Life Sciences Commons^™

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 …

Elucidating The Neural Circuitry Underlying Social Spacing In Drosophila Melanogaster Through The Lens Of Neuroligin 3, Abigail T. Bechard

Electronic Thesis and Dissertation Repository

Social interactions among animals can be complex, and abnormal social behaviours may result in negative fitness consequences for both the individual displaying them, and the entire group. To understand the neural basis of complex social behaviour, we can study simpler behaviours that precede and mediate them. Social spacing, the typical distance between individuals in a group, is an easily quantifiable behaviour in Drosophila melanogaster. Here, I investigated the neural circuitry underlying social spacing through the lens of Autism-candidate gene neuroligin 3. Based on the Nlg3 enrichment pattern in adult fly brains, I hypothesized that nlg3-expressing neurons, along …

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 …

Pillars Of Biology: 'The Genetical Evolution Of Social Behaviour, I And Ii'., Geoff Wild

Applied Mathematics Publications

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Selection Pressure On Surface Exposed Virus Proteins, Sareh Bagherichimeh

Electronic Thesis and Dissertation Repository

Viral infection requires the interaction between virus surface-exposed (SE) proteins and host cell receptors. This can result in an “arms race” that is assumed to drive accelerated rates of evolution, and some well known examples of diversifying selection involve surface pro- teins (HIV-1 env, influenza hemagglutinin). We conducted a systematic analysis to determine whether this is truly a distinctive feature of SE virus proteins, in comparison to non-SE proteins encoded by the same genomes.

We obtained reference and all neighbour genomes of 52 human viruses from the NCBI Viral Genomes database. The coding sequences (CDS) of each genome extracted by …

Investigation Of Neurotransmitter’S Knockdown Effect On Drosophila Melanogaster Female Aggression, Asil N. El Galad

Undergraduate Student Research Internships Conference

Drosophila melanogaster (D. melanogaster) is a popular model organism in neurobiology. While aggression has been studied heavily in male drosophila, there's minimal research on aggression in female drosophila. Neurotransmitters influencing aggressive behaviour in female D. melanogaster are poorly understood. Various neurotransmitters such as dopamine, octopamine, and serotonin influence aggression in D. melanogaster Further investigation of the role of neurotransmitters on aggression is thus important. The purpose of our experiment is to observe the effect of the knockdown of dopamine, octopamine and glutamate on aggression in female D. melanogaster.

Towards More Complete Metagenomic Analyses Through Circularized Genomes And Conjugative Elements, Benjamin R. Joris

Electronic Thesis and Dissertation Repository

Advancements in sequencing technologies have revolutionized biological sciences and led to the emergence of a number of fields of research. One such field of research is metagenomics, which is the study of the genomic content of complex communities of bacteria. The goal of this thesis was to contribute computational methodology that can maximize the data generated in these studies and to apply these protocols human and environmental metagenomic samples.

Standard metagenomic analyses include a step for binning of assembled contigs, which has previously been shown to exclude mobile genetic elements, and I demonstrated that this phenomenon extends to all conjugative …

Utilizing Rnai Technology To Develop Novel Agricultural Pesticides, Ethan B. Zand

Undergraduate Student Research Internships Conference

A general description of why RNAi based pesticide technology is a breakthrough technology able to target only pests while not harming others. This presentation gives a summary of RNAi technology, the current drawbacks of conventional pesticides, and our research on how RNAi can be used against the two-spotted spidermite; a polyphagic pest that causes significant financial damage to Canadian and worldwide agriculture

Rna-Interference As A Topical Pesticide, Eleanor Khochaba

Undergraduate Student Research Internships Conference

Tetranychus urticae are a species of pests that are very problematic in agricultural environments, as they cause lots of damage to a wide variety of crops. One step in figuring out a solution to this problem is through the use of RNA interference. After synchronizing the spider mites, they are treated with the dsRNA solution. The dsRNA is cleavage inside the mite, and the siRNA fragments then bind to a target gene in hopes of homologous sequence recognition leading to knockout/deregulation of the target gene. After treatment, the mites are observed for multiple variables, primarily fecundity and survival. If a …

Examining The Effect Of Knocking Down Dopamine Receptors In Fruitless Sexually Dimorphic Neurons On Social Spacing, Avneet A. Sahota

