New analytical and numerical approaches for calculating the gas holdup and drift velocity in bubble columns
SUPERVISOR: Dr. Siamak Elyasi (Chemical Engineering)
ABSTRACT: The two-phase flow phenomenon is of prime importance in the chemical, petroleum, nuclear and power industries. The two-phase flow encountered in nuclear and power industries is usually boiling and condensation two-phase flow while those practiced in the chemical and petroleum industries is essentially non-boiling in nature. The installation of two-phase flow lines has been proven economical in the oil and natural gas transportation system. The two-phase flow is used in chemical industry to enhance the mass transfer rate as in case of the bubble columns. Processes based on the contact between gas and liquid phases are commercially used for performing a variety of chemical reactions. Although different types of reactors are used for this purpose, bubble column reactors have received more attention during the past decade since they offer some unique advantages, such as ease of operation, high rates of heat and mass transfer, and lower maintenance costs due to the absence of moving parts. The design and scale-up of a bubble column reactor require a complete understanding of its complex hydrodynamics, which is influenced by the physical properties of the phases, the operating variables, and the design parameters. Current design procedures for bubble columns involve several steps of pilot-plant experimentation using equipment of different scales, which is expensive and time consuming. One of the critical unknown parameters involved in designing any gas–liquid system is the gas holdup which is the volume of space occupied by the gas. The gas holdup distribution varies dramatically throughout the flow regimes and determining its magnitude from input conditions in a given pipe is complicated as a result of the relative motion between the gas and the liquid phases. Owing to the complexity and lack of understanding of the basic underlying physics of the problem, the majority of the analyses were more inclined towards empirical correlations. The most of the empirical models have some form of restrictions attached to them. As a result, the design engineer is faced with the difficult task of choosing the right correlation. One of the most practical and accurate models for calculating void fraction is known as drift-flux theory. This model is of considerable importance because of its simplicity and easy applicability to a wide range of two-phase flow problems. Because of the practical significance, the focus of this study is on the drift-flux model as a simple and precise solution for determining the gas holdup.Furthermore, a numerical model to study the hydrodynamic behavior of a large-diameter bubble column, using the commercial CFD code ANSYS® FLUENT 16.2, was performed. The model was run for both semi-batch (stationary liquid) and continuous bubble columns. An Eulerian- Eulerian approach was used, and the effect of the superficial gas velocity on the flow regime, distribution parameter and drift velocity was examined. Based on the obtained results, a correlation was proposed to predict the gas holdup in semi-batch reactors. Verification of all simulation results and the suggested correlation against available experimental data showed a satisfactory agreement.A comprehensive literature study was carried out for the available drift-flux based gas holdup correlations and experimental data taken from researches conducted in different flow, operating and geometrical conditions. The performance of the existing correlations for the distribution parameter and drift velocity was evaluated against diverse data in small- and large-diameter bubble columns. Comparisons between the correlations showed that most of the developed correlations are very restricted in terms of handling a wide variety of data sets. A new approach to calculate the drift velocity that specifies the relative motion between phases was developed, and subsequently, a gas holdup correlation was proposed. A comparison of the models with various experimental data over various flow regimes and a wide range of flow parameters shows a satisfactory agreement.An analytical method was also developed for calculating the drift velocity. The model considers the axial flow through a fully developed bubble column. The reliability of this method was investigated and the evaluation of the results against the existing experimental data, considering different column diameters and flow conditions, indicated a reasonable prediction within ±20% error bands for large-diameter bubble columns while low accuracy for small ones.
Principles of superposition in viscoelastic finite-difference modelling
SUPERVISOR: Dr. Samuel Pichardo (formerly with Chemical Engineering)
ABSTRACT: The research presented in this thesis concerns the use of finite-difference time-domain (FDTD) numerical methods for use in modelling viscoelastic and acoustic wave propagation through complex media. FDTD models of viscoelasticity are widely used in the use of ultrasound for therapeutic applications, and this work includes two examples of the use of FDTD models for medical applications. The focus of the material presented is primarily concerned with a specific kind of numerical error - called the staircase error - which arises when boundaries between media do not align with the grid used in numerical simulations, and which require prohibitive amounts of time and computational resources to address. This thesis provides characterization of the numerical artifacts that arise due to the staircasing error, and presents an original numerical formulation to address these issues called the `Superposition Method'. The superposition method is characterized and shown to be an effective means of reducing these errors that does not become overly burdensome with computational time and resource requirements.
Pathogen-host omics analyses of human papillomavirus type 16 sub-lineages in a human epithelial organoid model
SUPERVISOR: Dr. Ingeborg Zehbe (Biology)
ABSTRACT: Pathogens such as human papillomaviruses (HPVs) have co-evolved with their hosts and form a molecular basis for common diseases. Persistent infection with the “high-risk” HPV type 16 (HPV16) is a potent cause of anogenital and oropharyngeal cancers. Taxonomic HPV16 sublineages, based on geographic origin of discovery, are noteworthy due to their variable tumourigenicity. In this dissertation, I present basic research and the resulting biotechnologies we developed, improved, and utilized to study their fascinating pathogen-host relationship with human stratified epithelia. A small number of variations in the E6 gene of HPV16, found in the D2 and D3 sub-lineages, lead to increased tumourigenic risk compared to the prototype A1 sub-lineage. Using an organotypic human epithelial model (or in vitro organoid) we recapitulated the viral life cycle and used “-omics” analyses to assess viral and host molecular differences due to sub-lineage variation. Sub-lineage variants of E6 were associated with host genome instability and viral integration into host DNA. Following these initial findings, I provide perspective on epithelial organoids, namely that the trade-off between model complexity and feasibility should be sensibly considered based on its utility for answering the biological research question at hand. Model applications and improvements are presented, including time-series epithelial stratification measurements, strategies for introducing full-length sub-lineage HPV16 genomes into host keratinocytes, and experiments to study innate immune evasion. These wet-lab works are accompanied by software to aid biologists in analyzing sequencing data. As well, we present current work using The Cancer Genome Atlas to test the association between HPV16 sub-lineage and integration. Overall, this interdisciplinary and interconnected collection has significance for basic researchers, providing insight on how a small number of natural viral variations can lead to increased tumourigenic risk, as well as for experimentalists to gain insight on organoid modelling and novel bioinformatics tools. More broadly, characterizing these molecular interactions between pathogen and host enables us to form a basis for diagnosis, treatment, and ultimately prevention of disease. Future research should aim to closely integrate biological and computational sciences for improving experimental approaches and our ability to make meaningful biological interpretations given the complexity and variability of biological systems.
Anaerobic membrane bioreactor for malting wastewater treatment and energy recovery under different temperature conditions
SUPERVISOR: Dr. Baoqiang Liao (Chemical Engineering)
ABSTRACT: In this dissertation, the novel application of anaerobic membrane bioreactor (AnMBR) technology for treating malting wastewater and resource recovery in the form of biogas under mesophilic (36±1 °C), room (23±1 °C), and psychrophilic (21-17 °C) temperature conditions is investigated. For each temperature condition, the effect of changing the organic loading rate (OLR) or the hydraulic retention time (HRT) on the biological treatment was evaluated by measuring the COD removal rate and efficiency, the effluent quality, as well as the biogas production yield and composition. During each study, the membrane performance was monitored by measuring the transmembrane pressure (TMP) and the permeate flux. Besides, the membrane fouling was characterized by comparing the permeability tests results, the scanning electron microscope (SEM) images, and the energy dispersive X-ray (EDX) spectra of the fouled membrane after cleaning to that of the virgin membrane. Additionally, the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) technique was employed to investigate the bacterial community shifts as a result of changing the OLR or the HRT for each temperature condition study. The OLR ranges, during the mesophilic, room, and psychrophilic temperature studies, were 1.3-3.2, 1.2-2.9, and 1.3-2.3 kg COD/m³/d, respectively. The results of this study demonstrated that treating malting wastewater and biogas production in an AnMBR under psychrophilic (21-17 °C) temperature conditions is feasible. The results of the psychrophilic study are comparable with the performance at mesophilic conditions. Also, the feasibility of treating malting wastewater and biogas production in an AnMBR under mesophilic and room temperature conditions was confirmed. For all studies, changing the OLR and fluctuations of the influent COD did not influence the COD removal efficiencies significantly. During all studies, the AnMBR adapted readily to the changes in OLR and HRT and the BOD₅ removal efficiency was above 99%. The COD removal efficiencies ranges of mesophilic and room temperature studies were 89-96% and 86-95%. Corresponding biogas yield for mesophilic and room temperature conditions were 0.31-0.34 and 0.14-0.29 L at STP/g CODremoved. Methane content of biogas varied between 68-73% for mesophilic and 54-73% for room temperature conditions. The AnMBR operation at psychrophilic conditions resulted in 86-94% COD removal efficiency, 0.22-0.30 L at STP/g CODremoved biogas yield, and 0.92-3.26 L/d biogas with 76-84% methane content. During room temperature and psychrophilic conditions the membrane permeability results, scanning electron microscope (SEM) images, and energy dispersive X-ray (EDX) spectra implied that cake layer was the predominant fouling mechanism. However, during room temperature study, when the membraned was reused and worked for an extended duration, irreversible fouling had happened. The results of the membrane permeability recovery for all studies suggested that physical and chemical cleanings were able to recover the permeability of the fouled membrane to a large extent.Furthermore, the PCR–DGGE results of all studies showed that the microbial community actively changed due to changes in OLR, HRT, as well as the progress of the process. The literature review of the kinetic studies of conventional anaerobic process and anaerobic membrane bioreactors (from 1992 to 2017) showed that the first-order kinetic model had been used for both substrate removal and biogas production in batch bioreactors. The Michaelis-Menten model, the Monod equation, the Stover-Kincannon model, and the Grau’s second-order model were used to describe the kinetics of continuous bioreactors. The Monod equation is the most popular model used for AnMBR systems and Contois model as well as Chen and Hashimoto model were occasionally used.
Development of nanostructured material-based electrochemical sensors for food safety and quality control
SUPERVISOR: Dr. Aicheng Chen (formerly with Chemistry)
ABSTRACT: The issue of foodborne related illnesses due to additives and contaminants poses a significant challenge to food processing industries. Electrochemical-based strategies offer simple and robust analytical tools, which are ideal for food safety and the quality assessment process, in contrast to conventional instrumentation methods. The development of nanomaterials based electrochemical sensors has garnered significant attention due to their capacity for accurate analytical quantification, which has strong potential toward the replacement of conventional techniques by offering advantages such as high sensitivity and selectivity, real-time monitoring, and ease of use. During my Ph.D. study, four distinct types of nanostructured materials were used to develop electrochemical sensors for the detection of food preservatives in food and beverage products. The consumption of excessive amounts of nitrite (NO2-) can be detrimental to the human body. In light of this, we developed an electrochemical sensor based on cobalt oxide nanosheets and gold nanoparticles (Co3O4/Au) for NO2- sensing. The nanomaterial was synthesized through the electrodeposition of gold (Au) on Co3O4 nanosheets. The Co3O4/Au/GCE was capable of electrooxidizing nitrite with a higher anodic peak current, and the sensor exhibited excellent linearity with a limit of detection (LOD) value of 0.11 µM. A nanoporous gold microelectrode was synthesized for the determination of contaminants (hydrazine, N2H4) and preservatives (sulfite (SO32-), nitrite (NO2-)). The fabricated microelectrode was characterized via scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDX). The nanoporous gold microelectrode exhibited excellent electrochemical performance for the simultaneous electrochemical oxidation of N2H4, SO32-, and NO2-. In addition, the nanoporous gold microelectrode possessed high selectivity and stability. The performance of the electrochemical sensor was further validated using actual samples such as water, wine, apple cider beer, and beef with good recovery rates, thereby confirming its potential for food safety and quality control applications. A novel electrochemical sensor was developed using fluorine-doped graphene oxide (F-GO) for the detection of caffeic acid (CA). The fabricated nanomaterial was systematically characterized using SEM and X-ray photoelectron spectroscopy (XPS). The electrochemical investigation of F-GO/GCE for CA oxidation revealed that it demonstrated high electrocatalytic activity compared with other electrodes (e.g., bare GCE and GO/GCE). The analytical quantitation of CA recorded with the F-GO/GCE produced a stable oxidation signal over the selected CA concentration range (0.5 μM to 100.0 μM, R2 = 0.9960) with a LOD value of 0.018 μM. The fabricated sensor successfully exhibited the capacity to directly detect CA in assorted wine samples without pretreatment. To further explore the applications of the F-GO, a nanocomposite material synthesized with Au and F-GO was employed for the development of an Au/F-rGO/GCE sensor for the detection of vanillin. The electrochemical performance and the analytical capabilities of this novel electrochemical sensor were investigated using electrochemical techniques such as CV and DPV. The excellent sensitivity, selectivity, augmented electrocatalytic activity, and reproducibility of these developed electrochemical sensors can be attributed to the high conductivity of the nanostructured materials. The dimensions and morphologies of the developed nanomaterials played a critical role in enhancing the electrochemical performance of these sensors.
