Dr. Sepideh Dadgar

Design and Evaluation of Novel Fluorescent Molecular Probes Targeting Cathepsin B 

SUPERVISOR: Dr. Wely Floriano
ABSTRACT: Lysosomal cysteine proteinase cathepsin B (CTB) is a member of the cysteine protease family known to participate in intracellular degradation processes and protein turnover in the lysosomes of healthy cells. Cathepsin B plays a crucial role in tumor invasion and progression by controlling extracellular degradation and participating in a proteolytic cascade activation (Gondi and Rao 2013). Its role in tumor invasion and progression makes CTB a promising biomarker and target for antibody-directed prodrug therapy (Dheer, Nicolas et al. 2019). The development of novel CTB-specific molecular probes opens new possibilities for image-based diagnostic methods for different types of cancers (Podgorski and Sloane 2003, Tan, Peng et al. 2013). Since aberrant expression of this protein has been an indicator of cancer development, detecting CTB expression and activity might be beneficial for the early detection of cancer or revealing aggressive lesions (Gondi and Rao 2013). Developing probes capable of binding with CTB is challenging due to binding site homology to other members of the cysteine cathepsin family (Turk, Stoka et al. 2012). In this study, to identify unique residues in human CTB compare to other members of the cathepsin family, amino acid sequences of these proteins were exposed to. multiple sequence alignments. Cathepsin B in humans has three active site residues critical for catalysis: Cys108; His278, and Asp298 (Ruan, Hao et al. 2015) were confirmed with multiple sequence alignment as fully conserved residues. The initial step in the development of a detection assay for CTB is finding appropriate fluorescent small molecules for enzyme binding. In this study, two ligand candidates CID8795 and CID535684 were identified and successfully conjugated to the dye ATTO680 and were tested for binding affinity and specificity to CTB. For CID535684ATTO680, no binding interaction was observed in the fluorescence polarization (FP) assay. CID8795ATTO680 demonstrated increases in fluorescence polarization assays in the presence of CTB with the half-maximal effective concentration (EC50) at 3.27 ± 1.27 nM. A third probe, Benzyloxycarbonyl (Cbz)-Lys-Lys-p-Aminobenzyl alcohol (PABA)-2’, 7’-dichloro-6’-methoxy-fluorescein (DCMF), was designed based on a known substrate scaffold for CTB. This novel substrate-based fluorescent probe was shown to be hydrolyzed by CTB having a specificity constant kcat/KM = 41.9 ± 0.07 mM-1 × s-1. Finally, we investigated single nucleotide polymorphisms (SNPs) within the coding region of the CTSB gene within the general population (random data from 2,504 samples) included in the 1,000 Genomes project. The mapping of SNPs onto the 3D structure of cathepsin B indicates that the active site of CTB is fully conserved among humans – as no SNPs were identified within the binding pocket of CTB. According to these results, probes that bind to the enzyme’s active site should be generally useful for detecting CTB in all populations studied in the 1,000 Genomes project.

Dr. Gabrielle Gaultier

Humoral immunity in patients with chronic kidney disease and their response to pneumococcal immunization

SUPERVISOR: Dr. Marina Ulanova
ABSTRACT: In northwestern Ontario the Indigenous population (First Nations, FN) have an increased prevalence of chronic kidney disease (CKD). There is an increased rate of infectious disease caused by Streptococcus pneumoniae (pneumococcus) and Haemophilus influenzae in the FN population. The 23-valent pneumococcal polysaccharide vaccine (PPV23) and 13-valent pneumococcal protein-polysaccharide conjugate vaccine (PCV13) are used to prevent pneumococcal disease in Canada. Since the implementation of H. influenzae type b (Hib) vaccine programs, invasive H. influenzae type a (Hia) disease has replaced invasive Hib disease in the FN population. However, there is no vaccine to prevent disease caused by non-Hib strains. To maintain immunological memory in adults, stimulation of pre-existing memory is achieved through immunization. To determine if patients with CKD have the ability to respond to conjugate vaccines specific for Hia or pneumococcus, naturally acquired humoral immunity against these pathogens must be evaluated. Immunization with PPV23 is recommended for adults with CKD, however PPV23 is suboptimal at inducing antibody responses in adults with CKD compared to healthy adults. PCV13 is recommended for certain immunocompromised adults, but only two studies evaluated the immunogenicity of PCV13 in adults with CKD with conflicting results. To determine if PCV13 is immunogenic in patients with CKD and the effect of previous PPV23 immunization on subsequent immunization with PCV13, quantification of Bcell subpopulations, pneumococcal specific B cells and their relationship with antibody responses to PCV13 must be evaluated.
The results suggest that the increased prevalence of invasive disease caused by pneumococcus and Hia in the FN population is not due to decreased numbers of B cells or concentrations of naturally acquired antibodies. The increased prevalence of diseases such as CKD causing immune dysfunction in a higher proportion of the FN population increases their risk for invasive disease. This suggests that a new Hia-conjugate vaccine may be immunogenic in adults with CKD, as it will potentially re-activate Hia specific immunological memory. The findings suggest that previous immunization with PPV23 has a negative effect on the humoral immune response that results in long-term changes in B-cell subpopulations and decreases antibody responses to subsequent immunization with PCV13 in patients with CKD. 
Dr. Ashley Nemec-Bakk

