Wilfred Arthur Jefferies


Relevant Thesis-Based Degree Programs

Affiliations to Research Centres, Institutes & Clusters


Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Deciphering non-coding driver mutations in prostate cancer (2023)

Androgen receptor (AR) mediated signalling is critical to the growth at all stages of prostate cancer (PCa). Hormone stimulated AR binds to tens of thousands cis-regulatory elements (CRE) to activate transcription of specific set of genes through enhancer activity. Intriguingly, there are 100X more enhancers compared to AR- regulated genes. How this complex network of ARenhancers regulate the AR transcriptome remains poorly understood. We have previously shown that ARBS have significantly higher rate of mutations in PCa compared to other TF- binding sites. Given their critical role, these mutations could alter the transcriptional landscape and influence the cancer growth and progression. However, while we can readily identify these non-coding mutations, they are extremely challenging to characterize due to poor functional annotation and limited understanding of how variants impact enhancer activity. In this thesis we developed an experimental and computational framework to stratify non-coding mutations at AR enhancers. Using massively multi-parallel enhancer assays, we functionally tested thousands of common clinical AR binding regions to create a map of enhancer activity for the first time. Using this functional map, we characterized the genomic features associated with active and inactive types of AR enhancers. Next, we developed a new statistical and computational tool to introduce clinically relevant mutations and measure their impact on enhancer activity. Using this system, we interrogated known PCa risk-associated loci and demonstrated that 35% of them harbour SNPs that significantly altered enhancer activity. We also provided a potential mechanism of action for 20 PCa GWAS risk regions. Lastly, we incorporated the enhancer quantification to single cell settings to better understand heterogenous enhancer usage for the first time. Overall, this work laid the foundation to functionally characterize non-coding ARBS variants in PCa at a nucleotide resolution level.

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Investigation of L-type calcium channels as potential targets for cancer therapy (2020)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Mechanism underlying dysregulated cerebral vessel growth in Alzheimer's disease (2019)

Alzheimer’s disease (AD), a neurodegenerative disorder of the elderly, causes loss of memory leading to dementia. The exact cause of this disease is still unknown, and the mechanism of pathogenesis highly debated. Amyloid beta (Aβ) peptide is central to the disease along with the cerebrovascular dysfunction and impaired cerebral blood flow (CBF). A strong link exitsts between brain vascular dysfunction and AD. This link was established due to evidence of reduced blood-brain barrier (BBB) integrity preceding various AD neuropathologies. Furthermore, BBB dysfunction could influence CBF, which in turn influences blood vessel growth. Current dogma holds that BBB leakiness is likely due to vascular deterioration. Inflammatory changes in the AD brain lead to up-regulation of mediators like, VEGF and Tie-2, that initiate angiogenesis. Studies indicate pathological angiogenesis and BBB disruption occur as a compensatory response to impaired CBF. Aβ-induced neuroinflammatory responses promote the generation of reactive oxygen species and further endothelial damage. Aβ is shown to be a modulator of blood vessel density and vascular remodeling via angiogenic mechanisms. Studies on the cerebrovascular integrity in AD model mice showed a significant increase in the incidence of disrupted tight junctions which is directly linked to an increase in microvascular density. This strongly supports amyloidgenesis-triggered angiogenesis as the basis of BBB disruption. This thesis aims in attempting to curb vascular damage seen in the brains of AD mice and improve cognition by treating them with anti-angiogenic drugs providing scope for these to modulate cerebral angiogenesis to ameliorate Aβ load, reduce vasculature damage and reverting cognitive decline. This may facilitate new preventive and therapeutic interventions for not only AD but also related vascular diseases such as small vessel disease. As a part of the study, a mechanism for vascular dysfunction in AD is proposed as follows: Aβ triggers dysregulated blood vessel growth via Angiopoietin-2 mediated activation of the Tie-2 receptor of the angiogenesis pathway. Also demonstrated in this thesis is that AD pathology can be established by a bone marrow transplant from an AD mouse model into an APP knockout mouse suggesting the role of soluble Aβ in AD pathogenesis.

