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  • 1.
    Asnake, Solomon
    Örebro University, School of Science and Technology.
    Interaction of brominated flame retardants with the chicken and zebrafish androgen receptors2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The survival of organisms depends on their ability to use different signaling pathways to adapt to the environment. The endocrine system consists of glands that release hormones to the blood stream. Male reproductive functions are regulated by androgens through interactions with the androgen receptor (AR). AR has been characterized in chicken and zebrafish where they use testosterone and 11-ketotestosterone as their primary androgens, respectively. AR function has been disturbed by different endocrine disrupting compounds (EDCs) present in the environment causing detrimental effects on avian and fish species. Brominated flame retardants (BFRs) are a group of EDCs that are ubiquitous in the environment. Molecular modeling techniques using computer simulations such as docking and molecular dynamics are a useful tool in the identification of EDCs. The capacity to test thousands of compounds at once has helped in the early identification of EDCs that interact with AR. Two groups of BFRs, the 1,2-dibromo-4- cyclohexane diastereomers (TBECH) and the compounds synthesized from 2, 4, 6-tribromophenol, allyl 2,4,6-tribromophenyl ether (ATE), 2-bromoallyl 2,4,6- tribromophenyl ether (BATE) and 2,3-dibromopropyl 2,4,6-tribromophenyl ether (DPTE) interact and alter AR activity in human in vitro studies. As models for avian and fish species, chicken and zebrafish were used to test these BFRs. TBECH diastereomers were able to bind to the AR, estrogen receptors and thyroid receptors in the chicken and to the AR in zebrafish. ATE, BATE and DPTE were also able to interact with the chicken AR and zebrafish AR. Activation studies using cell lines showed that TBECH diastereomers acted as agonists to the cAR and zAR while ATE, BATE and DPTE acted as antagonists. The BFRs also altered multiple signaling pathways such as the apoptotic, antiapoptotic, immune, drug metabolizing and DNA methylation systems and in vivo studies resulted in physiological effects on zebrafish.

    List of papers
    1. 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH)-mediated steroid hormone receptor activation and gene regulation in chicken LMH cells
    Open this publication in new window or tab >>1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH)-mediated steroid hormone receptor activation and gene regulation in chicken LMH cells
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    2014 (English)In: Environmental Toxicology and Chemistry, ISSN 0730-7268, E-ISSN 1552-8618, Vol. 33, no 4, p. 891-899Article in journal (Refereed) Published
    Abstract [en]

    The incorporation of brominated flame retardants into industrial and household appliances has increased their occurrence in the environment, resulting in deleterious effects on wildlife. With the increasing restraints on available compounds, there has been a shift to using brominated flame retardants that has seen the production of alternative brominated flame retardants such as 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH), which has been detected in the environment. In previous in silico and in vitro studies the authors have shown that TBECH can activate both the human androgen receptor (hAR) and the zebrafish AR (zAR) suggesting that it is a potential endocrine disruptor. The present study was aimed at determining the interaction of TBECH with the chicken AR (cAR). In the present study, TBECH bound to cAR, but in vitro activation assay studies using the chicken LMH cell line showed it had a potency of only 15% compared with testosterone. Sequence difference between ARs from different species may contribute to the different responses to TBECH. Further quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis showed that TBECH interacted with and altered the expression of both thyroid receptors and estrogen receptors. In addition, the qRT-PCR analysis showed that TBECH altered the transcription pattern of genes involved in inflammatory, apoptotic, proliferative, DNA methylation, and drug-metabolizing pathways. This demonstrates that TBECH, apart from activating cAR, can also influence multiple biological pathways in the chicken.

