| 11. |
- Bandmann, Nina, 1971-
(författare)
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Rational and combinatorial genetic engineering approaches for improved recombinant protein production and purification
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The bacterium Escherichia coli (E. coli) is in many situations an ideal host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve sufficiently high product quantities. However, there are several factors that may limit this host’s ability to produce large amounts of heterologous proteins in a soluble and native form. For many applications a high purity of the recombinant protein is demanded, which implies a purification strategy where the product efficiently can be isolated from the complex milieu of host cell contaminants. In this thesis, different strategies based on both rational and combinatorial genetic engineering principles have been investigated, aiming at improving and facilitating recombinant E. coli protein production and purification. One objective was to improve the PEG/salt aqueous two-phase system (ATPS) purification process of the lipase cutinase, by increasing the selectivity of the protein for the system top-phase. Peptide tags, with varying properties, were designed and genetically fused to the C-terminal end of ZZ-cutinase. Greatly increased partitioning values were observed for purified protein variants fused to tryptophan containing peptide tags, particularly a (WP)4 peptide. The partitioning properties of the ZZ-cutinase-(WP)4 protein were also retained when added to the ATPS directly from an E. coli total cell disintegrate, emphasizing the applicability of this genetic engineering strategy for primary protein purification in ATPSs. Further on, a combinatorial library approach using phage display technology was investigated as a tool for identification of peptide tags capable of improving partitioning properties of ZZ-cutinase in an ATPS. Repeated ATPS-based partitioning-selection cycles of a large phagemid (pVIII) peptide library, resulted in isolation of phage particles preferentially decorated with peptides rich in tyrosine and proline residues. Both a peptide corresponding to a phage library derived peptide sequence as well as peptides designed based on information of amino acid appearance frequencies in later selection rounds, were shown to improve partitioning several-fold when genetically fused to the C-terminal end of ZZ-cutinase. From the two- to four–fold increased production yields observed for these fusion proteins compared to ZZ-cutinase-(WP)4, it was concluded that the selection system used allowed for selection of desired peptide properties related to both partitioning and E. coli protein production parameters. Bacterial protein production is affected by several different mRNA and protein sequence-related features. Attempts to address single parameters in this respect are difficult due to the inter-dependence of many features, for example between codon optimization and mRNA secondary structure effects. Two combinatorial expression vector libraries (ExLib1 and ExLib2) were constructed using a randomization strategy that potentially could lead to variations in many of these sequence-related features and which would allow a pragmatic search of vector variants showing positive net effects on the level of soluble protein production. ExLib1 was constructed to encode all possible synonymous codons of an eight amino acid N-terminal extension of protein Z, fused to the N-terminal of an enhanced green fluorescent reporter protein (EGFP). In ExLib2, the same eight positions were randomized using an (NNG/T) degeneracy code, which could lead to various effects on both the nucleotide and protein level, through the introduction of nucleotide sequences functional as e.g. alternative ribosome binding or translation initiation sites or as translated codons for an Nterminal extension of the target protein by a peptide sequence. Flow cytometric analyses and sorting of library cell cultures resulted in isolation of clones displaying several-fold increases in whole cell fluorescence compared to a reference clone. SDS-PAGE and western blot analyses verified that this was a result of increases (up to 24-fold) in soluble intracellular ZEGFP product protein content. Both position specific codon bias effects and the appearance of new ribosomal binding sites in the library sequences were concluded to have influenced the protein production. To explore the possibility of applying the same combinatorial library strategy for improving soluble intracellular production of heterologous proteins proven difficult to express in E. coli, three proteins with either bacterial (a transcriptional regulator (DntR)) or human (progesterone receptor ligand binding domain (PRLBD) and 11-β Hydroxysteroid dehydrogenase type I (11-β)) origin, were cloned into the ExLib2 library. Flow cytometric sorting of libraries resulted in isolation of DntR library clones showing increased soluble protein production levels and PR-LBD library clones with up to ten-fold increases in whole cell fluorescence, although the product under these conditions co-separated with the insoluble cell material.
