PhD Exit Seminar -Nihar Bhattacharya-Tuesday, August 7, 2018
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”
Abstract
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.
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.
PhD Exit Seminar - Samantha Mahabir -Tuesday, August 7, 2018
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”
Abstract
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.
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.
PhD Proposal Exam-Colleen Gillon -Tuesday, June 12, 2018
PhD Proposal Exam
Tuesday, June 12, 2018 at 10:10am, MW 229- University of Toronto at Scarborough
Colleen Gillon (Richards Lab)
“How does the brain learn about the statistical structure of the environment?”
Abstract
Over the past decade, artificial intelligence has progressed at great speed, with impressive breakthroughs in fields like computer vision and speech processing using neural network algorithms. These can be broadly divided into two classes: (1) discriminative models, like feedforward, convolutional and recurrent neural nets, which learn to map inputs, like images, to specific outputs, like categories or classes and (2) generative models, like bidirectional Helmholtz machines, generative-adversarial networks and expectation maximization models, which are learn the underlying joint structure of the data. Studies of visual processing in the cortex strongly suggest that in learning to process environmental stimuli, our brains behave like generative models, developing internal models of the joint distribution over sensory stimuli in the environment. Thus, these algorithms could shed light on our brain’s remarkable ability to represent and process sensory information efficiently and accurately. We propose to investigate this by comparing how the brain and different algorithms trained on visual tasks process and adapt to major changes in the relationship between incoming visual stimuli and somatosensory or motor inputs. Specifically, we will record and analyse changes in the activity of layer 2/3 pyramidal neurons in primary visual cortex (V1) in response to a shift in the relationship between visual stimuli and sensory stimuli or motor commands. We predict that this shift will transiently increase activity in the apical dendrites, and alter the rate of apical trunk calcium spikes of these neurons while the system adapts. In parallel, we will train different generative algorithms on this same task, and analyze changes in network activity in order to identify those algorithms that show the greatest potential for explaining how our brains process sensory information.
PhD Exit Seminar -Hiwote Belay -Monday, June 4, 2018
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”
Abstract
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.
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.
PhD Proposal Exam - Klotilda Karkaj - Tuesday, May 22nd, 2018
PhD Proposal Exam
Tuesday, May 22nd, 2018 at 1:10 pm – CCT-4034, University of Toronto at Mississauga
Klotilda Narkaj (Zovkic lab)
“Histone Variant MacroH2A in Memory Formation”
Abstract
Epigenetic modifications are widely recognized for their role in memory formation. Although existing research has focused almost exclusively on DNA methylation and histone post-translational modifications (PTMs), we recently discovered that histone variant exchange, in which canonical histones are replaced by distinct variants, is a novel branch of epigenetics for regulating memory. Our initial work showed that binding of the histone variant H2A.Z is modified by learning, suggesting that the composition of histones that make up nucleosomes is subject to learning- and memory-related modification. Though H2A variants can replace one another in chromatin, which histones replace one another and how distinct variants influence memory is largely unknown. H2A.Z is one of several functionally diverse H2A variants that functions as a memory suppressor. For my thesis I will investigate another potential candidate for memory regulation, histone variant macroH2A (mH2A), its relationship with H2A.Z, and their interaction in memory formation. MacroH2A has a widely reported role in regulating gene expression, it is encoded by 2 genes, H2afy (encodes mH2A1) and H2afy2 (encodes mH2A2), both of which are expressed throughout the mouse brain, including the hippocampus, a brain region that is vital for memory formation. To explore the role of mH2A in memory, we use adeno-associated virus (AAV) to knock down either H2afy or H2afy2 in area CA1 and tested mice on an array of hippocampus-dependent memory tasks at the 24-hour and 7-day time points. We found that mice with depleted levels of both mH2A1 and mH2A2 had impaired fear memory 24 hours and 7 days after training, suggesting that both mH2A-encoding genes promote hippocampus-dependent memory formation. To identify the mechanism by which mH2A regulates memory, area CA1 was extracted 30 min after fear conditioning, exposed to mH2A chromatin-immunoprecipitation combined with next-generation sequencing, and compared to genome-wide gene-expression 1h after training, based on time points at which our lab previously found an association between H2A.Z dynamics and gene expression. To elucidate the relationship between H2A.Z and mH2A in memory, I will investigate binding of mH2A in chromatin in response to H2A.Z depletion, after learning. These data will explore involvement of histone variant exchange as a novel epigenetic regulator of behaviour and they are the first to show mH2A as a regulator of memory.
