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<p>Medical devices used for diagnosis and treatment often involve the exposure of the patient’s blood to biomaterials that are foreign to the body, and blood-material contact may trigger coagulation and lead to thrombotic complications. Therefore, the risk of thrombosis and the issue of blood compatibility are limitations in the development of biomaterials for blood-contacting applications. The objective of this research was to develop a dual strategy for surface modification of polydimethylsiloxane (PDMS) to prevent thrombosis by (1) grafting polyethylene glycol (PEG) to inhibit non-specific protein adsorption, and (2) covalently attaching an antithrombin-heparin (ATH) covalent complex to the distal end of the PEG chains to inhibit coagulation at the surface.</p> <p>Surface characterization via contact angle measurements confirmed reductions in hydrophobicity for the modified surfaces and x-ray photoelectron spectroscopy (XPS) indicated that heparin and ATH were present. The predisposition of PDMS to induce blood coagulation was investigated, and advantages of ATH over heparin in inhibiting coagulation on PDMS were demonstrated. Studies of protein interactions using radiolabelling and Western blotting demonstrated the ability of PEG-modified surfaces to resist non-specific protein adsorption, and the ability of ATH- and heparin-modified surfaces to specifically bind AT present in plasma, thereby providing anticoagulant activity. Through specific interactions with the pentasaccharide sequence on the heparin moiety, the ATH-modified surfaces bound AT more efficiently than the heparin-modified surfaces. Thromboelastography (TEG) was used to evaluate further the anticoagulant potential of the ATH-modified surfaces. It was found that coagulation occurred at a slower rate on the ATH-modified surfaces compared to unmodified PDMS, and the resulting clot was mechanically weaker. By creating a surface with bioinert and bioactive properties, non-specific protein adsorption was reduced and anticoagulation at the surface through specific protein binding was promoted. This dual PEO/ATH modification strategy may therefore offer an improved approach for the minimization of thrombosis on PDMS and biomaterial surfaces more generally.</p> / Master of Applied Science (MASc)

<p> Physical inactivity or deconditioning has been shown to be an independent risk factor for cardiovascular disease. Contrary to the previously demonstrated effects of exercise training on the cardiovascular system, the vascular adaptations that occur with a deconditioned state have not been adequately characterized within a young, healthy population. Thus, it was the interest of the present study to examine vascular adaptations to 12 days of unilateral lower limb immobilization (ULI) in young, healthy humans. Previous studies have used other models to mimic a deconditioned state such as paraplegia, simulated micro gravity and bed rest; however, such models are also associated with factors that are not physiologically applicable to normal deconditioning in the able-bodied population. Fifteen young, healthy participants [age: 20.6±0.51 (mean ± SEM)] participated in the 12-day knee-braced immobilization period that consisted of PRE and 12-DAY time point testing sessions. Measurements of supine common carotid, popliteal and common femoral artery cross sectional compliance as well as popliteal artery endothelial function (using flow mediated vasodilation (FMD)) were acquired prior to the 12-day immobilization (PRE) and on the 12th day of the immobilization (12-DAY). Arterial characteristics of the Immobilized legs (IMM) and NON-Immobilized (NIM) legs were assessed by echo Doppler ultrasound and applanation tonometry.</p> <p> Resting carotid artery cross sectional compliance and blood flow showed no change throughout the 12-day time period, (Compliance: PRE = 0.001209 ± 0.000067 mm^2/mmHg, 12-DAY= 0.001230 ± 0.00085 mm^2/mmHg; Blood Flow: PRE = 242.8±14.2 mL/min, 12-DAY = 226.0±14.27 mL/min). Popliteal artery cross sectional compliance decreased significantly over the 12 day time period in both legs (p<0.05) (IMM PRE = 5.7±0.4 10^-4mm^2/mmHg, IMM 12-DAY = 3.8±0.4 10^-4mm^2/mmHg; NIM PRE= 6.7±0.9 10^-4mm^2/mmHg, NIM 12-DAY = 5.5±0.6 10^-4mm^2/mmHg). Common femoral artery cross sectional compliance decreased in the immobilized leg but not in the non-immobilized leg (p<0.05) over 12 days of immobilization (PRE= 1.2±0.1 10^-4mm^2 /mmHg, 12-DA Y =: 0. 