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Characterizing Two Phage-Plasmids in the Context of the Bacterial Pathogen, Citrobacter rodentium
No full textCitrobacter rodentium is a bacterial pathogen adapted to the murine gastrointestinal tract. This pathogen is a member of the attaching and effacing (A/E) family of pathogens and is an important model in the study of the closely related human diarrheal pathogens, Enteropathogenic Escherichia coli (EPEC) and Enterohaemorrhagic E. coli (EHEC). Much complexity of this model remains unknown, including the interaction between phage and their role in mediating host physiological changes. Understanding the role of phage elements found in the genome of C. rodentium was motivated by an interest in how these elements contribute to the physiology of their host pathogen. Previous research has shown that C. rodentium strain DBS100 contains 10 prophage regions, while only two have been shown to be spontaneously active. Phage generally replicate through two cycles: lytic (responsible for host killing) and lysogenic (dormant in bacterial chromosome). This lytic-lysogenic switch is mediated by a complex protein network, however, some phage (P-Ps) are also capable of replicating as plasmid intermediates and are maintained in this state via conserved genes only found on other plasmid replicating elements. The purpose of this thesis overall, was to characterize the lifestyle preferences of two C. rodentium phage, phiNP and Shae_phiSM, suggesting a role in host physiology, if any. More broadly, we aimed to improve the standards for classification of P-P elements as the lines differentiating mobile genetic elements become blurred. Firstly, using genomic analysis and comparative alignments we found that both phage encode toxin-antitoxin addiction modules (phiNP- hicAB and Shae_phiSM-hok./sok) and highly conserved plasmid related (Shae_phiSM-parAB and repA) replication genes. This analysis confirmed that phage Shae_phiSM and phiNP encode the necessary plasmid replication and partitioning machinery in order to replicate through a plasmid-like lifestyle. We then investigated the phylogenetic relationships amongst related phage and found that Shae_phiSM has a direct evolutionary relationship to P2-like temperate phage and a distant relationship to P1-like P-Ps. Additionally, we found that phiNP is a novel phage with little homology to other phage sequences. Secondly, following bioinformatic characterization, we assessed basic parameters of the phage lifecycles using various assays in vitro. We found that both phage Shae_phiSM and phiNP have a Myovirus morphology, capable of infecting a narrow E. coli host range after adsorption to the bacterial lipopolysaccharide (LPS). This data supports previous reports on the infection and kinetics of these two phage. Thirdly, we aimed to detect the plasmid like state of these phage using qPCR experiments to assess their lifestyle preferences in vitro. When grown in an environment representative of a simulated gastric system, we found that Shae_phiSM largely prefers an integrative lysogenic state while phiNP may prefer episomal replication. Taken together, this supports the notion that P-Ps in general prefer lysogenic lifestyles where they form stable long- term associations with their host. Finally, we preliminarily utilized a CRISPRi knockdown system to generate C. rodentium strain backgrounds with dampened phage parA and hicA gene expression. Interestingly, we found that downregulated parA and hicA expression subsequently leads to increased phage abortive lysis, implicating these genes as important for plasmid maintenance. We unexpectedly hypothesize about a putative interaction between non-homologous plasmid regulatory genes parA and hicA encoded on Shae_phiSM and phiNP respectively. These plasmid regulatory systems seem to influence the stable maintenance of each other as a P-P via interaction between hicA and parA, though further research is required to confirm this interaction. Taken together, we have reason to reclassifying Shae_phiSM and phiNP as P-Ps capable of plasmid replication. Study of these P-Ps has expanded our view of phage diversity and demonstrates the complexity of phage biology, particularly the ever-evolving nature of phage. P-P evolution could be a large contributor to the hidden complexities underlying bacterial adaptation and lifestyle with one health related aspect of this being pathogenicity. Understanding the vast diversity of phage elements can provide us with one more lens to glimpse the beauty of divine creation and to appreciate both the integrated harmony as well as the incredible complexity within bacteriophage and their hosts
A Blooming ‘Canola Painting’ Beckons — Even to Pests
No full textEven a painting cannot escape the pests and diseases that plague canola.This image portrays a vibrant oil painting of blooming canola flowers, framed and mounted on the wall. Surrounding the frame are three common insect pests and invisible microscopic pathogens of canola. The image metaphorically illustrates a key challenge in Canadian canola production. However, while the threats loom around the painting, none have crossed into the frame. This design symbolizes the protective boundary created by Ualberta ongoing research into pest and disease resistance. The frame becomes a metaphorical wall of defense, built not of wood or metal, but of scientific understanding and innovation. It highlights the critical role that plant pathology and entomology research play in safeguarding canola health and ensuring sustainable canola production
Simulation-Optimization Framework for Short-term Planning Optimization of Open-Pit Mines with IPCC in the Presence of Haulage Uncertainties
No full textMining is a highly capital-intensive industry, with haulage being a critical and expensive component. In open-pit mines, material transportation costs increase significantly as mines deepen. Traditionally, diesel trucks have been the primary means of haulage, but In-Pit Crushing and Conveying (IPCC) systems have emerged as alternatives due to their lower operating costs and reduced greenhouse gas emissions compared to truck-shovel (TS) systems.
