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.

This study examined the concept of using a reactive blending process to develop new polymeric additive systems. The objective was to investigate the potential of using a reactive processing technique as a means to bond additives to functional polymers, to create “in situ” bonds between functional groups present on the polymers and those present on the additives. The work is reported in two parts; the first part studied the bonding of colorants to functional polyolefins and the second part investigated the bonding of UV stabilisers to functional polyolefins. The research was completed with the long term objective that the approach should offer alternative additives to conventional non-bonded systems for use in polypropylene. An ethylene ionomer was utilised for the bonding of dyes, this was chosen for its optical clarity and chemical functionality. Polyethylene methacrylic acid (EMAA) ionomers and methine dyes were blended in the melt phase using an internal mixer to produce bright intrinsically colored polymers. Fourier transform infrared spectroscopy (FTIR) in transmission mode was used to assess the bonding of the dye to the ionomer. Bonding resulted through electrostatic interactions between carboxylate groups on the ionomer and cations on the dye molecules. The reactive blending process also resulted in a change in the chromophoric structure of the dye. The bonded system was compared to a system whereby no bonding between the methine dye and polymer was expected. In the later system the methine dye was blended with polyethylene using an internal mixer. From FTIR results no interaction was observed between the dye and polyethylene in this system. This was supported by microscopic analysis that showed that the dye was present in the polyethylene as a dispersion. The second stage of research focussed on the UV stabilisation of polyolefins. A melt reaction was explored between polypropylene functionalised with maleic anhydride (PP-g-MA) and an alkoxyamine hindered amine light stabiliser (NOR-HALS) with hydroxyl functionality. The technology proposed is based upon the reaction between the carboxylic acid groups of maleated polypropylene and hydroxyl groups of a specific NOR-HALS (Tinuvin 152). The efficiency of the modification was assessed using FTIR to verify the esterification reaction between the NOR-HALS and the maleated polypropylene. This reaction resulted in the grafting of a pendant UV stabiliser to the polypropylene through an ester linkage. A twin-screw extruder (TSE) was used to complete this study. A larger quantity of material could be produced using a TSE compared to the colorant system where an internal mixer was used. Samples of the reactively blended materials were exposed to UV radiation for a maximum time period of three hundred hours to assess the resulting stability of the materials. Diffuse reflectance FTIR (DRIFT) spectroscopy and X-ray photoelectron spectroscopy (XPS) provided an effective means to study oxidative degradation. IR spectroscopic measurements were used to determine the effectiveness of HALS in inhibiting the photo-oxidation of maleic anhydride grafted polypropylene. The inhibition was quantified by measuring the formation of carbonyl groups, with and without HALS bonded to the polymer, at fixed exposure times of UV radiation. DRIFT and XPS analysis confirmed that stabilised samples oxidised less, as indicated by the lower carbonyl index values and O1s / C1s ratios. These findings were complemented by results from Charpy impact tests. The mechanical property results indicated that the longevity of the materials with UV stabilisers grafted to them exceeded the PPg- MA system where there was no stabiliser present. Visible spectrophotometry was used to assess the colour of the polymeric samples and change in colour following exposure to UV radiation. Samples with bonded HALS demonstrated greater colour stability than control samples. The microstructure of the polymer surfaces was viewed using scanning electron microscopy (SEM). The polymeric samples demonstrated resistance to crazing when the NOR-HALS were bonded to the polymer. For both the colorant and UV stabiliser areas of research, thermal properties of the materials were assessed using differential scanning calorimetry (DSC). It was found that increasing the additive concentration in the polymer resulted in an increase in the temperature of crystallisation (Tc). Melt flow index can indicate if any change in molar mass had occurred during processing. An increase in melt flow index values (MFI) was observed when additive loading increased which suggested that degradation of the polymer had occurred during processing. In summary, reactive processing showed considerable promise as a means to bond additives to a functional polypropylene.

