<p>Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections. These infections can be very serious and sometimes hard to get rid of, because of the many virulence factors the bacteria produce during infections.</p><p>This project was a research of the extracellular fibrinogen-binding protein, Efb, which is a 15.9 kDa protein that has been shown to be an important virulence factor during S. aureus infections.</p><p>The purpose with the project was to find out if the protein has more than one binding site to fibrinogen and if people produce antibodies against Efb.</p><p>This was performed with methods such as affinity chromatography, ELISA, coagulation test and western blot. It was shown that Efb has two binding sites to fibrinogen. One is placed on the C-terminal part of Efb and the other on the N-terminal. It was also shown that the production of antibodies against Efb rises significantly in people during an ongoing infection.</p>

<p>Rapid wound healing is important to regain the skins protective function after injury. Studies have shown that enamel matrix proteins (EMP) have many desirable effects which may accelerate wound healing [Bosshardt <em>et al.</em> 2008].</p><p> </p><p>Polymers (Polymer A, B and C) were formed into a mat form, with or without incorporated enamel matrix derivative (EMD) (Collaboration partner). The materials may be suitable for wound care and drug delivery systems.</p><p> </p><p>Protein release tests were performed on samples incubated in physiological-like solution using pyrogallol red staining, ultraviolet (UV) spectrophotometer and high-performance liquid chromatography (HPLC). Protein was detected in Polymer A material samples, compared to a reference material sample, using pyrogallol red staining. An in vitro experiment showed that normal human dermal fibroblasts (NHDF) cultivated with Polymer A material (with EMD) had significantly higher viability than NHDF cultivated with reference material (Polymer A without EMD) and comparable viability to fibroblasts grown with either 0.1 mg EMD in solution or with 10% fetal calf serum. Images taken of Polymer A material, with incorporated Fluorescein isothiocyanate- (FITC) labeled EMD, indicate a homogenous distribution of EMD peptides and/or EMD aggregates throughout the material. A dressing which contains an active substance may have clinical promise for wound care applications.</p>

<p>Påväxt på båtar och andra föremål som sätts ner i vattnet är ett stort och kostsamt problem världen över. I Sverige orsakar havstulpanen, Balanus improvisius, den största påväxten. Havstulpanen har många förstadier innan den blir en vuxen havstulpan. I det stadiet innan den sätter sig fast på olika platser, kallas den cyprid. Det tar mellan tre dagar och upp till några veckor innan cypriden valt plats att settla på. Den väljer plats mycket noggrant då den skall tillbringa resterade delen av sitt liv på denna.</p><p>Mycket forskning läggs ner på att finna ett alternativ till dagens giftiga båtbottenfärger. Tennorganiska föreningar, som använts för att motverka settling på båtbottnar, blir totalförbjudet 1 januari, 2008. Nu mer tillsätts allt oftare kopparföreningar istället vilket inte är någon större förbättring för miljön. Det finns idag många miljövänliga färger, som t.ex. silikonbaserad färg, men de fungerar inte lika bra som färgerna med metaller. Det forskas även på om ytan på båtbotten skall kunna förändras mekaniskt, för att motverka havstulpanens settling.</p><p>De substanser som är testade i denna rapport är selenmetionin och nitroglycerin. De testades i olika koncentrationsintervaller och i olika blandningar med varandra. Med en förhoppning om att selenmetioninen binder in i föreningar och då att hastigheten för reaktioner med tioler kan minska drastiskt och leda till gifteffekter, och att nitroglycerinen skall pacificera cypriden genom att binda till hemoglobinet istället för syre, skall dessa två substanser hålla cypriderna borta från båtbottnarna. Med föraningar om att substanserna har en synergistisk effekt då de blandas, skall lägre koncentrationer kunna användas. Då för höga koncentrationer är kostsamt och har troligen större negativ påverkan på miljön.</p><p>Selenmetionin visade en antydan till att minska settling men nitroglycerinen hade en mer främjande effekt på cypridens settling. Det visade sig även att substanserna hade en synergistisk effekt och därmed kan koncentrationerna för substanserna sänkas.</p> / <p>Biological growth on boats and other objects that are placed in water is a big and expensive problem over the entire world. In Sweden, the barnacle, Balanus improvisius, grows the most easily. This barnacle has many larvae stages before it becomes mature. During the larvae stage when it searching for a surface to settle on it is called cyprid. It takes between three days and a couple of weeks before a cyprid chooses a spot to settle on. It chooses the spot to settle on very carefully because it will spend the rest of its life on that exact place.</p><p>A lot of research goes into to finding a good alternative to today’s toxic boat paints. Tin organic compounds, as used to counteract settling of cyprids on boats, will be totally forbidden in January, 2008. As a replacement, copper compounds are being added more often to paints. These are not much better for the environment. Today there are many environmentally friendly paints, as for example silicon-based paint, but they do not work as well as the paints with metals. Research is also looking at mechanically changing a boat’s surface so that barnacles cannot attach as easily.</p><p>The substances that are tested in this report are selenomethionine and nitro glycerine. They were tested in different concentration intervals and in different mixes with each other with the hope of that selenomethionine will bound into compounds and then the reaction velocity with thioles will be reduced and give toxic effects. The nitro glycerine will make the barnacle passive through the idea that the NO is binding into a heme group instead of oxygen. It was hoped that the substances show a synergistic effect when they are mixed, allowing lower concentrations to be used. With too high concentrations in the paint, the paint becomes too expensive and likely has a bigger negative influence on the environment.</p><p>Selenomethionine showed an indication to reduce the likelihood that barnacles would settle on a pained surface, but nitro glycerine, on the other hand, caused an increased rate of cyprid settling. It was also shown that the substances had a synergistic effect and thus the concentrations for the substances can be lowered.</p>

