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Applying bioinformatic tools to better understand eye diseases

Singh, V ORCID: 0000-0003-4847-3643 2020 , 'Applying bioinformatic tools to better understand eye diseases', PhD thesis, University of Tasmania.

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The highly specialized cells of the eye function in concert to produce clear vision, one of the
most valued senses. Visual impairment or blindness can occur as a result of disease or trauma.
Age-related macular degeneration, glaucoma, cataracts and diabetic retinopathy are common
causes of vision loss in older individuals. This thesis explores the bioinformatics approaches
based on the central dogma as a model for exploring the experimental models for human eye
diseases including quality control of stem cells to detect chromosomal abnormalities;
epigenetic age prediction of ocular tissues; identification of novel genes in Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) knockout screening in uveal melanoma; and
RNA-Seq analysis to understand the molecular mechanisms in oxygen-induced retinopathy
and differentiation of dental pulp mesenchymal stem cells into trabecular meshwork cells.
In a virtual karyotyping study, I investigated the utility of a low-density genome-wide SNP
array for the karyotypic assessment of human pluripotent stem cells (hPSCs) using the Illumina
Infinium HumanCore BeadChip. Specifically, the resolution of these arrays in detecting
chromosomal aberrations and their ability to identify clonal variations was determined. It was
shown that the SNP array can detect chromosomal abnormalities when at least 25% of the cell
population is aberrant. Our data demonstrate that an array-based karyotyping offers an
economical and robust sampling method, in the genomic resolution, compared with standard
cytogenetic karyotyping of hPSCs. This approach could provide a reliable, rapid and costeffective
assessment of hPSCs clonality and virtual karyotype for large-scale generation and
maintenance of hPSCs.
To investigate the effects of aging among different tissues, we calculated the DNA methylation
age of whole peripheral blood and ocular tissue from the same individual and compared it with
the person’s chronological age. We found significant differences between chronological and
epigenetic ages (p<0.048). Our study showed that there is a significant difference (mean = 44.4
years) between chronological and epigenetic age in neurosensory retinal tissue and the same
pattern identified by various tools.
Through a CRISPR knockout screening study, we used the Human GeCKOv2 pooled library
in OCM1 cell lines (uveal melanoma) to identify novel genes that are associated with
tumorigenesis using the CRISPRAnalyzeR tool on next-generation sequencing data. Overall,
from our analysis, we found 15 genes that have relatively low expression in the passage 12 as
compared to the passage 0 and are associated with the metabolic process, cellular process,
primary metabolic process, cellular metabolic process, biological process and organic
substance metabolic process. Our study shows that these genes are crucial for cell proliferation;
however, further in-vitro and in-vivo validation is required.
In RNA-Seq studies, we investigated the miRNA expression in oxygen-induced retinopathy
(OIR) rat models through next-generation sequencing (RNA-Seq) data. Our RNA-Seq data
suggested that the expression of miR-143 dysregulated and mediated the regulatory networks,
leading to retinal neovascularization. Furthermore, miR-143 can influence cellular motion and
cell-matrix interaction during vascular formation. We also found that miR-126, miR-150 is
significantly down-regulated and directly involved in retinal neovascularization. We also
investigated the propensity of mesenchymal stem cells (MSC) populations (dental pulp-derived
MSCs) to differentiate into trabecular meshwork (TM) cells. We performed RNA sequencing
under two conditions: control (DPMSCs) and treated (with growth factors to differentiate into
the TM cells). Overall, we found over 8000 genes that are statistically significant and have an
association with tissue development, extracellular matrix development and related pathways,
and demonstrated the ability of DPMSCs to differentiate into TM cells. Using bioinformatics
techniques to develop models of blinding eye diseases can lead to new and better treatments to
preserve vision.

Item Type: Thesis - PhD
Authors/Creators:Singh, V
Keywords: Bioinformatics, RNA-Seq, Virtual Karyotyping, CRISPR Screening, Epigenetic clock
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Copyright 2019 the author

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