MSc Exit Seminar - Dominic Ludovice (Nambara lab)
MSc Exit Seminar
Thursday September 24th, 2:10 pm - Ramsay Wright Building, Rm. 432
Dominic Ludovice (Nambara lab)
"Engineering of the Plant Abscisic Acid Biosynthesic Pathway up to Zeaxanthin Epoxidase in Escherichia coli"
Abstract
Abscisic acid (ABA) is a phytohormone implicated in many aspects of plant growth development including the regulation of seed development dormancy and germination, and plant response to environmental stresses. ABA has potentially many industrial applications. Escherichia coli is an ideal platform to reconstruct the ABA biosynthetic pathway; because, the early part of the pathway, up to the precursor zeaxanthin, have been successfully reproduced in E. coli by Misawa and colleagues (1990). This was done by using the carotenoid biosynthesis gene cassette from Pantoea ananatis. In this study, A. thaliana genes was used to build the pathway starting from zeaxanthin. The next biosynthetic enzyme, AtZEP, was successfully expressed in E. coli and the new strain was able to produce violaxanthin efficiently. The next enzyme which utilizes violaxanthin, has yet to be identified, however. This system is now being used to identify missing genes of the pathway.
Ramsay Wright is a wheelchair accessible building.
MSc Exit Seminar - Abiramy Karunendiran (Stewart/Barzda labs)
MSc Exit Seminar
Tuesday September 22nd, 1:10 pm - HSC 332, University of Toronto at Mississauga
Abiramy Karunendiran (Stewart/Barzda labs)
"Measuring Changes in Membrane Structure and Rhodopsin levels in Drosophila Melanogaster Retinas using THG Microscopy"
Abstract
Third harmonic generation (THG) microscopy is a valuable imaging modality that can be used to reveal structural information in a biological system without staining. THG signal can be observed at an interface between two different refractive indices or third-harmonic susceptibilities and is augmented in the presence of molecules with long carbon chains. Previous studies have established that carotenoid compounds are ideal for THG microscopy. I therefore sought to determine whether THG from endogenous carotenoid-derived compounds, such as retinal in photoreceptors, could serve as a platform to develop THG microscopy for developmental studies. I used the developing compound eye of Drosophila melanogaster as a model system because of its well-known anatomy, developmental profile, and the availability of genetic tools. The Drosophila eye contains 750-800 ommatidia, each housing photoreceptor neurons that utilize the visual pigment rhodopsin, G-protein coupled receptor containing retinal, for photo-transduction. In the first series of observations THG microscopy was shown to be an effective tool for monitoring normal photoreceptor development. In a second series of experiments, using dietary restrictions or genetic alleles that perturb rhodopsin expression, the absence of rhodopsin during pupal development causes membrane atrophy, resulting in photoreceptor degeneration. The results of this thesis demonstrated that THG microscopy can detect rhabdomere degeneration earlier than fluorescence microscopy. THG intensities measured throughout pupal development showed a distinct age-dependent curve, which changes in the absence of rhodopsin. In conclusion, THG microscopy can be used to monitor retinal development and presents itself as a new technique to detect retinal degeneration. Further development of this technology could aid in the early detection of human retinal degeneration syndromes, particularly those linked to mutations of human rhodopsin.
MSc Exit Seminar - Jessica Schembri (Desveaux/Guttman labs)
MSc Exit Seminar
Tuesday September 22nd, 9:30 am - Earth Sciences Centre, Rm. 3087
Jessica Schembri (Desveaux/Guttman labs)
"Experimental evolution of Pseudomonas syringae pathovar phaseolicola 1448A in the non-host plant Arabidopsis thaliana"
Abstract
Plants use their innate immune system to recognize conserved molecular elicitors of microbes, known as pathogen associated molecular patterns, to contain most microbial infections. Successful microbes may evolve to overcome resistance by suppressing the immune system or avoiding recognition. In plant-pathogen systems, microbes that are not able to overcome the innate immune response are said to have non-host interactions. For example, in the model organism Arabidopsis thaliana Col-0, the bacterial phytopathogen Pseudomonas syringae pathovar phaseolicola 1448A (Pph1448A), a bean pathogen, exhibits a non-host resistance response where the bacteria do not increase in frequency but the infection is not cleared by the plant. Here, an experimental evolution approach was used to track the early stages of adaptation of Pph1448A to A. thaliana Col-0. After 30 serial passages, whole genome sequencing was used to identify single nucleotide polymorphisms that may be linked with Pph1448A’s adaptation to a new plant host. Though mutations seem to be accumulating in Pph1448A these mutations cannot yet be associated with any changes in phenotype. With continued passaging of Pph1448A through Col-0, mutations will likely continue to accumulate in Pph1448A that may, in future, be linked with a change in virulence or other phenotypic traits.
