PhD Exit Seminar - Heather Wheeler- Monday, November 20th, 2017

PhD Exit Seminar

Monday November 20^th, 2:05 pm – Ramsay Wright Building, Room 432

Heather Wheeler (Campbell lab)

Cellulose Biosynthesis has Extensive Impacts on Overall Plant Metabolism, Which are Mediated in Part by Pentaglycine, a Novel Peptide Signal

Abstract

As sessile organisms, plants must continually adapt to their environment. In order to allocate resources appropriately, plants have elaborate pathways in place for detecting and responding to changes in their environment and the integrity of their cells, particularly the cell wall. Cellulose is the major component of plant cell walls and a major sink for carbon in the plant body. Cellulose is central to plant structure and function, imparting sufficient strength and resilience to withstand turgor pressure, negative pressure, and gravity. It was therefore hypothesized that plants have broad, far-reaching metabolic responses to cellulose disruption.

To uncover the response to cellulose perturbation at the metabolic level, Comprehensive Multiphase-Nuclear Magnetic Resonance (CMP-NMR) was used in combination with a mutant analysis and pharmacological experimentation. This approach revealed several metabolic impacts of cellulose perturbation that were previously unknown, including decreased metabolism of seed lipid stores during germination, increased production of methanol and ethanol in seedlings, and the presence of pentaglycine in seedlings.

Another line of investigation revealed that a compound or compounds present in the cellulose synthase mutant eli1 was able to increase lignin content of wild-type seedlings. Pentaglycine had a similar effect on WT seedlings. Taken together with the increased abundance of pentaglycine in eli1 seedlings, the results suggest that pentaglycine is in part responsible for the increased lignin content that characterizes cellulose synthase mutants. A model is presented that integrates these components into a signaling pathway to monitor and respond to perturbation in the cellulosic component of cell wall integrity.

by Genna Zunde

PhD Exit Seminar- Shalabh Thankur (Guttman lab)

PhD Exit Seminar

Wednesday, September 13, 2017 at 2:10 pm, Earth Sciences Building, Room 3087

Shalabh Thakur (Guttman Lab)

“Comparative and Evolutionary Genomics of Pseudomonas syringae"

Abstract

The Pseudomonas syringae species complex comprises many genetically diverse strains ubiquitously found in both agricultural and non-agricultural environments. The species complex has a very broad host range; however, distinct strains show strong host specificity and are able to cause disease on limited crops. Although more popularly known as a plant pathogenic bacteria, many P. syringae strains are reported to be non-pathogenic and found in habitats linked to water sources, soil, and snow fields. Multi-locus sequence typing studies have sub-divided the P. syringae complex into at least 13 different subgroups referred as phylogroups. Given such extensive genetic and ecological diversity within the species complex, there is an ongoing debate over the species definition of P. syringae strains. My research work investigated whether strains within the P. syringae species complex belong to a single species population or if distinct phylogroups are in fact different species. We performed a whole-genome analysis of approximately 400 P. syringae strains using various comparative and evolutionary approaches to examine the extent of genetic cohesion within the P. syringae species complex due to various ecological and evolutionary mechanisms. The comparative genomics projects often face a computational challenge due to quadratic increase in time and the resources needed for the pairwise sequence comparisons with the increase in number of sequenced genomes. To overcome this challenge and facilitate large-scale sequence comparisons between hundreds of closely related prokaryotic genomes, we designed a novel comparative genomic pipeline named DeNoGAP. The DeNoGAP pipeline provides a robust computational pipeline for performing various comparative genomics tasks, such as gene prediction, ortholog prediction, functional annotation, and analysis of a pan-genome. DeNoGAP implements an iterative homolog clustering strategy to increase speed and accuracy for large-scale ortholog prediction analysis. Because of this strategy, DeNoGAP outperforms the efficiency of other ortholog prediction tools that implement traditional pairwise comparison algorithms. Our whole-genome comparative analysis of more than 400 strains shed insight into the P. syringae pan-genome. We found that the P. syringae pan-genome is big and diverse, comprising more than 79,000 gene families. We also found substantial diversity in the distribution of virulence-associated gene families, such as type III secreted effectors and toxins, across P. syringae strains. Evolutionary analyses of the gene families in the P. syringae pan-genome showed evidences of homologous recombination and positive selection across entire genomes of P. syringae strains. We found that the P. syringae strains in different phylogroups rarely exchange genes via homologous recombination. However, despite being rare, inter-phylogroup homologous recombination occurs disproportinately among virulence-associated and positively-selected genes that are essential for ecological adaptation and evolution of strains within the P. syringae species complex. Based on these findings, we hypothesized that P. syringae maintains genetic cohesion between its divergent strains due to an exchange of ecological and evolutionarily relevant genes. Together, my work provides a robust computational pipeline for large-scale comparative genomics projects and sheds insight into species definition of the P. syringae species complex based on strong evolutionary species concepts rather than molecular methods.

