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Antibiotic-resistant bacteria and their often-untreatable infections are increasing globally. The determinants that confer reduced susceptibility to antibiotics are encoded in the genetic information, or genes, of bacteria. Sequencing-based approaches to identify antibiotic resistance genes (ARGs) in diverse environments, such as the gut microbiome, are limited in throughput and inaccessible due to high costs. The microbiome and its associated ARGs are shaped by many factors throughout early life, which have yet to be fully characterized. In this thesis, I developed an approach to capture ARGs in the gut microbiome and study the effect of various early-life exposures. First, I describe a set of over 37,000 probes to target over 2,000 ARGs. This targeted sequencing approach was designed and validated against samples of DNA isolated from multi-drug resistant bacteria. This probeset proved superior to metagenomic shotgun sequencing in capturing the rare portion of ARGs from a human gut microbiome sample. Second, I investigated the potential benefits of providing probiotics to infants born preterm in reducing antibiotic resistance in their gut microbiomes. Preterm infants that received probiotics had a reduced burden of ARGs associated with potentially pathogenic bacteria at 5 months of age compared to non-probiotic-supplemented preterm infants. Finally, I explored the potential consequences of macrolide antibiotic exposure on ARGs in the gut microbiome of children with diarrhoea in Botswana, Africa. Compared to the standard treatment for diarrhoea, a three-day dose of azithromycin did not result in an increased selection for ARGs after 60 days. This thesis tackles the challenge of comprehensively detecting antibiotic resistance in the gut microbiome. A better understanding of the impact of early-life exposures, including probiotic and antibiotic treatments, in the prevention or maintenance of ARGs in the gut microbiome of children will help reduce unnecessary selective pressures and the persistence of antibiotic resistance into later life. Thesis Doctor of Philosophy (PhD) Infection rates associated with antibiotic-resistant bacteria are increasing. The traits that allow bacteria to survive antibiotics are encoded by their genetic information or genes. There is a wide diversity of antibiotic resistance mechanisms available to bacteria and comprehensive methods to detect them are needed. My work presents the development and validation of an approach to capture and sequence all known antibiotic resistance genes. With this tool, I investigated the early life exposures that shape antibiotic resistance in the diverse community of bacteria encompassing the human gut microbiome. First, I revealed the protective effects of providing beneficial bacteria, or probiotics, to preterm infants. Next, I uncovered a minimal impact of antibiotic treatment in selecting for antibiotic resistance genes in children with diarrhoea. Improved tools to understand antibiotic resistance in early-life and the factors that modulate its prevalence will help reduce the persistence of antibiotic-resistant bacteria in later life.

Antibiotics are one of the most important advances in medical science, but today, antibiotic-resistant bacteria threaten this legacy. We risk losing our ability to treat acute infections, perform invasive surgeries, and exploit immunosuppressive therapies like transplantation and cancer chemotherapy. The antibiotics we use today have ancient roots and have been produced by microbial denizens of the soil for millions of years before we adopted them in the 20th century. This history has modern consequences, as strategies to resist these compounds have evolved in concert for millions of years. The result is a vast reservoir of antimicrobial resistance that exists in environmental bacteria, which have the potential to be mobilized into human pathogens and cripple our antibiotic arsenal. Here, I set out to deepen our understanding of the environmental resistome, focusing on the rifamycin antibiotics. These compounds inhibit bacterial RNA polymerase and are frontline agents for treating tuberculosis. Environmental bacteria from the phylum Actinobacteria induce the production of resistance enzymes in response to these compounds. Although mechanistic questions remain, we demonstrate that this induction stems from the inhibition of RNA polymerase by rifamycins. The induction process is known to require a specific DNA motif; here, I identify additional sequences as part of this motif and use this information to map inducible rifamycin resistance across the entire phylum. The most common rifamycin-inducible gene was an uncharacterized family of proteins annotated as DNA helicases. I investigated these proteins and discovered that they bind to RNA polymerase and displace rifamycin antibiotics, a novel mechanism of rifamycin resistance. Lastly, we repurposed this inducible system to develop an assay to screen for novel RNA polymerase inhibitors. From this screen, we identified a rifamycin immune to a common environmental resistance enzyme and a new family of rifamycin antibiotics. Thesis Doctor of Philosophy (PhD) Our antibiotic arsenal consists primarily of metabolites produced by soil microbes, which humanity repurposed into life-saving medicines in the 20th century. As a direct result of the natural origin of antibiotics, resistant bacteria exist in these same environments, independent of human use. Individual genetic determinants from this reservoir can emerge in pathogenic bacteria without warning and render antibiotics ineffective. The aim of this work was to understand how environmental bacteria resist the rifamycin class of antibiotics. Firstly, I investigated the ability of some bacteria to sense the presence of rifamycins, and in response produce proteins to protect themselves. I discovered that this process requires specific DNA sequences nearby resistance genes. Using this DNA sequence as a guide I cataloged resistance genes in thousands of bacterial genomes and discovered a new mechanism of rifamycin resistance. Lastly, I exploited this rifamycin sensing system to discover new antibiotics from soil microbes.

Alzheimer’s Disease (AD) is the most common neurodegenerative disorder worldwide. The risk of developing AD is influenced not only by genetic factors, but also the environment. The multi-factored pathogenesis that leads to AD development poses a challenge for identifying causal factors that promote or protect against neurodegeneration. We use Caenorhabditis elegans as a model of AD to measure the impact of human microbiota species on AD-related phenotypes. The two hallmarks of AD are amyloid-β (Aβ) plaques and neurofibrillary tangles composed of the protein tau. Using a C. elegans model overexpressing Aβ, we identified a group of Enterobacteriaceae species that significantly reduce paralysis. To validate these findings, we used another C. elegans model that pan-neuronally expressed aggregate-prone tau (AP-tau). We observed decreased neurodegeneration in response to most of the same bacteria protective against Aβ-induced paralysis, providing additional evidence of microbiota-promoted neuroprotection. To explore the underlying host mechanisms, we examined gene expression changes in animals exposed to neuroprotective bacteria. Numerous biological processes were differentially regulated in response to the neuroprotective microbiota species, including innate immunity, stress responses, and protein phosphorylation. Several C. elegans orthologs of human tau tubulin kinase genes, TTBK1 and TTBK2, were downregulated in response to neuroprotective microbiota species. RNAi-mediated knockdown of C. elegans ttbks sufficiently induced neuroprotection in AP-tau animals. Further, Enterobacter caused decreased abundance of a tau species phosphorylated at S422, a TTBK1 direct phosphorylation site. These findings suggest that species from the human microbiota can reduce tau phosphorylation, and mediate neuroprotection through downregulation of ttbk. Overall, by studying the impact of the human microbiota on models overexpressing Aβ or AP-tau, we have uncovered a potential mechanism by which microbiota-mediated neuroprotection can occur. In doing so, we also gain a greater understanding of conserved pathways involved in gene-environment interactions promoting development of AD. Dissertation Doctor of Philosophy (PhD) Alzheimer’s Disease (AD) is the most common neurodegenerative disorder in the world. Causes and preventions of AD are difficult to find because many things affect neurodegeneration. For example, resident gut bacteria can change brain health. To study this, we use Caenorhabditis elegans worms as animal models that display traits of AD. C. elegans eat bacteria, have a nervous system, and share many genes with humans. We identified bacteria from the human microbiota that improved neuron health in C. elegans AD models. We examined C. elegans gene expression, and found a group of genes, tau tubulin kinases (TTBKs), were shut down by these bacteria. Removal of the TTBK gene from C. elegans improved nervous system health. Since we observed that TTBK is shut down in response to the microbiota, and loss of TTBK improves neuron health, we have found new evidence for how the microbiota can promote a healthier nervous system.

Recent studies have associated several pesticides, food packaging chemicals, and food processing chemicals with changes in microbiome composition and function. These agri-food chemicals are pervasive in modern food consumption, but no systematic approach has been taken to understand the extent of their direct impact at concentrations relevant to dietary exposure in the general population. I asked: what is the impact of agri-food chemicals on the growth and function of gastrointestinal microbiota? I screened 58 representative gastrointestinal bacteria species in the presence of 30 widely used agri-food chemicals at 1μM. I further characterized one observed growth interaction between Bisphenol S (BPS) and Bifidobacterium adolescentis by screening 31 B. adolescentis strains. Comparative genomics analysis of these strains was performed to identify enriched functions. A subset of seven chemicals and eight bacteria from our growth screen were also selected for RNA-seq to assess sub-inhibitory transcriptional response in the absence of growth inhibition. A Salmonella Typhimurium promoter library was screened to better characterized findings from the RNA-seq experiment. I observed 15% of bacteria were impacted by at least one agrochemical and 41% of agri-food chemicals impacted the growth of at least one bacteria. Azo food dyes and bisphenols were overrepresented in growth impacts among all compounds screened. Further screening with BPS and Bifidobacterium found that 52% of screened B. adolescentis were impacted. Comparative genomics analysis correlated the growth impact with functions relating to phage-associated and cell wall proteins. Transcriptomics of 56 different agrochemical-bacterial pairs found notable impacts of azo dyes, even in the absence of growth impacts. These impacts were found to relate to invasion and metabolic functions in random promoter library assays. These results characterize direct agri-food chemicals impacts on microbial growth and function, broaden our understanding of xenobiotic-microbiome interactions, and raise key questions regarding the widespread use of agri-food chemicals. Thesis Doctor of Philosophy (PhD) Humans use chemicals to grow, process, and package food. With every meal, humans consume small amounts of these chemicals. How these chemicals impact gut bacteria is not understood. Bacteria that live in the human gut are important for many normal functions, including nutrition, bowel function, and infection prevention. Here, I describe how I tested many gut bacteria with common food production, processing, and packaging chemicals. I show that there are a few bacteria affected in their growth by some chemicals, including some thought to be harmless to bacteria and humans. This growth inhibition can be specific to only some or very similar subsets of species of bacteria, such as with bisphenol S. I also show that even if they are not growing any different, food dyes can change how bacteria behave. This work helps us better understand how food chemicals impact our gut bacteria. What these findings mean for human health will require further studies.

