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ANALYSES OF STRUCTURE OF COLLAGEN FIBRES IN ARTERIAL WALL USING MODERN METHODS OF LIGHT MICROSCOPY
Turčanová, Michaela ; Kochová, Petra (referee) ; Hampl, Aleš (referee) ; Burša, Jiří (advisor)
The doctoral thesis deals with the analysis of the arrangement of collagen fibres in arteries and their correct evaluation and use in structurally motivated constitutive models of the material. The first part of the work is focused on the literature search of mechanical properties of arteries and on an overview of available methods for the detection of waviness, orientation and dispersion of fibres. Most works identify fibre angles as additional parameter from mechanical tests and thus degrade the structural nature of the model. The second part describes an automatic algorithm that can evaluate the local directions of fibres and their scattering from images from a polarizing microscope and structurebased hyperelastic constitutive models. Furthermore, there is an emphasis on choosing the most appropriate imaging method based on fluorescence microscopy, which will help us to distinguish the waviness and scattering of fibres. In the next part of the thesis, two experiments on porcine arteries are presented in order to determine the influence of different magnitudes of biaxial deformation on fiber orientation and dispersion. The last part of the work presents the evaluated structural parameters for porcine and human aortas, which were analyzed not only under polarized light, but also under a laser scanning confocal microscope, thanks to which the waviness of the fibers and their global direction were obtained.
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Neuroprotective effects of novel anorexigenic analogs of prolactin-releasing peptide (PrRP) in models of neurodegeneration in vitro and in vivo
Mengr, Anna ; Maletínská, Lenka (advisor) ; Sumová, Alena (referee) ; Hampl, Aleš (referee)
Alzheimer's disease (AD) is a progressive brain disorder characterized by extracellular beta amyloid (Aβ) plaques, intracellular neurofibrillary tangles formed by hyperphosphorylated Tau protein and neuroinflammation. Since obesity and type 2 diabetes mellitus (T2DM) have been established as risk factors for the development of neurological disorders, anorexigenic and antidiabetic peptides, such as prolactin-releasing peptide (PrRP) seem to be potential neuroprotective agents. In the first part of the study, the molecular mechanisms of action of natural PrRP31 and its lipidized analog palm11 -PrRP31 was studied in the human neuroblastoma cell line SH-SY5Y. Both compounds significantly activated the signaling pathways typical for insulin promoting cell survival and growth. Moreover, PrRP31 and palm11 -PrRP31 increased cell viability and suppressed apoptosis in methylglyoxal-stressed SH-SY5Y cells. The second part of the thesis was focused on the neuroprotective and anti-inflammatory effects of 2-month-long subcutaneous administration of palm11 -PrRP31 in the brains of APP/PS1 mice, model of Aβ pathology. Palm11 -PrRP31 significantly reduced the Aβ plaque load and microgliosis in the hippocampi, cortices, and cerebella. Furthermore, palm11 -PrRP31 increased the synaptogenesis and attenuated...
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Regeneration and structural changes of the nerve tissue after the extracellular matrix modification
Vallová, Jana ; Machová Urdzíková, Lucia (advisor) ; Hampl, Aleš (referee) ; Hubálek Kalbáčová, Marie (referee)
Regeneration and structural changes of the nerve tissue after the extracellular matrix modification. Abstract ECM modification may contribute to changes in nerve tissue plasticity. Therefore, 4- methylubulliferone is used in our study, which disrupted the structure of the perineuronal networks surrounding some types of neurons responsible for the formation of memory traces. Mice were fed a 4-MU diet (6.7 mg / g / day) for 6 months, which improved their memory skills in a spontaneous recognition test without a significant adverse effect on the kidneys, liver, or joints.In the next part of the study, an extracellular matrix (UC-ECM) was derived from fetal human umbilical cord tissue also generated as biomimetic hydrogel. Due to the generated UC-ECM's low stability and rapid degradation, the structure was stabilized by covalent genipin bonding. Stabilization with 1 mM genipine increased the biological stability of the material. UC-ECM as well as ECM/G didn't show toxicity in vitro in mesenchymal stem cell proliferation; axonal budding or neural stem cell growth and differentiation were not adversely affected. The biocompatibility of both materials was verified in vivo by applying the material to an intracortical photothrombotic rat lesion, where gelation and infiltration of the lesion and hydrogel by host...
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Manipulating the mammalian oocyte and embryo - Biological and epigenetic aspects
Fulková, Helena ; Hozák, Pavel (advisor) ; Hampl, Aleš (referee) ; Motlík, Jan (referee)
CONCLUSIONS . By antibodies against ďfferent covalent histone modifications and 5-methylcytosine, we have partialty characterised the epigenetic changes taking place during the oocyte mauration and in early mammalian embryogenesis in the mouse and pig, respectively. o We have also characterised thc epigenetic repogramming activities of cytoplasts derived from oocytes at different stages of maturation after somatic cell nuclear transfer. . We have evaluated the epigenetic effec$ of selected procedures that are currently used for embryo production. . Finally, we have developed a new cryopreservation scheme for oocyte nuclear material storage. orrr research is engaged in the development ofnew bíotechniques as well as elucidating and characterising the epigenetic pÍocesses that take place during normal and abnormal embryogenesis. Abnormal embryonic development is for example often observed in somatic cell nuclear transfer embryos. These techniques can also be potentially used not only in human medicine but also for valuable livestock and endangered species preservation Oy e.g. interspecies nuclear transfer). Especially in human meďcine, attention to the ethical issues associated with these techniques must be paid. It is also clear tbat many biological problems still do exist and these should not be...
