LMP student seminars: 11 March
Each week during term time, MSc and PhD candidates in the Department of Laboratory Medicine and Pathobiology present their research.
Anyone is welcome. No need to register.
Location: Medical Sciences Building, rooms 4171 or 4279, see below.
As part of the core research curriculum, students taking LMP1001/2/3: Graduate Seminars in Laboratory Medicine and Pathobiology will present their projects. Please see abstracts below.
4. Infectious Diseases, Inflammation, and Immunology
Location: MSB 4279
Sheng Han Li
- Title: Investigating the Association of Serum Metabolites with Psoriatic Arthritis Musculoskeletal Disease Activity and Patterns
- Supervisor: Dr. Vinod Chandran
1. Brain and Neuroscience
Location: MSB 4279
Raghav Sharma
- Title: Single-nucleus analysis of cell type-specific changes in the hippocampus due to C9orf72 haploinsufficiency in amyotrophic lateral sclerosis
- Supervisor: Dr. Janice Robertson
3. Cardiovascular, Physiology and Metabolism/ Molecular and Cell Biology and Regenerative Medicine
Location: MSB 4171
Ness Little
- Title: Adeno-Associated Virus (AAV) Mediated Expression of Bcl-xL Attenuates Apoptosis in Human Corneal Endothelial Cells
- Supervisor: Dr. Stephan Ong Tone
Andrew Song
- Title: Towards the formation of the nucleus pulposus component of a biological intervertebral disc replacement
- Supervisors: Dr. Rita Kandel, Dr. Paul Senterre
Abstracts
Sheng Han Li: Investigating the Association of Serum Metabolites with Psoriatic Arthritis Musculoskeletal Disease Activity and Patterns
Hypothesis: Psoriatic Arthritis (PsA) Musculoskeletal (MSK) disease patterns characterized by USG is associated with fatty acid and lipid metabolism dysregulation.
Materials and Methods: 76 PsA patients were assigned USG patterns via hierarchical clustering based on scores for synovitis, enthesitis, peritenonitis, and tenosynovitis determined by USG of joints and periarticular structures. Serum samples were prepared using SPME and analyzed via LC-MS. Machine Learning (ML) models were used to evaluate associations between metabolite levels and MSK disease patterns. Model performances were summarized using Area under Receiver Operater Characteristic curves (AUROC). Statistically significant metabolites were tentatively identified.
Results: Hierarchical clustering identified 3 different USG-based patterns. Cluster 1 (n=27) exhibit high synovitis, tenosynovitis, and peritenonitis scores with moderate enthesitis. Cluster 2 (n=6) exhibit high synovitis, moderate peritenonitis and tenosynovitis scores, and no enthesitis. Cluster 3 (n=43) exhibit high synovitis scores, and moderate scores in other domains. ML models with AUC > 0.7 are considered. The best performing ML models is a LogitBoost model with 20 features with AUC of 0.986 for USG-based patterns between cluster 2 and 3. The metabolites in best performing models were associated with dysregulation of fatty acid metabolism. Tentatively identified metabolites belong to compound classes, sphingomyelins, phospholipids, and diglycerides.
Conclusion: An untargeted metabolomics approach found associations between serum metabolome of PsA patients and USG-determined MSK disease pattern. Closer examination and validation of metabolites to reveal potential biomarkers predicting PsA disease activity is underway.
Raghav Sharma: Single-nucleus analysis of cell type-specific changes in the hippocampus due to C9orf72 haploinsufficiency in amyotrophic lateral sclerosis
Hexanucleotide repeat expansions in a non-coding region of C9orf72 (C9) is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). C9 repeat expansions downregulate C9orf72 and its protein product. Given the emerging role of C9 in synaptic membrane trafficking, we hypothesized that increased postsynaptic glutamate receptor levels observed in autopsied human tissue and mouse models results in susceptibility to glutamate excitotoxicity. We first characterized the transcriptomic changes at the single nucleus level in mouse hippocampus of both C9 heterozygous (C9-HET) and C9 knockout (C9-KO) mice compared with wild-type (WT). Here we found that both excitatory and inhibitory neurons subtypes in C9-KO hippocampus show dysregulation of genes in pathways related to pre-and post-synaptic membrane organization, glutamate transport, and synaptic adhesion. C9-HET mice also show up-regulation of GluA1 at the protein level, but many synaptic pathway genes are down-regulated across hippocampal cell types, specifically those involved in synaptic organization and transmembrane transport activity. Overall, we find changes associated in synaptic transmission and organization associated with the trafficking of ionotropic receptors at the synaptic interface. We aim to further recapitulate these changes in single nucleus assay for transposase-accessible chromatin to see if transcriptomic changes are captured at an epigenomic level, identify gene regulatory networks with differential motif analysis, and correlate these findings with a multiomic analysis of the mouse frontal cortex.
