Life Sciences Commons^™

Development Of An In Vitro Model Of Mitochondrial Dna Copy Number Depletion Via Stable Inducible Expression Of D1135a Mutant Dna Polymerase Gamma, Amanda L. Morin

Electronic Thesis and Dissertation Repository

Mitochondria are responsible for several crucial cellular processes and contain their own DNA (mtDNA) that exists in several copies. Variation of mtDNA copy number (mtDNA-CN) alters energy metabolism and can modify the epigenome and transcriptome. We hypothesized that inducible expression of polymerase-deficient D1135A dominant-negative DNA polymerase gamma (DN-POLG) would result in mtDNA-CN depletion. Here, an in vitro model expressing D1135A POLG was created using the Flp-InTM T-RExTM-293 stable inducible expression system. Stable integration was confirmed with PCR amplification and Sanger sequencing of post-integration genomic sequences. D1135A POLG expression was confirmed with Western blot of the FLAG-tag antibody. Induction of D1135A …

Advances In Phaeodactylum Tricornutum Nuclear Engineering, Mark Pampuch

Electronic Thesis and Dissertation Repository

The marine diatom Phaeodactylum tricornutum has the potential to become an excellent platform for the sustainable production of valuable compounds and pharmaceuticals, but currently large-scale engineering of this organism remains a challenge due factors like inefficient genetic transformation protocols and a lack of accurate genomic data. This thesis addresses these two bottlenecks by (i) optimizing an electroporation protocol to P. tricornutum and (ii) remapping genomic data from a scaffolded genome assembly to a telomere-to-telomere genome assembly. An optimized transformation protocol was developed that could consistently transform blunt-ended and DNA with overhangs and yielded up to 1000+ colony forming units per …

Characterization Of Etoxazole Resistance In Tetranychus Urticae Populations Collected From Commercial Greenhouses & Fields In Ontario, Jeremy Spenler

Electronic Thesis and Dissertation Repository

In this investigation, etoxazole resistance was monitored in two spotted spider mite (TSSM) populations collected from commercial greenhouses in Ontario. The frequency of an etoxazole resistant, recessive target-site mutation, I1017F, in chitin synthase I (CHS1) was tracked within populations over the course of the study. Cytochrome P450 (P450) activity emerged as a possible alternative mechanism to resist etoxazole exposure in one population.

Using genetic crossing between highly resistant mite populations and a susceptible lab population, filial (F1) hybrid populations were created. The recessive I1017F target-site mutation was abolished in F1 hybrids highly susceptible to etoxazole, while elevated P450 …

Recovery From Social Isolation In Drosophila: The Role Of Dopamine And The Autism-Related Gene Nlg3., Ryley T. Yost

Electronic Thesis and Dissertation Repository

Within a group, individuals establish their preferred distance from each other, or social space, a form of social behaviour. The resulting distance depends on the exchange of social cues from others that needs to be perceived and integrated within the organism’s neural circuitry. In humans, social spacing can be impaired in neuropsychiatric disorders such as autism and schizophrenia. When organisms are subject to social isolation, profound changes in social behaviour are observed in a variety of species from insects to mammals, including social space. However, the genetic and molecular mechanisms modulating a behavioural response to isolation and possible recovery remain …

Exploration Of The Immune Landscape Of Ebv-Associated Gastric Cancers, Mikhail Salnikov

Electronic Thesis and Dissertation Repository

Epstein–Barr virus (EBV) is a gammaherpesvirus associated with 9% of all gastric cancers (GCs). EBV-associated GCs (EBVaGCs) are pathologically and clinically distinct entities from EBV-negative GCs (EBVnGCs), with EBVaGCs exhibiting differential molecular pathology and patient prognosis. The purpose of this thesis is to investigate the tumor microenvironment (TME) of EBVaGCs, which has not been explored in-depth. We hypothesize that EBVaGCs and EBVnGCs are also distinct in terms of the molecular immune landscape. We employed over 400 stomach adenocarcinoma (STAD) samples from The Cancer Genome Atlas (TCGA), as well as a single cell dataset, for the construction of a web suite …