by Genna Zunde

PhD Exit Seminar - Raphaël Brisset Di Roberto

PhD Exit Seminar

Tuesday August 29^th, 1:10 pm – Ramsay Wright Building, Room 432

Raphaël Brisset Di Roberto (Peisajovich/ Chang lab)

“Evolutionary alternatives on the road to a new specificity in a G protein-coupled receptor”

Abstract

Cells sense change in their external environment and react appropriately through the action of signaling pathways. This process is initiated by receptor proteins with a high degree of specificity for a particular stimulus. G protein-coupled receptors form the largest family of membrane protein receptors and their ability to sense a broad variety of ligands is unparalleled despite their common ancestral origin. How GPCRs evolved their unique specificities is unknown, although ligand binding affinity is often given a central role. The goal of this thesis was to assess the possible contributions of secondary mechanisms of specificity, namely ligand efficacy and downstream signaling regulation, to changes in ligand recognition. Through directed evolution, we generated a yeast pheromone receptor with an altered specificity in two steps. First, promiscuous receptors were obtained through either improved binding affinity or weaker molecular interaction with a negative regulator of signaling. Second, a ligand-discriminating receptor was obtained from a promiscuous variant solely by reducing the efficacy of the native pheromone. These findings demonstrate the importance of assessing GPCRs’ pharmacological profiles in their native context, where signaling trumps binding affinity in significance.

by Genna Zunde

PhD Exit Seminar - Sophie St-Cyr (McGowan lab)

PhD Exit Seminar

Wednesday August 16^th, 3:00 pm – SW 403 – University of Toronto at Scarborough

Sophie St-Cyr (McGowan lab)

“Maternal Programming of Adult Rodent Integrative Phenotype by Prenatal Exposure to Predator Odour"

Abstract

Prenatal stress mediated through the mother can program the long-term phenotype of the offspring. The capacity for adaptation to adversity in early life depends in part on the life history of the animal. It is therefore likely that an early life ethologically relevant psychogenic stressor that has been present over evolutionary times could prime responses to an environment containing this stress via epigenetic mechanisms such as DNA methylation modifications. Pregnant C57BL/6 mice and, separately, Long-Evans rats were exposed daily to unpredictable and inescapable predator odors or distilled water control over the second half of pregnancy. I examined the effect of the prenatal predator odour exposure on the integrative phenotype of adult offspring at the level of behaviour, physiology, endocrinology, transcription and DNA methylation. Prenatally predator odour-exposed offspring exhibited an overall increase in stress-related behaviours on a variety of commonly-used and semi-naturalistic assessments of the response to stress, as well as modifications of energy consumption at baseline and under stress. These changes were accompanied by a sex-specific increase in endocrine responses to stress and an increase in circulating thyroid hormone. Additionally, I observed modifications in stress-related transcript abundance at birth and in adulthood accompanied by DNA methylation modifications in adulthood. Overall, assessments of the integrative phenotype of prenatal predator odour-exposed animals indicate a persistent increase in stress responsiveness across a variety of experimental conditions and phenotypic levels in the two rodent species. This phenotype supports the hypothesis that maternal programming allows developmental forecasting that shapes the individual developmental trajectory. Prenatal predator odour is therefore a potent programming stressor.

by Genna Zunde

PhD Exit Seminar - Stephanie Marie Prezioso (Christendat lab)

PhD Exit Seminar

Tuesday May 2^nd, 2:10 pm - Ramsay Wright Building, Rm. 432

Stephanie Marie Prezioso (Christendat lab)

"Identifying the Functions and Regulatory Mechanisms of Shikimate Dehydrogenase Homologs in Bacteria"

Abstract

The shikimate pathway is required for synthesis of the aromatic amino acids as well as other aromatic compounds in bacteria. The prototypical member of the shikimate dehydrogenase (SDH) enzyme family, AroE, catalyzes the fifth step in this pathway; the reduction of dehydroshikimate to shikimate. In addition to AroE, four other SDH family members have been identified: YdiB, Ael1, RifI, and SdhL. However, the metabolic functions of Ael1, RifI, and SdhL are poorly characterized, and few studies focus on the regulation of SDH enzymes. This thesis investigates the biological functions of the SDH family of enzymes, and explores the systems that govern their expression in bacteria.

