Molecular Biology and Biochemistry - Theses, Dissertations, and other Required Graduate Degree Essays

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The microbial diversity of watermelon snow

Author: 
Date created: 
2021-08-20
Abstract: 

During summer months in alpine systems around the world patches of green, orange and red snow appear. The phenomenon is sometimes called watermelon snow and is caused by a bloom of microalgae. The resulting microbiome is teeming with life, including algae, fungi, metazoans, other protists and bacteria. Here, I present data documenting the biological diversity in the ephemeral snow algae microbiome, which grows on snows threatened by global warming. I began by asking: what microbes are found in snow algae blooms? I focussed my work in the southwestern Coastal Mountain Range in B.C., Canada. The data I present detail the algal, bacterial, fungal, metazoan and other protist diversity in blooms and their distribution across the region. These data included sequences from undescribed algal species, with some potentially belonging to species names with no DNA data available. I therefore did an analysis, including five novel algal isolates, to clarify the taxonomy of Raphidonema and its sister genera, using genetic data. I was able to identify my five isolates as R. sempervirens, and in the process name two novel species: R. catena and R. monicae. As bacteria are commonly important mutualistic symbionts of microalgae, I next described their communities living alongside snow algae. I found that, unlike algae, the bacterial community composition does not change with elevation, and instead there are regionally widespread bacteria. I therefore wanted to learn more about the metabolic capabilities of these bacteria common to snow algae blooms. Using a shotgun metagenomics approach, I analyzed the bacterial metagenome, and metagenomically assembled genomes. These data included representative from the widespread bacterial families found during metabarcoding, and I furthered that analysis by describing their metabolic genes related to: nitrogen and sulfur cycling as well as biosynthesis of osmolytes/cryoprotectants, B-vitamins, phytohormones, and xanthophyll pigments. These data act as observations to form hypotheses on the biogeochemistry and microbial ecology of snow algae microbiomes.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Lynne Quarmby
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Novel secondary structures of DNA; and development of a sensitive methodology for capturing DNA/RNA G-quadruplexes from living Drosophila salivary glands

Author: 
Date created: 
2021-08-10
Abstract: 

Work reported in this thesis, from three independent projects, highlights: first, a novel DNA secondary structure fold from a neurodegenerative disease-linked repeat sequence; second, a new approach for assembling and reversing a long and 1- dimensional DNA nanostructure. The third and most substantial project reports the development of and biological results from a highly selective and sensitive approach for in vitro and in vivo tagging of DNA and RNA G-quadruplexes. In the first project, a wholly novel higher-order fold of DNA, named as “iCD-DNA”, was discovered and characterized. iCD-DNA was found to be formed uniquely by a hexanucleotide repeat expansion sequence, d(C2G4)n, located at the 5’ UTR of the C9orf72 gene, causally linked to multiple neurological disorders such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). It was found that incubating d(C2G4)n under mildly acidic conditions and in the presence of non-quadruplex supporting cations (e.g. Li+, Mg2+) gave rise to a distinctive higher order structure whose most striking feature was an inverted circular dichroism (CD) spectrum, distinguishable from the inverted CD spectra of either a left-handed duplex (“Z-DNA”) or a left-handed G-Quadruplex (“Z-GQ”). On the basis of CD spectroscopy, gel mobility and chemical footprinting, structural models were proposed for iCD-DNA. In the second project, a new strategy for creating a long (~200-300 nm) and reversible 1-Dimensional DNA nanostructure/ nanowire (1DDN), named “(TQs)n”, was designed and carried out. “(TQs)n” incorporates a hybrid of DNA triple and quadruplex helices. In this design, a novel approach for joining together DNA helices (called guanine-rich “glue junctions”) was proposed and demonstrated. In the third project, a highly specific and sensitive methodology for uniquely biotin-tagging DNA/RNA G-quadruplexes (by way of their intrinsic peroxidase activity while complexed with heme) was deeply characterised, first, in vitro, and then applied to tag and pull down G-quadruplex forming RNAs and DNAs from living Drosophila larval salivary glands. Preliminary-sequencing data, so obtained, provided initial insights for the potential occurrence of G-quadruplexes in living cells but needs detailed future investigation.

