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Title: Proteomic and metabolomic analysis of the effects of UV-A radiation on the cyanobacterium nostoc punctiforme ATCC 29133
Author: Wase, Nishikant V.
ISNI:       0000 0004 2724 8575
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2010
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Over the last two decades, thinning of the ozone layer has raised serious concerns throughout the world, since it allows penetration of harmful ultraviolet radiation (UV-R) onto the Earth. Ultraviolet radiation (UV-R, 280-400 nm) has both direct and indirect effects on living organism. Both UV-A and UV-B have deleterious effect on living organisms. Numerous studies were undertaken in the past that have attempted to elucidate the biological effect of UV-B on photosynthetic organisms such as higher plants and microbes such as green algae and cyanobacteria, but there is only very limited knowledge about the effect of UV-A radiation on photosynthetic organisms. In the context of climate change and ozone depletion, it is clearly beneficial to understand the physiological and underlying molecular mechanism of UV-A response of cyanobacteria. This thesis primarily serves to generate understanding of response of cyanobacterium at the proteome and metabolome level in response to UV-A radiation. During this PhD project, a cyanobacterium was selected as an organism of choice because it has a geological past that can traced back to the Precambrian era, long before the production of the present ozone layer. Because of their geological past, cyanobacteria were thought to be well equipped in sustaining UV-R. In the present study, the filamentous cyanobacterium Nostoc punctiforme ATCC 29133 was used as a model organism to understand the effect of UV-A radiation on the physiology and the underlying molecular response. In order to assess the effects that environmental UV-A perturbation has on regulatory networks and pathways of N. punctiforme, a quantitative proteomics investigation was performed using soluble proteome sample. A total of 572 unique proteins were found in both studies constituting 8.41 % of the total proteome. Effect of short-term exposure (4h, 12h, 24h) and long-term exposure (20 days) was elucidated with the recent and powerful mass spectrometry based approach using iTRAQ (isobaric tag for relative and absolute quantification) methodology. Approximately, 32 and 61 proteins were found significantly changed in abundance during short-term and long-term exposure respectively. Abundance of some of the metabolically important proteins (13) were assessed using pSRM (pseudo selected reaction monitoring) and a strong correlation with the iTRAQ dataset was observed. Further, using HPLC, it was observed that UV-A has strong effect on photosynthetic accessory pigments (UV-A treatment period 4, 8, 12, 24 hours, 3, 5, and 7 days). An initial increase in carotenoids, ~-carotene, astaxanthin, and zeaxanthin was observed which later on decreased. An induction of scytonemin production during an acclimatization phase (4 hrs) was also observed. Using GC-MS, 62 compounds of known chemical structures were identified. Statistically significant elevated levels of glycine, alanine, tyrosine, proline, malate and succinic acid was observed (treatment interval periods 1, 3, 5, 7, 9, and 11 days), indicating a possible role of these metabolites during UV-A stress. Under prolonged exposure, UV-A not only substantially retarded the growth of N. punctiforme but a lowered abundance of photosynthetic apparatus and phycobilisomes was also observed. A number of targets (3 proteins) that are believed to have a strong role in the UV-sensing and signal transduction were also identified. Additionally, it was observed that long-term exposure causes the induction of increased protein scaffolding, redox rebalancing, and DNA repairing. In contrast, short-term exposure causes an immediate response from both the primary photosynthetic machineries, and also the secondary pigmentation apparatus. Additiona"y increased abundance of heat shock proteins was observed in both short-term and long-term treatment condition. Fina"y, a comparison of iTRAQ and pSRM data reaffirms the caveat regarding underestimation of quantitative measurements using iTRAQ. As a thesis closure, a comprehensive proteomics and metabolomics characterization of effect of UV-A stress in a model cyanobacterium N. puncfiforme ATCC 29133 is presented. The compilations of established analytical tools used to infer both qualitative and quantitative biological observations can also be adapted to other systems. Suggestions for future work are made.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available