A novel metadata analysis approach for analyzing and understanding wood-decaying mechanisms exhibited by fungi
SUPERVISOR: Dr. Wensheng Qin (Biology)
ABSTRACT: Fuel has become an essential commodity in our day to day life. Increase in global population and decreasing fuel reserves have forced mankind to look for other fuel alternatives. Forest biomass serves as a potential renewable resource for substituting the conventional fossil-based fuels. In the last few decades, several chemical, physical and microbial based methods were being developed for the breakdown and conversion of lignocellulosic components to commercially valuable products including bioethanol and other platform chemicals. The separation of lignocellulosic biomass plays a significant role in conversion of lignocellulosic biomass to ethanol and other valuable products respectively. Naturally, lignocellulosic components are arranged in intricate networks leading to its high recalcitrance nature. Over years research groups around the world have isolated and characterized several lignocellulose degrading microorganims. Naturally, fungi play a crucial role in maintaining the geo-carbon cycle by decaying all the dead organic matter on the earth’s surface. Majority of the wood-decaying fungi are grouped under Basidiomycota division. Based on their decay patterns the basidiomycetous fungi were classified into white-rot, brown-rot and soft-rot fungi. Understanding these natural fungal decaying mechanisms will benefit the growing biofuel, biorefining and bioremediation industries. Next generation sequencing techniques have significantly enhanced our present day’s knowledge about various biological mechanisms. Phanerochaete chrysosporium was the first basidiomycetous fungi with complete annotated genome sequence, which has inspired the whole-genome sequencing studies of different wood-decaying fungi. Increasing whole genome sequencing studies have led to the enrichment of public repositories especially JGIMycoCosm, 1000 fungal genome project, Hungate 1000 projects have played a significant role in supporting these sequencing projects. As of today, there are 443 published and completely annotated fungal genome sequences in the JGI-MycoCosm repository. Thus, the availability of whole annotated genome sequences has significantly helped in designing genome-wide transcriptomic studies for understanding the molecular mechanisms underlying the process wood-decay. A total of 11 genome-wide transcriptomic studies of P. chrysosporium were reported and are publicly available under the NCBI-GEO repository. However, transcriptomic studies give a snapshot of gene expression at a given growth conditions and time period. Thus, we have developed a robust and efficient metadata analysis approach for re-analyzing gene expression datasets of P. chrysosporium and Postia placenta for understanding the common significant gene expression patterns of this model white and brown rot fungi cultured on different growth substrates (simple customized synthetic media and natural plant biomass media). We have reported a significant list of genes encoding for various lignocellulolytic enzymes significantly expressed among all the gene expression datasets. Based on the common gene expression patterns obtained from our analysis we have tentatively derived the molecular network of genes and enzymes employed during the breakdown and conversion of lignin, cellulose, hemicellulose components of plant biomass. For the first time, we have reported, and classified lignin degrading genes expressed during the ligninolytic conditions of P. chrysosporium. It is well-known that fungi experience a significant amount of stress during the process wood-decay. Plant secondary metabolites such as quinones, tannins, stilbenes, flavonoids and other phenolic compounds exhibits a fungicidal activity. Thus, wood-decaying fungi wood decaying fungi would have developed an efficient detoxification and stress responsive mechanisms for sustaining this effect. Using the results obtained in our study we have reported and classified as phase-I and phase-II metabolic genes involved in detoxification and stress responsive mechanisms respectively. Compared to P. chrysosporium, P. placenta lacks several copies of genes encoding for cellulolytic, hemicellulolytic, ligninolytic and pectinolytic enzymes. However, our gene expression metadata analysis has reported that P. placenta is strongly dependent on Fenton’s reaction for the degradation of lignocellulosic components. We have also observed that genes encoding for several hemicellulolytic enzymes were differentially expressed even during cellulolytic conditions. Based on the present metadata analysis we have also tentatively developed cellulose and hemicellulose metabolic mechanism. In the last few decades, genome sequencing studies of wood-decaying fungi have been extensively reported. Presently, the JGI-MycoCosm database resides 1165 whole genome sequences of fungi, out of which about 443 fungal genome sequences are published. It is hard to choose a particular fungus specifically for the degradation of plant biomass. Thus, we have developed an efficient metadata analysis pipeline for comparing and understanding the genome-wide annotations of fungi. The metadata analysis pipeline reported in our study can be used for selecting a wood-decaying fungus for the Invitro degradation of studies. We have compared the genome-wide annotations of about 42 wood-decaying basidiomycetous fungi (white-rot, brown-rot and soft-rot fungi) and reported a tentative comparison method explaining the total cellulolytic, hemicellulolytic, ligninolytic and pectinolytic abilities. Similarly, we have also specifically compared and analyzed the genome wide annotations of anaerobic fungi belonging to Neocallimastigomycota division fungi. This study has reported the complete genetic makeup of these peculiar fungi and their carbohydrate degrading abilities (plant cell wall carbohydrates), as they completely lack lignin degrading enzymes. We have also delineated and compared the genes coding for structural and functional components of cellulosomes and hydrogenosomes. We have also performed an extensive homology modeling and protein docking study of white-rot, brown-rot and soft-rot fungal laccases protein sequences using 6 different types of lignin model compounds. This study has revealed the structural and functional variations of white, brown and soft rot fungal laccases. Results obtained in this study reported that white and brown rot fungal laccases reported higher catalytic efficiencies compared to that of soft rot fungal laccases. This study also reported that soft rot fungal laccases exhibited small but significant variations in its structural and physicochemical properties. However, further molecular dynamic simulation and high throughput proteomic studies must be performed to understand the structural and functional properties of these laccases. The metadata analysis work frame reported in this thesis can be extended to understand the natural wood-degrading mechanisms of various microorganisms (e.g. fungi, bacteria). The highly reactive significant list of proteins obtained in this study can be used in vitro for developing highly resistant enzyme mixes used for the breakdown and conversion of various organic compounds including plant biomass. The above reported strategies also can be used as a preliminary analysis for designing large scale experiments.
The application of personalized medicine approaches to human papillomavirus 16-related cancers
SUPERVISOR: Dr. Ingeborg Zehbe (Biology)
ABSTRACT: High-risk types of human papillomavirus (HPV), including HPV16, are perhaps some of the most ubiquitous of the infectious agents now recognized to cause cancer. Through persistent infection, they initiate malignant changes in epithelial cells of the genital and oral mucosa. Epidemiological evidence has demonstrated that they are responsible for nearly every reported case of cervical cancer, cancers of other ano-genital regions such as the vagina, anus, and penis, as well as a rapidly increasing number of male oropharyngeal cancers. Accordingly, the main body of this thesis is comprised of a collection of publications which each apply personalized medicine approaches that aim to improve: 1) our understanding of why only a small proportion of HPV16-infected individuals develop cancer, and 2) the development of therapeutic molecules that specifically target HPV16-infected cells, reducing the general morbidity associated with the current standards-of-care. These include characterization of the functional properties of the commonly identified E-T350G E6 protein variant, investigation into the use of small interfering RNA (siRNA) to silence E6 expression, and isolation of the first potential camelid single-domain antibody candidates against the HPV16 E6 protein. Along with discussion of future research directions, this thesis concludes by anticipating how the outcomes of such studies may one day allow healthcare providers to predict which HPV16- infected individuals require prompt treatment of early precursor lesions and administer that treatment with minimal damage to surrounding tissues. The added benefit which can be derived from interdisciplinary collaboration is also demonstrated through the presentation of complementary side-projects which explore the use of sonoporation to localize the intracellular delivery of anti-E6 therapeutic molecules to specific regions of HPV-infected tissue.
Magnetic resonance imaging guidance of high intensity focused ultrasound: Optimizing guidance on pre-clinical models
SUPERVISOR: Dr. Laura Curiel (formerly with Chemical Engineering)
ABSTRACT: Magnetic resonance guided focused ultrasound (MRgFUS) is a treatment modality capable of administering mild or ablative hyperthermia. This technique has been used for many years improving patient outcomes for cancer and other ailments. Focused ultrasound (FUS) has the advantages of being non-invasive, highly precise, and inexpensive compared to other cancer treatments. Magnetic resonance imaging (MRI) is uniquely suited to guide FUS due to its high spatial resolution that matches the pinpoint accuracy of FUS. In addition, MRI is capable of monitoring temperature increases made by FUS leading to an accurate thermal dose. MRgFUS has been extensively studied and been used to treat various cancers. The work presented in this thesis aims to increase the efficacy and adoption of MRgFUS as a primary treatment modality by providing pre-clinical tools that facilitate its use and increase its specificity in targeting. The first part of the work, custom RF coils were developed to be used within a MRgFUS platform that allowed for accurate targeting and careful control of the thermal energy deposited into tissues. This work also explored the use of functional imaging biomarkers to aid in the targeting of MRgFUS. A biologically specific MRI contrast agent was developed and characterized in a mouse model of prostate cancer. The use of biomarkers may reduce patient side effects following prostate cancer treatments by increasing the differentiability of healthy tissue from malignant tissue. In addition to the development of contrast agents, the visibility of FUS targets using optimal pulse sequence parameters was demonstrated. Overall, interest in FUS to treat various ailments has sparked research groups around the world to build FUS systems. The work presented in this thesis developed tools to facilitate the use of MRgFUS as a tool for investigating new therapeutic applications in preclinical models. Using these tools, future applications of FUS may be explored.
Exploration of inflammasomes as targets for therapy of Pseudomonas aeruginosa infection
SUPERVISOR: Dr. Marina Ulanova (NOSM, Biology adjunct)
ABSTRACT: Pseudomonas aeruginosa is a common Gram-negative opportunistic bacterial pathogen capable of infecting humans with compromised natural defenses and causing severe pulmonary disease. It is the major cause of severe chronic pulmonary disease in cystic fibrosis (CF) patients subsequently resulting in progressive deterioration of lung function. Interaction between P. aeruginosa and host induces a number of marked inflammatory responses and is associated with complex therapeutic problems. NOD-like receptors (NLRs) can recognize a variety of endogenous and exogenous ligands and its activation initiate inflammasome formation that induces maturation of the proinflammatory cytokine interleukin (IL)-1β through activation of caspase-1. Through a literature search, no prior research on mutant strains as well as clinical isolates of P. aeruginosa from CF patients at different stages of infection has been conducted to explore NLR-mediated innate immune responses to this bacterial infection. All the work presented in this thesis focuses on the exploration of inflammasomes as targets for therapy of P. aeruginosa infection. We hypothesized that genetic alterations of P. aeruginosa affect the innate immune response of human monocytes. THP-1 human monocytic cells were infected with clinical P. aeruginosa isolates from CF patients, or with P. aeruginosa mutant strains lacking flagella, pili, lipopolysaccharide, or pyocyanin. The overall involvement of NLRs in innate immune recognition of P. aeruginosa was addressed through demonstrating of NLR-mediated caspase-1 activation or P. aeruginosa-induced IL-1β secretion. Our findings suggest that P. aeruginosa, which lost certain virulence factors during pulmonary infection, may fail to induce caspase-1 III activation and secretion of IL-1β in the process of host-pathogen interactions. This may reveal novel mechanism of the pathogen adaptation to avoid detection by NLR(s). As P. aeruginosa infections are characterized by strong inflammation of infected tissues anti-inflammatory therapies in combination with antibiotics have been considered for the treatment of associated diseases. Spleen tyrosine kinase (SYK), a non-receptor tyrosine kinase, is an important regulator of inflammatory responses. Several studies have highlighted SYK as a key player in the pathogenesis of a multitude of diseases. Inhibition of SYK activity was explored as a therapeutic option in several inflammatory conditions; however, this has not been studied in bacterial infections. We used a model of an in vitro infection of human monocytic cell line THP-1 and lung epithelial cell line H292 with both wild type and flagella-deficient mutant of P. aeruginosa strain K, as well as with clinical isolates from CF patients, to study the effect of a small molecule SYK inhibitor R406 on inflammatory responses induced by this pathogen. The role of SYK in regulation of inflammasome activation was also determined by evaluating the effect of SYK inhibitor on innate immune responses in P. aeruginosa infected cells. The results suggest that SYK is involved in the regulation of inflammatory responses to P. aeruginosa, and R406 may potentially be useful in dampening the damage caused by severe inflammation associated with this infection.
