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Tissue engineering is a multidisciplinary field dealing with the fabrication of artificial tissue substitutes for regenerative medicine. Current regenerative medicine uses various types of tissue grafts, which have different advantages and disadvantages depending on their origin, such as insufficient amount of replacement tissue when using autologous grafts or immunogenicity of allogeneic or xenogeneic grafts. An alternative could be artificial tissue replacement. Artificial tissue constructs may consist of a non-living matrix and a cellular component. The cellular component may remodel the construct, form a functional part of the construct, or help integrate the construct into the host body. A significant problem in the formation of such replacements is sufficient vascularization. It is essential to keep cells in larger tissue constructs alive. Vascularization can be enhanced by the addition of vascular endothelial cells that can form capillaries independently within the construct. Vascular formation can also be aided by angiogenic growth factors by their direct application to the construct or by their formation, e.g. in stem cells cultured in the construct. Another approach is 3D bioprinting, allowing direct placement of specific cell types, growth factors or biomaterials in the construct. This...

Retinal degenerative diseases are recognized worldwide as one of the predominant causes underlying irreversible visual impairment and even blindness. The current standard treatment has helped in both the prevention and supportive treatment of several pathologies, especially if they relate to the retina. However, many of these methods are rather invasive in terms of procedure with the subsequent possibility of infection, inflammation or even retinal detachment with the potential risk of vision loss. These restrictions represent a significant cost to patients' quality of life and also have an economic impact on the healthcare system, so new treatment options are being sought. Mesenchymal stem cells (MSCs) are a promising cell therapy tool for the treatment of many previously untreatable defects and diseases. In addition to MSCs, the use of nanoparticles and nanofibrous carriers is currently being studied in the diagnosis and therapy of retinal diseases. Combining the immunomodulatory and regenerative properties of MSCs and nanotechnologies offers a promising therapeutic strategy for a number of diseases. This thesis is therefore devoted to the current state of the use of nanomaterials, MSCs and their combination that may provide new opportunities in the treatment of retinal degenerative diseases.

The genes of the major histocompatibility complex are located on the short arm of chromosome 6 and encode surface glycoproteins (HLA glycoproteins), which ensure the presentation of self and foreign peptides on the cell surface. These glycoproteins are subsequently recognized by T-lymphocytes and by other cells of the immune system. When the HLA-peptide complex is recognized as foreign, T-lymphocytes and other components of the immune system are activated, and the foreign cell is destroyed. Therefore, in hematopoietic stem cell transplantation (HSCT), HLA incompatibility between donor and recipient causes a strong immune response against the transplanted cells, and is therefore a major criterion in selecting suitable stem cell donors. This work briefly summarizes the current knowledge about the structure and function of HLA class I and class II antigens. The work focuses on HLA typing techniques to help understand the HLA system, which include serological typing methods, as well as modern molecular typing methods based on PCR and next-generation sequencing, and their relevance for HSCT. We also focus on HSCT processes and preparatory therapy, but the main emphasis is on the importance of HLA incompatibilities between stem cell recipients and donors and their effect on HSCT outcome.

Stem cell differentiation is controlled by coordinated regulation of gene transcription. One of the regulatory factors is the loosening of chromatin and the accessibility of DNA to transcription factors. Chromatin remodeling is mediated by remodeling complexes. The ISWI chromatin remodeling ATPase Smarca5 (S5) is an important factor of remodeling complexes. It is a highly conserved chromatin-remodeling factor forming a catalytic subunit that can be found in several oligosubunit complexes. In these complexes, it actively regulates nucleosome structure and remodeling during DNA replication, repair and transcription. S5 has been identified as a key protein in embryonic development. Its deficiency leads to defects in hematopoiesis and male genital development. In the presented study, we focused on the role of S5 in hematopoiesis and spermatogenesis. Using a mouse model with transgenic expression of S5, co-immunoprecipitation and mass spectrometry, we identified S5 complexes in hematopoietic and testicular cells. We also studied the phenotypic consequences of S5 deficiency in mouse testes and found that it leads to impaired sperm development and male sterility. Using transcriptomic and proteomic analysis, we identified several molecular programs that could lead to reproductive disorders. Our work...

