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  • 1
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  • 2
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    Publication Date: 2021-06-15
    Description: Prostate cancer is one of the most common causes of death in men. In this work a siRNA screen of around 1500 cancer-relevant genes was performed using 3 different cell lines (VCaP, LAPC-4, RWPE-1). A novel technique, the living cell array, was initiated in order to obtain information about the biology of Androgeninduced growth in prostate tumour cell lines. This technique is based on the principal of reverse transfection [1] and genes are knocked down by siRNAs. The cells on the living cell array were set under stress by reduction of the androgens in the media while the proliferation and apoptosis were quantified. The statistical analysis of the data implicates the success of the screen and shows that this method is suitable for large-scale experiments.
    Description: Prostatakrebs ist neben Lungenkrebs eine der häufigsten Todesursachen bei Männern. In dieser Arbeit wurden 1500 potenziell tumorrelevante Gene in drei Zelllinien (VCaP, LAPC-4, RWPE-1) gescreent. Dafür wurde eine neue Technik, die Lebendzellarrays, genutzt, um Informationen über die Biologie Androgen-unabhängiger Prostatazellen zu gewinnen. Die Lebendzellarrays basieren auf dem Prinzip der reversen Transfektion [1], und in Folge werden die mRNAs der gewünschten Gene durch spezifische »silencer RNAs« (siRNAs) ausgeschaltet. Die Zellen wurden parallel sowohl in normalem Medium untersucht als auch mit androgen-reduziertem Medium unter Stress gesetzt. Das Wachstum der Prostatazellen wurde mittels Markern für Proliferation und Apoptose beobachtet. Die Daten der Screens wurden mit Hilfe statistischer Verfahren evaluiert. Die Lebendzellarrays konnten erfolgreich für einen umfangreichen SiRNA-screen angewendet werden.
    Keywords: ddc:620
    Language: English
    Type: article , doc-type:article , doc-type:article
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  • 3
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    Publication Date: 2021-06-15
    Description: Die Messung von Zytokinen in Stimulationsexperimenten zur Bestimmung von Stärke und Umfang einer Immunreaktion werden in der Praxis häufig an kryokonserviertem Zellmaterial durchgeführt. Bisherige Untersuchungen zum Einfluss der Kryokonservierung auf die Zytokinexpression sind widersprüchlich. In den hier durchgeführten Experimenten wurden die Genexpression und/ oder Sekretion der Zytokine IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, IFN-γ und TNF-α in frisch isolierten und kryokonservierten Immunzellen aus Blut (PBMC) eines gesunden Spenders bestimmt. Diese wurden mit den Immunsystem-aktivierenden Stoffen OKT-3 und Concanavalin A (Con A) stimuliert und für 4 bzw. 24 h kultiviert. Ein Vergleich der frisch-isolierten und kryokonservierten PBMC in Stimulationsexperimenten zeigt, (1) dass die Messung der Genexpression genauere Einblicke zu Beginn der einsetzenden Immunreaktion verschafft, als die Messung der ausgeschütteten Zytokine, (2) dass durch Einfrieren die Immunreaktion insbesondere zu diesem Zeitpunkt beeinflusst wird und (3) dass zu späteren Zeitpunkten die Konzentrationsbestimmung der Zytokine im Zellkulturüberstand das Mittel der Wahl ist.
    Description: The measurement of cytokines in stimulation experiments with the aim to determine the strength and the complexitiy of an immune reaction is commonly carried out on cryopreserved cells. Previous studies, investigating the impact of cryopreservation on the cytokine expression, are inconsistent in their findings. Experiments conducted in this study determine the gene expression and/or secretion of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, GM-CSF, IFN-γ and TNF-α in freshly isolated and cryopreserved PBMC of a healthy donor. The cells were stimulated with the substances OKT-3 and Concanavalin A (Con A) and cultured for 4h and 24h. The comparison of freshly isolated and cryopreserved cells in stimulation experiments showed (1) that the measurement of the gene expression offers a more accurate insight into the immune-reaction at early timepoints than the measurement of the secreted cytokines; (2) that freezing of the cells affect the immune response at this early stage and (3) that at later points the measurement of secreted proteins is the method of choice.