Development Of Reverse Genetics Tools In The Two-Spotted Spider Mite Tetranychus Urticae, Nivitha Bhaskar

Electronic Thesis and Dissertation Repository

Tetranychus urticae, commonly known as the two-spotted spider mite, poses a significant threat to agriculture due to its ability to feed on a diverse range of plant hosts and its strong detoxification abilities in overcoming xenobiotic response. With global warming projected to increase spider mite infestations, it is vital to study the detoxification genes that enable the mite to adapt and survive. The spider mite genome sequence reveals a unique set of detoxification genes that can be studied using RNAi as a promising reverse genetic tool. However, the current genetic toolkit requires improvement. This study examined the effectiveness of …

Mouse Genotyping Array-Based Analysis Of Variation At Single Nucleotide Polymorphic Loci In The Context Of Macro- And Micro-Environments, Freda Qi

Electronic Thesis and Dissertation Repository

Comparing genetic variation between populations can provide insight into selective pressures in the environment. The Mouse Diversity Genotyping Array (MDGA) is a low-cost, high-throughput tool developed to capture and analyze variation at single nucleotide polymorphic (SNP) loci in inbred mouse strains. However, its ability to assess genetic variation in non-inbred mice and associate SNPs with the environment has not been extensively studied. First, SNPs between laboratory inbred and wild-caught mice were compared. No SNPs were associated with either macroenvironment. Instead, SNPs reflected genealogy and inbreeding strategies, consistent with the MDGA’s intended use. Second, mutations at SNP loci were compared between …

Using Conservation Genetics To Inform Reintroduction Of The Endangered Mottled Duskywing (Erynnis Martialis), Shayla Kroeze

Electronic Thesis and Dissertation Repository

Habitat loss and climate change have caused declines in species diversity and abundance globally, including in butterflies which are important components of many ecosystems. Reintroductions are increasingly used to reverse diversity loss but are most effective when informed using genetics. I developed 24 microsatellites and characterized genetic structure and diversity of the endangered Mottled Duskywing (Erynnis martialis) in Ontario and neighbouring provinces and states. These were used to inform a planned reintroduction in Ontario. Populations had moderate levels of genetic diversity, however all but the largest populations may be subject to appreciable levels of genetic drift. Populations more …

Identification Of Dna Methylation Episignatures For Classification And Phenotype/Genotype Correlation In Mendelian Neurodevelopmental Disorders, John Reilly

Electronic Thesis and Dissertation Repository

ABSTRACT: Diagnosis for neurodevelopmental disorders poses numerous challenges, related to the lack of specific findings and limited understanding of clinical impact of the majority of genetic variation. Epigenomics mechanisms involve chemical modifications in DNA that involve a range of cellular mechanisms. DNA methylation is an epigenetic mechanism involving addition and removal of methyl groups to cytosine residues. These methylation signals form episignatures; patterns of methylation that can be used as biomarkers capable of differentiating neurodevelopmental disorders. EpiSigns have enabled molecular diagnosis of a number of genetic conditions, classification of variants of unknown significance, and provided insights into the pathophysiology of …

The Genetics Of Pain: An Exploration Of Gene-By-Environment Interactions And Their Effects On Pain, Mohamad F. Fakhereddin

Electronic Thesis and Dissertation Repository

The findings presented in this dissertation are part of the bigger SYMBIOME project which aims to use the biopsychosocial model of pain to develop a prognostic clinical phenotype for people that experience musculoskeletal (MSK) trauma. Chapter 2 presents an exploratory analysis to assess the relationships between genetic polymorphisms and pain severity and interference. Early childhood trauma was also explored as a moderator between genetic polymorphisms and pain outcomes. For pain severity, major allele carriers (A/A and G/A) of FKBP5 rs9394314 reported significantly higher scores than minor allele carriers (G/G). Further, major allele carriers who had at least one adverse childhood …

