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Aberrant glycosylation patterns of membrane-bound proteins have been observed to be a universal feature of malignant cells. Members of the mucin family of glycoproteins form one of the major carriers of aberrant glycosylation, where there in breast tissue cells and other carcinomas have been found an upregulation of the Mucin1 (MUC1) protein with an increase in several cancer-associated antigens such as the ST (alpha-Neu5Ac(2,3)beta-Gal(1,3)-GalNAc-Ser/Thr), T (beta-Gal(1,3)-GalNAc-Ser/Thr), Tn (GalNAc-Ser/Thr) and STn (alpha-Neu5Ac(2,6)-GalNAc-Ser/Thr) antigens. The presence of the different antigens are not fully understood, but an increase of sialic acid-glycostructures have been found in breast cancer cells. The galactose binding family of lectins called galectins have been found to be present both extracellularly and intracellularly. A member of the beta-galactose binding family, Galectin-3(Gal3), have further been associated with processes such as acute inflammation as well as cancer increase and metastasis. Due to its important role in cancer development, the binding specificity of Gal3 is of interests. Gal3 have further been observed to interact with MUC1(T), but not with the MUC1(Tn). In a study published in 2007, interaction between MUC1(ST) and Gal3 was reported not to occur, but an unpublished study from London revealed informal information about a possibility of an interaction to occur. The experimental data presented in this master thesis were recorded by using a dual beam setup of the sensitive force probe Optical Tweezers. The aim of this study was to provide new information concerning the interaction between the different MUC1-antigens and Gal3. The results obtained provide evidence that MUC1(ST) and Gal3 interact with a rupture strength of 11-31 pN when investigated using loading rates in the range of 39-72 pN/s. The energy landscape of the interactions gave a k(off)-range from 1.3-2.9 1/s and x(beta)-value of 0.54 nm. A rupture frequency analysis was included in order to estimate the occurrence of interaction. MUC1(ST) and Gal3 showed a main rupture frequency estimate of 42.8 % +/- 26. Parallel, interaction between the positive control MUC1(T) and Gal3 gave a rupture frequency estimate of 32.0 % +/- 16. The negative controls MUC1(Tn) and MUC1(Na) showed no specific interaction with Gal3. A parallel study was performed by another masterstudent at NTNU, Ragna Emilie Bugge, where she also reported interaction between MUC1(ST) and Gal3 to occur by use of the sensitive force probe atomic force microscopy(AFM).

Konservering av mat både bevarer og øker holdbarheten til mat. Vanlige konserveringsmetoder er salting, tørking, røyking og syrekonservering, men i de siste årene har det dukket opp nye teknologier for å øke holdbarheten på mat. Forbrukernes vaner har også endret seg, og stiller krav til ferskere produkter og til mindre bruk av konserveringsmidler. Dette byr på en rekk utfordringer når flere hinder/barrierer som skal sørge for mattrygghet hos forbrukerne fjernes. Målet med denne oppgaven var å se på effekt av kjølemetode (superkjøling og vanlig kjøling) og saltemetode (lakesalting og tørrsalting) på saltinnhold i laks etter røyking. I tillegg om beskyttende kulturer kan øke holdbarheten til laks ved biokonservering og om de beskyttende kulturene overlever superkjøling. Det ble utført to forsøk. I det første forsøket var målet å se på effekt av kjølemetode (superkjøling og vanlig kjøling) og saltemetode (lakesalting og tørrsalting) på saltinnhold i laksen etter røyking. Det ble analysert tørrstoff, vann- og saltløselige proteiner, og saltinnhold. Det ble ikke funnet noen signifikante forskjeller mellom tørrsaltet og lakesaltet prøver i tørrstoff, vanninnhold, eller vann- og saltløselige proteiner. Det ble derimot funnet en signifikant forskjell i saltinnhold hos lakesaltete og tørrsaltete prøver. De tørrsaltede prøvene (2,22% ± 0,23) innehold mer salt enn prøvene som var lakesaltet (1,6% 0,49) I det andre forsøket ble laks dyppet i beskyttende bakteriekulturer av melkesyrebakteriene, Lactococcus piscium (PC1), Leuconostoc gelidium (PC2), Carnobacterium ssp (PC3) og Vagococcus fluvialis (PC4) for biokonservering. Det ble undersøkt om melkesyrebakterien klarte å hemme den spesifikke kvalitetsforringelses flora som er med på å redusere holdbarheten til laksen. Allerede fra start var kimtallet for aerobe bakterie i overkant høyt og fortsatte å øke etter 4, 11 og 17 lagringsdager. Utviklingen av mengden melkesyrebakterier i prøvene økte også jevnt. Det ble også påvist H2S produserende bakterier i alle prøver (Kontroll gruppe, PC1, PC2, PC3 og PC4) etter 4 dager lagring. Valg av saltemetode vil påvirke saltopptaket i laks, mens biokonservering med melkesyrebakterier har potensial, men effekten vil være minimal om prøvene allerede er høyt kontaminert av bakterier.

Hyperaccumulator plant species accumulate exceedingly high concentrations of metals in aerial parts and are increasingly studied because of their potential for cleaning up land contaminated with heavy metals. One such hyperaccumulator, Noccaea caerulescens has been recognized as a model species for its remarkable Zn, Cd and Ni hyperaccumulation capacity and shares 88% sequence similarity with non-hyperaccumulator Arabidopsis thaliana in the coding region sequence. HIPPs (Heavy metal-associated isoprenylated plant proteins), are characterized by the presence of one or two HMA (Heavy metal associated) domain and a C terminal isoprenylation motif, present in both plants which have a possible role in hyperaccumulation. In the current study transcript levels of HIPP25 along with its closely related gene HIPP26 and HIPP27 were monitored in roots, leaves, flower and siliques of A. thaliana and root and leaves of N. caerulescens when exposed to Ni, Cu, Zn and Cd for 14 days (long term) and 24 hours (short term). Effect of metal treatment under long term condition was performed with two concentrations. Furthermore, the effect of these metal treatments on the HIPP25 protein level was also determined. Under long term treatments, in leaves of A. thaliana HIPP25 gene expression was higher with Cd, Zn and Cu exposure as compared to the control plants. HIPP25 gene in A. thaliana is possibly involved in the accumulation of Zn and Ni homeostasis as the expression of HIPP25 was higher in Zn treated leaves and lowered in the Ni treated leaves at both gene and protein level. In N. caerulescens, under short term treatments, Cu decreased HIPP25 and increased HIPP26 gene expression in roots. Under long term treatments, higher concentration of Ni reduced whereas lower concentration induced the HIPP26 gene expression in roots. From the comparative study on the protein level, HIPP25 proteins were expressed more in N. caerulescens than A. thaliana under both long term and short term treatments. Apart from this, changes in glucosinolate content in both plants exposed to Zn under both long term and short-term treatments were also monitored where the glucosinolate content in leaves of A. thaliana increased upon lower concentration under long term treatments whereas decreased upon higher concentration under both long and short-term treatments. In N. caerulescens under both concentrations in long term treatment, the glucosinolate content decreased and remains unchanged for short term treatments.

Keberhasilan dalam akuakultur sangat dipengaruhi oleh kemampuan membentuk ekosistem yang mampu mendukung kehidupan ikan yang dipelihara dan penyediaan pakan berkualitas dengan harga terjangkau. Pada ekosistem perairan, khususnya lingkungan budidaya ikan, mikroorganisme memiliki peran sangat kompleks dan vital. Seiring meningkatnya taraf pengetahuan masyarakat dan kesadaran budidaya berkelanjutan, pola pikir pembudidaya mulai bergeser ke arah optimalisasi peran mikroorganisme sebagai sahabat, bukan semata sebagai jasad penyebab masalah yang harus dimusnahkan dari ekosistem budidaya. Hal tersebut tentunya dapat dicapai apabila pembudidaya mampu mengelola jasad renik tersebut secara benar. Kemampuan mikroorganisme menghasilkan enzim pencernaan dan mengkonversi limbah pertanian menjadi protein juga sangat potensial untuk dimanfaatkan dalam menyediakan bahan baku pakan alternatif dengan harga terjangkau. Pemanfaatan bioteknologi berbasis mikroorganisme secara intensif dalam mengatasi permasalahan limbah budidaya, agen biokontrol, pakan alami, dan agen fermentasi pakan diharapkan mampu mendongkrak produktivitas budidaya perikanan nasional.

