Iron nanoparticles occur naturally in the environment, but their exposure increases dramatically due to the field of nanotechnology and –medicine. It is poorly understood how the intracellular cooperative mechanisms of submicron particles and microorganisms function on mammalian immune system. In this study, superparamagnetic iron oxide (SPIO) submicron particles will be used to benefit the research within environmental diseases, addressing the biocompatibility of these particles. The size-dependent effects in the immune system of two carboxyl coated SPIO particles with stated sizes 100 nm and 1 µm will be studied in vitro. It would be interesting to determine whether these particles were able to activate the inflammasome, but still, the precise molecular mechanisms for the activation remain unknown. In order to reveal the biocompatibility of these particles, tests were performed as a function of particle concentration ranging from 0.01 to 100 µg/mL using both whole blood and peripheral blood mononuclear cells (PBMC) isolated from healthy donors. The monocytes were first primed with Lipopolysaccharide from Escherichia coli 0111:B4 strain, followed by stimulation with increasing concentrations of the submicron particles. Flow cytometry on whole blood samples identified up-regulation of CD11b monocytes and granulocytes by the particles. In addition, Terminal Complement Complex analyses proved activation of the complement system. It is possible that the particles have been coated with C3b by the complement and phagocytized by the monocytes through CD11b/CD18 receptor. Cytokine secretion from monocytes and whole blood was measured with sandwich ELISA and Bio-plex. The smaller particles seemed to induce higher inflammatory responses than the larger ones. It was, however, interesting to find that the particles themselves caused secretion of active IL-1β without being primed in advance. The mechanisms of the NLRP3 inflammasome activation might be explained by ROS production due to iron imbalance in the cytoplasm. Toxicity of the particles was seen at 10 µg/mL, suggesting their potentially low biocompatibility above this concentration. However, it is suggested better biocompatibility of the silica coated 1 µm particles than the polysaccharide coated 100 nm particles.

En transdermal leveringsform gir store fordeler framfor oral, intramuskulær og intravenøs levering, men byr også på noen utfordringer. Den største utfordringen er at kun et begrenset antall molekyler kan administreres på denne måten. Hudens øverste lag, stratum corneum, er en effektiv barriere, som bare noen molekyler kan penetrere. Disse molekylene er karakterisert som lavmolekylære (≤ 500Da) og lipofile (hydrofobe). Det er gjennomført forsøk på fullskala hud fra mennesker, for å undersøke om vannløselige (hydrofile) molekyler som fiskegelatinpeptider og G-blokk vil kunne penetrere huden og diffundere ned i dermis. G-blokk og fiskegelatin ble først fraksjonert for å lage mest mulig monodisperse prøver, og deretter fluorescensmerket med alexa 488/532 fluorokrom. De fluorescensmerkede prøvene ble løst i 60 % dimetyl sulfoksid (DMSO), før de ble påført epidermis-siden av hudbiter montert i Franz-celler. Huden ble først forbehandlet med mikronåler. Konfokal laser skanning mikroskop ble benyttet for å undersøke fluorescensintensiteten i vevet, og dermed penetrasjonsevnen til de ulike G-blokkene og fiskegelatinpeptidene. Ved hjelp av hyperspektral avbildning ble det også undersøkt fluorescensintensitet i tillagde hudfantomer. I oppgaven er det lagt vekt på hvordan penetrasjonsevnen til ulike molekyler avhenger av molekylvekt, merkningsgrad, forbehandling (mikronåler og DMSO) og individuelle forskjeller mellom donorer. Det ble også undersøkt hvor lang tid det tar før det observeres en betydelig mengde fiskegelatinpeptider i vevet.Noen G-blokker og fiskegelatinpeptider ble funnet til å kunne penetrere huden. Det ble påvist at en forbehandling med mikronåler og 60 % DMSO som vehikkel, øker permeabiliteten til huden. I tillegg ble det observert store variasjoner i penetrasjonsevnen til prøvene når forsøk ble utført på samme eller ulike donorer. Fiskegelatinpeptider ble vist til å begynne å penetrere forbehandlet hud allerede etter 8 timer, men først etter 18 timer inkubasjonstid ble det observert en betydelig mengde fiskegelatinpeptider i vevet. Det ble funnet en sammenheng mellom fraksjon høymolekylære peptider i fiskegelatinprøvene og fluorescensintensitet i vevet etter endt inkubasjonstid (24 timer). Det ble observert høyere fluorescensintensitet i vevet etter påførte prøver med høyt innhold av lavmolekylære peptider, enn for prøver med flere høymolekylære peptider. Det ble også observert en sammenheng mellom merkningsgrad og detektert penetrasjonsevne. Der prøver med høy merkningsgrad ga høyere fluorescensintensitet i vevet og fluorescens i reseptorfasen, enn prøver med lavere merkningsgrad. I tillegg ble det observert lite eller ingen elektrostatisk binding av fluorescensmerkede prøver av fiskegelatin og G-blokk til pappilær dermis, mens stratum corneum ble observert som den store barrieren i huden ved at den hadde høyest fluorescensintensitet. Fiskegelatinprøvene ble funnet veldig polydisperse selv etter fraksjonering ved hjelp av dialysemembraner med ulik størrelse. Det er observert liten sammenheng mellom molekylvekts cut-off på dialysemembran, og faktisk størrelse av peptider som trekker ut.Hyperspektral avbildning ble funnet til å fungere for hudfantomer med mer enn 25 mg kovalent merket gelatin.

