Previous work in our laboratory indicates that the diameter of brain arteries and arterioles can be increased by N-methyl-D-aspartate (NMDA) receptor activation. We looked for expression of NMDA receptors and endothelial cell responses to NMDA receptor agonists and antagonists in the mouse brain endothelial cell line bEnd.3. Using RT-PCR and Western blotting we found evidence supporting the presence of NMDA receptor subunits NR1 and NR2C. Treatment of bEnd.3 cells with combinations of 100 μM glutamate and D-serine significantly increased intracellular calcium. However, we saw no direct evidence that NO was produced in response to NMDA receptor activation using the Griess method. We did observe an NMDA receptor-dependent increase in protein nitrosylation. This increase is unlikely related to enhanced NO levels since it was not correlated with NO production and was not inhibited by the endothelial NO synthase inhibitor L-NIO.

No description available.

The motivation for this work is the weak level of confidence in forecasting the temperature profile for the Martian atmosphere over the aerobraking region. This limitation comes mainly because of a misrepresentation of the atomic oxygen distribution in atmospheric models, which is a result of lack of measurements. One effective way to observe atomic oxygen and temperature remotely in the 50 to 100 km altitude region is through airglow measurements. The first part of the thesis involves the development of an airglow model to simulate four O2 emissions: the Herzberg I, Herzberg II, and Chamberlain band systems, as well as the Infrared Atmospheric emission at 1.27 um. The model predictions are compared with available observations from both the Mars and Venus atmospheres to exploit the consistency in the photochemistry of these two CO2-dominated atmospheres. Using composition from 3-D global circulation models of the Mars and Venus atmospheres, simulations are performed with realistic dynamical variations. Previous studies used 1-D photochemical models only. Hence, this novel approach allows in-depth investigation of the influence of dynamics and circulation on the airglow behaviour. A sensitivity study is conducted to understand the impact of the different photochemical parameters available in the literature and to recommend a set of parameters to be used in future model predictions. This approach also provides an understanding of the impact of atmospheric conditions, like temperature, dust load, water concentrations, etc., on the vertical structure of the emissions. A retrieval algorithm is also developed to perform a partial inversion of the recorded signal to extract the NO airglow emission in the Mars atmosphere from the SPICAM instrument. The method is tested with one year of stellar occultation measurements and validated with observations from SPICAM in a limb-viewing geometry and with airglow model predictions. This work identified and quantified, for the first time, localised variations in the NO nightglow, providing insights into the factors influencing the distribution of the oxygen species other than the general circulation and the photochemistry. The method proved to be a useful tool to build a climatology of the NO emission in the Mars atmosphere.

Severe influenza, recognized as a clinical syndrome characterized by hyper-induction of pro-inflammatory cytokine production, results in approximately 250–500 thousand deaths annually worldwide. Current influenza research is focused on therapeutics to target the influenza virus or modulate influenza virus-induced inflammation as potential treatment options to improve clinical outcome in experimental influenza A (H1N1) virus infection. The goals of this work were: (1) to evaluate the utility of inhaled nitric oxide (iNO) for decreasing influenza virus production in the lungs, and (2) investigate the use of mesenchymal stromal (stem) cells (MSCs) for mitigating deleterious host responses to influenza infection. Here, we report that MSCs and iNO, administered alone either prophylactically or post-influenza virus infection, fail to modulate host inflammation, fail to improve acute lung injury, fail to dampen lung viral load, and fail to improve survival of infected mice.

The motivation for this work is the weak level of confidence in forecasting the temperature profile for the Martian atmosphere over the aerobraking region. This limitation comes mainly because of a misrepresentation of the atomic oxygen distribution in atmospheric models, which is a result of lack of measurements. One effective way to observe atomic oxygen and temperature remotely in the 50 to 100 km altitude region is through airglow measurements. The first part of the thesis involves the development of an airglow model to simulate four O2 emissions: the Herzberg I, Herzberg II, and Chamberlain band systems, as well as the Infrared Atmospheric emission at 1.27 um. The model predictions are compared with available observations from both the Mars and Venus atmospheres to exploit the consistency in the photochemistry of these two CO2-dominated atmospheres. Using composition from 3-D global circulation models of the Mars and Venus atmospheres, simulations are performed with realistic dynamical variations. Previous studies used 1-D photochemical models only. Hence, this novel approach allows in-depth investigation of the influence of dynamics and circulation on the airglow behaviour. A sensitivity study is conducted to understand the impact of the different photochemical parameters available in the literature and to recommend a set of parameters to be used in future model predictions. This approach also provides an understanding of the impact of atmospheric conditions, like temperature, dust load, water concentrations, etc., on the vertical structure of the emissions. A retrieval algorithm is also developed to perform a partial inversion of the recorded signal to extract the NO airglow emission in the Mars atmosphere from the SPICAM instrument. The method is tested with one year of stellar occultation measurements and validated with observations from SPICAM in a limb-viewing geometry and with airglow model predictions. This work identified and quantified, for the first time, localised variations in the NO nightglow, providing insights into the factors influencing the distribution of the oxygen species other than the general circulation and the photochemistry. The method proved to be a useful tool to build a climatology of the NO emission in the Mars atmosphere.