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Differentiation of Human Embryonic Stem Cells into Endothelial and Smooth Muscle Cells as a Model for Vascular Development
Obrtlíková, Petra ; Trněný, Marek (advisor) ; Mokrý, Jaroslav (referee) ; Hampl, Aleš (referee)
Aims of the study: We hypothesized that the optimal source of cell for vascular regeneration will be the progenitor cells derived from human embryonic stem cells (ESCs) which can differentiate both into endothelial cells (ECs) as well as vascular smooth muscle cells (SMCs). We propose to test if the population of human ESCs, H9 cell line, can serve this role. Material and methods: Human ESCs were cocultured with stromal cells S17, M2-10B4 or Wnt1 expressing M2-10B4 cell line to generate a CD34+ cell population. After that, CD34+ cells were sorted and cultured in media containing specific cytokines to generate ECs. To induce SMC differentiation from ECs, culture conditions were changed to media containing platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-b1). Phenotypic and functional characteristics of these populations were demonstrated by flow cytometry, immunohistochemistry, QRT- PCR, tube formation assay, and response to calcium signaling agonists. Results: CD34+ vascular progenitor cells derived from human ESCs give rise to ECs and SMCs. These two populations express cell specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when cocultured in Matrigel. Wnt1 overexpressing stromal cells...
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Cytokine expression in chemically-induced senescence
Nováková, Zora ; Hodný, Zdeněk (advisor) ; Hampl, Aleš (referee) ; Dvořák, Michal (referee)
AbstractJanderova-Rossmeislova L., Novakova Z., Vlasakova J., Philimonenko V., Hozak P., Hodny Z., 2007. PML protein association with specific nucleolar structures differs in normal, tumor and senescent human cells. J. Struct. Biol. 159, 56-70. Cellular senescence, widely recognized as a potent suppressor of tumorigenesis, represents response to cellular stress and DNA damage which results in irreversible cell growth arrest. Growing evidence signalizes crucial role of cytokine production in senescence phenomenon. Recently, many research groups have efforted to define exact character of senescence-associated secretory phenotype and its function in senescence development and maintentance. Factors secreted by senescent cells, mainly of proiflammatory character, were found to have pronounced effects on their environment as well as on its own producer. These observations were obtained preferentially on models of replicative senescence and oncogene-induced senescence. Vlasakova J, Novakova Z, Rossmeislova L, Kahle M, Hozak P, Hodny Z. 2007. Histone deacetylase inhibitors suppress IFNalpha-induced up-regulation of promyelocytic leukemia protein. Blood 109: 1373-80 Novakova Z, Man P, Novak P, Hozak P, Hodny Z. 2006. Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis....
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New markers of colon cancer evolution in ulcerative colitis
Švec, Jiří ; Kment, Milan (advisor) ; Keil, Radan (referee) ; Hampl, Aleš (referee)
Background: Long-standing ulcerative colitis (UC) has an increased risk of evolving into colorectal cancer (CRC) and upregulated expression of cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), survivin, telomerase catalytic subunit (TERT), integrin-linked kinase (ILK) and transcription factors c- MYB and TCF-4, has been implicated in the development and progression of sporadic colorectal cancer. Nevertheless much less is known about their role in the process of UC-associated colon carcinogenesis. Methods: We analyzed the gene expression of these markers during the transition of colonic mucosa from chronic inflammation to epithelial neoplasia in biopsies of UC patients using quantitative real-time polymerase chain reaction and immunohistochemistry, and compared the expression profiles of this gene panel in samples of patients with CRC and in human tumor xenografts of SW620 malignant colonic cells. Additionally, we determined the expression of these genes in mouse models of sporadic and colitis-associated CRC in A/J and ICR mouse strains using quantitative RT-PCR and laser microdissection. Results: The transcript levels of survivin, c-MYB, COX-2, iNOS, and TCF-4 showed statistically significant increase during neoplastic transformation of UC-patient colonic mucosa, whereas TERT and ILK...
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Messenger RNA stability and microRNA activity in mouse oocytes
Flemr, Matyáš ; Svoboda, Petr (advisor) ; Motlík, Jan (referee) ; Hampl, Aleš (referee)
The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...
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Development of extracellular-matrix scaffolds for CNS repair
Výborný, Karel ; Kubinová, Šárka (advisor) ; Hampl, Aleš (referee) ; Vandrovcová, Marta (referee)
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....
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