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Identification of the epigenetic machinery regulating the differentiation block in acute myeloid leukemia. 

David Bellamy, Biochemistry, Supervised by Dr. Yang Shi
 

Although certain subtypes of leukemia have experienced a striking improvement in their prognosis over the past several decades, many remain resistant to current methods of treatment and require novel therapeutic interventions to improve patient outlook. One such example is the acute myeloid leukemia (AML) group of subtypes, which accounts for approximately 80% of all adult leukemias. It has been established that AML symptoms are largely due to an abnormal accumulation of immature, undifferentiated progenitor cells in the myeloid lineage along the hematopoietic differentiation cascade. As a result, treatments that induce the differentiation of these progenitor cells into more mature forms have strong therapeutic potential. Promising results have already been observed in the treatment of the acute promyelocytic leukemia (APL) subtype of AML with the use of the retinoid known as all-trans retinoic acid (ATRA) in combination with induction chemotherapy. Unfortunately, non-APL AML patients have proven to be mostly resistant. Since the process of differentiation, through which a cell changes its identity, is an epigenetically controlled process, past studies have attempted to identify chromatin-associated proteins (referred to as readers, writers, and erasers) that unlock this elusive myeloid differentiation blockage. Perhaps most notably of which was the discovery that the mono- and di-methyl lysine demethylase, LSD1 (also KDM1A), markedly increases ATRA responsiveness of human non-APL AML. In this study, we present the findings of a high-throughput RNAi screen of 380 chromatin readers and the differentiation phenotypes observed for the top candidate epigenetic machinery. SND1, for example, generated the largest, most robust differentiation phenotype and is being characterized further with the potential of becoming a novel therapeutic target for differentiation therapies of non-APL AML.

 

 

Understanding the effects of the G9a protein inhibitor UNC0638 on the retroviral transgene HSC1 inserted into mouse E14 stem cells.

Jason CS Kwan, Chemical Biology/Stem Cells, Supervised by Dr. Sylvie Rival-Gervier 

Jason CS Kwan, Flora Girard, Bertrand Pain, Sylvie Rival-Gervier

In embryonic stem cells, retroviral vectors are silenced by epigenetics mechanisms, which have been interpreted as a way for pluripotent cells to be protected from retroviral invasions. Silencing of retroviruses still remains a major concern for many applications such as gene and cell therapies; recombinant protein productions; or human disease models using stem cells. It has been shown that methylation of lysine 9 of histone H3 is associated to retroviral silencing. Two enzymes, G9a and Setdb1 are known to drive these epigenetic modifications and both work to maintain silencing. Currently, mechanisms involved in the establishment of silencing remain poorly understood since silencing is unpredictable and its kinetics is dependent on the integration site of the vector and the level of cell pluripotency. In the lab, a vector where silencing is inducible is used (Rival-Gervier S et al., 2013). Our goal here is to use this system to analyze whether G9a is involved in the establishment of SIN retroviral vector (HSC1) silencing. This inducible silencing retroviral vector was inserted into mouse embryonic stem cells (E14Tg2A). After inducing silencing, the cells were cultured in the presence or absence of UNC0638, a specific G9a inhibitor. Two concentrations of 150 and 300 nM were added to the media to determine if any effects were dose dependent. Flow cytometry was performed to determine the frequency and level of expression of green fluorescent protein (GFP), reporter gene contained within the retroviral vector and the expression of cell surface marker SSEA1, specific to pluripotent stem cells. Within all population, GFP expression decreased over a period of time. However, in comparison to the control, cells that are exposed to UNC0638 have a greater decrease in level of expression of GFP. Results also show an inverse relationship exists between level of expressions of SSEA1 and GFP in cells exposed to UNC0638. The results suggest that the inhibition of G9a with UNC0638 causes an increase in the rate of establishment of silencing that could be explain by an increase in the level of pluripotency in E14Tg2A cells. If these results are confirmed, UNC0638 could be a suitable chemical to improve gene therapy and to help in enerating pluripotent stem cells.

 

 

Extraction du fluor dans l’eau des milieux arides à l’aide de la précipitation. 

Laurie-Anne Roy, Chimie, Supervisé par Dr. David L. Bryce et Mme Karine Major

 

Dans certaines régions du monde, la concentration de fluor dans l'eau potable atteint 20 mg/L. La consommation prolongée de cette eau, souvent la seule à la portée des populations locales, pose problème en ce qui concerne la santé dentaire et bien plus sérieusement en ce qui a trait à la santé des os. La fluorose osseuse -ce trouble qui peut être causé par le fluor- peut mener jusqu'à l’invalidité. Certaines initiatives permettent d'extraire ce fluor, mais elles sont malheureusement peu accessibles à la population qui a souvent trop peu de ressources afin de mettre en place une méthode pour filtrer l'eau de puit. C'est pourquoi cette recherche vise à trouver un moyen simple et efficace d'extraire le fluor de l'eau en le faisant précipité avec un autre composé (CaCl2) et en inventant une méthode d'utilisation qui permettrait à des personnes n’ayant aucune connaissance scientifique de l'utiliser en toute sécurité. Les données en lien avec cette méthode ont été compilées et stipulent que le rendement est de 66% lors de l'utilisation de la concentration présente au Maghreb (soit 9,5 mg/L) selon le rapport Défluoruration des eaux par dialyse de Donnan et électrodialyse. Cela permet de demeurer en-dessous de la plus base dose toxique enregistrée par le RTECS (7 mg /L). La prochaine étape sera d'évaluer le rendement selon différentes températures et aussi de s'assurer que les autres composantes de l'eau locale n'affectent pas le processus.

