Experiment-based study: The 5’-untranslated region (5’-UTR), and the DNA region corresponding to it, have been shown to have a significant influence on the expression of genes both at transcriptional and translational level. Since transcription and translation are two independent mechanisms a 5’-UTR sequence will probably not be optimal for both. The suggested solution presented in this study is to design a long 5’-UTR composed of one transcription stimulating and one translation stimulating region. The stimulating regions consist of 5’-UTR variants independently identified as transcription or translation stimulating by screening for the desired trait, as well as translation stimulating 5’-UTR variants designed using a bioinformatics tool.The results indicated that 5’-UTR fusions tend to introduce limiting factors yielding a reduced gene expression. However, some 5’-UTR fusions successfully resulted in high gene expression and one variant surpassed both of its components showing a possible additive effect of stimulating both transcription and translation in the form of 5’-UTR fusions. This indicates that testing a relatively small number of different sequences gives a good chance of success. The method also proved viable to increase the expression of low expressive 5’-UTR variants while maintaining low uninduced expression. In addition 5’-UTR fusions containing in silico designed translation stimulating regions have the potential of reaching expression levels on par with the levels reached by fusions containing 5’-UTR variants identified through screening. Literature-based study: The nucleotide sequence at the gene 5’-end has a great influence on the expression level of genes, being the location of central mechanisms like transcription and translation initiation. Because of this the 5’-end sequence is an important target when designing genes for recombinant expression. This review will focus on recent research trends, covering the traits of the 5’-end that influence gene expression, as well as on approaches and tools targeting this region that have been utilized or show potential to be used to achieve desired recombinant expression levels in E. coli. In recent years it have become evident that the entire 5’-untranslated region as well as the initial coding sequence has great influence on gene expression, showing that there is more to designing genes for recombinant expression then picking a strong promoter and an optimal SD sequence.

In industry today standard oxidative quality parameters are based on measurements of primary and secondary oxidation products, measured by PV and AV respectively. These methods are all prone to limitations and weaknesses, and their suitability for application on marine oils is not well documented. An increase in fish oil products with added flavor, color compounds, antioxidants and vitamins has entered the market in recent years. However, no documentation on the effect of these additives on the oxidation parameters has been found. The aim of this thesis was therefore to study the effect of variations in procedures in an attempt to highlight weaknesses and further to establish the most suitable procedures for each method when performing measurements on marine oils. In addition, the effect of antioxidants and additives on the oxidation parameters in cod liver oil has been evaluated.In this thesis, PV measurements by iodometric titration and by the ferric thiocyanate method were used to measure primary oxidation products, and AV and TBARS measurements were used to measure secondary oxidation products. Uncertainty of the methods was determined by performing n -measurements at different stages of the oxidation process. Measurement by the iodometric titration method was found to have a lower detection limit of PV >2.0 mEq peroxide kg-1 oil, with an uncertainty of ± 2%. Measurement by the ferric thiocyanate method was found to have a lower detection limit of PV ≥3.6 mEq peroxide kg-1 oil, with an uncertainty of ±10%. Measurement by the AV method was found to have a lower detection limit of AV≥ 1.3, with an uncertainty of ±5%. Measurements by the TBARS method was found to have a lower detection limit of 0.7 μM TBARS /g sample, with an uncertainty of ±12%. The published method of the International Dairy Federation (IDF) for PV determination was evaluated by comparison with a modified version of the method. Factors such as type of solvent used, deaeration of reagents, premixing of reagents and addition of antioxidant were differences between the methods. It was observed significant difference in absorbance in the two methods, and it was therefore concluded that the varied factor had an influence on the method. It is necessary to perform further experiments to determine which of the varied factors that cause variations in the absorbance measurements.PV measurements by the iodometric titration method were found to be influenced by the stirring method, reagent reaction time and oxygen removal. Stirring by magnetic stirring was found to give a higher PV compared to gentle stirring. The importance of the 1 minute reagent reaction time was strengthened as the PV was found to rapidly increased at prolonged reagent reaction times. It was demonstrated that this to a higher degree is important for marine oils compared to vegetable oils, as new hydroperoxides are formed more rapidly in the unstable marine oils. A significant influence of oxygen removal in reagents was detected in cod liver oil. The findings in this thesis suggests that stirring by magnetic stirring, 1 minute reagent reaction time and deaeration of all reagents should be standard procedure when PV is determined in marine oils by the iodometric titration method.Among eight investigated antioxidants and additives, Q10, tocopherol, vitamin K1, lemon – and peppermint extract was found to significantly elevate the PV measured by iodometric titration. For PV determination by the ferric thiocyanate method, lemon extract was found to significantly elevate the PV. Rosemary extract was found to significantly lower the AV measurement, while lemon extract to a very high degree elevated the AV measurement. In measurements by the TBARS method only lemon extract was found to significantly interfere with the method, leading to an elevated TBARS value. Both methods for PV detection were influenced by several of the investigated antioxidants and additives. Clearly there is need for reevaluation of the methods is use today and development of new methods. New methods for measurements of secondary decomposition products are especially needed for fish oils with added lemon extracts as today’s measurements by the AV and TBARS method give highly unreliable results.

