<p>Prion diseases are a group of fatal neurodegenerative diseases affecting almost all mammals including humans. The diseases are caused by formation of the misfolded isoform of the cellular prion protein (PrP^C) to the disease causing PrP^Sc. The focus on this work has been to characterize molecular changes in persistently scrapie-infected murine neuronal cells possibly contributing to prion-induced neurodegeneration. PrP^C is localized to lipid rafts in the plasma membrane and this is also the place where it is suggested that the conformational change into PrP^Sc occurs. This work shows an increased expression of active Src kinase in scrapie-infected cells resulting in an increased overall tyrosine phosphorylation of several proteins. Additionally, an increase in the specific tyrosine kinase activity of Fyn is shown. Interestingly, the membrane distribution of Fyn from non-raft to raft-domains followed that of PrP^Sc in scrapie-infected cells as analyzed by immunoblotting of flotation-fractions after ultracentrifugation of Triton X-100 extracted cell lysates. This indicates a persistent Fyn activation, probably due to clustering of intracellular Fyn kinases due to PrP^Sc accumulation in lipid rafts. In addition to an increased Src family kinase activity in scrapie-infected cells these cells also express an increased number of insulin receptor (IR)/insulin-like growth factor-1 receptor (IGF-1R) hybrid receptors, and these receptors display an altered protein glycosylation of the IR subunits. Additionally, ScN2a cells do not respond to LPS-stimulation with NO production, putatively due to the lack of CD14 mRNA. Together, these findings may have pathological implications leading to neuronal cell death in prion diseases via several mechanisms which are discussed in this thesis.</p>

<p>Two independent endosymbiotic events gave rise to mitochondria and chloroplasts. Despite the fact that both organelles have their own small genome the majority of organellar proteins are encoded in the nucleus, synthesized in the cytosol and imported into the organelles. The targeting information for most organellar proteins is located in an N-terminal extension called a targeting peptide. Targeting peptides are cleaved off after import by organellar processing peptidases. The cleaved targeting peptides are toxic to organellar functions and are degraded by the PreP peptidasome, the metalloendopeptidase which is the main topic of this thesis.</p><p>We have overexpressed, purified and determined the first structure of a plant mitochondrial targeting peptide, the F1β presequence from Nicotiana plumbaginifolia, by NMR in a membrane mimetic environment. The structure showed that the targeting peptide formed two helices separated by an unstructured domain. The N-terminal helix being amphipatic. The F1β targeting peptide has been used as a model substrate for the mitochondrial and chloroplast PreP peptidasome. In Arabidopsis thaliana the PreP peptidasome is present as two isoforms, AtPreP1 and AtPreP2. We have shown that both forms are expressed and dually targeted to mitochondria and chloroplasts. Both AtPreP1 and AtPreP2 degrade targeting peptides and other non-related unstructured peptides up to 65 amino acid residues. Substrate specificity studies showed that both PreP isoforms have a preference for positively charged amino acid residues in the P1′ position and small uncharged residues in the P1 position. Mapping of cleavage sites revealed unique cleavage sites for both isoforms. We have generated and characterized both single and double AtPreP1 and AtPreP2 knockouts in A. thaliana. AtPreP1 was shown to be the major isoform. The double knockout exhibited a chlorotic phenotype with altered mitochondrial and chloroplast morphology. Furthermore,mitochondria were partially uncoupled. Throughout the development there was a slower growth rate and 40% lower biomass production. These results show that the PreP peptidasome is important for efficient organellar functions and normal plant development.</p>

