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Gradual Molecular Changes in Primary Porcine Cells Expressing Mutated Huntingtin

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Title:
Gradual Molecular Changes in Primary Porcine Cells Expressing Mutated Huntingtin
Title (in czech):
Postupné molekulární změny v primárních prasečích buňkách exprimujících mutovaný huntingtín
Type:
Dissertation
Author:
Mgr. Petra Šmatlíková, Ph.D.
Supervisor:
prof. MVDr. Jan Motlík, DrSc.
Opponents:
Mgr. Kateřina Trejbalová, Ph.D.
RNDr. Milan Reiniš, CSc.
Consultant:
Zdeňka Ellederová
Thesis Id:
115186
Faculty:
Faculty of Science (PřF)
Department:
Department of Cell Biology (31-151)
Study programm:
Developmental and Cell Biology (P1529)
Study branch:
-
Degree granted:
Ph.D.
Defence date:
11/06/2019
Defence result:
Pass
Language:
English
Keywords (in czech):
Huntingtonova choroba, mutovaný huntingtín, velký zvířecí model, primární fibroblasty, MSCs
Keywords:
Huntington's disease, mutated huntingtin, large animal model, primary fibroblasts, MSCs
Abstract (in czech):
ABSTRAKT / SÚHRN PRÁCE Huntingtonova choroba (HCH) je fatálne dedičné ochorenie spôsobené CAG tripletovými expanziami v géne huntingtínu, čo vedie k expresii mutovanej formy tohto proteínu (mtHtt). Hlavnými príznakmi HCH sú neurodegenerácia, osteoporóza, testikulárna degenerácia, strata svalového tkaniva a poruchy srdcového svalu, strata hmotnosti, metabolické zmeny a poruchy spánku. Vzhľadom k tomu, že proteín huntingtín (Htt) má úlohu vo viacerých biologických procesoch, mnoho molekulárnych mechanizmov ako oxidačný stres, mitochondriálna dysfunkcia, poškodenie DNA a iné, je ovplyvnených mtHtt. Avšak, patogénne mechanizmy mtHtt v HCH ešte nie sú veľmi dobre pochopené. Transgénny model miniprasaťa pre HCH (TgHD) ponúka možnosť izolácie neobmedzeného počtu primárnych buniek, a na rozdiel od primárnych buniek získaných od pacientov s HCH v neskorších štádiách ochorenia, TgHD miniprasací model umožňuje sledovať molekulárne zmeny vyskytujúce sa postupne s vekom a progresiou ochorenia. Takže TgHD model miniprasaťa a primárne bunky izolované z neho hrajú dôležitú úlohu pri skúmaní a pochopení základnej mechanickej príčiny HCH. Zamerali sme sa na molekulárne a bunkové zmeny v primárnych bunkách izolovaných z TgHD miniprasiat a ich wild type (WT) kontrol v rozličných vekových skupinách (24, 36 a 48 mesiacov). V mezenchymálnych kmeňových bunkách (MSCs) z TgHD miniprasiat sme zistili negatívny vplyv mtHtt na adipogénnu diferenciáciu týchto buniek. V primárnych fibroblastoch z TgHD miniprasiat v pre-symptomatickom štádiu (vo veku 24 – 36 mesiacov) je fragmentačný proces mtHtt sprevádzaný vyšším poškodením jadrovej DNA. Neskôr, s nástupom prvých neurologických príznakov u TgHD miniprasiat vo veku 48 mesiacov, fibroblasty vykazujú spolu s miznúcimi fragmentmi mtHtt zníženú permeabilitu a následnú dereguláciu bunkového cyklu a abnormálne kratšie G2/M oneskorenie, čo vedie k postupnej akumulácii oxidačného stresu v týchto bunkách. Tieto zmeny, ktoré sa postupne vyskytujú v primárnych fibroblastoch z TgHD miniprasiat v rozličnom veku, navrhujú nový pohľad na postupný účinok mtHtt, ktorý ešte doposiaľ nebol popísaný.
Abstract:
ABSTRACT Huntington´s disease (HD) is inherited fatal disorder caused by CAG triplet expansions in the huntingtin gene resulting in the expression of mutated huntingtin protein (mtHtt). The main symptoms of HD are neurodegeneration, osteoporosis, testicular degeneration, loss of muscle tissue and heart muscle malfunction, weight loss, metabolic changes, and sleeping disturbances. Since huntingtin protein (Htt) has a role in several biological processes, many molecular mechanisms, like oxidative stress, mitochondrial dysfunction, DNA-damage, and others, are affected by mtHtt. However, its exact pathogenic mechanisms in HD are still not well understood. Transgenic minipig model of HD (TgHD) serves an opportunity to isolate unlimited number of primary cells and unlike primary cells obtained from HD patients, often in the late stages of the disease, the TgHD minipig model allows to monitor molecular changes occurring gradually with age and progression of the disease. Thus, TgHD minipig model and primary cells isolated from it play an important role in investigating and understanding the underlying mechanistic cause of HD. We focused on molecular and cellular changes in primary cells isolated from TgHD minipigs and their wild type (WT) controls at different ages (24, 36, and 48 months). In mesenchymal stem cells (MSCs) from TgHD minipigs, we detected the negative impact of mtHtt on the adipogenic differentiation of these cells. In primary fibroblasts from TgHD minipigs at the pre-symptomatic stage (24 – 36 months old animals), the fragmentation process of mtHtt is accompanied by the higher nuclear DNA damage. Later, with the onset of first neurological symptoms in TgHD minipigs at the age of 48 months, the fibroblasts exhibit along with faded mtHtt fragments, decreased permeability followed by dysregulation of the cells cycle and aberrantly shorter G2/M delay leading to gradual accumulation of oxidative stress in these cells. These changes occurring gradually in primary fibroblasts from TgHD minipigs at different age, suggest new insight on the progressive action of mtHtt, which has not been described yet.
Documents
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Download Text of the thesis Mgr. Petra Šmatlíková, Ph.D. 1.13 MB
Download Attachment to the thesis Mgr. Petra Šmatlíková, Ph.D. 19.45 MB
Download Abstract in czech Mgr. Petra Šmatlíková, Ph.D. 89 kB
Download Abstract in english Mgr. Petra Šmatlíková, Ph.D. 59 kB
Download Autoreferat / doctoral thesis summary Mgr. Petra Šmatlíková, Ph.D. 285 kB
Download Supervisor's review prof. MVDr. Jan Motlík, DrSc. 59 kB
Download Opponent's review Mgr. Kateřina Trejbalová, Ph.D. 253 kB
Download Opponent's review RNDr. Milan Reiniš, CSc. 673 kB
Download Defence's report 91 kB