Alginates are a class of natural unbranched linear polysaccharides, and consist of the monomers β-D-mannuronic acid and α-L-guluronic acid. The inherent physical properties, relative ease of modification, wide availability and good biocompatibility of alginates have gained a great deal of attention with regards to therapeutic applications. Heparin is a highly negatively charged linear glycosaminoglycan that is widely used as an anticoagulant. The presence of carboxyl groups and several sulfate groups gives heparin its negative charge, while iduronic acid moieties confer a high degree of flexibility to the polysaccharide by being able to assume different stable conformations. Heparin shows diversity in molecular weight, monomer sequence and modification pattern, resulting in a vast range of biological effects. When administered therapeutically, this can in cause an unpredictable dose response and potentially severe adverse effects in certain patients. The main objective of this study was to create a structural analog of heparin exhibiting a more regular structure and distribution, through chemical sulfation of alginate using chlorosulfonic acid. Other important aims were to characterize the analog in terms of structure, distribution and sulfation degree, and assess protein binding and anticoagulating properties of the sulfated alginates in comparison with heparin and the unmodified alginate templates. Sulfation was performed using chlorosulfonic acid in formamide on a polymannuronic acid (poly-M) and a polyalternating alginate with a guluronic acid fraction of FG = 0.46 (poly-MG), introduced through enzymatic epimerization. FTIR, elemental analysis with HR-ICP-MS and carbon NMR were employed to detect the attached sulfate groups on the alginate. The average molecular weights and the mass distributions of the alginate samples were studied using SEC-MALLS. Elemental analysis was used to estimate the sulfation degrees of the alginates, and 13C NMR was employed to study substitution patterns, provide additional DS estimates and assess sample purity. The protein binding properties of the sulfated alginates were evaluated by studying their ability to release hepatocyte growth factor and osteoprotegerin bound to myeloma cells. Anticoagulating properties were studied by measuring prolongation of plasma coagulation time as a result of sulfated alginate supplementation. The alginates were successfully sulfated and exhibited different degrees of sulfation obtained by varying the chlorosulfonic acid concentration used (1 - 10 %), as estimated by elemental analysis. The poly-MG alginate showed increased solubility during the sulfation reaction, resulting in a higher estimated DS at lower chlorosulfonic acid concentrations compared with poly-M. No apparent degradation of the alginates as a result of the sulfation was observed, although preliminary acid hydrolysis resulted in a molecular weight disparity between poly-M and poly-MG samples. Analysis of carbon NMR spectra allowed characterization of novel peaks and secondary DS estimations for the sulfated poly-M samples, while the complexity of the sulfated poly-MG spectra prevented confident characterization of the structures. Sulfation resulted in a profound improvement of the protein binding properties of the alginates, and showed prolongation of the plasma coagulation time at high treatment concentrations.

