Microalgae have been so far identified as the major producers of organic matter through their photosynthetic activities. In the present work, Nannochloris sp. and Amphora sp., two marine microalgae, have been investigated for proteins and lipids production. Protein fraction was quantified using Bicinchoninic acid (BCA) assay. Protein content in Nannochloris sp. was 16.69 ±4.07 % of dry mass and in Amphora sp. it was 39.89 ±2.09 % of dry mass. Enzyme assays were conducted spectrophotometrically. Nannochloris sp. had malate dehydrogenase, peroxidase and catalase activities. Amphora sp. exhibited malate dehydrogenase, catalase and cytochrome C oxidase activities. These enzymes have several valuable applications in some metabolic pathways and as antioxidant nutrition additives. Besides, lipid extraction was conducted using methanol/ chloroform solvent extraction. Crude lipid extract was analyzed using gas chromatography-mass spectrometry. Lipid contents were 8.14 ±3.67 % in Nannochloris sp. and 10.48 ±1.26% on dry basis in Amphora sp., respectively. Nannochloris sp. fatty acids were composed of C16:0 and C18:0 that are valuable for biodiesel production, and É-3 C18:3, É-6 C18:2, É-6 C16:2 having great nutritional values. In Amphora sp., the fatty acids consisted of C14:0, C16:0 and C16:1 shown to be valuable for biodiesel production and É-3 C22:6 having high nutritional values. Furthermore, a single step conversion of microalgal oil to fatty acid methyl esters was carried out starting directly from lyophilized microalgae. This promising process, in situ transesterification, led to better yields of methyl esters as compared to conventional lipid extraction followed by separate transesterification. / Master of Science

Leptinotarsa decemlineata (Say) is the most important defoliating pest of potato Solanum tuberosum L., in North America and Europe.  Management of this pest relies heavily on chemical control and insecticide resistance is a persistent problem.  This phenomenon has increased the need for developing novel insecticides, resistance evaluation, and the development of alternative control strategies regarding this insect pest.  From 2010 to 2013, field and lab experiments were conducted to evaluate the efficacy of a novel insecticide tolfenpyrad on L. decemlineata.  In leaf-dip assays, tolfenpyrad was highly toxic to L. decemlineata with LC50 values of 0.013 and 0.164 g ai/L for larvae and adults, respectively.  Tolfenpyrad was also toxic to eggs with 0% hatching after being dipped in a field rate concentration.  In field efficacy trials, potato plots treated with tolfenpyrad at rates as low as 153 g ai/ha effectively controlled L. decemlineata. In 2012, populations of L. decemlineata were collected from the Eastern Shore of VA and subjected to toxicity assays to determine current susceptibility to permethrin and oxamyl.  The toxicity assays indicated an increase in toxicity to permethrin in L. decemlineata larvae (LC50 = 3.931 g ai/L) and an increase in toxicity to oxamyl in adult beetles (LC50 = 9.695 g ai/L) compared with LC50 values previously reported in 1990.  In 2012, populations of L. decemlineata from Cheriton, VA, New Church, VA, Painter, VA, and Plymouth, NC were also evaluated for enzyme activity after exposure to sub-lethal concentrations of permethrin, oxamyl, and tolfenpyrad.  Adult beetles were subjected to enzyme assays to measure the activity of cytochrome P450 mono-oxygenase (P450), glutathione-S-transferase (GST), general esterases, and protein content.  Results from the enzyme assays indicated significantly greater esterase activity in beetles from Painter, VA exposed to permethrin [±-naphthol (F= 11.66, df= 4, 20, P<0.0001) and "-naphthol (F= 11.86, df= 4, 20, P<0.0001)], oxamyl [±- naphthol (F= 10.64, df= 4, 20, P<0.0001) and "-naphthol (F= 6.94, df= 4, 20, P=0.0011)], tolfenpyrad [±- naphthol (F= 407.62, df= 1, 8, P<0.0001) and "- naphthol (F= 28.15, df= 1, 8, P= 0.0007)], and the untreated control [±- naphthol (F= 28.14, df= 3, 16, P<0.0001) and "- naphthol (F= 28.86; df= 3, 16, P<0.0001)] compared to most of the other populations tested.  GST activity was significantly greater in tolfenpyrad exposed beetles compared to the non-treated beetles from Painter VA (F= 17.66, df= 5, 24, P< 0.0001). Through laboratory assays and field experiments in potato, the efficacy of a new bio-pesticide derived from the bacterium Chromobacterium subtsugae was evaluated for the control of L. decemlineata.  Results from the laboratory assays showed L. decemlineata feeding was inhibited by the bio-pesticide derived from C. subtsugae.  However, field efficacy trials in 2010, 2011, and 2012, indicated no control of L. decemlineata. Methyl salicylate is an organic compound produced by potato and other plants in response to insect herbivory.  Abundance of predatory arthropods and L. decemlineata life stages were measured in plots treated with and without 5 g slow-release packets of methyl salicylate (95% methyl salicylate (Predalure")).  Methyl salicylate treatment had no impact on predator recruitment or cumulative mortality of L. decemlineata in potatoes. This research has provided us with a new tool for L. decemlineata management, as well as more information about resistance trends and alternative control strategies from which we can build on to reduce resistance development in L. decemlineata and ultimately formulate a stronger integrated pest management strategy for this insect pest. / Ph. D.

