VictorJuiz/thesis huggingface dataset:
Structure and Function of Multimeric BTB Proteins and Cullin3 Substrate Adaptors

Oligomerization and ubiquitylation are fundamental to the function of many proteins. Protein oligomerization confers numerous advantages including allosteric regulation, increased coding efficiency, and reduced transcriptional and translational error rates. Ubiquitylation is a widespread post-translational modification that targets modified proteins to a variety of fates including degradation by the 26S proteasome. Dysfunctional protein degradation is a common mechanism shared by a diverse range of human diseases. Many members of the BTB domain superfamily have roles centered on protein oligomerization and ubiquitylation. The BTB domain can facilitate self-assembly to form dimers, tetramers, and pentamers. In addition, many BTB proteins provide substrate specificity for the Cullin3 (Cul3) E3 ubiquitin ligase. Dysfunctional BTB/Cullin3 ubiquitinylation complexes are involved in certain forms of prostate cancer, medulloblastoma, hereditary hypertension and other diseases. Thus, understanding the details of BTB protein oligomerization and Cul3 interaction is important towards many aspects of human disease. The research presented in this thesis addresses several areas of BTB/Cul3 biology. The mechanism of dimeric BTB protein to Cul3 association was expanded to include the contributions from the BACK domain observed in the crystal structure of KLHL3/Cul3. Several KLHL3 mutations associated with familial hypertension were found to disrupt Cul3 binding. Next, the structure and oligomeric state of several KCTD family BTB proteins were investigated and found to be pentameric instead of tetrameric. The Cul3 binding properties of several KCTD proteins were characterized and found to be heterogeneous in contrast to other BTB protein families. The structural and functional properties of several KCTD proteins were used to propose a mechanism of KCTD/Cul3 association and the determinants for Cul3 binding. Finally, the structure of the KCTD5/Cul3/Gβγ complex was characterized and the binding affinities of Cul3 and Gβγ to KCTD5 were determined. Overall, these results provide insight into the assembly of multimeric BTB/Cul3 ubiquitylation complexes.

Keywords: BTB proteins
Cullin3
Protein structure
Ubiquitin
Advisor: Prive, Gilbert G
Environmental Chemistry of Perfluoroalkyl and Polyfluoroalkyl Substances in Aqueous Film Forming Foams

While aqueous film forming foam (AFFF) release has been known to result in environmental contamination with perfluoroalkyl and polyfluoroalkyl substances (PFASs) for over fifteen years, identities of individual PFASs in AFFFs have been largely unknown, proprietary information. Using mixed-mode ion exchange solid phase extraction (SPE), high resolution mass spectrometry (HR-MS), quadrupole time-of-flight (qTOF) tandem mass spectrometry (MS/MS), and liquid chromatography (LC)-MS/MS, 103 individual PFASs were identified in AFFFs and surfactant concentrates, including multiple chain-length congeners. Aerobic wastewater treatment plant sludge biodegradation of two AFFF components, 6:2 fluorotelomer sulfonamide alkylbetaine (FTAB) and alkylamine (FTAA), was investigated revealing biodegradation to short-chain perfluoroalkyl carboxylates (PFCAs) and known PFCA precursors. Additional degradation products were also identified, including 6:2 fluorotelomer sulfonamide (FTSAm) as a degradation product of both 6:2 FTAB and 6:2 FTAA, and six additional degradation products of 6:2 FTAA. A survey of selected Canadian surface waters for AFFF-related PFASs using SPE and LC-MS/MS revealed that several AFFF-related PFASs could be detected in urban and AFFF-impacted surface waters. These included FTABs and fluorotelomer betaines (FTBs), which were the fluorotelomer PFASs with the highest maximum concentrations. Other PFASs, such as perfluorohexane sulfonamide (FHxSA), 6:2 FTSAm, and 6:2 fluorotelomer mercaptoalkylamido sulfonate sulfone (FTSAS-SO2) were identified in some samples. Extraction and analysis of sediment from an AFFF-impacted river and batch sorption experiments with 6:2 FTAA and 6:2 FTAB in an agricultural soil provide preliminary insights into sorption behaviour of AFFF components, where longer chain-length FTBs were apparently more sorptive than shorter chain-lengths and 6:2 FTAA was more sorptive than 6:2 FTAB. This work is significant in identifying numerous PFASs found in AFFF that require further research in regards to their environmental fate and toxicology, demonstrating the presence of AFFF-related PFASs in Canadian environmental samples, and investigating biodegradation forming PFCAs and sorption of selected PFASs found in AFFFs.

