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  • 101.
    Båge, Tove
    et al.
    Karolinska Inst, Div Pediat Dent.
    Lindberg, Johan
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Modéer, Thomas
    Karolinska Inst, Div Pediat Dent.
    Yucel-Lindberg, Tülay
    Karolinska Inst, Div Pediat Dent.
    Microarray analysis of the regulation of TNFa-stimulated PGE2 production in gingival fibroblasts: special reference to intracellular signal transduction pathwaysArticle in journal (Other academic)
  • 102. Båge, Tove
    et al.
    Lindberg, Johan
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Modéer, Thomas
    Yucel-Lindberg, Tülay
    Signal pathways JNK and NF-kappa B, identified by global gene expression profiling, are involved in regulation of TNF alpha-induced mPGES-1 and COX-2 expression in gingival fibroblasts2010In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, p. 241-Article in journal (Refereed)
    Abstract [en]

    Background: Prostaglandin E-2 (PGE(2)) is involved in several chronic inflammatory diseases including periodontitis, which causes loss of the gingival tissue and alveolar bone supporting the teeth. We have previously shown that tumor necrosis factor a (TNF alpha) induces PGE(2) synthesis in gingival fibroblasts. In this study we aimed to investigate the global gene expression profile of TNF alpha-stimulated primary human gingival fibroblasts, focusing on signal pathways related to the PGE(2)-synthesizing enzymes prostaglandin E synthases (PGES), as well as the upstream enzyme cyclooxygenase-2 (COX-2) and PGE(2) production. Results: Microarray and western blot analyses showed that the mRNA and protein expression of the inflammatory induced microsomal prostaglandin E synthase-1 (mPGES-1) was up-regulated by the cytokine TNF alpha, accompanied by enhanced expression of COX-2 and increased production of PGE(2). In contrast, the expression of the isoenzymes microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNF alpha treatment. Using oligonucleotide microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, differentially expressed genes in response to TNF alpha treatment were identified. Enrichment analysis of microarray data indicated two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-kappa B (NF-kappa B). To evaluate their involvement in the regulation of mPGES-1 and COX-2 expression, we used specific inhibitors as well as phosphorylation analysis. Phosphorylation analysis of JNK (T183/Y185) and NF-kappa B p65 (S536) showed increased phosphorylation in response to TNF alpha treatment, which was decreased by specific inhibitors of JNK (SP600125) and NF-kappa B (Bay 11-7082, Ro 106-9920). Inhibitors of JNK and NF-kappa B also decreased the TNF alpha-stimulated up-regulation of mPGES-1 and COX-2 as well as PGE(2) production. Conclusion: In the global gene expression profile, the enrichment analysis of microarray data identified the two signal transduction pathways JNK and NF-kappa B as positively regulated by the cytokine TNF alpha. Inhibition of these TNF alpha-activated signal pathways reduced the expression of mPGES-1 and COX-2 as well as their end product PGE(2) in gingival fibroblasts. The involvement of the signal pathways JNK and NF-kappa B in the regulation of PGE(2) induced by TNF alpha may suggest these two pathways as possible attractive targets in the chronic inflammatory disease periodontitis.

  • 103.
    Calles, Karin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Regulation of productivity in Trichoplusia ni and Spodoptera frugiperda Sf9 serum-free cultures2005Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium.

    Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex.

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  • 104. Calles, Karin
    et al.
    Eriksson, Ulrika
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Häggström, Lena
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures.2006In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 3, p. 653-659Article in journal (Refereed)
    Abstract [en]

    The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.

  • 105. Calles, Karin
    et al.
    Svensson, Ingrid
    Lindskog, Eva
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Häggström, Lena
    Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity2006In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 2, p. 394-400Article in journal (Refereed)
    Abstract [en]

    The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Low passage (LP) cells at passages 20-45 were compared to high passage (HP) cells at passages > 100. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. LP cells passed a switch in growth kinetics, characterized by a shorter lag phase and a higher growth rate, after 30-40 passages. After this point, CM lost its stimulating effect on proliferation. HP cells displayed a still shorter lag phase and reached the maximum cell density 24-48 earlier than LP cells. HP cells also exhibited higher specific productivity of recombinant protein compared to LP cells, when infected with baculovirus during the initial 48 h of culture. The specific productivity of LP cells was decreased by 30-50% by addition of 20% CM or 10 kDa filtrated CM, whereas addition of CM to cells having passed the switch in growth kinetics had no negative effect on productivity. Cell cycle analysis showed that a large proportion of HP cells, >60%, was transiently arrested in G2/M after inoculation. In LP cultures this proportion was lower, 40-45%, and addition of CM decreased the arrested population further. This correlated to the cell size, the HP cells being the largest: HP cells > LP > LP + 20% CM > LP + 20% 10 kDa filtrated CM. Since the degree of synchronization in G2/M correlated to the productivity, yeastolate limitation was used to achieve 85% G2/M synchronized cells. In this culture the specific productivity was maintained during a prolonged production phase and a 69% higher volumetric yield was obtained. The results suggest that a decreasing degree of synchronization during the course of culture partly explains the cell-density-dependent drop in productivity in Sf9 cells.

  • 106. Carville, N. Craig
    et al.
    Neumayer, Sabine M.
    Manzo, Michele
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Gallo, Katia
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum Electronics and Quantum Optics, QEO.
    Rodriguez, Brian J.
    Biocompatible Gold Nanoparticle Arrays Photodeposited on Periodically Proton Exchanged Lithium Niobate2016In: ACS BIOMATERIALS SCIENCE & ENGINEERING, ISSN 2373-9878, Vol. 2, no 8, p. 1351-1356Article in journal (Refereed)
    Abstract [en]

    Photodeposition of silver nanoparticles onto chemically patterned lithium niobate having alternating lithium niobate and proton exchanged regions has been previously investigated. Here, the spatially defined photodeposition of gold nanoparticles onto periodically proton exchanged lithium niobate is demonstrated. It is shown that the location where the gold nanoparticles form can be tailored by altering the concentration of HAuCl4. This enables the possibility to sequentially deposit gold and silver in different locations to create bimetallic arrays. The cytocompatibility of photodeposited gold, silver, and bimetallic ferroelectric templates to osteoblast-like cells is also investigated. Gold samples provide significantly greater cell biocompatibility than silver samples. These results highlight a potential route for using photodeposited gold on lithium niobate as a template for applications in cellular biosensing.

  • 107.
    Cengic, Ivana
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Synthetic biology approaches for improving production of fatty acid-derived compounds in cyanobacteria2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The environmental consequences associated with the use of fossil-sourced fuels and chemicals have brought with it a realization that future development must move in a more sustainable direction. Currently available biofuels or renewably produced chemical, such as bioethanol or biodiesel, are produced from microbial fermentation of sugar-rich crops or by chemical conversion of natural oils or fats. However, these strategies are not sustainable in the long run as fuel and chemical production competes with food supply and arable land usage. Instead of relying on photosynthetic feedstocks that require further conversion, one can engineer photosynthetic cyanobacteria to produce a product of interest directly from CO2 and sunlight. The first part of this thesis aimed to develop new synthetic biology tools for the model cyanobacteria Synechocystis sp. PCC 6803. The second part of the thesis focused on evaluating the regulation of fatty acid synthesis in cyanobacteria, and the production of fatty acid-derived chemicals in Synechocystis.

    In paper I, fusion of small affinity proteins (Affibodies) to the major type IV pili protein was shown to mediate successful surface display of the affibody. This surface display strategy was further shown to allow inter-species binding between Synechocystis and Escherichia coli or Staphylococcus carnosus displaying complementary polymerizing affibodies.

