<p> </p><p>Platelets play a key role in primary haemostasis but are also related to the pathogenesis of arterial thrombosis. Thrombin is the most effective agonist inducing platelet activation. Human platelets express two G-protein coupled thrombin receptors (GPCRs), called protease activated receptor (PAR)1 and PAR4. The aim of this study was to clarify differences in the activities of PAR1 and PAR4, especially focusing on their resistance towards the platelet inhibitor nitric oxide (NO) and their ability to undergo desensitization. For this, PAR1- and PAR4- activating peptides (APs) (SFLLRN and AYPGKF, respectively) were used. Different aspects of platelet activities were studied: aggregation and the rise in intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Aggregation was analyzed with lumiaggregometry, and [Ca²⁺]_i were studied using the fura-2 method. PKC substrate phosphorylation and the expression of PAR1 surface receptors were also analyzed, using Western blot and flow cytometry, respectively. The results from this study showed that NO exerted similar inhibitory effects on the two thrombin receptors. However, PAR1 and PAR4 differed in their ability to undergo desensitization. In cumulative dose-response studies, a low concentration of PAR1-AP induced desensitization of platelets towards higher PAR1-AP concentrations. This was not the case when studying PAR4-AP. The mechanism behind the desensitization of PAR1 to some part involved PKC, at least when studying the mobilization of intracellular Ca²⁺. PAR1 desensitization did not seem to involve receptor internalization and neither did it affect the activity of PAR4. This thus suggests that PAR4 might be a more suitable therapeutic target in the future management of thrombosis.</p><p> </p>

Context: Several recovery strategies have been used, with limited effectiveness, to reduce the muscle discomfort or pain and the diminished muscle performance following a bout of unaccustomed physical activity, a condition known as delayed onset of muscle soreness (DOMS). Muscle damage in this condition is associated with mechanical disruption of the muscle and connective tissue and inflammation and increased oxidative stress. Low frequency, low intensity, whole body periodic acceleration (WBPA) that increases nitric oxide (NO) release from vascular endothelium into the circulation through increased pulsatile shear stress offers a potential solution. This is because endothelial derived nitric oxide has anti-inflammatory, antioxidant and anti-nociceptive properties. Objective: The purpose of this study was to examine the effects of WBPA on the pain and diminished muscle performance associated with DOMS induced by unaccustomed eccentric arm exercise in young male subjects. Design: Longitudinal. Setting: University Exercise Physiology Laboratory. Participants: Seventeen active men, 23.4 +/- 4.6 yr of age. Intervention: Subjects made six visits to the research facility over a two-week period. On day one, the subject performed a 1RM elbow flexion test and was then randomly assigned to the WBPA or control group. Criterion measurements were taken on Day 2, prior to and immediately following performance of the eccentric exercise protocol (10 sets of 10 repetitions using 120% of 1RM) and after the recovery period. During all subsequent sessions (24, 48, 72, and 96 h) these data were collected before the WBPA or passive recovery was provided. Main Outcome Measures: Isometric strength (MVC), blood markers (CPK, MYO, IL-6, TNF-alpha and Uric Acid), soreness, pain, circumference, and range of motion (ROM). Results: Significantly higher MVC values were seen for the WBPA group across the entire 96 h recovery period. Additionally, within group differences were seen in CPK, MYO, IL-6, soreness, pain, circumference, and ROM showing a smaller impact and more rapid recovery by the WBPA group. Conclusion: Application of WBPA hastens recovery from DOMS after eccentric exercise. Given the lack of other potential mechanisms, these effects appear to be mediated by the increased NO release with WBPA.

