Citrus fruits are widely consumed and can offer various health benefits. One enzyme found in grapefruits, Citrus paradisi flavonol specific 3-O-glucosyltransferase (CP3GT), catalyzes the addition of glucose to one specific flavonoid class and at only one site. These flavonoids are plant secondary metabolites that can be used in a variety of plant functions including signaling and protection. The only class of flavonoids that CP3GT glucosylates is flavonols, and this specificity is of interest to study for potential benefits in biotechnology and enzyme modeling. In order to study this enzyme and its structure, a variety of mutants were created using site-directed mutagenesis. One mutant, P297F, exhibited a loss of function. This mutant was previously studied by inserting a thrombin cleavage site, extracting the plasmid expressing the mutation and sequencing it. The gene sequence was previously verified to be in frame and contain the needed thrombin cleavage site to remove tags used for protein purification and identification. The plasmid was then linearized, and transformed into yeast. After this, conditions for protein expression were tested over a 72-hour period. The protein was found to have optimal expression at 50 hours with a constant temperature of 28 °C and methanol concentration of 0.5 %. However, numerous protein expression experiments indicated very low protein expression. For this reason, the P297F gene was amplified through colony PCR, extracted and sent for sequencing to verify the transformation of the gene into yeast and identify possible reasons for low protein production. Analysis of this sequencing data showed a single nucleotide addition early in the tag sequence causing a frameshift after this location. Reanalysis of the previous plasmid sequencing data showed this same mutation, indicating improper conclusions were drawn. Efforts should be made to identify a plasmid without the mutation or correct the frameshift mutation so that the tag sequence produces the correct amino acids.

The thesis investigates the concept of hybrid architecture as a juxtaposition of multiple architectural factors, including environmental, cultural, structural, and functional ones. The work examines the Najd region of Saudi Arabia and its historical and contemporary contexts concerning functional utility, cultural frameworks, tectonic architectural elements, and site-based environmental forces. The thesis proposes that architectural hybridity can enhance the human experience. The design project, a building combining a water purification facility with a cafe, exhibition hall, and other community functions, integrates traditional and contemporary cultural aesthetics by purposefully juxtaposing and relating: unlikely functions, temporally varied cultural forms, structural and ornamental tectonic elements, and contrasting environmental qualities. / Master of Architecture / The thesis explores the idea of hybrid architecture, combining various factors such as environment, culture, structure, and function. Focused on the Najd region in Saudi Arabia, it analyzes historical and modern aspects related to functionality, cultural context, architectural elements, and environmental influences. The main argument suggests that blending different architectural elements can enrich the human experience. In the design project, a facility merging water purification with a cafe, exhibition hall, and community spaces, traditional and contemporary cultural aesthetics are harmonized by purposefully juxtaposing and connecting: unexpected functions, culturally diverse forms across time, structural and decorative elements, and diverse environmental characteristics.

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As part of the ongoing loss of global biodiversity, amphibian populations are experiencing declines and extinctions. A primary factor in these declines is the skin disease chytridiomycosis, which is caused by the fungus Batrachochytrium dendrobatidis (Bd). Recent research suggests that the amphibian skin microbiota has anti-Bd activity and may be an important factor in host disease resistance. However, little is known about the basic ecology of this host-microbe symbiosis, such as how much variation there is in microbial symbionts among host species and populations, and the nature of symbiont transmission, culturability, and function. My dissertation research addressed these basic questions in microbial ecology, as well as used a novel system to examine the long-standing ecological theory of community structure-function relationships. First, host-specificity, population-level variation and potential environmental transmission of the microbiota were examined by conducting a field survey of bacterial communities from bullfrogs, newts, pond water, and pond substrate at a single pond, and newts from multiple ponds. There was variation among amphibian host species and populations in their skin symbionts, and, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment. Second, the culturability of amphibian skin bacteria was assessed by directly comparing culture-dependent and -independent bacterial sequences from the same individuals. Although less than 7% of the amphibian skin microbes were captured using R2A medium, most of the dominant bacteria were represented in our cultures, and similar patterns of diversity among four amphibian species were captured with both approaches. Third, the relationship between microbial community structure and function and selective forces shaping structure and function were examined in bullfrogs by tracking microbial community structure and function following experimental manipulation of the skin microbiota and pathogen exposure. Results of this study demonstrated that Bd is a selective force on cutaneous bacterial community structure and function, and suggest that beneficial states of bacterial structure and function may serve to limit infection and negative fitness consequences of Bd exposure. Using a combination of observational and experimental approaches, my dissertation contributes to understanding structure-function relationships of these complex symbiotic communities of vertebrates. / Ph. D.