SINTEF har en konsernsatsing innenfor systembiologi og dette omfatter blant annet forståelse og bekjempelse av biofilmer. Biofilm kan forårsake skader innenfor medisin, matindustri, havbruk og industri. På en annen side kan biofilmer utnyttes positivt innenfor for eksempel biologisk vannrensing.Formålet med denne oppgaven har vært å etablere relevante teknikker for å studere biofilm. Dette har omfattet utprøving av ulike dyrkingmetoder, samt studier av effekt av dyrkingsbetingelser på biofilmdannelse. Pseudomonas aeruginosa benyttes som modellorganisme i SINTEFs prosjekt, og arbeidet i denne oppgaven har vært utført med den samme organismen. Biofilm ble dyrket i brønnplater og i reaktorer. I tillegg ble det etablert metode for å kvantifisere mengde biofilm, samt etablert metoder for å studere biofilm ved hjelp av konfokalmikroskopi og for studier av genuttrykk ved RT-PCR analyser.Videre ble det valgt å studere produksjon av sekundærmetabolitten pyocyanin i P. aeruginosa. Det ble undersøkt hvilke vekstbetingelser som gir lav og høy produksjon. Disse betingelsene ble videre benyttet til å undersøke uttrykk av gener involvert i pyocyaninsyntese, både planktonisk (frie celler) og i biofilm. I tillegg har oppgaven inkludert undersøkelse av mediets effekt på biofilmdannelse, produksjon av pyocyanin og uttrykk av utvalgte gener. Disse genene involverer gener som inngår i biofilmdannelse, pyocyaninproduksjon og qourum sensing. Det ble valgt å benytte stammene P. aeruginosa PAO1 og P. aeruginosa PA14 i oppgaven. Stammene ble sammenlignet med hensyn på pyocyaninproduksjon, planktonisk vekst og biofilmdannelse.Etablering av teknikker for å studere P. aeruginosa biofilm viste at det ble mulig å studere biofilm i brønnplater og reaktor, men at forbedring og optimalisering av metodene er nødvendig. Det ble vist at P. aeruginosa PAO1 hadde større veksthastighet enn P. aeruginosa PA14. P. aeruginosa PA14 produserte mest pyocyanin og hadde høyest uttrykk av gener involvert i syntese av pyocyanin (phzM og phzS), både planktonisk og i biofilm. Forskjeller i biofilmdannelse ble vist ved at slimdannelse ble sett hos P. aeruginosa PAO1, mens hos P. aeruginosa PA14 ble aggregatdannelse sett.Både forsøk med planktoniske celler (frie celler) og biofilmforsøk viste at fosfatbegrensning inntrer tidligere ved dyrking av P. aeruginosa PAO1 enn ved dyrking av P. aeruginosa PA14. Fosfatbegrensning ga økt pyocyaninproduksjon, men nedsatt biofilmdannelse. Det ble vist at pyocyaninproduksjon initieres i tidlig stasjonærfase for vekst av planktoniske celler og i tidlig biofilmutviklingstrinn. RT-PCR-resultater har vist at genet phzS, som inngår i siste trinn i syntese av pyocyanin, varierer mer med dyrkingsbetingelser, enn det phzM, som inngår i tidligere trinn gjør.

AbstractWith the emergence of the nanotechnology industry, there has been a rapid expansion of different types and numbers of nanomaterials to be used in various applications. However, little is known of their potential to cause harmful effects on human health. Among other nanomaterials, carbon nanotubes, are found to harbor attractive characteristics that can be used in many applications. However, the same properties may cause harmful effects on human health that has raised serious concerns. The main route of exposure to carbon nanotubes is through the respiratory system with subsequent deposition in the lungs. Carbon nanotubes in contrast to other nanomaterials have fiber-like structures similar to the asbestos fibers. Exposure to asbestos has been associated with serious lung diseases such as fibrosis and cancer.A common type of carbon nanotubes called multiwalled carbon nanotubes has many valuable properties and several potential applications in different nanoproducts, manufactured commercially. It seems that the toxicological effects differ from product to product. Through this study potential harmful effects of two multiwalled carbon nanotubes designated as MWCNT-NO (produced in Norway) and MWCNT-JP (produced in Japan) have been investigated in vitro in normal human lung cells. The well known crocidolite asbestos was included to compare the effects with nanotubes. Hydrogen peroxide, a well known oxidative agent was also included since it has been hypothesized that carbon nanotubes may exert their effects through oxidative stress mechanisms. Cellular responses such as cytotoxicity, apoptosis and changes in expression of some inflammatory and apoptotic genes were studied. The results of the cytotoxicity assays (WST-8 assay) indicated a reduction of cell viability for carbon nanotubes and crocidolite asbestos, depending on the dose and time of exposure. However, MWCNTs, especially MWCNT-JP, were more toxic than crocidolite asbestos. For the hydrogen peroxide, the reduction in cell viability tended to depend only on the dose. Little differences between the two cell-lines were observed. Hoechst/PI staining revealed that cell death occurred essentially by necrosis and not apoptosis following exposure. Exposure to MWCNT-NO resulted in an increased expression of the IL1B, IL8 and IL6 inflammatory genes (qRT-PCR). This differed from the MWCNT-JP type where little changes in the expression were observed. Some differences between the two cell lines were also observed. The overall potential of the tested carbon nanotubes to cause harmful effects in normal human lung cells needs further verifications with improved particle characterization.

