Disruption of endogenous tidal rhythms of larval release linked to food supply and heat stress in an intertidal barnacle

The timing of larval release may greatly affect the survivorship and distribution of pelagic stages and reveal important aspects of life history tactics in marine invertebrates. Endogenous rhythms of breeding individuals and populations are valuable indicators of selected strategies because they are free of the neutral effect of stochastic environmental variation. The high-shore intertidal barnacle Chthamalus bisinuatus exhibits endogenous tidal and tidal amplitude rhythms in a way that larval release would more likely occur during fortnightly neap periods at high tide. Such timing would minimize larval loss due to stranding and promote larval retention close to shore. This fully explains temporal patterns in populations facing the open sea and inhabiting eutrophic areas. However, rhythmic activity breaks down to an irregular pattern in a population within the São Sebastião Channel subjected to large variation of food supply around a mesotrophic average. Peaks of chl a concentration precede release events by 6 d, suggesting resource limitation for egg production within the channel. Also, extreme daily temperatures imposing mortality risk correlate to release rate just 1 d ahead, suggesting a terminal reproductive strategy. Oceanographic conditions apparently dictate whether barnacles follow a rhythmic trend of larval release supported by endogenous timing or, alternatively, respond to the stochastic variation of key environmental factors, resulting in an erratic temporal pattern.

Chronic unpredictable mild stress alters an anxiety-related defensive response, Fos immunoreactivity and hippocampal adult neurogenesis

Previous results show that elevated T-maze (ETM) avoidance responses are facilitated by acute restraint. Escape, on the other hand, was unaltered. To examine if the magnitude of the stressor is an important factor influencing these results, we investigated the effects of unpredictable chronic mild stress (UCMS) on ETM avoidance and escape measurements. Analysis of Fos protein immunoreactivity (Fos-ir) was used to map areas activated by stress exposure in response to ETM avoidance and escape performance. Additionally, the effects of the UCMS protocol on the number of cells expressing the marker of migrating neuroblasts doublecortin (DCX) in the hippocampus were investigated. Corticosterone serum levels were also measured. Results showed that UCMS facilitates ETM avoidance, not altering escape. In unstressed animals, avoidance performance increases Fos-ir in the cingulate cortex, hippocampus (dentate gyrus) and basomedial amygdala, and escape increases Fos-ir in the dorsolateral periaqueductal gray and locus ceruleus. In stressed animals submitted to ETM avoidance, increases in Fos-ir were observed in the cingulate cortex, ventrolateral septum, hippocampus, hypothalamus, amygdala, dorsal and median raphe nuclei. In stressed animals submitted to ETM escape, increases in Fos-ir were observed in the cingulate cortex, periaqueductal gray and locus ceruleus. Also, UCMS exposure decreased the number of DCX-positive cells in the dorsal and ventral hippocampus and increased corticosterone serum levels. These data suggest that the anxiogenic effects of UCMS are related to the activation of specific neurobiological circuits that modulate anxiety and confirm that this stress protocol activates the hypothalamus-pituitary-adrenal axis and decreases hippocampal adult neurogenesis.

Evidence on the best chlorhexidine concentration to perform oral hygiene: meta-analysis

Although scientific literature has demonstrated the relevance of oral hygiene with chlorhexidine in preventing ventilation-associated pneumonia, there is a wide variation of concentrations, frequency and techniques when using the antiseptic. The aim of this research was to assessthe best chlorhexidine concentration used to perform oral hygiene to prevent ventilation-associated pneumonia. A systematic review followed by four meta-analysis using chlorhexidine concentration as criterion was carried out. Articles in English, Spanish or Portuguese indexed in the Cochrane, Embase, Lilacs, PubMed/Medline and Ovid electronic databases were selected. The research was carried out from May to June 2011. The primary outcome measure of interest was ventilation-associated pneumonia. Ten primary studies were divided in four groups (Gl-4), based on chlorhexidine concentration criterion. Gl (5 primary studies, chlorhexidine 0.12%) showed homogeneity among studies and the use of chlorhexidine represented a protective factor. G2 (3 primary studies, chlorhexidine 0.20%) showed heterogeneity among studies and chlorhexidine did not represent a protective factor. G3 (2 primary studies, chlorhexidine 2,00%) showed homogeneity among studies and the use of chlorhexidine was significant. G4 (10 primary studies with different chlorhexidine concentrations) showed homogeneity among studies and the common Relative Risk was significant. Statistic analyses showed a protective effect of oral hygiene with chlorhexidine in preventing ventilation-associated pneumonia. However, it was not possible to identity a standard to establish optimal chlorhexidine concentration.