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ABCF1 is a novel E2 ubiquitin-conjugating enzyme that controls Toll-like receptor-mediated innate immune responses and cytokine storm during sepsis (2018)

Immune responses are tightly controlled mechanisms that serve as the primary means by which invading pathogens are eradicated. These mechanisms are governed by the action of several proteins that, when activated, regulate a cascade of downstream effectors in immune cells that help control and, often eliminate the infection. We have discovered a protein, ABCF1, which regulates this immune response cascade through Toll-like receptor (TLR) signaling and helps maintain cellular homeostasis. ABCF1 is the only known member of the ATP-Binding Cassette (ABC) superfamily that lacks a trans-membrane domain and thus doesn’t function as a transporter. Previous studies on ABCF1 indicate a role for this protein in DNA sensing mechanisms and in the pathophysiology of rheumatoid arthritis and autoimmune pancreatitis.We have discovered that ABCF1 is the sole member of the ABC superfamily possessing E2 ubiquitin-conjugating enzyme activity and exclusively regulates TLR4 endocytosis in murine macrophages. In our studies, ABCF1 was found to target Spleen Tyrosine Kinase (SYK) and TNF Receptor Associated Factor (TRAF3) proteins for K63-linked polyubiquitination, thereby regulating the shift from MyD88-dependent to TRIF-dependent signaling. We also observed that ABCF1 negatively regulates MyD88-dependent pro-inflammatory cytokine production in TLR2 and TLR9 signaling, thereby controlling macrophage polarization to the M2 phenotype.ABCF1 was also found to control viral immune responses by regulating OAS1a enzyme activity and negatively regulating ABCE1 thereby modulating RNase L activation. ABCF1 also seemed to control FcγR II-mediated phagocytosis through phosphorylation of SRC Family Kinases (SFKs).Our data also revealed that ABCF1 negatively regulates cytokine storm during the inflammatory phase of sepsis. It associates with TRAF3 and targets TRAF3 for K63-linked poly ubiquitination, thereby controlling the shift from inflammatory phase to immune compromised phase during sepsis. ABCF1 haploinsufficiency was found to trigger lethal renal circulatory dysfunction, which drastically reduced survival rates during the inflammatory phase of sepsis.We herein report the discovery of a novel E2 enzyme, ABCF1, whose E2 ubiquitin-conjugating activity controls TLR-mediated inflammation and cytokine storm during sepsis in murine macrophages.

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Mutation of an L-type calcium channel gene leads to a novel primary cellular immunodeficiency in mice and humans (2018)

Human primary immunodeficiencies are inherited diseases that can provide valuable insight into our immune system. Calcium (Ca²⁺) is a vital secondary messenger in T cells that regulates a vast array of important events including maturation, homeostasis, activation, and apoptosis. The proper orchestration of Ca²⁺ signalling is essential to prevent immune related diseases. Upon antigen binding to the T cell receptor, extracellular Ca²⁺ enters the cell through CRAC, TRP and CaV channels. Here we describe a mutation in the L-type Ca²⁺ channel CaV1.4 leading to a novel immunological disorder. Three CaV1.4-deficient siblings presented with X-linked incomplete congenital stationary night blindness as well as recurrent infections, autoimmunity and pro-inflammatory cytokine production. The subjects uniformly exhibited a T cell memory phenotype and T cell exhaustion as well as chronic activation of their B cells. Moreover, their T cells but not B cells exhibited a reduced Ca²⁺ flux, compared to healthy control donors. This is the first example where the mutation of any CaV channel causes a primary immunodeficiency in humans and establishes their physiological importance in the immune system. In parallel, we detected a remarkably similar phenotype in a CaV1.4-deficient mouse model. In a separate set of experiments, a commercially available C57BL/6 mouse strain harbouring an undescribed mutation in Dock2, was introduced into our breeding stock resulting in some mice with a double deficiency of DOCK2 and CaV1.4. This provided the opportunity to assess the compound phenotype. We found that the double-deficient mouse model exhibited severe splenic cytopenia as well as chronic B cell activation but impaired BCR-induced activation / Ca²⁺ mobilization.

