This investigation assesses the role of poly(ADP-ribose) polymerase in ethanol-mediated hepatotoxicity using the untransfected HepG2 hepatocellular carcinoma line, an established, well-characterized toxicological model. HepG2 cells were treated with ethanol at concentrations between 100 mM and 800 mM, and assessed for markers of cytotoxicity. PARP-1 activity in total cell protein lysates was quantified as a proxy of apoptotic induction at six hours. Our results demonstrated a 1.43-fold AST activity increase in culture medium isolates of cells exposed to 800 mM without significant effect on cellular viability. PARP-1 activity varied greatly and results for enzyme activity remained inconclusive. The results suggest a high degree of insensitivity to ethanol toxicity and nuclear enzyme activity, demonstrating the metabolic irrelevance of untransfected HepG2 in ethanol toxicosis. There is a need to characterize phase 1 metabolic enzyme expression profiles relevant to ethanol for CYP2E1 and ADH pathways to facilitate comparisons across toxicological models using transfected, as well as the untransfected HepG2 model.

RNA interference (RNAi) is an evolutionarily conserved process that regulates gene expression. Host cells and viruses interact in many ways, including through miRNAs and RNAi. Viral miRNAs are encoded when viruses, specially including the the polyoma and herpes families, are transcribed in the nucleus. Some viral miRNAs function to regulate host or viral gene expression. Most viral miRNAs’ functions are not known, however, in great detail. A miRNA can be encoded late during infection, as it is by SV40, a model polyomavirus. This downregulates early viral gene expression by directing mRNA RISC-mediated cleavage. As more polyomaviruses are discovered that are associated with human disease, it becomes more important to understand their function and to uncover whether these emerging viruses encode miRNAs. The work presented here shows the discovery of several viral miRNAs in human polyomaviruses—JCV, BKV, and MCV. In addition, I found that viral miRNAs have the evolutionarily conserved function of negatively regulating viral early gene transcripts at a late stage in the infection. During viral replication, viruses utilize the miRNA components of RNAi. However, in invertebrate organisms RNAi also actively defends against viral infection. It is still being debated, though, whether RNAi plays an antiviral role in mammalian cells. Should it be true that RNAi is an antiviral response in mammalian cells, then what is predicted by such a scenario is inconsistent with my studies. I have found that RNAi is strongly inhibited in the early stages after viral infection. Studies with a chemical mimic of viral infection (poly I:C) imply that the innate cellular immune response is responsible for this inhibition. I investigated the molecular changes, in response to viral infection, (e.g. poly ADP-ribosylation of Ago2) in the RNA-induced silencing complex (RISC). I determined that the inhibition of RNAi is brought about by components of the innate response. Completion of this study details a previously unknown “cross talk” between RNAi and the host innate immune response in mammalian cells. Furthermore, I found mir-17 family attenuates a subclass of interferon-stimulated genes. An understanding of viral miRNA and RNAi offers a clue as to we can use molecular intervention for viral infections. / text

The polymer materials industry is heavily dependent on the use of petroleum based plastics. This poses a problem, as the world is facing ongoing petroleum supply problems. A need exists for a bio-carbon based polymer material that has the performance and cost of currently used petroleum plastics. However, the overall performance of current bio-based plastics indicate that they must be somehow supplemented to achieve the properties of that of petroleum-based polymers. The low impact strength and thermal stability of poly(lactic acid), PLA are targets for improvement. One option is for development is through blending with acrylonitrile butadiene styrene (ABS). The viability and efficacy of using these two polymers as blending partners is investigated. The PLA used in these studies has unique and interesting crystallization properties. These have been examined and detailed in part 1. The second part of study includes neat polymer properties, miscibility analysis, and large scale process results. This results in an optimized blending ratio on which to go forward with development. The mechanical, thermal, and morphological properties are investigated in these studies. Significance of this research and development is widespread, as the material developed has the potential to reduce the use of petroleum-based carbon in plastics. / The financial support from the 2010 Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA)/University of Guelph -Bioeconomy for Industrial Uses Research Program, Natural Sciences and Engineering Research Council (NSERC) AUTO21 NCE project and Grain Farmers of Ontario (GFO), to carry out this research is gratefully acknowledged.

