Identifier

etd-11162005-100643

Degree

Master of Science in Biological and Agricultural Engineering (MSBAE)

Department

Biological and Agricultural Engineering

Document Type

Thesis

Abstract

The objective of this study is to characterize the photobiological and thermal effects of UVA light on cell cultures. While near-ultraviolet light has been widely used to photoactivate fluorophores and caged compounds in cells, little is known of the long-term biological effects of this light. UVA (320-400nm) photoactivating light has been used for studying fast kinetic processes and is now being employed in higher doses to target longer duration phenomena (e.g. gene expression and silencing). Photoexposure experiments using 365nm light will examine how longer duration and increased UV doses affect photoactivation cell studies. Apoptotic and cellular injury assays have been used to determine the nature and threshold of this light-induced effect. UVA light sources of low and high intensity have been used for light doses up to 23.85 J/cm2. HeLa cells exposed to staurosporine, high intensity UVA, and equivalent UVB served as positive UVA-induced and UVB-induced apoptotic controls. Cells were stained with annexin V-Cy5 and propidium iodide for apoptosis analysis with a conventional flow cytometer. Cell cultures at lower densities had higher percentages of apoptotic and dead cells, and were also more susceptible to UVA damage than more dense cell seedings. The dose to induce apoptosis and death in 50% of the cells (dose1/2) was determined for two different commercially available UVA light sources: 7.6 J/cm2 for the GreenSpot photocuring system and 2.52 J/cm2 for the BlakRay lamp. No significant cellular responses were found for doses below 1.6 J/cm2 from the GreenSpot light source. A temperature control and measurement system was used to determine direct heating from the UVA sources and also the effect that cooling culture dishes has on minimizing cell damage. Photoduration was also found to be significant in determining UVA photoactivation doses. Together, these results show that many of these parameters are important for optimizing photobiological studies.

Date

2005

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

W. Todd Monroe

DOI

10.31390/gradschool_theses.3505

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