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Studies on immune escape mechanism in cancer (2017)

Metastatic cancer is the leading cause of death in Canada. The personalized medical framework considers each patient’s genetic profile as a background to develop a specific treatment approach. Due to the success of early diagnostics, the mutational landscape of a tumour is mainly based on the sequencing data collected from early resected primary tumours, which do not necessarily reflect the mutational heterogeneity of the metastatic form of the disease and/or local recurrences underestimating tumour adaptational variations, challenging biomarker development and hindering therapeutic strategies of personalized medicine. This study has been designed to define the changes in metastatic potential of a developing tumour highlighting the immunological tumour properties, as one of emerging cancer hallmark. Microarray profiling of two separate paired cell lines of murine lung and prostate carcinomas allowed the detection of IFI44 and IL-33 as possible regulators of immunological properties within tumours. Significant down-regulation of these genes allows cancer cells with high metastatic potential to acquire capabilities to avoid destruction by the immune system via suppression of MHC-I expression. The immune-evasive phenotype allows tumour to obtain biological advantages resulting in the evasion of eradication by the immune system, which is a significant barrier for tumour growth and progression. Further study found that the overexpression of these selected genes reverses the antigen presentation deficiency in murine metastatic lung carcinoma and makes the tumour recognizable to the immune system. A parallel human study demonstrated that the expression of IL-33 is also co-regulated with HLA-I levels in human prostate cancer. Moreover, IL-33 by itself may be used as an immune prognostic biomarker for recurrence and survival in human prostate and kidney renal clear cell carcinomas. This new link in cancer biology allowed for the development a novel immunotherapeutic strategy for cancer-free survival via IL-33/ILC2 axis and the use of adoptively-transferred ILC2s. Testing this strategy on ILC2-deficient animals and animals that received ILC2s via adoptive transfer showed the importance of IL-33 and ILC2s in reducing tumour growth rate and metastatic spread to distal organs. Collectively, this thesis demonstrates a new mechanism for tumour immune escape and suggests a novel immunotherapeutic approach for anti-cancer treatment.

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The creation of an avian vaccine for West Nile virus (2012)

West Nile Virus (WNV) arrived in North America in 1999 and has since caused significant morbidity and mortality, mainly in birds but also in horses and humans. Many families of birds, especially corvids, are highly susceptible to WNV, with infections often resulting in fatalities. Avian species susceptible to WNV infection also include endangered species, such as the Greater Sage-Grouse (Centrocercus uropbasianuts) and the Eastern Loggerhead Shrike (Lanius ludovicianus migrans). Although WNV is now endemic throughout the continent, to date there is no veterinary vaccine available for birds. This thesis focuses on the use of a recombinant adenovirus to construct vaccines against WNV, that would contain either the envelope or the NS3 ‘genes’ from WNV. To assist in assessing the vaccines, work was undertaken to assess to what extent avian antibody reagents could be used in an avian species for which the antibody was not created. The duck specific CD8 antibody, Du-CD8-1 and the chicken/turkey specific CD4 antibody, CT4, bound to Japanese Quail T cells. The CD4 and CD8 antibody reagents were used to analyse Japanese quail T cell populations, establishing the proportions of CD4+ and CD8+ cells, and discovering a previously unreported population of CD4/CD8 double positive cells. An anti bird IgG antibody was found to bind to chicken, House Sparrow and Japanese Quail IgY; the anti-bird IgG antibody was able to detect IgY from these three species when used as part of a serum ELISA assay. Results from initial vaccine testing in Japanese Quail (Coturnix japonica), indicated that the vaccines activated more T cells and triggered production of higher levels of antibodies in vaccinated birds compared to unvaccinated controls. This was achieved using an intracellular interferon gamma (IFN-γ) assay to assess T cell activation and a serum ELISA to measure levels of WNV specific antibodies. During a challenge assay, using a wild population of House sparrows (Passer domesticus) following infection with WNV, vaccinated birds showed overall reduced levels of viremia compared to unvaccinated controls.

