Studium modelových membrán, proteinů a protein-membránových interakcí pomocí různých fluorescenčních technik

Membránové rafty (také často uváděny jako nanodomény) jsou membránové struktury, které jsou zodpovědné za spoustu buněčných procesů. Jejich charakterizace je velice složitá, protože nanodomény jsou opticky průhledné, jejich velikost je pod rozlišením optických mikroskopů a dynamika je veliká. Navíc vysoká různorodost buněk ještě více komplikuje jejich studium. Pro zjednodušení buněk jsou velice často používány membránové modelové systémy jako obrovské unilamelární vezikuly (GUVs) nebo podporované fosfolipidové dvojvrstvy (SPBs). V mé disertační práci byly vyvinuty, testovány a vylepšovány různé fluorescenční techniky ke studiu těchto membránových domén v modelových systémech. Fázorová analýza, metoda pro analýzu dat dob dohasínání fluorescence, byla teoreticky a experimentálně testována a následně použita k charakterizaci membránových nanodomén v GUVs. Nejdříve jsme využili fázorovou analýzu v lipidových vezikulech ke studiu procesů excitovaných stavů, jako je jev relaxace rozpouštědla a Försterův rezonanční přenos energie (FRET). Dále jsme aplikovali fázorovou analýzu k charakterizaci interakcí mezi proteiny a ligandy, ke studiu sbalování a denaturace proteinů. Nakonec jsme použili fázorovou analýzu dat z FRET v kombinaci s fluorescenční korelační spektroskopií (FCS) k charakterizaci membránových...Membrane rafts (also referred as nanodomains) are membrane structures responsible for many cell processes. Their characterization is challenging because of the transparency, dynamics and small size of those structures. Moreover, high variability of cells makes their study even more complicated. In order to simplify the studies of membrane processes including the formation of those rafts often model membranes like Giant Unilamellar Vesicles (GUVs) and Supported Phospholipid Bilayers (SPBs) are used. In this Thesis new fluorescent tools for studying such membrane processed were developed, tested, or improved. Specifically, the phasor plot an approach applicable to the analysis of the fluorescence lifetime data, was theoretically and experimentally tested and afterwards applied to the characterization of the membrane nanodomains in GUVs. First, we introduced the phasor plots to the excitation state processes like solvent relaxation and Förster resonance energy transfer (FRET) in lipid vesicles. We also employed the phasor plots in protein-ligand interaction, protein folding and denaturation studies. Finally, the phasor plot analysis of FRET data in combination with Fluorescence Correlation Spectroscopy (FCS) was used in characterization of membrane nanodomains in terms of the size, mobility and...Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Challenging Intercultural Communicative Competence: Culture as an Emergent Phenomenon

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Molecular rheometry: direct determination of viscosity in L-o and L-d lipid phases via fluorescence lifetime imaging

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol–sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts

Recent Developments in Fluorescence Correlation Spectroscopy for Diffusion Measurements in Planar Lipid Membranes

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support

“A Very Remarkable Piece of Iron”: Towards a Theory of Material Imagination in Virginia Woolf’s “Solid Objects”

This article examines the supposed lack of “humanity” in Woolf’s short stories and novels by identifying its source in the sphere of “solid objects” and in the way these “objects” destabilize the coherence of what the western philosophical tradition typically refers to as “subject” (in the Cartesian sense). Referring to Moore’s direct realism as well as James’s and Mach’s radical empiricism, the discussion focuses on specific states of heightened perceptive intensity in which the perceiving subject stumbles on the verge of collapse and “mixes” itself with what it perceives. By considering these limit cases, this paper tries to demonstrate the way in which Woolf’s fiction might in fact be understood as illustrative of the process of de-humanizing de-centralization and dispersion of the already fluid consciousness and its blending with the impersonal material objects, resulting in a complete loss of one idea of “the human” (an idea based on the intellectual autonomy and sovereignty of a unified subject) and pointing towards a post-human and post-modern condition in which human becomes defined by the ever-widening circle of its own outsid

The study of model membrane systems, proteins and protein-membrane interactions using various fluorescence techniques

Membrane rafts (also referred as nanodomains) are membrane structures responsible for many cell processes. Their characterization is challenging because of the transparency, dynamics and small size of those structures. Moreover, high variability of cells makes their study even more complicated. In order to simplify the studies of membrane processes including the formation of those rafts often model membranes like Giant Unilamellar Vesicles (GUVs) and Supported Phospholipid Bilayers (SPBs) are used. In this Thesis new fluorescent tools for studying such membrane processed were developed, tested, or improved. Specifically, the phasor plot an approach applicable to the analysis of the fluorescence lifetime data, was theoretically and experimentally tested and afterwards applied to the characterization of the membrane nanodomains in GUVs. First, we introduced the phasor plots to the excitation state processes like solvent relaxation and Förster resonance energy transfer (FRET) in lipid vesicles. We also employed the phasor plots in protein-ligand interaction, protein folding and denaturation studies. Finally, the phasor plot analysis of FRET data in combination with Fluorescence Correlation Spectroscopy (FCS) was used in characterization of membrane nanodomains in terms of the size, mobility and..

