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  • 1. Agaton, C.
    et al.
    Galli, J.
    Guthenberg, I. H.
    Janzon, L.
    Hansson, M.
    Asplund, A.
    Brundell, E.
    Lindberg, S.
    Ruthberg, I.
    Wester, K.
    Wurtz, D.
    Hoog, C.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Ponten, F.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues2003In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 2, no 6, p. 405-414Article in journal (Refereed)
    Abstract [en]

    Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.

  • 2.
    Agaton, Charlotta
    KTH, Superseded Departments, Biotechnology.
    Transcriptome and Proteome Analysis using Signature Tags2003Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    With the full sequence of the human genome now available, anexciting era in biomedical research has started. The sequenceprovides information about all our genes and greatly increasesthe scope to compare genetic activities in different cells, toanalyze genetic variation between individuals and betweendifferent species and, most importantly, to investigatesystematically the whole genome in a gene-by-gene manner, andthus increase our understanding of gene function.

    This thesis describes studies in which developments weremade in several areas of functional genomics. Messenger RNAlevels were analyzed by the use of an amplification procedure,in which the 3´-ends of the transcripts were selected inorder to amplify the mRNA population in an unbiased fashion. Bysonicating cDNA originating from expressed mRNA, uniformlysized representatives of the transcripts,“signaturetags”, were obtained. The mRNA levels in the original mRNApopulation correlated well with the levels in the amplifiedmaterial, as verified by microarray analysis and realtimequantitative PCR. The expressed transcripts can be identifiedusing pyrosequencing, by comparing the obtained sequenceinformation from the signature tags to information contained invarious sequence databases. In one of the articles, the use ofpyrosequencing is illustrated by efforts to find genes involvedin the disease progression of atherosclerosis.

    More challenging than the study of mRNA levels is to analyzewhen, where and how proteins fulfill their wide-ranging rolesin all the various cellular processes. Proteins are morecomplex biomolecules than mRNA, each having unique properties.Current techniques for studying proteins need much improvement,and are often limited to investigations of a specific portionof the proteome. One approach for studying the whole proteomeis to systematically generate reagents with specific affinityfor the proteins encoded by the genome, one by one. Theaffinity reagents can be used as flags for their targets,providing a flag-specific detection system, so that the targetproteins can be sub-cellularly localized in the majority ofhuman tissues in an array format. One of the articles includedin the thesis presents a pilot project for large-scale affinityreagent production. The aim was to provide a sound basis forwhole proteome studies, but as a pilot study this investigationwas limited to the proteins encoded by human chromosome 21. Allputative genes on the chromosome were subjected to antibodygeneration in a systematic manner. Small, uniform, and easilyproduced representative portions of the full-length proteinswere expressed. These were denoted“Protein EpitopeSignature Tags”and were designed to be unique for theirfull-length counterparts. The antibodies were produced inrabbits and two of the articles in the thesis discuss differentapproaches for affinity purification of the antibodies toachieve the highest possible specificity towards the targets.The resulting“mono-specific”, but still“multi-epitope”, antibodies can be used for a widerange of additional biochemical studies, such as protein arrayand protein pull-out analyses.

    Keywords:functional genomics, 3´-end signaturetags, pyrosequencing, amplification, PrEST, chromosome 21,polyclonal antibodies, dual expression, affinitypurification.

  • 3.
    Agaton, Charlotta
    et al.
    KTH, Superseded Departments, Biotechnology.
    Falk, Ronny
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts2004In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1043, p. 33-40Article in journal (Refereed)
    Abstract [en]

    A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

  • 4.
    Agaton, Charlotta
    et al.
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments, Biotechnology.
    Genome-based proteomics2004In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 9, p. 1280-1288Article in journal (Refereed)
    Abstract [en]

    Protein-protein interactions play crucial roles in various biological pathways and functions. Therefore, the characterization of protein levels and also the network of interactions within an organism would contribute considerably to the understanding of life. The availability of the human genome sequence has created a range of new possibilities for biomedical research. A crucial challenge is to utilize the genetic information for better understanding of protein distribution and function in normal as well as in pathological biological processes. In this review, we have focused on different platforms used for systematic genome-based proteome analyses. These technologies are in many ways complementary and should be seen as various ways to elucidate different functions of the proteome.

  • 5.
    Agaton, Charlotta
    et al.
    KTH, Superseded Departments, Biotechnology.
    Unneberg, Per
    KTH, Superseded Departments, Biotechnology.
    Sievertzon, Maria
    KTH, Superseded Departments, Biotechnology.
    Holmberg, Anders
    KTH, Superseded Departments, Biotechnology.
    Ehn, Maria
    KTH, Superseded Departments, Biotechnology.
    Larsson, Magnus
    KTH, Superseded Departments, Biotechnology.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Gene expression analysis by signature pyrosequencing2002In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 289, no 1-2, p. 31-39Article in journal (Refereed)
    Abstract [en]

     We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.

  • 6. Agerberth, B
    et al.
    Gunne, H
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Kogner, P
    Boman, H G
    Gudmundsson, G H
    FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis.1995In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 92, no 1, p. 195-9Article in journal (Refereed)
    Abstract [en]

    PR-39, a proline/arginine-rich peptide antibiotic, has been purified from pig intestine and later shown to originate in the bone marrow. Intending to isolate a clone for a human counterpart to PR-39, we synthesized a PCR probe derived from the PR-39 gene. However, when this probe was used to screen a human bone marrow cDNA library, eight clones were obtained with information for another putative human peptide antibiotic, designated FALL-39 after the first four residues. FALL-39 is a 39-residue peptide lacking cysteine and tryptophan. All human peptide antibiotics previously isolated (or predicted) belong to the defensin family and contain three disulfide bridges. The clone for prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues. We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the putative peptide. In basal medium E, synthetic FALL-39 was highly active against Escherichia coli and Bacillus megaterium. Residues 13-34 in FALL-39 can be predicted to form a perfect amphiphatic helix, and CD spectra showed that medium E induced 30% helix formation in FALL-39. RNA blot analyses disclosed that the gene for FALL-39 is expressed mainly in human bone marrow and testis.

  • 7.
    Ahmadian, Afshin
    KTH, Superseded Departments, Biotechnology.
    Approaches for analysis of mutations and genetic variations2001Doctoral thesis, monograph (Other academic)
    Abstract [en]

    Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations.

    The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed.

    Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method.

    The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms.

    A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained.

    Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase.

  • 8.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    Gustafsson, A. C.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Single-nucleotide polymorphism analysis by pyrosequencing2000In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 280, no 1, p. 103-110Article in journal (Refereed)
    Abstract [en]

    There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic: alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

  • 9.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gharizadeh, B.
    O'Meara, D.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Genotyping by apyrase-mediated allele-specific extension2001In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 29, no 24Article in journal (Refereed)
    Abstract [en]

    This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.

