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Biology

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  • ItemOpen Access
    The Diversity and Distribution of Avian Communities in Relation to the Shrubs Ephedra Californica and Larrea Tridentata in the Central California Desert
    (2023-12-08) Hillier-Weltman, Zoe Emma; Lortie, C. J.
    Interactions between key landscape features in desert ecosystems can influence avian community assembly. Bird species may use resources provided by shrubs, including as thermal refuges and as a food source. Citizen science data, such as eBird, is broadly accessible and has been underutilized in the study of fine-scale avian populations and distributions. eBird data offers opportunities for examining avian diversity and abundance across ecological gradients. Using citizen science data, I tested the hypothesis that shrub density and aridity predict the abundance and diversity of bird communities throughout the Central California desert. Shrub density and aridity were important predictors of avian diversity and abundance, but this effect was not constant across species. eBird data offers promise for testing predictions at fine spatial scales, but limitations in the quality and availability of data across locations must be taken into consideration.
  • ItemOpen Access
    The stress response of pokeweed and phylogeny of a defense gene family in plants
    (2023-12-08) Dougherty, Kyra; Hudak, Katalin A.
    Pokeweed (Phytolacca americana L.) is a non-model plant known for its resistance to a variety of biotic and abiotic stressors. Part of the reason for this is the presence of ribosome inactivating proteins (RIPs), which can hydrolyze adenine bases from nucleic acids. These proteins are upregulated in pokeweed by jasmonic acid, a plant hormone involved in stress response. The goal of this research was to gain a better understanding of how plants respond to stress and so two different, but complementary, approaches were taken. Firstly, an in-depth look at the diversity and evolution of RIPs in plants was undertaken by curating a dataset of RIPs from publicly available data and using computational approaches to characterize their domain architecture, identify conserved amino acids, and construct a gene tree. This research revealed that despite the damage that RIPs can potentially cause to the plant’s own nucleic acids, RIPs are common among plants and their diversity indicates the potential for a multi-faceted impact on plant defense. Looking more closely at how pokeweed responds to stress, we applied jasmonic acid to leaves and analyzed changes in gene expression, through RNA sequencing (RNA-Seq). Identification of gene clusters involved in defense was aided by the generation of a pokeweed genome assembly. This research revealed that there is a variety of strategies that plants can implement to respond to stress, and that these strategies are applied differently by different species. Overall, this research contributes to a better understanding of the diversity and nuance present in the ways plants defend themselves.
  • ItemOpen Access
    The role of the environment on wild bee microbiomes
    (2023-12-08) Nguyen, Nhu Phuong; Rehan, Sandra
    Wild bees are important pollinators that are facing the effects of changing environments due to anthropogenic activities. Particularly sensitive to changes in environmental surroundings are bee microbiomes, or the microorganisms forming symbiotic relationships with their bee host. This thesis examines the role of environmental factors such as land use, microclimates, and pesticide residues on the microbiota within a small carpenter bee, Ceratina calcarata. Chapter I provides a review of the literature on bee microbiomes and environmental effects on community composition. In Chapter II, urban land use gradients were examined to characterize the microbiome of wild bees in Toronto. In Chapter III, urban and rural landscapes were studied on a broader scale, finding that bees and their pollen provisions from the city harbour different microbes from their agricultural counterparts. This research provides important implications on how anthropogenic activities may be disrupting bee microbiota and causing dysbiosis potentially harmful to pollinator health.
  • ItemOpen Access
    An Analysis of the Affinity and Stability of Base Pair Modifications of the Glucose-Binding and Cocaine-Binding Aptamers
    (2023-12-08) Osborne, Meghan Taylor; Johnson, Philip E.
