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.

<p>There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria <i>Acholeplasma laidlawii</i> and <i>Streptococcus pneumonie</i> and in the plant <i>Arabidopsis thaliana</i>, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures.</p><p>Here we have determined the genes encoding the main glycolipids of <i>A. laidlawii</i> and <i>S. pneumonie</i>. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of <i>Escherichia coli</i> lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of <i>A. laidlawii</i> and two analogous genes from <i>A. thaliana</i> yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in <i>E.coli</i> membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer <i>in vivo</i>.</p>

No description available.

<p>As a consequence of the presence of both mitochondria and chloroplasts in plant cells there is a higher sorting requirement in a plant cell than that in a non-plant cell. Reflecting this, protein import to mitochondria and chloroplasts has been shown to be highly specific. However, there is a group of proteins which are encoded by a single gene in the nucleus, translated in the cytosol and targeted to both mitochondria and chloroplasts. These proteins are referred to as dual targeted proteins. The first protein shown to be dual targeted was pea glutathione reductase (GR). The focus of this thesis is the targeting properties of the dual targeted protein glutathione reductase.</p><p>In order to overcome the limitations with traditional in vitro import systems we have developed an import system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The chloroplastic precursor of the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) was mis-targeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual system. The dual GR reductase precursor was targeted to both mitochondria and chloroplasts in both the single and dual import system.</p><p>We have investigated the targeting and processing properties of the GR targeting signal. Using N-terminal truncations we have demonstrated that the GR targeting signal has a domain organisation. Our results show that GR has evolved a dual targeting signal with the C-terminal part being sufficient for chloroplast import, the internal part required for the mitochondrial import and the N-terminal part housing a “fine-tuning” function. Furthermore, we have constructed a range of point mutations on the GR signal sequence changing positive amino acid residues and stretches of hydrophobic amino acid residues. Overall single mutations had a greater effect on mitochondrial import compared to import into chloroplasts. We have also shown that the recognition of the GR processing site differs between MPP and SPP. Single amino acid substitutions in the vicinity of the processing site clearly affected processing by MPP while processing by SPP showed low sensitivity to single mutations.</p>

<p>The aim of this examination project work was to develop one new sports drink for consumption during prolonged exercise. Most existing sports drinks contain carbohydrates and electrolytes but the drink developed during this project work aimed to contain protein/amino acids and other substances that potentially may help the athlete to perform better compared with sports dinks containing only carbohydrates and electrolytes. Since it is unclear if whole proteins, oligopeptides or free amino acids are preferred, three different sports drink where developed.</p><p>All three sports drinks contain 25 mmol Na+/l, 5.5 mmol K+/l, 240 mg caffeine (per serving), high molecular weight glucose polymer (7%), aromas, beta-carotene and other substances supposed to improve the taste of the drinks. The three drinks contain different sources of amino acids: 0.47% branched-chain amino acids (BCAA) in the BCAA-sports drink, 2% whey protein in the Whey protein-sports drink and 0.8% hydrolyzed casein (oligopeptides) in the Peptopro®-sports drink. The serving size of each sports drink is 0.75 l.</p><p>As the drinks should taste well and be easy to drink during exercise blinded tasting tests were performed. During these tests prototypes and the final versions of all three sports drinks were compared to a placebo drink containing no amino acids or caffeine.</p><p>The results from the taste-tests show that none of the test drinks or the placebo drink exhibited significantly better taste (p>0.05). However, the BCAA-sports drink, the Whey protein-sports drink and the placebo drink was shown to taste better than the Peptopro®-sports drink.</p><p>To investigate the effects of the sports drinks on performance, two elite trained cyclists were supplemented with the test drinks and the placebo drink during interval-trials on a cycle ergometer equipped to measure power output during blinded tests. The power output was compared to the heart rate of each test participant. The placebo drinks contained 8% carbohydrates but no caffeine or amino acids. The results indicate that all three sports drinks either were considered equal, or improved the performance, as compared to the placebo drink. All drinks were considered to taste well during exercise. These findings indicate that the three sports drinks developed during this project work, improved performance and tasted well. However, it is desired to improve the taste of both the Peptopro®-sports drink and the Whey protein-sports drink. Alternative drinks containing no caffeine should be developed in order for the athlete to intake large quantities of the developed sports drinks during prolonged exercise</p> / <p>Sportdrycker är i regel kolhydratbaserade i syfte att skapa goda förutsättningar för idrottslig prestation. De sportdrycker som vid detta arbete utvecklats innehåller substanser utöver kolhydrater som möjliggör en förbättrad prestation vid långtidsuthållighetsidrott relativt kolhydratsbaserade sportdrycker. Sportdryckerna skulle vara välsmakande och lätta att konsumera i stora volymer under arbete. Syftet var att utveckla en sportdryck, innehållande en protein-/aminosyra-källa, men då det mot bakgrund av aktuell forskning var oklart om helprotein, oligopeptider eller fria aminosyror var att föredra, utvecklades tre olika sportdrycker. Detta resulterade i följande tre sportdrycker: BCAA-sportdrycken, som innehåller 0.47 % grenade aminosyror (BCAA), Vassleprotein-sportdrycken, som innehåller 2 % vassleprotein och Peptopro®-sportdrycken som innehåller 0,8 % hydrolyserat kasein (i form av oligopeptider). Alla tre har en serveringsstorlek på 0,75 l. Övrigt innehåll i sportdryckerna är 240 mg koffein (per servering), 25 mmol/l Na+, 5,5 mmol/l K+, 7 % glukospolymer av hög molekylvikt (Vitargo®) samt smakämnen och betakaroten.</p><p>De tre sportdryckerna jämfördes vid enkelblindade tester relativt en etablerad sportdryck innehållande 8 % kolhydrater (Vitargo®), både genom smaktester utförda på tränad smakpanel och genom prestationstester utförda på unga elitcyklister. Även osmolalitet och pH mättes på färdiga drycker. Resultaten från smaktesterna visade att både BCAA-sportdrycken och Vassleprotein-sportdrycken var välsmakande, men att Peptopro®-sportdrycken ej var färdig smakmässigt. Ingen signifikant skillnad i smak uppmättes mellan sportdryckerna och kontrolldrycken (p>0,05).</p><p>Resultaten från prestationstesterna indikerade att samtliga testdrycker var likvärdiga med eller förbättrade prestationen under tidsintervallen relativt kontrolldryck med annorlunda elektrolytsammansättning samt utan koffein och aminosyror. Samtliga drycker uppfattades här som välsmakande och lätta att konsumera under arbete. Sammanfattningsvis bedöms samtliga produkter vara såväl prestationsmässigt som smakmässigt bra alternativ till etablerade sportdrycker för uthållighetsidrottare. Ytterligare smakförbättringar är önskvärda för såväl Vassleprotein-sportdrycken som Peptopro®-sportdrycken.</p><p>2008:L1</p>

