Ulm, Univ., Diss., 2003.

In 2010, comet 103P/Hartley 2 was observed pre- and post-perihelion using the George and Cynthia Mitchell Integral Field Spectrometer on the 2.7-m telescope at McDonald Observatory in Texas. Data for gaseous radicals C2, C3, CH, CN, and NH2 were collected over six nights from 15 July to 10 November. The spectral data were used to create coma maps for each of the observed species, and the maps were processed using radial and azimuthal mean division techniques to create enhanced images of the coma, revealing subtle morphological features. 340 enhanced coma images were created for each observation and species. Visual inspection reveals that the coma is heterogeneous between the five detected radicals, and statistical analyses verify this result. To compliment the ongoing investigation of Hartley 2 as studied by the EPOXI flyby mission, findings from other researchers (Belton et al., 2012; Syal et al., 2012; and Thomas et al., 2012) are used to characterize the nucleus spin state and identify dust jet locations on the nucleus. With rotational period measurements from EPOXI, dust jet vectors on the nucleus surface are rotated to relevant observation times in November to compare the computed jet directions with the radical densities in the coma. Dust jet sites on the smaller nucleus lobe show a stronger correlation with high radical concentrations than the dust sites on the larger nucleus lobe. Production rates for potential parentage of radical species are calculated using the radial outflow Haser model (Haser, 1957), which are compared to mixing ratios relative to water from separate campaigns to constrain parentage. NH3 is likely the sole producer of NH2, whereas CN may be produced from a combination of HCN, C2N2, and CH3CN. Traditional parentage of C2, C3, and CH do not yield acceptable fits or suitable mixing ratios with the Haser model, and it is possible that extended coma ices having relatively short scale lengths greatly contribute to production of these radicals. These results provide further evidence that the Hartley 2 nucleus is heterogeneous in composition, and the rotational analysis indicates that specific jet sites are correlated with certain radical species.

No / Phortress is a novel, potent, and selective experimental antitumor agent. Its mechanism of action involves induction of CYP1A1-catalyzed biotransformation of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) to generate electrophilic species, which covalently bind to DNA, exacting lethal damage to sensitive tumor cells, in vitro and in vivo. Herein, we investigate the effects of DNA adduct formation on cellular DNA integrity and progression through cell cycle and examine whether a relevant pharmacodynamic end point may be exploited to probe the clinical mechanism of action of Phortress and predict tumor response. Single cell gel electrophoresis (SCGE) was applied to quantify DNA damage and cell cycle analyses conducted upon 5F 203 treatment of benzothiazole-sensitive MCF-7 and inherently resistant MDA-MB-435 breast carcinoma cells. Following treatment of xenograft-bearing mice and mice possessing hollow fiber implants containing MCF-7 or MDA-MB-435 cells with Phortress (20 mg/kg, i.p., 24 hours), tumor cells and xenografts were recovered for analyses by SCGE. Dose- and time-dependent DNA single and double strand breaks occurred exclusively in sensitive cells following treatment with 5F 203 in vitro (10 nmol/L¿10 ¿mol/L; 24¿72 hours). In vivo, Phortress-sensitive and Phortress-resistant tumor cells were distinct; moreover, DNA damage in xenografts, following treatment of mice with Phortress, could be determined. Interrogation of the mechanism of action of 5F 203 in silico by self-organizing map-based cluster analyses revealed modulation of phosphatases and kinases associated with cell cycle regulation, corroborating observations of selective cell cycle perturbation by 5F 203 in sensitive cells. By conducting SCGE, tumor sensitivity to Phortress, an agent currently undergoing clinical evaluation, may be determined.