    Place, publisher, year, edition, pages
    Hoboken: Wiley-Blackwell, 2014
    Keywords
    Endocrine disruptor, Diastereomer, Enantiomer, Quantitative polymerase chain reaction (qPCR), Gene regulation
    National Category
    Environmental Sciences
    Research subject
    Enviromental Science; Biology
    Identifiers
    urn:nbn:se:oru:diva-34941 (URN)10.1002/etc.2509 (DOI)000333538700020 ()2-s2.0-84897431931 (Scopus ID)
    Funder
    Swedish Research Council
    Note

    Funding Agency:

    Örebro University

    Available from: 2014-05-05 Created: 2014-05-05 Last updated: 2017-12-05Bibliographically approved
    2. The brominated flame retardant TBECH activates the zebrafish (Danio rerio) androgen receptor, alters gene transcription and causes developmental disturbances
    Open this publication in new window or tab >>The brominated flame retardant TBECH activates the zebrafish (Danio rerio) androgen receptor, alters gene transcription and causes developmental disturbances
    2013 (English)In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 142, p. 63-72Article in journal (Refereed) Published
    Abstract [en]

    Tetrabromoethylcyclohexane (TBECH) is a brominated flame retardant that has been shown to be a potent agonist to the human androgen receptor (AR). However, while it is present in the environment, it is not known if it interacts with AR from aquatic species. The present study was therefore aimed at improving our understanding of how TBECH affects aquatic animals using zebrafish as a model organism. In silica modeling demonstrated that TBECH diastereomers bind to the zebrafish androgen receptor (zAR) and in vitro and in vivo data showed that TBECH has androgenic properties. Deleterious effects of TBECH were studied on embryonic and juvenile zebrafish and qRT-PCR analysis in vitro and in vivo was performed to determine TBECH effects on gene regulation. TBECH was found to delay hatching at 1 mu M and 10 mu M doses while morphological abnormalities and juvenile mortality was observed at 10 mu M. The qRT-PCR analysis showed alterations of multiple genes involved in chondrogenesis (cartilage development), metabolism and stress response. Thus, TBECH induces androgenic activity and has negative effects on zebrafish physiology and therefore its impact on the environment should be carefully monitored. (C) 2013 Elsevier B.V. All rights reserved.

    Keywords
    Androgens, Endocrine, Endocrine disruptor, Gene regulation
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:oru:diva-32902 (URN)10.1016/j.aquatox.2013.07.018 (DOI)000328093900007 ()23958786 (PubMedID)
    Funder
    Knowledge Foundation
    Available from: 2014-01-02 Created: 2014-01-02 Last updated: 2017-12-06Bibliographically approved
    3. The brominated flame retardants TBP-AE and TBP-DBPE antagonize the chicken androgen receptor and alter gene expression in chicken LMH cells
    Open this publication in new window or tab >>The brominated flame retardants TBP-AE and TBP-DBPE antagonize the chicken androgen receptor and alter gene expression in chicken LMH cells
    (English)Manuscript (preprint) (Other academic)
    Keywords
    EDC, Avian, signaling pathways, ATE, BATE, DPTE, TBECH
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:oru:diva-43909 (URN)
    Available from: 2015-03-27 Created: 2015-03-27 Last updated: 2017-10-17Bibliographically approved
    4. In silico and biological analysis of anti-androgen activity of the brominated flame retardants ATE, BATE and DPTE in zebrafish
    Open this publication in new window or tab >>In silico and biological analysis of anti-androgen activity of the brominated flame retardants ATE, BATE and DPTE in zebrafish
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    Brominated flame retardants, stereoidgenesis, gene regulation, hatch, teratogenesis
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:oru:diva-43911 (URN)
    Available from: 2015-03-27 Created: 2015-03-27 Last updated: 2017-10-17Bibliographically approved
  • 2.
    El Marghani, Ahmed
    Örebro University, School of Science and Technology.
    Regulatory aspects of innate immune responses2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Activation of innate immunity is regulated by a variety of signaling molecules within the immune cells. The present thesis was aimed to improve our understanding of innate signaling mechanism and their possible use as bio-indicators of exposure and disease. The first part of the thesis deals with the involvement of TOM1L1 (Target of Myb1 like 1) in innate immune signaling and regulation of inflammatory cytokines in immune cells (study I and II). The initial event of T-cells activation depend on the recruitment of Src family kinases Fyn and Lck, leading to interleukine-2 (IL-2) production in T cells. Understanding the regulatory aspects of IL-2 induction in T-cells is of importance as IL-2 is a key regulator for T-cell proliferation and survival. Interaction screening indicated the ability of TOM1L1 protein to interact with Fyn, and Lck, that is important for IL-2 production in Jurkat T-cells. TOM1L1 silencing decreased the levels of CD3/CD28 dependent induction of IL-2 in Jurkat T-cells, and LPS dependent induction of TNF-α in THP-1. Furthermore, overexpression of TOM1L1 in Jurkat T-cells causes an increase of STAT3 expression. This was accompanied by an increase in the levels of IL-1β dependent induction of IL-6 and TNF-α in THP-1 cells. These results indicate that TOM1L11 participate in regulation of innate immune response. The second part of the thesis deals with development of innate immune signaling responses used as a diagnosis tools for disease and exposure (study III and IV). Inflammatory diseases are associated with innate immune reactions. In response to inflammation, the immune cells release inflammatory cytokines such as IL-1-β, IL-2, IL-6, IL-10, TNF-α and CXCL8. These cytokines are regulated by stress related kinases include MAP kinase proteins such as ERK1-2, JNK, and MAPK p38, through activation of transcription factors AP-1, ATF-2, and NF-AT. In a clinical study, it was observed that activated MAPK p38 has a potential role in the regulation of IL-10 expression in intermittent claudication. However, expression of IL-10 and MAPK p38 was opposed in stable angina group. Therefore, targeting MAPK p38 in inflammatory disease such as cardiovascular diseases, diabetes, and rheumatoid arthritis might be useful in development of treatment strategies. Innate immune reactions can also be used to monitor stress related inflammatory responses following environmental exposure of immune cells. Inflammatory responses of exposure were studied by in vitro exposure to waters from sewage treatment works and recipient waters. The analysis shows that exposure to inland waters can result in activated immune responses and that these responses are both site dependent and vary over time.