Advanced lignin-based flocculent or dispersant for wastewater treatment
SUPERVISOR: Dr. Pedram Fatehi (Chemical Engineering, Biorefining Research Institute)
ABSTRACT: The wastewaters produced from different industries contain fine and charged suspended particles and other impurities. Today, the removal of these colloidal particles from the wastewater becomes a serious challenge for industry. Flocculation of the fine particles using polymers followed by settling is one popular technique in industry. Synthetic flocculants have been used in wastewater treatment systems, which are not biodegradable and eco-friendly. Therefore, natural based flocculants have been attracting wide interest of researchers because they have the advantages of biodegradability and environmentally friendly. In this study, kraft lignin derived from black liquor of kraft pulping process, was copolymerized with acrylamide (AM) and (2-methacryloyloxyethyl) trimethyl ammonium chloride (DMC) in an aqueous solution in the presence of K2S2O8 as an initiator to produce a water-soluble lignin-based copolymer. The influence of the reaction conditions on the charge density and solubility of resultant lignin copolymers were investigated. The resultant lignin copolymer was characterized by Fourier transform infrared (FTIR) spectrophotometry, nuclear magnetic resonance (HNMR), thermogravimetric analyzer (TGA), molecular weight and elemental analyses. The applications of the resultant copolymer as a flocculant in kaolin and bentonite suspensions were systematically assessed. The flocculation studies allowed for correlating the polymer characteristics, namely the charge density and molecular weight, with its adsorption affinity as well as the zeta potential and relative turbidity of kaolin and bentonite suspensions. This study showed that a highly charged cationic lignin adsorbed more than low charged ones, and an increase in the molecular weight of cationic lignin enhanced its adsorption. Thus, cationic flocculants with higher molecular weights and charge densities were more effective in reducing the turbidity of clay suspensions. One of the important findings of this work was that both polymer bridging and charge neutralization mechanisms facilitated the destabilizing of the colloidal particles to form flocs. An improved reflocculation ability of cationic polymers was observed as the molecular weight and charge density of the polymers increased. The flocculation studies also confirmed that the flocculation efficiency of these cationic lignin polymers depended on the adsorbed amount of polymer on kaolin and bentonite particles, but not on the unadsorbed amount present in the suspensions. In this study, the flocs size and structure of cationic lignin in kaolin suspension was determined by a focused beam reflectance measurement (FBRM) and the results were correlated with flocs properties obtained by small-angle laser light scattering technology (SALLS). The results showed that the flocs produced were larger and more porous as the polymer's charge density and molecular weight increased. Also, the flocs strength decreased as the flocs size increased. A strong correlation between the size of flocs and sedimentation behavior of kaolin suspension was established by a vertical scan analyzer. The results demonstrated that the maximum rate of settling increased with the increase in floc size. The effect of solution pH and salt concentration on the dispersant performance of anionic kraft lignin in kaolin suspensions was also studied. The adsorbed anionic kraft lignin on kaolin particles induced electrostatic repulsion between the particles at a more basic pH and thereby improved the dispersibility of suspensions. The results showed that the adsorption of lignin polymers decreased with pH increase, but increased with ionic strength increase. In this study, the mechanism of self-assembly of kraft lignin-based polymers in aqueous solutions was investigated using dynamic light scattering (DLS) and the results were correlated with conformation and viscoelastic properties of the adsorbed polymer layers on particles via Quartz crystal microbalance with dissipation (QCM-D) analysis. The results showed that a higher molecular weight lignin polymer was adsorbed in a greater quantity, and that more mass interacted as the molecular weight increased. The results in this work provided insights into the fundamental understanding of the flocculation, dispersion and self-assembly behavior of kraft lignin-based polymers in various systems. These results can help establish the criteria for selecting and developing kraft lignin based flocculants or dispersants for altered applications. The results of this thesis contributed to knowledge on the chemical modification and characterization of lignin products and to the fundamentals associated with the performance analysis of flocculation and dispersion systems.
Bioconversion of crude glycerols to biofuels and value-added bioproducts
SUPERVISOR: Dr. Wensheng Qin (Biology)
ABSTRACT: Biodiesel-derived crude glycerol, a core by-product of biodiesel production process is becoming of great environmental and economical concern for growth of biodiesel industries due to its ever-growing over supply problem. Therefore, biotransformation of crude glycerol to renewable energy would lead to both environmental and economic dividends of biodiesel plant. In this study, numerous bacterial strains isolated from environmental samples were screened for their capability of converting glycerol to 2,3-BD, a high worth green product uses as a liquid fuel or fuel additive. Moreover, high productions of 2,3-BD were reported using two mutant strains of newly isolated Klebsiella variicola SRP3 and K. pneumoniae SRP2 respectively. However, Real-time qPCR and glycerol dehydrogenase (GDH) enzyme activity assay revealed that the overexpression of GDH gene resulted in an increased GDH enzyme activity, led to a markedly boosted 2,3-butanediol (2,3-BD) production.
Kinetic and thermodynamic studies on pyrolysis of waste HDPE polymers
SUPERVISOR: Dr. Siamak Elyasi (Chemical Engineering)
ABSTRACT: Pyrolysis is a promising technology for converting waste plastic into high-value hydrocarbons, which can help to protect the environment and improve the waste management industry. Available methods for finding kinetic parameters and heat of solid state reactions are not compatible with the complexity of pyrolysis reactions, and do not give reliable parameters to design an industrial pyrolysis reactor. This work developed two new techniques to determine kinetic parameters and heat of solid state reactions with high-certainty. The proposed kinetic study method is a differential isoconversional technique that finds activation energy and pre-exponential factor at different extents of reaction using isothermal Thermogravimetric Analysis (TGA) datasets. Employing this method, kinetic parameters of pyrolysis of high-density polyethylene (HDPE) were determined at different reaction conversions. The obtained apparent activation energy values were obtained in the range of 270 to 290 kJ/mol. The developed method for finding heat of reaction employs Differential Scanning Calorimetry (DSC) technique at constant temperatures. This method involves a new procedure to find heat loss from the DSC instrument as function of temperature and sample weight. The method was employed to find heat of cracking of HDPE at constant temperature of 400, 410, 420 and 430 °C, and the average heat of reaction was determined to be 1375±233 kJ/kg. Based on available recommendations in the literature for using ZSM-5 catalysts in polymer pyrolysis, catalytic cracking of high-density polyethylene was studied using three ZSM-5 catalysts with different Si/Al ratios of 25, 38 and 80. Using a TGA instrument and altering the variables such as temperature and the catalyst to HDPE ratio, catalytic activity of the catalysts was investigated, and proper operating conditions were estimated. ZSM-5 catalysts with Si/Al ratios of 25 and 38 at constant temperatures of 330, 340, 350, 360 and 370 °C, and cat/HDPE ratio of 15 % III showed considerably high catalytic activity in cracking of HDPE (especially the ZSM-5 with Si/Al ratio of 25). Using the developed kinetic study method, kinetic parameters of catalytic cracking of HDPE were determined at the aforementioned conditions, and apparent activation energy values were dropped dramatically to the range of 20 to 90 kJ/mol. In addition, catalytic activity, deactivation behavior, regenerability and reuse of the ZSM-5 catalyst with Si/Al ratio of 25 in consecutive cracking tests were also investigated. When activity of the used catalyst dropped to 20% of its initial value, a catalyst regeneration at 480 °C for 5 h was conducted; however, due to dealumination reactions occurred in the regeneration step, the initial catalytic activity could never be recovered. After catalyst regeneration, the regenerated catalyst was used in the same cracking tests. With 10 regeneration cycles, the ZSM-5 was used in 54 cracking tests. The effect of calcination temperature on the activity of ZSM-5 in cracking of high-density polyethylene was then explored. Calcination at 600 and 700 °C reduced acidity and activity of the ZSM-5 mainly due to catalyst dealumination. On the contrary, no drop in activity of the 500 °C-calcined catalyst was detected. Overall, the findings of this study can be employed to design an industrial reactor for pyrolysis of waste polymers. Additionally, the methods developed in this study for obtaining kinetic parameters and heat of pyrolysis can be used in any other solid state reactions.
Conversion of Crude Glycerol from the Biodiesel Industry to Value-added Products
SUPERVISOR: Dr. Sudip Rakshit (Chemical Engineering)
ABSTRACT: Crude glycerol is a major by-product of the biodiesel industries. For every 100 kg of biodiesel produced, approximately 10 kg of the byproduct glycerol is generated. With large increase in biodiesel production, there is a glut in the glycerol produced. Presently crude glycerol is purified to its purer marketable form, burnt as a fuel or mixed with animal feed. However, none of these options contribute considerable revenues to the concerned biodiesel industry. Additionally, some of these routes are not environmentally friendly. It has thus become imperative to find ways to convert crude glycerol to some value-added products. Bioconversion of crude glycerol to microbial lipids is one possible way to valorize it. However, impurities like methanol, salts and soap present in crude glycerol inhibit the growth of microbes used for such conversions. The research work carried out in this thesis addressed these issues and developed tangible alternatives to overcome these problems. Initially the possible use of a heterogeneous catalyst Calcium oxide (CaO) attached to support alumina (Al2O3) for the production of biodiesel was studied. We found that the use of such a catalyst improves the purity of biodiesel and the glycerol produced. Crude glycerol obtained using such insoluble catalysts contained lower levels of impurities and can be converted relatively easily to other useful products. With CaO anchored on Al2O3 as catalyst, the purity of biodiesel and glycerol were found to be 97.66% and 96.36% respectively. The unanchored heterogeneous catalyst CaO resulted in purities of 96.75% and 92.73% respectively. As the byproduct glycerol containing smaller amount of impurities, the use of anchored heterogeneous catalyst is recommended. The potential use of ash from various sources as a cheap alternative heterogeneous catalyst was also studied. With the use of ash from birch bark and fly ash from wood pellets as catalysts, biodiesel and glycerol with purity in the ranges of 88.06%-99.92% and 78.18%-88.23% respectively were obtained. Since such catalysts are cheap and reusable, their application can reduce expenses and the use of environmentally unsafe compounds. The crude glycerol used in all experiments was obtained from a biodiesel producer in Ontario (Canada). It was found to contain 44.56 wt.% glycerol and many impurities including 13.86 wt.% methanol, 32.97 wt.% soap and 4.38 wt.%. After the characterization of the samples it II was converted to microbial lipids using an oleaginous yeast Rhodosporidium toruloides ATCC 10788. When this strain was grown on crude glycerol, double the biomass (21.16 g/L) and triple the lipid concentration (11.27 g/L) was obtained compared to growth on pure glycerol media. The capacity of this strain to grow on crude glycerol with high levels of impurities and produce large amounts of lipids proves its robustness. Investigation of the effect of individual components on the lipid production ability of this strain showed it to be capable of using soap as a sole carbon source. This was also the reason for enhanced lipid production even in the presence of other impurities present in crude glycerol. The lipids obtained were rich in oleic acid (47.16%), a mono-unsaturated fatty acid (MUFA). Feedstock rich in MUFA are considered suitable for biodiesel production. Thus, the process of conversion of crude glycerol to microbial lipids can be integrated to existing biodiesel plants. This will help in the management of crude glycerol produced during biodiesel production, save transportation and disposal costs and contribute to the revenues of such industries. The possible applications of microbial lipids obtained from oleaginous (oil producing) microbes depends on its fatty acid composition. Most reports suggest the potential use of microbial lipids for biodiesel production. However, tailoring lipids obtained from these microbes, by changing the fatty acid composition, can make it suitable for use in various biotechnology, pharmaceutical and food industries. The possible use of essential oils from plant sources to change the fatty acid profile of the lipids obtained from R. toruloides ATCC 10788 was studied. The addition of seven types of essential oils into the growth medium resulted in lipids with seven different fatty acid profiles. Most of the essential oils tested enhanced the stearic acid content of the lipids. The latter has numerous applications in food, pharmaceuticals and cosmetic industries. Additionally, specific levels of these essential oils in the media changed the metabolic pathways of lipids production resulting in unique fatty acids compositions. For instance, use of 2 g/L of orange essential oil in the growth medium produced lipids with fatty acid composition similar to mahua butter which has a number of food, cosmetic and medical applications. Addition of origanum (0.3 g/L) or pine essential oil (3 g/L) into the media improved the oleic acid content of the lipid by 17.90% and 14.35% respectively. This study also provides an insight into the ability of these essential oils to affect the activity of enzymes involved in fatty acid biosynthesis metabolism of R. toruloides ATCC 10788. Most of the essential oils used were found to inhibit Δ9 and Δ12 desaturase enzymes present in the fatty acids biosynthesis metabolic pathways of this strain. This in turn increased the amounts of stearic acids in most of the lipids obtained. Further investigations showed that mono-terpenoids in the essential oils played a major role in bringing about such changes in the fatty acids composition and that the effect of essential oils are specific to the microbial species under study. Subsequently, the possible use of microbial lipids obtained from crude glycerol for polyol production was studied. Polyol is a precursor to the production of polyurethane foams. In order to produce large quantities of lipids, fermentation of crude glycerol was carried out in a 1L bioreactor. This resulted in 27.48 g/L of biomass and 18.69 g/L of lipids at the end of 168 h. The lipids obtained were then converted to polyol using epoxidation and oxirane ring opening reactions. For comparison, polyols from commonly used vegetable oils (i.e. canola and palm oil) were also produced under similar reaction conditions. The hydroxyl numbers of polyols from canola, palm and microbial oil were found to be 266.86, 222.32 and 230.30 (mg KOH/g of sample) respectively. These values are within the range required for polyurethane production. We subsequently converted the polyols obtained from these oils into rigid and semi-rigid types of polyurethanes. Polyurethanes produced can potentially be used for various commercial applications. In this way, we have been successful in demonstrating the possible use of microbial lipids in biopolymer industries. The use of a robust microbial strain capable of growing on crude glycerol and the conversion of the lipids obtained to bioplastics can not only help fix the carbon produced (leading to reduction in GHG emission obtained by its combustion), but also potentially bring revenues to industries that integrate such processes into existing facilities.