No / BACKGROUND: The possibility that oligozoospermic men may have elevated levels of genetic damage in their sperm is of particular concern as they could transmit defects to their offspring. METHODS: Sperm samples were obtained from 12 infertile, oligozoospermic patients and 12 healthy normozoospermic volunteers. Fluorescence in-situ hybridization (FISH) was used to determine aneuploidy rates in sperm and inverse restriction site mutation (iRSM) assay to determine gene mutations; defective chromatin packaging was quantified by sperm chromatin structure assay (SCSA) and DNA strand breaks by the Comet assay. RESULTS: FISH analysis showed a significant increase in gonosomal X,Y,18 (P < 0.01) disomy and diploid sperm with X,Y,18,18 (P < 0.05) in the infertility patients compared with the controls. A significant increase (P < 0.01) in disturbed sperm chromatin was found in the infertility patients compared with the control group using the SCSA assay. In the Comet assay, a significant increase (P < 0.01) in the tail moment was found in the infertility patients compared with the control group, indicating significantly high levels of DNA strand breaks. There was no significant increase in point mutations detected by iRSM assay. CONCLUSIONS: The data indicate that infertile oligozoospermic men have an elevated level of XY aneuploidy and XY diploidy in the germ-line, as well as elevated levels of sperm chromatin disturbances and sperm DNA strand breaks. These data demonstrate that oligozoospermic infertility patients show several different types of genetic damage in their sperm. Thus, such men appear to have defects at a variety of levels of spermatogenesis and their infertility may not just be a result of the oligozoospermia.

Yes / The comet assay is a sensitive method used to detect DNA damage, measuring DNA breaks and alkali labile lesions in eukaryotic cells. Here, the use of whole blood in the alkaline gel electrophoresis method is described. Two hundred and seventy blood samples from individuals were examined: 120 healthy individuals, 65 suspected or pre-cancerous individuals and 85 cancer patients. Each sample was divided into two identical volumes in different falcon tubes. The blood was prepared and stored by adding the same amount of RPMI medium and 10% DMSO. Using the Student’s t-Test, the data showed a p value = 0.59 for Olive tail moment (OTM) and 0.16 for % tail DNA, and no statistically significant differences between the two methods, with or without treatment. In conclusion, using whole blood instead of isolated lymphocytes saves time, is still very sensitive and requires less than 20 µL of blood from each individual.

Comets are important for solar system studies because their interiors hold evidence of the conditions in which they formed in the outer solar system. However, the coma obscures the nucleus from view when observations are most easily performed, thus it is important to understand the nature of cometary comae. This study examines the spatial distribution of CN radicals in the coma of comet Encke and determines the likelihood that CN is a photodissociative daughter of HCN in the coma. Observations of CN were obtained from October 22-24, 2003, using the 2.7 m Cassegrain telescope at McDonald Observatory in Fort Davis, TX. The classical vectorial model was modified by introducing a fan-like feature in order to explain Encke’s aspherical coma. The results are consistent with HCN being the photodissociative parent of CN, based on the OH/CN ratios and the physical parameters used to match the model profiles with the observations.