    In paper II, a CRISPR-interference tool was successfully implemented in Synechocystis for inducible gene repression. Further, its multiplexing ability was proven by simultaneous repression of up to four aldehyde reductase/dehydrogenase genes. In paper III, this established CRISPRi tool was used to target and repress native pathways competing with heterologous fatty alcohol production in Synechocystis. Repressing the gene encoding the PlsX phosphate acyltransferase allowed re-direction of carbon-flux from membrane lipids to fatty alcohol production, with a final best strain producing 10.4 mg g-1 DCW octadecanol and hexadecanol.

    In paper IV, the transcriptional response towards perturbations within the fatty acid synthesis pathway was evaluated for the two model cyanobacteria Synechocystis and Synechococcus elongatus PCC 7942. Preliminary results indicate that blocking fatty acid synthesis initiation/elongation causes a transcriptional response of the involved pathway genes only in S. elongatus PCC 7942, indicating differential transcriptional responses in these two strains.

    In paper V, metagenomically sourced aldehyde deformylating oxygenase (Ado) variants were evaluated for their alka(e)ne synthesizing ability. Several of these novel Ado enzymes outperformed the generally well-performing Ado from S. elongatus when relating alka(e)ne production to the soluble enzyme amount.

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  • 108.
    Cengic, Ivana
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton P.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Surface Display of Small Affinity Proteins on Synechocystis sp Strain PCC 6803 Mediated by Fusion to the Major Type IV Pilin PilA12018In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 200, no 16, article id e00270-18Article in journal (Refereed)
    Abstract [en]

    Functional surface display of small affinity proteins, namely, affibodies (6.5 kDa), was evaluated for the model cyanobacterium Synechocystis sp. strain PCC 6803 through anchoring to native surface structures. These structures included confirmed or putative subunits of the type IV pili, the S-layer protein, and the heterologous Escherichia coli autotransporter antigen 43 system. The most stable display system was determined to be through C-terminal fusion to PilA1, the major type IV pilus subunit in Synechocystis, in a strain unable to retract these pili (Delta pilT1). Type IV pilus synthesis was upheld, albeit reduced, when fusion proteins were incorporated. However, pilus-mediated functions, such as motility and transformational competency, were negatively affected. Display of affibodies on Synechocystis and the complementary anti-idiotypic affibodies on E. coli or Staphylococcus carnosus was able to mediate interspecies cell-cell binding by affibody complex formation. The same strategy, however, was not able to drive cell-cell binding and aggregation of Synechocystis-only mixtures. Successful affibody tagging of the putative minor pilin PilA4 showed that it locates to the type IV pili in Synechocystis and that its extracellular availability depends on PilA1. In addition, affibody tagging of the S-layer protein indicated that the domains responsible for the anchoring and secretion of this protein are located at the N and C termini, respectively. This study can serve as a basis for future surface display of proteins on Synechocystis for biotechnological applications. IMPORTANCE Cyanobacteria are gaining interest for their potential as autotrophic cell factories. Development of efficient surface display strategies could improve their suitability for large-scale applications by providing options for designed microbial consortia, cell immobilization, and biomass harvesting. Here, surface display of small affinity proteins was realized by fusing them to the major subunit of the native type IV pili in Synechocystis sp. strain PCC 6803. The display of complementary affinity proteins allowed specific cell-cell binding between Synechocystis and Escherichia coli or Staphylococcus carnosus. Additionally, successful tagging of the putative pilin PilA4 helped determine its localization to the type IV pili. Analogous tagging of the S-layer protein shed light on the regions involved in its secretion and surface anchoring.

  • 109.
    Charoenrat, Theppanya
    KTH, School of Biotechnology (BIO).
    Process design for production of Thai rosewood β-glucosidase in Pichia pastoris2005Licentiate thesis, comprehensive summary (Other scientific)
  • 110.
    Charoenrat, Theppanya
    et al.
    KTH, School of Biotechnology (BIO).
    Ketudat-Cairns, M.
    Stendahl-Andersen, Helle
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes2005In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 27, no 6, p. 399-406Article in journal (Refereed)
    Abstract [en]

    An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q(O)/q(O,max)), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90-95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.

  • 111.
    Charoenrat, Theppanya
    et al.
    KTH, School of Biotechnology (BIO).
    Ketudat-Cairns, Mariena
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 122, no 1, p. 86-98Article in journal (Refereed)
    Abstract [en]

    Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST 1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

  • 112. Chaudhary, Himanshu
    et al.
    Meister, Sebastian
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Zetterberg, Henrik
    Löfblom, John
    Lendel, Christofer
    Dissecting the structural organization of multiprotein amyloid aggregates using a bottom-up approach2020In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193Article in journal (Refereed)
    Abstract [en]

    Deposition of fibrillar amyloid β (Aβ) in senile plaques is a pathological signature of Alzheimer's disease. However, senile plaques also contain many other components, including a range of different proteins. Although the composition of the plaques can be analyzed in post mortem tissue, knowledge of the molecular details of these multiprotein inclusions and their assembly processes is limited, which impedes the progress in deciphering the biochemical mechanisms associated with Aβ pathology. We here describe a bottom-up approach to monitor how proteins from human cerebrospinal fluid associate with Aβ amyloid fibrils to form plaque particles. The method combines flow cytometry and mass spectrometry proteomics and allowed us to identify and quantify 128 components of the captured multiprotein aggregates. The results provide insights in the functional characteristics of the sequestered proteins and reveal distinct interactome responses for the two investigated Aβ variants, Aβ(1-40) and Aβ(1-42). Furthermore, the quantitative data is used to build models of the structural organization of the multiprotein aggregates, which suggests that Aβ is not the primary binding target for all the proteins; secondary interactions account for the majority of the assembled components. The study elucidates how different proteins are recruited into senile plaques and establishes a new model system for exploring the pathological mechanisms of Alzheimer's disease from a molecular perspective.

  • 113. Cheeseman, J. D.
    et al.
    Tocilj, A.
    Park, Seongsoon
    KTH, Superseded Departments, Biochemistry and Biotechnology. Department of Chemistry, McGill University, Canada .
    Schrag, J. D.
    Kazlauskas, R. J.
    Structure of an aryl esterase from Pseudomonas fluorescens2004In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 60, no 7, p. 1237-1243Article in journal (Refereed)
    Abstract [en]

    The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 Å by X-ray diffraction and shows a characteristic α/β-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 Cα atoms between PFE and its five closest structural neighbors averaging 0.8 Å. PFE has far less similarity (r.m.s. deviation in 218 Cα atoms of 5.0 Å) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.

  • 114. Chen, G.
    et al.
    Ning, Z.
    Ågren, Hans
    KTH, School of Biotechnology (BIO), Theoretical Chemistry and Biology.
    Nanostructured solar cells2016In: Nanomaterials, ISSN 2079-4991, Vol. 6, no 8, article id 145Article in journal (Refereed)
    Abstract [en]

    We are glad to announce the Special Issue “Nanostructured Solar Cells”, published in Nanomaterials. This issue consists of eight articles, two communications, and one review paper, covering major important aspects of nanostructured solar cells of varying types. From fundamental physicochemical investigations to technological advances, and from single junction solar cells (silicon solar cell, dye sensitized solar cell, quantum dots sensitized solar cell, and small molecule organic solar cell) to tandem multi-junction solar cells, all aspects are included and discussed in this issue to advance the use of nanotechnology to improve the performance of solar cells with reduced fabrication costs.

