Proteome analysis techonologies are commonly employed for discovery-based biomarker identification studies. This thesis aims to help bridge the gap between analytical technology development and clinical application by improving and appling a proteomics workflow for biomarker discovery in congenital urinary tract obstruction (UTO). By accentuating the importance of experimental design, and evaluating the biological relevance of quantitative proteome analyses, the results of this research provide confidence in a number of identified candidate biomarkers of UTO. A sensitive method for quantification of proteome samples was developed using temperature controlled reversed-phase liquid chromatography (TPLC). The TPLC system provides high recovery (> 90 %), as well as high accuracy and precision in estimating the concentration across a number of protein sample types (CV < 10 %). The need for extensive fractionation strategies coupled with LC-MS analysis challenges the throughput of the overall experiment. Development of a dual column LC-MS interface reduced the total analysis time by a factor of 2 over conventional single column LC-MS systems. The system was applied to a quantitative proteome analysis of proximal tubule cells exposed to mechanical stretch, mimicking the conditions they experience during UTO and a urinary exosomal proteome analysis for candidate biomarker identification of this disease. A total of 1636 proteins were identified in the whole cell proteome analysis, of which 317 were found to be significantly altered in abundance. Analysis of the urinary exosomal proteome yielded 318 proteins, of which 189 were found to be altered in abundance due to obstruction. Western blot confirmation of a few select proteins provided backing to the quantitative proteome analysis, while gene ontology and KEGG pathway analysis yielded functional information. The results from the quantitative analyses of the urinary exosomes and proximal tubule cells identified candidates for both diagnosis and prognosis of UTO. In addition, activation of a novel pathway was identified, presenting a potential drug target which could be exploited to improve recovery of children following relief of UTO. This thesis therefore contributes useful technological and methodological advancements towards routine proteome analysis, as well as providing candidate biomarker identification for the leading cause of renal functional loss in children.

Development of temperature controlled packaging (TCP) systems involves a significant lead-time and cost as a result of the large number of tests that are carried out to understand system performance in different internal and external conditions. This MPhil project aims at solving this problem through the development of a transient spreadsheet based model using lumped system approach that predicts the performance of packaging systems under a wide range of internal configurations and dynamically changing environmental thermal conditions. Experimental tests are conducted with the aim of validating the predictive model. Testing includes monitoring system temperature in a wide range of internal configurations and external thermal environments. A good comparison is seen between experimental and model predicted results; increasing the mass of the chilled phase change material (PCM) in a system reduces the damping in product performance thereby reducing the product fluctuations or amplitude of the product performance curve. Results show that the thermal mathematical model predicts duration to failure within an accuracy of +/- 15% for all conditions considered.

The typical differential extraction procedure utilized by the forensic science community to extract male deoxyribonucleic acid (DNA) from the sperm cells of the perpetrator separately from female DNA from the epithelial cells of the victim is both time-consuming and labor-intensive. This has contributed greatly to the backlog of unanalyzed sexual assault evidence collection kits (SAECK) seen in many laboratories today and has encouraged research in new methods that are more efficient and more effective in achieving better sperm DNA recovery. The Cotton Lab has developed a Temperature-Controlled Differential Extraction (TCDE) procedure geared towards attaining better sperm recovery and better distribution of male DNA in the sperm fraction (SF) to generate a single source or distinguishable male profile. The TCDE protocol is a direct-lysis procedure that utilizes highly temperature-controlled enzymes, or enzymes that are active at or near their optimal temperatures. This procedure has been previously shown to decrease extraction time significantly and to extract samples that are suitable for downstream analysis. This research specifically attempted to modify the TCDE procedure in the hopes of obtaining higher sperm DNA recovery and eliminating previous concerns of too much sperm being retained by the cotton swab material. It also compared a slightly modified TCDE procedure where the material fraction (MF) and SF are kept as separate fractions (the Separate Method) and a method that results in a recombined MF and SF (Recombined Method) to see if there was a greater distribution of the total male DNA eluted into the SF. Preliminary experimentation with swabs prepared with semen was performed to help make effective modifications. Then, vaginal swabs from eight different female donors were prepared with semen to mimic forensic casework samples and extracted using the Separate and Recombined Methods for comparison of the two extraction methods. Despite unusual epithelial cell lysis results for some samples, the quantitation of the fractions by quantitative polymerase chain reaction (qPCR) showed that for approximately half of the samples extracted using the Separate Method, a majority of total male DNA was eluted into the SF. For these samples, a single source or distinguishable male profile can be generated. However, it was also demonstrated that even with good separation, a very small proportion of the female DNA in the SF still overwhelms the male DNA that is present in much smaller amounts, particularly for the Recombined Method where there are only two fractions. Though further experimentation is necessary, these modifications proved effective in achieving high sperm recovery in the SF and generating a distinguishable male profile when extracting samples using the Separate Method. This research has confirmed that the TCDE procedure can be faster and less labor intensive while still producing clean DNA profiles in downstream analysis, and thus has the potential to be implemented in forensic laboratories after some of the concerns are addressed.

Environmental conditions such as temperature inevitably impact the long term performance, strength and deformation characteristics of most materials in infrastructure applications. The mechanical and durability properties of geosynthetic materials are strongly temperature dependent. The interfaces between geotextiles and geomembranes as well as between granular materials such as sands and geomembranes in landfill applications are subject to temperature changes due to seasonal temperature variations as well as exothermic reactions occurring in the waste body. This can be a critical factor governing the stability of modern waste containment lining systems. Historically, most laboratory geosynthetic interface testing has been performed at room temperature. Information today is emerging that shows how temperatures in the liner systems of landfills can be much higher. An extensive research study was undertaken in an effort to investigate temperature effects on interface shear behavior between (a) NPNW polypropylene geotextiles and both smooth PVC as well as smooth and textured HDPE geomembranes and (b) sands of different angularity and smooth PVC and HDPE geomembranes. A temperature controlled chamber was designed and developed to simulate elevated temperature field conditions and shear displacement-failure mechanisms at these higher temperatures. The physical laboratory testing program consisted of multiple series of interface shear tests between material combinations found in landfill applications under a range of normal stress levels from 10 to 400 kPa and at a range of test temperatures from 20 to 50 °C. Complementary geotextile single filament tensile tests were performed at different temperatures using a dynamic thermo-mechanical analyzer (DMA) to evaluate tensile strength properties of geotextile single filaments at elevated temperatures. The single filament studies are important since the interface strength between geotextiles and geomembranes is controlled by the fabric global matrix properties as well as the micro-scale characteristics of the geotextile and how it interacts with the geomembrane macro-topography. The peak interface strength for sand-geomembrane as well as geotextile-geomembrane interfaces depends on the geomembrane properties such as hardness and micro texture. To this end, the surface hardness of smooth HDPE and PVC geomembrane samples was measured at different temperatures in the temperature controlled chamber to evaluate how temperature changes affect the interface shear behavior and strength of geomembranes in combination with granular materials and/or geotextiles. The focus of this portion of the experimental work was to examine: i) the change in geomembrane hardness with temperature; ii) develop empirical relationships to predict shear strength properties of sand - geomembrane interfaces as a function of temperature; and iii) compare the results of empirically predicted frictional shear strength properties with the results of direct measurements from the interface shear tests performed at different elevated temperatures.