<p> </p><p>The Thioredoxin (Trx) and Glutaredoxin (Grx) system are two major antioxidant redoxsystems in the cells that prevent and respond to oxidative stress, which is a well known factor in aging and causing several diseases, like neurodegenerative disorders, diabetes and cancer. Oxidative stress also can be described as an imbalance between production of reactive oxygen species (ROS) and the cellular protection. Thioredoxin Reductase (TrxR), a selenoproteine located both in the cytosol and the mitochondria, functioning as an electron donor for Trx, and also its mithochondrial form (TrxR2) it can be an electron donor for Grx2.</p><p> </p><p>Lately, several studies has been showing Selenite, a highly oxidized form of selenium, a well known essential tracemineral and antioxidant,  to be a potential therapeutic drug in cancer treatment by inhibiting tumor growth and induce apoptosis in cytostatic drug-resistant malign cell-lines. Gold, and gold-containing drugs have a long history in medicine, and has been validated as potent TrxR inhibitors.</p><p>In this study we have combined Selenite and a Goldcompound in treatment of Grx2-overexpressing HeLa cell-lines, and investigated the inhibiting effect on the Thioredoxin and Glutaredoxin system, and also how Grx2 would function without its electron donor, TrxR2.</p><p>By using a quantitative polymerase chain reaction and for this, we have successfully optimized a TrxR2-primer, measured and compared the expression of mRNA levels of TrxR1, TrxR2, Grx2 and Grx2tot.We have also investigated the enzyme kinetics of Grx1 with selenocysteine, selenodiglutathione and selenomethylselenocystine, to see/ and found that  they could be a substrate for Grx1.</p>

<p>Probiotics is a special group of health products on the market. The most common type of  these is live cultures of lactic acid fermenting bacteria from raw milk . These include, among others, yogurt and various trademarks such as Hälsofil ®, Acidophilus ®, Dofilus ® and Kefir ®. Such products are often used by patients who are treated with antibiotics to combat the intestinal flora eliminated, thus causing digestive problems. The present work deals with yet another type of health product, viz. kefir of Tibetan type. A literature review of mainly Russian sources has been made to identify the nutrients and potential health benefits of kefir consumption in humans. The study sought to find data on the nutritional values of this very particular culture of a certain fungus, <em>Zoogloea ramigera</em> and some species of the genus Lactobacillus. However, this has not been found other than the data on the product, which is on the Swedish market under the brand Kefir, which is a product of a different nature than the Tibetan form of kefir. One source indicates, however, that among other things, it contains vitamin D, which would otherwise break down in this type of product. Part of the work is a study of the characteristics of the kefir culture for an intended technological process of production. The study shows that culture can be stored while maintaining viability by freezing to -70 ⁰ C. The culture is not affected by <em>Gentamycin</em>, which is a common antibiotic. Kefir culture tends to grow in the form of granules or lumps, which may be separated and reused for production. It is sensitive to metal ions, so that process equipment, intended for contact with kefir must be manufactured from other materials such as enamel or polymer-coated steel sheets. A study of cultural interaction with three different strains of intestinal bacteria showed very little impact on the cultivation on agar plate. In batch fermentation the pH of the kefir culture decreases to 3.4, which is slightly higher than the pKa of lactic acid. This pH is unfavorable for disease-causing bacteria and can be expected to limit growth of those in the human gastrointestinal tract upon ingestion.</p>

<p>In Sweden about 26000 people are affected by myocardial infarction (MI) every year. Coronary vascular diseases and cerebrovascular diseases with blood flow disruption caused by thrombi are actually the most common cause of death. A contributing mechanism may be if the fibrinolytic system is uncapable to dissolve clots and it might in turn be related to the high amount of plasminogen activator inhibitor 1 (PAI-1) released from platelets. This study investigated if there is any difference between the thrombin activation of the platelet protease activated receptors (PAR) PAR1 and PAR4 concerning PAI-1 secretion. An ELISA assay was modified in order to measure PAI-1 released from stimulated platelets. Concerning PAI-1 secretion this study concluded that PAR1 activation is slightly more sensitive to PAI-1 secretion than PAR4. Both thrombin receptors are really potent in this respect, activating only one of PAR1 or PAR4 is enough to get a strong response. This study could also confirm that platelet PAR1 undergoes desensitization when stimulated with a low dose of agonist. Despite platelets poor protein synthesis machinery it seems like they have the ability to synthesize large amounts of PAI-1.</p>