  • 115.
    Chen, Shan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Land, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Berglund, Per
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Svedendahl Humble, Maria
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Stabilization of an amine transaminase for biocatalysis2016In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 124, p. 20-28Article in journal (Refereed)
    Abstract [en]

    The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a well-known enzyme to achievechiral amines of high enantiomeric excess in laboratory scales. However, the low operational stabilityof Cv-ATA limits the enzyme applicability on larger scales. In order to improve the operational stabilityof Cv-ATA, and thereby extending its applicability, factors (additives, co-solvents, organic solvents anddifferent temperatures) targeting enzyme stability and activity were explored in order to find out how tostore and apply the enzyme. The present investigation shows that the melting point of Cv-ATA is improvedby adding sucrose or glycerol, separately. Further, by storing the enzyme at higher concentrations and inco-solvents, such as; 50% glycerol, 20% methanol or 10% DMSO, the active dimeric structure of Cv-ATAis retained. Enzyme stored in 50% glycerol at −20◦C was e.g., still fully active after 6 months. Finally,the enzyme performance was improved 5-fold by a co-lyophilization with surfactants prior to usage inisooctane.

  • 116.
    Chen, Zhi-Hui
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. Taiyuan University of Technology, China; Beijing University of Posts and Telecommunications, China .
    Wang, Yang
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Yang, Yibiao
    Qiao, Na
    Wang, Yuncai
    Yu, Zhongyuan
    Enhanced normal-direction excitation and emission of dual-emitting quantum dots on a cascaded photonic crystal surface2014In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 6, no 24, p. 14708-14715Article in journal (Refereed)
    Abstract [en]

    Large normal-direction excitation and emission of dual-emitting quantum dots (QDs) are essential for practical application of QD sensors based on the ratiometric fluorescence response. We have numerically demonstrated an all-dielectric four-layer cascaded photonic crystal (CPC) structure (alternating TiO2 and SiO2/SU8 layers with two dimensional nanoscale patterns in each layer) which is capable of providing normal-direction high Q-factor leaky modes at excitation wavelengths of QDs and two low Q-factor leaky modes coinciding with the two emission peaks of a dual-emitting QD. Normal-direction excitation and far-field emission of the dual-emitting QDs are enhanced significantly when QDs are distributed on/in the top TiO2 layer of the CPC structure, especially in the spatial distribution areas of the resonant leaky modes. QDs can be positioned differently depending on the applications. Positioning QDs on the top TiO2 layer will improve the signal-to-noise ratios of QD biomedical/chemical/temperature sensors, while embedding QDs in the top TiO2 layer will increase the light extraction from the QD light emitting device, making our CPC a versatile optical coupling structure. Our CPC-QD structure is experimentally feasible and robust against the parameter perturbation in real fabrication.

  • 117.
    Cheng, Kimberley
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Single-particle cryo-electron microscopy of macromolecular assemblies2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In this thesis, single-particle cryo-electron microscopy (cryo-EM) was used to study the structure of three macromolecular assemblies: the two hemocyanin isoforms from Rapana thomasiana, the Pyrococcus furiosus chaperonin, and the ribosome from Escherichia coli.

    Hemocyanins are large respiratory proteins in arthropods and molluscs. Most molluscan hemocyanins exist as two distinct isoforms composed of related polypeptides. In most species the two isoforms differ in terms of their oligomeric stability, and thus we set out to investigate the two Rapana thomasiana hemocyanins (RtH) in order to explain this behaviour. Our findings showed that the two RtHio forms are identical at the experimental resolution. Furthermore, three previously unreported connections that most likely contribute to the oligomeric stability were identified.

    Chaperonins are double-ring protein complexes that assist the folding process of nascent, non-native polypeptide chains. The chaperonin from the hyperthermophilic archaea Pyrococcus furiosus belongs to Group II chaperonins, and unlike most othergroup II chaperonins it appears to be homo-oligomeric. The 3D reconstruction of the Pyrococcus furiousus chaperonin revealed a di-octameric structure in a partially closed/open state, something in between the closed folding-active state and the open substrate-accepting state.

    The ribosome is the molecular machine where protein synthesis takes place. In bacteria there is a unique RNA molecule called transfer-messenger RNA (tmRNA) that together with its helper protein SmpB rescues ribosomes trapped on defective messenger RNAs (mRNAs) through a process called trans-translation. tmRNA is about 4 times the size of a normal tRNA, and it is composed of a tRNA-like domain (TLD) that is connected to the mRNA-like domain (MLD) by several pseudoknots (PKs) and RNA helices. During trans-translation, tmRNA utilize its TLD to receive the incomplete polypeptide from the peptidyl-tRNA in the ribosomal P site of the stalledribosome. Subsequently, its MLD is used to tag the incomplete polypeptide with adegradation signal. When tmRNA enters a stalled ribosome the MLD and pseudoknots form a highly structured arc that encircles the beak of the small ribosomal subunit. Byutilizing maximum-likelihood based methods for heterogeneity analysis we could observe the Escherichia coli ribosome in a number of different tmRNA·SmpB-boundstates. The cryo-EM map of the post-accommodated state revealed that the TLD·SmpBpart of the tmRNA·SmpB complex mimics native tRNAs in the A site of stalled ribosomes. The density map also showed that the tmRNA arc remains well structuredand that it is still attached to the beak of the small ribosomal subunit. Thereconstructions of the double-translocation tmRNA-bound ribosome complex showed that the pseudoknots of tmRNA still form an arc, and that they are located at positions similar to the ones assigned for the pseudoknots in the post-accommodated state. In addition, the tmRNA arc exists in two states; one stable and highly structured and another more flexible and disorganized.

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  • 118.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ivanova, Natalia
    Biomedicinskt centrum, Uppsala.
    Scheres, S.H.W
    Pavlov, Michael
    Biomedicinskt centrum.
    Carazo, J.M.
    Herbert, Heinz
    Ehrenberg, Måns
    Lindahl, Martin
    KTH, School of Technology and Health (STH).
    Structural analysis of double translocated tmRNA on the 70S ribosome indicates flexibility of the tmRNA structure.Manuscript (preprint) (Other academic)
  • 119.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ivanova, Natalia
    Biomedicinskt centrum, Uppsala.
    Scheres, Sjores
    CSIC, Natl Biotechnol Ctr, Biocomp Unit, E-28049 Madrid, Spain .
    Pavlov, Michael Y
    Biomedicinskt centrum, Uppsala.
    Maria Carazo, Jose
    Lund Univ, Mol Biophys KILU.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ehrenberg, Måns
    Biomedicinskt centrum, Uppsala.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    tmRNA-SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes2010In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 169, no 3, p. 342-348Article in journal (Refereed)
    Abstract [en]

    Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 A resolution structure of the tmRNA-SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA-SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation. (C) 2009 Elsevier Inc. All rights reserved.

  • 120.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology (Closed 20130701).
    Karlström, M
    Purhonen, P
    Ladenstein, R.
    Herbert, Hans
    Koeck, Philip J.B.
    Low resolution structure and apparent melting temperature of the chaperonin from Pyrococcus furiosusManuscript (preprint) (Other academic)
  • 121.
    Cheng, Kimberley
    et al.
    Department of Biosciences at NOVUM, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, S-141 57 Huddinge, Sweden.
    Koeck, Philip J. B.
    Department of Biosciences at NOVUM, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, S-141 57 Huddinge, Sweden.
    Elmund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ternström, Tomas
    Schwarz, Heinz
    Idakieva, Krassimira
    Parvanova, Katja
    Rapana thomasiana hemocyanin (RtH): Comparison of the two isoforms, RtH1 and RtH2, at 19 Å and 16 Å resolution2006In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 37, no 6, p. 566-576Article in journal (Refereed)
    Abstract [en]

    Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 angstrom and 16 angstrom resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar-wall connection and one of the arch-wall connections are positioned between two individual subunit dimers, while the second arch-wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections.

