The white gene and locomotor recovery from anoxia in Drosophila melanogaster

Locomotor recovery from anoxia is complicated and little is known about the molecular and cellular mechanisms regulating anoxic recovery in Drosophila. For this thesis I established a protocol for large-scale analysis of locomotor activity in adult flies with exposure to a transient anoxia. Using this protocol I observed that wild-type Canton-S flies recovered faster and more consistently from anoxia than the white-eyed mutant w1118, which carries a null allele of w1118 in an isogenic genetic background. Both Canton-S and w1118 are commonly used controls in the Drosophila community. Genetic analysis including serial backcrossing, RNAi knockdown, w+ duplication to Y chromosome as well as gene mutation revealed a strong association between the white gene and the timing of locomotor recovery. I also found that the locomotor recovery phenotype is independent of white-associated eye pigmentation, that heterozygous w+ allele was haplo-insufficient to induce fast and consistent locomotor recovery from anoxia in female flies, and that mini-white is insufficient to promote fast and consistent locomotor recovery. Moreover, locomotor recovery was delayed in flies with RNAi knockdown of white in subsets of serotonin neurons in the central nervous system. I further demonstrated that mutations of phosphodiesterase genes (PDE) displayed wild-type-like fast and consistent locomotor recovery, and that locomotor recovery was light-sensitive in the night in w1118. The delayed locomotor recovery and the light sensitivity were eliminated in PDE mutants that were dual-specific or cyclic guanosine monophosphate (cGMP)-specific. Up-regulation of cGMP using multiple approaches including PDE mutation, sildenafil feeding or specific expression of an atypical soluble guanylyl cyclase (Gyc88E) was sufficient to suppress w-RNAi induced delay of locomotor recovery. Taken together, these data strongly support the hypothesis that White transports cGMP and promotes fast and consistent locomotor recovery from anoxia.

Stress Inversion Using LiDAR Tunnel Deformation Measurements

Far-field stresses are those present in a volume of rock prior to excavations being created. Estimates of the orientation and magnitude of far-field stresses, often used in mine design, are generally obtained by single-point measurements of stress, or large-scale, regional trends. Point measurements can be a poor representation of far-field stresses as a result of excavation-induced stresses and geological structures. For these reasons, far-field stress estimates can be associated with high levels of uncertainty. The purpose of this thesis is to investigate the practical feasibility, applications, and limitations of calibrating far-field stress estimates through tunnel deformation measurements captured using LiDAR imaging. A method that estimates the orientation and magnitude of excavation-induced principal stress changes through back-analysis of deformation measurements from LiDAR imaged tunnels was developed and tested using synthetic data. If excavation-induced stress change orientations and magnitudes can be accurately estimated, they can be used in the calibration of far-field stress input to numerical models. LiDAR point clouds have been proven to have a number of underground applications, thus it is desired to explore their use in numerical model calibration. The back-analysis method is founded on the superposition of stresses and requires a two-dimensional numerical model of the deforming tunnel. Principal stress changes of known orientation and magnitude are applied to the model to create calibration curves. Estimation can then be performed by minimizing squared differences between the measured tunnel and sets of calibration curve deformations. In addition to the back-analysis estimation method, a procedure consisting of previously existing techniques to measure tunnel deformation using LiDAR imaging was documented. Under ideal conditions, the back-analysis method estimated principal stress change orientations within ±5° and magnitudes within ±2 MPa. Results were comparable for four different tunnel profile shapes. Preliminary testing using plastic deformation, a rough tunnel profile, and profile occlusions suggests that the method can work under more realistic conditions. The results from this thesis set the groundwork for the continued development of a new, inexpensive, and efficient far-field stress estimate calibration method.