PhD Exit Seminar - Abiramy Karunendiran (Stewart Lab)
Sarcomere Structure in Drosophila Musculature Studied with Polarimetric Second Harmonic Generation Microscopy
Thursday, September 10, 2020 at 1:10pm
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https://utoronto.zoom.us/j/99127382979
Meeting ID: 991 2738 2979
Abstract
Nonlinear optical microscopy is a powerful imaging modality that enables label free imaging with endogenous signal generated from the protein arrays that constitute biological structures and can be used to reveal structural and functional information in a system. Second Harmonic Generation (SHG) Microscopy is a parametric process that does not require photon absorption for signal generation, providing the added benefit of having a significantly reduced photo-bleaching effect. SHG signal is observed in non-centrosymmetric molecules like myosin and can be used to directly visualize muscle structure. Additionally, polarimetric SHG imaging allows for the quantification of the microstructure of the sample. Muscle function depends on highly ordered protein complexes. UNC45 is a USC chaperone necessary for myosin incorporation into thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental
times have not yet been studied. The focus of my thesis is to utilize both traditional and nonlinear optical microscopy to investigate changes in myosin filament organization and interactions between protein complexes and how it relates to muscle function. UNC45 was knocked down with RNAi in a way that permitted survival to the pupal stage. Immunofluorescence and electron microscopy imaging showed diminished expression of UNC45 and disorganized myosin filaments and Z-disk proteins. Associated changes in synaptic physiology and impaired locomotor behavior were also observed. These observations were confirmed and further analyzed on a whole larval scale using the newly developed polarimetric wide-field SHG microscopy, revealing changes in the striated pattern of somatic muscles and a reduced signal intensity. Polarimetric parameters were used to study chiral and achiral properties of larval muscles. Respectively R- and C-ratios were determined using scanning SHG microscopy. R-ratio values were higher in the mutant compared to control, suggesting that not only myosin, but also a second protein structure was producing a second harmonic response. CD image and C-ratio revealed the bimodal distribution of myosin filaments and changes in chiral structure in UNC45 knockdown larva. Overall, this technique provides insight on how second harmonic properties changes with myosin filament structure. This opens new perspective to study the dynamic properties of muscle structure during contraction.
PhD Exit Seminar Samantha Lauby (McGowan lab)
Interactions of Early-Life Temperature Exposure, Offspring Genotype and Maternal Care on Later-Life Maternal Care Provisioning in Female Rats
Monday, September 14th, 2020 at 11:10am
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https://utoronto.zoom.us/j/97489004431
Meeting ID: 974 8900 4431
Passcode: 081620
Abstract:
The early-life maternal environment has a profound effect on offspring behaviour, including the transmission of maternal care across generations. Variations in rat maternal care provisioning are associated with alterations in the oxytocinergic and dopaminergic systems in the maternal brain and, in turn, maternal care received by pups can alter oxytocinergic and dopaminergic systems in the rat pup brain. Though there has been progress in elucidating the biological mechanisms underlying the developmental programming of maternal care, the mechanisms that link maternal care received to alterations in the dopaminergic and oxytocinergic systems in the offspring remains to be an active area of research. Previous work has studied the transmission of pup licking across generations, but there are other relevant factors that could interact with pup licking on later-life maternal care provisioning. In this thesis, I review early-life temperature exposure and offspring genotype as important factors also involved in the developmental programming of maternal care. One project directly manipulated early-life temperature exposure and levels of supplemental licking-like tactile stimulation. The second project investigated the main effects of observed early-life inter-individual maternal licking received and interactions with naturally occurring genetic variants in dopamine-related genes. I hypothesized that early-life temperature exposure and offspring genotype would interact with licking-like tactile stimulation or pup licking to alter the oxytocinergic and dopaminergic systems in the offspring and also influence their later-life maternal care provisioning. I found that 1) early-life temperature exposure influenced the epigenetic regulation of the oxytocin gene in week-old female pups with changes in oxytocin transcript abundance and 2) that both early-life temperature exposure and supplemental tactile stimulation affected later-life maternal care provisioning. In addition, I found that a single nucleotide polymorphism in the dopamine receptor 2 gene interacted with inter-individual maternal licking received on 3) later-life performance on dopamine-related tasks and 4) maternal licking provisioning. Moreover, the association between maternal licking received and maternal licking provisioning was mediated by dopamine levels in the nucleus accumbens of the maternal brain. These findings suggest novel biological mechanisms of the developmental programming of maternal care that could be involved with the transmission of maternal care across generations.
