Die Bedeutung des Telomerasepromotors in der Tumorgentherapie

Die Intention der vorliegenden Promotionsarbeit war, mit Hilfe des Telomerase-Promotors (TERT) eine tumorselektive Gentherapie zu entwickeln. Wir wählten den Telomerasepromotor, weil die katalytische Untereinheit der Telomerase bei über 85% der Tumoren stark exprimiert ist. In gesunden differenzierten Zellen ist die Telomerase jedoch inaktiv. Lediglich in einigen speziellen Zellarten wie Keimzellen oder Stammzellen ist Telomeraseaktivität nachweisbar. Nach Auswahl einiger Tumorzellinien wurde zunächst die hTERT-Expression (humane Telomerase Reverse Transkriptase) der Tumorzellen mit Hilfe von RT-PCR untersucht. hTERT ist die enzymatisch aktive, der Regulation unterliegende Untereinheit der humanen Telomerase Reverse Transkriptase. Die hTERT-Expression korreliert mit der Aktivität des Telomerasepromotors. Als weitere Untereinheit benötigt die Telomerase die ubiquitär vorliegende, nicht der Telomerase-Regulation unterliegende Telomerase-RNA, welche als Vorlage für die Telomerverlängerung dient. Die Telomeraseaktivität in den Zellinien wurde mit Hilfe des TRAP-Assays (Telomeric Repeat Amplification Protocol) nachgewiesen. Nach Untersuchung der hTERT-Expression und der Telomeraseaktivität in den ausgewählten Tumorzellinien wurden weitere Untersuchungen zur Charakterisierung dieser Zellinien durchgeführt. Als DNA-Vektoren wurden zwei unterschiedliche adenovirale Konstrukte verwendet. Die Vektoren enthielten zum einen das Luciferase Reportergen unter Kontrolle des hTERT-Promotors (Ad-hTERT-Luc) und zum anderen das Reportergen unter Kontrolle des Cytomegalievirus-Promotors (Ad-CMV-Luc). Die Luciferaseexpression in den Zellen ist quantitativ messbar und korreliert mit der jeweiligen Promotoraktivität. Nach in vitro- Nachweis der hTERT-Expression in den Tumorzellinien wurden die Vektoren in vivo im Hinblick auf die Tumorspezifität des Telomerasepromotors getestet. Die Versuche wurden mit NMRI- Nude Mäusen durchgeführt. Als Xenotransplantatmodell wählten wir die etablierte Zellinie H1299 eines humanen kleinzelligen Bronchialkarzinoms. Diese Tumorzellinie zeigte bei den in vitro- Versuchen eine hohe Telomerasepromotoraktivität im Vergleich zum CMV- Promotor und ist zur Tumorinduktion in vivo gut geeignet. Nach Tumorinduktion wurde den Mäusen Virussuspension in die Schwanzvene injiziert. Nach 72 Stunden wurden Organe und Gewebe präpariert. Bei den Untersuchungen wurde Virus- DNA in sämtlichen Geweben in unterschiedlichen Konzentrationen nachgewiesen. Es zeigte sich jedoch, dass die durchaus hohe Telomerase- bzw. Luciferaseaktivität sich nicht nur auf Tumorgewebe beschränkte, sondern in anderen Geweben ebenfalls sehr hoch war. Die Aktivität in der Leber war sogar höher als im Tumorgewebe, was durch den natürlichen Hepatotropismus der Adenoviren begründet sein kann. Beim Vergleich der Tumor-Leber-Aktivitätsverhältnisse von Ad-hTERT-Luc und Ad-CMV-Luc konnte man erkennen, dass die Aktivität in der Leber bei Ad-hTERT-Luc etwa das 50 fache und bei Ad-CMV-Luc das 100 fache von der Aktivität im Tumorgewebe betrug. Die Aktivitäten in einigen anderen Geweben ergaben ebenfalls sehr hohe Werte. Die Aktivitätsmuster der beiden Konstrukte Ad-CMV-Luc und Ad-hTERT-Luc zeigten außer den unterschiedlichen Aktivitätsmaxima kaum Unterschiede, insbesondere war keine Tumorspezifität zu erkennen. Diese Ergebnisse zeigen, dass mit dem hTERT Promotor im gewählten Ansatz keine Tumorselektivität erzielt werden kann

Evaluation of the in vitro elution characteristics of carboplatin-impregnated calcium sulfate beads