Alginate scaffolds show good promise for bone tissue engineering using stem cells. This is due to the fact that alginate is biocompatible, non-immunogenic, and in addition may direct differentiation of stem cells into a given phenotype. Finally, due to their ability to gel at physiological conditions, living and functional tissue are easily encapsulated into alginate beads. Alginate beads can be modified in a range of ways, not only to enhance the matrix stiffness and stability, but also to promote cell adhesion and direct differentiation towards a given phenotype. A method currently used to encourage bone growth is to mineralize the alginate beads, thus mimicking the structure of bone in vivo. Recently, Xie et al. (2011) demonstrated that enzymatic mineralization of alginate beads serves as a potent method for mineralizing beads as lower concentrations of CaCl2 is needed, which is beneficial for cell viability. In addition, the enzymatic method produces alginate beads with a uniformly distribution of calcium phosphate (CP) and stiffer mechanical properties. Mesenchymal Stem cells (MSCs) have the potential to differentiate into a variety of tissues, including cartilage, adipose, muscle and bone. MSCs extracted from bone marrow seem to possess the greatest potential for bone tissue engineering, as they are more easily differentiated into osteogenic phenotypes when compared with MSCs from e.g. adipose tissue. The main objective in the present study was to encapsulate MSCs into alginate beads mineralized with alkaline phosphatase (ALP) and study cell survival, as well as their potential to differentiate into mature osteoblasts inside the beads. To mineralize beads (ALP) was added to the alginate solution, whilst the precursors were added to the growth medium. The mineralization medium was changed every 3 hours (12 hours over night) for the first 2 days post encapsulation.Cell viability was surveyed by live/dead assay and imaging by Confocal Laser Scanning Microscopy (CLSM), and metabolic activity by Alamar Blue (AB) and colorimetric techniques. Examination of cell morphology was accomplished by phalloidin/DRAQ5 staining. Moreover, to investigate cell differentiation, PCR analysis on selected RNA candidates was performed. Quantification of mineral content was accomplished by running Alizarin Red-S (ARS-S) assay. ALP activity was determined using ALP assay. Finally, further investigation of cell- and alginate matrix structure was accomplished using scanning electron microscopy (SEM). To study capsule properties and cell survival, osteosarcoma cells were utilized as model cells. The main objective was to study bead stability, and how the beads and the cells within were affected by the enzymatic mineralization method. Alginate beads proved to be unstable and needed addition of CaCl2 for stabilization purposes. Furthermore, in order to recover cells, citrate was added to the cell/bead suspension. Initially, results were unsatisfactory, as little, or no, RNA was recovered. After optimizing the citrate treatment it was discovered that a sequential method needed to be utilized. Results demonstrated a successful recovery of cells, and RNA of excellent quality.Enzymatic mineralization of alginate beads was found to be a cell friendly way of mineralizing alginate beads. Mineralization of beads was observed in the light microscope, by visual inspection, as well as by SEM. Cell viability remained high when using a concentration of 0.25mg/ml ALP. The osteosarcoma cells proved to be good for optimizing the enzymatic mineralization method, but behaved differently compared with mesenchymal stem cells in terms of viability, and mineralization activity. Furthermore, mineralization of beads by addition of 0.25 mg/mL ALP compared with 0.5mg/mL appeared to be beneficial as image acquisition on CLSM was facilitated and slightly higher viability of cells was observed.In the second part of the present study, human MSCs were encapsulated into alginate beads. After initial experiments the optimal condition for cell survival, and bead stability was determined. Consequently, in the final experiment 16 million MSCs were encapsulated in beads containing 0.25mg/mL ALP, together with a sample without addition of ALP. At day 2 post encapsulation both samples were divided into two batches, one cultured in regular medium, and one in differentiation medium. All samples were stabilized with 7.5 mM of CaCl2.Observations