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Determining The Effects Of Mistranslating Transfer Rna Variants On Drosophila Melanogaster, Joshua R. Isaacson

Electronic Thesis and Dissertation Repository

Transfer RNAs (tRNAs) play a central role in translation as adaptor molecules between mRNA and protein. Variant tRNAs can cause the misincorporation of an amino acid into a growing polypeptide. Mistranslating tRNA variants are surprisingly common in humans but the effects of mistranslating tRNA variants on eukaryotic biology are poorly understood. My thesis aimed to create a model of tRNA-induced mistranslation using the fruit fly Drosophila melanogaster and characterize the effects of mistranslating tRNA variants on eukaryotic biology.

I first integrated a gene encoding a serine tRNA variant that induced proline-to-serine (P>S) mistranslation into the fly genome. Proteins isolated …

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 …

The Role Of The Leucine-Rich (Leur) Domain Of Rho Guanine Nucleotide Exchange Factor (Rgnef) In The Regulation Of Amyotrophic Lateral Sclerosis (Als) Associated Protein Tar Dna-Binding Protein Of 43 Kda (Tdp-43), Hind Amzil

Electronic Thesis and Dissertation Repository

The presence of neuronal cytoplasmic inclusions (NCIs) composed of RNA-binding proteins (RBPs) and neurofilaments is considered to be ALS’s neuropathological hallmark. RGNEF has been previously shown to interact with TDP-43 and to have a regulatory effect on the expression levels of NEFL mRNA and NFL protein in vitro. Here, I examined the mechanism of the RGNEF N-terminus, leucine-rich domain (LeuR) domain’s interaction with TDP-43. I observed that the minimal domain required is 110 amino acids (LeuR110), that the Ankyrin domain adjacent to LeuR110 does not participate, and that LeuR110 forms of a high molecular weight complex with TDP-43 in …

The Role Of Histone Demethylases In Learning And Memory In The Mushroom Body Of Drosophila Melanogaster, Crystal Keung

Electronic Thesis and Dissertation Repository

Intellectual disability (ID) is a neurodevelopmental disorder associated with many epigenetic regulators and chromatin modifying enzymes like histone lysine methyltransferases (KMTs) and demethylases (KDMs). Here, I systematically investigate the role of 7 KDMs: Su(var)3-3, KDM2, Lid, CG2982, UTX, KDM4B, JHDM2, and 1 KMT: trr in the context of learning and memory using Drosophila melanogaster. Genetic knockdown of each gene in the mushroom body (MB) of flies are tested for short- and long-term memory impairment using courtship conditioning. Knockdown of 6 KDMs and trr resulted in memory loss. MB morphology was analyzed to determine potential cause of memory loss. …

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 …

The Role Of H3k4 Methyltransferases In Drosophila Memory, Nicholas Raun

Electronic Thesis and Dissertation Repository

Gene transcription required for long-term memory requires the modification of histones. However, there are still many uncertainties about the identity and spatial expression of genes regulated by histone modifications during memory related processes. In this project I examined the role of Drosophila melanogaster methyltransferases Set1 and trx in courtship memory. Genetic knockdown of Set1 and trx in the mushroom body (MB) revealed that Set1 was necessary for short- and long-term memory, while trx was only required for long-term memory. Transcriptional profiling of MBs following trx-knockdown revealed expression changes in MB-enriched genes and genes involved in RNA processing. Among the …

Gene Expression Changes In The Mushroom Body Of Drosophila Melanogaster During A Time Course Of Long-Term Memory Formation And Maintenance, Spencer G. Jones

Electronic Thesis and Dissertation Repository

Long-term memory (LTM) requires gene transcription. However, there is still much to learn about which genes are transcriptionally regulated during LTM and the biological roles they play. Here, gene expression changes were characterized in Drosophila melanogaster over a time course of LTM formation and maintenance in neurons of the mushroom body (MB), a structure required for normal learning and memory. I identified 120 genes differentially expressed (q < 0.2, fold change > 1.3) 24h after LTM induction. Among these were 13 potential downstream targets for RNA localization by the known memory genes pumilo, staufen and oskar, several genes encoding chromatin regulators and seven genes with …

Role Of The Swi/Snf Chromatin Remodelling Complex In The Axon Development Of The Drosophila Mushroom Body, Melissa C. Chubak

Electronic Thesis and Dissertation Repository

The SWI/SNF complex is an evolutionarily conserved ATP-dependent chromatin remodelling complex that has been implicated in the aetiology of intellectual disability (ID). Among the dominant ID genes, the SWI/SNF complex is the most highly enriched protein complex. However, its role in the nervous system is not yet understood. I systematically investigated the role of this complex in the development of the Drosophila mushroom body (MB), a complex brain structure required for learning and memory. Gross MB morphology was assessed using confocal microscopy to identify morphological defects following RNAi-mediated knockdown of the 15 individual SWI/SNF genes in the MB. Knockdown of …

Regulation Of Learning And Memory By The Drosophila Melanogaster Swi/Snf Complex, Max H. Stone

Electronic Thesis and Dissertation Repository

The SWI/SNF complex is a highly-conserved ATP-dependent chromatin remodelling complex that is important in the etiology of intellectual disability (ID). I systematically investigated the overall and adult-specific roles of each of the 15 Drosophila melanogaster SWI/SNF complex components in memory. Flies with RNAi-mediated knockdown of individual SWI/SNF genes in the mushroom body (MB) were tested for short- and long-term memory impairment using courtship conditioning. Knockdown of several SWI/SNF genes through development, including brahma, Bap60, Snr1, and e(y)3, caused loss of memory. Adult-specific knockdown of SWI/SNF genes caused some loss of memory phenotypes, indicating an acute role in adult MB activity. …