Alimenti Funzionali e Componenti Nutraceutici come Biomodulatori

Recent knowledge supports the hypothesis that, beyond meeting nutrition needs, diet may modulate various functions in the body and play beneficial roles in some diseases. Research on functional foods is addressing the physiologic effects and health benefits of foods and food components, with the aim of authorizing specific health claims. The recognition that oxidative stress plays a major role in the pathophysiology of cardiac disorders has led to extensive investigations of the protective effects of exogenous antioxidants, but results are controversial. A promising strategy for protecting cardiac cells against oxidative damage may be through the induction of endogenous phase 2 enzymes with the enhancement of cellular antioxidant capacity. Sulforaphane (SF), a naturally occurring isothiocyanate abundant in Cruciferous vegetables, has gained attention as a potential chemopreventive compound thanks to its ability to induce several classes of genes implicated in reactive oxygen species (ROS) and electrophiles detoxification. Antioxidant responsive element (ARE)-mediated gene induction is a pivotal mechanism of cellular defence against the toxicity of electrophiles and ROS. The transcription factor NF-E2-related factor-2 (Nrf2), is essential for the up-regulation of these genes. We investigated whether SF could exert cardioprotective effects against oxidative stress and elucidated the mechanisms underpinning these effects. Accordingly, using cultured rat neonatal cardiomyocytes as a model system, we evaluated the time-dependent induction of gene transcription, the corresponding protein expression and activity of various antioxidant and phase 2 enzymes (catalase, superoxide dismutase, glutathione and related enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase, NAD(P)H: quinone oxidoreductase 1 and thioredoxine reductase) elicited by SF. The results were correlated to intracellular ROS production and cell viability after oxidative stress generated by H2O2, and confirmed the ability of SF to exert cytoprotective effects acting as an indirect antioxidant. Furthermore, to get better insight into SF mechanism of action, we investigated the effect of SF treatment on Nrf2 and the upstream signalling pathways MAPK ERK1/2 and PI3K/Akt, known to mediate a pro survival signal in the heart. The use of specific inhibitors of ERK1/2 and Akt phosphorylation demonstrated their involvement in phase 2 enzymes induction. The concentration of SF tested in this study is comparable to peak plasma concentration achieved after dietary exposure giving clear relevance to our data to support dietary intake of Cruciferous vegetables in cytoprotection against oxidative stress, a common determinant of many cardiovascular diseases.

Modulation of epidermial growth factor receptor signaling in colon adenocarcinoma

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, monoclonal antibodies (mAbs) and EGFR tyrosine kinase inhibitors (TKIs) seemed to be the most promising. However they have demonstrated low utility in therapy, the former being effective at toxic doses, the latter resulting inefficient in colon cancer. This thesis work presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphtoquinone core as shikonin, an agent with great anti-tumor potential. In HT-29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 μM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer. In addition, surface plasmon resonance (SPR) investigation of the direct EGF/EGFR complex interaction using different experimental approaches is presented. A commercially available purified EGFR was immobilised by amine coupling chemistry on SPR sensor chip and its interaction to EGF resulted to have a KD = 368 ± 0.65 nM. SPR technology allows the study of biomolecular interactions in real-time and label-free with a high degree of sensitivity and specificity and thus represents an important tool for drug discovery studies. On the other hand EGF/EGFR complex interaction represents a challenging but important system that can lead to significant general knowledge about receptor-ligand interactions, and the design of new drugs intended to interfere with EGFR binding activity.

