The Faroe Islands in the North Atlantic are part of the sub-Arctic region, a remote region far from industrial activity. In spite of this remoteness, the Islands are not a sanctuary: exposures and effects of environmental pollutants mar its natural beauty and wildlife. In the Arctic regions, fish, sea mammals and seabirds have shown to contain elevated levels of the classical persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), as well as more recent POPs such as the polybrominated diphenyl ethers (PBDEs). Human populations living in the Arctic regions are usually highly dependent on seafood and seabirds as food sources, and diet becomes their major source of exposures to POPs. As reported in the 1980’s, residents of the Faroe Islands were shown to have high concentrations of organohalogen substances (OHS) in their breast milk. Long-finned pilot whales (Globicephala melas) blubber and meat have been shown to be a major source of OHS exposure for some of the Faroe Islanders. The main objective of this thesis is to investigate the sources and concentrations of some POPs and their metabolites for the Faroese population. First, human milk and serum from pregnant women (mothers) and children were analyzed for PBDEs, PCBs, and polychlorinated biphenylols (OH-PCB), the major PCB metabolites. Second, POPs were measured in seabirds, i.e. PCBs in fulmars (Fulmarus glacialis) and guillemots (Uria algae), and PBDEs in fulmars to search for other potential sources of POPs exposure. The results reinforce previous findings that part of the Faroe Island population is highly exposed to OHS. Median concentrations (430 ng/g lipid weight (l.w.) of CB-153) in maternal serum (1994-95) are among the highest in the world. Serum concentrations of CB-153 in children (age 7, samples collected in the early 2000’s) were approximately 90% of those in the mothers, sampled 1994-95. Similarly high CB-153 concentrations (380 ng/g l.w.) were measured in samples of mother’s milk, collected in 1999. The OH-PCB concentrations were also high in segments of the population, with 2.9 ng/g fresh weight as the sum of five OH-PCBs. Except for 4-OH-CB107, concentrations of OH-PCBs were generally lower in children than in mothers. The ΣPBDE median concentrations in maternal serum and human milk (1999) are at the higher end of those reported in Europe, with levels of 9.5 and 8.2 ng/g l.w. respectively. ΣPBDE levels increase in human milk samples collected at three different time points (1987-1999), mainly due to increasing BDE-153 concentrations. The range of serum ΣPBDE concentrations in mothers and children are similar, although the congener patterns show differences. BDE-47 is the dominant congener in maternal serum, while BDE-153 is the major congener in children. The differences seen in PBDE congener patterns may arise differences in dates of sampling (7 years) for the two populations, maternal serum sampled in 1994-95 and children serum sampled in 2000-01, rather than from differences in uptake/metabolism or in contemporary exposures. PCB concentrations in fulmars and pilot whales show similar ranges. In contrast, PBDE concentrations are 100 times higher in pilot whales than in fulmars. Consequently, Faroese may be especially exposed to PCBs via consumption of fulmars and fulmar eggs, while the exposure to PBDEs is less pronounced. Results from this thesis highlight the pronounced exposures to PCBs, OH-PCBs, and PBDEs among residents of the Faroe Islands, a remote region in the Northern Atlantic far away from industrial and urban sources of pollution.

Obesity is today a problem that has reached epidemic proportions. One of the causes of obesity is the over-consumption of energy. Fat is the most energy-dense nutrient, where the quality seems to be more important for the development of the metabolic diseases than the quantity. The fatty acid composition in serum lipid fractions can be used to mirror the dietary fat quality. Stearoyl-CoA-desaturase (SCD) is an enzyme that converts saturated to monounsaturated fatty acids. A surrogate measure of SCD activity can be estimated as a fatty acid ratio; 16:1/16:0 (palmitoleic acid/palmitic acid) and 18:1/18:0 (oleic acid/stearic acid). The aim of this project was to investigate the intra-day variation in the SCD-ratio in humans eating a standardized diet. The results showed that triacylglycerol and nonesterified fatty acid fractions in serum lipids had a significant variance in the 16:1/16:0 ratio during the day, whereas 18:1/18:0 ratio in the same fractions did not exhibit the same pattern. In this study 16:1/16:0 ratio also seems to be a better marker than 18:1/18:0 ratio for estimating SCD activity. For further evaluation of the intra-day variation there need to be a more long-term study of the SCD-activity for a larger group of subjects.

Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth crisis, resulting in the loss of genomically normal cells prior to the appearance of established, aneuploid cell lines. I used the technique of serum-free cell culture to develop a serum-free mouse embryo (SFME) cell line in which serum was replaced by a set of defined supplements. SFME cells, cultured in a nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin, have maintained a diploid karyotype with no detectable chromosomal abnormalities for more than 200 generations. The cells did not undergo growth crisis and remain in culture today. SFME cells were dependent on EGF for survival and were reversibly growth inhibited by serum or platelet-free plasma. Treatment of SFME cells with serum or transforming growth factor beta led to the appearance of glial fibrillary acid protein (GFAP), a specific marker for astrocytes, identifying SFME cells as proastroblasts. Following the derivation of SFME cells my research focussed on (1) defining more precisely the growth response of SFME cells to medium supplements, (2) investigating the relationship between the nonsenescent nature of SFME cells and their responses to serum and EG1., and (3) applying the serum-free cell culture methods to the multipassage culture of human embryonic astrocytes. SFME cells in serum-containing medium arrested in the G1 phase of the cell cycle with greatly reduced DNA replication activity. A portion of the inhibitory activity of serum was extracted by charcoal, a procedure that removed steroid and thyroid hormones. However, the effect of serum on untransformed SFME cells could not be prevented by addition of antiglucocorticoid, and ras-transformed clones of SFME cells, which are not growth inhibited by serum, retained inhibitory responses to glucocorticoid and thyroid hormone T3. These results suggest that glucocorticoid or thyroid hormones may contribute to the inhibitory activity of serum on SFME cells, but additional factors are involved. SFME cell death resulting from EGF deprivation exhibited characteristics associated with apoptosis or programmed cell death. Ultrastructural analysis showed cells became small and vacuolated, with pyknotic nuclei. The cultures contained almost exclusively G1- phase cells. Chromatin exhibited a pattern of degradation into oligonucleosome-length fragments generating a regularly spaced ladder. I applied the serum-free approach used to derive SFME cells to the multipassage culture of human embryonic astrocytes. Cells were cultured in nutrient medium supplemented with insulin, transferrin, EGF, HDL, fibronectin, basic fibroblast growth factor and heparin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized GFAP. / Graduation date: 1992

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The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

The objectives of these studies were (1) evaluate the relationship between temperament, blood parameters, and performance in Brahman calves (n = 300); (2) evaluate the relationship between residual feed intake (RFI) and reproductive development in Brahman bulls (n = 41). Serum was collected at 24 h and d 21 to 24, and analyzed for total protein (TP) immunoglobulin G (IgG), and cortisol (CS). Calves were weighed at 24 h, weighed and evaluated for temperament using exit velocity (EV) at d 21 to 24, and at 28 d intervals thereafter. Beginning 28 d prior to weaning, and at 28 d intervals through 56 d post-weaning calves were evaluated for pen score (PS) used to calculate temperament score (TS = (EV+PS)/2). The average TS from 28 d prior to weaning and weaning was used to generate temperament groups; calves 1 SD below the mean being calm, those 1 SD above the mean being temperamental and all remaining classified as intermediate. Calf TS influenced WW (P = 0.04) and ADG from birth to weaning (P = 0.03). Serum TP at 24 h affected (P < 0.05) WW and ADG from birth to weaning. Serum IgG at 24 h affected (P = 0.03) WW. Brahman bulls (n = 41) were evaluated for RFI, insulin-like growth factor I (IGF-I), temperament, reproductive development, and ultrasound carcass traits. Serum was collected at d 0 and d 70 of the feeding trial and analyzed for IGF-I. Bulls were classified as efficient, intermediate, or inefficient (RFI classification method I) and as efficient or inefficient (RFI classification method II). Bulls were evaluated for temperament at weaning using TS. Temperament influenced (P < 0.05) IGF-I concentrations at d 0. Reproductive development was not affected (P > 0.05) by TS. Residual feed intake classification did not influence (P > 0.05) age at reproductive milestones. Ultrasound carcass traits were not affected by TS or RFI. Serum TP at 24 h was a viable indicator of future growth performance. Temperamental animals had lower growth rates in both studies. Reproductive development was not affected by RFI. BW at reproductive milestones was lower in temperamental bulls.

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