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

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(C2G4)n Repeat Expansion Sequences from the C9orf72 Gene Form an Unusual DNA Higher-Order Structure in the pH Range of 5-6

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-06-18
Abstract: 

Massive expansion of a DNA hexanucleotide sequence repeat (C2G4) within the human C9orf72 gene has been linked to a number of neurodegenerative diseases. In sodium or potassium salt solutions, single-stranded d(C2G4)n DNAs fold to form G-quadruplexes. We have found that in magnesium or lithium salt solutions, especially under slightly acidic conditions, d(C2G4)n oligonucleotides fold to form a distinctive higher order structure whose most striking feature is an “inverted” circular dichroism spectrum, which is distinguishable from the spectrum of the left handed DNA double-helix, Z-DNA. On the basis of CD spectroscopy, gel mobility as well as chemical protection analysis, we propose that this structure, which we call “iCD-DNA”, may be a left-handed Hoogsteen base-paired duplex, an unorthodox G-quadruplex/i-motif composite, or a non-canonical, “braided” DNA triplex. Given that iCD-DNA forms under slightly acidic solution conditions, we do not know at this point in time whether or not it forms within living cells.

Document type: 
Article
File(s): 

Interactions among Dam, SeqA and mismatch repair proteins in Escherichia coli

Author: 
Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2019-03-31
Abstract: 

The accuracy of DNA replication is very important, and organisms have several proofreading and repair systems to prevent mutations from occurring. Lesions can be introduced by errors during replication, chemical mutagens, UV or ionizing radiation. In Escherichia coli, mismatches are detected by MutS and MutL which together activate MutH to initiate repair. Repair is dependent on GATC hemi-methylation signals on the DNA which is added by DNA adenosine methylase (Dam). SeqA acts as a regulator of DNA replication, sequestering the origin and preventing reinitiation. We hypothesize that 1) Dam and SeqA are coordinated by MutL, and (2) persistent mismatches caused by lack of polymerase proofreading will increase mismatch repair activity. Results show that Dam binds to both SeqA and MutL, and no significant increase in mismatch repair activity was detected when the error prone polymerase was induced. These data suggest the importance of temporal coordination of methylation and/or interaction of Dam and MutL in preparation for mismatch repair. Our data is consistent with previous literature that shows mismatch repair primarily works against transitions and is inefficient at preventing transversions.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Claire Cupples
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Quantitative analysis of the coding capacity of C. elegans using RNA-Seq data

Author: 
Date created: 
2018-11-30
Abstract: 

Annotating the genome of the nematode Caenorhabditis elegans has been an ongoing challenge for the last twenty years. Studies have leveraged high-throughput RNA-sequencing (RNA-Seq) to uncover evidence for thousands of novel splicing events, indicating that the current annotations are far from complete. Yet, there is some uncertainty whether the many rare events represent functional transcripts, or simply biological noise. We developed a method that leverages the wealth of publicly available RNA-Seq data to perform a quantitative evaluation of the completeness of the current C. elegans genome annotation. We identified 134,949 and 204,812 novel high-quality introns and exons, respectively. We find that many introns and exons are rarely expressed overall, but strongly expressed at specific developmental stages suggesting a functional role. We assembled a high-quality set of 72,274 protein-coding transcripts to show that only a fraction of the coding transcriptome of C. elegans is represented in the current genome annotation.

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

Advances in fluorogenic RNA aptamer systems for live cell imaging: Towards orthogonality and multicolour applications

Author: 
Date created: 
2018-10-25
Abstract: 

Recent developments in the field of RNA biology continue to demonstrate the importance of RNA in regulating cellular processes. However, directly imaging biologically important RNAs has been hindered by a lack of live cell fluorescent tools. As such, aptamers that bind and enhance the brightness of fluorogenic dyes are promising tools to improve fluorescent RNA imaging. The Unrau laboratory developed RNA Mango I, a small, 39-nt aptamer that binds to a modified thiazole orange fluorophore (TO1-Biotin) with nanomolar affinity. This binding is accompanied by an 1,100-fold increase in its green channel fluorescence. To further improve the Mango aptamers, in collaboration with the Ryckelynck laboratory, we used microfluidics-based selection methods to isolate three brighter, high affinity RNA Mango fluorogenic aptamers (Mango II, III and IV). Together with the Rueda laboratory, we show that these new Mangos can accurately image the sub-cellular localization of three small non-coding RNAs in fixed and live mammalian cells. These new Mangos are unique in structure. Unlike Mango I and II, Mango III rigidly connects its ligand binding core to an external helix. As the Spinach/Broccoli aptamer family, which binds GFP-like chromophores (DFHBI, DFHBI-1T), also share this property, the Broccoli/DFHBI-1T aptamer complex was used as a FRET donor paired with the far red-shifted Mango III/YO3-Biotin complex as a FRET acceptor. Interestingly, the high affinity Mango I, II, III aptamers can discriminate between TO1/YO3-Biotin and DFHBI/DFHBI-1T by at least a 102-fold difference in affinity. In contrast, Spinach binds many fluorophores indiscriminately and weaker. With this, concentrations could be determined to obtain appropriate binding for Mango III/YO3-Biotin and Broccoli/DFHBI-1T when in the same system. FRET efficiency was measured using an RNA duplex of variable length between the two aptamers. FRET signal depended on the length of the duplex, and oscillated in intensity precisely with the predicted twist of the helix, demonstrating strong orientation dependence. While this pair of aptamers enable in vitro FRET studies, there are no truly orthogonal fluorescent aptamer systems. To that end, I discuss an in vitro selection to develop orthogonal aptamers for a red fluorophore, TO3-Biotin, that can potentially be paired with existing Mango aptamers.

