Naked mole rat glutamatergic receptors do not contribute to the control of ventilatory responses to acute or chronic hypoxia.

Sharn Gill, Biology Comparative Physiology with Dr. Matthew Pamenter

 

In most adult rodents hypoxia induces a reflex increase in ventilation (i.e. the hypoxic ventilatory response: HVR) and a decrease in metabolic rate (i.e. the hypoxic metabolic response: HMR). With chronic sustained hypoxia (CSH) a second time-dependent increase in ventilation occurs, termed ventilatory acclimatization to hypoxia (VAH). In most adult mammals, VAH is partially mediated by plasticity of glutamatergic receptors (NMDARs and AMPARs) in the ventilatory control circuits. Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals identified but their ventilatory responses to CSH have not been explored. To address this knowledge gap, we treated NMRs in room air or CSH (8% O2) for 8-10 days and measured their HVR before and after systemically administering AMPAR or NMDAR antagonists. We found that 1) when exposed to 7% O2 NMRs exhibited a ~ 64% decrease in ventilation, which was matched by a ~ 70% decrease in metabolic rate, 2) following acclimatization to CSH, NMRs did not exhibit VAH, 3) AMPAR or NMDAR antagonism had no effect on the HVR or HMR in any condition. Our results indicate that, contrary to all other adult rodents studied, glutamatergic synaptic transmission is not involved in the acute HVR or VAH of NMRs.

 

 

Extreme conditions studies of azides for the synthesis of single bonded nitrogen.

Spencer Sterling, Physics with Dr. Serge Desgreniers

Life on Earth has been defined by the usage of nitrogen-rich energetic materials. From the TNT in the excavation of our highways to the C-4 in our military warheads and the nitroglycerin in our hospitals, we have found many practical applications for these double-bonded nitrogen compounds. However, they can be unstable and environmentally toxic in addition to functioning at a much less powerful oxidation. In contrast, the single bond in nitrogen possesses three times the potential energy as the double bond. As such, nitrogen-rich energetic materials with nitrogen single bonds should exhibit highly modifiable qualities with much higher energetic capabilities. They should also combust to produce molecular nitrogen almost exclusively, providing a significant environmental advantage over the current organo-nitro explosives. With the theory that it is possible to synthesize polymeric nitrogen (pN) crystals with a sufficiently large hysteresis to be retained at lower pressures, this research observes the behaviour of high nitrogen azide (N3- ion) compounds under extreme pressures applied by Diamond-Anvil Cells (DAC) and explores the many experimental possibilities therein. The DACs consist of a microscopic sample cell between two diamonds which were forced together to produce pressures into the 40GPa range, with the potential to reach above 100GPa. X-Ray diffraction was performed on the cells at the Canadian Light Source in Saskatoon at intervals of increasing pressures until a phase change was observed. At this point, pressure was similarly relieved by taking diffraction images at pressure intervals. Diffraction image analysis provided the structures and structural properties of the samples to further develop their phase diagram and strategize a way to extract pN from the samples.

 

 

Deferasirox: Applications in the treatment of GLUT1 Deficiency Syndrome.
Mahdi Hassan, Cellular Molecular Medicine with Dr. Alex MacKenzie

GLUT1 Deficiency Syndrome (G1D) is a rare autosomal dominant encephalopathy that causes affected infants to suffer from seizures, movement disorders and intellectual impairment. G1D patients are refractory to anti-seizure medications and while in many cases their seizures are controlled to some extent by adhering to a ketogenic diet, it is ineffective therapy against the other disease symptoms. Moreover, some patients are unresponsive to this approach altogether and for these reasons new drug interventions are needed. G1D is genetically caused by mutations in the SLC2A1 gene that lead to a 50% loss of GLUT1 protein function (haploinsufficiency). Therefore if the root cause of the disease, insufficient gene/protein dosage, could be corrected one would predict that this would alleviate the disease symptoms. A drug screen of FDA approved compounds conducted by our lab identified the clinically-approved iron-chelator Deferasirox as a possible therapeutic agent that could upregulate GLUT1. The screen, performed in healthy human fibroblasts showed that Deferasirox caused up to a 4-fold upregulation of SLC2A1 transcript. The goal of the present work is to determine whether the Deferasirox-induced upregulation of the SLC2A1 gene translates to upregulation of the GLUT1 protein. Western blot analysis was used to determine the level of GLUT1 protein in response to Deferasirox treatment in healthy and patient human fibroblasts and in wild-type mice. Deferasirox was found to show a robust upregulation of transcript AND protein in vitro in some cell lines, but only a modest upregulation in protein in vivo when the drug was given for a longer period. Further investigation is warranted to confirm the effect of Deferasirox on GLUT1 protein.

