Introduction: Cancer is a group of about 200 different types of diseases. Cancer can appear in different cells and tissues in the body when abnormal cells grow in an uncontrolled manner and invade other tissues. A multistep process that occurs over a long period eventually leads to the onset of cancer. Kinases are enzymes that catalyze phosphorylation, that is, the reaction in which a phosphate group from ATP is transferred to another molecule. Kinase phosphorylation is an important mechanism for modifying proteins and thus regulating various cellular functions. Overactivity of kinases can lead to loss of control of signal transducer cascades controlled by kinases and it produces harmful effects such as cardiovascular disease, diabetes and cancer. Chronic myeloid leukemia (CML) is a rare blood cancer in which an abnormal BCR-ABL fusion protein occurs in neoplastic cells. This causes leukocytes to be produced abnormally and damages the bone marrow and blood. Dasatinib is a tyrosine kinase inhibitor indicated for the treatment of CML. Dasatinib inhibits the activity of the BCR-ABL kinase, but since many kinases are similar, it also binds to other kinases. Some of the kinases that dasatinib inhibits and that we analyzed in this study are ABL1, PTK6, MYT1 and STK10. Purpose: The purpose of this study was to explain with the help of the structures of the proteins ABL1, PTK6, MYT1 and STK10 why dasatinib binds well to some proteins but less well to others. Methods: Using the structures of the proteins, an analysis of interactions between dasatinib and the selected proteins has been performed. We then built a model for each protein with only the amino acids that binds to the drug and finally used these structures to estimate the binding energy ΔGb with the PM7 theoretical method. The PM7 method was used to calculate the energy of molecules. The results correspond to the enthalpy of formation ΔfH. The Gibbs energy is estimated by including the solvent, which also accounts for entropy changes, most of which are mediated by the solvent. The equation used to calculate Gibbs energy for protein-drug binding was: ΔGb = ΔfG(complex) - [ΔfG(drug) + ΔfG(protein)]. Results: The results show that there is a link between the number of dasatinib-protein interactions and Kd. Some proteins had several different structures in the same crystal. In such cases there was an extensive variation in the number of interactions between the different structures and dasatinib. The connection between the number of interactions and Kd was not perfect. Using the number of interactions we were able to distinguish between the proteins that bind best to dasatinib, which were ABL1 and PTK6, and the proteins that bind worst which were MYT1 and STK10. We could however not explain which protein binds best based on the number of interactions. The theoretical calculation of ΔGb follows the same trend (the more interactions there are in the structures, the lower is ΔGb). Discussion and conclusion: A relatively quick and easy calculation using a built model (dasatinib with protein binding interaction) could be used to distinguish between the best and worst binders. When it comes to the difference between ABL1 and PTK6, our analysis failed. A large difference between the calculated ΔGb values ​​for different structures of the same protein makes it difficult to choose the right structure for an analysis. A factor that could explain the discrepancy in the analysis is that the structure of the protein changes when it binds to the drug and such a change requires an investment of Gibbs energy. In general, experimental binding energies were higher than the calculated ones. For example, experimental ΔGb for MYT1 was -9 kcal/mol but the calculated ΔGb was -33.2 kcal/mol. This may be because the calculation with PM7 is too simplified.

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The nucleotide-binding oligomerization domain–like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a group of multi-protein complex that mediates immune responses through the production of biologically active IL-1β and IL-18. Dysregulation of NLRP3 inflammasome has been linked to various diseases associated with infection, inflammation, and cancer. However, the molecular mechanisms of ligand binding that result in the formation of the NLRP3 inflammasome are not fully understood. Potential therapeutics for NLRP3 inflammasomes related diseases are relatively nonspecific, have low efficacy, and may cause unexpected side effects. Therefore, this study aimed to understand if BABAM1 can serve as a potential target for treating NLRP3 inflammasome related diseases. This was done by attempting to knock out BABAM1 gene in THP-1 monocyte-like cell line using the CRISPR-Cas9 gene-editing technology. The cDNA prepared from THP-1 cells in which an attempt was made to knock out BABAM1 gene were amplified using qPCR. The result showed no biologically relevant difference in BABAM1 gene expression level between the target and control THP-1 cells. Additionally, this study found that the expression of the reference gene, ACTB, was not stable as the cycle threshold values for untransfected cells were lower when compared to cells transfected the plasmid DNA. In conclusion, successful attempts were made in this study to understand the role of BABAM1 in regulating the activation of NLRP3 inflammasome and that further research are needed if BABAM1 is to be considered as a potential target for treating NLRP3 inflammasome related diseases.

