PhD Exit Seminar - Laura Junker (Ensminger lab)
PhD Exit Seminar
Wednesday December 7th, 2:10 pm – Room CCT 2130, University of Toronto at Mississauga
Laura Junker (Ensminger lab)
"Photoprotective isoprenoids as indicators for stress responses in forest trees"
Abstract
For long-lived forest tree species, intraspecific variation among populations in their response to environmental conditions can reveal their ability to cope with and adapt to climate change. Plants constantly adjust the pigment composition of the photosynthetic apparatus to environmental conditions. Under abiotic stress conditions (e.g. drought), plants induce isoprenoid-mediated photoprotective mechanisms such as non-photochemical quenching (NPQ) and enhanced formation of antioxidants to minimize photooxidative stress. This thesis investigated the isoprenoid metabolism in Douglas-fir (Pseudotsuga menziesii) provenances that originated from contrasting habitats. Furthermore, the influence of foliar photosynthetic pigments on leaf optical properties was studied using senescing sugar maple (Acer saccharum) leaves. First, I established a simple and cost-effective protocol for the rapid analysis of isoprenoids using high-performance liquid chromatography (HPLC) (Chapter 2). Three experiments were conducted to evaluate 1) the adjustments of the isoprenoid metabolism when photosynthesis is limited in response to environmental conditions; 2) provenance-specific adjustments of photoprotective isoprenoids in response to drought; and 3) the influence of senescence-associated changes in isoprenoid levels on leaf optical properties in sugar maple. In chapter 3, photosynthesis and photosynthetic pigments in seedlings of two Douglas-fir provenances were compared under controlled drought conditions. In chapter 4, intraspecific variation in photosynthesis and photosynthetic pigments in response to changing environmental conditions were studied in mature trees of four provenances over the course of two years. Both experiments revealed that the more drought-tolerant interior provenances exhibit enhanced carotenoid-chlorophyll ratios, and larger pools of xanthophyll cycle pigments and β-carotene compared to coastal provenances from mesic habitats. This provenance-specific variation demonstrated the importance of the isoprenoid metabolism for the adaptation of provenances to drought. In chapter 5, the leaf optical properties of senescing sugar maple leaves were studied. The degradation of photosynthetic pigments as indicator for the progress of senescence was reflected by spectral reflectance measurements and digital image analysis. Isoprenoid metabolism may thus be a potential trait for selection of provenances for future forest management and indicator for remote-sensing of the plant physiological status.
PhD Exit Seminar - Thomas DeFalco (Yoshioka lab)
PhD Exit Seminar
Friday December 2nd, 10:10 am - Ramsay Wright Building, Rm. 432
Thomas DeFalco (Yoshioka lab)
"Regulation of Cyclic Nucleotide-Gated Channels and Plant Calcium Signaling"
Abstract
Plants must coordinate developmental processes and responses to environmental stimuli via signaling at the cellular level. Ca2+ serves a common second messenger in many signal transduction pathways, wherein increases in cellular Ca2+ levels are interpreted by a suite of downstream Ca2+-binding sensor proteins, such as calmodulin (CaM). While Ca2+ is known to mediate responses to diverse stimuli in plants, including biotic stress, comparably little is known regarding the channels involved in Ca2+ signaling or their regulation. Cyclic nucleotide-gated channels (CNGCs) represent one of the major classes of Ca2+-permeable channels thought to mediate Ca2+ flux in plants, and several isoforms of the 20-member Arabidopsis CNGC family, including CNGC12 and CNGC2, have been implicated in immune responses. In this thesis, I present a thorough analysis of the regulation of Arabidopsis CNGCs by CaM, and demonstrate that CNGC12 function is both positively and negatively regulated by CaM-binding to multiple, distinct sites at the cytosolic termini of the channel. My results with CNGC12 indicate that plant CNGCs are likely subject to complex regulation by Ca2+, suggesting that CaM is required for both channel function and feedback inhibition.
My findings with CNGC12 are expanded to the entire 20-member CNGC family, and I have shown that Arabidopsis CNGCs possess diverse CaM-binding sites, both in terms of number and mode of CaM-binding. In particular, the IQ motif appears to be broadly conserved across the CNGC family, and in most members, mediates permanent association with CaM via Ca2+-independent interaction with the CaM C-lobe. These data indicate that CaM functions as a Ca2+-sensing subunit of CNGC complexes in plants.