Bone cells in vivo reside in a dynamic environment exposed to constant chemical and mechanical stimuli caused by the interstitial fluid flow. It is hypothesized that perfusion of the medium through the scaffold increases the mass transport and creates at the same time shear stress, thereby in vitro simulating the interstitial fluid effects and bone tissue formation conditions. This work examined the effects of perfusion flow rates on cell viability, proliferation, migration and osteogenic differentiation of human mesenchymal stem cells within cell-seeded 3D poly-ε-caprolactone scaffolds. Scaffolds were perfused for 21 days at flow rates 1, 3 and 5 mL/min and were compared to the scaffolds from static culture. Cells were most viable, had upregulated expression of osteogenic markers collagen type I and highest alkaline phosphatase activity under flow rate 1 mL/min when compared to their static counterparts. Cells proliferated the most under flow rate 3 mL/min when compared to their static counterparts. Flow rate 5 mL/min did not significantly differ from the static culture in any of the examined parameters. Cell migration into the scaffold was not improved upon exposure to perfusion. This data confirms that medium perfusion may benefit cell proliferation and osteogenic differentiation by enhancing...

Microchimerism is defined as the presence of small amount of genetically distinct cells in one individual. It most often arises during pregnancy and breastfeeding. It can also arise from blood transfusion or organ transplantation. During pregnancy there occurs a bidirectional migration of cells through the placenta between the mother and the fetus. Cells which are transferred during pregnancy and breastfeeding can persist in the offspring until adulthood. During breastfeeding, a big number of immune cells is transferred to the offspring via mother's milk. These cells protect the offspring against pathogens and are involved in modulation of its immune system. Fetal cells persist in mother organism even decades after giving birth and can have long-lasting effect on mother's health condition. These fetal cells can help to regenerate mother's damaged tissues, but they can also contribute to the development of serious autoimmune diseases.

Therapeutic resistance of tumours represents an important clinical issue. We can classify the therapeutic tumour resistance in two ways. According to the clinical course, tumours can behave either as primary resistant, i.e. from the very beginning not responsive, or they can display a secondary (also called acquired) resistance, whereby an initial clinical response is lost and the tumour develops into chemo-, radio- or immunoresistant disease. An alternative classification distinguishes cell autonomous resistance mechanisms from resistance that relies on complex interactions within the context of tumour microenvironment. From the research perspective, modelling therapeutic resistance frequently involves experimental treatment of sensitive cancer cells and selection of daughter resistant cell lines. The Ph.D. thesis includes derivation of two unique models of urothelial bladder carcinoma therapeutic resistance. The first model involves newly established urothelial carcinoma cell lines BC44 and BC44DoxoR, which resulted from a prolonged doxorubicin exposure of the mother cell line. The daughter chemoresistant cell line exhibits multidrug resistant phenotype, which extends beyond the selecting drug - doxorubicin - to four additional chemotherapeutic drugs (cisplatin, methotrexate, vinblastine, and...

This bachelor's thesis is dedicated to donating umbilical cord blood. The thesis consists of a theoretical and practical part. The theoretical part contains a description of the placenta and its functions, umbilical cord blood, stem cells. Furthermore, the theoretical part contains a description of the technique of cord blood collection, contraindications of the collection and processing of umbilical cord blood. It also describes the education of midwives on this issue. Umbilical cord blood banks and legislative provisions are listed here. The aim of the bachelor's thesis was to find out how knowledgeable pregnant women are about this issue. A quantitative research survey was used in the practical part of this work. The questionnaire created contained 18 closed questions. The research team was pregnant women. This set consisted of 244 respondents. Of these, 155 (64%) first-time parents 89 (36%) multi-parents. Of these, 41.39% of respondents know the use of umbilical cord blood. Only 18.03% of respondents know what they need to do to be able to take umbilical cord blood. 19.67% of respondents reported the correct time of collection of umbilical cord blood. 13.52% of respondents reported that the maximum storage period of donated umbilical cord blood is 20 years. Two hypotheses were established in this work. The first hypothesis focused on whether pregnant women are given more information about this issue by a midwife or gynecologist. 5.70% of respondents received information from a midwife. 3.70% of respondents received information from a gynecologist. Statistical processing of the hypothesis has shown us that there is no difference in who gives information. Women are informed from the midwife as well as from the gynecologist. The second hypothesis was whether multi-parents are more informed about umbilical cord blood donation than first-time parents. Multi-parents accounted for 36%, and first-time parents 64%. 46.06% of multi-parents and 38.71% of first-time parents said they knew the use of umbilical cord blood. 20.22% of multi-parents and 19.35% of first-time parents know the time of collection of umbilical cord blood. 50.56% of multi-parents and 39.35% of first-time parents reported that the donation was not risky for newborns. We confirmed the second hypothesis. The results of this work show that women have very little information on the issue of umbilical cord blood donation. The midwife is a competent person, so women could be more education about this issue. This work can be used in seminars for midwives.