This study examined the concept of using a reactive blending process to develop new polymeric additive systems. The objective was to investigate the potential of using a reactive processing technique as a means to bond additives to functional polymers, to create “in situ” bonds between functional groups present on the polymers and those present on the additives. The work is reported in two parts; the first part studied the bonding of colorants to functional polyolefins and the second part investigated the bonding of UV stabilisers to functional polyolefins. The research was completed with the long term objective that the approach should offer alternative additives to conventional non-bonded systems for use in polypropylene. An ethylene ionomer was utilised for the bonding of dyes, this was chosen for its optical clarity and chemical functionality. Polyethylene methacrylic acid (EMAA) ionomers and methine dyes were blended in the melt phase using an internal mixer to produce bright intrinsically colored polymers. Fourier transform infrared spectroscopy (FTIR) in transmission mode was used to assess the bonding of the dye to the ionomer. Bonding resulted through electrostatic interactions between carboxylate groups on the ionomer and cations on the dye molecules. The reactive blending process also resulted in a change in the chromophoric structure of the dye. The bonded system was compared to a system whereby no bonding between the methine dye and polymer was expected. In the later system the methine dye was blended with polyethylene using an internal mixer. From FTIR results no interaction was observed between the dye and polyethylene in this system. This was supported by microscopic analysis that showed that the dye was present in the polyethylene as a dispersion. The second stage of research focussed on the UV stabilisation of polyolefins. A melt reaction was explored between polypropylene functionalised with maleic anhydride (PP-g-MA) and an alkoxyamine hindered amine light stabiliser (NOR-HALS) with hydroxyl functionality. The technology proposed is based upon the reaction between the carboxylic acid groups of maleated polypropylene and hydroxyl groups of a specific NOR-HALS (Tinuvin 152). The efficiency of the modification was assessed using FTIR to verify the esterification reaction between the NOR-HALS and the maleated polypropylene. This reaction resulted in the grafting of a pendant UV stabiliser to the polypropylene through an ester linkage. A twin-screw extruder (TSE) was used to complete this study. A larger quantity of material could be produced using a TSE compared to the colorant system where an internal mixer was used. Samples of the reactively blended materials were exposed to UV radiation for a maximum time period of three hundred hours to assess the resulting stability of the materials. Diffuse reflectance FTIR (DRIFT) spectroscopy and X-ray photoelectron spectroscopy (XPS) provided an effective means to study oxidative degradation. IR spectroscopic measurements were used to determine the effectiveness of HALS in inhibiting the photo-oxidation of maleic anhydride grafted polypropylene. The inhibition was quantified by measuring the formation of carbonyl groups, with and without HALS bonded to the polymer, at fixed exposure times of UV radiation. DRIFT and XPS analysis confirmed that stabilised samples oxidised less, as indicated by the lower carbonyl index values and O1s / C1s ratios. These findings were complemented by results from Charpy impact tests. The mechanical property results indicated that the longevity of the materials with UV stabilisers grafted to them exceeded the PPg- MA system where there was no stabiliser present. Visible spectrophotometry was used to assess the colour of the polymeric samples and change in colour following exposure to UV radiation. Samples with bonded HALS demonstrated greater colour stability than control samples. The microstructure of the polymer surfaces was viewed using scanning electron microscopy (SEM). The polymeric samples demonstrated resistance to crazing when the NOR-HALS were bonded to the polymer. For both the colorant and UV stabiliser areas of research, thermal properties of the materials were assessed using differential scanning calorimetry (DSC). It was found that increasing the additive concentration in the polymer resulted in an increase in the temperature of crystallisation (Tc). Melt flow index can indicate if any change in molar mass had occurred during processing. An increase in melt flow index values (MFI) was observed when additive loading increased which suggested that degradation of the polymer had occurred during processing. In summary, reactive processing showed considerable promise as a means to bond additives to a functional polypropylene.

