Ph.D. afhandling, Roskilde universitetscenter 2003.

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.

Kitin er en langkjedet, nitrogenholdig polymer som er bygget opp av N-acetyl-glukosamin-enheter (GlcNAc), og finnes i blant annet det ytre skallet til leddyr, og i celleveggen til sopp. Biosyntese av kitin er katalysert av enzymet kitin syntase som overfører GlcNAc til den ikke-reduserende enden av den voksende polymeren via substratet UDP-GlcNAc. Når kitin deacetyleres dannes det kitosan, som består av en blanding av GlcNAc og GlcN. Kitin og kitosan har en rekke egenskaper som gjør at de har mange ulike bruksområder. De er blant annet biokompatible og biodegraderbare, noe som gjør de svært godt egnet til medisinske og farmasøytiske bruksområder, og kitosan er en effektiv flokkulant på grunn av positivt ladete aminogrupper, og kan brukes til rensing av drikkevann og avløpsvann. Studier av genomsekvensen til E. carotovora har vist at det inneholder en genklynge hvor et gen (ECA2046) koder for en potensiell kitin syntase. Det kan se ut til at flere av genene i samme genklynge, inkludert ECA2046 tilhører samme operon. Formålet med denne oppgaven var å aktivere transkripsjon av ECA2046 ved hjelp av den induserbare PmDI-8 promotoren, og undersøke om dette førte til produksjon av bakteriell kitin. Det var også ønskelig å forsøke å aktivere hele det antatte operonet dersom produktene fra de andre genene er nødvendig for produksjon av en aktiv kitin syntase. Det ble konstruert to ulike rekombineringsvektorer, pKNH25 og pKNH35. I disse vektorene er henholdsvis ECA2046 og ECA2048 klonet inn nedstrøms for PmDI-8. ECA2048 er det første genet i det antatte operonet. Vektorene ble overført ved konjugering fra E. coli S17.1 til E. carotovora. De overførte plasmidene ble integrert i genomet til E.carotovora ved homolog rekombinering. Mutantene ble dyrket videre for å danne ønskede mutanter som hadde fått inkorporert PmDI-8 og xylS, og mistet resten av plasmidet ved videre rekombinering. Det ble funnet en ønsket mutant etter rekombinering med pKNH25, denne ble kalt E.carotovora KNH25, og PmDI-8 i denne mutanten vil kontrollere transkripsjon av ECA2046 og eventuelt nedstrømsgenet ECA2045 som koder for an mulig NAD avhengig epimerase/dehydratase. Ingen ønskede mutanter etter videre rekombinering av muntanten som hadde fått integrert pKNH35 ble funnet, og det var derfor ikke mulig å undersøke om aktivering av transkripsjon av hele det antatte operonet ville føre til produksjon av bakteriell kitin. E.carotovora KNH25 ble dyrket i medium tilsatt induseren m-toluat for å aktivere transkripsjon fra PmDI-8. Analyser av flokkuleringsaktiviteten viste ingen aktivitet, og derfor ingen merkbar produksjon av bioflokkulanter. 1H NMR-analyser ga heller ingen tydelig indikasjon på at det var produsert noe kitosanlignende produkt. Ut ifra NMR-spekteret kan det se ut som det er en blanding av flere stoffer, og grunnet tydelige topper mellom 3,5 og 4 ppm kan det tyde på at det er en annen polymer til stede siden dette område ofte indikerer protoner på sukkermonomerne i polysakkarider. Ved dyrking av både villtypen og mutanten i samme medium, med og uten induser, ble det produsert like store mengder stoff som kan felles med etanol uavhengig av tilstedeværelse av induser. Siden verken målingene av flokkuleringsaktiviteten eller 1H NMR-spekteret gir noen indikasjon på at det er kitosan til stede, og det heller ikke viser seg noen økning i produsert stoff som felles med etanol ved aktivering av PmDI-8 promotoren i E.carotovora KNH25, er det lite som tyder på at aktivering av transkripsjon av kun ECA2046 og eventuelt ECA2045 fører til produksjon av en aktiv kitin syntase, eller et annet aktivt enzym som katalyserer dannelsen av en eventuell polymer som kan felles med etanol.