Neuroanatomical Characterization Of Fruitless P2 Neurons And Assessment Of Their Role In Female Mate Receptivity, William Yeung

Electronic Thesis and Dissertation Repository

In behaviourally isolated species, preferential mate choice for conspecifics over heterospecifics is a primary isolating barrier for reducing interspecific gene flow. Drosophila males court heterospecific females more frequently than females are sexually receptive to courting heterospecific males, emphasizing the importance of female mate preference in reproductive isolation. The neurogenetic bases of female mate preference have been elusive historically, but recent research identified the D. melanogaster fruitless (fru) P2 exon to influence both conspecific and heterospecific female receptivity. I have expanded on these findings by creating a transgenic line that expresses Gal4 under fru^P2 regulation. Driving a fluorescent …

Developing A Toolbox To Engineer Quantitative Trait Variation In Legume Species Using Crispr/Cas Technologies., Petar Miletic

Electronic Thesis and Dissertation Repository

The impact of intensive agriculture on the environment is immense. This is especially dire with regard to the natural nitrogen (N) cycle, where the human driven interference, primarily associated with industrial fertilization, has reached unsustainable levels. Unlike cereals, legumes, such as soybean, alfalfa and common bean, have the ability to use atmospheric nitrogen, which limits the need for industrial fertilization. A more wide-spread use of legumes could alleviate some of the negative impacts on the biogeochemical cycle while also providing a useful alternative to meat consumption, an important factor in sustainability. To reach this goal, further improvements of legume crops …

Establishment Of A Pesticide Resistance Monitoring Tool For The Two-Spotted Spider Mite, Tetranychus Urticae, Hanna Varonina

Electronic Thesis and Dissertation Repository

Two-spotted spider mite (TSSM), or Tetranychus urticae (Koch), is a major agriculture pest known for its rapid development of resistance to pesticides. The analysis of spider mites’ pesticide resistance demonstrated that resistance patterns and frequencies vary between T. urticae strains collected from different geographic locations and host plants. This research aims at characterization of pesticide resistance patterns in mite populations present in greenhouses in the Southwestern Ontario by identifying genetic and metabolic markers of their pesticide resistance. The establishment of these markers lays the basis for development of a pesticide resistance diagnostic tool that will enable prediction of population resistance …

Functional Characterization Of Arogenate Dehydratase Isoforms In Soybean, Ramtin Sirjani

Electronic Thesis and Dissertation Repository

Phenylalanine flux is partitioned between phenylpropanoid and protein synthesis. The mechanisms behind the metabolic channeling of phenylalanine are largely unknown. Arogenate dehydratase (ADT) enzymes, which catalyze the last and rate-limiting step in the synthesis of phenylalanine in plants, have been shown to interact with the isoflavonoid metabolon in the cytosol. Cytosolic phenylalanine, however, can only be synthesized through prephenate dehydratase (PDT) activity. In this study, putative soybean ADTs (GmADTs) were characterized for their ADT and PDT activity. This was done using complementation assays with two different knockout yeast strains, aro8aro9 and pha2, which lack prephenate aminotransferase and PDT activity, …

Dynamics Of The Soil Microbiome In Ginseng Gardens, Megan E. Lambert

Electronic Thesis and Dissertation Repository

Ginseng Replant Disease (GRD) is a syndrome in which ginseng cannot be cultivated in soil previously used to grow ginseng. Since GRD can persist for decades, it severely impacts the Ontario ginseng industry. To better understand the origin of GRD, the impact of ginseng cultivation on the soil microbiome was investigated in bulk soil of three newly planted ginseng gardens in Ontario, from seeding through two years of cultivation. While specific trends in species richness, diversity and composition were unclear, PERMANOVA analyses confirmed that they changed over time. Known GRD-related pathogens, including Ilyonectria mors-panacis and Fusarium oxysporum, were detected in …

