The most common cancer among women in Europe and the United States is breast cancer. However, treatment remains a major challenge. If prediction of prognosis and treatment response become more reliable, treatment can be improved. An approach to overcome this challenge is to establish a technique for identification of subtypes. Metabolite profiling of breast cancer has shown potential to identify subtype. The aim was divided into three parts, including metabolite extraction and profiling and isotope labelling of metabolites.The first aim was to optimise a method for extraction of polar and non-polar metabolites for mass spectrometry-based analysis. To achieve the aim, beads-based homogenisation of xenograft tissue in 60% methanol solution and in chloroform was performed in a series of experiments. Requirements were fulfilled, including complete extraction, simple performance and high reproducibility. Method was therefore stated successfully optimised.The second aim was to obtain and compare metabolite profiles of luminal-like and basal-like subtypes of breast cancers. To achieve the aim, polar metabolites were extracted from xenograft tissue using the optimised method prior to gas chromatography mass spectrometry (GC-MS) analysis. Profiles comprising more than 30 metabolites and their concentration were obtained. For comparison of subtypes, data analysis including log2 ratios, principal component analysis (PCA) and Student's t-test were used. All data analyses indicated differences in metabolite concentrations between subtypes. 15 metabolites were found by Student's t-test to significantly differ in concentration between subtypes, including lactate, glycine, citrate, lysine and aspartate. Therefore, metabolite profiling is a potential tool for identification of subtype. Furthermore, metabolites shown to significantly differ may provide insight into metabolic changes in breast cancers that remain poorly understood. The third aim was to investigate metabolic pathways in luminal-like subtype, possessing reduced tumour growth rates due to treatment. To achieve the aim, 13C-labelled glucose was injected into xenograft models prior to tumour excision, extraction, GC-MS analysis and calculation of summed fractional labelling (SFL). 8.3% lactate, 2.2% citrate and 1.6% fumarate were found labelled using SFL. 13C labelling was therefore shown retained throughout glycolysis, to enter the tricarboxylic acid cycle (TCA) cycle and to give rise to TCA intermediates.

Breast cancer is a complex disease comprising subtypes with varying clinical behavior, biological features and treatment response. Breast cancer heterogeneity characterized by different subtypes, is responsible for the high mortality among breast cancer patients, since patients with identical diagnosis can have very different prognosis. Metabolite profiles and biomarkers can hopefully be used for the improved diagnosis and optimal therapeutic treatment.The scope of this study was split in two parts. The first aim was to develop an optimal method for the complete extraction of polar and non-polar metabolites, from invasive ductal carcinoma xenografts. The optimization experiments were performed with tissue samples of a basal-like xenograft model (MAS98.12), and a Precellys 24 homogenizer equipped with a cooling unit. Polar metabolites were detected by absolute quantification analysis by gas-chromatography triple quadrupole mass spectrometry (GC-QqQ-MS) after methyl chloroformate (MCF) derivatization. Non-polar metabolites were detected as fatty acid methyl esters (FAMEs) by quantitative analysis by GC-Q-MS (single quadrupole GC-MS). TMS derivatization was also evaluated, but MCF derivatization was concluded to be a more sensitive method for the polar extracts than TMS derivatization. The complete extraction was achieved after three homogenization rounds with methanol and chloroform, respectively.The second aim was to use the optimal method in metabolite profiling experiments of luminal-like (MAS98.06) and basal-like (MAS98.12) xenografts, in addition to metabolite profiling of basal-like xenografts treated with a cancer drug called MK-2206. Polar metabolite profiles were obtained by absolute quantitative MCF GQ-QqQ-MS and compared by principle component analysis (PCA) and Student`s t-tests. The statistical analyses showed that the MAS98.12 xenograft has significant higher concentrations of lactate and glycine compared to the MAS98.06 xenograft, while the MAS98.06 xenograft has significant higher concentrations of O-acetyl-L-serine and aspartate. The untreated MAS98.12 xenograft has significant higher concentration of lactate than the MK-2206 treated MAS98.12 xenograft. Classification of breast cancer subtypes can therefore be made based on the polar metabolite profiles. Non-polar metabolite profiles were not found after flow injection (FIA) MS of a non-polar extract, much due to a limited research time. Both the extraction method and the metabolite profiles need further validation in the search for biomarkers, that can serve as prognostic tools from the development of breast cancer and determination of prognosis to the establishment of personalized treatment.