Alginate hydrogel made from alginate and crosslinking divalent ions is a natural biomaterial that is biocompatible, has low toxicity, is relatively cheap and has mild gelation chemistry. It is a porous material that allows diffusion of small molecules. Alginate hydrogel is a polymeric network that contains 95-99% water and it does in many ways resemble the natural extracellular matrix (ECM) that surrounds cells in the body. It is also hydrophilic, which reduces friction in body fluids and minimizes protein adsorption and it is easily stored and sterilized.Alginate is produced by both algae and bacteria, and it is initially synthesized as mannuronan (M) with 100% M-residues. Guluronic acid residues (G) are introduced in a post-polymerization step by enzymes called mannuronan C-5 epimerases that catalyze conversion of M into G without breaking the glycosidic bond. Seven different mannuronan C-5 epimerases have been sequenced, cloned and produced recombinantly, and these enzymes introduce MG-blocks, G- blocks or both in the alginate chains. With the use of these mannuronan C-5 epimerases it is now possible to engineer alginate with desired and known structure. It is also possible to covalently modify alginates with coupling of cell specific adhesion molecules to the carboxylic group in the monomers. An example is the RGD peptide (arginine-glycine-aspartic acid) that is commonly found in collagen and fibronectin in the ECM. The RGD peptide is the smallest sequence that integrin receptors can recognize and bind to.Central nervous system (CNS) damage is still one of the major causes of both death and disability, despite intense research efforts to achieve neurogenesis and restore functional synaptic connection of CNS neurons. None of he current therapy strategies promote regeneration or regrowth of neural cells or axons. In vitro and in vivo studies has shown that CNS axons can regenerate when located in a permissive environment and it is known that on-going neurogenesis occurs in certain areas of the adult brain, such as the olfactory bulb. Olfactory ensheathing cells (OECs) are found in the olfactory mucosa and olfactory bulb and secrete neurotrophins, provide necessary ECM molecules and substrates for axon elongation and myelination. They do not activate and induce inhibitory molecules or hypertrophy in astrocytes, and are therefore believed to be a promising candidate for cell-mediated repair of the CNS.The major aims of this study was to investigate whether encapsulation of OECs in different types of alginate matrices would improve cell viability over time and induce change of cell morphology, as a future goal is to transplant OECs into the CNS. Viability of OECs up to 14 days in 1.8% UP-LVG capsules have been reported by Kristin Karstensen (Karstensen, 2010), and similar results were achieved in an experiment in this project. Indications of cell concentration dependency on viability were observed in this experiment, with higher viability in capsules with low cell concentration (1.5 mil cells/mL alginate, 3.0 and 5.0 mil/mL). It was decided to conduct an encapsulation of high and low OEC concentration (4.0 mil/mL and 1.0 mil/mL) in 1.0% UP-LVG Ca2+/Ba2+ alginate, with the aim of examining whether reduced alginate concentration would improve cell viability. The results were promising, with a live cell percentage of 50% in the low cell concentration batch after 51 days. The high cell concentration batch was discarded after 22 days with estimated 30% live cells. This result strengthened the hypothesis that lower cell concentration enhanced cell viability, and confirmed that lower alginate concentration improved cell viability notably. These indications were supported by the results of a second encapsulation with similar settings. High and low concentrations (1.5 mil/mL and 5.0 mil/mL) of OECs were encapsulated in 1.0% epimerized Ca2+ alginate with and without 0.2 % RGD peptide graft. The experiment did not show an effect of the RGD peptide on cell viability or morphology. The viability of the cells was extended with one week and viable cells could be observed for 22 days, but in this experiment increased viability as a result of lower cell concentration was less pronounced. This experiment was therefore inconclusive in terms of improved viability connected to cell concentration, but indicated that a lower alginate concentration had a beneficial impact on cell viability. Star shaped channels were observed inside all capsules in this experiment, and a large fraction of dead cells were found