Influence of particle size, density and concentration on bend erosive wear in pneumatic conveyors

Particle concentration is a principal factor that affects erosion rate of solid surfaces under particle impact, such as pipe bends in pneumatic conveyors; it is well known that a reduction in the specific erosion rate occurs under high particle concentrations, a phenomenon referred to as the “shielding effect”. The cause of shielding is believed to be increased likelihood of inter-particulate collisions, the high collision probability between incoming and rebounding particles reducing the frequency and the severity of particle impacts on the target surface. In this study, the effects of particle concentration on erosion of a mild steel bend surface have been investigated in detail using three different particulate materials on an industrial scale pneumatic conveying test rig. The materials were studied so that two had the same particle density but very different particle size, whereas two had very similar particle size but very different particle density. Experimental results confirm the shielding effect due to high particle concentration and show that the particle density has a far more significant influence than the particle size, on the magnitude of the shielding effect. A new method of correcting for change in erosivity of the particles in repeated handling, to take this factor out of the data, has been established, and appears to be successful. Moreover, a novel empirical model of the shielding effects has been used, in term of erosion resistance which appears to decrease linearly when the particle concentration decreases. With the model it is possible to find the specific erosion rate when the particle concentration tends to zero, and conversely predict how the specific erosion rate changes at finite values of particle concentration; this is critical to enable component life to be predicted from erosion tester results, as the variation of the shielding effect with concentration is different in these two scenarios. In addition a previously unreported phenomenon has been recorded, of a particulate material whose erosivity has steadily increased during repeated impacts.

Streptococcus agalactiae adapts to glucose stress conditions by modulating gene expression profile

Diabetes mellitus is considered a risk factor for Group B Streptococcus (GBS) infections. Typically, this pathology is associated to high glucose levels in the bloodstream. Although clinical evidences support this notion, the physiological mechanisms underlying GBS adaptation to such conditions are not yet defined. In the attempt to address this issue, we performed comparative global gene expression analysis of GBS grown under glucose-stress conditions and observed that a number of metabolic and virulence genes was differentially regulated. Of importance, we also demonstrated that by knocking-out the csrRS locus the transcription profile of GBS grown in high-glucose conditions was profoundly affected, with more than a third of glucose-dependent genes, including the virulence factor bibA, found to be controlled by this two-component system. Furthermore, in vitro molecular analysis showed that CsrR specifically binds to the bibA promoter and the phosphorilation increases the affinity of the regulator to this promoter region. Moreover, we demonstrated that CsrR acts as a repressor of bibA expression by binding to its promoter in vivo. In conclusion, this work by elucidating both the response of GBS to pathological glucose conditions and the underlined molecular mechanisms will set the basis for a better understanding of GBS pathogenesis.

Bedeutung von Rho-GTPasen bei der Regulation zellulärer Antworten auf genotoxischen Stress

SAPK/JNK regulieren nach genotoxischem Stress eine Vielzahl von Zielsubstraten, die bedeutsam für Reparatur und Überleben der Zelle sind, somit nehmen sie Einfluss auf das zelluläre Schicksal der Zelle. Ob DNA-Schäden eine Phosphorylierung von Stress-Kinasen nach sich ziehen ist bisher noch wenig untersucht. Mit reparaturdefizienten Zellen wurde der Einfluss von DNA-Schäden, durch Cisplatin/Transplatin/UV-C, auf die SAPK/JNK Aktivierung untersucht. Die Aktivierung der Stress-Kinasen erfolgte agenzspezifisch und abhängig von verschieden Reparaturfaktoren. Die Aktivierung korrelierte in reparaturdefizienten Zellen teilweise mit dem späten Auftreten von DNA-Strangbrüchen, war jedoch unabhängig von erhöhten initialen DNA-Schäden. Diese Befunde zeigten, dass die späte Aktivierung der SAPK/JNK DNA-schadensabhängig verläuft und das Cisplatin und Transplatin bei Verwendung von äquitoxischen Dosen zu einer vergleichbaren Aktivierung von SAPK/JNK führten. Die Hemmung der Rho-GTPasen sowohl durch Statine als auch mittels Clostridium difficile Toxin B zeigte weiterhin, dass Rho-GTPasen möglicherweise die späte DNA-schadensabhängige Aktivierung der Stress-Kinasen vermitteln. Die Hemmung von Rho-GTPasen durch physiologisch relevante Konzentrationen von Statinen führte in primären humanen Endothelzellen (HUVECs) zu einer Protektion vor IR-Strahlung und Doxorubicin. In beiden Fällen konnte eine Hemmung des pro-apoptotischen Transkriptionsfaktors p53 sowie der Chk1, welche einen Zellzyklusarrest reguliert, mit der Statin-Behandlung erreicht werden. Effektor-Caspasen wurden dabei durch den HMG-CoA-Reduktase Hemmer nicht beeinflusst. Ausschließlich bei dem Statin-vermittelten Schutz vor Doxorubicin kam es zu einer Reduktion von initialen DNA-Schäden, in Form von DNA-Strangbrüchen. Die IR-induzierten Strangbrüche in der DNA blieben von der Statin-Inkubation hingegen unbeeinflusst. Aufgrund ihrer protektiven Eigenschaften gegenüber IR- und Doxorubicin-induzierter Zytotoxizität in Endothelzellen und ihrer pro-apoptotischen Wirkung auf Tumorzellen könnten Statine möglicherweise die unerwünschten Nebenwirkungen von Zytostatika und einer Strahlentherapie günstig beeinflussen