in both light microscope and CLSM revealed that only beads given ALP were mineralized before reaching day 21. At day 21 the sample receiving no ALP, cultured in differentiation medium also appeared mineralized.Mesenchymal stem cells receiving differentiation medium were observed to differentiate into mature osteoblasts in the beads. This was verified by gene expression analysis, cell morphology studies, the presence of collagen in beads seen by SEM and analysis of ALP activity. Metabolic activity measurements confirmed little cell proliferation, nor cell death. However, an increased metabolic activity was observed for encapsulated cells cultured in regular medium relative to cells in differentiation medium. Cell morphology in differentiated samples was recognized by showing elongated actin filaments, compared with the ones cultured in regular medium, which appeared round in shape. The elongated filaments suggest that the cells are able to interact with the alginate matrix and/or minerals. The occurrence of collagen fibers in SEM images further confirmed presence of mature osteoblasts. Samples cultured in regular medium with or without added ALP both showed an increase in osterix expression until day 21 when the study was ended. This was surprising, as it inferred that the alginate matrix itself might influence differentiation of MSCs into osteoblasts, and that the minerals have little effect on differentiation. Runx2 expression was detected in all samples, including unencapsulated hMSCs. The expression of runx2 was at its maximum on day 21, when the study was ended.Encapsulating mesenchymal stem cells into alginate beads mineralized by the enzymatic method is cell friendly, and allows the cells to differentiate into mature osteoblasts when cultured in differentiation medium. Alginate without minerals seems to influence differentiation to a certain extent, suggesting that minerals are not needed for differentiation to occur. The minerals do, nonetheless, speed up the continuing mineralization process.

Saccharomyces cerevisiae, commonly known as Baker’s yeast, is a eukaryotic model organism widely used in biotechnology research. Its genome has a high degree of similarity to humans, and research done on S. cerevisiae can give us a better understanding of the mechanisms involved and the cellular responses to anti-cancer drugs. Yeast is therefore usefool in increasing the effectiveness of anti-cancer drugs.The main goal for this master thesis was to investigate the metabolic response of S. cerevisiae to DNA damaging compounds. Methods for cultivation of S. cerevisiae in shake flasks or in a bioreactor, for sampling and extraction of cells, and for analysis of the sample using GC – MS were developed. A custom Deconvolution Reporting Software library was created, and used to analyze the metabolic response of S. cerevisiae to 5-fluoruracil (5FU), a widely used anti-cancer drugs. The publicly available software FiatFlux 1.6X was used for metabolic flux analysis of S. cerevisiae grown in medium containing labeled [U-13C]-glucose and in the presence of 5-fluorouracil or methyl methanesulfonate (MMS), a model alkylating agent.An overall decrease in the amount of intracellular metabolites was observed as a response to addition of a growth-inhibiting dose of 5FU, while no clear response was observed for a lower, more tolerable dose. Growth of S. cerevisiae under the presence of 5FU resulted in a different cellular response than growth under the presence of MMS. The presence of both DNA-damaging agents resulted in a decrease in the ratio of the fluxes leading to mitochondrial acetyl-CoA compared to an unstressed culture, possibly indicating a reduction in the activity of the TCA cycle. The 5FU stressed cells showed an increase in conversion of fumarate to oxaloacetate and the subsequent export out of the mitochondrion, while the MMS stressed cells showed a decrease of this flux.The experimental methods can be further refined to obtain data of higher quality, and thus allow a more complete metabolic profile to be created. The conditions for derivatization can be optimized to ensure a complete derivatization of all metabolites, the DRS library can be expanded to include a wider range of metabolites, and experiments of stressing the cells performed in a chemostat can eliminate any effects the differences in specific growth rates might have on the metabolome or fluxome of S.cerevisiae.

