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.

Lite er kjent i forhold til proteiners interaksjon med alginat, noe som er viktig å ha kjennskap til ved bruk av biopolymeren som immobiliseringsmateriale for celler og proteiner. Denne oppgaven er derfor en studie av alginat – protein interaksjoner. En interaksjon med albumin, fibrinogen og insulin ble undersøkt, da proteinene har relevans i forhold til release systemer av proteiner, og en immunrespons mot alginatkuler ved celletransplantasjon. Lysozym ble brukt som positiv kontroll, ettersom proteinet er kjent å interagere med alginat.Ettersom alginat er negative ladet er det naturlig å tro at elektrostatiske krefter har betydning for polymerens vekselvirkning med andre molekyler. For å undersøke elektrostatiske krefters innflytelse på alginat – protein interaksjoner, ble proteinenes elektrostatiske overflatepotensial modellert ved hjelp av Adopter Poisson-Boltzmann Solver (APBS). Isoterm titreringskalorimetri (ITC) ble videre brukt for å måle vekselvirkningsvarmen ved interaksjon mellom proteiner og alginat i løsning. Proteinbinding til alginatkuler ble kvantifisert ved konsentrasjonsmålinger på nanodrop og mikroplateleser, og visualisert i mikroskop. Det ble tatt utgangspunkt i fysiologiske betingelser (pH = 7 og ionestyrke = 160mM), slik at forholdene samsvarte med det alginatkuler opplever ved transplantasjon.Ved ITC interagerte proteinene med alginat etter synkende styrke: lysozym (positivt ladet) >> albumin (negativt ladet) > fibrinogen (negativt ladet). Albumin og fibrinogen viste å interagere svakt med alginat, til tross for deres negative ladning. Dette kan forklares ved at proteinene har en stor overflate, med enkelte positive regioner selv ved pH > pI. Den høye ionestyrken brukt i studiet bidrar til å redusere de tiltrekkende kreftene mellom molekylene. Ingen målbar interaksjon ble observert mellom alginat og insulin (negativt ladet), noe som kan forklares ved at proteinet har en liten overflate med få positive regioner. Proteinbinding til alginatkuler viste samme trend som ITC, noe som indikerer at alginatets elektrostatiske egenskaper i gel og løsning er den samme. Binding av lysozym ga kulekollaps ved høye proteinkonsentrasjoner på grunn av kondensering til gelnettverket i alginat. Den svake bindingen av albumin og fibrinogen påvirket ikke kulenes stabilitet.Studiet indikerer at elektrostatiske tiltrekkende krefter har betydning ved alginat – protein interaksjoner, og at proteinenes overflateladning har betydning for deres interaksjon med alginat. APBS viste å gi et bra bilde på proteinenes mulige interaksjon med alginat, ettersom det var bra korrelasjon mellom antatt og observert interaksjon. Dette kan forklares ved at APBS implementerer eksperimentelle forhold (pH, ionestyrke etc.) som har betydning for proteinenes elektrostatiske overflatepotensial. De eksperimentelle metodene brukt i studiet ga i flere tilfeller bare et kvalitativt mål på proteinenes interaksjon med alginat. Dette skyldes mest sannsynlig den svake (nanodrop og plateleser) og uspesifikke (ITC) interaksjonen som ble observerte.I videre arbeid vil det være interessant å se nærmere på adsorpsjon og absorpsjon av proteiner til alginatkuler eksponert for helblod, et modellsystem som samsvarer mer med det alginatkuler opplever ved transplantasjon. Hvordan proteinsammensetningen på kuleoverflaten endres over tid er også av interesse, ettersom proteinbinding ikke er en statisk prosess.

Doxorubicin is a topoisomerase-targeting anthracycline that is one of the most effective anticancer drugs currently known. However, its clinical use is restricted by cardiotoxicity and the development of drug resistance. The main goal of this thesis has been to increase the knowledge of doxorubicin mechanism in addition to evaluate if predictive biomarkers for doxorubicin response could be identified. A total of 128 tumor samples collected from breast cancer patients before and after neoadjuvant treatment with doxorubicin were studied. mRNA expression level in tumor tissue was assessed using whole-genome mRNA microarray analysis (Agilent Human GE 4x44K microarray).More than 5000 genes were found to be up- and down regulated following doxorubicin treatment. The molecular and cellular functions as well as canonical pathways found to be enriched in the list of genes up regulated after doxorubicin exposure were involved in among other cardiovascular system development and function, cellular movement and immune responses. p53 was found to be the transcription factor regulating the highest number of target molecules within the list of up regulated genes. RNA processing, splicing and translation were shown to be overrepresented in the list of down regulated genes. The association between doxorubicin response and changes in gene expression revealed several genes such as CTGF, ITGB4 and IGF1 to be up regulated in the samples collected from patients characterized with a partial response to doxorubicin compared to those with minimal change and/or stable disease following treatment. In addition, the gene expression profiles between samples having wild type compared to mutated TP53 were studied, and a lower induction of expression were found for several genes such as FGF9 and COL11A2 in the samples having a mutated p53. This study showed that the gene expression profile in breast cancer tumors is altered as a response to doxorubicin exposure. Identifying genes significantly altered after therapy and associate their change with response to treatment may help identify the subgroup of patients benefitting from doxorubicin treatment. Patients with little or no effect of treatment could receive alternative therapy and be spared unnecessary treatment and risk of side effects.