    Aptamers are selected to bind to their ligands, usually with high affinity and selectivity for targets. An aptamer was previously selected to bind glucose with a Kd of 10mM which is physiologically relevant as the blood glucose concentration typically falls between 4-11 mM. NMR spectroscopy is suited to study weak aptamer-ligand interactions and was used to investigate the affinity of sequence modified glucose-binding aptamers to try and determine a sequence with a greater binding affinity. Three of thirteen modifications bound to glucose, the change of a G-T base pair to a G-C base pair (Glumod-7) with a Kd of 2.9mM±0.3mM, the alteration of the terminal triloop from a C-T-C to a G-A-A (Gumod-8) with a Kd of 12mM±3mM, and the truncation by 3 base pairs (Glumod-12) which was too weak to determine a Kd. Glumod-7 was the only sequence which had a comparable Kd and would require further investigation in terms of stability using NMR thermomelts. Cocaine-binding aptamers are intriguing as they are composed of three stems centered around two mismatch A-G base pairs and are very well studied in terms of secondary structure and affinity. The thermostability of cocaine-binding aptamers as a function of number of base pairs in stem one was investigated by DSC. There was a trend observed of increasing in stability from 1 base pair to 6 base pairs with a decrease seen at 7 base pairs. The most thermodynamically stable aptamer was determined to be MN4, a preformed structure.
  • ItemOpen Access
    Identification of Regions Required for CDCA7 Interaction with DNA Damage Repair Machinery
    (2023-12-08) Jaff, Shaina Rachel; Scheid, Michael
    CDCA7 (Cell Division Cycle Associated Protein 7) is a transcription factor protein that binds to DNA and histone modifying enzymes supporting DNA methylation and contributes to repair of double stranded breaks in DNA. Mutations of the cdca7 gene cause ICF (Immunodeficiency, centromeric instability, and facial abnormalities) syndrome. CDCA7 has been shown to bind with HELLS (Helicase, lymphoid specific) as a bipartite nucleosome remodeller to allow for de novo methylation by DNMT3b (DNA methyl transferase 3b). Additionally, CDCA7 associates with Ku70 and Ku80, proteins essential for DNA damage repair via the Non-Homologous End Joining (NHEJ) pathway, and -H2AX, whose accumulation is facilitated by Ku proteins and is a biomarker of DNA damage. I show here that CDCA7 requires a putative leucine zipper for association with HELLS, while the binding of 14-3-3 at a phosphorylated residue in CDCA7 regulates Ku70/80 and -H2AX association. This study further elucidates the mechanism of how CDCA7 plays a crucial role in maintaining genomic stability by participating in various DNA repair processes and DNA methylation.
  • ItemOpen Access
    Quantitatively Investigating the Genetic Response of the Euryhaline Sailfin Molly (Poecilia latipinna) to Manipulations of Environmental and Dietary Magnesium
    (2023-12-08) Melissis, Jacob Tyler; Bucking, Carol
    Magnesium (Mg2+) plays vital roles including aiding in DNA replication, cell signaling, hormone production, and musculoskeletal health. To date, research into the function and regulation of genes suspected to be involved in Mg2+ homeostasis has lacked a holistic approach. I therefore sought to quantitatively investigate the regulation of a multitude of genes including solute carriers (slc) slc41a1, slc41a2, cyclin m3 (cnnm3), transient receptor potential melastatin 7 (trpm7), as well as two Na+/K+-ATPase (nka) isoforms nka-1α, nka-2α in the gills, intestines, and kidneys of euryhaline sailfin mollies (Poecilia latipinna) during manipulations of environmental salinity and dietary Mg2+. I further investigated how these manipulations would affect plasma Mg2+ levels using Ion Selective Microelectrodes (ISME) to better understand the function of these genes and the responses to salinity in this species. All Mg2+ transporters were ubiquitously expressed across all tissues examined, regardless of environmental salinity. Furthermore, saltwater acclimation alone did not affect plasma Mg2+ levels, but downregulation of slc41a1, cnnm3, and trpm7 was observed in the gills and intestines, and downregulation of cnnm3 and trpm7 was observed in the kidneys. Increasing dietary Mg2+ led to acute elevation of plasma Mg2+ that was quickly normalized in both salinities. Freshwater-acclimated fish seemed to adapt to dietary manipulations by decreasing intestinal absorption, whereas saltwater-acclimated fish seemed to favour increased renal transport and decreased intestinal transport. This study was the first of its kind to quantitatively investigate the integrated roles of these genes in cellular Mg2+ homeostasis across multiple ionoregulatory epithelia in a euryhaline fish and highlight the need for more wholistic investigations in other species.