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Characterization of cerebral vascular abnormalities in an Alzheimer's disease mouse model (2011)

Alzheimer’s disease (AD) patients suffer progressive neurodegenerative loss of memory and other intellectual abilities leading to Dementia, the exact cause of this disease is still unknown. A central pathological hallmark of AD is the presence of an aggregated amyloid-beta peptide (abeta). A strong link between brain vascularity dysfunction and AD exits due to evidence of reduced blood-brain barrier (BBB) integrity preceding other AD neuropatholgies. Furthermore, BBB dysfunction could influence cerebral blood flow, which could in turn influence blood clotting mechanisms during AD. Current dogma holds that AD BBB leakiness is likely due to vascular deterioration and apoptosis. I propose an alternative hypothesis: angiogenesis and hypervascularization underlie increased vascular permeability in AD. Cerebrovascular integrity was characterized in Tg2576 AD model mice by examining the expression of tight junction (TJ) proteins (occludin and ZO-1) with markers of apoptosis and angiogenesis. In aged AD mice, a significant increase in the incidence of disrupted TJs was directly linked to an increased microvascular density, but not apoptosis, which strongly supports hypervascularity as a basis for BBB dysfunction. My results demonstrate that AD related BBB disruption is due to neoangiogenesis, resulting in the redistribution of TJs that maintain the barrier thus providing a new paradigm for connecting vascular remodelling with AD. Unique atypical nonvascular TJ expression was also noted in the aged Tg2576 mice including “halos” of ZO-1 expression surrounding dense-core abeta plaques and occludin expression on a subset of astrocytes sometimes associated with plaques. The observed brain TJ-related pathologies appeared to be linked to the presence of abeta. This argument wasiiistrengthened by observations of Tg2576 mice treated with abeta immunotherapy, which showed reduced brain abeta levels. Tg2576 mice actively immunized with abeta had normal BBB TJ morphology, no apparent occlusions, normal angiogenesis and lacked the unique atypical nonvascular TJ expression. An examination of the general status of the clotting abilities of the Tg2576 mouse was examined. Whole blood from aged Tg2576 mice clotted faster than controls. However, the biochemical components of the clotting cascades were normal, suggesting potential platelet involvement. Taken together, the observed BBB abnormalities will provide new entry points for therapeutic intervention.

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The function of ABCF1 in immunity and mouse development (2011)

ABCF1 is an ABC (ATP binding cassette) transporter protein that lacks trans-membrane domains. Gene expression of ABCF1 has been shown to increase upon tumour necrosis factor alpha (TNFα) stimulation [1]. This is significant as TNFα is a pro-inflammatory cytokine produced by macrophages and T cells and has a number of functions in the immune response [2]. ABCF1 is thought to function in translational initiation by interacting with the eukaryotic initiation factor 2 (eIF2) [3]. We have determined that ABCF1 is an essential gene in development by the generation and characterization of mice that have a gene trap insertion in the ABCF1 gene, which terminates the expression of the ATP binding cassettes. Homozygous ABCF1 knock-out (ABCF1-/-) mice are embryonic lethal at 3.5 days post coitus (dpc) while heterozygous ABCF1 knock-out (ABCF1+/-) mice appear to be developmentally normal. This thesis utilizes the ABCF1 gene trapped mouse model which contains a β-geo (β-galactosidase /neomycin) reporter gene in the ABCF1 gene to examine the endogenous ABCF1 promoter expression. This allows us to concurrently observe the activity of the ABCF1 promoter in all tissues through sectioning and X-Gal staining. This analysis provides further insight into the physiological function of ABCF1 by identifying the tissues and cell types that have the highest levels of promoter activity. Interestingly, ABCF1 appeared to be expressed in the marginal zone of the spleen and areas surrounding the lymphoid follicles.Macrophages, which were isolated from spleens of ABCF1+/- mice, were found to be hyper-responsive to stimulation by TLR ligands, particularly LPS, while macrophages derived from the bone marrow were found to be hypo-responsive. When challenged with LPS, ABCF1+/- mice produced altered cytokine production compared to their wild-type controls. Upon challenge with Listeria monocytogenes, ABCF1+/- mice succumbed to infection sooner than their ABCF1+/+ littermates. Taken together, these data indicate that ABCF1 is necessary for survival and development and likely has a role in the regulation of cytokines. Thus ABCF1 may play a significant role in regulating inflammation and pathogen induced “cytokine storm”.

