Sequencing of serum hepatitis B virus (HBV) RNA as a novel method for the genome analysis of HBV

The genome of hepatitis B virus (HBV) can be assessed by sequence analysis of HBV DNA in serum. In most chronic HBV infected patients treated with potent nucleos(t)ide analogues (NAs) this approach however is limited by the fast decrease of serum HBV DNA during NA treatment. In contrast, HBV RNA was shown to persist in serum of some HBV infected individuals receiving NA treatment. In this study, we established the sequencing of serum HBV RNA as a method for the monitoring of HBV variants during NA treatment after the decrease of serum HBV DNA to undetectable levels. Using this approach, we studied the evolution of HBV variants in follow-up serum samples (n=156) of 25 patients treated with the potent polymerase inhibitor tenofovir (TDF) as second or third-line treatment. In our cohort, specific reverse transcribed full-length and truncated HBV RNA remained detectable with real-time PCR for long periods in most serum samples, also after the decline of HBV DNA during treatment with TDF. The HBV genome could be analyzed based on serum HBV DNA sequencing in 61 serum samples for a mean duration of 6.0 ± 4.5 (0 – 13) months. After this, sequencing of reverse transcribed serum HBV RNA allowed the analysis of the HBV genome for an additional mean duration of 33.9 ± 12.7 (16 - 65) months in 68 serum samples. The comparison of serum HBV DNA and serum HBV RNA derived sequences showed a high homology. In most patients, acquired HBV resistance variants in the reverse transcriptase (rt) region of the polymerase gene were detectable on HBV DNA and HBV RNA basis. Serum HBV RNA sequencing further revealed a long persistence of these variants during TDF treatment (mean duration of 26.5 ± 15.8 (0 – 50) months), which indicates a high conservation in the cccDNA of the infected individuals. Also HBV stop mutations in the small surface (s) gene, which were discussed in the pathogenesis of hepatocellular carcinoma (HCC), were present at baseline in 5 patients and remained detectable on HBV RNA basis during follow-up. In this study, we demonstrated that sequencing of reverse transcribed HBV RNA from patient serum is a suitable method to assess HBV variants during NA treatment. We further provided insights into the evolution of HBV variants during strong suppression of the viral replication with the polymerase inhibitor TDF. Future studies should investigate more comprehensively the clinical application of the here presented method of serum HBV RNA sequencing for the early detection of resistant HBV variants during NA treatment and the observation of HBV s gene variants related to HCC development.:Table of content I Table of content 3 II Abbreviations 6 1 Introduction 10 1.1 HBV 11 1.1.1 Classification 11 1.1.2 HBV virion structure and genomic organization 11 1.1.3 HBV proteins 12 1.1.4 HBV replication cycle 15 1.2 Chronic HBV infection 17 1.2.1 