Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, in vitro approaches for functional assessments have been relatively limited. Herein, we describe the guidance of primary endothelial cells (ECs) in an agarose hydrogel scaffold that is chemically patterned with an immobilized concentration gradient of vascular endothelial growth factor 165 (VEGF165) using multiphoton laser patterning of VEGF165. This is the first demonstration of this patterning technology to immobilize proteins; and the first demonstration of immobilized VEGF165 to guide endothelial cell growth and differentiation in 3D environments. It is particularly compelling that this 3D hydrogels provide an excellent biomimetic environment for stem cell niche, thereby offering a new approach to study stem cell biology. In this thesis, we focused on the retinal stem cell niche, investigating cellular interactions between retinal stem and progenitor cells (RSPCs) and endothelial cells (ECs). By using this 3D in vitro model, we demonstrated the synergistic interactions between RSPCs and ECs wherein RSPCs migrated into 3D gels only in the presence of ECs and RSPCs stabilized EC tubular-like formations. Moreover, we characterized the contact-mediated effects of ECs on RSPC fate in terms of proliferation and differentiation.

Anaerobic digestion of excess biological wastewater treatment sludge (WAS) from pulp mills has the potential to reduce disposal costs and to generate energy through biogas production. The organic matter in WAS is highly structured, which normally hinders biogas production. This study investigated three methods of pretreating WAS from two different pulp mills before anaerobic digestion to improve biogas yield and production rate. The three pretreatment methods tested were: i) thermal pretreatment at 170oC, ii) caustic pretreatment at 140oC and pH 12, and iii) sonication at 20 kHz and 1 W/mL. Thermal pretreatment proved to be the most effective, increasing biogas yield by 280% and 50% and increasing production rates 300-fold and 10-fold for the two samples, respectively. Caustic pretreatment showed similar results, but resulted in the formation of soluble non-biodegradable compounds. Sonication was the least effective pretreatment and did not substantially increase biogas yield, but increased biogas production rate.

The objective of this thesis is to better understand the impact of paper and printer types on xerographic print quality. To achieve this objective, commercially printed samples comprising of ten different paper substrates printed using three different xerographic printers were examined. The print quality of these samples was assessed in terms of print microgloss and its nonuniformity, print density, print and gloss mottle, print roughness, and visual ranking. This study showed that print mottle conducted by Fast Fourier Transform produced the best correlation with visual ranking at the size range of 0.1 - 1mm, while print gloss mottle was found to affect print quality regardless of the mottle size. Brightness, opacity, basis weight, gloss 75, and roughness of these paper substrates were found to have the most significant effect on print quality. All of the optical properties of paper included in this analysis showed a strong correlation to print quality.

Embryogenesis occurs within a complex and dynamic cellular environment that influences cell fate decisions. Pluripotent stem cells (PSCs) are a valuable tool for research into disease models as well as a resource for cell therapy due to their capacity to self-renew and differentiate into all cell types. Mimicking aspects of the embryonic microenvironment in vitro impacts the resultant functional cells. The aim of this work was to develop a controlled and scaleable process for the generation of hematopoietic progenitor cells (HPCs) from embryonic stem cells (ESCs). We demonstrated with bioreactor-grown embryoid bodies (EBs) that increased HPC generation can be elicited by decreasing the oxygen tension by a mechanism where vascular endothelial growth factor receptor 2 (VEGFR2) activation is controlled through competition with the ligand decoy VEGFR1. This is important as it demonstrates the inherent responsiveness of the developing hematopoietic system to external forces and influences. We also established a serum-free system that facilitates directed differentiation, determining 5 ng/ml bone morphogenetic protein-4 (BMP4) with 50 ng/ml thrombopoietin (TPO) could generate 292 ± 42 colony forming cells (CFC)/5 x 10^4 cells with early VEGF treatment (25 ng/ml, day 0-5). We also controlled aggregate size influencing relative endogenous and exogenous growth factor signaling and modulating mesodermal differentiation; CFC output was optimal when initialized with 100 cell aggregates. For the first time, we demonstrated efficacy of local growth factor delivery by producing HPCs with gelatin microparticles (MP). Overall, these design components generate HPCs in a controlled and reproducible manner using a serum-free bioprocess that couples size controlled aggregates containing gelatin MPs for localized growth factor release of BMP4 and TPO with hypoxia to induce endogenous VEGF production. These strategies provide a tunable platform for developing cell therapies and high density growth, within a bioreactor system, can be facilitated by hydrogel encapsulation of the aggregates.