Mine planning is categorized into long-term and short-term planning. Long-term planning focuses on maximizing the net present value (NPV) over the mine’s lifespan, while short-term planning optimizes operational activities to meet production targets. The short-term planning horizon can range from monthly to daily schedules.
This thesis presents a simulation-optimization framework for short-term mine planning, applicable to both TS and IPCC systems. The framework combines a mixed integer linear programming (MILP) model for shovel allocation with a Monte Carlo Simulation (MCS) model to address operational uncertainties. The MILP model generates monthly production schedules by optimally allocating shovels to mining cuts, and the simulation evaluates the impact of uncertainties such as equipment performance on these schedules. This integration ensures production deliverability and reliable performance assessment.
Optimal utilization of equipment is critical for minimizing costs. The primary objective of this research is to maximize NPV while reducing haulage costs. This PhD research is structured into three phases. First, an MILP model for shovel allocation was developed and verified with an iron mine. Second, a Monte Carlo haulage simulation model was implemented in MATLAB to capture operational uncertainties. Finally, the shovel allocation model was integrated with the haulage simulation, allowing direct input of schedules into the simulation model. The integrated model was validated using the same iron ore mine case study.
The proposed MILP model optimally allocates shovels to mining faces to meet production requirements, reduce haulage costs, and maximize periodical profit. The case study presents two scenarios: one with semi-mobile IPCC and one with traditional truck haulage, comparing overall profit and haulage costs in each scenario. Variations in scheduling and extraction sequences based on shovel allocation demonstrate the impact of IPCC installation on mine planning from an operational perspective.
The Monte Carlo Simulation (MCS) model captures the hauling process and operational uncertainties associated with trucks, shovels, and the IPCC system. Due to the nature of MCS, which simulates stochastic inputs and outputs without modeling event-based sequences or discrete time steps, time is not explicitly represented. Instead, the model captures total production and performance indicators for the entire planning period.
Integration of the MILP model with the simulation model is achieved through an Excel database, which reads and stores the optimal production schedules generated by the MILP. The integrated framework evaluates total production across scenarios, comparing IPCC and traditional TS haulage systems under uncertainty. This comparison assesses how well each system adheres to the MILP-generated production schedules, focusing on metrics such as production output, reliability, and tonnes per gross operating hour (TPGOH).
Comparing In-Pit Crushing and Conveying (IPCC) with TS haulage in short-term mine planning is complex, influenced by factors such as cost structure, production needs, and environmental and safety considerations. This thesis undertakes a detailed comparative analysis of the two systems, emphasizing their cost structures and ability to meet production requirements.
This research can be marked as one of the pioneering works on short-term planning of open pit mines with IPCC because short-term planning with IPCC is an underexplored area of research. The research questions addressed by this thesis are: How does IPCC impact short-term mine sequencing? Does IPCC provide better haulage performance than pure TS haulage in the presence of operational uncertainties?