Analogous to the extracellular matrix (ECM) of natural tissues, properties of a tissue engineering scaffold direct cell behavior and thus regenerated tissue properties. These include both physical properties (e.g. morphology and modulus) and chemical properties (e.g. hydrophobicity, hydration and bioactivity). Notably, recent studies suggest that scaffold properties (e.g. modulus) may be as potent as growth factors in terms of directing stem cell fate. Thus, 3D scaffolds possessing specific properties modified for optimal cell regeneration have the potential to regenerate native-like tissues. Photopolymerizable poly(ethylene glycol) diacrylate (PEG-DA)-based hydrogels are frequently used as scaffolds for tissue engineering. They are ideal for controlled studies of cell-material interactions due to their poor protein adsorption in the absence of adhesive ligands thereby making them “biological blank slates”. However, their range of physical and chemical properties is limited. Thus, hydrogel scaffolds which maintain the benefits of PEG-DA but possess a broader set of tunable properties would allow the establishment of predictive relationships between scaffold properties, cell behavior and regenerated tissue properties. Towards this goal, this work describes a series of unique hybrid inorganic-organic hydrogel scaffolds prepared using different solvents and also in the form of continuous gradients. Properties relevant to tissue regeneration were investigated including: swelling, morphology, modulus, degradation rates, bioactivity, cytocompatibility, and protein adhesion. These scaffolds were based on the incorporation of hydrophobic, bioactive and osteoinductive methacrylated star polydimethylsiloxane (PDMSstar-MA) [“inorganic component”] into hydrophilic PEG-DA [“organic component”]. The following parameters were varied: molecular weight (Mn) of PEG-DA (Mn = 3k & 6k g/mol) and PDMSstar-MA (Mn = 1.8k, 7k, 14k), ratio of PDMSstar-MA to PEG-DA (0:100 to 20:80), total macromer concentration (5 to 20 wt%) and utilizing either water or dichloromethane (DCM) fabrication solvent. The use of DCM produced solvent induced phase separation (SIPS) resulting in scaffolds with macroporous morphologies, enhanced modulus and a more homogenous distribution of the PDMSstar-MA component throughout. These hybrid hydrogel scaffolds were prepared in the form of continuous gradients such that a single scaffold contains spatially varied chemical and physical properties. Thus, cell-material interaction studies may be conducted more rapidly at different “zones” defined along the gradient. These gradients are also expected to benefit the regeneration of the osteochondral interface, an interfacial tissue that gradually transitions in tissue type. The final aspect of this work was focused on enhancing the osteogenic potential of PDMS via functionalization with amine and phosphonate. Both amine and phosphonate moieties have demonstrated bioactivity. Thus, it was expected that these properties will be enhanced for amine and phosphonate functionalized PDMS. The subsequent incorporation of these PDMS-based macromers into the previously described PEG-DA scaffold system is expected to be valuable for osteochondral tissue regeneration.

Current synthetic vascular grafts have high failure rates in small-diameter (<6 mm) applications due to inadequate cell-material interactions and poor matching of arterial biomechanical properties. To address this, we have developed a multilayer vascular graft design with a non-thrombogenic inner layer that promotes endothelial cell (EC) interactions and a reinforcing layer with tunable biomechanical properties. The blood-contacting layer of the graft is based on a Streptococcal collagen-like protein (Scl2-1). Scl2-1 has the triple helical structure of collagen, but it is a non-thrombogenic protein that can be modified to have selective cell adhesion. For this application, Scl2-2 has been modified from Scl2-1 to contain integrin binding sites that promote EC adhesion. We have developed the methodology to incorporate Scl2 proteins into a poly(ethylene glycol) (PEG) hydrogel matrix. PEG-Scl2 hydrogels facilitate optimization of both bioactivity and substrate modulus to offer unique control over graft endothelialization. However, scaffold properties that promote endothelialization may not be consistent with the mechanical properties necessary to withstand physiological loading. To address this issue, we have reinforced PEG-Scl2-2 hydrogels with an electrospun polyurethane mesh. This multilayer vascular graft design decouples requisite mechanical properties from endothelialization processes and permits optimization of both design goals. We have confirmed the thromboresistance of PEG-Scl2-2 hydrogels in a series of whole blood tests in vitro as well as in a porcine carotid artery model. Additionally, we have shown that the electrospun mesh biomechanical properties can be tuned over a wide range to achieve comparable properties to current autologous grafts. Traditional acrylate-derivatized PEG (PEGDA) hydrogels were replaced with PEG diacrylamide hydrogels with similar properties to increase biostability for long-term implantation. These findings indicate that this multilayer design shows promise for vascular graft applications. As vascular graft endothelialization can significantly improve success rates, the ability to alter cell-material interactions through manipulations in PEG-Scl2-2 hydrogel properties was studied extensively. By reducing Scl2-2 functionalization density and utilizing a biostable PEG functionalization linker, Acrylamide-PEG-I, significantly improved initial EC adhesion was achieved that was maintained over 6 weeks of swelling in vitro. Additionally, increases in Scl2-2 concentration and in hydrogel modulus provided increased EC interactions. It was found that PEG-Scl2-2 hydrogels promoted enhanced EC proliferation over 1 week compared to PEG-collagen gels. In summary, we have developed a vascular graft with a biostable, non-thrombogenic intimal layer that promotes EC adhesion and migration while providing biomechanical properties comparable to current autologous grafts. This design demonstrates great potential as an off-the-shelf graft for small diameter arterial prostheses that improves upon current clinically available options.

