Allergy, as one of the worldwide most frequent pathologies, belongs to illnesses with constantly growing incidence among young children. In genetically predisposed individuals, dendritic cells are able to polarize the immune response of Th2 in contact with the allergen. Postnatal probiotic supplementation could be one the preventive measure to prevent the development of allergic diseases. It has been shown that introduction of selected probiotic strains or mixtures can prevent development of allergy. In this diploma thesis, the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to support maturation of dendritic cells (DC) and polarization of immune responses was tested. Introduction of this probiotic vaccine called Colinfant Newborn appears to be suitable preventive measure, lowering allergy incidence in children with predisposition to development of allergy. The aim of this diploma thesis was to observe capacity of E. coli O83 to support maturation of the two main subpopulations of dendritic cells (myeloid dendritic cells - mDC and plasmacytoid dendritic cells - pDC) in cord blood of newborns of healthy mothers (children with relatively low risk for allergy development) and allergic mothers (children with relatively high risk for allergy development). To achieve this goal,...

Allergy as one of the most frequent pathologies worldwide belongs to illnesses with constantly increasing incidence even amongst young children. It develops in genetically predisposed individuals whose dendritic cells (DC) are, after contact with allergen, able to polarize the immune response predominantly to Th2, while Th1 response is supressed. One of the possible preventive measures to avoid an allergic disease developement could be an early postnatal supplementation of chosen probiotic bacterial strains or their mixtures. One of them is a well characterized strain Escherichia coli O83:K24:H31 (E. coli O83). Administering of this probiotic vaccine called Colifant Newborn is showing to be an effective prophylaxis to decrese the incidence of allergies in children with predisposition to their developement. The aim of my diploma thesis was to experimentally confirm the capacity of E. coli O83 to support maturation of two main subpopulations of newborn DC available from cord blood: myeloid dentritic cells - mDC and plasmacytoid dentritic cells - pDC. This DC subpopulations were isolated from cord blood of children born to healthy (non-allergic) mothers who had a low risk of allergy development or from children of allergic mothers who had an increased risk of allergy development. Subsequently, after...

Allergy, as one of the worldwide most frequent pathologies, belongs to illnesses with constantly growing incidence among young children. Identification of prognostic markers pointing to increased risk of allergy development, allows introduction of early preventive measures. Probiotic supplementation could be one the preventive measure. It has been shown that introduction of selected probiotic strains or mixtures can prevent development of allergy. In this diploma thesis, the capacity of probiotic strain Escherichia coli O83:K24:H31 (E. coli O83) to support maturation of dendritic cells and polarization of immune responses was tested. Introduction of this probiotic vaccine called Colinfant Newborn appears to be suitable preventive measure, lowering allergy incidence in children with predisposition to development of allergy. The capacity of E. coli O83 to support maturation of the two main subpopulations of dendritic cells (myeloid dendritic cells - mDC and plasmacytoid dendritic cells - pDC) in cord blood of newborns of healthy mothers (children with relatively low risk for allergy development) and allergic mothers (children with relatively high risk for allergy development) was measured by flow cytometry. The presence of cytokines and transcription factors characteristic for particular...

Spinal cord injury (SCI) is complicated injury with serious socioeconomic consequences for the patient and his whole family. Big difficulty cause also extremely high living expenses for the patient with this type of injury. That's why there is a need for therapeutic methods which would help patients after SCI to recover the lost functions and be able at least partially to return to their normal life. Different therapeutic methods are being used for SCI treatment. In this study we used four various types of stem cells: human bone marrow stem cells (hBM-MSCs), human umbilical cord mesenchymal stem cells (hUC-MSCs), neural precursors derived from induced pluripotent stem cells (iPS-NPs) and neural stem cell line derived from human fetal spinal cord tissue (SPC-01). These cells have been transplanted intrathecally or intraspinally 7 days after induction of the experimental model of SCI in the rat. We studied expressions of genes related to neurogenesis, growth factors and inflammation 10 and 28 days after SCI. Our analysis showed significant changes in gene expression 10 days after SCI. Significant up-regulation in expression of vascular endothelial growth factor (Vegf), ciliary neurotrophic factor (Cntf) and interferon regulatory factor 5 (Irf5) were found after transplantation of hBM-MSCs and hUC-...

Hormonal aspects of antler growth regulation Erika Kužmová Abstract Deer antlers are the only mammalian organ that completely regenerates and therefore they became an object of rising interest as a potential model for bone growth and development. In recent years, it has been confirmed that annual regeneration of the antler is initiated from the stem cell niche localised in the pedicle periosteum. Antlers grow to the length at the tip. Only a little is known about endocrine stimulation of antler growth and some discrepancy has arisen between in vivo and in vitro studies over the decades. As the secondary sexual character, the antler cycle timing and growth are linked to seasonal levels of testosterone. Since the levels are at their minimum during the antler growth phase, according to many mainly in vitro studies, insulin-like growth factor-1 (IGF-1) tends to be accepted as the "antler stimulating hormone". Since the conclusion about the role of IGF-1 was contradictory to previous opinions and also in contrast with our own experience, we aimed to verify the role of IGF-1 in vitro. Our ex- periments were based on existing in vivo studies demonstrating the importance of testosterone, even in its low levels, and on the hypothesis that testosterone should be the "antler stimulating hormone". We performed in vitro...

