Acute promyelocytic leukemia associated with the PLZF-RARA fusion gene: two additional cases with clinical and laboratorial peculiar presentations

Acute promyelocytic leukemia (APL) is characterized by the presence of the t(15;17) and PML-RARa rearrangement, with good response to treatment with retinoids. However, few cases of variant APL involving alternative chromosomal aberrations have been reported, including t(11;17)(q23;q21) (Wells et al. in Nat Genet 17:109-113, 1; Arnould et al. in Hum Mol Genet 8:1741-1749, 2) t(5;17)(q35;q12-21), t(11;17)(q13;q21) (Grimwade et al in Blood 96:1297-1308, 3) and der(17) (Rego et al. in Blood (ASH Annual Meeting Abstracts)114:Abstract 6, 4), whereby RARa is fused to the PLZF, NPM, NuMA, and STAT5b genes, respectively, have been described. These cases are characterized by distinct morphology, clinical presentation, and in respect to PLZF, a lack of differentiation response to retinoids leading to the need of different approaches concerning diagnostic methods and therapeutics. This paper describes two cases of APL associated with the PLZF-RARA fusion gene enrolled in the IC-APL trial that is a non-randomized, multicenter study conducted in Brazil, Mexico, Chile and Uruguay with the aim to improve the treatment outcome of APL patients in developing countries. These cases, although rare, offer a challenge to its early recognition and proper conduction.

Planetary transit candidates in the CoRoT LRa01 field

Context. CoRoT is a pioneering space mission whose primary goals are stellar seismology and extrasolar planets search. Its surveys of large stellar fields generate numerous planetary candidates whose lightcurves have transit-like features. An extensive analytical and observational follow-up effort is undertaken to classify these candidates. Aims. We present the list of planetary transit candidates from the CoRoT LRa01 star field in the Monoceros constellation toward the Galactic anti-center direction. The CoRoT observations of LRa01 lasted from 24 October 2007 to 3 March 2008. Methods. We acquired and analyzed 7470 chromatic and 3938 monochromatic lightcurves. Instrumental noise and stellar variability were treated with several filtering tools by different teams from the CoRoT community. Different transit search algorithms were applied to the lightcurves. Results. Fifty-one stars were classified as planetary transit candidates in LRa01. Thirty-seven (i.e., 73% of all candidates) are "good" planetary candidates based on photometric analysis only. Thirty-two (i.e., 87% of the "good" candidates) have been followed-up. At the time of writing twenty-two cases were solved and five planets were discovered: three transiting hot-Jupiters (CoRoT-5b, CoRoT-12b, and CoRoT-21b), the first terrestrial transiting planet (CoRoT-7b), and another planet in the same system (CoRoT-7c, detected by radial velocity survey only). Evidence of another non-transiting planet in the CoRoT-7 system, namely CoRoT-7d, was recently found as well.

Evaluation of cytotoxic, genotoxic and antigenotoxic potential of Solanum lycocarpum fruits glicoalkaloid extract in V79 cells

