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Early phase of anti-Leishmania immunity in the host skin
Máčalíková, Bára ; Leštinová, Tereza (advisor) ; Kolářová, Iva (referee)
Leishmania parasites are parasitic protozoans that cause disease called leishmaniasis, which primarily affects mammals. Throughout evolution, Leishmania has adapted to the host's immune system, using it to its advantage. This bachelor's thesis describes the relationship between Leishmania and early immune components in the host's skin, as well as the parasite's ability to inhibit the microbicidal activities of cells. The infection begins with the inoculation of infectious promastigotes into the skin, and before reaching their target cells, Leishmania primarily interacts primarily with the complement system, keratinocytes, fibroblats, eosinophils, neutrophils, mast cells and dendritic cells. Understanding the mutual interaction between the host and the parasite is essential for vaccine development and the treatment of leishmaniasis. KEYWORDS: leishmania, skin, early imunity, complement system, keratinocytes, fibroblasts, eosinophils, neutrophils, mast cells, dendritic cells
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The co-culture of keratinocytes and fibroblasts on a multi-layered polyester nanofibrous membrane enriched with platelet lysate
Blanquer, Andreu ; Filová, Elena ; Jenčová, V. ; Brož, Antonín ; Kuželová Košťáková, E. ; Lisnenko, M. ; Procházková, R. ; Bačáková, Lucie
The prevalence of chronic wounds is increasing due to the population ageing and specific illnesses like diabetes mellitus and vascular diseases. Nanofibrous membranes fabricated using synthetic polymers are promising materials to enhance skin wound healing. PCL and PVA membranes are being studied to be used as scaffolds for skin tissue engineering and hydrogels for controlled drug delivery, respectively. The present study considers the development of a multi-layered membrane made of PCL and PVA loaded with platelet lysate (PL). PCL nanofibers allowed cell adhesion and growth, whereas PVA acted as a hydrogel that releases the bioactive compounds of platelet lysate. The cytocompatibility of the membranes containing PL and without it was demonstrated on two cell types involved in wound healing, i.e. keratinocytes and fibroblasts. Both cell types were able to adhere and proliferate on the membranes. In addition, the membrane containing PL enhanced the proliferation of fibroblasts. A co-culture study was also performed by seeding each cell type on one side of the membrane. The cells were co-cultured for 7 days and the results showed that PL increased the proliferation of cells achieving a monolayer of keratinocytes or fibroblasts on each side of the membrane. Thus, the beneficial effect of PCL-PVA+PL membranes on monocultures and co-cultures of skin cells was demonstrated, and these membranes can be considered potential scaffolds for treatment of chronic wounds.
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Interactions of skin and stem cells with polymer nanofibres for construction of skin substitutes
Tomšů, Júlia ; Bačáková, Lucie (advisor) ; Sedmera, David (referee) ; Jendelová, Pavla (referee)
The skin is the largest organ of a human body with a crucial role in the maintenance of homeostasis; therefore any extensive skin injury leads to severe complications. Since the application of auto-, allo- and xeno-grafts is accompanied by severe problems like the source limitation and the graft rejection, a bioengineered skin substitute seems to be one of the promising healing approach. This work is focused mainly on the construction of a pre- vascularized skin substitute consisting of a collagen hydrogel reinforced by a biodegradable nanofibrous membrane. Another strategy described in this work is the development of temporary cellulose-based wound dressings. For both research strategies, various cell types were utilized, i.e. normal human dermal fibroblasts (NHDFs), human keratinocytes (hKs), adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). In order to enhance the cell adhesion and growth, the synthetic nanofibrous membranes were improved by protein nanocoatings. It was found out that NHDFs and ADSCs preferred fibrin nanocoatings, mainly thin fibrin homogeneous mesh on the surface of the membrane. Keratinocytes rather adhered and stratified on collagen substrates. These observations further motivated the construction of the bi-layered construct, where...
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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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Mitochondria as a target of anticancer therapy.
Dvořák, Aleš ; Ježek, Petr (advisor) ; Poučková, Pavla (referee) ; Vecka, Marek (referee)
Mitochondrial isocitrate dehydrogenase 2 (IDH2) catalyzes reductive carboxylation (RC, reverse Krebs cycle pathway) and 2HG synthesis (2HG) - metabolite of which many scientists are interested. 2HG may be concurrently synthetized in cytosol by IDH1. RC is involved in anabolic reactions necessary for cell proliferation - produces citrate, fatty acid precursor - especially in hypoxia. IDH2 and IDH1 are not the only enzymes that are involved in 2HG synthesis. Recently, several enzymes, which participate in 2HG production, have been discovered. 2HG is useful in cancer diagnostics due to its overproduction by transformed cells. Moreover, 2HG may cause epigenetic changes via inhibition of 2-oxoglutarate dependent dioxygenase. In this work, the importance of RC and 2HG synthesis in cancer and healthy cells was investigated by gas chromatography with mass spectrometry detection as well as IDH2 influence. We found that IDH2 significantly participates in reverse RC and 2HG synthesis in breast cancer cell lines and uses glutaminolysis as a supplementary anaplerotic pathway. RC is increased by hypoxia, inhibition of respiration, and decreased by activation of respiration or hypocapnia. We confirmed 2HG synthesis and RC in healthy cells (fibroblasts, breast epithelial cells etc.) as well as in cancer cells....
