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Browsing by Author "Silva, Anita Ferreira Triguinho da"

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  • Study of BUB1 functions in the neurodevelopment
    Publication . Silva, Anita Ferreira Triguinho da; Carvalhal, Sara; Calado, Sofia
    Budding uninhibited by benzimidazole 1 (BUB1) is a kinase protein essential for proper chromosome segregation. Recently, we identified two biallelic BUB1 mutations linked to primary microcephaly, a rare condition causing reduced head size. The transmission pattern of BUB1’s individuals was consistent with the Autosomal Recessive Primary Microcephaly 30 (MCPH30). The present thesis aimed to understand how the BUB1 protein functions during human neurodevelopment, and how, when affected, it leads to microcephaly. We successfully generated a new induced pluripotent stem cells (iPSCs) line from patient 2’s fibroblasts. Two clones - clone 4 (C4) and clone 7 (C7) - were characterised by standard norms. Both showed pluripotency and the ability to differentiate in cells from all germ layers. However, while C4 had a diploid karyotype, C7 held trisomy on chromosome 8. To evaluate BUB1’s role in neurodevelopment, wild-type iPSCs were differentiated in neuro progenitor cells (NPCs) and then submitted to differentiation and maturation steps. Our findings reveal that when NPCs were treated with the BUB1 inhibitor drug, BAY1816032, they could not eliminate the signal for the main substrate of the BUB1 kinase, the phosphorylation on H2A-T120 (pH2A-T120), as demonstrated in somatic cells. However, pH2A-T120 levels on forebrain cells were sensible to BAY1816032. These cells hold several morphological modifications. In addition, diploid C4 iPSCs were differentiated into NPCs. However, C4-NPCs exhibited poor stability under multipotent conditions. Additionally, C4-forebrain cells also displayed morphological changes. Previous lab results showed that BAY1816032-treatment induced microcephaly in chicken embryos. However, our preliminary study revealed that microcephalic size depends on the developmental stage. This project successfully developed a new cell line for studying BUB1, made available to the community (Ferreira et al., 2024). And, despite preliminary, our overall results support BUB1's crucial role in NPCs maintenance and proper forebrain neurodevelopment.
    2025-03-19Master thesis Embargoed access Show more