Keywords: aqueous film forming foam (AFFF)
biodegradation
mass spectrometry
perfluoroalkyl and polyfluoroalkyl substances (PFAS)
Advisor: Mabury, Scott A.
Assessing Functional Impacts of Human Coding Variants

Advances in sequencing technology have made it routine to determine all coding variation in an individual human genome. A pressing challenge in the post-genomic era is to functionally characterize these variants, particularly within the disease-associated genes. Within the realm of cancer genome research, a critical problem that remains is how to separate the ‘driver’ from ‘passenger’ mutations and to further understand the functional mechanisms and consequences of driver mutations. I analyzed the missense somatic tumor mutations from 71 whole-genome or whole-exome sequencing studies across 21 cancers. I identified cancer-type-specific mutated domains and mutational hotspots. In some cases, I identified shifts in mutation and domain position between cancer types (but within a given gene product). I also provided clues to mutations’ functional effects. In addition to this, I identified different domain-centric mutational distribution patterns between oncoproteins and tumor suppressor proteins. The systematic correlation of mutations and cancer types at the domain level has the potential to guide more precise cancer treatments. Predictive models were also developed to quantify the impact of antigenicity on the spectrum of tumor missense somatic mutations. I found that somatic mutations are significantly depleted in peptides that are predicted to be displayed by MHC class I proteins, and characterized the dependence of this depletion on expression level. My results indicate that HLA class I alleles are, in general, incompletely dominant. I developed a model that produces an ‘antigenicity score’ for any input somatic coding mutation. These antigenicity scores could guide immunotherapy or aid in developing personalized cancer vaccines. In another collaborative effort to characterize human variation, I developed yeast-based functional assays to assess the functionality of the disease-associated coding variants. I evaluated the ability of wild-type human disease-associated genes to rescue homologous yeast mutants. Complementation between homologous human and yeast genes could often be found in the absence of annotated orthology, and these complementation relationships were of similar value as orthologous relationships for detecting human disease-causing variation. Finally, I found that the ability to detect pathogenic variation from complementation assays was not limited to variants which occur within the aligned region of human and yeast homology.

Keywords: cancer
functional impacts
human disease
mutation
yeast
Advisor: Roth, Frederick
Therapeutic Targeting of Tumour Hypoxia through the Unfolded Protein Response and Autophagy Pathways

Low oxygenation, or hypoxia, is prevalent in human solid tumours and is a known contributor to therapy resistance. Pancreatic cancer is characterized by a poorly perfused, hypoxic microenvironment which contributes to poor treatment response and dismal patient survival rates. We have interrogated the role of key cellular pathways that have been implicated in the survival of cancer cells in hypoxia, and their potential as new therapeutic targets. The unfolded protein response (UPR) is a three-arm adaptive signalling pathway activated in response to endoplasmic reticulum stress, which is induced by conditions of extreme hypoxia. Using genetic and pharmacological approaches, we demonstrated that the PERK signalling arm of the UPR was uniquely responsible for the protection of hypoxic cells, and that inhibition of this pathway caused selective cytotoxicity of hypoxic cells. We also demonstrated that PERK signalling led to the induction and activation of the mammalian autophagy protein kinase ULK1 during hypoxia. Autophagy is a pro-survival molecular mechanism that degrades cellular components and provides nutrients to cells exposed to stressful environments. ULK1 is essential for autophagy initiation and we demonstrated that ULK1 was essential for the survival of pancreatic cancer cell lines. ULK1 depletion led to an expansion of the endoplasmic reticulum (ER), induction of ER stress, and cell death under hypoxic environments. Furthermore, we uncovered a mechanism through which ULK1 can modify steady state oxygenation levels within tumours by regulating the mitochondrial content and oxygen consumption. We found that loss of ULK1 led to a progressive increase in mitochondrial content, and subsequent increased oxygen consumption. This consequential rise in oxygen demand exacerbated the development of hypoxia in tumours. However, at the same time ULK1 loss sensitized cells to hypoxia leading to increased cell death. Increased hypoxia sensitivity and tumour hypoxia resulted in dramatic tumour regression of pancreatic cancer xenografts. Inhibition of ULK1 patient-derived tumour organoids, using genetic and pharmacologic means, significantly decreased organoid formation efficiency in hypoxia. Taken together, this data established ULK1 as a potential therapeutic target in pancreatic cancer.

