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Evaluation of mitochondrial activity in living cells using fluorescence methods
Fedorov, Vasilii ; Mézl, Martin (referee) ; Čmiel, Vratislav (advisor)
Monitoring mitochondrial dynamics can help in the therapeutic treatment of serious diseases and slowing down the aging process. This work was parted into a theoretical part and a practical part. In the theoretical part, research was carried out in the field of mitochondria and methods of activity evaluation. In the practical part in the Python environment, an automatic algorithm for evaluating mitochondrial activity based on the analysis of morphological signs was implemented. The algorithm included image preprocessing, localization of mitochondria, segmentation, and calculation of morphological features such as number of mitochondria, total area, average area, circularity index, eccentricity index. We were able to get and perform an analysis on 16 microscopic images and confirm the success of an algorithm that could be used for drug discovery and testing purposes.
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3D rekonštrukcia povrchu buniek hypokotylov Arabidopsis thaliana so zmenenými hladinami cytokinínov
Hubinský, Marcel
Plants are multicellular organisms, which tightly control their growth and morphogene-sis to successfully accomplish the final reproductive individual plant. At the cellular level, plant development consists of two main processes, cell proliferation and cell ex-pansion. Differences in the timing, rate and spacing of these developmental processes determine the final size and structure of plants organs, such as hypocotyl. Cytokinins (CKs) play a key role during many aspects of plant development, including hypocotyl development. We confirmed the important role of CK during expansion phase of hypocotyl cells. Hypocotyls with increased levels of CK growing under low light conditions were formed by elongated epidermal cells whereas hypocotyls lacking CK produced smaller cells. Using advanced confocal microscopy methods and transgenic plants with GFP-labeled cortical microtubules (proCaMV35S::MBD-GFP) enabled to design recon-struction of hypocotyl surface with altered levels of CK. The final 3D model of hypocotyl epidermal cells shed a light on detail morphological changes upon CK alterations
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Exprese proteinu NS5 viru klíšťové encefalitidy v lidských neurálních buňkách
JAKLOVÁ, Kateřina
This study focuses on the detection of tick-borne encephalitis virus (TBEV) NS5 protein in infected and NS5-transfected DAOY HTB-186 human neural cells. TBEV NS5 protein was shown to localize mainly on the membranes of the endoplasmic reticulum. An interesting finding was also nuclear localization, which is supported by the obtained data from both, confocal microscopy and subcellular fractionation.
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Fluorescent Methods in Research of Eukaryotic Cells
Chmelíková, Larisa ; Babula, Petr (referee) ; Pešl,, Martin (referee) ; Provazník, Ivo (advisor)
Tato práce zkoumá aplikaci fluorescenčních metod používaných v in vitro studiích v oblasti regenerace srdeční tkáně. Konfokální fluorescenční mikroskopie je vhodnou mikroskopickou technikou pro výzkum v této oblasti, protože umožňuje vizualizaci 3D struktur a distribuce buněk ve 3D modelech. Používané fluorescenční markery by měly být dlouhodobě stabilní, biokompatibilní a netoxické pro živé buňky. V současné době je použití nanočástic jako superparamagnetické nanočástice oxidu železa (SPION) velmi populární; velké množství studií ukazuje, že jsou vhodné pro dlouhodobé experimenty. Tento výzkum využívá superparamagnetické maghemitové nanočástice svázaným rhodaminem na jejich povrchu (SAMN-R) a popisuje jejích excitační a emisní spektrum, velikost a lokalizaci vbuňkách. Stanovení toxicity bylo provedeno měřením reaktivních forem kyslíku (ROS) a nekvantitativním měřením pomocí fluorescenční mikroskopie bylo zjištěno, že hodnota dávky 20 µg·cm-2 je optimální pro aplikaci na živé buňky. Dále byl zkoumán vliv aplikace SAMN-R na buněčnou proliferaci a motilitu, kdy ve studii buněčné proliferace a scratch assay byla použita buněčná linie fibroblastů 3T3. Poté byla studována migrace jednotlivých buněk s použitím mezenchymálních kmenových buněk (MSCs), izolovaných zlidské tukové tkáně. Následná statistická analýza nepotvrdila, že by aplikace SAMN-R měla významný vliv na buněčnou proliferaci, kolektivní migraci nebo na migraci jednotlivých buněk. Lze tedy předpokládat, že SAMN-R jsou vhodným fluorescenčním markerem pro výzkum živých buněk, včetně experimentů voblasti regenerace tkáně. MSC buňky izolované z tukové tkáně mají velký potenciál v regeneraci srdeční tkáně. Jejich interakce s buněčnou linií srdečních svalových buněk HL-1 byly studovány pomocí scratch assay, kdy se tento model jeví jako nadějný a vhodný pro studium buněčných kontaktů a jejich roli přiregeneraci buněk.
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Detection of cells in confocal microscopy images
Hubálek, Michal ; Štursa, Dominik (referee) ; Škrabánek, Pavel (advisor)
The goal of the thesis was to create an application that automatically detects healthy cardiomyocytes from images captured by a confocal microscope. The thesis was created based on the specific needs of researchers from the Slovak Academy of Sciences.The application will facilitate and increase the efficiency of their research,because until now they have to evaluate the images and search for suitable cells manually. The RetinaNet convolutional neural network is used for detection and has been implemented in a user-friendly desktop application. The application also automatically records and stores coordinates of detected cells which can be used for capturing cells in higher image quality. Another advantage of the developed application is its versatility, which allows to train detection on other data, making it applicable to other projects. The result of this work is a functional, standalone and intuitive application that is ready to be used by researchers.
