Wilfred Arthur Jefferies

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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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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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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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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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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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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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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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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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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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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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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