Anxiety is a highly prevalent comorbidity in coronary artery disease (CAD) and confers increased risk of subsequent cardiac events and mortality. However, biological mechanisms of this relationship are not well understood. Ceramides are sphingolipids involved in inflammatory signaling and cell viability in the periphery and nervous system, and are implicated in pathophysiological mechanisms associated with anxiety. This study aimed to investigate relationships between plasma ceramide concentrations and anxiety symptomology as assessed by the Spielberger State-Trait Anxiety Inventory trait subscale (STAI-T) in CAD patients with linear regressions. High performance liquid chromatography coupled electrospray ionization tandem mass spectrometry was used to assay sphingolipid species. Plasma C22:0 ceramide (β=-0.232, p=0.018) concentrations and 8 other species of sphingolipids (SM18:0, SM20:1, C18:0, C20:0, C18:1, DHC22:0, LacC22:0, LacC24:1) were negatively correlated with STAI-T score when controlling for gender, BMI, and CES-D. Findings suggest specific sphingolipids to be potential markers for anxiety severity in CAD.

Anxiety is a highly prevalent comorbidity in coronary artery disease (CAD) and confers increased risk of subsequent cardiac events and mortality. However, biological mechanisms of this relationship are not well understood. Ceramides are sphingolipids involved in inflammatory signaling and cell viability in the periphery and nervous system, and are implicated in pathophysiological mechanisms associated with anxiety. This study aimed to investigate relationships between plasma ceramide concentrations and anxiety symptomology as assessed by the Spielberger State-Trait Anxiety Inventory trait subscale (STAI-T) in CAD patients with linear regressions. High performance liquid chromatography coupled electrospray ionization tandem mass spectrometry was used to assay sphingolipid species. Plasma C22:0 ceramide (β=-0.232, p=0.018) concentrations and 8 other species of sphingolipids (SM18:0, SM20:1, C18:0, C20:0, C18:1, DHC22:0, LacC22:0, LacC24:1) were negatively correlated with STAI-T score when controlling for gender, BMI, and CES-D. Findings suggest specific sphingolipids to be potential markers for anxiety severity in CAD.

Senescence is a cell stress program characterized by a stable cell cycle arrest and thus aims to protect against replication of potentially harmful cells. In oncogene-induced senescence (OIS) the cell cycle arrest is brought about by activation of an oncogene. This in turn initiates a DNA damage response and subsequently, the DDR induces p53-p21 and RB tumour suppressor pathways. The metabolism of senescent cells is highly altered, notably there is increased secretion of proteins and increased functional activity of certain metabolic enzymes. There have been many recent studies investigating the role of specific metabolic pathways in OIS and how they may be targeted for therapeutic benefit. This thesis aims to identify novel metabolic regulators of OIS, by combining high throughput RNAi screening and LC-MS based methods. This thesis has identified and validated 17 essential OIS metabolic genes; in this list, there was enrichment for genes involved in lipid biosynthetic processes. Lipid metabolism was an attractive focus for this thesis as it has not been extensively studied in current literature. Next, ceramide synthase 4 (CERS4) was extensively validated as a key enzyme for both OIS and replicative senescence. Using LC-MS based lipidomics, CERS4-driven rewiring of lipid metabolism in OIS was revealed and this corresponded with an accumulation of ceramides due to increased de novo ceramide synthesis. It was then confirmed OIS-related ceramide is mechanistically linked to cell cycle via the PP1-RB-E2F axis. Ceramide activates PP1, which physically binds to RB in a CERS4-dependent manner. PP1 is then able to dephosphorylate and activate RB, which inhibits transcription of E2F targets (cell cycle genes). Overall, this thesis identifies a metabolic checkpoint that links altered lipid metabolism with OIS.

