Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T

DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE By SANDAMALI AMARASINGHA EKANAYAKA December 2013 Advisor: Dr. Tamara L. Hendrickson Major: Biochemistry Degree: Doctor of Philosophy The enzyme glycosylphosphatidylinositol transamidase (GPI-T) mediates the attachment of a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of specific proteins to produce GPI anchored proteins. This post-translational modification is essential for viability of eukaryotic organisms. However, very little is known about GPI-T and its catalytic activity. Thus, the research described in this abstract was conducted to develop an in vitro assay to monitor GPI-T. A high-throughput assay for GPI-T will facilitate innumerable new experiments to study this complicated enzyme. The three core subunits of GPI-T (Gpi8, Gpi16, and Gaa1) were co-purified from a GPI8 knockout Saccharomyces cerevisiae strain containing a plasmid that expresses Gpi8 with an appended glutathione-S-transferase (GST) domain. Peptide substrates for GPI-T were synthesized and modified to contain a pair of chromophores suitable for the development of a fluorescence resonance energy transfer (FRET) assay. GPI-T activity was observed as a time-dependent increase in fluorescence by incubating peptides with pure, solubilized GPI-T in the presence of hydroxylamine, a small GPI anchor mimic. A FRET assay was developed and optimized to monitor GPI anchoring activity in vitro. The assay was used to investigate various aspects of GPI-T, including the importance of the C-terminal hydrophobic region in peptide substrates, the identity of the residue at the site of modification, substrate selectivity, and the effect of cofactors, co-substrates and inhibitors for GPI-T .To date no one has demonstrated robust GPI-T activity with pure solubilized GPI-T. Thus, this new FRET assay represents the first high-throughput method to quantitatively analyze GPI-T activity in vitro

Gender differences of young drivers on injury severity outcome of highway crashes

Problem: Gender differences of young drivers involved in crashes and the associated differences in risk factors have not been fully explored in the United States (U.S.). Accordingly, this study investigated the topic, where the Odds Ratios (OR) were used to identify differences in crash involvements between male and female young drivers. Method: Logistic regression models for injury severity of young male drivers and young female drivers were also developed. Different driver, environmental, vehicle, and road related factors that have affected young female drivers’ and young male drivers’ crash involvement were identified using the models. Results: Results indicated that some variables are significantly related to female drivers’ injury risk but not male drivers’ injury risk and vice versa.Variables such as driving with valid licenses, driving on weekends, avoidance or slow maneuvers at time of crash, non-collision and overturn crashes and collision with a pedestrian were significant variables in female driver injury severity model but not in young male driver severity model. Travel on unleveled roadways, travel on concrete surfaces, travel on wet road surfaces, collision with another vehicle, rear-end collisions were variables that were significant in male driver severity model but not in female driver severity model. Summary: Factors which increase young female drivers’ injury severity and young male drivers’ injury severity were identified. Some factors are significantly related to female drivers’ injury risk but not male drivers’ injury risk and vice versa. This study adds detailed information about gender differences and similarities in injury severity risk of young drivers

Modeling Injury Severity of Young Drivers Using Highway Crash Data from Kansas

Young drivers have higher motor vehicle crash rates compared to other drivers, and understanding the reasons for this would help to improve safety. This study, therefore, investigated characteristics and contributory causes of young-driver crashes and developed multinomial logit models to identify severity affecting factors. It was found that teen drivers were more likely to be involved in crashes due to failure to give time and attention and falling asleep. Among other factors, alcohol involvement, not wearing a seat belt, driving without a valid license, having restrictions on driver’s license, and involvement in off-roadway crashes were factors that increased young-driver injury severity. Based on identified factors, countermeasure ideas for improving safety have also been suggested

Modeling Frequency of Truck Crashes on Limited-Access Highways

Freight can be efficiently transported between most locations in the U.S. using large trucks. Involvement of large trucks in crashes can cause much damage and serious injuries, due to their large sizes and heavy weights. The purpose of this study was to identify the relationships between large truck crashes and traffic and geometric characteristics on limited access highways. Crash and traffic and geometric-related data for Kansas were utilized to develop a Poisson regression model and a negative binomial regression model for understanding the relationships. Based on model-fitting statistics, the negative binomial model was found to be the better model, which was used to identify the important characteristics. By addressing identified factors, safety could be promoted through introduction of appropriate engineering improvements

Traffic Signal Controller Optimization Through VISSIM to Minimize Traffic Congestion, CO and NOx Emissions, and Fuel Consumption

In developing countries with heterogeneous traffic, such as Sri Lanka, it is possible to observe severe traffic congestion at intersections and traffic corridors. The main objective of this study was to demonstrate the optimization of traffic signal controllers using VISSIM microsimulation software. It aimed to minimize traffic congestion, emissions, and fuel consumption. This study focused on developing a traffic signal controller optimization program for a congested traffic corridor which consisted of a three-legged signalized intersection, a four-legged unsignalized intersection, and a three-legged unsignalized intersection. The entire corridor was modeled here, and the already signalized three-legged intersection was optimized. Traffic signal controller optimization was done separately through the built-in optimization features in VISSIM and Webster’s Method. The results showed that emissions and fuel consumption were reduced by 14.89 % in VISSIM optimization and 14.11% in optimization using Webster’s Method. Through the comparison between the VISSIM optimized signal timing and manually calculated signal timing, it was found that the signal timing optimization provides much more improved results than the manual signal timing calculations. Using the proposed methodology, the traffic signal controllers can be optimized within a short duration in very few steps without any iterations compared to the existing traffic signal controller optimization techniques. Therefore, the proposed methodology is a good alternative method to optimize the traffic signal controllers

