The development of plants challenged by environmental stress alters plant architecture through several pathways, including those involving plant hormone responses and reactive oxygen species (ROS) production. Auxin, a phytohormone associated with every aspect of development, and abscisic acid (ABA), a phytohormone involved in abiotic stress responses, both interact with ROS. These ROS are used as secondary messengers to activate transcription of abiotic stress genes, and also in developmental responses such as cell elongation. To understand the mechanisms involved in the abiotic stress response and how the response intersects with auxin, ABA, and ROS, I examined COPPER INDUCED IN LEAVES 1 (<i>CIL1</i>) from <i>Brassica carinata</i> and its Arabidopsis orthologue, AUXIN INDUCED IN ROOTS 12 (AIR12). Expression of both genes increases in response to auxin and recent work has placed both <i>CIL1</i> and AIR12 within a family of plant-specific cytochrome b561 proteins thought to be involved with transmission of ROS signals. This suggests a link between auxin and ROS production resulting from abiotic stress. Antisense <i>CIL1 B. carinata</i> plants produced fewer lateral roots and were resistant to salinity stress during vegetative growth. Mutant air12 plants showed a 50% reduction in lateral root number, lateral root length, and H2O2 root distribution. Growth in the presence of H2O2 was able to restore lateral root length to control levels. In silica analysis of the <i>CIL1</i> and AIR12 amino acid sequences detected an attachment site for glucosylphosphatidylinositol, predicting that the protein is targeted to the extracellular leaflet of the plasma membrane where it could be cleaved and released into the apoplast. Subcellular localization using p35S::GFP-CIL1 and p35S::GFP-AIR12 translational fusions confirmed that CIL1 and AIR12 localize to the plasma membrane and are released into the apoplast. Organ localization of AIR12 using the pAIR12::GFP-AIR12 construct in stably transformed Arabidopsis showed fusion protein accumulation in the apex of the primary root and in the vascular tissue. Fusion protein also localized to cells flanking emerging lateral roots. Investigation of pAIR12::GUS Arabidopsis showed GUS accumulation in the apex of elongating lateral roots. I demonstrate that AIR12 is an extracellular protein and that air12 seedlings are susceptible to salt stress, but not osmostic stress and display increased and decreased sensitivity to ABA during germination and primary root elongation, respectively, suggesting that AIR12 acts downstream of abiotic stress recognition.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

It is well established that hypertension decreases endothelium-dependent vasomotor function, partially by excessive generation and reduced scavenging of reactive oxygen species (ROS). Nevertheless, at appropriate levels, some ROS can act as signaling molecules in the vasculature and contribute to endothelium-dependent dilation. Recent evidence in healthy resistance arteries suggests that the ROS species hydrogen peroxide (H2O2) acts as an endogenous endothelium-dependent dilator through a non-nitric oxide, non-prostaglandin (3NP) pathway. The aim of this study was to investigate the role of endogenous H2O2 in 3NP-mediated endothelium-dependent dilation of rat mesenteric arteries, and the changes that occur in these vessels with essential hypertension. 18-20wk old male spontaneously hypertensive rats (SHR; n=24) had an elevated systolic blood pressure of 198±6mmHg compared to 93±4mmHg (p<0.001) in the age matched normotensive Wistar-Kyoto rat (WKY; n=22). Isolated mesenteric arteries were preconstricted with norepinephrine (NEPI), followed by exposure to increasing doses of the endothelium-dependent dilator acetylcholine (ACh), which revealed vasomotor dysfunction in the SHR (maximal dilation: WKY: 94.8±1.3% vs. SHR: 75.2±2.9%, p<0.001). Incubation of the vessels with the non-specific cyclooxygenase (COX) inhibitor indomethacin (INDO) restored the ACh response in the SHR to the level of the WKY control (area under the curve: WKY: 354.6±8.6 vs. SHR INDO: 350.2±12.2, p>0.05) indicating that the release of constrictory prostaglandins from COX contribute to endothelial vasomotor dysfunction. Co-incubation of vessels with INDO and the nitric oxide synthase inhibitor Nω-nitro-L-arginine (LN) inhibited dilation in SHR (46.2±4.8%, p<0.001) but not in WKY (98.3±1.5%, p>0.05), indicating an elevated 3NP component in WKY over SHR. Further co-incubation with the H2O2 scavenger catalase (CAT), LN, and INDO inhibited the 3NP component