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Title: Comparative transcriptional profiling of the human limbal epithelial crypt
Author: Kulkarni, Bina
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2013
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Limbal stem cell deficiency (LSCD) is one of the most prominent causes of cornea related blindness in the world with an incidence of approximately 240 new cases per year in the UK alone. Symptoms of LSCD include reduced vision, pain, watering, redness, photophobia and blepharospasm due to chronic inflammation and breakdown of the corneal epithelium. LSCD mainly results in loss of barrier between the corneal and conjunctival epithelium with overgrowth of conjunctival epithelium over the cornea resulting in corneal vascularisation and scarring. Therefore, LSCD not only causes debilitating blindness depending on the severity of the condition but also leads to long term morbidity. Despite the availability of advanced treatment options for LSCD such as limbal tissue grafting and ex-vivo corneal epithelium transplantation, basic research questions such as the identification of limbal stem cell markers (LSC) for effective isolation of a pure population of LSCs from the corneal epithelium remains unanswered. Anatomical studies on the ocular surface have identified a new structure at the limbus termed Limbal Epithelial Crypt (LEC). It is a solid cord of cells arising from the undersurface of palisades of Vogt and extends into the subepithelial stroma of the limbus. Immunohistochemical and electron microscopic studies have demonstrated the presence of SCs in this region. The primary aim of this research is to confirm the hypothesis that LEC is a corneal SC niche (SCN) and to identify any SC related genes of interest in the LEe. The transcriptional profiling of the ocular surface (OS) epithelial regions was performed with the in-house manufactured spotted oligonucleotide and the Gene 1.0 ST arrays platform. Comparison of the results of these two microarray studies facilitated validation of the results and also contributed to the gene database of the as epithelial regions. Methods Frozen tissue blocks of corneoscleral buttons were cryosectioned to obtain serial tissue sections. Good quality tissue sections comprising of as epithelial regions were transferred onto PALM slides for laser microdissection. Extracted RNA samples were amplified & hybridised to the in-house manufactured 30,000k spotted human oligonucleotide microarray chips and Gene LOST arrays. Primary analysis of raw data of both the microarrays studies was performed to filter and normalise the microarray data for removal of the noise and to smoothen the raw data . Secondary analysis of the data involved statistical methods such as ANOVA, principal component analysis unpaired t-test and Significance Analysis of Microarrays (SAM). A differentially expressed gene list for each as epithelial region was generated with these statistical analysis methods. The gene lists in both the microarray studies were further analysed at tertiary level for pathway analysis with Ingenuity Pathway Analysis and geneontology with Database for Annotation, Visualisation, and Integrated Discovery (DAVID) software respectively. The genes of interest in the microarray studies were validated with quantitative gene expression analysis (qPCR) and immunohistochemistry.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available