Finally, I developed transgenic Nicotiana benthamiana and Nicotiana tabacum (tobacco) lines expressing the fluorescent Ca2+ indicator, GCaMP3, which can be used to visualize and measure Ca2+ signals in response to diverse abiotic or biotic stimuli. I examined the dynamics of Ca2+ signaling in response to stimuli including cold-shock, mechanical wounding, and pathogen-associated molecular patterns (PAMPs). These plants represent an excellent tool to dissect the molecular components of Ca2+ signaling via gain- or loss-of-function studies.
Ramsay Wright is a wheelchair accessible building.
PhD Exit Seminar - Derrick Groom (Welch lab)
PhD Exit Seminar
Wednesday October 19th, 11:10 am – Room SW 403, University of Toronto at Scarborough
Derrick Groom (Welch lab)
"Hummingbird Hovering Energetics and Flight Capacities Across Elevational Gradients"
Abstract
High elevations are characterized by reductions in environmental parameters, including oxygen availability and air density. This can be problematic for volant species, as sustaining flight in hypodense air is more energetically demanding relative to normodensity. Animals meet these challenges by increasing wingbeat kinematics and mechanical power output, necessitating increased metabolic input, while at the same time aerobic metabolism is constrained by hypoxic conditions associated with increasing elevation. These limits may restrict the range of behaviours that can be aerobically sustained. This thesis explores the kinematic and aerobic capacities for exercise across an elevational gradient, and examines possible morphological adaptations associated with life at high elevations. Using hummingbirds, a group of animals with high hovering metabolic rates and wide elevational distributions, I examine the flight capacities and aerobic scopes across an 1800-m elevational gradient, as well as any morphological adaptations that permit high elevation flight. The angular velocity of the wings during hovering flight varies across elevations during hovering flight challenges, with higher elevations associated with higher angular velocities. Subsequently, the mechanical and metabolic power output of the muscles increases with rising elevation and challenge. Nonetheless, there is no relationship between aerobic scope and elevation. Overall, this suggests that hummingbirds are not metabolically limited and maintain the capacity to perform aerobic activities across their native ranges. Examination of morphology has previously indicated that high elevation species may have disproportionately larger wings than their lower elevation counterparts, hypothesized to be compensatory for high elevations. My analysis of hovering metabolic rate scaling has demonstrated that there is no relationship between elevation and morphological characteristics; there does not appear to be a compensatory effect of morphological variation upon hovering flight metabolic rates. However, there is a significant relationship between mass and mechanochemical efficiency of the flight muscles during hovering. Given that larger species tend to be overrepresented at higher elevations, greater efficiency may have permitted larger species to expand upwards because of their capacity to economize oxygen use. Overall, hummingbirds do not vary in flight capacity across elevations, and many species are able to maintain large elevational ranges without apparent morphological or functional adaptation.
PhD Exit Seminar - Gianni Castiglione (Chang lab)
PhD Exit Seminar
Wednesday October 12th, 3:10 pm - Ramsay Wright Building, Rm. 432
Gianni Castiglione (Chang lab)
"The Evolution of Rhodopsin Structure and Function in Vertebrates"
Abstract
The visual perception of light is a complex phenomenon, achieved through sophisticated biological systems ranging from the molecular interactions of visual system proteins, to computational processing within retinal neuronal circuits, to the higher cortical synthesis of visual inputs. Through photon absorption, the activation of the rod visual pigment rhodopsin forms the first step in the visual transduction cascade, ultimately mediating all of dim-light organismal visual sensitivity. Natural selection has molded vertebrate rhodopsin into a remarkable molecular machine, with a stunning sensitivity to single photons and an ultra-fast, femtosecond photoproduct formation. These evolutionary innovations are underpinned by the elegant structural and functional features of rhodopsin, which have served as the basis for our understandings of other class A G protein-coupled receptors. The importance of rhodopsin for vision across a diversity of environments makes it an ideal candidate for investigating how natural selection has shaped the molecular evolution of a complex sensory protein’s structure and function. Following a general introduction, Chapter II presents the first evidence for the adaptation of rhodopsin structure and function to cold temperatures, using high altitude Andean catfishes (Siluriformes) as a model system to identify the molecular targets of natural selection. These targets are shown to be highly similar between high altitude Tibetan Plateau and Andean catfish rhodopsins, with evidence in Chapter III supporting the phenotypic convergence of rhodopsin kinetics at high altitudes. In Chapter IV a novel structural feature of rhodopsin is revealed, functioning as a compensatory unit to modulate both rhodopsin spectral sensitivity and light-activated kinetics. This thesis identifies novel features of rhodopsin structure and function, and contributes to the emerging understanding that the structure and function of proteins are inextricably linked to shifts in ecology, environment and the dynamics of molecular evolution.