Pilot Study: Objective: To characterize the elution of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CSH) beads in vitro. Sample: Sixty carboplatin-impregnated CSH beads and 9 CSH beads without added carboplatin (controls) Procedures: Carboplatin-impregnated CSH beads (each containing 4.6 mg carboplatin [2.4 mg platinum]) were placed into separate 10 mL plastic tubes containing 5 mL of PBS in groups of 1, 3, 6, or 10; 3 control beads were placed in a single tube of PBS at the same volume. Experiments were conducted in triplicate at 37 °C and pH 7.4 with constant agitation. Eluent samples were collected at 1, 2, 3, 6, 12, 24, and 72 hours. Samples were analyzed for platinum content by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Results: The mean concentration of platinum released per carboplatin-impregnated bead over 72 hours was 445.3 mg/L. Cumulative concentrations of platinum eluted increased as the number of beads per tube increased. There was a significant difference in platinum concentrations over time, with values increasing over the first 12 hours and then declining for all tubes. There was also a significant difference in percentage of total incorporated platinum released into tubes with different numbers of beads: the percentage eluted of platinum was higher in tubes containing 1 or 3 beads than in those containing 6 or 10 beads. Conclusions and Clinical Relevance: Carboplatin-impregnated CSH beads eluted platinum over 72 hours. Further studies are needed to determine whether implantation of carboplatin-impregnated CSH beads results in detectable levels of platinum systemically and whether the platinum concentrations eluted locally are toxic to tumor cells. Experiment 2: Objective: To characterize the long-term elution of platinum from carboplatin-impregnated CSH beads in vitro using two distinct sampling methods. Sample: Carboplatin-impregnated CSH beads containing 4.6 mg carboplatin/bead Procedures: Method 1: Three carboplatin-impregnated CSH beads were placed into 10 mL plastic tubes with 5 mL of PBS at 37 C and pH 7.4 with constant agitation. PBS was sampled by evacuation of all 5 mL eluent at 1, 2, 3, 6, 9, and 12 hours and 1, 2, 3, 6, 9, 12, 15, 18, 22, 26, and 30 days. The fluid was then replaced with 5 mL of fresh PBS at each time point. Method 2: Tubes corresponding to each sampling time were established at time zero, with each tube containing 3 carboplatin-impregnated CSH beads and 5 mL PBS. PBS was sampled from only the assigned tubes at each time point by evacuation of all 5 mL eluent. Control beads without carboplatin were also evaluated using both methods. Samples were analyzed for platinum concentration by ICP-MS. Results: Platinum was released from carboplatin-impregnated CSH beads for 22-30 days. There were significant differences in platinum concentrations and percentage of total incorporated platinum released over time and between methods. Conclusions and Clinical Relevance: Carboplatin-impregnated CSH beads eluted platinum for 22-30 days. Sampling method significantly affected platinum release from carboplatin-impregnated CSH beads at nearly all time points. Results from sampling Method 1 and Method 2 provide estimations of the minimum and maximum platinum concentrations expected to elute from carboplatin-impregnated CSH beads in vivo

Carrying Jeremy Safran into sessions: relying on internal representations of researchers to facilitate emotion regulation, clinical intervention, and self-efficacy

Relying on positive internal representations facilitates our ability to feel safe and secure when taking risks and provides a road map to guide us during interpersonal exchanges. Although most graduate programs encourage students to engage in research, we rarely link participating in research as directly influencing positive internal representations that can influence treatment. We used a qualitative method to examine how watching videos of Jeremy Safran, coding therapy sessions using his model, and reading his articles on ruptures and repairs influenced students’ ability to self-soothe, take risks, and engage when patients confront them or withdraw. Results revealed that students often thought of Jeremy Safran and his colleagues during a session and recalled how he addressed ruptures in the videos they watched. When they were anxious during a session, they reported relying on the video coding training to facilitate emotion regulation during sessions. Having the research experience increased their clinical skills and overall clinical self-efficacy. Implications of our findings and future recommendations are discussed

Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status. RESULTS: We have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity. CONCLUSIONS: We identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification

Interobserver reliability and diagnostic performance of Chiari II malformation measures in MR imaging—part 2

