The effects of podocyte-specific IRAK on NOX5-mediated ROS formation in progressive renal dysfunction
Awais Rahman, Nephrology, Supervised by Dr.Christopher R.J. Kennedy

 

NADPH Oxidase 5 (NOX5)-mediated reactive oxygen species (ROS) formation has been implicated in forms of renal disease and contributes to glomerular epithelial cell (podocyte) dysfunction. Toll-Like receptors (TLRs) have similarly been implicated in podocyte dysfunction by increasing NOX-mediated ROS formation. Additionally, TLRs interact directly with NOX4; however, the interaction between TLRs and NOX5 remains to be elucidated. The aim of this study was to assess if TLRs interact with NOX5 through Interleukin-1 Receptor-Associated Kinase (IRAK)—an essential component of the TLR pathway. It was hypothesized that IRAK increases NOX5 expression and NOX5-mediated ROS formation upon TLR stimulation in podocytes, thereby assisting in progressive renal damage. QRT-PCR results showed detectable IRAK1 and IRAK4 mRNA expression in human podocytes (hPODs). We established a suitable method for TLR stimulation in hPODs—6 hour lipopolysaccharide (LPS) treatment at 25 μg/mL—to assess changes in gene expression; our data suggest that this stimulation protocol does not induce a significant increase in NOX5, IRAK1 and IRAK4 mRNA expression. Lastly, we assessed if TLR stimulated ROS production was NOX5-dependent by transducing mouse podocytes (mPODs)—which normally lack NOX5—with an adenovirus to express NOX5; our preliminary data suggest that TLR stimulated increased ROS production may be NOX5-dependent. Taken together our data suggest that TLRs may be involved in NOX5-mediated ROS formation. Future in vivo studies will be aimed at addressing the role of IRAK in this pathway and consequently IRAK or NOX5 may surface as novel therapeutic targets for reducing the progression of nephropathy.

 

 

The Role of Podocyte-Specific EP/PGE2 Activity in Microparticle Formation During Diabetic Nephropathy

Zakirullah Sanaye, Nephrology, Supervised by Christopher R.J. Kennedy

 

Podocyte injury is caused by enhanced glomerular capillary pressure (Pgc) and hyperglycemia during diabetic nephropathy (DN). Microparticles (MPs) are small sub-cellular membranous vesicles that are released from cells in response to stress/injury. Podocyte MPs are increased in DN, suggesting MPs as putative biomarkers of pre-albuminuric podocyte injury. Although the molecular mechanisms by which DN increases podocyte MP formation remain to be determined, the role of podocyte-specific PGE2/EP1 activity in progression of DN suggests its involvement in podocyte MP formation. It was hypothesized that enhanced podocyte PGE2 synthesis and EP1 activity leads to an increase in podocyte MP formation. Cultured human podocytes were treated with a range of PGE2 concentrations with and without HG for 24hrs and 48hrs and media was analyzed to assess podocyte MP concentrations through sizing and enumeration. After 24hrs of 10μM PGE2 treatment, data suggested an increase in podocyte MP concentrations compared to control; this was only seen under diabetic conditions (HG)—suggesting that PGE2 plays a role in podocyte MP formation only under those conditions; however, whether it is through EP1 or EP4 remains to be determined. After 48hr, 10μM PGE2, HG treatment, preliminary data showed an increase in podocyte MP concentrations compared to control. Urine was collected from wild type (WT), EP1 knockout (EP1-/-), OVE26, and OVE26/EP1-/- mice and analyzed using podocyte MP specific markers via flow cytometry. OVE26 mice showed a significant increase in urinary podocyte MPs compared to WT whereas OVE26/EP1-/- mice did not; moreover, there was no change in EP1-/- mice compared to WT. Taken together, these findings suggest that podocyte specific EP1 receptor activity, under diabetic conditions, exacerbates podocyte MP formation. Through further validation of these findings, podocyte MPs will be further solidified as biomarkers of progressive renal damage before irreversible renal injury.