Epidemiology 17 1.2.2 Natural course of chronic HBV infection 17 1.3 Treatment of chronic HBV infection with nucleos(t)ide analogues 18 1.3.1 Nucleos(t)ide analogues 18 1.3.2 Treatment goals 18 1.3.3 Response to nucleos(t)ide analogue treatment 20 1.3.4 Treatment with nucleos(t)ide analogues and liver disease 21 1.4 Evolution of HBV variants during antiviral treatment 22 1.4.1 HBV resistance mutations in the pol gene 22 1.4.2 Resistance rates to treatment with nucleos(t)ide analogues 25 1.4.3 HBV variants in the s gene 26 1.5 HBV RNA in serum of chronically infected patients 29 1.5.1 HBV RNA molecules 29 1.5.2 HBV RNA packaging and release 29 1.5.3 HBV RNA as serum marker 31 1.6 Aim of the study 31 2 Materials and methods 33 2.1 Materials 33 2.1.1 Chemicals 33 2.1.2 Devices 33 2.1.3 Laboratory materials 34 2.1.4 Cycler 34 2.1.5 Kits 35 2.1.6 Buffers and solutions 36 2.1.7 Primers 36 2.1.8 Data analysis 39 2.2 Methods 39 2.2.1 Patient set and sample selection 39 2.2.2 Extraction of nucleic acids from serum samples 40 2.2.3 Reverse transcription of HBV RNA 40 2.2.4 Quantification of HBV serum DNA and HBV RNA by real-time PCR 41 2.2.4.1 Real-time PCR 41 2.2.4.2 Quantification of serum HBV DNA 43 2.2.4.3 Quantification of serum HBV trRNA and HBV flRNA 45 2.2.5 Sequencing of serum HBV DNA and HBV RNA 47 2.2.5.1 Primer design 47 2.2.5.2 Amplification by PCR 48 2.2.5.3 Purification of amplification products 50 2.2.5.4 Sanger sequencing of PCR fragments 51 2.2.6 Quantification of serum HBsAg and HBeAg 53 2.2.7 Cloning of HBV variants 53 2.2.8 Data analysis 55 2.2.8.1 Serum HBV DNA and HBV RNA quantities 55 2.2.8.2 Analysis of HBV DNA and HBV RNA sequences 55 3 Results 59 3.1 Composition of the patient set 59 3.2 Quantification of HBV DNA and HBV RNA in serum samples 59 3.2.1 Quantitative courses of serum HBV DNA 60 3.2.2 Quantitative courses of serum HBV flRNA and HBV trRNA 61 3.3 Sequencing of HBV DNA and HBV RNA 64 3.3.1 Method 64 3.3.2 Follow-up with sequencing of HBV DNA and HBV RNA 67 3.3.3 Genotyping of baseline samples 67 3.4 Evolution of HBV variants in the rt region 68 3.4.1 HBV resistance mutations in the rt region at baseline 68 3.4.2 HBV resistance mutations in the rt region during antiviral treatment 70 3.5 HBV variants in the s gene 77 3.5.1 HBV s gene variants at baseline 77 3.5.2 HBV s gene variants during antiviral treatment 80 4 Discussion 82 4.1 Patient cohort 82 4.2 Quantification of serum HBV DNA, HBV flRNA and HBV trRNA 82 4.3 Quantitative courses of serum HBV DNA, HBV flRNA and HBV trRNA 83 4.4 Sequencing of serum HBV RNA as novel method for HBV genome analysis 85 4.5 Evolution of HBV variants in the rt region 89 4.6 Evolution of HBV stop mutations in the s gene 91 4.7 Conclusion 92 III Summary 94 IV References 96 V List of Figures 104 VI List of Tables 105 VII Supplement 106 VIII Erklärung über die eigenständige Abfassung der Arbeit 107 IX Curriculum Vitae 108 X Publications 110 XI Acknowledgment 11

