MSc Exit Seminar -Paige O’Leary
Induction of Heat Shock Proteins in Differentiated Human SH-SY5Y Neuronal Cells by Caffeine and Cafestol
Abstract:
During aging, protein quality control fails, leading to the accumulation of misfolded, aggregation-prone proteins which are characteristic of neurodegenerative diseases for which few effective therapies exist. Enhancement of protein quality control mechanisms is a potential treatment strategy, specifically upregulation of heat shock proteins (Hsps) that function to counteract misfolded proteins. Non-toxic inducers are required that can be ingested regularly and cross the blood-brain barrier to trigger Hsp induction in neurons that are affected in neurodegenerative diseases. Caffeine is a non-toxic compound taken daily that crosses the blood-brain barrier. The effects of two coffee components namely, caffeine and cafestol, are examined on the induction and intracellular localization of Hsps in differentiated human neuronal cells grown in tissue culture. Cafestol, but not caffeine, induced HSPA1A (Hsp70-1) and the little studied HSPA6 (Hsp70B’) which localized to the nucleolus where these Hsps could protect ribosome biogenesis, a critical feature of neuronal protein synthesis.
MSc Exit Seminar- Alicia Harracksingh (Senatore lab)
Exploring Protein Interactions between CaV2 Calcium Channels and Pre-synaptic Scaffolding Proteins MINT and RIM in Invertebrates
During synaptic transmission, voltage-gated calcium type-2 (CaV2) channels mediate transient influxes of Ca2+ into the cytoplasm. This event activates synaptic vesicle (SV) calcium sensors, which facilitates docking, priming and fusion at the plasma membrane, and the subsequent release of SV contents into the synaptic cleft. A direct interaction has been proposed to tether CaV2 channels and SVs in nanometer proximity, primarily mediated by scaffolding proteins that bind to the distal C-terminus of CaV2 at a conserved PDZ-ligand domain. Here, we explored interactions between CaV2 and the scaffolding proteins RIM and MINT in vitro in the arthropod Drosophila melanogaster and the cnidarian Nematostella vectensis to understand the evolution of the synapse through conservation of CaV2 channel protein structure- function. We found that interactions between these proteins are conserved and occur at similar domains and ligand domains, which suggest that the CaV2-MINT and CaV2-RIM interactions serve as important evolutionary adaptations for the emergence of synapses.
MSc Exit Seminar- Yan Ling Iris Chiu (Chang Lab)
Functional characterization and molecular evolutionary analyses of dim-light adaptations in visual pigments and interactions with arrestin
Abstract:
The visual system of dim-light dwelling/nocturnal species are known be to highly adaptive due to the importance of vision for survival. Rhodopsin and cone opsins are responsible for dim-light and colour vision, respectively, and mediate photon absorption which results in isomerization of the retinal chromophore and activation of the visual pigment. This triggers the downstream visual transduction cascade which is subsequently inhibited by the binding of the signaling protein arrestin. Here, I investigated different aspects of dim-light adaptation using a combination of computational and experimental approaches. The first study investigated arrestin-rhodopsin complex formation relevant to a hypothesized photoprotection mechanism in dim-light vertebrates. Putatively adaptive mutations from dim-light animals identified in comparative analyses were found to enhance the ability of arrestin-rhodopsin complexes to sequester toxic retinal chromophore when experimentally assayed in vitro. The second study analyzed the selective pressures across the visual pigment gene RH2 of teleost fishes and found a positively selected site with unusual amino acid substitutions in the deep-sea northern lampfish. When assayed experimentally, substitutions at this site showed altered kinetics, with implications for better photosensitivity and cold adaptation. Overall, my findings provide insight into the less-well studied aspects of dim-light adaptation and show the significance of investigating the visual system as an interdisciplinary study involving evolution, biochemistry and molecular biology.