Severe influenza, recognized as a clinical syndrome characterized by hyper-induction of pro-inflammatory cytokine production, results in approximately 250–500 thousand deaths annually worldwide. Current influenza research is focused on therapeutics to target the influenza virus or modulate influenza virus-induced inflammation as potential treatment options to improve clinical outcome in experimental influenza A (H1N1) virus infection. The goals of this work were: (1) to evaluate the utility of inhaled nitric oxide (iNO) for decreasing influenza virus production in the lungs, and (2) investigate the use of mesenchymal stromal (stem) cells (MSCs) for mitigating deleterious host responses to influenza infection. Here, we report that MSCs and iNO, administered alone either prophylactically or post-influenza virus infection, fail to modulate host inflammation, fail to improve acute lung injury, fail to dampen lung viral load, and fail to improve survival of infected mice.

Glutathione (GSH), a 3-amino acid compound is ubiquitously expressed in eukaryotic cells and is the most abundant low molecular weight thiol. The importance of GSH is highlighted by its multitude of effects. Within the vascular wall GSH plays a crucial role as an intracellular antioxidant and it possess the ability to act as a signalling intermediate and store for nitric oxide (NO). The importance of NO and its role in vascular wall homeostasis is well recognized. Within the coronary circulation, NO is the primary dilator of many of the large arteries and the smaller arterioles. In addition to controlling coronary vascular tone, the importance of NO is highlighted by its antithrombotic, antihypertrophic, and antriproliferative effects. During instances of cardiovascular disease and normal aging, increases in the production of reactive oxygen species occur. A portion of the deleterious vascular effects of reactive oxygen species are believed to be due to reduction in NO bioavailability as a result of increased ROS-mediated destruction of NO. Altered GSH production in humans has been demonstrated to reduce endothelial function. Conversely, supplementation with GSH augments endothelium-dependent dilation. The mechanisms by which these alterations in GSH influence vasomotor function have not been resolved. The purpose of the studies within this thesis was to examine the impact of chronic and acute GSH modulations on coronary vascular resistance (CVR) and endothelium dependent dilation. In all experiments vascular reactivity was assessed in the isolated perfused rat heart. The advantage of this technique is that it allows the global coronary vasomotor functioning to be examined. Hearts were allowed to stabilize for 30 minutes to allow for the development of spontaneous coronary vascular resistance, followed by a bradykinin (BK) dose-response curve to assess endothelium-dependent dilation. The coronary circulation was then maximally dilated using an endothelium-independent agonist. In all cases BK-mediated dilation is expressed as a percentage of the endothelium-independent dilation. Chapter 2 of this document examines the chronic nature of GSH depletion and examines whether GSH depletion augments the influence of natural aging. Animals (mean age 33 and 65 weeks) were randomized to receive L-Buthionine-(S,R)-sulphoximine (BSO) in the tap water in order to inhibit GSH synthesis, or regular tap water (normal controls). Following 10 days of BSO treatment, ventricular GSH content was reduced in the BSO group compared to the control (0.182±0.021 vs 2.022±0.084 nmol/mg wet weight, p<0.05) and there was increased ventricular H2O2 content (1.345±0.176 vs 0.877±0.123 pmol/µg PRO, p<0.05). Baseline CVR was significantly reduced in the older animals compared to the adult animals (3.92±0.34 vs 4.76±0.20 and 3.67±0.24 vs 5.12±0.37 mmHg/ml×min-1 in the control and BSO treated groups, p<0.05). Conversely, in the presence of LNAME there was a significant increase in CVR in the adult BSO group (14.15±0.99, p<0.05) compared to all other groups. In the absence of LNAME, maximal dilation (percent endothelium-independent response) was reduced in the older animals compared to the adult animals (77±10.3% vs 95.0±1.0% for older and adult control and 92.7±4.5% vs 98.6±0.6% for the older and adult BSO, main effect of age). In the presence of LNAME the adult BSO group had a significantly reduced sensitivity (EC50) compared to all other groups (-7.39±0.09 