Nanomaterials-based electrochemical approaches for biosensing and bacterial disinfection
SUPERVISOR: Dr. Aicheng Chen (formerly with Chemistry)
ABSTRACT: Electrochemical approaches to myriad medical and environmental challenges are highly attractive due to their strong potential for extensive and green applications. Point of care diagnostics through the electrochemical monitoring of clinically and environmentally relevant molecules are gaining attraction due to their low cost and simple fabrication procedures. The development of highly stable and sensitive electrochemical sensors/biosensors, for a wide variety of biomolecules in actual samples, makes these methods alternative analytical tools in different pharmaceutical and hospital laboratories. Electrochemical biocatalysis is an additional promising area to address the removal of bacteria for the generation of safe potable water. As the world’s population is dealing with lack of access to safe drinking water, photoelectrocatalysis has been investigated as a very efficient technique for the destruction of pathogenic bacteria in water. Nanomaterials with dimensions of less than 100 nm have great potential to enhance the performance of electrochemical methods, due to their excellent electronic, mechanical, and thermal properties. These materials have the capacity to greatly enhance biocatalytic activity, and thus greatly improve the performance of electrochemical sensors/biosensors. This remarkable improvement in bacterial catalysis has been studied using a novel synergistic approach, which incorporates both photocatalysts and electrocatalysts. For my PhD thesis, we designed a high-performance electrochemical sensor based on graphene for the sensitive detection of acetaminophen, valacyclovir, and mixtures thereof. This sensor was fabricated through the concurrent electrochemical reduction and deposition of graphene oxide (GO) onto a glassy carbon electrode (GCE) using cyclic voltammetry (CV). The electrocatalytic properties of the electrochemically reduced graphene (ERG) for the oxidation of acetaminophen were analyzed via cyclic voltammetry (CV), differential pulse voltammetry, (DPV) and chronoamperometry. For comparison, various ERG/GCEs were prepared under different electrodeposition cycles to optimize the required quantity of ERG. Our experimental results indicated that the optimized ERG/GCE possessed robust activity toward the electrochemical oxidation of acetaminophen, valacyclovir, and their mixture, leading to the development of a highly sensitive electrochemical sensor for its detection. An extremely low detection limit of 2.13 nM for acetaminophen, and 1.34 nM for the exclusive detection of valacyclovir was achieved. A wide linear detection range of from 5.0 nM to 800 μM was II achieved via the combination of an amperometric technique and DPV. The developed electrochemical sensor was further employed for the determination of acetaminophen, valacyclovir, and their mixture in human serum, with excellent recovery, ranging from 96.08% to 103.2%. The fabricated electrochemical sensor also demonstrated high selectivity, stability and reproducibility. We investigated a novel enzyme entrapment approach utilizing a cationic polymer for the detection of ethanol. Entrapment is one of the primary approaches for enzyme immobilization; however, it suffers from a few critical drawbacks, including leakage and high mass transfer resistance to substrates. To address these challenges, herein we report on a new facile and effective enzyme entrapment platform utilizing a special cationic polymer, poly(2-(dimethylamino)ethyl methacrylate) (MADQUAT) on a nanohybrid single-wall carbon nanotube/reduced graphene oxide (SWCNT–rGO) thin film. The ethanol biosensor developed in this study exhibited rapid response, wide linearity range, high sensitivity (26.27 μA mM−1 cm−2), a remarkably low limit of detection (0.16 μM), high selectivity, and high stability. The optimized biosensor was further tested with actual samples including wine, beer, and blood alcohol, and demonstrated promising analytical and biomedical applications. The increasingly serious issue of lack of access to clean potable water globally is of great concern. The high costs associated with the purchase and operation of water treatment facilities hinders access to clean water for entire populations. Herein, we report on an efficient and cost-effective approach for the remediation of E. coli (as model organism), through a combination of photochemistry and electrochemistry enabled by a bifunctional electrode. The bifunctional electrode utilizes titanium (Ti) as the substrate, nanoporous TiO2 as a photocatalyst, and RuO2 nanoparticles as an electrocatalyst. A high disinfection rate at 0.62 min-1, with >99.999% of bacterial removal within 20 min has been achieved using this TiO2/Ti/RuO2 bifunctional electrode. Complete bacterial disinfection was attained within a 30 min electrochemical treatment, as assessed by a spread plate method. Further, the bifunctional disinfection mechanism was investigated through protein concentration leakage, total organic carbon mineralization, and metabolomics analysis. A major loss of vital metabolites occurred over a period of 20 to 30 min, which suggested the mass inactivation of E. coli within this treatment duration. The novel strategy for the integration of advanced electrochemical oxidation and photochemical degradation via the bifunctional electrode system developed in this study has III strong potential to be utilized as an environmentally compatible technology for water purification and wastewater treatment. Simple and robust electrochemical approaches utilizing novel nanomaterials have been developed for the sensing of biomolecules and water disinfection. Carbon based nanomaterials and nanocomposites were employed to develop sensitive electrochemical sensor/biosensors, and nanoporous TiO2/RuO2 bifunctional materials were utilized for the design of a high-performance bacterial disinfection technology.
New generation of dispersants by grafting lignin or xylan
SUPERVISOR: Dr. Pedram Fatehi (Chemical Engineering, Biorefining Research Institute)
ABSTRACT: Synthetic dispersants are commonly used in the stabilization of various colloidal suspensions. However, their non-biodegradable and toxic natures hamper their industrial use. The use of natural polymers as dispersants for various colloidal suspensions has been reported in the past, but the incentive for producing highly efficient natural polymeric dispersants is high. Lignin and xylan are the most abundant and sustainable natural polymers on earth, which are produced as by-products of pulping and cellulosic ethanol industries. These chemicals can be considered as the raw materials for producing value added products such as dispersants. In this dissertation, the anionic modification of kraft lignin and xylan via carboxymethylation, sulfomethylation and oxidation were comprehensively investigated and the applications of the products as dispersants in kaolin and coal suspensions were systematically assessed. The influence of reaction conditions on charge density and solubility of kraft lignin or xylan were systematically investigated. The anionic products obtained were characterized using NMR, FTIR, TGA, molecular weight and elemental analyses. The adsorption of anionic lignin or xylan on kaolin and coal particles and their effect on zeta potential of the suspensions was comprehensively assessed. The relative turbidity and viscosity analyses of colloidal suspensions confirmed the better dispersion performance of the products compared with commercial ones. The impact of dispersant dosage, pH of the suspension and time of mixing on dispersant performance of anionic lignin or xylan in kaolin and coal suspensions was also studied. The dispersion efficiency of suspensions found to increase with anionic lignin or xylan dosage and time of mixing. The dispersion efficiency of anionic lignin or xylan was at maximum under neutral conditions. The influences of molecular weight and charge density on the dispersant performance of anionic lignin in kaolin suspensions were also studied. The results showed that a highly charged anionic lignin adsorbed more compared with the low charged ones, and an increase in the molecular weight of anionic lignin showed no significant effect on its adsorption performance. Both charge density and molecular weight had an influence on dispersant activity of anionic lignin in kaolin suspensions. The dispersion studies of anionic kraft lignin or xylan in kaolin and coal suspensions showed that the increase in the dispersibility of these colloidal suspensions depends on adsorbed amount of anionic lignin or xylan on kaolin and coal particles, but not on the unadsorbed amount present in the suspensions. In the presence of anionic lignin or xylan dispersants, the zeta potential of kaolin or coal particles cannot determine directly the stability of colloidal suspensions. The adsorbed anionic lignin or xylan on kaolin and coal particles induced electrostatic repulsion between the particles and thereby improved the dispersibility of suspensions. The results obtained in this work confirms the possibilities for use of kraft lignin or xylan as dispersants in kaolin and coal suspensions and provides more insights on how the dispersability of these suspensions will be impacted by the properties of the produced dispersants. The results of this thesis contributed to knowledge on the chemical modification of lignin and xylan, characterization of modified products and to the fundamentals associated with the performance analysis of dispersion systems.
H2S S-sulfhydration of pyruvate carboxylase in gluconeogenesis and its regulation by Trx1
SUPERVISOR: Dr. Rui Wang (formerly with Biology)
ABSTRACT: Hydrogen sulfide (H2S), regarded as the third gasotransmitter, plays diverse physiological and pathological roles in the body along with another two gasotransmitters, including nitric oxide (NO) and carbon monoxide (CO). S-sulfhydration, a novel post-translational modification of proteins, is now considered as an important mechanism of H2S effects under various physiological and pathological conditions. In the liver, cystathionine gamma-lyase (CSE) is the main enzyme to generate H2S. The functions of H2S in the liver are largely unknown. Pyruvate carboxylase (PC), a mitochondrial protein, is an enzyme in the first step of gluconeogenesis in the liver. PC plays a critical role in tricarboxylic acid (TCA) cycle in mitochondria and in gluconeogenesis in the liver. PC is also involved in diverse metabolic pathways, such as lipogenesis and the biosynthesis of neurotransmitters. Here we found that H2S regulates gluconeogenesis through PC S-sulfhydration. First, PC activity and glucose production were decreased in liver of CSE-knockout (KO) mice comparing to the liver of wild type (WT) littermates. PC S-sulfhydration was lower in the liver tissue of CSE-KO mice than WT mice. NaHS treatment induces glucose production and PC activity in the primary liver cells. PC S-sulfhydration was increased by NaHS treatment in a time–dependent manner. Through mutation study, we further confirmed that the cysteine residue 265 is responsible for H2S S-sulfhdyration of PC. In cysteine 265 mutant-transfected cells, PC activity was significantly decreased and PC S-sulfhydration was disappeared. Thioredoxin 1 (Trx1), a well-known redox protein, is reported to be involved in transnitrosylation and/or denitrosylation. Here we first demonstrated that Trx1 acts as a S-desulfhydrase. Overexpressed Trx1 diminished H2S induced S-sulfhydration of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and PC proteins. The S-desulfhydration activity of Trx1 was inhibited by PX12, an inhibitor of Trx1, and DNCB, an inhibitor of thioredoxin reductase (TrxR). Furthermore, the cysteine 32 in Trx1 is required for the direct interaction of Trx1 with GAPDH or PC, and mutation of cysteine 32 abolished the S-desulfhydration activity of Trx1. Taken together, our studies demonstrated that H2S induces the gluconeogenesis through PC S-sulfhydration, and Trx1 acts as a S-desulfhydrase. Our findings will help to understand the mechanisms of H2S regulation of metabolic pathways via S-sulfhydration. These understandings will also extend the knowledge of physiological and pathological roles of H2S in both health and diseases.