Undergraduate Student Research Internships Conference

Social behaviour is an individual's response to others within a group. From humans to mice to even fruit flies, social behaviour involves interactions with others and their ability to exchange information. Social spacing is a type of social behaviour which measures the distance between two organisms. It is a balance of attractive and repulsive cues which are influenced by genetics and by the external environment. However, individuals with disorders such as autism spectrum or Williams syndrome display abnormal social spacing as they are unable to understand social and non-social cues. Also, dopamine is a neurotransmitter that is a contributing factor …

Illuminating Transfer Rna Variants As Genetic Modifiers In Models Of Human Disease, Jeremy T. Lant

Electronic Thesis and Dissertation Repository

Transfer RNAs (tRNAs) physically link the genetic code to an amino acid sequence, by recruiting amino acids to three-nucleotide codons in messenger RNAs. To ensure that the genetic code is translated as intended, tRNAs must be accurately aminoacylated and faithfully recognize codons in the ribosome during protein synthesis. Given the critical function of tRNAs, it has often been assumed that mutations in human tRNA genes would be either lethal to cells or not significantly impair tRNA function. My goal was to rigorously test this assumption in mammalian cell models, prompted by the recent discovery of unprecedented variation in human tRNA …

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 …

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 …

The Role Of Pannexin 3 In Forced Exercise Bone Remodelling And Its Potential Implications For Osteoarthritis, Justin Tang

Undergraduate Student Research Internships Conference

Exercise such as running has been hypothesized to be a factor of osteoarthritis development. However, the relationship is unclear as differences in genetic backgrounds may influence the risk of osteoarthritis (OA). The gene Panx3 codes for the Pannexin 3 protein (PANX3), a mechanically sensitive channel that allows for the passage of various signalling molecules in both chondrocytes and osteoblasts (Boassa et al., 2007; Penuela et al., 2013; Zhang et al., 2021). We have previously shown PANX3 to be protective in traumatic osteoarthritis, however beneficial in age-induced osteoarthritis (Moon et al., 2021), suggesting that PANX3’s role in osteoarthritis development …

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, …

Evaluation Of Two Mouse Models Of High Genetic Variation For Suitability To Test A Heterozygote Instability Hypothesis, Hailie Pavanel

Electronic Thesis and Dissertation Repository

Characterization of genetic variation underlying complex phenotypes is incomplete yet critical to understanding mutational mechanisms and phenotypes. Heterozygote Instability (HI) is a new, poorly understood source of mutations needing models for mechanistic study. Two models ideal for characterizing HI-associated mutational mechanisms are outbred mice and mouse basal cell carcinoma (BCC). Both have discontinuous landscapes of heterozygosity essential to assess HI-induced mutations. Here, heterozygosity and copy number variants (CNVs) in two outbred mouse stocks are characterized with 1690, and 3935 autosomal CNVs detected. A positive correlation exists between chromosomal heterozygosity and CNV occurrence (R² = 0.14 and 0.09), and 41 …

Genetic Approaches For The Study Of Complex Human Diseases, Julieta Lazarte

Electronic Thesis and Dissertation Repository

The field of human genetics has evolved from its initial narrow focus on single-gene Mendelian disorders, which largely affect children, to our current understanding that for most diseases there is continuum of rare to common variants which can exert a range of phenotypic effects. Despite advances in sequencing capabilities and our overall understanding of diseases, there remains a large proportion of heritability unexplained. Through the use of next-generation sequencing technologies and DNA microarray, I have explored a spectrum of genetic variations from rare, single and structural variants to common variants in individuals with i) “lone” atrial fibrillation; ii) familial hypercholesterolemia; …

The Role Of Gsk3 Alpha And Beta In Embryonic Craniofacial Development, Harman Jassar

Electronic Thesis and Dissertation Repository

Background: The GSK-3 genes (Gsk3a and Gsk3b) have been known to affect many cellular processes and signaling pathways some of which are implicated in the growth and development of the craniofacial skeleton.

Aim: The purpose of this study was to assess the effect of chondrocyte-specific deletion of Gsk3a and Gsk3b on the size of the mandible and craniofacial skeleton in embryonic mice.