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| 12. |
- Berglund, Björn, 1983-
(författare)
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Deliberations on the impact of antibiotic contamination on dissemination of antibiotic resistance genes in aquatic environments
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The great success of antibiotics in treating bacterial infectious diseases has been hampered by the increasing prevalence of antibiotic resistant bacteria. Not only does antibiotic resistance threaten to increase the difficulty in treating bacterial infectious diseases, but it could also make medical procedures such as routine surgery and organ transplantations very dangerous to perform. Traditionally, antibiotic resistance has been regarded as a strictly clinical problem and studies of the problem have mostly been restricted to a clinical milieu. Recently, non-clinical environments, and in particular aquatic environments, have been recognised as important factors in development and dissemination of antibiotic resistance. Elevated concentrations of antibiotics in an environment are likely to drive a selection pressure which favours resistant bacteria, and are also believed to promote horizontal gene transfer among the indigenous bacteria. Antibiotic resistance genes are often located on mobile genetic elements such as plasmids and integrons, which have the ability to disseminate among taxonomically unrelated species. The environmental bacteria can thus serve as both reservoirs for resistance and hot spots for the development of new antibiotic resistance determinants.There is still a lack of data pertaining to how high antibiotic concentrations are necessary to drive a selection pressure in aquatic environments. The aim of this thesis is to determine the effect of high and low concentrations of antibiotics on environmental bacterial communities from different aquatic environments. In the studies performed, antibiotics were measured using liquid chromatography-mass spectrometry. Bacterial diversity and evenness were assessed using molecular fingerprints obtained with 16S rRNA gene-based denaturing gradient gel electrophoresis, and antibiotic resistance genes and class 1 integrons were quantified using real-time PCR.Water and sediment samples were collected from different rivers and canals in Pakistan. The environments differed in anthropogenic exposure from undisturbed to heavily contaminated. A general trend could be observed of high concentrations of antibiotics correlating to elevated concentrations of antibiotic resistance genes and integrons. Extremely high concentrations of antibiotic resistance genes and integrons were found in the sediments downstream of an industrial drug formulation site, which likely correlated to the high load of antibiotics found in the water. Antibiotic and antibiotic resistance gene concentrations were also shown to increase downstream of Ravi river, which flows through Lahore, a city of more than 10 million inhabitants. Rivers not impacted by anthropogenic contamination were found to contain antibiotics and resistance gene concentrations of similar levels as in Europe and the U.S. Similar measurements were performed in the Swedish river Stångån. The concentrations of antibiotic resistance genes and class 1 integrons were shown to increase in the river after it had passed, and received urban wastewater effluent from the city of Linköping.A series of constructed wetlands were exposed to a mixture of different antibiotics at environmentally relevant concentrations over a few weeks. The antibiotic exposure did not observably affect the bacterial diversity or integron concentrations. Antibiotic resistance genes were found at low background concentrations, but the antibiotic exposure did not observably affect the concentrations. The constructed wetlands were also found to reduce most antibiotics at levels comparable to conventional wastewater treatment schemes, suggesting that constructed wetlands may be useful supplementary alternatives to conventional wastewater treatment.To investigate the effect of antibiotics on an uncontaminated aquatic environment in a more controlled setting, microcosms were constructed from lake water and sediments and subsequently exposed to varying concentrations of antibiotics (ranging from wastewater-like concentrations to 1,000 times higher). The water and sediments were gathered from the lake Nydalasjön, near Umeå, which is not exposed to urban waste. While antibiotic resistance genes and class 1 integrons were found in the lake sediments, no increase in the concentrations of these genes could be observed due to the antibiotic additions.In conclusion, although antibiotic resistance genes and integrons are part of the environmental gene pool, low concentrations of antibiotics do not seem to immediately impact their prevalence. However, aquatic environments exposed to anthropogenic waste do exhibit elevated levels of antibiotic resistance genes and integrons. Aquatic environments heavily polluted with antibiotics also clearly display correspondingly high concentrations of antibiotic resistance genes and integrons. These results clearly indicate the necessity to keep down pollution levels as well as the need to establish the range of antibiotic concentrations which do promote resistance. This must be done in order to enable risk assessments and to establish acceptable levels of antibiotic pollution. It should also be stressed that more research is required to elucidate what effect low levels of antibiotic exposure has on environmental bacterial communities.