Ekonomi global saat ini sedang pada titik puncak perubahan besar yang sebanding besarnya dengan munculnya revolusi industri pertama atau perkembangan perakitan produksi, atau bahkan penemuan mikrocip. Kemajuan teknologi memungkinkan terjadinya otomatisasi hampir di semua bidang. Sementara itu, kepemilikan perangkat pintar di berbagai bagian dunia mengarah pada tingkat keterkaitan satu sama yang lain yang tak terbayangkan sebelumnya. Di antara berbagai tantangan yang sedang dihadapi dunia saat ini, mungkin yang paling besar adalah bagaimana membentuk Revolusi Industri keempat (disebut juga sebagai Industri 4.0) yang dimulai pada permulaan abad ini. Teknologi dan pendekatan baru yang menggabungkan dunia fisik, digital, dan biologi dengan cara yang fundamental akan mengubah umat manusia. Ada banyak pendapat bahwa sektor kesehatan dan bioteknologi sangat diuntungkan oleh transformasi ini. Sejauh mana transformasi ini akan berdampak positif bergantung pada bagaimana kita menavigasi risiko dan peluang yang muncul di sepanjang jalan.

Uncontainable Life: A Biophilosophy of Bioart investigates the ways in which thinking through the contemporary hybrid artistico-scientific practices of bioart is a biophilosophical practice, one that contributes to a more nuanced understanding of life than we encounter in mainstream academic discourse. When examined from a Deleuzian feminist perspective and in dialogue with contemporary bioscience, bioartistic projects reveal the inadequacy of asking about life’s essence. They expose the enmeshment between the living and non-living, organic and inorganic, and, ultimately, life and death. Instead of examining the defining criteria of life, bioartistic practices explore and enact life as processual, differential, and always already uncontainable, thus transcending preconceived material and conceptual boundaries. In this way, this doctoral thesis concentrates on the ontology of life as it emerges through the selected bioartworks: “semi-living” sculptures created by The Tissue Culture and Art Project and the performance May the Horse Live in Me (2011) by L’Art Orienté Objet. The hope is that such an ontology can enable future conceptualisations of an ethico-politics that avoids the anthropocentric logic dominant in the humanities and social sciences. Otyglat liv: Biokonst och biofilosofi undersöker hur biofilosofisk praktik och biokonst, alltså tänkande genom samtida hybrida konstnärliga-vetenskapliga praktiker, kan bidra till en mer nyanserad förståelse av liv än vad vi vanligtvis möter i akademiska diskurser. Med utgångspunkt i ett feministiskt deleuzianskt perspektiv, och i dialog med samtida biovetenskap, pekar biokonstnärliga projekt på det otillräckliga i att ställa frågor om livets innehåll. Projekten tydliggör istället hur det levande och det icke-levande, det organiska och oorganiska, precis som liv och död, är sammanflätade. Istället för att sätta upp fasta kriterier för liv undersöker och framställer biokonstnärliga praktiker liv som en differentiell process, i sig omöjlig att fastställa och därmed något otyglat, som överskrider uppsatta gränser mellan det materiella och föreställda. Följaktligen fokuserar föreliggande avhandling på livets ontologi så som den framträder i ett urval av biokonstnärliga arbeten: ”semi-levande” skulpturer skapade av The Tissue Culture and Art Project, samt performance-konstverket May the Horse Live in Me (2011) av L’Art Orienté Objet. Förhoppningen är att en sådan ontologi kan möjliggöra framtida begreppsliggöranden av en etisk politik som undviker den antropocentriska logik som dominerar humaniora och samhällsvetenskap idag.

Due to negative health effects of high sodium intake, it is recommended to reduce the daily salt intake by around 50%. To reduce the sodium content, sodium salts can be exchanged with potassium or magnesium salts. The effect of sodium, potassium, and magnesium chlorides on extractability of proteins from fresh and frozen haddock muscle and minces was studied. Salting with KCl and MgCl2 instead of NaCl changed protein extractability. The highest solubility of the proteins was achieved using Na+. However, at low concentrations, extractability in K+ and Mg2+ is on the same level as Na+, showing that partial substitution of NaCl with KCl or MgCl2 is possible. Freezing affected the structure of tissue and protein properties, resulting in decreased amount of salt soluble proteins. This is an [Accepted Manuscript] of an article published by Taylor & Francis in [Journal of Aquatic Food Product Technology] on [06 Mar 2015], available at http://www.tandfonline.com/doi/full/10.1080/10498850.2013.879241

Rapporten omhandler ressurser og resultater innenfor bioteknologisk FoU i norsk universitets- og høgskolesektor (UoH-sektor) og instituttsektor i 2013. Datamaterialet stammer fra NIFUs kartlegging av bioteknologisk FoU tilknyttet den ordinære FoU-statistikken. Dette er den sjette kartleggingen av bioteknologisk FoU i Norge som har vært gjennomført annethvert år siden 2003. Næringslivet omfattes ikke av kartleggingen, men noen hovedtall fra den ordinære FoU-undersøkelsen i næringslivet er tatt med for å vise omfanget av total bioteknologisk FoU i Norge.

Several recent studies show European university scientists contributing far more frequently to company-owned patented inventions than they do to patents owned by universities or by the academic scientists themselves. Recognising the significance of this channel for direct commercialisation of European academic research makes it important to understand its response to current Bayh-Dole inspired reforms of university patenting rights. This paper studies the contribution from university scientists to inventions patented by dedicated biotech firms (DBFs) specialised in drug discovery in Denmark and Sweden, which in this respect share a number of structural and historic characteristics. It examines effects of the Danish Law on University Patenting (LUP) effective January 2000, which transferred to the employer university rights to patents on inventions made by Danish university scientists alone or as participants in collaborative research with industry. Sweden so far has left property rights with academic scientists, as they also were in Denmark prior to the reform. Consequently, comparison of Danish and Swedish research collaboration before and after LUP offers a quasi-controlled experiment, bringing out effects on joint research of university IPR reform. In original data on all 3,640 inventor contributions behind the 1,087 patents filed by Danish and Swedish DBFs 1990–2004, Difference-in-Difference regressions uncover notable LUP-induced effects in the form of significant reductions in contributions from Danish domestic academic inventors, combined with a simultaneous substitutive increase of non-Danish academic inventors. A moderate increase in academic inventions channelled into university owned-patents does appear after LUP. But the larger part of the inventive potential of academia, previously mobilised into company-owned patents, seems to have been rendered inactive as a result of the reform. As a likely explanation of these effects the paper suggests that exploratory research, the typical target of joint university-DBF projects in drug discovery, fits poorly into LUP’s requirement for ex ante allocation of IPR. The Pre-LUP convention of IPR allocated to the industrial partner in return for research funding and publication rights to the academic partner may have offered more effective contracting for this type of research. There are indications that LUP, outside the exploratory agenda of drug discovery, offers a more productive framework for inventions requiring less complicated and uncertain post-discovery R&D.

The 1998 announcement by American researcher Richard Seed that he intended to clone a human person for reproductive reasons created a large amount of journalistic attention and controversy in the Danish mass media. Developing a theoretical framework inspired by Bruno Latour, this paper analyzes the mass mediated articulation of this announcement as an exploration of the socially viable interpretations of human cloning within the controversial field of biotechnology. An inductive analysis of scripts employed by four national newspapers identifies four main scripts: scientific education, pragmatic regulation, absolute resistance and fatalistic irony. All scripts generally reject the idea of human cloning, but they are found to represent distinctively different forms of social response corresponding to the classification of different cultural dialogues on risk.

The promises and pitfalls of agricultural biotechnology have long been debated. The public is often thwarted because consumers frequently perceive as risky, and therefore undesirable, those advances that they do not understand or of which they are unaware. A comparative study of the introduction of a biotechnology innovation in the United States (US) and in the European Union (EU) is a case in point. In the US, despite concerns of consumer protection and environmental groups that the use of genetically produced growth hormone in milk-producing cows will adversely impact the safety of the milk supply, scientific evidence and governmental findings appear to indicate that milk from treated cows is identical in quality, taste and nutritional value to milk from untreated cows. Experience to date in the US demonstrates some consumer resistance to milk from those cows that have received the growth hormone, which typically leads to a 10% increase in milk production. In fact, if there is no perceived differentiation between the two forms of milk, the issue offers little choice to consumers at large, and may result in economic benefit only to selected dairy farmers, as well as the producers of the genetically produced growth hormone. This situation in the US is an example of dysfunctional technology transfer, with perceived desirable benefits to a few, and perceived undesirable benefits to society-at-large. The information suggests that the US may have reacted hastily in approving the use of bovine growth hormone in milk-producing cows. The EU has taken a divergent approach by enacting moratoriums against its use. The differences identified in this study, contrasting responses to the bovine somatotrophin issue in the US and EU – driven in part by general consumer attitudes towards biotechnology – may provide insights into the issues and challenges that will be faced by both advocates and opponents of global proliferation of certain advances in biotechnology.