Viral pathogens impose a significant burden on global public health. Given the increasing frequency of epidemics/pandemics, emphasis should be placed on improving current vaccines and developing next generation immunization strategies. The significant challenges observed in generating an efficacious seasonal influenza virus vaccine highlight the need for such advancements. Influenza results in approximately 650,000 deaths annually, most of which occur in pediatric, elderly, and immunocompromised patient populations. Currently approved influenza vaccines primarily elicit antibodies targeting the highly variable head domain of the hemagglutinin (HA) protein. Acquisition of point mutations in this region render antibodies elicited by seasonal vaccines less effective in subsequent seasons. Furthermore, seasonal vaccines do not provide protection from a pandemic influenza strain. Immense research efforts have, therefore, focused on the generation of a ‘universal’ influenza vaccine. Identifying optimal vaccine platforms is an ongoing challenge for influenza vaccine development. Vaccine delivery route and platform have been shown preferentially elicit different antibody isotypes. We demonstrated that stalk IgA antibodies elicit unique Fc-effector functions in neutrophils which may contribute to protection in vivo. As such, platforms which stimulate IgA should be considered for universal vaccines. Additionally, immune imprinting and pre-existing immunity to influenza impact both strain-specific and broadly-protective responses. In children, inactivated vaccines may skew immune responses to conserved regions of HA head shared by previously-encountered antigens. Induction of antibodies to conserved stalk epitopes is desired for a universal vaccine. We observed varied induction of stalk antibodies across vaccine platforms. Finally, seasonal vaccines can be of particular benefit to high-risk populations, such as patients with cardiovascular conditions, by preventing potentially life-threating exacerbations of their underlying conditions. We showed that inactivated seasonal vaccines were immunogenic and effective in these patients. These findings strengthen the observation that vaccine mediated protection helps to reduce cardiac events in vaccinated individuals. Together, this dissertation sheds light on factors affecting the elicitation and function of antibodies against influenza virus, which has implications for improving both seasonal and universal vaccine development. While the studies presented here focus on influenza virus, many of the findings may also be applied to other viral pathogens that cause respiratory infection, and are prone to antigenic drift, such as SARS-CoV-2. Thesis Doctor of Philosophy (PhD) The COVID-19 pandemic and repeated influenza virus pandemics over the past century emphasize the need for improved vaccines. Current seasonal influenza vaccines are limited by the narrow specificity of induced responses and varied efficacy across populations and seasons. Given these shortcomings, this dissertation explores various aspects of the human antibody response to influenza vaccination. However, these findings can be readily applied to other pathogens of interest. This body of work expands our understanding of antibody functions beyond neutralization and explores the role of vaccine type on immune imprinting and implications for universal vaccine platform selection. Furthermore, we explored the immunogenicity of seasonal vaccines in a heart-failure patient cohort to support the observation that vaccination reduces cardiac events. Investigating these, and other, features of the immune response to influenza vaccination will help to progress more effective strategies to combat viral pathogens.

Multiple myeloma is an aggressive plasma cell cancer that consistently acquires multi-drug resistance and relapses despite initial treatment successes. Patients may go through greater than 10-lines of therapy, highlighting the need for more effective treatment options. Immunotherapies are the latest evolution in targeted cancer treatments, and thus far have displayed impressive results in several hematological cancers, including multiple myeloma. T cells possess robust anti-tumor functions which can be harnessed and refined for the treatment of cancers. Genetic engineering of T cells to express a chimeric antigen receptor (CAR) confers antigen-specific tumor-targeting, and adoptive transfer of patient-derived CAR-engineered T (CAR T) cells has been efficacious in relapsed/refractory multiple myeloma. Despite the high efficacy, CAR T cell therapy for myeloma is associated with serious adverse events, which limits dose levels and patient eligibility. We have developed a novel synthetic antigen receptor platform, called the T cell antigen coupler (TAC) receptor, which has shown comparatively higher efficacy with a reduced pro-inflammatory profile compared with CAR T cells in pre-clinical models. The TAC receptor was purpose-built to co-opt the natural T cell activation machinery and lacks the costimulatory signaling typically incorporated in CAR designs. This thesis investigates strategies to augment TAC T cell function against for multiple myeloma through the evaluation of ancillary pharmacological and protein stimuli that would complement the anti-tumor functions of TAC T cells without modifying the TAC receptor design. In chapter 2, I investigated a strategy combining TAC T cells with the SMAC mimetic LCL161 to provide transient costimulatory effects. While LCL161 boosted TAC T cells survival and proliferation, the drug also enhanced susceptibility of TAC T cells to apoptosis and offered no advantage to the TAC T cells when challenged with myeloma. In chapter 3, I engineered TAC T cells to secrete IL-27 in an attempt to modulate the myeloma microenvironment and support T cell cytolytic function. IL-27 did not enhance the anti-tumor activity of TAC T cells but forced expression of IL-27 led to a reduction in the production of pro-inflammatory cytokines without altering cytotoxicity. In appendix I, I describe the process of optimizing CRISPR/Cas9 editing of primary TAC T cells. This methodology was required for much of the work in chapter 2. Ph.D. Thesis – Arya Afsahi McMaster University – Biochemistry and Biomedical Sciences v In appendix II, I describe an assessment of mRNA-engineering as a method to produce TAC T cells. This approach proved to be therapeutically futile and was not pursued beyond the work described herein. The work presented here highlights methods of combining TAC T cells with a clinically relevant SMAC mimetic, or the cytokine IL-27, and provides insights into the biological mechanisms that are affected by these approaches. Thesis Doctor of Philosophy (PhD)

Brain metastases (BM) have a 90% mortality rate within 4-12 months of diagnosis because they are mainly treated palliatively. The Singh Lab uses patient-derived BM samples in clinically relevant xenograft models of BM that recapitulate BM progression as seen in human patients. The objectives of my thesis were twofold: to understand whether targeting pre-metastatic cells could intercept BM formation, and whether macro-metastatic BM tumors could respond to immunotherapy as an alternative to palliative care. To intercept BM formation, I mined a Connectivity Map analysis on pre-metastatic BM gene expression signatures and identified inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo GTP synthesis pathway, as a key regulator of the pre-metastatic process. Pharmacological and genetic perturbation of IMPDH attenuates BMIC proliferation in vitro and slows down the formation of BM in vivo, highlighting a targetable metabolic vulnerability in BM. This work has inspired a preclinical drug development program focused on creating IMPDH inhibitors with enhanced brain permeation for clinical translation. To identify whether macro-metastatic tumors respond to immunotherapy, we used a chimeric antigen receptor (CAR) T-cell to target the glycoprotein CD133, whose expression correlates with disease progression, recurrence, chemo- and radio-resistance and overall poor prognosis. We saw significant impediment in tumor progression and significant survival advantage in tumor-bearing mice after a single dose. Together, I propose that prophylaxis should be the focus of BM research moving forward and that immunotherapies should be considered where BM diagnosis precedes the chance for interception. The development of a drug that can eradicate pre-metastatic cells has the potential to prevent BM in at-risk cancer patients, while the development of a CD133-targetting immunotherapy can dramatically improve the prognosis of patients who would otherwise be limited to palliative care. Thesis Doctor of Philosophy (PhD) Brain metastases have a 90% mortality rate within one year of diagnosis because they are largely untreatable. The objectives of this research were twofold: to understand whether we can prevent BM, and to understand whether developed metastatic brain tumors respond to immunotherapy. We discovered that inhibition of the enzyme inosine monophosphate dehydrogenase (IMPDH) prevents the growth of brain metastases cells. This inspired an ongoing drug development program aiming to develop IMPDH inhibitors as drugs that prevent metastatic brain cancer. To identify whether metastatic brain tumors respond to immunotherapy, we used a CAR-T cell immunotherapy to target the glycoprotein CD133 and found that tumor growth was delayed or completely blocked after a single dose. Based on these results, we propose that therapeutics that prevent the formation of brain metastases are a promising strategy to combat this deadly disease. Furthermore, we have shown that immunotherapies are a viable strategy for the treatment of brain metastases.

Graduate students are socialized into three key domains of academia throughout their studies – research, service, and teaching. The outcome of this socialization is impacted by a student’s disciplinary affiliation, training environment, and supervisory relationship. This multi-disciplinary dissertation represents a scholarly examination of three examples of disciplinary research, service, and teaching. For disciplinary research, we explore characterizing the DNA damage response of ataxin-1, the disease-causing protein of the neurodegenerative triplet-repeat disorder Spinocerebellar Ataxia type 1. For service, we examine the positive impact of a knowledge translation platform for ataxia research ataxia patient and family member readers, as well as its volunteer writers and editors. For teaching, we investigate how the COVID-19 pandemic has impacted graduate students' and postdoctoral fellows' development due to laboratory closures. Further, we return to this examination of the influence of COVID-19 on academia through the exploration of disparities in publication pressure reported by scholars in Canada. Though seemingly disparate research topics, each line of inquiry is grounded within research pragmatism, namely the identification of practical solutions through a clear understanding of a phenomenon. This breadth of research would not be possible without interdisciplinary graduate training, which develops scholars adept at creating innovative solutions to complex or ill-defined problems. Overall, this dissertation offers a snapshot of the opportunities and challenges of interdisciplinary research training within a biomedical research department. Dissertation Doctor of Philosophy (PhD) Graduate students generally receive three types of training at university – how to do research, how to give back to their communities, and how to teach. However, how well a student is trained in each of these tasks is impacted by who teaches them and their learning environment. Not only do students learn skills, but they also pick up on biases and stereotypes connected to their subject area. This dissertation combines three subject areas. First, biomedical research on the rare, fatal brain disorder Spinocerebellar Ataxia type 1. Second, science communication research on how an ataxia research news website benefits ataxia patients, family members, and even the volunteers to write for the website. Third, education research about how COVID-19 has affected research trainees who work in labs. Doing this many types of research in one dissertation can be challenging, but it trains you how to be a better problem solver.

Glycans, carbohydrate-based macromolecules, are present on the surfaces of all prokaryotes and eukaryotes cells. They play critical roles in cell-cell interactions, and host glycans also provide both binding sites and nutrition for commensal microbes. Many bacteria use glycoside hydrolases (GHs) on host-generated glycans and dietary glycans for nutrition and to modulate attachment sites and host protein function. The microbiota found in a single human gut sample has greater than 10,000 GH genes, and many of these gene products have specificity for host glycans. In eukaryotes, glycosylation of cell surface receptors is critical for proper localization and function, including receptors for innate immune signalling. The hypothesis of this project is that human-associated microbiota can express and secrete GHs that can alter the functioning of host immune signalling pathways. In this work, I measure GHs enzyme activity, use bacterial cell-free supernatant and purified enzymes to determine if they can modulate host cell signalling pathways, and examine glycans cell surface modifications by fluorescence microscopy using fluorescent-labelled lectins. Thesis Master of Science (MSc) Glycans are sugars that cover cells of animals, plants, and microbes. These sugars on the external surface of the cell help them interact with and protect them from the environment. For our microbiomes – the microbes that live on or in us- glycans on both microbe and our cells are important for interactions between them and us. Many bacteria produce enzymes (such as glycoside hydrolases) that change or remove sugars from the cell surface to obtain nutrients and manipulate host proteins. These enzymes can be attached to the bacterial cell surface or released into the environment, where they can interact with glycans attached to mucosal surface and immune cells. The modification of cell glycans from the immune system can modulate inflammatory responses that could change the way gut cells react to certain kinds of food, such as legumes and wheat. My work focuses on understanding how human gut microbiome can use these tools to reshape glycans and their effects on our health.