This thesis describes the development of a new type of sensor for nitric oxide (NO) that can be used in a variety of combustion diagnostics and control applications. The sensor utilizes the absorption of ultraviolet (UV) radiation by the NO molecule to determine the concentration via optical absorption spectroscopy. UV radiation at 226.8 nm is generated by sum frequency mixing the outputs from a 395-nm external cavity diode laser (ECDL) and a 532-nm diode-pumped, intracavity frequency doubled Nd:YAG laser in a beta-barium borate (BBO) crystal. This radiation is used to probe the (v'=0, v"=0) band of the Α*Σ+ - Χ*π electronic transition of NO. The ECDL is tuned so that the UV radiation is in resonance with a specific energy level transition, and it is then scanned across the transition to produce a fully resolved absorption spectrum. Preliminary experiments were performed in a room-temperature gas cell in the laboratory to determine the accuracy of the sensor. Results from these experiments indicated excellent agreement between theoretical and experimental absorption line shapes as well as NO concentrations. Further experiments were performed at two actual combustion facilities to demonstrate the operation of the sensors in realistic combustion environments. Tests on a gas turbine auxiliary power unit (APU) at Honeywell Engines and Systems and on a well-stirred reactor (WSR) at Wright-Patterson Air Force Base produced excellent results despite the harsh temperatures and vibrations present. Overall, the sensitivity was estimated to be 0.8 parts per million (ppm) of NO (at 1000 K) for a 1 meter path length and the measurement uncertainty was estimated to be ±10%.

We hypothesized that the interaction between tumor necrosis factor alpha(TNF)/nuclear factor-kappaB (NFkB) via activation of IKK may amplify one anotherresulting in the evolution of vascular disease and insulin resistance associated withdiabetes. The interaction between TNFa and monocyte chemoattractant protein-1 (MCP-1) may contribute to the evolution of vascular inflammation and endothelial dysfunctionin coronary arterioles in type 2 diabetes. To test this hypothesis, endothelium-dependent(ACh) and –independent (SNP) vasodilation of isolated, pressurized coronary arterioles(40-100 μm) from mLeprdb (heterozygote, normal), Leprdb (homozygote, diabetic) andLeprdb mice null for TNF (dbTNF-/dbTNF-) were examined. Although dilation of vesselsto SNP was not different between Leprdb and mLeprdb mice, dilation to ACh was reducedin Leprdb mice. The NFkB antagonist, MG-132, IKK inhibitor, sodium salicylate(NaSal), or Anti-MCP-1 partially restored endothelium-dependent coronary arteriolardilation in Leprdb mice. Protein expression of IKK and IKK were higher in Leprdb thanin mLeprdb mice. The expression of IKK, but not the expression of IKK was increasedin dbTNF-/dbTNF- mice. Leprdb mice showed increased insulin resistance, but NaSal improved insulin sensitivity. Protein expression of TNFa, NFkB, phosphorylation ofIKK and JNK were greater in Leprdb mice, but NaSal attenuated protein expression ofthem in Leprdb mice. The ratio of phosphorylated IRS-1 at Ser307 (pIRS-1)/IRS-1protein expression was elevated in Leprdb mice; both NaSal and JNK inhibitor SP600125reduced pIRS-1/IRS-1 in Leprdb mice. MG-132 or neutralization of TNF reducedsuperoxide production in Leprdb mice. Anti-MCP-1 attenuated superoxide productionand protein expression of nitrotyrosine (N-Tyr), which is an indicator of peroxynitriteproduction, in isolated coronary arterioles of Leprdb mice. Immunostaining resultsshowed that expression of MCP-1 and vascular cellular adhesion molecule-1 (VCAM) isco-localized with endothelial cells and macrophages. Anti-TNFa or anti-MCP-1markedly reduced macrophage infiltration and the number of MCP-1 positive cells.Neutralization of TNFa or anti-MCP-1 reduced the expression of adhesion molecules. Inconclusion, our results indicate that the interaction between NFkB and TNFa signalinginduces activation of IKKb. In addition, TNFa and TNFa-related signaling, includingthe expression of MCP-1 and adhesion molecules, further exacerbates oxidative stressleading to endothelial dysfunction in type 2 diabetes.