<p>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®.</p><p>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.</p>

<p>Cytochrome P450 (CYP) constitutes a superfamily of heme- containing enzymes that catalyze the oxidative biotransformation of structurally diverse xenobiotics including pharmaceuticals and drugs. Cytochrome P450 3A4 is not only the most abundant isoform in human liver but is also responsible for metabolizing approximately 60% of therapeutic drugs. This feature makes CYP3A4 highly susceptible to both reversible and irreversible, such as mechanism-based, inhibition. Mechanism-based inhibition is characterized by being time dependent as well as NADPH and concentration dependent, when some drugs are converted by CYPs to reactive metabolites. The inactivation of CYP3A4 can be due to chemical modification of the heme, the protein, or both by covalent binding of modified heme to the protein. Compared to reversible inhibition, mechanism-based inhibition of CYP3A4 more frequently causes unfavourable drug- drug interactions (DDI), as the inactivated CYP3A4 has to be replaced by newly synthesized CYP3A4 protein. DDI can lead to higher exposure of co-administered drugs, sometimes leading to toxicity. For these reasons, drug metabolism groups within pharmaceutical companies need a well established screening assay to assess mechanism-based inactivation of major human P450 enzymes by new chemical substances that are being developed by the company. Historically, adverse drug interactions were found in clinical trials or after the drugs were commercially released. That caused pharmaceutical companies large economical losses, since a large portion of development cost was in vain.Medivir AB is a small pharmaceutical company that would like to set up a screening method to be used to test candidate drugs for CYP3A4 mechanism-based inhibition. The central aim of this master degree project was to set up a screening assay to test irreversible inhibition of CYP3A4 and validate it with known inhibitors. Using the validated assay, a number of in-house project compounds will be measured for inhibition potential and the results analyzed for structure-activity correlations. Relationships between some functional groups and mechanism-based inhibition can provide insight for improvement of drug candidates and inhibition liability. The assay was based on microsomes containing recombinant human CYP3A4 and activity measured by conversion of the substrate dibenzylfluorescein into a fluorescent product. The product was quantified by measurement of fluorescence in a 96-well plate reader. Optimization was achieved by determining the reaction linearity with time and enzyme concentration. When possible the Km for the probe substrate was also determined. The effect of different backgrounds was studied to settle on a compensation for the enzyme activity. The effect of DMSO on CYP3A4 mediated metabolism of the substrate was studied to determine the acceptable solvent concentration. The concentration responsible for 50% inhibition (IC50) was also determined for several known inhibitors and compared with literature data.</p> / The master thesis was performed at Medivir AB, a small pharmaceutical company in Stockholm.

<p>Detoxification of dilute-acid lignocellulose hydrolysates by treatment with Ca(OH)₂(overliming) efficiently improves the production of fuel ethanol, but is associated with drawbacks like sugar degradation and CaSO₄ precipitation. In factorial designed experiments, in which pH and temperature were varied, dilute-acid spruce hydrolysates were treated with Ca(OH)₂, NH₄OH or NaOH. The concentrations of sugars and inhibitory compounds were measured before and after the treatments. The fermentability was examined using the yeast <em>Saccharomyces cerevisiae</em> and compared with reference fermentations of synthetic medium without inhibitors. The treatment conditions were evaluated by comparing the balanced ethanol yield, which takes both the degradation of sugars and the ethanol production into account. Treatment conditions resulting in excellent fermentability and minimal sugar degradation were possible to find regardless of whether Ca(OH)₂, NH₄OH or NaOH was used. Balanced ethanol yields higher than those of the reference fermentations were achieved for hydrolysates treated with all three types of alkali. As expected, treatment with Ca(OH)₂ gave rise to precipitated CaSO₄. The NH₄OH treatments gave rise to a brownish precipitate but the amounts of precipitate formed were relatively small. No precipitate was observed in treatments with NaOH. The possibility that the ammonium ions from the NH₄OH treatments gave a positive effect as an extra source of nitrogen during the fermentations was excluded after experiments in which NH₄Cl was added to the medium. The findings presented can be used to improve the effectiveness of alkali detoxification of lignocellulose hydrolysates and to minimize problems with sugar degradation and formation of precipitates.</p>