  • ItemOpen Access
    The Effects of Daily Exercise Duration on Cardiac Responses and Atrial Fibrillation
    (2023-12-08) Gorman, Renee Ann; Backx, Peter
    Atrial fibrillation (AF) is a supraventricular tachyarrhythmia strongly associated with cardiovascular disease (CVD) and sedentary lifestyles. Despite the abundant benefits of regular exercise, AF incidence for professional endurance athletes is proportionate to CVD patients. To assess exercise dose and AF, we compared the effects of strenuous endurance training on mice by varying daily swim durations (120, 180 and 240 minutes). After receiving the same cumulative work while swimming (estimated from O2 consumption), all exercised groups showed similar elevations (P<0.04) in skeletal muscle mitochondria content and ventricular hypertrophy (P<0.02). By contrast, inducible AF increased (P<0.04) progressively with daily swim dose without markedly affecting atrial refractoriness (P>0.05). Associated with a dose dependency is pronounced (P<0.0001) bradycardia, (P<0.003) hypertrophy, (P<0.0007) fibrosis and (P<0.0001) macrophage accumulation in the atria, that is not observed in the ventricles. Our results demonstrate that prolonging daily swim exercise promotes progressively adverse atrial-specific remodelling leading to increased AF susceptibility.
  • ItemUnknown
    Cytometry of Reaction Rate Constant (Crrc): Optimization of the Technique and Development of a Novel Protocol to Assess Cell Population Heterogeneity Based on Aldh1a1 Activity in Vitro
    (2023-12-08) Nebbioso, Giammarco; Krylov, Sergey N.
    Cytometry of Reaction Rate Constant (CRRC) is a novel analytical technique which aims to study cellular heterogeneity based on the activity of enzymatic reactions. In the past, CRRC was able to assess cellular heterogeneity in vitro by investigating the activity of ABC-transporter enzymes. However, CRRC showed poor robustness to highly motile cells. Here, I report on the development of a workflow to make CRRC robust to cell motility. The novel workflow was used to develop a protocol for in vitro CRRC studies of aldehyde dehydrogenase 1A1 (ALDH1A1)-based cell heterogeneity. The data collected suggested a potential positive correlation between the activity of ALDH1A1 and the age of a cell line. Finally, I demonstrated the robustness of the new CRRC ALDH1A1 assay to a 20% change in the initial substrate concentration. Overall, these studies confirm the potential for CRRC to become a reliable analytical tool for studies of reaction-based cell heterogeneity.
  • ItemUnknown
    Investigating substrate discrimination by the fission yeast La protein during stress
    (2023-12-08) Sinagoga, Vanessa Nicole; Bayfield, Mark
    The La protein is an RNA-binding protein first identified as an autoantigen in Lupus patients. La’s best characterized function is to protect the 3’ end of pre-tRNAs from degradation by binding their characteristic UUU-3’OH tails. La also facilitates pre-tRNA folding through a poorly understood RNA chaperone mechanism. We hypothesized that the Schizosaccharomyces pombe La protein (Sla1p) discriminates among RNA substrates based on fold, selectively binding misfolded RNAs for chaperone intervention during cellular stresses predicted to disrupt RNA structure. High-throughput sequencing of Sla1p-associated RNAs did not reveal altered interaction between Sla1p and pre-tRNAs during stress, suggesting a lack of substrate discrimination by Sla1p. Northern blotting revealed novel perturbations to pre-tRNA processing in S. pombe during oxidative stress and nutrient starvation. Overall, this work has added to our understanding of La’s chaperone activity, and seeded future work toward uncovering further details of stress-dependent alterations to pre-tRNA processing in fission yeast.
  • ItemOpen Access
    Transcription Factor FKH-9 Regulates Oxidative Stress Responses in Caenorhabditis Elegans
    (2023-12-08) Libertucci, Alessia Marie; Kubiseski, Terrance J.
