@masterthesis{Giebel, type = {Bachelor Thesis}, author = {Giebel, Alice}, title = {Cloning Of Human Secretory Gelsolin In Pichia Pastoris}, url = {http://nbn-resolving.de/urn:nbn:de:hbz:1383-opus4-6585}, school = {Hochschule Rhein-Waal}, pages = {74}, abstract = {The production of recombinant proteins plays an important part in biotechnology for over 30 years. Their applications range from pharmaceutical practice up to industrial (non-medical) purposes and, therefore, show great importance in today´s life. Most recombinant proteins are being produced by genetic modification of microorganisms to either generate those intracellularly or to secrete the desired protein into their surroundings. Dependent of the expression mechanism, the production can be influenced and improved to achieve high yields and protein purity. One aim of this study was to determine and optimise the most favorable cloning strategy for the expression and secretion of the plasma form of the actin binding protein gelsolin from Pichia pastoris. Gelsolin has two isoforms of which one of them gained particular attention due to its application in research projects concerning the current COVID-19 pandemic. For this project, a variety of oligonucleotide primers were designed to amplify the whole sequences and various parts of both gelsolin isoforms during PCR approaches. The accuracy and efficacy of primers is determined by several factors such as their length and exact structure. Also, many factors depending on the sequence of interest are influencing the outcome of the PCR. Therefore, the second aim of this study was to design and validate primer pairs with most preferable conditions to amplify the sequence for the expression of gelsolin and optimise the PCR conditions for this purpose.}, language = {en} } @masterthesis{Babenko, type = {Bachelor Thesis}, author = {Babenko, Anastasiia}, title = {Development of an ESI-MS/MS-based method for the quantification of acylcarnitines in murine tissue}, school = {Hochschule Rhein-Waal}, abstract = {Acylcarnitines serve as transporters of activated fatty acids into mitochondria for β-oxidation - the major source of the cell's energy and are considered as indicative markers for various metabolic disorders. The investigation of acylcarnitines in cell and tissue samples is a particularly complicated task due to their low abundance and high heterogeneity. This requires applications of sophisticated lipid separation methods. The purpose of this work was the development of an ESI-MS/MS method for the quantification of acylcarnitines with high specificity and reliability for the identification of low abundant acylcarnitine species in complex samples. General aspects of acylcarnitine analysis, including several types of solid-phase extraction, and derivatization with liquid extraction were investigated using commercially available acylcarnitine standards. The established experimental workflows, including an optimized cation-exchange extraction and derivatization with liquid extraction, were successfully applied to quantify acylcarnitine species in samples of biological origin. Consequently, murine brain tissue samples were quantitatively analyzed using two extraction procedures. The method developed in the study can be used for the qualitative and quantitative analysis of acylcarnitines in different biological samples.}, language = {en} }