Over the past ten years, the development of High Throughput (HT) synthetic chemistry techniques has allowed the rapid preparation of libraries of hundreds to thousands of compounds. These tools are now extensively used for drug and material discovery programmes. The subsequent development of analytical capabilities to carry out qualitative and quantitative assessment of the compounds generated by HT synthesis as well as their HT screening has led to a dramatic broadening of the scope of HT techniques, ranging from image based analysis techniques to mass spectrometry (MS). Based on the latter, a range of solid phase and solution phase analytical constructs was developed to enable the qualitative and quantitative assessment of mixtures of small compounds, using positive electrospray MS as the sole analytical tool. A version of the construct allowed HT reactivity profiling to be carried out on a range of ten carboxylic acids, ten aldehydes and ten isonitriles in the Ugi 4-component condensation reaction. The effect of various parameters such as the concentration of the monomers on the reactivity was investigated. The elaboration of a HT Hammett parameter assessment method was made possible by the development of an electrophilic version of the construct. The value of the Hammett value was afforded by means of combinatorial Hammett plots and values were successfully evaluated in a HT mode for around thirty anilines with substituents in the meta and para position of the aromatic ring. Finally, analytical constructs were used in an attempt to evaluate enzyme reaction kinetics via the labelling of peptides and small drug fragment with coded constructs, to afford affinity determinations between the enzyme (protease) and peptidic or fragment based substrates.

Pesticides are an important component of New Zealand’s primary production sectors. Infestation of pests and diseases can affect crop yield, crop value and damage the country’s export reputation, resulting in economic losses. Repeat applications of pesticides, however, can result in contamination of land and water. Therefore, it is important to understand the fate of pesticides in the environment. Factors which can affect pesticide persistence include soil properties (pH, SOM, CEC), leaching and run-off, volatilisation and co-contamination with heavy metals. Many soils in New Zealand contain high levels of copper from historical applications of copper-based pesticides. Co-contamination of soils may lead to the persistence of some synthetic organic pesticides. An investigation was undertaken to determine the effects of co-contamination with copper on the biodegradation of atrazine and indoxacarb in a New Zealand soil. A Templeton sandy loam soil was spiked with CuSO₄ to achieve concentrations of 0, 100, 250, 500 and 1000 mg kg⁻¹ Cu. The spiked soils were field aged for six months prior to pesticide spiking with either atrazine or indoxacarb. The aged Cu-spiked soils were spiked with either atrazine or indoxacarb at a rate of 2 mg kg⁻¹. A glasshouse study was conducted to determine if copper inhibited the degradation of the pesticides. The pesticide-spiked soils were sampled at the time of spiking (t₀), at the estimated half-lives (t₁) and at twice the estimated half-lives (t₂) of the individual pesticides. The estimated half-lives were based on literature values. The bioavailability and subsequent adverse effects of copper on the soil microbial community was investigated. Total and bioavailable copper concentrations, phosphatase and urease enzyme activities, microbial biomass, and pesticide residue concentrations were all measured in the experimental soil. Methods were developed for the extraction of atrazine, atrazine metabolite and indoxacarb residues from the experimental soil. Total copper concentrations extracted ranged from 4–1060 mg kg⁻¹ in the experimental soils and were consistent throughout the pesticide degradation studies. The bioavailability of copper was a maximum of 2% of the total copper concentration. Bioavailable copper concentrations were positively correlated to total copper (p<0.01). Soil biological properties were investigated to determine the effects of copper on the soil microbial community. Phosphatase and urease enzyme activities, as well as microbial biomass concentrations, were negatively correlated with total copper (p<0.05). Total copper was a better indicator of effects on microorganisms than bioavailable copper. The soil biological properties began showing adverse effects above a total copper concentration of 100 mg kg⁻¹. This concentration also corresponds to New Zealand’s copper limit in biosolids, which is protective of human, plant