Ramsay Wright is a wheelchair accessible building.
PhD Exit Seminar - Aya Sasaki (Erb Lab)
PhD Exit Seminar
Friday October 7th, 1:45 pm – Earth Sciences Centre, Rm. 3056
Aya Sasaki (Erb lab)
"Influence of maternal high fat diet, stress and cocaine on neural mechanisms of reward and anxiety in rat offspring"
Abstract
Maternal obesity has important health consequences for the mother and her offspring. Experiments presented in this dissertation explored the role of maternal overnutrition with a high fat diet (HFD) on several aspects of offspring phenotype: reward- and stress-related behaviours, the endocrine stress response, and associated neural gene expression. First, maternal HFD effects on offspring phenotype in stress-related brain regions were examined. Maternal HFD was associated with altered expression of stress-related genes, a heightened endocrine stress response and increased anxiety behaviour in adult offspring. Genes central to stress and drug addiction (tyrosine hydroxylase and corticotropin-releasing factor) were upregulated in HFD offspring, suggesting that maternal HFD alters neural systems underlying related processes. Second, the role of maternal HFD on offspring phenotype following chronic cocaine exposure was investigated. Maternal HFD increased anxiety in saline-treated control females, reduced anxiety in cocaine-treated females, but did not interact with cocaine-primed locomotor activity or neural gene expression. These findings suggest that maternal HFD modulates offspring anxiety behavior with chronic cocaine exposure. Third, the role of maternal HFD and maternal stress on offspring phenotype given acute cocaine exposure was investigated. Maternal HFD did not interact with cocaine at the level of behavior or gene expression. However, there was an increase in locomotor activity in males exposed to maternal HFD, and with maternal stress at a high dose of cocaine. These findings suggest that, overall, maternal HFD and stress increase cocaine-induced locomotor activity in offspring through common but not identical neural mechanisms. Finally, in parallel I investigated the role of pre-gestational cocaine on offspring phenotype, and demonstrated an effect on the locomotor activating effects of cocaine in adult male offspring, as well as dopamine receptor D1 expression in the medial prefrontal cortex. These findings suggest increased sensitivity to cocaine in the male offspring of mothers given pre-gestational cocaine. The collective findings are discussed within a framework of maternal influences on cocaine sensitivity in offspring, wherein maternal HFD and pre-gestational cocaine confer increased sensitivity of stress- and reward-related responses in offspring.
PhD Exit Seminar - Kewei Xu (Harrison lab)
PhD Exit Seminar
Thursday September 29th, 10:10 am – Room SW 403, University of Toronto at Scarborough
Kewei Xu (Harrison lab)
"Cytoskeletal changes during classical activation of macrophages"
Abstract
As effector cells of the innate immune response, macrophages are capable of recognizing and eliminating pathogens during inflammation. Macrophages achieve maximal immunological potential by undergoing cellular activation. Classical activation of macrophages (M1) with interferon-γ (IFNγ) and lipopolysaccharide (LPS) greatly enhances the immune response against intruding pathogens. We have previously revealed that stathmin association with microtubules (MTs) is considerably reduced in M1 macrophages using proteomics approaches. In this thesis, we demonstrate a LPS-dependent stathmin protein reduction in M1 macrophages. We also explored the functional roles of stathmin down-regulation by generating stable cell lines overexpressing stathmin-GFP. Stathmin-GFP overexpression impaired MT integrity and reduced activation-associated phenotypes. Furthermore, overexpressing stathmin-GFP inhibited complement receptor 3 (CR3)-mediated phagocytosis and cellular activation, implicating its pivotal inhibitory role in classical activation of macrophages.