Les émissions ultraviolettes des atmosphères de Vénus et de Mars, couramment appelées airglows, sont des phénomènes lumineux qui proviennent de relaxations d'atomes et de molécules passant d'un état excité à un état d'énergie inférieure. Ceux-ci nous renseignent sur les densités des constituants intervenant dans les réactions chimiques des atmosphères, ainsi que sur la dynamique des mésosphères respectives. Cette thèse traite de l'analyse détaillée de ces phénomènes dont le signal a été détecté entre 118 et 320 nm par les instruments SPICAV et SPICAM à bord des satellites Venus Express et Mars Express. Afin de bien comprendre le sujet abordé, les deux planètes, les différents airglows analysés et les instruments sont abordés dans une introduction constituant le premier chapitre de ce travail. Le chapitre 2 est consacré aux outils numériques qui servent à l'analyse et à la modélisation des airglows. Les trois chapitres suivants sont dédiés à leur caractérisation en intensité, à leur répartition en altitude et à leur localisation statistique. En particulier, nous étudions dans le chapitre 3 l'airglow de la molécule NO présent dans l'atmosphère nocturne de Mars à l'aide des données provenant de l'instrument SPICAM. Dans ce chapitre, nous utilisons un modèle chimique et diffusif unidimensionnel afin de confronter la théorie aux observations. Dans le chapitre 4, la même émission provenant de l'atmosphère de Vénus est analysée à l'aide du modèle unidimensionnel et des données enregistrées par l'instrument SPICAV. Les émissions de CO Cameron et du doublet CO2+ de l'atmosphère éclairée de Mars sont abordées dans le chapitre 5 à l'aide des données SPICAM. Ces émissions sont également comparées à un modèle stochastique afin de quantifier leurs processus de formation. Le chapitre 6 expose quelques perspectives de travail qui concernent ces phénomènes et qui permettrons à l'avenir d'acquérir de nouvelles informations à partir des données récoltées par SPICAV et SPICAM. Finalement, nous concluons en rassemblant les résultats clés des trois chapitres formant le corps principal de cette thèse.

Melanophores are dark brown pigment cells located in the skin of fish, amphibia, reptiles, and many invertebrates. The color of the animal can change via rearrangement of pigment granules, melanosomes, in the cells. The dark melanophores can either hide colorful cells so that the animal appears dark, or let through colors from underneath. The animal regulates its colors and patterns via communicating nerve cells and hormones in the blood stream. It is nowadays well established that melatonin-stimulation of melanophores results in aggregation of melanosomes to the cell center and that the evident outcome is more transparent cells. It has previously been shown that the activity of serine and threonine kinases as well as phosphatases regulates the distribution of melanosomes in the cells. We wanted to study if tyrosine phosphorylations were involved in the regulation of melanosome aggregation. Melatonin-stimulated signaling in the African clawed frog, Xenopus laevis, melanophores was examined. Melansome aggregation was accompanied by tyrosine phosphorylation as shown by immunoblots. Inhibition of tyrosine phosphorylation reduced melanosome aggregation by melatonin, and the phosphorylation most likely regulated pigment aggregation. Tyrosine phosphorylation of the protein was mediated via a Gi/o protein coupled receptor, probably the melatonin receptor Mel1c. The phosphorylation was most likely not a result of the classical Gi/o protein pathway, as Src-kinase and mitogen-activated protein kinase seemed required for phosphorylation and melanosome aggregation. Two candidates for the phosphorylated protein were presented, talin and β-spectrin. The possible involvement of nitric oxide in melanosome aggregation by melatonin was investigated. Nitric oxide appeared to be necessary for melanosome aggregation. The effect of nitric oxide synthase inhibition on melanosome aggregation was not mediated via changes in the tyrosine-phosphorylated protein. We speculated that nitric oxide could affect melanosome distribution via modifications of the actin cytoskeleton. The use of recombinant melanophores as a biosensor has also been examined. A human G protein coupled receptor, opioid receptor 3, was inserted into melanophores by electroporation. The transfected melanophores responded dose-dependently to opioids and an inhibitor of opioid receptors reduced the aggregation response. Future melanophore biosensors migh detect a variety of substances, such as narcotics, pheromones, odors, and tastes.

