Apr 22, 2024  |  3:00pm - 4:00pm
Student research presentation

LMP student seminars: 22 April

Agile education

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.

1. Brain and Neuroscience

Location: MSB 4279

Narisa Dhupar

  • Title: Spatial transcriptomics of the human corneal endothelium in Fuchs endothelial corneal dystrophy
  • Supervisor: Dr. Stephan Ong Tone

Victor Pham Truong

  • Title: Functional analysis of axon guidance programs in developmental photoreceptor connectivity
  • Supervisor: Dr. Valerie Wallace

3. Cardiovascular, Physiology and Metabolism/ Molecular and Cell Biology and Regenerative Medicine

Location: MSB 4171

Brock Kent Hoard

  • Title: Essential Role of PSI Domain in β3 Integrin Expression and Function
  • Supervisor: Dr. Heyu Ni

Suejean Park

  • Title: Extracellular vesicles as circulating biomarkers of cerebrovascular dysfunction in heart failure
  • Supervisor: Dr. Jason Fish


Narisa Dhupar: Spatial transcriptomics of the human corneal endothelium in Fuchs endothelial corneal dystrophy

Background: The corneal endothelium (CE) is composed of a monolayer of corneal endothelial cells (CECs) that rest on Descemet’s membrane. Fuchs endothelial corneal dystrophy (FECD) is characterized by progressive CEC loss and guttae formation. There is evidence that the CE is not a homogenous population, and that spatial differences exist with differential gene expression patterns. We performed a transcriptomics study to characterize CEC populations isolated from central and peripheral spatial domains in healthy and FECD CE using bulk RNA sequencing.

Methods: The central 8-mm and peripheral rims from healthy and FECD human donors were dissected for RNA isolation. A list of differentially expressed genes (DEGs) was generated for comparison between normal central and peripheral CE, and normal and FECD CE. DEGs were grouped in categories by molecular function and further analyzed by an over-representation test using a PANTHER Go-Slim molecular function annotation dataset.

Results: 369 total DEGs (130 upregulated, 239 downregulated) were found between FECD and healthy controls, and 167 DEGs (85 upregulated, 82 downregulated) were found between normal central and peripheral CE. Differential expression analysis of transcriptomic profiles demonstrated an enrichment of genes involved in collagen degradation and integrin cell surface interactions between the central and peripheral CE, and collagen formation and extracellular matrix (ECM) organization between healthy and FECD tissues. The dysregulation of ECM-associated pathways suggests a change in the ECM environment in the CE spatially and between healthy and FECD tissues. Conclusion: The CE is composed of a heterogeneous population of CECs, which become altered in FECD.

Victor Pham Truong: Functional analysis of axon guidance programs in developmental photoreceptor connectivity

Visual perception relies on the light sensing neurons of the retina, rod, and cone photoreceptors (PRs). Neurons in the mammalian retina are unable to regenerate, therefore, PR degeneration in diseases like macular degeneration or genetic retinopathies result in permanent visual deficits. Cell replacement therapies like PR transplantation are reported to rescue visual function in mouse models, however limited donor cell connectivity remains a key challenge for therapeutic applications. A better understanding of how developing PRs target their axons and form synapses could improve therapeutic connectivity. We hypothesize that a temporally regulated program of axon-related molecules mediates the targeting of PR axons. To identify axonal regulators, we developed a candidate gene approach using RNA-sequencing databases of the retina to screen for genes involved in axon extension, guidance, and synapse formation that were expressed during early rod development. We further validated the temporal and spatial expression of the selected candidates using RNAscope. Finally, we performed gain and loss of function studies in vivo to characterize the function of each candidate. Ncam1, a cell adhesion molecule, was reported to have three major isoforms that were reciprocally expressed in PRs over time. Functional characterization of Ncam1 in PR development suggests that it may be involved in the maintenance of synapse morphology, with some indicators that suggest PR process trajectory is impaired when Ncam1 is silenced. Further temporal and 3D analysis of PR processes during early development may reveal more severe targeting defects that support the role of Ncam1 in PR axon targeting.