Produksjon av aminosyrer ved hjelp av industriell bioteknologi er et voksende marked. I dag finnes det ingen kjente metylotrofe bakterier som benyttes til industriell produksjon av aminosyrer, men en attraktiv kandidat for framtidig produksjon er den termofile og metylotrofe bakterien Bacillus methanolicus.For å kunne utvikle en konkurransedyktig produksjonsstamme av B. methanolicus er det nødvendig med genetiske verktøy. Et viktig verktøy er et reportergen som blant annet kan utnyttes for å analysere promotere. Et av hovedformålene med denne oppgaven var å teste reportergenet lacZ som stammer fra den termotolerante bakterien B. coagulans, og deretter utvikle et assay for bestemmelse av aktiviteten til genproduktet β-galaktosidase. Dette reportergenet ble også brukt til å analysere styrken til ulike promotere i B. methanolicus MGA3. Promotorene som ble undersøkt (som også stammet fra B. methanolicus) var promotere til viktige gen i metanolassimileringen (mdhp og pfkp) og den potensielle promoterregionen til et metanoldehydrogenase-gen (mdh2p). I tillegg ble det testet en kobberinduserbar promotor (Cup) fra Lactobacillus sakei. I denne oppgaven ble også det antatte replikasjonsproteinet til den potensielle vektoren pBM69 undersøkt. pBM69 ble oppdaget i B. methanolicus MGA3 etter at genomet ble sekvensert. Det ble konstruert vektorer hvor de aktuelle Promotorene var lokalisert oppstrøms for reportergenet lacZ, deretter ble B. methanolicus transformert med vektorene. Den første bekreftelsen på at reportergenet var aktivt i B. methanolicus kom under transformasjon, hvor B. methanolicus transformert med vektorene pTH1mdhp-lacZ og pTH1Cup-lacZ ga opphav til blå kolonier da kulturene ble platet ut på fast medium tilsatt X-gal. B. methanolicus transformert med pTH1mdh2p-lacZ og pTH1pfkp-lacZ ga ikke blå kolonier.Utvikling av assayet ble utført med celleekstrakt fra B. methanolicus MGA3 transformert med pTH1mdhp-lacZ. Aktiviteten ble undersøkt ved ulike betingelser for å optimere assayet. Det ble testet ulike pH-verdier for bufferen og det ble undersøkt hvordan preinkubering av enzymet påvirket aktiviteten. Den optimale pH-verdien for assayet viste seg å være rundt 7 og det ble funnet en signifikant økning i enzymaktiviteten når enzymet ble preinkubert i fem minutter ved temperaturene 45, 50 og 60 °C. Enzymaktiviteten holdt seg også stabil etter preinkubering med de gitte temperaturene i inntil 30 minutter. Etter etablering av assay ble det ved alle målingene benyttet buffer med pH 7,0 og enzymet ble preinkubert i 5 minutter ved 50 °C.Den kobberinduserbare promotoren var aktiv i B. methanolicus MGA3. For å undersøke hvordan ulike konsentrasjoner av induseren CuSO4 påvirket veksten til B. methanolicus pTH1Cup-lacZ, ble det utført forsøk hvor kulturene ble indusert med konsentrasjoner fra 0 µM til 200 µM av CuSO4. Det var tydelig at veksten ble påvirket med økende konsentrasjon av CuSO4 og ved 200 µM døde cellene. De resterende kulturene ble høstet etter hvert som OD600 = ca 1,5 og enzymaktiviteten til β-galaktosidase i hvert celleekstrakt ble bestemt. Aktiviteten for hver kultur økte med økt konsentrasjon av induser, men det var vanskelig å sammenligne aktivitetene ettersom kulturene ble høstet etter ulik inkuberingstid. I celleekstrakt fra B. methanolicus transformert med vektorene pTH1pfkp-lacZ og pTH1mdh2p-lacZ var det ikke mulig å detektere aktiviteten til β-galaktosidase. Reportergenet lacZ som ble undersøkt i denne oppgaven er det første reportergenet som har gitt gode resultater i B. methanolicus. Det etablerte assayet kan blant annet benyttes til å undersøke nye promotere i B. methanolicus og dermed øke kunnskapen om regulering av transkripsjon. Av Promotorene som ble undersøkt var mdhp den sterkeste, noe som var forventet på bakgrunn av tidligere eksperimenter. Kobberpromotoren var aktiv, men ga opphav til enzymaktivitet selv om den ikke var indusert. Det må dermed utføres flere forsøk med denne promotoren for å avgjøre hvor godt den er egnet til kontrollert ekspresjon i B. methanolicus. Replikasjonsproteinet til pBM69 ble undersøkt, men på grunn av ustabilt DNA som førte til at deler av den amplifiserte sekvensen ble slettet etter transformasjon, var det ikke mulig å bekrefte om pBM69 faktisk er et plasmid.