to be located inside these channels. This experiment was later repeated with another source of epimerized alginate grafted with ≈ 0.4% RGD peptide with comparable results in terms of cell viability and morphology.Two encapsulations of low cell concentration in 1.0% UP-LVG Ca2+/Ba2+ alginate mixed with three different concentrations of gelatin (0.5%, 1.0% and 2.0%) were carried out, with the aim of observing capsule stability and cell viability. In first experiment the capsule stability appeared to be inversely proportional with gelatin concentration. This was not confirmed when the experiment was repeated, as the batch with the middle gelatin concentration was perceived as most stable. The cell viability was overall high for both encapsulations. Finally, four batches of 1.5 mil/mL OECs were encapsulated in 0.9% UP-LVG Ca2+/Ba2+ alginate gel with one type of ECM molecule mixed with the alginate per batch to yield a concentration of 1.0 mg/mL. Sulphated MG alginate was mixed with 0.9% UP-LVG Ca2+/Ba2+ alginate to a final concentration of 1.0 mg/mL, and included in the experiment. The experiment was terminated at day 28, with varying cell viabilities in the different batches. Common for all was overall lower cell viability compared with the viability observed for cells with similar concentration encapsulated in pure 1.0% UP-LVG, but the capsules proved to be relatively stable. In conclusion, reducing the alginate concentration from 1.8% to 1.0% had notable positive effect on cell viability. High cell concentration in the alginate capsules also proved to have a negative impact on cell viability, but this effect was most evident in the UP-LVG alginate gels. The negative effect on cell viability related to high cell concentration was not as profound in the epimerized alginate gels.RGD peptide grafted onto alginate did not show any unambiguous effect on cell viability and no effect on cell morphology, regardless of 0.2 % peptide graft or ≈ 0.4% peptide graft. The gelatin-1.0% UP-LVG alginate mixes also failed to induce morphology change in the OECs, and neither did any of the ECM molecule-1.0% UP-LVG alginate mixes or the sulphated alginate-1.0% UP-LVG alginate mix. The cells encapsulated in gelatin-alginate mix capsules displayed an overall high viability, while the cells encapsulated in ECM molecule- alginate mix and sulphated alginate- alginate mix displayed lower viability than cells encapsulated in pure UP-LVG alginate. All capsule varieties displayed generally good stability in culture, with the exception of the gelatin-alginate mix capsules that progressively dissolved in culture.

The mucus layers on the internal surfaces of the human body serve as an important barrier against foreign material, but it also create restrictions regarding drug delivery. Discovering methods to overcome this barrier would lead us closer to an efficient delivery of larger drugs and nanoparticles. Recent studies have shown that alginate G-block polymers can modify the physical properties of mucus, and that the G-blocks make the mucin network more open and increase particle transport through mucus. This shows that G-blocks are an interesting candidate, for use in drug delivery across mucosal barriers, but further work to understand the mechanisms by which the G-blocks interact with mucus is still desirable. Studies have shown that neutral particles diffuse more easily through mucus than charged particles. In this thesis the interactions between nanoparticles with different surface structures and mucus components were compared by dynamic light scattering and the effect of G-block on these interactions was established. The diffusions of all particle types were compared in pig gastric mucin (PGM) from Sigma, by the use of confocal microscopy and multiple particle tracking (MPT). Then alginate G-blocks were added and the diffusion of the particle types was compared. The results showed that G-blocks can reduce the amount of mucin components accumulating on to positively charged surfaces but not to negatively charged particles. The MPT showed that the surface charge of the nanoparticles is the primary determining factor when it comes to diffusion through Sigma mucin, and that the effect on the diffusions caused by G-blocks is relatively small, most probably due to the matrix of Sigma mucin, lacking the large networking polymers on which G-blocks previously have shown their effect. It was found that G-blocks make the distribution of trajectories more homogenous, but that this did not affect the mean displacements.