<p>This thesis is focused on the heparan sulphate (HS) structure from blind mole rat liver. HS is a glycosaminoglycan that is produced as a proteoglycan, in which linear polysaccharide chains are attached covalently to a protein core. Proteoglycans are widespread molecules in the body and have many important physiological functions. HS is synthesized as a polymer of alternating glucuronic acid and N-acetylglucosamine units. Parts of the polymer are subsequently modified by N-deacetylation /N-sulphation of the glucosamine units, C-5 epimerization of glucuronic acid to iduronic acid and O-sulphation at various positions.</p><p>The mole rats are from Israel and are of the Spalax ehrenbergi superspecies. Spalax Judaei (S60) has 60 chromosomes and Spalax Galili (S52) has 52 chromosomes. They are both completely blind and spend their entire life underground in hypoxic conditions. Spalax Galili (S52) inhabits the cool-humid Upper Galilee Mountains and Spalax Judaei (S60) inhabits the warm-dry southern regions. There is no current information about the heparan sulphate structure of these animals.</p><p>The two blind mole rats (S52 and S60) were metabolically labelled with [3H] Glucosamine. The animals were sacrificed and the organs were taken and frozen. The liver was chosen for the purpose of my project.</p><p>The HS structure was studied using various chromatographic methods such as ion-exchange and gel filtration. Structural analysis of HS indicated that the size of HS from the liver was the same in both species. However, the domain structure differed between the two animals, particularly with regard to sample S52(1) which had obvious differences. This leads to the study of the heparanase cleavage sites. Disaccharide composition analysis identified varying proportions of disaccharide species in S52 and also the possibility of an unknown disaccharide species.</p>

As a consequence of the presence of both mitochondria and chloroplasts in plant cells there is a higher sorting requirement in a plant cell than that in a non-plant cell. Reflecting this, protein import to mitochondria and chloroplasts has been shown to be highly specific. However, there is a group of proteins which are encoded by a single gene in the nucleus, translated in the cytosol and targeted to both mitochondria and chloroplasts. These proteins are referred to as dual targeted proteins. The first protein shown to be dual targeted was pea glutathione reductase (GR). The focus of this thesis is the targeting properties of the dual targeted protein glutathione reductase. In order to overcome the limitations with traditional in vitro import systems we have developed an import system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The chloroplastic precursor of the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) was mis-targeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual system. The dual GR reductase precursor was targeted to both mitochondria and chloroplasts in both the single and dual import system. We have investigated the targeting and processing properties of the GR targeting signal. Using N-terminal truncations we have demonstrated that the GR targeting signal has a domain organisation. Our results show that GR has evolved a dual targeting signal with the C-terminal part being sufficient for chloroplast import, the internal part required for the mitochondrial import and the N-terminal part housing a “fine-tuning” function. Furthermore, we have constructed a range of point mutations on the GR signal sequence changing positive amino acid residues and stretches of hydrophobic amino acid residues. Overall single mutations had a greater effect on mitochondrial import compared to import into chloroplasts. We have also shown that the recognition of the GR processing site differs between MPP and SPP. Single amino acid substitutions in the vicinity of the processing site clearly affected processing by MPP while processing by SPP showed low sensitivity to single mutations.