Spinal cord injury is a very serious, complex, and life changing injury for which today's medicine still does not have an efficient treatment. It is only possible to mitigate the consequences of this injury and the pathological processes associated with it. Neural stem cell transplantation has immunosuppressive effects in the pathology of spinal cord injury and promotes regeneration. mTOR kinase is a member of the crucial intracellular PI3K/Akt/mTOR signalling pathway, making it a suitable target for therapeutic intervention and immunosuppressants such as rapamycin. mTOR signalling is important for neural stem cells and in the pathology of spinal cord injury. The aim of this study was to investigate the role of the mTOR pathway in differentiation of stem cells into neuronal phenotype. Rapamycin was applied to in vitro culture of neural progenitors. Immunocytochemistry and immunoblotting techniques were used to study the effect of this inhibition on the cell phenotype and on the activity of the mTOR pathway. Using the rat model of spinal cord injury in vivo, immunohistochemistry and immunoblotting techniques were used to evaluate the impact of rapamycin inhibition on the mTOR pathway, autophagy, and cytokine production by cells in the damaged tissue. The results show that the mTOR pathway plays role...

Výborný, Karel. Vývoj materiálů na bázi extracelulární matrix pro léčbu centrálního nervového systému. [Development of extracellular-matrix scaffolds for CNS repair]. Praha, 2020. 105 stran, 3 přílohy. Dizertační práce (Ph.D.). Univerzita Karlova, 2. lékařská fakulta, Ústav experimentální medicíny, AV ČR v.v.i. Abstract Brain and spinal cord injury are a serious traumata and despite intensive research, there is still no effective treatment for patients. One of the studied approaches is to use various biomaterials to repair the damaged neural tissue. The aim of this thesis is to study the regenerative and neurotrophic effects of injectable extracellular matrix (ECM) hydrogels prepared by decellularization from porcine tissue (brain, spinal cord, bladder) and human umbilical cord (UC) in reconstruction of damaged neural tissue of the brain and spinal cord in rats. We characterized ECM hydrogels in terms of mechanical and rheological properties, structure and composition, cell adhesion, migration and proliferation. In a model of spinal cord hemisection, we compared the regenerative effect of ECM hydrogels derived from porcine spinal cord and urinary bladder. We found no benefits of tissue specific ECM prepared from the tissue of neural origin in terms of its neurotrophic properties in vitro or in vivo....

Part I: Endothelial cells form the posterior layer of the cornea and are important for maintaining its transparency. Dysfunctional endothelium can only be restored by transplantation. The global shortage of donor corneas requires the search for alternative treatments. The preparation of the graft by tissue engineering methods is complicated by low proliferative capacity of endothelium. To date, no endothelium-specific marker has been defined and the existence of endothelial stem cells has not been confirmed yet. We have prepared a protocol for culturing endothelial cells from research-grade tissue - corneoscleral rims obtained after transplantation or corneas excluded from the transplant process. We monitored localization of selected proteins, including stem cell markers, in native tissue and in primary cell cultures. We prepared up to 6.4 cm2 of endothelium from one cornea/rim, which had cellular features comparable to the native endothelium. This approach can increase the amount of endothelium for research or transplantation purposes. Using indirect immunohistochemistry, we showed that none of the previously proposed endothelial molecular markers is specific for these cells. We detected the expression of stem cell markers throughout the endothelial layer. In the porcine cornea model, we monitored...

Haematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analysed the cell cycle and cell production rate in HSPCs in murine haematopoiesis. The labelling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled the determination of the cell cycle flow rate into the G2-phase, the duration of the S-phase and the average cell cycle time in Sca-1+ and Sca-1- HSPCs. The determination of cells with 2n DNA content and labelled during the preceding S-phase was used to establish the cell flow rates in the G1-phase. Our measurements revealed a significant difference in how Sca-1+ and Sca-1- HSPCs self-renew and differentiate. The division of Sca-1+ progenitors led to the loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. In contrast both Sca-1- progenitors, arising from mitotic cell division, entered a new round of the cell cycle. This corresponds to symmetric self-renewing cell division. The novel data also enabled us to estimate the cell production rates in the Sca-1+ and in three subtypes of Sca-1- HSPCs. We focused on adult murine erythroid differentiation in the next part of our study. We introduced an original flow cytometry approach for identifying and studying erythroid...

Cultivation of cells in bioreactors with mechanical load simulates the physiological conditions to which cells in the body are exposed. This technology has been used to induce the differentiation of stem cells from adipose tissue towards the phenotype of vascular smooth muscle cells, which can further serve to form vascular replacements. At present, there is no established strategy for cultivating stem cells while being exposed to mechanical stress. The main aim of this work was therefore to optimize the cultivation strategy and determine the ideal load parameters. Differentiation was analyzed by immunofluorescence of specific smooth muscle cell markers, α-actin and h1-calponin, which were quantified by Western blot. Extracellular matrix production was also detected by immunofluorescence staining. The outcome of this work is the establishment of ideal conditions of cell culture in a bioreactor with mechanical load, during which they differentiate into smooth muscle cells. Three types of scaffolds were used for cultivation; plasma treated glass, fibrin-coated glass and decelularized pericardium. Preliminary results show that smooth muscle differentiation was succesfully induced in human and porcine adipose tissue stem cells. Cells were analyzed after 3 and 7 days of culture. Developing a stem cell...

Relevance of tumor infiltrating lymphocytes as a prognostic factors in patients with portal vein embolisation (PVE) and patients with PVE and administration of autologous stem cells Background: low future liver remnant volume (FLRV) is the cause of why 75% of patients with colorectal liver metastases (CLM) are primarily inoperable. Portal vein embolisation (PVE) helps to increase FLRV and so increase the operability. But PVE fails in almost 40 % of patients. Usage of stem cells (SCs) could be the way how to support the effect of PVE. Currently, there are studies of interactions of the immune system and malignancies. We do not know about papers focused on relations of the immune system and CLM in patients treated by PVE. There were not described interactions of ABC transporters and CLM at patients after PVE was performed too. Aims: the aim of this dissertation was to verify the effect of PVE and intraportal administration of SCs on the growth of FLRV and progression of the CLM. Other aims were to evaluate the tumor infiltrating lymphocytes, ABCC10 and ABCC11 transportes in patients treated by surgery for CLM after PVE and their clinical relevances. Methods: intraportal administration of SCs after PVE and their effect was explored in a group of 63 patients (43 patients with PVE alone, 20 in the group PVE with...

This thesis deals with the issue of stem cell research and its legal aspects. The issue raises many ethical questions, especially due to its closed connection to the topics of embryonic research and right to life. In the first introductory chapter the thesis explains the topic of stem cells from biological point of view and defines and classifies stem cells by their origin and differential potential. In the second chapter the thesis briefly summarizes the history of stem cell research and deals with the ethical questions raised by closed connection of the topics of embryonic stem cell research and in vitro fertilization to the right to life. The second chapter also notes some recent possibilities and medical methods connected with stem cells, such as gene therapy and diagnostics and new eugenics. In the following chapters the thesis concentrates on the legal aspects of the topic on both national and international level. Specifically, the third chapter puts the issue of stem cells research to the contexts of the two most important conventions - the Convention for the Protection of Human Rights and Fundamental Freedoms and the Convention on Human Rights and Biomedicine. The fourth chapter concentrates on legislation of Czech Republic regulating the legal status of embryo and its protection in the...