  • 10.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    A brief history of genetic variation analysis2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 5, p. 1122-+Article, review/survey (Refereed)
    Abstract [en]

    As the human genome sequence is determined, there is an emerging need for the analysis of human sequence variations as genetic markers in diagnosis, linkage and association studies, cancer research, and pharmacogenomics. There are several different techniques and approaches for detecting these genetic variations, and here we review some of these techniques and their application fields. However, all the techniques have advantages and disadvantages, and factors such as laboratory instrumentation, personnel experience, required accuracy, required throughput, and cost often have to be taken into account before selecting a method.

  • 11.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Ronaghi, M.
    Analysis of the p53 tumor suppressor gene by pyrosequencing2000In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 28, no 1, p. 140-+Article in journal (Refereed)
    Abstract [en]

    Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one ol both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Further-more, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.

  • 12.
    Ahmadian, Afshin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Russom, Aman
    KTH, Superseded Departments, Biotechnology.
    Andersson, Helene
    KTH, Superseded Departments, Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Stemme, Göran
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    SNP analysis by allele-specific extension in a micromachined filter chamber2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 4, p. 748-754Article in journal (Refereed)
  • 13. Alfredsson, M.
    et al.
    Hermansson, Kersti
    KTH, Superseded Departments, Biotechnology.
    OH frequency calculations for the hydroxylated MgO(001) surface2002In: Molecular Simulation, ISSN 0892-7022, E-ISSN 1029-0435, Vol. 28, no 07-jun, p. 663-681Article in journal (Refereed)
    Abstract [en]

    We have performed periodic Hartree-Fock calculations for OH groups adsorbed on the MgO(001) surface considering different surface coverages. Six types of OH groups are discussed: OH-, OH, H+, H and hydrogen-bonded OH and H. It is found that when both OH and H are present on the surface, the two groups are best described as OH-. We suggest that the highest-frequency fundamental band (similar to3750 cm(-1) in the experimental OH spectrum) is assigned to OH- groups adsorbed on top of Mg2+, while H+ adsorbed on top of O2- give rise to the broader band at similar to3550 cm(-1).

  • 14. Alfredsson, M.
    et al.
    Hermansson, Kersti
    KTH, Superseded Departments, Biotechnology.
    Dovesi, R.
    Periodic ab initio calculations of the spontaneous polarisation in ferroelectric NaNO22002In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 4, no 17, p. 4204-4211Article in journal (Refereed)
    Abstract [en]

    We present periodic ab initio calculations for ferroelectric NaNO2. The spontaneous polarisation (P-s) has been calculated with three different models based on: (i) point charges; (ii) a multipolar expansion of the charge ;distribution; and (iii) the Berry phase approach. Both the Hartree-Fock and LDA Hamiltonians were employed. Within the Hartree-Fock scheme, at the optimised geometry, we obtain P-s values of 20.3, 13.0 and 16.4 muC cm(-2) with the three models, compared to the experimental value of similar to11.9 muC m(-2). The Berry-phase approach at the Hartree-Fock level gives a value very close to experiment ( namely 12 muC cm(-2)) when the experimental structure is used. At the optimised LDA structure, the LDA P-s values are 16.8, 10.0 and 16.9 muC cm(-2) with models (i)-(iii). The optimised lattice parameters at the Hartree-Fock level are slightly shorter (between 0 and 4%) than those determined experimentally from X-ray-diffraction, while the lattice parameters of the LDA-optimised structure are up to 10% smaller than the experiment. The calculated lattice energies are 679 and 964 kJ mol(-1) at the HF and LDA levels, compared with 729 kJ mol(-1) from the experiment. Charge densities, Mulliken charges and dipole moments are discussed. Finally, we have studied the mechanism for the phase transformation in-between the ferroelectric and paraelectric crystal structures; a rotation of the NO2 group around the c-axis gives the lowest energy barrier.

  • 15. Amira, S.
    et al.
    Spangberg, D.
    Hermansson, Kersti
    KTH, Superseded Departments, Biotechnology.
    Derivation and evaluation of a flexible SPC model for liquid water2004In: Chemical Physics, ISSN 0301-0104, E-ISSN 1873-4421, Vol. 303, no 3, p. 327-334Article in journal (Refereed)
    Abstract [en]

    Molecular dynamics simulations of a new flexible water model are presented. The potential function is based on the simple point charge (SPC) model combined with an accurate experimental quartic intramolecular potential (CCL). This potential not only reproduces the key structural, dynamical and thermodynamical properties of liquid water, but also generates the correct values for both the absolute O-H vibrational frequency and the gas-to-liquid frequency shift. Thus, the model yields a value of ca. 3420 cm(-1) for the peak maximum of the anharmonic OH stretching band for liquid water (experiment 3400 cm(-1)) and a corresponding gas-to-liquid downshift of -300 cm(-1) (experiment -310 cm(-1)). The paper also emphasizes the importance of parameterizing correctly both the harmonic and anharmonic force constants in the gas-phase when developing flexible water models to be used in the liquid state.

  • 16. Amira, S.
    et al.
    Spangberg, D.
    Probst, M.
    Hermansson, Kersti
    KTH, Superseded Departments, Biotechnology.
    Molecular dynamics simulation of Fe2+(aq) and Fe3+(aq)2004In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 108, no 1, p. 496-502Article in journal (Refereed)
    Abstract [en]

    Molecular dynamics simulations of single-ion Fe2+(aq) and Fe3+(aq) solutions have been performed with two rigid-water models (SPC and SPC/E) and a newly constructed SPC-based flexible-water model (SPC+CCL). The SPC+CCL water model in combination with effective Fe2+ and Fe3+ ion-water potentials manages to reproduce many experimental structural and dynamical properties of the solutions. Special attention is given to the large ion-induced frequency shifts of the OH stretching bands, which are also well reproduced by the SPC+CCL model.