and microorganism health. Phosphate buffer extraction methods were developed for the analysis of atrazine and indoxacarb residues in the experimental soil by HPLC-UV. Elevated copper concentrations did not inhibit the degradation of atrazine or indoxacarb in the experimental soil. The half-lives of both atrazine (≤19.4 d) and indoxacarb (≤18.8 d) were lower in the spiked experimental soils than the means reported in previous New Zealand and international studies, but were within the reported ranges. This study provided the first data on the fate of indoxacarb in New Zealand. Hydroxyatrazine was the only metabolite detected in the atrazine-spiked experimental soils. Significant differences between the control (Cu-1) and copper levels above 100 mg kg⁻¹ were observed for hydroxyatrazine at t₂. Significant negative correlations were observed between hydroxyatrazine and the microbiomass at t₁ and phosphatase activity at t₂ (p<0.05). These significant relationships suggest that elevated copper concentrations may alter the degradation of this metabolite in the experimental soils due copper toxicity of the soil microbial community. The results of this thesis indicate that elevated levels of copper above 100 mg kg⁻¹ negatively impact the soil microbial community and may reduce the overall health of the soil. Biodegradation is a key mechanism for the degradation of atrazine and indoxacarb in the soil, so it is important that the health of the soil microbial community is maintained. Therefore, it is recommended that atrazine and indoxacarb are only applied to soils with a total copper concentration less than 100 mg kg⁻¹. This will protect the health of the soil microbial community and prevent the potential adverse effects of copper on the degradation of pesticide metabolites in the soil.

Pesticides are an important component of New Zealand’s primary production sectors. Infestation of pests and diseases can affect crop yield, crop value and damage the country’s export reputation, resulting in economic losses. Repeat applications of pesticides, however, can result in contamination of land and water. Therefore, it is important to understand the fate of pesticides in the environment. Factors which can affect pesticide persistence include soil properties (pH, SOM, CEC), leaching and run-off, volatilisation and co-contamination with heavy metals. Many soils in New Zealand contain high levels of copper from historical applications of copper-based pesticides. Co-contamination of soils may lead to the persistence of some synthetic organic pesticides. An investigation was undertaken to determine the effects of co-contamination with copper on the biodegradation of atrazine and indoxacarb in a New Zealand soil. A Templeton sandy loam soil was spiked with CuSO₄ to achieve concentrations of 0, 100, 250, 500 and 1000 mg kg⁻¹ Cu. The spiked soils were field aged for six months prior to pesticide spiking with either atrazine or indoxacarb. The aged Cu-spiked soils were spiked with either atrazine or indoxacarb at a rate of 2 mg kg⁻¹. A glasshouse study was conducted to determine if copper inhibited the degradation of the pesticides. The pesticide-spiked soils were sampled at the time of spiking (t₀), at the estimated half-lives (t₁) and at twice the estimated half-lives (t₂) of the individual pesticides. The estimated half-lives were based on literature values. The bioavailability and subsequent adverse effects of copper on the soil microbial community was investigated. Total and bioavailable copper concentrations, phosphatase and urease enzyme activities, microbial biomass, and pesticide residue concentrations were all measured in the experimental soil. Methods were developed for the extraction of atrazine, atrazine metabolite and indoxacarb residues from the experimental soil. Total copper concentrations extracted ranged from 4–1060 mg kg⁻¹ in the experimental soils and were consistent throughout the pesticide degradation studies. The bioavailability of copper was a maximum of 2% of the total copper concentration. Bioavailable copper concentrations were positively correlated to total copper (p<0.01). Soil biological properties were investigated to determine the effects of copper on the soil microbial community. Phosphatase and urease enzyme activities, as well as microbial biomass concentrations, were negatively correlated with total copper (p<0.05). Total copper was a better indicator of effects on microorganisms than bioavailable copper. The soil biological properties began showing adverse effects above a total copper concentration of 100 mg kg⁻¹. This concentration also corresponds to New Zealand’s copper limit in biosolids, which