We use Pseudomonas putida to investigate the functions of the SDH family of enzymes, as it has one representative homolog of each family member. The differences in expression profiles observed among these SDH homologs in P. putida, along with existing kinetic data, suggest that each homolog performs a distinct biological role related to the shikimate pathway. Additionally, we identified multiple novel metabolic disturbances due to SDH disruptions in P. putida that allow us to propose putative roles for the SDH enzymes in bacterial metabolism.

The quinate utilization operons in Listeria monocytogenes encode two SDH proteins among other enzymes that act in the quinate/shikimate utilization metabolic pathway. These operons are activated by a LysR Type Transcriptional Regulator (LTTR), called QuiR1, in response to shikimate. The structure of the QuiR1 effector binding domain with shikimate bound reveals overall LTTR structural conservation as well as the architecture of the shikimate binding site. This work sheds light on the mechanism of transcriptional regulation of the genes encoding enzymes involved in metabolism of the plant derived compound quinate, as well as shikimate, in L. monocytogenes. This intricate regulation may allow for an optimized metabolic response to changing environmental conditions.

Ramsay Wright is a wheelchair accessible building.

by previous undergrad

PhD Exit Seminar - Francisco Rodrigues (Harris lab)

PhD Exit Seminar

Thursday January 19^th, 1:10 pm - Ramsay Wright Building, Rm. 432

Francisco Rodrigues (Harris lab)

"The Arf GAP Asap regulates cleavage furrow biosynthesis in the early Drosophila embryo"

Abstract

Plasma membrane (PM) growth is a common feature of eukaryotic cells. For cells to grow and divide newly synthesized membrane needs to be inserted into their existing PM. Failure to do so may lead to developmental disorders and disease in animals. In the early Drosophila embryo, ingression of cleavage furrows during syncytial divisions is fueled by the insertion of new membrane into the apical domain via the biosynthetic secretory pathway. The highly conserved small G protein Arf1 regulates biosynthetic trafficking at the Golgi. Arf1 activity is regulated by Arf guanine nucleotide exchange factors and GTPase activating proteins (GAPs). However, the essential roles of Arf GAPs during development remain unclear. Here I demonstrate that the Arf GAP Asap is required for cleavage furrow ingression in the early Drosophila embryo. Asap primarily promotes Arf1 function at the Golgi. Asap is required and sufficient for Arf1 accumulation at the Golgi, and a conserved Arf1-Asap binding site is required for Golgi organization and output. Interestingly, Asap shifts localization during the cell cycle, and relocalizes to the nuclear region at metaphase. This shift coincides with a mild reorganization of the Golgi, and a natural phase of furrow regression. Together these data reveal an Asap-Arf1 regulatory pathway required for Golgi-dependent cleavage furrow biosynthesis. Asap may recycle Arf1 to the Golgi from post-Golgi membranes, providing optimal Golgi output for specific stages of the cell cycle. The results of this study may further our understanding of Asap function in cancer and other diseases.

Ramsay Wright is a wheelchair accessible building.

by previous undergrad

PhD Exit Seminar - Rex Shun Chiu (Gazzarrini lab)

PhD Exit Seminar

Friday January 13^th, 9:10 am – Room SW 403, University of Toronto at Scarborough

Rex Shun Chiu (Gazzarrini lab)

"Regulation of Seed Germination at High Temperature in Arabidopsis thaliana"

Abstract

Correct timing of the transition from embryonic to post-embryonic development, which in plants coincides with the break of seed dormancy and the onset of germination, is critical for plant survival. Endogenous signals and environmental cues dictate the dormancy state of the seed and the timing of germination. Two hormones play a pivotal role in regulating this phase transition; the stress hormone abscisic acid (ABA) promotes dormancy and inhibits germination, while gibberellins (GA) stimulate germination. Previously, it was shown that unfavorable environmental conditions, such as supra-optimal temperature, stimulate ABA metabolism to induce secondary dormancy and inhibit germination. In this study, we characterized the mechanisms that regulate seed responses to high temperature. Microarray analysis revealed that the seed transcriptome is highly altered during prolonged heat stress. Genes involved in hormone metabolism and signaling, stress tolerance and seed maturation were enriched, a subset of which are targets of B3 domain transcription factors such as FUSCA3 (FUS3). We showed that FUS3, a master regulator of seed maturation and dormancy, is degraded during germination at optimal temperature. However, FUS3 degradation is inhibited in seeds imbibed at high temperature due to the high ABA/GA. Overexpression of FUS3 increases seed sensitivity to high temperature by inducing de novo ABA synthesis, and confers greater seedling survival upon exit from heat stress.