Document type: 
Thesis
Supervisor(s): 
Dipankar Sen
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Explorative analysis of the mechanisms of Phaeocystis globosa blooms in the Beibu Gulf using amplicon sequencing data

Author: 
Date created: 
2021-05-12
Abstract: 

Phaeocystis is an ecologically important cosmopolitan genus with several species that form harmful algal blooms. Previous studies of the mechanisms of Phaeocystis blooms have been hindered by the small size of Phaeocystis cells and the complex Phaeocystis life cycle, which includes multiple free-living stages and a colonial stage that dominates during blooms. In this thesis, I apply 16S amplicon sequencing to explore the mechanisms underlying a P. globosa bloom in the Beibu Gulf. Using the spatial-temporal dynamics of P. globosa, bacteria, archaea, phytoplankton and environmental variables, I develop a model for the development and progression of the P. globosa bloom. After, I identify bacteria that interact with P. globosa during the bloom by studying the P. globosa colony microbiome. While P. globosa colonies had different bacterial compositions compared to seawater samples collected from the same locations, I did not find evidence for a core P. globosa colony microbiome.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Nansheng (Jack) Chen
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Discovery and characterization of novel non-coding 3′ UTR mutations in NFKBIZ and their functional implications in diffuse large B-cell lymphoma

Author: 
Date created: 
2021-08-16
Abstract: 

Diffuse large B-cell lymphoma (DLBCL) is a very heterogenous disease that has historically been divided into two subtypes driven by distinct molecular mechanisms. The activated B-cell (ABC) subtype of DLBCL has the worst overall survival and is characterized by activation of the NF-κB signaling pathway. Although many genetic alterations have been identified in DLBCL, there remain cases with few or no known genetic drivers. This suggests that there are still novel drivers of DLBCL yet to be discovered. In this thesis I aimed to leverage whole genome sequencing data to identify novel regions of the genome that were recurrently mutated, with a specific focus on non-coding regions. Through this analysis we identified numerous novel putative driver mutations within the non-coding genome. One of the most highly recurrently mutated regions was in the 3′ untranslated region (UTR) of the NFKBIZ gene. Amplifications of this gene have been previously discovered in ABC DLBCL and this gene is known to activate NF-κB signaling. Therefore, we hypothesized that these 3′ UTR mutations were acting as drivers in DLBCL. The remaining portion of this thesis is focused on the functional characterization of NFKBIZ 3′ UTR mutations and how they drive DLBCL and contribute to treatment resistance. To this end, I induced NFKBIZ 3′ UTR mutations into DLBCL cell lines and determined that they cause both elevated mRNA and protein expression. These mutations conferred a selective growth advantage to DLBCL cell lines both in vitro and in vivo and overexpression of NFKBIZ in primary germinal center B-cells also provided cells a growth advantage. Lastly, I found that NFKBIZ-mutant cell lines were more resistant to a selection of targeted therapeutics (ibrutinib, idelalisib and masitinib). Taken together, this thesis highlights the importance of surveying the entire cancer genome, including non-coding regions, when searching for novel drivers. I demonstrated that mutations in the 3′ UTR of a gene can act as driver mutations conferring cell growth advantages and treatment resistance. This work also implicates NFKBIZ 3′ UTR mutations as potentially useful biomarkers for predicting treatment response and informing on the most effective treatment options for patients.

Document type: 
Thesis
Supervisor(s): 
Ryan Morin
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Multi-omics characterization of pancreatic neuroendocrine neoplasms

Author: 
Date created: 
2021-08-18
Abstract: 

Pancreatic neuroendocrine neoplasms (PNENs) are biologically and clinically heterogeneous neoplasms in which pathogenic alterations are often indiscernible. Treatments for PNENs are insufficient in part due to lack of alternatives once current options are exhausted. Despite previous efforts to characterize PNENs at the molecular level, there remains a lack of molecular subgroups and molecular features with clinical utility for PNENs. In this work, I describe the identification and characterization of four molecularly distinct subgroups from primary PNEN specimens using whole-exome sequencing, RNA-sequencing and global proteome profiling. A Proliferative subgroup with molecular features of proliferating cells was associated with an inferior overall survival probability. A PDX1-high subgroup consisted of PNENs demonstrating genetic and transcriptomic indications of NRAS or HRAS activation. An Alpha cell-like subgroup, enriched in PNENs with deleterious MEN1 and DAXX mutations, bore transcriptomic similarity to pancreatic α-cells and harbored proteomic cues of dysregulated metabolism involving glutamine and arginine. Lastly, a Stromal/Mesenchymal subgroup exhibited increased expression and activation of the Hippo signaling pathway effectors YAP1 and WWTR1 that are of emerging interest as potentially actionable targets in other cancer types. Whole-genome and whole-transcriptome analysis of PNEN metastases identified novel molecular events likely contributing to pathogenesis, including one case presumably driven by MYCN amplification. In agreement with the findings in primary PNENs, four of the metastatic PNENs displayed a substantial Alpha cell-like subgroup signature and all harboured concurrent mutations in MEN1 and DAXX. Collectively, the identified subgroups present a potential stratification scheme that facilitates the identification of therapeutic vulnerabilities amidst PNEN heterogeneity to improve the effective management of PNENs.