Molecular Identification And Characterization Of Viral Pathogens Infecting Sweet Cherry, Aaron J. Simkovich

Electronic Thesis and Dissertation Repository

Stone fruits are a valuable crop grown worldwide, however pathogens such as viruses threaten fruit production by reducing tree health and fruit yield. In an orchard within the Niagara region of Ontario, symptoms typical of viral infection such as chlorosis and leaf deformation were seen on sweet cherry (Prunus avium L.) trees. Next generation sequencing was performed on symptomatic and asymptomatic leaves and four viruses were identified. On the tree displaying the most severe symptoms, Prune dwarf virus (PDV), was the only virus detected. A survey conducted during this work showed 42% of cherry trees on a single …

Characterization Of Mechanotransduction In Annulus Fibrosus Cells, Min Kyu M. Kim

Electronic Thesis and Dissertation Repository

IVD degeneration is a multifactorial pathological process associated with back pain. While biomechanical factors are important regulators of IVD homeostasis, mechanical loading also contribute to the onset of IVD degeneration. Importantly, the mechanotransduction pathways that mediate cell type-specific responses to mechanical loading are not well understood. Transient receptor potential vanilloid 4 (TRPV4) is a multimodally activated cell surface cation channel implicated as a mechanoreceptor regulating the mechano-response in other musculoskeletal cell types. Using both in vitro and in vivo models, the current study aimed to characterize the role of TRPV4 in annulus fibrosus (AF) cell mechanotransduction. Using a mechanically dynamic …

Rates And Patterns Of Indels In Hiv-1 Gp120 Within And Among Hosts, John Lawrence Palmer

Electronic Thesis and Dissertation Repository

Insertions and deletions (indels) in the HIV-1 gp120 variable loops modulate sensitivity to neutralizing antibodies and are therefore implicated in HIV-1 immune escape. However, the rates and characteristics of variable loop indels have not been investigated within hosts. Here, I report a within-host phylogenetic analysis of gp120 variable loop indels, with mentions to my preceding study on these indels among hosts.

We processed longitudinally-sampled gp120 sequences collected from a public database (n = 11,265) and the Novitsky Lab (n=2,541). I generated time-scaled within-host phylogenies using BEAST, extracted indels by reconstructing ancestral sequences in Historian, and estimated variable loop indel rates …

Regulators Of Ectopic Calcification In A Mouse Model Of Dish: A Multi-Omics Perspective, Matthew A. Veras

Electronic Thesis and Dissertation Repository

Diffuse idiopathic skeletal hyperostosis (DISH) is a non-inflammatory spondyloarthropathy and the second most common form of arthritis characterized by formation of ectopic mineral along the spine. Pathological findings in DISH include regional calcification of the anterior longitudinal ligament, paraspinal connective tissues, and annulus fibrosus (AF) of the intervertebral disc (IVD). Clinical symptoms of DISH include increased spine stiffness, decreased spinal range of motion, and in severe cases dysphagia and spinal cord/nerve root compression. The molecular pathways responsible for DISH have not been delineated and as such, there are no disease-modifying treatments. Clinical treatment for DISH is limited to surgical resection …

B Cell Acute Lymphoblastic Leukemia Is Driven By Activating Janus Kinase Mutations Cooperating With Spi1 And Spib Deletions In A Murine Model, Michelle Lim

Electronic Thesis and Dissertation Repository

B cell acute lymphoblastic leukemia (B-ALL) is caused by genetic lesions in developing B cells that function as drivers for accumulation of additional mutations in an evolutionary selection process. We investigated secondary drivers of leukemogenesis and their mechanism(s) of arising in a mouse model of B-ALL driven by PU.1/Spi-B deletion (Mb1-CreDPB). Whole exome sequencing revealed recurrent mutations in Jak3 (encoding Janus Kinase 3) and Jak1. Mutations with high variant allele frequency (VAF) were dominated by C->T transition mutations that were compatible with AID, whereas the majority of mutations, with low VAF, were dominated by C->A transversions associated with …

