Regulation of proto-oncogenic Pim-1 kinase and miR-17-92 microRNAcluster in the human leukemia cell line K562

In this thesis the regulation of the oncogenic kinase Pim-1 and the microRNA cluster miR-17-92 has been investigated using the leukemia cell line K562 as a model system. Pim-1 and miR-17-92 have been reported to be highly expressed in the context of leukemia cells as well as in other cancer cell lines, albeit little is known about mechanisms leading to their overexpression. The aim of the work presented here was to evaluate the functions of Pim-1 in the promotion of tumorigenesis and to clarify its regulation, with the goal of exploring its potential for therapeutic interference. Several targets of the miR-17-92 encoded miRs have been identified in different cellular contexts, and probably more will be found in the future. Nevertheless, many open questions concerning the regulation of the cluster still remain unanswered. For those reasons, we focussed our efforts on transcriptional regulation of the cluster in K562 cells, as well as on the clusters impact on one of its targets, the cell cycle regulator p21. High expression levels of the Pim-1 kinase are characteristic for K562 cells. At the protein level, degradation of Pim-1 is promoted by the phosphatase PP2A. Inhibition of PP2A thus increases Pim-1 levels. Within the context of this work, it was shown by our group that inhibition of PP2A by ocadaic acid (OA) leads to a temporary increase of Pim-1 followed by a complete downregulation. This was accompanied by an upregulation of p21 which under normal growth conditions is not found at the protein level in this cell line. The cellular phenotype during OA-treatment changed from proliferation to apoptosis and siRNA-targeting of p21 delayed the onset of this apoptotic response, indicating that downregulation of p21 in K562 cells contributes to their anti-apoptotic and proliferative phenotype. Interestingly, p21 protein is not found at normal growth in K562 cells, although substantial amounts of its mRNA can be detected. This implicates a posttranscriptional silencing mechanism. Here we found that p21 is a target of miR-17-5p and miR-20a, both miRs being encoded in the miR-17-92 cluster. We could prove this in two ways. First, we cloned the p21 3-UTR into a reporter vector. Mutation of the two predicted miR-binding sites in the respective constructs revealed regulation by those miRs as inferred from an increase of reporter activity. Second, we interfered with microRNA target-binding by application of antisense molecules directed against mature miR-17-5p and miR-20a. For this, we used locked nucleic acids (LNAs), as those modified oligonucleotides bind with largely increased affinity to their complementary strands. Current AntimiR approaches mostly rely on the use of 2´-O-Methyl-RNA molecules for targeting of mature miRs, as this modification stabilizes the molecules against nucleases compared to unmodified RNA. AntimiRs are designed to be complementary over the whole length of the targeted miR (22-24 nt). Here it was established in a proof of principle experiment that LNAs of only 14 nucleotides could efficiently abolish miR-function in vivo. For this, luciferase reporter constructs containing a let-7a binding site were generated. In K562 cells let-7a is abundantly expressed, so constructs were silenced when transfected, but not so in co-transfection experiments with respective LNAs targeting let-7a. LNA-AntimiRs, 8, 10, 12 or 14 nucleotides in length, were tested, and even the 8-mer showed some let-7a-specific derepression effect. With this tool at hand, we targeted miR-17-5p and miR-20a to evaluate the effects on cellular p21 protein levels. In Western blot analysis we found an induction of p21 protein upon transfection with LNA 14-mers against miR-17-5p and miR-20a. By this experimental approach, we were able to demonstrate the importance of translational downregulation of the p21 protein in K562 cells that in combination with Pim-1 overexpression is supposed to contribute to the observed proliferative phenotype..

