A redução eletroquímica de íons complexos do tipo trans-[Ru(NH3)4LNO]2+, foi investigada com o intuito de verificar a possibilidade desses complexos atuarem como doadores de nitroxil (HNO/NO-). Neste trabalho foi dada uma ênfase especial no estudo da espécie em que L = P(OEt)3. Aplicando-se as técnicas de voltametria cíclica e de pulso diferencial, voltametria de onda quadrada e espectroeletroquímica, foi possível verificar que a espécie trans-[Ru(NH3)4P(OEt)3NO]2+ sofre redução monoeletrônica gerando o íon complexo trans-[Ru(NH3)4P(OEt)3NO]+. Foram analisadas as estabilidades relativas das espécies possivelmente formadas, trans- [Ru(NH3)4P(OEt)3NO]+ e trans-[Ru(NH3)4P(OEt)3HNO]2+, baseadas na Teoria de Densidade Funcional (DFT). Após a transferência eletrônica, de acordo com a equação abaixo, a espécie formada sofre isomerização de spin, ocorrendo posteriormente a dissociação do grupo NO O processo apresenta-se como irreversível no espectro voltamétrico devido às restrições de spin. A reação do íon complexo trans-[Ru(NH3)4P(OEt)3NO]3+ com o alfacetoglutarato (Ered = -0,90 V), composto capaz de agir como redutor de dois elétrons por transferência de elétrons de esfera externa, foi monitorada eletroquímica e espectrofotometricamente utilizando a metmioglobina, Mb - Fe(III), captador seletivo de nitroxil. Os resultados obtidos sugerem o complexo supracitado como um possível doador de niroxil. / The electrochemical reduction behavior of the complexes trans-[Ru(NH3)4P(OEt)3NO]2+ was investigated in order to determine the possibility of these complexes to serve as nitroxyl donors (HNO/NO-). Special emphasis was given when L = P(OEt)3. Using cyclic voltammetry and differential pulse, square wave voltammetry and spectroelectrochemistry techniques was possible to infer that the ion complex trans-[Ru(NH3)4P(OEt)3NO]2+ suffers an one electron reduction generating the ion complex trans-[Ru(NH3)4P(OEt)3NO]+. Analyzing the behavior of the cyclic voltammetry spectra, is possible to conclude that the process is irreversible by spin restrictions. The reaction of the ion complex trans-[Ru(NH3)4P(OEt)3NO]+ with the reductor agent alpha-ketoglutarate (Ered = -0,90V), a two electron reductor by outersphere pathway, was monitored by electrochemistry and UV-vis spectroscopy techniques using Mb-Fe(III) as a selective nitroxyl scavenger. The results obtained with these experiments confirm the original purpose of this study.

Many illnesses and infections are exacerbated and/or caused by biofilms. Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently asymptomatic in women, which can lead to persistent infection. Persistent infection can result in pelvic inflammatory disease, tubo-ovarian abscesses, infertility, and ectopic pregnancy. N. gonorrhoeae has been shown to form biofilms over glass, primary and immortalized cervical cells, and during natural cervical infection. Asymptomatic infection occurs in only 1% of infected males, and the infection site is subject to periodic rapid fluid flow, which may limit biofilm formation. Thus, biofilm formation may specifically play an important role in the infection of women and could contribute to the infrequent occurrence of symptoms. Prior to work presented in this dissertation, little was known about biofilm formation by N. gonorrhoeae. Therefore, we elected to compare the transcriptional profiles of biofilms to their planktonic counterparts, to identify genetic pathways involved in biofilm formation and maintenance. We found that 3.8% of the genome was differentially regulated, and that genes involved in anaerobic metabolism and oxidative stress tolerance were up-regulated in biofilm, while genes involved in aerobic metabolism were down-regulated. We determined that expression of aniA , ccp, and norB is required for robust biofilm formation over glass and human cervical cells, and anaerobic respiration occurs in the substratum of gonococcal biofilms. Disruption of the norB gene resulted in severe attenuation of biofilm formation. We determined that the accumulation of nitric oxide (NO) contributes to the phenotype of a norB mutant and can retard biofilm formation when present at sublethal concentrations. However, higher concentrations of NO can enhance biofilm formation in the absence of nitrite. NO enhances biofilm formation in an aniA mutant, but cannot completely restore biofilm formation, suggesting that NO can support anaerobic growth, although nitrite is preferred. We determined that the majority of the genes involved in gonococcal oxidative stress tolerance are required for normal biofilm formation, as mutations in the following genes resulted in biofilm attenuation over cervical cells and/or glass: oxyR, gor, prx, mntABC, trxB, and estD. Overall, biofilm formation may represent an adaptation for coping with the stresses present in the female genitourinary tract.