The major contributions of this thesis to the research community in mining applications are: a novel simulation-optimization approach for uncertainty-based short-term planning that captures production deliverability and haulage operational details with IPCC or TS haulage, ⅱ) a deterministic MILP model for short-term planning of open-pit mines through optimal shovel allocation, applicable to both IPCC and TS haulage as a standalone tool, and ⅲ) a comparative tool to evaluate the performance of the IPCC system against TS haulage for maximizing haulage cost savings. The study shows that IPCC reduced the ore haulage cost by 45% and the ore truck requirement by 60% compared to pure truck-shovel haulage
The Design and Synthesis of Small Molecule Inhibitors of the DNA Repair Enzymes ERCC1-XPF and PNKP; and the Design and Synthesis of Antiviral Agents with Broad-Spectrum Activity
No full textSince the dawn of the antibiotic era, the number of deaths worldwide that have been attributable to infectious diseases has fallen dramatically. However, as humans live longer, they become more susceptible to age-related illnesses. Principal among these age-related diseases is cancer, a wide range of diseases characterized by abnormal and uncontrolled cell growth and the subsequent invasion of surrounding tissues by those cells. The opening chapter of this work aims to provide an overview of our understanding of cancer: the causes, types, the methods by which these rogue cells evade elimination, and a brief history and classification of treatments. The intimate link between cancer, DNA, DNA damage, and cancer treatments is explored and leads to a discussion of the mechanisms that cells employ to repair damaged DNA in the second part of the opening chapter.
The basis for chapters 2 and 3 is the discovery of compounds that can inhibit the activity of enzymes involved the repair of damaged DNA. The rationale for this is simple: since the majority of cancer treatments target DNA, and specifically the DNA of replicating cells, it follows that temporarily inhibiting enzymes involved in the repair of that damage will lead to more effective cancer treatments at lower doses. Furthermore, the development of PARP inhibitors that are effective monotherapies for BRCA1 and BRCA2 deficient cancers has proven that exploiting the synthetic lethality that is observed with the disruption of DNA repair mechanisms is an attractive target for the treatment of cancer.
In chapter 2 our efforts to inhibit the heterodimeric protein complex ERCC1-XPF, an endonuclease that plays a role NER and ICL repair, are described. ERCC1-XPF is a target for inhibition that has had some success in the past in the West group and building on this previous success we identified the FDA approved antimalarial compound pyronaridine as a potential inhibitor. The testing of pyronaridine and the synthesis of derivatives that incorporate key features in our previously disclosed ERCC1-XPF inhibitors and pyronaridine are described. Additionally, chapter 2 describes the development of a new synthetic route to a previously disclosed ERCC1-XPF inhibitor after a key starting material in the synthesis was discontinued. This new synthetic route was scaled up to provide additional material for the testing of that ERCC1-XPF inhibitor in animal models.
Efforts towards the inhibition of the DNA end-processing enzyme PNKP are described in chapter 3. PNKP plays a central role in multiple DNA repair pathways, and the synthetic lethality of PNKP inhibition with SHP-1 and PTEN deficient tumours has been previously demonstrated. We attempted to develop inhibitors based on hits in high throughput screening assays. Unfortunately, this work was plagued by false positives by pan assay interfering compounds and only one of the identified compounds was a true hit. Investigations of the mechanism of action and the attempted derivatization of this compound are described.
