Chinten James Lim

Acute lymphoblastic leukemia (ALL), a malignancy of the bone marrow that is mainly caused by the uncontrolled proliferation of B- or T-lymphoblasts, is the most common form of pediatric cancer in Canada. Pediatric ALL is currently highly treatable due to advances in contemporary risk-directed ALL chemotherapy. However, some patients will be unresponsive to treatment and prognosis remains poor for patients undergoing relapse. Adverse side effects from drug toxicity used in chemotherapy also remain a major challenge. Recently, the lectin chaperones calreticulin (CALR) and calnexin (CANX), along with their co-chaperone, PDIA3, are increasingly implicated in the biological processes of various human cancers, along with their main role of facilitating protein folding in the endoplasmic reticulum (ER). Here, I investigated the role of CALR and PDIA3 in the promotion of pro-survival JAK/STAT signaling in human T-lymphoblasts. Previously, PDIA3 was found to be required for CALR localization in the cytosol. I generated CALR-/- and PDIA3-/- T-lymphoblasts using CRISPR-Cas9 to assess the effects of deficiency in total and cytosolic CALR on JAK/STAT signaling. CALR-/- and PDIA3-/- cells exhibited reduction in STAT3 phosphorylation compared to wildtype cells. This suggested that cytosolic CALR played a role in the regulation of STAT3 phosphorylation. Unexpectedly, I found that STAT5b mRNA was reduced in CALR-/- and PDIA3-/- Jurkat T-lymphoblasts, leading to an observed deficiency in STAT5 protein expression. This further emphasized the biological significance of CALR and PDIA3 in their extra-ER functions. To assess the clinical significance of CALR, CANX and PDIA3 expression in ALL, I analyzed publicly available transcriptome databases of pediatric ALL, which revealed the potential clinical utility of CALR and CANX mRNA expression in diagnostic tumour samples as biomarkers for predicting treatment response. Elevated CALR and CANX was correlated with the presence of minimal residual diseases (MRD) at the end of induction treatment in T-ALL and B-ALL respectively. As MRD remains a powerful prognostic indicator for risk stratification, the linkage between diagnostic tumour CALR and CANX mRNA expression and MRD status may provide important insights in treatment response for physicians at the time of diagnosis.

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Acute lymphoblastic leukemia (ALL) is the most commonly diagnosed childhood cancer in Canada. Even though childhood ALL has a high overall survival rate, therapeutic options remain limited for those experiencing relapse; therefore, understanding the causes for treatment failure is vital. Adhesion of leukemic cells to bone marrow and the extracellular matrix provides chemotherapy protection to ALL, leading to drug resistance. In this thesis, I investigated the involvement of an endoplasmic reticulum (ER) chaperone, calreticulin (CRT), in integrin mediated cell adhesion. I show that integrin-mediated adhesion of Jurkat T-lymphoblasts is disrupted in CRT-/- cells, and CRT re-expression restores the wildtype phenotype. To determine the requirement of cytosolic CRT for integrin-mediated cell adhesion, I used ERp57-/- cells, previously characterized to express no CRT in the extra-ER compartment. Interestingly, ERp57-/- cells have decreased adhesion compared to wildtype cells, indicating the involvement of cytosolic CRT in integrin-mediated cell adhesion.To evaluate the requirement of LRP1 as a surface receptor for CRT during immunogenic cell death (ICD), I utilized CRISPR-Cas9 technology to generate LRP1-/- lymphoblasts. I show that drug-treated stimulation of surface CRT presentation is lost in LRP1-/- cells, a result consistent with LRP1 as a cis-acting receptor for CRT during ICD. I utilized CRT-/- HEK cells expressing various CRT constructs that are localized in different cellular compartments. All constructs successfully rescued the adhesion defect of CRT-/- cells. Furthermore, I found that mutant variants of CRT that lack the C-terminal KDEL ER retention motif were proteolytically truncated, and that the N-terminal truncated fragment can be detected on the cell surface.

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Cell migration plays an important role in development and the immune system, and the signaling pathways governing this process are regulated in a spatial and temporal manner. One important signaling molecule is cAMP-dependent Protein Kinase (PKA), which has been shown to be a key regulator of migration. In migrating cells, PKA activity exists in a gradient that is highest at the leading edge, where it phosphorylates proteins that promote migration. Although the functions of PKA in migration have been identified, upstream regulators of PKA-mediated cell migration have yet to be defined. A candidate for regulation of PKA during migration is the chemokine receptor CXCR4, which has been shown to induce cell migration and signal through PKA-phosphorylatable proteins upon ligation with stromal derived factor-1 (SDF-1). I have created a novel CXCR4 deficient Jurkat cell line, JC4, and show that Jurkat cell migration towards SDF-1 is CXCR4-dependent. As well, we show through biochemical studies that the SDF-1/CXCR4 signaling pathway is an upstream regulator of PKA activation in CHO-CXCR4 cells, J774 macrophages, and Jurkat T-cells. Furthermore, the SDF-1/CXCR4 signaling pathway is an upstream regulator of PKA activation in migrating cells. This is supported by the observation that there is a CXCR4-dependent increase in PKA-phosphorylated substrates at the periphery of J774 macrophages and protrusions of Jurkat T-cells upon SDF-1 stimulation. Furthermore, SDF-1/CXCR4 signaling establishes a PKA activity gradient in migrating Jurkat cells that is highest at the leading edge. These studies define a regulatory role for the SDF-1/CXCR4 signaling pathway in promoting PKA-mediated cell migration.