  • 122. Cheon, Seungwoo
    et al.
    Kim, Hye Mi
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Lee, Sang Yup
    Recent trends in metabolic engineering of microorganisms for the production of advanced biofuels2016In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 35, p. 10-21Article in journal (Refereed)
    Abstract [en]

    As climate change has become one of the major global risks, our heavy dependence on petroleum-derived fuels has received much public attention. To solve such problems, production of sustainable fuels has been intensively studied over the past years. Thanks to recent advances in synthetic biology and metabolic engineering technologies, bio-based platforms for advanced biofuels production have been developed using various microorganisms. The strategies for production of advanced biofuels have converged upon four major metabolic routes: the 2-ketoacid pathway, the fatty acid synthesis (FAS) pathway, the isoprenoid pathway, and the reverse β-oxidation pathway. Additionally, the polyketide synthesis pathway has recently been attracting interest as a promising alternative biofuel production route. In this article, recent trends in advanced biofuels production are reviewed by categorizing them into three types of advanced biofuels: alcohols, biodiesel and jet fuel, and gasoline. Focus is given on the strategies of employing synthetic biology and metabolic engineering for the development of microbial strains producing advanced fuels. Finally, the prospects for future advances needed to achieve much more efficient bio-based production of advanced biofuels are discussed, focusing on designing advanced biofuel production pathways coupled with screening, modifying, and creating novel enzymes.

  • 123.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Perfusion Processes2015In: Animal Cell Culture / [ed] Mohamed Al-Rubeai, Switzerland Springer: Springer , 2015, p. 407-443Chapter in book (Refereed)
    Abstract [en]

    The interest for perfusion is increasing nowadays. This new focus has emerged from a synergy of a demand for disposable equipment and the availability of robust cell separation device, as well as the need for higher flexibility and lower investment cost. The cell separation devices mostly used today are based on filtration, i.e. alternating flow filtration, tangential flow filtration, spin-filter, or acceleration/gravity, i.e. inclined settler, centrifuge, acoustic settler. This paper gives an introduction to the basic concepts of perfusion and its practical implementation. It reviews the actual cell separation devices and describes the approaches used in the field to develop and optimize the perfusion processes.

  • 124.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Diana, Rafael
    Ge Healthcare Bio-sciences Ab.
    Kaisermayer, Christian
    Ge Healthcare Bio-sciences Ab.
    Lindskog, Eva
    Ge Healthcare Bio-sciences Ab.
    Robinson, Craig
    Ge Healthcare Bio-sciences Ab.
    Rucker, Jimmie L.
    Ge Healthcare Bio-sciences Ab.
    Walsh, Kieron D.
    Ge Healthcare Bio-sciences Ab.
    Flexible bag for cultivation of cells2011Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    An inflatable bioreactor bag for cell cultivation, which comprising a top and a bottom sheet of flexible material, joined together to form two end edges and two side edges, wherein one baffle or a plurality of baffles extend from the bottom sheet in a region where the shortest distance to any one of the two end edges is higher than about one fourth of the shortest distance between the two end edges.

  • 125.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Jiang, Yun
    Biovitrum/SOBI, Sweden.
    westin, Jeanette
    Biovitrum/SOBI, Sweden.
    Dahlenborg, K
    Biovitrum/SOBI, Sweden.
    Sjöblom-Hallén, A
    Biovitrum/SOBI, Sweden.
    Svensson, Erik
    Biovitrum/SOBI, Sweden.
    Öberg, Mikael
    Biovitrum/SOBI, Sweden.
    Development of a fed-batch process for the production of a recombinant protein X in CHO-GS system: Case study from the cell to reactor process ready for pilot scale cultivation2010In: Cells and Culture: Proceedings of the 20th ESACT / [ed] Noll T, Springer Science+Business Media B.V., 2010, p. 723-725Conference paper (Other academic)
    Abstract [en]

    A new cell line was created using CHO-GS system. The most promising clones were adapted to different base cultivation media leading to the selection of one medium. The fed-batch process development was performed in spinner, shake flask and bioreactor scale. It included the selection of a feed medium, the choice of the feed strategy and the optimisation of the glucose feeding. The process was then simplified by using a single feed including the feed medium and the glucose feed. Finally up-scaling parameters like aeration and CO2 stripping were studied in 3 L and 15 L bioreactors in preparation for pilot scale operation. This process proved to be robust, reproducible and suitable for large and commercial scale operation.

  • 126.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Lindqvist, Anna
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Study of the effect of high pH and alkali addition in a cultivation of Chinese Hamster Ovary cell2012In: Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT) / [ed] Jenkins, Nigel; Barron, Niall; Alves, Paula, Springer Science+Business Media B.V., 2012, p. 323-326Conference paper (Refereed)
    Abstract [en]

    This work aimed at studying the impact of alkali addition in a Chinese Hamster Ovary cell culture. Two phenomena were studied, the kinetic rate of direct cell death in presence of high pH and the effect of transitory single contact of high pH on cell viability and growth. Contact with pH 11 or 10 did not provoke immediate cell lysis. The cells survived several minutes to such conditions. Contact with pH 11 during 2 minutes, with pH 10 during 5 minutes, with pH 9 during 5 minutes or 10 minutes did not affect the viability. In these conditions, the growth was not affected except after 5 minutes contact at pH 10 or 10 minutes contact at pH 9 for which the growth was slowed down the first day only. As expected, NaOH addition affected the cells more than Na2CO3 addition. This was due to a higher pH but could be even observed at the same pH (10).

  • 127.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO).
    Wåhlgren, Caroline
    Biovitrum/SOBI, Sweden.
    Jiang, Yun
    Biovitrum/SOBI, Sweden.
    Svensson, Erik
    Biovitrum/SOBI, Sweden.
    Process for cultivating animal cells comprising the feeding of plant-derived peptones2005Patent (Other (popular science, discussion, etc.))
    Abstract [en]

    A process for cultivating animal cells producing complex proteins, wherein one plant-derived peptone or a combination of plant-derived peptones is fed to the cell culture, as well as a method for reducing the toxic effect of over-feeding amino acids during a fed-batch process for cultivating animal cells producing complex proteins.

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  • 128.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wåhlgren, Caroline
    Biovitrum/SOBI, Sweden.
    Pettersson, Helena
    Biovitrum/SOBI, Sweden.
    Effect of Peptones and Study of Feeding Strategies in a CHO Based Fed-batch Process for the Production of a Human Monoclonal Antibody2007In: Cell Technology for Cell Products: Proceedings of the 19th ESACT Meeting, Harrogate, UK, June 5-8, 2005 / [ed] Smith R, Dordrecht, The Netherlands: Springer Netherlands, 2007, p. 371-374Conference paper (Other academic)
    Abstract [en]

    Eight commercial peptones, derived from plants, were studied for their ability of improving the cell growth and the productivity of a CHO cell line producing a human monoclonal antibody. They were also compared to yeast, lactalbumin and meat derived peptones. Seven plant peptones were selected and further studied in combination by Design of Experiment. The best three peptones were then tested in combinations in fed-batch cultivation. The fed-batch process was based on low concentrations of glucose and glutamine with feeding of amino acids, peptones and feed medium including vitamins, metal traces and biosynthesis precursors. This process was based on Biovitrum protein-free proprietary medium for the base medium and the feeding medium. Different feeding strategies, different peptone combinations and phosphate feeding were studied for their ability to improve the cell density, the cell specific productivity and the cultivation longevity