This paper examines the organization of distributed innovation shaped by the major discontinuity in the life sciences and their associated technologies that has unfolded over the past three decades. While most studies have focused on its effects on pharmaceutical R&D, this paper studies food processing technologies, taking biotech exploitation of the ubiquitous micro-organism of Lactic Acid Bacteria as its example. Patents provide most of the data. Although highly distributed forms of innovation emerge from 1980 onwards, incumbents introduce virtually all innovations in this field, while the role of dedicated biotechnology firms (DBFs) remains negligible. Public research organizations contribute significantly to distributed R&D, and to a limited extent they also take on the role of economic actors. To explain the organizational characteristics of this distributed innovation this paper suggests a distinction between definition and solution of innovation problems. Extending Simon's analysis of complex problems, it is argued that definition and problem solving in innovation need not have the same levels of decomposability. By implication, the US model for biotech growth, emphasizing the market mechanisms of DBF formation, venture capital and scientist entrepreneurs, should not incautiously be pursued in all contexts and for all applications of biotechnology. Low decomposability of problem definition in innovations may preclude the emergence of these vehicles for market-driven growth, and in such cases distributed innovation must take other forms, including not least an active role of public science.

Eukaryotic algae and cyanobacteria are a promising source of future biofuels, having many important advantages over 1st and 2nd generation biofuels. Synechococcus sp. PCC7002 is the fastest growing oxygen producing photoautotroph known at present, and is capable of surviving a large wide of environments. As such, it is a very interesting organism for the production of biofuels. This is the first work that involves Synechococcus sp. PCC7002 at NTNU. In order to develop this organism into a hydrogen-producing organism, the native and inefficient [NiFe]-hydrogenase should be deleted and replaced by an energetically poised efficient [FeFe]-hydrogenase. A codon-optimized gene for expressing a [FeFe]-hydrogenase was designed. A plasmid was designed to allow for easy transformation and expression of exogenous genes, including the codon-optimized [FeFe]-hydrogenase, in Synechococcus. It contained a high expression promoter, capable of driving expression of proteins in a related cyanobacteria to 15% of the cell's dry weight. A temporary marker gene, mRFP1, was used to verify transformation. However, due to time-constraints, this overexpression system was not evaluated in Synechococcus. In addition, a plasmid was constructed that could delete the native [NiFe]-hydrogenase in Synechococcus. Unfortunately, Synechococcus could not be succesfully transformed with this deletion plasmid. Further, using the gene expression plasmid, a different hydrogenase could be expressed in this strain. Modifications to the Synechococcus transformation protocol are discussed, to help future researchers at NTNU.

Lung cancer is the number one cause of worldwide cancer deaths for men, and second cause of cancer deaths for women. Tobacco smoking is strongly associated with lung cancer, and carcinogenic compounds inhaled when smoking cause DNA damage of lung tissue when they are metabolized in the cells. Epithelial-mesenchymal transition (EMT) is a process in tumorigenesis where the cells gain an invasive and migratory phenotype, and it mediates metastasis of the primary tumor. EMT is characterized by specific markers expressed from the mesenchymal cells. A possible connection between steroid receptor pathways and carcinogens has been proposed, and transcription factors (FOXA family) regulating these pathways may therefore play an important part in regulating carcinogen metabolism. FOXA1 and FOXA2 are downregulated in human bronchial epithelial cells (HBECs) that are exposed to cigarette smoke condensate. As these cells also have gained a mesenchymal phenotype, this downregulation supports the notion that FOXA factors may be involved in EMT. Ectopic expression of FOXA1 in the transformed HBECs is an interesting way of studying whether the factor alone plays a significant part in EMT. In this thesis, we aimed to 1) Develop a method for controlled integration of FOXA1 into transformed HBEC cells 2) Restoring close to normal expression levels in order to study the role of FOXA1 in regulating mesenchymal markers and phenotype To achieve these goals, we used CRISPR-Cas9 system for RNA guided site-specific genome engineering, and designed an expression construct to be integrated into the target genome at a genomic safe harbor target locus. The CRISPR-Cas9 system creates a double strand break at a desired locus, and this break may be repaired by homologous recombination using a repair construct containing the gene of interest (FOXA1). The FOXA1 construct was chemically synthesized as three gene fragments (gBlocks) of ~2000 bp to be assembled using the Gibson Assembly method. After assembly, the construct would be inserted into the AAVS1 safe harbor locus, a site widely used for gene knock-in with no adverse effects reported. In the course of this thesis, assembly of the gBlocks into a FOXA1 construct could not be achieved, and this was probably caused by the overlapping regions needed for Gibson Assembly being too long, and by unspecific products in the PCR-amplified gBlocks inhibiting the reaction. The two custom guide RNA-sequences required for the CRISPR-mediated double strand break were designed, and one of them (gRNA2) was successfully cloned into a CRISPR delivery plasmid vector. The remaining steps required to investigate the role of FOXA1 in EMT is to redesign and successfully assemble the FOXA1 construct, clone gRNA1 into a CRISPR delivery vector, and co-transfect HBEC cells with the FOXA1 construct and guide RNA-containing CRISPR vectors so FOXA1 can be expressed in the cells. Once established, the in vitro site-specific knock-in system will provide a versatile tool for future knock-in studies in HBEC cells and other human in vitro models.

This project aimed to create new fluorescent protein (FPs) variants using the GFPmut3b, EYFP and mRFP1 through fusing two FPs together. The proximity of these fused FPs was expected to result in altered light absorption and fluorescence emission spectra compared to the single FPs. In order to fuse the Fps, plasmids were generated by genetically fusing the coding sequences of two FPs into the expression vector pSB1AK3 with a lacI IPTG-inducible promoter. Gibson cloning and CPEC cloning were used to genetically link the FP. Sequence analysis of the generated constructs, however, indicated that two identical FPs were integrated into the expression vector. Characterization of the linked FPs for light absorption and fluorescence emission showed that their spectral features were identical to the original FPs with absorption maxima at 501 nm, 514 nm, 584 nm and fluorescence emission peaks at 511 nm, 527 nm and 607 nm, for GFPmut3b, EYFP and mRFP1 respectively. The maturation of expressed single FPs and linked FPs was also characterized by inhibiting protein expression with the inhibitor chloramphenicol.

Bacteria in the water interacts with the fish and can affect their health and survival. A stable microbial community in the rearing water can promote a good rearing environment for the fish. The purpose of this study was to examine the microbial community dynamics in three recircu-lating aquaculture systems (RAS) that were run at different salinities. This was performed by comparing the microbial community structure of samples from biofilm carriers, biofilter water and rearing water that were sampled at different dates over a period of 5 months from the three RAS that run salinities at 12 , 22 and 32 . Total DNA were extracted from the samples and PCR (polymerase chain reaction) was used to amplify the variable region 3 of the highly conserved and universal bacterial gene 16S rRNA. The DGGE (denaturing gradient gel electrophoresis) method was used to analyze the resulting PCR products, and to characterize the microbial communities. The results were ana-lyzed statistically to examine variations between communities of samples from the different systems and dates. It was found that the different salinities gave different microbial communities, which indicate that the salinity had an impact on the microbial communities in RAS. Both the biofilter and rearing tank water communities showed significant differences when compared between sys-tems and within systems at different dates. The results indicate that the microbial communities of the biofilm carriers were less affected by the salinity than the water communities. The mi-crobial communities in biofilm carriers developed to become more similar to the communities of the biofilm carriers from the other RAS over time, even though they were sampled from different salinities. The biofilm communities were also found to be more stable than water communities over time.

