Title Standardization and Characterization of Neural Cellular Models and Biomarker Assay for Rare Genetic Diseases
Title (croatian) Standardizacija i optimizacija neuralnih staničnih modela i biomarker eseja za rijetke genetičke bolesti
Author Leonarda Vučić
Mentor Barbara Mlody (mentor)
Mentor Igor Jurak (komentor)
Mentor Matthias Berg (komentor)
Committee member Barbara Mlody (predsjednik povjerenstva)
Committee member Igor Jurak (član povjerenstva)
Committee member Anđelka Radojčić Badovinac (član povjerenstva)
Committee member Duško Čakara (član povjerenstva)
Committee member Miranda Mladinić Pejatović (član povjerenstva)
Granter University of Rijeka (Faculty of Biotechnology and Drug Development) Rijeka
Defense date and country 2023-10-26, Croatia
Scientific / art field, discipline and subdiscipline BIOTECHNICAL SCIENCES Interdisciplinary Technical Sciences
Abstract Niemann-Pick Disease Type C (NPCD) is a rare neurodegenerative disorder affecting 1 in 120,000 live births, characterized by complex alterations in sphingolipid metabolism. While cholesterol accumulation is a prominent feature, the situation is more intricate in the brain, where primary accumulating lipids are sphingolipids such as GM2 and GM3 gangliosides. So far, the only approved therapy to date, Miglustat, targets sphingolipid pathways and is used to prevent disease progression. The objective of this study is to improve an existing ganglioside assay for its utilization in cellular models. The optimization process encompasses the careful selection of the most effective extraction buffers, optimizing the protocol steps to enhance efficiency, and selecting appropriate sample sizes for cellular pellets. This study aims to develop a universally applicable assay that can be used across a wide range of cell lines. The assay validation involves the utilization of differentiated astrocytes and neural progenitor cells derived from fibroblast lines harboring NPC1-heterozygous and NPC1-homozygous variants. Quantification of gangliosides is performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).The findings of this investigation demonstrated a notable disparity in the buildup of GD3 ganglioside in fibroblast cell lines that were homozygous for the NPC1 gene mutation. Conversely, neural progenitor cells and astrocytes exhibited a reduction in GD3 ganglioside levels. Furthermore, it was observed that NPC1-homozygous fibroblasts exhibited increased concentrations of complex GD1a and GT1b gangliosides. Additional investigation is required to effectively address the inquiries that have been prompted by these results. This assay holds significant potential, hence redefining the application of cellular models in in vitro experiments related to lysosomal storage diseases. Upon optimization, the ganglioside assay will function as a valuable instrument for capturing the accumulation pattern of gangliosides that are linked to rare diseases.
Abstract (croatian) Niemann-Pickova bolest tipa C (NPCD) rijedak je neurodegenerativni poremećaj koji pogađa 1 od 120 000 živorođene djece, a karakteriziraju ga složene promjene u metabolizmu sfingolipida. Dok je nakupljanje kolesterola glavno obilježje ove bolesti, situacija je složenija u mozgu gdje se primarno nakupljaju sfingolipidi kao što su GM2 i GM3 gangliozidi. Do sada jedina odobrena terapija, Miglustat, inhibira sintezu sfingolipida i koristi se za sprječavanje progresije bolesti. Cilj rada je prenamijeniti postojeći esej gangliozida za njegovu upotrebu u staničnim modelima. Proces optimizacije obuhvaća pažljiv odabir otopine za ekstrakciju, optimiziranje koraka protokola za povećanje učinkovitosti i odabir odgovarajuće veličine uzorka za stanične pelete. Cilj ove studije je razviti univerzalno primjenjivi test koji se može koristiti u širokom rasponu staničnih linija. Validacija testa uključuje korištenje diferenciranih astrocita i neuralnih progenitorskih stanica diferenciranih iz linija fibroblasta koje sadrže NPC1-heterozigotne i NPC1-homozigotne varijante. Kvantifikacija gangliozida provodi se pomoću tekućinske kromatografije-tandem masene spektrometrije (LC-MS/MS). Rezultati ovog istraživanja pokazali su primjetnu razliku u nakupljanju GD3 gangliozida u linijama fibroblasta koje su bile homozigotne za mutaciju gena NPC1. Suprotno tome, neuralne progenitorske stanice i astrociti pokazali su smanjenje razine GD3 gangliozida. Nadalje, primijećeno je da NPC1-homozigotni fibroblasti pokazuju povećane koncentracije kompleksnih GD1a i GT1b gangliozida. Dodatna istraživanja su potrebna kako bi se učinkovito odgovorilo na upite koje su potaknute ovim rezultatima. Ovaj test ima značajan potencijal, stoga redefinira primjenu staničnih modela u in vitro eksperimentima koji se odnose na lizosomske bolesti nakupljanja. Nakon optimizacije, analiza gangliozida funkcionirat će kao vrijedan instrument za mjerenje gangliozida koji su povezani s rijetkim bolestima.
Keywords
Gangliosides
Niemann-Pick disease type C
Rare diseases
LC-MS/MS
Keywords (croatian)
gangliozidi
Niemann-Pickova bolest tip C
rijetke bolesti
LC-MS/MS
Language english
URN:NBN urn:nbn:hr:193:301580
Study programme Title: Medicinal chemistry Study programme type: university Study level: graduate Academic / professional title: magistar/magistra medicinske kemije (magistar/magistra medicinske kemije)
Type of resource Text
File origin Born digital
Access conditions Embargoed access Embargo expiration date: 2028-10-26
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Created on 2023-10-27 18:26:20