Prior to this project, a high throughput assay was developed in order to perform automated RNAi screens with siRNA- libraries targeting potential regulators of the G2 checkpoint. The libraries were covering the human kinases, phosphatases and DNA repair. The aim of this project was to validate the candidate hits from the phosphatome screen as possible G2 checkpoint regulators. To validate the candidate hits, esiRNAs were applied in order to down regulate the target proteins, and G2 checkpoint abrogation was assayed by flow cytometry analysis. To confirm that the assay did work, the effects of inhibiting WEE1 by the small molecule inhibitor, MK1775, were tested in both U2OS and U2OSp53 cells. Both the early and the late G2 checkpoint were tested for.WEE1 was a hit in the kinome screen, and a well-known regulator of the G2 checkpoint. The samples treated with MK1775, and 6Gy were showing a dose-dependent abrogation of the G2 checkpoint when stained for H3-p and analyzed by flow cytometry.In optimization experiments for esiRNA transfection, Effectene was chosen over Oligofectamine as the preferred transfection reagent. The transfection conditions which were decided to be the most efficient in down regulating the target protein were 5μL esiRNA and 10 μL Effectene. For the validation experiments it was focused on the two phosphatases SSH3 and PTPN7. Western blotting analysis showed that the protein level of SSH3 was reduced to less than 50% at two days following transfection with SSH3 esiRNA.The validation experiment did not show any abrogation of the G2 checkpoint by neither SSH3 nor PTPN7 in irradiated cells. More repetitions of the experiment are needed in order to validate- or reject these as false hits. However, the results from a whole-genome DNA damage response siRNA screen were recently published. Neither PTPN7 nor SSH3 were scoring in this screen. These facts are supporting the results that SSH3 and PTPN7 are false hits.

Dette prosjektet ble satt i gang på grunnlag av et ønske om en ny metode for analyse av flyktige forbindelser i marine oljer. En ønsker å se på hvilke flyktige forbindelser som finnes i oljen under normale forhold. Det er da viktig at det ikke dannes degraderingsprodukter som følge av metoden. Ved Short-Path Distillation, SPD, opereres det med lavt trykk, helt ned til 0,001 mbar. Dette gir en lavere fordampningstemperatur slik at flere, og tyngre forbindelser kan destilleres av oljen ved en lavere temperatur. Muligheten for å separere oksidasjonsprodukter fra marine oljer ved SPD og innvirkning av fødehastighet og oppholdstid på mengde forbindelser som destilleres av oljen er studert. Som råstoff ble sildolje produsert ved ulik prosess (termisk behandlet og hydrolysert) benyttet, i tilegg til en fiskeolje bestående av 12 til 15 % sild. I forprosjektet til denne oppgaven ble antioksidanten BHT identifisert i sildoljen produsert ved hydrolyseprosess. Sildoljen var ikke tilsatt antioksidanter. Det er derfor lagt vekt på å studere hvordan denne forbindelsen er havnet i oljen og i hvilken konsentrasjon den er tilstede. Råoljen samt retentat, destillat og kondensat etter separasjon i SPD er analysert ved mengde konjugerte forbindelser, peroksidverdi, anisidinverdi og GC-MS. Ut fra resultatene oppnådd i denne oppgaven kan det konkluderes med at Short-Path Distillation egner seg som metode for å separere de flyktige forbindelsene fra marine oljer. I tilegg til tyngre flyktige forbindelser kan muligens lettere forbindelser fanges ved å utstyre kolonnen med en mer effektiv kuldefelle og/eller en adsorbent før utgangen til vakuumpumpa. For kolonnen brukt i dette forsøket tyder resultatene på at oppholdstiden bør holdes mellom 16 og 22 sekunder. Dette tilsvarer fødehastighet på mellom 7.1 ml/min og 2,8 ml/min. De flyktige forbindelsene som er identifisert er hovedsakelig tyngre flyktige forbindelser (fra 9 til 27 karbon). Pristan utgjør den største toppen på GC-MS kromatogram i sildoljen. Denne forbindelsen samt fytol stammer mest sannsynelig fra degradering av α-tokoferol og karotenoider i fytoplankton og inkorporeres i lipidlaget til sild gjennom næringskjeden.Kilden til BHT i oljen er ikke funnet. Det er imidlertidig avkreftet av BHT kommer fra forhold under selve separasjonen eller enzymblanding tilsatt ved hydrolyse av oljen. Det er beregnet at sildolje produsert ved hydrolyseprosess inneholder minst 0,0017 mM BHT, dette tilsvarer 0,37 mg BHT/L olje. Mengden BHT i oljen er under den lovlige grensen for tilsetning av BHT i animalske oljer og fett.

