LMP student seminars: 11 November

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.

3. Cardiovascular, Physiology and Metabolism

Location: MSB 4171

Anca Maglaviceanu

• Title: Elucidating the effects and mechanisms of action of delta-9-tetrahydrocannabinol on knee osteoarthritis
• Supervisor: Dr. Mohit Kapoor

Anisha Hundal

• Title: Deciphering the tubulin code in nuclear envelope-mediated DNA double-strand break repair
• Supervisor: Dr. Karim Mekhail and Dr. Razq Hakem

5. Infectious Diseases, Inflammation and Immunology

Location: MSB 4279

Ream Abdullah

• Title: Investigating mismatch repair deficiency (dMMR) and KCNQ1 as gastric adenocarcinoma biomarkers for treatment efficacy & prognosis
• Supervisor: Dr. Carol Swallow

Sumiha Karunagaran

• Title: TBA
• Supervisor: TBA

Abstracts

Anca Maglaviceanu: Elucidating the effects and mechanisms of action of delta-9-tetrahydrocannabinol on knee osteoarthritis

Objectives: Osteoarthritis (OA) involves pathological joint changes and signalling at the dorsal root ganglia (DRG) that contribute to chronic pain. Some OA patients use cannabis to alleviate symptoms. ∆⁹-tetrahydrocannabinol (THC), a prominent phytocannabinoid, can signal in joint cells and in neurons. In this study, I investigated the effects and signalling mechanisms of THC on pain and joint degeneration in pre-clinical models of knee OA.

Methods: Destabilization of the medial meniscus (DMM) and monosodium iodoacetate (MIA; 0.5 mg) mice were given THC (0, 5, or 10 mg/kg) orally 5 days/week for 9 or 2 weeks, respectively. Von Frey tests were used to evaluate pain. DMM mouse joints were assessed for cartilage degeneration/synovitis (OARSI scoring) and Ki67/αSMA expression [immunohistochemistry (IHC)]. Plasma isolated from DMM mice administered THC 10-weeks post-surgery was analyzed by targeted metabolomics. RNA sequencing was performed on THC-treated (1 μM) human OA FLS and chondrocytes from synovium and cartilage, respectively, to determine differentially expressed genes (DEGs) and DEG-enriched pathways. Ipsilateral L3-L5 DRG were collected 3-weeks post-MIA injection and single nucleus RNA sequencing (snRNAseq) was performed to determine THC-induced transcriptomic changes in distinct cell populations. Computational and bioinformatics was used to identify enriched pathways and inferred intercellular communications.

Results: 10 mg/kg THC provided the most significant pain reduction in DMM and MIA mice  (n=15/group). In DMM mice, all THC doses reduced cartilage degeneration, with 10 mg/kg THC reducing synovitis (n=9-10/group) and decreasing αSMA but not nuclear Ki67 synovial expression (n=6/group). Metabolomic analyses identified serotonin, carnosine, and 5-oxoproline to be reduced upon DMM surgery and rescued with THC administration (n=10/group). In vitro, RNA sequencing identified 73 DEGs in OA FLS and 21 DEGs in OA chondrocytes after 1 μM THC treatment (n=4). Extracellular matrix (ECM) organization and cholesterol biosynthesis pathways were enriched in upregulated and downregulated genes, respectively, in both cell types.  snRNAseq analyses identified transient receptor potential melastatin 8 (Trpm8)-expressing neurons, peptidergic nociceptors (Pep), and neurofilament (NF)-expressing neurons as having the highest DEGs in response to THC of all cells identified in the DRG (n=3/group). DEGs of Trpm8 neuronal cells were enriched for neuronal transmission pathways, immune system- and lipid-related pathways. Cell communication analyses determined a putative decrease in ligand-receptor signaling between Trpm8 neurons and Pep/NF neurons after THC administration.

