Previous studies have shown that pullulan films are able to stabilize enzymes and other labile molecules from thermal and oxidative degradation. Solutions made with commercially available pullulan are extremely viscous and difficult to process limiting the ability to use low-cost printing systems, such as inkjet printers, to format pullulan-containing. In this work, we show that pullulan can be made printable by decreasing its chain length by acid hydrolysis. The acid hydrolysis reaction was modelled using statistical software; the molecular weight of pullulan decreased with increasing reaction time, temperature and acid concentration. Interactions between time and temperature, and temperature and acid concentration were determined to be significant to the reaction as well. The mechanical properties and oxygen permeability of films made from pullulan with different molecular weights were also measured. The films were found to have similar tensile properties and oxygen permeabilities to each other and to those obtained using native pullulan. Using a thermally unstable enzyme (acetylcholinesterase) and an easily oxidizable small molecule (indoxyl acetate) as test materials, it was found that these films have the same ability to stabilize the enzyme and to serve as an oxygen barrier, as the films made with native pullulan. It was also found that pullulan is inkjet printable as long as the molecular weight is 56 kDa. Poor jetting and clogging of the printhead was observed when pullulan with a molecular weight higher than this threshold was used. Microarray printing was also demonstrated by a printing acetylcholinesterase/pullulan in nano-sized volumes using a Dimatix inkjet printer and showing activity of the enzyme after printing and storage at ambient conditions. Proof of concept of microarray printing opens up the potential for future applications of pullulan in other high throughput applications. / Thesis / Master of Applied Science (MASc)

Cement stabilized material used for subbase or base pavement layers has been a widely accepted practice by many state Departments of Transportation (DOTs); particularly, for DOTs with limited access to quality crushed aggregates for pavement construction. Despite over 100 years of use, construction specifications governing cement stabilized pavement layers have largely remained the same and are primarily method based specifications (i.e. individual components evaluated and construction methods prescribed) rather than evaluating or testing mechanical properties of the end product. With the recent emergence of the Plastic Mold compaction Device (PM Device), multiple agencies are looking to depart from method based soil-cement specifications by implementing the PM Device for design and construction quality control and quality assurance (QC/QA) testing. Prior to this dissertation, PM Device protocols have been validated under lab conditions but only limited field validation had been performed. Additionally, time delay between initial mixing and compaction of cement stabilized soils is a known issue, which can affect compaction of PM Device specimens as well as construction target density values determined through AASHTO T134 Proctor testing. The main objectives of this dissertation are to investigate time delay effects on cement stabilized soil compactability during Proctor testing, develop a nationally recognized Standard Practice for PM Device specimen fabrication, and perform PM Device field evaluations for QC/QA testing. Lab experiments were conducted to investigate time delay effects and finalize PM Device Standard Practice protocols. Five field projects were evaluated to validate PM Device QC/QA applications and Standard Practice protocols in a construction environment. Time delay was observed to have a notable detrimental influence on compactability during AASHTO T134 Proctor testing and PM Device specimen fabrication. Recommended guidance was provided to characterize compaction delay effects. AASHTO PP92-19 was developed and published by AASHTO's Committee on Materials and Pavements to standardize specimen fabrication for the 3x6 inch and 4x8 inch versions of the PM Device. The PM Device fared well for construction activities when benchmarked relative to density, strength, and modulus of cores taken from constructed cement stabilized pavement layers. The PM Device was recommended for implementation consideration by state DOTs and other agencies.