Keywords: autophagy
cancer
hypoxia
metabolism
pancreatic cancer
therapeutics
Advisor: Wouters, Bradly G
Data Visualization Tools for Large Biological Data Sets

Researchers have access to an ever-growing volume of data available at multiple levels of biological analysis. Many visual analytic tools have been developed to display a variety of biological data types but many of these tools are challenging to use and only examine one biological level of analysis at a time. The development and testing of hypotheses is difficult when the information is hard to integrate and laborious to interpret. The application of data visualization principles and user experience design best practices could improve systems biology research workflows by providing visual analytic tools with what is known in the information visualization community as a “transparent” user interface. This thesis consists of four papers that explore two central questions: 1) What is the best way to represent biological information at different levels of analysis? and 2) How do we enable researchers to explore and interact with their data as naturally and intuitively as possible? The first paper describes, ePlant, a tool for visualizing multiple levels of data that was developed using an agile process that included several rounds of user testing. The second paper presents Gene Slider, a tool for visualizing the conservation and entropy of orthologous DNA and protein sequences using a data visualization paradigm that takes better advantage of preattentive visual processing than current methods. The third paper describes Topo-phylogeny, a tool for visualizing phylogenetic relationships using a topographic map visualization paradigm that requires less cognitive processing to interpret than traditional tree diagrams. The final paper demonstrates the importance of user testing when developing a “rapid serial visual presentation” interface for identifying genes of interest using electronic fluorescent pictographs. Together these papers illustrate the complexities and benefits of applying data visualization principles and user experience design best practices to building data visualization tools for the analysis of large biological data sets. Given that hypothesis generation is fundamentally a creative process, any tools or techniques that can help researchers consider their data at a deeper level should be valuable to the scientific community.

Keywords: Arabidopsis
Bioinformatics
Data Visualization
Plant Biology
User Experience Design
Visual Analytics
Advisor: Provart, Nicholas J
The Mechanism of Mammalian Pexophagy

The mechanism of mammalian pexophagy is poorly understood. Although ubiquitination is thought to be involved in the process, the upstream and downstream effectors of ubiquitination remain elusive. Specifically, the E3 ligase responsible for mediating ubiquitination is not known, the site(s) of ubiquitination on the peroxisomal membrane are not known, and the key autophagy receptor which binds ubiquitin at the surface of the peroxisome is not known. In the first chapter of this thesis, I introduce the peroxisome and the key players in general autophagy, as well as in the selective autophagy of the peroxisome. In the second chapter, I explain the reagents and techniques used in this thesis. In the third chapter, I identify and characterize the peroxisomal E3 ubiquitin ligase PEX2 as the causative agent for mammalian pexophagy, identify two peroxisomal membrane proteins which are ubiquitinated by PEX2, and identify NBR1 as the primary receptor in PEX2-mediated pexophagy. In the fourth chapter, I examine the regulation of PEX2 and demonstrate how a key metabolic stimulus, protein restriction, initiates signaling through the mTORC1 pathway which leads to pexophagy in cultured cells as well as in a mouse model. Finally, I conclude my thesis with a discussion on my findings and some general thoughts on the current state of the field.