H2S as a novel biomarker and therapeutic target for asthma
SUPERVISOR: Dr. Rui Wang (formerly with Biology)
ABSTRACT: Asthma is a chronic inflammatory disease with hyper-responsive bronchoconstriction and airway remodeling, leading to extensive airway narrowing. The pathogenic mechanisms for asthma remain unclear. Studies in the literature have shown that cystathionine gamma lysase (CSE)/ hydrogen sulfide (H2S) system participates in the regulation of airway contractility and immune response. In this PhD thesis study, we found that CSE was a major enzyme responsible for endogenous H2S synthesis in the lung and spleen as CSE gene knock out (CSE-KO) dramatically decreased H2S production rates in these two organs. In asthma model established via ovalbumin (OVA) sensitization and challenge, lung resistance of CSE-KO mice (12-16 weeks old), in response to aerosolized methacholine (MCh) at 12.5 mg/ml, was two times higher than that of wild type (WT) mice (12-16 weeks old). CSE-KO mice also developed more peri-bronchial inflammation and had higher levels of type 2 helper T cell (TH2) cytokines in bronchoalveolar lavage fluid (BALF). As allergic asthma is more prevalent among children than in adults, we next used young (3-4 weeks old) and old (7-8 months old) mice to observed if CSE/H2S were involved in the onset of childhood asthma. With the same intensity and duration of OVA treatments, WT young mice developed much more severe asthma with greater lung resistance, higher levels of eosinophils and TH2 cytokines in BALF, and more peri-bronchial inflammation than did WT old mice. This age-dependent propensity of immunoreaction and asthma development resulted from lower levels of CSE expression and H2S production in splenocytes from young mice, which was reversed by H2S donor treatment. Human umbilical cord blood mononuclear cells also had lower level of CSE proteins than that of peripheral blood mononuclear cell from adult people. CSE-KO mice had more severe asthma than WT mice but without the age-dependent asthma propensity. Lower endogenous level of CSE/H2S in WT young mice and in CSE-KO mice at all ages than in old WT mice promoted the differentiation of splenocytes into type 2 helper T cytokines-generating cells, which was suppressed by H2S donor. CSE/H2S-induced inhibition of type 2 immunity is not mediated by STAT-6 activation. Instead, H2S caused S-sulfhydration of GATA3 in spleen cells and decreased GATA3 nuclear translocation, leading to the inhibition of type 2 immunity. We also found that CSE expression in the airway of WT mice was increased in an age-dependent manner. Lower abundance of CSE in young WT mice or absence of CSE in young/old CSE-KO mice aggravated airway responsiveness to MCh challenge, in the absence of allergen exposure, by more than two times compared to old WT mice. In conclusion, CSE/H2S in peripheral lymph tissues and the lung suppresses allergen-induced type 2 immunity, airway responsiveness and the consequential asthma. Lower activity of CSE/H2S pathway renders higher incidence of allergic asthma in childhood.
Functional imaging of the lungs using magnetic resonance imaging of inert fluorinated gases
SUPERVISOR: Dr. Mitchell Albert (Chemistry)
ABSTRACT: Fluorine-19 (19F) magnetic resonance imaging (MRI) of the lungs using inhaled inert fluorinated gases can potentially provide high quality anatomical and functional images of the lungs. This technique is able to visualize the distribution of the inhaled gas, similar to hyperpolarized (HP) helium-3 (3He) and xenon-129 (129Xe) MRI. Inert fluorinated gases have the advantages of being nontoxic, abundant, and inexpensive compared to HP gases. Due to the high gyromagnetic ratio of 19F, there is sufficient thermally polarized signal for imaging, and averaging within a single breath-hold is possible due to short longitudinal relaxation times. Since inert fluorinated gases do not need to be hyperpolarized prior to their use in MRI, this eliminates the need for an expensive polarizer and expensive isotopes. Inert fluorinated gas MRI of the lungs has been studied extensively in animals since the 1980s, and more recently in healthy volunteers and patients with lung diseases. This thesis focused on the development of static breath-hold inert fluorinated gas MR imaging techniques, as well as the development functional imaging biomarkers in humans and animal models of pulmonary disease. Optimized ultrashort echo time (UTE) 19F MR imaging was performed in healthy volunteers, and images from different gas breathing techniques were quantitatively compared. 19F UTE MR imaging was then quantitatively compared to 19F gradient echo imaging in both healthy volunteers and in a resolution phantom. A preliminary comparison to HP 3He MR imaging is also presented, along with preliminary 19F measurements of the apparent diffusion coefficient (ADC) and iv gravitational gradients of ventilation in healthy volunteers. The potential of inert fluorinated gas MRI in detecting pulmonary diseases was further explored by performing ventilation mapping in animal models of inflammation and fibrosis. Overall, interest in pulmonary 19F MRI of inert fluorinated gases is increasing, and numerous sites around the world are now interested in developing this technique. This work may help to demonstrate that inert fluorinated gas MRI has the potential to be a viable clinical imaging modality that can provide useful information for the diagnosis and management of chronic respiratory diseases.
Bioremediation of contaminated soils from mine sites using native plants in Northwestern Ontario
SUPERVISOR: Dr. Peter Lee (Biology)
ABSTRACT: Practical and scientific importance can be found in this research topic since the results directly apply to remediation of industrial and mined lands in the boreal forest region. Plants suitable for phytostabilization of As, Mo and Sb are identified as well as two hyperaccumulators of Zn. Using phytostabilization practices, metals are immobilized by the below ground components of the plants therefore restricting the flow into the ecosystem and lessening the impacts of metal pollution to the surrounding area. As long as there is little disturbance of the soil physically or chemically, the plants will continue to stabilize the metal in the organic portion of the deceased plants. The ease of replanting a site could incorporate successional ecosystem in the region by focusing on trees and shrubs that are earlier in the revegetation process after a disturbance. The addition of woodbark to the reestablishment of the top soil increases potential nutrients, organic matter, water holding potential as well as diluting potential harmful metal content of the soil and providing a mulching effect. Some concerns exist by using agronomic plant species as the sole part of revegetation as they have the potential to impact the wildlife in the region through excess Mo. Results from this thesis could be helpful for future mine closure plans and in the rehabilitation of other industrial sites.
Hydrogen sulfide modulates gluconeogenesis and mitochanodrial biogenesis in mouse primary hepatocytes
SUPERVISOR: Dr. Lingyun Wu (formerly with Biology)
ABSTRACT: Among many endogenous substances that regulate hepatic energy production is the gasotransmitter hydrogen sulfide (H2S). In the liver, H2S production is largely catalyzed by cystathionine γ-lyase (CSE) and, to a lesser degree, by cystathionine β-synthase. We previously showed that H2S stimulates glucose production in an immortalized carcinoma liver cell line (HepG2 cells) as well as induce ATP generation in isolated vascular smooth muscle cells (VSMCs). Furthermore, we found that H2S upregulates peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression in rat VSMCs. PGC-1α is a crucial regulator of hepatic gluconeogenesis and mitochondrial biogenesis. Both of these PGC-1α-mediated energy processes are pivotal to maintain whole-body energy homeostasis, whereby their sustained disturbance may lead to the development of type 2 diabetes and metabolic syndrome. Therefore, we investigated the regulation of gluconeogenesis and mitochondrial biogenesis by CSE-generated H2S under physiological conditions in isolated mouse hepatocytes. We found that CSE-knockout (KO) mice had a reduced rate of gluconeogenesis, which was reversed by administration of NaHS (an H2S donor) (i.p.). Interestingly, isolated CSE-KO hepatocytes exhibited a reduced glycemic response to chemical-induced activation of the cAMP/PKA and glucocorticoid pathways compared to wild-type (WT) hepatocytes. Treatment with the inhibitors for PKA (KT5720) or glucocorticoid receptor (RU-486) significantly reduced H2S-stimulated glucose production from both WT and CSE-KO mouse hepatocytes. NaHS treatment upregulated the protein levels of key gluconeogenic transcription factors, such as PGC-1α and CCAAT-enhancer-binding proteins-β (C/EBP-β). Moreover, exogenous H2S augmented the S-sulfhydration of the rate-limiting gluconeogenic enzymes and PGC-1α and increased their activities, which were lower in untreated CSE-KO hepatocytes. Finally, knockdown of PGC-1α, but not C/EBP-β, significantly decreased NaHS-induced glucose production from the primary hepatocytes. After determining that H2S stimulates hepatic glucose production through the PGC-1α signaling pathway, we focused on whether or not H2S induces hepatic mitochondrial biogenesis. We found that CSE-KO hepatocytes produced less mtDNA compared to WT hepatocytes. Mitochondrial content was decreased in CSE-KO hepatocytes compared to normal hepatocytes, which was restored with NaHS treatment. CSE-KO hepatocytes exhibited lower levels of mitochondrial transcription factors and the mitochondrial transcription coactivator, peroxisome proliferator-activated receptor-γ coactivator-related protein (PPRC) compared to WT hepatocytes. Interestingly, NaHS administration upregulated PPRC, yet downregulated PGC-1β protein level in mouse hepatocytes. Moreover, exogenous H2S induced the S-sulfhydration of PPRC, which was lower in untreated CSE-KO hepatocytes, but not that of PGC-β. Finally, knockdown of either PGC-1α or PPRC significantly decreased NaHS-stimulated mitochondrial biogenesis in hepatocytes, where knockdown of both genes were required to completely abolish NaHS-induced mitochondrial biogenesis. Overall this thesis demonstrates the stimulatory effect of endogenous H2S on liver glucose production and reveals four underlying mechanisms. 1) H2S upregulates the expression levels of PGC-1α and PEPCK via glucocorticoid receptor pathway. 2) H2S upregulates the expression level of PGC-1α through the activation of the cAMP/PKA pathway, as well as PGC-1α activity via S-sulfhydration. 3) H2S upregulates the expression and the activities (by S-sulfhydration) of G6Pase and FBPase. 4) H2S augments the protein expression level and activity (via S-sulfhydration) of PPRC. By stimulating the combined activities of PPRC and PGC-1α, H2S induces mitochondrial biogenesis, thereby supplying energy to support its induction of hepatic glucose production. This study may offer clues to the regulation of hepatic energy homeostasis under physiological conditions and its dysregulation in insulin-resistance diseases.
Modeling the molecular mechanisms of biocompatibility of artificial materials
SUPERVISOR: Dr. Oleg Rubel (formerly with Chemistry)
ABSTRACT: One of the most common reasons for implant failure is immune rejection. Implant rejection leads to additional surgical intervention and, ultimately, increases health cost as well as recovery time. Within a few hours after implantation, the implant surface is covered with host proteins. Adsorption of fibrinogen, a soluble plasma glycoprotein, is responsible in triggering the immune response to a given material and, subsequently, in determining its biocompatibility. The work presented here is focused on modeling the interaction between artificial surfaces and plasma proteins at the microscopic level by taking into account the physico-chemical properties of the surfaces. Carbon-based nanomaterials are chosen as a model system due to their unique bioadhesive and contradictory biocompatible properties as well as the possibility of functionalization for specific applications. Graphene and its derivatives, such as graphene oxide and reduced graphene oxide, demonstrate controversial toxicity properties in vitro as well as in vivo. In this study, by covalently adding chemical groups, the wettability of graphene surfaces and the subsequent changes in its biocompatibility are being examined. An empirical force field potential (AMBER03) molecular dynamic simulation code implemented in the YASARA software package was utilized to model graphene/biomolecule interactions. The accuracy of the force field choice was verified by modeling the adsorption of individual amino acids to graphene surface in a vacuum. The obtained results are in excellent agreement with previously published ab initio findings. In order to mimic the natural protein environment, the interaction of several amino acids with graphene in an explicit solvent was modeled. The results show that the behaviour of amino acids in aqueous conditions is drastically different from that in vacuum. This finding highlights the importance of the host environment when biomaterial-biomolecule interfaces are modeled. The surface of Graphene Oxide (GO) has been shown to exhibit properties that are useful in applications such as biomedical imaging, biological sensors and drug delivery. An assessment of the intrinsic affinity of amino acids to GO by simulating their adsorption onto a GO surface was performed. The emphasis was placed on developing an atomic charge model for GO that was not defined before. Next, the simulation of a fibrinogen fragment (D-domain) at the graphene surface in an explicit solvent with physiological conditions was performed. This D-domain contains the hidden (not expressed to the solvent) motifs (PI 7190-202 and P2 7377-395, and specifically P2-C portion 7383-395) that were experimentally found to be responsible for attracting inflammatory cells through CDllb/CD18 (Mac-1) leukocyte integrin and, consequently, promoting the cascade of immune reactions. It was hypothesized that the hydrophobic nature of graphene would cause critical changes in the fibrinogen D-domain structure, thus exposing the sequences and result in the foreign body reaction. To further study this issue, molecular mechanics was used to stimulate the interactions between fibrinogen and a graphene surface. The atomistic details of the interactions that determine plasma protein affinity modes on surfaces with high hydrophobicity were studied. The results of this work suggest that graphene is potentially pro-inflammatory surface, and cannot be used directly (without alterations) for biomedical purposes. A better understanding of the molecular mechanisms underlying the interaction between synthetic materials and biological systems will further the ultimate goal of understanding the biocompatibility of existing materials as well as design of new materials with improved biocompatibility.