Bacterial outbreak publications outline the key factors involved in uncontrolled spread of infection. Such factors include the environments, pathogens, hosts, and antimicrobial resistance (AMR) genes involved. Individually, each paper published in this area gives a glimpse into the devastating impact drug resistant infections have on healthcare, agriculture, and livestock. When examined together, these papers reveal a story across time, from the discovery of new resistance genes to their dissemination to different pathogens, hosts, and environments. My work aims to extract this information from publications by using the biomedical deep-learning language model, BioBERT. BioBERT is pre-trained on all abstracts found in PubMed and has state-of-the-art performance with language tasks using biomedical literature. I trained BioBERT on two tasks: entity recognition to identify AMR-relevant terms (i.e., AMR genes, taxonomy, environments, geographical locations, etc.) and relation extraction to determine which terms identified through entity recognition contextualize AMR genes. Datasets were generated semi-automatically to train BioBERT for these tasks. My work currently collates results from 204,094 antimicrobial resistance publications worldwide and generates interpretable results about the sources where genes are commonly found. Overall, my work takes a large-scale approach to collect antimicrobial resistance data from a commonly overlooked resource, i.e., the systematic examination of the large body of AMR literature. Thesis Master of Science (MSc)

While antibiotics have saved the lives of millions of people since the discovery of the first β-lactam, penicillin, their continued effectiveness is being increasingly threatened by resistant bacteria. Bacterial resistance to β-lactams is mainly achieved through the production of serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs). Although both types of β-lactamases are commonly isolated in clinical settings, MBLs represent the greatest threat to public health since they are resistant to SBL inhibitors and most β-lactams. However, aspergillomarasmine A (AMA), a fungal natural product synthesized by Aspergillus versicolor, was shown to be a rapid and potent inhibitor against two clinically relevant MBLs: NDM-1 and VIM-2. In bacteria possessing these enzymes, AMA could rescue the activity of meropenem, a broad-spectrum β-lactam that is usually reserved for the treatment of the most severe bacterial infections. However, many questions remain revolving around AMA's inhibitory potency and spectrum. Therefore, the activity of AMA in combination with six β-lactams from three subclasses (carbapenem, penam, cephem) was explored against 19 MBLs from three subclasses (B1, B2, B3). After determining that AMA activity was linked to MBL zinc affinity and that AMA was more potent when paired with a carbapenem, the efficacy of an AMA/meropenem combination was evaluated with and without avibactam, a potent SBL inhibitor. This study used ten Escherichia coli and ten Klebsiella pneumoniae laboratory strains as well as 30 clinical strains producing at least one MBL and one SBL. Once establishing that the AMA/avibactam/meropenem combination was effective against carbapenemase-producing Enterobacterales, new Acinetobacter and Pseudomonas shuttle vectors were created. With these shuttle vectors, it was determined that the AMA/avibactam/meropenem combination was effective against some of the bacteria topping the World Health Organization’s priority pathogen list. Thesis Doctor of Philosophy (PhD) Bacteria are all around us. While some bacteria can promote human health, others can cause serious infections. These infections are typically treated with antibiotics. β-Lactam antibiotics, such as penicillins and cephalosporins, are especially important to medicine. Unfortunately, an increasing number of bacteria employ enzymes, known as β-lactamases, which negate the effects of β-lactam antibiotics. Previous studies demonstrated that a natural product, known as aspergillomarasmine A (AMA), could inhibit some β-lactamase enzymes. Consequently, the inhibitory power of AMA was further explored against a larger number of β-lactamase enzymes and in combination with different β-lactam antibiotics. After discovering that AMA had more inhibitory power when combined with a β-lactam antibiotic known as meropenem, the efficacy of the AMA/meropenem pairing was evaluated against resistant bacteria in the presence and absence of avibactam, another β-lactamase inhibitor. The AMA/avibactam/meropenem combination was shown to be effective against some of the world’s most antibiotic-resistant bacteria.

Autism spectrum disorder (ASD) encompasses a broad range of neurodevelopmental disorders, with two core symptoms: deficits in social communication, and restrictive interests and repetitive behaviors. Genetics is thought to play a large role in ASD and currently there are hundreds of associated genes. We first studied the thousand and one amino acid kinase gene (TAOK2), which plays an important role in neurodevelopment. We found that loss of TAOK2 causes deficits in neuron development and activity, leading to morphological changes in various mouse brain regions and ASD-related behaviors. We studied the impact of de novo mutations identified in TAOK2, which caused aberrant neuron dendritic arborization and formation of synapses. To elucidate how TAOK2 regulates neuron development we used a proximity-labeling proteomics technique (BioID) to identify its protein-protein interaction (PPI) network. We applied this same methodology to a total of 41 ASD-risk genes and observed multiple convergent biological processes, including the less-studied mitochondrial and metabolic pathways. ASD-risk genes, including TAOK2, associated with mitochondrial proteins were found to have altered cellular respiration. The shared ASD-risk gene PPI network enriched for other ASD-risk genes and was used to group genes based on their shared PPI networks. These gene groups showed correlation between the clinical behavior scores of individuals that had mutations within the distinct gene groups. Lastly, we identified changes in the PPI networks of multiple ASD-risk genes through BioID, which we validated with various functional assays. In summary, we developed a proximity-labeling proteomics method that identified multiple convergent biological pathways associated with ASD. Studying the function of TAOK2 revealed multiple disease-relevant pathologies associated with the disorder, however proximity labeling has the potential to categorize multiple ASD-risk genes and elucidate their shared signaling pathways, which together, can advance the development of robust treatments for ASD. Thesis Doctor of Philosophy (PhD) Autism spectrum disorder (ASD) is a group of brain disorders that affect more than 1% of children. Genetic variants are thought to cause ASD pathology, however there are currently hundreds of genes that have not been studied. We studied how disruption of one of those genes, TAOK2, alters brain development in mice and identified TAOK2 variants in multiple children with ASD. We then used BioID to find the shared disease-related mechanisms between multiple ASD-risk genes, and found that mitochondrial function and activity were connected to many of these genes. We showed that BioID can be used to study the effect of mutations in multiple ASD-risk genes simultaneously. Last, we could group children with ASD with similar behavior test scores based on the shared mechanisms of ASD-risk genes. Together our findings could be used to advance the development of robust treatments or new diagnostic tools for ASD.

Acute myeloid leukemia (AML) is an aggressive cancer of the blood and bone marrow, affecting 1,100 Canadians annually. Older patients make up 75% of cases yet have the lowest survival rates due to the lack of tolerable treatments. Recently, the combination of Venetoclax and Azacitidine (Ven/Aza) has shown great therapeutic promise, however, chemoresistance has become a growing concern. Current evidence points towards a chemoprotective role from the bone marrow (BM) microenvironment, specifically by BM-derived mesenchymal stromal cells (BMSCs) and adipocytes. AML cells can manipulate BMSCs and adipocytes to create a niche that supports its own growth and evades chemotherapy. However, the role of the microenvironment in Ven/Aza chemoresistance has yet to be studied. Our objective was to study the ability of the microenvironment cells to induce AML chemoresistance to Ven/Aza. We employed a 2-dimensional direct contact co-culture system between MOLM-13 AML cells and BMSCs or adipocytes in both the absence and presence of Ven/Aza to determine the effects on the AML cells. In the absence of Ven/Aza, adipocyte co-cultured AML cells showed a 47% reduction in proliferation, 10% reduction in viability, yet a 1.7-fold increase in Maximal respiration when compared to the monocultured cells. In the presence of Ven/Aza, adipocyte co-cultured AML cells showed a significant increase in both proliferation and viability. Preliminary work investigating the mechanism of action of this support points toward an anti-apoptotic mechanism mediated by the upregulation of MCL-1 upon co-culture with adipocytes. Combination of Venetoclax and Tapotoclax, an MCL-1 inhibitor, abrogated the chemoprotection provided by BMSCs and adipocytes. Overall, our data suggests a dual role of adipocytes, where their inhibition or support of AML is context dependent. Therapeutic targeting of mechanisms for adipocyte chemoprotection such as MCL-1 upregulation may re-sensitize AML cells to Ven/Aza, thereby improving patient outcomes. Thesis Master of Science (MSc) Acute myeloid leukemia (AML) is an aggressive cancer of the blood and bone marrow, affecting 1,100 Canadians annually. Older patients make up 75% of cases yet have the lowest survival rates due to the lack of tolerable treatments. A novel combination of Venetoclax and Azacitidine (Ven/Aza) has shown great therapeutic promise, however, chemoresistance remains an important concern. Previous studies have implicated fat cells, or adipocytes, in AML chemoresistance, however, their role in Ven/Aza treatment has yet to be studied. Here, we show that adipocytes reduce growth of AML cells, yet enhance their metabolism. In the presence of Ven/Aza, adipocytes induce chemoresistance. We show preliminary data that this chemoprotection may be mediated by the upregulation of mitochondrial MCL-1 protein as inhibition of this protein neutralized the protection. By understanding the relationship between adipocytes and AML chemoresistance, we can target this and re-sensitize AML to Ven/Aza, thereby improving older patient outcomes.

Type IV pili (T4P) are long adhesive surface filaments produced by bacteria and are a key virulence factor for many pathogens. T4P are produced by a dynamic intracellular nanomachine that facilitates the assembly (extension) and disassembly (retraction) of pili. Pilus dynamics are enabled by the motor subcomplex of the nanomachine, where cytoplasmic ATPases power pilus assembly (PilB) and disassembly (PilT and PilU). In many, but not all, T4P expressing bacteria – including our model organism Pseudomonas aeruginosa – two retraction ATPases are required for functional retraction, which can be assessed by measuring twitching motility. Deletion of pilT results in loss of twitching and phage susceptibility (another hallmark of pilus function) while deletion of pilU results in loss of twitching but retention of phage susceptibility, indicating pili can still be retracted. We hypothesized that PilU adds to the force of pilus retraction, facilitating disassembly when the fiber is under tension. We mutated ΔpilU and pilU::Tn5 strains with ethyl methanesulfonate and screened for gain-of-twitching mutants. Whole genome sequencing revealed multiple point mutations in the major pilin protein PilA or the pilus adhesin, PilY1. These point mutations were recapitulated in a ΔpilU strain and restored twitching to varying degrees. Complementation of pilA point mutants with pilU in trans influenced the twitching zone of only one mutant, and in trans expression of wild-type pilA resulted in a significant reduction in twitching in most. The contribution of PilU to the force of pilus retraction was further investigated by a polyacrylamide micropillar assay, where no pulling events could be detected for either ΔpilT or ΔpilU mutants. Exopolysaccharide production, a proxy for surface sensing, was uncoupled from twitching motility in the pilA point mutants. These results are a significant step forward to understanding what PilU does and, provides insight to the dynamics of the pilus fiber. Thesis Master of Science (MSc) Pseudomonas aeruginosa is a bacterium that causes serious infections. P. aeruginosa uses adhesive, “grappling hook” filaments called Type IV pili (T4P) to stick to its hosts. T4P can be repeatedly extended and retracted, allowing the bacteria to crawl on surfaces (twitching) but making them susceptible to bacteriophages, viruses that attach to pili then kill the bacterial cells. The motor proteins PilT and PilU are required for twitching, but only PilT is essential for phage killing, implying that pili are retracted even when PilU is missing. Here we hypothesized that PilU is important for twitching because it helps generate force for retraction when pili are under tension. We isolated multiple mutations in pilus components that restored twitching in the absence of PilU, and propose that these mutations allow for easier retraction of pili. This information helps us understand how T4P help the bacteria to spread during infection.