Keywords: autophagy
peroxisome
PEX2
pexophagy
Advisor: Kim, Peter Kijun
Enzymatic Depolymerization of Synthetic Polyesters by Microbial Carboxylesterases

Several types of biodegradable polyesters with different physical properties have emerged to replace traditional petroleum-based polymers, including polylactic acid (PLA). However, the lack of a sustainable recycling strategy for most biodegradable plastics and their accumulation in aquatic environments necessitate the design of ecologically benign polymers with improved life cycles. Enzymatic degradation of polyesters may not only serve as an energy-efficient approach to convert and re-use post-consumer plastics, but will also pave the way to design plastics with improved biodegradability. Although a number of polyesterase enzymes have been identified, the growing global demand for novel plastics and polymers has stimulated an interest in biocatalytic approaches for plastics recycling technologies. The discovery of novel polyesterases with high activity against a broad range of complex synthetic polyesters remains a key challenge for the development of enzyme-based plastic recycling. In this work, over 200 purified uncharacterized hydrolases from different microbial genomes and metagenomes were screened for esterase activity against polylactic acid, polycaprolactone (PCL), and the model polyethylene terephthalate substrate 3PET producing 39 active enzymes. Direct, activity-based screening of purified uncharacterized hydrolases has revealed the presence of polyesterase activity in several uncharacterized hydrolases from diverse esterase families, whose activity could not have been predicted using current bioinformatics tools. In this work, 17 novel polyester-degrading enzymes were biochemically characterized using both mono-ester and polyester substrates. Some of the identified polyesterases catalysed complete or extensive hydrolysis of solid PLA with the production of soluble lactic acid oligomers and monomers. Agarose-based assays with 24 different synthetic polyesters demonstrated a broad substrate range of the six identified polyesterases. Moreover, the crystal structures of two polyester- hydrolases were determined and the active site residues critical for the enzymatic activity were identified using site-directed mutagenesis. The crystal structure of the RPA1511 polyesterase from Rhodopseudomonas palustris revealed the presence of a molecule of polyethylene glycol bound close to the catalytic Ser residue, which represents the first structure of the polyesterase-substrate mimic complex. The results presented in this thesis indicate that microbial carboxylesterases can hydrolyze a broad range of polyesters with high rates, making them attractive biocatalysts for plastics depolymerization and recycling.

Keywords: biochemical characterization
biodegradable plastic
enzyme discovery
polyesterase
protein structure
site-directed mutagenesis
Advisor: Yakunin, Alexander F.
Edwards, Elizabeth A.
Deconvolution of Leukemic Evolution Through Initiation, Progression and Relapse

Significant advancements in the study of the genomic architecture of B-cell acute lymphoblastic leukemia (B-ALL) have been made, but our understanding of the mechanisms of leukemic initiation and progression remain limited. To gain clarity on these processes, a deeper understanding of the co-operative role of genetic aberrations, the functional impact of clonal diversity and the cellular target of transformation are required. Human models of leukemogenesis are vital in answering these questions. Using primary human umbilical cord blood cells, transduced with leukemic variants in a xenograft assay, we uncovered the importance of novel transcriptional programs, including induced stem cell transcriptional programs, in leukemogenic transformation. Recent evidence has shown that in the majority of B-ALL patients, relapse arises from a minor ancestral clone present in the diagnosis sample. An important question therefore arises as to what functional properties these clones possess and whether therapy resistance is acquired following treatment or is an intrinsic property of pre-existing cells. Using paired diagnosis and relapse patient samples in combined genomic and functional assays, we determine that minor ancestral clones present at diagnosis display decreased therapeutic response and are thus fated to relapse. These findings emphasize the need for improved minimal residual disease monitoring (MRD) and the development of therapeutic strategies that eliminate all genetic clones equally to prevent disease recurrence. Chromosomal translocations in B-ALL are known to be early initiating events occurring prenatally in some pediatric B-ALL patients, but there is conflicting data on their cellular origin. Using paired umbilical cord blood and leukemic samples, we found evidence that bi-lineage progenitors may act as the reservoir of pre-leukemia in pediatric B-ALL. Collectively, our work sheds light on the origin of disease and disease recurrence in B-ALL. By deepening the understanding of the forces at play leading to selection and expansion during disease evolution, novel therapeutic strategies can be developed to improve patient outcomes.