Cardiac oxidative stress and antioxidant status in response to radiation and monocrotaline-induced cardiac dysfunction
SUPERVISOR: Dr. Neelam Khaper (NOSM, Biology adjunct)
ABSTRACT: Cardiovascular disease is the leading cause of death and disability worldwide. Oxidative stress has been implicated in many types of cardiovascular disease. Chronic cardiac stress conditions have been shown to be associated with an increase in myocardial oxidative stress following myocardial infarction, which in turn may lead to depressed contractile function, myocardial remodeling and heart failure. Antioxidants play a protective role against oxidative stress damage through the removal of free radical intermediates and inhibition of oxidation reactions. An imbalance of free radicals and antioxidants results in cellular and sub-cellular damage. Therefore, treatment with non-enzymatic antioxidants may provide protection against high levels of free radicals. We investigated, in three different studies, if treatment with antioxidants can protect the heart under conditions of oxidative stress. In the first study, a complex dietary supplement composed of numerous antioxidants and anti-inflammatory components was given to C57BL/6 mice that received a whole body radiation dose of 5 Gy to investigate potential cardioprotective effects by measuring cardiac antioxidant status and apoptosis. In the second study, the same complex dietary supplement was given to Thy1-GFP mice that received a radiation dose of 10 Gy to the head to investigate the abscopal effect on the heart by measuring cardiac inflammation and fibrosis. In the final study, the potential cardioprotective effects of secoisolariciresinol diglucoside, a compound found in flaxseed, was investigated in a Wistar rat model of pulmonary arterial hypertension by correlating cardiac functions with oxidative stress. We have shown that treatment with antioxidants may offer some protection to the heart in these models of oxidative stress though it is important to consider the extent of oxidative stress and when developing a antioxidant treatment protocol.
Physiological adaptations contributing to stress survival in the foodborne pathogen Campylobacter jejuni
SUPERVISOR: Dr. Heidi Schraft (Biology)
ABSTRACT: In spite of being considered fragile and fastidious, the zoonotic pathogen Campylobacter jejuni remains the leading cause of foodborne bacterial gastroenteritis in the developed world. Lacking many of the stress responses common to other enteric pathogens, C. jejuni employs the survival strategies, biofilm formation and entry into the viable but non-culturable (VBNC) state, which have not been well characterized. Recent studies have indicated that these strategies are likely related at the molecular level. The purpose of this thesis was threefold: 1) to characterize entry into the VBNC state for planktonic and biofilm cells of C. jejuni with starvation at 4°C; 2) to evaluate a novel PMAqPCR method to quantify viable cells (both culturable and viable but non-culturable) in planktonic and biofilm cells of C. jejuni during starvation at 4°C; and 3) to investigate changes in gene expression of selected genes involved in biofilm formation and entry into the VBNC state. The three strains C. jejuni NCTC 11168 V1, C. jejuni NCTC V26 and C. jejuni 16-2R were included in all studies to compare variation based on strains. Cells were considered VBNC when there was no growth with enrichment, but cells scored as viable based on membrane integrity. Biofilm cells which became VBNC in some cases after 10 days of stress were found to enter the VBNC state earlier than planktonic cells by 10 to 50 days. Additionally, no significant reductions occurred in viable cell counts over the course of the experiments, confirming that the loss of culturability was not due to cell death (p<0.05). To date, no methods have been used to quantify viable but non-culturable biofilm cells of C. jejuni. The novel method PMAqPCR which has been successful for the enumeration of planktonic C. jejuni as well as for biofilm cells of other species was validated for quantifying C. jejuni biofilm cells in late log phase (20 h) and once cells had entered the VBNC state. The genes that affect both entry into a VBNC state and the ability to form biofilm in C. jejuni were upregulated during biofilm formation. Gene expression prior to stress treatment was 5 to 37 fold higher in biofilm cells than in their planktonic counterparts for all three strains IV (p<0.001). For the planktonic samples, only one of the 3 strains showed significant changes in gene expression during the transition to the VBNC state. In this case, all 4 target genes were significantly upregulated 4-6 fold just prior to cells becoming VBNC (p<0.05). At present food and drinking water safety in Canada continues to be assessed primarily using culture-based methodology. As validated in this thesis, the ability to quantify both culturable and viable but non-culturable C. jejuni cells in both planktonic and biofilm forms will allow for improved evaluation of quality control methods in both research and industries where these pathogens are a concern. Also, the understanding of the interaction between biofilm formation and entry into the VBNC state at the molecular level described herein provides information which can be used to develop appropriate interventions and reduce the incidence of campylobacteriosis.
Adventures in immunochemistry: Towards a fully synthetic vaccine
SUPERVISOR: Dr. Zi Hua Justin Jiang (Chemistry)
ABSTRACT: Lipid A, a unique disaccharide glycolipid, is the active principle of Gram-negative bacterial lipopolysaccharide in activating the innate immune response via Toll-like receptor 4 (TLR4). Given the important role that TLR4 plays in innate immunity, and ultimately, the development of an adaptive immune response, ligands that can modulate TLR4-mediated signalling have great therapeutic potential as both vaccine adjuvants, and anti-sepsis agents. In attempting to develop novel ligands which can successfully modulate TLR4-mediated signalling in a well defined fashion, simplified structures which aim to mimic the natural lipid A structure have shown great promise. The notion of cancer immunotherapy, in which the vast power of the immune system is tapped to prevent and/or eradicate the disease has begun to garner considerable attention. Tumour associated carbohydrate antigens, carbohydrate containing epitopes which are either unique of over-expressed by cancer cells, are viable targets of said immunotherapy. A major limitation, however, is the low antigenicity displayed by these carbohydrate epitopes. Studies have shown that the inclusion of adjuvant structures, especially when directly chemically conjugated to the antigen, improve the success of anti-cancer vaccination efforts. The primary goal of this study has been aimed at the development of novel vaccine adjuvants, specifically the design of novel molecular frameworks to mimic the structure of lipid A in the activation of TLR4. A secondary goal of this study has aimed at the application of successful novel lipid A mimics as the immunostimulatory component of self-adjuvanting carbohydrate antigens for use in therapeutic cancer vaccines. One novel molecular framework that has been designed and synthesized employs a flexible, acyclic diethanolamine-based scaffold to mimic one of the sugar moieties natural to the lipid A disaccharide. Several structural variations of this framework were generated for structure-activity relationship studies in an effort to maximize immunostimulatory potency. The mimics were evaluated in vitro for their ability to induce TLR4-mediated cytokines. All variations showed confirmed TLR4 stimulatory activity, the potency of which was dependent on the functionalization of the terminal ethanol moiety of the diethanolamine-based acyclic scaffold. In vivo studies evaluating the adjuvant potential of this novel family of lipid A mimics are currently underway. As part of an industrial partnership aimed at the development of novel vaccine adjuvants, a second lipid A mimic framework was designed and synthesized, in which an aromatic residue has been incorporated into the structural backbone. Two structural variations of the framework were generated which vary in the functionalization of the phenolic hydroxyl of the aromatic-based backbone. Several in vivo studies have shown that both mimics exhibit potent TLR4 immunostimulatory activity, and successful adjuvant properties. In an effort to construct a fully synthetic, self-adjuvanting tumour associated carbohydrate antigen for eventual use in therapeutic cancer vaccines, the immunostimulatory activity of the diethanolamine-based lipid A mimic framework designed herein, was tapped. As such, a conjugate structure in which the lipid A mimic framework and the Thomsen-Friedenreich carbohydrate antigen are directly linked via a flexible chemical linker was designed and synthesized. Future studies will determine the ability of the conjugate to induce an effective antibody response towards the carbohydrate epitope.
H2S as a novel biomarker and therapeutic target for Asthma
SUPERVISOR: Dr. Rui Wang (formerly with Chemistry)
ABSTRACT: Asthma is a chronic inflammatory disease with hyper-responsive bronchoconstriction and airway remodeling, leading to extensive airway narrowing. The pathogenic mechanisms for asthma remain unclear. Studies in the literature have shown that cystathionine gamma lysase (CSE)/ hydrogen sulfide (H2S) system participates in the regulation of airway contractility and immune response. In this PhD thesis study, we found that CSE was a major enzyme responsible for endogenous H2S synthesis in the lung and spleen as CSE gene knock out (CSE-KO) dramatically decreased H2S production rates in these two organs. In asthma model established via ovalbumin (OVA) sensitization and challenge, lung resistance of CSE-KO mice (12-16 weeks old), in response to aerosolized methacholine (MCh) at 12.5 mg/ml, was two times higher than that of wild type (WT) mice (12-16 weeks old). CSE-KO mice also developed more peri-bronchial inflammation and had higher levels of type 2 helper T cell (TH2) cytokines in bronchoalveolar lavage fluid (BALF). As allergic asthma is more prevalent among children than in adults, we next used young (3-4 weeks old) and old (7-8 months old) mice to observed if CSE/H2S were involved in the onset of childhood asthma. With the same intensity and duration of OVA treatments, WT young mice developed much more severe asthma with greater lung resistance, higher levels of eosinophils and TH2 cytokines in BALF, and more peri-bronchial inflammation than did WT old mice. This age-dependent propensity of immunoreaction and asthma development resulted from lower levels of CSE expression and H2S production in splenocytes from young mice, which was reversed by H2S donor treatment. Human umbilical cord blood mononuclear cells also had lower level of CSE proteins than that of peripheral blood mononuclear cell from adult people. CSE-KO mice had more severe asthma than WT mice, but without the age-dependent asthma propensity. Lower endogenous level of CSE/H2S in WT young mice and in CSE-KO mice at all ages than in old WT mice promoted the differentiation of splenocytes into type 2 helper T cytokines-generating cells, which was suppressed by H2S donor. CSE/H2S-induced inhibition of type 2 immunity is not mediated by STAT-6 activation. Instead, H2S caused S-sulfhydration of GATA3 in spleen cells and decreased GATA3 nuclear translocation, leading to the inhibition of type 2 immunity. We also found that CSE expression in the airway of WT mice was increased in an age-dependent manner. Lower abundance of CSE in young WT mice or absence of CSE in young/old CSE-KO mice aggravated airway responsiveness to MCh challenge, in the absence of allergen exposure, by more than two times compared to old WT mice. In conclusion, CSE/H2S in peripheral lymph tissues and the lung suppresses allergen-induced type 2 immunity, airway responsiveness and the consequential asthma. Lower activity of CSE/H2S pathway renders higher incidence of allergic asthma in childhood.