archives@tulane.edu / Experimental stroke in endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) knockout mice showed diverse effects on brain injury. nNOS and eNOS have been shown to uncouple in pathological conditions to produce superoxide. Oxidative stress is believed to be the underlying cause of several cardiovascular diseases including ischemic stroke. However, the role of eNOS and nNOS uncoupling in ischemic stroke is not well studied. Our objective of the study was to determine the effect of eNOS and nNOS inhibition on reactive oxygen species (ROS), NO, viability and mitochondrial bioenergetics in rat brain microvascular endothelial cells (BMECs) and rat cortical neurons following oxygen-glucose deprivation-reoxygenation (OGD/R). We found that non-specific inhibition of NOS in endothelial cells reduced ROS levels in BMECs but increased ROS levels in neurons under normoxia. This suggests that a pool of uncoupled NOS exists in the BMECs whereas the dominant functional NOS in neurons produces NO. We observed increased levels of ROS following OGD/R that is sensitive to NOS inhibition in both BMECs and neurons indicating eNOS and nNOS uncoupling during OGD/R. Furthermore, NOS inhibition reduced mitochondrial respiration while it improved cell survival rate in both BMECs and neurons following OGD/R. Thus, it is possible that decreased mitochondrial respiration in the immediate aftermath (4 hours) of OGD/R could be protective against reoxygenation injury. Moreover, we identified the expression of nNOS in BMECs from rat, human, and mouse. We observed that the nNOS in the BMECs constitutively produces superoxide under physiological conditions instead of NO. In contrast, nNOS in the neurons produces NO and doesn’t contribute to ROS. We also confirmed the nNOS expression and its function in freshly isolated rat brain microvessels. In addition, we developed a novel method to measure mitochondrial respiration in freshly isolated mouse brain microvessels using Seahorse XFe24 Analyzer. We validated the method by demonstrating impaired mitochondrial respiration in cerebral microvessels isolated from old mice compared to young mice. In summary, the present doctoral research investigated the distinct role of NOS isoforms in BMECs and Neurons leading to the identification of novel functional variant of nNOS in BMECs and brain microvessels. / 1 / RAMARAO SVNL

Accurate knowledge of heat transfer to materials in recombining plasmas is needed to improve heat shield designs. A lack of understanding of the chemical component of surface heating motivates the use of conservative assumptions with regards to surface catalysis in the design of thermal protection systems (TPS) that detrimentally impact payload capability. Chemical heating is the release of potential energy from recombining reactive species on the surface to form molecules. For a stable surface interacting with partially-dissociated air, the chemical heating component is due to surface-catalyzed recombination reactions of atomic O and N to produce molecular O2, N2, and NO. Unfortunately, heat flux measurements provide no fundamental information about the surface recombination pathways involved, or how the energy reaches the surface. Rather, they give a total heating rate. This work has taken steps to advance the current poor understanding about the chemical energy transport to and from material surfaces in high-temperature, recombining plasmas. A combination of spatially resolved laser-based diagnostics and emission spectroscopy was used to measure the number densities and gradients of the reactants (N, O), the products (NO, N2) and the energy distribution of recombined molecules (NO, N2) in the boundary layer adjacent to a plasma heated material. Laser excitation can probe individual species by electronic state (atoms) and by electronic, vibrational and rotational states (molecules). Emission can probe the radiative emission for a range of species and electronic, vibrational and rotational states of both atoms and molecules. These measurements of spatial variations in species concentrations through the boundary layer are directly related to near-surface gas-phase chemistry and energy exchange and have provided experimental information that was not currently available. Results provide the initial steps to determine recombination rates and the energy deposited on the surface due to surface catalyzed recombination of atomic nitrogen and oxygen in air plasma.

共著者あり。共著者名:Hizawa Nobuyuki, Yamaguchi Etsuro, Jinushi Eisei, Nishimura Masaharu. / Hokkaido University (北海道大学) / 博士 / 医学

<p>Reduksjon av gassbobler i blodbanen: metoder for å forebygge ugunstige effekter av dekompresjon.</p><p>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.</p><p>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.</p><p>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.</p><p>Kandidat: Andreas Møllerløkken</p><p>Institutt: Institutt for sirkulasjon og bildediagnostikk</p><p>Veileder: Professor Alf O. Brubakk</p><p>Finansieringskilder: Statoil, Norsk Hydro, Phillips Petroleum Company Norway og Petroleumstilsynet gjennom programmet forskning og utvikling innen dykking, kontraktsnr. 4600002328 med Norsk Undervannsintervensjon (NUI).</p><p>Ovennevnte avhandling er funnet verdig til å forsvares offentlig</p><p>for graden Philosophia Doctor i medisinsk teknologi</p><p>Disputas finner sted i Auditoriet, Medisinsk teknisk forskningssenter</p><p>Tirsdag 15.01.08 , kl. 12.15</p>

<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.