PhD Exit Seminar - Steven Tran - (Gerlai Lab)
PhD Exit Seminar
Wednesday July 6th, 11:10 am – Room DV 3130, (Council Chambers) University of Toronto at Mississauga
Steven Tran (Gerlai lab)
"Alcohol induced locomotor activity in zebrafish is mediated by activation of the dopaminergic system"
Abstract
Alcohol addiction is a major unmet medical issue with current pharmacological treatment options being limited. Alcohol’s locomotor stimulant effect in animals is thought to parallel the euphoric effects of alcohol consumption in humans, yet the mechanisms underlying this behavioral response is not completely understood. Using zebrafish, we demonstrated a positive correlation between alcohol induce locomotor activity and whole-brain dopamine levels. We found that zebrafish developed tolerance to alcohol's effect on locomotor and dopaminergic activity when continuously housed in alcohol, highlighting the importance of the dopaminergic system. We correlated the alcohol induced increase in dopamine levels with increased tyrosine hydroxylase activity and protein expression, the rate-limiting enzyme in dopamine synthesis. We also found selective inhibition of phosphorylated tyrosine hydroxylase to attenuate the alcohol induced increase in locomotor activity and abolished the increased dopamine levels. Examination of dopamine receptor activation during alcohol exposure revealed that antagonism of dopamine D1-like receptors inhibited locomotor and dopaminergic activity, although independent of alcohol’s effect. Conversely, antagonism of dopamine D2-like receptors attenuated alcohol’s locomotor stimulant effect suggesting an important role for this family of receptor subtypes regulating alcohol induced locomotor activity. Finally, since alcohol is known to alter stress and anxiety in mammals, we examined the interaction between stress and alcohol in zebrafish. We found that stress and anxiety potentiated the locomotor stimulant effect of alcohol and abolished the alcohol induced increase in dopamine. Although counterintuitive, the abolished dopaminergic response suggests stress potentiated the release and breakdown of dopamine. Overall, these studies suggest the pharmacological aspects of alcohol in mammals including humans are evolutionarily conserved in zebrafish. Given the translational relevance of our findings, future attempts to investigate novel mechanisms regulating alcohol induced behavioral changes in zebrafish may provide key insights into alcohol addiction in humans.
PhD Exit Seminar - Laura Sedra (Lange lab)
PhD Exit Seminar
Friday June 3rd, 1:30 pm – Room IB 250, University of Toronto at Mississauga
Laura Sedra (Lange lab)
"FMRFamide-like peptides and their role in reproduction in the Chagas vector, Rhodnius prolixus"
Abstract
Insect reproductive systems are tightly modulated by neurotransmitters via direct innervation, by neurohormones circulating in the haemolymph and by neuromodulators locally released from endocrine cells in the periphery. FMRFamide-like peptides (FLPs) are large families of neuropeptides with conserved RFamide C-termini and have been implicated in vertebrate and invertebrate neuroendocrinology. This thesis examines the differing roles that members of the FLP superfamily have in the adult female reproductive system of Rhodnius prolixus. The entire female reproductive tract is composed of different arrangements of striated muscles. Two members of the N-terminally extended FM/L/IRFamides have been shown to stimulate ovariole, ovary, oviduct and bursal muscle contraction in a dose-dependent manner; whereas the myosuppressin, RhoprMS, does not have any myogenic effect on the reproductive tract. The RhoprNPF (neuropeptide F) and RhoprNPF receptor (RhoprNPFR) genes have been cloned from the R. prolixus central nervous system (CNS) and phylogenetic analysis implies possible receptor-ligand co-evolution. RhoprNPFR has been classified as a G-protein coupled receptor (GPCR) containing 7 transmembrane domains and a conserved 8th α-helix that is a classic characteristic of rhodopsin-type GPCRs. FMRFamide-like immunoreactivity (FLI) was observed in cells and processes in the adult CNS and processes on the reproductive tract; moreover a specific subset of median neurosecretory cells (MNSCs) in the brain, as well as small cell bodies along the muscle fibers of the lateral oviduct, express the RhoprNPF transcript. The RhoprNPFR transcript is also expressed in putative nurse cells in the germarium and pre-follicular cells of the R. prolixus telotrophic ovariole. RhoprNPFR transcript appears to be supplied to the developing oocyte during vitellogenesis and the receptor most likely aids in the differentiation of pre-follicular cells into mature follicle cells surrounding the growing oocyte and helps regulate the supply of nutrients. Screening members of the FLP family in an egg-laying assay showed that N-terminally extended FM/L/IRFamides and short NPF stimulate oogenesis, whereas MS inhibits it. Although RhoprNPF does not have a myogenic effect on lateral oviduct muscle, we have shown that it potentially aids in ovulation. Sulfakinins exhibit no effect on egg-laying. In summary, this thesis demonstrates the importance of FLPs in the control and regulation of the female R. prolixus reproductive system.