The neural crest is a transient structure formed during the neurulation which undergoes change of phenotype in the process of epithelial-mesenchymal transition. Subsequently, neural crest cells delaminate and migrate collectively and individually to their place of destination, where they differentiate into a broad repertoir of mesenchymal and non-mesenchymal cell types. This thesis aims to examine true differential potential of these cells based on crucial in vitro experiments. Neural crest cells show not only high migration potential, but also stem cells characteristics like multipotency and self-renewal capacity. I also provide answers to questions about cellular potency at the level of neural crest population and along anterior-posterior axis. It was necessary to clarify the essence of events leading to induction, specification, epithelial-mesenchymal transition and migration of neural crest cells, since they are crucial for their differentiation potential. A major role in differentiation as well as in previous processes plays the gene regulatory network which is comprised of mutually affecting signalling pathways. Studying the behaviour of migratory and post-migratory neural crest cells is important for the research of regenerative medicine and even cancer and neurocristopathic treatment.

Stem cells have a huge therapeutic potential due to their ability to differentiate in multiple tissues. They could be used for neurodegenerative diseases treatment, which are typical for loss of specific groups of neurons, progressive course and lack of effective treatment due to their complicated pathophysiology, only therapies for elongation and simplification of patients' life are available. This thesis summarizes results of completed clinical studies and informs about ongoing studies, in which stem cell treatments are used for selected neurodegenerative diseases. Stem cell therapy for multiple sclerosis and amyotrophic lateral sclerosis have a long history, some of the studies has proven therapeutic efficiency of stem cells. We don't know much about effect of stem cell treatment for patients with Alzheimer's and Parkinson's diseases, because first clinical studies were finished recently. But all clinical trials have proven safety of stem cell treatment. Replacement of damaged neurons haven't been reached yet, just protection of remaining neurons by neurotrophic and immunomodulatory factors secreted by stem cells. Keywords: neurodegenerative diseases, stem cells, clinical trials, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease

Vertebrate dentition is a dynamic structure, which continuously renews its elements, the teeth. Continuous tooth renewal is enabled thanks to the presence of epithelial and mesenchymal dental stem cells. Epithelial stem cells are located in the dental lamina, which, together with the adjacent mesenchyme, takes part in regulation of the stem cells through a complicated signalling network. This network is responsible for the positioning, correct amount, inactivity, proliferation and differention of the stem cells. Vertebrate dentitions are morphologically diverse. However, their development is, to a certain extent, controlled by shared evolutionarily conserved mollecular mechanisms. For example, epithelial stem cells of all vertebrate groups examined so far express the transcription factor Sox2 and are shown to be regulated by signalling pathways, such as Wnt/β-catenin, Shh, Fgf and Bmp. Due to the rich diversity in dental lamina morphologies, the locations of presumptive stem cells correspondingly differ among vertebrates. This thesis summarizes current knowledge on dental stem cells in each lineage to identify shared and derived aspects of vertebrate dentitions.

Human induced pluripotent stem cells (hiPSCs) play roles in both disease modeling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. I focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as H2AX) and p53-binding protein 1 (53BP1) in multiple lines of hiPSCs, their source cells, human embryonic stem cells (hESCs) and cells differentiated from hiPSCs. I measured spontaneously occurring DSBs throughout the process of reprogramming and during long-term in vitro culture and differentiation process. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by gamma irradiation and the decrease over time was analyzed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. Source cells contained a low number of non-replication-related foci, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging or differentiation. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages but fail to recognize a substantial proportion of DSBs at high passages. Observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, with long-term hipSCs culture reparation capacity decreases.

Diabetic retinopathy is retinal disease causing irreversible cell damage and consequently a loss of vision. Current treatment protocols have many limitations and are associated with serious site effects. A possible treatment options for retinal degenerative diseases is a use of stem cells. There are different types of stem cells. These include embryonal stem cells, induced pluripotent stem cells and cells from an adult organism, among which we can include mesenchymal stem cells (MSCs). MSCs can be found in almost all tissues of the adult organism. MSCs can migrate to the site of damage, regulate development of inflammation in retina, suppress the formation of fibrovascular scars and replace damaged cells such as nerve cells, photoreceptors and epithelial pigment cells. Application of MSCs could be a promising treatment for degenerative retinal diseases.