Effects of Global Warming on Berry Composition of cv. Sangiovese: Biochemical and Molecular Aspects and Agronomical Adaptation Approaches

Wine grape must deal with serious problems due to the unfavorable climatic conditions resulted from global warming. High temperatures result in oxidative damages to grape vines. The excessive elevated temperatures are critical for grapevine productivity and survival and contribute to degradation of grape and wine quality and yield. Elevated temperature can negatively affect anthocyanin accumulation in red grape. Particularly, cv. Sangiovese was identified to be very sensitive to such condition. The quantitative real-time PCR analysis showed that flavonoid biosynthetic genes were slightly repressed by high temperature. Also, the heat stress repressed the expression of the transcription factor “VvMYBA1” that activates the expression of UFGT. Moreover, high temperatures had repressing effects on the activity of the flavonoids biosynthetic enzymes “PAL” and “UFGT”.Anthocyanin accumulation in berry skin is due to the balance between its synthesis and oxidation. In grape cv. Sangiovese, the gene transcription and activity of peroxidases enzyme was elevated by heat stress as a defensive mechanism of ROS-scavenging. Among many isoforms of peroxidases genes, one gene (POD 1) was induced in Sangiovese under thermal stress condition. This gene was isolated and evaluated via the technique of genes transformation from grape to Petunia. Reduction in anthocyanins concentration and higher enzymatic activity of peroxidase was observed in POD 1 transformed Petunia after heat shock compared to untrasformed control. Moreover, in wine producing regions, it is inevitable for the grape growers to adopt some adaptive strategies to alleviate grape damages to abiotic stresses. Therefore, in this thesis, the technique of post veraison trimming was done to improve the coupling of phenolic and sugar ripening in Vitis vinifera L. cultivar Sangiovese. Trimming after veraison showed to be executable to slow down the rate of sugar accumulation in grape (to decrease the alcohol potential in wines) without evolution of the main berry flavonoids compounds.

Lipid quality and oxidative stability in food products from animal origin, as affected by breeding factor

Nowadays it is requested more investigations on alternative rearing systems that are able to improve poultry welfare and to warrant high-quality and safe meat products. This thesis work was focused on the evaluation of the oxidative stability of poultry meats, obtained with different rearing systems, diets (supplemented with bioactive compounds), and packaging conditions. The thesis work was divided into the following parts: - Evaluation of the effects of different rearing systems on the quality, fatty acid composition and oxidative stability of poultry thigh and breast meat belonging to different product categories (“rotisserie” and “cut-up” carcasses); - Evaluation of the effects of different rearing systems and packaging conditions on the shelf-life of poultry thigh meat stored at 4°C for 14 days, and the effects of feed supplementation with thymol (control diet and diet with 2 different concentration of thymol) and packaging conditions on lipid oxidation of poultry thigh meat shelf-life (stored at 4°C for 14 days). The oxidative stability of poultry meat was studied by means of the spectrophotometric determinations of peroxide value and thiobarbituric acid reactive substances. - Evaluation of anti-inflammatory effects of different flavonoids (thymol, luteolin, tangeretin, sulforaphane, polymethoxyflavones, curcumin derivates) to detect their biological activity in LPS-stimulated RAW 264.7 macrophage cells in vitro, in order to study more in depth their action mechanisms. It was evaluated the cell vitality (MTT assay), nitrite concentration and protein profile. The study was focused on the identification of potential dietary bioactive compounds in order to investigate their biological activity and possible synergic effects, and to develop new suitable strategies for long-term promotion of human health, in particular against cancer.