Reduksjon av gassbobler i blodbanen: metoder for å forebygge ugunstige effekter av dekompresjon. Når en dykker returnerer til overflaten etter dykking, kan det dannes gassbobler i kroppen som følge av overmetning av gasser. Slike gassbobler kan igjen føre til trykkfallsyke, men det gjenstår fremdeles å finne alle mekanismene bak denne sammenhengen. Gassbobler er derimot gode indikatorer på risiko for trykkfallsyke, og den gjennomgående arbeidshypotesen i denne avhandlingen har vært at gassbobler i blodbanen er den bakenforliggende årsaken til alvorlig trykkfallsyke. Det å redusere mengden gassbobler vil dermed øke sikkerheten for dykkeren. Avhandlingen består av tre studier som på forskjellige måter forsøker å redusere boblemengden ved trykkreduksjon. Alle arbeidene er gjennomført med bruk av gris som forsøksdyr, og alle dykkene er simulert i trykk-kammer spesielt laget for slike studier. For å måle gassbobler har vi benyttet ultralydavbildning, samt at vi har tatt ut kar for å måle eventuelle funksjonelle endringer i disse i etterkant av dykkene. Den første studien demonstrer en ny metode for å redusere gassbobledannelsen ved dekompresjon. Ved kortvarig å øke trykket under pågående trykkreduksjon kan boblemengden signifikant reduseres, resultatene viser at en modell som tar hensyn til bobledannelse beskriver resultatene bedre enn en tradisjonell modell som bare tar hensyn til overmetningen. I den andre studien har vi for første gang vist at gassbobler i blodbanen kan påvirkes medikamentelt også hos store dyr under dekompresjon fra metning. Ved å gi nitrater umiddelbart før dekompresjonen startet, ble mengden gassbobler signifikant redusert sammenlignet med kontrollene som ikke fikk tilført nitrater. Studien åpner veien for videre studier av biokjemiske prosesser involvert i både dannelsen av og effektene av gassbobler. I den siste studien undersøkte vi om en behandlingsprosedyre for trykkfallsyke til bruk når et trykk-kammer ikke er tilgjengelig ville være effektiv om behandlingstrykket ble redusert fra 190 kPa til 160 kPa med pusting av ren oksygen. Vi viste her at trykket var tilstrekkelig for å fjerne boblene etter dykket, men vi forhindret ikke skader på blodkarene. Kandidat: Andreas Møllerløkken Institutt: Institutt for sirkulasjon og bildediagnostikk Veileder: Professor Alf O. Brubakk Finansieringskilder: Statoil, Norsk Hydro, Phillips Petroleum Company Norway og Petroleumstilsynet gjennom programmet forskning og utvikling innen dykking, kontraktsnr. 4600002328 med Norsk Undervannsintervensjon (NUI). Ovennevnte avhandling er funnet verdig til å forsvares offentlig for graden Philosophia Doctor i medisinsk teknologi Disputas finner sted i Auditoriet, Medisinsk teknisk forskningssenter Tirsdag 15.01.08 , kl. 12.15

Human platelets are constantly regulated by activating and inhibitory effectors. Thrombin,the most potent platelet agonist, induces signaling through the protease-activated receptors(PARs) 1 and 4 which in turn convey their signal by coupling to G-proteins. Nitric oxide (NO)is a potent platelet inhibitor continuously formed by the endothelium exerting its effect byincreasing cGMP through activation of soluble guanylyl cyclase (sGC). The purpose of thiswork has been to investigate how NO would affect platelets already activated by PARagonists.To examine the different contributions of the PAR1- and PAR4-signals, the selectiveagonist peptides SFLLRN and AYPGKF-NH2 were utilized. Aggregation, Ca2+-mobilization andphosphorylation of threonine 696 in myosin phosphatase target subunit 1 (MYPT1) wereanalyzed. Intriguingly PAR1-, but not PAR4-, agonist provoked aggregation was rapidlyreversed upon NO exposure. PAR-agonist induced Ca2+-mobilization was markedly reducedafter exposure to NO, however this Ca2+-suppression did not cause the disaggregation ofPAR1-agonist evoked platelet aggregation. The reversal of aggregation was suspected to becaused by a cGMP-mediated inhibition of the Rho-kinase/ROCK-signaling pathway. This wassupported by Westen blot analysis where a marked decrease of MYPT1 phosphorylationcompared to basal levels could be observed. In conclusion, NO was found to reverse humanplatelet aggregation evoked by PAR1-activation by inhibition of a Rho-kinase/ROCK-signalingpathway.