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The requirement for competent antigen presenting dendritic cells and poised T cells for immune responses (2011)

Immune responses are initiated by dendritic cells (DCs) that cross present exogenous antigen to naïve T cells. DC cross presentation is essential for generating primary immune responses, yet the mechanistic details remain undefined. Using a CD74⁻/⁻ mouse model, a CD74-MHC I association was shown to mediate trafficking of MHC I from the endoplasmic reticulum to endolysosomal compartments for antigenic loading. These studies describe a novel CD74-mediated cross presentation pathway in DCs that plays a major role in the generation of cytolytic T lymphocyte (CTL) responses against viral and cell-associated antigens. Viruses such as Human Immunodeficiency Virus (HIV) have evolved mechanisms to interfere with immune activation allowing persistence in the host. HIV can infect DCs so the HIV virulence factor, Nef, which interacts with MHC I, has the potential to interfere with MHC I trafficking in the cross presentation pathway. Using a Nef-expressing DC line, Nef was shown to downregulate surface MHC I by inhibiting Golgi-to-surface transport of newly synthesized MHC I and by increasing the recycling of surface MHC I. Coordinately, Nef was shown to inhibit both direct and cross presentation of viral and soluble antigen. Similarly, in a Nef transgenic mouse, Nef was shown to inhibit CTL responses to bacterial and viral infections. This unique immunosubversion mechanism likely contributes to immunodeficiency associated with Acquired Immunodeficiency Syndrome. Peripheral pools of naïve T cells capable of responding to DC stimulus are maintained through homeostatic cues including TCR signalling. Key to this is the second messenger calcium (Ca²⁺); however, the identity of the components regulating intracellular Ca²⁺ concentrations is unclear. Through examination of a knock-out mouse model, the Ca²⁺ channel, CaV1.4, was shown to play a cell-intrinsic role in naïve T cell development and survival. CaV1.4 is critical for regulation of intracellular Ca²⁺ stores and for TCR-induced increases in cytosolic Ca²⁺, which impacts Ras/ERK and NFAT activation. The CaV1.4 deficiency causes a loss of naïve T cells and results in immunodeficiency. These studies reveal a critical function for CaV1.4 in naïve T cell homeostasis. Collectively, this thesis demonstrates the importance of cross presenting DCs and maintenance of T cells for functional immunity.

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The cellular and functional role of melanotransferrin in melanomas (2009)

Melanoma, one of the deadliest forms of skin cancers, is a malignant tumorderived from abnormal proliferation of epidermal melanocytes, and its formation involvesa series of events leading to altered cellular properties and rapid proliferation. Highcellular iron content has been connected to the development of cancers in human. Oneway iron may affect cancer development and progression is by altering cell growth andproliferation. Iron has been proposed to promote progression from G₁ to S phase of thecell cycle through activation of ribonucleotide reductase during DNA synthesis. Iron isnormally absorbed by the enterocyte of the duodenum where it is transported throughoutthe body via serum transferrin. However, the existence of transferrin-transferrin receptorindependent iron transport pathways has been shown. Such pathways are thought to playcritical roles in non-transferrin bound iron overload in diseases such as hemochromatosisand Alzheimer’ s disease, though the molecular details of these pathways remain obscure.Many genes are up-regulated to aid the abnormal proliferation of cancer cellsand subsequent invasion of tissues. In the case of melanoma, the expression ofmelanotransferrin (p97), an iron binding molecule, is elevated. In this thesis, the cellularand functional role of glycosyiphosphatidylinositol (GPI)-anchored p97 in melanoma wasinvestigated. The first aim of this thesis was to perform detailed investigation of theinternalization pathway of GPI-anchored p97 in melanoma cells. Although many GPI anchored proteins have been studied previously, there is still an on-going debate whether caveolae-dependent or clathrin-dependent pathways are involved. Using confocal immunofluorescence microscopy and quantitative immunoelectron microscopy, iron bound GPI-anchored p97 was shown to be internalized via a caveolae vesicles dependent endocytotic pathway. In addition, endosomal disruption studies demonstrated that theintracellular trafficking of the GPI-anchored p97 in melanoma cells is endosomal dependent. The studies performed in this thesis demonstrate that GPI-anchored p97 protein can mediate the iron uptake in melanoma cells. As GPI-anchored p97 is highly expressed in melanoma cells, this thesis also examined the functional role of GPIanchored p97 in melanoma cells and tumors. The expression of GPI-anchored p97 in melanoma cells through over-expression and down-regulation techniques. The results demonstrated that the GPI-anchored p97 promote melanoma cell proliferation, melanin production and secretion in vitro and melanoma tumor growth in vivo. Taken together, this thesis sheds new light on the relationship between GPI-anchored p97, melanomadevelopment and cellular iron uptake.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Characterizing ocular pathology in an animal model of Alzheimer's disease (2021)