Tracking Behavioural And Neuronal Responses To Social Pheromones: Insights From A Drosophila Model, Justin R. Croft

Electronic Thesis and Dissertation Repository

If eusociality evolved through modification of pre-social mechanisms for regulating personal reproduction, then even insects like Drosophila may be vulnerable to latent effects of 'queen' pheromone. Here, I test if male fruit flies respond to a eusocial queen bee pheromone. I found that male flies were attracted to queen bee pheromone, and pheromone-treated males raised the intensity of their courting towards conspecific females. These novel observations from Drosophila suggest that male flies have the capacity to respond to queen pheromone in a manner that is comparable to the native response from male (drone) bees. I therefore optimized a nuclear factor …

The Effect Of Age On Social Behaviour In Drosophila Melanogaster And The Progeny Of Aged Parents, Dova Brenman

Electronic Thesis and Dissertation Repository

Recent studies have linked neuropsychiatric disorders to older parents. These disorders often include changes in social behaviours like the social spacing between neighbouring individuals, which can be modeled in organisms such as Drosophila melanogaster. I investigated the effects of aging on the social space between neighbouring D. melanogaster and how aging impacts the next generation. To achieve this, I used the social space assay and found that individuals become less social with age and that this effect is passed on to the first generation only. Additionally, accelerating the physiological process of aging via increased rearing temperatures or exposure to …

A Genetic Test Of A Model For Two Activities Of Fushi Tarazu Protein In Drosophila Melanogaster, Alaa Briek

Electronic Thesis and Dissertation Repository

The important pair-rule segmentation gene fushi tarazu (ftz) encodes a homeodomain (HD)-containing protein involved in the establishment of even-numbered parasegments during embryonic development. The D. melanogaster ftz is a derived homeotic selector (Hox) gene which lost its homeotic function during the evolution of arthropods. Genetic analyses have shown that FTZ has two distinct activities required during development: HD-dependent and HD-independent FTZ activities. The aim of this study was to test the interaction of the two FTZ activities proposed by Hyduk and Percival-Smith (1996), by generating site-specific mutant ftz alleles for intragenic complementation. CRISPR-mediated homology directed repair (HDR) was used to …

Investigating Hox Protein Requirement For Tarsus Determination In Drosophila Melanogaster, Samantha Koot

Electronic Thesis and Dissertation Repository

Generally, all bilaterans examined have similar conservation of HOX protein structure, function, expression, and requirement. However, at the level of being the same, it is unknown whether the HOX protein, Antennapedia, is required for tarsus determination in Drosophila melanogaster as in Tribolium casteneum, or whether the requirement of HOX proteins in determination of body parts diverges in insects. I proposed to use a heat shock-inducible nanobody (UAS- NSlmb-vhhGFP4 driven by hsp-GAL4) activated during the third larval stage in all cells to degrade thoracically expressed HOX proteins (Sex combs reduced, Antennapedia, and Ultrabithorax) tagged with green fluorescence protein …

Determination Of The Phylogenetic Range Of Sex Combs Reduced Activity In Drosophila Melanogaster, Laura E. Garofalo

Electronic Thesis and Dissertation Repository

The homeotic selector (Hox) genes are required for body patterning in bilaterians. Sex combs reduced (SCR) is a HOX protein in Drosophila melanogaster with two activities: SCR^T1 and SCR^lab activity required for patterning the prothorax (T1) and labia, respectively. SCR^T1 is proposed to be conserved throughout bilaterians while the phylogenetic range of functional conservation of SCR^lab is comparatively unknown. The goal of this work was to elucidate the evolutionary time point at which SCR activity changed. CRISPR/Cas9 transgenesis was used to incorporate ɸC31 integrase recombination sites in Drosophila Scr. The ɸC31 integrase could …

Investigating The Functional Significance Of Evolutionarily Conserved Protein Motifs Of The Drosophila Melanogaster Hox Protein, Sex Combs Reduced, Lovesha Sivanantharajah

Electronic Thesis and Dissertation Repository

Bilaterans share a common anterior-posterior (A-P) axis that is patterned by the Homeotic selector (Hox) genes. In Drosophila melanogaster, Hox gene expression in spatially restricted domains along the A-P axis of the embryo determines segmental identity. Identifying the genetic mechanisms of HOX control of development is essential for understanding body patterning in animals. I identified and characterized the role of evolutionarily conserved protein domains of the HOX protein, Sex combs reduced (SCR), in protein function. SCR is required for establishing the identity of both the labial and prothoracic segments. To identify regions of functional importance, 15 Scr …

How Does Gene Flow Limit Local Adaptation At A Species Range-Edge? An Artificial Selection Drosophila Model, Justin P. Saindon

Electronic Thesis and Dissertation Repository

Gene flow has the potential to create species range limits by impeding adaptation to selective pressures at the range-edge, but it is unclear whether there is a threshold level of gene flow that causes this effect. This gene swamping hypothesis was tested using laboratory populations of Drosophila melanogasterunder selection for desiccation resistance, and subject to a gradient of migration from unselected populations. Desiccation tolerance was impeded across the entire migration gradient, and populations receiving intermediate levels of migration exhibited no tolerance for desiccation stress, following twelve selection events. Female, but not male, flies increased desiccation tolerance following selection by …