PhD Transfer Exam - Urfa Arain (Erclik lab)
PhD Transfer Exam
Thursday May 18th, 2:30 pm – Rm. DV 3138, University of Toronto at Mississauga
Urfa Arain (Erclik lab)
"Mechanisms underlying neuronal migration in the Drosophila optic lobe"
Abstract
Our ability to see the world around us relies on the precise assembly of neurons into complex circuits in the visual centers of our brain. The Drosophila medulla, which comprises the largest structure of the optic lobe, is an excellent model to explore neural circuit assembly due to its precise organization of 40,000 neurons across 800 columns. These neurons can be subdivided into two major classes: uni-columnar and multi-columnar. Uni-columnar neurons are born throughout the entire medulla and have small dendritic arborizations that contact a single column. In contrast, multi-columnar neurons are fewer in number and have large arborizations that contact several columns. During development, the majority of multi-columnar neurons are born in a restricted region and express the transcription factor Vsx1. Subsequently, they migrate up to 60 microns from where they are born to cover the entire medulla. The goal of my research is to identify the mechanisms that underlie the migration of Vsx1-derived multi-columnar neurons during development. I have found that migration occurs as a two-step process that begins 20 hours after puparium formation (APF) and continues until 50 hours APF. Surprisingly, the cell bodies of multi-columnar neurons initially migrate with a ventral bias that persists for at least 10 hours of pupal brain development, after which they cover the entire dorsoventral axis of the medulla. Additionally, the axons of Vsx1-derived multi-columnar neurons target locally in the developing neuropil prior to migration, indicating that their movement is likely independent of axonal targeting. Remarkably, the steroid hormone Ecdysone plays a key role in the process; transgenic flies expressing a dominant-negative form of the Ecdysone receptor in Vsx1 neurons show a cluster of cell bodies that fail to migrate in the adult medulla. In this PhD, I propose to describe the development of an individual multi-columnar neuron using genetic and live-imaging techniques. I will also investigate the role played by Ecdysone in migration by generating mutant MARCM clones and following them developmentally. Lastly, I intend to explore whether heterotypic signals from adjacent cell types or homotypic signals within the same subtype affect the migration of multi-columnar neurons through attractive or repulsive cues. It is anticipated that the results of this project will inform our understanding of neuronal development, neural circuit assembly and neurodevelopmental disorders in which these processes are disrupted, such as autism.
PhD Transfer Exam - Derek Seto (Desveaux lab)
PhD Transfer Exam
Thursday May 11th, 2:10 pm – Earth Sciences Centre, Rm. 3087
Derek Seto (Desveaux lab)
"Recognition of the Pseudomonas syringae type III effector HopF2a by the Arabidopsis R protein ZAR1"
Abstract
The plant pathogen Pseudomonas syringae uses effector proteins to disrupt plant immune pathways, allowing for successful colonization. However, plants have evolved resistance (R) proteins that detect the presence of some effectors, and subsequently trigger an immune response that suppresses pathogen growth. Recognition of effectors by R proteins often involves monitoring another host protein for effector-induced molecular perturbations; upon detection of these modifications, an immune response is triggered. The effector HopF2a from P. syringae pv. aceris M302273PT has been found to trigger immunity in Arabidopsis. Recognition of HopF2a has been shown to require the R gene ZAR1, as well as ZRK3, which encodes for a kinase. The R protein ZAR1 is also involved in the recognition of two other unrelated T3SEs: HopZ1a from P. syringae pv. syringae and AvrAC from Xanthamonas campestris pv. campestris. These also require kinases from the same family as ZRK3 for their recognition: ZED1 and ZRK1, respectively. In addition to this, AvrAC requires the PBL2 kinase for its recognition as well. The objectives of this research are to identify other potential genetic requirements for HopF2a recognition in Arabidopsis, and to understand how these components are involved in the HopF2a recognition mechanism. The results of this project will show how a single R protein is able to recognize several unrelated effectors by associating with different members of a diverse family of kinases. In turn, this can demonstrate how plants can potentially defend themselves against a wide variety of pathogens.