During supra-optimal temperature imbibition, the activity of the ubiquitin proteasome system (UPS) is suppressed in an ABA-dependent manner; both protein ubiquitination, and proteasome activity are strongly decreased. Similarly, UPS function is inhibited in seeds exposed to ABA at optimal temperature, but only within 2 days post-germination, a developmental window that governs seed sensitivity to ABA. Inhibition of proteasome activity arrested germination of excised, matured embryos, suggesting that protein degradation is required for germination. Supra-optimal temperature or ABA treatment do not alter transcription of most UPS genes, instead the ratio of various proteasomal complexes changes under these conditions, suggesting that ABA and high temperature affect proteasome activity posttranslationally.

This study shows that supra-optimal temperature reshapes the transcriptome of imbibed seeds to induced stress and seed maturation genes, and changes in hormones metabolism leading to an increase in ABA level. A high ABA level inhibits UPS activity, leading to the accumulation of dormancy and stress regulators, ultimately inducing thermoinhibition. These findings provide a novel perspective on how hormones integrate environmental cues to regulate germination through modulation of UPS function.

by previous undergrad

PhD Exit Seminar - Jamie Waese (Provart lab)

PhD Exit Seminar

Wednesday January 11^th, 2:10 pm – Earth Sciences Centre, Rm. 3087

Jamie Waese (Provart lab)

"Data Visualization Tools for Large Biological Data Sets"

Abstract

Researchers have access to an ever-growing volume of data available at multiple levels of biological analysis. Many visual analytic tools have been developed to display a variety of biological data types but many of these tools are challenging to use and only examine one biological level of analysis at a time. The development and testing of hypotheses is difficult when the information is hard to integrate and laborious to interpret. The application of data visualization principles and user experience design best practices could improve systems biology research workflows by providing visual analytic tools with what is known in the information visualization community as a “transparent” user interface. This thesis consists of four papers that explore two central questions: 1) What is the best way to represent biological information at different levels of analysis? and 2) How do we enable researchers to explore and interact with their data as naturally and intuitively as possible? The first paper describes, ePlant, a tool for visualizing multiple levels of data that was developed using an agile process that included several rounds of user testing. The second paper presents Gene Slider, a tool for visualizing the conservation and entropy of orthologous DNA and protein sequences using a data visualization paradigm that takes better advantage of preattentive visual processing than current methods. The third paper describes Topo-phylogeny, a tool for visualizing phylogenetic relationships using a topographic map visualization paradigm that requires less cognitive processing to interpret than traditional tree diagrams. The final paper demonstrates the importance of user testing when developing a “rapid serial visual presentation” interface for identifying genes of interest using electronic fluorescent pictographs. Together these papers illustrate the complexities and benefits of applying data visualization principles and user experience design best practices to building data visualization tools for the analysis of large biological data sets. Given that hypothesis generation is fundamentally a creative process, any tools or techniques that can help researchers consider their data at a deeper level should be valuable to the scientific community.

by previous undergrad

PhD Exit Seminar - Alex Sin (Harrison lab)

PhD Exit Seminar

Tuesday December 13^th, 2:10 pm – Room SW 403, University of Toronto at Scarborough

Alex Sin (Harrison lab)

"Growth of the Mammalian Golgi Apparatus During Interphase"

Abstract

The Golgi apparatus is an important processing station in all eukaryotic cells, where proteins and lipids are glycosylated and sent to their final destinations. For this reason, it is expected that the Golgi apparatus must be duplicated during the cell cycle to ensure subsequent daughter cells have sufficient Golgi content following mitosis. In mammalian systems, extensive research has been focused on the Golgi apparatus’ inheritance process during mitosis, yet research investigating its growth during interphase has been limited. Challenges in monitoring mammalian Golgi growth have been attributed to its elaborate ribbon structure, thus any theories of its growth have mostly been based on observations in lower eukaryotes. The aims of my thesis were to definitively characterize the growth of the mammalian Golgi apparatus during interphase, and elucidate the underlying mechanisms regulating this process. To circumvent the challenges associated with the intricacies of the mammalian Golgi structure, I employed a combination of flow cytometry, fluorescent microscopy and transmission electron microscopy to measure different aspects of its growth. Here I demonstrate the growth of the mammalian Golgi apparatus in its protein content and volume during interphase. Through ultrastructural analyses, physical growth of the mammalian Golgi apparatus was revealed to occur by cisternal elongation of the individual Golgi stacks contrary to previous speculations. By examining the timing and regulation of Golgi growth, I establish that Golgi growth starts after passage through the cell growth checkpoint at late G1 phase and continues growing in a manner highly correlated with cell size growth. Finally, by identifying S6 kinase 1 as a major player in Golgi growth, I reveal the coordination between cell size and Golgi growth via activation of the protein synthesis machinery in early interphase.

by previous undergrad