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

A conserved interdomain linker of CCT mediates allosteric communication between regulatory and catalytic domains

Author: 
Date created: 
2018-12-03
Abstract: 

Folding landscapes of linkers between ligand-binding and functional domains evolved to facilitate transmission of inter-domain signals. I investigated the structure/function of a conserved linker between the catalytic and membrane-binding (M) domains of CCT, which regulates phosphatidylcholine synthesis and activates upon membrane binding. The activity of CCT is very sensitive to mutations in the linker. Recent molecular dynamics simulations revealed that upon removal of auto-inhibitory contacts between the M domain and the active site, the αE helix adjacent to the linker bends toward the active site, bringing the catalytic domain close to the membrane surface. Tryptophan fluorescence quenching revealed that the linker lies superficially on the membrane surface. FRET between engineered tryptophans and vesicles containing Dansyl-Phosphatidylethanolamine support a bent αE helix conformation that is dependent on the native linker sequence. The data suggests that the linker may communicate membrane binding signals to enhance CCT activity by directly stabilizing a bent αE.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Rosemary Cornell
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Gfat1 and Gfat2 encode functionally equivalent enzymes in Drosophila melanogaster: a molecular, genetic, and evolutionary analysis

Author: 
Date created: 
2018-10-26
Abstract: 

The hexosamine biosynthesis pathway (HBP) diverts 2-5% of glucose from glycolysis, ultimately producing uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important substrate in protein glycosylation. This pathway is of particular importance in Drosophila melanogaster because chitin, the primary component of insect cuticle, is composed of N-acetylglucosamine polymers. I report that the rate-limiting enzymes of the HBP, GFAT1 and GFAT2 (glutamine:fructose-6-phosphate aminotransferase), are functionally equivalent by genetic rescue using cDNA transgenes of both genes. I also report that neither transgenically upregulating the enzymes of the HBP, nor those of the hexosamine signalling pathway (HexSP), extends the lifespan of D. melanogaster. Evolutionary analyses using fluorescence in situ hybridization in six species of Drosophila support a model that would place the Gfat1 relocalization event from euchromatin to heterochromatin after the melanogaster group diverged from the rest of Drosophila.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Barry Honda
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Investigating the role of HIV-1 Nef during reactivation of latent viral reservoirs

Author: 
Date created: 
2018-09-21
Abstract: 

A major barrier to development of a cure for HIV-1 is the ability of the virus to establish latent infection that reactivates to cause disease if antiretroviral therapy is discontinued. The HIV-1 protein Nef displays multiple in vitro and in vivo functions, including the ability to modulate T cell signaling events that may alter the activation status of infected cells. Nef is critical for viral pathogenesis, but its role during latency remains unclear. To investigate this, I generated a novel panel of latent T cell clones (C-Lat) harbouring a single integrated copy of HIV-1 encoding functional or defective nef genes. By assessing the location and genomic features of the proviral DNA integration sites in these clones, I observed that the presence of functional Nef variants was associated with a broader repertoire of inducible latent T cell lineages. By characterizing the reactivation phenotypes of these clones following stimulation with latency reversing agents (LRAs), I observed higher early and late viral protein expression in C-Lat clones encoding functional nef compared to those encoding defective nef. I confirmed these observations by disrupting the functional nef gene in C-Lat clones using CRISPR/Cas9 strategies. Variable viral reactivation phenotypes were observed in Nef knock-out (NefKO) clones following stimulation with LRAs, but the efficiency of early and late viral protein expression was consistently lower in NefKO clones compared to their corresponding parental isolates. My research highlights the ability of Nef to modulate HIV-1 reactivation from latency. Results indicate that Nef may play an important role in determining the breadth and diversity of inducible viral reservoirs following infection. If so, a better mechanistic understanding of Nef’s impact may uncover new strategies to enhance viral reactivation from latency that are clinically beneficial.