Bakgrunnen for prosjektet var behovet for å etablere en metode for høykapasitetsanalyser (mange prøver – få målinger) av proteiner, på innsamlet materiale fra norske biobanker.Norsk mikromatrisekonsortiums node ved NTNU har tidligere utført en serie piloteksperimenter for å avklare om mikromatriseformatet kan brukes til dette formålet. Resultatene var lovende, og gav et grunnlag for videreutvikling. Grunntanken var å trykke (“spotte”) serumprøver på mikromatriseslides og deretter merke disse med et antistoff for å registrere dette med et reportermolekyl. Dersom en på samme slide trykte prøver/standarder med kjent mengde antigen ville disse kunne fungere som en standardkurve og en fikk et kvantitativt mål på mengden av antigen i de ukjente serumprøvene. Målet var å kunne trykke opptil 20.000 serumprøver per mikromatrise og på denne måten analysere et stort antall prøver til en lav kostnad.Det ble valgt å bruke Prostata spesifikt antigen (PSA) som serumprotein. Dette fordi PSA er viktig i sammenheng med screening for prostatakreft, og var en utfordring da det er såpass lite av proteinet i serumet til menn. Dersom en fikk etablert mikromatrisebaserte målinger av PSA ville svært mange andre proteiner som finnes i høyere konsentrasjoner sannsynligvis være lett tilgjengelig for metoden.De nåværende resultatene viser en lineær kurve for gjennomsnittsverdiene av signal for hver PSA-konsentrasjon, men metoden er ikke presis nok. På dette tidspunktet er det ikke mulig å se forskjell på prøver med forhøyede PSA konsentrasjon og prøver med PSA konsentrasjon i normalområdet. Den dynamiske rangen må forbedres ved å minske egenfluorescensen og få sterkere prøvesignaler. For å få til dette er det ønskelig å fjerne amplifiseringstrinnene da amplifisering medfører en usikkerhet som gjør det vanskelig å vurdere resultatene. Metoder som kan gjøre dette mulig er bruk av flere antistoffer som er rettet mot forskjellige epitoper i en kombinasjon med for eksempel Quantum dots.

Lung cancer is the leading cause of cancer related deaths world wide. The major cause of lung cancer is tobacco smoking. Tobacco smoke contains many different carcinogens including polycyclic aromatic hydrocarbons (PAH), which are known to cause DNA damage and initiate tumourigenesis.The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix protein of the PER-ARNT-SIM (PAS) family of transcription factors, which is involved in many biological processes including development, cell cycle regulation, cell differentiation, cell-cell signalling, cellular growth, and circadian rhythms. Most of the known function of AHR, however, is regarding its role in transcriptional induction of xenobiotic metabolis- ing enzymes (XMEs). In the lung, PAHs bind and activate the AHR, leading to transcriptional induction of members of the cytochrome P450 (CYP) enzyme family that are involved in activation of pro-carcinogenic PAHs to carcinogenic metabolites.MicroRNAs (miRNAs), a group of short non-coding RNA molecules, mediate sequence specific silencing of messenger RNAs (mRNAs) and are important in regulation of gene expression. The deregula- tion of certain miRNAs has been observed in several types of cancer, including lung cancer. Like mRNAs, miRNAs belong to the class II type of genes, and transcription of these types of RNA can therefore be regulated by the same transcription factors.Little is known regarding the role of miRNAs in environmental toxicology. The aim of this thesis was to identify miRNAs regulated by the AHR, and consequently may be important in mediating toxicity of tobacco smoke carcinogens.Expression levels of 750 miRNAs in mouse lung of Ahr wild-type and knockout animals were pro- filed by RT-qPCR arrays. Fifteen miRNAs were found to be differentially expressed between the groups, of which only one was up-regulated. The biological pathways in which these miRNAs may be involved were studied by an bioinformatic approach. Four miRNAs from the mouse experiment, whose expression were most significantly divergent, together with three miRNAs previously reported to be regulated by the Ahr in murine cell lines, were studied in an RNA interference (RNAi) experiment in immortalised human lung epithelial cell lines. Two of these miRNAs, both selected from the literature, were found to have altered expression in AHR knockdown cells compared to control cells. Discrepancies between AHR regulated miRNAs identified in the in vivo and in vitro experiments were observed. Taken together this suggest a difference between effect of the AHR in vivo and in vitro, rather than differences between species.In conclusion, it was found that expression of several miRNAs possibly is regulated in an AHR- dependent manner in lung. Discrepancies between in vivo and in vitro experiments suggest that care must be taken when extrapolating miRNA expression data from cell culture studies to whole organs or organisms.