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It is estimated that about 26 million metric tons of oil palm oil residues are produced annually in Malaysia. The oil palm residues contain high ash content, high alkali metals and low lignin content. Ash and alkali metals lead to slagging in a combustion reactor and fouling of surfaces in convective tubes of heat exchangers. Low lignin content leads to disintegration of pellets during pellet handling and storage. This research thesis investigated the pelletization performance and the ash reduction on Empty Fruit Bunches (EFB) and Palm Kernel Shell (PKS) of oil palm residues by two different pre-treatment processes: steam explosion and water leaching. Steam explosion for increasing binding ability was performed by reacting EFB/PKS with saturated steam ranging from 120 to 220℃ for 5 minutes in a batch reactor. Water leaching for reducing the ash and alkali metals of EFB and PKS was investigated by immersing the samples in water from 25 to 55℃. The immersion time ranged from instantaneous to 240 minutes. Single pellets were made from water treated and steam treated samples. A significant ash reduction from 5.47 to 2.47 % was found for the EFB pellet due to water leaching. A slight reduction of pellet’s porosity and a significant reduction in ash content suggested that water leaching may be used to improve the quality of EFB as a biofuel. On the other hand, pre-treatment processes were not necessary for PKS, considering low binding ability and no ash reduction after pre-treatments. Kinetic models of EFB and PKS leaching were developed. A mass balance of cross flow process was investigated for evaluating the number of stages for a continuous leaching system of EFB and PKS. The techno-economic analysis showed that the additional leaching process costs $25.75 per tonne in a pellet making line.

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Integrins are heterodimeric, cell-surface receptors that play a role in adhesion and chemoresistance. Integrins can be found in one of two states: an inactivated state where integrin-mediated adhesion is not supported, or in an activated state where integrin-mediated adhesion is possible. CD47 is a cell surface receptor that is said to regulate integrin functions by interacting with integrins and regulating their activation state. A thrombospondin C-terminal motif, RFYVVMWK, has been found to be responsible for cell adhesion and was subsequently shown to be specific for CD47. The 10-mer peptide derived from this sequence, 4N1K, has since been used as the prototypic ligand for CD47 and this interaction has been shown to decrease integrin-mediated adhesion and induce cell aggregation and apoptosis. However, the role of the 4N1K/CD47/integrin axis in chemoresistance has not been investigated. My thesis investigated the consequences of CD47 ligation by 4N1K on integrin-mediated functions.I found that the 4N1K peptide induces binding of a variety of antibodies, including non-specific control antibodies, to the surface of cells. In addition, cells that were deficient in CD47 expression were able to bind substrate-immobilized 4N1K as efficiently as their CD47-expressing parental cells. 4N1K was also found to block cell adhesion and induce cell aggregation in a manner that was independent on CD47 expression. These results suggest that 4N1K may produce artifacts in assays that use antibodies as reporters of integrin activation due to its hyper-adhesive nature, resulting in 4N1K binding to a variety of Ig-containing proteins, such as antibodies and cell-surface proteins. In addition, non-specific binding of 4N1K on cell surfaces and homotypic 4N1K interactions appear to be responsible for many of the observed phenotypes on cell adhesion and aggregation, and may explain the reported CD47-independent effects of 4N1K. As such, I propose that the 4N1K peptide not be used as a ligand to assess the role of thrombospondin/ CD47 interactions on cell functions, since the non-specific adherent nature of 4N1K could lead to erroneous interpretations of experimental data.

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Hygroscopicity and dust generation are among major challenges to the safe and cost effective use of wood pellets. Wood pellets can rot and disintegrate during storage due to the moisture adsorption from humid environment. In British Columbia, pellets are transported by rail from inland manufacturing plants to a shipping port and stored in silos. At the ports, pellets are loaded from the silo on to the ocean vessel for transport to overseas. Loading is stopped during rain because wood pellets disintegrate when they come into contact with water. The lost revenue from loading shutdown during rain can be large. Furthermore, breakage of wood pellets during handling and storage causes dustiness. Dust and fines may cause adverse health effect, fire, and explosion in storage and silos. Encapsulation of wood pellets with a hydrophobic membrane or surface modification can be a good way to increase water repellent capacity of wood pellets, and avoid dust generation. In this research, commercial wood pellets were coated with a wax solution, linseed oil, cellulose acetate, canola oil, etc. The treated pellets were either dipped in water or exposed to humid environment. The results showed that the investigated liquid coatings increased the durability of wood pellets in water. However, the tested liquid coatings did not decrease water vapor adsorption of pellets significantly. Surface treatments with O₂ etching and CF₄ plasma were applied to render wood pellets hydrophobic. It was demonstrated that CF₄ plasma treatment increased water repellency of wood pellets while O₂ etching without CF₄ deposition step made pellets more hydrophilic. Surface modification with O₂ etching combined with CF₄ deposition created the most hydrophobic surface when pellets were dipped in water. However this combined plasma treatment did not decrease water vapor adsorption from humid environment.

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