Stem cell-based therapy represents a perspective approach for the treatment of many so far incurable diseases. Mesenchymal stem cells (MSC) are currently the most studied stem cells. They are able to differentiate into different cell types, to produce growth and trophic factors and can suppress the functions of cells of the immune system. During the study of the immunomodulatory properties of MSC, we focused on their effect on B cells. The mechanism of impact of interferon-γ (IFN-γ) on MSC and their effect on the production of interleukin 10 (IL-10) by B cells was analysed. We have demonstrated that MSC-treated with IFN-γ inhibit production of IL-10 by activated B cells via the cyclooxygenase-2 involving pathway. Due to their regenerative and immunomodulatory properties, MSC can be for treatment of many diseases. In this study we focused on the disease and damage of the eye. The limbal stem cells (LSC) are used for the treatment of damaged ocular surface, however their isolation is difficult and they can not be used in all cases of damage. Appropriate candidates in these cases are MSC. Therefore we have decided to compare the therapeutic potential of LSC and MSC isolated from bone marrow or adipose tissue. The study have shown that MSC isolated from bone marrow have a similar regenerative effect on...

Microchimerism is the presence of small population of cells with a different genetic information within the organism, which can result from bidirectional transfer of the cells between the mother and fetus during pregnancy. It is very studied phenomenon whose biological role is not clear yet. The presence of fetal cells in mother's body is associated with both positive and negative effects on maternal health. Microchimerism plays a role in cancer or autoimmune disease and it is implicated in development of tolerance mechanisms during pregnancy. Microchimerism could be used in prenatal diagnostics for aneuploidies or in prediction of complications during pregnancy.

We consider a finite planar segment process in a circle, having a density with respect to the Poisson process. This density involves unknown parameters and a reference length distribution which is not observed. The aim is to estimate these quantities semiparametrically. The segment process is inhomogeneous, but it is isotropic. Combining the relation between the observed and reference length distribution and the maximum pseudolikelihood method we suggest an estimation procedure. Its properties (bias and variability) are investigated in a simulation study. In the last part we present two more complex models. The motivation is to model stress fibers observed in cultured stem cells.

The work deals with looking at the status of the human embryo. The structure of the work is divided into several chapters that deal with a particular topic. The first chapter deals with the historical view of the adoption of a human embryo, in the second chapter is devoted to philosophical adopting the human embryo and view individual philosophers on this issue. The third chapter is devoted to the ethical acceptance of the issue. Here is discussed an ethical perspective on human embryo status and access two main streams which occur in this area. The next part is devoted to the present, both in terms of the ethical-philosophical, and in terms of health. In the medical section includes prenatal communication between mother and child, and research on embryonic stem cells, which is associated with a number of ethical issues.

Introduction: The reason for the inability of performing the liver resection for colorectal carcinoma metastasis is usually insufficient remnant liver parenchyma after liver resection (future liver remnant volume - FLRV). The current standard method of increasing FLRV is the embolization of the branch of portal vein (portal vein embolization - PVE) on the side of the tumor, and then suspended after hypertrophy of the non-embolised lobe liver resection. Unfortunately, there are some patients who do not increase liver volume despite perfectly executed PVE. Besides that, FLRV occurs during the time necessary for hypertrophy progression of metastatic disease. Therefore, we are trying to find the appropriate way to encourage the growth of remaining liver parenchyma and accelerate hypertrophy of the contralateral liver lobe. From our previous experience (IGA MZ NS 10240), it is possible to be optimistic that there hope is the way of hematopoietic progenitor cells (HPC - adult stem cells) after previous PVE to non-embolised branches of the portal vein. These cells do not only accelerate liver regeneration, but are also able to improve its function (function of the liver) which is especially important for patients after neoadjuvant chemotherapy (steatohepatitis or steatofibrosis), and for patients with...