ATP-sensitive potassium (K+) channels (KATP) in vascular smooth muscle cells (VSMC) play a major role in the regulation of vascular tone by coupling cell contractility and K+ fluxes to cellular metabolism. They are composed of the regulatory sulphonylurea receptors (SUR) and the pore-forming inwardly rectifying K+ (Kir) channels. SUR subunits interact closely with Kir subunits by conferring their sensitivity to nucleotide or sulphonylurea. However, the modulatory mechanisms of KATP channels in VSMC are largely unknown. In particular, the effects of hydrogen sulfide (H2S) and hydroxylamine (HA) on KATP channels and underlying mechanisms have not been addressed in VSMC of resistance arteries. The combined approaches including molecular biology, biochemical assays, and patch-clamp techniques were applied. The electrophysiological and pharmacological features of native KATP channels in VSMC and cloned KATP channels in HEK-293 cells, and the modulation of KATP channels by H2S and HA in single freshly isolated VSMC from rat mesenteric arteries were characterized. In the present study, only small conductance KATP channels of 13 pS were found in rat mesenteric artery VSMC. The recorded macroscopic and unitary KATP currents were activated by nucleoside diphosphate in the presence of magnesium and K+ channel openers, inhibited by a specific KATP channel blocker glibenclamide, but were insensitive to ATP inhibition. The reversal potential shifted rightward in response to the elevation of extracellular K+ and matched the calculated K+ equilibrium potential, indicating the basal currents in both VSMC and HEK-293 cells are carried by K+ ions. Heterologous expression of Kir6.1 with SUR2B in HEK-293 cells formed functional channels and elicited whole-cell K+ currents, which shared some similar biophysical characteristics of native KATP channels in VSMC. Basal KATP currents and resting membrane potential in VSMC were reduced by glibenclamide, demonstrating that KATP channels contribute to background K+ conductance and in the setting of resting membrane potential in this resistance artery. Exogenous H2S enhanced macroscopic and unitary KATP currents with an EC50 of 116 ± 8.3 µM and hyperpolarized membrane potential. H2S activated KATP channels by increasing the open probability of single channels, but not single channel conductance. The reduced endogenous H2S production by D, L-propargylglycine resulted in the attenuation of KATP currents. H2S-induced activation of KATP channels and resultant hyperpolarization were not mediated by cGMP signaling pathway. HA enhanced reversibly KATP currents in a dose-dependent fashion with an EC50 of 54±3.4 µM and also hyperpolarized the cell membrane. HA-stimulated KATP currents were blocked by free radical scavengers (superoxide dismutase and N-acetyl-L-cysteine), and KATP channels were stimulated by a free radical generating system (hypoxanthine/xanthine oxidase), indicating the involvement of superoxide (O2-) in HA effects. Sodium nitroprusside and 8-Br-cGMP did not affect basal KATP currents and HA-stimulated KATP currents, disproving the involvement of NO-sGC-cGMP-mediated signaling pathway in the HA effects. Therefore, HA-induced KATP channel activation and hyperpolarization are likely due to the generation of O2-. In conclusion, KATP channels in resistance artery VSMC serve as the regulatory targets of H2S and HA. These two endogenous molecules modulate KATP channels via different mechanisms. H2S may directly act on KATP channel proteins while HA oxidized them via the formation of O2-, leading to the activation of KATP channels.

The lymphatic system plays an important role in fluid and protein balance within the interstitial spaces. Its dysfunction could result in a number of debilitating diseases, namely lymphedema. Lymphatic vessels utilize both intrinsic and extrinsic mechanisms to pump lymph. Intrinsic pumping involves the active contraction of vessels, a phenomenon that is regulated in part by nitric oxide (NO) produced by lymphatic endothelial cells (LECs). NO production by arterial endothelial cells has been shown to be sensitive to both shear stress and stretch. Therefore, because of the unique mechanical environment of the LECs, we hypothesize that mechanical forces play an important role in regulation of the lymphatic pumping. Parallel-plate flow chambers and indenter-based cyclic stretch devices were constructed and used to apply mechanical loads to LECs. In addition, high-throughput micro-scale channels were developed and tested for shear experiments to address the need to increase the productivity and high- resolution imaging. Twenty-four hours treatment of LECs with different shear stress conditions showed a shear-dependent elevation in NO production. Moreover, 2.5 folds increase in cumulative NO was observed for stretched cells compared to the unstretched cells over six hours period. In conclusion, the upregulation observed in NO production under mechanical stimuli suggest new regulatory mechanisms that can be pharmaceutically targeted. These results provide an unprecedented insight into lymphatic pumping mechanism.