PhD Exit Seminar - Duncan Holbrook-Smith (McCourt lab)
PhD Exit Seminar
Monday September 26th, 1:10 pm - Ramsay Wright Building, Rm. 432
Duncan Holbrook-Smith (McCourt lab)
"Chemical Genetic Interrogation of Strigolactone Receptors in Arabidopsis and Striga hermonthica"
Abstract
Strigolactones are a class of terpenoid plant hormones that regulate various areas of plant growth and development. They are best known as suppressors of axillary growth, but have also been implicated in areas as diverse as leaf shape, hypocotyl length, and seed germination. However, parasitic plants of the genera Striga, Orobanche, and Phelipanche have evolved to use strigolactones released from host plant roots as a cue for germination and parasitism. Because these parasitic plants cause billion-dollar yield losses in the developing world each year, considerable research efforts have been dedicated towards understanding the mechanism of strigolactone perception.
The alpha/beta hydrolase HTL is the receptor for strigolactones in the seed. HTL interacts with effector proteins to elicit a strigolactone response. HTL-dependent signaling leads to increased germination and reduced hypocotyl length. We decided to use a chemical genetic approach to both probe HTLs while also developing Striga control technologies. Separately, we screened approximately 4000 compounds from a chemical library to identify small molecules that could be agonists and antagonists for Arabidopsis HTL. We were able to show that many of the compounds we isolated from the chemical screen were able to directly bind to HTL, and their action was specific to strigolactone signaling. These compounds were also able to stimulate Striga hermonthica germination, showing they have promise as leads for Striga control technology development.
In order to identify new genes involved in strigolactone signaling, we screened a collection of overexpression lines for the ability to resist the effects of an HTL antagonist on germination. We found that the overexpression of the splicing factor U2AF35B was sufficient to suppress the effect of an antagonist on germination, and could partially suppress phenotypes associated with loss-of-function alleles of HTL. This suggests that U2AF35B plays a role in strigolactone signaling at or downstream of HTL.
Ramsay Wright is a wheelchair accessible building.
PhD Exit Seminar - Brandy Velten (Welch lab)
PhD Exit Seminar
Friday August 26th, 11:10 am – Room SW 403, University of Toronto at Scarborough
Brandy Velten (Welch lab)
"Comparative Identification and Characterization of Myosin Heavy Chain Isoforms in Avian Skeletal Muscle"
Abstract
Organisms require muscles capable of meeting the functional demands of locomotion. Many of the contractile properties of muscle are tightly correlated to the expression of specific myosin heavy chain (MHC) isoforms. While the identification and characteristics of mammalian MHC isoforms have been well documented, comparatively little is known about avian MHC expression. The diversity of locomotor styles and life histories observed in birds suggests that the locomotor muscles of these species must operate over a wide range of contractile conditions that may be achieved, in part, by the differential expression of MHC isoforms. Specifically, it was hypothesized that, due to their very rapid wingbeat frequencies, the MHC expression of hummingbird flight muscle would differ from that of larger-bodied species to enable rapid muscle shortening. Further, the unique contractile requirements associated with different locomotor and life history strategies across avian species would lead to varying MHC isoform expression both intra- and interspecifically. The aim of this thesis was to explore the MHC expression in avian skeletal muscle across species, muscle groups, and life history stages. While MHC expression appeared to vary across muscle groups tasked with performing different locomotor activities, the MHC expressed by the flight muscle initially appeared relatively conserved across species. However, analysis of pectoral MHC isoform(s) in a larger array of species revealed greater diversity, including the presence of several characteristically distinct avian isoforms. Analysis of characteristics that may influence MHC expression revealed that the migratory predisposition of a species corresponded with MHC expression in small-bodied passerine species. Examining the pectoral MHC expression of one migratory passerine species, the white-crowned sparrow (Zonotrichia leucophrys), across three life stages demonstrated that MHC expression of the flight muscle altered with the migratory status of the species. Thus, the avian MHC family of proteins appears to much more diverse than previously anticipated, with expression associated, in part, with meeting the mechanical demands associated with migration in some species. Continued research into the MHC expression and gene families of avian species will further our understanding of the evolutionary and functional implications of this observed diversity.
