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.
PhD Transfer Exam reminder - Stuart Macgregor
PhD Transfer Exam
Tuesday, April 17, 2018 at 1:30 pm – Earth Science Building, Room 3087
Stuart Macgregor (D. Goring lab)
“Investigating the role of secretion in the Arabidopsis thaliana compatible pollen response pathway”
Abstract
The acceptance of compatible pollen in the Brassicaceae is tightly regulated through interactions between the pollen and the pistil. Secretion in the stigmatic papillae is proposed to be key to this interaction to provide resources to the pollen for hydration and germination. The objective of this proposed research is to investigate components of the Arabidopsis thaliana secretory pathway machinery for their requirement in compatible pollen acceptance. Fluorescently-tagged markers that identify different compartments in the endomembrane system will be examined to gain a fuller understanding of the secretory activity that occurs following compatible pollinations. This includes tracking of secretory markers from the initiation of secretion at the endoplasmic reticulum to the final site of vesicle release at the papillar plasma membrane under the pollen contact site. The requirement of SNARE complex subunits, which are implicated in vesicle fusion and cargo release, will also be investigated through loss-of-function mutants. This will be accomplished by using a combination of SNARE T-DNA insertion mutants and generating CRISPR/Cas9 gene editing mutants when needed. This proposed research will provide a better understanding of the stigmatic papilla’s secretory system, and how this system is employed in the acceptance of compatible pollen.
PhD Proposal Exam - Luís Eduardo Abatti - Thursday, January 25th , 2018
PhD Proposal Exam
Tuesday, January 25th, 2018 at 10:10 am – Ramsay Wright Building, Room 432
Luís Eduardo Abatti (Mitchell lab)
“Investigating the SOX2 transcriptional network in estrogen-responsive and estrogen-resistant breast cancer cells”
Abstract
Breast cancer is a multifactorial disease characterized by aberrant gene expression. The sex-determining region Y box2 (SOX2), a key transcription factor associated with pluripotency, is often overexpressed in breast cancer cells, where it has been linked to epithelial-mesenchymal transition (EMT) and hormone resistance. In mouse embryonic stem cells, Sox2 is regulated by a wide transcription factor network that interacts with its distal enhancer. However, the SOX2 transcriptional network in breast cancer cells remains unknown. Mammary epithelial cells rely on the estrogen receptor alpha (ESR1) and its cofactors – FOXA1 and GATA3 – to properly respond to estrogen stimulation, while breast cancer cells frequently display a dysfunctional estrogen response. My hypothesis is that SOX2 is normally downregulated by the repressive action of ESR1, FOXA1 and GATA3 at a distal enhancer. Once the estrogen pathway is disrupted in hormone-resistant cells, the repressive effect of estrogen over SOX2 expression is abolished, and SOX2 recruits the RNA Polymerase II transcriptional complex at multiple genomic targets. To better understand the role and regulation of SOX2 in this scenario, I propose three objectives: first, to identify the SOX2 transcriptional network in breast cancer cells; second, to investigate SOX2 cis- and trans-regulatory elements in MCF-7 cells; and third, to understand SOX2 upregulation in hormone-resistant MCF-7 cells. This SOX2 functional investigation will elucidate how breast cancer cells rely on this transcription factor to maintain their tumourigenesis and how its upregulation is linked to hormone resistance.
PhD Transfer Exam - Nawar Alwash - Thursday, January 25th, 2018
PhD Transfer Exam
Thursday, January 25th, 2018 at 1:10pm –University of Toronto at Mississauga - CCT2134
Nawar Alwash (Levine lab)
“The role of foraging gene (for) in Drosophila melanogaster social interaction networks (SINs)”
Abstract
Drosophila melanogaster display social behaviours such as courtship, mating, aggression and foraging in groups. Recent studies have shown that different strains of D. melanogaster form social interaction networks (SINs) with different properties, suggesting that genes influence network phenotypes. The foraging gene (for) regulates food-related behaviours in several species including D. melanogaster. There are two naturally occurring alleles of the for gene: rover and sitter, where the rover flies are characterized with higher mobility in the presence of food. However, the role of the two variants in the formation of social networks remains unknown and that will be the focus of my research. I hypothesize that the for gene influences the formation of SINs and thus manipulating the for gene would lead to formation of networks with different SIN properties. I have shown that SINs formed by rover females have different properties than those formed by sitter females. I have also shown an effect of for copy number that is reflected in SIN phenotypes. To investigate this further, I aim to characterize the role of the for gene on social structure by investigating the effect of for on the formation of SINs during different developmental stages. I will also examine the allelic dominance pattern of the rover/sitter variant of for in the formation of SINs. Furthermore, I will explore the effect of external factors, such as stress and social experience, on SIN formation of the rover/sitter variants. This research will be the first to identify a specific gene influencing social network structure in D. melanogaster. In addition, understanding the role of for in the formation of SINs could potentially provide an insight into understanding the role of this gene in SIN formation of other organisms.