Trace element status in serum and follicular fluid in assisted reproduction

Das Polyzystisches Ovar Syndrom (PCOS) ist die häufigste Endokrinopathie bei Frauen im reproduktiven Alter, die in 75% mit Infertilität einhergeht. Allein in Europa werden 900.000 Zyklen künstlicher Befruchtung jährlich durchgeführt, wobei die Schwangerschaftsrate pro Embryotransfer weiterhin niedrig ist. Zur Verbesserung der Schwangerschaftsrate erfolgt die Auswahl der Eizelle anhand morphologischer Kriterien. Während die Relevanz von Spurenelementen für Fertilität gezeigt wurde, ist der Einfluss auf Eizell-/Embryoqualität weitgehend unbekannt. In der vorliegenden Arbeit wurden Kupfer, Selen und Zink in Follikelflüssigkeiten (FF) als potenzielle Biomarker für Eizell-/Embryoqualität untersucht. Die erweitere Selenstatuserfassung mittels Glutathionperoxidase 3 (GPX3) und Selenoprotein-P (SELENOP) stellte einen Schwerpunkt dar. Unsere Hypothese lautete, dass höhere Spurenelementkonzentrationen mit verbesserter Eizell-/Embryoqualität einhergehen. Hierzu untersuchten wir Proben (n=400 FF; n=40 Serum) von n=40 Frauen, unter Hormontherapie mit anschließender FF- und Eizellgewinnung. Eingeschlossen wurden dabei n=20 Frauen mit PCOS. Zur Qualitätsbeurteilung der Eizellen-/Embryonen wurden Gardner/Schoolcraft-Kriterien verwendet. Die FF der Eizellen-/Embryonen wurden fünf Qualitätsgruppen zugeordnet. Die Spurenelementkonzentrationen in den Proben wurden über Totalreflexions-Röntgenfluoreszenzanalyse ermittelt. Die Probengewinnung erlaubte die Spurenelementkonzentrationen der einzelnen FF mit jeweils zugehörigen Serumproben zu vergleichen. Zur Berechnung der Konzentrationsdifferenzen der Spurenelemente zwischen den verschiedenen FF, wurden die Spurenelementkonzentrationen in Gruppe 1 (Follikel ohne Eizelle) als Basiswert verwendet und Konzentrationsdifferenzen zwischen FF der Gruppen 2 bis 5 abzüglich Gruppe 1 berechnet. Über alle Proben hinweg zeigten sich niedrigere Spurenelementkonzentratio-nen in FF verglichen mit Serum. Der direkte Vergleich der bis zu n=10 FF pro Frau zeigte deutliche Konzentrationsschwankungen zwischen den Proben. Es konnte eine Tendenz zu niedrigeren Spurenelementkonzentrationen in FF der niedrigsten Qualitätskategorie festgestellt werden, sowie kürzere Synchronisationszeiten der Embryonen höchster Qualität. Es zeigten sich keine starken Unterschiede bezüglich der Spurenelementverteilung zwischen der PCOS- und Kontroll-Gruppe. GPX3 und SELENOP waren in FF nachweisbar und korrelierten positiv mit Selen. Da FF mit der schlechtesten Qualität tendenziell niedrigere Spurenelementkonzentrationen aufwiesen, kommen wir zu dem Ergebnis, dass ein Spurenelementmangel unter reproduktionsmedizinischer Therapie mit einer schlechteren Eizell-/Embryoqualität assoziiert sein könnte. Die intraindividuellen Variationen weisen, neben der Beeinflussung durch Serumkonzentrationen, auf eine individuelle Feinabstimmung je Follikel hin. Die Arbeit könnte von unmittelbarer Bedeutung für die optimierte Auswahl einer Eizelle sein.In Europe, 900,000 IVF cycles are performed per year, reflecting the high de-mand for assisted reproduction. Infertility affects around 72 million couples worldwide. Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age and is associated with infertility in 75%. The pregnancy rate per em-bryo transfer is low at 27-58%. A key factor in improving pregnancy rate is the selec-tion of the oocyte/embryo, which is based on morphological and morphokinetic criteria. While the relevance of trace elements for fertility has been shown, the influence on oocyte/embryo quality is still unknown. In this present study copper, selenium, and zinc in follicular fluid (FF) were in-vestigated as potential biomarkers for oocyte/embryo quality. The determination of the seleniumstatus incorporated three complementary biomarkers : selen, glutathione pe-roxidase (GPX3) and selenoprotein-P (SELENOP). Our hypothesis is that higher trace element concentrations are associated with improved oocyte/embryo quality. To this end, we analyzed samples (n=400 FF; n=40 serum) from n=40 women undergoing hormone stimulation therapy followed by FF and oocyte retrieval. Included were n=20 women with PCOS. Gardner/Schoolcraft criteria were used to assess oo-cyte/embryo quality. The FF of the oocytes/embryos were assigned to five groups. Trace element concentrations in the samples were determined using total reflection X-ray fluorescence. The sample collection allowed to compare the trace element con-centrations of the individual FF with respective serum samples. We considered the dif-ferences of the FF samples within a woman. To calculate the differences in trace ele-ment concentrations between the different FF samples, the trace element concentra-tions in group 1 (follicle without oocyte) were used as a baseline value and concentra-tion differences between FF of groups 2 to 5 minus group 1 were calculated. Within the whole cohort, lower concentrations of trace elements were found in FF compared to serum. The direct comparison of up to n=10 FF per woman showed strong variations in the concentration of trace elements between the samples. A ten-dency towards lower trace element concentrations in FF of the lowest quality category was observed, as well as shorter synchronization times of the highest quality embryos. There were no strong differences in trace element distribution between the PCOS and control groups. GPX3 and SELENOP were detectable in FF and correlated with sele-nium. Since FF with the poorest quality tended to have the lowest trace element con-centrations, we conclude that trace element deficiency under reproductive medicine therapy may be associated with poorer oocyte/embryo quality. The intraindividual var-iations indicate, besides influences of serum concentrations, an individual fine-tuning in single follicles. The results could be of direct relevance for the optimized selection of an oocyte. Larger studies are needed to confirm the assumptions