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    Chotteau 2007 Effect of Peptones and Study of Feeding Strategies in a CHO Based Fed-batch Process for the Production of a Human Monoclonal Antibody
  • 129.
    Chotteau, Veronique
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Clincke, Marie-Francoise
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Very High Cell Density in Perfusion of CHO Cells by ATF, TFF, Wave Bioreactor, and/or CellTank Technologies: Impact of Cell Density and Applications2014In: Continuous Processing in Pharmaceutical Manufacturing / [ed] Ganapathy Subramanian, Germany Weinheim: Wiley-VCH Verlagsgesellschaft, 2014, p. 339-356Chapter in book (Other academic)
  • 130.
    Chotteau, Véronique
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hjalmarsson, Håkan
    KTH, School of Electrical Engineering (EES), Automatic Control. KTH, School of Electrical Engineering (EES), Centres, ACCESS Linnaeus Centre.
    Tuning of dissolved oxygen and pH PID control parameters in large scale bioreactor by lag control2012In: Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT), 2012, p. 327-330Conference paper (Refereed)
    Abstract [en]

    A method has been developed to tune the DO and pH controller PID parameters for pilot / large scale mammalian cultivation. Our approach is to identify a model of the variable to be controlled (e.g. DO, pH) and to design several possible PID controllers based on this model. The controllers were first tested in computer simulations, followed by wet simulation and finally the best controller was tested on the real process. The approach is developed for the tuning of the DO controller of a 50 L bioreactor using microbubble continuous oxygen flow. The method, called lag control here, is based on a lead lag control design using Bode analysis where the prediction part is omitted. Experiments show that the approach results in a highly satisfactory DO control. The oxygen microbubbles were almost completely consumed before reaching the liquid surface so the oxygen flow used to maintain the DO gave an excellent indication of the cellular oxygen consumption. The control system was robust against all the perturbations, i.e. cell growth, cell bleed, addition of air-saturated fresh medium, DO set point change and a second gas sparger used to strip out the carbon dioxide. This approach was also successfully used for the tuning of a 400 L bioreactor DO controller and pH controller.

  • 131. Chouhan, Dimple
    et al.
    Thatikonda, Naresh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Widhe, Mona
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Mandal, Biman B.
    Recombinant Spider Silk Functionalized Silkworm Silk Matrices as Potential Bioactive Wound Dressings and Skin Grafts2018In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 10, no 28, p. 23560-23572Article in journal (Refereed)
    Abstract [en]

    Silk is considered to be a potential biomaterial for a wide number of biomedical applications. Silk fibroin (SF) can be retrieved in sufficient quantities from the cocoons produced by silkworms. While it is easy to formulate into scaffolds with favorable mechanical properties, the natural SF does not contain bioactive functions. Spider silk proteins, on the contrary, can be produced in fusion with bioactive protein domains, but the recombinant procedures are expensive, and large-scale production is challenging. We combine the two types of silk to fabricate affordable, functional tissue-engineered constructs for wound-healing applications. Nanofibrous mats and microporous scaffolds made of natural silkworm SF are used as a bulk material that are top-coated with the recombinant spider silk protein (4RepCT) in fusion with a cell-binding motif, antimicrobial peptides, and a growth factor. For this, the inherent silk properties are utilized to form interactions between the two silk types by self-assembly. The intended function, that is, improved cell adhesion, antimicrobial activity, and growth factor stimulation, could be demonstrated for the obtained functionalized silk mats. As a skin prototype, SF scaffolds coated with functionalized silk are cocultured with multiple cell types to demonstrate formation of a bilayered tissue construct with a keratinized epidermal layer under in vitro conditions. The encouraging results support this strategy of fabrication of an affordable bioactive SF-spider silk-based biomaterial for wound dressings and skin substitutes.

  • 132.
    Chung, Hannah
    et al.
    Korean Adanced Institute of Science and Technology (KAIST).
    Yang, Jung Eun
    Korean Adanced Institute of Science and Technology (KAIST).
    Ha, Ji Yeon
    Korean Adanced Institute of Science and Technology (KAIST).
    Chae, Tong Un
    Korean Adanced Institute of Science and Technology (KAIST).
    Shin, Jae Ho
    Korean Adanced Institute of Science and Technology (KAIST).
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lee, Sang Yup
    Korean Adanced Institute of Science and Technology (KAIST).
    Bio-based production of monomers and polymers by metabolically engineered microorganisms2015In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 36, p. 73-84Article, review/survey (Refereed)
    Abstract [en]

    Recent metabolic engineering strategies for bio-based production of monomers and polymers are reviewed. In the case of monomers, we describe strategies for producing polyamide precursors, namely diamines (putrescine, cadaverine, 1,6-diaminohexane), dicarboxylic acids (succinic, glutaric, adipic, and sebacic acids), and ω-amino acids (γ-aminobutyric, 5-aminovaleric, and 6-aminocaproic acids). Also, strategies for producing diols (monoethylene glycol, 1,3-propanediol, and 1,4-butanediol) and hydroxy acids (3-hydroxypropionic and 4-hydroxybutyric acids) used for polyesters are reviewed. Furthermore, we review strategies for producing aromatic monomers, including styrene, p-hydroxystyrene, p-hydroxybenzoic acid, and phenol, and propose pathways to aromatic polyurethane precursors. Finally, in vivo production of polyhydroxyalkanoates and recombinant structural proteins having interesting applications are showcased.

  • 133. Cifuentes, Carolina
    et al.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Emons, Anne Mie C.
    Biosynthesis of Callose and Cellulose by Detergent Extracts of Tobacco Cell Membranes and Quantification of the Polymers Synthesized in vitro2010In: J INTEGR PLANT BIOL, ISSN 1672-9072, Vol. 52, no 2, p. 221-233Article in journal (Refereed)
    Abstract [en]

    The conditions that favor the in vitro synthesis of cellulose from tobacco BY-2 cell extracts were determined. The procedure leading to the highest yield of cellulose consisted of incubating digitonin extracts of membranes from 11-day-old tobacco BY-2 cells in the presence of 1 mM UDP-glucose, 8 mM Ca2+ and 8 mM Mg2+. Under these conditions, up to nearly 40% of the polysaccharides synthesized in vitro corresponded to cellulose, the other polymer synthesized being callose. Transmission electron microscopy analysis revealed the occurrence of two types of structures in the synthetic reactions. The first type consisted of small aggregates with a diameter between 3 and 5 nm that associated to form fibrillar strings of a maximum length of 400 nm. These structures were sensitive to the acetic/nitric acid treatment of Updegraff and corresponded to callose. The second type of structures was resistant to the Updegraff reagent and corresponded to straight cellulose microfibrils of 2-3 nm in diameter and 200 nm to up to 5 mu m in length. In vitro reactions performed on electron microscopy grids indicated that the minimal rate of microfibril elongation in vitro is 120 nm/min. Measurements of retardance by liquid crystal polarization microscopy as a function of time showed that small groups of microfibrils increased in retardance by up to 0.047 nm/min per pixel, confirming the formation of organized structures.

