Hair is important for thermoregulation, physical protection, sensory activity, seasonal camouflage and social interactions. Hair is produced in hair follicles (HFs), complex mini-organs in the skin devoted to this task. HFs are formed during embryonic development (morphogenesis) and new hair is continuously generated throughout life since the postnatal HF goes through cycles of regression (catagen), quiescence (telogen) and growth (anagen). The transcriptional regulation of this process is not well understood. The LIM-homeodomain transcription factor Lhx2 has previously been shown to be critically involved in epithelial-mesenchymal interactions during development of various organs and a potent regulator of stem cell function. We therefore elucidated the expression pattern and function of Lhx2 during hair formation. Lhx2 is expressed during both morphogenesis and anagen in cells scattered in the outer root sheath and in a subpopulation of the matrix cells in the proximal part of the hair bulb. Matrix cells are proliferating progenitor cells that differentiate into the components of the HF including the hair shaft. Expression is turned off during telogen, however Lhx2 expression reappears in the secondary hair germ immediately prior to initiation of the anagen stage. In contrast to previously published results Lhx2 appears to be expressed by progenitor cells distinct from those in the stem cell niche in the bulge region. The developmental-, stage- and cell-specific expression pattern of Lhx2 suggests that Lhx2 is involved in the generation and regeneration of hair. To test our hypothesis we used different genetically modified mouse strains. First we studied the effect of over-expression of Lhx2 in the HFs using a mouse model where transgenic Lhx2 expression could be induced in dorsal skin. Using this model we could show that Lhx2 expression is sufficient to induce anagen. To analyze the consequence of lack-of-function of Lhx2 we developed a mouse model where it is possible to conditionally inactivate Lhx2 and a mouse strain harbouring a hypomorphic allele of Lhx2. Mice where Lhx2 was conditionally inactivated in postnatal HFs were unable to regrow hair on a shaved area whereas all controls did regrow their hair. The mutant HFs initiated anagen but were unable to produce normal hair shafts. Thus Lhx2 is required for postnatal hair formation. We used the mouse strain carrying a hypomorphic allele of Lhx2 to study the role of Lhx2 during HF morphogenesis. Embryos homozygous for the hypomorphic allele form significantly less HFs compared to control embryos, and the HFs that do form in the mutant embryos appear to be developmentally arrested. These results suggest that Lhx2 is also important during HF morphogenesis. Thus, Lhx2 is an essential positive regulator of hair generation and regeneration.

Large-scale culturing of algae with the aim of production of economically valuable substances is a growing industrial sector. A common problem of such industrial algae cultures is that they are prone to suffer from fungal infections causing growth inhibition of the algae and reduced production of the target substances. Having sensitive and target specific methods for the early detection of these infections are of great economic importance since in later stages infections may be very hard to treat. There are several potential methods that can be used for infection detection such as visual detection through microscopy or DNA amplification based methods like PCR. This report presents a novel protocol for the early detection of the parasitic fungi Paraphysoderma sedebokerense infecting the green micro algae Haematococcus pluvialis. DNA from infected frozen algae cultures collected from the Gustavsberg production plant of Astareal AB was extracted using ZR Fungal /Bacterial DNA MiniPrep kit. Conventional PCR and SYBR Green quantitative PCR were further compared to each other and optimized to be more sensitive in detecting parasitic DNA. The annealing temperature and primer concentration were optimized. In addition, two different SYBR Green PCR kits and the use of fresh versus frozen algae cultures were compared. The quantitative PCR method was able to detect as low as 300 DNA copies accurately but occasionally was capable to give a signal corresponding to as little as 4 DNA copies in a reaction. Meanwhile the conventional PCR method could accurately detect as low as 600 DNA copies and occasionally 60 DNA copies. This report shows the possibility of improving the detection of P. sedebokerense infection in industrial H. pluvialis cultures by PCR technique, emphasizes the differences between conventional PCR and quantitative PCR and presents a working protocol for the quantitative PCR based monitoring of P. sedebokerense infections.

The natural occurrence of the non-mevalonate (also called MEP after the compound methyl-erythriol phosphate) pathway in the model cyanobacterium Synechocystis sp. PCC 6803 allows for biosynthesis of various high-value terpenoid compounds. An important co-factor of this pathway is thiamine pyrophosphate (TPP), coenzyme to the 1- deoxy-D-xylulose-5-phosphate synthase (DXS) reaction in the initial step of the MEP pathway. Concurrently, TPP biosynthesis derives partially from 1-deoxy-D-xylulose phosphate, the product of DXS. This makes TPP a potentially significant measure of MEP pathway activity, and thus terpenoid productivity. The implementation of a molecular biosensor for TPP could be a promising approach towards on-line assessment and feedback regulation of MEP pathway activity and this application is therefore investigated in this work. Riboswitches have been suggested as versatile RNA-based tools for biotechnological applications in bacteria, including various cyanobacterial species. However, TPP-responsive riboswitches have not been addressed in cyanobacteria thus far. This project therefore aims at the evaluation and implementation of TPP-responsive riboswitches in Synechocystis, using a yellow fluorescent reporter protein as quantitative readout of translational regulation. Native putative OFF-switches from two cyanobacterial species are investigated along with one synthetic ON-switch, originally based on the native riboswitch from E. coli. The induction effects are assessed on both RNA and protein level for both TPP and its precursor thiamine. The synthetic riboswitch is found to be effective in Synechocystis and is further examined for its dynamic range. Several protocols for fluorescence and transcript level experiments are developed. Several continuation experiments are suggested, including further investigation of the cyanobacterial OFF-switches.

SAD-C, a gene belonging to the small short-chain alcohol dehydrogenase-like protein (SAD) gene family, is up-regulated in Pisum sativum (pea) when the plant is exposed to UV-B (280-320 nm) radiation. SAD-C has a molecular weight of about 28 kDa and adopts a tetrameric structure. The aim of this work was to purify the protein SAD-C from Pisum sativum when overexpressed in E. coli strain BL21 StarTM (DE3) One Shot®. The purification was facilitated by the presence of a His-tag consisting of six histidine residues at the C-terminal end of the protein. The purification trials of SAD-C were faced with problems since the sample fractions contained several other proteins as well. Several purification steps seem to be necessary for future trials. A crystallization trial was still set up and crystals were formed, but the crystals formed were probably not of SAD-C.

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.

No description available.

No description available.

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.