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Effects of the mutation in MCM3AP gene on neuromuscular junction formation

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Title: Effects of the mutation in MCM3AP gene on neuromuscular junction formation
Author(s): Sinkko, Matilda
Contributor: University of Helsinki, Faculty of Medicine
Degree program: Master's Programme in Translational Medicine
Specialisation: Cross-disciplinary translational medicine
Language: English
Acceptance year: 2021
Abstract:
In this master’s thesis, in vitro neuromuscular junction (NMJ) model was set up using microfluidic devices. Additionally, the effect of R878H/R878H mutation in MCM3AP gene that causes an early-onset peripheral neuropathy on NMJ formation and maintenance was studied. To study human NMJs that significantly differ from other mammal NMJs is challenging and new models to study the function of these complex and highly specialized structures are needed. Induced pluripotent stem cells (iPSC) and motor neurons were characterized with gene expression studies using qRT-PCR and with immunocytochemistry studies using commonly known markers for pluripotency and motor neurons. NMJs were studied in 2D co-cultures and with microfluidic devices. Gene expression studies were conducted from 2D co-cultures and co-cultures in microfluidic devices provided detailed information of the localization and morphology of NMJs. Expression of essential genes for NMJ formation together with immunocytochemistry results with alpha-bungarotoxin (BTX) staining showed that NMJs were formed in both control and R878H/R878H mutant cell line co-cultures. There was a trend of lower gene expression levels of NMJ essential genes in the R878H/R878H mutant line compared to the control line and also immunocytochemistry results indicated impairment in NMJ formation in the mutant line, but further studies are needed to validate the effect of R878H/R878H mutation on the NMJ formation. In future, functional studies could be conducted to investigate whether these NMJs are functional and the information from the motor neuron terminal is conveyed to the muscle membrane.
Keyword(s): neuromuscular junction peripheral neuropathy motor neuron induced pluripotent stem cell co-culture myoblast microfluidic device


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