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Title: Vibrational spectroscopy for the interrogation of mechanisms for cellular transformation and differentiation
Author: Patel, Imran Iqbal
Awarding Body: Lancaster University
Current Institution: Lancaster University
Date of Award: 2012
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Vibrational spectroscopy occurs through the interaction of specific frequencies of light with molecular chemical bonds. Both Raman and FTIR spectroscopy detect frequency change or absorbance respectively. Application to biological samples derives a vibrational spectrum of the biomolecular components present in the interrogated area. Subtle alterations in spectra can be determined through sophisticated multivariate approaches. In this PhD, a number of studies show the potential of vibrational spectroscopy in determining disease related biomolecular alterations prior to morphological manifestation, and its potential as a diagnostic tool. Raman microspectroscopy was employed to interrogate zones of the prostate with varying susceptibility to malignancy. Point spectra were acquired from glandular epithelial cells and stroma from central, peripheral and transitional zones. Exploratory analysis using principa component analysis (PCA) significantly discriminates spectra between all zones of the prostate. Wavenumber-intensity relationships primarily identified biomolecular alterations in DNA of epithelial cells between the peripheral zone (PZ) to less susceptible transitional zone (TZ) and central zone (CZ). These alterations may facilitate functional roles to the susceptibility of cancer. In addition to zone specific susceptibility to malignancy, incidence of prostate cancer varies worldwide amongst different demographical populations. A seven-fold difference incidence is present between UK and India populations. Multiple vibrational spectroscopy techniques were utilised to acquire spectra from prostate TZ glands and stroma between India and UK cohorts. Application of multivariate approaches PCA and principal component analysis-linear discriminant analysis (PCALDA) significantly discriminating spectra between both populations. Protein secondary structure alterations were present between cohorts with DNA alterations exclusively located in epithelial layers. These imply underlying alterations potentially contributing to differential incidence of prostate cancer worldwide. The application of vibrational spectroscopy as an in-vivo imaging technique is being established within the biomedical field. Contrast and resolution of tissue structures in images can be limited depending on the sophisticated computational analysis utilised to distinguish morphology. Such requirements are vital during surgical resections when a high degree of confidence is needed and to retain as much normal functioning tissue. We employed Raman microspectroscopy imaging of endometrial tissues and tested the applicability of a number of sophisticated computational approaches for biochemical image reconstruction. Comparison of results with histological images of tissue identified multivariate curve resolution-alternating least squares (MCR-ALS) as a superior computational analysis for image re-construction than PCA and Hierarchical Cluster Analysis (HCA). Cancer epidemiological studies suggest that cancers may arise decades after initial exposure to a carcinogen. The complexity for diagnosis of these latent cancers is due to the lack of biomarkers for identification. A by-product of chronic oxidative stress, 4-hydroxy-2-nonenal (4HNE) could provide a potential diagnostic biomarker. Synchrotron radiation-Fourier transform infrared spectroscopy (SR-FTIR) was employed to interrogate terminal ductal lobular units (TDLU) of breast identified as 4HNE positive or negative. Significant discrimination between TDLUs was achieved, with DNA (VsP02-) as tlie major biomolecular alteration present between epithelial cells of positive and negative TDLU. Such alterations were not present in inter or intra-lobular stroma. This suggests that DNA-damaging influences in early-life breast epiu~elium may confer alterations that may underlie subsequent later-life progression. Adult stem cells are vital for the maintenance, repair and regeneration of many tissues in the human body. Tracking of these stem cells has been limited to labelling techniques which has proven difficult as there is no definitive biomarker of adult stem cells. SR-FTIR microscopy was used to image map human inter-follicular epidermis as a non-labelling approach to stem cell identification in the epidermis. Focal plane array (FP A) imaging was also applied for sub-cellular spatial resolution and wider imaging areas. Primary exploratory analyses using peA was applied prior to application of linear discriminant analysis (LDA). Asymmetric and symmetric phosphate (P02) was identified as discriminating biomolecular markers between cells which are hypothesized to be stem, transit-amplifying (TA) and terminally differentiated (TD) cells. The basal layers of epidermal regions were identified to contain cells with signatures which are likely to represent stem cells approximately every 5 to 6 cells. Spectrally similar cells, likely to be adult stem cells were also identified in the dermis in close proximity to rete pegs possibly related to the region of the follicular bulge. This PhD utilises the potential of vibrational spectroscopy techniques coupled with computational analyses to determine biomolecular alterations that can assist in diagnosis and understanding the underlying mechanisms of a range of cancers. -
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available