Keywords: B acute lymphoblastic leukemia
cancer heterogeneity
cell of origin
relapse
xenografts
Advisor: Dick, John E
The development of executive functions and working memory in children and adolescents with Autism Spectrum Disorder: A longitudinal study of brain and behaviour

Children with Autism Spectrum Disorder (ASD) exhibit social and communicative problems, repetitive and restrictive patterns of behaviour, and a range of cognitive deficits, including executive functioning (EF) impairments. Literature in ASD has focused mostly on the behavioural phentotype of the disorder and less research has investigated the cognitive and EF profiles in this population. The objective of this thesis was to gain a greater understanding of EF deficits, their longitudinal development, their underlying neural correlates using neuroimaging techniques, and their impact on critical outcomes in children with ASD compared to typical developing (TD) individuals. Structural and functional brain disturbances were evident in children and adolescents with ASD, relative to controls. In a diffusion tensor imaging (DTI) study, significant white matter differences were found in tracts essential for higher order cognitive processing, such as EF, and general information processing in children with ASD (ages 7-15 years). White matter developmental trajectories did not appear to differ between groups. Functional neural systems associated with EF, specifically working memory, were also impaired in youth ASD (ages 7-13 years) relative to controls, particularly in parietal and temporal regions. In this functional magnetic resonance imaging (fMRI) study, children with ASD showed inadequate modulation of neural activity in response to increasing cognitive demands, which did not improve over two years. Last, children with ASD (ages 7-14 years) showed marked EF impairments in everyday settings as observed by parents, which persisted over time compared to TD children. Early EF deficits predicted later symptoms of co-morbid psychopathology and social difficulties in ASD. Findings suggest that children with ASD exhibit structural and functional neural deficits associated with EF across childhood that make them vulnerable to the increasing complexity of environmental demands as they mature. Results support EF as potential target in autism interventions for improving executive development, and also for preventing co-morbid psychopathology and promoting social competence in ASD.

Keywords: Autism Spectrum Disorder
DTI
executive function
fMRI
working memory
Advisor: Taylor, Margot J
Balance of Forces in Bitumen-particle-water Systems

The oil sands in Canada are an abundant source of energy, but must be utilized in a way that bitumen can be extracted economically while it is environmentally responsible. This thesis focuses on three distinct studies on the interaction of bitumen-particle-water systems from a fluid dynamic perspective. The goal of this work is to understand the role of viscosity, interfacial tension, shear rate, shearing time, bitumen chemistry, and other process variables on separation processes related to bitumen production. These processes include the separation of oil from particles, the separation of particles from oil-particle clusters, and the separation of water from a water-in-bitumen emulsion. Results are quantified using different image analysis techniques, and characterized using dimensionless numbers, including capillary and Reynolds numbers, to understand the fundamentals of the governing forces and their mutual interactions. The first part of this thesis describes the separation of oil from an oil-coated sphere falling through an aqueous solution. Results reveal that maximum separation takes place when the capillary number is close to unity. The experiments are then extended to a more complicated geometry, where we examined the breakup and separation of oil-particle clusters in a simple shear flow. At high solid volume fraction, the oil that binds the particles together governs the dynamics of the cluster deformation, and therefore the critical capillary number for cluster breakup is the same as those already published in the literature for oil droplet breakup. Also, at capillary numbers above unity, clean particles separate from the cluster. Results from both studies show that maximum separation takes place when the oil-to-water viscosity ratio is between 0.1 and 1. An analytical solution shows that at those viscosity ratios, both normal and tangential stresses at the oil-water interface contribute to greater separation. In the final study, the formation of water in diluted bitumen emulsions was evaluated as a function of hydrodynamic conditions, pH, dilution ratio, and bitumen composition. A bimodal population of drop sizes was identified. The viscosity ratio was found to be a determining factor in the fraction of sub-micron droplets. A process of dilution and shear was introduced to mitigate this sub-micron fraction.

Keywords: bitumen
capillary number
emuslion
oil-particle cluster
oil sands
separation
Advisor: Bussmann, Markus
Acosta, Edgar J