Biocatalysts are important tools for harnessing the potential of wood fibres since they can perform specific reactions with low environmental impact. Challenges to bioconversion technologies as applied to wood fibres include low accessibility of plant cell wall polymers and the heterogeneity of plant cell walls, which makes it difficult to predict conversion efficiencies. White-rot fungi are among the most efficient degraders of plant fibre (lignocellulose), capable of degrading cellulose, hemicellulose and lignin. Phanerochaete carnosa is a white-rot fungus that, in contrast to many white-rot fungi that have been studied to date, was isolated almost exclusively from fallen coniferous trees (softwood). While several studies describe the lignocellulolytic activity of the hardwood-degrading, model white-rot fungus Phanerochaete chrysosporium, the lignocellulolytic activity of P. carnosa has not been investigated. An underlying hypothesis of this thesis is that P. carnosa encodes enzymes that are particularly well suited for processing softwood fibre, which is an especially recalcitrant feedstock, though a major resource for Canada. Moreover, given the phylogenetic similarity of P. carnosa and P. chrysosporium, it is anticipated that the identification of pertinent enzymes for softwood degradation can be more easily conducted. In particular, this project describes the characterization of P. carnosa in terms of the growth conditions that support lignocellulolytic activity, the effect of enzymes secreted by P. carnosa on the chemistry of softwood feedstocks, and the characterization of the corresponding secretome using proteomic techniques. Through this study, cultivation methods for P. carnosa were established and biochemical assays for protein activity and quantification were developed. Analytical methods, including FTIR and ToF-SIMS were used to characterize wood samples at advancing stages of decay, and revealed preferential degradation of lignin in the early stages of growth on all softwoods analyzed. Finally, an in depth proteomic analysis of the proteins secreted by P. carnosa on spruce and cellulose established that similar sets of enzyme activities are elicited by P. carnosa grown on different lignocellulosic substrates, albeit to different expression levels.

The reduction of mill effluent discharge as a result of stringent environmental legislations can have a significant impact on sodium (Na) and sulfur (S) balances in the kraft pulping process. In order to maintain a proper balance of Na and S, kraft mills may need to adopt different makeup strategies. For this purpose, a dynamic model was developed to predict the Na and S balance in the kraft recovery cycle, as well as the accumulation of undesirable non-process elements such as chlorine (Cl) and potassium (K). The model was developed using the CADSIM software and was validated using data obtained from a Brazilian bleached kraft pulp mill. The calculated data from the model showed good agreement with mill data with respect to all parts of the mill simulated. Dynamic tests designed to calculate the white liquor sulfidity over specific periods of time also presented good agreement. The result indicates that the model is able to describe the balance of chlorine, potassium, sodium and sulfur in the kraft process. A study conducted to evaluate the Cl and K accumulation agrees with the expected behaviour observed in mill data. The presence of ash treatment systems allow to reduce Cl and K contents in recovery boiler precipitator ash from 4.2 mol% Cl(Na+K) to 1.25 mol % and from 2.25 mol % K/(Na+K) to 0.8 mol% for 100% ash treated. The tests performed for Na and S balances focused in the makeup requirement for two situations: ash purging and ash treatment to control Cl and K levels. The use of ash treatment systems reduced Na and S makeup requirement from 5 to 50% depending on the amount of ash treated. A simple mathematical model was then used to estimate the Cl balances around the recovery cycle. Given that the proper simplifications are applied, the CADSIM model and the CSTR model presented good agreement in estimating the Cl balances. This result provided not only another method for the CADSIM model to be validated but also a way to calculate a rough estimate for Cl balance.

A study was carried out into the computational fluid dynamic simulation of bio-oil combustion. Measurements were taken in an empirical burner to obtain information regarding the flow behaviour. A surrogate fuel was developed to mimic the unique chemical and physical properties of bio-oil combustion. The resulting computational model of the burner domain and surrogate fuel was compared with empirical data. The bio-oil model displayed a good agreement with the data in terms of the combustion behaviour, but was limited by the uncertain flow solution associated with the burner used.