PhD Transfer Exam - Sara Pintwala (Peever lab)
PhD Transfer Exam
Thursday May 11th, 10:10 am – Ramsay Wright Building, Rm. 432
Sara Pintwala (Peever lab)
"Central nervous system cell transplants: a new model for disordered sleep"
Abstract
Narcolepsy is an autoimmune disorder affecting three million people worldwide. This disorder is characterized by degeneration of 85-95% of neurons in the lateral hypothalamus producing orexin, a neurotransmitter essential for arousal stability. This results in undetectable levels of orexin in the central nervous system (CNS) and the onset of debilitating symptoms such as excessive daytime sleepiness and a sudden loss of postural muscle tone, termed cataplexy. Presently there is no cure for narcolepsy. By the nature of this disorder it could be reasoned that reinstating orexin neurotransmission will lead to a recovery of function and behavior. Cell transplant experiments to cure disease have before proven successful, but are dependent on the availability of neurons of identical phenotype to the cells lost. Thus, the goal of my project is to investigate the character of three novel hypothalamic cell lines and to determine the consequences of transplantation of these neurons into the CNS in a mouse model of narcolepsy. First, I will characterize three novel hypothalamic, and putatively orexinergic, cell lines. Second, I will determine survival of these cultured neurons in the CNS post-transplantation. Thirdly, I will demonstrate reversal of the behavioural abnormalities associated with narcolepsy. Lastly, I will demonstrate the recovery of orexin neurotransmission in the CNS with microdialysis. I hypothesize that the hypothalamic cell lines described here are of an orexinergic phenotype, will survive transplantation into the CNS and reverse narcoleptic symptomatology. If these novel hypothalamic cell lines possess characteristics and morphology similar to that of their in vivo counterparts, their transplant and survival in the CNS will represent a novel therapeutic target for narcolepsy and cure this disorder. This proposal has significant implications for clinical interventions for narcolepsy, but also provides significant insight into the field of cell transplantation.
Ramsay Wright is a wheelchair accessible building.
PhD Transfer Examination - Dennison Trinh (Nash lab)
PhD Transfer Exam
Thursday May 4th, 2:10 pm – Ramsay Wright Building, Rm. 432
Dennison Trinh (Nash lab)
"Validation of Sirtuin 3 as a Potential Disease-Modifying Agent in Parkinson’s Disease"
Abstract
With an aging population that becomes more susceptible to Parkinson’s Disease (PD), the lack of a treatment that can halt or reverse the progression of the disease results in hardships for the affected individual, their families, and their communities. Physical symptoms of PD include tremor, rigidity, and posterior instability, with aggregates of misfolded α-synuclein and ubiquitin forming as Lewy bodies in the substantia nigra pars compacta (SNc) region of the brain leading to dopaminergic neuron loss. While PD can be caused through familial and environmental factors, central to its pathology is the dysfunction of mitochondria. Dopaminergic neurons, which are high energy demanding cells, require proper mitochondrial health to regulate energy production and stress responses that rely on protein acetylation. Sirtuin 3 (SIRT3), a member of the Sirtuin family of NAD+ dependent proteins, is the main deacetylase in the mitochondria. Due to the link between protein deacetylation and mitochondrial health, the role of SIRT3 as a potential disease-modifying agent in the pre-formed fibril (PFF) model of PD in rats was examined. Through evaluations using the cylinder test to determine forelimb asymmetry and unbiased stereology to quantify the number of dopaminergic neurons in the SNc, the current data suggests that SIRT3 has a neuroprotective role in the Parkinsonian rat model. SIRT3 overexpressing rats showed a reduction in unimpaired forelimb bias and a protective effect on dopaminergic neurons (-2.737 ± 5.318 % asymmetry and 2093 ± 637.5 cells compared to -7.212 ± 9.539 % asymmetry and 1562 ± 278 cells in parkinsonian animals). The beneficial effects of SIRT3 were lost in rats overexpressing deacetylation deficient SIRT3, where forelimb bias and the number of dopaminergic neurons was comparable to parkinsonian animals (35.96 ± 8.871 % asymmetry and 1320 ± 379.4 cells). Currently, SIRT3’s neurorestorative capabilities are being studied, which would provide crucial support for SIRT3 being able to reverse the progression of PD. Based on these observations, the next steps are to investigate the mechanism in which SIRT3 acts as a therapeutic agent and to test the use of high-intensity focused ultrasound (HIFU) as a less invasive gene delivery tool for SIRT3 overexpression. These studies would therefore lay the foundation for SIRT3 to be developed into a potential therapeutic agent in PD patients in the future.