Dr. Young Jun Ju
H2S S-sulfhydration of pyruvate carboxylase in gluconeogenesis and its regulation by Trx1
SUPERVISOR: Dr. G. Yang (formerly in Chemistry)
ABSTRACT: Hydrogen sulfide (H2S), regarded as the third gasotransmitter, plays diverse physiological and pathological roles in the body along with another two gasotransmitters, including nitric oxide (NO) and carbon monoxide (CO). S-sulfhydration, a novel post-translational modification of proteins, is now considered as an important mechanism of H2S effects under various physiological and pathological conditions. In the liver, cystathionine gamma-lyase (CSE) is the main enzyme to generate H2S. The functions of H2S in the liver are largely unknown. Pyruvate carboxylase (PC), a mitochondrial protein, is an enzyme in the first step of gluconeogenesis in the liver. PC plays a critical role in tricarboxylic acid (TCA) cycle in mitochondria and in gluconeogenesis in the liver. PC is also involved in diverse metabolic pathways, such as lipogenesis and the biosynthesis of neurotransmitters. Here we found that H2S regulates gluconeogenesis through PC S-sulfhydration. First, PC activity and glucose production were decreased in liver of CSE-knockout (KO) mice comparing to the liver of wild type (WT) littermates. PC S-sulfhydration was lower in the liver tissue of CSE-KO mice than WT mice. NaHS treatment induces glucose production and PC activity in the primary liver cells. PC S-sulfhydration was increased by NaHS treatment in a time–dependent manner. Through mutation study, we further confirmed that the cysteine residue 265 is responsible for H2S S-sulfhdyration of PC. In cysteine 265 mutant-transfected cells, PC activity was significantly decreased and PC S-sulfhydration was disappeared. Thioredoxin 1 (Trx1), a well-known redox protein, is reported to be involved in transnitrosylation and/or denitrosylation. Here we first demonstrated that Trx1 acts as a S-desulfhydrase. Overexpressed Trx1 diminished H2S induced S-sulfhydration of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and PC proteins. The S-desulfhydration activity of Trx1 was inhibited by PX12, an inhibitor of Trx1, and DNCB, an iii inhibitor of thioredoxin reductase (TrxR). Furthermore, the cysteine 32 in Trx1 is required for the direct interaction of Trx1 with GAPDH or PC, and mutation of cysteine 32 abolished the S-desulfhydration activity of Trx1. Taken together, our studies demonstrated that H2S induces the gluconeogenesis through PC S-sulfhydration, and Trx1 acts as a S-desulfhydrase. Our findings will help to understand the mechanisms of H2S regulation of metabolic pathways via S-sulfhydration. These understandings will also extend the knowledge of physiological and pathological roles of H2S in both health and diseases.
Dr. Andrew Gibson
Characterizing the microbial degradation of Kraft lignin and lignin-derived compounds
SUPERVISOR: Dr. Lada Malek (Biology)
ABSTRACT: Analytical methods for characterizing the microbial degradation of Kraft lignin and lignin-derived compounds were utilized with the goal of biologically generating demethylated lignin for subsequent industrial applications. Selected ion flow tube mass spectrometry (SIFT-MS) technology was used for the first time with both bacterial and fungal cultures growing on lignin as a sole carbon source. Methanol and other volatile compounds were evaluated using this method and lignin-derived compounds were identified. Methanol oxidation products were found in the headspace of seven microbial cultures, as well as several unknown products not present in the SIFT-MS compound library. An assay was then developed to both confirm the results obtained by the SIFT-MS and help to understand the nature of the microbial demethylation reactions. The Ti(III)-NTA assay was found to be an economical method for rapidly determining the relative degree of lignin demethylation by cultures of microorganisms and their enzymes. Using the Ti(III)-NTA assay, some fungal cultures were found to degrade lignin monomers completely and others to metabolize methanol. Four cultures were then selected for growth optimization; to both maximize vicinal diol generation and methanol formation. By altering variables such as induction day, incubation length, culture agitation, hydrogen peroxide concentration and micronutrient concentrations (known to promote enzyme production), the effect on four fungal species was investigated. Induction with vanillin after 1 week of growth on glucose resulted in the highest demethylation activity. In the final study, culture media from the fungus Absidia cylindrospora and the bacterium Sphingobium sp. SYK-6 were used to partially purify demethylating activity. The fungal enzyme had higher specific activity than the bacterial III enzyme, but was much less abundant. Further research is needed to purify these enzymes responsible for demethylation.
Relating gene expression profiles to behavioural and neurophysiological responses to standard chemosensory cues using wild yellow perch (Perca Flavescens)
SUPERVISOR: Dr. Greg Pyle (formerly with Biology)
ABSTRACT: To play their ecological role in an ecosystem, a fully functioning olfactory system is vital for fishes. A fish's olfactory system is very sensitive to low concentrations of contaminants. A great deal of research has been conducted on the toxicity of metals on the olfactory systems of fish. While tightly-controlled laboratory studies are necessary to reveal the mechanism of toxicity, field-based studies using more realistic conditions are also needed to produce more environmentally relevant data for regulatory needs. This research has four main areas of focus: 1) to examine the effect of copper on gene transcription in olfactory tissues of yellow perch (Perca flavescens); 2) to test if a modified diet can protect olfaction against copper toxicity in rainbow trout (Oncorhynchus mykiss); 3) to examine chemosensory-mediated behaviours and gene transcription profiles in wild yellow perch from metal contaminated lakes; and 4) to investigate the potential recovery of yellow perch with contaminant-induced chemosensory function from metal contaminated lakes. We used gene transcription (a recently developed custom made micro-array for yellow perch and real-time PCR), neurophysiological testing (electro-olfactography (EOG)), and behavioural assays (avoidance from alarm cues). In this research, yellow perch were exposed to elevated concentrations of copper for 3 and 24 hours. While 3 hours of exposure did not influence gene expression, 24-hour exposures to copper elicited a differential expression of 71 genes. Of these 71 genes, differential expression of two subunits of Na/K-ATPase was further explored using real-time PCR in a time-series study. To investigate if increased dietary sodium can protect fish's olfaction against copper-induced olfactory impairment, rainbow trout were fed with diets having elevated concentrations of sodium. While fish exposed to 10 μg/L Cu and fed with a normal sodium diet had an impaired EOG response to standard olfactory cues, olfaction in Cu-exposed fish fed with Na-supplemented food (regardless of low or high levels of supplementation) remained intact. However, subsequent feeding trials found no evidence to support that Cu-exposed fish preferentially chose high-Na food. In addition, in this study olfactory impairment of fish from one clean and two metal contaminated lakes were compared using gene expression, EOG and behavioural I-maze choice experiments. While behavioural testing and EOG confirmed the impairment of olfaction in fish from metal contaminated lakes, the micro-array was not able to detect differential gene expression. To investigate if impaired olfaction of fish from metal contaminated lakes has recovery potential, EOG and behavioural testing methods were employed. Yellow perch from metal contaminated lakes with impaired olfaction kept for 24 hours in water from clean lakes. The results showed that impaired EOG responses of fish from metal contaminated lakes can recover quickly (within 24 hours) in water from a clean lake. When the behavioural testing methods were employed the results showed that olfactory mediated behaviours of fish from a moderately contaminated lake recovered after 24 hours of holding in clean water. However, olfactory mediated behaviours of fish from a severely contaminated lake did not recover after 24 hours of exposure to clean water. The data produced by this thesis significantly improves our knowledge regarding the protection against metal induced olfactory toxicity as well as the recovery potential for impaired olfaction in fish. These results could be used to draft ecologically-relevant regulations that will protect fish inhabiting sensitive fresh water ecosystems.
Dr. Zaid Altany
The cross-talk of hydrogen sulfide and nitric oxide in vascular endothelial cells
SUPERVISOR: Dr. G. Yang (formerly adjunct in Biology)
ABSTRACT: Analytical methods for characterizing the
Dr. William Dew
The effects of copper, calcium, and nickel on the olfactory response of Fathead minnows: From neurophysiology to behaviour
SUPERVISOR: Dr. Greg Pyle (formerly in Biology)
ABSTRACT: Analytical methods for characterizing the
Dr. Ling Zhang
Cystathionine gamma-lyase/hydrogen sulfide system and glucose homeostasis
SUPERVISOR: Dr. Rui Wang (formerly in Chemistry)
Dr. Miranda Maki
Development of bacterial systems for large-scale production of cellulase and bioethanol
SUPERVISOR: Dr. Wensheng Qin (Biology)
ABSTRACT: The wide varieties of extant bacterial species are often resistant to various environmental stresses. This demonstrates their frequent ability to adapt to and thrive in challenging environments. One such adaptation in wood-degrading species that may be exploited to produce a product of high value to humans is a more efficient cellulase activity, which may help to overcome current challenges in biofuel production. In this study, 18 efficient cellulase-producing bacteria were isolated from organic fertilizers and paper mill sludges and characterized for consideration in large scale biorefining. All cellulase positive isolates were further characterized to identify those with the greatest cellulase activities for potential industrial application. Six of these isolates produced greater cellulase activity on soluble cellulose in 48 h than the positive control (Cellulomonas xylanilytica). Phylogenetic analysis of a portion of the 16S rDNA gene revealed genera belonging to two major phyla of Gram positive bacteria: Firmicutes and Actinobacteria. Additionally, isolates E2 and E4 (Paenibacillus species) displayed qualitative cellulase activities towards filter paper under limited oxygen condition. When total cellulase activities of E2 and E4 were examined, it was shown that 1% (w/v) carboxymethyl cellulose (CMC) could induce total cellulase activities of 1652 ± 62 and 1457 ± 31 nM of glucose equivalents that were 8- and 5.6-fold greater than total cellulase activities induced by filter paper for E2 and E4, respectively. The genus Paenibacillus includes many highly-expressing cellulase producing strains, and E2 and E4 represent excellent candidates for further cellulase activity analysis and characterization. Cellulose hydrolysis is only one of the rate-limiting steps in the industrial production of biofuels which can be improved by isolation and characterization of novel enzymes. In addition, pretreatment of lignocellulosic biomass is a costly hurdle which can be improved by the application of bacteria capable of producing a greater variety of enzymes. The potential use of lignocellulosic biomass for biofuel production has been hampered due to the complexity of its composition and the lack of microorganisms capable of modifying or decomposing the different components. Thus, CMC-containing agar was used to isolate and characterize 20 cellulase-producing bacterial strains from peat and municipal wastes that belonged to four major phyla: Firmicutes, Actinobacteria, Proteobacteria and Bacteriodetes. Seven of the cellulase positive isolates also exhibited filter paper activities, while 13 exhibited activities towards xylan. Moreover, 10 of the isolates were capable of surviving 21 days incubation with 1% black liquor. Five strains increased the absorbance of black liquor by greater than 10-fold. Similarly, these five strains could also increase the absorbance of lignin at 280 nm when grown with 0.1% pure lignin. Additionally, although FTIR analysis of 1% barley straw treated for 21 days with these 5 strains showed a preference for consumption of hemicelluloses over lignin, a change in lignin was observed. Two isolates, 55S5 and AS1, a Bacillus sp. and Pseudomonas sp., respectively, have the highest lignocellulase activity, that is activities towards cellulose, hemicellulose and lignin, and possess the greatest potential for industrial use because of their concomitantly high cellulase activities, including filter paper activity and in addition, xylanase activity. The anaerobic, thermophilic and ethanogenic bacterium Clostridium thermocellum has great potential for use in consolidated bioprocessing for a more cost effective production of biofuels. However, its application is still hindered by such obstacles as end-product inhibition, i.e. feedback inhibition to cellulase activity by cellobiose. To increase cellulase activity and ethanol production, the copy number of β-glucosidase A (bglA) in C. thermocellum 27405 was increased using shuttle vector pIBglA to lower the end-product inhibition of cellulase. Using a modified electrotransformation protocol, C. thermocellum transformant (+MCbglA) harbouring pIBglA was successfully produced. The β-glucosidase activity of +MCbglA was 2.3- and 1.6-fold greater than wild-type (WT) during late log and stationary phases of growth, respectively. Similarly, total cellulase activity of +MCbglA was shown to be 1.7-, 2.3- and 1.6-fold greater than WT during, log, late log and stationary phases of growth. However, there was no significant correlation found between increased cellulase production and increased ethanol titres for +MCbglA compared to the WT, perhaps due to the accumulation of toxic end-products (i.e. ethanol). We successfully increased total cellulase activity by increased expression of bglA and thereby increased the productivity of C. thermocellum during the hydrolysis stage in consolidated bioprocessing. Our work also provides insight into the complex metabolism of C. thermocellum for future further improvement of this strain. The co-culture of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum has great potential in the production of biofuels because it will consolidate the hydrolysis and fermentation steps and potentially increase bioethanol titres. However, there is little knowledge of the industrial application of this kind of co-culture such as substrate conditions and the number of generations for stable co-culture in addition to the effect of ethanol titres. The goal of this study was to develop a stable co-culture of C. thermocellum 27405 and T. saccharolyticum 31097 which can produce greater ethanol titres than mono-cultures in batch fermentation. Comparison of C. thermocellum and T. saccharolyticum growth in reducing sugar (1% (w/v) cellobiose and 0.5% (w/v) xylose) and polysaccharide (1% (w/v) Avicel and 0.5% (w/v) cellobiose) media, showed that T. saccharolyticum could grow 2-fold faster in reducing sugar medium compared to C. thermocellum, while C. thermocellum grew to 2.3-fold greater turbidity in polysaccharide medium in mono-cultures. Subsequent co-culture batch cultures revealed that both strains could only co-exist for complete cell culture in reducing sugar medium, as confirmed by biomarker genes (bglA and xylB, respectively) detected by PCR, while in the subsequent subcultures only T. saccharolyticum was detected. In polysaccharide medium, both strains were detected continuously for 4 generations in batch culture trials, using the same biomarker genes. After the fourth continuous subculture, the co-culture required re-establishing or further media optimization due to growth inhibition of strains. Additionally, the ethanol titres also increased by 2.01-fold in the first and second subcultures compared to the mono-cultures. However, third and fourth subcultures did not have significantly different ethanol titres. Nonetheless, C. thermocellum and T. saccharolyticum co-culture has potential application if added during the hydrolysis stage of complex polysaccharides but not if added to simple sugars such as short poly- and oligo-saccharides produced during the fermentation stage. All of the work presented here in this thesis, focuses on the potential exploitation of bacteria to improve the economic feasibility of biofuels from stages of pretreatment, to hydolysis and fermentation. Due to the large variety and extreme environmental resistance, as well as genetic advances in prokaryotic systems the potential to improve existing bacterial systems or isolate new strains for industrial application is immense.