  • 17.
    Andersson, Anders
    et al.
    KTH, Superseded Departments, Biotechnology.
    Keskitalo, J.
    Sjödin, A.
    Bhalerao, Rupali
    KTH, Superseded Departments, Biotechnology.
    Sterky, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Wissel, K.
    Tandre, K.
    Aspeborg, Henrik
    KTH, Superseded Departments, Biotechnology.
    Moyle, R.
    Ohmiya, Y.
    Brunner, A.
    Gustafsson, P.
    Karlsson, J.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Nilsson, O.
    Sandberg, G.
    Strauss, S.
    Sundberg, B.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Jansson, S.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    A transcriptional timetable of autumn senescence2004In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 5, no 4, p. R24-Article in journal (Refereed)
    Abstract [en]

    Background: We have developed genomic tools to allow the genus Populus ( aspens and cottonwoods) to be exploited as a full-featured model for investigating fundamental aspects of tree biology. We have undertaken large-scale expressed sequence tag ( EST) sequencing programs and created Populus microarrays with significant gene coverage. One of the important aspects of plant biology that cannot be studied in annual plants is the gene activity involved in the induction of autumn leaf senescence. Results: On the basis of 36,354 Populus ESTs, obtained from seven cDNA libraries, we have created a DNA microarray consisting of 13,490 clones, spotted in duplicate. Of these clones, 12,376 (92%) were confirmed by resequencing and all sequences were annotated and functionally classified. Here we have used the microarray to study transcript abundance in leaves of a free-growing aspen tree ( Populus tremula) in northern Sweden during natural autumn senescence. Of the 13,490 spotted clones, 3,792 represented genes with significant expression in all leaf samples from the seven studied dates. Conclusions: We observed a major shift in gene expression, coinciding with massive chlorophyll degradation, that reflected a shift from photosynthetic competence to energy generation by mitochondrial respiration, oxidation of fatty acids and nutrient mobilization. Autumn senescence had much in common with senescence in annual plants; for example many proteases were induced. We also found evidence for increased transcriptional activity before the appearance of visible signs of senescence, presumably preparing the leaf for degradation of its components.

  • 18. Andersson, C.
    et al.
    Hansson, M.
    Power, U.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column2001In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 34, p. 25-32Article in journal (Refereed)
    Abstract [en]

    The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

  • 19. Andersson, C.
    et al.
    Liljestrom, P.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Power, U. F.
    Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen2000In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 29, no 4, p. 247-253Article in journal (Refereed)
    Abstract [en]

    Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.

  • 20. Andersson, C.
    et al.
    Sandberg, L.
    Wernérus, Henrik
    KTH, Superseded Departments, Biotechnology.
    Johansson, M.
    Lovgren-Bengtsson, K.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation2000In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 238, no 02-jan, p. 181-193Article in journal (Refereed)
    Abstract [en]

    We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-MS-M, ABP-MS-MI, ZZ-MS-M and ZZ-MS-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce MS-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.

  • 21.
    Andersson, Christin
    KTH, Superseded Departments, Biotechnology.
    Production and delivery of recombinant subunit vaccines2000Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated.

    Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived fromStaphylococcus aureusprotein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of thePlasmodium falciparumblood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies forin vivoorin vitrolipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived fromToxoplasma gondii. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation.

    Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product.

    Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy.

    We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against theP. falciparumPf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein.

    Keywords: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,Staphylococcus aureusprotein A, Subunit vaccine,Toxoplasma gondii

  • 22.
    Andersson, Christin
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wikman, Maria
    KTH, Superseded Departments, Biotechnology.
    Lövgren-Bengtsson, Karin
    Lundén, Anne
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation2001In: Journal of Immunological Methods, ISSN 0022-1759, Vol. 255, no 1-2, p. 135-148Article in journal (Refereed)
    Abstract [en]

    We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment ΔSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-ΔSAG1 and His6-ABP-ΔSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-ΔSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, ΔSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that ΔSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

  • 23.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    Griss, P.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Expandable microspheres - surface immobilization techniques2002In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 84, no 2-3, p. 290-295Article in journal (Refereed)
    Abstract [en]

    In this study, a novel component for microfluidics is introduced. Expandable microspheres have been studied for their application in microfluidics. Two methods for selective immobilization of expandable microspheres without the use of mechanical barriers on silicon, including patterning by photolithography and self-assembly based on surface chemistry have been shown. After the immobilization step the microspheres were expanded thermally. The expansion is irreversible and the volume of the microspheres increases more than 60 times. Patterns of microspheres with features as small as 15 pm have successfully been generated by photolithography. By using self-assembly the microspheres can conveniently be immobilized in monolayers. Future applications of the expandable microspheres can be as fluidic components, such as one-shot valves or micropumps, positioning other microcomponents or to enlarge the surface area.

  • 24.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    Jonsson, C.
    Moberg, Christina
    KTH, Superseded Departments, Chemistry.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Consecutive microcontact printing - ligands for asymmetric catalysis in silicon channels2001In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 79, no 1, p. 78-84Article in journal (Refereed)
    Abstract [en]

    Consecutive microcontact printing ( mu CP) has been developed to enable multiple functionalization of silicon surfaces, such as the immobilization of chiral ligands. The technique involves two subsequent printing steps using unstructured poly(methylsiloxane) stamps. The pattern is already defined on the substrate, consisting of etched channels. Hence, no precise alignment is needed between the two printing steps. A carboxylic acid group containing reagent was initially printed onto the silicon oxide surface and transformed to an anhydride. hi the second printing step an ester bond was formed with the hydroxy-functionalized ligand. The formed molecular layers were evaluated by contact angle measurements, scanning electron microscopy (SEM) and electron spectroscopy for chemical analysis (ESCA), indicating that the consecutive mu CP was successful. Initially, printing was performed on planar silicon surfaces but to realize a flow-through microfluidic device for high throughput screening a mu CP technique was developed for etched channels. To verify the technique, hydrophobic valves consisting of octadecyltrichlorosilane were formed using mu CP in deep reactive ion etched channels (50 mum wide and 50 mum deep). The printed hydrophobic patches were visualized by SEM and functioned well. Finally, the consecutive mu CP technique was applied to immobilize the ligand in the channels. The channels were then sealed with a low-temperature bonding technique using an adhesive PDMS film, which does not destroy the printed ligand. In this study mu CP is used in a novel manner. It enables a convenient method for performing complex surface modification of etched structures, which is a frequently appearing problem in biochemical microfluidic systems.

  • 25.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    Jonsson, C.
    Moberg, Christina
    KTH, Superseded Departments, Chemistry.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Patterned self-assembled beads in silicon channels2001In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 22, no 18, p. 3876-3882Article in journal (Refereed)
    Abstract [en]

    A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 mum wide and 50 mum deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 mum, has also been generated on the bottom of a 500 mum wide and 50 mum deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.

  • 26.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    Jonsson, C.
    Moberg, Christina
    KTH, Superseded Departments, Chemistry.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Self-assembled and self-sorted array of chemically active beads for analytical and biochemical screening2002In: Talanta: The International Journal of Pure and Applied Analytical Chemistry, ISSN 0039-9140, E-ISSN 1873-3573, Vol. 56, no 2, p. 301-308Article in journal (Refereed)
    Abstract [en]

    A technique for generating a general screening platform consisting of dots of immobilized beads on silicon has been developed via self-sorting and -assembly of different kinds of beads. The dots are defined by a teflon-like film, which due to its hydrophobic characteristics also prevents cross-contamination of liquid from different dots. To enable functionalization of individual dots with different target molecules simultaneously a new way of microcontact printing has been explored where different target solutions are printed in parallel using one stamp. In order to show that this platform can be designed for both biochemical assays and organic chemistry, streptavidin-, amino- and hydroxy-functionalized beads have been self-sorted and -assembled both on separate and common platforms. The self-sorting and -arrangement are based on surface chemistry only, which has not previously been reported. Beads of different sizes and material have successfully been immobilized in line patterns as narrow as 5 mum. Besides silicon, quartz and polyethylene have also been used as substrates.