The primary purpose of this study is to model the water vapor flow produced by a comet impact on the Moon using the Direct Simulation Monte Carlo (DSMC) method. Toward that end, our DSMC solver was modified in order to model the cometary water from the time of impact until it is either destroyed due to escape or photodestruction processes or captured inside one of the lunar polar cold traps. In order to model the complex flow induced by a comet impact, a 3D spherical parallel version of the DSMC method was implemented. The DSMC solver was also modified to take as input the solution from the SOVA hydrocode for the impact event at a fixed interface. An unsteady multi-domain approach and a collision limiting scheme were also added to the previous implementation in order to follow the water from the continuum regions near the point of impact to the much later rarefied atmospheric flow around the Moon. The present implementation was tested on a simple unsteady hemispherical expansion flow into a vacuum. For these simulations, the data at the interface were provided by a 1D analytical model instead of the SOVA solution. Good results were obtained downstream of the interface for density, temperature and radial velocity. Freezing of the vibrational modes was also observed in the transitional regime as the flow became collisionless. The 45° oblique impact of a 1 km radius ice sphere at 30 km/s was simulated up to several months after impact. Most of the water crosses the interface under 5 s moving mostly directly downstream of the interface. Most of the water escapes the gravity well of the Moon within the first few hours after impact. For such a comet impact, only ~3% of the comet mass remains on the Moon after impact. As the Moon rotates, the molecules begin to migrate until they are destroyed or captured in a cold trap. Of the 3% of the water remaining on the Moon after impact, only a small fraction, ~0.14% of the comet mass, actually reaches the cold traps; nearly all of the rest is photo-destroyed. Based on the surface area of the cold traps used in the present simulations, ~1 mm of ice would have accumulated in the polar cold traps after such an impact. Estimates for the total mass of water accumulated in the polar cold traps over one billion years are consistent with recent observations. / text

Beim Zigarettenrauchen als der häufigsten Form des Tabakkonsums stellt das respiratorische Epithel des oberen und unteren Aerodigestivtraktes das primäre Kontaktorgan der zyto- und genotoxischen Inhaltsstoffe dar. Nikotin, das Hauptalkaloid des Tabaks, ruft nicht nur eine starke Abhängigkeit hervor, sondern kann in Anbetracht früherer Studien auch zum Tabak assoziierten Krebsrisiko beitragen. Neben tumorproliferativen Effekten wie etwa der Angioneogenese, der Zellproliferation oder einer Apoptoseinhibition ist die Rolle der tumorinitiierenden Wirkung von Nikotin durch eine direkte Schädigung der DNA noch unzureichend untersucht. Ziele dieser experimentellen Arbeit waren deshalb, Nikotin induzierte DNA-Schäden an frisch isolierten sowie rekultivierten humanen Lymphozyten mit Hilfe des alkalischen Einzelzell-Mikrogelelektrophorese (Comet) Assays darzustellen, den Nachweis dieser Schäden durch Koinkubation mit dem Reparaturenzyminhibitor Aphidicolin (APC) zu sensitivieren sowie oxidativ geschädigte Basen durch die Formamidopyrimidin-Glykosylase (Fpg) aufzuzeigen. Durch Koinkubation mit Epibatidin, einem Subtyp spezifischen und kompetitiven Agonisten am nikotinergen Acetylcholinrezeptor (nAChR), wurde die Rolle der rezeptorvermittelten Mechanismen Nikotin induzierter DNA-Schäden an Lymphozyten und nasalen Mukosazellen untersucht. Auch der Frage, ob Rauchen zu einer erhöhten basalen Schädigung an nasalen Mukosazellen führe, wurde nachgegangen. Nach der Zellisolierung der humanen nasalen Schleimhautzellen und peripheren Lymphozyten erfolgte zum Ausschluss zytotoxischer Effekte