PhD Proposal Exam - Anna Van Weringh (Provart lab)
PhD Proposal Exam
Wednesday April 27th, 2:10 pm – Earth Sciences Centre, Rm. ESC3087
Anna van Weringh (Provart lab)
"Characterization of Drought Responsive Transcriptional Programs in Guard Cells"
Abstract
During drought, guard cells (GCs) are signaled to close stomatal pores to reduce water loss, necessary for survival during prolonged dry periods. As the site of gas exchange for photosynthesis, stomatal aperture is also a key determinant of plant productivity and water use efficiency. Thus, GCs are a topic of great study for crop improvement. My PhD research aims to tackle the gene regulatory programs induced within the GCs of Arabidopsis thaliana during drought by profiling the dynamic transcriptomic and epigenomic changes in GCs over the course of a drought, using RNA-seq and ATAC-seq respectively. The INTACT (Isolation of Nuclei Tagged in Cell-Types) system expressed under a GC-specific promoter will be used to sample nuclei from GCs during a time course during mild, moderate and severe drought. To identify the drought responsive transcription factors driving these changes, a YIH approach will be taken to determine which transcription factors (TF) can bind the promoters of differentially expressed genes. Y1H experiments provide TF-DNA interactions outside of the native context, yet chromatin accessibility shapes which target genes of a TF are activated at a given moment. Therefore, the network of Y1H connections will be filtered by chromatin accessibility and genes expressed at each time point. The functional contribution of TFs to GC behaviour during drought will be assessed by generating Arabidopsis plants with GC-specific knock-down or overexpression of identified TFs, and regulatory models will be tested by examining gene expression patterns in these mutants.
PhD Proposal Exam - Zahra Dargaei (Woodin lab)
PhD Proposal Exam
Wednesday April 27th, 10:10 am – Ramsay Wright Building, Rm. RW432
Zahra Dargaei (Woodin lab)
"Aberrant Chloride Homeostasis and Inhibitory Synaptic Transmission in Huntington's Disease"
Abstract
Proper GABAA-mediated synaptic inhibition requires low levels of neuronal Cl- that is mainly achieved by the K+-Cl- cotransporter, KCC2. When KCC2 expression decreases, the neuronal Cl- gradient collapses and there is a profound reduction in synaptic inhibition. Reduced inhibition can reduce the inhibition-excitation balance, which can contribute to the development and symptoms of neurological disorders such as epilepsy. Huntington's disease (HD) is a progressive hereditary brain disorder. It is a devastating disease for which there is currently no effective treatment. Early signs of the disease include depression, uncontrolled movements, and loss of memory. The disease is caused by mutations in the Huntingtin protein (Htt). Recent studies have screened for Htt interacting proteins and demonstrated a map of the Htt protein’s cellular partners. Interestingly, the proteomic interactome of Htt revealed that the KCC2 encoding gene, Slc12a5, is highly enriched in the Htt proteome, and this interaction appears to decrease when Htt is mutated. However, despite the strong correlation between KCC2 and Htt, this interaction and the possible role of KCC2 in HD has not been examined experimentally. Based on the critical role of KCC2 in neurophysiological function, and the reported decease in KCC2 interaction with Htt in HD, I propose that KCC2 function is compromised in HD brain. Thus, the focus of my PhD project is to determine whether KCC2 interacts with Htt and investigate whether this interaction is associated with HD pathology using a combination of biochemical, imaging, and electrophysiological assays in both mammalian cell expression systems and transgenic mice. Characterizing KCC2: Htt interaction will be the first study to demonstrate a clear link between KCC2 and neurodegenerative disease and could reveal novel molecular targets that can be used therapeutically to enhance KCC2 function and possibly restore aberrant synaptic transmission in HD brain.
Ramsay Wright is a wheelchair accessible building.
PhD Proposal Exam - Gurdeep Singh (Mitchell lab)
PhD Proposal Exam
Monday April 25th, 2:10 pm – Ramsay Wright Building, Rm. RW432
Gurdeep Singh (Mitchell lab)
"Identifying Tissue Specific Transcriptional Regulatory Elements in Mammalian Genomes"
Abstract
The non-coding region in mammalian genomes ranges between 97-99% and much of its function is yet unknown. Enhancers are one of the major components of the complex non-coding genome which regulate gene expression in a tissue specific manner. These regulatory elements are challenging to identify and characterize their function because of their variable locations, tissue-specific on-off status and inefficiency of indirect methods leading to thousands of inactive enhancer-like regions. STARR-seq is a genome-wide active enhancer identification and strength validation method, primarily explored on the Drosophila genome so far. My analysis suggests that regions bound with higher number of transcription factors (TFs) from a specific set, have higher enrichment of enhancer features, H3K27ac & p300 and drive higher expression of target genes. I hypothesize that identification of a positive set of tissue-specific enhancers by STARR-seq will lead to the identification of a high confidence set of tissue-specific TFs and features for better enhancer prediction modeling. The proposed objectives to address my hypothesis are first, to optimize the STARR-seq method for enhancer identification in mouse embryonic stem cell (mES); second, to apply it in a genome-wide manner to identify positive enhancers active in 3 cell types namely mES, mouse trophoblast stem cells (mTS) and mouse embryonic fibroblasts (mEFs); and third, to use these positive enhancers to identify the set of TFs binding them using available ChIP-seq and TF binding motif data. Furthermore, ChIP-seq of suspected novel TFs involved in mES pluripotency will be performed to upgrade the mES specific TF set for determining MTLs. My preliminary data from a mammalian STARR-seq experiment in mES using mouse 38 BACs suggests that it gives higher background signal as compared to Drosophila STARR-seq. This is because of strong basal activity of SCP1 promoter used in mammalian STARR-seq and hence will be replaced with minimal promoter for STARR-seq optimization. The identification of active enhancers and novel TFs in general is essential to further our understanding of genome function across diverse cell types and to better understand development, disease and species evolution.