Solanum lycocarpum St.-Hil (Solanaceae) is a hairy shrub or small much-branched tree of the Brazilian Cerrado, popularly known as "fruit-of-wolf". Considering that the induction of chromosomal mutations is involved in the process of carcinogenesis, and that S. lycocatpum is often used in folk medicine, it becomes relevant to study its effect on genetic material. In this sense, the aim of present study was to determine the possible cytotoxic, genotoxic and antigenotoxic potentials of S. lycocarpum fruits glycoalkaloid extract (SL) in Chinese hamster lung fibroblasts (V79 cells). The cytotoxicity was evaluated by the colony forming assay, apoptosis and necrosis assay. Trypan blue exclusion dye method and mitotic index. Genotoxic and antigenotoxic potential were evaluated by comet and chromosomal aberrations assays. Four concentrations of SL (4, 8, 16 and 32 mu g/mL) were used for the evaluation of its genotoxic potential. The DNA damage-inducing agent methyl methanesulfonate (MMS, 221 mu g/mL) was utilized in combination with extract to evaluate a possible protective effect. The results showed that SL was cytotoxic at concentrations above 32 mu g/mL by the colony forming assay. For apoptosis and necrosis assay, the concentration of 64 mu g/mL of SL showed statistically significant increase in cell death by apoptosis and necrosis, while the concentrations of 128 and 256 mu g/mL of SL demonstrated statistically significant increase in cell death by necrosis, compared with the control group. Analysis of cell viability by Trypan blue exclusion indicated >96% viability for treatments with concentrations up to 32 mu g/mL of SL No significant differences in MI were observed between cultures treated with different concentrations of 51 (4, 8, 16 and 32 mu g/mL) alone or in combination with MMS and the negative control, indicating that these treatments were not cytotoxic. The comet and chromosomal aberrations assays revealed that SL does not display genotoxic activity. Moreover, the different concentrations of SL showed protective effect against both genomic and chromosomal damages induced by MMS. (C) 2012 Elsevier Ltd. All rights reserved.

Biomonitoring genotoxicity and cytotoxicity of Microcystis aeruginosa (Chroococcales, Cyanobacteria) using the Allium cepa test

Water pollution caused by toxic cyanobacteria is a problem worldwide, increasing with eutrophication. Due to its biological significance, genotoxicity should be a focus for biomonitoring pollution owing to the increasing complexity of the toxicological environment in which organisms are exposed. Cyanobacteria produce a large number of bioactive compounds, most of which lack toxicological data. Microcystins comprise a class of potent cyclic heptapeptide toxins produced mainly by Microcystis aeruginosa. Other natural products can also be synthesized by cyanobacteria, such as the protease inhibitor, aeruginosin. The hepatotoxicity of microcystins has been well documented, but information on the genotoxic effects of aeruginosins is relatively scarce. In this study, the genotoxicity and ecotoxicity of methanolic extracts from two strains of M. aeruginosa NPLJ-4, containing high levels of microcystin, and M. aeruginosa NPCD-1, with high levels of aeruginosin, were evaluated. Four endpoints, using plant assays in Allium cepa were applied: rootlet growth inhibition, chromosomal aberrations, mitotic divisions, and micronucleus assays. The microcystin content of M. aeruginosa NPLJ-4 was confirmed through ELISA, while M. aeruginosa NPCD-1 did not produce microcystins. The extracts of M. aeruginosa NPLJ-4 were diluted at 0.01, 0.1, 1 and 10 ppb of microcystins: the same procedure was used to dilute M. aeruginosa NPCD-1 used as a parameter for comparison, and water was used as the control. The results demonstrated that both strains inhibited root growth and induced rootlet abnormalities. The strain rich in aeruginosin was more genotoxic, altering the cell cycle, while microcystins were more mitogenic. These findings indicate the need for future research on non-microcystin producing cyanobacterial strains. Understanding the genotoxicity of M. aeruginosa extracts can help determine a possible link between contamination by aquatic cyanobacteria and high risk of primary liver cancer found in some areas as well as establish water level limits for compounds not yet studied. (C) 2012 Elsevier B.V. All rights reserved.

Voice disorders in children and its relationship with auditory, acoustic and vocal behavior parameters