Ramsay Wright is a wheelchair accessible building.
PhD Transfer Examination - Trisha Mahtani (Treanor lab)
PhD Transfer Exam
Friday January 27th, 3:10 pm – Room SW 403, University of Toronto at Scarborough
Trisha Mahtani (Treanor lab)
"The role of TRPV5 in B cell activation and signaling"
Abstract
B cells are the precursors to plasma cells which secrete antibodies that target foreign pathogen (antigen) within the body. This differentiation into plasma cells and antibody secretion begins with the activation of B cells through binding of B cell receptor (BCR) to specific antigen, which leads to formation of BCR clusters and initiation of downstream signaling cascades. One of the key second messengers is calcium, which activates many calcium dependent proteins involved in activation. Calcium ions enter the cell through ion channels on the plasma membrane, of which the best characterized are Calcium Release-Activated Calcium (CRAC) channels. However, B cells deficient in CRAC channel components mount normal antibody responses, suggesting that alternative channels may be important. Several members of the Transient Receptor Potential (TRP) ion channel family are calcium permeable, yet the function of many members of this family in the immune cell activation has not been characterized. To determine the members of the TRPV subfamily that are expressed in B cells, we performed a quantitative qPCR screen and found that TRPV2, TRPV4 and TRPV5 are expressed. TRPV5 is selectively permeable to calcium ions and has been shown to shuttle to the plasma membrane upon stimulatory cues in other cell types. We investigated whether this phenomenon occurs in B cells and found that upon BCR activation this channel polarizes to the side of the cell where BCR clusters localize. To examine whether TRPV5 is involved in calcium signaling during B cell activation, we used siRNA knockdown to reduce protein expression and found that this impaired signaling. Thus, our findings suggest that TRPV5 may play a role in the initial events of B cell signaling during activation. To further explore this novel finding, this project aims to characterize a TRPV5 knockout generated by CRISPR-Cas9. This work will help us understand the role of a novel regulator in B cell activation.
PhD Transfer Examination - Ian (Shen Yen) Hsu (Moses lab)
PhD Transfer Exam
Thursday December 1st, 10:10 am – Earth Sciences Centre, Room 3087
Ian (Shen Yen) Hsu (Moses lab)
"Understanding the Evolution and Mechanism of Pulsatility"
Abstract
Several transcription factors involved in important signaling pathways have been, recently, reported to show pulsatility. This phenomenon includes unsynchronized and aperiodic promoter activation of downstream targets via rapid shuttling of transcription factors between nuclei and cytoplasm. Different from a constant nuclear localization, the dynamics of these transcription factors show pulses across time. The similarities and differences between pulsatile transcription factors (PTFs) are unclear. Although the profiles of pulses of each PTF look intuitively different from each other, in principle PTFs sharing a common signaling pathway function may have similar pulse profiles. I hypothesize that the profile of pulses can be quantified and classified to a signalling pathway function. Crz1 is a PTF that responds to a stronger calcium stress with higher pulsing frequency (1). However, the genetic circuit that encodes the intensity of environmental stress into pulsing frequency is unclear. My preliminary results suggest that vacuolar calcium transporters are involved in regulating pulse frequency, and that the localization of Crz1 is regulated by a positive feedback and then a negative feedback. To explore the circuit further, I propose to screen for genes involved in the positive and negative feedbacks. I also hypothesize that the feedbacks are mediated by only post-translational modification because Crz1 pulses with a duration within a 10-minute scale, and this hypothesis predicts that truncated Crz1 can pulse without DNA binding domains. Two motifs that are essential for Crz1 to pulse are in a disordered region, a sequence of polypeptide that does not have secondary or tertiary structure and goes through rapid evolution. Finally, I hypothesize that the intrinsically disordered region of Crz1 plays a role in Crz1 pulsatility, and I propose to ectopically express Crz1 orthologs that contain diverged disorder regions in S. cerevisiae in order to investigate how the frequency and profile of pulses is affected. This work will facilitate our understanding of the mechanism and the evolution of pulsatility.
