Mehanizmi vezikulacije in stabilnost vezikularnih struktur v vzorcih humane krvne plazme in morskih mikroalg : doktorska disertacija

Božič, Darja (author), Kralj Iglič, Veronika (mentor), Kogej, Ksenija (comentor)

Z namenom preučevanja mehanizmov nastanka in stabilnosti submikronskih veziklov v bioloških vzorcih smo analizirali male delce v krvi in kulturah treh vrst morskih mikroalg: Tetraselmis chuii (T. chuii), Dunaliella tertiolecta (D. tertiolecta) in Phaeodactylum tricornutum (P. tricornutum). V krvi in različnih pripravkih krvne plazme, pridobljenih s postopkom centrifugiranja, ter izolatih malih delcev, pripravljenih iz različnih krvnih frakcij s postopkom diferencialnega centrifugiranja in ultracentrifugiranja, smo identificirali dva mehanizma nastanka malih delcev med procesiranjem vzorcev – ehinocitno transformacijo eritrocitov s cepitvijo veziklov s koncev brstov ter trganje trombocitov pod vplivom strižnih sil pri centrifugiranju. Razvili smo matematični model za opis dinamike prerazporeditve delcev v krvi pri centrifugiranju. Ta omogoča teoretično oceno optimalnega časa centrifugiranja za pridobitev krvne plazme s kar najvišjo koncentracijo trombocitov in malih delcev, ter njihov visok izplen iz krvi; pri čemer je protokol mogoče prilagoditi posamezniku na osnovi znane vrednosti hitrosti sedimentacije eritrocitov iz hemograma. Pri mikroalgah T. chuii, D. tertiolecta in P. tricornutum smo identificirali tri različne mehanizme nastanka malih delcev, ki jih najdemo v izolatih, pripravljenih z metodami diferencialnega centrifugiranja in filtracije s tangentnim pretokom: odcepitev bička in sproščanje gradnikov v medij pri mikroalgi T. chuii, razpad unduliranih protruzij in bičkov pri mikroalgi D. tertiolecta in tvorbo virusu podobnih membranskih nanodelcev pri mikroalgi P. tricornutum. Z metodo dinamičnega sipanja svetlobe smo preverili občutljivost izolatov malih delcev za razgradnjo s surfaktantom Triton X-100 ter njihovo stabilnost pri spreminjanju osmolarnosti in pH ter spremljali njihove spremembe pri segrevanju.

• Language: Slovenian
• Keywords: biološki vzorci, biološki nanomaterial, brstenje membran, diferencialno centrifugiranje, krvna plazma, morske mikroalge, sedimentacija, zunajcelični vezikli
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2021
• PID: 20.500.12556/RUL-128899
• COBISS.SI-ID: 83836163
• Publication date in RUL: 13.08.2021

BOŽIČ, Darja, 2021, Mehanizmi vezikulacije in stabilnost vezikularnih struktur v vzorcih humane krvne plazme in morskih mikroalg : doktorska disertacija [online]. Doctoral dissertation. [Accessed 7 June 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=128899

Secondary language

• Language: English
• Title: Mechanisms of vesiculation and stability of vesicular structures in samples of human blood plasma and marine microalgae : doctoral dissertation

Abstract

Aiming to study genesis and stability of submicron vesicles in biological samples we analysed small particles in samples derived from human blood and cultures of three marine microalgae: Tetraselmis chuii (T. chuii), Dunaliella tertiolecta (D. tertiolecta), and Phaeodactylum tricornutum (P. tricornutum). In blood and various blood plasma preparations obtained by centrifugation, and in isolates of small particles prepared from different blood fractions by differential centrifugation and ultracentrifugation we identified two mechanisms of small particle formation during sample processing – echinocyte transformation of erythrocytes with subsequent budding of vesicles from the tip of spicules, and fragmentation of platelets under the influence of shear forces during centrifugation. We developed a mathematical model to describe the dynamics of redistribution of particles in blood during centrifugation. This model allows a theoretical estimation of the optimal centrifugation time to obtain blood plasma with the highest possible concentration of platelets and small particles and their high recovery from the blood; and wherein the protocol can be adjusted for an individual blood sample, based on haemogram. In the microalgae T. chuii, D. tertiolecta and P. tricornutum, we identified three different mechanisms of formation of small particles found in isolates prepared by tangential flow filtration and by differential ultracentrifugation. These mechanisms include the shedding of flagella and the release of flagellar hairs and scales into the medium in T. chuii, the disintegration of undulating protrusions and flagella in D. tertiolecta and formation of virus-like nanoparticles in P. tricornutum. Using the dynamic light scattering method, we also assessed the solubilisation of the small particles in the isolates by surfactant Triton X-100, their stability in media with different osmolarity, and pH and their changes upon heating.

• Keywords: biological nanomaterial, biological samples, blood plasma, differential centrifugation, extracellular vesicles, marine microalgae, membrane budding, sedimentation