Lung cancer is the leading cause of cancer related deaths worldwide. Several lines of evidence are confirming that air pollution significantly increase the risk of lung cancer. Exhaust from diesel engines is a component of air pollution and categorized as a human carcinogen by IARC. Diesel exhaust particles and the associated complex mixture of organic pollutants, such as PAHs and their derivatives are major components of diesel emissions, have been under surveillance to exert adverse effects on human health. The aim of the thesis was to identify DEP induced changes on the expression of genes and miRNAs over short period of time and with increasing concentrations of DEP (SRM 2975). An in vitro human bronchial epithelial cell (HBEC3-KT) model was set up and cells were exposed for a short span of time (6-72 h) with 100 μg/ml of DEP or with different concentrations of DEP (25-400 μg/ml) for a fixed time of forty eight hours. Additionally, variations in gene/miRNA expression when the HBEC3-KT cells were exposed for long term (27 weeks) to DEP (100 μg/ml) and for short term (6-72 h) exposure to DEP & B[a]P (100 μg/ml and 3μM respectively) were analyzed. Gene expression analysis was carried out on selected candidate genes and miRNAs, which are known to be involved in lung cancer and/or environmentally induced carcinogenesis. The analyses included CYP1A1, COX2, CDH1, IL1B, HO1, NRF2, miR17, miR21, miR27, miR31, miR146a and miR146b, respectively. In addition, secretion of IL-1-β to the cell culture medium was analyzed. Expression of genes and miRNA s was measured by RT-qPCR, while secretion of IL-1-β was analyzed by ELISA. CYP1A1, which is a phase 1 metabolizing enzyme involved in biotransformation of xenobiotic compounds, was found to be induced by the exposure of DEP both in the time course experiments (maximum at 48 h) and over a wide range of DEP concentrations. Heme oxygenase 1 (encoded by HO1) is involved in many biological processes such as regulation of angiogenesis, cell death, response to oxidative stress and many others. We found that HO1 expression might be up-regulated when HBEC3-KT was exposed to DEP (and benzo[a]pyrene; B[a]P), which gives 4 indications of oxidative stress on the cells. DEP has the ability to induce inflammatory response in cells. Interleukin-1beta (IL-1-β) is a key pro-inflammatory cytokine, which regulates the expression of several genes involved in inflammation. We found significant induction of IL1B by the exposure to DEP and B[a]P. Similarly, IL-1-β secretion from the cells was stimulated by DEP (and B[a]P) exposure, but no clear time or concentration patterns were observed. COX2 is a key enzyme involved in prostaglandin production and prostaglandin function as attractant of inflammatory cells. The COX2 gene was possibly induced by the DEP exposure to HBEC3-KT cells. In contrast, expression of CDH1, NRF2 and the tested miRNAs were found not to be significantly altered by the exposure to DEP. In conclusion, these data indicate that exposure of HBEC3-KT to DEP may affect genes involved in xenobiotic metabolism, induction of inflammatory response, and response to oxidative stress. However, further studies are needed for solid conclusions regarding several of the gene/miRNAs.

The r/K concept is a theoretical concept that can serve as a tool in evaluating the risk of invasion by pathogenic bacterial species in rearing of marine fish larvae. In rearing of marine fish larvae, a dominance of opportunistic, fast-growing r-strategists can result in poor survival of the fish. Dominance of slow-growing K-strategists have been shown to increase the survival and growth. The r-strategists generally exhibits many copies of the 16S rRNA gene in their genomes, whereas the K-strategists have one or a few. Moreover, the maximum specific growth rate also correlates with the maximum RNA content of a cell. Fast-growing r-strategists thus have a higher maximum RNA content per cell, than the slow-growing K-strategists. The aim of this study was to test two methods based upon this fact, as possible methods for determining the fraction of r-or K-strategists in seawater communities. One quantitative real-time PCR (rtPCR) method was used to quantify the number of genomic 16S rRNA genes. Maximum cellular RNA content was determined by a flow cytometry analysis of single cells after a nutrient pulse, to induce the cells to grow at maximum specific growth rate. The methods were established in pure cultures and then in intact microbial communities where a shift from K- to r-selection was established, with gradually a succession towards K-selection. The study in pure bacterial strains confirmed the hypothesis of a correlation between maximum specific growth rate and 16S rRNA gene copy numbers per genome. Moreover, the maximum RNA content of bacterial cells correlated with maximum specific growth rate. In addition, the fast-growing r-strategist was found to divide within 8 hours incubation with nutrients at 15°C, whereas slow-growing K-strategist did not. The r-selected communities showed considerably higher maximum RNA content and higher 16S rRNA gene copy numbers per genome, than K-selected communities. Of the two methods evaluated, the flow cytometry method was best suited to determine the fraction of r- or K-strategists within seawater microbial communities, e.g. rearing water for marine fish larvae. However, the method is based on a nutrient pulse to achieve maximum cellular RNA content, which is a disadvantage, because the optimal growth conditions for uncultivable cells are not identified. The rtPCR method lacked a precise normalization, and an accurate estimate of the 16S rRNA gene copy number per genome was thus not achieved. Ideal normalization could be achieved by using a single-copy protein gene and designing universal bacterial PCR primers targeting the gene. If a successful normalization by a single-copy gene is achieved, the rtPCR method, or possibly the proposed digital droplet PCR (ddPCR) method could be alternative methods to determine the fraction of r- or K-strategists in microbial communities. A method like this relies on analysis on DNA level, and would be cultivation independent, as opposed to the flow cytometry method.

The goal of this thesis is to develop concepts to separate photosynthetic hydrogen production from oxygen production. For this the strains will be generate and with deficient in the photosystems (PSI and PSII) that mediate oxygen production and hydrogen production, respectively. The potential to interfacing these two strains via artificial and natural electron transport systems will be evaluated. The growth experiment will be estimated for mutants on different iron and manganese sources, with light or dark condition. Cyanobacteria are large and diverse group of prokaryote. It is composed of two main membrane, cell wall and cytoplasmic membrane. Within the membrane the photosynthetic apparatus is incorporated where photosynthesis take place. Photosynthesis is the most important process on Earth, by which the oxygen gas is produced and released. The photosynthesis began with light been absorbed by the pigment on the surface of the photosystem II. Energy from the absorbed electron then is used for water oxidation. The electron from water molecule together with electron from the light are transferred to cytochrome b6f complex. Cytochrome b6f complex catalyzes the transfer of electrons from plastoquinol to plastocyanin. It mediates the electrons from photosystem II complex to photosystem I complex. The electron is re-energies and then used to drive synthesis ATP. The Synechocystis sp. PCC6803 was used to create mutants with deficient photosystems (PSI and PSII). The mutant with deficient in PSI was created by deletion slr1834-1835 gene, that is responsible for production P700 apoprotein subunit Ia and Ib. The PSII deficient were create by deletion slr0906 gene, which produce core light harvesting protein. The growth experiments were performed with different iron and manganese sources and exposed to the dark condition and light condition. The results showed that all mutants possess ability to growth on all the media that was made for this experiment. However, there are small differences in growth rate on different media. The Δslr1834-1835 showed increased growth on media that were supplemented with manganese sources exposed to dark condition, compared to the Δslr0906 mutant. The Δslr0906 showed increased growth rate on media supplemented with iron and manganese sources exposed to dark condition. The strains grown on media exposed to light condition showed increased growth rate compared to the growth under the darkness. The strains that were grown under the light condition showed the most increased growth rate over the time. The interfacing two strains via artificial and natural electron transport systems will not be evaluated. This is due to lack of time.

Methionine is a sulfur-containing amino acid which is susceptible to oxidation by Reactive Oxygen Species (ROS). Oxidation of methionine could lead to protein denaturation; with methionine itself turning into methionine sulfoxide (MetSO), a radical which further damages other cellular components. To prevent these damages, the cell uses antioxidizing enzymes which reduce MetSO back to methionine. These enzymes are called methionine sulfoxide reductase (MSR). MSR is divided into two types which correspond to each MetSO diastereomers. The first type is MSRA which shows specificity towards S-MetSO, while the second is MSRB to R-MetSO. These MSR genes are highly conversed and widely distributed amongst the animal and plant kingdom, suggesting their importance in the repair of oxidative damage. The cyanobacterium, Synechocystis PCC 6803 (PCC 6803), contains two MSRA (slr1795 and sll1394) and one MSRB. The function of these MSRs for this organisms' physiology, however, is still poorly defined. The main objective of this research was to therefore characterize MSRs functionality in PCC 6803. In order to do so, two mutant strains lacking either the slr1795 or sll1394 MSRA were generated. These mutant strains were then exposed to moderate and high-light intensity experiments with ROS promoting plate conditions to induce oxidative stress. Parameters such as growth rate and doubling time, degree of pigmentation, chlorophyll and carotenoid levels, and colonial morphology were quantified to determine the effect of MSRAs partial absence on the cells' physiology. Analysis of the experiments revealed that partial absence of MSRA did not necessarily affect the cells overall survivability against oxidative damage; mainly because of the cells many layers of defense against ROS. However, the data gathered from the experiments seem to indicate that the mutant strains were experiencing more stress in comparison to the wild type. In particular, the Δsll1394 strain displayed elevated stress levels on two experiments; the first one being in the high-light experiment, and the second on chlorophyll and carotenoid measurements. Strains exposed to high-light and grown on glucose displayed a decrease in cell density and enter a lag time after 100 hours of incubation, before being able to recover again. Deletion of the sll1394 gene seem to prolong this lag time, suggesting slower repair of the damaged done by ROS. In addition, the Δsll1394 strain also had higher levels of carotenoid in comparison to the other two strains, indicating more sensitivity towards high-light. In summary, the gathered results only gave an initial insight of MSRAs repair and protective functionalities in PCC 6803. Further experiments, such as generation of a double deletion strain, are needed to give a more detailed analysis.