Materials & Methods: Mice were bred to generate cartilage-specific Gsk3a and Gsk3b KO mice. On embryonic day 18.5 (E18.5) the offspring were gathered by caesarian section. Whole mount skeletal staining was completed on the specimens using …

The Genetic Determinants Of Complex Lipid And Lipoprotein Phenotypes, Jacqueline S. Dron

Electronic Thesis and Dissertation Repository

Cardiovascular disease (CVD) is the primary cause of death globally and is estimated to cause one-third of deaths in Canada. Each year, millions of Canadians are affected by CVD despite ongoing efforts to reduce risk through lifestyle modifications and pharmacological therapies. With the expected rise in CVD prevalence due to the obesity epidemic, we need to better understand the genetic basis of heritable, modifiable risk factors, including levels of high-density lipoprotein (HDL) cholesterol and triglyceride, for insights into future therapeutic treatments and risk prediction. Through the use of a targeted next-generation sequencing panel designed specifically to study lipid and metabolic …

Genetic Basis Of Hybrid Sterility Between Drosophila Pseudoobscura And D. Persimilis, Alannah J.P. Mattice

Electronic Thesis and Dissertation Repository

Speciation is the underlying process that leads to formation of new species, and therefore is the basis of biodiversity. Genes involved in each stage of speciation, such as those involved in interspecies sterility, remain elusive. Male hybrid sterility and postzygotic isolation between Drosophila pseudoobscura and D. persimilis was examined in this study through backcrossing of female hybrids into each parental line (introgression), selecting for a sterile sperm phenotype, needle-eye sperm. Sperm phenotypes did not separate through backcrossing; instead, males presented with multiple sperm phenotypes. A relationship between the phenotypes observed and the potential genes involved was examined through whole genome …

Estimating Partial Body Ionizing Radiation Exposure By Automated Cytogenetic Biodosimetry, Ben Shirley, Peter Rogan

Biochemistry Publications

Purpose: Inhomogeneous exposures to ionizing radiation can be detected and quantified with the dicentric chromosome assay (DCA) of metaphase cells. Complete automation of interpretation of the DCA for whole-body irradiation has significantly improved throughput without compromising accuracy, however, low levels of residual false positive dicentric chromosomes (DCs) have confounded its application for partial-body exposure determination.

Materials and methods: We describe a method of estimating and correcting for false positive DCs in digitally processed images of metaphase cells. Nearly all DCs detected in unirradiated calibration samples are introduced by digital image processing. DC frequencies of irradiated calibration samples and those exposed …

Estimating Partial Body Ionizing Radiation Exposure By Automated Cytogenetic Biodosimetry, Peter Rogan

Biochemistry Publications

Purpose: Inhomogeneous exposures to ionizing radiation can be detected and quantified with the Dicentric Chromosome Assay (DCA) of metaphase cells. Complete automation of interpretation of the DCA for whole body irradiation has significantly improved throughput without compromising accuracy, however low levels of residual false positive dicentric chromosomes (DCs) have confounded its application for partial body exposure determination.

Materials and Methods: We describe a method of estimating and correcting for false positive DCs in digitally processed images of metaphase cells. Nearly all DCs detected in unirradiated calibration samples are introduced by digital image processing. DC frequencies of irradiated calibration …

Epigenetics A Decolonizing Science, Wade Paul

Electronic Thesis and Dissertation Repository

Epigenetics is the study of gene expression that does not entail alterations to the actual DNA. Decolonization is a theoretical and political movement that seeks to deconstruct colonial institutions and ideologies and reconstruct new and balanced approaches that accept and respect Indigenous worldviews. This project studies the decolonizing potential of epigenetics. Using genealogy as the method, the study establishes a long history of reductionist and deterministic thought that shaped the study of genetic science. Particular instances like thrift gene theory are explored to highlight how genetic explanations have been detrimental to the health and wellbeing of Indigenous people and illustrate …

Advanced Phenotyping Of Otosclerosis In An Ontario Population And Two Large Newfoundland Families, Matthew B. Lucas

Electronic Thesis and Dissertation Repository

Otosclerosis is a relatively common hearing loss disorder characterized by abnormal bone growth in the otic capsule leading to stapes fixation. In approximately half of cases, otosclerosis is inherited as an autosomal dominant trait. Typically, gene discovery efforts rely on surgical confirmation, audiometry and occasionally acoustic reflexes to identify affected cases of otosclerosis within families, requiring that the otosclerosis was at an advanced stage to be detected. This makes it difficult to identify individuals with early otosclerosis. The use of advanced phenotyping to identify cases of otosclerosis was tested in an Ontario otosclerotic population as well as in two large …

Generation Of Conditional Ku70 Knockouts In Human Cell Lines Using Crispr/Cas9 And Dual-Nuclease Crispr/Tevcas9, Gursimran Parmar