  • 134.
    Clincke, Marie-Francoise
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mölleryd, Carin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Zhang, Ye
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Lindskog, Eva
    GE Healthcare, Uppsala, Sweden.
    Walsh, Kieron
    GE Healthcare, Westborough, MA, USA.
    Chotteau, Veronique
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Study of a recombinant CHO cell line producing a monoclonal antibody by ATF or TFF external filter perfusion in a WAVE Bioreactor™2011In: BMC Proceedings, 2011, Volume 5, Supplement 8, P105, BioMed Central, 2011, p. 105-Conference paper (Refereed)
    Abstract [en]

    Major advantages of perfusion are high cell numbers and high total production in a relatively small size bioreactor. Moreover, perfusion is optimal when the product of interest is unstable or if the product yield is low. On the other hand, disadvantages are for example technical challenges originating from non-robust cell separation devices as well as sterility concerns from the more complex set-up needed.

    In the present work, the use of a WAVE Bioreactor™ system 20/50 in perfusion mode with10 L disposable Cellbag™ bioreactors customized with two dip tubes in combination with disposable hollow fiber filters as external cell separating devices were investigated. A comparison between Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) was performed using a recombinant CHO cell line producing a monoclonal antibody (mAb) as a model system. 

  • 135. Colombani, A.
    et al.
    Djerbi, Soraya
    KTH, Superseded Departments, Biotechnology.
    Bessueille, L.
    Blomqvist, Kristina
    KTH, Superseded Departments, Biotechnology.
    Ohlsson, Anna
    KTH, Superseded Departments, Biotechnology.
    Berglund, Torkel
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula
    KTH, Superseded Departments, Biotechnology.
    Bulone, V.
    In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen2004In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 11, no 3-4, p. 313-327Article in journal (Refereed)
    Abstract [en]

    The aim of this work was to optimize the conditions for in vitro synthesis of (1 --> 3)-beta-D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula x tremuloides) cells grown as suspension cultures. Callose was the only product synthesized when CHAPS extracts were used as a source of enzyme. The optimal reaction mixture for callose synthesis contained 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 8 mM Ca2+, and 20 mM cellobiose. The use of digitonin to extract the membrane-bound proteins was required for cellulose synthesis. Yields as high as 50% of the total in vitro products were obtained when cells were harvested in the stationary phase of the growth curve, callose being the other product. The optimal mixture for cellulose synthesis consisted of 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 1 mM Ca2+, 8 mM Mg2+, and 20 mM cellobiose. The in vitro beta-glucans were identified by hydrolysis of radioactive products, using specific enzymes. C-13-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (1-->3)-beta-D-glucan systematically had a microfibrillar morphology, but the size and organization of the microfibrils were affected by the nature of the detergent used for enzyme extraction. The discussion of the results is included in a short review of the field that also compares the data obtained with those available in the literature. The results presented show that the hybrid aspen is a promising model for in vitro studies on callose and cellulose synthesis.

  • 136.
    Costa Felicissimo, Viviane
    KTH, School of Biotechnology (BIO), Theoretical Chemistry (closed 20110512).
    Principles of Infrared - X-ray Pump-probe Spectroscopy2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using these two techniques combined in pump-probe experiments. Four main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding in the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; the development of theory, with sample applications, for infrared X-ray pump-probe spectroscopy; and molecular dynamics simulations of light-induced fragmentation of water clusters.

    Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found out that the X-ray photoelectron spectrum of the water dimer differs dramatically from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy.

    The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-Ray Pump-Probe Spectroscopy - are examined in detail using the wave packet technique in three applications; the NO molecule and the dynamics of proton transfer in core ionized water dimer and glyoxalmonoxime. It is found out that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential, which results in a phase dependence of the X-ray pump-probe spectra. A proper choice of the delay time of the X-ray pulse allows the direct observation of the X-ray transition in the proton transfered well of the core excited potential. It is found out that the glyoxalmonoxime molecule possesses an important feature; proton transfer accompanied by core hole hopping. Special attention is paid to the quantum control of the populations of vibrational level which is of crucial importance to shape the wave packet of desirable size.

    The wave packet technique becomes computationally very expensive when the number of nuclear degrees of freedom is large. Molecular dynamics is used instead in studies of light-induced nuclear kinetics in the water hexamer cluster. We predict a novel mechanism of the mechanical action of light on atoms and molecules. This mechanism is based on the rectification of the Lorentz force, which gives a unique opportunity of direct site selective mechanical action of light on atoms and molecules inside large systems like clusters or biomolecules.

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  • 137.
    Cuvilas, Carlos Alberto
    et al.
    Swedish University for Agricultural Sciences (SLU), Department of Energy and Technology, Uppsala, Sweden .
    Jirjis, R.
    Swedish University for Agricultural Sciences (SLU), Department of Energy and Technology, Sweden .
    Lucas, C.
    Energy situation in Mozambique: A review2010In: Renewable & sustainable energy reviews, ISSN 1364-0321, E-ISSN 1879-0690, Vol. 14, no 7, p. 2139-2146Article, review/survey (Refereed)
    Abstract [en]

    The need to increase energy security and promote development, especially in rural areas has forced many developing countries in southern Africa, like Mozambique to take several actions toward development of several infrastructures and legislations for production and use of liquid biofuels. The main objective of this study is to present the energy situation in Mozambique and assess the potential for energy generation from widely available renewable sources including residues from agricultural crops and forest industry. The country is endowed with great potential for biofuels, solar, hydro and wind energy production. The energy production today is, however, far from fulfilling energy needs of the country, and the majority of people are still not benefiting from these resources. The potential of total residues from agricultural sector and forest industry is estimated to be around 128 PJ. This amount of energy covers almost half of the combined production of charcoal and firewood which amounted to approximately 298 PJ in 2006. However, such amount of energy resources is wasted and is not visible on national energy statistics.

  • 138. Danmark, Staffan
    et al.
    Finne Wistrand, Anna
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Wendel, Mikael
    Arvidson, Kristina
    Albertsson, Ann-Christine
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Mustafa, Kamal
    Osteogenic Differentiation by Rat Bone Marrow Stromal Cells on Customized Biodegradable Polymer Scaffolds2010In: Journal of bioactive and compatible polymers (Print), ISSN 0883-9115, E-ISSN 1530-8030, Vol. 25, no 2, p. 207-223Article in journal (Refereed)
    Abstract [en]

    In this report, poly(L-lactide-co-epsilon-caprolactone), poly(LLA-co-CL) and poly(L-lactide-co-1,5-dioxepan-2-one), poly(LLA-co-DXO) were evaluated and compared for potential use in bone tissue engineering constructs together with bone marrow stromal cells (BMSC). The copolymers were tailored to reduce the level of harmful tin residuals in the scaffolding. BMSC isolated from Sprague-Dawley rats were seeded onto the scaffolds and cultured in vitro for up to 21 days. Cell spreading and proliferation was analyzed after 72 h by scanning electron microscopy and thiazolyl blue tetrazolium bromide (MTT) conversion assay. Osteogenic differentiation of BMSC was evaluated by real-time PCR after 14 and 21 days of culture. Hydrophilicity was significantly different between poly(LLA-co-CL) and poly(LLA-co-DXO) with the latter being more hydrophilic. After 72 h, both scaffolds supported increased cell proliferation and the mRNA expression of osteocalcin and osteopontin was significantly increased after 21 days. Further investigation of these constructs, with lower levels of tin residuals, are being pursued.