<p>Silver was used for its antimicrobial effect by the ancient Greeks, long before the existence of microorganisms were first suspected. Nowadays a wide range of antimicrobial dressings containing silver, either incorporated within or applied on the dressings, are available for clinical use. This type of dressings is designed to provide the antimicrobial activity of silver in a more convenient application.</p><p>The aim with this master thesis was to evaluate if silver release and antimicrobial effect of nine silver containing dressings are dependent on the test medium and if there is any relation between silver release and antimicrobial effect.</p><p>Release of silver and antimicrobial effect was evaluated by using a 6-well co-culture system, with inoculated test medium in the wells and dressing pieces in the culture well inserts. Three different test media with increased complexity and nutrient value were inoculated with either</p><p>Results show that release of silver depends on the test fluid used; for phosphate buffered saline (PBS), the silver concentration was as most 1.2 ppm, but for a complex media containing calf serum (SWF), it varied from 9 ppm to 134 ppm. The viable counts in PBS were reduced by at least 3 log units for all dressings and bacteria, whereas in SWF there were no reduction and instead growth was observed. In general, a high release resulted in less bacterial growth. Results also indicated that kinetics of silver release affect the antimicrobial effect. It is likely to assume that it is important for a dressing to release silver quickly.</p><p>It has previously not been possible to correlate silver release of wound care dressings and antimicrobial effect, since the two factors have been measured in different test systems and in different media. Since both factors depend on test medium and method used, it is shown in the present study that it is important to use relevant test medium for in-vitro evaluation. When measuring silver release and antimicrobial effect in the same test system, a relation is found.</p>

<p>Bakgrund: Bioteknikbranschen är en bransch som präglas av stor osäkerhet. Många företags fortsatta existens beror på om företagets projekt eller produkt får godkänt av läkemedelsverket, vilket gör en värdering av bolaget under utvecklingsperioden mycket svår. Den vanligaste metoden vid värdering av bioteknikföretag idag är kassaflödesmetoden. Denna metod anses dock av vissa aktörer på den finansiella marknaden vara otillräcklig. Istället förespråkas en metod med hjälp av reala optioner som de anser tar bättre hänsyn till den osäkerhet som omgärdar bioteknikföretag.</p><p>Syfte: Syftet med denna uppsats är att utveckla en real optionsmodell för värdering av Bioteknikföretag samt att pröva denna på ett fallföretag.</p><p>Genomförande: För att uppfylla syftet utgicks från existerande företagsvärderingsmodeller och befintlig optionsteori. Vidare studerades reala optioner, och vilka av dessa som på ett bra sätt kunde förknippas med den osäkerhet som existerar i bioteknikföretag. Genom hela processen med utvecklandet av värderingsmodellen användes ett fallföretag, Diamyd Medical AB, och dess finansiella historia samt framtidsprognoser. Därutöver har information om bioteknikmarknaden och de förutsättningar företagen inom denna bransch ställs inför använts.</p><p>Slutsats: Den skapade värderingsmodellen med hjälp av reala optioner visade sig på vårt fallföretag vara ett mycket användbart redskap och som på ett bra sätt klarar av att hantera den osäkerhet som omgärdar många bioteknikföretag och andra forskningsintensiva företag.</p> / <p>Background: The Biotech industry is an industry that is characterized by great uncertainty. Many companies future existence depends on if the company’s project or product gets an approval from the deciding authority, which makes a valuation of the company during the developing period very difficult. The most utilized method when valuing biotech firm today is the cash flow method. This method is nevertheless considered inadequate by some actors at the financial market. Instead a method using real options is recommended by some experts, which they think takes better concern to the uncertainty that surrounds biotech firms.</p><p>Purpose: The purpose with this thesis is to develop a real option model to use when valuing biotech firm, and to try it on a case company.</p><p>Implementation: To attain the goal for the thesis, the authors started out with the traditional methods of valuation and the existing theory surrounding financial options. Furthermore the authors studied real option and which of them that could be applied on the complex nature of biotechnology firms. Throughout the whole development process of the valuation model the authors used Diamyd Medical AB as a case company and in addition to that they also used more general information about the biotechnology sector.</p><p>Conclusion: The valuation model containing real options proved to be a useful instrument to establish a monetary value on biotechnology firms.</p>