PhD Exit Seminar - Jordan Silver (Tepass lab)
PhD Exit Seminar
Thursday May 12th, 2:10 pm - Ramsay Wright Building, Rm. 432
Jordan Silver (Tepass lab)
"Yurt and Cysts Link Adherens Junction Stability to Actomyosin Remodeling in Drosophila Embryogenesis"
Abstract
The integrity of epithelial cells relies on the stability of adherens junctions (AJs) during development. This dissertation focused on how remodeling of the actomyosin cytoskeleton can stabilize AJs.
The FERM domain protein Yurt acts as a basolateral polarity regulator without any known interaction partners. I showed that Yurt genetically interacts with myosin II and DE-cadherin (DEcad) to support epithelial integrity in the Drosophila embryo. I found that a pool of Yurt localizes to AJs and physically interacts with myosin II and members of the cadherin-catenin complex. Loss of Yurt leads to fragmentation of AJs and disruption of myosin II throughout the ectoderm. In dorsal closure, I demonstrated that Yurt is essential for the recruitment of DEcad to the leading edge (LE) -amnioserosa interface and assembly of the LE multicellular myosin II cable. Yurt appears to support the LE cable through the organization of apical Crumbs and aPKC. In wound healing, we showed that Yurt can be recruited de novo to the site of myosin II cable assembly. I propose a model where an apical pool of Yurt acts to link the cadherin-catenin complex and the actomyosin cytoskeleton to stabilize AJs.
Next, I characterized a novel RhoGEF, Cysts (previously CG10188), that is essential for epithelial polarity in the Drosophila embryo. RhoGEFs can regulate actomyosin effectors through targeting of small Rho GTPases. I found that Cysts is required for epithelial cell polarity. Depletion of Cysts leads to a progressive reduction in myosin II in the early embryonic ectoderm, followed by a disruption of AJs and apicobasal polarity, ultimately leading to cellular apoptosis. Structure-function analysis demonstrated that the RhoGEF domain and the C-terminal region of Cysts are essential for function. We showed that the C-terminal region of Cysts facilitates recruitment to apical junctions, whereas genetic and biochemical experiments suggested that Cysts predominantly activates Rho1 rather than Cdc42 or Rac. I propose a model whereby Cysts acts through Rho1 to maintain a critical level of myosin II at AJs in support of junctional and epithelial integrity.
Ramsay Wright is a wheelchair accessible building.
