No description available.

Platelet-derived growth factor receptors (α and β) are included in the family of receptor tyrosine kinases (RTKs). The dimeric PDGF ligands bind to the PDGF receptors and this results in receptor dimerization, autophosphorylation, and thus activation. The autophosphorylated receptor dimer activates numerous signaling pathways. The responses of the cell to the activation of these pathways include proliferation, migration and survival. After ligand stimulated activation, PDGF receptors are internalized mainly via clathrin-coated pits and subsequently degraded in the lysosomes. However, the proteasomes have also been reported to play a role in PDGFRβ degradation. The main aim of this project was to study the mechanism of ligand-stimulated cleavage of PDGFRβ and to explore its possible consequences. By using antibodies that recognize the extracellular and intracellular parts of the receptor, respectively, we showed that ligand stimulation of PDGFRβ leads to the formation of an extracellular (~130 kDa) and an intracellular (~70 kDa) fragment. These fragments were shown to be specific fragments of the receptor located at the plasma membrane and not newly synthesized receptor. The presence of calcium ions intracellularly was found to be essential for cleavage. Interestingly, we observed that with the use of the proteasomal inhibitor called bortezomib, receptor cleavage was inhibited. On the other hand, lysosomal inhibition did not inhibit PDGFRβ fragmentation. Subsequently, we showed that cleavage occurs after the receptor has been endocytosed. Then, we examined whether blocking PDGFRβ cleavage had any effect on the downstream signaling of the receptor. We revealed an increase in receptor, PLCγ and STAT3 phosphorylation and a decrease in Erk phosphorylation upon bortezomib treatment. Moreover, we observed that PDGFRβ undergoes another type of cleavage upon ligand stimulation in fibroblasts, known as ectodomain shedding. Finally, we showed that receptor internalization is decreased upon bortezomib treatment, suggesting that bortezomib blocks receptor cleavage by inhibiting its internalization. The cleavage site, responsible enzyme, as well as potential functional consequences of ligand-stimulated PDGFRβ cleavage, remain to be determined.

No description available.

Ribosome biogenesis is the maturation and accurate assembly of ribosomal RNA and proteins to form functional ribosomes competent to enter the translation cycle, facilitated by the action of ribosome biogenesis factors. RbfA and KsgA are two factors involved in late stages of the 30S subunit biogenesis, yet before translation initiation. Despite the extensive research on both factors, their precise mechanisms remain unclear in translation initiation. Initiation factors constitute a key component of the translation initiation system, especially IF3 that proofreads the codon-anticodon interaction of the mRNA and initiator tRNA to maintain the accuracy of the initiation step. However, defective ribosomes with knockout RbfA (ΔrbfA) or KsgA (ΔksgA) appear to weaken IF3 binding affinities and subsequent proofreading. Previous results suggest that when defective ribosomes enter in vivo translation with cognate (AUG) or even near-cognate (AGG) start codons could bypass the fidelity checkpoints created by initiation factors, leading to the inaccurate initiation of translation. This study aimed to characterize the assembly defective ΔrbfA and ΔksgA ribosomes in in vitro initiation assays on cognate and near-cognate codons and with the interplay of initiation factors for the rate and accuracy in translation initiation. The two assays, BOP-Met-tRNAfMet binding and subunit association, reveal that ΔrbfA ribosomes are defective in both rate and accuracy in translation initiation, which largely contribute to its growth defect at 37°C. In contrast, ΔksgA ribosomes are not defective in the initiation step of translation when comparing to the wildtype ribosome. Therefore, RbfA seems to have the primary role in ribosome biogenesis compared to KsgA. Further, the results show that the initiation factors, in particular IF3, adds to the fidelity of initiation by dissociating the initiator tRNA on near/non-cognate codons, with no major difference between Wt and mutant ribosomes in selection against near/non-cognate codons. Therefore, we do not see any loss of fidelity for the ΔrbfA and ΔksgA ribosomes in in vitro initiation based assays, contrary to previous in vivo based results.