Log M, p<0.05). Additionally, adult BSO treated animals had an increase in eNOS protein content. These results demonstrate that chronic thiol depletion resulted in an increased reliance on NO in the adult BSO group only. In chapter 3 the beneficial effects of GSH supplementation on BK mediated dilation were examined. Acute GSH was administered in the perfusate at either 0 (control) or with 10 µM for 2 reasons, 1) this concentration does not reduce basal coronary vascular resistance, allowing for a similar baseline CVR across conditions and 2) the 10 µM concentration is a physiologically relevant concentration of plasma/extracellular fluid GSH. The sensitivity to the endothelial agonist bradykinin was enhanced in the presence of GSH (-8.70±0.16 vs -7.94±0.06 LogM, p<0.01). The GSH effect was not dependent on NO production or utilization by soluble guanylate cyclase (sGC) as the enhanced dilation in the GSH group was maintained despite NOS (LNAME) and/or sGC inhibition. When the hearts were supplemented with a ROS scavenger TEMPOL, enhanced dilation was seen in the control group, but was not further enhanced in the GSH group. The requirement for ROS was best demonstrated when both the CON and GSH groups were supplemented with both TEMPOL and LNAME. This condition resulted in similar sensitivity (-7.76±0.19 vs -7.75±0.17 LogM, p>0.05) and area under the curve (182.33±12.70 vs 170±13.86, p>0.05) between GSH and CON. Thus, it was concluded that the effects of GSH administration requires the presence of ROS and exerts its effect in the microvasculature. The study presented in chapter 4 examined the effects of acute thiol modulation (depletion) on CVR and endothelium-dependent dilation. Previous reports have suggested that a reduction in intracellular GSH causes impaired NO production, and functional data support this contention. However, a majority of the data regarding the effects of thiol manipulation are from endothelial-removed vessels. The following agents were used to reduce GSH: the glutathione reductase inhibitor, BCNU; the thiol oxidizing agent, diamide; the thiol conjugating agent, ethacrynic acid (EA); and a thioredoxin inhibitor (CDNB). Preliminary data revealed that only CDNB (11.46±0.71 mmHg/ml×min-1) and EA (8.61±0.36 mmHg/ml×min-1) caused an elevation in CVR compared to the control (6.73±0.24 mmHg/ml×min-1). Conversely, Diamide and BCNU did not significantly affect baseline CVR, or the BK mediated responses. In the presence of EA, there was an overall blunting of the BK-response curve as observed by reduced EC50 (-7.85±0.07 Log M) and maximal dilation (90.8±1.8 %, percent endothelium-independent dilation) compared to the control group (-8.42±0.08 Log M and 97.7±1.6%). In the presence of CDNB the maximal dilation was 74.4±1.9% and the EC50 was -8.83±0.28 Log M. In addition to altering BK mediated responses, acute thiol depletion with all agents resulted in an increased minimal CVR with significant increases observed in the presence of CDNB and EA. There was a significant correlation with GSH:GSSG ratio and baseline (-0.547, p<0.05) and minimal CVR (r=-0.581, p<0.05). This study demonstrates that modulation of the GSH:GSSG ratio using a variety of agents with diverse mechanisms elicits differential responses within the vasculature. Specifically conjugation of GSH and inhibition of thioredoxin significantly alters BK mediated response, where as BCNU and dimaide did not. These results suggest that a modulation in the GSH:GSSG ratio impairs endothelium-dependent dilation and alters total dilatory capacity (baseline-minimal CVR) and thus may have implications for adequate tissue perfusion. Across all studies there was significant correlation between GSH and GSSG with both baseline and minimal CVR. Therefore it is likely that changes in overall glutathione content plays a role in determining baseline and minimal coronary vascular resistance. These results demonstrate the complexity that manipulations of GSH have on both CVR and endothelium-dependent dilation, and provide mechanistic insight into how changes in GSH alter coronary vascular resistance and endothelium-dependent dilation.

No description available.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph.D.)--Tufts University, 2001. / Adviser: Juliet A. Fuhrman. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 42-54). Access restricted to members of the Tufts University community. Also available via the World Wide Web;