Acute myeloid leukemia (AML) is a highly heterogeneous cancer with a poor prognosis. Clinical presentation is characterized by the abundant non-functional immature hematopoietic cells (blasts) in the bone marrow (BM) and peripheral blood (PB) of patients. Untreated, the rapid proliferation of these blasts contributes to hematopoietic system failure within months, leading to death. Although the standard chemotherapy regimen has remained relatively unchanged for decades and has proven to be effective at achieving initial remission induction, most patients succumb to relapse forming the basis of poor long-term survival. Incomplete mechanistic understandings of disease initiation, progression and maintenance of AML have impeded advances in therapy required for the improvement of long-term patient survival rates. This suggests that innovative and new model systems are required to understand the earliest initiation processes of AML disease towards more effective targeted therapy development. The ability to generate induced pluripotent stem cells (iPSCs) from human somatic cells provided a breakthrough in biomedicine to ‘capture’ diseased cells and their genome in a self-renewing state. Patient-derived human induced pluripotent stem cells (hiPSCs) have the theoretical ability to develop patient-specific (autologous) cell-based therapies and/or produce an endless number of specialized disease-associated cells, allowing replication of pathological characteristics of human disease in vitro. Despite the technical challenge of reprogramming human cancer cells due to the high inefficiency of the process compared to healthy samples, whereby iPSCs are often skewed in favor of residual normal cells over cells of the premalignant or malignant clone, I propose to develop a library of AML patient-derived iPSCs. Moreover, I hypothesize that pluripotent reprogramming can provide a unique alternative approach to dissect heterogeneity and molecular basis of AML that cannot be captured and studied in existing in vitro or in vivo patient-derived xenograft models. Additionally, I seek to investigate the ability of human iPSCs in the derivation of HSCs as a proof of concept for autologous cell-based therapies. Dissertation Doctor of Philosophy (PhD) A diagnosis of Acute Myeloid Leukemia (AML) varies from patient to patient, as there are many different subtypes of this cancer. This is referred to as ‘patient cancer heterogeneity’. AML represents one of the most heterogeneous types of cancers, and unfortunately, only 20% of AML patients survive past five years from diagnosis. AML is a cancer of the blood system that causes the bone marrow to fill up with immature cells that are unable to develop into functional blood cells. The ability to reprogram AML patient cells into induced pluripotent stem cells (iPSCs), which can give rise to any cell in a dish, provides an opportunity to study this disease. Our research proposal is to develop a diverse library of AML patient-derived iPSCs providing a unique alternative approach to interrogate the therapeutic potential, heterogeneity, and molecular basis of AML that “drive” the disease to continue which cannot be captured and studied in existing in vitro or animal models of the disease.

The collection of microbes that inhabits the human gastrointestinal tract is known as intestinal microbiota, and an enormous body of work has shown that their activities contribute to health and disease. Ulcerative colitis (UC), which is a type of inflammatory bowel disease, is considered to arise due to a disruption in the balance between the immune system and microbiota. However, there is little consensus on the mechanism of action and microbes involved in the disease manifestation. In this work, I applied culture-enriched metagenomics (CEMG) to characterize the dynamics of gut microbiota in healthy individuals and UC patients. I showed that CEMG provides a higher resolution to study these microbial communities, and we used this approach to understand microbial colonization after fecal microbiota transplantation (FMT) therapy in UC patient. I showed that sequencing approaches alone did not reveal consistent engraftment across FMT responders. Using CEMG and a collection of bacterial whole-genome sequences, I showed patient-specific microbial strain transfer and a signature of commonly engrafted genes only in patients who responded to FMT. In this work, I also investigated the dynamics of a highly abundant bacteriophage, crAssphage, in an FMT donor and implemented a new method to detect bacteriophage engraftment post-FMT using SNP analysis. Finally, it has been suggested that antibiotic treatment before FMT may increase the efficacy of FMT. However, in this work, I show that while antibiotics alter the microbiome, there was no difference in the composition of the microbiome of antibiotic vs placebo group post-FMT. This is consistent with the randomized controlled trial results that shows pretreatment with antibiotics does not improve FMT outcome. Together, this work demonstrate the importance of in-depth microbiome analysis applied to culture-dependent and -independent sequencing to characterize microbial changes post-FMT. Dissertation Doctor of Philosophy (PhD) Many bacteria reside in the human gut, and they are essential in our health and in disease. It is evident that these bacteria are associated with inflammatory bowel disease, but we do not yet know how and what bacteria are involved in this disease. In this work, I describe a method to study these bacteria from stool that relies on growing them and investigating their DNA. I showed that our approach helped us recover a greater diversity of these bacteria and their genetic content in healthy individuals and patients with inflammatory bowel disease compared to methods that use only DNA based approaches. Using this method, we could better understand why some patients responded to a treatment consisting of transferring stool content from healthy donor to patient. I also investigated a group of viruses that infect bacteria and implemented a new computational method based on DNA sequencing to test whether these viruses transfer to the patient after receiving the fecal therapy. We also found that antibiotic treatment before fecal therapy in patients with inflammatory bowel disease does not improve the patient’s recovery.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse drug reaction to the anticoagulant heparin.1,2 HIT is characterized by pathogenic antibodies that form immune complexes with platelet factor 4 (PF4) and heparin, which can cause platelet activation and thrombosis.1,3-6 Prompt diagnosis is crucial because HIT patients experience a 5-10% daily increased risk of thrombotic episodes, which are fatal in up to 30% of patients.2,6-10 The immune response in HIT is polyspecific, resulting in the production of anti-PF4/heparin antibodies that either can (pathogenic) and/or cannot (non-pathogenic) activate platelets.10-12 Differentiating between pathogenic and non-pathogenic antibodies remains a significant diagnostic challenge in HIT, exacerbated by the low specificity and limited availability of current laboratory assays.10,14,15 Previously it was shown that platelet-activating immune complexes form when pathogenic antibodies bind PF4 at a localized region that is distinct from non-pathogenic antibodies.13 In this work, high affinity inhibitors of pathogenic anti-PF4/heparin antibodies were designed using a murine monoclonal antibody (named KKO) that binds to a region on PF4 overlapping with pathogenic antibodies.16 These inhibitors were used to develop a novel diagnostic assay that can detect pathogenic antibodies with improved specificity compared to most standard tests for HIT (specificity ~50-70%).2,15 Enzyme immunoassays (EIAs) are widely used for HIT diagnosis, but have high false-positive rates because they cannot differentiate between pathogenic and non-pathogenic antibodies.10,14,15 This study found that incorporating inhibitors to block clinically significant antibody binding sites on PF4 improved the specificity (90%) of a diagnostic EIA for HIT without sacrificing sensitivity (90-100%). Unlike most high-sensitivity and -specificity diagnostic tests for HIT, such as platelet-activation assays, this EIA can also be performed with technical ease in non-specialized laboratories. Thesis Master of Science (MSc)

The bone marrow (BM) microenvironment (the niche) plays a critical role in regulating normal hematopoietic process and is dysregulated in bone and marrow-related malignancies such as Acute Myeloid Leukemia (AML). AML is an aggressive blood cancer affecting the myeloid lineage of the hematopoietic hierarchy, and occurs in mostly individuals of older age. The goal in recent years has been to prolong the survival of these elderly patients, who show morbidity and early induction mortality in the face of intensive treatments. Disease relapse also poses a major hurdle to overcome following the supposed eradication of the leukemic disease, as current disease monitoring strategies used to detect minimal residual disease (MRD) are insufficient and may vary between patients. Many turn to look at the hematopoietic niche for the development of non-cell autonomous AML therapies in the hopes of more targeted and tolerable therapies; however, these strategies focus solely on the elimination of leukemic cells, and remain ignorant to the deterioration of the niche stemming from the disease. As yet, studies on niche cells, including mesenchymal stem cells and their progenies, are riddled with discrepancies and remain challenging to perform as methods used in the field to investigate mesenchymal populations rely primarily on ex vivo assays which are subject to extraneous factors that can lead to biased interpretation. Here, we used an in vitro mesenchymal CFU assay to interrogate the BM niche of AML patients and identify aberrations to the diseased niche throughout progression of the leukemic disease. Our experimental findings showed that while BM adipocyte and osteoblast populations appeared to be equally impaired in AML patients, the reduction in adipogenesis capacity of BM mesenchymal cells seemed to be unique in relapsed patients. We further identified a novel adipogenesis-inducing molecule, Isoginkgetin, that can successfully correct this deficiency in AML BM-MSCs and promote BM adipogenesis in vivo. Thesis Master of Health Sciences (MSc)

The type VII secretion system is a protein export pathway linked to diverse phenotypes in both Actinobacteria and Firmicutes. The Actinobacterial subtype of the T7SS, referred to as T7SSa, has been shown to play a critical role in various aspects of Mycobacterial life including virulence, conjugation, and metal homeostasis. The T7SSb of Firmicutes bacteria on the other hand has similarly been shown to influence virulence but by the direct growth inhibition of competitor bacteria. Structure-function analyses of the T7SSa apparatus as well as various effectors and chaperones have begun to build a more mechanistic understanding of how T7SSa functions. In contrast, we know little of how the T7SSb functions despite its noted importance to both pathogens and environmental bacteria such as Bacillus, Staphylococcus, Enterococcus, and Streptococcus. During my thesis work, I have addressed several gaps in our understanding of T7SSb function. The three major questions that I have studied are: (1) how do T7SSb immunity proteins inhibit the toxicity of their cognate toxins, (2) how does the T7SSb export effectors through the thick Gram-positive cell wall, and (3) what is the role of chaperone proteins in facilitating T7SSb effector export? Thesis Doctor of Philosophy (PhD) Bacteria require space and various nutrients to survive and grow and must therefore compete against other bacteria for access to these resources. To gain advantage over their competitors, many bacteria have developed molecular weapons that target and kill other closely related bacteria. Some of these weapons take the form of protein secretion machines that export antibacterial toxins. Gram-positive bacteria use the type VIIb secretion system (T7SSb) to inhibit the growth of other Gram-positive bacteria. In this work, I explore several aspects of T7SSb including: (1) how toxins are inhibited by immunity proteins, (2) how toxins are secreted through the cell envelope, and (3) how toxins are recognized by the secretion apparatus. The goal of this work is to better understand how T7SSb functions at the molecular level.

Bacteria frequently encounter changes in their environment and must adapt accordingly. When these changes are predictable, there is evidence of anticipatory gene regulation. For example, the model enteric pathogen Salmonella has a well-defined natural history, typically only encountering iron in the lumen of the gut. However, this bacterium responds to the presence of iron not by upregulating genes needed to thrive in the lumen, but rather the iron-deplete epithelium, the subsequent environment it encounters. This may be similar to Pavlovian conditioning, a type of associative learning that involves pairing two unrelated stimuli and anticipatory behavioural changes. Since conditioning has not been well explored in bacteria, we are investigating whether Salmonella can learn new conditioned responses by pairing two unrelated carbon sources, citrate and maltose. We leveraged a prototype of the Microbial Evolution and Growth Arena (MEGA)-plate motility assay to define a new natural history. By pairing stimuli across a physical landscape, we can select for bacteria that learn to use citrate to anticipate maltose and can quickly deplete the second carbon source. Time-series imaging of bacteria as they swim across the plate shows evidence of emerging variants capable of swimming faster through maltose and unique swimming behaviours through repeated passaging. This approach selects for the fastest swimming bacteria, not necessarily bacteria that have acquired anticipatory regulation. As such, further genetic and transcriptional analysis of the variants are necessary. Similar passaging of Salmonella in broth allowed us to compare anticipatory regulation across a physical and non-physical landscape. Learning to anticipate environmental changes will provide a bacterium with a selective advantage, allowing it to outcompete its conspecifics which are slower to respond. From this investigation, we hope to provide insight into the learning capacity of bacteria and further understand how bacteria exploit memory to problem-solve. Thesis Master of Science (MSc) Bacteria that encounter predictable changes in their environment can acquire anticipatory gene regulation. This may be similar to Pavlovian conditioning, a type of associative learning that involves pairing two unrelated stimuli and anticipatory behavioural changes. Since conditioning has not been well explored in bacteria, we investigate whether Salmonella can learn new behaviours by pairing two unrelated carbon sources, citrate and maltose. We leveraged a motility plate assay to define a new natural history. By pairing stimuli across a physical landscape, we can select for bacteria that learn to use citrate to anticipate maltose. Time-series imaging shows evidence of variants capable of swimming faster through maltose and unique swimming behaviours through repeated passaging. Similar passaging of Salmonella in broth allowed us to compare anticipatory regulation across physical and non-physical landscapes. From this investigation, we hope to further understand the learning capacity of bacteria and how bacteria exploit memory to solve problems.