As a part of cancer research and therapy, it is important to study metabolic responses caused by DNA damaging agents. Whereas earlier studies have focused on changes at protein levels caused by DNA damaging agents, this project focuses on establishment of sampling and cultivation protocols for metabolome analysis, using Saccharomyces cerevisiae as a model organism growing in exponential phase. Sampling and cultivations protocols were optimized before cultures were stressed by DNA damaging agent. At the time of sampling, cells and extracellular media were separated by filtration prior to quenching and extraction in 75% boiling ethanol. Solvent evaporation under reduced pressure and ambient temperature were used for metabolite concentration. Yeast cultures were exposed to osmotic stress and two concentrations of the DNA damaging agent 5-fluorouracil. Samples were taken prior to stress exposure, at the time of exposure and in intervals after stress exposure. Metabolite extracts were analyzed by GC-MS single quadrupole in respect of amino acids, citric acids cycle intermediates and fatty acids. Generally, citric acid cycle intermediates were most influenced by stress agents, followed by amino acids and fatty acids. GC-MS single quad was the only analytical method applied. For a more complete metabolic profiling, including other groups of metabolites, LC-MS/MS should be applied. This analytical method will cover groups, such glycolytic intermediates, which can not be analyzed by GC-MS, due to their volatility.

Multiple myeloma (MM) is a malignancy of antibody producing plasma cells located in the bone marrow. MM is also called bone marrow cancer and is the second most common hematologic cancer. The exact cause for this type of cancer is still unknown and the disease is today generally thought to be incurable. One hallmark of MM is the degradation of bone. Bone morphogenetic proteins (BMP) are a group of signaling molecules that have multiple roles in normal and malignant cells. BMPs regulate growth, differentiation and apoptosis in myeloma cells, but also have a role in the induction of bones. This means that BMPs have the potential to inhibit the growth of cancer cells and reduce the degradation of bones. In this thesis BMP-9 was studied by looking at how it affects apoptosis and proliferation in different types of human myeloma cell lines (hMCLs). Experiments were also performed to see if any of the common BMP antagonists had an effect on this protein. Efforts were also made to identify by which receptors BMP-9 signals in myeloma cells and which downstream signaling pathways are activated. It was found that BMP-9 induced apoptosis and/or inhibited proliferation in several hMCLs. The SMAD pathway was activated by BMP-9 in myeloma cells, and BMP-9 most likely uses ALK-2 as Type I receptor and ACVR2A or ACVR2B as Type II receptor in these cells. The antagonists CHL-1 and Tsg, the Type III receptor endoglin and the SMAD inhibitor DMH1 were found to interfere with BMP-9 signaling. Another interesting discovery was that CpG-ODN did not have the same inhibitory effect on BMP-9 as has been shown on other BMPs. Further experiments have to be done to investigate what makes BMP-9 different from other BMPs in this regard.