Chapter 4 of this work stands alone as it describes the urgent need for the development of antiviral agents with broad spectrum activity. A series of ester linked polygallate compounds that inhibited a range of unrelated viruses was previously developed in the West group. We sought to investigate the necessary interactions in this series of inhibitors; to achieve this, several control compounds were synthesized and tested against the same viruses that the pollygallate inhibitors were tested against. Furthermore, we attempted to synthesize compounds that did not contain the biologically labile ester linkage. Despite some success in the synthesis of these compounds, the final deprotection steps in the synthesis of ketone linked polygallates was unsuccessful. Further work utilizing different protecting groups for the synthesis of these ketone linked polygallate inhibitors is required
Updating the Monro-Kellie Doctrine: A Translational Exploration of Brain Tissue Compliance and Intracranial Pressure Dynamics Following Acute Stroke in Rodents
No full textStroke is a leading cause of death and disability, with a profound impact on the lives of patients, caregivers, and families. Intracerebral hemorrhage (ICH), a stroke subtype caused by bleeding into the brain due to ruptured cerebral vasculature, constitutes a smaller proportion of all stroke cases (~20%), yet disproportionately accounts for stroke mortality and functional impairment. Ischemic stroke, which makes up the other ~80% of stroke cases, occurs due to impeded cerebral blood flow, leading to brain tissue death and dysfunction in affected areas. Following ICH, the extravasation of blood into the brain parenchyma and subsequent edema (e.g., mass effect) can result in elevated intracranial pressure (ICP), a situation that places the patient at a greater risk of mortality and poor functional outcome. A similar rise in ICP can occur following severe ischemic stroke. Due to the fixed volume of the cranium, the added mass of a bleed and edema requires displacement of other intracranial components, such as cerebrospinal fluid (CSF) or blood. When these intracranial compliance reserves are inadequate, ICP rises, which can ultimately result in additional brain injury, brain herniation, and brainstem compression- a common cause of early mortality, especially following ICH.
These intracranial pressure-volume relationships were first described several centuries ago by the Monro-Kellie doctrine, which asserts that an increase in one intracranial component (e.g., brain, CSF, and blood) must be offset by a decrease in another to prevent elevated ICP. Modern interpretations of the Monro-Kellie doctrine acknowledge that CSF and blood provide dynamic intracranial compliance reserve, but brain tissue volume has historically been considered inelastic. However, in the past decade, mechanosensitive cellular signalling has gained increasing recognition for its role in a diverse array of neurological processes, with the capacity to operate over rapid timescales. Despite these advancements, the longstanding assumption that brain tissue largely does not contribute to acute intracranial compliance reserves in the context of mass effect and elevated ICP has largely remained unchallenged. Spurred on by our lab’s initial serendipitous observations, this thesis challenges the traditional view that brain tissue volume itself is relatively inelastic in response to elevated ICP. Using preclinical rodent models, we demonstrated that the brain parenchyma undergoes volumetric and cellular organizational changes within distal uninjured regions to accommodate the acute mass effect of ischemic and hemorrhagic stroke, a phenomenon we term “tissue compliance.”
In Chapter 2, we used hemorrhagic and ischemic rodent stroke models to investigate the spatial distribution of tissue compliance over time within distal uninjured brain regions, and examined its relationship to elevated ICP. We found that tissue compliance only occurred in the context of elevated ICP, following a similar time course to the ICP elevations observed in preclinical stroke models. Chapters 3, 4, and 5 explored the impact of age, strain, and comorbidities on tissue compliance and ICP dynamics following rodent ICH, revealing that these factors differentially modulate intracranial compliance reserves (including tissue compliance) and influence post-stroke ICP responses. Chapter 6 retrospectively analyzed ICP and edema data collected in our past preclinical studies, establishing the collagenase ICH model as better suited for modelling ICP. This analysis also challenged the assumed linear relationship between edema and ICP, underscoring the importance of intracranial compliance reserves in shaping post-stroke ICP dynamics, at least in preclinical models. In Chapter 7, we employed a multimodal approach to begin characterizing the ionic and molecular basis of tissue compliance, using synchrotron-based X-ray fluorescence microscopy and Fourier-transform infrared imaging, both highly precise spatial mapping techniques. We found that distal uninjured tissue compliance regions (such as contralateral hippocampus) exhibited ion dyshomeostasis following ICH, occurring in the absence of pronounced blood-brain barrier dysfunction, cell death, or oxidative stress- perhaps driven by mechanosensitive signalling.