  • 139.
    de Almeida, Katia Júlia
    KTH, School of Biotechnology (BIO), Theoretical Chemistry.
    Optical and Magnetic Properties of Copper(II) Compounds2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    This thesis encloses quantum chemical calculations and applications of a response function formalism recently implemented within the framework of density functional theory. The optical and magnetic properties of copper(II) molecular systems are the main goal of this work. In this work, the visible and near-infrared electronic transitions, which have shown a key role in studies on electronic structure and structure-function relationships of copper compounds, were investigated in order to explore the correlation of the positions and intensities of these transitions with the geometrical structures and their molecular distortions. The evaluation of solvent effects on the absorption spectra were successfully achieved, providing accurate and inedit computational insight of these effects for copper(II) complexes. Electron Paramagnetic Resonance (EPR) parameters, that is, the electronic g tensor and the hyperfine coupling constants, are powerful spectroscopic properties for investigating paramagnetic systems and were thoroughly analysed in this work in different molecular systems. Relativistic corrections generated by spin-orbit interactions or by scalar relativistic effects were taken into account in all calculations. In addition, we have designed a methodology for accurate evaluation of the electronic g tensors and hyperfine coupling tensors as well as for evaluation of solvent effects on these properties. It is found that this methodology is able to provide reliable and accurate results for EPR parameters of copper(II) molecular systems. The spin polarization effects on EPR parameters of square planar copper(II) complexes were also considered, showing that these effects give rise to significant contributions to the hyperfine coupling tensor, whereas the electronic g tensor of these complexes are only marginally affected by these effects. The evaluation of the leading-order relativistic corrections to the electronic g tensors of molecules with a doublet ground state has been also taken into account in this work. As a first application of the theory, the electronic g tensors of dihalogen anion radicals X$_2^-$ (X=F,~Cl,~Br,~I) have been investigated and the obtained results indicate that the spin--orbit interaction is responsible for the parallel component of the g tensor shift, while both the leading-order scalar relativistic and spin--orbit corrections are of minor importance for the perpendicular component of the g tensor in these molecules

    since they effectively cancel each other. Overall, both optical and magnetic results show quantitative agreements with experiments, indicating that the methodologies employed form a practical way in study of copper(II) molecular systems including those of biological importance.

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  • 140.
    Dev, Apurba
    et al.
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics. Uppsala University, Sweden.
    Horak, J.
    Kaiser, A.
    Yuan, X.
    Perols, A.
    Björk, P.
    Karlström, A. E.
    Kleimann, P.
    Linnros, Jan
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Material Physics, MF.
    Electrokinetic effect for molecular recognition: A label-free approach for real-time biosensing2016In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 82, p. 55-63Article in journal (Refereed)
    Abstract [en]

    We present a simple and inexpensive method for label-free detection of biomolecules. The method monitors the changes in streaming current in a fused silica capillary as target biomolecules bind to immobilized receptors on the inner surface of the capillary. To validate the concept, we show detection and time response of different protein-ligand and protein-protein systems: biotin-avidin and biotin-streptavidin, barstar-dibarnase and Z domain-immunoglobulin G (IgG). We show that specific binding of these biomolecules can be reliably monitored using a very simple setup. Using sequential injections of various proteins at a diverse concentration range and as well as diluted human serum we further investigate the capacity of the proposed technique to perform specific target detection from a complex sample. We also investigate the time for the signal to reach equilibrium and its dependence on analyte concentration and demonstrate that the current setup can be used to detect biomolecules at a concentration as low as 100 pM without requiring any advanced device fabrication procedures. Finally, an analytical model based on diffusion theory has been presented to explain the dependence of the saturation time on the analyte concentration and capillary dimensions and how reducing length and inner diameter of the capillary is predicted to give faster detection and in practice also lower limit of detection. © 2016 Elsevier B.V.

  • 141.
    Dias Batista, Edgard
    KTH, School of Industrial Engineering and Management (ITM), Energy Technology, Energy and Climate Studies, ECS.
    Etanolens hållbarhet – en litteraturöversikt2012Report (Other (popular science, discussion, etc.))
    Abstract [sv]

    Etanol är ett omdebatterat bränsle bland miljödebattörer och politiker, men även bland forskare. I denna studie har 192 vetenskapligt granskade artiklar om etanol studerats och slutsatserna kvantifierats. Slutsatserna har delats in i sex frågeområden, utöver de övergripande slutsatser om etanol som hållbart drivmedel. Litteraturstudien visar att en majoritet av artiklarna har slutsatser som är positiva till att använda etanol som biodrivmedel. Sammanställningen över artiklarnas övergripande slutsatser, som är den viktigaste frågan i studien, visar att 65 procent av slutsatserna är positiva eller mycket positiva, 10 procent av artiklarna är neutrala eller ofullständiga, medan summan av de negativa och helt negativa slutsatserna är 25 procent. Inom samtliga sex områden är andelen positiva slutsatser högre än de negativa. Frågan där fördelningen är jämnast gäller den om etanolens inverkan på livsmedelspriser och livsmedelsproduktion där 54 procent av artiklarna var positiva eller mycket positiva till använda etanol som drivmedel. 43 procent var negativa eller mycket negativa.

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    Etanol - Edgard
  • 142.
    Dincbas-Renqvist, Vildan
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lendel, Christofer
    KTH, School of Biotechnology (BIO).
    Dogan, Jakob
    KTH, Superseded Departments, Biotechnology.
    Wahlberg, Elisabet
    KTH, Superseded Departments, Biotechnology.
    Härd, Torleif
    Göteborgs Universitet.
    Thermodynamics of folding, stabilization, and binding in an engineered protein-protein complex2004In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 36, p. 11220-11230Article in journal (Refereed)
    Abstract [en]

    We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA-1) affibody and it's Z domain binding partner as a model. Free Z(SPA-1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA-1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA-1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA-1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA-1) complex is associated with a large change in conformational entropy that opposes binding.

  • 143.
    Ding, Haozhong
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Altai, Mohamed
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Gräslund, Torbjörn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, S-75123 Uppsala, Sweden..
    Incorporation of a Hydrophilic Spacer Reduces Hepatic Uptake of HER2-Targeting Affibody-DM1 Drug Conjugates2019In: Cancers, ISSN 2072-6694, Vol. 11, no 8, article id 1168Article in journal (Refereed)
    Abstract [en]

    Affibody molecules are small affinity-engineered scaffold proteins which can be engineered to bind to desired targets. The therapeutic potential of using an affibody molecule targeting HER2, fused to an albumin-binding domain (ABD) and conjugated with the cytotoxic maytansine derivate MC-DM1 (AffiDC), has been validated. Biodistribution studies in mice revealed an elevated hepatic uptake of the AffiDC, but histopathological examination of livers showed no major signs of toxicity. However, previous clinical experience with antibody drug conjugates have revealed a moderateto high-grade hepatotoxicity in treated patients, which merits efforts to also minimize hepatic uptake of the AffiDCs. In this study, the aim was to reduce the hepatic uptake of AffiDCs and optimize their in vivo targeting properties. We have investigated if incorporation of hydrophilic glutamate-based spacers adjacent to MC-DM1 in the AffiDC, (Z(HER2:2891))(2) -ABD-MC-DM1, would counteract the hydrophobic nature of MC-DM1 and, hence, reduce hepatic uptake. Two new AffiDCs including either a triglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-3-MC-DM1, or a hexaglutamate-spacer-, (Z(HER2:2891))(2)-ABD-E-6-MC-DM1 next to the site of MC-DM1 conjugation were designed. We radiolabeled the hydrophilized AffiDCs and compared them, both in vitro and in vivo, with the previously investigated (Z(HER2:2891))(2)-ABD-MC-DM1 drug conjugate containing no glutamate spacer. All three AffiDCs demonstrated specific binding to HER2 and comparable in vitro cytotoxicity. A comparative biodistribution study of the three radiolabeled AffiDCs showed that the addition of glutamates reduced drug accumulation in the liver while preserving the tumor uptake. These results confirmed the relation between DM1 hydrophobicity and liver accumulation. We believe that the drug development approach described here may also be useful for other affinity protein-based drug conjugates to further improve their in vivo properties and facilitate their clinical translatability.