This thesis consists of two chapters, each of which is a unique research project. Chapter 1 is focused on the synthesis and biological evaluation of novel sideromycin antibiotics. Sideromycins are bifunctional “Trojan Horse” molecules that have an iron chelator “siderophore” moiety covalently bound to an antibiotic. Such molecules exploit existing bacterial mechanisms for obtaining iron from their environment. Antibiotics that would typically not pass Gram-negative membranes are allowed access via siderophore transporter proteins. This project utilized a siderophore that has not previously been used in this capacity. The synthesis and biological evaluation of multiple sideromycin conjugates is reported. Chapter 2 describes the chemical synthesis of coumarin natural products using a synthetic process recently developed in the Magolan laboratory that enables the efficient prenylation of phenols. These natural products are molecules of biological interest in various capacities but are rare and difficult to isolate from their plant sources. They have also previously been cumbersome to make via chemical synthesis. The chemistry described herein constitutes an inexpensive and efficient process to produce these compounds that is superior to previously known methods. Thesis Master of Science (MSc) This thesis is divided into two chapters. The first chapter is focused on the development of new sideromycin antibiotics. Sideromycins are “Trojan Horse”-like antibiotics that exploit the mechanisms of Gram-negative bacteria for obtaining iron, an essential nutrient, to enable antibiotic entry. This chapter details the synthesis of molecules that attach functionalities called “siderophores” to antibiotics, enabling them to be “smuggled” into Gram-negative microbes. This project uses a siderophore not previously utilized in sideromycin research. The second chapter is focused on the chemical synthesis of rare natural products that are phenols with prenyl substituents. Many such compounds are plant-derived and have potential for biomedical use. However, difficulty in isolating them makes them prohibitively expensive in the purity and quantity required for research. They are also challenging to make synthetically. This chapter details the application of a recently discovered process in the Magolan laboratory to synthesize coumarin-containing prenylated phenolic natural products.

The outer membrane (OM) is a formidable barrier that has made antibiotic drug discovery in Gram-negatives exceedingly difficult. Many antibiotics which are effective against Gram-positive bacteria cannot permeate the Gram-negative OM to reach their intracellular targets. Thus, it is important to explore unconventional approaches to overcome the intrinsic resistance conferred by the OM. Herein, we used both genetic and chemical means to compromise OM integrity to potentiate the activity of large-scaffold antibiotics against Escherichia coli. First, we mapped the genetic interaction network of OM biosynthetic genes using synthetic genetic arrays (SGAs) to reveal permeability determinants of the E. coli OM. This led to the creation of a publicly accessible dataset of ~155,400 double deletion strains with growth data in the presence of the large-scaffold antibiotics rifampicin and vancomycin. Investigations of a subset of synthetic sick interactions revealed connectivity in the context of permeability between lipopolysaccharide (LPS) inner core biosynthetic genes and an enigmatic gene involved in enterobacterial common antigen (ECA) regulation. Second, we leveraged a chemical screening platform based on the observation that disruption of the E. coli OM leads to antagonism of vancomycin activity at cold temperatures to uncover molecules that potentiate Gram-positive-targeting antibiotics at 37 ºC. Two of these compounds, liproxstatin-1 and MAC-0568743, were characterized to bind to LPS and disrupt OM integrity specifically without impacting the inner membrane (IM). Third, we performed genetic and chemical screening to unearth targets capable of potentiating the activity of Gram-positive-targeting antibiotics against E. coli. This validated the OM as a valuable target for antibiotic adjuvants and led to the discovery of two membrane active compounds and an inhibitor of lipid A biosynthesis. Overall, this thesis emphasizes the importance of elucidating biological factors contributing to OM permeability and the attractiveness of the OM as a target for antibiotic potentiators. Thesis Doctor of Philosophy (PhD)

Salmonella enterica is a facultative intracellular pathogen capable of eliciting severe, systemic disease necessitating antibiotic intervention. Systemic infection is facilitated by intracellular replication within host immune cells, which is enabled by complex regulatory networks governed by two-component systems (TCSs). Intracellular-active TCSs sense antimicrobial chemical cues in the microenvironment and respond adaptively through transcriptional regulation to support intracellular survival. SsrA/SsrB and PhoQ/PhoP are two essential TCSs that elicit a robust defense against host immunity by regulating clusters of virulence genes and integrating novel targets to support regulon expansion and enhance pathogenicity. Metabolic adaptation is critical to bacterial survival and can initiate host-pathogen interactions that influence infection outcome. Further, mitigation of host immunity by manipulation of arginine metabolism has been documented in intracellular pathogens. Herein, I investigated TCS-mediated regulatory evolution pertaining to arginine metabolism, hypothesizing that adaptations to metabolic regulation might confer a fitness advantage to Salmonella replicating intracellularly. I explored intracellular regulation of de novo biosynthesis and extracellular import of arginine, establishing PhoP-mediated regulation of arginine transport. I determined that arginine transport contributes to bacterial fitness in macrophages and began to investigate the mechanism by which arginine importation enriches for intracellular replication. This work informs on evolutionary mechanisms that serve to enhance virulence in Salmonella and provides further insight into our understanding of the intracellular lifestyle of infection. Thesis Master of Science (MSc) Salmonella enterica is an intestinal pathogen that survives within host immune cells and causes systemic disease. These bacteria replicate within antimicrobial cells by using sensory networks to detect harmful immune factors and respond adaptively by eliciting change in gene expression to defend against immune-based killing. The amino acid arginine is an important component of host immunity, as well as bacterial antimicrobial defenses; therefore, I hypothesized that bacterial metabolism might be adapted to the host immune cell environment in order to mitigate arginine-dependent antimicrobial activity. Here, I establish that arginine metabolism is controlled by intracellular-specific sensory networks, and demonstrate that this regulation is important for bacterial survival. This work provides evidence for the importance of this amino acid in Salmonella infection, which informs on our overall understanding of systemic disease.

Glioblastoma (GBM) remains the most aggressive and prevalent malignant primary brain tumor in adults. Unchanged since 2005, standard of care (SoC) consists of surgical resection, followed by radiation therapy (RT) with concurrent and adjuvant chemotherapy with temozolomide (TMZ). Despite these therapeutic efforts, patients succumb to recurrent disease with a median overall survival of 14.6 months and a five-year survival rate of 5.5-6.8%. Therapeutic failure is largely explained by ITH and the presence of treatment-resistant GBM stem-like cells (GSCs). Given the lack of understanding of recurrent GBM and absence of second line therapies patients, I hypothesize that genome-scale functional genetic interrogation will unravel recurrent GBM-specific tumor biology and inform development of novel therapeutics. First, I compared primary and recurrent GBM at the genetic, transcriptomic, proteomic and functional genetic levels. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 repress axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and exploit a genetic dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. Given the genetic dependency on ROBO signaling and enrichment of ROBO1 expression in GBM tissues, I undertook a campaign to evaluate ROBO1 as a therapeutic target in recurrent GBM and develop anti-ROBO1 chimeric antigen receptor T (CAR-T) cells using camelid single-domain antibodies targeting human ROBO1. I optimized the design of anti-ROBO1 CAR-T cells and tested the anti-tumor activity of these modalities in in vitro using patient-derived recurrent GBM lines and orthotopic patient-derived xenograft models. I present data to expand the repertoire of GBM-enriched antigens suitable for effective CAR-T cell therapy. Given that resistance to SoC and disease relapse are inevitable for GBM patients, pre-clinical and clinical advancement of immunotherapeutic modalities, combined with recent insights into the tumor immune microenvironment, are poised to improve clinical outcomes for this patient population. Thesis Doctor of Philosophy (PhD) Glioblastoma remains the most lethal and prevalent primary brain tumor in adults. Standard of care for patients remains unchanged since 2005, consisting of surgery to remove visible tumor at diagnosis (primary tumor), followed by radiation therapy and chemotherapy to treat remaining tumor cells. Despite these therapeutic efforts, tumor relapse (recurrent tumor) is inevitable with no standardized second-line therapy. Patients succumb to recurrent disease with a median overall survival of 14.6 months and only 5.5-6.8% of patients survive five years post diagnosis. Therapy failure and tumor relapse are explained by immense diversity among tumor cells at the DNA and protein levels, giving rise to a subset of tumor cells with abilities to resist therapy and seed the recurrent tumor. Previous studies have presented evolution of tumor cells through therapy, with recurrent tumor cells harboring novel changes at the DNA and protein levels. However, the impact of these changes on tumor cell function has not been evaluated. In this thesis, we developed and applied a genetic screening technique to determine the functional role of thousands of genes in primary and recurrent tumor cells from the same patient. This analysis revealed numerous genes that exhibit differential effects on survival of primary and recurrent tumor cells, including genes that drive recurrent tumor cell growth but are dispensable in primary tumor cells. Functional remodeling of these genes and pathways revealed a new functional role of multiple proteins belonging to a process called axonal guidance in recurrent tumor cells. To evaluate the therapeutic potential of these findings, we deeply interrogated the mechanism by which axonal guidance drives recurrent tumor cells and targeted crucial molecular players using chemical and immunological therapies. Using models that predict clinical effectiveness, we engineered and tested a novel therapy that redirects immune cells to target recurrent tumor cells driven by dysfunctional axonal guidance activity. The goal of this thesis was to discover the functional differences between primary and recurrent tumor cells, thereby leveraging this information to engineer candidate therapies for treatment of recurrent glioblastoma.

Obesity-induced inflammation is a factor involved in the risk and progression of type 2 diabetes and non-alcoholic fatty liver disease. These diseases are associated with changes in gut microbiota composition and bacterial metabolites. Bacterial components with known innate immune receptors can cooperate with non-immunogenic metabolites derived from the gut microbiota to alter host immunity. Gut bacteria produce almost all D-lactate in the host, whereas L-lactate is host-derived. It is known that microbial-derived D-lactate in the portal circulation programs liver-resident macrophages to help combat bacterial infections. It was unknown how D-lactate and L-lactate alter cell-autonomous inflammation in macrophages exposed to low levels of bacterial cell wall muropeptides or lipopolysaccharide (LPS). It was also unknown if macrophage co-stimulation altered inflammation in macrophages exposed to bacterial metabolites and cell wall components. In vitro models showed that D-lactate had no effect (or a very small effect) on multiple markers of inflammation in the absence of interferon-gamma (IFN-γ) co-stimulation. Nevertheless, initial experiments on clonal macrophages discovered that equimolar and physiological levels of D-lactate increased Nos2 expression compared to L-lactate. Bone marrow-derived macrophages (BMDMs) co-stimulated with IFN-γ revealed a larger effect of D-lactate on immunity. Compared to L-lactate, D-lactate increased nitric oxide (NO) production and increased activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in BMDMs co-stimulated with IFN-γ and exposed to LPS. Further, D-lactate prevented L-lactate-induced lowering of NO production and NLRP3 inflammasome-mediated release of IL-1β from BMDMs co-stimulated with IFN-γ and LPS. Identifying appropriate cell models and defining conditions that reveal the effect of D-lactate versus L-lactate on immune responses in isolated cells was a major contribution of this work. Future work should characterize the interaction of macrophages and hepatocytes. This research may lead to the identification of gut microbiota-based approaches to limit liver inflammation in obesity and metabolic disease. Thesis Master of Science (MSc) Gut bacteria can influence both immunity and metabolism in the liver. It is not clear how bacteria in the gut communicate to the liver. Lactate is a key metabolite that can fuel liver metabolism and alter immunity. There are 2 types of lactate. L-lactate is host-derived, whereas D-lactate is mainly produced by gut bacteria. Bacterial-derived D-lactate can alter immune responses mediated by immune cells in the liver, including macrophages. Bacterial metabolites such as D-lactate and bacterial components that cause inflammation are increased in the blood during obesity. Our findings show that D-lactate can increase inflammation in macrophages when combined with bacterial components and that D-lactate increases inflammation more than L-lactate. Understanding the role of D-lactate may help us understand how bacteria contribute to insulin resistance, higher blood glucose and the risk of type 2 diabetes and fatty liver disease.