The uptake and incorporation of zink by a Si-, Co- , and Zn starved Thalassiosira pseudonana culture was examined. Two experiments were conducted in which a pre-determined amount of a zinc sulfate solution and sodium metasilicate nonahydrate solution were delivered to an exponentially grown fed-batch algae culture yielding the wanted start concentrations in the medium. Experiment 1 and 2 had final zinc concentration of 8.3 μM (high dose) and 1.7 μM (low dose), respectively. The experiments were conducted in batch mode for 48 hours. Frequent sampling during the first part of the experiments were performed in order to examine the biological uptake of zinc and silicate. The biological response to zinc addition was followed throughout the experiment by measuring photo-physiological parameters (Ft og QY), organic carbon and nitrogen content, cell density, optical density, and nutrient concentrations (PO43- and NO2-). The concentration development of silicate and zinc was followed in the uptake assay samples through ICP-MS analysis. The structural and elemental composition of the diatom frustules were later examined through the use of Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy, respectively. The cellular zinc development followed a two-phase sorption process in which first phase consisted of rapid metal binding to cellular surfaces followed by a gradual desorption of zinc from the cells in the second phase. High initial pH-increase followed by precipitation of zinc as hydroxides is assumed to be the main reason explaining the observed sorption trend, explicitly through the attachment of particulate zinc to cellular surfaces. Other possible explanations are assigned to complexation of zinc by organic exudates excreted from actively photosynthesizing and growing cells. Reduced biological availability of zinc may have led to decreased metal uptake by the algae. However, a higher fraction of zinc was present in the experimental frustules as compared with frustules from untreated algae culture. The highest amount of zinc was detected in the low-dose experiment corresponding to a weight-fraction relative to silicate of 5.16% for the 24 h-sample and 0.146% for the 48 h-sample. Due to the high inter-sample difference in zinc fraction combined with possible sample-contamination from chemicals or equipment through the cleaning process (H2O2) used on the frustule samples from this experiment, these fractions are connected with high uncertainty. No explicit biological response to zinc addition was observed in the algae cultures in either of the two experiments, and the reduction of the photo-physiological condition observed by the end of the experiments were related to silicate-depletion of the culture medium.

