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

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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.

Genetic and molecular characterization of paediatric endemic and sporadic Burkitt lymphoma

Author: 
Date created: 
2019-12-03
Abstract: 

Though generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in over 90% of cases in malaria-endemic regions and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumour EBV status or geographic origin. In this thesis, I describe an integrative analysis of whole genome and transcriptome sequencing data generated from a large cohort of endemic and sporadic paediatric BL patients. This analysis revealed that the mutational landscape of BL genomes is primarily shaped by four different processes, and that at least two of them—aberrant somatic hypermutation and defects in DNA mismatch repair—appear associated with the presence of EBV. After identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, I explored the incidence of mutations affecting genes and pathways involved with BL pathogenesis and found that EBV-positive tumours had significantly fewer driver mutations, especially among genes with roles in apoptosis, and that this difference did not exist when comparing geographic subtypes of BL. I also identified a subset of immunoglobulin variable region genes encoding clonal B-cell receptors (BCRs) that were disproportionally used in the tumours, including IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Many of these results suggest that tumour EBV status defines a specific BL entity irrespective of geographic origin with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply potential improvements that could be brought to BL therapy. This includes the rational use of DNA-damaging chemotherapy in some BL patients and targeted agents such as the CDK4/6 inhibitor palbociclib in others. The importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk and Src family kinases among these patients. Lastly, the identification of USP7 as a tumour-suppressor gene in BL highlights the potential clinical utility of MDM2 inhibitors in treating patients with otherwise wild-type TP53.

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

HCN channel regulation by the cAMP-binding fold is facilitated by interdomain interactions

Author: 
Date created: 
2019-11-07
Abstract: 

Hyperpolarization and Cyclic Nucleotide-gated (HCN) channels regulate electrical activity in rhythmically firing cells of the brain and heart. Their voltage-gating is mediated by a transmembrane (TM) region and also by direct binding of cAMP to a cyclic nucleotide-binding (CNB) fold in the cytoplasmic C-terminal region. In its unliganded form, the CNB fold mediates autoinhibition, a mechanism that thermodynamically and kinetically stabilizes the closed channel state; cAMP binding relieves this inhibition. The CNB fold mediates two additional regulatory mechanisms called Open-State Trapping (OST) and Quick-Activation (QA), which can govern the kinetic stability of the open and closed states, respectively. Structural studies have shown direct interactions of the TM region with the cytoplasmic C-linker which connects the TM region to the CNB fold, but the functional role of these interdomain interactions is poorly understood. In this work I found functional evidence for interdomain interactions facilitating autoinhibition, OST and QA, using domain replacements and site directed mutagenesis combined with voltage-clamp assays of channel gating. First, I replaced the TM region of HCN2 with that of HCN4, which augmented the magnitude of autoinhibition by the unliganded CNB fold. This implies that HCN2 channels have an interdomain interaction that limits the magnitude of autoinhibition. The augmentation of autoinhibition was also associated with disruption of the OST and QA mechanisms which are characteristic of HCN2. This resulted in autoinhibition becoming the dominant mechanism contributed by the C-terminal region determining kinetics for both activation and deactivation. Second, I used site-directed mutagenesis to identify a C-linker residue (E457) which stabilizes the open state but exerts different effects on thermodynamics and kinetics, most likely through an electrostatic interaction with the HCN4 TM region. A charge reversal mutation (E457R) limited the thermodynamic effect of autoinhibition while augmenting the kinetic effect of autoinhibition. Notably, in full-length autoinhibited channels E457 undergoes a conformational change such that it no longer stabilizes the open state. Overall, my work suggests that the molecular mechanisms of autoinhibition, OST, and QA must include participation of TM region structures and provides functional evidence for interdomain interactions that facilitate HCN channel regulation by CNB fold-mediated mechanisms.