  • 27.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    van den Berg, A.
    Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities2004In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 4, no 2, p. 98-103Article, review/survey (Refereed)
    Abstract [en]

    An introductory overview of the use of microfluidic devices for tissue engineering is presented. After a brief description of the background of tissue engineering, different application areas of microfluidic devices are examined. Among these are methods for patterning cells, topographical control over cells and tissues, and bioreactors. Examples where microfluidic devices have been employed are presented such as basal lamina, vascular tissue, liver, bone, cartilage and neurons. It is concluded that until today, microfluidic devices have not been used extensively in tissue engineering. Major contributions are expected in two areas. The first is growth of complex tissue, where microfluidic structures ensure a steady blood supply, thereby circumventing the well-known problem of providing larger tissue structures with a continuous flow of oxygen and nutrition, and withdrawal of waste products. The second, and probably more important function of microfluidics, combined with micro/nanotechnology, lies in the development of in vitro physiological systems for studying fundamental biological phenomena.

  • 28.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    van den Berg, A.
    Microfluidic devices for cellomics: a review2003In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 92, no 3, p. 315-325Article, review/survey (Refereed)
    Abstract [en]

    A review of microfluidic devices for cellomics is presented. After a brief description of the historical background of Lab-on-Chip (LOC) devices, different areas are reviewed. Devices for cell sampling are presented, followed by cell trapping and cell sorting devices based upon mechanical and electrical principles. Subsequently, a popular type of cell sorters, flow cytometers, is considered, followed by a chapter describing devices for cell treatment: cell lysis, poration/gene transfection and cell fusion devices. Finally a number of microfluidic devices for cellular studies are reviewed. The large amount of very recent publications treated in this review indicates the rapidly growing interest in this exciting application area of LOC.

  • 29.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    van der Wijngaart, Wouter
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Enoksson, P.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Micromachined flow-through filter-chamber for chemical reactions on beads2000In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 67, no 1-2, p. 203-208Article in journal (Refereed)
    Abstract [en]

    A new flow-through micromachined device for chemical reactions on beads has been designed, manufactured, and characterized. The device has an uncomplicated planar design and microfabrication process. Both nonmagnetic and magnetic beads can be collected in the reaction chamber without the use of external magnets. The sample flow-through volume of liquid or gas is adjustable and unlimited. The device is sealed with Pyrex to allow real time optical detection of the chemical reactions. At a constant pressure of 3 kPa at the inlet the flow rate for water is about 3.5 mu l/min without beads in the filter chamber, for all the designs. The smallest reaction chamber has a volume of 0.5 nl and can collect approximately 50 beads with a diameter of 5.50 mu m. At a constant pressure of 3 kPa at the inlet, the flow rate for water is about 2.0 mu l/min when the reaction chamber is completely packed with beads. Hence, the flow rate decreases with about 40% when the reaction chamber is packed with beads. The flow-through microfluidic device is not sensitive to gas bubbles, and clogging of the filter is rare and reversible. The beads are easy to remove from the reaction chamber making the micromachined flow-through device reusable. A new and simple technique for fluid interconnection is developed.

  • 30.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    van der Wijngaart, Wouter
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Griss, P.
    Niklaus, Frank
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Hydrophobic valves of plasma deposited octafluorocyclobutane in DRIE channels2001In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 75, no 1-2, p. 136-141Article in journal (Refereed)
    Abstract [en]

    The suitability of using octafluorocyclobutane (C4F8) patches as hydrophobic valves in microfluidic biochemical applications has been shown. A technique has been developed to generate lithographically defined C4F8 hydrophobic patches in deep reactive ion-etched silicon channels. Some of the advantages of this process are that no specific cleaning of the substrate is required, C4F8 is deposited on the sidewalls and the bottom of the channels, a standard photoresist mask can be used to define the patches, and that it is a fast and convenient dry chemical process performed by a standard inductively coupled plasma etcher using the Bosch process. Different patch lengths (200-1000 mum) of C4F8 were deposited in 50 mum wide channels to evaluate which size is most suitable for microfluidic biochemical applications. The valve function of the hydrophobic patches was tested for the following liquids: DD water, acetone, propanol, bead solution and a mixture used for pyrosequencing of DNA. Patch lengths of 200 mum of C4F8 successfully stopped each solution for at least 20 consecutive times. The C4F8 film resists water for at least 5 h. The hydrophobic valve also resists very high concentrations (25%) of surfactants (Tween 80). C4F8 shows a much higher resistance towards water and surface active solutions than previous hydrophobic patches. However, 50% Tween 80 was not stopped at all by the hydrophobic patch. An applied pressure of 760 Pa at the inlet was needed for water to over-run the hydrophobic patch.

  • 31.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    van der Wijngaart, Wouter
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Enoksson, P.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    A valve-less diffuser micropump for microfluidic analytical systems2001In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 72, no 3, p. 259-265Article in journal (Refereed)
    Abstract [en]

    The suitability of valve-less micropumps in biochemistry has been shown. Fluids encountered in various biochemical methods that are problematic for other micropumps have been pumped with good performance. The pump is fabricated as a silicon-glass stack with a new process involving three subsequent deep reactive ion etching steps. Some of the main advantages of the valve-less diffuser pump are the absence of moving parts (excluding the pump diaphragm), the uncomplicated planar design, and high pump performance in terms of pressure head and flow rare. In addition, the micropump is self-priming and insensitive to particles and bubbles present in the pumped media. The results show that the valve-less micropump successfully pumps fluids within the viscosity range of 0.001-0.9 N s/m(2). The micropump is not sensitive to the density, ionic strength, or pH of the pumped media. Effective pumping of solutions containing beads of different sizes was also demonstrated. Living cells were pumped without inducing cell damage and no cell adhesion within the pump chamber was found. No valve-less micropump has previously been reported to pump such a wide variety of fluids.

  • 32.
    Andersson, Helene
    et al.
    KTH, Superseded Departments, Biotechnology.
    van der Wijngaart, Wouter
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Stemme, Göran
    KTH, Superseded Departments, Signals, Sensors and Systems.
    Micromachined filter-chamber array with passive valves for biochemical assays on beads2001In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 22, no 2, p. 249-257Article in journal (Refereed)
    Abstract [en]

    The filter-chamber array presented here enables a real-time parallel analysis of three different samples on beads in a volume of 3 nL, on a 1 cm(2) chip. The filter-chamber array is a system containing three filter-chambers, three passive valves at the inlet channels and a common outlet. The design enables parallel sample handling and time-controlled analysis. The device is microfabricated in silicon and sealed with a Pyrex lid to enable real-time analysis. Single nucleotide polymorphism analysis by using pyrose-quencing has successfully been performed in single filter-chamber devices. The passive valves consist of plasma-deposited octafluorocyclobutane and show a much higher resistance towards water and surface-active solutions than previous hydrophobic patches. The device is not sensitive to gas bubbles, clogging is rare and reversible, and the filter-chamber array is reusable. More complex (bio)chemical reactions on beads can be performed in the devices with passive valves than in the devices without valves.