Fyzický a psychický prostor v anglické modernistické literatuře

Souhrn (Abstrakt) v českém jazyce Práce se zabývá problematikou vztahu mezi fyzickým a psychologickým prostorem v díle D. H. Lawrence, Virginie Woolfové a Wyndhama Lewise v kontextu filozofie a estetiky počátku dvacátého století s ohledem na změny ve ztvárnění prostoru, místa a lidské osobnosti. Diskuse se zaměřuje na problematiku jednoty lidské subjektivity v povídkách a románech Virginie Woolfové a D. H. Lawrence a sleduje vztah mezi změnou stavů lidské mysli a její interakce s vnějším světem a jeho proměnlivými strukturami. Diskuse se dále zaměřuje na alternativní způsoby "prostorového" vyjádření procesu decentralizace lidské subjektivity, její prolínání s materiálním světem, hmotou a prostorem a studuje proměnlivý vztah mezi subjektem a jeho vnějškovostí. Zásadním přínosem k diskutované problematice je filozofie konzervativního myslitele, malíře a spisovatele Wyndhama Lewise. Lewis ve svém díle zastává opačnou pozici a trvá na autonomním, soběstačném a centralizovaném pojetí lidské subjektivity, nezávisle na vlivech vnějšího prostředí. Diskuse se dále opírá o srovnání s vybranými estetickými problémy a teoriemi výtvarného umění, především o italský futurismus a Cézannův postimpresionismus a zkoumá jejich vliv na dílo D. H. Lawrence a Wyndhama Lewise.The thesis discusses affinities between physical and psychical spaces in selected works of D. H. Lawrence, Virginia Woolf and Wyndham Lewis in connection with the main philosophical and aesthetic problems posed by the changes in modernist representation of character with respect to space and place. In doing so, the argument assesses the "in-human humanism" of D. H. Lawrence and Virginia Woolf which manifests itself in the interrelation between states of mind and material universe, the way in which the consciousness accommodates various material "admixtures" and how subjectivity "escapes" from subject to its own outside. Using the conservative thought of Wyndham Lewis as a vital source of comparison, the thesis examines how the interaction of these newly constructed modernist subjectivities with space changes and challenges traditional ideas of unity of self, personal identity and autonomous agency. Drawing on a number of themes from visual arts, the discussion connects these psychical factors with the notions of solidity and fluidity/stability and instability of material reality and individual objects, moving bodies or things in space. As a part of this, the thesis incorporates a detailed discussion of Italian Futurism, especially F. T. Marinetti's and Umberto Boccioni's theories of physical...Department of Anglophone Literatures and CulturesÚstav anglofonních literatur a kulturFilozofická fakultaFaculty of Art

Desing of supplementary coding switching unit

Téma diplomové práce je z oblasti řídicí a zabezpečovací techniky pro železniční dopravu. Řeší se zde problém návrhu a praktické realizace funkčního vzorku přepínací jednotky napájecího konce kolejového obvodu. Přepínací jednotka umožňuje činnost liniového vlakového zabezpečovače. Osnovu návrhu tvoří výkonový spínač ovládaný mikroprocesorem. Z důvodu zvýšení bezpečnosti jednotka obsahuje ještě kontrolní mikroprocesor, který kontroluje správnou činnost pracovního mikroprocesoru a bezporuchový stav výkonového spínače.The diploma thesis is dealing with the area of controlling and safety technology for the railway transportation. The problems of the design and practical realization of an functional sample of switching unit supplying the ends of a track circuit are solved here. The switching unit enables the operation of the train control system. The outline of the design is formed by a power switch operated with a microprocessor. In addition, for the increase in safety the switching unit contains a control processor, which is controlling the right function of a work microprocessor and the failure-free state of a power switch.

Physical and Psychical Spaces in Modern English Literature

The thesis discusses affinities between physical and psychical spaces in selected works of D. H. Lawrence, Virginia Woolf and Wyndham Lewis in connection with the main philosophical and aesthetic problems posed by the changes in modernist representation of character with respect to space and place. In doing so, the argument assesses the "in-human humanism" of D. H. Lawrence and Virginia Woolf which manifests itself in the interrelation between states of mind and material universe, the way in which the consciousness accommodates various material "admixtures" and how subjectivity "escapes" from subject to its own outside. Using the conservative thought of Wyndham Lewis as a vital source of comparison, the thesis examines how the interaction of these newly constructed modernist subjectivities with space changes and challenges traditional ideas of unity of self, personal identity and autonomous agency. Drawing on a number of themes from visual arts, the discussion connects these psychical factors with the notions of solidity and fluidity/stability and instability of material reality and individual objects, moving bodies or things in space. As a part of this, the thesis incorporates a detailed discussion of Italian Futurism, especially F. T. Marinetti's and Umberto Boccioni's theories of physical..