Improving safety of teenage and young adult drivers in Kansas

Doctor of PhilosophyDepartment of Civil EngineeringSunanda DissanayakeYoung drivers have elevated motor vehicle crash rates compared to other drivers. This dissertation investigated characteristics, contributory causes, and factors which increase the injury severity of young driver crashes in Kansas by comparing them with more experienced drivers. Crash data were obtained from the Kansas Department of Transportation. Young drivers were divided into two groups: 15-19 years (teen) and 20-24 years (young adult) for a detailed investigation. Using data from 2006 to 2009, frequencies, percentages, and crash rates were calculated for each characteristic and contributory cause. Contingency table analysis and odds ratios (OR) analysis were carried out to identify overly represented factors of young-driver crashes compared to experienced drivers. Young drivers were more likely to be involved in crashes due to failure to yield-right-of way, disregarding traffic signs/signals, turning, or lane changing, compared to experienced drivers. Ordered logistic regression models were developed to identify severity affecting factors in young driver crashes. According to model results, factors that decreased injury severity of the driver were seat belt use, driving at low speeds, driving newer vehicles, and driving with an adult passenger. The models also showed that alcohol involvement, driving on high-posted-speed-limit roadways, ejection at the time of crash, and trapping at the time of crash can increase young drivers’ injury severity. Based on identified critical factors, countermeasure ideas were suggested to improve the safety of young drivers. It is important for teen drivers and parents/guardians to gain better understanding about these critical factors that are helpful in preventing crashes and minimizing driving risk. Parents/guardians can consider high-risk conditions such as driving during dark, during weekends, on rural roads, on wet road surfaces, and on roadways with high speed limits, for planning teen driving. Protective devices, crash-worthy cars, and safer road infrastructures, such as rumble strips, and forgiving roadsides, will particularly reduce young drivers’ risk. Predictable traffic situations and low complexity resulting from improved road infrastructure are beneficial for young drivers. The effectiveness of Kansas Graduated Driver Licensing (GDL) system needs to be investigated in the future

Optimization of VISSIM Driver Behavior Parameter Values Using Genetic Algorithm

Modeling effective vehicular traffic is a highly contested topic, especially in developing countries like Sri Lanka, which has a wide range of driving conditions. VISSIM microsimulation software is currently used by Road Development Authority (RDA) and relevant authorities to perform traffic management solutions in Sri Lanka. However, it is required to do modifications to the existing driver behavior parameter values to effectively reflect the realistic traffic conditions observed in the real-world in the simulated model. The main purpose of this study is to calibrate the VISSIM driver behavior parameter values using a genetic algorithm (GA). The methodology and results of the VISSIM model’s sensitivity analysis and calibration, which was developed for the Malabe three-legged signalized intersection, are presented in this study. A sensitivity analysis was used to find the most sensitive driver behavior parameters. Using the multi-objective GA optimization tool in the MATLAB software's optimization toolbox, the optimum driver behavior parameter values for these identified most sensitive driver behavior parameters were determined. The findings revealed that GA optimization is effective in reducing the difference between observed and simulated results

Knowledge, Attitudes and Practices on Critical Results Management among the Medical Laboratory Technologists in Southern Province, Sri Lanka

Critical results require immediate medical intervention. The study aimed to determine knowledge, attitudes and practices on critical results management among Medical Laboratory Technologists (MLTs) in southern province, Sri Lanka. A descriptive cross-sectional study was conducted using a self-administered, pre-tested questionnaire with the participation of 85 MLTs. The results were analysed using SPSS software version 21. 32.9% of the participants stated that there is a critical result management system in the laboratory and 42.4% stated that there is no critical result management system in the laboratory. Among the participants, 23.5% were not aware whether there is a critical result management system in their laboratory. Study participants were categorized based on their knowledge and practice scores. The mean (SD) knowledge score and practice score of the participants were 42.20(±11.67) and 43.39(±10.66). MLTs exceeding ten years of experience had a significantly higher knowledge score (50.38±10.51) compared to MLTs with less than ten years of experience (40.67±11.07, p=0.008). The MLTs with more than 30 years of experience had a significantly higher practice score (58.25±3.95) compared to MLTs with less than one year of experience (39.30±7.57, p=0.002). There was no statistically significant difference in knowledge score and practice score between MLTs with reference to gender, age or education. The overall knowledge and practice of MLTs on critical results management is not satisfactory. The overall attitudes of MLTs on critical results management are satisfactory. The study emphasizes the value of conducting educational and training programs on critical results management and the evaluation of their effectiveness.   KEYWORDS:    Attitudes, Critical Results, Knowledge, Medical Laboratory technologists, Practice

Modification of the o-cresolphthalein complexone method by improving stability of reagents for determination of serum total calcium

Objective: Measurement of total calcium level in serum plays a main role in the diagnosis of many diseases such as osteomalacia, primary hyperparathyroidism and bone metastasis of carcinoma. Commonly used WHO recommended o-cresolphthalein complexone method has some disadvantages such as poor reagent stability, inconvenience in reagent preparation and higher reagent wastage. Therefore this study was performed to develop a new method to measure the total serum calcium by avoiding the weaknesses of the currently employed method. Methods: A comparative study was performed using in-ward adult patient’s serum samples (n=100) received at the Department of Chemical Pathology, National Hospital, Sri Lanka for calcium analysis. They were analyzed by using 4 methods namely; developed method, validated kit method, WHO method and WHO (2000) method. Results: The developed method exhibited linear results of calcium concentration from 2.5 to 15 mg/dl. No significant change in reagent performance over 60 days from preparation was observed. Recovery range was very high in the developed method. The results of the developed method correlated well with the WHO method and other two methods. Conclusion: It was concluded that the developed method for the serum calcium analysis benefits over currently used methods in the Medical Laboratory practice due to the stability of the reagent performance.