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.