jeweils vor und nach der einstündigen Fremdstoffinkubation mit Nikotin (1 µM bis 1000 µM), APC (2,5 µg/ml), Epibatidin (1 µM bis 100 µM) und MMS (100 µM) die Bestimmung der Zellvitalität mit dem Trypanblau-Ausschlusstest. Durch Inkubation mit Fpg nach der Lyse zellulärer Membranen erfolgte die Augmentation oxidativ geschädigter Basen. Potentielle DNA-Schäden in Form von Einzelstrangbrüchen und alkalilabilen Stellen der DNA wurden mit dem Comet Assay erfasst. An frisch isolierten Lymphozyten konnte nach ein-stündiger Inkubation mit Nikotin ab 100 µM ein signifikanter DNA-Schaden festgestellt werden. Mit dem Einsatz von Fpg kam es ab 10 µM Nikotin zu einem signifikanten Anstieg der DNA-Fragmentierung. An rekultivierten Lymphozyten konnte nach Kryokonservierung bei einstündiger Inkubation mit Nikotin bereits ab 1 µM eine signifikante DNA-Schädigung nachgewiesen werden, die sich ebenfalls bei Koinkubation mit APC ab 1 µM darstellte. Durch Koinkubation von Nikotin (1000 µM) mit Epibatidin in aufsteigender Konzentration konnte an frisch isolierten Lymphozyten nur in einer Konzentration (10 µM) die Nikotin induzierte DNA-Fragmentierung gesenkt werden. Hierbei zeigte Epibatidin selbst einen DNA-Schaden in niedriger Konzentration (1 µM und 10 µM). An nasalen Mukosazellen konnte der Nikotin induzierte DNA-Schaden durch die Koinkubation mit Epibatidin nicht gesenkt werden. Auch an nasaler Mukosa rief Epibatidin ab 1 µM einen signifikanten DNA-Schaden hervor. Bezüglich einer Einflussgröße durch das Rauchen auf die Ergebnisse im Comet Assay konnte kein Unterschied der basalen als auch der durch Nikotin induzierten DNA-Fragmentierung zwischen der Gruppe der Raucher und Nichtraucher festgestellt werden. Nikotin verursachte bereits bei einer einstündigen Expositionsdauer DNA-Schäden an humanen Lymphozyten und nasalen Mukosazellen. Der Nachweis oxidativ geschädigter Basen an Lymphozyten zeigt auf eine Generierung von reaktiven Sauerstoffspezies (ROS) durch Nikotin hin. Die Aktivierung des homomeren α7 nAChR durch Nikotin soll hierbei eine wichtige Rolle als Auslöser der intrazellulären Signaltransduktion der Radikalbildung spielen. Epibatidin als ein starker Agonist am α7 Rezeptor führte bereits in geringen Konzentrationen zu einer signifikanten DNA-Fragmentierung. Bei fehlender Reparatur dieser DNA-Schäden und einer ausbleibenden Elimination der geschädigten Zelle können diese Mutationen akkumulieren und zur Tabak assoziierten Krebsentstehung beitragen. Eine Substitutionstherapie mit Nikotin zur Raucherentwöhnung muss bei solchen Ergebnissen äußerst kritisch betrachtet werden. / Respiratory epithelia of the upper and lower aero digestive tract are in first contact with cyto- and genotoxic components of tobacco smoke. Nicotine, being the main alkaloid of tobacco, is responsible for addiction to tobacco and contributes to tobacco carcinogenesis, too. While mechanisms of tumor proliferation caused by nicotine, such as stimulation of angiogenesis and cell proliferation or inhibition of apoptosis, are well accepted, the role of direct DNA damage by nicotine as a potentially tumor initiating factor is in focus of the present study. This experimental study demonstrates DNA damage in freshly isolated and cryo-preserved human lymphocytes after exposure to nicotine with the alkaline single-cell microgel electrophoresis (comet) assay. Co-incubations with aphidicolin (APC), an inhibitor of repair enzymes, and formamidopyrimidine-glycosylase (Fpg), an enzyme to detect oxidative damaged bases, were performed. Epibatidine as a subtype-specific and competitive agonist of the nicotinic acetylcholine receptor (nAChR) correspondent to nicotine was implemented to evaluate the role of receptor associated mechanisms of DNA damage. Furthermore, the influence of the smoking-status on the DNA damage of nasal mucosa cells was evaluated. Cytotoxic effects were investigated before and after one hour of