Pharmaceuticals have routinely been detected in the environment resulting in a growing concern about whether these drugs could elicit effects on aquatic organisms. The concerns are centered on the highly conserved nature of mammalian therapeutic targets in fish. These pharmaceuticals are found at very low levels in the environment, which can result in sub-lethal effects in aquatic organisms. Therefore, 28 d early-life stage studies were conducted on six pharmaceuticals to assess their impacts on survival and growth fathead minnow larvae. Two pharmaceuticals tested, carbamazepine and fenofibrate, resulted in no alterations to survival and growth. However, amiodarone, clozapine, dexamethasone, and levonorgestrel (LNG) reduced survival at concentrations tested with LNG being the most potent at 462 ng/L. Survival was increased with amiodarone and clozapine; however LNG significantly decreased growth at 86 ng/L. Therefore, the most potent pharmaceutical tested was the synthetic progestin LNG with survival and growth impacts at concentrations less than 1 μg/L. Further analysis was conducted by measuring specific endocrine related mRNA transcript profiles in FHM larvae following the 28 d ELS exposure to LNG. Transcripts of 3β-HSD, 20β-HSD, and FSH were significantly down-regulated following 28 d exposure to both 16.3 and 86.9 ng/L LNG. Also, CYP19a expression was significantly down-regulated at 86.9 and 2392 ng/L LNG. Subsequently, a second study examined time periods that may be most sensitive (e.g., windows of sensitivity) for FHM larvae exposed to LNG. Larvae were exposed to a single concentration of LNG (i.e. LOECgrowth of 86.2 ng/L as determined in the 28 d ELS study) for different time periods starting with fertilized egg through 28 dph. Growth and mRNA expression of the four differentially expressed transcripts from the first study were measured. Regardless of the duration of exposure, LNG significantly decreased growth in fathead minnow larvae at day 28. For both 20β-HSD and CYP19a, mRNA expression was decreased following exposure to LNG; however, these transcripts returned to baseline levels after removal of LNG. 3β-HSD and FSH showed similar trends after exposure to LNG with 7-14 d and 14-28 d exposures exhibiting a decrease in expression; however, FSH expression returned to baseline once removed for LNG exposure. Based on these data, 3β-HSD was the only transcript to remain down regulated after LNG exposure. Together these data suggest LNG can negatively impact FHM larval survival and growth, with significant alterations in endocrine related responses. However, these changes in endocrine related responses may not directly correlate to the changes in growth demonstrated with LNG exposure to fathead minnows. Therefore, additional research is warranted to ascertain additional mechanisms, either endocrine related or non-endocrine functions, related to changes in growth of larval fathead minnows.

Puberty and adolescence are periods of brain-driven physiological development that display increased incidences of depression development. Adolescents display significant alterations to their stress response signaling, sleep patterns, and metabolism when compared to pre-pubescents. Increased exposure to stress, sleep disturbances, and impaired energy acquisition is typical during puberty and adolescence and similarly increases the likelihood of developing depression. A promising avenue of limiting the deleterious effects of stress and sleep disruption on pubertal and adolescent depressive behaviour is the use of treatments that blunt underlying metabolic impairments associated with depression. Treatments that directly or indirectly increase availability of the glucose metabolite L-lactate are associated with depression reduction. The investigations included in this dissertation evaluate the usability of L-lactate treatments in reducing depression development in pubertal CD-1 male and female mice. This work first examines a previously proposed oral lactate solution, its effect on energy substrate concentration and drowsiness, and its efficacy as a safe oral L-lactate treatment (Chapter 2). Subsequent research aimed to identify a pubertal model of depression that would allow future testing of L-lactate antidepressant treatment. Pubertal male and female mice exposed to chronic sleep disruption were evaluated for stress reactivity and depressive behavior and were identified as a model for antidepressant testing (Chapter 3). In the final study, we evaluated the effects of chronic sleep disruption on the expression of energy metabolites like L-lactate and glucose within the brain, its effect on neurotransmitters associated with depression, and changes to sleep architecture in relation to depression behaviour. Sleep disrupted and depressed animal models were administered L-lactate producing probiotics and were evaluated for improvements to energy substrate concentration, neurotransmitter expression, sleep recovery, and depression reduction (Chapter 4). The present thesis provides groundwork for the use of L-lactate therapies in depressed pubertal and adolescent groups and provides initial evaluations of probiotic intervention as a prevention strategy for juvenile depression.