  • 144.
    Djerbi, Soraya
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cellulose synthases in Populus- identification, expression analyses and in vitro synthesis2005Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Cellulose is a biopolymer of great relevance in the plant cell walls, where it constitutes the most important skeletal component. Cellulose is also an important raw material in the pulp- and paper, forest, and textile industries, among others. Cellulose biosynthesis in particular, and xylogenesis in general are processes which are currently poorly understood. Yet, research in cellulose synthesis is progressing and different applications of cellulose, mainly cellulose derivatives for e.g. pharmaceuticals and coatings, are constantly emerging. This thesis depicts how cellulose synthase (CesA) genes in Populus were identified and characterized by gene expression- and bioinformatics analyses. Within an EST database of more than 100,000 clones from wood forming tissues of three different Populus taxa, ten CesA genes were identified in Populus tremula x tremuloides. Subsequent gene expression analyses by using microarrays and real-time PCR experiments in woody tissues, revealed distinct regulation patterns among the genes of interest. This enabled proper classification and characterization of the secondary cell wall related CesA genes, in particular. Bioinformatic analyses of the genome sequence of Populus trichocarpa further provided a complete picture of the number of putative CesA genes retained after several duplication events during tree evolution. In contrast to the previously reported set of ten 'true' CesA genes in many other plant species, the genome of P. trichocarpa encodes 18 putative proteins, which could be assembled into nine groups according to their sequence similarities. Interestingly, studies in the EST database suggested that paralogs within at least two groups have corresponding orthologs in P. tremula x tremuloides, which are furthermore transcribed. This implies that at least some of the duplicated genes have remained functional, or may have acquired a modified function.

    By focusing on the CesA genes associated with secondary cell wall formation, cellulose synthesis was also studied in poplar cell suspension cultures. Selection of CesA enriched material was performed by determining expression intensities of the CesA genes using RT-PCR, whereupon membrane protein extraction was initiated. CesA proteins are part of large cellulose synthesizing complexes in the plasma membrane. Subsequent proteomic approaches comprised partial purification of these cellulose synthesizing complexes from protein enriched culture material and in vitro cellulose synthesis experiments. De novo synthesized material was successfully characterized and the acquired yields were as high as 50% cellulose (compared to previously reported yields of 30% in other plant systems) of the total in vitro synthesized product. Elevated CesA gene expression levels can thus be correlated to increased protein activity in poplar cell suspension cultures. In addition, antibodies raised against CesA antigens were used in Western blot analyses comprising samples along the protein extraction- and purification procedure. Proteins with corresponding molecular weight to the theoretical 120kDa of CesA proteins were recognized by a range of different specific antibodies. The study demonstrates that poplar cell suspension cultures can provide a valuable model system for studies of cellulose synthesis and different aspects of xylogenesis.

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  • 145.
    Djerbi, Soraya
    et al.
    KTH, Superseded Departments, Biotechnology.
    Aspeborg, Henrik
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Blomqvist, Kristina
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tuula
    KTH, Superseded Departments, Biotechnology.
    Identification and expression analysis of genes encoding putative cellulose synthases (CesA) in the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.)2004In: Cellulose (London), ISSN 0969-0239, E-ISSN 1572-882X, Vol. 11, no 3-4, p. 301-312Article in journal (Refereed)
    Abstract [en]

    Cellulose is synthesized in plant cell walls by large membrane-bound protein complexes proposed to contain several copies of the catalytic subunit of the cellulose synthase, CesA. Here we report identification of 10 distinct CesA genes within a database of 100,000 ESTs of the hybrid aspen, Populus tremula (L.) x P. tremuloides (Michx.). Expression analyses in normal wood undergoing xylogenesis and in tension wood indicate xylem specific expression of four putative CesA isoenzymes, PttCesA1, PttCesA3-1, PttCesA3-2 and PttCesA9. Both the protein sequences and the expression profiles of PttCesA3-1 and PttCesA3-2 are very similar, and they may thus represent redundant copies of an enzyme with essentially the same function. Further, one of the generally more constitutively expressed CesA genes, PttCesA2, seems to be activated on the opposite side of a tension wood induced stem, while PttCesA6 appears to be more specific for leaf tissues. The rest of the hybrid aspen CesA genes were found to be relatively evenly expressed over the poplar tissues hereby studied.

  • 146.
    Djerbi, Soraya
    et al.
    KTH, School of Biotechnology (BIO).
    Lindskog, Mats
    KTH, School of Biotechnology (BIO).
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Sterky, Fredrik
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula
    KTH, School of Biotechnology (BIO).
    The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes2005In: Planta, ISSN 0032-0935, E-ISSN 1432-2048, Vol. 221, no 5, p. 739-746Article in journal (Refereed)
    Abstract [en]

    The genome sequence of Populus trichocarpa was screened for genes encoding cellulose synthases by using full-length cDNA sequences and ESTs previously identified in the tissue specific cDNA libraries of other poplars. The data obtained revealed 18 distinct CesA gene sequences in P. trichocarpa. The identified genes were grouped in seven gene pairs, one group of three sequences and one single gene. Evidence from gene expression studies of hybrid aspen suggests that both copies of at least one pair, CesA3-1 and CesA3-2, are actively transcribed. No sequences corresponding to the gene pair, CesA6-1 and CesA6-2, were found in Arabidopsis or hybrid aspen, while one homologous gene has been identified in the rice genome and an active transcript in Populus tremuloides. A phylogenetic analysis suggests that the CesA genes previously associated with secondary cell wall synthesis originate from a single ancestor gene and group in three distinct subgroups. The newly identified copies of CesA genes in P. trichocarpa give rise to a number of new questions concerning the mechanism of cellulose synthesis in trees.

  • 147.
    Dogan, Jakob
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Structural and thermodynamical basis for molecular recognition between engineered binding proteins2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA).

    In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity.

    A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation.

    A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization.

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  • 148.
    Dogan, Jakob
    et al.
    KTH, School of Biotechnology (BIO).
    Lendel, Christofer
    KTH, School of Biotechnology (BIO).
    Härd, Torleif
    Göteborgs Universitet.
    NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex2006In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 36, p. (Electronic publication ahead of print Feb. 6; doi:10.1007/s10858-005-5350-8)Article in journal (Refereed)
  • 149.
    Dogan, Jakob
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lendel, Christofer
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Härd, Torleif
    Thermodynamics of folding and binding in an affibody:affibody complex2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 359, no 5, p. 1305-1315Article in journal (Refereed)
    Abstract [en]

    Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K-d = 0.1 mu M. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T-M similar to 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with Delta H degrees(bind) = -9.0(+/- 0.1) kcal mol(-1). The heat capacity change Delta C-P degrees,(bind) = -0.43(+/- 0.01) kcal mol(-1) K-1 is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-T Delta S degrees(bind) = -0.6(+/- 0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.

  • 150.
    Doverskog, Magnus
    et al.
    KTH, Superseded Departments, Biotechnology.
    Bertram, Eva.
    KTH, Superseded Departments, Biotechnology.
    Ljunggren, Jan
    Öhman, Lars
    Sennerstam, Roland
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Cell cycle progression in serum-free cultures of Sf9 insect cells: Modulation by conditioned medium factors and implications for proliferation and productivity2000In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 16, no 5, p. 837-846Article in journal (Refereed)
    Abstract [en]

    Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (mu(N,max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining mu(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase mu(N,max), decrease the time to reach mu(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean;population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.

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