Background and objective: Removal of boron is one of the main challenges in the purification of metallurgical grade silicon (MG-Si) for solar cells and a simple low cost method is therefore needed. Boron removal by slag treatment is today regarded as the most promising method, but the efficiency of the refining method is relatively low. Slag refining as a method for boron removal can be improved by optimization of the slag composition by changing the components and/or the composition of the slag. Another method for improving the slag refining efficiency is to remove boron containing species from the slag by evaporation. The aim of this work is to study the refining properties of the Al2O3-MgO-SiO2 slag and the CaO-SiO2-TiO2 slag, respectively. Furthermore, the possibility of evaporating boron from a CaO-SiO2 slag when refined in a hydrogen containing atmosphere will be investigated. Methods: In all, 12 experiments were performed at 1600˚C for determination of the distribution- and mass transfer coefficient of boron in the Al2O3-MgO-SiO2 and CaO-SiO2-TiO2 slag, respectively. In the study of boron evaporation from CaO-SiO2 slag in hydrogen atmosphere 14 experiments were performed. Boron evaporation was investigated in the temperature range 1500-1600˚C, the refining time was 0-6 hours and the slag/silicon mass ratio was varied between 0.25 and 1.5. Results: The distribution- and mass transfer coefficient of boron in the Al2O3-MgO-SiO2 (29%-23%-48%) slag was found to be 1.9 and 2.3x10-6 m/s, respectively. The amount of TiO2 in the CaO-SiO2-TiO2 slag was found to rapidly decrease due to reduction of TiO2 by Si. The distribution- and mass transfer coefficients found for the system were therefore not the same as for the original system. A decrease in the boron content in the slag was observed with increasing refining time in a hydrogen atmosphere at 1600˚C. This indicated that boron evaporated from the system, but the evaporation rate was found to be low. Temperature was not found to significantly influence the evaporation rate of boron in the temperature range 1500-1600˚C. When varying the slag/silicon mass ratio, a considerable increase in the refining efficiency compared to conventional slag refining was observed for the lower slag/silicon mass ratios. The effect diminished for the higher slag/silicon mass ratios. Conclusion: The kinetic and thermodynamic properties of the Al2O3-MgO-SiO2 slag evaluated in this thesis were shown to be in the same range as comparable slags in previous studies. In the CaO-SiO2-TiO2 slag most of the TiO2 was reduced during the refining process which makes it difficult to use this compound as a part of a slag for refining of silicon. When slag refining was performed in a hydrogen atmosphere the refining efficiency compared to conventional slag refining was shown to increase.