Material Issues in Layered Forming

A brief overview of key issues in layered thermal processing is given. Incremental sintering and layered fusion ofpowder and molten droplets are discussed. The criteria for remelting the solid substrate are derivedfrom a one dimensional heat transfer model. Temperature gradients which occur during solidification and subsequent cooling. are responsible for the build up of internal stresses which can be estimated through establishing an elastic beam model. The difficulties as well as opportunities regarding the generation of multi-layer multi-material structures are also described in this article.Mechanical Engineerin

PEI-F25-LMW als Transfektionsreagenz für DNA und siRNA sowie seine Anwendung im Targeting von VEGF mittels siRNA in Prostatakarzinomzellen

Vor allem in der Krebstherapie wird nach Alternativen zu viralen Gentherapie-Vektoren gesucht, da diese immunogene und tumorigene Nebenwirkungen entwickeln können. Polyethylenimine (PEIs) sind kationische Polymere mit der Fähigkeit Nukleinsäuren zu komplexieren. Durch diese Eigenschaft konnte gezeigt werden, dass sie sich als Transfektionsreagenz in vitro und in vivo eignen. Mit PEIs können Zellen in vitro und in vivo transfiziert werden, wenn auch nicht so effizient wie mit viralen Vektoren. Gezeigt wurde dies für DNA- bzw. siRNA-PEI-Komplexe, die meist in NaCl- oder Glucose-Lösung angesetzt und frisch transfiziert wurden. Die Transfektionen in vitro finden dabei oft in serumfreiem Zellkulturmedium statt, um eine ausreichende Transfektionseffizienz zu gewährleisten. In dieser Arbeit wurde das Polyethylenimin PEI-F25-LMW charakterisiert und gezeigt, dass sich damit komplexierte DNAs bzw. siRNAs einfrieren und lagern lassen, ohne dadurch an Transfektionseffizienz zu verlieren. Weiterhin konnte nachgewiesen werden, das der Serumgehalt von Zellkulturmedium für in vitro Transfektionen keinen Einfluß auf die Transfektionseffizienz von DNA- oder siRNA-PEI-F25-LMW-Komplexen hat. Damit ergeben gute Möglichkeiten für in vivo Anwendungen, bei denen es zwangsläufig zu Interaktionen der Komplexe mit Serumproteinen kommt. Durch PEI-F25-LMW-vermitteltes siRNA Targeting gegen VEGF an PC-3 und Du145 Prostatakarzinom-Zellen in vitro wurde gezeigt, dass dieses System eine effiziente Herrunterregulation des VEGF-Proteins ermöglicht und somit die Untersuchung klinisch relevanter Fragestellungen erlaubt. Das Targeting wurde dabei durch QT-RT-PCR und VEGF-ELISA nachgewiesen. Die Auswirkungen auf das Zellwachstum wurden mit Proliferations- und Soft-Agar-Assays untersucht. Trotz effizienter Herrunterregulation von VEGF wurde jedoch kein Effekt auf die Zellproliferation beobachtet. Damit spielt VEGF zumindest in vitro mit großer Wahrscheinlichkeit keine Rolle als autokrin wirkender Wachstumsfaktor. Für Untersuchungen in vivo wurde ein Du145-Tumor-Modell etabliert. Dazu wurde die notwendige Zellzahl zur Initiation von subkutanen Du145 Tumoren an athymischen Nacktmäusen ermittelt, wobei sich eine Zellzahl von 5 Mio als sinnvoll erwies

Performance of an Innovative Bio-Based Wood Chip Storage Pile Cover—Can It Replace Plastic Tarps?

There is currently great general interest in reducing the use of fossil-based materials. Fossil-based tarps are still widely used as cover for wood chip storage piles, causing additional waste or requiring further waste treatment in the supply chain. This study aimed to investigate the performance of an innovative bio-based wood chip pile cover compared to conventional treatments (plastic-covered and uncovered) in eastern Finnish conditions. The experiment evaluated the drying process during the storage of stemwood chips during 5.9 months of storage. It included the developments of temperature, moisture content, heating value, energy content, basic density, particle size distribution, and the dry matter losses of a total of six piles. As a result, the forest stemwood chips dried by 11%, with dry-matter losses of 4.3%, when covered with the bio-pile cover. Using the plastic covering, the forest stemwood chips dried by 22%, with dry matter losses of 2.9%. At the end of the experiment, the energy content in plastic-covered piles was 6.1% higher than uncovered piles and 3.1% higher than bio-pile-covered piles. While differences in the key drying performance parameters can be observed, the differences between uncovered piles and those covered with plastic tarps, as well as between the bio-based and the uncovered piles, were not statistically significant. We conclude that the bio-based cover, under the studied conditions, do not render better storage conditions than in current practices. However, our study indicates possible fossil-substitutional benefits by using a bio-based cover, which calls for further R&D work in this matter