Previous studies have characterized common pathological hallmarks of Alzheimer’s disease such as amyloid accumulation and plaque consolidation in the brain. The difficulty in accessing the brain in vivo has remained a barrier to the diagnosis or measuring the progression of the disease. Studies have shown that, in addition to amyloid deposition, angiogenesis and increased blood-brain barrier permeability are present in the brain of Alzheimer’s patients and animal models. Our study investigated whether these pathologies could be recapitulated in a more accessible organ associated with the central nervous system: the eyes. Using the Tg2576 model of Alzheimer’s disease, a transgenic mouse model which expresses human amyloid, an increase in amyloid was seen in the eyes of these mice and it was determined that the amyloid coalesced primarily around retinal blood vessels. This link between amyloid and ocular vasculature prompted a pilot study using a proteome profiler and found increased proangiogenic factors in the eyes and brains of the Tg2576 mouse, 20 proteins of which were upregulated in both organs. Performing further analyses of functional pathways on these findings revealed several reoccurring proteins in several pathways across both organs, including chemokines and fibroblast growth factors. Additional evidence of angiogenic processes was determined via the increased expression of CD105, a marker of neoangiogenesis, in whole eye homogenates of Tg2576 mice. When the distribution of the increased vasculature was examined, while the Tg2576 retina did not have increased amounts of CD105⁺CD31⁺ microvessels, the choroid did have more CD31⁺ vessels. Importantly, expression of tight junction proteins zonula occludens-1 (ZO-1) and occludin was decreased in the Tg2576 retinal pigment epithelia and in whole eye homogenates, respectively. Along with evidence of an increased presence of albumin in whole eye homogenates, these results suggest that there is increased permeability in the Tg2576 eyes. Our findings provide new insights into concurrent pathologies exhibited in a commonly used Alzheimer’s mouse model, suggesting that the eyes may be used as a proxy for brain pathology.

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Elucidating the mechanism of type 2 innate lymphoid cell-mediated tumour elimination (2020)

Besides amplifying type 2 immune responses, the role of type 2 innate lymphoid cells (ILC2s) in cancer remains ambiguous. While some studies suggest that ILC2s exert immunosuppressive functions, others present evidence they enhance anti-cancer immunity. However, their successful isolation from mammal tissues and in vitro expansion continues to pose challenges. This is partly due to their scarcity compared to other leukocyte populations, but also because our current knowledge on ILC2 biology is incomplete. In this study, we focused on ST2+ IL-25Rlo lung resident ILC2s and demonstrate for the first time how mouse type 2 innate lymphoid cells can be cultured, grown and expanded in serum-free media containing interleukin-33 (IL-33), TSLP, IL-2 and IL-7. Moreover, we report that ILC2s from the lungs of mice with primary tumours are able to cross-present the ovalbumin peptide OVA257-264 and effectively cross-prime a population of OT-I cells. Our data also demonstrates that ILC2s from naïve mice are less efficient at antigen cross-presentation, suggesting a plastic cellular identity in the presence or absence of a tumour. To further our understanding of ILC2s harvested from mice with primary tumours, our single-cell RNA sequencing analyses reveal a highly heterogeneous population with several subsets. One of them downregulates markers associated to type 2 identity such as GATA-3, IL-5 or IL-13, but also begins to express pro-inflammatory markers like granzyme B, IFNγ or interferon-induced membrane proteins. We hypothesize that systemic inflammation or a tumour environment can trigger differentiation of a portion of the lung resident ILC2 population into a pro-inflammatory subset. The existence of these cells challenges current paradigms of ILC2 biology and implies the participation of a distinct agent in the process of cancer immune surveillance.