    Keywords: ddc:620
    Language: German
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  • 4
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    Publication Date: 2021-06-15
    Description: Mit Hochdurchsatz-Kultivierungs- und -Screeningmethoden können viele Proben parallel, miniaturisiert und kostengünstig bearbeitet werden. Für phototrophe Organismen wie Mikroalgen und Cyanobakterien sind Hochdurchsatz-Kultivierungsverfahren jedoch bis heute kaum etabliert. Im Rahmen dieser Arbeit wurden diese Verfahren beispielhaft für das Cyanobakterium Synechocystis sp. PCC 6803 etabliert. Die benötigte technische Automatisierung wurde hierbei durch den Einsatz eines Tecan Genesis RSP 150 Pipettierroboters erreicht. Die Kultivierung erfolgte in Deepwell-Mikrotiterplatten innerhalb einer speziell angefertigten Kammer mit programmierbaren Schüttlern, einstellbarer Belichtung und CO2-Atmosphäre. Die in diesem System erreichten Wachstumsraten sind vergleichbar mit publizierten Kultivierungsmethoden. Das Hochdurchsatz-Screening wurde mit Hilfe eines in den Roboter integrierten Tecan Genios Plus Plattenreaders durchgeführt. Es wurden beispielhaft Methoden zur Bestimmung von optischer Dichte und Chlorophyllgehalt etabliert. Die hier vorgestellte Plattform kann vielseitig zur Analyse phototropher Organismen eingesetzt werden und ist durch entsprechende Assays leicht zur Messung anderer Parameter erweiterbar.
    Description: High-throughput cultivation and screening methods allow a parallel, miniaturized and cost efficient processing of many samples. These methods however, have not been generally established for phototrophic organisms such as microalgae or cyanobacteria. In this work we describe and test high-throughput methods with the model organism Synechocystis sp. PC6803. The required technical automation for these processes was archived with a Tecan Genesis RSP 150 pipetting robot. The cultivation was performed in deepwell microtiter plates within a specially constructed cultivation chamber. The chamber is outfitted with programmable shaking conditions, variable illumination and an adjustable CO2 atmosphere. The growth rates archived within this system are comparable to those achieved with established methods such as bioreactors. The high-throughput screening was achieved with a Tecan Genios Plus plate reader integrated within the pipetting robot. Methods for determination of optical density and amount of chlorophyll were established within the scope of this work. The presented platform can be used for a variety of analyses of phototrophic organisms and is easily expandable with further assays to screen for additional targets.
    Keywords: ddc:660
    Language: German
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  • 5
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    Publication Date: 2021-06-15
    Description: Die medizinischen Blutegel, Hirudo medicinalis und Hirudo verbana, werden wegen ihrer kurativen Wirkung in der Humanmedizin angewandt. Während des Blutsaugens injizieren sie über ihren Speichel eine Vielzahl bioaktiver, derzeit noch unbekannter Moleküle. Eine vollständige Aufklärung aller Inhaltsstoffe mit Wirkmechanismen ist für die Entwicklung von neuen Pharmaka von großem Interesse. Vor diesem Hintergrund wurden verschiedene Organe beider Arten auf ihre Besiedlung durch symbiontische Bakterien untersucht. Dazu wurden die Bakterien zunächst unter geeigneten Bedingungen kultiviert und mittels biochemischer Methoden charakterisiert. Die Identifizierung der Symbionten erfolgte durch Polymerasekettenreaktion (PCR) und Sequenzierung der 16S rDNA. Die biochemischen Tests ergaben, dass die kultivierbaren Bakterien Amylase positiv, Gram negativ und Ornithin Decarboxylase negativ sind. Mit Hilfe von datenbankgestützten Analysen der 16S rDNA-Sequenzen konnte Aeromonas veronii biovar sobria nachgewiesen werden. Hochdurchsatzsequenzierungen der gesamtgenomischen DNA des Bakteriums aus H. medicinalis zeigten deutliche Abweichungen zum Referenzgenom von Aeromonas veronii B565.
    Keywords: ddc:570
    Language: German
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  • 6
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    Publication Date: 2021-06-15
    Description: Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum.
    Keywords: ddc:570
    Language: English
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  • 7
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    Publication Date: 2021-06-15
    Description: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.
    Keywords: ddc:570
    Language: English
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  • 8
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    Publication Date: 2021-06-15
    Description: Tardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress.
    Keywords: ddc:570
    Language: English
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  • 9
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    Publication Date: 2021-06-15
    Description: Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.
    Keywords: ddc:570
    Language: English
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  • 10
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    Publication Date: 2021-06-15
    Description: DNA sequencing continues to evolve quickly even after 〉 30 years. Many new platforms suddenly appeared and former established systems have vanished in almost the same manner. Since establishment of next-generation sequencing devices, this progress gains momentum due to the continually growing demand for higher throughput, lower costs and better quality of data. In consequence of this rapid development, standardized procedures and data formats as well as comprehensive quality management considerations are still scarce. Here, we listed and summarized current standardization efforts and quality management initiatives from companies, organizations and societies in form of published studies and ongoing projects. These comprise on the one hand quality documentation issues like technical notes, accreditation checklists and guidelines for validation of sequencing workflows. On the other hand, general standard proposals and quality metrics are developed and applied to the sequencing workflow steps with the main focus on upstream processes. Finally, certain standard developments for downstream pipeline data handling, processing and storage are discussed in brief. These standardization approaches represent a first basis for continuing work in order to prospectively implement next-generation sequencing in important areas such as clinical diagnostics, where reliable results and fast processing is crucial. Additionally, these efforts will exert a decisive influence on traceability and reproducibility of sequence data.
    Keywords: ddc:570
    Language: English
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