PhD Transfer Examination - Roxanne Fournier (Harrison lab)
PhD Transfer Exam
Tuesday October 11th, 10:10 am – Room SW 403, University of Toronto at Scarborough
Roxanne Fournier (Harrison lab)
"Osteocyte Form and Function in Loading and Unloading Environments"
Abstract
Bone is a dynamic tissue composed of a variety of specialized cells such as osteocytes, which are known to regulate bone remodeling due to their mechanosensitive abilities. In space, mechanical stimuli normally sensed by these cells are absent which is highly unfavourable for their functioning. We propose that spaceflight inhibits the osteocyte’s ability to sense mechanical forces through morphological changes to its cytoskeleton and functional changes to its mechanosensory pathways. This may support why astronauts experience greater bone loss than controls subjected to bed-rest on Earth. The mechanisms involved in this unique disease have not been elucidated and will therefore be the focus of this research. To achieve this objective, osteocytes will be studied in a 3D bone-like matrix in vitro to determine gene, protein and morphological changes in conditions simulating microgravity and will be compared to simulated bed-rest controls and dynamic loading controls. Additionally, transcriptome analyses of these three conditions will be assessed for novel therapeutic gene targets. As a result, we will have a better understanding of astronaut osteoporosis which can be used to establish mitigation strategies for future space missions.
PhD Transfer Examination - Amanda Miles (Tropepe lab)
PhD Transfer Exam
Tuesday July 26th, 10:10 am – Ramsay Wright Building, Rm. RW 432
Amanda Miles (Tropepe lab)
"Investigating the function of dmbx1 and znf644 through epigenetic interactions in the developing zebrafish retina"
Abstract
The developing zebrafish retina initially consists entirely of proliferating retinal progenitor cells (RPCs), which are multipotent cells capable of differentiating into any of the 6 main retinal cell types. The transition from a RPC to post-mitotic, differentiated retinal cell requires RPCs exit the cell cycle, and repress the expression of progenitor and cell cycle genes, including vsx2 and cyclinD1, respectively. However, we lack a detailed understanding of the molecular basis of how this transition is regulated. Two genes implicated in controlling RPC cell cycle exit are the homeobox transcription factor, dmbx1 and the atypical zinc finger protein, znf644. Research in our lab has shown that knockdown of either gene results in a similar phenotype, showing prolonged RPC proliferation, concomitant reduction of retinal differentiation, and upregulated expression of vsx2 and cyclinD1. Evidence indicates that znf644 represses these genes by physically interacting with the histone methyltransferase, g9a, to deposit the repressive H3K9me2 on the gene promoters. However, whether dmbx1 interacts and functions together with this epigenetic machinery remains unknown. My preliminary data, through combinatorial gene knockdown of dmbx1 with znf644 or g9a supports a genetic interaction among these genes. Additionally, znf644 and dmbx1 knockdown similarly show a reduction of H3K9me2 marks in the retina indicating that loss of either gene impairs epigenetic gene silencing. I hypothesize that dmbx1, znf644 and g9a genetically and physically interact to form a repressive complex that silences RPC genes to promote cell exit. To test this hypothesis my objectives are aimed at (1) understanding whether dmbx1 and znf644 function cooperatively during retinal development (2) characterizing the chromatin modification during retinal development in wildtype and dmbx1/znf644 gene loss of function embryos and (3) identifying protein-protein interaction and direct transcriptional targets of dmbx1. This knowledge will provide insight into the molecular basis of retinal development and into the aetiology of retinal disorders, since human DMBX1 and ZNF644 have been linked to pathological retinal growth defects.
