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.

no / Purpose To determine whether the structure–function relationship in glaucoma can be strengthened by using more precise structural and functional measurements combined with individualized structure–function maps and custom sector selection on the optic nerve head (ONH). Design Cross-sectional study. Participants One eye of each of 23 participants with glaucoma. Methods Participants were tested twice. Visual fields were collected on a high-resolution 3° × 3° grid (164 locations) using a Zippy Estimation by Sequential Testing test procedure with uniform prior probability to improve the accuracy and precision of scotoma characterization relative to standard methods. Retinal nerve fiber layer (RNFL) thickness was measured using spectral-domain optical coherence tomography (OCT; 4 scans, 2 per visit) with manual removal of blood vessels. Individualized maps, based on biometric data, were used. To customize the areas of the ONH and visual field to correlate, we chose a 30° sector centered on the largest defect shown by OCT and chose visual field locations using the individualized maps. Baseline structure–function correlations were calculated between 24-2 locations (n = 52) of the first tested visual field and RNFL thickness from 1 OCT scan, using the sectors of the Garway-Heath map. We added additional data (averaged visual field and OCT, additional 106 visual field locations and OCT without blood vessels, individualized map, and customized sector) and recomputed the correlations. Main Outcome Measures Spearman correlation between structure and function. Results The highest baseline correlation was 0.52 (95% confidence interval [CI], 0.13–0.78) in the superior temporal ONH sector. Improved measurements increased the correlation marginally to 0.58 (95% CI, 0.21–0.81). Applying the individualized map to the large, predefined ONH sectors did not improve the correlation; however, using the individualized map with the single 30° ONH sector resulted in a large increase in correlation to 0.77 (95% CI, 0.47–0.92). Conclusions Using more precise visual field and OCT measurements did not improve structure–function correlation in our cohort, but customizing the ONH sector and its associated visual field points substantially improved correlation. We suggest using customized ONH sectors mapped to individually relevant visual field locations to unmask localized structural and functional loss.

yes / Purpose: We evaluated variability and conviction in tracing paths of retinal nerve fiber bundles (RNFBs) in retinal images, and compared traced paths to a computational model that produces anatomically-customized structure–function maps. Methods: Ten retinal images were overlaid with 24-2 visual field locations. Eight clinicians and 6 naïve observers traced RNFBs from each location to the optic nerve head (ONH), recording their best estimate and certain range of insertion. Three clinicians and 2 naïve observers traced RNFBs in 3 images, 3 times, 7 to 19 days apart. The model predicted 10° ONH sectors relating to each location. Variability and repeatability in best estimates, certain range width, and differences between best estimates and model-predictions were evaluated. Results: Median between-observer variability in best estimates was 27° (interquartile range [IQR] 20°–38°) for clinicians and 33° (IQR 22°–50°) for naïve observers. Median certain range width was 30° (IQR 14°–45°) for clinicians and 75° (IQR 45°–180°) for naïve observers. Median repeatability was 10° (IQR 5°–20°) for clinicians and 15° (IQR 10°–29°) for naïve observers. All measures were worse further from the ONH. Systematic differences between model predictions and best estimates were negligible; median absolute differences were 17° (IQR 9°–30°) for clinicians and 20° (IQR 10°–36°) for naïve observers. Larger departures from the model coincided with greater variability in tracing. Conclusions: Concordance between the model and RNFB tracing was good, and greatest where tracing variability was lowest. When RNFB tracing is used for structure–function mapping, variability should be considered.

yes / Purpose: We have developed customized maps that relate visual field and optic nerve head (ONH) regions according to individual anatomy. In this study, we aimed to determine feasible map resolution for research use, and to make a principled recommendation of sector size for clinical applications. Methods: Measurement variability in fovea–ONH distance and angle was estimated from 10 repeat OCT scans of 10 healthy people. Errors in estimating axial length from refractive error were determined from published data. Structure–function maps were generated, and customized to varied clinically-plausible anatomical parameters. For each parameter set (n = 210), 200 maps were generated by sampling from measurement/estimation error distributions. Mapped 1° sectors at each visual field location from each parameter set were normalized to difference from their mean. Variation (90% ranges) in normalized mapped sectors represents the precision of individualized maps. Results: Standard deviations of repeated measures of fovea–ONH distance and angle were 61 μm and 0.97° (coefficients of variation 1.3% and 12.0%, respectively). Neither measure varied systematically with mean (Spearmans's ρ = 0.26, P = 0.47 for distance, ρ = −0.31, P = 0.39 for angle). Variation (90% ranges) in normalized mapped sectors varied across the visual field and ranged from 3° to 18° when axial length was measured accurately, and from 6° to 32° when axial length was estimated from refractive error. Conclusions: The 90% ranges represent the minimum feasible ONH sector size at each visual field location. For clinical use an easily interpretable scheme of 30° sectors is suggested.

No / To explore the performance of patient-specific prior information, for example, from structural imaging, in improving perimetric procedures. Computer simulation was used to determine the error distribution and presentation count for Structure–Zippy Estimation by Sequential Testing (ZEST), a Bayesian procedure with prior distribution centered on a threshold prediction from structure. Structure-ZEST (SZEST) was trialled for single locations with combinations of true and predicted thresholds between 1 to 35 dB, and compared with a standard procedure with variability similar to Swedish Interactive Thresholding Algorithm (SITA) (Full-Threshold, FT). Clinical tests of glaucomatous visual fields (n = 163, median mean deviation −1.8 dB, 90% range +2.1 to −22.6 dB) were also compared between techniques. For single locations, SZEST typically outperformed FT when structural predictions were within ± 9 dB of true sensitivity, depending on response errors. In damaged locations, mean absolute error was 0.5 to 1.8 dB lower, SD of threshold estimates was 1.2 to 1.5 dB lower, and 2 to 4 (29%–41%) fewer presentations were made for SZEST. Gains were smaller across whole visual fields (SZEST, mean absolute error: 0.5 to 1.2 dB lower, threshold estimate SD: 0.3 to 0.8 dB lower, 1 [17%] fewer presentation). The 90% retest limits of SZEST were median 1 to 3 dB narrower and more consistent (interquartile range 2–8 dB narrower) across the dynamic range than those for FT. Seeding Bayesian perimetric procedures with structural measurements can reduce test variability of perimetry in glaucoma, despite imprecise structural predictions of threshold. Structural data can reduce the variability of current perimetric techniques. A strong structure–function relationship is not necessary, however, structure must predict function within ±9 dB for gains to be realized.

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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