This thesis describes the improvement of two radically different cellulose materials, paper and artificial blood vessels, constructed from two diverse cellulose sources, wood pulp and Acetobacter xylinum. The improvement of both materials was possible due to the natural affinity of the hemicellulose xyloglucan for cellulose. Chemical and mechanical pulps were treated with xyloglucan in the wet-end prior to hand sheet formation or by spray application of dry hand sheets, loading a comparable amount of xyloglucan. The tensile strength increases for the wet-end treatment and spray application were 28% and 71% respectively for bleached soft wood, compared to untreated sheets (20.7 Nm/g). The corresponding strength increases for hand sheets made of thermo-mechanical pulp were 6% and 13% respectively compared to untreated sheets (42.4 Nm/g). The tendency for chemical pulp to be superior to mechanical pulp with respect to strength increase was valid even for tear strength and Scott-Bond. These results suggest, in agreement with other studies, that adhesion of xyloglucan to wood fibres is dependent on their degree of surface lignification. Also, a method was developed to increase the blood compatibility of artificial blood vessels constructed of bacterial cellulose. Xyloglucan was covalently linked to the endothelial cell adhesion motif (Arg-Gly-Asp). To obtain this, new solid-phase coupling chemistry was developed. Xyloglucan oligosaccharides (XGO) were transformed into XGO-succinamic acid via the corresponding XGO--NH2 derivative prior to coupling with the N-terminus of the solid-phase synthesised Gly-Arg-Gly-Asp-Ser peptide. The resin-bound glyco-peptide was then cleaved and enzymatically re-incorporated into high molecular weight xyloglucan. The glyco-peptide was further adsorbed onto bacterial cellulose scaffolds, increasing the adhesion and proliferation of endothelial cells and therefore blood compatibility. / QC 20101102

Enzymes are successfully employed in the synthesis of different types of polymers. Candida antarctica lipase B is a highly efficient catalyst for the synthesis of polyesters by ring opening polymerization. ω-Pentadecalactone is an interesting lactone due to the unique proprieties of its polymer (poly-pentadecalactone). These polymers have not been applied in any industrial application due to the difficulties to reach them by chemical polymerization. Enzymatically, poly-pentadecalactone macromonomers can be obtained to high conversion. In this investigation we synthesized difunctionalized poly-pentadecalactone with different functional groups. Taking advantage of the selectivity of Candida antarctica lipase B, we introduced different functional end groups. α,ω-Difunctionalized poly-pentadecalactone macromonomers with two thiol ends, two (meth)acrylate ends or with one thiol and one acrylate end were obtained with a high degree of functional ends. We have improved the difunctionalization procedure to a single-step route for the synthesis of α,ω-functionalized poly-pentadecalactones. This procedure has a great potential for industrial applications due to the simplicity of the process and the clean products afforded. Macromonomers with functionalized ends can be used to obtain new polymer architectures with novel proprieties. We also show how the use of enzymes could have some limitations when using an initiator with a cleavable ester bond. 2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the ring opening polymerization (eROP) of ε-caprolactone and ω-pentadecalactone aiming for methacrylate functional polyester. However, the lipase catalyzed not only the ring opening polymerization but also the cleavage of the HEMA moiety resulting in a mixture of polymer products with various end groups. A kinetics study of the eROP and the transesterification processes when using HEMA showed that the transesterification processes occurs at moderate frequency at low monomer concentration, it becomes dominant at longer reaction times. We showed that fully difunctionalized polymers can be obtained when using HEMA as initiator for the eROP of lactones by adding a proper end capper. / QC 20101124

As life expectancy increases globally, so does the prevalence of age-related diseases, some of which are more difficult to adapt to and accommodate for than others. In particular, neurodegenerative disorders are among those for which adaptations are more complex, often requiring long-term care. Alzheimer’s disease is a neurodegenerative disorder linked with atrophy of certain cognition related brain regions, causing severe memory, and cognitive function loss. A major hypothesis behind Alzheimer’s disease, upon which most pharmaceutical therapies are based, proposes its cause as the degeneration of cholinergic neurons. Nerve growth factor is a biomolecule found to stimulate the generation, protection, and regeneration of cholinergic neurons. Synthesis of nerve growth factor has been found to be promoted by hericenones and erinacines, bioactive compounds originally extracted from the mycelium of the Lion’s Mane Mushroom (Hericium erinaceus); however, direct supplementation with H. erinaceus has also yielded positive results. In animal models H. erinaceus has enhanced Nerve growth factor levels, increased neuronal survival, promoted hippocampal neurogenesis, decreased amyloid plaque build-up, and improved behavioural outcomes. Human trials showed improvements in cognitive function scores, short-term memory, and visual contrast sensitivity. Phytotherapeutic remedies such as these have long been used across a multitude of cultures, however, now with quantitative scientific evidence supporting their benefits, their implementation into clinical therapies is being explored. Though there is still room for further research, H. erinaceus shows a promising future as a potential pharmaceutical therapy for Alzheimer’s disease and other cognitive impairments.

