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Calcium signalling in astrocytes under physiological and pathological conditions
Svatoňová, Petra ; Anděrová, Miroslava (advisor) ; Kolář, David (referee)
Calcium signalling in astrocytes represents an important component, which enables proper neuronal functioning under physiological conditions. Alterations in Ca2+ signalling, accompanied by an increase in intracellular calcium levels is a hallmark for numerous pathological states of central nervous system, such as traumatic and ischemic brain/spinal cord injuries, epilepsy as well as neurodegenerative diseases, such as Alzheimer's disease and psychiatric disorders, such as schizophrenia. The research analyzing the molecular components of astrocytic Ca2+ signalling can help us understand the control mechanisms used in calcium signalling and thus be greatly beneficial for further therapeutic research. Powered by TCPDF (www.tcpdf.org)
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Nervous tissue regeneration following ischemic injury in adulthood - the role of glial cells
Kamenická, Monika ; Anděrová, Miroslava (advisor) ; Kletečková, Lenka (referee)
Ischemic stroke (ischemia) is one of the most common causes of death in the world. The consequences of this disease are enormous and markedly affect the lives of patients who often are unable to live a full quality of life as before. Therefore, the current research is focused on elucidating new mechanisms that could mitigate the effects of ischemia and better regeneration of nerve tissue. This theses aims to summarize the current knowledge about neurogenesis/gliogenesis in the nerve tissue under physiological conditions, after ischemic injury and subsequent regeneration. The first chapter is focused on neurogenesis in the nervous tissue of adults. The two main neurogenic regions are described, such as subventricular zone and the gyrus dentatus in hippocampus. The following is a brief description of cells that are located in neurogenic regions and their function under physiological conditions. The second chapter focuses on gliogenesis in adult nervous tissue and describes the glial cells responsible for numerous functions in CNS. Furthermore, the functions of individual types of glial cells are listed. The third chapter gives the overview about pathophysiology of ischemia. The author tries to explain what is happening in the brain tissue during and following ischemia, what types of ischemia are...
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Astrocyte volume regulation during aging
Eliášová, Barbora ; Anděrová, Miroslava (advisor) ; Vargová, Lýdia (referee)
Astrocytes, as one of the glial cell types, have many important functions in healthy functioning of the central nervous system (CNS) but also in its pathology. Since they play a key role in maintenance of ionic, neurotransmitter and water homeostasis in CNS, they possess the ability to regulate their volume. Hypo- or hyperosmotic stress can trigger regulatory volume decrease or increase in astrocytes in order to stabilize their volume. During aging, astrocytes undergo many changes together with the rest of the brain. In order to determine whether these alterations involve also regulatory volume mechanisms, we employed three dimensional morphometry, which comprises confocal microscope scanning of fluorescently labelled astrocytes in brain slices of EGFP/GFAP mice and quantification of astrocyte volume during different pathological stimuli. Time-dependent volume changes of hippocampal astrocytes were recorded while applying either hypoosmotic solution or solution with high extracellular potassium concentration. In the four different age groups studied in the experiment, several differences in volume changes were discovered together with some sex-dependent alterations in astrocyte volume. Additionally, in accordance with previous studies, two subpopulation of astrocytes were identified using...
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Circadian system in astrocytes
Ľalíková, Kristýna ; Bendová, Zdeňka (advisor) ; Honc, Ondřej (referee)
The circadian system affects almost all cells in the mammalian body. These cells include astrocytes, which together with microglia and oligodendrocytes represent the main types of glial cells found in the brain. The first chapter of this thesis presents a summary of circadian system characteristics and focuses mainly on the molecular mechanism underlying its functioning. The second chapter is devoted to astrocytes, astrocyte calcium signaling, and the process of gliotransmission. The third and last chapter connects both topics and discusses the circadian system in astrocytes. It presents evidence of astrocytic circadian oscillations existence and physiological consequences of its action. Great attention is paid to circadian rhythms in gliotransmission, with a focus on gliotransmitters ATP and glutamate. As the most impressive output of the circadian system of astrocytes is presented the participation in maintaining the rhythmic activity of the main circadian oscillator located in the suprachiasmatic nucleus of the hypothalamus. Key words: circadian system, clock genes, astrocytes, gliotransmission, calcium signaling, glutamate, ATP
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The immune response of naïve mice infected with the neuropathogenic schistosome Trichobilharzia regenti
Macháček, Tomáš
Helminth neuroinfections represent a serious health issue, but the mechanisms of the host immune response often remain neglected despite the fact they might contribute to pathogenesis. This is partly due to the unavailability of clinical samples and the lack of suitable laboratory models. Herein, I focused on the characterization of several aspects of the immune response of mice infected with the neuropathogenic avian schistosome Trichobilharzia regenti. After the percutaneous infection of mice (accidental hosts), most T. regenti schistosomula are entrapped and eliminated in the skin, but the parasite antigens initiating the protective immune reaction are not known. Our in vitro experiments revealed that T. regenti cathepsin B2, a cysteine peptidase used for the skin penetration, activates bone marrow-derived dendritic cells much stronger than the parasite homogenate, suggesting its role in initiating the mixed type1/2 host immune response. However, some schistosomula manage to escape from the skin and continue their migration to the spinal cord. Here they crawl preferentially within the white matter which we demonstrated by the robust 3D imaging techniques, ultramicroscopy and micro-CT. The invasion of the spinal cord is accompanied by striking hypertrophy of astrocytes and microglia. We showed...