Collectively, the experiments that constitute this thesis challenge long-standing assumptions made under the Monro-Kellie doctrine, revealing a role for brain tissue compliance in accommodating acute mass effect following stroke. Tissue compliance appears to be an adaptive response to elevated ICP, with potential therapeutic value, though further functional and mechanistic investigation is required to refine possible treatment targets. By emphasizing both experimental and translational rigour, we have ensured that our findings are robust and clinically relevant, laying the groundwork for future strategies aimed at manipulating tissue compliance and mitigating elevated ICP
The Blurring of Imperfect Pieces
Get PDFThis image portrays a fragmented face embraced by the blurry backdrop of nature. The two sides of the face are joined together by a tree trunk weaving its way through the middle of the canvas, and creating an umbrella of leaves above the head. My research focuses on the ways in which trauma-sensitive practices support one’s journey towards wholeness. This art piece aims to capture the paradoxical relationship between the fragmented parts of self and the unified cycle of life to which each person belongs. The
mosaic of jagged felt pieces against the blurred spirals of acrylic paint resists the ‘perfect portrait’ of self that capitalist societies often encourage individuals to pursue. Importantly, the eyes form the focal point of the image, symbolizing a key aspect of healing, which involves the willingness to see oneself and the vulnerability to be seen as imperfect
Degradation Assessment and Maintenance Decision-making for Mechanical and Electrical Assets Driven by Condition Data
Get PDFCondition-based maintenance (CBM) is a proactive maintenance strategy that utilizes condition data to inform maintenance decisions, aiming to prevent critical asset failures, reduce maintenance costs, and enhance system safety. Despite significant advancements in prognostic techniques and the widespread adoption of sensors for continuous data collection, integrating these heterogeneous data into effective maintenance decision-making remains challenging. In terms of methodology, developing degradation models that accurately reflect system health is both critical and complex. This complexity arises from the diverse data types and intricate failure mechanisms associated with various assets, such as electrical distribution systems (EDS), wind turbines incorporating load and supervisory control and data acquisition (SCADA) data, and rotating machinery with multiple failure modes. From the application aspect, practical implementation faces obstacles including stakeholders’ specific asset requirements, cost-effective monitoring decisions, and the necessity for targeted maintenance policies rather than generic strategies. For example, stakeholders may have differing benefits and priorities, as seen between maintenance contractors and wind farm owners. Making cost-effective monitoring decisions involves determining which assets require sensor deployment. Furthermore, the implementation of targeted maintenance policies is crucial, such as the adoption of contractor-oriented maintenance strategies for wind farms and opportunistic maintenance (OM) strategies for large-scale electrical distribution systems. Addressing these challenges is essential for optimizing maintenance practices in mechanical and electrical assets.
To address these gaps, the overarching objective of this thesis is structured around four key topics, focusing on integrating precise degradation modeling with practical maintenance decision-making frameworks for targeted mechanical and electrical assets. In the first topic, an opportunistic CBM is proposed for EDS to address the complexity of large-scale asset management. Maintenance actions are triggered based on the health status observed during inspections. In the second topic, a contractor-oriented maintenance strategy is developed for both onshore and offshore wind farms, aiming to maximize the maintenance contractor profits. This strategy utilizes prognostic information from both monitorable and non-monitorable components, constructing a degradation-related efficiency model that quantifies wind turbine efficiency losses due to component degradation and integrates this into the maintenance decision-making process. Moreover, the third topic introduces an asset-criticality-guided maintenance strategy, which incorporates machine criticality and sensor deployment into the decision-making framework. This approach provides practical insights for identifying asset-specific criteria and aims to maximize the expected revenue of the mechanical systems. Finally, the fourth topic explores the feasibility of implementing additional load monitoring for wind turbine degradation assessment. A cost-effective load sensor system is designed to collect the load data, and a novel degradation assessment method is proposed to incorporate the load data with a nonlinear dynamic state-space neural network model to extract the degradation information of a wind turbine more efficiently.