Chitosans are cationic polymers desirable for use in nucleic acid delivery due to its biocompability, biodegradable and low toxic nature. The P-glycoprotein (P-gp) pump is a member of membrane transporters in brain endothelial cells which recognizes xenobiotics, extruding them from the cells. P-gp is a highly clinically relevant target as it also limits accessibility of drugs from the blood to the brain.The overall aim of this work was to investigate the feasibility of using chitosan as a delivery vehicle for siRNA-mediated knockdown of P-glycoprotein (P-gp) efflux pump in an in vitro model of blood brain barrier. The human lung cancer cells (H1299) stably expressing GFP was used for screening and optimization of the delivery system and conditions. Lung cancer is often represented among multidrug resistant cell lines and is therefore also a suitable target for knockdown of transporters. The main objective was to implement this knowledge to efficiently downregulate the P-gp pump residing in the rat brain endothelial cell line RBE4. The approach was to first establish suitable physiochemical characteristics of the chitosans like DPn and the N/P ratios of chitosan/siRNA complexes by studying exogenous GFP and endogenous GAPDH expression and uptake of fluorescently labeled siRNA by flow cytometry. Cytotoxicity profiling of the chitosans and polyplexes was preformed with AlamarBlue, PI-staining, Cell counting and BCA assay. We further investigated the effect of chitosan mediated mdr1a-siRNA on drug efflux in the RBE4 cells using two functional assays; Rhodamine 123 efflux by flow cytometry and visualization of the anticancer drug Doxorubicin by CLSM.We demonstrated that chitosan can be used as an efficent delivery vehicle for siRNA in both H1299 and RBE4 cells with negligible cytotoxicity in vitro. The formulated chitosan/siRNA polyplexes was found to dependent on DPn and N/P ratios in order to exert maximal effect on both uptake and knockdown. Specifically, we managed to downregulate the P-gp expression in RBE4 cells using chitosans with a high DPn complexed with mdr1a-siRNA in a low N/P ratio. Together these results elucidate the potential of chitosan mediated siRNA downregulation of the P-gp pump in RBE4 cells and further development for in vivo applications.

The increase of obesity that we have experienced during the last decades and its association with insulin resistance, type 2 diabetes and other metabolic diseases has resulted in an enormous interest for understanding the mechanisms underlying these disorders. Tissue inflammation triggered by food with a high glycemic index has been suggested to be an important mediator in the development of insulin resistance. Despite great research efforts lately, more research is needed in order to understand how nutrients interact with the genetic factors that control and triggers the inflammatory responses. The composition of macronutrients in a diet influences the levels of insulin secretion in the body. Besides controlling the blood glucose concentration, insulin also regulates a range of inflammatory processes. Inflammation is largely dependent on some small cell-signaling molecules called cytokines, as these activate a wide range of inflammatory-related genes. The objective of this study is to explore the regulatory effects of insulin and cytokines on the transcription of the following selected genes related to inflammation; D5D, D6D, SCD and FOXO3A. In addition, expression of TRAIL, BTG1 and TWIST1 is studied as they all are target genes for FOXO3A, and related to inflammatory processes and/or glucose metabolism. Quantitative-PCR was used to study mRNA expression of relevant genes in THP-1 cells treated with insulin and cytokines. As the investigation was performed on THP-1 monocytes, it was necessary to optimize the in vitro conditions in order to obtain a maximal response from the insulin and cytokine treatments. The concentration of insulin was an important factor in this study, because the regulation of FOXO3A and desaturases (D5D, D6D and SCD) mRNA expression seemed to be dose-dependent. The treatment period was also critical, as a set of time-course experiments revealed that FOXO3A and the desaturases were regulated by insulin and cytokines at different time-points. In this study, THP-1 cells treated with insulin and/or cytokines revealed significant regulations of the relevant genes. Gene expression of D5D, D6D and SCD was significantly up-regulated in response to insulin. Furthermore, mRNA expression of the transcription factor FOXO3A was significantly down-regulated by insulin, IL-1β and TNF-α. However, neither FOXO3A nor the desaturases were cooperatively regulated by these stimulating factors. TRAIL, TWIST and BTG1 on the other hand, were significantly up-regulated in a synergistic manner when cells were treated with a combination of insulin, IL-1β and TNF-α. The observed regulation of gene expressions in THP-1 monocytes treated with insulin and cytokines suggests that insulin may affect the regulation of inflammatory related genes in circulating human monocytes. As insulin is secreted in the bloodstream followed by elevated levels of glucose after a meal, these results may reflect possible diet-induced changes in gene expression.