>Magister Scientiae - MSc / Apoptosis or programmed cell death is vital to the development of organisms as they maintain the balance between cell death and cell growth. Failure to activate apoptosis has been implicated in carcinogenesis and often results from the over expression of anti–cancer proteins such as the X–linked inhibitor of apoptosis protein (XIAP). XIAP is over expresses in certain cancers and is a potent inhibitor of the initiator caspase 9 and effector caspases 3 and 7. The increased expression of XIAP in cancer cells result in the resistance to apoptosis. The control of XIAP is therefore considered as a target for anti–cancer drug development. Embelin or 2,5–dihydroxy–3–undecyl–1,4–benzoquinoine is a dihydroxyquinone compound that was previously shown to inhibit XIAP. This drug was discovered by structure based computational screening. The binding of embelin to XIAP displaces XIAP from caspases, consequently eliminating the inhibitory effect of XIAP on apoptosis. The objective of this study was to develop a gold nanoparticle that can be used for the targeted delivery of embelin to cancer cells thereby enhancing pro–apoptotic effects of the pro–apoptotic drug, ceramide. XIAP expression levels were investigated by Western blot analysis in a panel of human cancer cell lines available in the laboratory to identify two cell lines that can be used as low and high XIAP expression controls. Gold nanoparticles were synthesized and conjugated with embelin and a cancer targeting peptide with the amino acid sequence LTVSPWY. The biconjugated nanoparticles were used to co–treat MCF7 and HepG2 cells with ceramide. Apoptosis was quantified using flow cytometry. The uptake of gold nanoparticles was investigated using HR–TEM and ICP–OES. This study showed that gold nanoparticles conjugated with the LTVSPWY peptide is specifically targeted to and taken up by cancer cells. Gold nanoparticles conjugated with embelin promoted ceramide induced apoptotic cell death of cancer cells. However, it was observed that gold nanoparticles biconjugated with the LTVSPWY peptide and embelin failed to enhance the pro–apoptotic effects of ceramide. iii This study successfully demonstrated that gold nanoparticles conjugated with embelin could be used to enhance the effects of anti–cancer drugs using ceramide as an example.

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Ceramides are sphingolipids that serve as important second messengers in an increasing number of stress-induced pathways. Ceramide has long been known to affect the mitochondria, altering both morphology and physiology. Lipopolysaccharide (LPS) is a prevalent circulating inflammatory agent in obesity, potentially mediating some of the pathologies associated with weight gain. Given previous findings of TLR4-mediated ceramide accrual and ceramide-mediated mitochondrial disruption, we questioned whether ceramide is necessary for LPS-induced mitochondrial disruption. We found that LPS treatment increased gene transcript levels of ceramide synthesis enzymes and mitochondrial fission proteins and increased ceramide content in cultured myotubes and in mouse tissue. Mitochondrial respiration from permeabilized red gastrocnemius was reduced from animals receiving LPS injections when compared with those receiving vehicle (PBS). However, respiration from mice receiving both LPS and myriocin, a ceramide inhibitor, (0.3 mg/kg) was similar to PBS-injected animals. We treated murine myotubes with similar LPS conditions. These cells demonstrated increased ceramide synthesis and increased levels of mitochondrial fission with LPS treatment; these effects were mitigated with the addition of myriocin. However, in contrast to the whole gastrocnemius response in animals receiving LPS, respiration from myotubes was increased with LPS alone, and even higher with both myriocin alone and myriocin with LPS. We also sought to assess the impact of ceramide on skeletal muscle mitochondrial structure and function. A primary observation was the rapid and dramatic division of mitochondria in ceramide-treated cells. This effect is likely a result of increased Drp1 action, as ceramide increased Drp1 expression and Drp1 inhibition prevented ceramide-induced mitochondrial fission. Further, we found that ceramide treatment reduced mitochondrial O2 consumption (i.e., respiration) in cultured myotubes and permeabilized red gastrocnemius muscle fiber bundles. Ceramide treatment also increased H2O2 levels and reduced Akt/PKB phosphorylation in myotubes. However, inhibition of mitochondrial fission via Drp1 knockdown completely protected the myotubes and fiber bundles from ceramide-induced metabolic disruption, including maintained mitochondrial respiration, reduced H2O2 levels, and unaffected insulin signaling. These data suggest that the forced and sustained mitochondrial fission that results from ceramide accrual may alter metabolic function in skeletal muscle, which is a prominent site not only of energy demand (via the mitochondria), but also of ceramide accrual with weight gain.