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Spontaneous Regression of Melanoma in Pigs of the MeLiM Strain
Plánská, Daniela ; Horák, Vratislav (advisor) ; Smetana, Karel (referee) ; Bartůňková, Jiřina (referee)
Melanoma is a skin tumour arising from melanocytes - skin cells bearing pigment melanin. Melanoma belongs among immunogenic tumours, which is probably associated with a relatively high incidence of partial spontaneous regression (SR). Melanoma-bearing Libechov Minipigs (MeLiM) represent a specially bred animal model that is mostly affected by nodular melanomas, which fully regressed in about 2/3 of the affected animals. Our interest was to examine immune response (associated with melanoma cell destruction) and the role of proteins related to the extracellular matrix (reflecting tissue remodeling) during SR of MeLiM melanoma. We performed an extensive time-lapse study of skin melanomas taken from individuals of the MeLiM strain at 3, 4, 6, 8, 10, 12, 20 and 32 weeks (5-10 samples in each age category) in which we immunohistochemically detected the expression of collagen IV, laminin, fibronectin, tenascin C, as well as MMP-2 and we monitored the proportion of basic immune subpopulations in blood and tumour by flow cytometry. The higher expression of collagen IV, laminin and MMP-2 positively correlated with the appearance of melanoma cells. The expression of collagen IV and laminin indicates a possible survival of tumour cells due to the interaction with these proteins, the presence of MMP-2 in these...
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The effect of carbon nanostructures on human cell behavior and the role of fetal bovine serum in cell adhesion
Verdánová, Martina ; Hubálek Kalbáčová, Marie (advisor) ; Brábek, Jan (referee) ; Smetana, Karel (referee)
Graphene (G) and nanocrystalline diamond (NCD) are carbon allotropes and promising nanomaterials with an excellent combination of their properties, such as high mechanical strength, electrical and thermal conductivity, possibility of functionalization and very high surface area to volume ratio. For these reasons, G and NCD are employed next to electronics in biomedical applications, including implant coating, drug and gene delivery and biosensing. For a fundamental characterization of cell behavior on G and NCD, we studied osteoblast adhesion and proliferation on differently treated G and NCD. Generally, both G and NCD exhibited better properties for osteoblast cultivation than control tissue culture polystyrene. Better cell adhesion but lower cell proliferation were observed on NCD compared to G. The most surprising finding was that hydrophobic G with nanowrinkled topography enhanced cell proliferation extensively, in comparison to hydrophilic and flat G and both NCDs (hydrophobic and hydrophilic) with slightly higher roughness. Promoted cell proliferation enables faster cell colonization of G and NCD substrates, meaning faster new tissue formation which is beneficial in biomedical applications. Furthermore, it was shown that osteoblast adhesion was promoted in the initial absence of fetal bovine...
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Anti-tumor activity and toxicity of HPMA copolymer conjugates bearing cytostatic drug
Tomalová, Barbora ; Kovář, Marek (advisor) ; Smetana, Karel (referee) ; Špíšek, Radek (referee)
7 ABSTRACT In this study, we addressed the biological activity and pharmacological features of selected HPMA copolymer-based drug conjugates. We determined their cytostatic activity in vitro as well as toxicity in vivo and therapeutic effcicacy in mouse tumor models. Assessment of maximum tolerated dose (MTD) of two structurally different HPMA copolymer-based conjugates bearing doxorubicin (DOX) attached via pH-sensitive hydrazon bond (HPMA- DOXHYD ) showed that high molecular weight non-degradable star HPMA-DOXHYD conjugate possesses relatively low MTD ~22.5 mg DOX/kg, while linear HPMA-DOXHYD has MTD ~85 mg DOX/kg. Thus, MTD of linear conjugate is 3.7 times higher than that of the star conjugate. Subsequently, we reported that linear conjugate proved to be more efficient in case of treatment of solid tumor EL4 lymphoma and star conjugate to be superior in case of BCL1 leukemia treatment. We also compared biological activity of star and linear HPMA copolymer-based conjugates bearing docetaxel (DTX) attached via pH-sensitive hydrazon bond (HPMA-DTXHYD ). MTD of star conjugate (~160 mg DTX/kg) was proved to be 4 times higher than MTD od free DTX (40 mg/kg). We were not able to determine MTD of linear conjugate as it exceeded 200 mg DTX/kg (the highest soluble dose we were able to administer as a bolus)....
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Studies of intercellular interactions in tumours
Jechová, Alžběta ; Smetana, Karel (advisor) ; Skalníková, Helena (referee) ; Masařík, Michal (referee)
Beside tumor cells themselves, tumors consist of many non-malignantly transformed cellular elements and an extracellular matrix. This so-called tumor microenvironment, or stroma, significantly influences the biological properties of the tumor through intercellular interactions. In this thesis I have focused on the study of tumor-associated fibroblasts in squamous cell carcinomas of the head and neck, malignant melanoma and glioblastoma. The data show the presence of cells with mesenchymal characteristics, present even in the glioblastoma stroma, which could potentially have a positive effect on proliferative activity and invasiveness of glioblastoma cells. In malignant melanoma, the presence of keratinocytes should also be considered, as they are the major cells of the epidermis influencing tumor melanocytes. The conditioned medium from UVB irradiated keratinocytes and non-irradiated fibroblasts stimulates the invasion of malignant melanoma cells. Targeting the tumor stroma may be a new direction in oncological therapy, so we have focused on the influence of synthetic polyamine on the formation of myofibroblasts, which are an active part of the population of tumor-associated fibroblasts. The tested polyamine prevents the formation of myofibroblasts but has no effect on those already formed nor on...