Magnetic oxide nanoparticles provide a fascinating tool for biological research and medicine, serving as contrast agents, magnetic carriers, and core materials of theranostic systems. Although the applications rely mostly on iron oxides, more complex oxides such as perovskite manganites may provide a much better magnetic performance. To assess the risk of their potential use, in vitro toxicity of manganite nanoparticles was thoroughly analysed and compared with another prospective system of Mn-Zn ferrite nanoparticles. Magnetic nanoparticles of La0.63Sr0.37 MnO3 manganite were prepared by two distinct methods, namely the molten salt synthesis and the traditional sol-gel route, whereas nanoparticles of Mn0.61Zn0.42Fe1.97O4 ferrite, selected as a comparative material, were synthesized by a new procedure under hydrothermal conditions. Magnetic cores were coated with silica and, moreover, several samples of manganite nanoparticles with different thicknesses of silica shell were prepared. The size-fractionated and purified products were analysed using transmission electron microscopy, dynamic light scattering, measurement of the zeta-potential dependence on pH, IR spectroscopy, and SQUID magnetometry. The silica-coated products with accurately determined concentration by atomic absorption spectroscopy were subjected to a robust evaluation of their cytotoxicity by four different methods, including detailed analysis of the concentration dependence of toxicity, analysis of apoptosis, and experiments on three different cell lines. The results, comparing two manganese-containing systems, clearly indicated superior properties of the Mn-Zn ferrite, whose silica-coated nanoparticles show very limited toxic effects and thus constitute a promising material for bioapplications. Magnetické oxidové nanočástice poskytují fascinující nástroj pro biologický výzkum a medicínu, slouží jako kontrastní látky, magnetické nosiče a materiály jádra teranostických systémů. Pro vyhodnocení rizika jejich potenciálního použití, in vitro toxicita manganitových nanočástic byla důkladně analyzována a porovnána s jiným potenciálním systémem Mn-Zn feritových nanočástic. Magnetické nanočástice La0,63Sr0,37MnO3 manganitu byly připraveny dvěma různými metodami, a to syntézou z taveniny a tradiční sol-gel cestou, zatímco nanočástice Mn0,61Zn0,42Fe1,97O4 feritu vybrané jako srovnávací materiál, byly syntetizovány novým postupem za hydrotermálních podmínek. Magnetická jádra byla obalena silikou a navíc několik vzorků manganitových nanočástic s různými tloušťkami silikové slupky bylo připraveno. Tyto silikou obalené produkty byly podrobeny důkladnému vyhodnocení jejich cytotoxicity různými metodami zahrnujícími podrobnou analýzu koncentrační závislost toxicity, analýzu apoptózy a experimentů na třech různých buněčných linií. Výsledky porovnání dvou systémů obsahujících mangan jasně indikovaly vynikající vlastnosti Mn-Zn feritu, jehož silikou obalené nanočástice vykazují velmi omezené toxické účinky, a tudíž představují slibný materiál pro bioaplikace.

The plot of this PhD thesis is dedicated to investigation of the molecular pathways and events and their disruptions in the gastrointestinal tract (further abbreviated as GIT). The major role in this part plays the Wnt signaling pathway. This marvelous multipurpose machinery is responsible for epithelia renewal from stem cells (SCs) in the stomach and intestine, and for correct zonation and function of hepatic units. Of note, the Wnt pathway directs also development of embryo as well as homeostasis of many tissues apart from GIT in all metazoans, thus its flawless function is indispensable from one's origin to death. The main part of the thesis follows canonical Wnt signaling in its physiological condition and, in contrast, with pathological disturbances. This issue can be taken by variety of means as it is described in attached publications. The first publication deals with searching for new participants of Wnt signaling and their functions and describing unique markers of SCs in the intestine. Troy, the member of tumor necrosis factor receptor (TNFR) superfamily, was identified as a novel marker of intestinal SCs by probing microarray data from chromatin immunoprecipitation obtained in cultured colorectal cancer cell lines. Moreover, we found that Troy is a Wnt target gene inhibiting the...

The thesis deals with an issue of legal aspects of stem cell research with a special emphasis on the use of embryonic stem cells which is connected with the most of ethical and legislative controversies. At first, there are presented biological and ethical aspects of the topic necessary for its understanding. The thesis further analyses related legislation relevant for the research conducted in the Czech Republic. With respect to international law, the thesis focuses mainly on the Council of Europe system of protection of human rights. Then, the thesis presents regulation in primary and secondary European Union law. In the two final chapters, there is presented Czech regulation in the constitutional order (in the Charter of Fundamental Rights and Freedoms) and laws, which is then viewed in European context as moderately permissive and relatively well prepared. Its greatest weakness is the lack of discussion about the topic in the Czech public.