<p>Life science sysselsätter i Uppsala 4500 personer varav 60% arbetar på de fyra största företagen. Branschen är forskningsintensiv och producerar bland annat läkemedel och produkter till läkemedelsbolag och akademiska institutioner. Produkterna är nischade och företagen måste ofta vara globala redan från start. Denna studie undersöker med hjälp av teorier om internationalisering tre fallföretag inom life science från Uppsala. Genom intervjuer och årsrapporter dras slutsatsen att fallföretagen främst har internationaliserat sin försäljning med olika strategier för att penetrera internationella marknader. Gemensamt för samtliga företag är att den personliga erfarenheten inom internationaliseringar  anses  mycket  viktig,  vare  sig man använder  sig av egna försäljningsorganisationer, agenter eller konsulter. Ingen strategi för internationell försäljning föredras framför den andra utan vilken strategi som används är olika från marknad till marknad och från produkt till produkt. Att vara nära kunderna med egna försäljningsorganisationer är särskilt viktigt då kunderna är läkemedelsbolag och akademiska institutioner, eftersom det ger möjlighet till återkoppling från dessa. Därigenom får man information om vilka produkter dessa efterfrågar. För läkemedelsbolag tycks det inte lika viktigt eftersom deras produkt genomgått ingående tester innan de lanseras. Egna försäljningsorganisationer är dock dyrt varför andra strategier också väljs. Enbart det största företaget Biotage har egna säljorganisationer på flera marknader.</p>

Tidigare forskning har genom att studera alla tillgängliga företag eller företag med udda branscher exkluderade identifierat vissa nyckeltal som bra indikatorer på finansiell kris. I denna studie undersöks om dessa nyckeltal även ger ett samstämmigt resultat för en udda bransch. Med utgångspunkt från bioteknikbranschen identifieras genom logistisk regression nyckeltalen korta skulder genom eget kapital och avkastning på investerat kapital som indikatorer på finansiell kris. Avkastning på investerat kapital överensstämmer med tidigare resultat, medan korta skulder genom eget kapital bör, för denna bransch, tolkas tvärt emot jämfört med tidigare studier.

Conjugated polymers have been found useful in a wide range of applications such as solar cells, sensor elements and printed electronics, due to their optical and electronic properties. Functionalization with charged side chains has enabled water solubility, resulting in an enhanced interaction with biomolecules. This thesis focus on the emerging research fields, where these conjugated polyelectrolytes (CPEs) are combined with biomolecules for biological sensing and DNA nanowire assembling. CPEs have shown large potential in biomolecular detection where the optical read out is due to the geometrical alternation in the backbone and aggregation state. This thesis focused on transferring the biomolecular detection to a surface of CPEs. The characterization of the CPE layer show that a hydrogel can be formed, and how the layer can undergo geometrical changes upon external stimulus such as pH change. A selective sensor surface can be created by imprinting ssDNA or an antibody in the CPE layer. The discrimination for complementary DNA hybridization and specific antibody interaction can be monitored by surface plasmon resonance or quartz crystal microbalance. We have also taken the step out from the controlled test tube experiments to the complex environment of the cell showing the potential for staining of compartments and structures in live and fixed cell. Depending on the conditions and CPE used, cell nuclei, acidic vesicles and cytoskeleton structure can be visualized. Furthermore, the live staining shows no sign of toxic effect on cultured fibroblasts. CPEs can also be a valuable element when assembling electronics in the true nano regime. I have used DNA as building template due to its attractive size features, with a width of around 2 nm and a length scale in the µm regime, and the inbuilt base-paring recognition elements. This thesis shows how DNA can be decorated with CPEs and stretched on surfaces into a model for aligned semiconducting nanowire geometries. Not only making the template structures is of importance, but also how to place them on the correct surface position, i.e. on electrodes. Strategies for positioning DNA nanowires using transfer printing and surface energy patterning methods have therefore been developed in the thesis. The stretched DNA decorated with CPE also offers a way to further study the molecular binding interaction between the two molecules. Single molecular spectroscopy in combination with polarization has given information of the variation of the CPE binding along a DNA chain.

QC 20100628

Proteins are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue protein A-derived Z domain scaffold. In the first paper, solid phase peptide synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo.   In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an Affibody molecule that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl-glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by amino acid substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity.   Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications. / QC 20100719