MSc Exit Seminar-Thomas Dodsworth
Characterization of the Teneurin C-terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line
Teneurin C-terminal associated peptide (TCAP) is an ancient and conserved bioactive peptide that is evolutionarily related to corticotropin releasing factor (CRF). Recently, synthetic TCAP-1 was shown to increase cellular energy availability and alter contractile performance in rodent skeletal muscle. However, the exact receptor signalling mechanism through which this occurs is unknown. Based on evidence of their interaction in vitro, I hypothesized that TCAP-1 signals through latrophilins—a family of adhesion G-protein coupled receptors—to elicit its effects in muscle. To test this, I knocked-down and knocked-out latrophilins in the immortalized mouse myoblast C2C12 cell line by small interfering RNA (siRNA) and CRISPR/Cas9 gene editing methods, respectively, and examined the efficacy of TCAP-1 in knockdown and knockout cells. I determined that latrophilin-1 is necessary for TCAP-1-mediated increases in intracellular calcium, NADH turnover, and PGC-1a expression. This establishes, for the first time, that the TCAP-latrophilin ligand-receptor pair has a functional role in skeletal muscle.
MSc Exit Seminar- Paige O’Leary (Brown Lab)
Induction of Heat Shock Proteins in Differentiated Human SH-SY5Y Neuronal Cells by Caffeine and Cafestol
During aging, protein quality control fails, leading to the accumulation of misfolded, aggregation-prone proteins which are characteristic of neurodegenerative diseases for which few effective therapies exist. Enhancement of protein quality control mechanisms is a potential treatment strategy, specifically upregulation of heat shock proteins (Hsps) that function to counteract misfolded proteins. Non-toxic inducers are required that can be ingested regularly and cross the blood-brain barrier to trigger Hsp induction in neurons that are affected in neurodegenerative diseases. Caffeine is a non-toxic compound taken daily that crosses the blood-brain barrier. The effects of two coffee components namely, caffeine and cafestol, are examined on the induction and intracellular localization of Hsps in differentiated human neuronal cells grown in tissue culture. Cafestol, but not caffeine, induced HSPA1A (Hsp70-1) and the little studied HSPA6 (Hsp70B’) which localized to the nucleolus where these Hsps could protect ribosome biogenesis, a critical feature of neuronal protein synthesis.
Neuropeptide Mimetics: The Physiological Effects of Kinin and CAPA Analogs in Rhodnius prolixus
Neuropeptide Mimetics: The Physiological Effects of Kinin and CAPA Analogs in Rhodnius prolixus
In the Chagas disease vector Rhodnius prolixus, the kinin and CAPA neuropeptides modulate a host of feeding and diuresis-related behaviours that are implicated in disease transmission. CAPA and kinin neuropeptide analogs have been developed to elicit potent changes in physiology, to be later incorporated in novel pest-control strategies. Here, the effects of kinin and CAPA analogs were investigated on feeding and diuresis-related tissues, with the kinin and CAPA analogs inducing physiological changes in vivo and in vitro. Within the hindgut, novel intracellular interactions were uncovered between RhoprCAPA, Rhopr-kinin, and serotonin [5- hydroxytryptamine (5-HT)]. Following identification and sequencing of the Rhopr-kinin receptor, the receptor transcript was observed throughout the gut, with RNA interference (RNAi)-mediated knockdown of the receptor causing reductions in hindgut contractions and increasing the size of blood meal consumed. Overall, these findings highlight the role of kinin and CAPA within R. prolixus, and the promise of their neuropeptide analogs to be used as lead compounds in pest- control strategies.
MSc Exit Seminar- Gabriele Nandal
MSc Exit Seminar
Friday, July 19th, 2019 at 10:10am - Ramsay Wright Building, Room 432
Gabriele Nandal (Buck Lab)
Characterization of Sirtuin 3 and Targets in the Western Painted Turtle
The western painted turtle (Chrysemys picta) is a champion anaerobe capable of surviving months with little to no oxygen. This species’ anoxia tolerance is enabled by many physiological and biochemical adaptations; metabolic depression is perhaps one of the most useful mechanisms that enables the turtle’s survival. In response to anoxia the turtle can suppress its metabolic rate by 90%, this tolerance is partly achieved through downregulation of both protein synthesis and activity in order to minimize ATP demand. Cellular functioning can then be modulated through an ATP-inexpensive, reversible, and rapid post-translational protein modifications. Cellular metabolism is highly regulated by reversible-acetylation of the mitochondrial proteome, however it is relatively unexamined in the anoxia-tolerant turtle. In the mitochondria, Sirtuin 3 (SIRT3) is the global deacetylase, and is involved in many cellular processes such as metabolic regulation and stress resistance. This thesis shows that while the turtle exhibited increases mitochondrial acetylation during early anoxia in the brain and liver, concomitantly SIRT3 protein levels were also elevated. In addition, Cyclophilin D levels, a direct target of SIRT3, were also shown to be elevated during anoxia. Another target of SIRT3, p65 subunit of NF-kB exhibited increased deacetylation during anoxia and reoxygenation in the brain. While no difference in prevalence or activity of manganese superoxide dismutase, an antioxidant target of SIRT3, was observed either during anoxia or reoxygenation. Overall, this thesis provides the first evidence that SIRT3 and mitochondrial proteome acetylation may play a role in the regulation of anoxia in the painted turtle.