Objective: Parameters to distinguish normal from deviant voices in early childhood have not been established. The current study sought to auditorily and acoustically characterize voices of children, and to study the relationship between vocal behavior reported by teachers and the presence of vocal aberrations. Methods: One hundred children between four and 6 years and 11 months, who attended early childhood educational institutions, were included. The sample comprised 50 children with normal voices (NVG) and 50 with deviant voices (DVG) matched by gender and age. All participants were submitted to auditory and acoustic analysis of vocal quality and had their vocal behaviors assessed by teachers through a specific protocol. Results: DVG had a higher incidence of breathiness (p < 0.001) and roughness (p < 0.001), but not vocal strain (p = 0.546), which was similar in both groups. The average F-0 was lower in the DVG and a higher noise component was observed in this group as well. Regarding the protocol used "Aspects Related to Phonotrauma - Children's Protocol", higher means were observed for children from DVG in all analyzed aspects and also on the overall means (DVG = 2.15; NVG = 1.12, p < 0.001). In NVG, a higher incidence of vocal behavior without alterations or with discrete alterations was observed, whereas a higher incidence of moderate, severe or extreme alterations of vocal behavior was observed in DVG. Conclusions: Perceptual assessment of voice, vocal acoustic parameters (F-0, noise and GNE), and aspects related to vocal trauma and vocal behavior differentiated the groups of children with normal voice and deviant voice. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Meiotic segregation and interchromosomal effect in the sperm of a double translocation carrier: a case report

Abstract Background Infertility is a natural mechanism of selection intended to prevent the delivery of a child with malformations or mental retardation. Male infertility is closely related to chromosomal abnormalities. This study was focused on the analysis of meiotic segregation involving a Robertsonian translocation, 45,XY,der(13;13) [56]/45,XY,der(13;14) [44] and the evaluation of possible interchromosomal effects. Results Hybridisation with LSI 13q14 and subtelomere 14q probes and WCP13 SpectrumGreen and WCP14 SpectrumOrange probes showed a high proportion of unbalanced gametes, corresponding to 71.2% of the spermatozoa. The disomic frequencies of the sexual chromosomes and chromosome 18 of the patient were higher (5.28% and 2.55%, respectively) than those of the control (0.6% and 0.59%, respectively). Conclusion Meiotic segregation studies in sperm are an important tool for genetic counselling of chromosomal aberrations, allowing for a prediction of the risks and consequent implications for the reproductive life. The patient with this rare translocation exhibited meiotic segregation fidelity, and a high rate of unbalanced gametes with disomic spermatozoa.

Mechanisms of ring chromosome formation, ring instability and clinical consequences

Background The breakpoints and mechanisms of ring chromosome formation were studied and mapped in 14 patients. Methods Several techniques were performed such as genome-wide array, MLPA (Multiplex Ligation-Dependent Probe Amplification) and FISH (Fluorescent in situ Hybridization). Results The ring chromosomes of patients I to XIV were determined to be, respectively: r(3)(p26.1q29), r(4)(p16.3q35.2), r(10)(p15.3q26.2), r(10)(p15.3q26.13), r(13)(p13q31.1), r(13)(p13q34), r(14)(p13q32.33), r(15)(p13q26.2), r(18)(p11.32q22.2), r(18)(p11.32q21.33), r(18)(p11.21q23), r(22)(p13q13.33), r(22)(p13q13.2), and r(22)(p13q13.2). These rings were found to have been formed by different mechanisms, such as: breaks in both chromosome arms followed by end-to-end reunion (patients IV, VIII, IX, XI, XIII and XIV); a break in one chromosome arm followed by fusion with the subtelomeric region of the other (patients I and II); a break in one chromosome arm followed by fusion with the opposite telomeric region (patients III and X); fusion of two subtelomeric regions (patient VII); and telomere-telomere fusion (patient XII). Thus, the r(14) and one r(22) can be considered complete rings, since there was no loss of relevant genetic material. Two patients (V and VI) with r(13) showed duplication along with terminal deletion of 13q, one of them proved to be inverted, a mechanism known as inv-dup-del. Ring instability was detected by ring loss and secondary aberrations in all but three patients, who presented stable ring chromosomes (II, XIII and XIV). Conclusions We concluded that the clinical phenotype of patients with ring chromosomes may be related with different factors, including gene haploinsufficiency, gene duplications and ring instability. Epigenetic factors due to the circular architecture of ring chromosomes must also be considered, since even complete ring chromosomes can result in phenotypic alterations, as observed in our patients with complete r(14) and r(22).

Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation.

Clinicopathological and molecular characterization of gastroesophageal junction (GEJ) adenocarcinoma before age of 40 years

Gastroesophageal junction (GEJ) adenocarcinoma are uncommon before age of 40 years. While certain clinical, pathological and molecular features of GEJ adenocarcinoma in older patients have been extensively studied, these characteristics in the younger population remain to be determined. In the recent literature, a high sensitivity and specificity for the detection of dysplasia and esophageal adenocarcinoma was demonstrated by using multicolor fluorescence in situ hybridization (FISH) DNA probe set specific for the locus specific regions 9p21 (p16), 20q13.2 and Y chromosome. We evaluated 663 patients with GEJ adenocarcinoma and further divided them into 2 age-groups of or= 50 years, rispectively. FISH with selected DNA probe for Y chromosome, locus 9p21 (p16), and locus 20q13.2 was investigated with formalin fixed and parassin embedded tissue from surgical resections of 17 younger and 11 older patients. Signals were counted in > 100 cells with each given histopathological category. The chromosomal aberrations were then compared in the 2 age-groups with the focus on uninvolved squamous and columnar epithelium, intestinal metaplasia (Barrett's mucosa), glandular dysplasia, and adenocarcinoma. Comparisons were performed by the X2 test, Fisher's exact test, Student's t-test and Mann-Whitney U-test as appropriate. Survival was estimated by the Kaplan-Meier method with univariate analysis by the log-rank. Significance was taken at the 5% level. There was no difference in the surgical technique applied in both age groups and most patients underwent Ivor Lewis esophagectomy. Among clinical variables there was a higher incidence of smocking history in older patient group. We identified a progressive loss of Y chromosome from benign squamos epithelium to Barrett's mucosa and glandular dysplasia, and, ultimately, to a near complete loss in adenocarcinoma in both age groups. The young group revealed significantly more losses of 9p21 in both benign and neoplastic cells when compared to the older patients group. In addition, we demonstrated an increase in the percentage of cells showing gain of locus 20q13.2 with progression from benign epithelium through dysplasia to adenocarcinoma with almost the same trend in both the young and the older patients. When compared with the older age-group, younger patients with GEJ adenocarcinoma possess similar known demographics, environmental factors, clinical, and pathologic characteristics. The most commonly detected genetic aberrations of progressive Y chromosomal loss, 9p21 locus loss, and 20q13 gains were similar in the younger and older patients. However the rate of loss of 9p21 is significantly higher in young patients, in both the benign and the neoplastic cells. The loss of 9p21, and possibly, the subsequent inactivation of p16 gene may be one of the molecular mechanisms responsible for the accelerated neoplastic process in young patients.

Sviluppo di un tomografo multi-energy per lo studio pre-clinico di nuove metodiche diagnostiche finalizzate al riconoscimento precoce della patologia tumorale

A new multi-energy CT for small animals is being developed at the Physics Department of the University of Bologna, Italy. The system makes use of a set of quasi-monochromatic X-ray beams, with energy tunable in a range from 26 KeV to 72 KeV. These beams are produced by Bragg diffraction on a Highly Oriented Pyrolytic Graphite crystal. With quasi-monochromatic sources it is possible to perform multi-energy investigation in a more effective way, as compared with conventional X-ray tubes. Multi-energy techniques allow extracting physical information from the materials, such as effective atomic number, mass-thickness, density, that can be used to distinguish and quantitatively characterize the irradiated tissues. The aim of the system is the investigation and the development of new pre-clinic methods for the early detection of the tumors in small animals. An innovative technique, the Triple-Energy Radiography with Contrast Medium (TER), has been successfully implemented on our system. TER consist in combining a set of three quasi-monochromatic images of an object, in order to obtain a corresponding set of three single-tissue images, which are the mass-thickness map of three reference materials. TER can be applied to the quantitative mass-thickness-map reconstruction of a contrast medium, because it is able to remove completely the signal due to other tissues (i.e. the structural background noise). The technique is very sensitive to the contrast medium and is insensitive to the superposition of different materials. The method is a good candidate to the early detection of the tumor angiogenesis in mice. In this work we describe the tomographic system, with a particular focus on the quasi-monochromatic source. Moreover the TER method is presented with some preliminary results about small animal imaging.