Breast cancer is the most common cancer type amongst women, and closer to 3000 women in Norway will be diagnosed with this disease in 2012. Although major improvements have been achieved in the treatment, and thus the outcome, of breast cancer patients in the past years, little has been accomplished for those with an advanced disease. B7-H3 is an immunoregulatory protein, and its overexpression has been associated with advanced disease and poor prognosis in breast cancer. A previous study has shown that B7-H3 silencing increased Paclitaxel sensitivity in B7-H3 expressing breast cancer cell lines. Resistance to treatment is a general challenge in systemic management of advanced breast cancer, and increased knowledge about the molecules and pathways involved in this process is important in order to improve the outcome for these patients.To further study the function of B7-H3 and its putative involvement in lack of treatment response in breast cancer, we compared the efficacy of 22 different anti-cancer drugs in two B7-H3 expressing triple negative metastatic breast cancer cell lines, MDA-MB-435 and MDA-MB-231, and their B7-H3 silenced counterparts. In particular two drugs targeting the PI3K/Akt pathway, API-2 and Everolimus, showed a significantly better efficacy in the B7-H3 silenced cells. To elucidate the cellular mechanisms involved in the observed sensitization in the B7-H3 knockdown cells, we performed Western blot analysis on several proteins in the PI3K/Akt/mTOR pathway. The cells that did not express B7-H3 had lower levels of both phospho-Akt and the downstream signaling molecule phospho-p70S6K following drug exposure, indicating B7-H3 associated the regulation of proteins in this pathway. This, together with the previously observed relationships between B7-H3 expression in metastasis and chemoresistance, suggest that this protein might be a therapeutic marker to increase the effect of current anti-cancer treatment, although the specific roles of B7-H3 in this context need to be investigated further.

Recombinant protein production in Escherichia coli provides a cheap and efficient way of producing medically and industrially relevant proteins. Sequence features of individual genes and especially their 5’ terminal coding sequences act on the efficiency of gene expression by complex regulatory mechanisms which are still not fully understood. This study aimed to investigate the features of the 5’ coding region of recombinant mRNAs, and to optimize them for increased expression in E. coli. A previous study had found that a synonymous change of the bla reporter gene 2nd codon leads to an increased expression, and accordingly a synonymous library in the 5’ bla coding sequence was created by a directed evolution approach building on this feature. Variants conferring up to three-fold increases in active enzyme amounts were identified, and the increased expression was shown to stem from increased transcriptional efficiency. The effect of changing the 2nd codon synonymously was further investigated by synonymous substitutions of the 2nd codons of the bla and two other reporter genes, phoA and celB. These experiments showed that the effect of 2nd codon changes on the gene expression is determined by the sequence context, as changes in expression levels appeared to be gene specific. All the coding sequences of the study were also analysed in silico, and an application for calculating the tRNA adaptation index was programmed in Python and made freely available online.As the synonymous codon changes did not lead to a great improvement in protein amounts and any sequence features affecting the expression were hard to pinpoint, an alternative strategy involving 5’ terminal gene fusions was investigated. Combinatorial mutagenesis coupled to an effective screening technique was applied to further optimize a 5’ terminal fusion partner, previously shown to improve expression of several eukaryotic genes. The application of the best identified fusion partner candidate yielded a 3.8-fold improvement in IFN-α2b protein amounts over the original fusion, and showed twice as high protein amounts than a pelB-IFN-α2b fusion previously proven to give industrial expression amounts. The developed peptide fusion is thus an eligible candidate for further development for use in heterologous protein production.

Oral delivery is considered a convenient route for administration of pharmaceuticals. Great effort has been made to optimize oral delivery vehicles to increase the bioavailability of the pharmaceutical, and enhance patient compliance to ease swallowing. Emulsion-based gelled matrices have shown promising features as delivery systems. They are soft chewable matrices that are easy to swallow, and have the ability to entrap the pharmaceutical, providing prolonged, and controlled release to avoid fast dissolution in the stomach. The purpose of this study was divided into two main objectives. The first aim was to study emulsion-based gelatin matrices intended for oral drug delivery by investigation of the influence of gelatin type and/or oil content on the matrix properties. The second aim was to investigate the properties of multiple emulsions, regarding their potential as oral drug delivery systems for water-soluble pharmaceuticals, and controlled drug delivery. Emulsion-based matrices stabilized by 160g Bloom gelatins, either type A or B, and containing various amounts of corn oil (0, 10, 30 and 50 wt %) were subjected to rheological characterization and in vitro dissolution studies at simulated gastric conditions. The results showed an increase in viscosity, storage modulus and gelling and melting temperatures in line with increased oil content in the matrix, and to a larger extend for matrices with type A gelatin compared to type B. Longitudinal deformations of the gelled matrices did show a trend of slightly increasing Young’s modulus when oil was added to the matrix, but no clear trend was observed for force and strain at break. A correlation between rheological matrix properties and dissolution time was observed: An increase in dissolution time with higher fractions of oil and prolonged dissolution time for matrices with type A gelatin compared to type B. Overall the results showed that different oil contents and gelatin types changed the physical properties of the matrices, providing a possibility to tailor matrices to obtain suitable delivery systems for various pharmaceuticals. Water-in-oil-in-water double emulsions, stabilized by either 226g Bloom gelatin type B or tween80 was examined by long time stability studies, and by in vitro lipolysis studies simulating small intestinal conditions. The water-soluble marker tartrazine, was entrapped in the inner water phase of the emulsions. The release of tartrazine was measured during a period of 78 days and both double emulsions with gelatin and tween80 were found to possess long-term stability at room temperature. In vitro lipolysis of gastric stable double emulsions stabilized by gelatin was conducted in a dissolution medium containing bile extracts, with or without lipases. A complete release of tartrazine was obtained; both in the presence and absence of lipases, while a faster release was observed when lipases were present. Although the release mechanism was not completely determined, the results indicate that release of drugs can occur in the small intestines due to lipolysis. The double emulsions thus offer great potential in delivery of gastric unstable pharmaceuticals.

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.