Chemical Communication In Songbirds, Leanne A. Grieves

Electronic Thesis and Dissertation Repository

Avian chemical communication has been understudied due to the misconception that olfaction is unimportant or even lacking in birds. Early work focused on the olfactory foraging capabilities of seabirds because of their ecology (open ocean foraging) and large olfactory bulbs. In contrast, olfaction in passerine birds, comprising over half of all extant avian taxa, was long overlooked due to their relatively small olfactory bulbs. It is now well established that passerines can smell, and their olfactory acuity is comparable to that of macrosmatic mammals such as rats. Much of our theory on communication and mate choice has involved studying visual …

Practical Applications And Future Directions Of Genetic Code Expansion: Validation Of Novel Akt1 Substrates And The Design Of A Synthetic Auxotroph Strain Of B. Subtilis, Mcshane M. Mckenna

Electronic Thesis and Dissertation Repository

In Chapter 1, site-specifically phosphorylated variants of the oncogene Akt1 were made in Escherichia coli using the orthogonal translation system that enable genetic code expansion with phosphoserine. The differentially phosphorylated variants of Akt1 were used to validate newly predicted Akt1 substrates. The predicted target sites of the peptide substrates were synthesized and subjected to in vitro kinase assays to quantify the activity of each Akt1 phosphorylated variant towards the predicted peptide. A previously uncharacterized kinase-substrate interaction between Akt1 and a peptide derived from RAB11 Family Interacting Protein 2 (RAB11FIP2) was validated in vitro. Chapter 2 describes the preliminary development of …

Thyroxine-Dependent And -Independent Effects On Premature Aging And Myelination In Atrx Mutant Mice, Megan E. Rowland

Electronic Thesis and Dissertation Repository

ATRX is an ATP-dependent chromatin remodeler required to safeguard genomic integrity. Conditional deletion of Atrx in the mouse embryonic forebrain and anterior pituitary in Atrx^Foxg1Cre mice phenocopies mouse models of progeria which display increased DNA damage, coupled with reduced lifespan, growth and subcutaneous fat. These mice also have severely low circulating levels of insulin like growth factor 1 (IGF-1) and (T4) which have been reported in models of premature aging. Based on evidence that Igf1 is activated by the ligand-bound thyroid hormone receptor, I tested whether T4 supplementation could restore IGF-1 levels and ameliorate premature aging phenotypes in Atrx …

Behavioural And Molecular Consequences Of Postnatal Stress In A Mouse Model Of Fetal Alcohol Spectrum Disorder, Bonnie Alberry

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Fetal alcohol spectrum disorders (FASD) are caused by prenatal alcohol exposure (PAE) and affect 1‑5% of the North American population. Children born with FASD often face maternal separation throughout childhood. How this early life stress (ELS) affects the severity of FASD-related deficits is poorly understood. Using a mouse model, this dissertation establishes that behavioural deficits accumulate following prenatal alcohol exposure and early life stress, assessed using tests for activity, anxiety-like behaviour as well as learning and memory. Hippocampal gene expression was evaluated using RNA-seq followed by clustering of expression profiles through weighted gene co-expression network analysis (WGCNA). A set of …

Identifying Brachypodium Distachyon Proteins Interacting With Histone Deacetylase Bdhd1, Alberto Giovanni Torrez

Electronic Thesis and Dissertation Repository

Current evidence has revealed the involvement of epigenetic mechanisms, including histone deacetylases (HDACs), in plant stress responses. In Arabidopsis thaliana, HDA19, belonging to the RPD3/HDA1 class, interacts with transcription factors to form repressor complexes. HDAC research mainly exists for dicotyledons, whereas research on monocotyledons is limited. Brachypodium distachyon is used as a model plant to investigate questions unique to monocot crops. BdHD1 is the closest homologous gene to HDA19 in B. distachyon. This study investigated potential protein-protein interactions between BdHD1 and each of BdMYB22, BdWRKY24, and BdHOS15. Protein interactions were investigated using yeast two-hybrid (Y2H) and bimolecular fluorescence …