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Flow cytometry in the diagnostics and characterization of congenital disorders of glycosylation
Veselá, Šárka ; Hansíková, Hana (advisor) ; Hodek, Petr (referee)
Congenital disorders of glycosylation (CDG) are rare multisystem metabolic diseases and their number has rapidly grown in recent years. The clinical manifestation includes very broad spectrum of symptoms. In most of all cases CDG are caused by mutations in genes encoding the enzymes of glycosylation pathway. Based on the type of defect, CDG are divided into the following groups: disorders of N-glycosylation or O-glycosylation of proteins, defects in modification of proteins by GPI anchor, disorders of lipid glycosylation and defects that impact multiple glycosylation pathways. The aim of the thesis was to find new biochemical analyses suitable for diagnostics and characterization of CDG patients. The experimental conditions were optimized for selected markers (Sambucus Nigra (SNA) lectin, proaerolysin (FLAER), antibodies to proteins CD55 and CD59) and the staining was applied to cultivated skin fibroblasts from controls and patients diagnosed with CDG by whole-exome sequencing (ATP6AP1-CDG, PIGN-CDG, SLC10A7-CDG, PISD deficiency). The experiments were performed using flow cytometry (FACS) and fluorescent microscopy (FM). The detection of sialylation by SNA lectin and analysis of the mitochondrial membrane potential changes by a fluorescent labelled probe JC-1 with FCCP simulation of mitochondrial...
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Mitochondria as a target of anticancer therapy.
Dvořák, Aleš
RK se podílí a prolifera i ádorový h u ěk ve stíže ý h zkrá e é Kre sově klu v tváří reverz í reak í z OG itrát, který ůže ýt e portová do tosolu, kde slouží jako prekurzor ast ý h k seli a další h olekul. )ároveň IDH při RK spotře ovává stej ě tak i při s téze HG NADPH. RK se tak z výzku u zdá ještě zají avější, e oť ůže ovliv it produk i ROS a íru o idač ího stresu. HG ůže ýt s tetizová a IDH i ěkolik další h e z ů. HG regulač í olekul součas é do ě je v užívá Bývá oz ačová jako ož ý i hi itor α DD , jež se pod í a růz ý h epige eti ký h z ě á h a zv šují alig itu rakovi ého fe ot pu spoje ou se z ě ou prolifera e u ěč ý h li ií h kar i o u prsu a další h uňká h, včet ě pri ár í h potka í h fi ro lastů a aktivitě respira e. de o strují, že HG ádorový h uňká h, a aví že ve zdravý h uňká h z ě hladi ovlivňují prolifera i. elkově jeví jako vhod ý íl proti ádorové terapie, zej é a ve světle edáv ý h o jevů, ve který h se ukázalo, že utova á íře s tézu α Klíčová slova: MS, uňk prs ího 6, fi ro last , HG/ OG po ěr, C izotopi ké z ače í, prolifera e fi ro lastů, h po ie
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The role of new pro-inflammatory and/or pro-fibrotic molecules in the pathogenesis of systemic sclerosis.
Tomčík, Michal ; Bečvář, Radim (advisor) ; Hrnčíř, Zbyněk (referee) ; Horák, Pavel (referee)
Introduction: Systemic sclerosis (SSc) is a generalized connective tissue disease affecting the skin and internal organs. The pathogenesis of SSc is characterized by inflammation, vasculopathy and fibrosis. To date, none of the tested drugs have demonstrated convincing efficacy in the treatment of SSc. S100A4 is involved in the regulation of cell motility, proliferation, apoptosis, angiogenesis and remodeling of the extracellular matrix. It was originally described as a promoter of metastasis in tumors, however, its pro-inflammatory properties have recently been demonstrated in inflammatory rheumatic diseases. The aim of this study was to assess the role of S100A4 in pathological activation of fibroblasts in SSc and in experimental models of dermal fibrosis. Results: The expression of S100A4 was increased in the skin of SSc patients, in SSc fibroblasts and in experimental fibrosis in a TGF-β / Smad dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, inhibition of S100A4 or its complete deficit abrogated the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4 knock-out mice (S100A4-/- ) were protected from bleomycin-induced skin fibrosis with reduced dermal thickening,...
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The effect of carbon nanostructures on human cell behavior and the role of fetal bovine serum in cell adhesion
Jannová, Martina
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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