Document type: 
Thesis
Supervisor(s): 
Sharon Gorski
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Decoding nutrient sensing and metabolic regulation in the Drosophila Hipk tumor model

Author: 
Date created: 
2020-06-29
Abstract: 

Sustaining proliferative signals and deregulating cellular energetics are two hallmarks of cancer. However, how oncogenic signals respond to nutrients and coordinate with metabolic states remains poorly understood. Here, using Drosophila melanogaster as a genetic model organism, we establish an in vivo tumor model with elevation of oncogenic fly Homeodomain-interacting protein kinase (Hipk). This tumor model features cell hyperproliferation, tumor invasion, and cellular changes reminiscent of epithelial-to-mesenchymal transition, including induction of matrix metalloproteinases and loss of E-cadherin. The tumor phenotypes arise from the redundant and/or synergistic effects of more than one perturbed oncogenic signaling pathway caused by elevated Hipk, underlying the need for targeting multiple signaling molecules to reduce tumor growth. To search for simpler therapeutic strategies, we examine the metabolic requirements of Hipk tumor growth.We find that high sugar potentiates the tumorigenic potential of Hipk. Mechanistically, nutrient sensors O-GlcNAc transferase (OGT) in the hexosamine signaling pathway and salt-inducible kinase 2 (SIK2) in the insulin signaling pathway physically bind to Hipk and induce covalent post-translational modifications of Hipk, namely O-GlcNAcylation and phosphorylation, respectively. Both nutrient sensors are required for Hipk protein expression and synergize with Hipk to drive tumor progression. Our works demonstrate two modes of nutritional regulation of Hipk, which can accelerate Hipk tumor growth in nutrient-rich conditions like diabetes. We further characterize the metabolic profile of the Hipk tumor model. The tumor cells display the oncogene Myc-induced aerobic glycolysis, which in turn functions to perpetuate Myc accumulation post-transcriptionally, forming a positive feedback loop. Disruption of the loop abrogates Hipk tumor growth. Downstream of the loop, the tumor cells harbor an accumulation of highly fused, functional mitochondria. Targeted inhibition of a Pd subunit of the respiratory complex I blocks the tumor growth. Our works reveal that both aerobic glycolysis and active mitochondrial metabolism are required to promote Hipk tumor growth.Taken together, using the Drosophila Hipk tumor model, we functionally characterize the nutrient sensing and metabolic crosstalk with cell signaling, and reveal potential metabolic vulnerabilities that could be exploited in cancer treatment.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Esther Verheyen
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Understanding Type IV pilus-mediated secretion in Vibrio cholerae

Author: 
Date created: 
2020-12-08
Abstract: 

Bacterial pathogens depend on the expression of virulence factors that aid host infection. A mechanistic understanding of bacterial virulence can provide insights into novel antimicrobial targets and therapies. One virulence factor is Type IV pili (T4P), long thin filaments found on bacterial surfaces with roles in adhesion, DNA uptake and exoprotein secretion. The T4P system is closely related to the Type II secretion (T2S) system where periplasmic “pseudo-pili” exhibit a piston-like motion for exoprotein export. My research aims to understand T4P-mediated exoprotein secretion in the simple T4P system of Vibrio cholerae. I show that the exoprotein’s flexible N-terminal segment is the export signal, which may bind to minor pilin at the pilus tip for delivery across the secretin channel.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Lisa Craig
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Cilium length regulation in Caenorhabditis elegans

Author: 
Date created: 
2019-09-26
Abstract: 

Nearly all vertebrate cells possess a primary cilium, akin to a cellular antenna, that plays essential roles in various physiological and developmental processes. Cilium length is tightly regulated to provide the optimal functions for each type of cell and tissue, such as the eye and kidney. Impairment of this regulation can result in cilium-associated disorders, collectively termed ciliopathies. The intraflagellar transport (IFT) system, involved in cilium assembly, and microtubule depolymerizing kinesins, which participate in cilium disassembly, play key roles in cilium length regulation. Additionally, several classes of kinases, including CDKL5, modify IFT components and/or depolymerizing kinesins to modulate ciliary length. We therefore hypothesized that the entire family of cyclin-dependent kinase-like (CDKL) proteins (CDKL1-5) may have similar ciliary functions. To test this hypothesis, we undertook studies in C. elegans. This nematode has one CDKL protein (CDKL-1) closely related to CDKL1-4 and more distantly related to CDKL5. We find that CDKL-1 localizes to cilia, including the transition zone (TZ), and negatively regulates cilium length by controlling IFT flux. Cilium length regulation by CDKL-1 is distinct from that of other kinases, namely DYF-18 (mammalian CCRK ortholog), DYF-5 (MAK) and NEKL-1 (NEK8/9). It also occurs independently from the depolymerizing kinesin-13 family, KLP-7 (KIF2A), which positively controls cilium length at the TZ. To query the molecular etiologies of human diseases caused by mutations in CDKL5 (epilepsy and atypical Rett syndrome) or KIF2A (brain malformations), we introduced corresponding patient mutations in C. elegans CDKL-1 and KLP-7, respectively. The mutations cause mislocalization and ciliary length defects. In addition, we find that disrupting C. elegans cdkl-1 results in sensory (CO2 avoidance) and developmental (body size) phenotypes, possibly resulting from anomalies in signaling pathway(s), including cGMP signaling. These data suggest that human ailments such as Rett syndrome and brain anomalies may arise from cilium length misregulation, and consequently, disruption of signaling pathways. In summary, our findings provide evidence that CDKL-1 works cooperatively with other kinases, and independently from a depolymerizing kinesin, to maintain correct ciliary length. Our work also suggests links between ciliary length control and potential ciliopathies, which provides potentially useful experimental avenues of exploration.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Michel Leroux
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Designing a high-throughput screen for small molecule modulators of the molecular motor KIF1A