to a greater extent in SHR (29.7±3.1%, p=0.062) than in WKY (91.6±2.5%, p<0.05). The responses of SHR and WKY mesenteric arteries to the endothelium-independent dilator sodium nitroprusside, the receptor-mediated constrictor NEPI, and the electrochemical constrictor KCl were no different between LN INDO and CAT LN INDO conditions. These data suggest that endogenous H2O2 has a greater role in mediating endothelium-dependent dilation in the mesenteric resistance arteries of SHR. Interestingly, in SHR, co-incubation with LN INDO improved dilation over LN alone (46.2±4.8% vs 23.3±3.2±, p=0.001), and CAT LN INDO decreased dilation from LN INDO to a similar extent, suggesting that COX-inhibition could be a source of H2O2 for endogenous vasodilation. Western blotting revealed a 54% increase in COX-1 protein expression in the SHR mesenteric arteries (WKY: 1.00±0.18 (n=9) vs. SHR: 1.54±0.17 (n=13), p<0.05), but no difference in the expression of the pro-oxidant enzyme p47phox, and the anti-oxidant enzymes CAT, SOD-1, and SOD-2. Administration of exogenous H2O2 to NEPI preconstricted mesenteric arteries revealed a dose-dependent dilation that was no different between SHR and WKY, and incubation of isolated WKY and SHR mesenteric arteries with CAT reduced the accumulation of H2O2 to a similar extent, as assessed by the H2O2-specific fluorescent dye Amplex Red. In conclusion, endogenous H2O2 is a vasodilator in the mesenteric arteries of SHR and WKY rats in the absence of nitric oxide and prostaglandins. In the SHR, COX-1 inhibition may allow endogenous H2O2 to become bioavailable for vasodilation. This study is the first to show a role for endogenous H2O2 in maintaining endothelium-dependent dilation in hypertensive rat resistance arteries, and provides evidence to support a role for COX-1-inhibition in the increased availability of H2O2 for dilation.

In Sweden more than 400 000 private households have not yet sufficient wastewater purification. These effluent is considered as an increasing problem and many onsite purification methods have been studied. In this investigation, the method of reactive bed filters have been tested by column and batch experiments. Five different kinds of filter materials with reactive surfaces have been studied concerning their capacity to absorb ammonium and phosphorus from wastewater. The materials that were examined are Filtra N, wollastonite Filtra P, blast furnace slag and Polonite®. The first two materials were examined for their capacity to remove ammonium, and the others for their phosphorus removal capacity. Ten columns were used, two for each material. A synthetic solution with the ammonium and phosphate concentration similar to that of domestic wastewater (NH4-N 30 mg/l; PO4-P 5 mg/l) were pumped to the columns under two flow regimes. Five columns were continuously saturated with solution and the other five columns were saturated under three one hour periods a day The solution was pumped three times a day to the columns at a volume equal to the pore volume of each material. The objective of the batch experiment was to find out how variations in pH-value and concentration of the nutrients influenced the sorption capacity of the materials. The result was then used for modelling in the computer program Visual Minteq to determine the probability of precipitation of known compounds. Results from the column experiment showed that sorption of phosphorus in both saturated columns and intermittently saturated columns were 96 % or more for blast furnace slag, Filtra P and Polonite®. Filtra N showed the best ammonium sorption with 92 %. Sorption of ammonium was much better in periodically saturated columns for the material wollastonite. The wollastonite used in this experiment showed a higher phosphorus sorption capacity than expected. A possible explanation could be that the easy weathering of Ca-silicate compounds favoured the sorption of phosphorus. Results from Visual Minteq modelling showed that the probability of calcite formation in Filtra P and Polonite® are very likely. The two materials had high pH-values and the calcite was most likely formed at values of pH &gt; 10. Hydroxyapatite is the most common precipitation when phosphorus sorption occurs and is suggested to be formed in wollastonite. / Mer än hälften av Sveriges enskilda avlopp bedöms inte uppfylla kraven i miljöbalken på längre gående rening än slamavskiljning. Dessa står för en betydande del av fosfor- och kväveutsläppen till hav, sjöar och vattendrag. Åtskilliga systemlösningar utreds, men i denna rapport har filter med fosfor- och kvävesorberande