doktorska disertacija
Iztok Urbančič (Author), Janez Štrancar (Mentor), Miha Škarabot (Co-mentor)

Abstract

Membrane živih celic opravljajo vrsto nujno potrebnih nalog: opredeljujejo notranjost celice, jo ščitijo pred zunanjimi vplivi, nadzirajo pretok snovi ter s tem vzdržujejo stalne notranje razmere, prevajajo signale, omogočajo ali pospešujejo kemijske reakcije idr. Pri tem poleg beljakovin, ki so donedavna veljale za glavno aktivno sestavino, ključno vlogo igrajo tudi lipidi, ki se v membrani lateralno razporedijo v t. i. membranske domene. Njihove vloge in lastnosti so še razmeroma nepoznane, saj so domene zaradi velikosti med nano in mikrometri ter življenjskega časa med nano in milisekundami današnjim merilnim metodam težko dostopne. . V okviru doktorskega dela smo razvili fluorescenčno mikrospektroskopijo (FMS), ki povezuje dve uveljavljeni metodi z različnimi okni občutljivosti: fluorescenčno mikroskopijo, ki omogoča slikanje vzorcev z krajevno ločljivostjo okoli 200 nm, ter spektroskopijo, s katero lahko spremljamo molekularne lastnosti neposredne okolice označevalcev na ravni nanometrov in nanosekund. Z izvirnim načinom zajemanja podatkov in uvedenim prilagajanjem modelnih funkcij smo spektralno ločljivost, značilno za spektroskopske meritve razsežnih vzorcev, prenesli na mikroskopski nivo. Hkrati smo odpravili vpliv bledenja fluorescence na izmerjene podatke, ki je doslej neobhodno popačilo obliko spektrov svetlobno neobstojnih barvil in zato omejevalo zanesljivost FMS. Z uvedenim pristopom smo razširili paleto uporabnih okoljsko občutljivih molekularnih označevalcev, ki jih lahko raziskovalci sedaj še bolje prilagodijo potrebam svojih raziskav. Bledenje fluorescenčnega signala smo z razvitim načinom merjenja in obdelave celo izkoristili za pridobivanje novih podatkov o molekularnem okolju, saj je hitrost bledenja nekaterih barvil odvisna od fizikalno-kemijskih lastnosti okolice označevalcev. Tako smo dopolnili nabor krajevno odvisnih informacij, kar je še posebno pomembno za raziskave pestrih bioloških sistemov, pri katerih za zadovoljiv opis potrebujemo čim več neodvisnih spremenljivk. S FMS smo pokazali, da lahko tako na modelnih membranskih sistemih kot na membranah živih celic zanesljivo spremljamo spremembe faz in domen pod vplivom temperature in biokemijske sestave. Izmerjen premik vrha fluorescenčnega spektra barvil NBD in Laurdan za 1 3 nm zaradi različnih lokalnih polarnosti je zadostoval, da smo razločili posamezne liposome v gelski, tekoči urejeni ali tekoči neurejeni lipidni fazi. Nedvoumnost rezultatov smo potrdili z opazovanjem faznega prehoda posameznih liposomov iz gelske v tekočo neurejeno fazo med segrevanjem vzorca z grelnim stekelcem ali infrardečim laserskim žarkom. Spektralna in krajevna ločljivost FMS sta ostali na podobni ravni tudi pri opazovanju kompleksnejših vzorcev, kot so npr. mešanice liposomov in celic, zaradi česar smo lahko razjasnili mehanizem dostave protitumorske učinkovine v celice raka dojke s pomočjo lipidnih nanodelcev. Iz razlik v izsevanih fluorescenčnih spektrih smo ugotovili, da se membrane liposomov zlivajo celičnimi, s čimer smo pripomogli k razvoju novih biomedicinskih pristopov za učinkovitejše zdravljenje najtežjih bolezni našega časa. Metodo FMS smo nadgradili z analizo polarizacije izsevane fluorescenčne svetlobe, ki je povezana z ureditvijo dipolov barvila v membranah in s tem s konformacijami označevalcev. Z združitvijo spektralnega in polariziranega zaznavanja smo dokazali, da eni najpogosteje uporabljenih fluorescenčnih označevalcev % fosfolipidi z barvilom NBD % v membranah zavzamejo različne konformacije na razdaljah pod optično krajevno ločljivostjo. Z razvitim matematičnim modelom smo ta stanja podrobneje opisali in določili njihove deleže. Na slednje je najmočneje vplivala visoka koncentracija holesterola v membrani, kar bi lahko v prihodnje izkoristili za raziskovanje nanoskopskih značilnostih zgradbe bioloških membran ter z njo povezanih biofizikalnih in biokemijskih procesih.

