advisor) ; Dřízhal, Ivo (referee) ; Říhová, Blanka (referee)
iii Abstract Glycobiology represents a very progressive subject of cell biology. Protein-saccharide interactions play not only supporting and cell organization role, but they also represent medium for information storage and its decoding. Galectins, group of animal lectins (saccharide-binding proteins), which have selective affinity to ß-galactosides, are multifactorial molecules. They participate in cell-cell and cell-matrix interaction, transmembrane signaling, apoptosis, pre- mRNA splicing and are also present in various types of carcinomas. High expression of galectin-1 has been detected in cancer stroma originated from squamous cell epithelium. In the previous study we established that the fibroblasts - myofibroblasts transition, apart from the known TGF- beta, is also induced by galectin-1. We compared relationship between galectin-1 expression, presence of myofibroblasts and gene expression in tissue samples from patients with head and neck squamous cell carcinoma. Cancer stroma with myofibroblasts was rich in galectin-1 expression in comparison with stroma without myofibroblasts. Moreover, we used microarray analysis (ILLUMINA) to compare the whole genome transcriptome from samples with and without presence of galectin-1. High expression of galectin-1 in tissue samples corresponded with expression...
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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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