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Modern optical in vivo methods in neurophysiological research
Tomáška, Filip ; Novák, Ondřej (advisor) ; Elsnicová, Barbara (referee)
Accurate visualization of structures and events at subcellular level is one of the major challenges of current neuroscience. Optical methods based on fluorescence imaging were optimized to record and control neural activity, thus presenting a powerful approach complementary to historically dominant electrophysiological techniques. The employment of two-photon excitation enabled in vivo imaging of neurons up to 1 mm from the sample surface without causing significant photodamage. The application of methods of molecular biology has yielded protein-based genetically targetable indicators of neural activity, possessing performance comparable to the traditional organic dyes. Moreover, heterologous expression of microbial opsins proved capable of light-induced neural excitation or silencing in a single-component manner. The combination of these optogenetic tools offers two-way control over neuronal populations with single cell resolution. If coupled with calcium or voltage fluorescent indicators and transgenic animal models, such systems represent a non- invasive, all-optical tool for simultaneous control and imaging of specific neuronal subtypes. Its application supported by electrical recordings may finally provide the data necessary for the uncovering of fundamental principles of neural functioning.
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Inducible promoters and their use in yeast cell manipulation
Přibáňová, Gabriela ; Palková, Zdena (advisor) ; Vopálenský, Václav (referee)
Promoters which can be regulated by different chemical or physical factors are often used in cell manipulations. This thesis focuses predominantly on promoter systems which use light as an inductor. There are two main approaches to controlling a promoter by light. The first one uses so-called "caged molecules", chemical inducers whose inducing activity is "masked" by a photolabile protecting group. The second approach includes optogenetic systems, which can regulate transcription in cells. These systems are encoded in the DNA of the organism, and light is the only external regulatory stimulus. Photoreceptors that need a specific cofactor (chromophore) are the main components of optogenetic systems. There are several groups of photoreceptors classified by the type of chromophore and photoactivation mechanism. This thesis gives an overview of optogenetic systems used for transcription regulation and focuses on different photoreceptors and induction mechanism used. The systems using photocaged molecules are described as well. Furthermore, the thesis deals with light- systems in yeast as a model organism as well as organism used for biotechnological purposes. Finally, some limitations of light inducible promoters are discussed, including the chromophore type, the wavelength of the light, and the...
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Dynamics of yeast colony differentiation: New approaches
Jurečková, Gabriela ; Palková, Zdena (advisor) ; Zimmermannová, Olga (referee)
The yeast Saccharomyces cerevisiae when growing on solid medium forms structured colonies. During its development, two subpopulations of cells are formed, termed as U cells ("upper cells") and L cells ("lower cells"). This nomenclature derives from their position within the colony. These cells differ from each other considering their morphology, metabolism, physiology and are characterized by varying degrees of resistance to stress. This diploma thesis deals with new methodological approaches that can be used in further research of yeast differentiation. The first part deals with yeast differentiation and de-differentiation. In the experimental part, the development of mechanically mixed yeast colonies was monitored. The development was monitored in situ in the colony section using fluorescence and "wide-field" microscopy. The ability to de-differentiate of already specialized cells was evaluated mainly according to the nature of the expression of the fluorescently labeled protein Ato1p, which serves as a protein marker of U cells. The second part deals with the optogenetic system EL222 and its possible use in yeast. Many optogenetic tools have been described to control various cellular processes using light. One of these optogenetic tools is the EL222 system, which allows the induction of...
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Inducible promoters and their use in yeast cell manipulation
Přibáňová, Gabriela ; Palková, Zdena (advisor) ; Vopálenský, Václav (referee)
Promoters which can be regulated by different chemical or physical factors are often used in cell manipulations. This thesis focuses predominantly on promoter systems which use light as an inductor. There are two main approaches to controlling a promoter by light. The first one uses so-called "caged molecules", chemical inducers whose inducing activity is "masked" by a photolabile protecting group. The second approach includes optogenetic systems, which can regulate transcription in cells. These systems are encoded in the DNA of the organism, and light is the only external regulatory stimulus. Photoreceptors that need a specific cofactor (chromophore) are the main components of optogenetic systems. There are several groups of photoreceptors classified by the type of chromophore and photoactivation mechanism. This thesis gives an overview of optogenetic systems used for transcription regulation and focuses on different photoreceptors and induction mechanism used. The systems using photocaged molecules are described as well. Furthermore, the thesis deals with light- systems in yeast as a model organism as well as organism used for biotechnological purposes. Finally, some limitations of light inducible promoters are discussed, including the chromophore type, the wavelength of the light, and the...
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Modern optical in vivo methods in neurophysiological research
Tomáška, Filip ; Novák, Ondřej (advisor) ; Elsnicová, Barbara (referee)
Accurate visualization of structures and events at subcellular level is one of the major challenges of current neuroscience. Optical methods based on fluorescence imaging were optimized to record and control neural activity, thus presenting a powerful approach complementary to historically dominant electrophysiological techniques. The employment of two-photon excitation enabled in vivo imaging of neurons up to 1 mm from the sample surface without causing significant photodamage. The application of methods of molecular biology has yielded protein-based genetically targetable indicators of neural activity, possessing performance comparable to the traditional organic dyes. Moreover, heterologous expression of microbial opsins proved capable of light-induced neural excitation or silencing in a single-component manner. The combination of these optogenetic tools offers two-way control over neuronal populations with single cell resolution. If coupled with calcium or voltage fluorescent indicators and transgenic animal models, such systems represent a non- invasive, all-optical tool for simultaneous control and imaging of specific neuronal subtypes. Its application supported by electrical recordings may finally provide the data necessary for the uncovering of fundamental principles of neural functioning.
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