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| 13. |
- Berglund, Björn, et al.
(författare)
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Detection and Quantification of Antibiotic Resistance Genes in Stångån River, Sweden
- 2014
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistant bacteria are an emerging global problem which threatens to undermine important advances in modern medicine. It is becoming increasingly clear that the dynamics of antibiotic resistance are not confined to clinical settings. The environment is likely to play an important role in dissemination of antibiotic resistance genes from and to both environmental and pathogenic bacteria. Wastewater treatment plants accumulate both chemical and biological waste from the surrounding urban milieu and have therefore been viewed as potential hotspots for dissemination and development of antibiotic resistance. To assess the effect of wastewater effluent on a river which flows through a Swedish city, sediment and water samples were collected from Stångån River, both upstream and downstream of an adjacent wastewater treatment plant over three months. Seven antibiotic resistance genes and the integrase gene on class 1 integrons were quantified in the collected sediment using realtime PCR. Furthermore, liquid chromatography-mass spectrometry was used to assess the abundance of ten different antibiotics in the water phase of the samples. The results showed an increase in ARGs and integrons downstream of the wastewater treatment plant as compared to upstream. The measured concentrations of antibiotics were low in the water samples from Stångån River, suggesting that selection for antibiotic resistance genes did not occur in the surface water. Instead, the downstream increase in antibiotic resistance genes is likely to be due to accumulation of genes present in the treated effluent discharged from the wastewater treatment plant.
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| 14. |
- Berglund, Erik, 1980-
(författare)
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Adherence to drug treatment and interpretation of treatment effects
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Suboptimal adherence to medical treatments is prevalent across several clinical conditions and can lead to treatment failure. Adherence is a far from fully explored phenomenon and there is little knowledge about how patients interpret treatment effects. Commonly used treatment evaluation measures are often relative measures, which may be difficult for lay people and patients to understand.The overall aim of this thesis was to investigate factors with relevance to adherence, to estimate treatment effects with the time-based Delay of Event (DoE) measure in anticoagulant preventive treatments, and to explore how lay people responded to the DoE measure, as compared with established measures, regarding treatment decisions and effect interpretation.A quantitative population-based cross-sectional design was used for Study I. Study II used data from the Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation (ARISTOTLE) clinical trial and estimated effects as DoEs. Studies III and IV were carried out as randomised survey experiments.The results showed that general adherence behaviour was associated with both environmental and social factors. Estimations of DoE showed that stroke or systemic embolism was delayed 181 (95% CI 76 to 287) days through twenty-two months of apixaban use, as compared with warfarin use. The delay of major and intracranial bleeding was 206 (95% CI 130 to 281) and 392 (95% CI 249 to 535) days, respectively, due to apixaban use for twenty-two months, as compared with warfarin use. Presenting preventive treatment effects as DoEs to lay people was associated with high willingness to initiate treatment and positive views on treatment benefits and willingness to pay for treatment.Non-optimal adherence was partly associated with modifiable factors and it might be possible to increase adherence by managing these factors. Estimations of DoEs in preventive treatments gave information on effects regarding delay of different outcomes; the estimation also provides tools that might be useful for interpreting and communicating treatment effects in clinical decision-making. Lay people seemed to react rationally to variations in DoE magnitude; a higher proportion accepted treatment when the magnitude was greater.
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| 15. |
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| 16. |
- Berglund, Johan C, 1978, et al.
(författare)
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Measuring strategies for smooth tool steel surfaces
- 2008
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Ingår i: Proceedings. - Aachen : Shaker Verlag. - 1610-4773. - 9783832269128 - 3832269126 ; , s. 110-119
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Comparisons between different measuring strategies were made on three types of smooth tool steel surfaces. Three replica materials were tested to study possibilities within replication techniques. An optical interferometer as well as a mechanical stylus was used to evaluate the surfaces. The results showed that the tested replica materials generated good representations of both the form and the surface roughness (Sq > 300 nm). The evaluated surfaces were quite homogeneous, thus, few measurements are needed to get representative results. However, it was found that caution must be taken regarding manually polished surfaces which can be less homogenous and therefore require more measurements to get representative results.