MSc Exit Seminar - Annik Yalnizyan-Carson (Richards lab)
MSc Exit Seminar
Monday September 21st, 10:10 am - Ramsay Wright Building, Rm. 432
Annik Yalnizyan-Carson (Richards lab)
"Modeling Ca2+-Dependent Regulation of KCC2 Phosphorylation as a Mechanism for Inhibitory Synaptic Plasticity"
Abstract
Inhibitory synaptic transmission in the mammalian central nervous system is chiefly mediated by the neurotransmitter γ-aminobutryic acid (GABA). The primary receptor for GABA is the Cl- -permeable ionotropic GABAA receptor. In mature neurons, maintenance of low intracellular Cl- by the K+-Cl- co-transporter KCC2 results in Cl- influx and subsequent hyperpolarization of the cell upon GABA binding. Sustained repetitive activation of pre- and postsynaptic neurons with a fixed temporal separation has been shown to alter the strength of synaptic signaling. These changes occur via Ca2+ -dependent mechanisms which are sensitive to precise pre- and post-synaptic spike times, a phenomenon known as spike timing-dependent plasticity (STDP). At GABAergic synapses, plasticity induction using “coincident” intervals between pre- and post-synaptic spikes (i.e. intervals < ±20ms) leads to depolarizing shifts in the GABA reversal potential (EGABA) and, upon subsequent stimulation of GABAergic synapses, the driving force for Cl- is reduced and hyperpolarization is diminished. The precise molecular mechanisms for this change have not been fully resolved. It has been shown that inhibitory STDP principally involves Ca2+ influx via specific voltage-gated Ca2+ channels (VGCCs), namely the L- and T-type VGCCs. Furthermore, the Ca2+-sensitive kinase PKC and phosphatase calcineurin have been shown to control the phosphorylation state of KCC2 at key residues involved in membrane stabilization and transport efficacy. It has been hypothesized that Ca2+ influx via L- and T-type VGCCs can affect the level of activation of PKC and calcineurin, thereby effecting the phosphorylation state of KCC2, and hence, its Cl- extrusion capacity. This thesis presents a computational model to explore Ca2+-dependent dephosphorylation of KCC2 as a plausible mechanism for the depolarizing shift in EGABA seen in inhibitory STDP. Using a kinetic model of KCC2 phosphorylation in a biophysically realistic neuron model, we demonstrate that dephosphorlation of KCC2 triggered by T-type mediated Ca2+ influx can cause a depolarizing shift in EGABA that is sensitive to precise spike times. These results suggest that Ca2+-dependent dephosphorylation of KCC2 is a plausible molecular mechanism for inhibitory STDP. This model predicts that T-type VGCCs should be particularly important for triggering changes in KCC2 phosphorylation in response to coincident activity.
Ramsay Wright is a wheelchair accessible building.
MSc Exit Seminar - Noushin Koulena (Desveaux lab)
MSc Exit Seminar
Friday September 18th, 10:10 am - Ramsay Wright Building, Rm. 432
Noushin Koulena (Desveaux lab)
"Functional Characterization of the HopF Family of Type III Effectors in Arabidopsis thaliana"
Abstract
Pseudomonas syringae is a Gram-negative bacterial phytopathogen that causes disease in economically important crops. An essential virulence strategy of P. syringae involves injecting type III secretion effectors (T3SEs) into host cells to inhibit immune signaling. Advances in genomic sequencing have led to an increase in the available number of sequences of plant bacterial pathogens, which has led to identification of divergent homologs of known T3SEs. The HopF family of T3SE contains over 40 diverse family members. In order to explore the functional diversification of HopF family, this thesis undertakes the functional characterization of HopF T3SE in Arabidopsis thaliana. Functional analysis revealed multiple members with novel immune responses in Arabidopsis. A reverse genetics approach was used to identify the mechanism underlying the novel immune responses triggered by HopF family of T3SE. The Arabidopsis R protein ZAR1 was required for recognition of a novel member of the HopF family.
Ramsay Wright is a wheelchair accessible building.
MSc Exit Seminar - Alexandra Menna (Guttman/Desveaux labs)
MSc Exit Seminar
Thursday September 17th, 2:30 pm - Ramsay Wright Building, Rm. 432
Alexandra Menna (Guttman/Desveaux labs)
"Environmental Requirements Underlying Effector-Triggered Immunity in Arabidopsis thaliana"
Abstract
Pseudomonas syringae is a Gram-negative bacterium that infects multiple plant species by manipulating cellular processes via injection of type three secreted effectors (T3SEs) into host cells. Nucleotide-binding leucine-rich repeat (NLR) resistance (R) proteins recognize specific T3SEs and trigger a robust immune response, called effector-triggered immunity (ETI). ETI is often associated with localized programmed cell death (PCD), known as the hypersensitive response (HR), which limits pathogen proliferation. In this project, we examined the influence of elevated temperature on ETI. Elevated temperature differentially affects ETI outputs examined in this study – namely, HR and pathogen virulence suppression. We observed that elevated temperature conditions capable of suppressing HR had minimal influence on ETI-associated virulence suppression, thereby uncoupling these two ETI responses. We have identified ecotypes of Arabidopsis that show suppression of ETI-associated virulence suppression at elevated temperature, highlighting the natural variation that exists in coping with elevated temperature. These results not only reinforce the influence of abiotic factors on plant immunity, but also emphasize the importance of carefully documented environmental conditions in studies of plant immunity.