This thesis presents two techniques in the field of biocatalysis: An enzyme immobilisation method based on the His6-tag for attachment on modified silica oxide beads, and it’s employment in aqueous and organic medium for synthesis applications. The method functions as a one step extraction and immobilisation protocol. An equilibrium displacement system which enables complete conversion in reactions with ω-transaminases where isopropylamine is the donor, a route for synthesis of pharmaceutically interesting enantiopure amines. Biocatalysis is predicted to be a paramount technology for an environmentally sustainable chemical industry, to which every newly developed method represents a small but important step. The work done here is aimed to be a part of this development. / I denna avhandling presenteras två tekniker inom ämnet biokatalys: En metod för immobilisering av His6-enzym på modifierad kiseloxid, och användning av detta konstrukt för kemiska synteser i vatten och organiska lösningsmedel. Detta system fungerar även som en snabb extraherings- och immobiliseringsmetod. Ett jämviksförskjutningssystem som möjliggör fullständig omsätt-ning i reaktioner med ω-transaminaser där isopropylamin är amino-donator, en syntesväg för tillverkning av farmakologiskt intressanta kirala aminer. Biokatalys förutspås att bli en ovärderlig teknologi i en miljömässigt hållbar kemisk industri, i vilken varje ny metod är en liten men dock viktig del. Detta arbete är menat som en del i denna utveckling. / QC 20100519

Cellulose is the most abundant polymer on earth. It is one of the main components in lignocellulosic biomass, which has great potential as a renewable energy source. To utilize the biomass, for instance in biofuel production, cellulose needs to be degraded. In nature there are microorganisms that are specialized on such degradation, and they produce interesting cellulose hydrolysing enzymes. Understanding the function of these enzymes can hence be one step towards a more sustainable future.  The aim of this project was to find out if the enzyme GtCel45A from Gloeophyllum trabeum could hydrolyse soluble oligosaccharides and produce mono- or disaccharides as products. The study was executed by cultivating Aspergillus nidulans A773 recombinantly expressing GtCel45A followed by a purification process consisting of anion exchange chromatography and size exclusion chromatography. From 1.4 liters of culture, grown for 8 days at 30°C, 9.9 mg of purified GtCel45A was obtained. Activity measurements using p-hydroxybenzoic acid hydrazide (PHBAH) reagent for reducing sugar showed that the enzyme is active against and does hydrolyse barley beta-glucan. However, no hydrolysis of cellohexaose, cellotetraose, cellotriose or cellobiose could be detected, even after 223 minutes of incubation with GtCel45A as shown by carbohydrate analysis with high performance anion exchange chromatography with pulsed amperiometric detection (HPAE-PAD). In addition, a number of crystallization trials were performed, which resulted in formation of crystals that could subsequently be used to solve the structure of the protein.

Lipases are one of the most important biocatalysts for biotechnological applications. They have high importance since lipases have a catalytic activity that is related to their hydrolysis and synthesis reactions to high regioselectivity and enantioselectivity. They also have an activity over a wide range of temperature, pH, and diverse substrates. The aim of this master thesis project was to apply computational calculations to understand the reaction mechanism of the hydrolysis of p-nitrophenyl butyrate performed by the cold-adapted lipase, Bacillus pumilus Lipase A (BplA). Cold-adapted lipases are enzymes that have evolved to perform catalysis in a colder environment. The methods used for performing the computational calculations in this thesis project can be divided into three sections. The structure preparation, molecular dynamic (MD) simulations using NAMD, and quantum mechanics combined with molecular mechanics (QM/MM) using ORCA. Through eight substeps with MD and QM/MM implementations potential energy surfaces (PES), minimum energy path (MEP) and frequency calculations for the enzymatic reaction were provided. The rate-limiting step is the formation of the tetrahedral intermediate, the nucleophilic addition during the deacylation. The largest free energy barrier provided from the results had an activation free energy of 20.3 kcal/mol. The barriers of the deacylation were larger than the one for the acylation process. By understanding the reaction mechanism, one can understand how cold-adapted enzymes could catalyze the reaction to create lower energy barriers at low temperatures.