Det har blitt estimert at 30 % av verdensuoppdagede gassreserver og 13 % av verdens uoppdagede oljereserver ligger i de polare områdene i nord. Man trenger derfor materialer som tåler lave temperaturer for å hente opp denne oljen og gassen. HSLA-stål har generelt lav omslagstemperatur, men dette kan endres under sveising. Sveisevarmen gjør at mikrostrukturen endres i den varmepåvirkede sonen (HAZ). Dette kan gjøre stålet sprøere. Den grovkornede varmepåvirkede sonen (CG HAZ) er den sprøeste sonen ved avkjøling fra austenittområdet. Målet med denne oppgaven er å undersøke forskjeller i mikrostrukturen og forskjeller i initieringsmekanismen til kløvningsfasetter i CG HAZ med to forskjellige avkjølingstider. Det blir også sett på hva som kan ha innvirkning på initieringen av fasettområdene. Prøver av et 420 MPa HSLA-stål fra Nippon Steel har blitt sveisesimulert for å oppnå mikrostrukturen til CG HAZ. Halvparten av prøvene hadde en avkjølingstid fra 800 til 500 ◦C (∆τ8/5) på 5 sekunder, og andre halvparten ∆τ8/5 på 15 sekunder. Prøvene undergikk avbrutt CTOD-testing. Det foregikk ved at prøvene ble bøyd ved -30 ◦C til det ble avgitt et akustisk emisjons-signal (AE-signal), for så å bli avlastet. Etter avlastning ble prøven utmattet videre til endelig brudd. Hensikten med avlasting og utmatting av prøvene er at man får et tydelig definert fasettområde, slik at det blir lettere å identifisere det i scanning elektronmikroskopet (SEM). Mikrostrukturen til prøven simulert med ∆τ8/5 på 5 sekunder hadde en høyere andel martensitt i bainittstrukturen enn prøven simulert med ∆τ8/5 på 15 sekunder. Det ble også funnet partikler i de to mikrostrukturene. I prøven simulert med ∆τ8/5 på 5 sekunder ble det funnet massive partikler inne i noen av bainittområdene. De ble antatt å være MA-partikler. Det ble i tillegg funnet runde partikler på noen av de tidligere austenittkorngrensene som ble antatt å være korngrenseferritt. I prøven simulert med ∆τ8/5 på 15 sekunder ble det funnet liknende massive partikler. Det ble i tillegg funnet avlange partikler inne i noen av bainittpakkene. De ble antatt å være MA-fase. De fleste prøvene simulert med ∆τ8/5 på 5 sekunder hadde fasettområder med partikler i nærheten av initieringspunktet. Disse partiklene hadde omtrent samme sammensetning som stålmatriks, men det ble i noen tilfeller funnet forhøyede verdier av silisium. Partiklene ble antatt å være MA-partikler. To av prøvene simulert med ∆τ8/5 på 15 sekunder hadde en partikkel i initieringspunktet som hadde omtrent lik sammensetning som stålmatriks, og disse ble også antatt å være MA-partikler. Arealet av alle fasettene i alle prøvene ble målt og sammenlignet med størrelsen på AE-signalet. Av dette ser man en sammenheng der størrelsen på AE-signalet øker med størrelsen på fasettene for prøvene simulert med ∆τ8/5 på 5 sekunder. For prøvene simulert med ∆τ8/5 på 15 sekunder er det flere som skiller seg ut fra denne sammenhengen. Dette forklares ved at disse fasettene oppstod under utmatting. Lengden fra sprekkspissen til initieringspunktet ble også målt og sammenlignet med CMOD-verdien. Det ser ut til å være en tilnærmet lineær sammenheng der økende CMOD-verdi gir bruddinitiering lengre bort fra sprekkspissen. Hovedforskjellen mellom de to avkjølingstidene er at fasettene i prøvene simulert med ∆τ8/5 på 5 sekunder ligger mye nærmere sprekkspissen og er mer topografiske enn prøvene simulert med ∆τ8/5 på 15 sekunder. Når det gjelder kimdanningsmekanisme ser det ut til at de fleste av fasettene i prøvene simulert med ∆τ8/5 på 5 sekunder initieres ved hjelp av spenninger fra partikler som ligger i nærheten av initieringsområdet. Det ble ikke bevist at partiklene er MA-partikler, og man kan derfor ikke si noe sikkert om MA-partiklenes innvirkning på bruddet. Det er vanskelig å si hva kimdanningsmekanismen er for prøvene simulert med ∆τ8/5 på 15 sekunder ettersom initieringspunktene er svært forskjellige.

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.

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.

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.

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.

Mixed ionic electronic conducting ceramic oxides are being investigated for use as membranes for oxygen separation from air and as electrodes in solid oxide fuel cells [1, 2]. Oxygen surface exchange rate is an important parameter controlling the oxy- gen permeation rate of these membranes [3]. In this work surface structuring by wet chemical etching is investigated as a possible route to improve the surface exchange rate of La0.2Sr0.8Fe0.8Ta0.2O3−δ (LSFT). Several different etchant solutions have been in- vestigated (HCl, HNO3, H2SO4, H2C2O4, NaHCO3, Na2CO3 and KOH) and evaluated with respect to etch rate, the resulting surface morphology, selectivity and special effects caused by the etchant or the nature of the sample being etched. The resulting surface structure after etching proved to be very dependent on microstructure, showing both el- evated grain boundary and mid grain etching. The mid grain etching was unexpected, and additional experiments were done to investigate the cause of this behaviour. The effect of surface structuring using wet chemical etching was investigated using electri- cal conductivity relaxation at 800◦C and 900◦C. This showed that an increase of the specific surface area 3.6 times gave an increase of the chemical surface exchange coef- ficient about 3.16 times at 800◦C. The influence of temperature and partial pressure of oxygen on the effect of surface structuring is discussed based on the obtained results. LSF-based membranes (La1−xSrxFe1−yMyO3−δ where M = transition metal cation) show limited stability towards CO2 containing atmospheres, forming surface layers of SrCO3 [4, 5]. It is suggested that a subsequent removal of this layer by i.e. chemical etching could lead to a fine surface structuring. In this work, exposure of LSFT-samples to CO2- rich atmospheres was done at 900◦C and 1000◦C without the formation of such a layer. Reasons for this are discussed and alternate experimental conditions are suggested.