Detoxification of dilute-acid lignocellulose hydrolysates by treatment with Ca(OH)2 (overliming) efficiently improves the production of fuel ethanol, but is associated with drawbacks like sugar degradation and CaSO4 precipitation. In factorial designed experiments, in which pH and temperature were varied, dilute-acid spruce hydrolysates were treated with Ca(OH)2, NH4OH or NaOH. The concentrations of sugars and inhibitory compounds were measured before and after the treatments. The fermentability was examined using the yeast Saccharomyces cerevisiae and compared with reference fermentations of synthetic medium without inhibitors. The treatment conditions were evaluated by comparing the balanced ethanol yield, which takes both the degradation of sugars and the ethanol production into account. Treatment conditions resulting in excellent fermentability and minimal sugar degradation were possible to find regardless of whether Ca(OH)2, NH4OH or NaOH was used. Balanced ethanol yields higher than those of the reference fermentations were achieved for hydrolysates treated with all three types of alkali. As expected, treatment with Ca(OH)2 gave rise to precipitated CaSO4. The NH4OH treatments gave rise to a brownish precipitate but the amounts of precipitate formed were relatively small. No precipitate was observed in treatments with NaOH. The possibility that the ammonium ions from the NH4OH treatments gave a positive effect as an extra source of nitrogen during the fermentations was excluded after experiments in which NH4Cl was added to the medium. The findings presented can be used to improve the effectiveness of alkali detoxification of lignocellulose hydrolysates and to minimize problems with sugar degradation and formation of precipitates.

A microorganism was isolated which could grow on unusually high concentrations of the toxic pollutant 4-chlorophenol. Taxonomic studies showed that the microorganism constituted a novel species within the genus Arthrobacter and it was named Arthrobacter chlorophenolicus A6. A. chlorophenolicus A6 was chromosomally tagged with either the gfp gene, encoding the green fluorescent protein (GFP), or the luc gene, encoding firefly luciferase. When the tagged cells were inoculated into 4-chlorophenol contaminated soil they could completely remove 175 µg/g 4-chlorophenol within 10 days, whereas no loss of 4-chlorophenol was observed in the uninoculated control microcosms. During these experiments the gfp and luc marker genes allowed monitoring of cell number and metabolic status. When A. chlorophenolicus A6 was grown on mixtures of phenolic compounds, the strain exhibited a preference for 4-nitrophenol over 4-chlorophenol, which in turn was preferred over phenol. Analysis of growth and degradation data indicated that the same enzyme system was used for removal of 4-chlorophenol and 4-nitrophenol. However, degradation of unbstituted phenol appeared to be mediated by another or an additional enzyme system. The luc-tagged A. chlorophenolicus A6 gave valuable information about growth, substrate depletion and toxicity of the phenolic compounds in substrate mixtures. The 4-chlorophenol degradation pathway in A. chlorophenolicus A6 was elucidated. The metabolic intermediate subject to ring cleavage was found to be hydroxyquinol and two different pathway branches led from 4-chlorophenol to hydroxyquinol. A gene cluster involved in 4-chlorophenol degradation was cloned from A. chlorophenolicus A6. The cluster contained two functional hydroxyquinol 1,2-dioxygenase genes and a number of other open reading frames presumed to encode enzymes involved in 4-chlorophenol catabolism. Analysis of the DNA sequence suggested that the gene cluster had partly been assembled by horizontal gene transfer. In summary, 4-chlorophenol degradation by A. chlorophenolicus A6 was studied from a number of angles. This organism has several interesting and useful traits such as the ability to degrade high concentrations of 4-chlorophenol and other phenols alone and in mixtures, an unusual and effective 4-chlorophenol degradation pathway and demonstrated ability to remove 4-chlorophenol from contaminated soil.