The main objective of this thesis was to develop an efficient transformation protocol for Nannochloropsis oceanica CCMP1779 by biolistic transformation using the linearized pSELECT100 plasmid which confers hygromycin B resistance. In addition the toxicity of several antibiotics for different Nannochloropsis species was determined. Nannochloropsis is the genus of unicellular photosynthetic microalgae in the class of Eustigmatophyceae, which have industrial significance due to their production of high amounts of lipids. The biolistic genetic transformation system of Nannochloropsis oceanica CCMP1779 is still not well established. In the current study, transformation of N. oceanica CCMP1779 was achieved by particle bombardment with the PDS-1000/He instrument using plasmid coated onto gold and tungsten microparticles. Results show that the type of microparticle is an important factor for the efficiency of transformation. N. oceanica is transformed more efficiently with 0.6 µm gold particles than with 0.7 µm tungsten particles. Using the optimized transformation condition, the transformation efficiency was 5-25 transformants per 1.3×109 recipient cells, which is more efficient comparing with those reported in this species transformation studies. Antibiotics are chemical substances that have antagonistic effects on the growth of other microorganisms. This study investigated the growth inhibiting effects of eleven antibiotics on three Nannochloropsis species: Nannochloropsis gaditana, Nannochloropsis limnetica and Nannochloropsis oceanica. Ampicillin and Kanamycin were not toxic at the tested concentrations (20 to 200 µg/mL) to the three species, whereas Zeocin and Cycloheximide were the most toxic to the three Nannochloropsis species (Resulted more than 65% growth reduction at 1 µg/mL). Chloramphenicol, Zeocin, Hygromycin B, Cycloheximide, and G418 neomycin had a promise as selective reagent for the three Nannochloropsis species.

The discovery of new antibiotics is one of the most urgent tasks facing scientists today. The escalating rate of resistant microbes creates a constant need for new or improved drugs. With the recent progress in genome sequencing a new world is opening up as researchers can explore bacterial antibiotic biosynthesis gene clusters that are normally silent under laboratory conditions. Waking up these silent gene clusters harbours the potential of novel drug discoveries to fight microbial infections, cancer or other major life-threatening conditions. In this work, genetic potential of Streptomyces bambergiensis, known producer of antibacterial antibiotic moenomycin, was investigated. Whole genome sequence of S. bambergiensis showed that it has, in addition to moenomycin cluster, 28 secondary metabolite biosynthetic gene clusters, including a giant polyketide synthase (PKS) gene cluster composed of 30 genes and spanning over 190 kb. The product of this gene cluster is unknown and likely not produced under laboratory conditions. First, moenomycin gene cluster was inactivated in S. bambergiensis by deletion in order to eliminate antibacterial background activity. Next, constitutive expression of the regulatory gene, a LuxR family transcriptional regulator, from the PKS gene cluster was accomplished, leading to the production of a compound with inhibitory activity against Bacillus subtilis. Preliminary analysis suggested that this may be a new compound that can be further studied as a potential antibiotic.

Excess of insulin in the circulating blood relative to the level of glucose (Hyperinsulinemia) may be linked to systemic low-grade inflammation and some chronic disease such as the metabolic syndrome, type 2 diabetes (T2D), and cardiovascular disease (CVD). The function of high levels of insulin in vascular smooth muscle cells (VSMCs), endothelial cells (ECs), and macrophages in relation to CVD has been investigated, but exact role of high insulin levels in monocytes, as an important cell type in the pathogenesis of CVD, is largely unknown. In order to better understand the physiological consequences of high levels of insulin in monocytes, we investigated the cellular effects of insulin in THP-1 human monocytic cell line by cell counting, resazurin assay, and flow cytometry cell cycle phase analysis in a dose- and time-dependent manner. We found that insulin induces dose- and time-dependent hyperproliferation of THP-1 cells, as observed by cell counting (significant and most evident increase in cell numbers by 23 % following 24 h at 100 nM insulin). Insulin did not change THP-1 monocytic morphology and color of culture medium, as monitored by microscopy. Insulin treatment also did not trigger cell adhesion to the bottom of culture flasks or clustering of cells. These findings are in agreement with a hyperproliferative effect of insulin in THP-1 monocytes as observations suggest that insulin may not promote differentiation of monocytes to macrophages. Insulin increases metabolic activity of monocytes in a cell density-, time- and dose-dependent fashion (significant increase in metabolic activity following 24 h with a seeding density of 20000 cells/well at 20 nM and 100 nM insulin, each by 5 %), as shown by resazurin bioassays. These stimulatory effects of insulin on cell proliferation and metabolic activity subsided following longer exposure periods. Finally, by flow cytometry, we demonstrated that insulin alters cell cycle phase distribution supporting that insulin may indeed be a dose- and time-dependent inducer of mitosis (significant and most evident alteration following 24 h at 100 nM insulin), hence this result validates the findings by cell counting and resazurin assay in favor of increased proliferation by insulin. In conclusion, the results of this study suggest that insulin increases monocyte proliferation, and hence may affect important aspects of monocyte function, for instance immune and vascular function.

Fucoidan is a sulphated heteropolysaccharide with muglicious nature. It has recently gained much attention because of its bioactive properties. The first objective of this research was to evaluate methods for qualitative determination of fucoidan. As laminaran is co extracted with fucoidan when using acid for extraction, main focus was to determine, to what extent glucose would interfere with the overall quantification for fucose. Two methods were chosen, the Dische method and an enzymatic method. The Dische method was cheap and rapid but it showed a limitation for quantitatively detecting fucose. The method worked precisely if the glucose concentration is 5 times or less, than the concentration of fucose within the samples. The enzymatic method was more sensitive, specific and reliable. The method worked efficiently even if glucose concentration was 40 times higher than fucose. The second objective was to optimize a method for extraction of fucoidan from L. hyperborean to obtain a quantitative yield of fucoidan. Different acid concentrations and extraction times were tested and the conditions were optimized for extraction with 0.1 molar HCl at 80oC for 2 hours. The third objective was to determine the release of fucoidan from whole live algae with damaged parts of the same algae i.e. old leaf, new leaf and stem. L. hyperborea were harvested form Storsteinan (Trondheim) at the end of February 2015 and analyzed for the content of fucoidan. Whole algae of L.hyperborea submerged in distilled water and seawater in darkness and at 4oC were found to release very low amounts. The release of total carbohydrate from plants in seawater was determined to increase with time, with a maximum of about 100 mg/kg wet weight of the algae. The release of fucoidan from the same whole algae was very low, less than what could be reliably detected with the method (< 2 mg fucose /kg wet weight of algae). For the corresponding experiment with whole plants in distilled water, the total carbohydrate content could not be reliably determined. The release of fucose from whole plants in distilled water was similar to in seawater. For the release of total carbohydrate from old leaf in distilled water could not be determined reliably, while the release of fucose from old leaf was found to increase up to about 400 mg/kg wet weight of algae. For the corresponding experiment with old leaf in seawater, both the total carbohydrate and fucose was found to increase about 400 mg/kg wet weight, i.e. almost all carbohydrate released is fucoidan. For the release of total carbohydrate from new leaf in both distilled water and seawater were found to increase to up to around 2500 mg/kg wet weight. The release of fucose from new leaf both in fresh water and seawater were found to increase up to around 120-140 mg/kg wet weight of algal i.e. very much lower that the total carbohydrate content. For the release of total carbohydrate and fucose from the stem in distilled water, the total carbohydrate was found to increase up to around 200 mg/kg wet weight of algae, while fucose increased up to 50-100 mg/kg wet weight of algae, i.e. about half of the carbohydrate released from the stem is fucoidan. Release of total carbohydrate from stem in seawater could not be reliable determined. The release of fucose from stem in seawater was found to increase to about 50 mg/kg wet weight of algae, was found to be not very different from the release of fucose from stem in distilled water. Overall, the release of carbohydrate and fucoidan from whole plants both in distilled water and seawater was very much lower (on the level of few percent), than the release from damaged parts of the plants. No large differences between the release from plants / plants in distilled water as compared to seawater could be found. Interestingly, the release of fucoidan from the individual parts of the plant was quite different, where old leaf released almost exclusively fucoidan, where new leaf released quite low amounts (around 10 %) and stem releasing about half of the total carbohydrates as fucoidan. Whole plant in distilled water, Old leaf in distilled water and stem is sea water could not be determined reliable for total carbohydrate content and gave a higher absorbance at 269 nm which is in the rang for the poly phenols. The hypothesis if fucoidan is produced by the plant in respond to damage could neither be proved nor disproved. Several dried algae samples were showing an increased content of fucoidan after the extraction but this also could have been caused by a faster release of other compounds e.g. laminaran, proteins, salts and phenols, resulting in a counterfeit increase of the fucoidan amount. The determined change in content was too small to answer that question precisely. So it will remain a topic of further research.