The Proper Motion of the Central Compact Object RX J0822-4300 in the Supernova Remnant Puppis A

Using the High Resolution Camera (HRC) aboard the Chandra X-ray Observatory, we have re-examined the proper motion of the central compact object RX J0822-4300 in the supernova remnant Puppis A. New data from 2010 August, combined with three archival data sets from as early as 1999 December, provide a baseline of 3886 days (more than 10 1/2 years) to perform the measurement. Correlating the four positions of RX J0822-4300 measured in each data set implies a projected proper motion of mu 71 \pm 12 masy. For a distance of 2 kpc this proper motion is equivalent to a recoil velocity of 672 \pm 115 km/s. The position angle is found to be 244 \pm 11 degrees. Both the magnitude and direction of the proper motion are in agreement with RX J0822-4300 originating near the optical expansion center of the supernova remnant. For a displacement of 371 \pm 31 arcsec between its birth place and today's position we deduce an age of (5.2 \pm 1.0) 10^3 yrs for RX J0822-4300. The age inferred from the neutron star proper motion and filament motions can be considered as two independent measurements of the same quantity. They average to 4450 \pm 750 yrs for the age of the supernova remnant Puppis A.Comment: Accepted for publication in the Astrophysical Journa

Prototype of hybrid technology chipper-D4.6

INFRES - Innovative and effective technology and logistics for forest residual biomass supply in the EU (311881) is a collaborative project co-funded under the European Commission's FP7 Work Programme 2012 (FP7-KBBE-2012.1.2-01)201

Management of a Type I Hypersensitivity Reaction to IV Etoposide in a Woman with a Yolk Sac Tumor: A Case Report

Type I hypersensitivity reactions to intravenous administration of etoposide are extremely rare. Etoposide is an essential component of several chemotherapy regimens used in gynecologic oncology, and discontinuation of this drug during a course of treatment should only be due to severe patient intolerance. We report the successful use of intravenous etoposide phosphate as a substitute drug in a patient with a yolk sac tumor who manifested a Type I hypersensitivity to intravenous etoposide. The patient ultimately completed all 4 cycles of bleomycin, etoposide, cisplatin (BEP) using etoposide phosphate as a substitute drug

Spatio-temporal prediction of soil moisture using soil maps, topographic indices and SMAP retrievals

Milder winters and extended wetter periods in spring and autumn limit the amount of time available for carrying out ground-based forest operations on soils with satisfactory bearing capacity. Thus, damage to soil in form of compaction and displacement is reported to be becoming more widespread. The prediction of trafficability has become one of the most central issues in planning of mechanized harvesting operations. The work presented looks at methods to model field measured spatio-temporal variations of soil moisture content (SMC, [%vol]) – a crucial factor for soil strength and thus trafficability. We incorporated large-scaled maps of soil characteristics, high-resolution topographic information – depth-to-water (DTW) and topographic wetness index – and openly available temporal soil moisture retrievals provided by the NASA Soil Moisture Active Passive mission. Time-series measurements of SMC were captured at six study sites across Europe. These data were then used to develop linear models, a generalized additive model, and the machine learning algorithms Random Forest (RF) and eXtreme Gradient Boosting (XGB). The models were trained on a randomly selected 10% subset of the dataset. Predictions of SMC made with RF and XGB attained the highest R2 values of 0.49 and 0.51, respectively, calculated on the remaining 90% test set. This corresponds to a major increase in predictive performance, compared to basic DTW maps (R2 = 0.022). Accordingly, the quality for predicting wet soils was increased by 49% when XGB was applied (Matthews correlation coefficient = 0.45). We demonstrated how open access data can be used to clearly improve the prediction of SMC and enable adequate trafficability mappings with high spatial and temporal resolution. Spatio-temporal modelling could contribute to sustainable forest management.publishedVersio