Several studies have suggested that the host genetics may influence the composition of gut microbiota, but few genes involved in host control have been proposed (Sekirov et al., 2010). Lipocalin 2 (Lcn2) prevents growth of bacteria that rely on catechol type siderophores for iron acquisition (Goetz, et al., 2003; Flo, et al., 2004). It was hypothesized that Lcn2 may impart a selection pressure on establishment of the gut microbiota and thus influence the commensal diversity. The aim of this study was to find out whether the antimicrobial protein, Lipocalin 2, has a determining effect on the colonization of the gut microbiota in mice. Two factors were investigated: genotype (Wt, Ht and Lcn2 KO) and habitation (single-housing and co-housing). The naturally developing gut microbiota of wild type mice (Wt), heterozygote mice (Ht) and lipocalin 2 deficient mice (Lcn2 KO) were studied, as well as re-established microbiota after antibiotic perturbation, by collecting stool samples. Microbial community profiles were generated by the use of PCR and denaturing gradient gel electrophoresis (DGGE). The gels were analysed by the software program gel2K (Norland, 2002) and band intensity profiles were compared by statistical analysis. The study of mice gut microbiota revealed differences in the microbial profiles between Wt-, Ht- and Lcn 2 KO-mice. The result showed that both the factor of genotype and habitation were significant factors for the observed differences. For the single-housed mice (mice of same genotype), a significant difference of gut microbiota was found between Wt/Ht-mice and Lcn 2 KO-mice, indicating that the genotype was the main factor for the observed differences. Lcn 2 thus seems to influence the natural colonization of the mice gut, as well as the re-establishment of the microbiota after a perturbation with antibiotics treatment.For the co-housed mice (mice of mixed genotypes) both the effect of genotype and maternity seemed to influence the composition of the microbiota, although the factor of maternity was not taken into account in the analysis. The experimental set-up was designed with the intention of comparing littermates in order to minimize other effects than the knockout of Lcn2. Unfortunately this design meant that the effect of genotype and maternity could not be differentiated.

In a complex system such as the Norwegian railway network, there are much thatcan affect a trains punctuality. The undertakers strive to achieve higher and higherpunctuality, while the infrastructure owner, Jernbaneverket, strive for minimaldowntime on the railway network. There is collected much data for analysis aboutthe trains run and the infrastructure, in order to achieve higher punctuality and lessdowntime. The users are able to track down the source of delays and find possibleimprovements on the infrastructure, by analyzing and comparing the different datasets collected.There are many users across both different companies and internal divisionsin a company that need to cooperate, due to the size and complexity of a railwaynetwork. The different users have different needs when studying the data sets. Aarea director have the need to see the big picture over time, while a segment directorwants to see every detail within its segment.In this thesis we demonstrate a system that is aware of the different stakeholdersrequirements when presenting data. The system also takes into consideration thestakeholders need for analyzing different types of data, and comparing these.Finally, we conclude how users should be defined within a domain in order tobe aggregated over.

Duplex stainless steel (DSS) is a common material in offshore pipe fittings. A manufacturing defect has altered the behavior of the components through an erroneous heat treatment, which has led to precipitation of 𝜎-phase. A report showing a strong correlation between precipitated 𝜎-phase and the reduction in impact toughness, initiated a great interest in the offshore industry for further studies of the effect of precipitated 𝜎-phase in DSS pipe fittings. In this thesis a numerical approach to simulate the behavior of duplex stainless steel with precipitated 𝜎-phase has been performed. The simulations have been done using the Gurson material model, which is implemented in the user defined material model in the SIMLab Metal Model. In addition the material model has been combined with a brittle fracture criterion. The results deviate from the observations done in the laboratory. The reasons for the deviations were found to be quite complex, but effects from mesh sensitivity and incorrect material optimization was found. The influence of the Gurson model is limited for the specimens acting in a brittle manner. It is therefore considered unnecessary to apply this material model for these problems.