For å undersøke faktorer som innvirker på alginat-produksjonen til Azotobacter vinelandii har det blitt laget et mutant-bibliotek med stammen A. vinelandii ATCC 12518 som utgangspunkt. Etter screeningsforsøk med henblikk på vekst- og alginatproduksjon av mutant-stammene ble det plukket ut et antall opp- og nedmutanter som ble undersøkt videre for å bestemme hvilke gener som var muterte og deres funksjon i forbindelse med alginatbiosyntesen i A. vinelandii.Denne oppgaven startet med å undersøke 17 nedmutanter fra screening-undersøkelse av biblioteket. Det ble satt opp vekstforsøk med alginatessay i to runder.Da utvalgte mutanter ble sekvensert for å finne ut hvilke gener som hadde transposoninsersjon viste det seg at transposon-vektoren pCAM140 var tilstede i alle stammene. Denne delen av oppgaven ble avbrutt og strategien for oppgaven noe endret. Det ble plukket ut to stammer som var kjente ned-mutanter fra screeningsforsøk og som allerede var sekvensert. Stammene 20A6 og 21T3C ble undersøk for å bekrefte fenotype og forsøkt komplementert for å bekrefte/undersøke videre funksjon av gener. 20A6 har mutasjon i genet for sucA (Avin_29770) som sannsynligvis kotranskriberes med sucB og lpdA. Denne stammen viste seg å ha både vill-type alleler og mutant-alleler tilstede, selv etter langvarig forsøk på seleksjon for mutant-alleler. Stammen 21C3T har mutasjon i Avin_13880, et gen som koder for en transkripsjonsregulator av IclR-klasse. Komplementering gav ikke stammene forventet alginat-produksjon tilbake. Det ble også gjort bioinfomatiske undersøkelser og litteraturstudier for å forsøke å klarlegge hvilken funksjon de ovennevnte genene har i alginatsyntesen i A. vinelandii.

The purpose of this thesis was to investigate the transfection efficiencies of chitosan- siRNA nanoparticles in the two rat brain-derived cell lines C6 and RBE4. Furthermore, the main goal was to silence P-glycoprotein (P-gp) expression in these cell lines and to assess the possibility of implementing them in a future glioma-blood- brain barrier (BBB) model in vitro. Finally, previously developed serum-stable chitosans were screened for transfection efficiencies in the RBE4 cell line to evaluate their potential in in vivo applications.Linear (LIN350) and self-branched (SB300) chitosans were tested for their transfection efficiencies in the C6 glioma cell line. Uptake of the nanoparticles was measured using fluorescent siRNA by flow cytometry. Knockdown of GAPDH was measured using a GAPDH protein activity assay, and P-gp knockdown was measured by flow cytometry using a Rhodamine123 (R123) efflux assay.The uptake efficiency was optimal at N/P ratio 30 and with a siRNA concentration of 100 nM. SB300 was more cytotoxic than LIN350, as indicated by the decrease in GAPDH protein activity when using negative control siRNA. It was found by reverse transcriptase PCR (RT-PCR) that the C6 cells did not initially express the Abcb1a gene, but expression was initiated after several passages in cell culture. The results show that the C6 cell line has the potential to be implemented in a future glioma-BBB model, however there is a need for optimization of the transfection when using chitosan-siRNA nanoparticles for P-gp silencing in this cell line.Rat Brain Endothelial (RBE4) cells were used as an in vitro BBB-model, and several chitosans were investigated in regards to transfection efficiencies in this cell line. LIN350 and SB300 mediated efficient transfection at N/P 30 and a siRNA concentration of 100 nM. An uptake kinetics experiment was also carried out showing that the siRNA is removed within two days from RBE4 cells when using these chitosans. From a P-gp knockdown kinetics experiment measuring R123 efflux, it was found that repeated transfection with LIN350-based nanoparticles prolonged the knockdown of P-gp. LIN350 was used for transfecting confluent RBE4 cells; mediating knockdown efficiencies of GAPDH comparable to non-confluent cells. This indicates that RBE4 cells could be used as endothelial cells in a future glioma- BBB model.In addition to LIN350 and SB300, several modified chitosans with increased serum stability ((DP85 (4AM), Fa=0 (2% PEG) and chitosans coated with hyaluronic acid (HA)), developed for efficient in vivo siRNA delivery were screened for their in vitro transfection efficiencies, measuring uptake, GAPDH protein activity and R123 efflux. The serum-stable chitosans mediated high uptake but did not produce RNA interference (RNAi), indicating that these chitosans need further modifications before they can be utilized for in vivo experiments.It was found by transmission electron microscopy (TEM) that nanoparticles with LIN350, LIN350 w/HA, DP85 (4AM) and Fa=0 (2% PEG) were internalized by the RBE4 cells in vesicles larger than 500 nm, indicating that macropinocytosis is a highly possible uptake mechanism utilized by these chitosans. LIN350 was the only chitosan among these formulations that mediated RNAi, and the TEM showed that this chitosan is taken up in large aggregates, possibly bursting the vesicles by osmotic swelling.