PhD Exit Seminar- Aimee Michelle Caron
Physiological Consequences of Sleep Loss in the Rat
In this thesis, I examined the homeostatic and physiological consequences of acute total sleep deprivation (TSD) and chronic sleep restriction (CSR) in the rat. I first developed a rat model of CSR using a walking wheel to confine sleep opportunities to designated times. I used this model to investigate the response of the sleep homeostat to a sleep deficit accumulated at different rates. I then compared the effects of CSR to the well-established acute ‘sleep deprivation syndrome’. Further, I considered a role for TSD in the production of reversible neuron damage. Finally, I investigated the impact of TSD and CSR on the integrity of neurons using an adapted model of mild traumatic brain injury (TBI). My research program led to the following conclusions:
- CSR results in selective attenuation of the non-rapid eye movement (NREM) sleep homeostat, while leaving the rapid eye movement (REM) sleep homeostat in-tact.
- CSR induces weight loss, body temperature and metabolic alterations that are qualitatively similar but quantitatively diminished compared to those of TSD.
- TSD does not result in neuron damage in the rat sensorimotor cortex as measured by classical dark neurons (DNs).
- Developing a sleep deficit from TSD or CSR prior to mild TBI does not cause exacerbation of neuron damage as measured by classical DN production.
- There may be two under-represented demonstrations of neuron damage in the rat cortex: non-compacted DNs and light compacted neurons. When these neuron types are accounted for, a sleep deficit developed from TSD reduces the significant neuron damage produced by mild TBI. A sleep deficit developed by CSR does not afford the same neuroprotection as TSD.
Supervisor: Prof. John Peever
PhD Exit Seminar- Afif Aqrabawi
An Olfactory Memory Circuit
Episodic memory is defined simply as memory for what happened, when, and where. The hippocampus mediates episodic memory and represents contextual information using the parameters of space and time, including where an event unfolded and the sequential order of related events. Episodic recollections are characterized by rich multisensory details, yet the mechanisms underlying the reinstatement of these non-spatiotemporal aspects of experience are unknown. In this thesis, we identified direct, topographically organized hippocampal projections to a poorly understood ring-like structure known as the anterior olfactory nucleus (AON). We demonstrated that manipulation of the hippocampal-AON pathway can influence odour perception and odour-guided behaviours. Selective inhibition of hippocampal-AON projections impaired mice in their ability to recognize odours associated with the spatial and/or temporal aspects of their environment. We also revealed that AON activity is generated by coincident olfactory and contextual inputs arriving from the olfactory bulb and hippocampus, respectively. Thus, we hypothesized that the AON acts as the physical repository for populations of neurons representing previously encountered odours within the context in which they occurred. The precise pattern of activity produced by the AON therefore composes the olfactory memory trace, or “odour engram”. To this end, we used a tamoxifen-inducible Cre recombinase system to control the timing of gene expression in the AON. In combination with chemo- and optogenetic tools, we manipulated tagged AON neuronal populations in a carefully designed set of behavioural paradigms. We found that AON activity is necessary and sufficient for driving the behavioural expression of specific odour memories, thereby establishing the AON as the long-term storage site for contextually-based odour engrams. This thesis represents the first demonstration of the neural substrate of odour memory in vertebrates, satisfying all criteria used for defining an engram. The ease and suitability of using olfaction will undoubtedly position the hippocampal-AON pathway as an ideal circuit model for investigating fundamental mnemonic and cognitive principles. Indeed, this model can become particularly important in translational research that may yet lead to the development of therapeutic targets for disorders of memory, such as Alzheimer’s disease.
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.
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.
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.
PhD Exit Seminar- Mithunah Krishnamoorthy - Tuesday, January 16, 2018
PhD Exit Seminar
Tuesday, January 16, 2018 at 2:10 pm, SW 403 – University of Toronto at Scarborough
Mithunah Krishnamoorthy (Treanor Lab)
“The Role of Novel Ion Channel TRPM7 in B Cell Development and Function”
Abstract
The channel-kinase Transient Receptor Potential Subfamily M7 (TRPM7) is known to regulate magnesium homeostasis and was first channel implicated in the survival of a B cell line. Our study is the first to show that B cells require TRPM7 for development in a murine model. By using a mouse model where TRPM7 is specifically deleted in B cells under the control of the mb1 promotor, we show that B cells are absent in all peripheral lymphoid tissues due to apoptosis of pre B cells. By using an in vitro stromal cell line system, we demonstrate that B cell development can be partially rescued by high levels of extracellular magnesium. Interestingly, the lack of B cells is accompanied by an expanded granulocyte population in the spleen. In addition to identifying TRPM7 as an essential factor for B cell development, we show that TRPM7 is also an important regulator of B cell activation. DT40 B cells lacking TRPM7 fail to contract and gather antigen when activated. To investigate the role of the kinase domain of TRPM7 we made use of B cells expressing a kinase dead point mutant. These cells were also unable to gather antigen, showing that the kinase domain is an important regulator of this process. We also show that the kinase domain may potentially interact with another important regulator of B cell activation, PLCγ2 to mediate antigen collection and cell contraction. Importantly, primary murine B cells expressing only one allele of TRPM7 or treated with a TRPM7 inhibitor both displayed defects in antigen gathering, confirming our results in the DT40 cell line. Lastly, we show that TRPM7 is essential for antigen internalization, a process that is important for the recruitment of T cell help and ultimately, antibody production.