    The biological process of energy production is an essential mechanism needed for the strength and survival of many living organisms. Although highly beneficial, energy production can also exert a negative effect through the creation of reactive oxygen species (ROS). ROS are highly reactive chemicals that are generated when oxygen molecules get reduced in the cell such as from mitochondrial oxidative metabolism. This production of ROS is a normal cellular event; however, its accumulation can cause oxidative stress by modifying compounds, interfering with signalling cascades, and damaging macromolecules. Therefore, the cell is known to have many detoxification systems that involve specific cellular signalling pathways. One pathway seen in C. elegans is the skinhead-1 (SKN-1) glutathione s-transferase-4 (GST-4) pathway. GST-4 is an important detoxification enzyme that helps reduce the accumulation of ROS in the cell, while the SKN-1 transcription factor regulates gst-4 at the promoter. However, under non-oxidative stress-inducing conditions SKN-1 is hypothesized to be indirectly held in the cytoplasm by the BRCA1 associated protein-2 (BRAP-2). Recently, our lab discovered elevated gst-4 levels in brap-2(ok1492) deletion mutants, however RNAi of the transcription factor forkhead-9 (fkh-9) in these mutants caused a decrease in gst-4 expression. I have confirmed that FKH-9 is needed for gst-4 expression levels in brap-2(ok1492) mutated worms. I have also uncovered its potential role in the signalling pathway as FKH-9 significantly increased SKN-1-mediated activity at the skn-1c promoter indicating a potential synergistic effect with SKN-1 to help increase SKN-1 transcription levels. Biological analysis of FKH-9 was also carried out and mutants were seen to have compromised longevity and survival under various stress-inducing conditions compared to wild-type (WT). Thus, my investigation ultimately presents an initial indication upon the role and function of FKH-9 in the C. elegans oxidative stress response.
  • ItemOpen Access
    FoxP1 represses MEF2A in striated muscle
    (2023-12-08) Steiman, Sydney; McDermott, John Charles
    Myocyte Enhancer Factor 2 (MEF2) is a transcription factor complex encoded by genes mef2a-d. MEF2 proteins belong to the MADS-box DNA binding protein superfamily involved in many developmental pathways including myogenesis and the survival of cardiomyocytes. MEF2 proteins interact with myogenic regulatory factors and modulate the expression of muscle-specific genes. Thus, unbiased characterization of the MEF2A interactome would expand our understanding of MEF2 functions. A GFP-nanotrap purification followed by LC-MS/MS proteomic analysis was employed to identify MEF2A interacting proteins. Following gene ontology analysis, we focused on an interaction between MEF2A and its novel interacting protein, FOXP1, in striated muscle. FOXP1 was found in the MEF2A-immunocomplex in muscle cells, and ectopic expression of FOXP1 delays myogenic differentiation. FOXP1 inhibited MEF2A activation on myogenic reporter genes driven by the regulatory regions of creatine kinase muscle and myogenin genes. Additionally, siRNA-mediated deletion of FOXP1 in myoblasts and cardiomyocytes enhances MEF2A transactivation properties. Lastly, various biochemical experiments indicate that FOXP1 antagonizes p38 MAPK activation of MEF2A through Threonine-312 phosphorylation. Collectively, we documented a novel repressive interaction of FOXP1 and MEF2A in proliferating striated muscle cells.
  • ItemOpen Access
    Intramolecular Inhibition of the Ptb Domain in the Aida1 Neuronal Scaffolding Protein
    (2023-12-08) Chakkal, Tanvir Kaur; Donaldso, Logan
    Scaffolding proteins serve key functions in signalling cascades through mediating protein-protein interactions. A prominent member of the neuronal post-synaptic density is AIDA1, which is known to bind amyloid precursor protein (APP), a contributor to Alzheimer’s Disease (AD) development. Among the isoforms of AIDA1, AIDA1b is the largest and is unique in that its phosphotyrosine binding (PTB) domain is autoinhibited; thus, it cannot bind APP until this inhibition is relieved. This thesis presents structural and functional studies on AIDA1b. Fluorescence anisotropy and biolayer interferometry assays confirmed that the deletion of Exon 14 relieves the autoinhibition of the PTB domain, allowing AIDA1b to bind APP. However, Exon 14’s removal does not significantly impact the thermostability of AIDA1b. Moreover, Exon 14 contributes an alpha helical structural, with 3D bead models exhibiting potential domain reorganization upon Exon 14’s removal. The predicted structure of Exon 14 is an amphipathic alpha helix that presumably contacts the PTB domain in cooperation with other intervening sequences. Overall, AIDA1b can play a potential role in AD pathogenesis, with potential for contribution to novel therapeutic development.