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5' Nucleotidase (5NU) in <i>Dictyostelium discoideum</i> is an enzyme that shows high substrate specificity to 5'AMP. The enzyme has received considerable attention in the past because of the critical role played by cyclic AMP in cell differentiation in this organism. Degradation of cAMP by cAMP phosphodiesterase (PDE) produces 5'AMP, the substrate of 5NU. Dictyostelium switches its genetic program from growth to cellular differentiation when nutrients become limited. During the time course of development, the activity of 5NU is high and becomes restricted to a narrow band of cells that form the interface between the prestalk/prespore zones. Understanding how this gene is regulated will provide knowledge underlying the process of cell differentiation. In order to understand the functional significance of the 5NU, I first purified of the 5NU protein using an artificial substrate p-nitrophenol phosphate (pNPP). An activity stain on non-denaturing gels with Nitro Blue Tetrazolium (NBT) and 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) as the substrate was also used. A polypeptide of approximately 90 kDa was associated with 5NU enzyme activity after gel filtration chromatography and denaturing gel electrophoreses. Protein sequence of this peptide was obtained from Mass Spectrometry and Edmund Degradation. Various databanks were searched for similar sequences, but no matches with high identity were obtained. However, a search of the sequences of an ongoing cDNA project at the University of Tsukuba in Japan revealed a clone that corresponded to the peptide sequence of 5NU. In addition, a clone was found that corresponded to the classical "alkaline phosphatase" found in several organisms. Analysis of the expression of the 5NU and AP during Dictyostelium development by Northern blotting determined that the 5NU is developmentally regulated while the AP is expressed at all stages of the life cycle. Southern blot analysis showed a single form of the gene for both 5NU and AP. Targeted gene disruption and knockout mutagenesis using the 5NU sequences flanking a blasticidin-resistant cassette was attempted. Analysis of the transformants showed the 5NU gene was not disrupted, and that the blasticidin-resistant cassette was randomly inserted into the genome. / Ph. D.

The pink hibiscus mealybug (PHM), Maconellicoccus hirsutus (Green) (Hemiptera: Pseudococcidae) was investigated with regard to damage caused to hibiscus by feeding, dispersal of nymphs, evaluation of management tactics, and the use of sex pheromone based monitoring in southern Florida from 2005 to 2008. Understanding the ability of PHM to locate and colonize new hosts, and the response of hosts is essential to optimized monitoring and management strategies. Investigation of the onset and severity of PHM feeding symptoms by Hibiscus rosa-sinensis L. showed that severity of symptoms differed among cultivars and that PHM were found on plants that did not exhibit feeding symptoms. Aerially dispersing PHM were predominantly first instars. Dispersal occurred with a diel periodicity that peaked between 14:00 and 18:00 h and was significantly influenced by mean wind speed. Initial infestation with 5, 10, or 20 PHM adult females had no significant affect on the number of dispersing individuals captured from hibiscus plants and PHM were captured at 50 m from infested source plants. The effects of mating disruption, the insecticide (dinotefuran), the parasitoid, Anagyrus kamali (Moursi), and the predator, Cryptolaemus montrouzieri (Mulsant) on PHM on hibiscus plants in screened field cages were evaluated. The total number of mealybugs captured in sticky band and pheromone traps during the study was reduced by dinotefuran and the predator. At the end of the study, the number of nymphs recovered from hibiscus terminals was reduced by the dinotefuran, predator and parasitoid treatments. Field experiments showed that the time of day during which male PHM were captured in pheromone traps in May and September was crepuscular, with most captures occurring from 18:00 to 21:00 h. Significantly more males were captured in traps placed in non-host trees at an elevation of 2 m above the ground than 6 m, and more males were captured in traps placed within host plants than in those 3 m upwind. Pheromone traps placed in hibiscus treated with soil applied dinotefuran or left untreated captured equal numbers of males during the 3 wk prior to treatment and during the 12 wk after treatment. Release of parasitoids at residential sites did not have a significant effect on the total number of males captured in sex pheromone traps over 18 mo. The number of mealybugs found at both parasitoid release and untreated sites were highly variable and corresponded with males captured in sex pheromone traps, as high and low levels of mealybugs corresponded with high and low levels of males captured. The number of males captured in pheromone traps during a two week survey at residential sites in May were a strong predictor of subsequent captures in 2006 (r2 = 0. 712), but not 2007 (r2 = 0.019). The relationship between PHM populations and males captured in sex pheromone traps was influenced by a multitude of factors that can impact the ability of traps to accurately reflect populations at a given location. Pheromone traps have the potential to provide meaningful information towards monitoring and mitigating risk from PHM. The contributions of this dissertation towards optimizing PHM sex pheromone monitoring, as well as facets of PHM monitoring that have yet to be resolved are discussed. / Ph. D.