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Use of small molecule activity in upregulation of Class I MHC and change in tumour cytokine profile in metastatic murine epithelial carcinoma (2019)

Cancer is a devastating disease that is responsible for the death of a quarter of Canadians. Cancer is the result of cells that gain genetic mutations or epigenetic changes that incur abnormal, uncontrolled proliferation. Oftentimes, such mutations give rise to the metastatic form of cancer, which is capable of moving throughout the body to distant sites. Adaptive immunity, including Cytolytic T cells (CTLs), are intended to kill tumours based on tumour specific antigen presented on Major Histocompatability Complex (MHC) Class I. However, metastatic tumours are capable of escaping such death through the downregulation of Antigen presentation machinery (APM), including MHC Class I. Here, we investigate the role of the small molecule Cannabigerol in upregulating class I MHC and altering cytokine signalling in metastatic tumours, and the consequent CTL tumour killing ex-vivo. Additionally, we determined the impact of Cannabigerol on immune infiltrates and tumour burden in vivo. Small molecules which enhance the efficacy of the CTL killing through upregulation of tumour-antigen bound MHC class I killing would be beneficial for therapeutic use through increased tumour killing, as well as reduced tumour metastasis, and would result in a prolonged host survival.

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Induction of the Antigen Presentation Machinery using Novel Small Molecules (2017)

The immune system is crucial in the prevention and eradication of cancer. However, cancer genomes mutate more frequently than healthy cells and a commonly acquired phenotype is reduced expression of the antigen presentation machinery (APM) that is required for immunosurveillance. This phenotype can allow cancer cells to become invisible to the immune system and metastasize with limited inhibition. Since this phenomenon is seen across a wide variety of cancers discovering methods to reverse this immune evasion could lead to the development of widely applicable therapeutics.A compound, S-(+)-curcuphenol was previously identified as a novel candidate for restoring expression of the APM, however, its synthesis is greatly hindered due to chirality. Therefore, I investigated the ability of curcuphenol analogues to induce expression of the major histocompatibility complex I (MHC-I) in a murine metastatic lung carcinoma cell line in vitro. Two derivatives of curcuphenol, P02-113 and P03-97-1, showed improved outcomes in vitro and I further evaluated them in vivo, as anti-cancer therapeutics. Both compounds reduced tumour burden and increased immune cell infiltration into tumours.To explore a potential mechanism of P02-113 and P03-97-1, I evaluated them for histone deacetylase (HDAC) inhibition based on structural similarity to established HDAC inhibitors. Upon examination I observed a completely novel effect of enhanced class I/II HDAC activity. Furthermore to identify if this effect was direct and to uncover which HDAC enzymes were being affected, I evaluated individual HDAC enzymes. I discovered that HDAC8 was inhibited by both P02-113 and P03-97-1, however, the activities of HDAC 5 and 10 were enhanced upon treatment.While the field of cancer immunotherapy has grown, few therapeutics have been identified to target loss of immunogenicity through the up-regulation of the APM. In this thesis two analogues of curcuphenol P02-113 and P03-97-1 have been identified that up- regulate the APM in vitro and reduce tumour burden in vivo. With these effects P02-113 and P03-97-1 hold great potential as future therapeutics for cancers exhibiting an immunodeficient phenotype through loss of the APM. As well these compounds presentiithe first class II HDAC enhancers, as they increase the enzymatic activity of HDAC 5 and 10.

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Altered clot formation and anticoagulation in a familial Alzheimer's disease mouse model (2013)

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading form of dementia. Its complex etiology is traditionally attributed to an increase in the production and aggregation of amyloid-beta (Aβ). However, vascular risk factors and related disorders are also associated with the development of AD. The mechanism by which these deficiencies lead to neurodegeneration remains unclear. An altered hemostatic state has increasingly been implicated in AD pathogenesis, with the majority of research focusing on the interaction between Aβ and fibrin. It was therefore of interest to assess previously uncharacterized components of coagulation and anticoagulation in a familial AD (FAD) mouse model. Clot formation was initially analyzed using platelet rich plasma, with aged AD mice exhibiting 40% shorter clotting times compared to age-matched controls. Thrombin generation revealed differences attributable to an altered clot integrity. AD mice may form clots composed of a dense network of thin fibrin strands, resistant to fibrinolysis. Correspondingly, antithrombin (AT) activity was also reduced. These changes were subject to age, occurring specifically in aged mice only. These data suggest a change in clot formation and integrity concomitant with the development of AD. The interaction between Aβ and AT was examined in vitro, with Aβ42 and Aβ40 decreasing AT activity in a concentration dependent manner. A clinical study evaluating prothrombotic markers revealed a trend towards lower AT activity in AD patients, though not statistically significant. The changes in clot formation and anticoagulation potentiating a prothrombotic state may be specific to the FAD mouse model, but may still contribute to the elucidation of AD pathogenesis.