förmågor testats eller s.k. reaktiva filter. Mineraliska filter som ingått i studien är Filtra N, wollastonit, Filtra P, hyttsand och Polonite®. De två första filtren studerades speciellt på deras förmåga att avskilja ammonium och de tre övriga främst på deras fosforavskiljnings förmåga. Kolonnförsök utfördes med tio kolonner där fem belastades med mättat flöde och de övriga med intermittent mättat flöde. Kolonnuppsättningen var två kolonner per filtermaterial med vardera olika flödesförhållanden. Belastningen på kolonnerna sattes till betydligt högre än traditionell infiltration/markbädd. Ett artificiellt avloppsvatten användes och tillreddes med en koncentration av kväve (NH4-N) på 30 mg/l och fosforkoncentration (PO4-P) på 5 mg/l. De valda koncentrationerna efterliknar ett vanligt hushållsspillvatten. Skakförsök utfördes för att undersöka sorptionsförmågan hos de olika mineraliska filtren där vikten låg på hur koncentrationen av näringsämnena och pH påverkar sorptionen. Resultaten från skakförsöken utvärderades med jämviktsmodellen Visual Minteq. Sannolikheten att kända utfällningar bildas studerades. Resultaten från kolonnförsöken visade att hyttsand, Filtra P och Polonite® gav bäst fosforavskiljning med över 96 % för båda flödesregimerna. Filtra N var den bästa ammoniumavskiljaren med över 92 %. Wollastonit hade en bättre avskiljning med intermittent mättat flöde med 65 % jämfört med 11 % för mättat flöde. En intressant iakttagelse var att wollastonit hade en bättre fosforavskiljning än ammoniumavskiljning med ca 60 % för båda flödena. Det kan bero på att filtret innehåller lättvittrade Ca-silikatföreningar som ökar fosfatavskiljningen. Skakförsöken resulterade i att för Filtra P, hyttsand och Polonite® sorberades all tillsatt fosfat. Filtra N visade samma resultat som i kolonnförsöken med över 92 % sorption. Endast fosfat sorberades vid försök med tillsättning av både fosfat och ammonium till hyttsand och wollastonit. Vid modellering i Visual Minteq finns det en viss sannolikhet att kalcit bildats i Filtra P och Polonite® p g a deras höga pH, kalcit fälls ut vid pH &gt; 10. Hydroxyapatit som är den vanligaste utfällningen av kalciumfosfat vid avskiljning av fosfor bildades mycket troligt i wollastonit.

This thesis explores the feasibility of high power impulse magnetron sputtering (HiPIMS) to synthesize hydrogenated amorphous carbon (a-C:H) thin films in Ar-hydrocarbon ambient and the relationship between process parameters, gas phase composition and film properties. To this purpose a stable process based on HiPIMS and direct current magnetron sputtering (DCMS) has been developed. Four series of amorphous carbon thin films were deposited by hybrid HiPIMS-DCMS and pure DCMS processes at 15 mTorr pressure using different Ar-acetylene compositions and a substrate bias from 0 to -350 V. The effect of Ar-acetylene compositions and depositions processes on the film properties was investigated by characterizing the films using scanning electron microscopy (SEM), x-ray reflectometry (XRR), nanoindentation and elastic recoil detection analysis (ERDA). Moreover the process characterization was done by recording the optical emission spectrum and current and voltage waveforms of the hybrid HiPIMS-DCMS discharge. The characterization of the films revealed that the hybrid HiPIMS-DCMS process is a powerful tool for controlling the amorphous carbon film properties such as density, deposition rate, hardness and hydrogen content.

We are working on the development and design of an approach to agents that can reason, react to the environment and are able to update their own knowledge as a result of new incoming information. In the resulting framework, rational, reactive agents can dynamically change their own knowledge bases as well as their own goals. An agent can make observations, learn new facts and new rules from the environment, and then update its knowledge accordingly. The knowledge base of an agent and its updating mechanism has been implemented in Logic Programming. The agent’s framework is implemented in Java. This aim of this thesis is to design and implement an architecture of a reactive, rational agent in both Java and Prolog and to test the interaction between the rational part and the reactive part of the agent. The agent architecture is called RR-agent and consists of six more or less components, four implemented in Java and the other two are implemented in XSB Prolog. The result of this thesis is the ground for the paper “An architecture of a rational, reactive agent” by P. DellAcqua, M. Engberg, L.M. Pereira that has been submitted.