Stess-induced fetal programming of adult C57BL/6 mice: Study of cardiac and hepatic glucose metabolism and oxidative stress

CO-SUPERVISORS: Drs. Neelam Khaper and Simon Lees
ABSTRACT: An adverse in utero environment can result in fetal programming that can manifest as diseases later in life. Fetal programming is the hypothesis that the fetal environment can cause genetic alterations and induce developmental changes depending on the type of stress and time of fetal development. This research investigates how ionizing radiation and stress hormone exposure during late gestation can affect development of disease in adulthood in a mouse model. Female and male C57BL/6J mice received 0, 50, 300, or 1000mGy of whole body gamma radiation on day 15 of gestation. Liver and cardiac oxidative stress was assessed by measuring redox sensitive markers and antioxidant protein content and activity. Cardiac metabolism was also assessed by measuring glucose uptake by using positron emission tomography (PET), glycogen content and insulin signaling. Hepatic oxidative stress markers were increased in only female 1000mGy radiation group compared to Sham irradiated mice. Cardiac metabolism was altered in both 300mGy and 1000mGy female hearts. 1000mGy radiation was shown to increase glucose uptake and glycogen storage in the heart but had no effect on Akt protein expression while the lower doses did not change these parameters. In another study, dexamethasone (Dex), a synthetic stress hormone, was administered to pregnant mice during day 15-17 of gestation. Hepatic and cardiac glucose metabolism and oxidative stress markers were also assessed in this study. Dex treatment did not alter plasma glucose or insulin levels and did not induce changes in liver metabolism. Cardiac glucose uptake and glycogen storage were not altered by Dex treatment. Cardiac and liver oxidative stress markers were not altered due to Dex treatment. Therefore, prenatal exposure to non-lethal doses of radiation resulted in programming of hepatic oxidative stress and cardiac metabolism but no significant changes were observed with prenatal exposure to Dex.

Dr. Harutyun Poladyan

Organ-specific Positron Emission Tomography Devices Based on SiPMs

SUPERVISOR: Dr. Alla Reznik
ABSTRACT: In the era of personalized medicine, development of medical imaging devices that are dedicated for specific applications are getting more and more mainstream adoption, and molecular imaging is no exception. Positron emission tomography (PET), in particular, sees prominent advancements in the field of organ-specific applications for breast, brain, heart and prostate imaging. The main driving force of this new paradigm is the benefits that organ specific imaging devices bring in comparison with whole-body general purpose systems. Advantages of organ-specific imagers include: (1) improved spatial resolution and higher sensitivity, which are achieved by placing the detectors closer to the organs of interest, (2) improved image contrast and shorter imaging time that are the result of noise reduction from other organs, and, of course, (3) the reduced cost of the imager, which could be the major limiting factor for its clinical adoption and wide-spread use. The goal of this PhD work is to design and evaluate the performance of a PET block detector to be used in organ-specific PET imagers. The main structural requirements of the block detector are modularity, so that imaging systems of specific shape and size can be assembled, and compactness that will allow integration into multimodality imagers. As for the operational characteristics, energy resolution of 15% and special resolution in the order of 2 mm were chosen as the nominal performance parameter requirements as clinically required for a large number of diagnostic procedures. Achievement of the targeted operational parameters and overall high performance of PET detectors is only possible with an optimized coordinate reconstruction algorithm that must be tuned for a particular detector configuration. This is vital for high spatial resolution, since the errors in coordinate calculations ultimately propagate into tomographic images, reduce spatial resolution and introduce image blur, thus negatively affecting imaging capabilities of the PET scanner. The results of this PhD study demonstrate that the developed position weighted coordinate reconstruction methods provide exceptional coordinate reconstruction accuracy, yielding uniform crystal flood maps and negating the field-of-view shrinkage effects caused by commonly used linear center of gravity methods.