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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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Interactions of vascular and bone cells with bioactive polymers for construction of tissue replacements
Pařízek, Martin ; Bačáková, Lucie (advisor) ; Smetana, Karel (referee) ; Hrubý, Martin (referee)
This work deals with the interaction of cells with surface-modified existing or newly created materials developed for vascular and bone tissue engineering, and also for controlled drug delivery into implants. In the first part of this work, we modified the surface of the polyethylene foil by Ar plasma, and then we grafted them with bioactive molecules (glycine, polyethylene glycol, albumin) and with C or Au nanoparticles. These modifications improved the chemical and physical characteristics of the material for the adhesion and growth of vascular smooth muscle cells (VSMC), and also for their phenotypic maturation towards the contractile fenotype. In future, these modifications can be also used for material currently used for fabrication of clinically used vascular prostheses in order to increase their biocompatibility. The aim of the second part of this work was to develop a perivascular drug-delivery system that would release the antiproliferative drug Sirolimus. This perivascular system is designed to be wrapped around a venous graft, implanted to the arterial position, such as in the case of the aortocoronary bypass. The system comprises a polyester mesh, which ensures the mechanical stability of the system and of the venous wall, and a copolymer of L-lactide and ε-caprolactone (Purasorb), serving as a...
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International legal standing of Saarland in 1918-1935
Smetana, Karel ; Císař, Jaromír (advisor) ; Seltenreich, Radim (referee)
106 Závěr: Sársko jako zvláštní mezinárodněprávní útvar bylo vytvořeno na Pařížské mírové konferenci v důsledku snahy Francie zajistit si vlastnictví sárských uhelných dolů při jejím neustálém koketování s myšlenkou vylepšení své východní hranice. Francouzští zástupci se na mírové konferenci museli utkat především s prezidentem Spojených států Woodrowem Wilsonem, který se k francouzským nárokům z principiálních důvodů stavěl zcela odmítavě. Francii se však do značné míry přesto podařilo prosadit svou, což vyústilo ve zřízení autonomního Sárska stojícího na patnáct let pod správou Společnosti národů. Řešení přijaté Velkou čtyřkou na Pařížské mírové konferenci bylo něčím zcela novým, správa Sárska byla na patnáct let odejmuta Německu a svěřena na stejnou dobu nově založené Společnosti Národů, v které z počátku Německo nemělo ani svého zástupce. Společnost Národů spravovala Sársko prostřednictvím mezinárodně obsazené pětičlenné Komise. V této Komisi nejdříve zcela převládal vliv Francie reprezentovaný jejím autoritativním prvním předsedou Francouzem Victorem Rautem. Následně, zhruba od druhé poloviny dvacátých let, však tento vliv Francie v Komisi i v celém Sársku ustupoval a Sársko pak bylo spravován Komisí - jakousi mezinárodní vládou, "v duchu Společnosti Národů". Tato změna byla především následkem...
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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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Immunocompetent cells and their importance in immunopathological conditions
Podrazil, Michal ; Bartůňková, Jiřina (advisor) ; Reiniš, Milan (referee) ; Smetana, Karel (referee)
Pokroky v současné imunologii vedou k lepšímu porozumění interakcí mezi imunitním systémem a nádory, což vzbuzuje zájem o využití imunitního systému v léčbě nádorových onemocnění. V úvodní části práce jsou shrnuty teoretické poznatky o mechanismech protinádorové imunitní odpovědi a jednotlivých složkách imunitního systému podílejících se na tomto procesu. Ústřední úlohu v této interakci reprezentují dendritické buňky. Právě jejich využitím v klinické medicíně se dlouhodobě zabývá naše pracoviště. V této práci se věnuji několika dílčím cílům. První z nich je analýza zastoupení jednotlivých subpopulací imunokompetentních buněk v imunoterapeutickém léčivém přípravku na bázi dendritických buněk proti karcinomu prostaty- DCVAC/PCa vyvinutého na Ústavu imunologie, dále praktickým aspektům přípravy této formy aktivní buněčné imunoterapie a její optimalizaci za podmínek správné výrobní praxe. Výsledky této části byly součástí farmaceutické dokumentace, která vedla ke schválení výrobního postupu i zahájení klinických studií u pacientů s karcinomem prostaty regulačními autoritami. Dalším krokem bylo následné praktické testování imunoterapie DCVAC/PCa nejprve v pilotní "first-in-men" aplikaci u pacienta s pokročilým metastatickým karcinomem prostaty a následně klinické hodnocení tohoto preparátu v rámci 2...
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