  • 33. Andersson, M.
    et al.
    Ronnmark, J.
    Arestrom, I.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Ahlborg, N.
    Inclusion of a non-immunoglobulin binding protein in two-site ELISA for quantification of human serum proteins without interference by heterophilic serum antibodies2003In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 283, no 02-jan, p. 225-234Article in journal (Refereed)
    Abstract [en]

    Measurement of human serum molecules with two-site ELISA can be biased by the presence of human heterophilic anti-animal immunoglobulin antibodies (HAIA) that cause false-positive signals by cross-linking the monoclonal (mAb) and/or polyclonal antibodies (pAb) used for the pre- (capture) and post-analyte steps (detection). To evaluate a novel ELISA format designed to avoid interference by HAIA, a target-specific non-immunoglobulin (Ig) affinity protein (affibody) was used to replace one of the antibodies. First, a human IgA-binding affibody (Z(IgA)) selected by phage display technology from a combinatorial library of a single Staphylococcus aureus protein A domain was used. The detection range of IgA standard using an ELISA based on Z(IgA) for capture and goat pAb against IgA (pAb(IgA)) for detection was comparable with that of using pAb(IgA) for both capture and detection. Secondly, another affibody (Z(Apo)) was combined with mAb and used to detect recombinant human apolipoprotein A-1. The affibody/antibody ELISAs were also used to quantify human serum levels of IgA and apolipoprotein A1. To verify that human serum did not cause false-positive signals in the affibody/antibody ELISA format, the ability of human serum to cross-link affibodies, mAb (mouse or rat) and/or pAb (goat) displaying non-matched specificities was assessed; affibodies and antibodies were not cross-linked whereas all combinations of mAb and/or pAb were cross-linked. The combination of affibodies and antibodies for analysis of human serum molecules represents a novel two-site ELISA format which precludes false-positive signals caused by HAIA.

  • 34. Andersson, T.
    et al.
    Borang, S.
    Larsson, M.
    Wirta, V.
    Wennborg, A.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Novel candidate genes for atherosclerosis are identified by representational difference analysis-based transcript profiling of cholesterol-loaded macrophages2001In: Pathobiology (Basel), ISSN 1015-2008, E-ISSN 1423-0291, Vol. 69, no 6, p. 304-314Article in journal (Refereed)
    Abstract [en]

    Objectives: To analyze the early gene expression in macrophages accompanying the phenotypic changes into foam cells upon exposure to oxidized low-density lipoprotein. To identify candidate genes and markers for further studies into the pathogenesis of atherosclerosis. Methods: Cells of the monocytic cell line THP-1 were activated by PMA and exposed to oxidized low-density lipoprotein. Gene expression profiles were investigated after 24 h, using a solid phase cDNA representational difference analysis (RDA) method and shotgun sequencing. Results were verified by microarray hybridization, and analyzed in the virtual chip display of a novel software tool for transcript profile exploration. Results: By comparing transcript profiles of exposed/unexposed cells, 1,984 transcript sequences, representing a total of 921 genes with altered expression levels in response to oxidized low-density lipoprotein exposure, were identified. Genes that are central to cell cycle control and proliferation, inflammatory response, and of pathways not previously implicated in atherosclerosis were identified. The data obtained is also made available on-line at http:// biobase.biotech.kth.se/thp1a for further exploration. Conclusion: The identification of new candidate genes for atherosclerotic disease through RDA-based transcript profiling facilitates further functional genomic studies in coronary artery disease. Candidate genetic polymorphism markers of potential clinical relevance can be identified by filtering information in genome variation databases through the virtual chip analysis of the transcript profiles and subsequently tested in association studies.

  • 35. Andersson, T.
    et al.
    Borang, S.
    Unneberg, P.
    Wirta, V.
    Thelin, A.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Shotgun sequencing and microarray analysis of RDA transcripts2003In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 310, p. 39-47Article in journal (Refereed)
    Abstract [en]

    Monitoring of differential gene expression is an important step towards understanding of gene function. We describe a comparison of the representational difference analysis (RDA) subtraction process with corresponding microarray analysis. The subtraction steps are followed in a quantitative manner using a shotgun cloning and sequencing procedure that includes over 1900 gene sequences. In parallel, the enriched transcripts are spotted onto microarrays facilitating large scale hybridization analysis of the representations and the difference products. We show by the shotgun procedure that there is a high diversity of gene fragments represented in the iterative RDA products (92-67% singletons) with a low number of shared sequences (<9%) between subsequent subtraction cycles. A non redundant set of 1141 RDA clones were immobilized on glass slides and the majority of these clones (97%) gave repeated good fluorescent signals in a subsequent hybridization of the labelled and amplified original cDNA. We observed only a low number of false positives (<2%) and a more than twofold differential expression for 32% (363) of the immobilized RDA clones. In conclusion, we show that by random sequencing of the difference products we obtained an accurate transcript profile of the individual steps and that large-scale confirmation of the obtained transcripts can be achieved by microarray analysis.

  • 36. Andersson, T.
    et al.
    Unneberg, P.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Odeberg, Jacob
    KTH, Superseded Departments, Biotechnology.
    Quackenbush, J.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Monitoring of representational difference analysis subtraction procedures by global microarrays2002In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 6, p. 1348-+Article in journal (Refereed)
    Abstract [en]

    Various approaches to the study of differential gene expression are applied to compare cell lines and tissue samples in a wide range of biological contexts. The compromise between focusing on only the important genes in certain cellular processes and achieving a complete picture is critical for the selection of strategy. We demonstrate how global microarray technology can be used for the exploration of the differentially expressed genes extracted through representational difference analysis (RDA). The subtraction of ubiquitous gene fragments from the two samples was demonstrated using cDNA microarrays including more than 32 000 spotted, PCR-amplified human clones. Hybridizations indicated the expression of 9100 of the microarray elements in a macrophage/foam cell atherosclerosis model system, of which many were removed during the RDA process. The stepwise subtraction procedure was demonstrated to yield an efficient enrichment of gene fragments overrepresented in either sample (18% in the representations, 86% after the first subtraction, and 88% after the second subtraction), many of which were impossible to detect in the starting material. Interestingly, the method allowed for the observation of the differential expression of several members of the low-abundant nuclear receptor gene family. We also observed a certain background level in the difference products of nondifferentially expressed gene fragments, warranting a verification strategy for selected candidate genes. The differential expression of several genes was verified by real-time PCR.