Dr. Mahdieh Samavi

Microbial production of value-added products from wood hemicellulose pre-hydrolysate

SUPERVISOR: Dr. Sudip Rakshit
ABSTRACT: Hemicellulose, the second most abundant polymer in nature, has the great potential to be used for the production of biochemicals under the concept of biorefining.As hemicellulose is water-soluble, it can also be easily obtained by the pre-hydrolysis of wood prior to the pulping processes such as Kraft delignification or during the production of dissolving pulp. Utilization of these byproduct streams can play an important role in the development of a circular bioeconomy as it helps in maintaining the materials and resources for a long period instead of disposing it as waste.In the biochemical conversion platform for biorefineries, it is crucially important to use all fermentable sugars of lignocellulosic biomass including both hexose (C-6) and pentose (C-5) sugars.The overall objective of this study was to produce bioplastic building blocks from hemicellulose streams. The specific objective of this work was to investigate the possibility of using wood-based hemicellulose for microbial production of value-added biochemicals including microbial oil, biopolyol and poly-γ-glutamic acid. The hemicellulose pre-hydrolysate used in this study was produced by a proprietary pretreatment process. Composition analysis of the hemicellulose pre-hydrolysate indicated that it had 143.89 ± 1.28 g/L of xylose along with smaller quantities of sugars like glucose and arabinose.Bioconversion of hemicellulose sugar to microbial oil is one possible way to valorize this industrial side stream. An oleaginous yeast Cryptococcus curvatus (ATCC 20509) was selected for this bioconversion as it is known to accumulate high content of lipids and is able to grow on complex lignocellulosic hydrolysates/pre-hydrolysates even in the present of impurities. Initially, the effect of xylose concentration and carbon to nitrogen ratio were investigated in order to maximize the lipid accumulation. The robust yeast strain used was able to produce 13.78 g/L of cell biomass and 5.13 g/L of lipid after 164 hr of fermentation using poplar wood pre-hydrolysate without detoxification. The obtained microbial oil was characterized to identify its fatty acid profile. Oleic acid (45.86 ± 0.69 wt%) was found to be the main fatty acid present. This fermentation was scaled up in a batch bioreactor with 1 L capacity. 16.54 ± 0.65 g/L of cell biomass and 6.97 ± 0.58 g/L of lipid were obtained in the reactor which had better control of environmental conditions.The microbial oil produced was then used as feedstock for production of bio-based polyol which has many applications in polymer industry and importantly serves as a precursor for polyurethane production. The bio-polyol was produced using a two-step approach: epoxidation followed by ring-opening reaction. Lipase enzyme produced by Candida antarctica (Novozyme 435) and immobilized on acrylic resin was used as an unconventional catalyst for in situ epoxidation of microbial oil.  84.55 ± 1.80% conversion was achieved after 12 hours.  Novozyme 435 was found to be very stable and can be reused up to 3 cycles efficiently. In the second step, Isopropanolamine was used to open the epoxy ring with the addition of hydroxyl group. Hydroxyl value and acid number of the microbial-based polyol were found to be 299.53 ± 1.24 mg KOH/g and 4.93 ± 1.07, respectively. Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) were used for structural confirmation of produced bio-polyol. Production of biobased monomers from renewable materials using enzyme catalysts can be considered clean and leads to energy saving processes. Therefore, synthesis of renewable polymers via enzymatic polymerizations of biobased monomers provides an opportunity for achieving green polymers and a future sustainable polymer industry.The production of another useful chemical, poly-γ-glutamic acid (PGA) was also studied. PGA is known to have a number of useful applications. In order to make this fermentation efficient, the detoxification of the pre-hydrolysate was carried out using a method previously developed in our lab. This detoxification method includes the combination of vacuum evaporation and solvent extraction procedures as a result of which acetic acid and hydroxy methyl furfural (HMF) were removed effectively with minimal loss of xylose. Bacillus subtilis (ATCC 23857), which is a glutamic acid-dependent strain, was used for this bioconversion. Initially, the concentration of pure xylose and L-glutamic acid were optimized by synthetic medium using response surface methodology (RSM). 65.40 g/L of xylose and 44.98 g/L of L-glutamic acid were found to be the optimal concentrations for maximum production of PGA. This strain was able to produce 12.93 ± 0.9 g/L of PGA after 96 hr of fermentation in pre-treated hemicellulose. Such studies on the production of PGA from renewable sources will contribute to further development of biorefineries and lead to commercial scale production of such products.Overall, the findings of this dissertation will contribute to the utilization of available hemicellulose streams through fully biobased processes that can lead to the development of a successful economically feasible circular bioeconomy.