Molecular analysis of special type breast carcinomas

The project was developed into three parts: the analysis of p63 isoform in breast tumours; the study of intra-tumour eterogeneicity in metaplastic breast carcinoma; the analysis of oncocytic breast carcinoma. p63 is a sequence-specific DNA-binding factor, homologue of the tumour suppressor and transcription factor p53. The human p63 gene is composed of 15 exons and transcription can occur from two distinct promoters: the transactivating isoforms (TAp63) are generated by a promoter upstream of exon 1, while the alternative promoter located in intron 3 leads to the expression of N-terminal truncated isoforms (ΔNp63). It has been demonstrated that anti-p63 antibodies decorate the majority of squamous cell carcinomas of different organs; moreover tumours with myoepithelial differentiation of the breast show nuclear p63 expression. Two new isoforms have been described with the same sequence as TAp63 and ΔNp63 but lacking exon 4: d4TAp63 and ΔNp73L, respectively. Purpose of the study was to investigate the molecular expression of N-terminal p63 isoforms in benign and malignant breast tissues. In the present study 40 specimens from normal breast, benign lesions, DIN/DCIS, and invasive carcinomas were analyzed by immunohistochemistry and RT-PCR (Reverse Transcriptase-PCR) in order to disclose the patterns of p63 expression. We have observed that the full-length isoforms can be detected in non neoplastic and neoplastic lesions, while the short isoforms are only present in the neoplastic cells of invasive carcinomas. Metaplastic carcinomas of the breast are a heterogeneous group of neoplasms which exhibit varied patterns of metaplasia and differentiation. The existence of such non-modal populations harbouring distinct genetic aberrations may explain the phenotypic diversity observed within a given tumour. Intra-tumour morphological heterogeneity is not uncommon in breast cancer and it can often be appreciated in metaplastic breast carcinomas. Aim of this study was to determine the existence of intra-tumour genetic heterogeneity in metaplastic breast cancers and whether areas with distinct morphological features in a given tumour might be underpinned by distinct patterns of genetic aberrations. 47 cases of metaplastic breast carcinomas were retrieved. Out of the 47 cases, 9 had areas that were of sufficient dimensions to be independently microdissected. Our results indicate that at least some breast cancers are composed of multiple non-modal populations of clonally related cells and provide direct evidence that at least some types of metaplastic breast cancers are composed of multiple non-modal clones harbouring distinct genetic aberrations. Oncocytic tumours represent a distinctive set of lesions with typical granular cytoplasmatic eosinophilia of the neoplastic cells. Only rare example of breast oncocytic carcinomas have been reported in literature and the incidence is probably underestimated. In this study we have analysed 33 cases of oncocytic invasive breast carcinoma of the breast, selected according to morphological and immunohistochemical criteria. These tumours were morphologically classified and studied by immunohistochemistry and aCGH. We have concluded that oncocytic breast carcinoma is a morphologic entity with distinctive ultrastructural and histological features; immunohistochemically is characterized by a luminal profile, it has a frequency of 19.8%, has not distinctive clinical features and, at molecular level, shows a specific constellation of genetic aberration.