Antagonistic interactions between competing species of bacteria are an important driver of bacterial community composition in the human gut microbiota. Of particular significance is the role of the type six secretion system (T6SS), which many species of Gram-negative bacteria use to kill competitor bacteria in a contact-dependent manner. T6SSs are syringe-like nanomachines that function to deliver antibacterial toxins into susceptible competitors. Many bacteria present in the human gut microbiota possess an extremely potent T6SS that is capable of rapidly eradicating nearby bacteria. Remarkably, however, species of beneficial bacteria that coexist in the gut are often resistant to T6SS attack by their neighbours. This resistance is mediated by bacterial immunity proteins that block the activity of the antibacterial toxins delivered by the T6SS. Intriguingly, past studies have shown that the widespread T6SS-mediated competition in the gut has led to the acquisition of repertoires of immunity genes across different bacterial strains. By examining available human gut metagenomes, I identified a putative immunity locus, named I2, in a species of gut bacteria. This locus is located downstream of its cognate T6SS toxin-encoding locus, E2, and I show when co-expressed with E2 in E. coli, it protects against E2 mediated-toxicity. Additionally, I show that four gut-derived I2 homologues bearing sequence identity levels to I2 ranging from 38% to 75% are equally capable of abrogating E2 toxicity. Using quantitative biophysical measurements, I also show that these I2 homologues physically bind E2 equally tightly pointing to the potential molecular mechanism of toxin neutralization. Lastly, through mutagenesis experiments, I found that the E2-I2 interaction is likely mediated by electrostatic forces between a small number of residues found in the interaction interface of the two proteins. Overall, these findings demonstrate that a human gut microbiome encoded type VI secretion system effector can be neutralized by divergent immunity proteins. Thesis Master of Science (MSc)

Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and their long-term integrity. While regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventative machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and when overexpressed endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127, and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis, and classifies PLAG1 among a rare set of bona fide regulators of mRNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit. Thesis Doctor of Science (PhD) Blood production (hematopoiesis) is a stem cell-driven regenerative system that can repair damaged blood systems and therefore offer lifesaving treatments for devastating malignancies and immune disorders. Realizing the full potential of hematopoietic stem cells (HSCs) is encumbered by our inability to maintain HSCs with long-term functionality in clinical settings. Enhanced fundamental insights into the properties that define human HSC identity and fates can thus inform the rational design and advancement of HSC-based therapies. We discovered a regulator, PLAG1, that is essential for the long-term blood production function of human HSCs. When elevated PLAG1 amplified the absolute number of human HSCs 15-fold and improved their maintenance and function in clinically-relevant culture and transplantation settings. Using genome-wide technologies paired with functional assays we elucidated that PLAG1 employs a multi-pronged strategy to rewire protein production rates as a means to enhance HSC preservation and function. Our findings highlight regulation of protein production as an untapped strategy that could be incorporated into clinical settings to improve patient outcomes.

The type VI secretion system (T6SS) is a widely distributed protein nano-machinery found in Gram-negative bacteria. T6SSs are a harpoon-like apparatus that delivers toxic effector proteins directly into target cells in a contact-dependent manner. These effectors are associated with a diverse range of functions such as cytoskeletal modification, biofilm formation, and bacterial competition. Effectors are loaded onto the T6SS by interacting with its structural components. Often, additional accessory proteins are needed for the secretion of these effector proteins. Despite their importance, the direct role of these accessory proteins in the assembly of the T6SS warhead or its subsequent ‘firing’ is unclear. One such example is the putative DUF2169 family of proteins. These proteins are required for effector secretion, however, the molecular function of DUF2169 proteins remains unknown. I have shown that DUF2169-encoding genes co-occur with the structural components of T6SS, such as VgrG and effectors that possess a specialized N-terminal PAAR-like domain. Using a Pseudomonas aeruginosa T6SS-associated gene cluster, I show the network of protein interactions with DUF2169 and these T6SS structural components. Furthermore, I was able to show that these gene clusters form a conserved synteny. Multiple proteins encoded within DUF2169 syntenies are predicted to mimic previously characterized proteins associated with T6SS function. Lastly, using X-ray crystallography I was able to solve the structure of DUF2169 from Vibrio xiamenensis as the first representative of the DUF2169 protein family. Additionally, using structural predictions I show the molecular interaction between DUF2169 and PAAR-like domains of effectors as a potential chaperone. Gaining structural insight into the role of this protein will not only enhance our understanding of T6SS function but also highlight the mechanistic differences between different T6SS warheads. Since T6SSs can deliver a variety of proteins into both prokaryotic and eukaryotic cells, understanding how this delivery mechanism works expands our knowledge of how bacteria interact with diverse cell types. Furthermore, uncovering a new mechanism for directly delivering proteins into different cell types has valuable potential for medical and industrial applications. Thesis Master of Science (MSc) Gram-negative bacteria can use a pathway called the type VI secretion system or T6SS to kill these competitor bacterial species. T6SS is a protein complex that facilitates the injection of toxic proteins, from attacking cells directly into target cells. The secretion of these toxic proteins requires additional accessory proteins, which recruit and stabilize the toxic proteins before secretion. In this work, I characterize an accessory protein family that functions as a chaperone for these toxic proteins. First, I showed that these accessory protein family genes are linked to genes that encode for the core T6SS apparatus proteins. Secondly, I was able to capture the first snapshot of this accessory protein family. This snapshot gives molecular details into the role of this accessory protein in T6SS secretion. Collectively, these results expand our knowledge of how bacteria use the T6SS to secrete toxic proteins. Uncovering a new mechanism for directly delivering proteins into different cell types has valuable potential for medical and industrial applications.

Obesity-related inflammation increases one’s risk of developing cardiovascular disease, type 2 diabetes, among many other disorders. Energy imbalance during obesity overwhelms the adipose tissue leading to immune cell infiltration, elevations in pro-inflammatory cytokines, and consequent local and systemic insulin resistance. Certain potent pro-inflammatory cytokines are produced by the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. This metabolic danger sensor is involved in a variety of autoimmune and inflammatory conditions, hence regulation of this node as a therapeutic approach is a topic of great interest. To date, there is no clinically approved inhibitor of the inflammasome, which makes indirect targeting through endogenous molecules an attractive area of research. Bruton’s tyrosine kinase has been shown to physically interact with NLRP3 inflammasome components and is required for its production of pro-inflammatory cytokine, IL-1β. However, the consequences of BTK inhibition on metabolic tissues like adipose tissue remain largely unstudied. In vitro experiments showed that pharmacological BTK inhibitors lower NLRP3 inflammasome priming and activation in macrophages. Bone marrow-derived macrophages (BMDMs) from a Btk-null mice strain treated with BTK inhibitors demonstrated lowering of lipopolysaccharide (LPS) -induced elevations in inflammatory cytokines. This effect was interestingly observed in both BMDMs derived from wild-type and Btk-null mice, postulating the idea that pharmacological inhibitors of BTK have anti-inflammatory properties that do not require the presence of the BTK gene product. Further, the optimized conditions for testing the influence of BTK inhibitors on adipose tissue insulin signaling ex vivo is outlined. Future work should focus on pinpointing off-targets shared between BTK inhibitors to identify the node responsible for the observed anti-inflammatory effects. This research may lead to the development of an alternative therapeutic approach to treating inflammatory and autoimmune diseases mediated by the NLRP3 inflammasome. Thesis Master of Science (MSc) Obesity overwhelms fat storage sites and causes inflammation, which can contribute to metabolic disease. An important part of this inflammation is the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. This sensor of stress detects danger signals and produces molecules involved in inflammation. However, overactivation of NLRP3 can cause inflammatory diseases. Blocking another protein, Bruton’s tyrosine kinase (BTK), can limit the activation of NLRP3 inflammasome. Our findings show that blocking BTK using drug inhibitors lowers inflammation caused by the NLRP3 inflammasome. Interestingly, we found that these drugs can lower inflammation without working on BTK, the protein that these drugs are designed to block. Understanding how these drugs are anti-inflammatory will help us understand the NLRP3 inflammasome, and how to lower inflammation during obesity and metabolic disease.

With the rise of antibiotic resistance, there is ongoing need to find new antibiotics. As bacteria develop resistance to the current classes of antibiotics available, it is imperative to discover new ways to target bacteria. In this thesis, I focused on one of the basic components that all bacteria need to survive: a source of carbon. Here, I explore whether we can exploit this aspect for drug discovery. For bacteria to colonize a host and cause an infection, it must first be able to meet its nutritional needs for growth. Different host infection sites will have different carbon sources available. Some sites, like the gut, will have commensal bacteria which will compete with invading pathogens for carbon sources. While we still lack understanding of the specific growth environment bacteria experience during infection, it is important to understand how bacteria grow when given different nutrients. For the first part of my work, I systematically probed the gene essentiality patterns of E. coli grown in different carbon sources. I generated a large dataset of growth phenotypes that I compiled into a user-friendly web-application, Carbon Phenotype Explorer (CarPE). I identified many poorly annotated genes, and further characterized the gene ydhC as an adenosine transporter. After characterizing how the growth of E. coli and the genes essential for survival change depending on each carbon source, I looked at whether antibiotic efficacy changed depending on the carbon source used. I found that growth in oxaloacetate alters the proton motive force and potentiates macrolide antibiotics. I also found that linezolid, a compound that does not work on gram-negative bacteria due to efflux, is more effective when adenosine is the carbon source. Together, this work forms a foundation for future research into studying how carbon sources can be exploited in the field of antibiotic discovery. Thesis Candidate in Philosophy There is an urgent need for new antibiotics. Previous antibiotic discovery has primarily been conducted on bacteria growing in nutrient rich laboratory conditions. This led to antibiotics that targeted the same few bacterial processes. However, since bacteria need to survive in a host to cause an infection, there are targets that may be viable during an infection that we miss by using standard laboratory media. Bacteria need a source of carbon to survive, and each infection site contains different chemicals that bacteria can use as a source of carbon. My work studies how bacteria grow in the presence of different carbon sources. First, I systematically tested which bacterial genes are required for E. coli to grow in 30 different carbon sources. I then examined the effectiveness of antibiotics on bacteria grown using these different carbon sources. Together, this work helps us understand how changing carbon sources in the growth media we use to cultivate bacteria can change which genes are required and how it may change how bacteria survive antibiotic stress. When we discover the specific compositions of host infection environments, we can leverage this knowledge to find antibiotics that target these carbon acquisition pathways in bacteria.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets. An uncharted area of antibiotic discovery can be explored by inhibiting nutrient biosynthesis. Herein, we investigate the potential of inhibiting biotin biosynthesis in monotherapy and combination therapy approaches to treat multidrug-resistant Gram-negative pathogens. In chapter 2, we validate biotin biosynthesis as a viable target for Gram-negative pathogens. Historically, biotin biosynthesis was overlooked as a target in Gram-negative pathogens as there was no observed fitness cost associated with its inhibition in standard mouse infection models. We discovered traditional mouse models do not accurately represent the biotin levels in humans. We developed an innovative mouse model to account for this discrepancy, validating biotin biosynthesis as an antimicrobial target in the presence of human-mimicking levels of biotin. Exploiting this sensitivity, we show that an inhibitor of biotin biosynthesis, MAC13772, is efficacious against Acinetobacter baumannii in a systemic murine infection model. In chapter 3, we continue to investigate the potential of targeting biotin biosynthesis in a combination therapy approach. In this work, we identify the ability of MAC13772 to synergize with colistin exclusively against colistin-resistant pathogens. The first committed step of fatty acid biosynthesis requires biotin as a cofactor; therefore, it is indirectly inhibited through the action of MAC13772. We propose that the inhibition of fatty acid biosynthesis leads to changes in membrane fluidity and phospholipid composition, restoring colistin sensitivity. The combination of a fatty acid biosynthesis inhibitor and colistin proved superior to either treatment alone against mcr-1 expressing Klebsiella pneumoniae and colistin-resistant Escherichia coli murine infection models. Together, these data suggest that biotin biosynthesis is a robust antibiotic target for further development in monotherapy and combination therapy approaches. Thesis Doctor of Philosophy (PhD)