Type 2 diabetes mellitus represents a metabolic disease reaching epidemic dimensions in the 21st century. Fatty acid-induced apoptosis of pancreatic β-cells significantly contributes to its pathogenesis. Saturated fatty acids (FAs) are strongly cytotoxic for β-cells, whereas unsaturated FAs are well tolerable by β-cells, they are even able to inhibit proapoptotic effects of saturated FAs when co-incubated. According to recent studies, FAs-induced apoptosis in pancreatic β-cells is partly regulated by autophagy, a catabolic process involved in the degradation and recyclation of cell components in lysosomes. The aim of this diploma thesis was to contribute to the clarification of the role of autophagy in FAs-induced apoptosis regulation. We induced apoptosis in human pancreatic β- cell line NES2Y by 1 mM stearic acid (SA) and inhibited it with 0.2 mM oleic acid (OA) co- incubated with SA. We revealed, that the saturated SA used in apoptosis-inducing concentration simultaneously inhibits the autophagic flux in pancreatic NES2Y cell line. When SA is co- incubated with unsaturated OA in concentration sufficient for inhibition of proapoptotic effect of SA, OA is also able to inhibit the block of autophagy induced by the effect of SA. Application of unsaturated OA alone in this concentration did not...

In this diploma thesis we are discussing about using 3D bioprinting in tissue engineering. We are discribing using biomaterials for construction scaffolder and aplication stem cells in 3D bioprinting. Last section of theoretical part deals with very often used techniques of 3D bioprinting and we are focused on extrusion technique. In the practical part we propose a method for print vasculars structures. We realized prototype of print head, her design and 3D printing of individual parts. To mechanical part we create a control system for printing control. At the end we visualize the organization of the cells using program modules.

Opioids are one of the oldest analgesics used to relieve pain. Besides their therapeutic properties, they also have negative side effects, which include impaired tissue regeneration. Therefore, it can be assumed that opioids also have a negative effect on stem cells which are responsible for tissue healing. One of stem cell populations involved in wound regeneration are mesenchymal stem cells (MSCs). MSCs are undifferentiated, multipotent cells that could be find in almost all tissues. They have immunoregulatory properties and they can migrate to the site of inflammation or injury where they contribute to healing of tissues. Therefore, the purpose of this thesis was to evaluate the effect of morphine and methadone on properties and migration of MSCs. Their effect on the metabolic activity of MSCs and also on the production of cytokines and growth factors was measured. The effect of these opioids on the immunoregulatory properties of MSCs acting on both innate and adaptive immune cells in vitro was studied. The effect of morphine on expression of adhesive molecules on MSCs was also examined. Furthermore, the effect of morphine on migration properties of systemically administered exogenous MSCs in vivo was investigated in mouse models. Distribution of MSCs to individual organs and to the site of...

Pre regeneráciu a rast poranených nervových vlákien bolo preskúmaných mnoho postupov, no výsledný rast axónov je často náhodný až dezorganizovaný a odráža sa na zložitejšom zotavovaní pacienta. V tejto práci boli vyrobené nové skafoldy s mikroštruktúrnymi a mechanickými vlastnosťami nervového skafoldu pomocou metódy freeze-casting. Konkrétne boli vyrobené biokeramické skafoldy na báze fosforečnanov vápenatých, oxidu titaničitého alebo oxidu zirkoničitého. Pomocou kontrolovaného rastu ľadu v jednom smere bola pripravená orientovaná mikroštruktúra. Pozorovanie pomocou skenovacej elektrónovej mikroskopie potvrdilo lineárne orientované póry (lamelárny systém), v ktorých priemerná veľkosť pórov klesala so zvyšujúcou sa rýchlosťou mrazenia. Skafoldy pripravené pomocou mrazenia v tekutom dusíku vykazovali vynikajúce mechanické vlastnosti, kde pevnosť v ohybe bola získaná v rozmedzí 10–17 MPa. Tie isté skafoldy mali vzdialenosť medzilamelamelárnych priestorov 10–30 µm, ktorých parametre sú vhodné pre nervové skafoldy. Biokompatibilita bola vyhodnotená pomocou Schwannových buniek in vitro, kde bola pozorovaná adhézia a rast v lamelárnom smere. Cytotoxické testy odhalili negatívny vplyv vyššej koncentrácie vápnika na prežitie Schwannových buniek. Pripravené skafoldy mali schopnosť tvorby apatitu na povrchu v podobe embryonálnych a nukleačných centier a apatitu samotného. Skafoldy na báze fosforečnanov vápenatých a oxidu titaničitého vykazovali sľubné regeneračné vlastnosti, konkrétne adhéziu a rast prostredníctvom pórovitej štruktúry a taktiež vynikajúce mechanické vlastnosti.

Recent breakthroughs in the RNA quantification of single cells are rapidly transforming the view on biology and medicine. Flexibility and sensitivity of reverse transcription quantitative PCR (RT-qPCR) make it an ideal method for quantification of single-cell material, but its limits had not been yet fully explored. In this thesis, various factors influencing RT-qPCR performance in single-cell application have been assessed, including conditions of sample collection and processing, importance of quality control, performance of reverse transcription, preamplification and role of qPCR assays. We showed that prolonged time for single cell collection as well as repeated freeze-thaw cycles had negligible effect on RT-qPCR data quality. Direct lysis routinely applied for RNA extraction from single cells may be scaled up to 256 cells. The comprehensive comparison of 11 reverse transcriptases in low RNA input conditions identified 2 best-performing enzymes. Decrease in preamplification volume as well as poor primer design resulted in the loss of sensitivity. Finally, the established workflow has been applied to profile gene expression of astrocytes in mouse model of amyotrophic lateral sclerosis (ALS) identifying important components of ALS-induced changes to astrocyte transcriptome. Altogether, the thesis...