  • 37.
    Andersson, Tove
    KTH, Superseded Departments, Biotechnology.
    Approaches to differential gene expression analysis in atherosclerosis2002Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Today’s rapid development of powerful tools for geneexpression analysis provides unprecedented resources forelucidating complex molecular events.

    The objective of this workhas been to apply, combine andevaluate tools for analysis of differential gene expressionusing atherosclerosis as a model system. First, an optimisedsolid-phase protocol for representational difference analysis(RDA) was applied to twoin vitromodel systems. Initially, The RDA enrichmentprocedure was investigated by shotgun cloning and sequencing ofsuccessive difference products. In the subsequent steps,combinations of RDA and microarray analysis were used tocombine the selectivity and sensitivity of RDA with thehigh-throughput nature of microarrays. This was achieved byimmobilization of RDA clones onto microarrays dedicated forgene expression analysis in atherosclerosis as well ashybridisation of labelled RDA products onto global microarrayscontaining more than 32,000 human clones. Finally, RDA wasapplied for the investigation of the focal localisation ofatherosclerotic plaques in mice usingin vivotissue samples as starting material.

    A large number of differentially expressed clones wereisolated and confirmed by real time PCR. A very diverse rangeof gene fragments was identified in the RDA products especiallywhen they were screened with global microarrays. However, themicroarray data also seem to contain some noise which is ageneral problem using microarrays and should be compensated forby careful verification of the results.

    Quite a large number of candidate genes related to theatherosclerotic process were found by these studies. Inparticular several nuclear receptors with altered expression inresponse to oxidized LDL were identified and deserve furtherinvestigation. Extended functional annotation does not liewithin the scope of this thesis but raw data in the form ofnovel sequences and accession numbers of known sequences havebeen made publicly available in GenBank. Parts of the data arealso available for interactive exploration on-line through aninteractive software tool. The data generated thus constitute abase for new hypotheses to be tested in the field ofatherosclerosis.

    Keywords:representational difference analysis, geneexpression profiling, microarray analysis, atherosclerosis,foam cell formation

  • 38.
    Aspeborg, Henrik
    KTH, Superseded Departments, Biotechnology.
    Discovery of fiber-active enzymes in Populus wood2004Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen.

    First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod.

    A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient.

    Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis.

    Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our ownPopulusESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes.

    Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified.

    Key words:Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase

  • 39. Augustsson, A.
    et al.
    Kashtanov, Stepan
    KTH, Superseded Departments, Biotechnology.
    Luo, Yi
    KTH, Superseded Departments, Biotechnology.
    Chang, C.L.
    Ågren, Hans
    KTH, Superseded Departments, Biotechnology.
    Rubensson, J.-E.
    Nordgren, J.
    Conformations and core-excitation dynamics liquid water.Manuscript (preprint) (Other academic)
  • 40. Backvall, H.
    et al.
    Stromberg, S.
    Gustafsson, Anna
    KTH, Superseded Departments, Biotechnology.
    Asplund, A.
    Sivertsson, Åsa
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Ponten, F.
    Mutation spectra of epidermal p53 clones adjacent to basal cell carcinoma and squamous cell carcinoma2004In: Experimental dermatology, ISSN 0906-6705, E-ISSN 1600-0625, Vol. 13, no 10, p. 643-650Article in journal (Refereed)
    Abstract [en]

    Foci of normal keratinocytes overexpressing p53 protein are frequently found in normal human skin. Such epidermal p53 clones are common in chronically sun-exposed skin and have been suggested to play a role in skin cancer development. In the present study, we have analyzed the prevalence of p53 mutations in epidermal p53 clones from normal skin surrounding basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Using laser-assisted microdissection, 37 epidermal p53 clones adjacent to BCC (21) and SCC (16) were collected. Genetic analysis was performed using a multiplex/nested polymerase chain reaction followed by direct DNA sequencing of p53 exons 2-11. In total, 21 of 37 analyzed p53 clones consisted of p53-mutated keratinocytes. The identified mutations were located in p53 exons 4-8, corresponding to the sequence-specific DNA-binding domain. All mutations were missense, and 78% displayed a typical ultraviolet signature. The frequency of p53 mutations was similar in skin adjacent to BCC compared to SCC. The presented data confirm and extend previous knowledge on the genetic background of epidermal p53 clones. The mutation spectra found in epidermal p53 clones resemble that of non-melanoma skin cancer. Approximately, 40% of the epidermal p53 clones lacked an underlying p53 mutation, suggesting that other genetic events in genes up- or downstream of the p53 gene can generate foci of normal keratinocytes overexpressing p53 protein.

  • 41. Baev, A.
    et al.
    Feifel, R.
    Gel'mukhanov, Faris
    KTH, Superseded Departments, Biotechnology.
    Ågren, Hans
    KTH, Superseded Departments, Biotechnology.
    Piancastelli, M. N.
    Bassler, M.
    Miron, C.
    Sorensen, S. L.
    de Brito, A. N.
    Bjorneholm, O.
    Karlsson, L.
    Svensson, S.
    Geometrical information on core-excited states obtained from interference quenching of vibrational states in resonant x-ray photoemission2003In: Physical Review A. Atomic, Molecular, and Optical Physics, ISSN 1050-2947, E-ISSN 1094-1622, Vol. 67, no 2Article in journal (Refereed)
    Abstract [en]

    An interference quenching of the m=1 final state vibrational line in the resonant Auger decay of N 1s-->pi(*) core-excited N-2 is observed and analyzed. The intensity ratio between the m=1 and m=0 vibrational levels of the X (2)Sigma(g)(+) final state shows a surprising nonmonotonic variation as a function of frequency detuning, going through a minimum with a complete suppression of m=1. We have developed a simple model which indicates a linear relation between the value of the detuning frequency for this minimum and the equilibrium bond distance of the core-excited state. This implies the possibility of determining the equilibrium bond distances for core-excited states to a high degree of accuracy. Simultaneously with the simple model we present a strict theory of the studied effect. This strict theory allows us to explore the accuracy of determining the bond length of the core-excited state from resonant Auger spectra. We obtain a weak influence of the core-hole lifetime on the determined bond length, whereas the number of intermediate vibrational states accounted for in the numerical simulations seems to be quite important.