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Cross presentation of oral antigens for induction of CD8z T cell responses (2012)

The biggest challenge of intestinal immunity is inducing tolerance towards harmless antigens derived from food and commensal bacteria while maintaining protection against dangerous pathogens. The dendritic cells (DCs) found in the intestine are crucial to maintaining intestinal homeostasis. DCs are constantly sampling antigens, such as food antigens, found in the intestine and present them to CD8⁺ T cells, which are generally known for killing infected cells. CD8⁺ T cells have been shown to be involved in pathogenesis of some cases of inflammatory bowel disease. Little is known about how these T cells respond towards the presentation of oral antigens derived from food. Therefore, it is important to understand the mechanisms involved in preventing the induction of harmful CD8⁺ T cell responses towards food derived antigens. For this purpose, CD8⁺ T cells with a transgenic T cell receptor that recognizes the food derived antigen ovalbumin were analyzed. It was demonstrated that ovalbumin given to mice orally induced CD8⁺ T cell activation and proliferation. Although the responding CD8⁺ T cells lacked cytolytic activity, they were induced to produce IFN-γ. This observed CD8⁺ T cell activation and proliferation is known to require cross presentation of oral antigens by DCs. Two cross presentation deficient mouse models, ΔYKb and CD74-/-, were used to assess mechanisms in the cross presentation pathway of oral antigens. The ΔYKb mice have a mutated endocytosis motif on MHC-I required for cross presentation and CD74-/- mice lack the CD74 chaperone protein. A low dose of oral ovalbumin resulted in reduced antigen specific CD8⁺ T cell proliferation and activation in the ΔYKb mouse strain only. Given the importance of the endocytosis motif for efficient cross presentation of oral antigens, human MHC-I alleles were examined for possible polymorphism in this motif. All analyzed human MHC-I alleles were conserved in the endocytosis motif. Collectively, this thesis demonstrates that cross presentation is an important pathway for antigen presentation and required for induction of CD8⁺ T cells activation and proliferation towards oral antigens. This mechanism might contribute to intestinal homeostasis and oral tolerance.

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The role of Cav1.4 calcium channel in T cell activation, proliferation, effector functions and death (2010)

T lymphocytes are an important part of the immune system that identify and destroy foreign antigens in the body as well as activate and deactivate other immune cells. In T lymphocytes, calcium is a secondary messenger that regulates activation and proliferation, effector function, survival and death. Although calcium release from the intracellular stores within T lymphocytes is well characterized, the calcium entry pathway from extracellular sources into T lymphocytes is unclear, despite contributing to the majority of elevated intracellular calcium ions during T lymphocyte activation. Preliminary studies have shown that L-type calcium channels play significant roles in the calcium influx pathways, mediating T lymphocyte activation and proliferation in vitro. Cav1.4 L-type calcium channel has been found to be expressed in both mouse and human T lymphocytes. To date, three Cav1.4 calcium channel splice variants have been identified with differential expression throughout the T lymphocyte proliferation process. I hypothesized that pore forming subunit of a L-type calcium channel, Cav1.4, regulates T lymphocyte activation and proliferation in vivo. To test this hypothesis, I used loss of function L-type calcium channel knock-out (KO) mice lacking the entire gene coding for this calcium channel and its splice variants to study its effects on T cell activation, proliferation, death, calcium uptake, and effector responses. From these studies, I predict that the lack of L-type calcium channels will cause T lymphocyte activation, proliferation and development to be impaired. These studies shed light on the mechanism of T lymphocyte activation and also enabled us to better understand how antagonistic drugs such as nifedipine may cause immunosuppressive effects.

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