Thermo Fisher Scientific in Uppsala manufactures in vitro tests for allergy diagnostics by producing recombinant allergens. However, the absence of an effective method for quantification of recombinant protein post-fermentation, challenges the production since protein yield cannot be determined. This study is made upon request by Thermo Fisher Scientific in Uppsala as a response to this issue with the aim to provide a recommendation of  a method to implement. Thermo Fisher Scientific uses histidine-tagged recombinant proteins in their production which allows purification through immobilized metal ion affinity chromatography (IMAC). Hence, this study mainly surveys quantification methods that utilize the histidine-tag. This project provides insight into the following methods: SDS-PAGE, Western blotting, ELISA, Simple Western and FluoroHis-PAGE. The methods have been evaluated based on three key aspects: cost, analysis time, and method performance.  The recommended method for implementation is FluoroHis-PAGE, as it was found to be the most appropriate method for Thermo Fisher Scientific’s current process. FluoroHis-PAGE utilizes fluorescent Ni-NTA or antibody conjugates, which demonstrated high performance in the quantification of His-tagged recombinant proteins at a low cost and analysis time.

Cancer utgör ett betydande globalt hälsoproblem och är den näst vanligaste dödsorsaken globalt sett. Transforming growth factor-beta (TGF-β) är en tillväxtfaktor som signalerar huvudsakligen via transkriptionsfaktorerna SMAD2 och SMAD3 som fosforyleras och aktiveras av TGF-β receptorerna. I normala celler fungerar TGF-β som tumörsuppressor men forskning har visat att mutationer i TGF-β- signaleringen ofta uppstår, vilket leder till motsatta effekter i senare stadier av cancer. I stället för att hämma tumörtillväxt kan TGF-β stimulera metastasering och invasion i senare stadier av cancer. Orsaken och mekanismer bakom detta är fortfarande oklara därför behövs det mer forskning inom TGF-β signalering för att kunna förstå orsaken bakom mutationerna som kan uppstå. Cdc42 är ett GTPase-protein som tillhör Rho GTPases-familjen och är en av de tre klassiska Rho GTPases i däggdjur celler. Flera studier har visat att detta GTPase-protein påverkar signaleringen av andra tillväxtfaktorer såsom VEGFA och EGF. Därför är syftet med denna studie att undersöka hur Rho GTPaset Cdc42 påverkar TGF-β SMAD-signalering i cancerceller. I denna studie odlades MDA-MB-231-celler och därefter transfekterades de med en Cdc42-specifik siRNA för att nedreglera Cdc42-uttrycket. Efter detta stimulerades cellerna med TGF-β och den relativa fosforyleringsnivån och aktiveringen av SMAD analyserades med western blot. Resultaten visade att nedreglering av Cdc42 inte påverkade nivån av fosforylering av SMAD2 i TGF-β-signaleringen jämfört med kontrollerna. Dock observerades en lägre fosforylering av SMAD3 vid nedreglering av Cdc42. Sammanfattningsvis visar denna studie ingen påverkan på fosforyleringen av SMAD2 i TGFβ-signaleringen, men det kan finnas en roll för fosforyleringen av SMAD3. Ytterligare forskning behövs för att bekräfta detta.

Loss of appetite is together with fever and malaise hallmarks of infection. Loosing appetite during an acute infection such as influenza does not result in any longlasting effects, but loosing appetite during chronic diseases such as cancer or AIDS constitutes a risk factor for mortality. Food intake regulation during inflammation is orchestrated by the brain in response to peripheral inflammatory signals. It is known that expression of the prostaglandin synthesizing enzyme cyclooxygenase 2 (COX-2) is crucial for the mechanisms underlying inflammation-induced anorexia, and that prostaglandin E2 (PGE2) is involved in anorexia induced by interleukin-1 beta (IL-1β). In this thesis I examined the prostaglandin-pathways proposed to be involved in anorexia. We show that acute anorexia is dependent on COX-2 expression, while cancer-induced anorexia is mediated by cyclooxygenase 1 (COX-1), at least in the initial stages, suggesting that the signaling pathways for chronic- and acute anorexia are distinct. We were able to demonstrate that the pathway underlying acute anorexia is distinct from that of fever, and that taste aversion is prostaglandin independent. We could also show that both acute and chronic anorexia-cachexia is dependent on expression of myeloid differentiation primary response gene (MyD88) in hematopoietic/myeloid cells. In summary, the findings presented in this thesis suggest that anorexia is a result of many different signaling pathways, as opposed to what is the case for several other inflammatory symptoms such as fever and malaise, where the pathways have been shown to be very exclusive. This provides new insight into the diversity of the pathways underlying inflammatory symptoms, which is fundamental for the ability to present potential, symptom-specific drug targets.