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

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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.

Discovery of a novel tumor suppressor from the autophagy regulatory network: LRRK2 in lung adenocarcinoma

Author: 
Date created: 
2019-08-14
Abstract: 

(Macro)autophagy captures, degrades and recycles cytoplasmic components and organelles, via fusion of double-membraned autophagosomes with lysosomes. Basal autophagy is homeostatic, while increased rates of autophagy are stress adaptive. Autophagy is a potent tumor cell survival mechanism during stress, while in pre-malignant cells, it can provide a barrier against transformation. Mechanisms of autophagic cytoprotection and tumor promotion are established in preclinical models; however, whether the autophagy pathway is a target for recurrent molecular alteration in patient tumors remains unknown. I present a survey of 211 human autophagy-associated genes for tumor-related alterations to DNA sequence and RNA expression levels and an examination of their association with patient survival outcomes, in multiple cancer types with publicly available sequence data from The Cancer Genome Atlas (TCGA) consortium. In general, the core autophagy machinery was not a target of recurrent mutation in patients; therefore, the pathway remains functional and exploitable for tumor cell survival. However, autophagy regulators were targets of recurrent mutation and dynamic expression ─ between specific patient groups, in select cancers. Hence, context-dependent autophagy regulation contributes to tumor heterogeneity in patients. I further established that knockout of LRRK2, a previously described autophagy modulator that appears transcriptionally repressed in TCGA lung adenocarcinoma (LUAD), increased tumor initiation in a murine model of carcinogen-driven lung cancer. LRRK2 is an overactive kinase in Parkinson’s disease (PD) and LRRK2 inhibition in primates produces immature lamellar bodies in a main LUAD cell-of-origin. Lamellar bodies are lysosome-related secretory organelles that exocytose pulmonary surfactant, a mixture of phospholipids and lipoproteins key to innate lung defence. In TCGA LUAD patients, reduced LRRK2 was associated with current smoking, worse overall survival, genomic instability and gene signatures of poor differentiation, reduced surfactant metabolism and immunosuppression. LRRK2 was recently identified as an alveolarization gene in mouse lung development. I identified shared transcriptional signals of increased proliferation concomitant with decreased surfactant metabolism, in LRRK2-low LUAD and postnatal alveolar septation in mice, suggesting aberrant activation of a cell-of-origin developmental program in these tumors. I conclude that LRRK2 has tumor suppressive properties in LUAD, warranting further consideration for LRRK2 inhibition strategies in PD.

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

Development of rapid and robust assays to test the functions of human PTEN Variants of Unknown Significance in Drosophila melanogaster

Author: 
Date created: 
2020-04-17
Abstract: 

Large-scale sequencing projects and sequencing of patient samples can reveal mutations or polymorphisms in many genes, but the functional consequences are not always apparent especially for single amino acid substitutions. My research project focussed on addressing the discrepancy between the amount of sequenced gene variants and the knowledge about their functionality in development and disease. We developed Drosophila genetic assays for rapid, inexpensive functionalization of human PTEN variants with unknown significance (VUS) in order to learn if individual mutations play a role in development of disease. We assayed the ability of PTEN variants to suppress phenotypes observed when the oncogenic phosphoinositide 3-kinase (PI3K) signalling pathway is activated in the developing Drosophila wing. Our assay was validated with a few previously studied variants followed by characterizing 100+ human PTEN VUS. Ultimately, knowing which PTEN variants are non-functional or functional is crucial for targeted therapeutic and personalized treatment of PI3K-dependent diseases and cancers.

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

C. briggsae genome annotation and comparative analysis with C. elegans using RNA-Seq data

Author: 
Date created: 
2020-04-06
Abstract: 

Complete genome annotations are essential for comparative genomics. Currently, the C. briggsae genome annotation is incomplete that limits its utility as a comparative platform for C. elegans. Using RNA-Seq data, we have generated a more complete C. briggsae genome annotation. We identified 20,660 novel introns, 35,635 novel exons, and 5,654 novel protein-coding transcripts, and generated improved databases consisting of 123,974 introns, 150,690 exons, and 28,129 protein-coding transcripts, respectively. The improved C. briggsae annotation together with comparative analyses revealed 132 novel ortholog relationships (between C. briggsae and C. elegans) and 2 novel C. elegans protein-coding genes. This has shown that despite limited data available for C. briggsae, the improved annotation has enhanced the utility of C. briggsae as a comparative platform for C. elegans. As more RNA-Seq data becomes available, this method can be used to further refine not only C. briggsae annotation but also C. elegans annotation.

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

Characterizing a novel interaction between ecdysone receptor and the AP-1 transcription factor in the regulation of gene expression during Drosophila dorsal closure

Author: 
Date created: 
2020-04-15
Abstract: 

Dorsal closure (DC) of the Drosophila embryo is a well-characterized model system for studying morphogenetic events in wound healing and other developmental fusions such as palate fusion and neural tube closure. Prior to DC, a hole occupied with an extraembryonic tissue called amnioserosa (AS) is naturally left at the dorsal side of the embryo. DC begins when the epithelial sheets migrate over a hole and fuse to form a continuous epidermis. A commonly used secretable signal is a member of the transforming growth factor β (TGFβ) family, such as Dpp in Drosophila. During DC, the leading edge cells secrete Dpp into the AS cells to produce the steroid hormone, ecdysone (20E), which then drives AS morphogenesis by triggering gene expression. Here, we provide evidence that ecdysone-mediated gene expression is achieved through a novel interaction between the ecdysone receptor (EcR) and a subunit of the JNK-activated AP-1 transcription factor, Jun. While steroid hormone receptor interactions with AP-1 have been described in vertebrates, to our knowledge they have not been described in invertebrates and our work suggests that these interactions are ancient, predating the split between the vertebrate and invertebrate lineages.

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

Quantitative analysis of dynamic Spliced Leader Trans-Splicing in Caenorhabditis elegans

Author: 
Date created: 
2020-04-08
Abstract: 

Spliced Leader Trans-Splicing (SLTS) is an important process in Caenohabditis elegans transcript maturation that is required for viability. However, the role it plays in development remains unclear. We explore the dynamic use of SLTS during C. elegans development. Using PacBio Iso-Seq data and WormBase annotations, we characterized SLTS Acceptor Sites (SLTS ASs) in full-length transcripts and predicted putative SLTS ASs for 98.8% of annotated protein-coding transcripts. By taking advantage of over 1000 publicly available RNA-seq datasets, we quantified the level of SL1 and SL2 SLTS and found evidence supporting SLTS for 70.3% of annotated protein-coding transcripts, which was consistent with previous research. We found cases of dynamics during embryogenesis, including those where the dominant SL changed, which suggests that SLTS is dynamic and may be regulated. This improves the current understanding of the role of SLTS in gene expression during development and provides insight into the dynamic nature of operons.

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