Electronic Thesis and Dissertation Repository

The Ku heterodimer, Ku70 and Ku80, plays a key role in DNA repair. Viable Ku70 knockouts exist in mice but not in human cell lines. The objective was to create Ku70 knockouts and evaluate knockout viability in human cells using CRISPR/Cas9 and TevCas9. Editing is achieved by Cas9 through one sequence-specific blunt cut accompanied by error-prone DNA repair. However, TevCas9, a novel fusion protein of Cas9 and I-Tev, creates two non-compatible DNA breaks and biases editing events towards a small deletion. Ku70 has five processed pseudogenes therefore intron-exon junctions were targeted by gRNA and a cell line stably transfected with …

Mushroom Body-Specific Gene Regulation By The Swi/Snf Chromatin Remodeling Complex, Kevin Cj Nixon

Electronic Thesis and Dissertation Repository

Over the lifetime of an organism, neurons must establish, remodel, and maintain precise connections in order to form neural circuits that are required for proper nervous system functioning. Disruptions in these processes can lead to neurodevelopmental disorders such as intellectual disability (ID) and autism spectrum disorder. Mutations in genes encoding subunits of the SWI/SNF chromatin remodeling complex have been implicated in ID, yet the role of this complex in neurons is poorly understood. In this project, I established cell-type specific methods to examine the effect of SWI/SNF subunit knockdowns on gene transcription and chromatin structure in the memory-forming neurons of …

Analysis Of The Chemotactic And Anti-Ovarian Response To Honeybee Queen Mandibular Pheromone (Qmp) And Its Constituent Components In Drosophila Melanogaster, Suhayr Solangi

Electronic Thesis and Dissertation Repository

Drosophila melanogaster can mimic qualities of honeybee altruism mediated by queen mandibular pheromone (QMP), a honeybee pheromone, which includes suppression of oogenesis and chemotactic attraction of males to sources of QMP. In this study, I assessed chemotaxis to the components of synthetic QMP (sQMP): the phenols; methyl-p-hydroxybenzoate (HOB) and 4-hydroxy-3-methoxyphenylethanol (HVA), and decenoic acids; 9-hydroxy-decenoic acid (9-HDA) and 9-oxo-decenoic acid (9-ODA). I found that the chemotactic response to these components is sexually dimorphic and they have complex interactions with one another. I conducted a screen to inhibit olfactory receptor neurons (ORN) using tetanus toxin (TeTx) and OrGAL4 drivers. Of the …

Rna Interference Of Worker Sterility Genes: Testing Mechanisms Of Reproductive Regulation In Apis Mellifera, Anthony J. Gallo

Electronic Thesis and Dissertation Repository

Social insects such as the honey bee (Apis mellifera) are well known for their reproductive division of labour and eusocial colony systems. Honey bee workers forgo reproduction in favour of being selfless in directing reproductive benefits towards their queen mother. This altruistic tendency is a result of both behavioural and physiological changes under control of the presence of the queen’s mandibular pheromone. The genetic mechanism that underlies this response is, however, not well understood. Here, this study used RNA interference to knock down the expression of single genes (fruitless and ftz-f1) that have been previously identified …

Assessing The Role Of Drosophila Melanogaster Neuroligin 3 On Social Spacing And Climbing Behaviour, J. Wesley Robinson

Electronic Thesis and Dissertation Repository

Autism spectrum disorders can be clinically defined in part by impairments of social interactions. Social interactions can be modeled in Drosophila melanogaster with behaviours such as social spacing. Here, I examined the effects of autism-related gene neuroligin 3 on fly social spacing. I hypothesized if neuroligin 3 is mutated or gene expression is targeted for knockdown, then flies will have altered social space in males and females at different ages. Using the social space assay, I found that different mutations to neuroligin 3 change the fly’s behavior, in a mutation and sex-specific manner. Using an antibody against Nlg3, I localized …

Cross-Species Utility Of The Mouse Diversity Genotyping Array In Assaying Single Nucleotide Polymorphisms, Rachel Kelly

Electronic Thesis and Dissertation Repository

In the study of genetic diversity in non-model species there is a notable lack of the low-cost, high resolution tools that are readily available for model organisms. Genotyping microarray technology for model organisms is well-developed, affordable, and potentially adaptable for cross-species hybridization. The Mouse Diversity Genotyping Array (MDGA), a single nucleotide polymorphism (SNP) genotyping tool designed for M. musculus, was tested as a tool to survey genomic diversity of wild species for inter-order, inter-family, inter-genus, and intra-genus comparisons. Application of the MDGA cross-species provides genetic distance information that reflects known taxonomic relationships reported previously between non-model species, but there …