A study was carried out into the computational fluid dynamic simulation of bio-oil combustion. Measurements were taken in an empirical burner to obtain information regarding the flow behaviour. A surrogate fuel was developed to mimic the unique chemical and physical properties of bio-oil combustion. The resulting computational model of the burner domain and surrogate fuel was compared with empirical data. The bio-oil model displayed a good agreement with the data in terms of the combustion behaviour, but was limited by the uncertain flow solution associated with the burner used.

Despite the increasing clinical demand in reconstructive, cosmetic and correctional surgery there remains no optimal strategy for the regeneration or replacement of adipose tissue. Previous approaches to adipose tissue engineering have failed to create an adipose tissue depot that maintains implant volume in vivo long-term (>3 months). This is due to inadequate mechanical properties of the biomaterial and insufficient vascularization upon implantation. Modular tissue engineering is a means to produce large volume functional tissues from small sub-mm sized tissues with an intrinsic vascularization. We first explored the potential of a semi-synthetic collagen/poloxamine hydrogel with improved mechanical properties to be used as the module biomaterial. We found this biomaterial to not be suitable for adipose tissue engineering because it did not support embedded adipose-derived stem cell (ASC) viability, differentiation and human microvascular endothelial cell (HMEC) attachment. ASC-embedded collagen gel modules coated with HMEC were then implanted subcutaneously in SCID mice to study its revascularization potential. ASC cotransplantation was shown to drive HMEC vascularization in vivo: HMEC were seen to detach from the surface of the modules to form vessels containing erythrocytes as early as day 3; vessels decreased in number but increased in size over 14 days; and persisted for up to 3 months. Early vascularization promoted fat development. Only in the case of ASC-HMEC cotransplantation was progressive fat accumulation observed in the module implants. Although implant volume was not maintained, likely due rapid collagen degradation, the key result here is that ASC-HMEC cotransplantation in the modular approach was successful in creating vascularized adipose tissue in vivo that persisted for 3 months. The modular system was then studied in vitro to further understand ASC-EC interaction. Coculture with ASC was shown to promote an angiogenic phenotype (e.g. sprouting, migration) from HUVEC on modules. RT-PCR analysis revealed that VEGF, PAI-1 and TNFα was involved in ASC-EC paracrine signalling. In summary, ASC-HMEC cotransplantation in modules was effective in rapidly forming a vascular network that supported fat development. Future work should focus on further elucidating ASC-EC interactions and developing a suitable biomaterial to improve adipose tissue development and volume maintenance of engineered constructs.

Pluripotent stem cells provide the opportunity to study human cardiogenesis in vitro, and are a renewable source of tissue for drug testing and disease models, including replacement cardiomyocytes that may be a useful treatment for heart failure. Typically, differentiation is initiated by forming spherical cell aggregates wherein an extraembryonic endoderm (ExE) layer develops on the surface. Given that interactions between endoderm and mesoderm influence embryonic cardiogenesis, we examined the impact of human embryonic stem cell (hESC) aggregate size on endoderm and cardiac development. We first demonstrated aggregate size control by micropatterning hESC colonies at defined diameters and transferring the colonies to suspension. The ratio of endoderm (GATA-6) to neural (PAX6) gene and protein expression increased with decreasing colony size. Subsequently, maximum mesoderm and cardiac induction occurred in larger aggregates when initiated with endoderm-biased hESCs (high GATA-6:PAX6), and in smaller aggregates when initiated with neural-biased hESCs (low GATA-6:PAX6). Additionally, incorporating micropatterned aggregates in a stirred suspension bioreactor increased cell yields and contracting aggregate frequency. We next interrogated the relationship between aggregate size and endoderm and cardiac differentiation efficiency in size-controlled aggregates, generated using forced aggregation, in defined cardiogenic medium. An inverse relationship between endoderm cell frequency (FoxA2+ and GATA6+) and aggregate size was observed, and cardiogenesis was maximized in mid-size aggregates (1000 cells) based on frequency of cardiac progenitors (~50% KDRlow/C-KITneg) on day 5 and cardiomyocytes (~24% cTnT+) on day 16. To elucidate a relationship between endoderm frequency and cardiac differentiation efficiency, aggregates were initiated with varying frequencies of ExE progenitors (SOX7-overexpressing hESCs). Maximum cardiomyocyte frequencies (~27%) occurred in aggregates formed with 10 to 25% ExE progenitors. These findings suggest a geometric relationship between aggregate size and ExE differentiation efficiency subsequently impacts cardiomyocyte yield, elucidating a mechanism for endogenous control of cell fate through cell-cell interactions in the aggregate.