Expression und Funktion der Paraoxonase-2 im Hinblick auf oxidativen Stress im Gefäßsystem

Erhöhte Spiegel von oxidativem Stress bedingen Atherosklerose, eine Krankheit die über 50% aller Todesfälle in der westlichen Welt ausmacht. Es ist entscheidend Mechanismen zur Abwehr dieser Krankheit zu ergründen.rnDa genetische Polymorphismen des körpereigenen Enzyms Paraoxonase 2 (PON2) mit kardiovaskulären Erkrankungen assoziiert sind, wurden ihre Regulation und potentiell antioxidativen Funktionen in vaskulären Zellen analysiert. Mittels verschiedener molekularbiologischer Methoden konnte ich erstmals zeigen, dass PON2 in vaskulären Zellen vornehmlich subzellulär im ER lokalisiert ist. Anhand verschiedener Experimente wurde PON2 als potenter Faktor zur Reduktion von ROS identifiziert. Erhöhte ROS-Spiegel führen zur Aktivierung eines als unfolded protein response (UPR) bekannten ER-Stress-Signalwegs. Dieser ist neben Atherosklerose in eine Vielzahl von Erkrankungen involviert und hat kritischen Einfluss auf das Überleben oder Absterben von Zellen. Durchgeführte Promoter-Reporter Studien bewiesen die Induktion der Protein-Expression von PON2 nach Aktivierung des UPR-Signalwegs, was als kompensatorischer Mechanismus der Zelle zur Vermeidung UPR-induzierter Apoptose verstanden werden könnte. PON2 wehrt oxidativen Stress und die UPR-induzierte Apoptose ab und ist ein protektiver Faktor vor Atherosklerose.rnIn einem Krebsmodell könnte PON2 aber als antiapoptotischer Faktor entscheidend am Überleben von Tumorzellen beteiligt sein. Gerade diese beiden gegensätzlichen Aspekte der antiapoptotischen Funktion des Proteins zeigen die Notwendigkeit für weitere Untersuchungen zu PON2 auf.rn

In vitro study of the osteocytes response to hypoxia and their regulation of bone homeostasis

Bone remodelling is a fundamental mechanism for removing and replacing bone during adaptation of the skeleton to mechanical loads. Skeletal unloading leads to severe hypoxia (1%O2) in the bone microenvironment resulting in imbalanced bone remodelling that favours bone resorption. Hypoxia, in vivo, is a physiological condition for osteocytes, 5% O2 is more likely physiological for osteocytes than 20% O2, as osteocytes are embedded deep inside the mineralized bone matrix. Osteocytes are thought to be the mechanosensors of bone and have been shown to orchestrate bone formation and resorption. Oxygen-deprived osteocytes seem undergo apoptosis and actively stimulate osteoclasts. Hypoxia and oxidative stress increase 150-kDa oxygen-regulated protein (ORP 150) expression in different cell types. It is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. It well known that ORP 150 plays an important role in the cellular adaptation to hypoxia, as anti-apoptotic factor, and seems to be involved in osteocytes differentiations. The aims of the present study are 1) to determine the cellular and molecular response of the osteocytes at two different conditions of oxygen deprivation, 1% and 5% of O2 compared to the atmospheric oxygen concentration at several time points. 2) To clarify the role of hypoxic osteocytes in bone homeostasis through the detection of releasing of soluble factors (RANKL, OPG, PGE2 and Sclerostin). 3) To detect the activation of osteoclast and osteoblast induced by condition media collected from hypoxic and normoxic osteocytes. The data obtained in this study shows that hypoxia compromises the viability of osteocytes and induces apoptosis. Unlike in other cells types, ORP 150 in MLO-Y4 does not seem to be regulated early during hypoxia. The release of soluble factors and the evaluation of osteoclast and osteoblast activation shows that osteocytes, grown under severe oxygen deprivation, play a role in the regulation of both bone resorption and bone formation.