  • ItemOpen Access
    Investigating the role of mitoNEET in iron overload-induced insulin resistance
    (2023-12-08) Tam, Eddie; Sweeney, Gary
    Excess iron, in a process termed iron overload (IO) is closely linked to cardiovascular and metabolic diseases. Previous research has already established a causal link between IO and insulin resistance in both cardiac and skeletal muscle setting. Building upon this knowledge, the potential for mitoNEET to offer protection against IO-induced insulin resistance was investigated. The potential mechanisms underlying the protective effects of mitoNEET, which included mitochondrial dynamics, oxidative stress, and mitophagy, was also examined in H9c2 cardiac and L6 skeletal muscle cells. Using various experimental approaches including quantitative polymerase chain reaction (qPCR), western blot, fluorescent microscopy, and reporter cell lines, mitoNEET was shown to be protective against IO-induced insulin resistance. In H9c2 cells, mitoNEET provided protection by regulating mitochondrial iron and reactive oxygen species (ROS) to prevent insulin resistance. In L6 cells, mitoNEET prevented insulin resistance via regulation of mitochondrial iron, ROS, and mitochondrial fission.
  • ItemOpen Access
    Measuring neotropical bat diversity using airborne eDNA
    (2023-12-08) Garrett, Nina; Clare, Elizabeth
    This thesis develops the novel technique of capturing airborne eDNA for the detection of tropical bat species. In chapter two, I use an artificial mixed species community to test three prototype samplers and validate airborne eDNA as a survey method. I demonstrate that airborne eDNA can accurately characterize a mixed species community with varying abundances and that the type of sampler does not impact DNA concentration or read count. In chapter three, I used airborne eDNA to survey 12 known or suspected bat roosts in Orange Walk District, Belize. I identified 23 taxa, 11 of which were bats. This thesis adds to the body of research that seeks to better understand airborne eDNA and its potential applications. Based on the data presented as well as those from other studies, airborne eDNA could be a valuable tool in the monitoring of biodiversity.
  • ItemOpen Access
    Mug Shots: Systematic Biases in the Perception of Facial Orientation within Pictorial Spaces
    (2023-12-08) Esser, Maxwell Jacob Rosenstein; Troje, Nikolaus
    Pictures are 2-D projections of a 3-D world, so pictorial spaces behave differently than the 3-D visual spaces we inhabit. For instance, the angular orientation of a face pictured in half-profile view is systematically overestimated by the human observer – a 35° view is estimated to be approximately 45°. What is the cause for this perceptual orientation bias? We tested three different hypotheses. (1) The phenomenon is specific to pictorial projections due to the twofoldness of the medium and does not occur in 3-D space. (2) It can be explained with the depth compression expected when the vantage point of the observer is closer to the picture than the point of projection. (3) The visual system uses a shape prior that does not match the elliptical horizontal cross section of a typical head. Our results support the third hypothesis, and this effect can be mitigated through adding geometric information through structure-from-motion.
  • ItemOpen Access
    Identifying Novel Interaction Between CDK8 Kinase Module and The Deubiquitinating Enzyme Ubiquitin Specific Protease 7 (USP7)
    (2023-12-08) Banyameen, Krstina Akram; Saridakis, Vivian
    Ubiquitin Specific Protease 7 (USP7) is a deubiquitinating enzyme that regulates and interacts with various cellular substrates, playing regulatory roles in various pathways such as DNA damage repair and epigenetics. This thesis explores the role of USP7 in regulating components of gene expression regulators. The gene expression of target genes is fine-tuned by the transcriptional co-activator, the Mediator complex. Our group has determined that Cyclin Dependent Kinase 8 (CDK8) and Mediator Complex Subunit 12 (MED12), components of the kinase module in the Mediator complex, contain consensus sequences for binding to the N-terminus of USP7. Using various co-immunoprecipitation, pulldowns, tissue culture, and western blotting related techniques, our findings show that USP7, using the DWGF motif at its N-terminus, interacts with and stabilizes protein levels of MED12 and CDK8. This provides insights onto the cellular role of USP7 in gene expression, giving such novel interactions therapeutic potential for diseases such as cancer.