Dr. Bruce Rosa
Improving novel gene discovery in high-throughput gene expression datasets
SUPERVISOR: Dr. Wensheng Qin (Biology)
Dr. Mehdi Dashtban
Engineering fungi for large-scale production of cellulase
SUPERVISOR: Dr. Wensheng Qin (Biology)
ABSTRACT: Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi produce large amounts of extracellular cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and β- glucosidases that work efficiently on cellulolytic residues in a synergistic manner. The ascomycete Hypocrea jecorina (anamorph Trichoderma reesei), an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. However, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for enzyme induction in this organism are not completely understood. In this study, the optimum conditions for cellulase production by T. reesei strains were investigated. Three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the latter group including 4 pure polysaccharides and 3 lignocelluloses. Maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred after 120 h of incubation, while RUT-C30 had the greatest cellulase activity after around 72 h. Maximum cellulase production was 0.035, 0.42 and 0.33 μmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production with the ability to grow on microcrystalline cellulose was positively correlated in QM9414 and negatively correlated in RUT-C30. Although T. reesei is widely used as an industrial strain for cellulase production, its low yield of β-glucosidase has limited its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, inhibiting further cellulase production. In order to improve β-glucosidase production and ultimately overall cellulase activity of T. reesei, a thermostable β-glucosidase gene from the fungus Periconia sp. was engineered into the genome of the T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher β-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high cellulase activity (about 39.0 FPU/mg) at 24 h of incubation, whereas the parent strain showed almost no cellulase activity at 24 h of incubation. The recombinant β- glucosidase was thermotolerant and remained fully active after two-hour incubation at temperatures as high as 60 °C. Additionally, it maintained about 88% of its maximal activity after a four-hour incubation at 25 °C across a wide range of pH values from 3.0 to 9.0. Furthermore, an enzymatic hydrolysis assay using untreated, NaOH- or Organosolv-pretreated barley straw or microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain. These features suggest that the transformants can be used for β-glucosidase production as well as improving biomass conversion using cellulases. Xylitol, a naturally occurring five-carbon sugar alcohol derived from D-xylose, is currently in high demand by industries for its sweetening and anti-microbial properties. Biotechnological methods can be used for large-scale xylitol production since its current industrial production relies on chemical methods which are costly and energy intensive. While T. reesei is capable of selectively using D-xylose for xylitol production as an intermediate metabolite, its production can be enhanced by genetic engineering of the metabolic pathway. In this study, two T. reesei mutant strains were used, including a single mutant in which the endogenous xylitol dehydrogenase gene was deleted (Δxdh1), and a double mutant in which L-arabinitol-4-dehydrogenase was additionally deleted (Δlad1Δxdh1). The widely available agricultural residue barley straw was used for xylitol production by the strains after its pretreatment using NaOH- and Organosolv-pretreatment methods. High xylitol production by both strains was achieved when barley straw (untreated or pretreated) was supplemented with 2% D-xylose, whereas the D-glucose supplementation did not increase production. The highest production of xylitol was 6.1 and 13.22 g/L obtained after 96 and 168 h of incubation, respectively, using medium supplemented with 2% Organosolv-pretreated barley straw and 2% D-xylose by single and double T. reesei mutant strains, respectively. Maximum saccharification of barley straw was observed after 120 h of incubation for NaOH-pretreated by single (692 ± 1.01 mg/g reducing sugars) and double T. reesei mutant (685 ± 11.9 mg/g reducing sugars) strains, respectively. Moreover, the significant increase of xylitol production by the T. reesei strains using medium containing Organosolv-pretreated barley straw supplemented with Dxylose suggests that the pretreatment in combination with the added sugar favored xylitol production. These results suggest that agricultural residues, such as barley straw, could be a suitable resource for bioconversion to produce value-added products such as xylitol.
Dr. Susanne Walford
Environmental considerations for wet mining peatlands in Northwerstern Ontario
SUPERVISOR: Dr. Peter Lee (Biology)
Dr. Weijue Gao
Submerged anaerobic membrane bioreators for wastewater treatment and membrane fouling characterization and control
SUPERVISOR: Dr. Baoqiang Liao (Chemical Engineering)
ABSTRACT: This thesis focused on effects of transient conditions (pH shocks, temperature variation and temperature shocks) on the performance and membrane fouling characterization and control of submerged anaerobic membrane bioreactors (SAnMBR) for pulp and paper effluent treatment (thermomechanical whitewater and pressate). A comprehensive characterization of cake layer formed on membrane surfaces was conducted using various techniques. Results show that a pH 8.0 shock had a minor impact, while pH 9.1 and 10.0 shocks exerted significant long-lasting negative impacts on performance of the SAnMBR. The SAnMBR system was highly resilient and could successfully tolerate the 5 °C/10 °C temperatures shocks at 37 °C and the temperature variations from 37 to 45 °C. The temperature shock of 5 °C and 10 °C at 45 °C led to slight disturbance and significant disturbance of the performance, respectively. A new insight was achieved on fundamental understanding of membrane fouling with regard to cake layer structure that changes significantly from the top layer to the bottom layer. Additionally, the freshly formed cake layers (2 days) were thinner, higher in EPS concentration, high in porosity, but much lower in particle size distributions (PSDs), as compared to the cake layers of 25 days at 55 °C. The elevated pH shocks, high operating temperatures and temperature shocks induced dispersion and breakage of large sludge flocs. Smaller flocs had a higher tendency to accumulate on membrane surfaces. PSDs act as a key factor to generate high fouling resistance. EPS, SMP, and colloidal particles contents in supernatant were increased with an increase in operating temperature and were positively correlated to the membrane filtration resistance that increased with an increase in operating temperature. PCR-DGGE study showed that there were significant differences in microbial community population density along the cake layer depth. Temperature not only affected the richness of the microbial populations but also the diversity of the microbial community. The results suggest that SAnMBR is a promising technology for pulp and paper effluent treatment and for system closure. However, membrane fouling, particularly cake layer formation, should be minimized. Operating SAnMBR at mesophilic temperatures is more attractive than that at thermophilic temperature, from a membrane fouling point of view.
Dr. Asieh Ahmadalinezhad
Nanomaterial-based electrochemical sensors for the detection of biological compounds
SUPERVISOR: Dr. A. Chen (formerly in Chemistry)
ABSTRACT: Electrochemical detection methods are highly attractive for the monitoring of glucose, cholesterol, cancer, infectious diseases, and biological warfare agents due to their low cost, high sensitivity, functionality despite sample turbidity, easy miniaturization via microfabrication, low power requirements, and a relatively simple control infrastructure. The development of implantable biosensors is laden with great challenges, which include longevity and inherent biocompatibility, coupled with the continuous monitoring of analytes. Deficiencies in any of these areas will necessitate their surgical replacement. In addition, random signals arising from non-specific adsorption events can cause problems in diagnostic assays. Hence, a great deal of effort has been devoted to the specific control of surface structures. Nanotechnology involves the creation and design of structures with at least one dimension that is below 100 nm. The optical, magnetic, and electrical properties of nanostructures may be manipulated by altering their size, shape, and composition. These attributes may facilitate improvements in biocompatibility, sensitivity and the specific attachment of biomaterials. Thus, the central theme of this dissertation pertains to highlighting the critical roles that are played by the morphology and intrinsic properties of nanomaterials when they are applied in the development of electrochemical biosensors. For this PhD project, we initially designed and fabricated a novel amperometric glucose biosensor based on the immobilization of glucose oxidase (GOx) on a Prussian blue modified nanoporous gold surface, which exhibited a rapid response and a low detection limit of 2.5 μM glucose. The sensitivity of the biosensor was found to be very high (177 μA/mM) and the apparent Michaelis–Menten constant was calculated to be 2.1 mM. Our study has demonstrated that nanoporous gold provides an excellent matrix for enzyme immobilization. To adopt these advanced properties, we fabricated a highly sensitive and mediator-free electrochemical biosensor for the determination of total cholesterol. The developed biosensor possessed high selectivity and sensitivity (29.33 μA mM−1 cm−2). The apparent Michaelis–Menten constant, of this biosensor was very low (0.64 mM), which originated from both the effective immobilization process and the nanoporous structure of the substrate. The biosensor exhibited a wide linear range, up to 300 mg dL−1, in a physiological environment (pH 7.4); making it a promising candidate for the clinical determination of cholesterol. The fabricated biosensor was tested further by utilizing actual food samples (e.g., margarine, butter and fish oil). The results indicated that it has the potential capacity to be employed as a facile cholesterol detection tool in the food industry and for supplement quality control. To enhance the stability of the biosensors in the continuous monitoring of glucose, we designed a novel platform that was based on buckypaper. The fabricated biosensor responded to glucose with a considerable functional lifetime of over 80 days and detected glucose with a dynamic linear range of over 9 mM with a detection limit of 0.01 mM. To investigate the effects of the physical dimensions of nanomaterials on electrochemical biosensing, we synthesized TiO2 nanowires with controllable dimensions via a facile thermal oxidation treatment of a Ti substrate. To improve the conductivity of the TiO2 nanowires and to facilitate the immobilization of enzymes, a thin layer of carbon was deposited onto the TiO2 nanowires via a chemical vapour deposition method. Upon the immobilization of glucose oxidase as a model protein, direct electron transfer was observed in a mediator-free biosensing environment. Our electrochemical studies have revealed that the electron transfer rate of the immobilized glucose oxidase is strongly dependent on the dimensions of the carbonized TiO2 nanowires, and that the designed glucose biosensor exhibits a wide linear range, up to 18 mM glucose, as well as high sensitivity and selectivity. Glucose measurements of human serum using the developed biosensor showed excellent agreement with the data recorded by a commercial blood glucose monitoring assay. Finally, we fabricated an enzyme-free glucose sensor based on nanoporous palladium-cadmium (PdCd) networks. A hydrothermal method was applied in the synthesis of PdCd nanomaterials. The effect of the composition of the PdCd nanomaterials on the performance of the electrode was investigated by cyclic voltammetry (CV). Amperometric studies showed that the nanoporous PdCd electrode was responsive to the direct oxidation of glucose with high electrocatalytic activity. The sensitivity of the sensor for continuous glucose monitoring was 146.21 μAmM−1cm−2, with linearity up to 10 mM and a detection limit of 0.05 mM. In summary, the electrochemical biosensors proposed in my PhD study exhibited high sensitivity and selectivity for the continuous monitoring of analytes in the presence of common interference species. Our results have shown that the performance of the biosensors is significantly dependent on the dimensions and morphologies of nanostructured materials. The unique nanomaterials-based platforms proposed in this dissertation open the door to the design and fabrication of high-performance electrochemical biosensors for medical diagnostics.