660.6 Biotechnologie
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Influenza A viruses are responsible for the outbreak of epidemics as well as pandemics worldwide. The surface protein neuraminidase of this virus is responsible, among other things, for the release of virions from the cell and is thus of interest in pharmacological research. The aim of this work is to gain knowledge about evolutionary changes in sequences of influenza A neuraminidase through different methods. First, EVcouplings is used with the goal of identifying evolutionary couplings within the protein sequences, but this analysis was unsuccessful. This is probably due to the great sequence length of neuraminidase. Second, the natural vector method will be used for sequence embedding purposes, in hopes to visualize sequential progression of the virus protein over time. Last, interpretable machine learning methods will be applied to examine if the data is classifiable by the different years and to gain information if the extracted information conform to the results from the EVcouplings analysis. Additionally to using the class label year, other labels such as groups or subtypes are used in classification with varying results. For balanced classes the machine learning models performed adequately, but this was not the case for imbalanced data. Groups and subtypes can be classified with a high accuracy, which was not the case for the years, continents or hosts. To identify the minimal number of features necessary for linear separation of neuraminidase group 1 subtypes, a logistic regression was performed at last, resulting in the identification of 15 combinations of nine amino acid frequencies. Since the sequence embedding as well as the machine learning methods did not show neuraminidase evolution over time, further research is necessary, for example with focus on one subtype with balanced data.
In dieser Arbeit geht es um die Grundlage einer grünen Alternative zur chemischen Aufbereitung gelöster Metallionen. Im Fokus stehen dabei zwei wirtschaftlich wichtige Elemente: Cobalt und Nickel. In einem vorangegangenen Praktikum wurden dafür bereits Peptide mittels Isothermer Titrationskalorimetrie (ITC) untersucht, welche in der Lage waren, diese beiden Metallionen zu binden. Diese Peptide wurden mittels Phagen Display selektiert. Da die zwei Peptide die gewünschten Affinitäten aufwiesen, wurden mit diesen Fusionsproteine konstruiert, wobei die Peptide als Insert, neben weiteren Funktionalitäten, hinzugefügt wurden. Somit entstanden Proteine, welche als Fusionsproteine (FP) bezeichnet werden. Ein Zusätzliches FP mit sieben Histidinen wurde als Positivkontrolle mitgeführt, da Histidin bekanntermaßen beide Elemente binden kann
Die vorliegende Bachelorarbeit beeinhaltet Untersuchungen zur Gewinnung leistungsverbesserter Mutanten des Produktionsstammes Penicillium verruculosum M28-9. Dazu wurde die chemische Mutation mittels N-Methyl-N-Nitro-Nitroso-Guanidin angewendet. Die Selektion von Mutanten mit partiell bzw. vollständig ausgeschalteter C-Katabolitrepression erfolgte mittels eines Submers-Screening.
Das nach der Mutation folgende Submers-Screening wurde unter Verwendung der Antimetabolite 2-Deoxy-D-Glukose sowie 1- oder 2-Thioglycerol in unterschiedlichen Konzentrationen durchgeführt. Die mutierten Konidien wurden kultiviert und konnten anschließend mittels Klonselektionierung auf Agarmedien mit amorpher Cellulose und dem Einsatz der wachstumsbegrenzenden Substanz Bengalrosa selektiert werden.
Im Rahmen der Untersuchungen konnten potentielle Mutanten auf einem Medium mit amorpher Cellulose und 25 g/l Glukose als Repressor erhalten werden.
The epithelial membrane proteins (EMP1-3), which belong to the family of peripheral myelin proteins 22-kDa (PMP22), are involved in epithelial differentiation. EMP2 was found to be a downstream target gene of the tumor suppressor gene HOPX, a homeobox-containing gene. Additionally, a dysregulation of EMP2 has been observed in various cancers, but the function of EMP2 in human lung cancer has not yet been clarified.
In this study, a real-time RT-PCR, Western blot and cytoblock analysis were performed to analyze the expression of EMP2. Gain-of-function was achieved by stable transfection with an EMP2 expression vector and loss-of-function by siRNA knockdown. Stable transfection led to overexpression of EMP2 at both mRNA and protein levels in the transfected cell lines H1299 and H2170.
Functional assays including proliferation, colony formation, migration and invasion assays as well as cell cycle analyzes were performed after stable transfection and it was found that the ectopic EMP2 expression resulted in a reduced cell proliferation, migration and invasion as well as a G1 cell cycle arrest. After the EMP2 gene was silenced by the siRNA knockdown, inhibition of the cell invasive property was observed. These phenomena were accompanied by reduced AKT, mTor and p38 activities.
Taken together, the data suggest that the epithelial membrane protein 2 (EMP2) is a tumor suppressor and exerts its tumor suppressive function by inhibiting AKT and MAPK signaling pathways in human lung cancer cells.
The aim of this bachelor thesis was to establish extracytoplasmic function (ECF) σ factors as synthetic genetic regulators for biotechnological and synthetic biology applications in the new emerging model organism Vibrio natriegens. Therefore, synthetic genetic circuits were engineered on plasmids as test set-up for the investigated ECFs and their target promoters. The resulting plasmid library consisted of the reporter plasmids with the target promoter, fused to a lux cassette, a set of high-copy ECF plasmids and a backup set of lower-copy ECF plasmids. First, the high-copy plasmids were transformed in V. natriegens to test them for their functionality upon different inducer levels, which yielded good inducibility for few, but showed too high ECF-expression in most strains. For this reason, the set of lower copy plasmids was used for combinatorial co-transformation, to investigate the ECFs for their cross-talk to unspecific ECF target promoters. The switching to the lower-copy plasmid-set seemed to be partly helpful, while still much room for fine-tuning of the circuits remains. The knowledge gained can be used to achieve higher success rates when engineering synthetic circuits for various applications in V. natriegens, by using the ECFs here recommended as suitable synthetic genetic regulators.