  • ItemOpen Access
    Maternal Exposure to Prostaglandin E2 Affects Hippocampal Synaptic Plasticity in Mice Offspring -A Link to Autism Spectrum Disorder
    (2023-08-04) Abdul Rahiman, Aisha; Crawford, Dorota Anna
    Prostaglandin E2 (PGE2) is a lipid signaling molecule involved in early healthy brain development. Exposure to environmental risk factors such as air pollutants, infections, and drugs such as acetaminophen during early pregnancy have shown to impact PGE2 levels and have all been linked to Autism Spectrum Disorders (ASDs). Our previous studies show that maternal exposure to PGE2 and the lack of the PGE2 producing enzyme Cyclooxygenase-2 (COX2) results in sex-specific abnormal dendritic morphology within the cerebellum and the hippocampus as well as ASD-like behaviors including motor deficits and anxiety in mice offspring. In this study, I investigated sex-dependent effects of prenatal PGE2 exposure on hippocampal electrophysiology in the C57bl/6 mice offspring at postnatal day 90-100. I measured Schaffer collateral long-term potentiation (LTP), paired-pulse facilitation (PPF), input/output (I/O) responses, the expression of glutamate receptor components NMDAR subunit 2A, AMPA subunit GluR1, beta-actin, and morphological characteristics such as primary dendrite length and cell soma size of pyramidal neurons in the hippocampus. I found that PGE2 exposure decreased LTP in males and I/O responses in females at higher stimulation intensities with no effect on PPF. PGE2 also increased the expression of NMDAR2A in males with no effect on GluR1 or β-actin. However, PGE2 did not affect pyramidal cell morphology. Overall, our data suggests that prenatal PGE2 exposure disrupts innate sex differences by reducing LTP maintenance in males, while impairing basal synaptic strength in females. I propose a model that PGE2-dependent upregulation of NMDAR2A observed in male offspring may reflect a neurotoxicity effect of PGE2 mediated by glutamate, subsequently leading to neuronal death, which could explain the corresponding decrease in LTP. In summary, this study adds further evidence that abnormal maternal PGE2 levels known to be influenced by many environmental risk factors may affect hippocampal function and contribute to specific deficits in ASDs in a sex-dependent manner.
  • ItemOpen Access
    Regulation of Viral Subgenomic mRNA-Mediated Gene Expression in Three RNA Plant Viruses
    (2023-08-04) Chkuaseli, Tamari; White, K. Andrew
    RNA virus genomes encode different viral proteins that are essential for establishing infections in their hosts. Expression of a subset of these proteins occurs from viral genome- derived subgenomic (sg) mRNAs that are transcribed during infections. The regulation of sg mRNA transcription and translation ensures that requisite amounts of each of these viral proteins are produced at specific stages of the infectious cycle. Higher-order RNA structures present within viral genomes and sg mRNAs are commonly used as molecular switches to achieve the necessary control. The goal of my dissertation was to investigate the structure and function of RNA elements involved in regulating sg mRNA-mediated gene expression in three plus-strand RNA plant viruses: carnation Italian ringspot virus (CIRV), pea enation mosaic virus 2 (PEMV2), and PEMV1. Structural and functional analyses, both in vitro and in cell infections, allowed for the delineation of distinct regulatory RNA elements in each virus. The RNA structures involved in activating sg mRNA transcription were investigated in CIRV and PEMV2. Activation of sg mRNA1 transcription in CIRV requires the formation of a large, complex, intragenomic higher-order RNA structure that assembles via a multistep folding pathway involving six long-distance RNA-RNA base-pairing interactions. In contrast, PEMV2 sg mRNA transcription involves a small RNA stem-loop that contains a self-complementary palindromic loop sequence. Transcription is activated by viral genome dimerization via an intergenomic kissing-loop interaction involving pairing of the palindromic sequences. In PEMV1, the RNA structure required to promote translation readthrough of a C-terminally extended capsid protein from its sg mRNA was investigated. A complex, non-contiguous RNA structure assembled by sequential formation of three long-distance RNA-RNA interactions was found to be required for this recoding event. Collectively, these results have uncovered several distinct regulatory RNA structures involved in controlling different aspects of sg mRNA-mediated viral gene expression and provide novel insights into RNA-based regulation in plus-strand RNA viruses.