 

 

The time course of recovery from chronic social stress in rainbow trout. 
Brett Culbert, Biology, Supervised by Dr. Katie Gilmour

When salmonid fish are held in pairs they interact and form a social hierarchy with one fish becoming subordinate. These interactions cause increases in circulating levels of the stress hormone cortisol in both fish. Upon hierarchy formation, the cortisol levels of the dominant fish return to baseline values. The subordinate, however, maintains characteristically high levels of circulating cortisol. It is well established that chronically elevated cortisol causes deleterious physiological effects that include altered energy mobilization, decreased growth and increased mortality, as well as behavioural effects such as reduced feeding, activity and aggression. What has not been studied is the ability of subordinate fish to recover from social stress. Using juvenile female rainbow trout, Oncorhynchus mykiss, length-matched pairs were allowed to interact for either two or four days. After this interaction period, the fish were separated by an opaque perforated divider and allowed to recover for zero, two, or four days. By measuring plasma cortisol, plasma glucose, and liver glycogen concentrations, along with several morphometric traits and behavioural observations, we were able to assess the ability of subordinates to recover from chronic social stress. Preliminary findings suggest that subordinates are capable of physiological recovery from social stress after both short and long interaction periods. Future research will assess the capacity of recovered subordinates to mount a cortisol response when facing an acute stressor, as well as their ability to become dominant when paired with a socially naïve fish. These results will provide insight into the time course of recovery, both behavioural and physiological, from chronic social stress in salmonids.

 

 

Investigating the potential role of novel protein c20orf27 in targeting proptein phosphatase 1 to n-linked glycosylation in the rough endoplasmic reticulum.

Vincent Nguyen, Cellular Molecular Medicine, Supervised by Dr. Laura Trinkle-Mulcahy

 

Protein phosphatase 1 (PP1) is a promiscuous phosphatase protein involved in hundreds of dephosphorylation pathways, thus regulating many functions of the cell. Novel protein c20orf27, unnamed and uncharacterized, has consistently been pulled down in our PP1 interactome screens. c20orf27 also has a specific amino acid motif that is notorious for binding to PP1's hydrophobic pocket, which lead us to believe the two were direct interactors. To investigate its potential role in the cell, we confirmed its direction interaction with PP1 using the bifluorescence complementation and fluorescence two hybrid assays by exploiting genetic cloning techniques that involve fluorescent marker tags, such as GFP and YFP (green and yellow fluorescent protein). Once confirmed, we mapped c20orf27's interactome and found that it pulled down the entire OST complex - the transmembrane complex found in the rough ER responsible for n-linked glycosylation (transferring a mannose sugar to an Asparagine residue on a growing nascent polypeptide chain). Our hypothesis speculates that c20orf27 targets PP1 to the rough ER to regulate the activity of n-linked glycosylation by the OST complex. Current and future directions include glycosylation biomarker assays to measure global glycosylation levels when c20orf27 is overexpressed and knocked down, as well as a near-neighbour bio-ID labelling approaches to map a sensitive c20orf27 interactome in hopes of getting a better picture of other proteins c20orf27 interacts with, such as potential players in other glycosylation pathways.

 

 

How useful is citizen science for science? A look into the advantages of including citizen science data in ecological research.

Peter Soroye and Najeeba Ahmed, Macroecology, Supervised by Dr. Jeremy Kerr

Citizen science (CS) programs, in which researchers recruit volunteers to collect data as part of a scientific inquiry, are becoming increasingly accessible and popular. For example, eButterfly – which monitors butterfly species across North America – has grown by about 48000 observations in the last year alone: an increase of 58%. While the value of these CS programs in terms of engaging the public in science is priceless, there is little study into the advantages of including CS data into scientific analyses to complement or replace the traditional, voucher-based datasets that are commonly used in research. Here, we compare a CS dataset (eButterfly) to a traditional dataset to find how often CS provides geographically or ecologically novel observations for a given species. By comparing species distribution in 100x100km quadrants across Canada from both datasets, we find that CS was able to provide geographically novel information for 205 of 262 species (78%) and was able to confirm observations made in the traditional database for 251 species (96%). On average, 15% of the observations provided by CS for a given species were in a previously unknown area (geographically novel), while 85% of observations confirmed traditionally-gathered observations. CS was also able to provide species observations in a previously unrecorded ecozone for 47 species (18%). As all eButterfly observations are verified by a regional expert, eButterfly’s ability to provide novel and confirmatory species observations indicates that CS could provide reliable, inexpensive and current information on species distributions. This would make CS data extremely useful in global change research, where current and spatially-extensive datasets are needed. Our results provide a tangible reason for researchers to use CS data and suggest that CS could revolutionize the study of the ecological impacts of land-use and climate change by providing valid, up-to-date, and continental-scale species data.