Workplace bullying : effects, prevention and legal consequences

Diese Bachelorarbeit befasst sich mit dem Thema Mobbing am Arbeitsplatz. Sie beschäftigt sich mit den Auswirkungen, der Prävention und den Rechtsfolgen von Mobbing. Zu Anfang betrachtet die Arbeit die Definition und den Entstehungsprozess von Mobbing. Dafür werden die Ursachen und Formen von Mobbing erläutert. Im darauffolgenden Kapitel werden die Auswirkungen von Mobbing auf das Mobbingopfer und sein Umfeld betrachtet. In den nachfolgenden Kapiteln werden die Bereiche Prävention, Rechtsgrundlagen und therapeutische Maßnahmen behandelt. Das nächste Kapitel befasst sich mit dem öffentlichen Umgang von Mobbing in den Medien. In der Arbeit veranschaulicht das Fallbeispiel eines Mobbingopfer, den Verlaufsprozess von Mobbing am Arbeitsplatz und seine Auswirkungen auf die betroffene Person. Zum Abschluss folgt eine Schlussbetrachtung, in der die Behandlung des Themas Mobbing am Arbeitsplatz in der gesellschaftlichen Sichtweise kritisch beurteilt wird. Das Ziel der Bachelorarbeit ist, die Folgen von Mobbing am Arbeitsplatz deutlich zu machen

A touching advantage:cross-modal stop-signals improve reactive response inhibition

The ability to inhibit an already initiated response is crucial for navigating the environment. However, it is unclear which characteristics make stop-signals more likely to be processed efficiently. In three consecutive studies, we demonstrate that stop-signal modality and location are key factors that influence reactive response inhibition. Study 1 shows that tactile stop-signals lead to better performance compared to visual stop-signals in an otherwise visual choice-reaction task. Results of Study 2 reveal that the location of the stop-signal matters. Specifically, if a visual stop-signal is presented at a different location compared to the visual go-signal, then stopping performance is enhanced. Extending these results, study 3 suggests that tactile stop-signals and location-distinct visual stop-signals retain their performance enhancing effect when visual distractors are presented at the location of the go-signal. In sum, these results confirm that stop-signal modality and location influence reactive response inhibition, even in the face of concurrent distractors. Future research may extend and generalize these findings to other cross-modal setups.</p

Authentication of Primary Murine Cell Lines by a Microfluidics-Based Lab-On-Chip System

The reliable authentication of cell lines is a prerequisite for the reproducibility and replicability of experiments. A common method of cell line authentication is the fragment length analysis (FLA) of short-tandem repeats (STR) by capillary electrophoresis. However, this technique is not always accessible and is often costly. Using a microfluidic electrophoresis system, we analyzed the quality and integrity of different murine cell lines by STR profiling. As a proof of concept, we isolated and immortalized hematopoietic progenitor cells (HPC) of various genotypes through retroviral transduction of the fusion of the estrogen receptor hormone-binding domain with the coding sequence of HoxB8. Cell lines were maintained in the HPC state with Flt3 ligand (FL) and estrogen treatment and could be characterized upon differentiation. In a validation cohort, we applied this technique on primary mutant Kras-driven pancreatic cancer cell lines, which again allowed for clear discrimination. In summary, our study provides evidence that FLA of STR-amplicons by microfluidic electrophoresis allows for stringent quality control and the tracking of cross-contaminations in both genetically stable HPC lines and cancer cell lines, making it a simple and cost-efficient alternative to traditional capillary electrophoresis