Ramsay Wright is a wheelchair accessible building.
MSc Exit Seminar - Rebecca Woelfle (Lovejoy lab)
MSc Exit Seminar
Thursday September 10th, 10:10 am - Ramsay Wright Building, Rm. 432
Rebecca Woelfle (Lovejoy lab)
"Linking the teneurin-latrophilin interaction with glucose metabolism: role of the teneurin C-terminal associated peptide (TCAP)-1"
Abstract
A newly discovered neuropeptide called teneurin C-terminal associated peptide (TCAP) exists in four isoforms, and is functionally related to its proprotein ‘teneurin’. TCAP-1 is expressed as a separate mRNA distinct from teneurin-1 and its mature peptide binds to the latrophilin (LPHN) G-protein coupled receptors (GPCRs) and associates with β-dystroglycan to activate a neurotrophic-like signal cascade, stimulating energy production and regulating cytoskeletal development. I found reduced levels of diacylglycerol (DAG), but not inositol triphosphate (IP3), in TCAP-1-treated hippocampal cells, indicating that TCAP-1 may downregulate the LPHN pathway. Immunocytochemical analyses show TCAP-1 co-localizes with LPHN-1 in hippocampal cells. I also confirm the endogenous mRNA expression of LPHN-1 and -3, and all four teneurins and TCAPs in mouse hippocampal and hypothalamic cells. An in vivo approach established that TCAP-1 significantly reduces serum glucose levels in diabetic rodents. Thus, the teneurin/TCAP-1-LPHN interaction may strengthen synapse formation and increase brain glucose utilization during stressful conditions.
Ramsay Wright is a wheelchair accessible building.
MSc Exit Seminar - Yan Li (Harris lab)
Thursday August 27th, 10:10 am - Ramsay Wright Building, Rm. 432
Yan Li (Harris lab)
"A RhoGEF that Promotes Par6-aPKC Pathway Activity for Establishing Epithelial Polarity in Drosophila"
Abstract
Apical-basal polarity is important for the integrity and function of Drosophila embryonic epithelia. Polarity is established when Cdc42, a Rho GTPase, activates the conserved Par6/atypical protein kinase C (aPKC) complex, and recruits them to the apical domain. However, the RhoGEF that activates Cdc42 in Drosophila is unknown. Through an shRNA screen, I isolated the RhoGEF CG10188 which when knocked down, produced similar defects as aPKC mutants. Furthermore, disruption of CG10188 and aPKC together enhanced the defects observed with either single disruption, demonstrating a genetic interaction between the two. However, aPKC localization appears to be independent of CG10188 regulation. Lastly, mammalian proteins that share high sequence identity with CG10188 have been previously shown to activate RhoA, another Rho GTPase, suggesting that CG10188 could be upstream of either Cdc42-Par6-aPKC or Drosophila RhoA (Rho1). Overall, my results suggest that CG10188 is a novel regulator of the polarity machinery that promotes epithelial development in the Drosophila embryo.
Ramsay Wright is a wheelchair accessible building.
MSc Exit Seminar - Charlene Lancaster (Harrison lab)
MSc Exit Seminar
Wednesday August 26th, 9:30 am - Room SW 403, University of Toronto at Scarborough
Charlene Lancaster (Harrison lab)
"Effects of Vitamin D, K1, K2 and Calcium on Bone Formation by Osteoblasts in Vitro"
Abstract
Bone loss is a major health problem that most people will face and thus research focusing on enhancing bone formation is of great importance. Although there have been many cell biology articles focusing on the effects of vitamin D, K1 or K2 on bone formation in vitro, there has yet to be a consensus amongst the literature. Through the use of several meta-analyses of past vitamin studies, we found that supplementation of vitamin D, K1 and K2, along with the combination of K2 + 1,25D, increased mineralization, while not consistently changing all of the other parameters associated with bone formation. In addition we quantified the area of von Kossa stained bone nodules in calcium, vitamin K1, vitamin K2, 25D and 1,25D treated human and mouse osteoblast cultures and found that mineralization levels varied depending on the presence of ascorbic acid or the organism from which the cell lines were derived.