Lipids from marine sources contain high amounts of omega-3 fatty acids which are known to have several beneficial effects on human health. Their use as food ingredients is however limited due to their high susceptibility to lipid oxidation resulting in development of rancidity. Liposomes prepared from marine phospholipids have previously been used as a model system to study lipid oxidation by measurement of oxygen consumption. It was of interest to study lipid oxidation by this method in oil-in-water emulsions which can be considered to be more similar to foods. Incorporation of antioxidants in foods is an approach to increase the stability of foods containing omega-3 fatty acids. Emulsions were used as a model system to study the influence of EDTA, citric acid, caffeic acid, propyl gallate, α-tocopherol, ascorbic acid, β-carotene and astaxanthin on iron catalyzed lipid oxidation. Crude herring oil was washed with water to remove impurities. Analyses showed that impurities including carbonyl compounds and carotenoids were removed by the washing, while the oxidation level of the oil slightly increased. The herring oil was used for preparation of emulsions with herring phospholipids as emulsifier by the use of a dispersing tool where increased dispersing time resulted in larger oil droplets with a wider size distribution.Iron catalyzed oxidation of lipids in the emulsions occurred at a lower rate than what has previously been measured in liposomes, but the initial reaction between lipid hydroperoxides and Fe2+ occurred at the same rate in the two systems. The use of soy lecithin as emulsifier inhibited oxidation of lipids in the emulsions. Interactions between iron and antioxidants had a major impact on oxidation in the emulsions. EDTA and citric acid completely inhibited the oxidation when they were added in twice the ratio to iron. Citric acid was not able to inhibit the initial reaction between lipid hydroperoxides and Fe2+ which was thought to be due to its inability to bind Fe2+. Caffeic acid and α-tocopherol enhanced the oxidation rates by reducing Fe3+ to the more catalytically active Fe2+. The prooxidative activity of caffeic acid was significantly greater than that of α-tocopherol. Caffeic acid, α-tocopherol and propyl gallate inhibited the initial reaction between lipid hydroperoxides and Fe2+ to a similar degree which was thought to be related to their free radical scavenging and metal chelating activities. Propyl gallate was also able to reduce the oxidation rates. Ascorbic acid was itself oxidized by Fe2+ and Fe3+ which resulted in increased initial consumption of oxygen, but not increased oxidation of the lipids. Ascorbic acid was able to decrease the prooxidative activity of α-tocopherol by regeneration of α-tocopherol from the α-tocopheroxyl radical. β-Carotene and astaxanthin showed only minor influences on lipid oxidation in the emulsions.

Nanotechnology is an emerging industry manufacturing engineered nanomaterials used in industry, general consumer products and medicine. Nanomaterials are made of nanoparticles which have at least one dimension less than 100 nm. The toxicological properties of nanoparticles are under increasing concern because of their nano size and unique physico-chemical properties. Carbon nanotubes (CNT) are a group of nanomaterials that are under extensive toxicological investigations due to their fiber-like structure and structural similarity with asbestos. Inhalation of fiber-like compounds such as asbestos has been shown to lead to several adverse health effects including fibrosis and cancer. Similar to asbestos, CNTs, in particular multi-walled CNTs (MWCNT), have been shown to induce biological responses such as oxidative stress, inflammation, DNA damage and cell death. However the effects have been observed to differ between different CNTs. It is also hypothesized that genetic factors may modulate the cellular responses following exposure to CNTs, especially genes involved in inflammation. IL-1 is such a gene, encoding an important pro-inflammatory cytokine. To investigate the effect of Il1 on the cellular responses following exposure to CNTs, an Il1 model system including a wild-type Il1 cell line and an Il1a/b (-/-) knock-out cell line were used. Two MWCNTs, one produced in Norway (MWCNT-NO) and one produced in Japan (MWCNT-JP) were investigated for cytotoxicity (WST-8 assay), apoptotic cell death (Hoechst/PI) and alterations in gene expression (qRT-PCR). The effects were then compared with cells exposed to Crocidolite asbestos and hydrogen peroxide. The results showed a dose and time dependent increase in toxicity for both MWCNT-NO and MWCNT-JP. MWCNT-JP was shown to be the most toxic at low doses and also induced a higher level of gene expression. MWCNT-NO, however, showed similar patterns to Crocidolite asbestos both concerning toxicity and gene expression after 24 hours in the Il1a/b KO cell line. A common property of MWCNT-NO and MWCNT-JP was the ability to induce expression of the Ptgs2 (COX-2) gene, an effect which was not seen for Crocidolite or H2O2. Il1 seemed to influence the biological response following MWCNT exposure, with increased toxicity in the knock-out cell line following MWCNT-JP exposure, and differential gene expression of Tnfa and Il6 between cell lines following MWCNT-NO exposure. Neither of the MWCNTs induced apoptotic cell death in the cell lines used. The reasons for the differences between particles in toxicity and inflammatory potential may be due to the higher length of the MWCNT-JP or different production method used, but several other factors may also be involved including differences in contaminations, surface charge and aggregation /agglomeration state. In summary, our results show that MWCNTs from two different producers affect cellular responses differentially. The changes in toxicity and gene expression following exposure varied between the tested MWCNTs, as well as between cell lines with different genetic background.