PURPOSE: Brain MR imaging is essential in the assessment of Chiari II malformation in clinical and research settings concerning spina bifida. However, the interpretation of MR images of the malformation is not always straightforward. Morphometric analyses of the extent of Chiari II malformation may improve the assessment. In an attempt to select appropriate morphometric measures for this purpose, we investigated the interobserver reliability and diagnostic performance of several morphometric measures of Chiari II malformation on MR images. METHODS: Brain MR images of 79 children [26 with open spinal dysraphism, 17 with closed spinal dysraphism, and 36 without spinal dysraphism; mean age 10.6 (SD 3.2; range, 6-16) years] were evaluated. All children had been assessed for Chiari II malformation (defined as cerebellar herniation in combination with open spinal dysraphism; n = 23). Three observers blindly and independently reviewed the MR images for 21 measures of the cerebellum, brainstem, and posterior fossa in three planes. The interobserver reliability was assessed by an agreement index (AI = 1 - RRE) and the diagnostic performance by receiver operating characteristic analyses. RESULTS: Reliability was good for most measures, except for the degree of herniation of the vermis and tonsil. Most values differed statistically significantly between children with and without Chiari II malformation. The measures mamillopontine distance and cerebellar width showed excellent diagnostic performance. CONCLUSIONS: Morphometric measures may reliably quantify the morphological distortions of Chiari II malformation on MR images and provide additional tools to assess the severity of Chiari II malformation in clinical and research settings

Parents' psychological adjustment in families of children with Spina Bifida: a meta-analysis

BACKGROUND: Spina Bifida (SB) is the second most common birth defect worldwide. Since the chances of survival in children with severe SB-forms have increased, medical care has shifted its emphasis from life-saving interventions to fostering the quality of life for these children and their families. Little is known, however, about the impact of SB on family adjustment. Reviewers have struggled to synthesize the few contradictory studies available. In this systematic review a new attempt was made to summarize the findings by using meta-analysis and by delimiting the scope of review to one concept of family adjustment: Parents' psychological adjustment. The questions addressed were: (a) do parents of children with SB have more psychological distress than controls? (b) do mothers and fathers differ? and (c) which factors correlate with variations in psychological adjustment? METHODS: PsycInfo, Medline, and reference lists were scanned. Thirty-three relevant studies were identified of which 15 were eligible for meta-analysis. RESULTS: SB had a negative medium-large effect on parents' psychological adjustment. The effect was more heterogeneous for mothers than for fathers. In the reviewed studies child factors (age, conduct problems, emotional problems, and mental retardation), parent factors (SES, hope, appraised stress, coping, and parenting competence), family factors (family income, partner relationship, and family climate), and environmental factors (social support) were found to be associated with variations in parents' psychological adjustment. CONCLUSION: Meta-analysis proved to be helpful in organizing studies. Clinical implications indicate a need to be especially alert to psychological suffering in mothers of children with SB. Future research should increase sample sizes through multi-center collaborations

Towards a new image processing system at Wendelstein 7-X: From spatial calibration to characterization of thermal events

Wendelstein 7-X (W7-X) is the most advanced fusion experiment in the stellarator line and is aimed at proving that the stellarator concept is suitable for a fusion reactor. One of the most important issues for fusion reactors is the monitoring of plasma facing components when exposed to very high heat loads, through the use of visible and infrared (IR) cameras. In this paper, a new image processing system for the analysis of the strike lines on the inboard limiters from the first W7-X experimental campaign is presented. This system builds a model of the IR cameras through the use of spatial calibration techniques, helping to characterize the strike lines by using the information given by real spatial coordinates of each pixel. The characterization of the strike lines is made in terms of position, size, and shape, after projecting the camera image in a 2D grid which tries to preserve the curvilinear surface distances between points. The description of the strike-line shape is made by means of the Fourier Descriptors

Forward modeling of collective Thomson scattering for Wendelstein 7-X plasmas: Electrostatic approximation

In this paper, we present a method for numerical computation of collective Thomson scattering (CTS). We developed a forward model, eCTS, in the electrostatic approximation and benchmarked it against a full electromagnetic model. Differences between the electrostatic and the electromagnetic models are discussed. The sensitivity of the results to the ion temperature and the plasma composition is demonstrated. We integrated the model into the Bayesian data analysis framework Minerva and used it for the analysis of noisy synthetic data sets produced by a full electromagnetic model. It is shown that eCTS can be used for the inference of the bulk ion temperature. The model has been used to infer the bulk ion temperature from the first CTS measurements on Wendelstein 7-X