This thesis advances the field of CBM by offering innovative, data-driven, and actionable strategies tailored to the specific needs of different stakeholders in mechanical and electrical asset management. The developed methods will contribute to significantly reducing operation and maintenance expenses while enhancing net revenue for mechanical and electrical assets across diverse engineering applications
Snow accumulation on plants: mountain ash
No full textThe fruit of mountain ash trees often persist through the winter. In this early spring snow storm, snow accumulated on
both the branches and fruit clusters
Exploring BMP2 and BMP7 in Cartilage Physiology: Mechanistic Insights from Temporomandibular and Nasal Cartilage through In Vivo and scRNA-seq Analysis
No full textObjectives: Previous research has demonstrated that Bone Morphogenetic Protein 7 (BMP7) plays a crucial role in regulating various aspects of cartilage biology, including its development, maintenance, and repair. Specifically, Baddam et al. (2021) highlighted BMP7’s ability to regulate the biomechanical and structural properties of nasal cartilage, influencing both extracellular matrix composition and cellular function. Given the importance of BMP7 in cartilage physiology, its potential role in other cartilaginous tissues, such as the temporomandibular joint (TMJ), has garnered increasing interest. TMJ is a critical joint that allows for jaw movement and is susceptible to various disorders, including temporomandibular joint disorders (TMD). These conditions often result in pain, inflammation, and cartilage degradation. Understanding how BMP7 impacts TMJ cartilage is essential for developing novel therapeutic approaches to restore joint function and repair cartilage damage. In this study, I investigated whether BMP7 exerts similar regulatory effects on TMJ cartilage as it does on nasal cartilage. I hypothesize that BMP7 controls mechanical properties and gene expression independent of cartilage type, thereby modulating cartilage properties in the TMJ. By exploring BMP7’s role in TMJ cartilage, we hope to uncover shared or distinct molecular mechanisms that could provide insights into cartilage repair strategies for TMJ disorders.
Methods: To investigate the role of BMP7 in TMJ development, I used a neural crest-specific BMP7 knockout mouse model. TMJ sections from 4-week-old mutant and control mice were analyzed using Safranin O/Fast Green staining, which clearly distinguishes cartilage and bone tissue by staining cartilage red and bone green. This method effectively highlights the structure of articular cartilage, subchondral bone, and other associated tissues. Following this, I analyzed the data from shotgun proteomics comparing wildtype (WT) and BMP7-deficient TMJ cartilage from 2- and 4-week-old mice. Protein interaction networks and gene ontology pathways were established, and differentially expressed proteins were validated through immunofluorescence. To further assess the impact of BMP7 on cartilage properties, chondrocytes were isolated from 1-week-old WT TMJ cartilage and cultured as micromass pellets (2.5 × 10^5 cells). These pellets were incubated for 21 days in differentiation media with or without 2.5 ng/ml recombinant human BMP7 (rhBMP7). Cartilage pellets were tested for mechanical properties using a microsquisher, and phenotypic analysis was performed via immunofluorescence. To deepen our understanding of BMP7’s effect on cartilage characterization, single-cell RNA sequencing(scRNA-seq) was conducted on micromass cultures after 21 days of incubation with and without rhBMP7. This provided further insights into BMP7’s influence on chondrocyte behavior and cartilage development at the molecular level.
Results: Safranin O staining revealed that BMP7-deficient mice exhibited posterior superficial cartilage depressions in the middle portion of the TMJ, accompanied by a tendency toward ossification and a lack of cartilage matrix in these depressed areas. Immunofluorescence analysis indicated increased expression of Elastin (ELN) and Fibrillin-1 (FBN1) in the depressed regions of the mutant cartilage, suggesting significant alterations in cartilage properties. Morphological comparisons showed that TMJ pellets were more rounded and had a less shiny appearance than nasal septum pellets. Notably, the addition of rhBMP7 did not affect the compressive mechanical properties of TMJ cartilage pellets, contrasting with findings from nasal cartilage. scRNA-seq revealed that the addition of BMP7 to the culture medium resulted in downregulation of numerous genes associated with ribosomal biological pathways. These findings were consistent with proteomics data and have been further validated through qPCR and immunofluorescence staining.
Conclusions: Our findings indicate that BMP7 plays a crucial role in regulating the properties of TMJ cartilage, as evidenced by altered structural characteristics and changes in protein expression in BMP7-deficient mice. While BMP7 influences cartilage properties similarly to nasal cartilage, the specific molecular changes observed in TMJ cartilage suggest unique adaptations. Notably, BMP7 treatment did not affect the compressive properties of TMJ cartilage pellets, highlighting potential differences in how BMP7 modulates cartilage across different tissues. The identified molecular signatures and gene expression patterns provide valuable insights for further research into BMP7’s role in cartilage biology and the mechanisms underlying TMJ disorders