Coronary artery disease (CAD) is one of the leading causes of mortality worldwide, and is driven by atherosclerosis development that can lead to occlusive thrombus formation and myocardial infarction. Platelets are known to contribute to atherosclerosis development and thrombosis. Scavenger receptor class B type 1 (SR-B1) deficiency in apolipoprotein E (ApoE) knockout or low-density lipoprotein receptor (LDLR) knockout mice results in occlusive coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis. In this thesis, we examine the effects of genetically inactivating neurobeachin-like 2 (NBEAL2), which has a prominent role in platelet function, on CAD development in SR-B1/ApoE double knockout mice. NBEAL2 deficiency in SR-B1/ApoE double knockout mice reduced aortic sinus and coronary artery atherosclerosis and platelet accumulation in atherosclerotic coronary arteries, but increased myocardial fibrosis levels and reduced survival. NBEAL2 deficiency in SR-B1/ApoE double knockout mice reduced neutrophil granularity and the proportion of myeloperoxidase-positive neutrophils in the myocardium, which may in part contribute to the increased myocardial fibrosis levels. Protease-activated receptor 4 (PAR4) also has a prominent role in platelet function. By using a novel pepducin (RAG8), we examine the effects of pharmacological inhibition of PAR4 on CAD development in high-fat, high-cholesterol, cholate (HFCC) diet-fed SR-B1/LDLR double knockout mice. RAG8 treatment did not alter aortic sinus atherosclerosis levels, but reduced coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis. These protective effects are not dependent on changes in circulating lipids, cytokines, and immune cells, but may be due to reduced vascular cell adhesion 1 (VCAM-1) protein expression in coronary arteries. We also examine the effects of aging on CAD development in HFCC diet-fed SR-B1 single KO mice. Older age in SR-B1 single KO mice did not alter susceptibility to HFCC diet-induced aortic sinus atherosclerosis, but increased susceptibility to HFCC diet-induced coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis. Older-aged SR-B1 KO mice exhibited reduced survival when fed the HFCC diet. These age-dependent changes are not driven by changes in plasma lipids, but likely driven by a combination of increased VCAM-1 protein levels in coronary arteries and circulating cytokines and neutrophils. This thesis demonstrates the potential effects of platelet inhibition and aging on CAD development in mice, and demonstrates that SR-B1 single KO mice may be a versatile and useful diet-inducible mouse model for atherosclerosis studies. Moreover, this thesis highlights the importance of analyzing atherosclerosis at multiple sites and utilizing mice that develop CAD and myocardial fibrosis when investigating the effects of interventions on atherosclerosis and/or CAD development. Dissertation Doctor of Philosophy (PhD)

Introduction: Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening adverse event that occurs in approximately 5% of patients following heparin administration to prevent clotting during cardiac as well as orthopedic surgeries, kidney dialyses and blood transfusions. HIT is mediated by antibodies that bind complexes of platelet factor 4 (PF4 or CXCL4) and heparin that cross-link Fc receptors on platelets to cause platelet activation, thrombocytopenia with or without thrombosis, potentially leading to death. Recently, a new anti-PF4 disorder has emerged in response to COVID-19 adenoviral-vector based vaccines against SARS-CoV-2 known as vaccine-induced immune thrombotic thrombocytopenia (VITT). VITT is a rare but serious adverse syndrome reported 5-30 days after administration of a COVID-19 adenoviral vector vaccine (Oxford-AstraZeneca ChAdOx1 nCoV-19 (AZ) and Johnson & Johnson/Janssen (J&J/Janssen)) and is characterized by thrombocytopenia and thrombosis in unusual locations, including cerebral sinus venous thrombosis (CVST). HIT and VITT are characterized by platelet-activating antibodies that bind to PF4 and often lead to thrombocytopenia and/or thrombosis. There are two types of platelet-activating antibodies that lead to HIT, antibodies that activate platelets only in the presence of heparin (heparin-dependent (HD)) and antibodies that can activate platelets in the absence of heparin (heparin-independent (HI)). In VITT, the two types of platelet-activating antibodies are ones that only activate platelets in the presence of PF4 (PF4-dependent) and antibodies that can activate platelets in the absence of PF4 (PF4-independent). We hypothesize that the binding sites of HIT and VITT antibodies on PF4 differ and that identifying these sites could be relevant to differentiate between both anti-PF4 disorders. Methods: All 70 PF4 mutants produced by alanine-scanning mutagenesis (ASM) were expressed and purified from E. coli BL21 (DE3) cells. ASM was performed using HIT (n=10) and VITT (n=14) patient samples to determine the binding sites of HIT and VITT antibodies on PF4. Clinical outcomes of CVST, DVT, PE incidences in VITT patients with PF4-dependent and PF4-independent antibodies were also analyzed. In addition, ASM was performed on the HIT-mimicking monoclonal antibodies (1E12, 1C12, 2E1, P4B1, P3D4, P3E10) to confirm the pathogenic antibody binding sites of HIT antibodies on PF4. Finally, all 70 PF4 mutants were screened against HIT (n=22), VITT (n=9) and non-HIT (n=12) patient samples and we identified 10 PF4 mutants that differentiate between HIT and VITT using an EIA. Results: We confirmed all 70 PF4 mutants were free of contaminants compared to bacterial lysate by visualizing the purity using gel electrophoresis. Using a cut-off of 50% or greater reduction in antibody binding to PF4 mutants compared to wild-type PF4, we determined that most monoclonal antibodies that mimic platelet-activating HIT antibodies, as well as antibodies purified from HIT patients bind to a site on PF4 like the KKO site, a known monoclonal antibody against PF4/heparin complexes that mimics platelet-activating HIT antibodies. While most VITT antibodies recognize a site on PF4 overlapping the heparin-binding site, we found a subset of VITT patients have antibodies that recognize an additional site on PF4 that is like the KKO site. By observing the incidences of clinical thrombotic characteristics in VITT patients, it was determined that CVST (6/14, 42.9%), DVT (4/14, 28.6%), and PE (4/14, 28.6%) were the most common types of thrombosis. This unusually high incidence of VITT is not typically seen in HIT. Information on the binding characteristics of HIT and VITT anti-PF4 antibodies have sparked interest on whether there are characteristics that can help differentiate between HIT and VITT. After screening the 70 PF4 mutants against HIT and VITT patient samples, we then identified 10 PF4 mutants (H23A, R20A, T25A, E28A, K46A, R49A, K50A, K62A, K65A, K66A) that help differentiate between HIT and VITT in an EIA. We also screened the 70 PF4 mutants against non-HIT patients, identified as individuals that have non-platelet activating anti-PF4/heparin antibodies. We found that 5 PF4 mutants (K14A, H23A, V29A, T38A, K50A, K66A) help differentiate HIT from non-HIT in an EIA. Conclusion: Purifying PF4 mutants reduced background reactivity likely caused by contaminants in assays using bacterial lysate. ASM using PF4 mutants revealed that there are 2 distinct binding sites on PF4 that are recognized by either HI and HD HIT or anti-PF4 VITT antibodies. We confirmed that the molecular characteristics of antibodies from VITT and HIT patients dictate the platelet activation profiles in these disorders and can be used to separate VITT from HIT antibodies in the EIA. Thesis Master of Science (MSc)

Cancer is the leading cause of death in Canada and one of the leading causes of death worldwide. Conventional cancer therapies such as chemotherapy often include severe side effects that can decrease the quality of life of patients undergoing treatment. Immunotherapy is designed to harness the host immune response and enhance its ability to seek out and kill cancer cells. Immunotherapy has gained traction in the past decade due to its improved safety and efficacy over conventional cancer therapies. However, there is room for improvement as most patients fail to respond to immunotherapy. The work described in this dissertation involves the development of therapeutic combination platforms that are designed to improve upon immunotherapy outcomes. Murine tumor models were used to develop a better understanding of biological processes associated with therapeutic efficacy. These findings can be used for the development of therapeutic strategies that can further improve the efficacy of cancer immunotherapy. Cancer immunotherapy has demonstrated immense promise in the past decade. Immune checkpoint therapy has shown unprecedented responses in many cancers; however most patients fail to respond to checkpoint therapy. This highlights the need to develop a better understanding of factors in the tumor microenvironment that can influence therapeutic outcomes. In this body of work, we have utilized oncolytic viruses (OVs) to enhance immunogenicity in the tumor and study the cellular mechanisms that enable a therapeutic response. We utilize a combination of OVs and low dose chemotherapy to further sensitize murine models of mismatch repair-deficient colorectal cancer to checkpoint therapy. Using a Clariom S transcriptome assay we found that the combination induced gene signatures associated with the recruitment and activation of myeloid subsets. When we assessed tumor infiltrates, we found that the combination induced the chemoattraction of several myeloid subsets, including type I conventional DCs (cDC1s) which are known for their role in antigen presentation. Using Batf3-/- mice, we demonstrated that the therapeutic efficacy of our combination platform was dependent on the presence of cDC1s. In this dissertation, we also studied the role of OV-induced type I IFN (IFNI) in enabling or suppressing antitumor immunity. We found that OVs induced the upregulation of PD-L1 in an IFN-I-dependent manner in cancer cells and circulating immune cells. Inhibition of IFN-I signaling using an anti-IFNAR monoclonal antibody partially prevented OV-induced upregulation of PD-L1. Furthermore, the combination of OV and v | P a g e IFNAR blockade enhanced the effector functions of tumor-specific T cells and led to better tumor control compared to OV monotherapy. Altogether, these findings demonstrate that OVs can be an effective agent for enhancing immunogenicity in the tumor and promoting the infiltration of inflammatory myeloid subsets. By combining OVs with checkpoint or IFNAR inhibitors, we prevent the onset of immunosuppression and enable a favorable therapeutic response. Thesis Doctor of Philosophy (Medical Science)