Ramsay Wright is a wheelchair accessible building.
PhD Transfer Exam - June (Jee) Bang (Kim lab)
PhD Transfer Exam
Wednesday May 25th, 2:10 pm – Ramsay Wright Building, Rm. RW 432
June (Jee) Bang (Kim lab)
"Characterization of hippocampal inhibitory circuitry of stress response and anxiety behavior using optogenetics"
Abstract
Stress initiates the release of glucocorticoid hormones (GCs) by activating hypothalamic-pituitary- adrenal (HPA) axis which then triggers diverse adaptive physiological and behavioral responses. During emotionally stressful experience, the ventral hippocampus (vHPC) is believed to attenuate the HPA-axis activity by indirectly inhibiting the paraventricular nucleus of the hypothalamus (PVN). While much effort has been made to demonstrate the inhibitory influence of vHPC on the HPA-axis during psychogenic stress, the underlying neural pathway has not been directly examined. Using the pathway specific optogenetic approach in mice, we activated vHPC inputs at the anterior hypothalamic nuclei (AHN) during a 30 min- physical restraint stress and examined its effects on stress-induced anxiety behaviours in the elevated plus maze, the successive alleys, and open field tests. We also tested whether this input can change basal anxiety level by activating the same pathway in freely moving mice with no prior stressor during the same anxiety tests. Our findings suggest that the predominantly GABAergic AHN is the functional intermediary structure through which vHPC exerts its inhibitory control on both anxiety behaviors and physiological responses such as respiration and heart rate. Together, these results show an important top-down modulation of stress response and anxiety level by the hippocampal-hypothalamic pathway as a key element in the central feedback of HPA-axis and inhibitory regulator of negative affect. We aim to further confirm our findings by measuring circulating corticosterone (CORT) level, optogenetically inactivating the vHPC-AHN pathway and by testing whether vHPC-AHN activation can rescue deleterious psychotic states such as anxiety disorder and depression-like behaviors manifested by chronic CORT exposure in mice.
Ramsay Wright is a wheelchair accessible building.
PhD Transfer Exam - Eliana Vonapartis (Gazzarini lab)
PhD Transfer Exam
Wednesday May 11th, 12:10 pm – Room SW 403, University of Toronto at Scarborough
Eliana Vonapartis (Gazzarrini lab)
"Investigating the Role of XERICO in GA-ABA Crosstalk During Germination and Stress Response"
Abstract
Hormones regulate plant growth and development, and allow for these sessile organisms to effectively sense changes in their environment. Complex hormone crosstalk networks modulate plant response to endogenous signals and external stressors. A well-known instance of crosstalk occurs between the two antagonistic hormones gibberellin (GA) and abscisic acid (ABA) during developmental phase transitions and unfavourable growth conditions. XERICO (XER) is a DELLA-induced putative RING E3 ubiquitin ligase that increases intracellular ABA levels through a yet unexplored molecular mechanism. The aim of my research is to understand the role of XER in GA-ABA crosstalk during stress response and germination, a phase transition that is tightly regulated to ensure seedling survival. To this end, putative XER substrates have been identified via a high-throughput yeast two-hybrid screen using a cDNA library of genes differentially regulated by ABA. I have also isolated potential upstream regulators from an Arabidopsis transcription factor library using a yeast one-hybrid approach. Preliminary data suggests that XER expression is regulated by stress-responsive transcription factors and that XER may modulate enzymes involved in hormone metabolism. Furthermore, as a crucial step in understanding the role of XER in planta, I analyzed its subcellular localization by tagging it with fluorescent proteins. Transient assays indicate that this protein is targeted to the cytoplasm, chloroplast envelope and possibly to other organelles. In addition, I have generated constitutive and inducible overexpression lines to study its function at the molecular level in future experiments. This project will provide important insight as to how a chloroplast-localized E3 ligase integrates the GA and ABA signaling pathways, and how plants maintain a delicate balance between growth and development to promote survival during instances of environmental stress.