  • ItemOpen Access
    An Investigation into Neuroendocrine Regulators of Excretory Organs in the Adult Disease-Vector Mosquito, Aedes aegypti
    (2023-08-04) Sajadi, Farwa; Paluzzi, Jean-Paul
    Maintenance of ionic and osmotic homeostasis in insects allows them to succeed in many ecological and environmental niches, while utilizing a variety of feeding strategies. When faced with extreme and variable conditions, most insects regulate the composition of their blood within narrow limits. Haematophagus insects, such as the female yellow fever mosquito, Aedes aegypti, ingest bloodmeals comparable to twice their body volume, resulting in considerable amounts of salts and water in excess, threatening the osmotic and ionic balance of their haemolymph. Like other insects, mosquitoes achieve strict regulation of their hydromineral balance through the neuroendocrine control of their excretory system, consisting of the Malpighian ‘renal’ tubules (MTs), which are responsible for formation of the primary urine, along with the hindgut, which functions as a primarily reabsorptive organ. While extensive studies have examined this process of hydromineral balance in A. aegypti focusing on diuretic regulation, the roles of anti-diuretic hormones remained largely elusive. This research sought to advance our understanding of the hormonal regulation of the excretory system in A. aegypti by (1) investigating the role of CAPA neuropeptides as anti-diuretic hormones in adult MTs; (2) identifying the signalling components leading to CAPA-induced inhibition of fluid secretion; and (3) elucidating the expression and putative functional roles of ITP and ITP-L neuropeptides. In adult MTs, AedaeCAPA-1 peptides elicit a selective anti-diuretic role, inhibiting DH31- and 5HT-stimulated secretion through the NOS/cGMP/PKG pathway. CAPA-mediated inhibition promotes V-type H+-ATPase (VA) disassembly, reducing the driving force of DH31-stimulated secretion. Post-blood feeding, DH31 peptides are immediately released into the female haemolymph to promote natriuresis and diuresis while CAPA peptides are released shortly after, hindering the effects of DH31. Lastly, examination of ITP and ITP-L neuropeptides in A. aegypti indicated enrichment of ITP in the brain and ITP-L in the abdominal ganglia. Novel observations of AedaeITP and AedaeITP-L in feeding/starvation, ionoregulation, and reproductive behaviour and success suggest a vital pleiotropic role for these neuropeptides. Together, these studies highlight the complexity of neuroendocrine control of excretory organs in adult A. aegypti mosquitoes, furthering our understanding of various diuretic and anti-diuretic signalling systems in this important human disease vector.
  • ItemOpen Access
    RNA methyltransferases Influence Noncoding RNA Biogenesis and Function Through Catalytic-Independent Activities
    (2023-08-04) Porat, Jennifer Gali; Bayfield, Mark
    Continued advances in the high throughput detection of post-transcriptional RNA modifications have enabled large scale, mechanistic studies into the importance of RNA modifications in regulating the structure, function, and stability of coding and noncoding RNAs. While modifications themselves have a major role in influencing the fate of an RNA, recent evidence from bacteria suggests that RNA modification enzymes can possess non-catalytic functions that nevertheless contribute to RNA functionality. This dissertation aims to expand this idea to eukaryotic RNA modification enzymes, with a focus on uncovering the catalytic-independent functions of the fission yeast RNA methyltransferases Bmc1 and Trm1. Bmc1 is a homolog of the human methyl phosphate capping enzyme (MePCE), which has been well studied for its role in catalyzing the addition of a 5 -monomethyl phosphate cap on select RNA Polymerase III transcripts such as the 7SK snRNA. In answer to the long-standing question as to the function of an MePCE homolog in fission yeast, an organism with no known 7SK, this work revealed that Bmc1 assumes a non-catalytic role in the fission yeast telomerase enzyme by promoting holoenzyme assembly and telomerase RNA stability. Further analysis demonstrated that Bmc1 also interacts with the U6 snRNA to direct 2-O-methylation and influence formation of a U6-containing snRNP, and that neither of these activities requires Bmc1 catalytic activity. Finally, this work shows that the fission yeast tRNA methyltransferase Trm1 promotes tRNA functionality and structural stability even in the absence of catalysis, suggesting its function as a tRNA chaperone. Collectively, these studies provide evidence supporting the multi-faceted nature of eukaryotic RNA modification enzymes and underscores their importance in many fundamental biological processes including splicing, protein translation, and maintaining genome integrity.