Bacterial specialized metabolites (SMs) have long interested scientists due to their diverse chemistries which can harbour antimicrobial activity. The discovery of these molecules experienced a period of exponential success in the mid-1900s and today is guided by next-generation sequencing and liquid chromatograph-mass spectrometry technology. Despite these technological advances, however, they remain under-leveraged. Now, merging disease and antibiotic resistance threaten the security provided by existing antimicrobial medicines. There is an urgent need for a more targeted approach in the discovery of novel SMs, which leverages the tremendous efforts of the past alongside modern big data analytics. To this end, I set out to develop a foundation that could guide SM discovery in the future. I began by bridging all available bacterial metabolism data into a common latent space. Using known metabolic pathways, I generated a library of biosynthetic units used in a novel program to encode metabolites. Each unit was connected to its requisite genes, which were leveraged to reverse engineer this platform by predicting the chemical structures of SMs directly from their encoding genes. Finally, deep learning models were used to annotate some of these chemical-genomic connections as being associated with a particular activity, in this case siderophore activity. This suite of software tools offers promising opportunities to pursue downstream applications, including the connection of unknown genes to metabolites and the identification of novel chemical-genomic connections. In order for these prospects to be realized, however, this intricately-connected network of data necessitates more sophisticated interpretation. Indeed, interconnected chemical, genomic and activity data lends itself particularly well to analysis by graph neural networks. The foundational work described in this communication builds the basis for this comprehensive analysis, which may uncover new insights into the bacterial metabolism we thought we knew. Thesis Master of Science (MSc)

Repair of two major forms of DNA damage, single strand breaks and base modifications, are dependent on XRCC1. XRCC1 orchestrates these repair processes by temporally and spatially coordinating interactions between several other repair proteins. Here we show that XRCC1 contains a central DNA binding domain (CDB, residues 219–415) encompassing its first BRCT domain. In contrast to the N-terminal domain of XRCC1, which has been reported to mediate damage sensing in vitro, we demonstrate that the DNA binding module identified here lacks binding specificity towards DNA containing nicks or gaps. Alanine substitution of residues within the CDB of XRCC1 disrupt DNA binding in vitro and lead to a significant reduction in XRCC1 retention at DNA damage sites without affecting initial recruitment. Interestingly, reduced retention at sites of DNA damage is associated with an increased rate of repair. These findings suggest that DNA binding activity of XRCC1 plays a significant role in retention at sites of damage and the rate at which damage is repaired.

Ongoing antibiotic drug discovery is vital as antimicrobial resistance continues to be a significant issue faced in the clinic. Natural products have long been a highly productive source to mine for new antimicrobials. While it has been challenging to discover new and unique antimicrobial natural products, numerous drugs have been derived from studying how natural products function as secondary metabolites. Previous studies suggested that screening natural product extract fraction libraries for antimicrobials can be more productive than screening crude extracts alone. These studies from large industrial enterprises are generally not directly portable to an academic setting due to significant infrastructure costs. We developed a screening platform consisting of low pressure reversed-phase chromatographic separation of methanolic extracts of bacteria and fungi to generate a prefractionated natural product library. This platform is suitable for academic labs to screen for antimicrobial compounds. A large growth inhibitor screen against multiple pathogens and lab strains of microbes was conducted to assess the validity of the advantages of screening fraction libraries versus crude extract libraries and to search for potential new drug-like compounds. Hits were investigated for reproducibility, isolated, and purified. One compound was discovered in an antifungal screen which may be a novel lipopeptide. Thesis Master of Science (MSc) To combat the growing antibiotic resistance crisis, new strategies for drug discovery must be investigated and implemented. Natural products from bacteria and fungi have long been a source of critically important drugs. Prior research suggests that looking for vital natural products in fraction libraries can be more productive than screening crude extracts. Here the development and assessment of a fractionation library suitable for an academic lab's ingrained limitations are described. Assessing the library indicates an increased hit rate on screening fractions compared to crude extract. Furthermore, pursuing these hits may have revealed a novel antifungal lipopeptide.

Tauopathies refer to a group of neurodegenerative diseases that are characterized by pathological aggregations of the microtubule-associated protein Tau (MAPT). Aberrant alternative splicing of Tau exon 10 leads to the imbalanced expression of Tau isoforms that contain either 3 or 4 microtubule binding repeats (3R-Tau or 4R-Tau) and this is sufficient to cause the formation of Tau inclusions. Nonetheless, the exact molecular mechanisms that regulate aberrant Tau exon 10 splicing regulation and subsequent Tau aggregation in tauopathies remain elusive. In my thesis research, I used RNA Antisense Purification by Mass Spectrometry (RAP-MS) to identify upstream regulators of Tau splicing events. Among the 15 identified novel protein candidates, I validated that hnRNPA2B1 and hnRNPC are required to promote 4R-Tau expression, whereas hnRNPH1 supports 3R-Tau expression. Separately, to elucidate the functional difference between 3R- and 4R-Tau isoforms, I performed proximity-dependent biotin identification (BioID2) for all six human central nervous system Tau isoforms in mouse primary neurons. Followed by tandem mass tag (TMT)-labeling proteomics and data analysis, I observed that 4R-Tau proximal proteins are highly enriched in endocytosis, whereas 3R-Tau proximal proteins show top enrichment in fatty acid metabolism. Through further biochemical validations, I found that MAT2A, a S-adenosylmethionine synthase, has higher binding affinity with 3R-Tau versus 4R-Tau. Overall, using novel proteomics methods, I discovered novel Tau splicing regulators and characterized the neuronal Tau isoform-specific proximity proteome networks. These proteins, once validated through future functional studies in cellular and animal models, can represent therapeutic and diagnostic targets for neurodegenerative tauopathies. Thesis Doctor of Philosophy (PhD)

Type IV pili (T4P) are hair-like adhesins involved in many processes, including surface attachment, twitching, DNA uptake, electron transfer, and pathogenesis. These flexible filaments are expressed in various pathogens, including the opportunistic pathogen Pseudomonas aeruginosa. The pilus fibre is primarily composed of the major pilin structural subunit, PilA, which is rapidly polymerized or depolymerized during pilus extension or retraction, respectively. The transcription of pilA is tightly controlled by the PilS-PilR two-component system, which responds to fluctuating levels of PilA in the inner membrane. In addition to pilA, the response regulator, PilR, also regulates a subset of other non-T4P related genes. Here, we used hyperactivating point mutants in the PilS-PilR two-component system, which induce hyperpiliation without loss of pilus function, to assess the effects of increased surface pili expression on virulence against Caenorhabditis elegans, and to identify additional non-T4P genes regulated by the PilS-PilR two-component system. We hypothesized that dysregulation of the PilS-PilR two-component system impacts the expression of pilA and other genes, which impacts both surface piliation and T4P dynamics, resulting in altered P. aeruginosa virulence. C. elegans slow killing assays revealed that hyperpiliation, independently of T4P function, reduces virulence of model P. aeruginosa strains PAK and PA14. We propose a model whereby a surfeit of pili reduces virulence, potentially through impeding effective engagement of contact-dependent antagonism systems, such as the type III secretion system. Transcriptomic analysis of the hyperactive PilR point mutant also identified a subset of 26 genes, including those related to phenazine biosynthesis, quorum sensing, and ethanol oxidation, regulated by the PilS-PilR two-component system. Last, a T4P cysteine-labelling system was implemented for P. aeruginosa, allowing for the visualization of real-time pilus dynamics. Together, this work provides new insights into the consequences of hyperpiliation and the scope of the PilS-PilR signalling network, as well as novel tools for investigating P. aeruginosa T4P dynamics in vivo. Thesis Master of Science (MSc) Pseudomonas aeruginosa is a major contributor to hospital-acquired infections and is of particular concern due to its intrinsic resistance to many frontline antibiotics. To aid in infection, Pseudomonas encodes an arsenal of virulence factors, including type IV pili (T4P), hair-like adhesins involved in many processes, such as twitching motility and surface attachment. T4P are primarily composed of the major pilin, PilA, whose expression is tightly regulated by the PilS-PilR two-component system. The sensor kinase, PilS, monitors the inner membrane PilA inventory and modifies activity of the response regulator, PilR, to regulate pilA transcription. Here, we demonstrate that P. aeruginosa virulence in a roundworm infection model is reduced when the amount of T4P expressed at the cell surface increases, regardless of the ability of the bacteria to twitch. We propose that inappropriate increases in surface T4P expression may impair pathogenicity-associated systems which require intimate host-cell contact. New genes in the regulon of the PilS-PilR two-component system were also identified. A tool to fluorescently label and image T4P in real-time using microscopy was established in the lab. This work highlights the consequences of increased surface T4P expression, providing potential new targets for antipseudomonal therapeutics which act on components involved in T4P expression and function.

Adoptive cell therapy (ACT) is a powerful cancer immunotherapy platform, where T lymphocytes are collected from cancer patients, expanded ex vivo, and infused back into the patient as a cellular drug. Genetic modification with chimeric receptors redirects T cell tumour-specificity. While ACT is a clinical success in many cancers, it is dependent on T cell proliferation following infusion, where robust expansion is a predictor of a positive outcome. Costimulation is the activation of a class of receptors which provide maximal stimulatory signals to T cells, resulting in an enhancement of proliferation, survival, and memory. Costimulatory receptor signaling occurs in cooperation with the T cell receptor, and is provided by several receptor families that converge on costimulatory signaling pathways. Chimeric receptors can be modified with costimulatory domains however, the non-canonical signaling can lead to overstimulation and serious toxicity in the clinic. We hypothesize that pharmacological costimulation can be used to boost T cell proliferation, where drug administration and dosage is controlled and the drug can cease to be administered should toxicities manifest. Small molecule costimulation may prove to be a useful approach to boost the efficacy of ACT as a cancer therapy. The objective of my research project is to screen and characterize for small molecules that enhance T cell proliferation. Specifically, I will investigate the ability small molecule drugs to enhance T cell expansion following antigen-specific stimulation of T cells engineered with T cell antigen coupler (TAC) receptors. Methods: I identified candidate small molecules with costimulatory effects on TAC T cells using two strategies. First, I generated a database of small molecules that were predicted to interact with costimulation pathways based on connectivity mapping, provided by the Broad Institute, using the transcriptomes of TAC T cells activated in the presence or absence of costimulation. As a second screening strategy, I developed a bioluminescent reporter system that was used for high throughput screening of small molecules libraries available at the Centre for Microbial and Chemical Biology. Following the identification of several hits, these compounds were tested in vitro for their modification to TAC T cell proliferation and function. Results: We have identified several chemical classes of compounds which contributed to a robust increase in TAC T cell proliferation in the absence of costimulation, and have evaluated the top three drugs in vitro by proliferation and RNA sequencing assays. Ferutinin, and protein kinase C activating compounds mezerein and phorbol 12,13-dibutyrate, were found to robustly increase TAC T cell proliferation. The significance of this project is to identify costimulatory small molecules to enhance the efficacy of T cell cancer therapy, and yield novel insight into the chemical space that modulates costimulation. Thesis Master of Science (MSc)