79±0.1 10^-4mm^2/mmHg). Neither popliteal nor common femoral artery mean blood flow changed throughout the 12 days of immobilization. Popliteal arterial mean diameter decreased significantly over time in both the IMM and NIM legs showing greater decreases in the IMM leg, while common femoral arterial mean diameter decreased in both the IMM and NIM legs through the 12 days (Popliteal: IMM PRE = 0.57±0.02 cm, IMM 12-DAY = 0.50±0.02 cm; NIM PRE = 0.59±0.02 cm, NIM 12-DAY = 0.55±0.02 cm; Common Femoral IMM PRE = 0.83±0.04 cm, IMM 12-DAY = 0.77±0.03 cm; NIM PRE = 0.81±0.03 cm, NIM 12-DAY = 0.77±0.03 cm). Popliteal artery endothelial function, calculated as both relative FMD and FMD normalized to shear stress, increased (p<0.05) throughout the 12 days in the immobilized leg while showing no change in the non-immobilized leg (Relative FMD: IMM PRE 6.0 ± 1.4%, IMM 12-DAY = 12.6 ± 2.7%; NIM PRE = 5.8 ± 1.4%, NIM 12-DAY = 8.3 ± 1.6 %; Normalized FMD: IMM PRE = 0.023 ± 0.007%/^sec-1, IMM 12-DAY = 0.037 ± 0.008%/^sec-1; NIM PRE = 0.016 ± 0.003%/^sec-1, NIM 12-DAY = 0.022 ± 0.004%/^sec-1).</p> <p> In conclusion, 12 days of deconditioning by ULI was able to cause structural and functional changes in the arteries of the immobilized leg, but not the central elastic artery in the neck in healthy young humans. Specifically in the legs, a decrease in arterial compliance, increases in mean blood velocity and increases in endothelial function were noted, with no change in volumetric blood flow. Surprisingly, our results suggest, with regards to endothelial function, that the vascular effects of deconditioning are not simply the inverse of exercise training which also shows increases in endothelial function. Thus the present study concludes that there exists a very short time course to arterial adaptations in healthy young humans with significant changes within the vasculature occurring within 12 days of deconditioning.</p> / Thesis / Master of Science (MSc)

<p> We aimed to determine the effectiveness of a low volume of high-intensity resistance exercise, alone (EX) or in combination with a whey protein supplement (WHY +EX), on prevention of muscle mass and strength loss following 14 days of knee immobilization in humans. Seventeen recreationally active (i.e., exercise ≤ 2-3 d·wk^-1) participants (23.9±5.0 yr; BMI = 25.4±3.6 kg·m^-2) were divided into three groups: exercise (EX; n=6), whey protein supplementation with exercise (WHY+EX; n=6), and control (CON; n=5). All subjects wore a knee-immobilization brace such that one leg was completely non-weight bearing for 14d. The resistance exercise (RE) were performed unilaterally and consisted of one set of ten repetitions of leg press (with plantar flexion at full extension), knee extension, and seated calf raises every other day during the 14d immobilization period, at 80% of one repetition maximum. Subjects in the WHY+EX group consumed two 30g boluses of whey protein daily while EX and CON consumed isocaloric carbohydrate beverages. Immobilization induced a significant reduction (p<0.05) in thigh cross-sectional area (CSA), isometric knee extensor strength, and isometric plantar flexion strength in CON but not in EX and WHY+EX. There were significant losses in lower leg CSA for all three groups, with a tendency for losses to be mitigated in both the EX and WHY+EX groups versus CON (p=0.065). The two constituent muscles of the triceps surae muscle group showed a differential response to the RE training with the gastrocnemius showing reductions in CSA almost uniformly across the three groups whereas soleus CSA was significantly reduced in the CON (p<0.05) but not in the EX and WHY+EX groups. We conclude that a relatively low volume of high-intensity resistance exercise is an effective countermeasure against atrophy of the thigh and the soleus muscle, as well as against knee extension and plantar flexion strength loss during 14d of leg immobilization. As a countermeasure to immobilization, there is no additional benefit of consumption of a daily whey protein supplement in combination with resistance exercise in maintaining muscle mass or strength.</p> / Thesis / Master of Science (MSc)

While the Canadian food supply is among the healthiest in the world, almost 4 million (1 in 8) Canadians are affected by food-borne illnesses, resulting in 11,600 hospitalizations and 238 deaths per year. Microbial pathogens are one of the major causes of foodborne sicknesses that can grow in food before or following packaging. Food distribution is an important part of the food processing chain, in which food supplies are at a higher risk of contamination due to lack of proper monitoring. Among myriad of research around biosensors, current devices focusing on packaged food monitoring, such as leakage indicators or time temperature sensors are not efficient for real-time food monitoring without separating the sample from the stock. Packaged food such as meat and juice are directly in touch with the surface of their containers or covers. Therefore, real-time sensing mechanisms, installed inside the food packaging and capable of tracing the presence of pathogens, are of great interest to ensure food safety. This work involves developing thin, transparent, flexible and durable sensing surfaces using DNA biosensors, which report the presence of a target bacterium in food or