Dr. O'Niell Tedrow

Development of response-predictive laboratory- and field-scale exposure scenarios to site-specific waters and sediments using wild rice

ABSTRACT: Four bioassays were used to expose wild rice (WR) to site-specific waters and sediments within the boundaries of site-specific conditions. Mesocosm- and microcosm- scale bioassays were developed to measure responses of WR to sediment exposures. In mesocosms, WR height (HT), dry weight biomass (DWB), and seed production (SP) were statistically lower for Cleaver and Unnamed Lake sediment-grown plants compared to Rat River Bay sediment-grown plants. An accelerated-growth microcosm-scale bioassay accurately represented the mesocosm-scale bioassay, while decreasing overall time, sediment, water, and space. WR developed to near reproductive maturity. Significant differences were not identified between mesocosm: microcosm ratios for WR DWB or SP, which appeared to be primarily influenced by sediment ammonia-nitrogen concentrations. Rafts were deployed in two select aquatic systems: three in the Seine River (non-industry-influenced); and two rafts in each of three legacy-mine influenced pits to determine the life stage (aerial, floating leaf, submerged) more sensitive to water depth and water depth increases. Based on data obtained during this study, floating leaf plants were determined more sensitive to depth increases; aerial stage was least sensitive to depth increases. WR developed to aerial stage in 20 and 40 cm water depth treatments in all legacy-mine influenced pits indicating no adverse responses to pit waters. Two flow-through WR paddies were constructed adjacent to separate legacy-mine influenced pits with elevated sulphate concentrations. Seeded WR in each paddy developed according to typical phenology during each of multiple successive growing seasons. In the Pit A paddy, no statistical decreases in HT or DWB were observed between growing seasons; average SP statistically increased in 2019; and seed DWB remained statistically similar between growing seasons. Over two successive growing seasons, WR stem density remained statistically similar in the Pit C paddy. Despite conditions potentially conducive to iron sulphide root coating formation, this was not identified via SEM-EDX characterization. No adverse WR responses observed throughout this study were determined resultant of Pit A or Pit C water exposures. Representativeness of natural WR areas is paramount to bioassay data defensibility. Bioassays described herein were designed to represent field conditions to the extent possible given the scale of the bioassay.

Dr. Meijia Zhang

Microalgal-Bacterial Membrane Photobioreactor (MB-MPBR) for Wastewater Treatment and Membrane Fouling Characterization

CO-SUPERVISORS: Drs. Baoqiang Liao and Kam Tin Leung
ABSTRACT: This thesis focused on the effects of transient conditions (combined hydraulic retention time (HRT) and nitrogen/phosphorus (N/P) ratio variation, and solo solids retention time (SRT) variation) on the biological performance and membrane fouling of microalgal-bacterial membrane photobioreactor (MB-MPBR) for wastewater treatment. The results showed that both HRT and N/P ratio significantly affected biomass production, nutrient removal, and biological micromorphology. The reason was attributed to the different nutrient loading rates resulted from the various combinations of HRT and N/P ratio. Controlling nutrient loading rate below the nutrients removal capacity threshold was critical to achieving superior effluent quality meeting the discharge standards. A lower N/P ratio of 3.9:1 led to a more quickly transmembrane pressure increase under the same HRT and total influent nitrogen concentration. Characterization of mixed liquor showed that smaller particle size under the lower N/P ratio was the primary contributor to the faster increase in membrane fouling. X-ray photoelectron spectroscopy, fourier transform infrared spectroscopy, and microscopic analysis demonstrated that the underlying reason for the decreased floc size was attributed to the strengthened competitiveness and overgrowth of microalgae at P-rich conditions. The application of MB-MPBR for the treatment of high strength anaerobic digestion effluent at three SRTs of 10, 20, and 30 d was evaluated. Longer SRT led to a higher biomass concentration and increased total phosphorus removal efficiency, which was attributed to the enhanced surface-adsorption under higher biomass concentration. The total nitrogen removal relied on microalgae assimilation, whereas they nonlinearly correlated to SRT. SRT had significant influences on the particle size distribution and microscopic morphology of microalgal-bacterial consortium. Membrane fouling rate in MB-MPBR nonlinearly correlated with SRT and the highest membrane fouling was observed at SRT of 20 d. It was mainly attributed to the higher concentration of extracellular polymeric substances and soluble microbial products. Environmental stress and fierce competition between microalgae and bacteria were considered as the underlying reason for the increased production of extracellular polymeric substances and soluble microbial products. The results suggest that MB-MPBR is a promising technology for simultaneous removals of organics and nutrients from wastewater. Optimizing the operating conditions to balance the microalgae and bacteria at an appropriate rate is the key to achieve high-quality effluent meeting the discharge standards and effective membrane fouling control in MB-MPBRs.