  • 42. Baev, A.
    et al.
    Gelmukhanov, Faris
    KTH, Superseded Departments, Chemistry.
    Kimberg, Viktor
    KTH, Superseded Departments, Biotechnology.
    Ågren, Hans
    KTH, Superseded Departments, Biotechnology.
    Nonlinear propagation of strong multi-mode fields2003In: Journal of Physics B: Atomic, Molecular and Optical Physics, ISSN 0953-4075, E-ISSN 1361-6455, Vol. 36, p. 3761-3774Article in journal (Refereed)
    Abstract [en]

    We develop a strict theory of nonlinear propagation of few interacting stronglight beams. The key idea of our approach is a self-consistent solution ofthe nonlinear wave equation and the density matrix equations of the materialbeyond the rotatory wave approximation. We assume a Fourier expansion ofthe density matrixwhich goes beyond the conventionalTaylor expansions of thepolarization over the field amplitudeswhich is inadequate for the field strengthsthat we are interested in. Two qualitatively different situations are considered,with and without phase matching. Unlike in our previous paper (Baev et al2003 J. Opt. Soc. Am. B at press) devoted to the three-photon (TP) absorptioninduced upconverted lasing, we obtain here a strict solution for the nonlinearinteraction between different light beams. The general theory is applied to anumerical study of the role of saturation in TP photoabsorption by an organicchromophore in solution.

  • 43. Baev, A.
    et al.
    Gel'mukhanov, Faris
    KTH, Superseded Departments, Biotechnology.
    Macak, P.
    Luo, Yi
    KTH, Superseded Departments, Biotechnology.
    Ågren, Hans
    KTH, Superseded Departments, Biotechnology.
    General theory for pulse propagation in two-photon active media2002In: Journal of Chemical Physics, ISSN 0021-9606, E-ISSN 1089-7690, Vol. 117, no 13, p. 6214-6220Article in journal (Refereed)
    Abstract [en]

    The propagation of laser pulses of different lengths in nonlinear media of organic absorbers is described starting out from a recently suggested dynamical theory for two-photon absorption (TPA) of molecules in solutions [J. Opt. Soc. Am. B 19, 937 (2002)]. The roles of saturation effects and pulse duration on the suppression of TPA are emphasized. The numerical simulations of the pulse propagation are performed for a two-photon active charge transfer molecule using molecular parameters obtained from first principle calculations.

  • 44. Baev, A.
    et al.
    Gel'mukhanov, Faris
    KTH, Superseded Departments, Biotechnology.
    Salek, Pawel
    KTH, Superseded Departments, Biotechnology.
    Ågren, Hans
    KTH, Superseded Departments, Biotechnology.
    Ueda, K.
    de Fanis, A.
    Okada, K.
    Sorensen, S.
    Doppler interference in dissociative resonant photoemission2002In: Physical Review A. Atomic, Molecular, and Optical Physics, ISSN 1050-2947, E-ISSN 1094-1622, Vol. 66, no 2Article in journal (Refereed)
    Abstract [en]

    Resonant photoemission involving dissociative core excited states has been the subject of a great number of experimental and theoretical investigations in recent time. The resonant decay of such dissociating systems has been shown to lead to semiatomic Auger electron emission spectra, with particular angular behavior. In the present paper a detailed theoretical analysis of dissociative resonant photoemission spectra of homonuclear diatomic molecules is presented. The theory addresses both fixed in space and randomly oriented homonuclear molecules and emphasizes the Doppler effect and the role of the interference between channels referring to the Doppler split atomic fragments. It is shown that peaks originating from decay in the atomic fragments can be asymmetric and structured due to the Doppler interference effect. The predicted strong non-Lorentzian behavior of the substructure on the top of the Doppler broadened atomiclike contribution is traced to the interplay between decay channels leading to gerade and ungerade final states. Simulations based on wave-packet theory are compared with experimental data for molecular oxygen. Our numerical simulations of the atomiclike resonance of fixed in space molecules show that the spectral profile is very sensitive to the shape of interatomic potentials of core excited and final states. It is shown that the Doppler effect in the decay spectra depends upon the symmetry of the core excited state.

  • 45. Baev, A
    et al.
    Salek, Pawel
    KTH, Superseded Departments, Biotechnology.
    Gelmukhanov, Faris
    KTH, Superseded Departments, Biotechnology.
    Ågren, Hans
    KTH, Superseded Departments, Biotechnology.
    de Brito, N
    Bjorneholm, O
    Svensson, S
    Picturing molecular femtosecond processes through an ultra-fast controllable X-ray shutter2003In: Chemical Physics, ISSN 0301-0104, E-ISSN 1873-4421, Vol. 289, no 1, p. 51-56Article in journal (Refereed)
    Abstract [en]

    We show that frequency detuning in a resonant X-ray scattering experiment acts as an X-ray camera shutter by regulating the duration time of the scattering process. The camera shutter can be used to select processes at different time scales for observation. This is illustrated by a resonant Auger study of the ultra-fast dissociation of the core-excited HF molecule. We present experimental results and first principle simulations of the molecular fraction in the resonant Auger spectra of HF which is a dynamical parameter that well illustrates X-ray shutter controlled dissociation.

  • 46.
    Baev, Alexander
    KTH, Superseded Departments, Biotechnology.
    Dynamics of optical and X-ray responses of molecules2002Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The aim of this licentiatethesis is to investigate by means of theoretical simulationsthe dynamics of light scattering by molecules, with specialemphasis on molecules in gas phase and in solutions.

    The first part of the thesisconcerns the two-photon absorption (TPA) process in the opticalwavelength region. A new dynamical theory for TPA of moleculesin solutions is developed. The roles of collisions with solventparticles, of pulse duration, of saturation effects and ofpropagation effects on the absorbing capacity of nonlinearmedia are analyzed which can promote a choice of new compoundsfor various applications.

    The second part addresses thesoft X-ray region of exciting light, namely resonant X-rayRaman scattering (RXS). It is shown that collisional dephasingin solutions changes the physics of X-ray scattering. Anadditional, incoherent contribution to the RXS cross sectioncomes into play, resulting in a doubling of the spectrallines.

    A theoretical analysis ofdissociative resonant photoelectron spectra of homonucleardiatomic molecules is performed. Special attention is paid tothe electron Doppler effect and to the role of the interferencebetween scattering channels on formation of an atomic-like partof the spectra.

    The quantum oscillatorybehaviour for the vibrational collapse in the resonant Augerdecay of core excited nitrogen molecule is explored. Aone-phonon model is presented which opens a new possibility todetermine the equilibrium bond distance for a core excitedstates.

  • 47.
    Baev, Alexander
    KTH, Superseded Departments, Biotechnology.
    Multi-Photon Interactions with a Time Structure2003Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The present thesis concerns aspects of the interaction ofmatter in gas, liquid and solid phases, with electromagneticradiation, ranging from the optical to the X-ray region. Overthe last decade the availability of ultrashort strong laserpulses as well as of high power synchrotron sources of tunableX-ray radiation has stimulated a rapid development of newexperimental techniques which makes it possible to analysedifferent physical, chemical and biological processes inunprecedented detail. All of this urges a concomitantdevelopment of adequate theoretical language and methodscombined with simulation techniques.

