Analiza difuzije integrina alfa5beta1 metodom praćenja na razini jedne molekule

master's thesis

• Title (english): Analysis of alpha5beta1 integrin diffusion with Single-Particle Tracking
• Author: Jelena Ivčević
• Mentor: Carlo Manzo (mentor)
• Mentor: Petra Korać (mentor)
• Committee member: Petra Korać (predsjednik povjerenstva)
• Committee member: Renata Šoštarić (član povjerenstva)
• Committee member: Duje Lisičić (član povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2019-09-26, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

U ovom radu implementirana je metoda proučavanja lateralnog gibanja integrina α5β1 na razini jedne
molekule. Integrini su transmembranski, glikoproteinski heterodimeri, najvećim dijelom odgovorni za
procese usidrenja, adhezije i migracije stanice. Lateralna difuzija integrina na membrani važan je aspekt
stanične adhezije i mobilnosti, a njen utjecaj na stanične biokemijske i biomehaničke procese još uvijek
nije detaljno istražen. Kvantificiranje gibanja proteina membrane stanice i razlučivanje njihove biološke
funkcije uvelike je unaprijeđeno razvojem metode praćenja na razini jedne molekule (SPT od engl.
Single-Particle Tracking). SPT se sastoji od obilježavanja ciljane molekule niskom koncentracijom
reportera, snimanja video-zapisa mikroskopom i obrade slika složenim algoritmima koji
nanometarskom preciznošću lociraju individualne molekule te povezuju njihovo gibanje u putanje.
Razumijevanje složenog gibanja na razini molekule potencijalno može doprinijeti razvijanju terapijskih
postupaka za stanja povezana s disfunkcijom integrina, poput kožnih rana ili metastaziranja tumora.
Glavni cilj ovog istraživanja je stoga bio implementirati i optimizirati SPT-metodu za mjerenje difuzije
integrina α5β1. Difuzija integrina obilježenih s reduciranim poluantitijelom uspoređena je s difuzijom
integrina obilježenih klasičnim cjelovitim antitijelom, kako bi se utvrdilo utječe li imunoobilježavanje
na gibanje integrina zbog dimerizacije i stvaranja umjetnih klastera. Utvrđena je stat

Abstract (english)

In this thesis, a method for studying lateral mobility of integrin α5β1 on a single-molecule level was set
up and applied. Integrins are transmembrane, heterodimeric glycoproteins, mainly responsible for the
processes of cell anchoring, adhesion, and migration. Lateral diffusion of integrins on the membrane is
an important aspect of the cell adhesion and mobility, but how it affects the cell’s biochemical and
biomechanical processes is still not thoroughly investigated. Quantifying movement of membrane
proteins and elucidating their biological function has greatly been improved with Single-Particle
Tracking (SPT). SPT is based on the sparse labeling of the target molecule, recording the movement of
particles in a series of videos by optical microscopy and analysis of videos by image-processing
algorithms that locate individual molecules with nanometric precision and establish a correspondence
between particles in each frame. Understanding integrin’s complex behavior at the single-molecule
level could potentially lead to the development of treatments for conditions linked to integrindysfunction
such as injury or cancer. The objective of this thesis was to implement and optimize the
SPT method for studying the diffusion of α5β1 integrin at the single-molecule level. Diffusion of
integrins labeled with the reduced half-antibody fragment was compared to those labeled with the whole
antibody to test if the immuno-labeling is influencing integrin movement through dimerization and
artificial clustering. A statistically significant difference in the distribution of two types of labeled
integrins was determined.

• Keywords: praćenje na razini jedne molekule; lateralna difuzija; integrini
• Keywords (english): Single-Particle Tracking; lateral diffusion; integrins
• Language: croatian
• URN:NBN: urn:nbn:hr:217:778153
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2021-09-26
• Created on: 2019-10-16 07:41:07