Date of Award

12-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering and Sciences

First Advisor

Vipuil Kishore

Second Advisor

Margaret Ahmad

Third Advisor

Chris Bashur

Fourth Advisor

Emily Ralston

Abstract

Cryptochromes (CRYs) are flavoproteins that absorb blue light and are associated with the perception of electromagnetic stimuli in a variety of organisms including animals and plants. When exposed to blue light, CRYs undergo flavin redox cycles and form radical pairs intermediates that are assumed to be magnetically sensitive as predicted by the radical-pair hypothesis. However, it is unknown if CRYs fulfill the criteria to act as biological magnetosensors. In the first aim, Arabidopsis thaliana seedlings were exposed to blue light and a time-dependent weak magnetic field at radiofrequency (RF) of 7 MHz as a diagnostic tool to test the radical-pair hypothesis. RF was used to disrupt the perception of the static magnetic fields (earth's magnetic fields). Results showed that the RF was able to negate the impact of blue light on the CRY receptor as evidenced by the decrease in CRY phosphorylation, a finding which is in support of the radical pair mechanism. Additionally, the CRY’s response to low-level static magnetic field (LLF) condition which nearly cancelled the external magnetic fields, was demonstrated, and also found to have a disruptive effect similar to RF. Aim 2 studies focused on investigating an ongoing disagreement regarding the magnetosensitive step, and the magnetically sensitive radical pairs of the CRY redox photocycle. Here, Arabidopsis thaliana seedlings were exposed to blue light pulses (5 min blue light, 10 min dark) and the static magnetic field was applied only during the light mode. Results showed that magnetic sensitivity of the CRY receptor was lost when the magnetic stimulus was applied exclusively in the blue-light intervals indicating that the CRY’s photocycle’s magnetically sensitive reaction step had to take place during flavin reoxidation (the dark step). Aim 3 studies investigated the impact of blue light exposure on mammalian cells by evaluating its role in osteoblast differentiation and mineralization. Human osteosarcoma cells (Saos-2) were cultured in 2D plates and exposed to two different blue light dosing conditions: 1) 4 J/cm2 every two days and 2) 1 J/cm2 twice a day. Dark condition was used as control. The impact of different blue light dosing conditions on Saos-2 cell viability, differentiation and mineralization was investigated. Results showed that exposing Saos-2 cells to blue light at energy density of 4 J/cm2 every two days significantly enhanced alkaline phosphatase activity and cell-mediated mineralization compared to the dark condition. No such effect was observed with the 1 J/cm2 twice a day condition indicating a dose-dependent effect of blue light on Saos-2 cells. Finally, reactive oxygen species (ROS) generation was investigated as a possible mechanism that drives enhanced osteoblast function in response to blue light exposure.

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