Bilberry (Vaccinium myrtillus) is native to Europe and North America and constitutes an important nutritional resource for both humans and animals. Over the years, a series of chemical analyses have revealed several health-beneficial compounds in bilberry, and with the current demand of the berries mainly covered by Sweden and Eastern Europe, there has become an increasing desire to cultivate bilberry in Norway. In order for such cultivation to be successful an increased knowledge about bilberry is seen as essential and thus several studies have investigated the chemical composition of the berry. However, the underlying genetic diversity and the variation between populations in biochemical compounds remain to be thoroughly investigated. Therefore, the aim of present study was to investigate the differences in biochemical composition between populations of bilberry distributed on a latitudinal gradient, and estimate the level of genetic variation within and among the populations. This in order to examine whether biochemical composition was reflected by observed levels of genetic variation.Bilberries from four Norwegian populations at three regions differing in latitude were analysed for content of total phenolics (TPH), total anthocyanins (ACY) and antioxidant activity (FRAP). Furthermore, metabolic profiling was performed by gas chromatography-mass spectrometry (GC-MS) to reveal biochemical differences between the populations in content of sugars, acids and some simple phenolics. Multivariate statistics were performed and revealed a clustering of samples from the two locations in Mid-Norway, and a clustering of the northern with the southern population. In addition, there were found significant differences in some of the compounds between the populations.Genetic analyses using four microsatellites were carried out to examine whether metabolic differences between populations were reflected by genetic differentiation. Despite the significant differences between populations in the concentrations of some metabolites no significant genetic differentiation was found. Thus, it seems that the variation in biochemical compounds discovered among populations could be environmentally induced differences on a similar genetic background. However, due to the limited number of working microsatellites and the fact that these molecular markers are neutral, there is still a possibility that the genetic differences causing compound concentrations to differ could be so minor as to remain undetected. Hence, further studies utilizing more microsatellite markers or new state-of-the-art molecular techniques are needed to determine whether this result holds and is valid also for genetic variation in coding parts of the genome.

In Norway today no commercial exploitation of the wild growing Norwegian bilberry (Vaccinium myrtillus) exists. The market in other regions of Europe, North- and South-America is based on the utilization of cultivated Vaccinium species. V.myrtillus shows generally a higher content of biochemical compounds with health-beneficial properties. As a consequence of an increasing demand for healthier food, the Nordic Bilberry project started in 2008 with the major goal to find superior clones adapted for different regions with effective production of phytochemicals. In addition, a 4-years Norwegian Bilberry project aiming at cultivation and yield potential aspects, was launched the same year (NFR project no. 184797). The presented master projected was affiliated to this project focusing on phytochemical and genetic diversity in Bymarka. It is known that life history traits of a plant species influence the clonal diversity and structure within populations. In this thesis different phytochemical methods were used for detection of total phenolics, anthocyanins and antioxidants in berry and plant material from 4 different areas in the geographic restricted area Bymarka, were a total of 80 individuals were collected. Average values detected for total phenols were 490 mg/100 g, 155 mg/ 100 g for anthocyanins and 4 mmol/ 100 g for antioxidants. The results showed little variation among clones in the restricted area. 16 primer pairs for 16 microsatellite loci were tested but only 4 (NA741, NA961, CA421 and CA483) turned out to be of good quality. These microsatellite loci were used to estimate genetic variability within and between populations. Little genetic variation was detected between the different plots, and populations had similar levels of within-population genetic variation. The highest diversity both genetic and phytochemically was found in plot D with berries without any wax layer. This plot was also more genetically different from the other populations than the berries in the same location/area/plot with a wax layer, though no significant differences was found in FST, Heterozygosity observed, Heterozygosity expected or allelic richness (P<0.05). The result from this thesis could be used further for improvement of breeding strategies and selection of cultivars with high phenolic contents for production of quality food.

African savannah elephants play a vital role in the Serengeti ecosystem with the opportunity to alter the entire ecosystem by its sheer number. Management of these animals are therefore of high importance, but little genetic research has been done thus far in the ecosystem. Their recent traumatic history of poaching serves as a template for intriguing evolutionary theories and further understanding of elephant behavior. In this study it was investigated on the genetic structure and spatial differentiation of the elephants in Serengeti using a mitochondrial DNA (mtDNA) marker. A widespread sample size of 55 elephants were collected in three zones of the Serengeti National Park (West, North and Seronera) and analyzed for genetic diversity. The results gave the impression of a slightly outbreeding population with no ongoing subdivision (FST = -0.04864, p = 0.92082). A total of 7 haplotypes were obtained with one clearly being dominant (78.2 %). All collected haplotypes were compared to earlier studies using the same mitochondrial marker and having a wider perspective, with samples ranging across the sub-Saharan Africa. Results show that there is a possibility that the elephants now inhabiting the Serengeti are primary descendants of Northern populations coming from Kenya and Uganda, with additional individuals giving the impression that the Serengeti elephants are a mixture of individuals with historical connections from all over sub-Saharan Africa.