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The Fruitless Gene Influences Female Mate Preference In Drosophila, Tabashir A. Chowdhury

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Species can arise as a result of reproductive barriers that prevent gene flow between diverging populations that force them to remain isolated from one another. Behavioural isolation is one of the earliest acting reproductive barriers determined by the evolution of mating preferences that prevent inter-specific matings. Several traits have been identified that contribute to behavioural isolation, but the genetic basis of interspecific female preference is yet to be determined. I used genetic mapping techniques to identify and confirm that the fruitless gene is affecting species-specific female rejection of interspecies males, contributing to the behavioural isolation between Drosophila melanogaster and D. …

The Group A Streptococci Bacteriocins Facilitate A Competitive Advantage During Nasopharyngeal Infection, Lana Estafanos

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Pathogenic streptococci have evolved specific systems to eliminate bacterial competitors within their biological niche. In microbial environments, niche competition is often driven by the production of short antimicrobial peptides called bacteriocins; this provides a mechanism by which Streptococcus pyogenes may compete for ecological stability and establish infection. Recent findings from our laboratory have identified two novel Class IIb bacteriocin systems – Streptococcus pyogenes bacteriocin (Spb) JK and MN in the M18 serotype S. pyogenes strain MGAS8232 – that may contribute to nasopharyngeal infection. Here, we show that galactose and CO₂ are distinct regulatory cues which induce antimicrobial activity. Under …

Investigating The Role Of Brachypodium Distachyon Cellulose Synthase 8 In Gluconacetobacter Diazotrophicus Colonization, Xuan Yang

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Nitrogen is an essential nutrient for plant growth. Significant amount of nitrogen fertilizer is applied to crop field to maintain high yield. Alternatives to chemical nitrogen fertilizer are needed to reduce the costs of crop production and offset environmental damage. Gluconacetobacter diazotrophicus is a nitrogen fixing bacterium that was originally isolated from sugarcane and has been proposed as a possible biofertilizer for monocot crop production. However, the colonization of G. diazotrophicus in most monocot crops is limited and deep understanding of the response of the host plants to G. diazotrophicus colonization is still lacking. In this study, research was conducted …

Spatial And Temporal Patterns Of Neutral And Adaptive Genetic Variation In The Alpine Butterfly, Parnassius Smintheus, Maryam Jangjoo

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Understanding how much genetic diversity exists in populations, and the processes that maintain that diversity, has been a central focus of population genetics. The evolutionary processes that determine patterns of genetic diversity depend on underlying ecological processes such as dispersal and changes in population size. In this thesis, I examine the influence of dispersal and population dynamics on neutral and adaptive genetic variation in a naturally occurring network of populations of the alpine butterfly, Parnassius smintheus.

My first objective was to determine the combined consequences of demographic bottlenecks and dispersal on neutral genetic variation within and among populations. Using …

Brachypodium Distachyon Histone Deacetylase Bdhd1: A Positive Regulator In Aba Sensitivity And Drought Tolerance, Jingpu Song

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Despite evidence that specific histone deacetylases (HDACs) play important roles in the abiotic stress responses of plants, their roles in the stress responses of monocot plants remain largely unexplored. I investigated a HDAC gene, Bradi3g08060 (BdHD1), in the monocot Brachypodium distachyon (Brachypodium). The Brachypodium BdHD1-overexpression (OE) plants displayed a hypersensitive phenotype to abscisic acid (ABA) and exhibited higher survival under drought conditions. Conversely, the BdHD1-RNAi plants were insensitive to ABA and had low survival under drought stress. Based on ChIP-Seq at the genome-wide level, overexpressing BdHD1 led to lower H3K9 acetylation at the …