The fat-soluble vitamin A, which is chemically referred to retinol (ROH), is known to be essential for the process of vision, the immune system but also for cell differentiation and proliferation. Recently, ROH itself has been reported to be involved in adipogenesis and a ROH transport protein, the retinol-binding protein 4 (RBP4), in insulin resistance and type 2 diabetes. However, there is still considerable scientific debate about this relation. With the increasing amount of studies investigating the relation of ROH in obesity and type 2 diabetes, basic research is an essential prerequisite for interpreting these results. This thesis enhances the knowledge on this relation by reviewing ROH metabolism on extra- and intracellular level. Aim 1: In the blood stream ROH is transported in a complex with RBP4 and a second protein, transthyretin (TTR), to the target cells. The levels of RBP4 and TTR are influenced by several factors but mainly by liver and kidney function. The reason for that is that liver and the kidneys are the sites of RBP4 synthesis and catabolism, respectively. Interestingly, obesity and type 2 diabetes involve disorders of the liver and the kidneys. Therefore the aim was to investigate factors that influence RBP4 and TTR levels in relation to obesity and type 2 diabetes (Part 1). Aim 2: Once arrived in the target cell ROH is bound to cellular retinol-binding protein type I (CRBP-I) and metabolised: ROH can either be stored as retinylesters or it can be oxidised to retinoic acid (RA). By acting as a transcription factor in the nucleus RA may influence processes such as adipogenesis. Therefore vitamin A has been postulated to be involved in obesity and type 2 diabetes. CRBP-I is known to mediate the storage of ROH in the liver, but the extra-hepatic metabolism and the functions of CRBP-I are not well known. This has been investigated in Part 2 of this work. Material & Methods: RBP4 and TTR levels were investigated by ELISA in serum samples of human subjects with overweight, type 2 diabetes, kidney or liver dysfunction. Molecular alterations of the RBP4 and TTR protein structure were analysed by MALDI-TOF mass spectrometry. The functions of intracellular CRBP-I were investigated in CRBP-I knock-out mice in liver and extra-hepatic tissues by measuring ROH levels as well as the levels of its storage form, the retinylesters, using reverse phase HPLC. The postprandial uptake of ROH into tissues was analysed using labelled ROH. The mRNA levels of enzymes that metabolize ROH were examined by real-time polymerase chain reaction (RCR). Results: The previous published results showing increased RBP4 levels in type 2 diabetic patients could not be confirmed in this work. However, it could be shown that during kidney dysfunction RBP4 levels are increased and that RBP4 and TTR levels are decreased during liver dysfunction. The important new finding of this work is that increased RBP4 levels in type 2 diabetic mice were increased when kidney function was decreased. Thus an increase in RBP4 levels in type 2 diabetes may be the effect of a reduced kidney function which is common in type 2 diabetes. Interestingly, during severe kidney dysfunction the molecular structure of RBP4 and TTR was altered in a specific manner which was not the case during liver diseases and type 2 diabetes. This underlines the important function of the kidneys in RBP4 metabolism. CRBP-I has been confirmed to be responsible for the ROH storage in the liver since CRBP-I knock-out mice had decreased ROH and retinylesters (the storage form of ROH) levels in the liver. Interestingly, in the adipose tissue (the second largest ROH storage tissue in the body) ROH and retinylesters levels were higher in the CRBP-I knock-out compared to the wild-type mice. It could be shown in this work that a different ROH binding protein, cellular retinol-binding protein type III, is upregulated in CRBP-I knock-out mice. Moreover enzymes were identified which mediate very efficiently ROH esterification in the adipose tissue of the knock-out mice. In the pancreas there was a higher postprandial ROH uptake in the CRBP-I knock-out compard to wild-type mice. Even under a vitamin A deficient diet the knock-out animals had ROH and retinylesters levels which were comparable to wild-type animals. These results underline the important role of ROH for insulin secretion in the pancreas. Summing up, there is evidence that RBP4 levels are more determined by kidney function than by type 2 diabetes and that specific molecular modifications occur during kidney dysfunction. The results in adipose tissue and pancreas of CRBP-I knock-out mice support the hypothesis that ROH plays an important role in glucose and lipid metabolism. / Vitamin A gehört zur Gruppe der fettlöslichen Vitamine und wird chemisch als Retinol bezeichnet. Es ist essentiell für den Prozess des Sehvorgangs und der Zelldifferenzierung und kann daher bestimmte Entwicklungsprozesse wie die Bildung des Fettgewebes beeinflussen. Aufgrund seiner Fettlöslichkeit muss Retinol im Blut (= extrazellulär) sowie in der Zelle (= intrazellulär) an sogenannte Transport-Moleküle, die Retinol-bindenden Proteine (RBPs) gebunden werden. Die zwei bekanntesten Vertreter der RBPs sind das Retinol-bindende Protein 4 (RBP4) und das intrazelluläre Retinol-bindende Protein Typ I (CRBP-I). RBP4 transportiert Vitamin A im Blut von der Leber zur Zielzelle und zum Abbauorgan für Vitamin A, der Niere. CRBP-I ist in der Leber für die Speicherung von Vitamin A zuständig. In den letzten Jahren wurden neben der Beteiligung des Retinols an der Bildung des Fettgewebes auch Studien veröffentlicht, in denen ein Zusammenhang zwischen erhöhten RBP4-Werte im Blut und Typ-2-Diabetes gezeigt wurde. Bis heute ist der mögliche Zusammenhang zwischen RBP4, CRBP-I und Übergewicht nicht ausreichend erforscht. Im ersten Teil der Arbeit war daher das Ziel, Einflussfaktoren, die zu Veränderungen der RBP4-Werte im Blut führen können, zu untersuchen. Dazu wurden Blutproben von Personen mit Übergewicht und/oder Typ-2-Diabetes und Patienten mit Nierenfunktionsstörungen oder mit Leberfunktionsstörungen analysiert. Es konnte gezeigt werden, dass bereits geringe Nierenfunktionsstörungen zu erhöhten RBP4-Konzentrationen im Blut führten. Bei Typ-2-Diabetikern, die sehr oft an Nierenfunktionsstörungen leiden, war eine Erhöhung der RBP4-Konzentration mit einer Abnahme der Nierenfunktion verbunden. Somit lässt sich zusammenfassen, dass nicht Typ-2-Diabetes sondern vielmehr die dabei auftretenden Nierenfunktionsstörungen zu einer Erhöhung der RBP4-Werte führen. Bei Lebererkrankten konnte ein Absinken der RBP4-Werte nachgewiesen werden, was der verminderten Bildung von RBP4 in der Leber bei diesen Patienten zuzuschreiben ist. Im zweiten Teil sollte der Frage nachgegangen werden, wie Retinol intrazellulär verstoffwechselt wird. Dabei lag der Fokus auf der Erforschung der bisher nicht bekannten Funktionen von CRBP-I im Fettgewebe und der Bauchspeicheldrüse. Zur Untersuchung der Funktionen von CRBP-I wurden Mäuse gezüchtet, bei denen das Gen für CRBP-I gelöscht wurde. Da CRBP-I für die Speicherung von Vitamin A in der Leber verantwortlich ist, zeigen diese Mäuse sehr geringe Vitamin-A-Speicher in der Leber. Das gleiche zeigte sich für die Bauchspeicheldrüse, die für die Sekretion von Insulin Vitamin A benötigt: In den Mäusen ohne CRBP-I waren die Retinol-Werte drastisch gesunken. Interessanterweise zeigte sich im Fettgewebe ein gegenteiliges Bild: Die Konzentrationen an Retinol und dessen Speicher waren in den Mäusen ohne CRBP-I höher im Vergleich zu den normalen Mäusen. Mit bestimmten Nachweismethoden konnte herausgefunden werden, dass Retinol im Fettgewebe an ein anderes RBP, das CRBP-III, gebunden wird und dadurch effektiver gespeichert werden kann als durch CRBP-I.