water samples by generating a fluorescence signal that can be detected by simple fluorescence detecting devices. The covalently-attached DNA probes generate the signal upon contact with the target bacteria with as low as 103 CFU/mL of Escherichia coli in meat and apple juice. The fabricated sensing surfaces remained stable up to several days under varying pH conditions (pH 5 to 9). In addition to detecting pathogens on packaged food or drinking bottles, these surfaces have the potential to be used for a variety of other applications in health care settings, environmental monitoring, food production chain, and biomaterials like wound dressing. / Thesis / Master of Science (MSc) / Microbial pathogens can grow in food following packaging and preceding consumption. Current biosensors are not efficient for post-packaging real-time food monitoring without separating the sample from the stock. Packaged food such as meat and juice are directly in touch with the surface of their containers or covers. Therefore, real-time sensing mechanisms, installed inside the food packaging, tracing the presence of pathogens, are much useful to ensure the food safety. Here we report on developing thin, transparent, flexible and durable sensing surfaces using DNA biosensors, which generate a fluorescence signal in the presence of a target bacterium in food or water samples. The covalentlyattached DNA probes can detect as low as 103 CFU/mL of Escherichia coli in meat, sliced apple and apple juice. The fabricated sensing surfaces remained stable up to several days under varying pH conditions (pH 5 to 9). In addition to pathogen monitoring in packaged food or drinking bottles, these surfaces are promising for a variety of other applications in health care settings, environmental monitoring, and biomaterials like wound dressing.

Pathogenic bacteria pose serious threats to public health and safety. They can cause illness, death, and substantial economic losses. The most widely used bacterial detection methods include cell culturing, antibody-based assays, and nucleic acid amplification techniques, such as polymerase chain reaction (PCR). Unfortunately, these techniques are not well suited for point-of-care application, especially in the resource-limited regions of the world, as they require highly trained personnel to perform the test, they take a long time to complete (especially culturing), and they require sophisticated lab equipment. Thus, there is a great need for simpler, faster, and more accurate methods for bacterial detection. In this thesis, we present a simple, low-cost assay for detecting pathogenic bacteria that is based on the immobilization of a bacteria-specific RNA-cleaving DNAzyme (DNAzyme) onto a surface. If the target bacteria is present, a fluorescently labelled piece of DNA (FDNA) is released through the activity of the DNAzyme; if the target bacteria is not present, the FDNA remains attached to the surface as part of the DNAzyme construct. This method allows untrained users to determine whether a target bacteria is present by simply monitoring the fluorescence intensity in the liquid phase with a hand-held fluorimeter. The first step in this work was to experimentally evaluate different surfaces (including reduced graphene oxide and different beads) onto which the DNAzyme could be immobilized. These tests determined that agarose beads, covered with streptavidin, were ideally suited for DNAzyme immobilization. Next, we conducted a comparative evaluation of the kinetics/activity of the DNAzyme that had been immobilized onto the beads and the free DNAzyme in solution; the results of this evaluation revealed virtually identical reaction rates for the two cases, suggesting no loss of activity after immobilization. Finally, we explored how the DNAzyme sequence length influenced the assay. Specifically, we analyzed a full-length DNAzyme (Full DNAzyme) sequence and a truncated alternative (Short DNAzyme) and found that the full-length construct resulted in faster signal generation. Therefore, it was determined that the long version should be used in the assays. When coupled with a filtration step, the immobilization of biotinylated DNAzymes onto the surface of streptavidin-coated agarose beads enabled the sensitive detection of E. coli in both water samples and complex matrices, such as milk and apple juice. The bead-based assay was able to produce a strong fluorescence signal readout in as little as 2.5 min following contact with E. coli, and it was capable of achieving a detection limit of 1,000 colony-forming units (CFUs) without sample enrichment. As DNAzyme probes can be generated through in vitro selection to react to different bacteria, the RNA-cleavage based detection mechanism described in this work can be adapted for the detection of a wide range of bacterial targets. Overall, this research has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that is highly attractive for field applications, especially in resource-limited regions. / Thesis / Master of Applied Science (MASc)