    The first part of the thesis addresses nonlinear propagationof strong optical pulses. This study is motivated by thebreakthrough in synthesis of novel organic materials possessingprespecified nonlinear optical properties and which has led toa multitude of potential applications such as, for example, 3Dimaging and data storage, optical limiting and photodynamiccancer therapy. In order to clarify the underlying physics, astrict solution has been derived of the density matrixequations of a material aiming at an explicit treatment of itsnonlinear polarization without addressing a conventional Taylorexpansion over field amplitudes. Such a formalism is developedfor many-level molecules, allowing to solve the coupledMaxwell's and density matrix equations for the propagation of afew interacting laser pulses through a nonlinear molecularmedium. The theory presented is capable to account formulti-photon processes of an arbitrary order and for differentsaturation effects. The theory is applied to simulations oftwo- and three-photon absorption as well as to upconvertedstimulated emission of organic molecules in solvents.

    The second part of the thesis is devoted to resonant X-rayRaman scattering from free molecules, solutions and polymerfilms. The temporal analysis of the spectral profiles isperformed using the technique of scattering duration whichallows to select physical processes with different time scales.The slowing-down/speeding-up of the scattering by frequencydetuning provides insight in the formation of the differentparts of the scattering profile like atomic and molecularbands, resonant and vertical scattering channels, anomalousenhancement of the Stokes doubling effect. The lifetimevibrational interference (LVI), playing a crucial role inresonant scattering, is found to strongly influence thedispersion of the Auger resonances of polymers in agreementwith experiment. An almost complete quenching of the scatteringcross section by LVI is observed for the N2molecule. It is found that the interferenceelimination of the scattering amplitude gives valuableinformation on molecular geometry. The electron Doppler effectis minutely studied making use of a wave packet technique. Thesimulations show an "interference burning" of a narrow hole onthe top of the Doppler broadened profile of the Auger spectraof molecular oxygen. For the SF6molecule the Auger Doppler effect is found to besensitive to the detuning due to the scattering anisotropy. Inall of these studies the temporal language was foundconstructive and enormously helpful for understanding theunderlying physical processes. Most theoretical predictionsmade have been verified by experiments.

  • 48.
    Baev, Alexander
    et al.
    KTH, Superseded Departments, Biotechnology.
    Gel'mukhanov, Faris
    KTH, Superseded Departments, Biotechnology.
    Rubio-Pons, Oscar
    KTH, Superseded Departments, Biotechnology.
    Cronstrand, Peter
    KTH, Superseded Departments, Biotechnology.
    Ågren, Hans
    KTH, Superseded Departments, Biotechnology.
    Upconverted lasing based on many-photon absorption: an all dynamic description2004In: Journal of the Optical Society of America. B, Optical physics, ISSN 0740-3224, E-ISSN 1520-8540, Vol. 21, no 2, p. 384-396Article in journal (Refereed)
    Abstract [en]

    A theory is developed for the propagation through a nonlinear medium of strong pump and amplifiedspontaneous-emission pulses. The theory is based on a solution of the density matrix equations that aims at providing an adequate treatment of the nonlinear polarization of the material without addressing the Taylor expansion over the powers of intensity. The theory has been applied for modeling of three-photon absorption induced upconverted stimulated emission of organic molecules in solvents. Numerical results are presented for the organic chromophore 4-[N-(2-hydroxyethyl)-N-(methyl)amino phenyl]-4'-(6-hydroxyhexyl sulfonyl) stilbene dissolved in dimethyl sulfoxide. The results are in good agreement with available experimental results.

  • 49.
    Baev, Alexander
    et al.
    KTH, Superseded Departments, Biotechnology.
    Rubio-Pons, Oscar
    KTH, Superseded Departments, Biotechnology.
    Gel'Mukhanov, Faris
    KTH, Superseded Departments, Biotechnology.
    Ågren, Hans
    KTH, Superseded Departments, Biotechnology.
    Optical limiting properties of Zinc- and Platinum-based organometallic compounds2004In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 108, no 36, p. 7406-7416Article in journal (Refereed)
    Abstract [en]

    Optical power limiting is theoretically studied using an approach that combines quantum electronic structure calculations of multiphoton excitations and classical calculations of dynamical wave propagation. We illustrate the capability of such a combined approach by presenting results for a couple of organometallic compounds; basic metal-base porphyrins, vinylphenylamine porhyrin, and the so-called type IVc platinum compound. A comparative analysis of their electronic properties related to nonlinear absorption of electromagnetic radiation and their optical limiting capability has been performed based on dynamical simulations of the nonlinear pulse propagation taking account of resonant as well as off-resonant effects. Several key features and rate-limiting steps in the transmission have been examined in relation to various characteristics of the pulse. It is found that the resonant vs off-resonant conditions, the saturation conditions and the dephasing play critical roles for the nonlinear transmission. The saturation effects are sensitive to the pulse duration, the inter-system crossing rate and the quenching of the higher triplet state. The inter-system crossing rate has to be comparable with the inverse pulse duration in order to boost the stepwise two-photon channel associated with singlet-singlet followed by triplet-triplet transitions. It is illustrated that structure-to-property relations of the rate-limiting steps serve as important criteria for choices of compounds suitable for the application of interest.

  • 50.
    Bandmann, Nina
    et al.
    KTH, Superseded Departments, Biotechnology.
    Collet, Eric
    KTH, Superseded Departments, Biotechnology.
    Leijen, J
    Uhlén, M
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 79, no 2, p. 161-172Article in journal (Refereed)
    Abstract [en]

    The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitioning of the enzyme in PEG-salt based aqueous two-phase bioseparation systems. Seven different cutinase lipase variants were constructed containing various C-terminal peptide extensions including tryptophan rich peptide tags ((WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positively ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified cutinase variants were stably produced in Escherichia coli as secreted to the periplasm from which they were efficiently purified by IgG-affinity chromatography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants. Partitioning experiments performed in a PEG 4000/sodium phosphate aqueous two-phase system showed that for variants containing either (WP)(2) or (WP)(4) peptide extensions, 10- to 70-fold increases in the partitioning to the PEG rich top-phase were obtained, when compared to the wild type enzyme. An increased partitioning was also seen for cutinase variants tagged with both tryptophans and charged amino acids, whereas the effect of solely charged peptide extensions was relatively small. In addition, when performing partitioning experiments from cell disintegrates, the (WP)(4)-tagged cutinase showed a similarly high PEG-phase partitioning, indicating that the effect from the peptide tag was unaffected by the background of the host proteins. Taken together, the results show that the partitioning of the recombinantly produced cutinase model enzyme could be significantly improved by relatively minor genetic engineering and that the effects observed for purified proteins are retained also in an authentic whole cell disintegrate system. The results presented should be of general interest also for the improvement of the partitioning properties of other industrially interesting proteins including bulk enzymes.

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