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| 17. |
- Berglund, Kerstin, 1961-
(författare)
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Straffrätt och kön
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis starts in feminist theory and the debate on sex/gender and knowledge. There are two lines of inquiry, one in criminal law and one in feminist theory that interconnect throughout the book. Their common grounds are the depiction of the individual. In criminal law “man” is found first of all on the theoretical level as part of legal theory. Criminal law is based on the traditional liberal ideal of the autonomous individual. When criminal law is applied legal reasoning also demands an idea of what a human being is. Legal reasoning is therefore always dependent on descriptions of both “man” and “reality”. Feminist theory, in turn, deals primarily with sex/gender issues. In an attempt to structure this field of research and to highlight important differences in feminist theory, three positions are presented. The three positions are defined in terms of the ideological aspects of the sex/gender debate, and the epistemological theories that can be related to these different ideological positions.Different descriptions of “man” and “reality” can lead to profoundly different conditions for legal reasoning. In the book it is the contradiction between the idea of sex as a role played by a neutral individual, and the idea of gender as a fundamental aspect of human life, that is used as a starting point for the analyses of legal arguments. One question that is raised is in what way the understanding of harm to the individual changes when the conditions for describing “man” are altered. In order to answer this question, selected committee reports on rape and physical abuse of women during the period 1958 to 2001, are analyzed. All of these are to various extents related to the question of harm to the individual. When judging harm in the given examples, the gendered individual is used as an alternative way of describing “man”. The study concludes that it is important for criminal law to recognize that women are sexually abused because they are women. This is fundamental to the way in which these crimes must be interpreted. But it is also important to stress that women are sexually abused in their capacity of being women. It is argued that this constitutes the very basis for understanding harm to the individual in these cases. Victims of sexual violence are always embodied, gendered and socially situated. It is therefore important to find ways to define harm to the gendered individual. In brief, the conflicts surrounding criminal law today can be understood as the dichotomy between the liberal ideal of the autonomous individual and the feminist ideology of difference. It is therefore argued that there is a need for an ethical theory that includes the gendered individual.
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| 18. |
- Berglund, Olof, et al.
(författare)
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Organochlorine accumulation and stable isotope ratios in an Atlantic salmon (Salmo salar) population from the Baltic Sea
- 2001
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Ingår i: Science of the Total Environment. - 1879-1026. ; 281:1-3, s. 141-151
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- We investigated the organochlorine accumulation (p,p′-DDE and ∑PCB) and stable isotope ratios (N and C) in a migrating salmon (Salmo salar) population in the Baltic Sea. ∂15N in the entire population ranged from 11.7 to 13.7‰ and ∂13C ranged from −22.0 to −19.6‰. In the entire population, ∂15N were weakly related to p,p′-DDE and ∑PCB concentrations (r2=0.20 and 0.14, respectively). However, lipid content was a better predictor of organochlorine (OC) concentrations (r2=0.48 and 0.50, respectively). Lipid adjusted ∑PCB concentrations were not related to ∂15N, and lipid adjusted p,p′-DDE concentrations were only weakly related (r2=0.12). We divided the salmon population into three groups representing reproductive strategies: small males <5 kg (SM); large males >5 kg (LM); and females (F). ∂15N varied between the three reproductive strategies. The mean ∂15N for SM was 12.7±0.3‰, for F was 12.9±0.3‰, and for LM equalled 13.2±0.3‰. Lipid adjusted OC concentrations differed between the three reproductive strategies, with the females having the highest concentrations. We conclude that the ∂15N varied between reproductive strategies indicating differences in prey choice, but the lipid content was better able to predict OC concentrations than the trophic position of the salmon.
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| 19. |
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| 20. |
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