Keywords

spektralno slikanje;obdelava spektrov;bledenje fluorescence;okoljsko občutljivi fluorescenčni označevalci;NBD;Laurdan;membranske domene;velikanski enoslojni liposomi;molekularne konformacije;disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UM FNM - Faculty of Natural Sciences and Mathematics
Publisher: I. Urbančič]
UDC: 543.423.3:535.372:577.352(043.3)
COBISS: 271289088 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Response of biomembrane domains to external stimuli
Secondary abstract: The membranes of living cells support diverse vital: they define and protect the interior of the cell, control the flux of matter to sustain desirable internal conditions, transmit signals, enable or promote chemical reactions, etc. Besides proteins, which had long been considered the main active component of biomembranes, lipids also play an important part by organizing themselves into lipid rafts or membrane domains. However, their roles and functions remain largely unclear due to their small size, ranging between nano- and micrometers, and short lifetimes on the order of nano to milliseconds, which makes them inaccessible to the experimental methods presently available. In the scope of this doctoral thesis, we set up fluorescence microspectroscopy (FMS), bridging two well established methods with different windows of sensitivity: fluorescence microscopy, which enables imaging of the samples with spatial resolution down to 200 nm, and fluorescence spectroscopy that provides molecular information of the environment at nanometer and nanosecond scale. Introducing a novel acquisition scheme and fitting the data with a mathematical model, we preserved the spectral resolution, characteristic for spectroscopic measurements of bulk samples, also at microscopic level. We have at the same time overcome the effects of photobleaching, which had previously distorted the measured spectral lineshape of photosensitive dyes and consequently hindered the reliability of FMS. Our approach has therefore greatly extended the range of applicable environment-sensitive probes, which can now be designed to better accommodate the needs of each experiment. Moreover, photobleaching of fluorescence signal can now even be exploited to obtain new information about molecular environment of the probes, as bleaching rates of certain probes also depend on physical and chemical properties of the local surroundings. In this manner we increased the number of available spatially localized parameters, which becomes invaluable when investigating complex biological systems that can only be adequately characterized by several independent variables. Applying the method FMS to model membrane systems as well as to living cells, we showed that we can reliably detect the differences in lipid phases and membrane domains upon changes of temperature or biochemical composition. A 1-3 nm spectral shift of probes NBD and Laurdan due to different local polarity was sufficient to distinguish individual vesicles in gel, liquid ordered, or liquid disordered lipid phase. The results were corroborated by observations of phase transition of individual liposomes from gel to liquid disordered phase upon controlled heating of the sample. The spectral and spatial resolution of FMS were preserved also when observing complex biological samples, such as mixtures of liposomes and cells, which allowed us to identify the delivery mechanism of a cancerostatic drug into human breast cancer cells by lipid nanoparticles. Small spectral shifts revealed that the membranes of drug-carrying liposomes fuse with cell membranes. Our findings pave the way towards more efficient treatment of the gravest maladies of our time. Furthermore, we upgraded FMS by analyzing the polarization of emitted fluorescence, which is related to the orientational order of dyes' dipoles in the membrane and therefore to molecular conformations of the probes. The combination of spectral and polarized detection revealed that some of the most widely used probes - NBD-labelled phospholipids - undertake various conformations that coexist at distances below optical spatial resolution. Developing a mathematical model, we additionally characterized these conformations and determined their relative portions. The latter were greatly affected by high concentration of cholesterol, which could be largely exploited to extend our insight into the nano-structure of biomembranes and associated biophysical and biochemical processes.
Secondary keywords: fluorescence microspectroscopy;spectral imaging;spectral analysis;fluorescence photobleaching;environment-sensitive fluorescent probes;NBD;Laudran;biomembranes;membrane domains;giant unilamellar vesicles;molecular conformations;dissertations;Biološke membrane;Disertacije;Biomembranske domene;Fluorescenčna mikrospektroskopija;
URN: URN:SI:UM:
Type (COBISS): Doctoral dissertation
Thesis comment: Univ. v Mariboru, Fak. za naravoslovje in matematiko, Oddelek za fiziko
Pages: 131 str.
ID: 8728556