PhD Exit Seminar - Tauqeer Ahmad (AbouHaidar lab)
PhD Exit Seminar
Thursday January 21st, 10:10 am - Earth Sciences Centre, Rm. 3087
Tauqeer Ahmad (AbouHaidar lab)
"Translocation of Virus-Derived Nucleic Acids to Chloroplasts and Mitochondria in Plants"
Abstract
Many studies have documented the role of transit peptides and viral proteins in targeting various viruses to specific subcellular regions of their hosts. However, knowledge hitherto about the specific sequence(s) and/or motif(s) involved in the trafficking and subcellular localization of viral RNAs and DNAs is very limited. In my thesis, I have demonstrated that a non-coding RNA sequence from potato virus X (PVX-RNA) as small as 127 nucleotides (located near the 3´end of 8 kDa and the start of CP genes as well as the non-coding intergenic region) is capable of translocating not only its own PVX-RNA sequence but also a reporter gene, GFP (green fluorescent protein) mRNA, into chloroplasts of the transgenic tobacco plants. This is the first evidence showing that a small viral RNA sequence (designated “RNA tractor”) is capable of translocating RNA sequences into the chloroplasts; however, the functional role of this mechanism remains elusive.
Begomovirus, the largest genus of the Geminiviridae family, consists of plant viruses with circular single-stranded DNA genomes. Begomoviruses infect a wide range of economically important plants. I investigated two begomoviruses on various Nicotiana species to assess the effects of their ploidy level on infectivity and symptomatology. For this purpose, infectious clones of Ageratum enation virus (AEV), a monopartite (DNA-A with Beta-satellite DNA particle) and Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite (DNA-A and DNA-B), begomoviruses were used. All plants inoculated with ToLCNDV were systematically infected and showed characteristic symptoms. However, in the case of AEV, all plants except N. tabacum cv. Xanthi were infected by the virus but remained symptomless. Taken together, these results indicate that there is no clear relationship between infectivity and ploidy levels; furthermore, symptomatology depends on the type of virus and/or plant species.
Another key question to answer was whether or not the genomes of the begomoviruses could be isolated from chloroplasts of the infected tobacco and tomato plants. PCR results confirmed the presence of only DNA-A of the AEV in the chloroplasts. Preliminary studies also clearly show that the “RNA tractor” sequence and AEV genome are incapable of targeting the mitochondria. These findings suggest that members from different viral families may be associated with the same organelle, but that members do not necessarily target the different organelles. Thus, the present study could be important for understanding the evolutionary importance of the interactions of viral genomes with different organelles of plant cells and their consequential pathological effects.
PhD Exit Seminar - Aaron Allen (Sokolowski lab)
PhD Exit Seminar
Tuesday December 15th, 1:10 pm - Ramsay Wright Building, Rm. 432
Aaron Allen (Sokolowski lab)
" Deciphering Pleiotropic Effects: A molecular characterization of the foraging gene in Drosophila melanogaster"
Abstract
Pleiotropy is defined as the manifold effects of a gene at the phenotypic level. Understanding the mechanisms of manifold gene action has important implications for many fields of biology ranging from evolution and to medicine. The foraging gene, in Drosophila melanogaster, has long been a pivotal example of a single gene with natural variants that affect feeding-related phenotypes. One possible mode of action for foraging’s pleiotropy is through independent regulation of its gene products. Characterization of the foraging gene revealed 4 distinct promoters that produce 21 transcripts, and 9 ORFs. A foraging null mutant of the locus was generated using Ends-out gene targeting. foraging null mutants had reduced foraging behaviour, reduced food intake behaviour, and increased lipid levels. A recombineered full genomic rescue of the gene rescued the effects of the null mutation. By comparing the effects of the null mutant with those of the natural variants, I showed that these feeding-related phenotypes were differentially regulated. A promoter manipulation strategy identified diverse, and non-overlapping expression patterns associated with the 4 foraging promoters. Expression was seen in the nervous and gastric systems of the larva and adult fly, as well as the reproductive systems of the adult fly. This expression suggests potential new roles for the foraging gene in the larva and adult fly. Characterizing the regulation of foraging's gene products will further our understanding of its role in behaviour, and shed light on the evolutionary origins of natural variants of the foraging. Not only will this study further our understanding of this gene's conserved function across taxa, but will help elucidate the role for differential transcriptional regulation in achieving multiple functions of a gene. This could further serve as fodder for investigations into the roles of neo-functionalization versus escape from adaptive constraint.