Critical infrastructure systems, such as transportation, energy, water and communication, are the backbones of sustainable economic and social development. The tragedies and catastrophic events in the past few years have motivated researchers to study the vulnerability of infrastructure systems to disastrous events. A number of existing studies address roadway networks where researchers have characterized the robustness and vulnerability of roadways to earthquakes, floods, and targeted attacks. However, extreme events with infrequent return periods are not very likely to occur in a 50-60 year analysis period of roadways, while many roadways are located in areas that are not even exposed to floods or earthquakes at all. On the other hand, roadway network endogenous characteristics such the condition and degradation over time not only increases the vulnerability of roadways to disastrous events, but also makes the roadway network vulnerable to disruptions that are caused by maintenance and repair activities on the roadways system. Nevertheless, the impacts of these endogenous network characteristics on roadway vulnerability have not been explicitly addressed in the existing studies. This dissertation introduces the concept of condition-based vulnerability assessment (CBVA) to capture the effect of roadway endogenous characteristics such as condition and condition uncertainties, roadway network deterioration over time, topological properties of roadways, and travel rate and travel pattern on the dynamics of roadway network vulnerably. First a methodological framework is developed and the method is applied to an illustrative roadway system. The results show that the vulnerability of roadway system is more affected by the average condition of the roadway network than by the condition of individual roads in the system. Moreover, the findings show that small uncertainties associated with the condition of individual roads can significantly increase the variance of the predicated vulnerability. This initial methodological framework is then enhanced to account for physical degradation of the network over time and network equilibrium, and is applied to a real highway system. For the network studied network degradation increases roadway system vulnerability in a nonlinear mode. The result also suggest that the network vulnerability pattern is not very sensitive to travel pattern and link topological properties when the average network disruption probability (representing average network condition) is less than about 0.5. In other words, at low values of average disruption probability, it does not matter what link has what disruption probability level or how the travelers move across the network. However with further network degradation and as the average network disruption probability increases, the dynamics of network vulnerability depends on travel pattern on the network as well as on the link topological properties. / Ph. D.

In recent years, rare catastrophic disasters have triggered pharmaceutical supply chain disruptions, resulting in product shortages and other problems that have negatively impacted healthcare delivery. Since the health sector is part of many nations’ critical infrastructure, ensuring minimal disruption in the supporting supply chains is an essential aspect of national security. While traditional risk management approaches have proven adequate for handling independent routine operational risks, these methods are inadequate for infrequent disruptive risks that propagate further risks, producing a domino effect in complex systems. Since today’s pharmaceutical supply chains have many characteristics of complex systems, elucidating the disruptive risks and required resilience capabilities, through knowledge gained from actual disasters, can be valuable in developing mitigation strategies. This research aims to investigate two catastrophic disasters that disrupted pharmaceutical supply chains in recent years. The first case explores the impact of a localized Category IV hurricane that hit Puerto Rico and disrupted the supply chain for saline, an essential product used in the treatment of many illnesses. The risk events that propagated from the disaster are identified and modeled using a Bayesian network to determine where resilience should be strengthened. In accordance with ISO 31000 risk management guidelines, Bayesian networks have been identified as appropriate tools for modeling complex system risks. The second case employs a qualitative research study on the vaccine supply chain supporting mass immunization during the COVID-19 pandemic. A content analysis is conducted to extract the insights of over 100 professionals on disruptors and resilience capability requirements of the COVID-19 vaccine supply chain. The results indicate that a large and diverse set of risk factors and resilience capabilities are pertinent to pharmaceutical supply chains during localized severe weather and global pandemic disasters. Furthermore, to mitigate the impacts of severe weather risk in the case of Puerto Rico, the predominant type of resilience required is absorptive capacity, while in the case of the mass immunization vaccine supply chain, adaptive capacity is key. These findings should be useful to national leaders, pharmaceutical supply chain and healthcare sourcing managers, disaster preparedness officers, and others charged with pharmaceutical supply chain resilience.