 

 

Plant-derived triterpenes as inhibitors of human recombinant monoacylglycerol lipase metabolism

Evan Trofimchuk, Phytochemistry and Ethnobotany, Supervised by Dr. Cory Harris

 

Stimulation of the endocannabinoid system via administration of both endogenous and exogenous cannabinoids can result in psychomodulatory effects. Moreover, the mammalian endocannabinoid system plays a central role in the mediation of mood and anxiety, and is therefore a promising target for the development of anxiolytic therapies, including medicinal plants and natural products. This study builds on previous research revealing that several triterpenes found in anxiolytic herbal products inhibit the activity of monoacylglycerol lipase (MAGL), a major enzyme responsible for 2-arachidonoylglycerol (2-AG) endocannabinoid degradation. Beginning with Souroubea sympetala and Platanus occidentalis plant extracts, we identified and characterized the inhibitory activity of five naturally occurring triterpenes (IC50 = 800-1800 nM) using human recombinant MAGL. We subsequently optimized a MAGL-specific bioassay and screened a library of semi-synthetic triterpene derivatives that inhibited MAGL catalytic activity more potently and more weakly than their parent structures. Preliminary structure-activity relationship modeling of MAGL inhibition will be presented. In-vivo inhibition of MAGL activity has been important in studying the physiological effects of varying 2-AG levels and has revealed its role in regulating various metabolic processes and its association with the onset of various diseases. Therefore, identifying a compound with a reversible inhibition mechanism and high specificity for MAGL would have significant therapeutic practicality.

 

 

Aminocarbonylation intermoléculaire à partir d'imines»

Philippe Lemire, Chimie, Supervisé par Dr. André Beauchemin
 

Les hétérocycles azotés sont prédominants parmi les composés bioactifs et ont donc un rôle capital dans les domaines pharmaceutique et agrochimique. Il est donc essentiel de développer des méthodes de synthèse efficaces et peu coûteuses. Des travaux antérieurs du groupe ont élaboré la génération in situ d’isocyanates et de thioisocyanates, permettant de contrôler la réactivité. Les réactifs de départ sont bloqués par divers groupements protecteurs (ex. : OPh, OMe, Ot-Bu, etc.), réduisant leur toxicité. S’étant déjà intéressé aux réactions de cycloaddition entre ces intermédiaires réactifs et des alcènes, la présente recherche étudie de façon similaire la réactivité d’imines avec des (thio)isocyanates N-substitués. Ces amino(thio)isocyantes sont des intermédiaires très rares dans la littérature et possèdent une intéressante réactivité amphotérique, d’où l’importance d’explorer les possibilités qu’ils offrent. Ce projet se penche donc sur l’aminocarbonylation et l’aminothiocarbonylation entre des amino(thio)isocyanates et des imines pour former des triazolidinones/triazolidine-3-thiones dipolaires et avoir accès à des dérivés, tels que des triazolones. L’efficacité réactionnelle de divers réactifs, présentant des profils électroniques et stériques variés, permettra d’établir la portée de cette réaction et d’avoir un aperçu sur le mécanisme réactionnel. Des résultats concluants permettraient, en somme, d’aboutir à une voie de synthèse rapide, bon marché et sans catalyseur métallique pour la production de telles molécules.

 

 

Beyond Histone Deacetylation: New Functions for Sirtuins in Yeast.

Jimmy Tseng, Cellular Molecular Medicine, Supervised by Dr. Michael Downey

 

Sirtuins are a highly conserved family of NAD+ dependent protein deacetylases that can be found in eukaryotes from yeast to humans. They are well known for their function in regulating gene transcription, which involves deacetylation of histone tails. Sirtuins in a number of organisms have also been implicated as pro-longevity factors and as regulators of cellular stress responses. Recent work demonstrating that sirtuins can also target non-histone proteins calls into question whether histones are the relevant targets involved in these functions. To gain insight into pathways regulated by sirtuins, the Downey lab screened a collection of 1500 yeast mutants for sensitivity to nicotinamide, a sirtuin inhibitor. In follow-up analyses, I confirmed that fpk1∆ mutants show very high sensitivity to this drug. FPK1 encodes a protein kinase that is proposed to be an upstream regulator for flippases, which are crucial for cell membrane construction and phospholipid asymmetry. I hypothesize that FPK1 and sirtuins function together to regulate membrane integrity. I used an insertional mutagenesis screen to isolate 7 suppressors of fpk1∆ hypersensitivity from a pool of ~4000 mutagenized yeast strains. Identification and characterization of these suppressors will give clues as to how sirtuins and FPK1 function together to regulate plasma membrane structure and function.