 

Sex differences in the growth rates of thoracic aortic aneurysms: role of aneurysm etiology

Katie Cheung, Cardiology with Dr. Thais Coutinho

 

Thoracic aortic aneurysm (TAA) is a deadly disease. Although TAAs are more common in men, women have higher incidence of aneurysm dissection/rupture and death. However, the reasons for worse prognosis in women remain unknown. Since TAA growth rate is directly associated with the risk of dissection/ rupture, we hypothesized that TAA growth would be greater in women than men. In addition, since hypertension is also a risk factor for aortic dissection/rupture, we hypothesized that disease etiology would modify the association of sex with aneurysm growth. 82 unoperated subjects with TAA were recruited and TAA etiologies were categorized as heritable (hTAA) or degenerative (dTAA). Aneurysm growth was calculated as mm/year, and then normalized for body surface area. The association of female sex with aneurysm growth rate was tested with multivariable linear regression analyses adjusted for age, body surface area, baseline aneurysm size, history of hypertension, diabetes, dyslipidemia and smoking, and beta blocker use. In addition, the interaction term sex*aneurysm etiology was tested. Mean age was 67.7±11.8 years, 74% were men, and 57% had dTAA. Mean baseline aneurysm size and follow-up time were 45.6±4.3 mm and 3.1±2.8 years, respectively. Aneurysm growth rate was twice as fast in women than men [0.65±0.58 vs. 0.29±0.32 (mm/year)/m2, P=0.01]. In multivariable analyses, female sex remained independently associated with faster aneurysm growth (β±SE: 0.20±0.06, P=0.003). The sex*aneurysm etiology interaction was significant (P=0.001), and we found that the association of female sex with faster aneurysm growth was present in subjects with dTAA (β±SE: 0.34±0.08, P=0.0001), but not in those with hTAA (β±SE: 0.002±0.11, P=0.99). In conclusion, TAA growth rates are greater in women than men, which was specific to women with dTAA. To further our evaluations, we will explore hemodynamic, histological and biochemical differences in men and women with TAA.

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Selection of DNA aptamers against human red blood cells toward a novel drug delivery system.

Zerin Mahzabin Khan, Bioanalytical Chemistry with Dr. Maxim Berezovksi

Current drug delivery systems have limitations like low blood residence times and inability for targeted delivery, which can be addressed by appropriate drug carriers. Red blood cells (RBC) can become drug carriers by coupling drugs to RBC surface using affinity ligands like antibodies. Since antibodies possess limitations in vivo, aptamers have emerged as promising alternatives. Aptamers binding malaria infected RBC at room temperature (RT) had been identified for diagnostics. Our group previously developed DNA aptamers binding normal RBC at RT for cell purification. Thus, there exists a need to develop aptamers binding normal RBC under physiological conditions for in vivo applications. This study optimized and employed a cell-SELEX protocol to identify aptamers binding normal RBC under physiological conditions, like 37°C. It was hypothesized these aptamers bind with higher affinity at 37°C than RT. Optimization results revealed phire II polymerase, 15 PCR cycles, and eliminating a gel purification step were optimal for isolating/enriching bound aptamers. An aptamer pool with known binding to RBC at RT was then subjected to three rounds of optimized cell-SELEX protocol comprising selection, amplification, exonuclease digestion, and gel purification to isolate aptamers binding RBC at 37°C. Binding affinities of three pools were screened with flow cytometry, and preliminary results indicated original pool’s affinity for binding at RT was changed successfully, as rounds two and three had higher propensity to bind at 37°C. Further rounds of cell-SELEX can be performed to develop higher affinity aptamers. Impact of these aptamers are significant, as aptamer-drug conjugates can be developed and coupled to RBC surface for a novel drug delivery system with longer circulatory half-lives and targeted delivery via bi-specific aptamers.

 

Understanding the role of Cyclooxygenase-2 on cell survival of mouse ovarian surface epithelial cells in the context of ovarian cancer.

Howard Wong, Cellular and Molecular Medicine with Dr. Barbara Vanderhyden

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The ovarian surface epithelium (OSE) is a monolayer of epithelial cells surrounding the ovary that ruptures during ovulation to allow release of the mature oocyte. After ovulation, this ovulatory wound is repaired, although the processes involved are poorly understood. The OSE is one origin of ovarian cancer and ovulation is the primary non-hereditary risk factor for this disease. Currently there are no reliable early detection methods for ovarian cancer. Increasing our understanding of risk factors may allow for the identification for novel early detection methods.
During ovulation, follicular fluid bathes the ovulatory wound site and the surrounding OSE. Our lab has previously reported that Transforming Growth Factor βeta 1 (TGFβ1), one component of the follicular fluid, may aid in ovulatory wound repair. To elucidate ways in which TGFβ1 may aid in ovulatory wound repair, our lab performed a TGFβ1 Signalling Targets PCR array to compare control OSE cells with OSE cells treated with TGFβ1. We identified an 8-fold increase in Cyclooxygenase-2 (Cox-2) with TGFβ1 treatment. In stress conditions such as hypoxia, Cox-2 overexpression was shown in colorectal tumor cells to reduce cell death but also induced Hypoxia-inducible factor 1 (HIF-1). HIF-1, a regulator of oxygen homeostasis, is released to aid with wound healing such as cell migration and cell survival. We hypothesize that during ovulation, Cox-2 is expressed to enhance OSE cell survival and promote ovulatory wound repair. Using constitutive Cox-2 overexpressing OSE cells, we assessed cell survival using various cell stressors and found Cox-2 overexpression enhances cell survival under hypoxic conditions. Future directions include identification of the molecular pathways underlying this phenotype. We hypothesize that an increase in OSE cell survival during ovulation not only promotes ovulatory wound repair, but may be a mechanism for accumulation of mutations over time and lead to tumorigenesis.