exposure to nicotine (1 µM to 1000 µM), APC (2,5 µg/ml), epibatidine (1 µM to 100 µM) and methyl methane sulfonate as control (100 µM) in the trypan blue exclusion test. After lysis of cellular membranes, oxidative damaged bases of DNA were detected by incubation with Fpg. DNA damage like single strand brakes and alkali labile sites of DNA were investigated with the aid of the comet assay. Freshly isolated lymphocytes showed at 100 µM nicotine a significant DNA damage. Applying Fpg, a significant increase in DNA fragmentation could already be detected at 10 µM nicotine. Cryopreserved lymphocytes showed a significant DNA damage at 1 µM nicotine both with and without the co-incubation of APC. The co-incubation of 1000 µM nicotine with epibatidine showed in a single concentration (10 µM) a reduction of nicotine induced DNA damage in lymphocytes. Epibatidine itself caused DNA damage at lower concentrations (1 µM and 10 µM) in lymphocytes. In nasal mucosa cells epibatidine did not reduce the nicotine induced DNA damage but caused itself DNA damage at 1 µM. The smoking-status of the participants had no influence on basal and nicotine induced DNA fragmentation. Nicotine induced DNA damage to cells of human blood and nasal mucosa just after one hour of exposure. The evidence of oxidative damaged bases of DNA in lymphocytes shows the development of reactive oxygen species (ROS) by nicotine. The activation of the homomeric α7 nAChR by nicotine seems to be relevant to the intracellular transduction for generating ROS. Epibatidine, as a strong agonist on the α7 nAChR, caused significant DNA fragmentation in low concentrations, already. By insufficient repair of the DNA damage and inhibited elimination of damaged cells, mutations can accumulate and contribute to tobacco carcinogenesis. Hence, nicotine in a replacement therapy should be considered very critically.

Rauchen stellt in den Industrienationen das bedeutendste vermeidbare Gesundheitsrisiko dar. Die Rolle des suchtauslösenden Alkaloids Nikotin in der Tabak assoziierten Kanzerogenese wird kontrovers diskutiert. Ziel dieser Arbeit war die Untersuchung genotoxischer Effekte von Nikotin in Zellen des oberen und unteren Aerodigestivtrakt sowie deren intrazellulärer Mechanismen. Dazu wurden Zellen aus humaner Nasenschleimhaut und humaner Bronchialschleimhaut enzymatisch isoliert sowie bronchiales Zelllinienepithel kultiviert und mit Nikotin unterschiedlicher Dosierungen für eine Stunde inkubiert. Zur Untersuchung beteiligter Signalkaskaden wurden Koinkubationen von Nikotin und dem nicht-kompetitiven nikotinergen Acetylcholinrezeptorblocker Mecamylamin und dem Antioxidans N-Acetylcystein durchgeführt. Die Erfassung Nikotin induzierter DNASchäden erfolgte mit Hilfe des Comet Assays. Zur Untersuchungen von Zellzyklusalterationen sowie Apoptoseinhibition durch Nikotin kam die Durchflusszytometrie zum Einsatz. Die Ergebnisse der Einzelzellgelelektrophorese zeigten eine dosisabhängige DNASchädigung im einstündigen Inkubationsversuch durch Nikotin. Diese Schäden waren gewebeabhängig ab einer Konzentration von 100μM in Zelllinienepithel (n=5) und 1mM in Nasenschleimhautzellen (n=8) signifikant. In humanem Bronchialzellepithel konnte bei dem Stichprobenumfang von n=4 keine signifikante DNA-Schädigung durch die getesteten Nikotindosierungen nachgewiesen werden. Durch eine Koinkubation mit dem Antioxidans N-Acetylcystein sowie dem nicht kompetitiven nACh Rezeptorblocker Mecamylamin konnte eine im Comet Assay nachweisbare Nikotin induzierte DNA-Schädigung verhindert werden. Durchflusszytometrische Untersuchungen zur Klärung einer möglichen Modulation der Apoptose durch Nikotin an bronchialem Zelllinienepithel zeigten keine signifikante Induktion oder Inhibition. Eine Beeinflussung des Zellzyklus durch Nikotin konnte in der Durchflusszytometrie nicht erfasst werden. Zusammenfassend induziert Nikotin DNA-Schäden in Epithelien des