New approaches for the treatment of SCI use advances in the fields of nanotechnology, biomaterial science and cell therapy. The results presented in this thesis showed that superparamagnetic iron oxide nanoparticles coated with a stable dopamine-hyaluronane associate can be used for the safe and effective labeling of MSC. Cell labeling efficiency, viability and the relaxivity of the tested particles were significantly better than those obtained with the commercial particles Endorem®. The DPA-HA coated nanoparticles can be used for the noninvasive monitoring of cell therapy using MRI. Furthermore, we showed that SPION can be used for the targeted delivery of MSC to the site of a spinal cord lesion. The labeled cells can be concentrated in the lesion area by means of a magnetic implant. The process of cell targeting depends on the physical characteristics of the magnetic implant as well as on the biological features of the cells and nanoparticles, as we described with a proposed mathematical model. It is possible to modify the properties of the magnetic system, e.g. by changing the shape or size of the magnet, thus tuning the magnetic force distribution and the gradient of the magnetic field necessary for effective cell targeting. A promising therapeutic strategy for the treatment of spinal cord injury is the...

Stem cells are unique thanks to their ability to differentiate into all cell types and self-renewal. They can be used for repairing and functional recovery of various tissues and organs. Regenerative medicine, progressive medical field, use these properties to give a chance to patients with yet incurable health complitations. These include spinal cord injury, which results in huge demyelination and disorders in neurological function. Mesenchymal stem cells contribute to recovery of damaged tissue thanks to its immunomodulatory abilities and production of a number of growth factors. Embryonic stem cells can differentiate into the functional nerve cells, however its use is connected with the posibility of tumorigenesis and also ethical problems. Promising results gives a combinatorial treatment using biomaterials as scaffolds for cells or growth factors. The knowledge of genetic modification of cells will be used in the future. The aim of this thesis is to sumarise current knowledge of the spinal cord treatment with emphasis on tissue specific stem cells. Key words: stem cells - spinal cord injury - tissue recovery - biomaterials

anglicky This thesis deals with legal aspects of stem cell research, which in the case of the Czech Republic remains quite disregarded by the general public. The text examines and analyzes the legislation regulating the aforementioned field of research at the international, European and Czech national level in a comprehensive manner. Particular emphasis is placed on legal aspects of embryonic stem cell research, which is also closely related to the legal status of embryo and its protection. The thesis also notes what types of regulatory approaches are adopted in various countries with respect to stem cell research and it subsequently demonstrates these conclusions by examples of Slovak Republic and the United Kingdom. Explanation is set in a broader framework through the introductory chapters that deal with biological and ethical aspects of stem cell research.

Immunogenicity of stem cells and their derivatives Nikola Doležalová Abstract Stem cells (SCs) have the potential to be used in regenerative medicine on the basis of their differentiation capacity and promising immunological properties, including low expression of histocompatibility antigens and costimulatory molecules, or secretion of suppressive cytokines. Their immunogenicity has often been ignored in the past but it is becoming clear that rejection of genetically incompatible SCs represents a very common issue. At present, SCs are extensively studied from the immunological point of view, since it represents a critical aspect of the safety of SC therapy. This thesis presents an overview of current knowledge about immunogenicity of SCs and their derivatives, including both pluripotent SCs (embryonic and induced pluripotent SCs) and adult SCs (mesenchymal, limbal, neural, haematopoietic and umbilical cord blood SCs). The expression of immunologically relevant molecules on their surface and interaction with the immune cells in vitro and in vivo will be discussed, together with suggestions for overcoming the immunological barriers for transplantation. Detailed analysis of these aspects necessarily has to precede the safe clinical translation of SC therapies.

The primary aim of this diploma thesis was to draw attention to the issue of bone marrow donation. The thesis focuses on the level of public awareness and people's significant doubts regarding bone marrow donation, as these doubts and apprehensions might be the main reason restraining them from entering the bone marrow donor register. The theoretical part provides a comprehensive summary of findings in this area. Substantial part addresses particular types of the hematopoietic cells taking process and the following donor treatment. The empirical part of this diploma thesis constitutes of a quantitative research, in which hundereds of respondents have been reached through an electronic questionnaire. Within exactly 10 days, 576 of them filed for the survey, 496 of them were valid and processed. The outcome of the research reflects strong need for raising factual public awareness in the area of hematopoietic cells donation, especially in respect to current donation methods. 79,51 % of respondents had no knowledge of peripheral blood stem cell (PBSC) donation method. 50,26 % of them admitted fear of the bone marrow donation procedure itself. Respondents taking part in the research showed great interest in the topic. 85,48 % of them would appreciate further education and 82,66 % assumes that...