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Introduktion: Mikrodialys används för att studera metabola förändringar i olika vävnader. Vid mikrodialys sätts en kateter i muskeln vilket inducerar en traumafas, som kan påverka frisläpp av substanser. En 120 minuters stabiliseringstid har föreslagits så att metabola förändringar p.g.a. traumat ska normaliseras och ej påverka resultatet. En lång stabiliseringstid leder dock till att dessa studier är tidskrävande och därför även dyra och svårgenomförda. Syfte: Att undersöka och fastställa lämplig stabiliseringsperiod för mikrodialys av serotonin och glutamat i massetermuskeln hos friska individer samt hos patienter med myofascial TMD. Material och metod: Intramuskulär mikrodialys utfördes och dialysat samlades in på 15 friska kontroller samt 15 patienter med myofascial TMD för analys av serotonin och glutamat. För att tillåta vävnaden återhämta sig från traumafasen utvärderades en 120-minuters stabiliseringsperiod, där de första 20 minuterna utgjorde ursprunglig baseline och de sista 20 minuterna stabiliserad baseline. Resultat: Ingen signifikant förändring av serotonin och glutamat observerades över tid för kontroll-gruppen (P>0,05). För TMD-gruppen sågs däremot såg en signifikant sänkning av serotoninhalten over tid (P<0,001) följt av en signifikant ökning mellan tidpunkten T100-120 och T120-140 (P<0,001). För glutamat sågs en reduktion vid tidpunkten T20-40 jämför med det ursprungliga baselinevärdet (P<0,05). Konklusion: Resultaten antyder att 20 minuters stabiliseringsperiod är tillräcklig för friska individer vid mikrodialys av serotonin och glutamat i masseter muskeln. Hos patienter med myofacial TMD är glutamat-nivåerna stabiliserade efter 40 minuter. Serotonin nivåerna är däremot inte stabiliserade efter 120 minuter, vilket tyder på en spontan ökning av intramuskulär serotonin 2 timmar efter införandet av katetern. / Introduction: Microdialysis is a technique used to study metabolic changes in tissues. When performing microdialysis, a catheter is inserted into the muscle inducing a trauma phase, which may affect the release of substances. A 2-hour stabilization period has been suggested to allow tissues to recover from metabolic changes following the trauma. A long stabilization period however, makes these studies time-consuming and thus expensive.Aim: To investigate the necessary stabilization period for microdialysis of serotonin and glutamate in the masseter muscle in healthy subjects and in patients with myofascial TMD.Material and Methods: Intramuscular microdialysis was carried out in 15 patients with myofascial TMD and 15 healthy controls to collect serotonin and glutamate. To allow the tissue to recover following the probe insertion, a 120-min stabilization period was evaluated where the first 20 min served as the zero baseline and the last 20 min as the stabilized baseline. Results: No significant alterations of serotonin or glutamate were observed over the 2-hour period for the controls (P>0.05). For the TMD group, a significant decrease of serotonin was observed over time (P<0.001) followed by a significant increase between T100-120 and T120-140 (P<0.001). For glutamate, a significant reduction was observed at T20-40 compared with the zero baseline (P<0.05). Conclusion: A 20-min stabilization period is sufficient for healthy subjects for microdialysis of serotonin and glutamate in the masseter muscle. In patients with myofascial TMD, glutamate levels are stabilized after 40 minutes. Serotonin levels are not stabilized after 2 hours indicating a spontaneous increase of serotonin.

Phospholipid bilayers constitute the primary structural element of biological membranes, and as such, they play a central role in biochemical and biophysical processes at the cellular level, including cell protection, intercellular interactions, trans-membrane transport, cell morphology, and protein function, to name a few. The properties of phospholipid bilayers are thus of great interest from both experimental and theoretical standpoints. Although experiments have provided much of the macroscopic functions and properties of biological membranes, insight into specific mechanisms at the molecular level are seldom accessible by conventional methods. To obtain a better understanding of biochemical and biophysical processes at the molecular level involving phospholipid bilayers, we apply molecular simulation methods to investigate the complexity of the membrane matrix using atomistic models. Here, we discuss three specific biological processes that are associated with biological membranes: 1) membrane stabilization, 2) membrane phase behavior, and 3) fatty acid-induced toxicity in cell membranes. For membrane stabilization, molecular dynamics studies were performed for mixed phospholipid bilayers containing two of the most prevalent phospholipids (phosphatidylcholine and phosphatidylethanolamime) in biological membranes. We presented structural and dynamics properties of these systems, as well as the effect of stabilizing agents, such as trehalose, on their properties. Furthermore, we performed a comprehensive analysis of the phase transition of lipid bilayers and investigated the interactions of stabilizing agents (glucose or trehalose) with lipid bilayers under dehydrated conditions to understand the mechanisms for preservation of cellular systems. For membrane phase behavior, a comprehensive study of the structural properties of saturated and monounsaturated lipid bilayers near the main phase transition were investigated using molecular dynamics simulations. In this study, we demonstrated that atomistic simulations are capable of capturing the phase transformation process of lipid bilayers, providing a valuable set of molecular and structural information at and near its transition state. Lastly, the third study investigated the mechanism for fatty acid-induced toxicity by integrating in vitro and in silico experiments to reveal the biophysical interactions of saturated fatty acid (palmitate) with the cellular membranes and the role of trehalose and unsaturated fatty acids (oleate and linoleate) in preventing changes to the membrane structure. Knowledge gained from this study is essential in the prevention and treatment of obesity-associated cirrhosis diseases. / Ph. D.