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According to the International Diabetes Federation (IDF), there were 371 million people in the age from 20 to 79 years worldwide affected by diabetes in 2012. This means diabetes has become a global epidemic disease and, therefore, the importace of insulin research still grows. Insulin is a protein hormone that plays a key role in regulating blood glucose level which has a widespread impact on whole metabolism. Insulin acts through binding of its monomeric form to the insulin receptor. It is clear that insulin monomer has to undergo structural changes upon binding to the insulin receptor as the residues which are crucial for the interaction are burried within the native form. According to studies of highly active hormone analogs and the new information about the insulin-insulin receptor complex, there is a strong evidence that the C-terminal part of the B-chain is a dynamic element in insulin activation and receptor binding. Probably, there is also a great importance of the B-chain N- terminus and the transition between T and R conformations of insulin. However, the exact significance of the T and R states of insulin still remains unclear. In this work, several new insulin analogs AibB3-insulin, AibB5-insulin, AibB8- insulin, N-MeAlaB8-insulin and D-ProB8-insulin were prepared for the purpose of...

The aim of this thesis is to understand RNA-RNA interactions steering cellular functions, as in the case of this thesis the structure of mRNA(Sirt1) in complex with microRNA-34a (miR-34a). MiR-34a regulates the cancer protein p53 via Sirt1 modulation. This work will be the basis for future drug design and the understanding of misguided regulation in cancer. The miR-34a binds to the mRNA(Sirt1) 3’ untranslated region (3’-UTR) and will either inhibit the translation of the protein Sirtuin 1 by capturing its mRNA or by degrading it. p53, a key activator of miR-34a, prevents cancer development by inducing programmed cell death (apoptosis) on cells with DNA damage. In contrast, the protein Sirtuin 1 (Sirt1) has been shown to help cells with DNA damage to survive by down regulating the activity of protein p53 and will therefore increase the risk of cancer development. Studying the interaction between the mRNA(Sirt1) and miR-34a can present valuable information on the structure of the complex as well as the mode miR-34a uses to inhibit translation of mRNA(Sirt1) leading to down regulation of protein Sirtuin 1 and therefore prevent cancer development. For the elucidation of this question different biochemical and biophysical methods were applied, such as in vitro transcription, gel electrophoresis, RNA purification with gel, crush & soak and Cicular Dichroism (CD) melting studies. For this thesis work, the target sequence in mRNA(Sirt1) was optimized and purified so melting studies could be carried out. For future structural characterization using Nuclear Magnetic Resonance (NMR) studies with the miR-34a also produced in the lab. The mRNA(Sirt1) target sequence was produced and purified with the final yield of 0.02%. The results show that the sequence is highly ATP dependent and suggest the ratio between the nucleotides ATP/CTP to be 1:2. Low yield of purified mRNA(Sirt1) was received and still contained some impurities, which imply that another method than crush & soak should be used when purifying. The results, indicate that High-Preformance Liquid Chromatography (HPLC) might be a better solution for the pufication process. The melting profiles done on mRNA(Sirt1) show that the secondary structures decrease with an increase in temperature. Accroding to the results, the mRNA(Sirt1) sequence is folded in room temperature, though not very stable. The wavelength which provided the best resolution was at 268 nm and the melting point of mRNA(Sirt1) was determined to 44 °C. This thesis also contains an educational part, where an educational material was provided and testing was conducted for the subject Chemistry 2 for students age 18 and the material was evaluated with qualitative methods together with pedagogical methods. The study showed that the student can develope the different abilities stated in the curriculum with the material created. The results also showed that the students preferably choose cultural arguments when dicussing socio scientific question, rather than economical, democratic or utility arguments. / Syftet med studien är att förstå RNA-RNAinteraktioner som styr cellulära funktioner, i detta fall mRNA(Sirt1) i komplex med microRNA-34a (miR-34a). MiR-34a reglerar cancerproteinet p53 via modulation av Sirt1. Detta arbete kommer lägga grund för framtida läkemedelsdesign vid reglering av cancer. MiR-34a binder till den 3’ otranslerade regionen i mRNA(Sirt1) och hämmar antingen translationen av protein Sirtuin 1 (Sirt1) genom att fånga dess mRNA eller genom att försämra det. p53 förhindrar utvecklingen av cancer genom att framkalla programmerad cell död (apoptosis) av celler med skadat DNA. Det har visats att proteinet Sirtuin 1 hjälper celler med skadat DNA att överleva, genom att sänka aktiviteten av p53. På så vis ökar risken för utveckling av cancer. Genom att studera interaktionen mellan mRNA(Sirt1) och miR-34a kan värdefull information kring komplexets struktur fås. Samt hur miR-34a hämmar translationen av mRNA(Sirt1), vilket leder till minskad aktivitet av protein Sirt1. För att klarlägga denna fråga har olika biokemiska och biofysiska metoder använts, såsom in vitro transkription, gelelektrofores, RNA rening med gel och Circular Dichroism (CD). För detta arbete har målsekvensen i mRNA(Sirt1) optimerats och renats så CD smältstudier med kunde genomföras. Resultatet visar att mRNA(Sirt1) sekvensen renats med ett utbyte på 0.02 %. Sekvensen är beroende av ATP och förhållandet mellan ATP/CTP nukleotider bör vara 1:2. Resutatet visar på ett lågt utbyte som visar på att High-Performance Liquid Chromatography (HPLC) kan vara en bättre metod än Crush & soak för reningen av mRNA(Sirt1). Ur de smältprofiler som gjorts visade det sig att de sekundära strukturerna av mRNA(Sirt1) minskade med ökande temperatur. I enlighet med resultaten visar det att mRNA(Sirt1) är veckat i rumstemperatur men är inte stabil. Den bästa upplösningen erhölls vid 268 nm och mRNA(Sirt1) har en smältpunkt runt 44 °C. Detta arbete innehåller även ett utbildningskapitel, där ett utbildningsmaterial har skapats och testats på 18-åriga kemi 2 studenter i åldern 18 år. Materialet har utvärderats med hjälp av kvalitativa metoder tillsammans med pedagogiska metoder. Studien visade att de flesta förmågorna för kemi 2 kan utvecklas med hjälp av denna typ samhällsfrågor i det naturvetenskapliga klassrummet (SNI-fall) förutom förmågan att planera och genomföra experiment. Det argument som eleverna helst väljer att använda då de diskuterar det skapade SNI-fallet är Kulturargument och det minst använda är Demikratiargument.