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Application of carbon based electrodes for electrochemical detection in flow system
Mika, Jan ; Dejmková, Hana (advisor) ; Fischer, Jan (referee)
Carbon is due to its electrochemical properties a favourite material for construction of electrodes suitable for detection in flow system. The two most often used techniques (coulometric and amperometric) will be discussed in this Thesis. Carbon paste electrodes (CPE) represent the amperometric detectors. CPE are unstable in the medium with high content of organic solvents, which might complicate their application in HPLC, where the mobile phase contains organic component. It is interesting, that glassy carbon paste electrodes are more stable under such conditions. The change of surface and response of the electrode caused by the effect of methanol are described in this Thesis along with the possible reason of stability of GCPE. Roughing of the surface exposed to methanol is demonstrated by confocal microscopy. This effect leads to increase of height of the peak, background current and noise and to the shift of peak potential to less positive values. These changes are dependent on the time of exposure and the concentration of methanol. Coulometric detectors work with high conversion effectiveness and sensitivity, but in case of their passivation, reactivation is a relatively complicated problem, often leading to the replacement of electrode material. Answer to this problem can be given by using...
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Actuoecology of testate amoebae in fresh water and soil environment in enteraction with fungi and their analysis with new microscopic techniques
Burdíková, Zuzana ; Holcová, Katarína (advisor) ; Jansa, Jan (referee) ; Hudáčková-Hlavatá, Natália (referee)
4 Abstract The present thesis focuses on testate amoebae (TA) and their relationship to their natural environment, as well as on relevant microscopic imaging methods. The bulk of the data has been published in original scientific papers and is compiled into three separate chapters (Pt I, Pt II and Pt III), each annotated by a brief introduction. (Pt I) The methods section is devoted to specialized microscopic techniques employed to broaden the scope of the ecological analyses. In particular, precise discrimination between live and dead individuals, biomass determination inside individual tests and a multi-modal visualization of the cytoplasm and organelles enhance the data. Laser scanning confocal microscopy and two-photon microscopy are the main imaging modalities employed to study TA morphology in detail. The data have implications for taxonomy and ecophysiology, including the use of TA as bioindicators of pollution. (Pt II) An actuoecological analysis focuses on the seasonal variability of TA species composition in a freshwater ecosystem, namely the Komo any ponds in Prague, during the course of the year. The species composition variation is correlated to simultaneously recorded limnological parameters such as temperature, pH, contamination by (heavy) metals (As, Cd, Mn, Ni, Fe, Pb), polycyclic aromatic...
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Study of quantitative parameters of Norway spruce needle structure under the effect of elevated CO₂ concentration and different irradiance
Kubínová, Zuzana ; Lhotáková, Zuzana (advisor) ; Urban, Otmar (referee) ; Gebauer, Roman (referee)
Zuzana Kubínová: Study of Quantitative Parameters of Norway Spruce Needle Structure under the Effect of Elevated CO2 Concentration and Different Irradiance, Doctoral Thesis, Prague 2019 Abstract Atmospheric concentration of CO2 is increasing, while its influence on plants is still not fully elucidated. Norway spruce (Picea abies L. Karst.) is an abundant conifer tree in European temperate and boreal forests, which behave as carbon sink in the global carbon cycle. The physiological response to elevated CO2 concentration may be interconnected with changes in leaf anatomy and morphology. Needle structure is also determined by other factors in addition to CO2 concentration, irradiance being the most important one. Thus, effect of irradiance was also included in our studies. The effects of elevated CO2 concentration and irradiance on Norway spruce needle structure were studied using new applications of well-established quantitative methods and novel methods enabling effective and unbiased analysis of needle structural traits. The General Procrustes analysis showed to be effective for needle shape on cross section comparison and the disector method proved to be suitable for chloroplast number estimates. The influence of elevated CO2 concentration and different irradiance on needle structure was studied at two...
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Mechanisms of invasiveness and transcription regulation in cancer cells
Tolde, Ondřej ; Folk, Petr (advisor) ; Kovář, Jan (referee) ; Brdička, Tomáš (referee)
The mechanisms of invazivity and regulation of transcription of cancer cells Cancer originates in cells that overcome the control mechanisms of the organism. Cancer cells can be eventually released from the site of origin and spread through tissues. Cancer cells can acquire certain mechanisms that enable them to more effectively invade surrounding tissue or layers of other cells. The research on the migration of cancer cells is important for the understanding of the origin and spreading of metastases and consequently for anticancer therapy. In my Ph.D. work, I participated in the research of the properties of invasive metastatic cells. We compared non-invasive rat sarcoma cell line with a higly metastatic cell line derived from it. We showed that cells of the invasive cell line use amoeboid mode of migration, have upregulated Rho/ROCK signaling, and have accumulated actin and myosin at the leading edge. It is at the leading edge where the cells generate their traction forces. Cells of non-invasive cell line use mesenchymal mode of migration and generate forces mainly at their retracting end. We also compared two breast cancer cell lines derived from a single carcinoma. We showed that the more invasive cell line, derived from its parental line by neoplastic transformation, displayed elevated cytoskeletal...
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Determination of the area ratio of tubular and surface membranes in cardiomyocytes
Hluší, Veronika ; Čmiel, Vratislav (referee) ; Chmelíková, Larisa (advisor)
This work contains a literature search regarding the function and structure of the tubular system of cardiomyocytes. Another theory includes the basic principle of fluorescence and a description of the fluorescence and confocal microscope. Next chapter describe the principle of isolation and subsequent staining of cardiomyocytes to obtain images of the tubular system is described. Next step is the implementation of a method for segmentation of the membrane and tubular system of cardiac cells and the application of this method to the obtained data.
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