LBT AO system: tests and calibration

An Adaptive Optic (AO) system is a fundamental requirement of 8m-class telescopes. We know that in order to obtain the maximum possible resolution allowed by these telescopes we need to correct the atmospheric turbulence. Thanks to adaptive optic systems we are able to use all the effective potential of these instruments, drawing all the information from the universe sources as best as possible. In an AO system there are two main components: the wavefront sensor (WFS) that is able to measure the aberrations on the incoming wavefront in the telescope, and the deformable mirror (DM) that is able to assume a shape opposite to the one measured by the sensor. The two subsystem are connected by the reconstructor (REC). In order to do this, the REC requires a “common language" between these two main AO components. It means that it needs a mapping between the sensor-space and the mirror-space, called an interaction matrix (IM). Therefore, in order to operate correctly, an AO system has a main requirement: the measure of an IM in order to obtain a calibration of the whole AO system. The IM measurement is a 'mile stone' for an AO system and must be done regardless of the telescope size or class. Usually, this calibration step is done adding to the telescope system an auxiliary artificial source of light (i.e a fiber) that illuminates both the deformable mirror and the sensor, permitting the calibration of the AO system. For large telescope (more than 8m, like Extremely Large Telescopes, ELTs) the fiber based IM measurement requires challenging optical setups that in some cases are also impractical to build. In these cases, new techniques to measure the IM are needed. In this PhD work we want to check the possibility of a different method of calibration that can be applied directly on sky, at the telescope, without any auxiliary source. Such a technique can be used to calibrate AO system on a telescope of any size. We want to test the new calibration technique, called “sinusoidal modulation technique”, on the Large Binocular Telescope (LBT) AO system, which is already a complete AO system with the two main components: a secondary deformable mirror with by 672 actuators, and a pyramid wavefront sensor. My first phase of PhD work was helping to implement the WFS board (containing the pyramid sensor and all the auxiliary optical components) working both optical alignments and tests of some optical components. Thanks to the “solar tower” facility of the Astrophysical Observatory of Arcetri (Firenze), we have been able to reproduce an environment very similar to the telescope one, testing the main LBT AO components: the pyramid sensor and the secondary deformable mirror. Thanks to this the second phase of my PhD thesis: the measure of IM applying the sinusoidal modulation technique. At first we have measured the IM using a fiber auxiliary source to calibrate the system, without any kind of disturbance injected. After that, we have tried to use this calibration technique in order to measure the IM directly “on sky”, so adding an atmospheric disturbance to the AO system. The results obtained in this PhD work measuring the IM directly in the Arcetri solar tower system are crucial for the future development: the possibility of the acquisition of IM directly on sky means that we are able to calibrate an AO system also for extremely large telescope class where classic IM measurements technique are problematic and, sometimes, impossible. Finally we have not to forget the reason why we need this: the main aim is to observe the universe. Thanks to these new big class of telescopes and only using their full capabilities, we will be able to increase our knowledge of the universe objects observed, because we will be able to resolve more detailed characteristics, discovering, analyzing and understanding the behavior of the universe components.

Bestimmung der Ionisationsenergie von Actinium und Ultraspurenanalyse von Plutonium mit resonanter Ionisationsmassenspektrometrie (RIMS)