Synthese und präklinische Evaluation von neuen antioxidativen Substanzen zur Neuroprotektion

Oxidativer Stress ist seit über 25 Jahren als ein Charakteristikum vieler pathologischer Prozesse bekannt. Helmut Sies beschrieb bereits in den 1980er Jahren oxidativen Stress als Störung in der prooxidativ – antioxidativen Balance zugunsten der prooxidativen Seite, wodurch es potentiell zu Schäden in verschiedenen Geweben kommt. Oxidativer Stress tritt sowohl bei neurodegenerativen Erkrankungen wie Morbus Alzheimer, Morbus Parkinson und zerebraler Ischämie, bei peripheren Erkrankungen wie Arteriosklerose, als auch beim Alterungsprozess per se auf und wird als Ursache oder zumindest als ein krankheitsfördernder Faktor diskutiert. Die in in vitro-Experimenten als vielversprechend antioxidativ getesteten Substanzen (meist phenolhaltig) ergaben in mehreren klinischen Studien keinen signifikanten Vorteil. Um die Ursachen dieser Ergebnisse näher zu analysieren, wurde in der vorliegenden Arbeit auf Basis des cytoprotektiven Phenothiazins, einem aromatischen trizyklischen Amin, der Einfluss von verschiedenen Substituenten im Hinblick auf Lipophilie, Radikalstabilisierung und Löslichkeit des Moleküls chemisch vorhergesagt. Anhand dieser in silicio Struktur-Wirkungs-Beziehung wurden anschließend neue Modellsubstanzen synthetisiert, welche sich systematisch in den drei zuvor genannten Parametern unterschieden. Dies wurde durch Substitution von unterschiedlich langen Fettsäureketten, von löslichkeitsbeeinflussenden funktionellen Gruppen, oder durch Anellierung zusätzlicher aromatischer Ringe erreicht. In den folgenden Versuchen zu antioxidativer Kapazität, zellulärem Überleben, Lipidperoxidation und Proteinoxidation zeigte sich, dass mit gesteigerter Stabilität der korrespondierenden Radikale und mit wachsender Lipophilie die antioxidativ cytoprotektive Aktivität der neuen Derivate bis zu einer gewissen Grenze (logP ≈ 7) signifikant zunahm; über diesen Wert hinaus sank die Effektivität wieder ab. Benzanellierte Phenothiazine entwickelten mit EC50-Werten von ungefähr 8-10 nM die höchste mittlere effektive Wirkkonzentration in oxidativ geschädigten, klonalen hippocampalen Neuronen (HT-22 Zellen). Dies entspricht einer etwa 20-fachen Verbesserung gegenüber α-Tocopherol, welches bisher als bestes natürliches lipophiles Antioxidans angesehen wurde. Im Vergleich zu Phenothiazin erreichen die neuen Antioxidantien immerhin eine höhere Effektivität um den Faktor 4. Folglich sind es sowohl Aspekte der Löslichkeit und der Distribution, welche die Potenz der gegenwärtigen Antioxidantien limitieren als auch Aspekte der Radikalstabilisierung, die Einfluss auf die primäre Wirksamkeit nehmen. Dieses Wissen sollte beim zukünftigen Design neuer, antioxidativ potenter Moleküle im Hinblick auf ihren langfristigen Einsatz bei neurodegenerativen Erkrankungen von Nutzen sein.

Effects of glucocorticoids on vascular NADPH oxidases and endothelial NO synthase