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The immune response of naïve mice infected with the neuropathogenic schistosome Trichobilharzia regenti
Macháček, Tomáš
Helminth neuroinfections represent a serious health issue, but the mechanisms of the host immune response often remain neglected despite the fact they might contribute to pathogenesis. This is partly due to the unavailability of clinical samples and the lack of suitable laboratory models. Herein, I focused on the characterization of several aspects of the immune response of mice infected with the neuropathogenic avian schistosome Trichobilharzia regenti. After the percutaneous infection of mice (accidental hosts), most T. regenti schistosomula are entrapped and eliminated in the skin, but the parasite antigens initiating the protective immune reaction are not known. Our in vitro experiments revealed that T. regenti cathepsin B2, a cysteine peptidase used for the skin penetration, activates bone marrow-derived dendritic cells much stronger than the parasite homogenate, suggesting its role in initiating the mixed type1/2 host immune response. However, some schistosomula manage to escape from the skin and continue their migration to the spinal cord. Here they crawl preferentially within the white matter which we demonstrated by the robust 3D imaging techniques, ultramicroscopy and micro-CT. The invasion of the spinal cord is accompanied by striking hypertrophy of astrocytes and microglia. We showed...
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The immune response of naïve mice infected with the neuropathogenic schistosome Trichobilharzia regenti
Macháček, Tomáš ; Horák, Petr (advisor) ; Bilej, Martin (referee) ; Schabussova, Irma (referee)
Helminth neuroinfections represent a serious health issue, but the mechanisms of the host immune response often remain neglected despite the fact they might contribute to pathogenesis. This is partly due to the unavailability of clinical samples and the lack of suitable laboratory models. Herein, I focused on the characterization of several aspects of the immune response of mice infected with the neuropathogenic avian schistosome Trichobilharzia regenti. After the percutaneous infection of mice (accidental hosts), most T. regenti schistosomula are entrapped and eliminated in the skin, but the parasite antigens initiating the protective immune reaction are not known. Our in vitro experiments revealed that T. regenti cathepsin B2, a cysteine peptidase used for the skin penetration, activates bone marrow-derived dendritic cells much stronger than the parasite homogenate, suggesting its role in initiating the mixed type1/2 host immune response. However, some schistosomula manage to escape from the skin and continue their migration to the spinal cord. Here they crawl preferentially within the white matter which we demonstrated by the robust 3D imaging techniques, ultramicroscopy and micro-CT. The invasion of the spinal cord is accompanied by striking hypertrophy of astrocytes and microglia. We showed...
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Identification of changes in membrane properties of astrocytes in a mouse model of amyotrophic lateral sclerosis
Vaňátko, Ondřej ; Turečková, Jana (advisor) ; Vlachová, Viktorie (referee)
Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder of the central nervous system characterized by loss of motor neurons and voluntary muscle degeneration. Astrocytes play a major role in regulation of the disease onset and progression due to their intimate association with neurons. Regulation of ionic homeostasis is one of their key functions and its failure has been linked to several neurological diseases. The aim of this thesis was to explore differences in membrane properties of astrocytes in ALS. To fulfill this aim, a double transgenic mouse strain with ALS-like phenotype and a specific expression of enhanced green fluorescent protein in astrocytes was generated. To phenotype this strain, two sensorimotor tests, wire grid hang test and rotarod test, were conducted. Immunohistochemistry was used to characterize the strain on a cellular level and to explore changes of specific ion channels. Functional properties of astrocytes were explored using the patch clamp technique. The double transgenic strain has the characteristic ALS-like phenotype and is comparable to the original strain with differences in symptom onset and progression between models and sexes. On the cellular level, there are characteristic ALS features, specifically loss of motor neurons and astrogliosis....
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Practical aspects of single-cell RT-qPCR analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Pavlínková, Gabriela (referee)
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...
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