Conclusions: Oral administration of 10 mg/kg THC reduced pain, cartilage degeneration, synovial inflammation, and synovial αSMA expression in DMM/MIA mouse knee joints. THC treatment induced systemic metabolic changes in DMM mice and gene expression/pathway/communication changes in DRG Trpm8 neuronal cells, implicating potential mechanisms for KOA pain modulation by THC. In local human OA FLS and chondrocytes, direct THC treatment modified gene expression associated with ECM organization and cholesterol biosynthesis. Next studies will focus on identifying mechanisms of action of THC in joint cells to uncover potential disease-attenuating mechanisms.

Anisha Hundal: Deciphering the tubulin code in nuclear envelope-mediated DNA double-strand break repair

The nuclear envelope is a dynamic membrane that separates the contents of the nucleus from the cytoplasm. Upon the induction of DNA double-strand breaks (DSBs), the nuclear envelope forms a network of tube-like structures (DSB-capturing nuclear envelope tubules or ‘dsbNETs’) that poke the nucleus and support DSB repair. Microtubules, which are polymers of tubulin, drive the formation dsbNETs. Microtubules can undergo various post-translational modifications (PTMs) collectively known as the ‘tubulin code’. One such modification is tubulin acetylation mediated by the enzyme alpha-tubulin acetyltransferase 1 (ATAT1), which increases in DNA damage conditions. We have shown that acetylated microtubules are required for the formation of dsbNETs. Interestingly, aggressive forms of breast cancer are known to have higher levels of acetylated tubulin as well as other types of modified microtubules. Moreover, we have shown that breast cancer cells form dsbNETs to respond to DNA damage. Therefore, we hypothesize that the tubulin code regulates dsbNETs and can be targeted to limit the growth and survival of breast cancer cells. To test this hypothesis, we will first investigate the signaling mechanisms involved in microtubule hyperacetylation upon DNA damage by assessing how ATAT1 may be activated when DNA damage is induced. We will also explore other tubulin PTMs to evaluate whether these PTMs and the enzymes responsible for them regulate the formation of dsbNETs. We will then use growth assays in cultured cells and mice to determine whether targeting tubulin-modifying enzymes limits the growth of breast cancer cells and increases their sensitivity to chemotherapies. Overall, this work can improve our understanding of DNA repair and uncover novel therapeutic strategies to target breast cancer.

Ream Abdullah: Investigating mismatch repair deficiency (dMMR) and KCNQ1 as gastric adenocarcinoma biomarkers for treatment efficacy & prognosis

Gastric cancer, also known as stomach cancer, is one of the deadliest cancers worldwide. Most cases are diagnosed in the later stages, where few treatment options exist. Those that are available are ineffective, and add heavy side effects. Upcoming treatments such as immunotherapy work well in some patients, but not others. New biomarkers are required to identify which patients may benefit and potentially identify new treatment options as well. I plan to identify and expand information on biomarkers that could stratify patients based on prognosis or treatment efficacy.

Using spatial transcriptomics, I plan to assess two groups of patients who underwent immune checkpoint blockade (ICB) treatment. Previous research has shown that patients with a mismatch repair deficiency (dMMR) benefit greatly from immunotherapy. Many have theorized that dMMR patients that benefit from ICB may have a greater number of immune-rich regions. My project aims to investigate this theory and spatially identify any genes that may be activated in the presence of these immune-rich regions.

For prognostic biomarkers, I aim to investigate an ion channel, KCNQ1, that may be associated with a tumour suppressor gene known as FAM46C. Relative mRNA expression of various EMT genes in immortalized cell lines with FAM46C shRNA knockdown in vivo and in vitro showed a difference in mesenchymal markers. In the future, I plan to explore the EMT mRNA and protein expression of KCNQ1 shRNA knockdown cells, along with increased pcDNA3.1 KCNQ1 cells in vitro. Growth, viability, migration and invasion will also be assessed.

Yuetong Song: TBA

TBA

Contact

No need to register.

Contact lmp.grad@utoronto.ca with any questions