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

Regulation of inducible nitric oxide synthase expression in human cells

Author: 
Date created: 
2018-06-28
Abstract: 

Nitric oxide (NO) is a bioactive gas that has multiple roles in innate and adaptive immune responses and dysregulated expression of its production by inducible nitric oxide synthase (iNOS) is implicated in the pathogenesis of various inflammatory diseases and cancer. The mechanisms by which the expression of iNOS is regulated in human cells is incompletely understood. I show that NO positively regulates iNOS expression through a positive feedback mechanism that involves S-nitrosylation and activation of the small Ras GTPase, which in turn activates downstream PI3K/Akt and mTOR pathways. This feedback mechanism acts in a post-translational manner to increase iNOS protein levels by reducing its ubiquitination and proteasomal degradation. In addition to examining the feedback regulation of iNOS protein expression, I also studied how gene expression of this enzyme is controlled. I focused on the hypoxia inducible factor (HIF) because of the central role of this transcription factor in controlling many aspects of cell biology. The human iNOS gene promoter has three predicted HIF binding sites and mutation of one of these sites at -4.9 kb reduced the induction of iNOS gene promoter activity by cytokines. Further, HIF-1a gene editing by CRISPR/Cas9 eliminated HIF activity and reduced the induction of iNOS mRNA and protein expression by cytokines. Altogether, I discovered two mechanisms whereby iNOS expression is positively regulated at the protein and gene expression levels in an inflammatory setting. The role of NO and HIF in augmenting iNOS expression may provide insight into new therapeutic strategies for inflammatory diseases and cancer.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Jonathan C. Choy
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Investigating the neuroprotective role of OGA inhibition by Thiamet-G against Alzheimer\'s disease

Author: 
Date created: 
2018-02-23
Abstract: 

The glycosylation of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is important for regulation of protein function and cellular signaling. Addition of GlcNAc monosaccharide unit to target proteins requires the enzyme O-GlcNAc transferase (OGT), and removal of O-GlcNAc depends on the enzyme O-GlcNAcase (OGA). Previous works have shown that OGA inhibitors and enhancers of autophagy both reduced cognitive impairment as well as Aβ and tau aggregation in Alzheimer\'s disease (AD) mouse models. Here, it was shown that OGA inhibition enhanced autophagy in neuro-2a cells and AD mouse brain through an mTOR independent pathway. These data suggest that OGA inhibition provides neuroprotection by promoting autophagy dependent clearance of protein oligomers. To investigate this relationship, we established inducible cellular models of tauopathy to show that OGA inhibition decreased levels of pathological tau species. These results suggest OGA inhibition is a possible therapeutic strategy against AD that may involve the enhancement of autophagy.

Document type: 
Thesis
File(s): 
Senior supervisor: 
David Vocadlo
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Directed evolution of a bacterial sialidase and characterization of mechanism based inactivation of glycosidases

Date created: 
2017-12-07
Abstract: 

Sialic acids are often found at the terminal positions on the glycan chains that adorn all vertebrate cells and glycoproteins. This prominent position confers an essential role to sialic acid residues in biology, evolution and disease propagation. The most widespread sialic acid family members are N-acetylneuraminic acid, N-glycolylneuraminic acid and Kdn, which is an abbreviation for 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid. Enzymes that catalyze the removal of carbohydrate linkages from biological molecules are called glycoside hydrolases (GHs). These enzymes have been categorized into more than 130 different families. Glycoside hydrolase family 33 (GH33) contains exo-sialidases (E.C. 3.2.1.18, neuraminidases), from both eukaryotes and prokaryotes, which catalyze the hydrolysis of sialic acid from glycoconjugates. Interestingly, subtle differences exist in both the structure of the particular sialic acid and its position of attachment to glycoconjugate chains between humans and other mammals. These differences are indicators of the unique aspects of human evolution, and are relevant to understanding an array of human conditions. The present thesis reports on routes that we explored to further unravel the importance of sialic acids. We developed tools to probe for various sialic acid structures such as Kdn. To this end, we constructed a random mutant library of the neuraminidase from the soil bacterium Micromonospora viridifaciens (MvNA) and identified a number of recurring mutations in the sialidase gene which lead to a more efficient hydrolysis of synthetic natural substrate analogues such as 8FMU α-Kdn-(2→6)- β-D-Galp. We also using the available structure of wild type MvNA bound to the natural inhibitor, DANA, to identify amino acids potentially involved in recognition and binding to acetylated sialic acids and generated genetic libraries which we used along with positive and negative evolutionary screens to identify several clones capable of hydrolyzing Kdn glycosides more efficiently than Neu5Ac substrates. Kinetic studies on these clones allowed for determination of enzyme efficiencies and specificities. We also report our study of covalent inhibition of α-glucosidase from Saccharomyces cerevisiae (GH13). The measured pH-rate profiles for inhibition and reactivation as well as the corresponding catalytic and inhibitory proficiencies suggested that inhibition results from the formation of carbenium ions in the active site that are trapped rapidly by an enzymatic residue.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Andrew Bennet
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.