It is well established that hypertension decreases endothelium-dependent vasomotor function, partially by excessive generation and reduced scavenging of reactive oxygen species (ROS). Nevertheless, at appropriate levels, some ROS can act as signaling molecules in the vasculature and contribute to endothelium-dependent dilation. Recent evidence in healthy resistance arteries suggests that the ROS species hydrogen peroxide (H2O2) acts as an endogenous endothelium-dependent dilator through a non-nitric oxide, non-prostaglandin (3NP) pathway. The aim of this study was to investigate the role of endogenous H2O2 in 3NP-mediated endothelium-dependent dilation of rat mesenteric arteries, and the changes that occur in these vessels with essential hypertension. 18-20wk old male spontaneously hypertensive rats (SHR; n=24) had an elevated systolic blood pressure of 198±6mmHg compared to 93±4mmHg (p<0.001) in the age matched normotensive Wistar-Kyoto rat (WKY; n=22). Isolated mesenteric arteries were preconstricted with norepinephrine (NEPI), followed by exposure to increasing doses of the endothelium-dependent dilator acetylcholine (ACh), which revealed vasomotor dysfunction in the SHR (maximal dilation: WKY: 94.8±1.3% vs. SHR: 75.2±2.9%, p<0.001). Incubation of the vessels with the non-specific cyclooxygenase (COX) inhibitor indomethacin (INDO) restored the ACh response in the SHR to the level of the WKY control (area under the curve: WKY: 354.6±8.6 vs. SHR INDO: 350.2±12.2, p>0.05) indicating that the release of constrictory prostaglandins from COX contribute to endothelial vasomotor dysfunction. Co-incubation of vessels with INDO and the nitric oxide synthase inhibitor Nω-nitro-L-arginine (LN) inhibited dilation in SHR (46.2±4.8%, p<0.001) but not in WKY (98.3±1.5%, p>0.05), indicating an elevated 3NP component in WKY over SHR. Further co-incubation with the H2O2 scavenger catalase (CAT), LN, and INDO inhibited the 3NP component to a greater extent in SHR (29.7±3.1%, p=0.062) than in WKY (91.6±2.5%, p<0.05). The responses of SHR and WKY mesenteric arteries to the endothelium-independent dilator sodium nitroprusside, the receptor-mediated constrictor NEPI, and the electrochemical constrictor KCl were no different between LN INDO and CAT LN INDO conditions. These data suggest that endogenous H2O2 has a greater role in mediating endothelium-dependent dilation in the mesenteric resistance arteries of SHR. Interestingly, in SHR, co-incubation with LN INDO improved dilation over LN alone (46.2±4.8% vs 23.3±3.2±, p=0.001), and CAT LN INDO decreased dilation from LN INDO to a similar extent, suggesting that COX-inhibition could be a source of H2O2 for endogenous vasodilation. Western blotting revealed a 54% increase in COX-1 protein expression in the SHR mesenteric arteries (WKY: 1.00±0.18 (n=9) vs. SHR: 1.54±0.17 (n=13), p<0.05), but no difference in the expression of the pro-oxidant enzyme p47phox, and the anti-oxidant enzymes CAT, SOD-1, and SOD-2. Administration of exogenous H2O2 to NEPI preconstricted mesenteric arteries revealed a dose-dependent dilation that was no different between SHR and WKY, and incubation of isolated WKY and SHR mesenteric arteries with CAT reduced the accumulation of H2O2 to a similar extent, as assessed by the H2O2-specific fluorescent dye Amplex Red. In conclusion, endogenous H2O2 is a vasodilator in the mesenteric arteries of SHR and WKY rats in the absence of nitric oxide and prostaglandins. In the SHR, COX-1 inhibition may allow endogenous H2O2 to become bioavailable for vasodilation. This study is the first to show a role for endogenous H2O2 in maintaining endothelium-dependent dilation in hypertensive rat resistance arteries, and provides evidence to support a role for COX-1-inhibition in the increased availability of H2O2 for dilation.