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Rapid development of bacterial resistance and multiresitance to conventional antibiotics has resulted in an intensive search for alternative antimicrobial agents. Antimicrobial peptides (AMPs) belong to promising anti-infective candidates since they do not development bacterial resistance. They kill microbes by disturbing or permeabilizing the cytoplasmic membrane, or may target putative key intracellular compartments. Their advantages include fast action and selectivity between prokaryotic and eukaryotic cells. We have isolated several novel AMPs from the venom of wild bees: halictines (HAL-1 and HAL-2) from Halictus sexcinctus, lasiocepsin (Las) from Lasioglossum laticeps and macropin (MAC-1) from Macropis fulvipes. They are active against Gram-positive and Gram- negative bacteria and against yeast Candida albicans. While halictines and macropin have moderate hemolytic activity, Las shows no hemolytic activity. A novel AMP was isolated also from the mucus of Xiphydria camelus. This AMP belongs to the category of insect defensins. It contains 55 amino acid residues, three disulphide bridges and its C-terminus is amidated. CD and NMR studies of HAL-1, HAL-2 and MAC-1 revealed propensity to form amphipathic α-helical structure in the presence of sodium dodecyl sulfate or trifluoroethanol. For the...

The group 2 late embryogenesis abundant (LEA) proteins, also known as the dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. In this work, we study the potential roles that dehydrins may have in stabilizing membranes and actin microfilaments during cold stress. We have cloned and expressed in E. coli two dehydrins from Thellungiella salsuginea, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). These proteins were expressed as SUMO-fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and for structural analysis by CD and Fourier transform infrared (FTIR) spectroscopy. We show using transmission-FTIR spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. The increase in secondary structure by membrane association is further facilitated by the presence of Zn2+. Lipid composition and temperature have synergistic effects on the secondary structure. Our single molecule force spectroscopy studies also suggest tertiary folding of both TsDHN-1 and TsDHN-2 induced by association with lipids. From Langmuir-Blodgett monolayer compression studies, and from topographic studies using atomic force microscopy at variable temperature, we conclude that TsDHN-1 stabilizes the membrane at lower temperatures. Finally, we show that the conformations of TsDHN-1 and TsDHN-2 are affected by pH, interactions with cations and membranes, and phosphorylation. Actin assembly by these dehydrins was assessed by sedimentation assays, and viewed by transmission electron and atomic force microscopy. Phosphorylation enabled both dehydrins to polymerize actin filaments, a phenomenon that may occur in the cytosols of plant cells undergoing environmental stress. These results support the hypothesis that dehydrins stabilize plant organellar membranes and/or the cytoskeleton in conditions of stress, and further that phosphorylation may be an important feature of this stabilization. / NSERC