Color in fish products is sometimes used as quality index. Behind the development of color in fish product are a series of chemical reactions as protein and lipid oxidation as well as Maillard reactions. Transition metals and the composition of the muscle influences can triggered such reactions. The complexity of such reactions is higher when the proteins suffer denaturation due to salting processes. Salt also influences in the degree of oxidation.In this project, changes in color of two fish products were studied: salted and smoked herring fillets and klippfish. Protein oxidation was measured as carbonyl groups. Lipid oxidation was measured as peroxide value (PV) in salted and dried herring and thiobarbituric acid quantification (TBARS) in fully salted cod. In order to determine discoloration, browning intensity was measured.In the salted and smoked herring fillets the process to obtain the desired color is very expensive. To achieve a golden brown color is necessary to smoke the fillets for a long period. It was added transition metals to the salting brine resulting in higher oxidation. The use of glucose and xylose as alternative to sucrose was investigated resulting in even higher browning intensity (xylose>glucose>sucrose). This study shows that the use of xylose or glucose as an alternative to the sucrose or the addition of prooxidants as transition metals can optimize the process reducing the time the fillets need to be at the smoking process.In the klippfish part, fully salted cod cubes were prepared to simulate the salted process. Cod cubes were divided in dark and white muscle. Copper and heme were added to the salting brine. The cod cubes were brined and then kench cured. Protein and lipid oxidation were measured after each salting process. The results show that copper and heme work as prooxidant and may be the responsible of discoloration of klippfish. Copper can be found in salt impurities, thus its importance. The other important factor in the klippfish process is removing all residues of blood or viscera.

Abstract BACKGROUND: Arthritis and other rheumatic conditions are a growing health problem, in terms of prevalence, disability and cost. Rheumatism is classified as chronic, systemic, and autoimmune diseases. Major symptoms are synovial inflammation and swollen joints, autoantibody production, deformation of cartilage and bone structures, and systematic features such as cardiovascular, pulmonary, psychological and skeletal disorders. The mortality rate in patients with rheumatoid arthritis (RA) is 1.5 – 1.6 compared to that of the general public, and cardiovascular diseases (CVD) account for 40-50% of the deaths. In addition, a five-fold increased CVD-risk, was observed in female patients with RA who were diagnosed at a young age. Scientific studies have shown that aerobic interval training of high intensity is effective in improving general physical status and cardiovascular health, but is not yet used as a treatment option for people with rheumatism. The aim of the present study was to find out if ten weeks of high intensity interval training was well tolerated by people with rheumatic diseases, and if it would improve activity of the disease, quality of life, and traditionally risk factors for cardiovascular diseases. METHODS: Eighteen women with RA and juvenile idiopathic arthritis (JIA), aged 20-50 years, were recruited to this cross-over study. Participants performed supervised interval training 2 times a week for 10 weeks on a spinning bike. The exercises consisted of four 4-minute intervals at ~90% of maximal heart rate (HRmax), interspersed with 3 minute recovery periods at ~70% of HRmax. Maximal oxygen uptake (VO2max), HRmax, one minute heart rate recovery (1-minHRR), blood pressure, body composition, blood analysis, molecular status (TNF-α, Interleukin-6 (IL-6), Human Cartilage Oligometric Matrix Protein (COMP) and Pentraxin-3), disease activity status, and questionnaires (Visual analogue scales (VAS), Modified Stanford Health Assessment Questionnaire (MHAQ), and SF-35) were measured before and after both exercise and control periods. Statistical analyses were performed using the software program SPSS. RESULTS: As a consequence of high intensity interval training (HIIT), the results showed that; VO2max increased by 12.2% (p < 0.001), 1-minHRR decreased by 3.7% (p = 0.02), BMI, body fat, visceral fat and waist circumference decreased by 1.2% (p= 0.04), 1.0% (p= 0.05), 0.2% (p= 0.08) and 1.6% (p=0.004), respectively, whereas muscle percentage increased by 0.6% (p=0.03). Significant differences were found for serum glucose (increased 6.3%, p=0.05), haemoglobin (decreased 2.2%, p= 0.03) and ferritin (decreased 24.0%, p= 0.006). High sensitive CRP (hsCRP) level decreased by 41.9% (p= 0.08). IL-6 mRNA increased by 32.6% (p=0.02), COMP levels increased by 12.6% (p=0.06), and Pentraxin-3 levels decreased by 27.3% (p=0.14). MHAQ score decreased by 24.6% (p=0.13), self reported global health VAS-score decreased by 33.8% (p=0.13), and pain VAS-score decreased by 34.8% (p=0.12). The participants scored themselves better for bodily pain and emotional role in SF-36. CONCLUSION: Ten weeks of HIIT was well tolerated by females with RA and JIA, and improved the disease activity, quality of life, and traditionally risk factors for cardiovascular diseases. HIIT might be recommended as a safe treatment for persons with RA and JIA.