I denne masteroppgaven har det blitt utviklet primerpar for 17 humane PLA2-isotyper. Ved hjelp av primerene har vi påvist uttrykk av 17 PLA2-isotyper i HaCaT-keratinocytter, hvorav 11 av disse representerer førstegangspåvisninger i HaCaT. Funnene representerer økt kunnskap om PLA2-familien og viser at HaCaT uttrykker et mangfold av PLA2-isotyper. Primerene ble også benyttet til å kartlegge PLA2-uttrykk gjennom keratinocytters differensiering. Resultatene viser at 13 av 17 isotyper uttrykkes gjennom hele differensieringsprosessen, som indikerer at PLA2-enzymer spiller sentrale roller i lipidmetabolisme i human epidermis. PLA2G2A viste, som den eneste av de studerte isotypene, sterk oppregulering under differensieringen, som vitner om at enzymet kan spille en viktig rolle i de øvre strata i epidermis.Sammendrag Under utviklingen av primerparene ble human postnatal placenta tatt i bruk som antatt positiv kontroll for uttrykk av samtlige PLA2-isotyper. 8 av 17 primerpar detekterte mRNA i placenta, hvorav PLA2 gruppe 2D, 4C, 4D og 10 representerer førstegangspåvisninger i human placenta. Sammendrag Primerene utgjør et verktøy som gjør det mulig å kartlegge PLA2-uttrykk i alle humane celler og vev. Da diverse PLA2-isotyper har blitt vist å spille en sentral rolle i inflammasjonssykdommer ved å katalysere hydrolysen av arakidonsyre fra glyserofosfolipider, vil det være intressant å kartlegge PLA2-uttrykk i friskt vev versus betent vev. Primerene vil legge grunnlaget for økt forståelse av de mange PLA2-enzymenes funksjoner i inflammasjonssammenheng, og potensielt legge grunnlag for utvikling av målrettede medisiner mot kronisk inflammatoriske sykdommer.

IDL6 and IDL7 are postulated peptides of the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) peptide family in the model organism Arabidopsis thaliana. The genes encoding the postulated peptides, IDL6 and IDL7, were investigated to potentially elucidate the function of the peptides and the possible relations to plant defense and stress tolerance in A. thaliana. A phenotypic characterization study of single and double knockout mutant lines, and over-expression lines of IDL6 and IDL7 was conducted to potentially find a phenotype linked to the genes by analyzing deviations in growth and development, compared to wild type (Wt) A. thaliana. Over-expression lines showed a tendency to have a higher amount of individuals reaching defined growth stages in seedling development, but this could not be concluded to be a phenotype linked to IDL6 or IDL7.The genes have been shown to be highly up-regulated in response to several different stress treatments, both abiotic and biotic in silico data. Several different transgenic lines of A. thaliana were subjected to different types of stress treatments to potentially verify the in silico data. Knockout lines, double knockout lines, over-expression lines and promoter:GUS lines of IDL6 and IDL7 were treated with abiotic stress factors (NaCl, mannitol, UV-B light, H2O2 and paraquat) and biotic stress factors (aphids, Pseudomonas syringae and flagellin22) to investigate differences in tolerance, gene expression and promoter activity in the respective transgenic lines and Wt A. thaliana.No absolute phenotype was detected in experiments related to stress tolerance, and great variations in promoter activity using promoter:GUS lines were observed. However, double knockout mutants of idl6 and idl7 showed a small trend to tolerate NaCl better than Wt A. thaliana and other transgenic lines. The great variation in GUS expression from GUS assay lead to a thorough screening and expression analyses of promoter:GUS lines. Results presented in this work, indicate that expression of the IDL6 and IDL7 genes may be subjected to extensive post transcriptional regulation through mRNA degradation, possibly governed by stress related environmental signals. A novel member of the IDA peptide family, IDL8, was also analyzed. Segregation analyses of knock out lines were conducted and they were genotyped to verify that mutant lines of idl8 were real knockouts. Results presented here show that one of the idl8 mutant lines had the T-DNA inserted to the promoter region of the gene and is postulated to be a real knockout. However, further expression analyses should be conducted to verify that the gene is not transcribed. Over-expression lines of IDL8 were successfully constructed through recombinant DNA technology and by T-DNA insertion using Agrobacterium tumefaciens.