ZusammenfassungDie Resonanzionisationsmassenspektrometrie (RIMS) verbindet hohe Elementselektivität mit guter Nachweiseffizienz. Aufgrund dieser Eigenschaften ist die Methode für Ultraspurenanalyse und Untersuchungen an seltenen oder schwer handhabbaren Elementen gut geeignet. Für RIMS werden neutrale Atome mit monochromatischem Laserlicht ein- oder mehrfach resonant auf energetisch hoch liegende Niveaus angeregt und anschließend durch einen weiteren Laserstrahl oder durch ein elektrisches Feld ionisiert. Die Photoionen werden in einem Massenspektrometer massenselektiv registriert.Ein Beispiel für die Anwendung von RIMS ist die präzise Bestimmung der Ionisationsenergie als fundamentale physikalisch-chemische Eigenschaft eines bestimmten Elements; insbesondere bei den Actinoiden ist die Kenntnis der Ionisationsenergie von Interesse, da es dort bis zur Anwendung der laser-massenspektroskopischer Methode nur wenige experimentelle Daten gab. Die Bestimmung der Ionisationsenergie erfolgt durch die Methode der Photoionisation im elektrischen Feld gemäß dem klassischen Sattelpunktsmodell. Im Experiment werden neutrale Atome in einem Atomstrahl mittels Laserlicht zunächst resonant angeregt. Die angeregten Atome befinden sich in einem äußeren, statischen elektrischen Feld und werden durch einen weiteren Laserstrahl, dessen Wellenlänge durchgestimmt wird, ionisiert. Das Überschreiten der Laserschwelle macht sich durch einen starken Anstieg im Ionensignal bemerkbar. Man führt diese Messung bei verschiedenen elektrischen Feldstärken durch und erhält bei Auftragen der Ionisationsschwellen gegen die Wurzel der elektrischen Feldstärke durch Extrapolation auf die Feldstärke Null die Ionisationsenergie.Im Rahmen dieser Arbeit wurde die Ionisationsenergie von Actinium erstmalig zu 43398(3) cm-1 º 5,3807(4) eV experimentell bestimmt. Dazu wurden Actiniumatome zunächst einstufig resonant mit einem Laser mit einer Wellenlänge von 388,67 nm auf einen Zustand bei 25729,03 cm-1 angeregt und anschließend mit Laserlicht mit einer Wellenlänge von ca. 568 nm ionisiert. Damit sind die Ionisationsenergien aller Actinoiden bis einschließlich Einsteinium mit Ausnahme von Protactinium bekannt. Als Atomstrahlquelle wird ein spezielles 'Sandwichfilament' benutzt, bei dem das Actinoid als Hydroxid auf eine Tantalfolie aufgebracht und mit einer reduzierenden Deckschicht überzogen wird. Das Actinoid dampft bei Heizen dieser Anordnung atomar ab. Bei den schwereren Actinoiden wurde Titan als Deckschicht verwendet. Um einen Actiniumatomstrahl zu erzeugen, wurde aufgrund der hohen Abdampftemperaturen statt Titan erstmals Zirkonium eingesetzt. Bei Protactinium wurde Thorium, welches noch stärkere Reduktionseigenschaften aufweist, als Deckmaterial eingesetzt. Trotzdem gelang es mit der 'Sandwichtechnik' nicht, einen Protactiniumatomstrahl zu erzeugen. In der Flugzeitapparatur wurde lediglich ein Protactinium-monoxidionensignal detektiert. Um ein erst seit kurzem verfügbares Fest-körperlasersystem zu explorieren, wurden zusätzlich noch die bekannten Ionisations-ener-gien von Gadolinium und Plutonium erneut bestimmt. Die gemessenen Werte stimmen mit Literaturdaten gut überein.Ferner wurde noch ein bestehender Trennungsgang für Plutonium aus Umweltproben auf die Matrices Meerwasser und Hausstaub angepasst und für die Bestimmung von Plutonium und dessen Isotopenzusammensetzung in verschiedenen Probenreihen mittels RIMS eingesetzt. Der modifizierte Trennungsgang ermöglicht das schnelle Aufarbeiten von großen Probenmengen für Reihenuntersuchungen von Plutoniumkontaminationen. Die ermittelten Gehalten an 239Pu lagen zwischen 8,2*107 Atome pro 10 l Meerwasserprobe und 1,7*109Atome pro Gramm Staubprobe.