PhD Transfer Exam reminder - Kevin Xue -Tuesday, May 22, 2018
PhD Transfer Exam
Tuesday, May 22, 2018 at 1:10 pm – Earth Science Building, Room 3087
Kevin Xue (D. Christendat lab)
“Elucidating the Protocatechuate Biosynthetic Pathway in Listeria monocytogenes and its Role in Microbial Interactions”
Abstract
Listeria monocytogenes is a ubiquitous bacterial saprophyte capable of causing fatal listeriosis in mammalian hosts. L. monocytogenes boasts a high tolerance to sanitation measures and persists in food processing environments. Although much investigation of its pathogen lifestyle has been conducted, its role as a saprophyte remains poorly understood. Our lab has identified and partially characterized two operons containing genes for the biosynthesis of protocatechuate, a compound derived from plant material degradation and industrial waste (Prezioso et al. 2018 and Bonfa et al. 2013). Though other microorganisms will produce and utilize protocatechuate to generate energy or produce protocatechuate type siderophores, L. monocytogenes lacks these pathways. L. monocytogenes produces protocatechuate when the LysR type transcriptional regulator (LTTR), QuiR, induces qui1 and qui2 with its coinducer and ligand, shikimate. Shikimate supplementation leads to accelerated growth of Listeria innocua followed by rapid cell density loss. I hypothesize that L. monocytogenes generates protocatechuate, which it will exchange mutualistically with other microorganisms. Alternatively, L. monocytogenes is utilizing protocatechuate to generate a novel metabolite. The former hypothesis is supported by the loss of cell density when protocatechuate accumulates in shikimate supplied monoculture. I plan to characterize the qui1 and qui2 operons by gene deletion to study their roles in protocatechuate biosynthesis. I will also study the effect of protocatechuate on Listeria growth. Furthermore, I plan to investigate the role of protocatechuate in microbial interactions by performing co-cultured growth analysis between Listeria and other bacterial species.
MSc Exit Seminar - Van Phan -Friday, May 11th, 2018
MSc Exit Seminar
Friday, May 11th, 2018 at 10:10am Earth Sciences Building, Room 3087
Van Phan (Yoshioka Lab)
Abstract:
Evaluation of GCaMP3 Protoplasts for the Analysis of Ca2+ Signaling in Plant Immunity
Cytoplasmic calcium [Ca2+]cyt elevation is an early event that occurs after recognition of various environmental stimuli. Utilization of Ca2+visualization tools such as genetically-encoded Ca2+ indicators has advanced our knowledge of the temporal and spatial nature of Ca2+ signals. GCaMP3 is a green fluorescence protein (GFP)-based genetically-encoded indicator that can be detected by a conventional fluorescence microscope and a plate reader system. In this study, the use of GCaMP3 to study plant Ca2+ signals in protoplasts was evaluated.
Firstly, Arabidopsis thaliana leaf mesophyll protoplasts derived from stable transgenic lines expressing GCaMP3 were evaluated in comparison to leaf discs. Using fluorescence microscopy and a plate reader, Ca2+ signals upon abiotic and biotic stimuli in protoplasts were assessed in a quantitative and qualitative manner. Ca2+signals upon various stimuli were detected by both microscope and a plate reader. Ca2+ signals detected in protoplasts were generally quicker and stronger than those in leaf discs. However, the signal patterns were fundamentally similar between protoplasts and leaf discs. Thus, it was concluded that protoplasts expressing GCaMP3 can be utilized to study Ca2+ signaling.
Next, the usage of the transient expression of GCaMP3 in protoplasts (by transfection) was tested. This can be a powerful tool to analyze Ca2+signals in various mutants. Several methods to detect Ca2+signals from GCaMP3-transfected protoplasts were evaluated utilizing the bacterial elicitor, flg22. However, the detection by a plate reader was not successful, likely due to the insufficient number of the cells expression GCaMP3 by transfection, while some signals could be observed in individual protoplasts under the microscope.
Taken together, protoplasts can be used for investigating Ca2+signals upon various stimuli using GCaMP3. However, further optimization of the protocol for transfection is required.