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

A mechanistic investigation of T cell receptor-mediated HIV control

Author: 
Date created: 
2019-09-24
Abstract: 

HIV remains a global pandemic. No vaccine or cure exists. Most infected individuals progress to AIDS in the absence of antiretroviral therapy, but a rare group of elite controllers (<0.5% of the infected population) suppresses viremia to an undetectable level. HIV control is often associated with a robust host immune response, mediated by selected HLA alleles that elicit T cells against more conserved HIV peptide epitopes. T cell recognition of an infected cell is determined by its unique T cell receptor (TCR), which binds a virus-derived peptide presented on the cell surface by an HLA protein. An individual’s repertoire of TCR clones is large, but finite, and varies even among those who express the same HLA alleles. TCR sequence differences between controllers and non-controllers have been associated with variation in the antiviral activity of T cells, but few studies have explored this question comprehensively. My thesis project aims to identify TCR features that contribute to HIV control. To do this, I examined CD8+ T cell responses against the immunodominant HIV Gag TL9 (TPQDLNTML) epitope. TL9 is presented by HLA-B*42 and B*81, but only B*81 is associated with HIV control. I sequenced TCR from TL9-specific T cells, including dual-reactive cells associated with HIV control in B*42 individuals that recognized TL9 presented by both B*42 and B*81, and then conducted functional and structural assessments of selected TCR clones. TL9-specific TCR from B*81 individuals and dual-reactive TCR from B*42 individuals were highly enriched for TRBV12-3 gene usage. Furthermore, dual-reactive TCR from B*42 individuals were dominated by shared (or public) clones. Comprehensive functional analyses revealed that TCR from B*81 individuals and dual-reactive TCR from B*42 individuals displayed greater capacity to recognize TL9 variants, including common HIV escape mutations. Structural analyses of two dual-reactive TCR clones demonstrated an unusual peptide binding conformation driven by TRBV12-3 germline residues. My results demonstrate that clonal differences in the ability of TCR to recognize TL9 variants are associated with HIV control. Functional and structural data provide mechanistic insight into key features of more effective TL9-specific TCR. By highlighting the impact of TCR clonotype on HIV control, my results will inform development of new vaccine and therapeutic strategies.

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

The immunological and vascular effects of IL-6 signalling in transplant arteriosclerosis

Author: 
Date created: 
2019-07-08
Abstract: 

Transplant arteriosclerosis (TA) is a pathological vascular condition that is a main cause of chronic rejection and eventual failure of cardiac transplants. TA is caused by the activation of T cells towards allogeneic antigens expressed by vascular cells, which results in T cell-mediated injury and dysfunction of allograft arteries. T cells also support the activation and secretion of donor specific antibodies (DSAs) by B cells that contribute to the pathogenesis of TA. Interleukin-6 (IL-6) is secreted within hours of surgical transplantation and plays an important role in activation of allogeneic immune responses and regulation of vascular processes that influences TA. There are two main mechanisms by which IL-6 can signal to cells, classic and trans, that are distinguished by whether it binds to membrane bound or soluble forms of its receptor (IL-6R). In classic signalling IL-6 binds to IL-6R on the surface of cells. In trans-signaling IL-6 binds to soluble IL-6R. In both scenarios, the IL-6/IL-6R complex associates with the signalling subunit gp130 on cell membranes to transduce intracellular signalling events. Interestingly, IL-6 classic and trans-signalling have distinct biological outcomes. I examined the mechanism by which IL-6 classic and trans signalling contributes to immune activation in TA and characterized the biological outcomes of IL-6 signalling in endothelial cells. IL-6 classic and trans signalling were redundant for the activation of peripheral T cells that cause TA. However, eliminating IL-6R expression in T cells significantly reduced the development of DSAs in the serum of graft recipients, suggesting that IL-6 classic signalling in T cells may be required for antibody-mediated pathology in TA. In addition to activating immune cells, IL-6 acts on endothelial cells to induce inflammation and protect the vasculature from injury. IL-6 trans signalling in ECs significantly induced STAT-3, ERK1/2 and Akt activation. This signalling mechanism was needed for ICAM upregulation and the secretion of inflammatory cytokines by IL-6, indicating that IL-6 trans-signaling drives inflammatory activation of ECs. IL-6 classic signalling induced ERK1/2 and Akt activation but not STAT3 and was sufficient to stimulate the secretion of IL-8 and to protect ECs from cell death caused by serum deprivation but did not induce other inflammatory processes in ECs. My results suggest that IL-6 trans and classic signalling differentially affect inflammatory and survival responses in endothelial cells, which may have implications for understanding the vascular effects of IL-6R blockade in patients. Overall, my findings provide new insight into the immune and vascular effects of IL-6 in transplantation.