Glukokortikoide (GCs) stellen wichtige Hormone in der Regulation der metabolischen Homöostase dar. Synthetische GCs, wie Dexamethasone (DEX), spielen eine essentielle Rolle in der Behandlung inflammatorischer Krankheiten. Jedoch sind unter einer Dexamethason-Therapie zahlreiche Nebenwirkungen bekannt, so z.B. auch die Entwicklung einer Hypertonie, in deren Pathogenese oxidativer Stress eine entscheidende Rolle spielt. Obwohl sich in den vergangenen Jahren zahlreiche Studien zum Ziel setzten die GC-induzierte Hypertonie (GC-HT) aufzuklären, sind die genauen Mechanismen bis heute unklar. Eine erhöhte Expression von NADPH Oxidasen (Nox) und eine Entkopplung der endothelialen NO Synthase (eNOS), die Hauptquellen reaktiver Sauerstoffspezies (ROS) im vaskulären System, tragen maßgeblich zur Pathogenese kardiovaskulärer Erkrankungen bei. Daher ist eine Beteiligung dieser Enzyme in GC-induziertem oxidativen Stress sehr wahrscheinlich. Folglich wurde die Hypothese aufgestellt, dass NADPH Oxidasen und eine entkoppelte eNOS die vielversprechendsten unter den zahlreichen involvierten pro- und anti-oxidativen Enzymen sind. Mit Fokus auf die oben genannten Systeme wurde in der vorliegenden Studie der Effekt von DEX mit Hilfe von in vivo (WKY Ratten) ebenso wie in vitro Experimenten (A7r5 und EA.hy 926 Zellen) untersucht. Dabei zeigte sich, dass Nox1, Nox4 und p22phox durch DEX unterschiedlich reguliert wurden. Nox1 wurde hoch-, Nox4 hingegen herunterreguliert, während p22phox unverändert blieb. Die Modufikation schien hierbei auf transkriptioneller und post-transkriptioneller Ebene stattzufinden. Durch die gegensätzliche Regulation von Nox1 und Nox4 bleibt die Nettowirkung der verschiedenen Nox Isoformen unklar. Immer mehr Studien bringen vaskulären oxidativen Stress mit der Pathogenese einer GC-HT in Zusammenhang, welche letztendlich zu einer verminderten Bioverfügbarkeit von Stickstoffmonoxid (NO) führt. Durch die eNOS produziertes NO stellt einen essentiellen Schutzfaktor der Blutgefäße dar. Eine verminderte NO-Bioverfügbarkeit könnte die Folge einer eNOS-Entkopplung darstellen, ausgelöst durch oxidativen Stress. Da die Verfügbarkeit von Tetrahydrobiopterin (BH4) entscheident ist für die Aktivität und enzymatische Kopplung der eNOS, beschäftigt sich die vorliegende Arbeit mit GC-induzierten Veränderungen in der BH4-Versorgung. Die Behandlung von EA.hy 926 Zellen mit DEX führte zu einer zeit- und konzentrationsabhängigen Herunterregulation von eNOS auf mRNA- und Proteinebene. Gleichzeitig wurde die Phosphorylierung an Serine1177 vermindert. Als maßgeblicher “Kopplungs-Schalter” kann BH4 endogen über zwei verschiedene Signalwege synthetisiert werden, welche durch die Enzyme GCH1 und DHFR reguliert werden. DEX führte zu einer zeit- und konzentrationsabhängigen Herunterregulation von BH4, BH2 und Biopterin, wobei ebenso das BH4 / BH2 -Verhältnis vermindert wurde. Beide Enzyme, GCH1 genauso wie DHFR, wurden auf mRNA- und Proteinebene herunterreguliert, was auf einen Effekt von GCs auf beide rnBH4-produzierenden Signalwege schließen lässt. Nach Behandlung mit DEX wurde die Produktion von NO in Endothelzellen maßgeblich vermindert. In ROS-Messungen zeigte sich eine Tendenz hin zu einer eNOS-Entkopplung, jedoch war es mit unserem experimentellen Aufbau nicht möglich, diese endgültig zu beweisen.rnZusammenfassend lässt sich sagen, dass die Behandlung mit GCs zu Veränderungen in beiden untersuchten Systemen, den NADPH Oxidasen ebenso wie dem eNOS-NO System, führte. DEX erhöhte die Expression von Nox1 in glatten Muskelzellen und reduzierte die Nox4-Expression in Endothelzellen. Gleichzeitig verminderte DEX die Verfügbarkeit von BH4 und inhibierte die Phosphorylierung / Aktivität von eNOS. Mithilfe weiterer Studien muss die endgültige Beteiligung von NADPH Oxidasen und einer eNOS-Entkopplung an oxidativem Stress in GC-HT abschließend aufgeklärt werden.rn

Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.