PhD Exit Seminar - Devrim Coskun (Kronzucker lab)
PhD Exit Seminar
Thursday August 25th, 11:10 am – Room SW 403, University of Toronto at Scarborough
Devrim Coskun (Kronzucker lab)
"On the Roles of Membrane Channels in Plant Mineral Nutrition and Toxicity"
Abstract
The study of plant mineral nutrition and toxicity has made major strides recently, particularly at the level of molecular genetics. Arguably, however, this has been at the expense of “classical” physiology, which is of concern because critical physiological examinations of cellular and molecular models in planta are needed if extrapolations of these models to “real-world” (field-level) conditions are to be made. This is particularly urgent in the face of increasing environmental degradation and global food demands. To this end, the preset work explores the physiological role of membrane channels in higher-plant nutrition and toxicology, building upon foundational work in nutritional physiology and applying the important new discoveries in the areas of molecular-genetics and electrophysiology. Combining radioisotopic (42K+ and 13NH3/13NH4+) flux kinetics and compartmentation analyses with techniques in electrophysiology, mutant analysis, gas exchange, fluorescence imaging and tracing, and tissue mineral-content analysis, this work investigates the involvement of inward- and outward-rectifying Shaker-like K+ channels (KIR and KOR, respectively), nonselective cation channels (NSCCs), and aquaporins (AQPs), in K+ and NH3/NH4+ transport under conditions of both stress (low K+, salinity, and ammonium toxicity) and non-stress, in the intact plant. Efflux analyses showed that KOR channels mediate K+ efflux in barley roots only at low external [K+] (<1 mM), above which transmembrane efflux ceased altogether, and demonstrated their lack of involvement in Na+-induced K+ efflux in rice roots, an important salinity-stress response. Influx analyses elucidated the involvement of KIR channels in high- and low-affinity K+ uptake in barley and Arabidopsis roots, demonstrating dramatic capacity and plasticity, as well as their relevance to salinity stress. By contrast, no evidence of NSCC activity was found under similar conditions. Lastly, rapid transmembrane cycling of NH3, likely mediated by AQPs, was demonstrated to underlie ammonium toxicity in barley roots, fundamentally revising the mechanistic model of low-affinity NH3/NH4+ transport.
PhD Exit Seminar - Cara Fiorino (Harrison lab)
PhD Exit Seminar
Thursday August 11th, 1:10 pm – Room EV 140, University of Toronto at Scarborough
Cara Fiorino (Harrison lab)
"Examining novel factors that influence contact-dependent osteoblast and osteoclast differentiation"
Abstract
A dynamic equilibrium between bone destruction by osteoclasts and bone formation by osteoblasts is responsible for the maintenance of bone integrity, mineral homeostasis and protection from bone-related disease. As such, the focus of this work was to examine novel factors contributing to the contact-dependent differentiation of osteoblasts and osteoclasts. Osteoblast differentiation and maturation is stimulated by multiple external factors, one of which being ascorbic acid (AA). Utilizing MC3T3-E1 cells and primary murine osteoblasts, we identified a novel role for EB1, a microtubule plus-end binding protein, during osteoblast differentiation. AA-stimulation strongly induced EB1 expression and EB1 knockdown significantly impaired the osteoblast differentiation program in AA-induced osteoblasts. Furthermore, we identified that EB1 function was important for the global stability of b-catenin, a major signaling molecule in osteoblasts. Lastly, the influence of E-cadherin, a cell-cell adhesion and recognition molecule, was investigated in AA-stimulated osteoblasts. Up-regulation of Cdh1 (E-cadherin) paralleled that of Catnb (b-catenin), and E-cadherin blocking antibody treatment dampened osteoblast-specific gene expression.
E-cadherin is also expressed in monocyte/macrophage cells, which are precursors to osteoclasts. Receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation involves a period of precursor expansion followed by multiple fusion events to generate a multinucleated osteoclast. Interestingly, our results indicated that E-cadherin participated in early precursor interaction/recognition rather than during periods of osteoclast fusion. In both RAW 264.7 cells and primary murine macrophages, E-cadherin expression and surface localization was highest during early osteoclast differentiation. Utilizing E-cadherin blocking antibodies prior to the onset of fusion delayed osteoclast-specific gene expression and significantly impaired multinucleated osteoclast formation. Long-term imaging revealed that blocking E-cadherin function prolonged the proliferative phase of the precursor population while concomitantly decreasing the proportion of migrating precursors; the lamellipodium and polarized membrane extensions were identified as principal sites of fusion, establishing migration as a requirement for osteoclast differentiation.
This work characterizes EB1 and E-cadherin function during osteoblast differentiation and the E-cadherin-mediated transition from proliferative to migratory activities during osteoclast differentiation. Taken together, both studies highlight the value of exploring the early intra- and intercellular events that direct osteoblast and osteoclast differentiation.