MSc Exit Seminar - Syed Saad Husainie -Friday, January 12th, 2018
MSc Exit Seminar
Friday, January 12th, 2018 at 10:10am Ramsay Wright Building, Room 432
Syed Saad Husainie (Godt Lab)
Analysis of the Function of Protein Kinase C δ in Regulating Collective Cell Migration during Drosophila Oogenesis through Live Imaging
Abstract:
Border cell cluster (BCC) migration during Drosophila oogenesis has provided an excellent model to study collective cell migration. We have previously demonstrated that the serine/threonine kinase Protein Kinase C δ (PKCδ) is a regulator of BCC motility and protrusion morphology. My project aimed at examining the effects of altered PKCδ expression on BCC motility through live imaging, focusing mainly on BCC migration behaviour and protrusion dynamics. Although known for its pro-apoptotic role, I found no evidence of cell death in BCCs with increased or decreased PKCδ expression. Live imaging revealed that BCCs require normal levels of PKCδ expression to migrate at a normal speed. Increasing PKCδ expression levels in BCCs delayed detachment from the epithelium and significantly slowed down migration. Decreased PKCδ expression caused BCCs to migrate slower and less linearly during the early phase of migration. In addition, PKCδ-deficient BCCs showed defects in cellular protrusion dynamics, including frequent bifurcations and multiple cycles of extension and partial retraction. In summary, my analysis contributed to a better understanding of the function of PKCδ in regulating migration and protrusion dynamics during collective cell migration.
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.
PhD Exit Seminar - Jason Wen- Friday October 6th, 2017
PhD Exit Seminar
Friday October 6th, 11:10 am – Ramsay Wright Building, Room 432
Jason Wen (Winklbauer lab)
“Endoderm internalization in Xenopus laevis, and the role of ephrinB1 in gastrulation”
Abstract
During animal development, the early embryo undergoes gastrulation to pattern the primary germ layers. The ancestral mode of vertebrate gastrulation can be studied using the African clawed frog Xenopus laevis. A core function of gastrulation is the movement of prospective endoderm from the surface to the embryo interior. In X. laevis, endoderm internalization is achieved through vegetal rotation, a morphogenetic process that has only been characterized at the tissue level. I have investigated the cell and molecular basis of endoderm morphogenesis in X. laevis gastrulae to connect vegetal rotation with common mechanisms of germ layer internalization in other organisms. I characterized the arrangement, shape change, and migration of cells in the endoderm and found that movement of vegetal endoderm cells located deep to the embryo surface represents an adaptation of mechanisms of epithelial internalization, particularly invagination and cell ingression, to the multilayered structure of the vegetal cell mass. Thus, this movement was termed “ingression-type migration”. Ingression-type migration integrates amoeboid-type motility, whereby cells propel forward using cell shape changes to generate locomotion, with cell tail retraction by macropinocytosis. In conjunction with differential migration — a mode of intercellular migration where cells move in a common direction but at different velocities with respect to neighbouring cells — this drives cell rearrangement during vegetal rotation. Despite their collective movement, endoderm cells are separated by vast gaps filled with extracellular material. Cells are interconnected by stitch-like contacts, and cell migration requires the adhesion molecule C-cadherin, and the matrix protein fibronectin. A basic requirement for migration is that cells must detach to complete rearrangement, and in this regard, the leading cell resolves its contact with the following cell through trailing edge retraction involving ephrinB1-dependent macropinocytosis and trans-endocytosis. Beyond the endoderm, ephrinB1 is also required for the modulation of ecto- and mesoderm tissue cohesion.
CSB Research Day 2017
Seminars to be held in RW 117; Poster exhibition in RW 010
Special Presentation: Prof. Leah E. Cowen, Dept. of Molecular Genetics
Refreshment & lunch provided.
Afternoon reception and awards ceremony in RW010 from 4:30-6:30
Registrants only please!