Untersuchung der Wirkung von Schwerionenstrahlen auf menschliche Hautfibroblasten unter besonderer Berücksichtigung chromosomaler Veränderungen

In der vorliegenden Arbeit wurden humane diploide Vorhaut-Fibroblasten u.a. auf Chromosomensch?den hin untersucht. Die konfluenten Zellen wurden mit d?nnionisierender R?ntgen-und Kohlenstoffstrahlung, sowie mit dichtionisierendenKohlenstoff- und Nickelionen bestrahlt und der chromosomaleSchaden in Intervallen bis zu 100 h nach Bestrahlungbestimmt. Dabei wurde nach dichtionisierender Strahlung ein deutlicher Anstieg in der Frequenz aberranter Zellen undAberrationen je Metaphase mit der Sammelzeit gefunden. Dieszeigt, dass gesch?digte Zellen von Zellzyklusverz'ogerungenst?rker betroffen sind als ungesch?digte Zellen.Durch Integration ?ber die Zeit wurde der genetische Gesamt-schaden in der proliferierenden Zellpopulation bestimmt. Dabei zeigte sich, dass ein Grossteil der Zellen nachTeilchenbestrahlung einen permanenten Zellzyklusarrest bzw.eine beschleunigte Differenzierung erf?hrt. Nur ein kleinerTeil erreicht die 1. Mitose nach Bestrahlung, so dass nurein geringer Teil der genetischen Sch?den auf die folgendenGenerationen ?bertragen wird. Der beobachtete Gesamtschadenist viel kleiner, als anhand von Daten aus Experimenten mitV79-Zellen abgesch?tzt wurde. Die direkte Extrapolation vonDaten etablierter Nagerzellen auf prim?re menschliche Zellenist demnach nicht m?glich. F?r die Beurteilung vonErgebnissen aus Tierexperimenten w?re es w?nschenswert zuwissen, ob die Unterschiede auf der Art der Zellen, alsoetablierten und prim?ren Zellen beruhen, oder von derSpezies abh?ngen.

Mechanismus der Apoptose, Gentoxizität und DNA-Reparatur in Herpesvirus-Thymidinkinase-exprimierenden Säugerzellen nach Behandlung mit Antiherpes-Virustatika vom Typ der Nukleosidanaloga

Um Cytotoxizität und Gentoxizität nukleosidischer Antiherpes-Virustatika zu untersuchen, wurden stabile CHO-Klone etabliert, die Thymidinkinase (TK) des Herpes simplex-Virus Typ 1 (HSV-TK) oder des Varicella zoster-Virus (VZV-TK) exprimieren. In HSV-TK-exprimierenden Zellen wurde das Purinanalogon Ganciclovir (GCV) effizient in die genomische DNA eingebaut, worauf in den nächsten Replikationsrunden DNA-Strangbrüche und Aberrationen entstehen und Apoptose ausgelöst wird. GCV-induzierte Apoptose wird hauptsächlich über den mitochondrialen Weg vermittelt, wobei das anti-apoptotische Protein Bcl-2 im Mittelpunkt steht. Nach GCV-Behandlung konnte eine Caspase-9-vermittelte post-translationale Spaltung von Bcl-2 nachgewiesen werden. Das 23 kDa-großes Bcl-2-Fragment wirkt im Gegensatz zum intakten Bcl-2-Protein pro-apoptotisch und verstärkt die Cytochrom C-Freisetzung und damit die Aktivierung der Caspase-9, die Bcl-2 spaltet, was zu einem positiven 'Amplifikationsloop' des mitochondrialen apoptotischen Weges führt. In weiteren Experimenten wurde gezeigt, daß in die DNA inkorporiertes GCV durch Basenexzisionsreparatur repariert wird, wobei die DNA-Polymerase ß eine entscheidende Rolle spielt. Diese Reparatur führte zu einer signifikanten Reduktion der Apoptose und Klastogenität und damit zur Resistenzsteigerung gegenüber GCV. In VZV-TK-exprimierenden Zellen wurde gezeigt, daß Brivudin (BVDU), gleichermaßen Apoptose und Nekrose induzierte. Für die BVDU-induzierte Cytotoxizität konnte die Hemmung der Thymidylatsynthetase als Ursache identifiziert werden. Im Gegensatz zur GCV-induzierten Apoptose war für die BVDU-induzierte Apoptose der Rezeptor (Fas/CD95/APO-1)-vermittelte Weg von vorrangiger Bedeutung.