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.

Flavonoids are of great interest for their antioxidant and health-promoting activities. Ascorbate (vitamin C) has antioxidant activities but also sometimes displays pro-oxidant activities <i>in vitro</i> and reportedly <i>in vivo</i>. This research investigated to what extent flavonoids moderate oxidative stress from vitamin C <i>in vivo</i>.<p>Dietary experiments were conducted in two phases using adult male Wistar rats. First, all animals were maintained for two weeks on a control flavonoid-free diet with the dietary requirement (27 IU) of vitamin E/kg diet. In the subsequent four weeks, the animals were treated in four groups (8 rats/group), being fed the following diets: flavonoid-free control (C), ascorbate-supplemented (7.55 mmol/kg diet) (A), flavonoid-supplemented (2.67 mmol/kg diet) (F) and flavonoids (2.67 mmol/kg diet) plus ascorbate (7.55 mmol/kg diet)-supplemented (T). Measurements were done on in vivo biomarkers of oxidative stress, tissue antioxidants and on tissue in vitro susceptibility to oxidative stress.<p>In the combined feeding of ascorbate plus flavonoids, endogenous thiobarbituric acid reactive substances (TBARS) increased in liver by 114%. No effects of dietary ascorbate or flavonoids were seen on endogenous TBARS in brain or heart, or on plasma thiols or erythrocyte fragility.<p><i>In vitro</i>, the susceptibility to TBARS formation of liver homogenate (incubated for 60 min at 37ºC in air) showed a significant 60% increase in ascorbate-fed animals compared to control, but no increase in animals fed ascorbate plus flavonoids, suggesting that the additional feeding of flavonoids helped to prevent the increase produced by ascorbate-feeding. Incubation of liver mitochondria with 300 µM ascorbate in vitro produced a large (2-7 fold) increase in TBARS, but there was no difference among mitochondria from the different feeding groups.<p>The ability of flavonoid-feeding in protecting against oxidative stress from ascorbate in vivo could not be demonstrated in this study, even showing pro-oxidant effects of flavonoids in combination with ascorbate in liver. However, in vitro tests in liver suggest a protective effect of flavonoid-feeding against susceptibility to oxidative stress from ascorbate. Further investigations are needed in order to resolve the differences observed in vitro and in vivo and to determine the endogenous effects of specific flavonoids under ascorbate-induced oxidative stress.

Therapeutic strategies of stroke can take two directions: to prevent brain damage from stroke or aid in its repair after a stroke. In this thesis, a rat stroke model, which mimics the human small vessel stroke, was used. Two potential repair strategies were investigated with this model, reduction of inflammatory processes with the aid of minocycline treatment and replacing necrotic neurons with new ones with the aid of neurogenesis of endogenous progenitor cells. <p>The stroke model is induced by disrupting the medium-size pial vessels within a 5mm-circular brain surface of adult Wistar rats. This leads to a cone-shaped cortical lesion. Therefore it mimics the clinical situation of lacunar infarction, the most frequent outcome of small vessel stroke. <p>Minocycline, a second-generation tetracycline, prevented cavitation and facilitated the repopulation of the lesion by reactive astrocytes. However, I could not identify the molecular target as the number of activated microglia, infiltrating leukocytes and CD3+ lymphocytes as well as interleukin-1β expression were not significantly altered. Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and immature neurons. After injury, DCX-positive cells appeared in the neocortex at the base of the lesion. These cells exhibit a morphology resembling differentiated post-migratory neurons with long branched processes. Some of the DCX-positive cells were also immunoreactive for βIII-tubulin, another marker of immature neurons. This might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE containing cells were found in the cortex underneath the lesion. These results suggest that the DCX-positive cells may not originate from neural precursors from the SVZ, but might be generated from local progenitor cells. In summary, using the PVD II model, which mimics the lacunar stroke, I found that neuroblasts appeared spontaneously near the lesion in the cerebral cortex and were attempting to upregulate neuronal properties. Reducing inflammation with post-stroke minocycline treatment prevented cavitation. I think both findings open up exciting new avenues for treatment of lacunar infarctions.