Antifungal proteins were isolated from the crude bacterial supernatant using ammonium sulfate salt precipitation followed by passage over DEAE -cellulose and Biogel P100 columns. The purified protein had an apparent molecular mass of 14 kDa. Its antifungal activity was retained even at 100°C, for 60 min. The results of protein identification using MALDI -TOF/TOF mass spectrometer suggested that the purified protein is indeed a chitin binding protein that has 206 acid amine containing chitin -bind -3 region with a relative molecular mass of 22230 Da. / Protein có hoạt tính kháng nấm được tinh sạch từ dịch ngoại bào chủng vi khuẩn Bacillus subtilis sau khi qua ba bước tinh sạch: tủa muối ammonium sulphate 30-70%, qua cột sắc ký trao đổi ion DEAE – cellulose và cột săc ký lọc gel Biogel P100. Protein tinh sạch có khối lượng phân tử đạt 22 kDa trên điện di SDS-PAGE. Hoạt tính kháng nấm của protein tinh sạch vẫn còn duy trì khi ủ ở 100°C trong 60 phút. Kết quả nhận dạng bằng khối phổ MALDI -TOF/TOF đã chỉ ra rằng protein bền nhiệt này là chitin binding protein được mã hóa bởi 206 acid amin cùng với khối lượng phân tử là 22230 Da. trị an toàn đối với Al và Atrazie trong môi trường nước tự nhiên về khía cạnh bảo vệ sức khỏe sinh thái.

Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.

No description available.

Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.