Ness Little: Adeno-Associated Virus (AAV) Mediated Expression of Bcl-xL Attenuates Apoptosis in Human Corneal Endothelial Cells
Introduction: Corneal transplantation is the standard-of-care treatment for severe corneal endothelial cell (CEC) loss, such as in Fuchs Endothelial Corneal Dystrophy (FECD), but is limited by the availability and suitability of donor corneas. Currently, there is a global shortage of donor corneas and new strategies are needed to increase the number of suitable donor corneas and reduce the need for future transplantations. Despite this need, limited studies have explored using AAVs to deliver the anti-apoptotic genes, such as Bcl-xL, to human CECs. This study aims to investigate AAV2/5-mediated expression of Bcl-xL to attenuate apoptosis in FECD CECs.
Methods: FECD CECs were transduced in-vitro with anti-apoptotic Bcl-xL2/5 (AAV2/5-CAG-eGFP-P2A-BCLXL) or control AAV2/5 (AAV2/5-CAG-eGFP) 24-hours after plating using an MOI of 200,000. Twenty-four hours post viral transduction, cells were treated with 10µg/mL etoposide and 0.5µM Caspase-3/7 Red dye. Apoptosis was quantified 24-hours post-etoposide treatment.
Results: BclxL2/5 and AAV2/5 were both able to transduce FECD CECs with high efficiency, with transduction rates of 70.47%±8.28 and 65.86%±3.54, respectively. Infection with BclxL2/5 resulted in significantly lower levels of etoposide-induced apoptosis (23.96%±11.54) compared to AAV2/5 (70.43%±14.35; p=0.000) and non-transduced FECD CECs (71.68%±18.70; p=0.000). In untreated FECD CECs, Bcl-xL2/5 resulted in lower levels of apoptosis (9.98%±5.95) compared to AAV2/5 (21.08%±16.43; p=0.264), and non-transduced FECD CECs (9.61%±7.87; p=0.999).
Conclusions/Discussion: Anti-apoptotic BclxL2/5 can transduce FECD cells with high efficiency and protect against etoposide-induced cell death. Future studies could explore using BclxL2/5 to extend the viability of donor corneas and increase the number of donor corneas available for transplantation.
Andrew Song: Towards the formation of the nucleus pulposus component of a biological intervertebral disc replacement
The intervertebral disc (IVD) consists of a central nucleus pulposus (NP) surrounded by annulus fibrosus integrated to the adjacent vertebral body through the cartilage endplate (CEP). IVD degeneration is associated with lower back pain (LBP), one of the most common causes of disability worldwide. Current treatments are incapable of restoring full function, so regenerative medicine strategies such as biological disc replacements are promising alternatives. Previously, our lab demonstrated that a biphasic NP-CEP construct could successfully be formed in-vitro on a porous bone substitute material. Thus, the hypothesis of this study is that a fully integrated NP-CEP-like component of a biological IVD (i.e. bone substitute and CEP on either side of the NP) can be formed. We investigated the effect of the age of cartilage on integration by seeding NP cells on top of cartilage tissue at various time points to form biphasic NP-CEP constructs. The age of cartilage seems to influence integration, as the most successful integration was observed after culturing cartilage tissue for a period of 5 days. Immunofluorescence staining confirmed the presence of matrix molecules in the in-vitro tissues. Notochordal cells, present in the NP, were quantified by cell counting following immunostaining for keratin. The cell numbers were similar in the NP tissue whether grown alone or in combined co-culture with cartilage. Future work will focus on growing these tissues in a specially designed 3D-printed holder to determine the optimal growth conditions to form CPP-CEP-NP-CEP-CPP constructs in-vitro. These findings will bring us closer to the development of a biological disc replacement.
Contact
No need to register.
Contact lmp.grad@utoronto.ca with any questions