Charged-Particle Thermonuclear Reaction Rates: III. Nuclear Physics Input

The nuclear physics input used to compute the Monte Carlo reaction rates and probability density functions that are tabulated in the second paper of this series (Paper II) is presented. Specifically, we publish the input files to the Monte Carlo reaction rate code RatesMC, which is based on the formalism presented in the first paper of this series (Paper I). This data base contains overwhelmingly experimental nuclear physics information. The survey of literature for this review was concluded in November 2009.Comment: 132 page

Capture of alpha Particles by Isospin-Symmetric Nuclei

The reaction rates for alpha capture processes on self-conjugate nuclei in the mass range A=20-40 have been investigated. The rates were calculated using the statistical model code NON-SMOKER taking into account isospin suppression rules. These theoretical predictions are compared with rates derived from the available experimental data about the alpha capture reactions taking also into account additional experimental information from different reaction channels populating the alpha unbound states of the self-conjugate compound nuclei.Comment: 29 pages including 7 figures; figures are revised in the new version; accepted for publication in Nuclear Physics

Comprehensive CRISPR-Cas9 screens identify genetic determinants of drug responsiveness in multiple myeloma

The introduction of new drugs in the past years has substantially improved outcome in multiple myeloma (MM). However, the majority of patients eventually relapse and become resistant to one or multiple drugs. While the genetic landscape of relapsed/ resistant multiple myeloma has been elucidated, the causal relationship between relapse-specific gene mutations and the sensitivity to a given drug in MM has not systematically been evaluated. To determine the functional impact of gene mutations, we performed combined whole-exome sequencing (WES) of longitudinal patient samples with CRISPR-Cas9 drug resistance screens for lenalidomide, bortezomib, dexamethasone, and melphalan. WES of longitudinal samples from 16 MM patients identified a large number of mutations in each patient that were newly acquired or evolved from a small subclone (median 9, range 1-55), including recurrent mutations in TP53, DNAH5, and WSCD2. Focused CRISPR-Cas9 resistance screens against 170 relapse-specific mutations functionally linked 15 of them to drug resistance. These included cereblon E3 ligase complex members for lenalidomide, structural genes PCDHA5 and ANKMY2 for dexamethasone, RB1 and CDK2NC for bortezomib, and TP53 for melphalan. In contrast, inactivation of genes involved in the DNA damage repair pathway, including ATM, FANCA, RAD54B, and BRCC3, enhanced susceptibility to cytotoxic chemotherapy. Resistance patterns were highly drug specific with low overlap and highly correlated with the treatment-dependent clonal evolution in patients. The functional association of specific genetic alterations with drug sensitivity will help to personalize treatment of MM in the future

Effect of Propranolol on Functional Connectivity in Autism Spectrum Disorder—A Pilot Study

A decrease in interaction between brain regions is observed in individuals with autism spectrum disorder (ASD), which is believed to be related to restricted neural network access in ASD. Propranolol, a beta-adrenergic antagonist, has revealed benefit during performance of tasks involving flexibility of access to networks, a benefit also seen in ASD. Our goal was to determine the effect of propranolol on functional connectivity in ASD during a verbal decision making task as compared to nadolol, thereby accounting for the potential spurious fMRI effects due to peripheral hemodynamic effects of propranolol. Ten ASD subjects underwent fMRI scans after administration of placebo, propranolol or nadolol, while performing a phonological decision making task. Comparison of functional connectivity between pre-defined ROI-pairs revealed a significant increase with propranolol compared to nadolol, suggesting a potential imaging marker for the cognitive effects of propranolol in ASD