Infrared film analysis, a method based on infrared spectroscopy in the mode of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), is demonstrated as a novel analytical method for monitoring enzymatic activity on surface-attached substrate films in the mid infrared range (400-4000 cm(-1)). The ATR-FTIR technique is sensitive to molecules within a distance of approximately 1 microm from the ATR sampling unit surface (a 7 cm(2) hydrophobic ZnSe crystal). Applying a 0.2-0.3 microm thick film on the ATR unit surface, any chemical changes within this film as well as at the interface can be continuously monitored, even having an aqueous phase on top of the film. Infrared film analysis is considered especially useful for studying detergent enzymes, which act on surface bound films consisting of food component like vegetable oils (triacylglycerols) and carbohydrates (e.g. starch). Experimental data are presented for hydrolysis of a triacylglycerol film (triolein) by use of a triacylglycerol lipase (cutinase), and starch film degradation by use of an alpha-amylase.

The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.

This paper is an attempt to unpack the emergence and dynamics of science-based technologies in conceptual forms that allow us to understand better when and how the social and economic organization of search and problem-solving matters. The evolution over two decades of a specific science-based technology is mapped with data from its 192 patents. For the five European countries generating the majority of patents, we identify the host organizations of all 275 inventors involved in the R&D behind the patents. Using network analysis we then map the evolution of separate innovation systems and their structural and evolutionary characteristics. The best performing system combines a cumulative pattern with frequent and shifting connections to non-system R&D partners while maintaining a small core of almost omnipresent inventor-organizations. The role of multinational corporations in orchestrating innovation systems is apparent.

Glucosinolate myrosinase system is well-established defense mechanism against insect herbivores feed on plants from Brassicaceae family. This defense mechanism is also known as the mustard oil bomb which bring myrosinase enzyme and glucosinolate together upon insect attack and tissue disruption and cause glucosinolate hydrolysis by myrosinase enzyme activity. Glucosinolate and its hydrolysis products have a deterring effect on herbivores, and protect plant parts from insect damage. The modified plants have been named MINELESS due to lack of myrosin cells. The aim of this study was to get an insight into defense responses of MINELESS and wild- type Brassica napus plants after being challenged by insects Delia radicum, Delia floralis, Mamestra brassicae and Meligethes aeneus. The hypothesis behind this work were due to lack of myrosin cells and the enzyme myrosinase, the glucosinolate-myrosinase system is modulated and MINELESS plants possess more glucosinolates in plant tissues. Glucosinolate have adverse and deterring effects on insect herbivores, and larvae of generalist herbivore, Mamestra brassicae preferred to feed on wild-type cotyledons of Brassica napus plants, which contain lower amounts of glucosinolates than those of MINELESS. Role of glucosinolates on attraction of pollen beetles, Meligethes aeneus, is not exactly known, however, pollen beetles are attracted by glucosinolates hydrolysis products, isothyocyanates. Wild-type plants were preferred by pollen beetles for feeding over MINELESS plants which possess high levels of glucosinolates, but low levels of isothiocyanates due to lack of myrosinase activity. Four glucosinolates were detected with the highest amount of indol-3-ylmethyl- (I3M) glucosinolate both in control and insect challenged aboveground tissues, with slightly higher amounts in MINELESS plants than wild-type. However total glucosinolate levels did not show exact difference between MINELESS and wild-type plants. I3M glucosinolates levels increased upon herbivore feeding in all treatment days. Increasing days of treatment increased levels of aliphatic glucosinolate, 5-methylsulphinylpentyl- (5MSOP) glucosinolate and decreased levels of indole glucosinolate, I3M glucosinolate. In both no-choice and free choice experiments, M. brassicae larvae consumed more wild-type cotyledons than those of MINELESS. In no-choice experiments consumed area of cotyledons increased in correlation with increasing number of days of feeding, and wild-type plants were consumed more than MINELESS plants. iii The ablation of myrosin cells and consequent lack of myrosinase activity did not affect feeding by pollen beetles in both feeding experiments. Wild-type buds were damaged more than MINELESS buds, however the difference was not significant. Also in Y-tube olfactometer experiment beetles moved mostly towards odour of oilseed rape flowers rather than fresh air. Due to the small number of individuals tested, we did not consider this result significant.

Perfluorooctanoic acid (PFOA) is a fluorinated compound used in many industrial and consumer products. It is environmentally persistent, widespread and bioaccumulative, and multiple toxicities have been reported in experimental models and wildlife. Trace amounts of PFOA have been detected in humans, animals and numerous environmental compartments, including drinking water sources. PFOA can be removed from drinking water by technically complex and energy intensive treatment processes. If the target compound is biodegradable, biological filtration could be a suitable alternative for removal of the contaminant from drinking water. Biofiltration is simple and robust, and represents a green treatment technology. Studies have demonstrated potential of microbial degradation of fluorinated compounds, however the published information is very limited. The short-term impact of environmental occurrence PFOA concentration on biofilm bacterial community dynamics in a biofilter for drinking water treatment was examined. Fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) were applied as molecular methods for characterization of bacterial community dynamics. Bacterial community structure changed during continuous PFOA exposure, as demonstrated by both FISH and PCR-DGGE. FISH analysis of α-, β- and γ-proteobacteria revealed a significant shift from γ- to β-proteobacterial dominance in the biofilter communities in response to the polluted synthetic surface water.

De siste årene har muligheten for å kartlegge det menneskelige arvestoffet utviklet seg i en eksplosiv fart. Utviklingen kan potensielt gi nye muligheter innenfor medisinsk behandling, forebygging og diagnostisering. For fullt ut å benytte disse mulighetene må man kartlegge og tolke menneskelig DNA. Medisinen beveger seg da fra one size fits all til skreddersøm tilpasset hver enkelt pasient. Slik bruk av det menneskelige arvestoffet reiser en rekke rettslige problemstillinger, og det er enkelte av disse som er temaet for denne avhandlingen.

Rapporten omhandler ressursinnsats og resultater innenfor bioteknologisk FoU i universitets- og høgskolesektoren (UoH-sektoren) og instituttsektoren i 2011. Rapporten er basert på en kartlegging NIFU har gjennomført og er tilknyttet den ordinære FoU-undersøkelsen. Tilsvarende kartlegginger er foretatt for årene 2003, 2005, 2007 og 2009. Næringslivet er ikke med i kartleggingen, men noen totaltall fra den ordinære FoU-undersøkelsen er tatt med for å gi et bilde av totalomfanget av bioteknologisk FoU i Norge.

Ada tiga faktor yang mempengaruhi hasil belajar siswa yaitu (1) sumber belajar dan proses belajar di sekolah, (2) kemampuan dan kecakapan guru, dan (3) kemampuan peserta didik. Dosen melalui empat pilar kompetensi dituntut untuk mendorong dan memotivasi mahasiswa dalam belajar. Hal ini dilakukan untuk meningkatkan keaktifan mahasiswa dalam belajar, sehingga dapat meningkatkan hasil belajar. Penelitian ini mengungkap hubungan persepsi dan tingkat keaktifan dengan hasil belajar mahasiswa biologi pada perkuliahan bioteknologi di Universitas Muhammadiyah Malang. Hasil penelitian menunjukkan ada korelasi yang signifikan antara tingkat keaktifan mahasiswa dengan hasil belajar bioteknologi (r = 0,789), sedangkan persepsi mahasiswa tidak memiliki korelasi dengan hasil belajar bioteknologi (r = 0,00).