Denne masteroppgaven ser på hvilken effekt et armeringsnett har i vegoverbygninger, samt erfaringer som er gjort fra forskjellige hold. I tillegg er det foretatt analyser av seks vegstrekninger i Sør-Trøndelag der armering har vært benyttet. Forskningsresultatene viser hovedsakelig at geonett har effekt for tynne overbygninger, og at desto dårligere undergrunnen er desto større effekt har geonettet. Flere forskningsrapporter viser også at man kan redusere overbygningstykkelsene ved hjelp av geonett, eventuelt forlenge levetiden til vegen. Statens Vegvesen er relativt reserverte for ideen om å redusere lagtykkelsene i overbygningen. De er også i mot å bruke «unaturlig» materiale i overbygningen. Fra et miljøperspektiv er ikke dette en unaturlig tankegang, men det er flere argumenter for at det i enkelte prosjekter kan være mer miljøvennlig å benytte geonett. Faktumet at det er kaldt i Norge på vinteren, gjør at argumentet om frostsikring av vegene er aktuelt. Reduserte lagtykkelser kan muligens føre til mer telehiv på vegene. Analysen av vegstrekningene (spor, IRI, bæreevne, G90-verdier og skadeomfang) viser at strekninger med geonett, total sett, har like bra eller bedre resultater som strekninger uten geonett. Samlede resultater viser at både skadenes utbredelse og alvorlighet er mindre der det ligger geonett. Dette selv om initialforholdene var verre, og lagtykkelsene redusert for strekninger med geonett. Strekninger med geonett har ikke like gode resultater som der det er masseutskiftet. Både bæreevnen og G90-verdiene antyder at det har vært en låsende effekt over tid i geonett. Generelt har bæreevnen til strekninger med geonett økt mer enn for strekninger uten geonett. Tilsvarende har G90-verdiene sunket mer for strekninger med geonett. For veger med dårlig undergrunn, tynn overbygning eller for forsterkningstiltak virker geonett som en god løsning. Om dette er en god løsning med tanke på frostsikring, miljøhensyn, økonomi og annet må diskuteres i hvert enkelt prosjekt. Alt i alt virker geonett som en god løsning der forholdene ligger til rette for det.

Bakgrunnen for denne masteroppgaven er at erfaringer viser at det er en utfordrende oppgave å gjennomføre kvalitetsvurderinger av gangfelt med etterfølgende forslag til tiltak, samt prioriteringer blant disse. Særlig bykommunene, med et meget stort antall gangfelt, står ovenfor betydelige utfordringer på dette området. I denne masteroppgaven er Trondheim benyttet som typisk eksempel på en større norsk by. Hensikten med denne masteroppgaven er å foreslå en metode for prioritering av hvilke gangfelt som bør utbedres først og hvilke tiltak som synes best egnet. Utvelgelsen baseres på en ulykkesanalyse over fotgjengerulykkene i Trondheim fra 2002 til 2011, samt kriteriene som stilles til gangfelt. Et sentralt grunnlag for arbeidet har vært å ivareta regjeringens mål om å bedre trafikksikkerheten og framkommeligheten på fotgjengernes premisser. Litteraturstudie, erfaringer fra andre byer, direkte observasjon og målinger, samt innsamling av eksisterende data er metoder som er benyttet for å besvare problemstillingen. Resultatet av ulykkesanalysen viser at barn er en utsatt trafikantgruppe. Fotgjengerulykkene foreslås derfor delt inn etter byens skolekretser. Samtlige gangfelt innenfor skolekretsene med flest fotgjengerulykker foreslås kvalitetsvurdert først. Gangfelt som ligger nær skole, som ikke er signalregulerte, har høyt fartsnivå, høy trafikkmengde, samt et høyt antall kryssende i makstimen anses som mest kritisk. Resultatet av kvalitetsvurderingene over gangfeltene som er valgt i denne masteroppgaven viser at samtlige gangfelt ikke tilfredsstiller kravene som stilles. Ulike problem som må løses er høy fart, lang kryssingsavstand, dårlig belysning, dårlig sikt, mangel på skilt/oppmerking, mangel på universell utforming, samt manglende vedlikehold. All erfaring og forskning viser det ubestridelige faktum om at nedsettelse av fartsgrensen gir størst effekt dersom målet er å bedre trafikksikkerheten og framkommeligheten på fotgjengerens premisser. For å oppnå ønsket fartsreduksjon anbefales utstrakt bruk av opphøyde gangfelt, da tiltaket skiller seg positivt ut. Det anbefales derfor at nedsettelse av fartsgrensene, på alle hoved- og samleveger i bolig- og sentrumsområder i Trondheim, gjennomføres.