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Plague bacteria (Yersinia pestis) are transmitted to susceptible mammals by fleas. At least 25 flea species found in North America have been identified as plague vectors. The most efficient flea vector is the Oriental rat flea Xenopsylla cheopis, while the cat flea Ctenocephalides felis is a poor vector. The factors that determine vector competence of different fleas are not known. The main obstacles that the bacteria must overcome in the flea gut are also unknown. Fleas' molecular responses to Y. pestis invading could be a determining factor to control the bacterial survival and growth. Good and poor vectors might have different gene expression patterns when they are infected with Y. pestis. To investigate this hypothesis, we constructed cDNA libraries of infected fleas (X. cheopis and C. felis) using Suppression Subtractive Hybridization at 1 and 2 days post-infection. The infection approaches were either hemocoel injection or oral infection. We measured expression of several of the genes using quantitative real-time PCR. The results indicated that changes in gene expression were modest. We observed that the route of infection (oral vs. hemocoel injection) had a strong effect on the genes that were upregulated, with hemocoel injection inducing more obvious immune-related genes than oral infection. We also saw that infected X. cheopis has different gene expression patterns than infected C. felis. Several of the genes from both species are predicted to be involved in production and removal of reactive oxygen species (ROS). Consistent with this observation, the levels of peroxide in X. cheopis midguts was higher following oral infection with Y. pestis, than in uninfected fleas, and Y. pestis grew differently in antioxidant-fed fleas, demonstrating that ROS production could be an important defense in fleas early after infection

Reactive oxygen species (ROS) such as H2O2 can damage cellular components and are formed as a byproduct of mitochondrial oxidative metabolism. Studies using the nematode C. elegans have found that increasing ROS during development or early adulthood can extend lifespan, while increasing ROS during later adulthood normally decreases lifespan. NADPH provides the reducing power for several cellular antioxidants and is synthesized in a two-step reaction from NAD+ with the first step being catalyzed by NAD kinase (NADK). In this study, it questioned the effects of knocking down C. elegans cytoplasmic NADK, nadk-1 globally or in a neuron or intestine-specific manner starting from early development on oxygen consumption and ROS levels. It was hypothesized that knocking down the cytoplasmic NADK in C. elegans alters NADP+/NADPH ratios. This led to the prediction that ROS and O2 consumption levels would be decreased, and thioredoxin reductase activity levels will be increased in global and tissue-specific knockdowns. Three different strains of nematodes were used for this study: N2 wild type, intestinal RNAi sensitive strain VP303, and neuronal RNAi sensitive strain TU3401. The nadk-1 RNAi was introduced to the nematodes through their food source. To measure ROS levels, nematodes were treated with a tert-butylhydroperoxide insult to induce ROS generation, along with H2DCFDA dye to fluoresce when oxidized by ROS, pipetted into a 96-well plate, and ran through a microplate reader. For the oxygen consumption measurements, the nematodes were run through a Clark O2 electrode. Finally, Thioredoxin Reductase activity levels were measured using an assay kit that would inhibit testing groups thioredoxin reductase to only measure glutathione reductase levels and subtract it from uninhibited testing groups. The nematodes were pipetted into a 96-well plate and ran through a microplate reader. All experiments were normalized to average protein levels of C. elegans and analyzed using a t-test. Whole body knockdown of nadk-1 to decrease cytoplasmic NADPH levels decreased oxygen consumption and tert-butyl hydroperoxide-stimulated ROS levels, which was phenocopied by intestine-specific or neuron-specific knockdown. Thioredoxin reductase measurements following nadk-1 knockdown showed a trend toward increased activity. These results establish that nadk-1 does have antioxidant properties but does not extend lifespan as is the case of other NADPH generating enzyme knockdowns. .

This thesis presents the research and development behind the integration of an autonomous propulsion system for a four-wheeled Ground Penetrating Radar (GPR) measurement unit, previously requiring manual operation. In order to ease the administration of the complex new system, Robot Operating System (ROS) 2 was used as middleware, where an implementation of Light Detection And Ranging (LiDAR) 3D-SLAM (Simultaneous Localization And Mapping) served to secure precise localization of the Unmanned Ground Vehicle (UGV) and mapping of its environment. This, with the ultimate goal of enabling accurate survey execution along paths optimized for various dynamic indoor- and outdoor environments. From a more general point of view, this work can also act as a hardware- and software selection guide for similar projects, especially if stricter physical limitations apply and the autonomous system is not considered the primary system, but the majority of the internal enclosed space of the UGV is reserved for higher purpose equipment or storage requirements. In this first prototype iteration, the mapping accuracy of the autonomous system reached centimeter precision and the execution of surveys in grid- and spiral patterns yielded position accuracies of 5(2) cm and 6(4) cm, respectively. These results are indeed very promising and show the proof of concept needed to enter the next development phase.

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To map the subsurface Ground Penetrating Radar (GPR) can be used in a non-invasive way. It is currently done manually by pushing a wheeled device on a handlebar. This thesis suggests an alternative method using an integrated autonomous solution. To ac- complice that: several sensors were fused to give the robot perception of the world, the ability to localize itself within it, and plan a path to reach the goal. Detecting algorithms were implemented and tested to ensure the robot could handle a dynamic and compli- cated world. The results showed that the robot could independently navigate in a grid pattern conducting GPR surveys while avoiding obstacles and finding a safe route. All this will allow for collecting GPR data with precise localization measurements and in paths more detailed than a human operator could. In addition, it enables the operator to be at a safe distance in dangerous environments and to search large areas.

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