Ramsay Wright is a wheelchair accessible building.
PhD Proposal Exam - Sahara Khademullah (Woodin lab)
PhD Proposal Exam
Thursday April 21st, 12:10 pm – Ramsay Wright Building, Rm. RW432
Sahara Khademullah (Woodin lab)
"Inhibitory Synaptic Transmission and KCC2 Function in the Motor Cortex of the Presymptomatic ALS Mouse"
Abstract
A balance between synaptic excitation and inhibition is essential for normal brain function. When this delicate balance is disrupted, it can lead to neuronal hyperexcitability, resulting in alterations in neuronal network activity and the onset of various neurological disorders, such as epilepsy, autism spectrum disorder and schizophrenia. Recent studies have shown the presence of an imbalance between excitation and inhibition in amyotrophic lateral sclerosis (ALS) as well. Thus, there is an intense need to determine how this equilibrium is maintained and what triggers its disarray. ALS (a.k.a. ‘Lou Gehrig's disease’) is a fatal neurodegenerative disease characterized by rapid progressive muscle weakness and atrophies that affects both upper and lower motor neurons. Following the initial onset of symptoms, approximately 50% of cases are fatal within the first 1.5 years and in about 20% of the remaining cases, the disease persists for approximately 5 to 10 years until the patient dies. Recent studies performed in the SOD1G93A familial mouse model of ALS have observed reductions in KCC2 membrane expression, a key regulatory protein of inhibition, in spinal cord motor neurons. Similar studies in the SOD1G93A mouse model have also found an increase in network hyperexcitability in presymptomatic cultured spinal cord motor neurons. Thus, the focus of my PhD project is to determine if hyperexcitability in the primary motor cortex (PMC), where many patients with ALS present predominant neurodegeneration, results in a reduction in the strength of inhibitory synaptic transmission. I will also investigate whether this reduction in synaptic inhibition is associated with KCC2 expression. Evidence from this study may provide novel insights into the pathogenesis of ALS.
Ramsay Wright is a wheelchair accessible building.
PhD Proposal Exam - Purva Karia (Yoshioka lab)
PhD Proposal Exam
Tuesday April 12, 10:10 am – Earth Sciences Building, Rm. ESC 3087
Purva Karia (Yoshioka lab)
"Investigation of the molecular mechanism of triphosphate tunnel metalloenzymes in Arabidopsis"
Abstract
Triphosphate tunnel metalloenzymes (TTMs) comprise a superfamily of enzymes that hydrolyze organophosphate substrates. Members of this superfamily are found across taxa, and the Arabidopsis thaliana genome encodes three TTM genes (AtTTMs). Unlike other members of this family, two of these isoforms, AtTTM1 and 2, hydrolyze pyrophosphate in vitro, making them the only TTMs characterized so far to possess pyrophosphatase activity. However, despite their high sequence identity, genetic analyses showed them to have different biological functions. AtTTM2 plays a role in pathogen resistance whereas AtTTM1 is involved in dark-induced leaf senescence. The objective for my PhD thesis project is to determine the molecular mechanism underlying the biological functions of AtTTMs. As a logical first step, I identified the involvement of AtTTM1 in natural senescence indicating that AtTTM1 plays a crucial role not only in dark-induced senescence but also in natural senescence signaling in Arabidopsis. To gain insight into their molecular mechanisms, AtTTM proteins were determined to likely localize at the mitochondrial outer membrane. Furthermore, we are exploring the connection between autophagy and AtTTM related phenotypes since autophagy is an important degradation process that regulates cell death during both senescence and pathogen resistance. More specifically, it is possible that AtTTMs are involved in mitophagy, a mitochondrial degradation process, given the subcellular localization of AtTTMs. Both transcriptional and phosphoproteomic data also suggest a connection between AtTTM1 and ABA responses. Thus, we are also investigating the connection between AtTTM1 and ABA signaling components. Finally, we are assessing whether the documented pyrophosphatase activity is required or sufficient for the in vivo function of AtTTMs. Overall, this project will provide insight into the molecular mechanisms governing AtTTMs functions and may establish novel connections between autophagy, ABA or pyrophosphatase activity and senescence and/or pathogen defense.
PhD Proposal Exam - Ahmed Hamam (Kronzucker lab)
PhD Proposal Exam
Thursday March 10, 12:10 pm - Room MW 229, University of Toronto at Scarborough
Ahmed Hamam (Kronzucker lab)
"A Critical Re-examination of Sodium Transport and Its Role in Salinity Stress in Plants"
Abstract
Soil salinity is a major threat to agriculture, affecting more than 6% of the world’s total land area. NaCl is the most soluble and widespread salt on earth, and at high cytosolic concentrations, is considered metabolically toxic. For decades, researchers have been investigating the pathways of Na+ transport and its distribution in plants, especially at the point of contact, the root surface, but the underlying mechanisms and pathways remain poorly resolved. According to the current proposed model, Na+ enters the roots passively down its electrochemical gradient at very high rates, and then actively pumped out of the cytosol at nearly the same rate. This model of rapid Na+ cycling has been called into question, as the energy requirement to mediate such high fluxes is unfeasible, and evidence from electrophysiological and radiotracer measurements are conflicting. The aim of this project is to critically re-examine the credibility of this model, by systematically comparing radiotracer and electrophysiological recordings in the model systems barley (Hordeum vulgare), rice (Oryza sativa), and Arabidopsis thaliana. Based on my progress to date, it would appear that most of the reported Na+ fluxes are a gross overestimation of symplastic flow, and more likely to be predominately apoplastic in nature.