is protective of human, plant and microorganism health. Phosphate buffer extraction methods were developed for the analysis of atrazine and indoxacarb residues in the experimental soil by HPLC-UV. Elevated copper concentrations did not inhibit the degradation of atrazine or indoxacarb in the experimental soil. The half-lives of both atrazine (≤19.4 d) and indoxacarb (≤18.8 d) were lower in the spiked experimental soils than the means reported in previous New Zealand and international studies, but were within the reported ranges. This study provided the first data on the fate of indoxacarb in New Zealand. Hydroxyatrazine was the only metabolite detected in the atrazine-spiked experimental soils. Significant differences between the control (Cu-1) and copper levels above 100 mg kg⁻¹ were observed for hydroxyatrazine at t₂. Significant negative correlations were observed between hydroxyatrazine and the microbiomass at t₁ and phosphatase activity at t₂ (p<0.05). These significant relationships suggest that elevated copper concentrations may alter the degradation of this metabolite in the experimental soils due copper toxicity of the soil microbial community. The results of this thesis indicate that elevated levels of copper above 100 mg kg⁻¹ negatively impact the soil microbial community and may reduce the overall health of the soil. Biodegradation is a key mechanism for the degradation of atrazine and indoxacarb in the soil, so it is important that the health of the soil microbial community is maintained. Therefore, it is recommended that atrazine and indoxacarb are only applied to soils with a total copper concentration less than 100 mg kg⁻¹. This will protect the health of the soil microbial community and prevent the potential adverse effects of copper on the degradation of pesticide metabolites in the soil.

Climate change is likely to alter plant species composition and interactions between plants and soil microbes that together dictate the quantity and quality of forage produced in pastures, the base of animal production in central Kentucky. This study assessed the seasonal dynamics of soil microbes and their response to increased temperature (+3oC) and growing season precipitation (+30% of the mean annual). Total soil microbial biomass, community composition, enzyme activities, potential carbon mineralization, and catabolic responses to selected substrates were measured seasonally in the different climate treatments. In this system, seasonal variability was a dominant driving factor for all the soil microbial characteristics that I investigated. Summer maxima and winter minima were identified in the active microbial biomass, while soil microbial community structure differed between each season. Extracellular enzyme activities were generally highest in either the spring or summer, while seasonal patterns for each substrate were unique across catabolic response profiles. Climate treatments produced few significant main or interactive effects on the soil microbial biomass and function. This resiliency, coupled with evidence of functional redundancy, suggests that central Kentucky pasture ecosystems may be well-equipped to handle future environmental stress associated with climate change and to maintain critical ecosystem services.

There is an unmet medical need in the treatment of gout. This type of inflammatory arthritis can be efficiently alleviated by the enzyme uricase. This enzyme breaks down uric acid, the causative agent of gout, so it can be flushed from the body. In humans and the other great apes, uricase is a pseudogene and as such is inactive. Research on therapeutic uricases has focused on using enzymes from naturally occurring sources; however, these foreign proteins can be very antigenic and present a potentially life-threatening safety risk to patients. We address the challenges of developing a safer uricase therapeutic by exploiting evidence that, while inactive, the human pseudogene is expressed in the human body and may be recognized as self by the immune system. To develop a ﾓhuman-likeﾔ? uricase we apply the hybrid computational and experimental approach of Ancestral Sequence Reconstruction to search functional sequence space of uricase proteins to engineer an enzyme with high sequence identity to the human pseudogene, and possessing therapeutic levels of activity for the breakdown of uric acid. This dissertation describes the development and characterization of several uricase leads. The most active ancestral uricase possesses both enhanced in vitro and in vivo stability (in healthy rats) when assayed head-to-head Pegloticase, the only FDA approved uricase for the treatment of gout.