PhD Exit Seminar - Zahra Dargaei - Thursday, December 14th, 2017
PhD Exit Seminar
Thursday, December 14th at 10:10am – Ramsay Wright Building, Room 432
Zahra Dargaei (Woodin lab)
Aberrant chloride homeostasis and inhibitory synaptic transmission in Huntington's disease
Abstract
Huntington’s disease (HD) is primarily characterized by progressive motor incoordination and involuntary movements that result from neurodegeneration of the striatum. However, cognitive impairments and learning and memory deficits involving the hippocampus emerge in the early stages of the disease and precede the motor impairments by ~15 years. Despite the critical role of GABAergic inhibition in learning and memory, inhibition has not been studied in detail in the HD hippocampi. In my PhD thesis, I have presented three novel pieces of evidence that collectively demonstrate the causative role of Cl- homeostasis in hippocampal-related learning and memory deficits in HD. First, the reduced expression of the Cl--extruding cotransporter KCC2, and the increased expression in the Cl--importing cotransporter NKCC1 together result in excitatory GABAergic transmission in the hippocampi of the R6/2 transgenic mouse model of HD. Second, inhibition of the Cl--importing transporter NKCC1 with the FDA-approved drug bumetanide restores hyperpolarizing GABAergic inhibition and rescues the performance of R6/2 mice on hippocampal-associated behavioral tests. Third, the strength of overall hippocampal inhibition is altered at both the presymptomatic and symptomatic stages of the disease, and involves the disruption of both presynaptic and postsynaptic components. Taken together, my PhD work not only significantly increases our understanding of a less recognized aspect of HD, but also for the first time describes the involvement of Cl- homeostasis in a neurodegenerative disease.
PhD Exit Seminar -Wilfred Carlo de Vega -Wednesday, November 22, 2017
PhD Exit Seminar
Wednesday, November 22, 2017 at 2:10 pm, SW 403 – University of Toronto at Scarborough
Wilfred Carlo de Vega (McGowan Lab)
“DNA Methylation Modifications Associated with Glucocorticoid Sensitivity and Clinical Subtypes of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome”
Abstract
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex chronic disease with an unknown etiology that is primarily characterized by the presence of a highly debilitating fatigue that fails to resolve after sufficient rest. Additionally, patients must present other nonspecific symptoms relating to pain, unrefreshing sleep, cognitive impairment, and autonomic dysregulation in order to be diagnosed with ME/CFS, resulting in a population with highly heterogeneous symptom profiles. Previous research has consistently observed long-lasting changes in the immune system and hypothalamic-pituitary-adrenal (HPA) axis, a neuroendocrine system that regulates stress response. As a prototypical medically unexplained disease, environmental factors are believed to play a major role in the onset and manifestation of ME/CFS symptoms. These enduring differences may be partly explained through epigenetic modifications, referring to heritable gene expression alterations in the absence of mutations, which are known to reflect environmental, genetic, and stochastic influences on gene expression. This thesis explored the epigenetic modifications associated with the immune and neuroendocrine differences in ME/CFS. Specifically, DNA methylation across the genome was examined in peripheral blood mononuclear cells of ME/CFS patients, and immune response was tested by measuring in vitro sensitivity to glucocorticoids, a class of hormones that serve as an HPA axis effector. Differential methylation in ME/CFS was significantly enriched in immune response and cellular signaling genes, and was localized to 4,699 sites, which may serve as potential biomarkers. Two ME/CFS immune subtypes were observed according to glucocorticoid sensitivity. Integration of methylation and clinical data via machine learning discovered 4 clinical subtypes that were differentiated by T cell response genes, physical functioning, and post-exertional malaise. These results suggest that DNA methylation modifications are a feature of ME/CFS pathology. The identified potential biomarkers and clinical subtypes may be implemented in future work to better understand and clarify the biological differences related to specific ME/CFS symptoms.