This study examined the concept of using a reactive blending process to develop new polymeric additive systems. The objective was to investigate the potential of using a reactive processing technique as a means to bond additives to functional polymers, to create “in situ” bonds between functional groups present on the polymers and those present on the additives. The work is reported in two parts; the first part studied the bonding of colorants to functional polyolefins and the second part investigated the bonding of UV stabilisers to functional polyolefins. The research was completed with the long term objective that the approach should offer alternative additives to conventional non-bonded systems for use in polypropylene. An ethylene ionomer was utilised for the bonding of dyes, this was chosen for its optical clarity and chemical functionality. Polyethylene methacrylic acid (EMAA) ionomers and methine dyes were blended in the melt phase using an internal mixer to produce bright intrinsically colored polymers. Fourier transform infrared spectroscopy (FTIR) in transmission mode was used to assess the bonding of the dye to the ionomer. Bonding resulted through electrostatic interactions between carboxylate groups on the ionomer and cations on the dye molecules. The reactive blending process also resulted in a change in the chromophoric structure of the dye. The bonded system was compared to a system whereby no bonding between the methine dye and polymer was expected. In the later system the methine dye was blended with polyethylene using an internal mixer. From FTIR results no interaction was observed between the dye and polyethylene in this system. This was supported by microscopic analysis that showed that the dye was present in the polyethylene as a dispersion. The second stage of research focussed on the UV stabilisation of polyolefins. A melt reaction was explored between polypropylene functionalised with maleic anhydride (PP-g-MA) and an alkoxyamine hindered amine light stabiliser (NOR-HALS) with hydroxyl functionality. The technology proposed is based upon the reaction between the carboxylic acid groups of maleated polypropylene and hydroxyl groups of a specific NOR-HALS (Tinuvin 152). The efficiency of the modification was assessed using FTIR to verify the esterification reaction between the NOR-HALS and the maleated polypropylene. This reaction resulted in the grafting of a pendant UV stabiliser to the polypropylene through an ester linkage. A twin-screw extruder (TSE) was used to complete this study. A larger quantity of material could be produced using a TSE compared to the colorant system where an internal mixer was used. Samples of the reactively blended materials were exposed to UV radiation for a maximum time period of three hundred hours to assess the resulting stability of the materials. Diffuse reflectance FTIR (DRIFT) spectroscopy and X-ray photoelectron spectroscopy (XPS) provided an effective means to study oxidative degradation. IR spectroscopic measurements were used to determine the effectiveness of HALS in inhibiting the photo-oxidation of maleic anhydride grafted polypropylene. The inhibition was quantified by measuring the formation of carbonyl groups, with and without HALS bonded to the polymer, at fixed exposure times of UV radiation. DRIFT and XPS analysis confirmed that stabilised samples oxidised less, as indicated by the lower carbonyl index values and O1s / C1s ratios. These findings were complemented by results from Charpy impact tests. The mechanical property results indicated that the longevity of the materials with UV stabilisers grafted to them exceeded the PPg- MA system where there was no stabiliser present. Visible spectrophotometry was used to assess the colour of the polymeric samples and change in colour following exposure to UV radiation. Samples with bonded HALS demonstrated greater colour stability than control samples. The microstructure of the polymer surfaces was viewed using scanning electron microscopy (SEM). The polymeric samples demonstrated resistance to crazing when the NOR-HALS were bonded to the polymer. For both the colorant and UV stabiliser areas of research, thermal properties of the materials were assessed using differential scanning calorimetry (DSC). It was found that increasing the additive concentration in the polymer resulted in an increase in the temperature of crystallisation (Tc). Melt flow index can indicate if any change in molar mass had occurred during processing. An increase in melt flow index values (MFI) was observed when additive loading increased which suggested that degradation of the polymer had occurred during processing. In summary, reactive processing showed considerable promise as a means to bond additives to a functional polypropylene.

