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

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Development of single-molecule assays for proteolytic susceptibility: Force-induced collagen destabilization

Author: 
Date created: 
2019-04-26
Abstract: 

Collagen’s unique triple helical structure allows for mechanical support and stability at all hierarchical levels including single molecules, fibrils, fibers and tissues. Surprisingly, the structural stability of the triple helix under force is not well known. I explored its response to applied force with single molecule proteolytic susceptibility assays. To perform the experiments I designed, developed and constructed a low-cost and accessible single-molecule force spectroscopy instrument called the Mini-Radio Centrifuge Force Microscope or MR.CFM. Centrifuge force microscopy allows for a wide dynamic force range, massively parallel single-molecule force measurements, and external field-free application of force. Collagen’s triple helix is resistant to proteolytic cleavage due to steric hindrance. By comparing trypsin’s cleavage rate of collagen with and without force in the MR.CFM, I monitored the triple helix’s force stability. I showed an increase of cleavage rate with force, suggesting a force-enhanced destabilization of the triple helix. One of the most challenging components of single-molecule force spectroscopy is surface chemistry; unsatisfied with available approaches I developed a surface passivation and chemical conjugation method based on an NHS end-labeled F127 Pluronic block copolymer, F127-NHS. I created an easy-to-use assay called the microsphere adhesion by gravity, inversion, then counting, or “MAGIC” assay, to quantitatively measure the interactions between a surface and microsphere. Guided by data from the MAGIC assay I reduced nonspecific interactions with a multistep labelling protocol for the microspheres. I achieved 94% of DNA microspheres singly tethered to the F127-NHS surface. In addition, I showed the surface’s utility in fluorescence and force spectroscopy assays. Taken together my work has enabled the advancement of accessible force spectroscopy and furthered the understanding of collagen’s response to force.

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

Examining the function of primary HIV-1 Nef isolates in the setting of viral control

Author: 
Date created: 
2014-04-24
Abstract: 

HIV is a global pandemic. While combination anti-retroviral therapy can block viral replication, HIV establishes latent reservoirs that persist for life. A rare group of HIV-infected individuals called “elite controllers” spontaneously suppress plasma viremia to clinically undetectable levels. Understanding how elite controllers contain HIV could assist in developing more effective interventions, such as vaccines or eradication strategies. The HIV Nef protein is critical for viral pathogenesis and progression to AIDS. Nef modulates cellular proteins to increase viral replication and to evade host immunity. Functional impairment of several HIV proteins has been seen in elite controllers, but prior to my thesis a systematic analysis of Nef had not been done. To explore this, I examined Nef clones from elite controllers and progressors. I observed that Nef clones from controllers displayed significantly lower activity for multiple functions. Furthermore, I identified natural polymorphisms in Nef that contributed to attenuated function and confirmed these by testing Nef mutants. In summary, I have shown that natural sequence variation in Nef results in substantial differences in protein function. Nef clones isolated from elite controllers displayed the poorest activity for multiple functions, indicating that attenuation of Nef may contribute to reduced pathogenesis in these cases. Therapeutic targeting of the Nef domains identified in my studies could contribute to HIV eradication strategies.

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

Cytogenetic evolution of genus Salmo

Author: 
Date created: 
2019-02-21
Abstract: 

The ancestral genome duplication that occurred around 80-100 MYA in the common ancestor of extant salmonids induced a stressed auto-tetraploid genome. This was followed by expansion of transposable elements which is believed to facilitate large-scale chromosomal rearrangements. The species radiation did not occur until around 40-50 million years later when earth’s climate was going into a cooling stage. As the result of the large-scale genome reorganization, present-day salmonids share large syntenic collinear blocks in a species-specific order. In the present study, our first aim was to visualize these rearrangements by taking advantage of genus Salmo (including two sole members, namely, Atlantic salmon (S. salar) and brown trout (S. trutta)) where species not only exhibit distinct karyotypes, but also intra-species chromosomal polymorphisms. Moreover, another aim of present study was to provide cytogenetic support for confirming the identity of sex determining gene, sdY, in Atlantic salmon by revealing its physical locations in both European and North American derived Tasmanian Atlantic salmon. In the genus of Salmo, large scale rearrangements including fusions, translocations, fissions and possible chromosome arm loss were evident in the present study, giving rise to the extant karyotypes of S. salar and S. trutta. The fact that no polymorphic karyotype of the S. trutta was observed in our study and other publications suggests a relative stable genomic background. In contrast, polymorphic karyotypes due to Robertsonian rearrangements of three pairs of chromosomes were found in Canadian subpopulations of S. salar. The fixation of the homozygous translocation in most Canadian populations indicates possible fixation of advantageous mutations and suggests a mild to near-neutral underdominance of this rearrangement in its ancestral heterozygous state. On the other hand, the observation of potentially deleterious Robertsonian tandem fusions in all Canadian populations indicate the initial rearrangement likely took place in a small effective population with subsequent spreading into other river populations through colonization. In our effort to visualize the physical locations of sdY, a single-copy gene, the gene was indeed found residing on the sub-telocentric region of Ssa02q in European Atlantic salmon, consistent with results from previous SEX mapping studies. In comparison, multiple locations including Ssa02q, Ssa03p and Ssa06p were shown to be locations of sdY in a North American derived Tasmanian population, supporting a previous study suggesting a jumping gene theory.

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

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

Author: 
Date created: 
2019-03-29
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.

(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.