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.
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.
MSc Exit Seminar - Wednesday, December 13, 2017 - Gordana Scepanovic
MSc Exit Seminar
Wednesday, December 13th, 9:10am –University of Toronto at Mississauga - CC-2150
Gordana Scepanovic (Stewart lab)
"Analysis of Drosophila Nervous System Development Following a Brief Exposure to Ethanol"
Abstract
Improper neuronal mapping predisposes many of the neurodevelopmental deficits that arise in patients of Fetal Alcohol Spectrum Disorders (FASD). Here I used Drosophila embryos as a model to investigate the neuronal defects that arise after exposure to ethanol. Drosophila is an excellent model for studying the mechanisms underlying FASD due to their vulnerability to the toxic effects of alcohol and the ease of manipulating their development. Using this model, I was able to provide detailed morphological analyses of the effects of a brief ethanol exposure on Drosophila neural development. I also examined the mechanisms that may regulate the FASD phenotype, specifically the serum response factor (SRF) signaling pathway. SRF signaling regulates numerous functions in the nervous system, including neuronal growth and migration. Through SRF manipulation in Drosophila, I was able to show that SRF is necessary for proper Drosophila axonogenesis, and that it may contribute to the FASD phenotype.
MSc Exit Seminar- Alexa Chioran - Tuesday, November 14, 2017
MSc Exit Seminar
Tuesday November 14th, 2:30 pm – Earth Sciences Centre, Rm. 3087
Alexa Chioran (Ringuette lab)
"SPARC promotes proper nucleation and assembly of collagen IV from Drosophila melanogaster embryonic haemocytes"
Abstract
Basement membranes (BMs) are a hallmark feature of all multicellular organisms in which they support essential developmental events. Collagen IV is the major structural component of the BM, however, its spatiotemporal assembly into stereotypic lattice networks is poorly understood. SPARC (Secreted Protein Acidic and Rich in Cysteine), a collagen IV-binding glycoprotein, is enriched in the BMs of Drosophila embryos and larvae. Fat body cells of Drosophila larvae and egg chamber follicular epithelial cells of the adult deficient in SPARC expression exhibit a fibrotic-like accumulation of collagen IV and other BM components in the pericellular space, resulting in larval lethality. Thus, SPARC is hypothesized to be an extracellular chaperone-like protein, whereby its association with collagen IV prevents aberrant collagen IV polymerization. In this study, I utilized molecular markers distinguishing secretory pathway compartments to demonstrate that SPARC and collagen IV co-localize primarily in the trans-Golgi network prior to secretion. Moreover, knockdown of SPARC expression in cultured embryonic haemocytes supports a role for SPARC in preventing premature nucleation of collagen IV molecules in embryonic haemocytes adherent to a laminin matrix. In order to gain further mechanistic insight on the functional relationship between SPARC and collagen IV, CRISPR gene editing technology was used to generate a sparc-null mutant. This genetic tool will be used in future experiments to unmask the structural domains of SPARC responsible for collagen IV assembly, maturation, and BM homeostasis.
MSc Exit Seminar - Cassandra Marie D'Amata - Wednesday, September 27th, 2017
MSc Exit Seminar
Wednesday, September 27, 2017 at 10:10 am, Ramsay Wright Building, Room 432
Cassandra Marie D’Amata (Tropepe Lab)
Characterizing the role of ice1 in maintaining zebrafish neural stem cells
Abstract:
Maintenance of neural stem cell (NSC) niches is required for the continued growth of the zebrafish retina and forebrain after embryogenesis. The zebrafish mutant kess564, which maps to the ice1 locus, exhibits reduced NSC niches. RNA polymerase II-dependent snRNA transcription requires the little elongation complex (LEC) for which ICE1 is an essential scaffolding component. Mutant NSCs which are normally active no longer express markers of cycling cells and become apoptotic. Furthermore, quiescent NSCs of the mutant retina are unable to proliferate in response to UV lesion. Whole-transcriptome analysis of ice1 mutant larvae show a downregulation of CNS and cell cycle genes, and an upregulation of splicing genes indicative of a possible compensatory mechanism. snRNA production appears to be unaffected in a subset of NSCs but reduced in differentiated neurons. This work demonstrates that ice1 is essential for NSC maintenance in an in vivo loss of function model.
MSc Exit Seminar - Stefan Vujadinovic - Monday, September 25th, 2017
MSc Exit Seminar
Monday, September 25, 2017 at 11:10 am, Ramsay Wright Building, Room 432
Stefan Vujadinovic (Tepass Lab)
The Okapi (Oka) FERM Domain Protein Regulates Somatic Stem Cell Numbers and Cell Intercalation during Drosophila Oogenesis
Abstract:
Okapi (Oka) is a Drosophila melanogaster FERM domain protein that is essential for oogenesis. oka (CG34347) null mutants are viable but display multiple defects during oogenesis. Mutant ovaries showed greatly enlarged and multilayered interfollicular stalks, compromised follicular epithelia, and germline disorganization, leading to reduced female fertility. I generated both endogenously and exogenously GFP-tagged Oka protein to examine the distribution of Oka.
Oka was present is all somatic cells of the ovary and localized to adherens junctions (AJs) in the germarium and follicles, including the cap cells, follicular epithelium, intercalating cells that form the interfollicular stalk, and in the migrating border cell cluster and centripetal cells. I also found that oka mutant ovaries contain an increased number of follicle stem cells, a defect that could explain the increased somatic cell numbers observed, and is reminiscent of the phenotypes of several overactive signalling pathways such as Hippo and Hedgehog. Examination of a series of five oka alleles that display different phenotypic strength suggest that interfollicular stalk formation is more sensitive to the reduction in Oka function than other observed defects. Together, my findings suggest that Oka is a component of AJs that negatively regulates follicle stem cell numbers and is required for the intercalation of interfollicular stalk cells.