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

Mechanism of eubacterial 6S RNA release and Development of RNA Mango toolkit to study Ribonucleoprotein complexes

Date created: 
2017-04-13
Abstract: 

Ribonucleic acids (RNA) perform diverse biochemical functions in cells ranging from being an intermediate in the flow of genetic information, catalyst performing crucial reactions and as regulators of several processes. In bacteria, 6S RNA is a global transcription regulator that binds and inhibits house keeping RNA polymerase holoenzyme (core polymerase + σ70) under low nutrient conditions and rescues transcription in high nutrient conditions by the synthesis of a short product RNA (pRNA) using itself as a template. I show that a kinetic intermediate containing 6S RNA:core enzyme complex, that is enhanced by the formation of a phylogenetically conserved ‘release’ hairpin arises during 6S RNA release. Using nucleotide feeding experiments to slow down the release and a 6S RNA mutant which precludes the hairpin formation, I found the release process involves intrinsic ‘scrunching’ type mechanism that is modulated by the ability to form a release hairpin during the process of 6S RNA release. Given the importance of RNA in regulating various cellular processes, a fluorescence tool to track RNA in real time is limiting as RNA lacks intrinsic fluorescence. The Unrau lab has in vitro selected RNA Mango aptamer that binds thiazole orange with nanomolar affinity while increasing its fluorescence up to 1100 fold. To elucidate how this small aptamer exhibits such properties, which make it particularly well suited for studying low-copy cellular RNAs, we, in collaboration with D′Amaré’s lab, determined its co-crystal structure, discovering a three-tiered G-quadruplex connected to a duplex through a GAAA tetraloop-like junction. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb) ligand I have created a Mango toolkit that simultaneously enables the purification and characterization of endogenous cellular RNPs in vitro.

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

Integration of transport pathways in Saccharomyces cerevisiae

Author: 
Date created: 
2017-02-28
Abstract: 

Plasma membrane (PM) homeostasis is essential for viability and depends on maintaining a constant balance between the amount of membrane material arriving at the cell cortex and the amount recycled. This membrane transport is mediated by two distinct mechanisms: (i) vesicular transport, utilizing membrane-enclosed vesicles for bulk transport of membrane proteins and lipids; and (ii) non-vesicular lipid transport. While the molecular mechanisms of these processes are well defined, they are generally considered independent events but how they are integrated is poorly understood. To gain insight into how these transport pathways are coordinated at the PM, three avenues of research were conducted using a combination of genetic, biochemical and live-cell microscopy assays. First, I showed that the Oxysterol-binding protein-related protein (ORP) Osh4p, implicated in non-vesicular sterol transfer, was found to associate with exocytic vesicles and formed complexes with regulators of polarized exocytosis, including the small GTPases Sec4p, Cdc42p, and Rho1p. Second, I tested the function of the evolutionarily conserved endoplasmic reticulum (ER)-associated protein Arv1p, also suggested to be involved in non-vesicular sterol transfer. Ultimately Arv1p was found to be dispensable for sterol exchange between the ER and PM but instead it was shown to play an important role in maintaining ER ultrastructure; Arv1p might be involved in regulating insertion of tail-anchored proteins into membranes. Finally, the essential yeast Rab GTPase Sec4p, principally known to be a key regulator of exocytosis, was shown to mechanistically couple polarized exocytosis with cortical actin polymerization, which activates Las17p (the yeast Wiskott–Aldrich syndrome [WASp])-dependent endocytosis. Las17p activation results in actin filament nucleation, which pulls the PM inward for endocytic vesicle biogenesis. Sec4p thereby represents the first direct regulatory link that couples exocytosis and endocytosis, which we termed \"yeast compensatory endocytosis.\" By identifying novel mechanisms that coordinate intracellular transport pathways, these studies provided important new insights into how PM homeostasis is regulated and maintained.

Document type: 
Thesis
File(s): 
S1 Video. Photobleaching enables single particle tracking of GFP-Sec4p at the cell cortex.
S2 Video. Las17p-RFP and GFP-Sec4p particles co-localize at cortical actin patches.
S3 Video. Sla1p-RFP and GFP-Sec4p co-localize at cortical actin patches.
S4 Video. GFP-Sec4p particles co-localize with Abp1p-RFP at cortical actin patches.
S5 Video. SEC4 and SEC2 function are required for normal actin patch polarization and dynamics.
Supervisor(s): 
Christopher Beh
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.