MSc Exit Seminar - Chun Hua Wei- Tuesday, August 7, 2018
MSc Exit Seminar
Tuesday, August 7, 2018 at 1:10 pm SW -403, University of Toronto at Scarborough
Chun Hua Wei (Hasenkampf Lab)
The role of HOP2 in Homologous Recombination in Arabidopsis thaliana
Abstract:
The purpose of this study was to investigate the role of HOP2 protein in non-meiotic cells in Arabidopsis. HOP2 is already known to be important to meiotic chromosome pairing and homologous recombination, yet the role of HOP2 outside of meiosis is far from being fully elucidated. My study focused on the mitotic chromosome events with and without the application of radiation. In the absence of radiation, no fragments and chromatin bridges were found in hop2-1 plants, but they did seem to experience a modest chromosome separation delay. When irradiated, both genotypes had significant decreases of mitotic indices and increases of bridges. The decreases in mitotic indices were comparable for the two genotypes, suggesting they accomplish repair at similar rates. Irradiated hop2-1 had significantly more mis-repaired breaks, as determined by the bridges. My findings suggest that HOP2 is also important for the fidelity of the exchange process in non-meiotic HR repair.
MSc Exit Seminar - Peilu Gan -Tuesday, May 29, 2018
MSc Exit Seminar
Tuesday, May 29, 2018 at 10:10pm, SW 403- University of Toronto at Scarborough
Peilu Gan (Hasenkampf Lab)
The Role of the Arabidopsis Hop2 Protein in Promoting Homologous Chromosome Interactions and Blocking Nonhomologous Interactions
Abstract:
The Homologous Pairing Protein 2 (Hop2) is important for its role in reciprocal genetic exchange in meiosis. It is thought to operate as a part of the double-strand break (DSB) repair pathway. Recent models give two potential roles for Hop2: it acts to promote interactions between homologous chromosomes, or it acts to block interactions between non-homologous chromosomes. The goal of my study was to see if the Hop2 protein acted to block non-homologous interactions by analyzing its role in haploid plants. Haploid hop2-1 mutants were analyzed by light and fluorescent microscopy and compared with haploid WT plants. Like WT haploids, hop2-1 mutants showed univalents in early meiosis. However, unlike WT haploid plants, hop2-1 haploid mutants showed large amounts of DNA fragmentation and chromosomal bridging in anaphase and metaphase for both Meiosis I and Meiosis II. This suggests that Hop2 acts to block non-homologous interactions.
MSc Exit Seminar - Delara Dadsepah -Tuesday, May 29, 2018
MSc Exit Seminar
Tuesday, May 29, 2018 at 2:10pm CCT -3000, University of Toronto at Mississauga
Delara Dadsepah (Levine Lab)
Anatomical and Behavioural Characterization of Dpr-Interacting Protein Beta in Drosophila melanogaster
Abstract:
The mammalian limbic system has many important biological functions. During development, the limbic-system associated membrane protein (LSAMP) plays a crucial role by ensuring proper neuronal connectivity within the system. Similarly, the LSAMP homologue in the Drosophila, the Dpr-interacting protein beta (DIP-β), is believed to assist in neuronal formation during the development of the fly central nervous system. Other data suggests that DIP-β even regulates social interactions. Researchers have only more recently begun investigating DIP-β however, and DIP-β remains to be extensively studied. Thus, the aim of this project was to fully characterize DIP-β expression in the brain and the behaviour of DIP-β mutants, to obtain a better understanding of DIP-β function. DIP-β’s predominant expression in the optic lobes and regions in the central brain, along with changes in behavioural rhythmicity observed in DIP-β mutants, suggests DIP-β may be associated with clock mechanisms.