Author: 
Date created: 
2020-12-07
Abstract: 

The neuron-specific kinesin, KIF1A, is involved in the transport of cargos critical for neuronal function such as synaptic vesicle precursors and dense-core vesicles. Notably, mutations in KIF1A are implicated in the pathogenesis of neurological disorders such as hereditary spastic paraplegia. Therapies that specifically target KIF1A do not currently exist. I developed a high-throughput, phenotypic screen for small molecule modulators of KIF1A. I used two-color fluorescent imaging to identify a cell-based, phenotypic assay that allows for characterization of the inactive, autoinhibited form of KIF1A and the active form of KIF1A. When expressed in COS-7 and SK-N-SH cells, wild-type KIF1A-GFP is diffusely distributed and does not co-localize with the microtubule marker EB3-mKate2. The hyperactive mutant V8M KIF1A-GFP does co-localize with microtubules and distributes peripherally. These findings support the feasibility of a functional assay to distinguish between the active and inactive forms of KIF1A in a high-throughput screening format.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Michael Silverman
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Autophagy machinery contributes to cell survival and small extracellular vesicle composition in triple-negative breast cancer cells

Author: 
Date created: 
2019-12-05
Abstract: 

Macroautophagy (hereafter autophagy) is a catabolic cellular process where double-membraned autophagosomes capture cytoplasmic cargos and fuse with lysosomes for content degradation. Basal autophagy maintains cellular homeostasis by removing long-lived proteins and damaged organelles. Autophagy can also be upregulated to promote cell survival in the presence of stressors such as starvation and oxidative stress. Autophagy can suppress tumorigenesis by maintaining genome stability in normal cells, or enable cancer cell survival during nutrient limitation, hypoxia or chemotherapy treatment. Therefore, inhibiting autophagy may improve chemotherapy efficacy. Triple-negative breast cancers (TNBC) are a subtype of breast cancers that do not over-express hormone receptors. Chemotherapy remains one of the few systemic treatment options for TNBC, making the development of chemotherapy resistance particularly problematic in disease management. This thesis describes cell-intrinsic and cell-extrinsic functions of autophagy machinery in cultured TNBC cells, and explores the potential utility of autophagy inhibition to enhance treatment response. Cytoprotective autophagy was induced in response to epirubicin treatment in TNBC cells. Autophagy inhibition reduced cell viability and improved efficacy of epirubicin in both drug-naïve and drug-resistant cells. Further investigation revealed cell-extrinsic roles of autophagy, in the form of its contribution to the composition of small extracellular vesicles (sEV), nano-sized vesicular entities with known roles in cell-cell communication. Lysosomal inhibition by chloroquine (CQ) induced co-localization of mammalian autophagy-related (ATG) 8 homologs with endolysosomal tetraspanins, and introduced significantly higher levels of ATG8s in TNBC-derived sEV. The concurrent increase in poly-ubiquitinated proteins and autophagy adaptors in sEV suggested a potential mechanism where degradative cargos are loaded into sEV by autophagy machinery and then expelled. CQ-induced enrichment of ATGs was limited to a subpopulation of sEV, highlighting the heterogeneity and context-dependency of sEV composition. Finally, CQ-mediated lysosomal inhibition was found to dampen the growth-promoting effects of sEV in recipient cells. Taken together, this work demonstrated cytoprotective roles of autophagy in TNBC cells, and the dynamic contribution of autophagy machinery to sEV composition, warranting further examination of autophagy inhibition as a potential therapeutic avenue in TNBC.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Sharon Gorski
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.