In vivo production of medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHA) containing a side chain carboxyl group from azelaic acid (AzA), a nine-carbon α,ω-dicarboxylic acid, was investigated using Pseudomonas citronellolis DSM 50332 in a phosphate (P)-limited chemostat. Co-feeding with nonanoic acid (NA) and inhibition of β-oxidation with acrylic acid (AA) were strategies that were used to stimulate the incorporation of carboxylated monomers, but both were unsuccessful. P. citronellolis DSM50332 was capable of growing on AzA as a sole source of carbon and energy, indicating that enzymes in β-oxidation utilized AzA and its derivatives. However, the MCL-PHA produced from AzA comprised 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) monomers, which was consistent with precursor supplied via the de novo fatty acid biosynthesis pathway. This evidence suggests that one or more of 3-ketoacyl-CoA reductase (FabG), enoyl-CoA hydratase (PhaJ) and PHA synthase (PhaC) of this organism do not have the low specificity required to utilize a carboxylated substrate. Future work involving mutations may broaden the substrate specificity of these key enzymes to overcome this obstacle. Two-stage high-cell density carbon (C)-limited chemostat cultivation of P. putida KT2440 was examined for MCL-PHA production from nonanoic acid (NA) at high intracellular polymer content and volumetric productivity. Growth conditions stimulating good PHA production were first established in single-stage chemostat, which yielded 63.1 wt% PHA containing 90 mol% C9 units and a productivity of 1.52 g L-1h-1 at a dilution rate of 0.30 h-1. This productivity was higher than any value reported in literature for continuous MCL-PHA production systems and comparable to the upper range of fed-batch results. Two-stage production yielded promising results, notably the increase in polymer content from the first to second stage. However, complications involving foaming and an unexplained decline in PHA content adversely affected system performance. The best PHA content and overall productivity were 58.5 wt% and 0.76 g L-1h-1, respectively. Nonetheless, the results demonstrate the potential to achieve high PHA content without the need for pure oxygen at high dilution rates, warranting further investigation focusing on the optimization of growth conditions. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-12-19 15:48:21.808

No description available.

Production of poly(3-hydroxybutyrate) (PHB) was done in a cyclone bioreactor using various culture methods; including batch (lab and pilot scale) fed-batch and self-cycling fermentation with and without starvation periods. Alcaligenes eutrophus DSM 545 was used to accumulate about 87% (wt/wt) PHB to a total of 6.2 g L$ sp{-1}$ PHB at the end of a 48 hour batch. Similar pilot scale experiments contained a maximum of 96% (wt/wt) PHB with 4.9 g L$ sp{-1}$ accumulated. Fed-batch culture of A. eutrophus produced 96% (wt/wt) PHB with a final PHB concentration of 22.2 g L$ sp{-1}$ after 54 h. Self-cycling fermentation (SCF) production of PHB resulted in an average of 35% (wt/wt) PHB without starvation periods with production rates reaching 0.24 g L$ rm sp{-1} hr sp{-1}.$ With starvation periods of 4, 6 and 8 h extended on the cycle times, production of PHB decreased except in the 8 hour starvation period which was 59% (wt/wt). However, the rates of production all decreased to below 0.13 g $ rm L sp{-1} h sp{-1}$ as the lengths of the starvation periods were increased.

Rapid creation of devices with microscale features is a vital step in the commercialization of a wide variety of technologies, such as microfluidics, fuel cells and self-healing materials. The current standard for creating many of these microstructured devices utilizes the inexpensive, flexible material poly-dimethylsiloxane (PDMS) to replicate microstructured molds. This process is inexpensive and fast for small batches of devices, but lacks scalability and the ability to produce large surface-area materials. The novel fabrication process presented in this paper uses a cylindrical mold with microscale surface patterns to cure liquid PDMS prepolymer into continuous microstructured films. Results show that this process can create continuous sheets of micropatterned devices at a rate of 1.9 in2/sec (~1200 mm2/sec), almost an order of magnitude faster than soft lithography, while still retaining submicron patterning accuracy.

Interleukin-10 (IL-10) is a pleiotropic cytokine which limits inflammatory responses by balancing the hosts immune response against infection. Mammalian Toll-like receptors (TLRs) are pattern recognition receptors that recognize specific molecular pattens on microbial pathogens and activate intracellular signaling via the transcription factors NF-B and IRF-3. In this study we evaluate the contribution of the TLR ligands Poly I:C, Pam3CSK4, LPS and LTA to IL-10 receptor dysfunction in murine peritoneal macrophages (PM). We examine how these ligands are able to alter IL-10 mediated STAT3 phosphorylation and CCR5 gene expression in PM. The ability of Poly I:C and Pam3CSK4 to alter the immunosuppressive activity of IL-10 in C2-ceramide stimulated PM is also examined. The results of our study indicate a delayed inhibition of IL-10 mediated activation of STAT3 by LPS, LTA, Poly I:C and Pam3CSK4. The CCR5 gene expression experiments demonstrate that LPS was able to down-regulate IL-10 induced CCR5 mRNA expression in PM.