Atemtraktes. An diesem Effekt sind oxidative sowie nAch-Rezeptor abhängige Stoffwechselschritte beteiligt. Vor dem Hintergrund einer potentiellen Beteiligung von Nikotin an der Tumorinitiation und -progression muss eine Nikotinersatztherapie besonders kritisch abgewogen werden. / Tobacco smoking is the single most preventable cause of the death in the world. The role of tobacco´s main alcaloide nicotine in smoking related cancer is still unclear. Aim of this study was to investigate the genotoxicity of nicotine in epithelia of the human aerodigestive tract and the intracellular pathways which lead to DNA-damage. The genotoxicity was assessed by the comet assay. To assess cell cycle alterations and inhibition of apoptosis flow cytometry was performed. Cells of human nasal epithelia and cells of human bronchial epithelia were encymatically isolated and afterwards incubated with nicotine in different dosages for one hour. For investigation of the cellular pathways of DNA-damage cells were co-incubated with Nicotine and either N-Acetylcysteine, a known antioxidative substance, or Mecamylamine, a nAch-receptor blocking agent. Results showed a dosage dependend DNA-damage in nasal epithelia after one-hour-treatment with nicotine. Flow cytometry showed no alterations in cell cycle and no influence on apoptosis in nicotine treated cells. Coincubation of nicotine and N-Acetylcysteine or Mecamylamine reduced the nicotine induced DNA-damage significantly. Nicotine induces DNA damage in epithelia of the human aerodigestive tract. This damage is caused by oxidative effects and nAch-receptor dependend pathways.

O objetivo do presente trabalho é estudar a correlação entre as taxas de produção gasosa e as magnitudes heliocêntricas do cometa C/1995 O1 (Hale-Bopp), tanto na fase pré-periélica como na fase pós-periélica. As evoluções da magnitude e das taxas de produção gasosa de H2O (água), OH (radical hidroxila) e CO (monóxido de carbono), ao longo da aproximação e do afastamento do cometa em relação ao Sol, são analisadas. Para essa análise, foram utilizadas 11.734 estimativas de magnitudes visuais, extraídas do ICQ (International Comet Quarterly) e 88 observações do monóxido de carbono (Biver, comunicação particular (2007); Disanti et al. 2001; Jewitt et al. 1996), cobrindo o intervalo de distâncias heliocêntricas de rh = 7,464 UA (na fase pré-periélica) até rh = 14,070 UA (na fase pós-periélica). É mostrado que a atividade do Hale-Bopp (temperatura média superficial ~ 110 K), além de 6,3 UA do Sol é controlada pela emissão do CO (temperatura de sublimação ~ 24 K), antes que pela emissão da H2O (temperatura de sublimação ~ 152 K). Esse resultado é consistente com as observações em ondas milimétricas de Biver et al. 1996 e Jewitt et al. 1996, realizadas em 6,5 UA. / The purpose of the present work is to study the correlation between the gas production rates and heliocentric magnitudes of comet C/1995 O1 (Hale-Bopp), in the pre-perihelion phase as well as in the post-perihelion phase. The evolutions of magnitudes and gas production rates of H2O (water), OH (hydroxil radical) and CO (carbon monoxide), along the approach to and leave of the comet from the Sun, are analyzed. For this analysis, we used 11,734 visual magnitude estimates, extracted from ICQ (International Comet Quarterly) and 88 observations of carbon monoxide (Biver, private communication (2007); DiSanti et al. (2001); Jewitt et al. (1996)), covering the range of heliocentric distances from rh = 7.464 AU (in the pre-perihelion phase) to rh = 14.070 AU (in the post-perihelion phase). It is shown that the activity of Hale-Bopp (average surface temperature ~ 110 K) beyond 6.3 AU from the Sun is controlled by CO emission (sublimation temperature ~ 24 K) rather than by H2O (sublimation temperature ~ 152 K). This result is consistent with millimeter-wave observations of Biver et al. (1996) and Jewitt et al. (1996), made at 6.5 AU.