Astma er en kronisk betennelsessykdom som er et resultat av inflammasjon i luftveiene og det er anslått at 300 millioner mennesker lider av denne sykdommen. Antallet stiger ettersom flere land blir urbanisert og det er anslått at tallet vil nå 400 millioner innen 2025. Astmatikere krever kontinuerlig medisinsk pleie og en nedgang i antall tilfeller vil ha stor effekt på verdensøkonomien. Kitin er en biopolymer som finnes i rikelige mengder i naturen, og årlig produseres det opp mot 100 milliarder tonn. Det er en lineær polymer bestående av repeterende β-(1,4)-N-acetylglukosamin [(GlcNc)n] som finnes som en strukturell polymer i cellevegger hos sopp, eksoskjelett hos insekter og skalldyr og hos helminter og artropoder. Kitin produseres og brytes ned av henholdsvis kitin syntaser og kitinaser. Kitinaser har vist å ha en rolle i antiparasittresponser i lavere livsformer, og mennesker produserer kitinaser som en del av immunforsvaret. Acidic mammalian chitinase (AMCase) er en av kitinasene i glykosyl hydrolase familie 18, og anses som en komponent i astma og allergiske sykdommer. Proteinet er oppregulert i lungevev og makrofager hos astmapasienter, noe som indikerer at medfødte og arvelige defekter i AMCase kan ligge bak luftveisbetennelse og allergisk respons. AMCase har 57 % sekvenslikhet med chitotriosidase (HCHT), den andre kitinasen som er funnet hos mennesker. De har begge flere aromatiske residuer i direkte kontakt med substratet, og disse antas å påvirke enzymenes prosessivitet. HCHT er en del av det medfødte immunforsvar og er assosiert med flere sykdommer, blant annet Gaucher sykdom og sarkoidose, og forskning på de mekaniske egenskaper til HCHT er svært interessant. Det antas at ettersom de fleste substitusjonene av residuer fra HCHT til AMCase befinner seg på proteinoverflaten, vil dette være tegn på tilpassing av enzymet og ikke en forandring som påvirker proteinets reaksjonsmekanisme. Hemming av HCHT og AMCase er av medisinsk interesse og innsikt i enzymenes preferanser er viktig. Spesielt hemming av AMCase har vist å ha en positiv effekt på sykdomsforløpet hos astmatikere. De bakterielle kitinasene kitinase A (ChiA) og kitinase B (ChiB) har store likheter med de humane kitinasene og forskning på ChiA og ChiB kan avdekke kunnskaper som også er relevante for de humane kitinasene. ChiA og ChiB er raskere å dyrke og isolere, noe som gjør forskning på disse mer tidseffektivt. De bakterielle kitinasene deler flere strukturelementer med de humane, blant annet har de aromatiske residuer i det aktive setet som bidrar til å øke prosessiviteten, evnen til å holde på substratet. Polare residuer nær det aktive setet bidrar til å forstyrre den krystallinske strukturen og øke tilgjengeligheten til substratet. Ved å mutere residuer i og nær det aktive setet, antas det at prosessivitet og transglykosyleringsaktivitet kan påvirkes. Dette er svært interessant og vil gi mye kunnskap og muligheter innen kitinaseforskning.

I Norge og spesielt i Oslo-området har det i de siste årene vokst frem et spennende miljø for bioteknologi. I denne oppgaven blir det sett nærmere på internasjonaliseringsprosessen til norske biotech Born Globals. Born Globals er kjennetegnet ved at de ikke internasjonaliserer i samsvar med tidligere teori om bedrifters internasjonaliseringsprosess. Internasjonaliseringsprosessen ble undersøkt ved å ta for seg flere faktorer som kan påvirke denne prosessen for et selskap. Faktorene ble identifisert i litteratur og empiri om hvordan Born Globals internasjonaliserer. Faktorene dannet grunnlaget for undersøkelsens forskningsspørsmål: - I hvilken grad påvirker entreprenør/ledelsens tidligere bakgrunn internasjonaliseringsprosessen? - Hvordan er muligheter i utenlandske markeder blitt oppdaget? - Hvordan ble mulighetene i det utenlandske markedet utnyttet? - Hvordan har det å være medlem av en næringsklynge påvirket internasjonaliseringsprosessen? Det ble gjennomført kvantitative dybdeintervjuer med 4 ledere i bioteknologiselskaper som oppfylte oppgavens utvalgskriterier for casebedrifter. Undersøkelsen viste at: - Alle bedriftene bærer preg av å inneha karakteristikker som er typiske for Born Globals. - Selskapenes entreprenør/ledelse bærer preg av å ha erfaring som kan påvirke internasjonaliseringsprosessen. - De brukte mange like verktøy når de søkte etter muligheter i utlandet. - Det var noe forskjell i inngangsmetodene bedriftene valgte, men forskjellene her bar preg av å ha en sammenheng med hvilke ressurser bedriftene hadde til rådighet. - Klyngemedlemskap hadde ikke påvirket internasjonaliseringsprosessen så langt for bedriftene, men det ble oppdaget faktorer som kan påvirke dette senere. Funnene viser at de fire undersøkte selskapene internasjonaliserer slik teori og empiri om Born Globals tilsier. In recent years an exciting environment has grown forward for biotechnology in Norway and especially in the Oslo region. This master thesis has taken a closer look on the internationalization process for Norwegian biotech Born Globals. A characterization of Born Globals is that they do not seem to follow the suggested internationalization path that earlier literature on the firm’s internationalization process suggests. The internationalization process was examined by identifying several factors that could influence this process. The factors were identified in literature about theory and previous research on how Born Globals internationalize. Those factors made up the foundation for the research questions. - To what degree does the entrepreneur/managements previous experience influence the internationalization process? - How are opportunities in foreign markets discovered? - How are the foreign opportunities taken advantage of? - How is being member of a cluster influence the internationalization process? It was conducted qualitative in dept interviews with four members of the management in biotechnological companies that meet the criteria for selection. The research found; - All of the companies have characteristics that are typical for Born Globals - The companies’ entrepreneurs/ management all had experience that could influence the internationalization process. - They used the same methods when searching for foreign opportunities - It was some difference in the mode of entry the companies choose, but that seem to be in relation to the resources each company had available. - Being member of a cluster had not affected the internationalization process to a noticeable degree thus far. But it was discovered factors that could influence the internationalization process at a later time. The findings in this thesis showed that the four examined companies internationalize in the same manner that the theory and empirical findings about Born Globals suggests.

The blood coagulation Factor XIII (FXIII) is a transglutaminase catalyzing γ-glytamyl ε-lysine crosslinks between various molecules. It is most known for its role in crosslinking fibrin and stabilizing the blood clot in the process of coagulation in wound healing. The Factor XIII is also essential in maintaining pregnancy, and recurrent spontaneous abortions are reported in FXIII deficient patients. The localization and the role of FXIII in the development of the placenta were investigated in this study. Preliminary, FXIII was found to be located in macrophages, and in this study we verified by immunoblotting and mass spectrometry that FXIII is present in the decidual part of the placenta. The effect of FXIII on trophoblastic invasion during placental development was also examined. The invasion process was studied with the Boyden chamber assay using both an immortalized trophoblast cell line (HTR-8/SVneo), and primary culture of extravillous trophoblasts. A significant effect of FXIII on inhibition of invasion using trophoblast cell line was found, but no effect was observed with the primary culture. The cause of the inhibition on invasion observed with the cell line was ruled out to be caused by other indirect effects of FXIII, such as cytotoxicity, effect on secretion of matrix metalloproteases and proliferation. The results showed unfortunately great variability; therefore better controls must be included in the assays in order to obtain more reliable results. Future perspectives are to optimize the existing method or to use another technique to study the more in vivo approach on invasion using primary culture. Later, it would be interesting to examine the proteomics of FXIII; to identify the molecules involved in crosslinking by FXIII in placental development. This would allow us to understand more of the physiology of FXIII and its role in placental development which could aid in recurrent abortion managements.