 

 

Toxicity of petroleum-derived naphthenic acids exposed to two Canadian frog species, wood frog (Lithobates sylvaticus) and leopard frog (Lithobates pipiens) tadpoles during late stages of metamorphosis

Erin Blake, Biology, Supervised by Dr. Vance Trudeau

E. Blake, M. Galus, J. M. Gutierrez-Villagomez, V. Trudeau
Naphthenic acids (NAs) are a toxic component of oil sands processed water produced by non-conventional petroleum extraction. The contaminated water is held in tailings ponds for long-term storage abiding by a current zero-discharge policy. However, these tailing ponds reportedly leak into surrounding waters, including the Athabasca River in Alberta, increasing potential ecological risk. There is little knowledge on the extent that NAs impact Canadian wildlife, especially on vulnerable amphibians in the region. The purpose of this research is to study the effects of an oil NA extract on two native Canadian frog species, wood frog (Lithobates sylvaticus) and leopard frog (Lithobates pipiens). Tadpoles were exposed to environmentally relevant concentrations (0.3-3 mgL-1) of a Merichem NA mixture. The tadpoles were exposed in the late stages of metamorphosis (Gosner 42-46). Tail regression, metamorphic rate and survival were assessed every 12 h for a 7 day period. . Preliminary results of the wood frog exposure have shown NAs impact survival, and slow metamorphic rate and tail regression. The transition from water to land is impeded in the highest concentration of NAs, which has important implications for juvenile frog survival and potential population level effects. The bioassay will also be replicated with leopard frogs for a comparative analysis. Overall, this study provides insight into the toxicity of NAs in Canadian frog species and raises concern for their population status within contaminated ecosystems.

 

 

In vitro disease modelling using patient-derived iPSCs with ABCA3 mutation.

Xin Xu, Regenerative Medicine, Supervised by Dr. Mehdi Shafa

 

Belonging to the ATP-binding cassette transporter family, ABCA3 in lung alveolar epithelial type II cells reside in membrane of lamellar bodies and is essential for surfactant protein maturation. Non-functional surfactant protein leads to alveolar collapse in the lung during expiration. This autosomal recessive disease results in fatal respiratory distress syndrome and patients with mutations for this lipid transporter are commonly preterm babies. The only available therapy today is lung transplantation, which in most cases, the body eventually rejects. There exist many different mutations that fall under this disease but it has yet to be well characterized. An in vitro model of a patient-specific null ABCA3 mutation was created using patient episomally derived induced pluripotent stem cell (hiPSCs) line, CCRM39Q. A wild type cell line, 168A3.8, was also grown and differentiated parallel as control to the disease cell line. Prior to differentiation, the hiPSCs were cultured using a chemically defined and xeno-free system. Differentiation lasted thirty days, allowing the hiPSCs to progress through the definitive endoderm stage, the anterior foregut endoderm stage, and lung progenitor stage until they enter the maturation stage to become alveolar epithelial type II cells. Immunofluorescent stains were performed to verify the state of the cells at the end of each stage. Other characterization of the disease model is in progress. Modelling this ABCA3 mutation in vitro with cells episomally derived from the patient creates the potential for safe and patient-specific drug screening and gene editing that may lead to cell replacement therapy.

 

 

Expanding the scope of olefin metathesis catalysts and comparing ligand effects.