This study examined the concept of using a reactive blending process to develop new polymeric additive systems. The objective was to investigate the potential of using a reactive processing technique as a means to bond additives to functional polymers, to create “in situ” bonds between functional groups present on the polymers and those present on the additives. The work is reported in two parts; the first part studied the bonding of colorants to functional polyolefins and the second part investigated the bonding of UV stabilisers to functional polyolefins. The research was completed with the long term objective that the approach should offer alternative additives to conventional non-bonded systems for use in polypropylene. An ethylene ionomer was utilised for the bonding of dyes, this was chosen for its optical clarity and chemical functionality. Polyethylene methacrylic acid (EMAA) ionomers and methine dyes were blended in the melt phase using an internal mixer to produce bright intrinsically colored polymers. Fourier transform infrared spectroscopy (FTIR) in transmission mode was used to assess the bonding of the dye to the ionomer. Bonding resulted through electrostatic interactions between carboxylate groups on the ionomer and cations on the dye molecules. The reactive blending process also resulted in a change in the chromophoric structure of the dye. The bonded system was compared to a system whereby no bonding between the methine dye and polymer was expected. In the later system the methine dye was blended with polyethylene using an internal mixer. From FTIR results no interaction was observed between the dye and polyethylene in this system. This was supported by microscopic analysis that showed that the dye was present in the polyethylene as a dispersion. The second stage of research focussed on the UV stabilisation of polyolefins. A melt reaction was explored between polypropylene functionalised with maleic anhydride (PP-g-MA) and an alkoxyamine hindered amine light stabiliser (NOR-HALS) with hydroxyl functionality. The technology proposed is based upon the reaction between the carboxylic acid groups of maleated polypropylene and hydroxyl groups of a specific NOR-HALS (Tinuvin 152). The efficiency of the modification was assessed using FTIR to verify the esterification reaction between the NOR-HALS and the maleated polypropylene. This reaction resulted in the grafting of a pendant UV stabiliser to the polypropylene through an ester linkage. A twin-screw extruder (TSE) was used to complete this study. A larger quantity of material could be produced using a TSE compared to the colorant system where an internal mixer was used. Samples of the reactively blended materials were exposed to UV radiation for a maximum time period of three hundred hours to assess the resulting stability of the materials. Diffuse reflectance FTIR (DRIFT) spectroscopy and X-ray photoelectron spectroscopy (XPS) provided an effective means to study oxidative degradation. IR spectroscopic measurements were used to determine the effectiveness of HALS in inhibiting the photo-oxidation of maleic anhydride grafted polypropylene. The inhibition was quantified by measuring the formation of carbonyl groups, with and without HALS bonded to the polymer, at fixed exposure times of UV radiation. DRIFT and XPS analysis confirmed that stabilised samples oxidised less, as indicated by the lower carbonyl index values and O1s / C1s ratios. These findings were complemented by results from Charpy impact tests. The mechanical property results indicated that the longevity of the materials with UV stabilisers grafted to them exceeded the PPg- MA system where there was no stabiliser present. Visible spectrophotometry was used to assess the colour of the polymeric samples and change in colour following exposure to UV radiation. Samples with bonded HALS demonstrated greater colour stability than control samples. The microstructure of the polymer surfaces was viewed using scanning electron microscopy (SEM). The polymeric samples demonstrated resistance to crazing when the NOR-HALS were bonded to the polymer. For both the colorant and UV stabiliser areas of research, thermal properties of the materials were assessed using differential scanning calorimetry (DSC). It was found that increasing the additive concentration in the polymer resulted in an increase in the temperature of crystallisation (Tc). Melt flow index can indicate if any change in molar mass had occurred during processing. An increase in melt flow index values (MFI) was observed when additive loading increased which suggested that degradation of the polymer had occurred during processing. In summary, reactive processing showed considerable promise as a means to bond additives to a functional polypropylene.

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Biochemistry. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 6 uppsatser.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Biochemistry. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 122-124). Also available on the Internet.