Brock Kent HoardYAP/TAZ-mediated suppression of chemotherapy-induced antiviral signaling in lung cancer

Integrin αIIbβ3 is the essential platelet receptor for hemostasis and thrombosis via intercellular and extracellular matrix interactions. We recently discovered that the plexin-semaphorin-integrin (PSI) domain of the β3 subunit possess endogenous thiol-isomerase activity through its two CXXC motifs. Inhibiting this activity reduced platelet aggregation in-vitro and attenuated thrombus formation in-vivo (Blood, 129(13), 1840, 2017). Interestingly, mutations of cysteines within the PSI domain have been associated with greater risk of hemorrhaging, known as Glanzmann’s Thrombasthenia, while the L33P polymorphism increases risk of cardiovascular disease. Saying this, the mechanism of which the mutations effect receptor function has yet to be explored. Here, we transfected cysteine mutated PSI domain constructs into HEK-293T cells and found decreased/abolished cell surface expression using flow cytometry and immunofluorescence staining. Interestingly, western blot analysis revealed comparable cellular protein levels. Additionally, recombinant L33P PSI domain (rL33P) protein exhibited increased thiol-isomerase activity in comparison to recombinant wild-type PSI (rPSI) in a scrambled and reduced RNase assay. Clot formation time was decreased for rL33P-treated blood compared to rPSI in thromboelastography and clot retraction assays. rL33P-treated platelet-poor-plasma displayed significantly faster clotting times in comparison to rPSI through a prothrombin time assay, an extrinsic coagulation pathway test. Activated partial thromboplastin time, an intrinsic pathway test, showed no significant difference between groups. Our results suggest that mutations of cysteines within PSI cause protein mis-folding leadings to lack of functional receptor expression, while the L33P polymorphism may increase risk of cardiovascular events due to enhancement of thiol isomerase activity and upregulation of blood coagulation.

Suejean ParkExtracellular vesicles as circulating biomarkers of cerebrovascular dysfunction in heart failure

Introduction: Cardiovascular disease is the leading cause of death globally with over 64 million individuals living with heart failure (HF). Patients that develop heart failure (HF) with reduced ejection fraction experience systemic inflammation, reduced cerebral microvascular perfusion, and cognitive impairment (CI). Despite worse prognosis and increased mortality rates for individuals presenting with HF and cognitive impairment, the connection between heart and brain health has not been well characterized and early diagnostics that can predict the risk of developing cognitive impairment are currently lacking. The blood-brain barrier (BBB) is impaired under inflammatory conditions and cerebral microvasculature dysfunction impacts cognitive function. Endothelial cells (ECs), a key component of the BBB, contribute to circulating pools of extracellular vesicles (EVs) which are released for intercellular communication. EV cargo derived from dysfunctional cerebral microvascular ECs could be utilized as an early circulating biomarker for assessing CI risk in HF patients.

Methods: Using ultracentrifugation, EVs were isolated from conditioned media of quiescent or inflamed (ie. TNFα treatment) ECs after 24 hours. EC phenotype was assessed by RT-qPCR and immunofluorescence. Nanoparticle tracking analysis characterized EV size and concentration. A myocardial infarction (MI) was surgically induced in 8-10 week old C57BL/6 mice. BBB integrity was assessed via injection of 10 kDa fluorescent-dextran at 3-days post-MI (d3) and 21-days post-MI (d21), reflecting acute and chronic HF. Leakage was assessed using light-sheet microscopy.

Results: ECs treated with TNFα showed upregulation of inflammatory markers and downregulation of endothelial markers. All endothelial vascular beds produced more EVs in response to TNFα stimulation. Due to insufficient resolution of light-sheet microscopy, at both d3 and d21, BBB leakage could not be observed in MI or sham-mice.

Conclusion: In conclusion, we have shown that ECs are activated in response to TNFα stimulus and cause increased production of EVs. Proteomics will be run on the isolated EVs to identify EV cargo unique to cerebral microvascular ECs. Excessive leakage after MI could not be observed by light-sheet microscopy due to low resolution of the technique. To provide higher resolution and assess BBB integrity, confocal microscopy will be used. Identified markers will be validated by assessing EV samples isolated from the plasma of our in vivo HF mouse model to explore the potential of EVs as a circulating biomarker of cerebral dysfunction in HF.


No need to register.

Contact lmp.grad@utoronto.ca with any questions