Nikita Panov, Chemistry, Supervised by Dr. Deryn Fogg

 

Contributing to Global Sustainability: Synthesis of a NEW Promising Z-Selective Olefin Metathesis Catalyst. Olefin metathesis is a powerful tool for the assembly of carbon-carbon double bonds; a chemical process that enables the synthesis of a wide array of advanced materials. Implementing organometallic metathesis catalysts in otherwise energy-demanding industrial processes is thus a means of contributing towards the achievement of a more sustainable future. A reliable access to Z-alkenes is a particular challenge, overcoming which requires a fine-tuned catalyst with exceptional efficiency and stereoselectivity. The purpose of this research project is to develop a synthetic pathway towards a new thiolate derivative of a 2nd generation Hoveyda-Grubbs catalyst (HII-Monothiolate) starting from well documented, commercially available metathesis catalysts. Based on existing scientific literature regarding this family of organometallic catalysts, there are reasons to believe that HII-Monothiolate has promising potential for Z-selective homocoupling of terminal olefins. Up to date, three synthetic pathways have been explored with extensive effort, resulting in the synthesis and characterization of two new organometallic complexes. All chemistry was performed using air/water sensitive techniques and characterization (1H/13C) was achieved via NMR spectroscopy. Scope of future work continues to be the development and optimization of a synthetic pathway towards HII-Monothiolate. In addition, supportive characterization such as elemental analysis and crystallographic data–as well as insight into metathesis capability–of the synthesized catalysts will be obtained shorty.

 

 

Ketone body supplementation induces NK cell proliferation, survival and effector function for anti-tumor immunity
Usaamah Gill, Immunology, Supervised by Dr. Seung-Hwan Lee

 

Natural Killer (NK) Cells are a key component of the body's defense mechanism, being particularly important in surveillance and defense against virally infected, as well as cancerous cells. They act by stimulation of a activating receptors, allowing recognition and killing of 'abnormal' target cells. Recently the potential usage of NK cells in therapeutic applications for treating cancer has been a hot research topic, and has seen small levels of success. In order to further advance the potential for NK cell based immunotherapy use in clinical settings, innovative methods to enhance and ensure safety are needed. To this end, we have observed that use of B-hydroxybutyrate (BHB) as a supplement in low glucose conditions promotes NK cell proliferation and survival, while simultaneously depriving tumors of abundant nutrients, weakening cancerous cells. This metabolically induced 'shift' in the tumor microenvironment, from a typically very immune hostile environment towards conditions more favourable for NK cells has significant implications for the balance between tumors and immunity. Furthermore, it is possible to induce these conditions in a mouse model, or in human patients with a combination of therapeutic fasting and oral delivery of a ketone ester of BHB, which proportionally raises blood BHB levels. This promising combination of altered metabolic conditions and NK cell mediated immunotherapy is exciting for it's potential to be clinically relevant, and relatively easy to implement. It could drastically change the way we manage cancers, making overall treatment more effective, safer, and quicker with remission.

 

 

Retrospective chart review for patients with ‘Cord Herniation'

Sara Omaiche, Radiology, Supervised by Dr. Santanu Chakraborty

 

‘Cord Herniation’: Are you sure?
Introduction: Idiopathic spinal cord herniation is an entity where the spinal cord is displaced anteriorly and herniates through a defect in the dura. It presents clinically with progressive myelopathy with a reported mean duration of symptoms of 40 months. Commonly found in the thoracic spine and in female patients, it may evolve into severe paraparesis. The condition is best seen on MRI or CT myelography. Almost all cases are diagnosed by the Radiologists while interpreting images for usually nonspecific symptoms or as an incidental finding. More recently 2 other differential diagnoses have been proposed for anterior displacement of the cord, one being posterior arachnoid web and the other a posterior thoracic arachnoid cysts. There is a high rate of misdiagnosis (up to 48% in some series) which may lead to unnecessary surgery. Also the surgical approaches are quite different for these pathologies. Most of the patients reported in the literature were treated surgically with limited evidence to predict which patients will benefit from surgery. In a metanalysis (2009) of 129 surgically managed patients; there was at least some improvement in motor function in 68%, stabilization in 19%, and permanent neurological deterioration in 7%. Objectives: To review the outcomes of patients who were described to have ‘cord herniation’ in thoracic spinal cord MRI or myelography.
Methods: Patients, with a diagnosis or description of ‘cord herniation’ will be identified from PACS search retrospectively. Using electronic medical records, we will obtain information about patient demographics, clinical symptoms and findings, treatment received, progress and follow-up. PACS images will be assessed by 2 neuroradiologists and diagnosis reclassified as ‘cord herniation’, arachnoid cyst or web. Specific features like degree of compression, displacement and signal changes will be correlated with symptoms and clinical improvement following surgery.