[27/2022/IGC/PSD] Announcement concerning recruitment to the Poznań Doctoral School of the...

RESEARCH PROJECT OBJECTIVES / RESEARCH HYPOTHESES

Numerous keratoconus (KTCN) loci, coding genes, and sequence variants were postulated as involved in the pathogenesis of KTCN. We HYPOTHESIZE that accumulation of both variants in regulatory elements (REs) near the previously described KTCN candidate genes (e.g. adjacent to genes with mutations, SNPs, indels, and/or genes with differential expression) and the changes in chromatin availability, substantially impact phenotypic alterations in the KTCN corneal epithelium (CE) and corneal stroma (CS). THE GOAL OF THIS PROJECT is to decipher the potentially complex connections by determining KTCN-specific chromatin alterations and regions containing the REs with non-coding sequence variants affecting gene function, taking into account the implications of previously identified coding DNA sequence variation and altered expression in dysregulated signaling pathways in KTCN.

STUDY DESIGN The study will be performed on human corneas. Characterization of morphologically altered corneal regions surrounding the KTCN cone apex and subsequent topographic regions will be carried out in KTCN CE and CS, comparing the findings to the corresponding CE and CS topographic regions in control corneas. Cell nuclei, DNA, and corneal cryosections will be prepared and processed in the multi-omic approach including 1) Assay for Transposase- Accessible Chromatin with Sequencing (ATAC-Seq), 2) Whole-Genome Sequencing (WGS), 3) Spatial transcriptomics, and 4) Two-dimensional proteomic assessment. Functional significance of selected SNVs that could possibly affect the gene expression or function of REs, chosen based on integrated data of the ATAC-Seq, WGS, spatial transcriptomics, and two-dimensional proteomic assessment, will be further determined using the reporter assays in the Human Corneal Epithelial (HCEpC) and Human Corneal Keratocytes (HCK) cell lines. Our data/material samples obtained during previous KTCN research in corneas will be used in the verification experiments, including Sanger sequencing, RT-qPCR, Western Blots, and immunohistochemistry. Finally, the performed bioinformatics integration analyzes will point to the KTCN-specific genomic, transcriptomic, and proteomic features, influencing the corneal morphological and structural alterations.