Ramsay Wright is a wheelchair accessible building.
PhD Exit Seminar - Jennifer Lapierre (Peever lab)
PhD Exit Seminar
Wednesday October 7th, 10:10 am - Ramsay Wright Building, Rm. 432
Jennifer Lapierre (Peever lab)
" Neurochemical Substrates of Unihemispheric Sleep in the Fur Seal"
Abstract
Several neuronal systems contribute to the generation and maintenance of sleep and waking. Knowledge pertaining to the neurotransmitters involved in sleep-wake regulation has been derived primarily from species that display bilaterally symmetric EEG states (e.g. cats, dogs, rats, and mice). Such studies, however, do not indicate which aspects of sleep these neurotransmitters are specifically linked to. Fur seals display both bilateral (BSWS) and asymmetrical sleep slow-wave sleep (ASWS); this unusual sleeping pattern provides a unique opportunity to determine which of the many physiological and neurochemical changes seen bilaterally in terrestrial mammals are linked to the cortical EEG changes and which may be linked to the motor and/or autonomic aspects of sleep. In studying this remarkable feature of the fur seal, we can dissociate the thalamocortical EEG activity in each hemisphere from aspects of behavioural state and study each independently as they relate to neurotransmitter release. The overall aim of my thesis was to examine, simultaneously in both hemispheres, the release of key neurotransmitters in the cortex across the sleep-wake cycle in the northern fur seal, and to specifically compare levels during BSWS and ASWS. In vivo microdialysis and high-performance liquid chromatography were used to monitor the release of neurotransmitters during polygraphically defined states. Neurotransmitters previously thought to contribute equally to the waking state, actually behave quite differently in a brain that is half awake and half asleep. Cortical acetylcholine (ACh) release was highly lateralized during ASWS, with greater release in the activated hemisphere, whereas cortical serotonin (5-HT), noradrenaline (NA), and histamine (HA) were symmetrically released during ASWS. Of the arousal systems studied so far, only ACh is involved in the lateralized cortical EEG activation manifested in asymmetric sleep. Bilaterally symmetric levels of the monoaminergic transmitters studied here are compatible with interhemispheric EEG asymmetry. Findings suggest distinct functional roles for ACh, 5-HT, NA, and HA in the regulation of wakefulness, with respect to cortical activation and behavioural arousal.
Ramsay Wright is a wheelchair accessible building.
PhD Exit Seminar - Jessica Pressey (Woodin lab)
PhD Exit Seminar
Thursday September 10th, 1:10 pm - Ramsay Wright Bldg., Rm. 432
Jessica Pressey (Woodin lab)
"Protein Interactions Regulate KCC2-mediated Chloride Homeostasis"
Abstract
KCC2 is the primary Cl- cotransporter which maintains low [Cl-]i required for inhibitory neurotransmission. Despite intense investigation, the underlying mechanisms governing KCC2 regulation remain poorly understood. In my thesis I have presented two novel pieces of evidence providing further insight into KCC2 regulation. First, KCC2 membrane expression and function require the expression of the kainate-type glutamate receptor subunit GluK2. Second, KCC2 membrane recycling can be increased by increasing PKC-mediated phosphorylation of GluK2. Together, modification of KCC2 by GluK2 interaction and PKC-mediated phosphorylation functionally interact to synergistically increase KCC2 expression and function. This is the first demonstration of protein interaction and protein phosphorylation which create a functional cross-talk which regulates KCC2 surface expression and function. Elucidating the mechanisms which regulate KCC2 function are highly coveted as KCC2 down-regulation is commonly observed in neuropathophysiological conditions such as epilepsy and neuropathic pain. Taken together, my results present novel findings regarding the mechanisms which regulate KCC2 function and surface expression and critically regulate neuronal Cl- homeostasis and inhibitory neurotransmission.
Ramsay Wright is a wheelchair accessible building.