The knee joint is a diarthrodial joint that rotates in the flexion-extension axis to provide individuals mobility. A limitation in the passive range of motion (ROM) is detrimental for function and this limitation is termed a joint contracture. A commonly shared characteristic between conditions that lead to contracture formation is prolonged periods of immobilization. However, the etiology of immobility-induced joint contractures is not well described and requires quantitative data on anatomical structures limiting knee mobility to design new interventions aimed at restoring function. In turn, our research group has developed an experimental animal model to study the temporal pathophysiology of knee immobilization and reversibility through unassisted remobilization. With durations of immobilization ranging from 1 to 32 weeks and remobilization up to 48 weeks, our experimental design provides a comprehensive temporal overview on the various stages of contracture formation: initiation, progression, and severity. A combination of muscles and articular structures are involved in the pathophysiology of knee flexion contractures, but the posterior joint capsule is of particular interest. Through histomorphological analysis, we provided quantitative data on the contribution of the reduced posterior capsule length in the limitation of knee extension and increased joint stiffness. Moreover, elucidation of synoviocyte profiles within the synovium of the capsule provided insights to potential mechanisms of capsule shortening. Our novel measurable outcome of mechanical joint stiffness revealed distinct temporal differences with ROM measurements after joint immobilization and remobilization, suggesting that alterations in the biomechanical properties of articular tissue structures are also contributing to the limitation in function. Malleability of the dynamic reciprocal relationship between trabecular bone loss and accumulation of marrow adipose tissue (predominately through adipocyte hyperplasia) after knee immobilization underscores the sensitivity of the bone marrow microenvironment in response to mechanical stimuli and lack thereof. Remobilization of the knee joint is limited in its capacity to reverse detriments induced by extended periods of joint immobilization. Findings from this work point to the temporal changes detected in different musculoskeletal tissues during knee immobilization and emphasizes the contribution of the joint capsule in limiting joint mobility.

The production of hydrogen from low-cost abundant renewable biomass would be vital to sustainable development. Cell-free (in vitro) biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it is essential to produce (purified) enzymes at low costs and stabilize them for long periods to decrease biocatalyst costs. Thermophilic recombinant enzymes as building blocks were discovered and developed: fructose 1,6-bisphosphatase (FBP) from Thermotoga maritime, phosphoglucose isomerase (PGI) from Clostridium thermocellum, triose phosphate isomerase (TIM) from Thermus thermophiles and fructose bisphosphate aldolase (ALD) from T. maritima and T. thermophilus. The recombinant proteins were over-expressed in E. coli, purified and characterized. For purification and stabilization of enzymes, one-step, simple, low-cost purification and immobilization methods were developed based on simple adsorption of cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose. Also, a simple, low-cost purification method of thermophilic enzymes was developed utilizing a combination of heat and ammonium sulfate precipitation. For development of cascade enzymes as building modules (biocatalyst modules), it was discovered that the presence of other enzymes/proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme (e.g., PGI) due to the in vitro macromolecular crowding effect. And substrate channeling among CBM-tagged self-assembled three-enzyme complex (synthetic matabolon) immobilized on the easily-recycled cellulose-containing magnetic nanoparticles can not only increase cascade reaction rates greatly, but also decrease enzyme cost in cell-free biosystems. The high product yield and fast reaction rate of dihydrogen from sucrose was validated in a batch reaction containing fifteen enzymes comprising a non-natural synthetic pathway. The yield of dihydrogen production from 2 mM of sucrose was 96.7 % compared to theoretical yield at 37 °C. The maximum rate was increased 3.1 fold when the substrate concentration was increased from 2 to 50 mM in a fed-batch reaction. The research and development of cell-free biosystems for biomanufacturing require more efforts, especially in low-cost recombinant thermostable enzymes as building blocks, efficient cofactor recycling, enzyme and cofactor stabilization, and fast reaction rates. / Ph. D.