PhD Exit Seminar
Tuesday, August 7, 2018 at 12:10pm, DV 3130 – University of Toronto at Mississauga
Samantha Mahabir (Gerlai Lab)
“The effect of embryonic alcohol exposure on brain function and behavior in zebrafish strains”
The biological mechanisms that underlie fetal alcohol spectrum disorder (FASD) are complex and poorly understood. This thesis aims to investigate potential underlying mechanisms of FASD by using zebrafish as a model organism. The research question asked is how does embryonic alcohol exposure alter brain function and behavior in different zebrafish strains? My first experiment explored the influence of environmental factors salinity and olfactory cues on zebrafish behavior. This was conducted to reduce experimental error variation and create more sensitive behavioral paradigms. My second experiment focused on characterizing the development of shoaling behavior and correlated neurochemicals in the absence of embryonic alcohol in order to establish baseline behavior. Next, I examined the effect of embryonic alcohol exposure on neurochemicals dopamine, serotonin and their metabolites and found embryonic alcohol exposure to disrupt the dopaminergic and serotonergic systems in the developing fish; as well I discovered these effects to be strain- dependent. I found that the specific development time point, concentration and short duration of alcohol exposure used in my experiments do not alter amino acid neurotransmitters glutamate, glycine, aspartate, taurine and GABA. Lastly, I have investigated apoptosis and have adapted the labeling TUNEL assay, for zebrafish. I found that mild alcohol exposure during development results in an increase in apoptosis and that these early responses result in long-lasting changes in neuronal markers and number of cells in specific brain areas. I included results on different zebrafish strains in some of my studies. Strain differences will facilitate the discovery of molecular mechanisms underlying changes in alcohol-related genes and will also allow researchers to choose the more appropriate strain for drug or mutation screening all of which will facilitate a better understanding of FASD.
PhD Exit Seminar
Tuesday, August 7, 2018 at 9:30am, Ramsay Wright Building, Room 432
Nihar Bhattacharya (Chang Lab)
“CHARACTERIZING VERTEBRATE RHODOPSIN NATURAL VARIATION IN EVOLUTION, FUNCTION, AND DISEASE”
Vertebrate dim light vision is mediated by the rod visual pigment, rhodopsin, a member of the G protein-coupled receptor (GPCR) superfamily of proteins. In the dark, rhodopsin is covalently bound to a vitamin A-derived 11-cis chromophore, which acts as an inverse agonist to stabilize the inactive state of rhodopsin. When exposed to light of a maximal wavelength (λmax), the 11-cis retinal chromophore isomerizes to an all-trans conformation, initiating a series of structural shifts to the light-activated state of rhodopsin. This results in a signalling cascade within the rod photoreceptor cell and, ultimately, the perception of light. The goal of this thesis is to investigate natural variation in rhodopsin function in the context of evolutionary adaptation, chromophore usage, and disease mutations. Following a general introduction, in Chapter II, I characterize the visual system of the diurnal colubrid snake Pituophis melanoleucus using immunohistochemistry of retinal sections and spectroscopy of purified visual pigments expressed in vitro, revealing an unusual rhodopsin with cone opsin properties found in cone-like rod photoreceptors. In Chapter III, I investigate the effects of the rare vertebrate chromophore, 11-cis 3,4 dehydroretinal (A2), on the spectral and non-spectral properties of rhodopsin. In Chapters IV and V, I study the effects of pathogenic mutations in rhodopsin that cause the retinal degenerative disease retinitis pigmentosa (RP). In Chapter IV of my thesis, I identify the phenotype of RP mutations found in the extracellular loop 2 of rhodopsin and assess the effects of functional rescue using two different approaches. Finally, in Chapter V, I characterize three novel RP mutations to investigate the relationship between the in vitro and clinical disease phenotypes. The investigations in this thesis expand our understanding of snake retinal evolution, the role of the chromophore in rhodopsin function, and the effect of pathogenic mutations on rhodopsin structure and function. This thesis combines data from non-model organisms, non-mammalian chromophores, and non-wildtype pathogenic mutations to significantly increase our understanding of the scope of rhodopsin functionality.
PhD Exit Seminar
Monday, June 4, 2018 at 10:10am, Ramsay Wright Building, Room 432
Hiwote Belay (Sokolowski Lab)
“GENETIC VARIATION IN THE timeless GENE MEDIATES METABOLIC STATES OF Drosophila melanogaster IN RESPONSE TO PHOTOPERIOD”
Genetic variations in the circadian clock may regulate photoperiod-induced anticipatory metabolic adjustments that allow organisms to meet the changes in energetic demands associated with different seasons. Both mammalian and Drosophila studies have shown that perturbed circadian feeding rhythm and abberant light cycles result in disruptions in fat and glucose metabolism. In this thesis, Drosophila melanogaster was used to investigate the effect of genetic variation in the circadian system on the regulation of feeding and metabolic responses to photoperiod.
Here, we analyzed the metabolic responses of two naturally occurring variants of the Drosophila timeless (tim) gene to changes in photoperiod. We found that ls-tim variants, which are known to have attenuated light-sensitivity and are more responsive to diapause, display metabolic traits that are associated with enhanced energy stores and reduced energy expenditure in response to a short-day. Analysis of tim RNA levels in the fat body revealed that it is elevated in ls-tim in response to a short-day suggesting that altered regulation of the clock in the fat body of ls-tim may mediate these enhanced metabolic adjustments to short-day. To examine the role of the foraging gene as a mediator of metabolic outputs regulated by the clock, we analyzed the circadian feeding pattern of foraging variants. Genetic variation in the foraging gene, which encodes cGMP dependant protein kinase (PKG), is known to regulate feeding behavior and energy homeostasis in Drosophila. Our results suggest that foraging regulates the frequency and daily distribution of meals.
These findings demonstrate that genetic variations in the circadian system are important in mediating photoperiodic responses to feeding and metabolic state. Characterization of a role of genetic variations in clock genes on the regulation of feeding and metabolism by abberant light cycles is important in identifying candidate pathways involved in metabolic perturbations associated with shift-work and Seasonal Affective Disorder.