MSc Exit Seminar -Paige Homme (McMillen Lab)
MSc Exit Seminar
Thursday, September 14, 2017 at 12:10 pm, DV3129- University of Toronto at Mississauga
Paige Homme (McMillen Lab)
“Whole-cell regenerating ‘microbead' to capture disease antibodies using the yeast species, Pichia pastoris”
Abstract:
Guiding appropriate medical treatment and population surveillance are a few examples of the crucial functions of diagnostics in health care [1]. Antibody based assays are routinely employed, however, they often require substantial resources limiting their use in low-resource settings. Richard Kil and others in Prof. David McMillen’s lab proposed a synthetic biology solution: using yeast as `microbeads' to detect Chagas disease and dengue antibodies. The yeast diagnostic combines yeast surface display and agglutination. Surface display engineers yeasts natural cell wall linked proteins to display antigens. The cells are placed in a u-shaped well settle into a dot at the bottom by default. Disease specific antibodies bridge the cells causing them to agglutinate; distinguishable by the naked eye. Preliminary work utilized Saccharomyces cerevisiae and I rebuilt the biosensor using an alternative yeast species, Pichia pastoris. It is an industrially viable yeast species that thrives in high-density bioreactors [2]; I investigated the viability of using P. pastoris.
MSc Exit Seminar - Adam Isaac Kramer (Godt Lab)
MSc Exit Seminar
Monday June 12th, 1:10 pm – Ramsay Wright Building, Rm. 432
Adam Isaac Kramer (Godt Lab)
" Protein Kinase C δ Regulates Cellular Protrusions and Filamentous Actin Organization during Drosophila Border Cell Migration"
Abstract
The migrating border cell cluster (BCC) of Drosophila ovarian follicles is an excellent model for studying collective cell migration. My project aimed at gaining mechanistic insight into the function of the serine/threonine kinase Protein Kinase C δ (PKCδ) in the migrating BCC. Analysis of mosaic BCCs containing PKCδ mutant or PKCδ-overexpressing cells indicates that normal PKCδ expression is important for a cell in the cluster to lead migration. Increasing or eliminating PKCδ caused a reduction of non-muscle myosin II in cellular protrusions and along the cell cortices of the BCC perimeter. Tagged transgenic PKCδ enriched in the leading protrusion in a complementary distribution with F-actin, where it appears to antagonize F-actin induced by the actin elongation factor Enabled. Inhibition of Enabled activity suppresses the effects of loss of PKCδ on F-actin organization. These findings suggest that PKCδ is a negative regulator of F-actin and functions in an Enabled-dependent process.
Ramsay Wright is a wheelchair accessible building.
MSc Exit Seminar - Nina Kirischian (Guttman/Desveaux lab)
MSc Exit Seminar
Tuesday June 27th, 2:00 pm – Earth Sciences Centre, Rm. 3087
Nina Kirischian (Guttman/Desveaux lab)
“Identification of novel resistance specificity in Arabidopsis thaliana against Pseudomonas syringae strains”
Abstract
Pseudomonas syringae is a plant pathogen that infects a diverse set of hosts by injecting type III secreted effectors (T3SEs) into plant cells to cause disease. Plants like Arabidopsis thaliana have evolved resistance (R) proteins to recognize these effectors and trigger robust immunity. Co-evolutionary pressures have led to effector and R protein diversification as plants and pathogens struggle for survival. To date, there are >60 T3SE families in P. syringae and ~160 predicted R proteins in Arabidopsis, yet, less than a dozen R protein-effector pairs are characterized. To tackle this problem, we took 2 approaches. 1) We screened Arabidopsis ecotypes for resistance to PtoDC3000. We identified the Uod-1 ecotype and propose HopM1 as a potential T3SE triggering immunity. 2) We screened P. syringae strains for those capable of triggering immunity in the Arabidopsis Col-0 ecotype. We identified two strains with immune-eliciting effectors that have yet to be characterized.
MSc Exit Seminar - Michael Fana (Cheng Lab)
MSc Exit Seminar
Wednesday, July 19, 2017 at 2:00 pm, DV3129- University of Toronto at Mississauga
Michael Fana (Cheng Lab)
“The Regulatory Function of Raf-1 Kinase Inhibitory Protein in Adult Hippocampal Neurogenesis”
Abstract
Adult hippocampal neurogenesis is the generation of new neuronal cells within the dentate gyrus of mammals. The process avails the hippocampus in its innate function of memory and notwithstanding any aberration, shows consistency throughout early adulthood until depreciating in old age. It begins with neural stem cells which undergo activation and division, mature over several weeks, and finally (of those that survive), integrate into existing neural circuitry. Several possible cellular pathways exist to mediate this neurogenic process, including the MAPK pathway. RKIP, a novel and elusive protein, is known to inhibit this pathway and regulate others, such as NF-kB, GSK3β, and GRK2. Owing to this expansive function is the finding of its role in promoting a neuronal fate in neural progenitor cells (NPCs). Here, I looked to delineate the role of RKIP throughout the entirety of the neurogenic process in mice. With voluntary exercise employed for neurogenesis induction, I discovered RKIP as functioning to reduce NPC proliferation while maintaining proper cell cycle kinetics. Furthermore, RKIP did not affect the overall formation of mature neuronal cells despite promoting a neuronal fate early in development.