The ErbB family of receptors are involved in signalling relating to cell proliferation and differentiation through activation of pathways such as PI3K and MAPK. Their overexpression is often found in different cancer types and therefore, their expression is under tight regulation. The ErbB family includes, EGFR, ErbB2, ErbB3, and ErbB4 where all but ErbB3 has a kinase domain, making ErbB3 a pseudokinase. Upon activation of the receptors, they are endocytosed through the formation of a clathrin-coated pit and are degraded in the lysosome. Interestingly, researchers have found that newly synthesised ErbB3 can also be degraded in the proteasome by protein Nrdp1. Suggesting that ErbB3 might work in a ligand-independent manner and needs additional regulatory mechanisms. ACK1 is a non-receptor tyrosine kinase that has a reported effect on EGFR by promoting receptor degradation in the autophagosome. However, their role in EGFR regulation is still debated. Therefore, this information alludes to the fact that ACK1 might influence other ErbB family members as well.   This report aims to investigate whether ACK1 influences ErbB3 levels. Through RNAi mediated knockdown of ACK1 in MCF10A cells, a novel role of ACK1 acting as a regulator of ErbB3 is hinted at. Surprisingly, these results also show that ACK1 seems to act specifically on ErbB3 and not on its family members, EGFR and ErbB2. Moreover, this report shows that ErbB3 expression is linked to cell density.

Bakgrund: Platelet derived growth factor receptorn (PDGF receptorn) är en receptor i kroppen som tillhör typ III av Recetor tyrosine kinas (RTK) familjen. PDGF-β receptorn är en typ av dessa receptorer som enligt en studie klyvs i två delar efter ligandbindning med PDGF-BB ligand: till en extracellulär del och en intracellulär del. Hypotesen är att den extracellulära delen går till lysosomen medan den intracellulära delen går till proteasomen efter klyvningen. Syfte: Att undersöka rollen för proteasomen i ligandinducerad fragmentation av PDGF-β receptorn och se om det finns ko-lokalisation mellan receptorns extracellulära del och proteasomen. Dessutom ska det undersökas hur eventuell ko-lokalisering av PDGF receptorns extracellulära fragment med proteasomet påverkas när proteasomerna i cellerna inhiberas. Metod: Bj-hTERT celler stimulerades med PDGF-BB ligand under fyra olika stimuleringstidspunkter (0, 30, 60 och 90 min) och med hjälp av immunofluorescens undersöktes det om det fanns ko-lokalisering mellan proteasomen och PDGF-β receptorns extracellulära del. I ett annat experiment inhiberades även proteasomens aktivitet med inhibitoren MG132 och ko-lokalisationen mättes och jämfördes med kontrollceller behandlat med DMSO för de fyra stimuleringstidspunkterna. Resultat: För det första ko-lokalisationsexperimentet visades ingen större ko-lokalisering mellan proteasomen och receptorns extracellulära del för alla fyra tidspunkter. För det andra ko-lokalisationsexperimentet visades ingen skillnad i ko-lokalisationen mellan proteasomen och receptorns extracellulära fragment för cellerna vars proteasomaktivitet inhiberats och kontrollcellerna. Det visade sig däremot att proteasomen spelade roll för internaliseringen av receptorns extracellulära del in i cellen.

To maintain a healthy growth rate of living cells, protein synthesis must take place accurately and efficiently. Translation, the core step decoding the genetic information, governs both fidelity and efficiency. For keeping a low error level, translation mainly relies on the codon-anticodon basepairing of mRNA and tRNA, which is known as ‘initial selection’. Apart from that, nature also evolved proofreading to reduce errors in protein synthesis. The current study focuses on initial selection of the correct tRNA.  EF-Tu, Elongation Factor Thermo-unstable, is an essential housekeeping GTPase factor responsible for transferring aa-tRNA to the ribosome. EF-Tu contributes to accuracy of initial selection although the exact mechanism is unknown. Here we have characterized and compared two sequence reconstructed ancestral EF-Tus, which are 1.3 and 3.3 billion years old respectively. Using dipeptide formation assay, we obtained the Michaelis-Menten parameters for Leu-tRNALeu on a near-cognate codon. Comparing the specificity parameter kcat/KM for the near-cognate vs. cognate we determine the accuracy of tRNA selection. My results show lower efficiency but higher accuracy using ancestral EF-Tus supporting the theory of trade-off between efficiency and accuracy. We envisage that during evolution EF-Tu sacrifices some accuracy to achieve higher efficiency as seen with modern EF-Tus.  My results demonstrate that EF-Tu can coordinate both the fidelity and the efficiency of translation.