Encapsulation of pancreatic islets in alginate microbeads and microcapsules show great promise for the treatment of Type 1 diabetes mellitus. Significant progress has been made in developing a biocompatible capsule that allows sufficient exchange of nutrients and products with the encapsulated cells, while at the same time maintaining a barrier to immune cells and preventing rejection of the transplanted cells. However, a truly biocompatible capsule has, as yet, not been developed, and implanted capsules often trigger low levels of inflammation leading to fibrosis, diminished insulin secretion, and sometimes death of the encapsulated cells. A lepirudin-based human whole blood model was used to demonstrate the inflammatory potential of a set of different alginate microcapsules and microbeads. This was performed to elucidate the effect of different capsule and bead parameters, such as the effect of a hollow versus solid inner core, polycation type, polycation concentration, alginate type, and capsule and bead diameter. Complement activation after incubation of capsules in whole blood was measured as sTCC generation. In addition, the secretion of chemokines, inflammatory cytokines, antiinflammatory cytokines, and growth factors was analyzed by ELISA and Bio-plex. Leukocyte activation as measured by CD11b expression was detected using flow cytometry. Finally, Confocal Laser Scanning Microscopy (CLSM) was used in order to screen for a set of plasma proteins and observe what proteins adsorbed to the capsule surface. TAM alginate microbeads did not trigger complement activation, secretion of cytokines, or up-regulation of CD11b expression, and thus appeared to have a minimal inflammatory potential. In addition, the protein adsorption assay showed no apparent protein surface deposition on the microbeads after 6 hours of incubation in plasma of the proteins screened for (complement protein C3, complement regulatory proteins factor H, factor I, C1 inhibitor, and vitronectin, as well as coagulation cascade proteins fibrinogen, plasminogen, and HMWK). Solid alginate APA microcapsules containing poly-L-lysine (PLL), on the other hand, showed an increase in complement component sTCC levels, in chemokine levels (IL-8, MCP-1, and MIP- 1α), in inflammatory cytokine levels (IL-6, IL-1β, and TNFα), in anti-inflammatory cytokine levels (IL-1RA and IL-10), and in growth factors levels (PDGF, HGF, and VEGF), as well as a decrease in cytokine IP-10 levels. In addition, the capsules also stimulated leukocyte activation by up-regulating the expression of CD11b. The solid APA micrcapsules showed heavy C3 adsorption, coupled with vitronectin and factor H surface deposition, indicating increased complement activity on these capsules. Hollow APA microcapsules with PLL triggered a rapid and strong sTCC response, as well as significantly increased secretion of the chemokine MCP-1. At the same time, a significant decrease in secretion of chemokines (IL-8 and MIP-1α) and inflammatory cytokines (IL-1β and TNFα), as well as a decrease in secretion of growth factor VEGF, and cytokine MIF, and an increase in cytokine IP-10 was observed. All these cytokine levels except the chemokine MCP- 1 and the complement complex sTCC suggested reduced inflammatory potential for hollow APA capsules. It was proposed that these capsules adsorbed the anaphylatoxins C3a and C5a, thus preventing the complement mediated activation of leukocytes. No increased surface adsorption of C3 was detected on hollow APA capsules compared to solid APA capsules. Conversely, the C3 adsorption was higher on solid APA capsules, thereby not reflecting the increased sTCC generation seen for hollow APA capsules. One explanation for this might be that the hollow capsules secreted some soluble molecule capable of triggering sTCC generation. No apparent change in inflammatory potential could be observed by exchanging the polycation PLL with PLO (poly-L-ornithine), except for abolishing the strong sTCC response observed for hollow APA capsules with PLL as well as lowering the MCP-1 response. It was suggested that this observation could be the result of PLO reducing the permeability of the capsules, thus preventing the diffusion of the hypothesized soluble trigger of sTCC. Increased sTCC was detected with increasing PLL concentration in High G alginate APA capsules. The same could not be observed for High M alginate capsules, however, the chemokine IL-8 and the inflammatory cytokines IL-1β and TNFα increased with increasing PLL concentration, suggesting increased inflammation with increasing PLL concentration. No change in inflammatory potential could be detected with varying alginate microbead diameter. Nor could any change in inflammatory potential be observed by the addition of HEPES in the gelling solution. TAM alginate microbeads appear to have the lowest inflammatory potential of the capsules tested, and are therefore the most suited for in vivo application from an inflammatory aspect, as demonstrated by the whole blood assay. A recent study in Type 1 diabetes patients however showed increased fibrosis when encapsulating human islet cells in barium alginate microbeads [61]. Further studies where incubation of TAM microbeads with isolated monocytes are co-cultured with fibroblasts could further elucidate the mechanisms of fibrosis on the microbeads. In addition, continued screening of protein adsorption on the bead surface should be performed.

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.

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.

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.

The aim of this experiment was to monitor the microbial communities in two biogas reactors and evaluate the efficiency of denaturing gradient gel electrophoresis (DGGE) as a technique for visualizing shifts in the microbial compositions. The reactors were followed from September 2011 to May 2012. The first reactor is a pilot scale upflow anaerobic sludge blanket (UASB) reactor situated at Foss farm outside of Porsgrunn, running on cow manure. The second reactor is lab scale and situated at Telemark University College, running on pig manure. Samples were taken from the reactors at regular intervals. DNA was extracted from the samples and amplified by polymerase chain reaction (PCR). The primers were 338f and 518r, targeting the 16S rDNA sequence. Changes in the microbial diversity were detected by DGGE in both reactors. Some bands appeared and other disappeared during the period. These changes could not be correlated to changes in operating conditions. This was probably because DGGE reflects cell amounts and not microbe activity levels. DGGE is a highly reproducible and consistently performing fingerprinting technique. It is capable of reflecting long term shifts in the microbial communities and several samples can be compared in one gel. This makes DGGE an effective method for monitoring reactors over time. Several DGGE bands were excised and sequenced, but the results were either negative, or of too poor quality, for further analysis. The probable cause was insufficient separation of bands leading to multiple sequences in the extracted DNA. This may be overcome by using a more specific primer set to reduce the amount of bands.

Breast cancer develops through multiple stages from hyperplasia to invasive and finally metastatic disease. Ductal carcinoma in situ (DCIS) is an abnormal proliferation of epithelial cells within the milk ducts in the breast without invasion beyond the basement membrane. The incidence of DCIS accounts for about 20-25% of newly diagnosed breast cancer cases. Some in situ lesions are believed to rapidly transit to invasive ductal carcinoma (IDC), while others remain unchanged or disappear. Nowadays, women who would never experience invasive breast cancer are undergoing unnecessary and potentially harmful treatment. Studies have revealed that the invasive phenotype of breast cancer is determined at the preinvasive stages of the tumor. Molecular studies of DCIS are therefore important in order to identify those lesions that have a greater risk of developing into invasive disease. The objective of this thesis was to characterize in situ and invasive breast carcinomas by gene expression profiling. Differences in gene expression within DCIS and between DCIS and invasive breast carcinomas were examined to gain insights about molecular mechanisms underlying tumor progression and to identify potential progression markers. 58 tumor tissues from 37 pure DCIS and 21 pure invasive cancers were subjected to microarray gene expression analysis using Agilent One-Color Microarray 8times 60K. Hierarchical clustering proved that the samples related more to subtype than diagnosis. The most significant genes separating the invasive cancers from DCIS were found to be involved in functions related to the extracellular matrix and tumor-stromal interaction. A subgroup of eight DCIS tumors separated from the other DCIS by high expression of genes characteristic of the invasive tumors. These genes could be potential progression markers if validated in other studies. Heterogeneity was observed among the DCIS patients and two subgroups of in situ lesions were clearly differentiated based on upregulated immune response. Elevated levels of immune signaling were found in HER2+, basal-like, normal-like and luminal B subtypes, but were completely absent in luminal A tumors. The suppressing role of the immune system compared with the promoting role needs to be further investigated, and could potentially increase our knowledge concerning the progression of in situ lesions to invasive breast cancer.

The diagnosis of chronic kidney disease (CKD) by examination of the urine has the potential to improve patients outcome by means of earlier detection. Due to the fact that the urine contains metabolic signatures for many biochemical pathways, this biofluid is ideal for metabolomics. A feature unique to diseases of the kidney is that the components of the kidney excrete urine. On the basis of this, analysis of urine have great potential for discovering new biomarkers for renal failure. The aim of this study was therefore to compare urine samples obtained from CKD patients with healthy volunteers, in order to observe differences in metabolite concentration. Four different methods were applied for metabolite analysis. The three first methods used targeted analysis with gas chromatography coupled with single and triple quadrupole mass spectrometry and two different derivatization techniques were evaluated, alkylation and silylation respectively. The fourth method used untargeted analysis with hydrophilic interaction liquid chromatography coupled to a time-of-flight mass spectrometer. The combination of these techniques covers a large part of the urine metabolome by enabling detection of amino- and nonamino acids, sugars, sugar alcohols, purines, pyrimidines etc. The first method identified 36 amino- and nonamino acids in the in-house library as well as finding one unidentified compound present in the samples. The second method identified 59 metabolites using silyaltion as derivatization techniques and identified metabolites which are not amino- and nonamino acids, hypoxanthane and uracil respectively. The third method identified 46 amino- and nonamino acid with absolute quantification. The fourth method using mass profiler professional for feature selection algorithm found 6 accurate masses higher represented in the CKD group, however later it was found that these masses were present in both groups. The results from this study showed differences in metabolite concentration between the CKD group and the control group, where the excretion of almost all components into urine was decreased for the chronic kidney disease subjects. However, some compounds such as benzoate and proline were observed to be at higher concentration. Finally, the results were comparable with previous studies as well as observing metabolite variations between the two groups. However, there is still a long way to go before this can be applied in clinical settings. Future work needs to be performed on a larger group where the patients are with same diagnosis and off medications.

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.

Kartleggingen omfatter ressursinnsatsen innenfor bioteknologisk FoU i universitets- og høgskolesektoren og instituttsektoren. Arbeidet er en oppfølging av tilsvarende kartlegginger for 2003, 2005 og 2007. Ved hjelp av FoU-statistisk materiale belyses ressursinnsats til bioteknologisk FoU i form av utgifter og personale, samt data om resultater, kommersialisering og samarbeid om bioteknologisk FoU. Norges forskningsråd

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.