    List of papers
    1. TOM1L is involved in a novel signaling pathway important for the IL-2 production in Jurkat T cells stimulated by CD3/CD28 CoLigation
    Open this publication in new window or tab >>TOM1L is involved in a novel signaling pathway important for the IL-2 production in Jurkat T cells stimulated by CD3/CD28 CoLigation
    2009 (English)In: Mediators of Inflammation, ISSN 0962-9351, E-ISSN 1466-1861, p. 416298-Article in journal (Refereed) Published
    Abstract [en]

    TOM1L (target of Myb-1 Like) was identified as a binding partner for the full length and catalytically-active Lck in a yeast 2-hybrid screening assay. Here we show that in Jurkat T cells stimulated by CD3/CD28 coligation where the expression of TOM1L is reduced by lenti virus mediated-siRNA results in a dramatically lower IL-2 production. The production of IL-2 in siRNA treated cells stimulated with PMA/ionomycin was not affected indicating an involvement of TOM1L in a pathway proximal of TCR and CD28. The coexpression of Fyn with TOM1L increased the level of the phosphorylated form of Fyn indicating that TOM1L has the ability to activate Fyn. The ability of TOM1L to activate Fyn was further shown in a kinase assay using angiotensin II as a substrate. By confocal microscopy, we show that the expression of TOM1L in non-treated HeLa and SK-N-SH cells colocalizes with the mitochondrial membrane but not with lysosomal compartments or the trans-Golgi network. Furthermore, we show that the over-expression of TOM1L in Jurkat cells causes an increase of the STAT3 expression. Based on our results, we here propose that TOM1L is involved in a novel signaling pathway that is important for the IL-2 production in T cells. Copyright (C) 2009 Ahmed Elmarghani et al.

    Place, publisher, year, edition, pages
    Hindawi Publishing Corporation, 2009
    National Category
    Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-21385 (URN)10.1155/2009/416298 (DOI)000276144100001 ()2-s2.0-77949305423 (Scopus ID)
    Available from: 2012-01-27 Created: 2012-01-27 Last updated: 2017-12-08Bibliographically approved
    2. Involvement of TOM1L1 in cytokine regulation in THP-1 cells
    Open this publication in new window or tab >>Involvement of TOM1L1 in cytokine regulation in THP-1 cells
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-21459 (URN)
    Available from: 2012-02-02 Created: 2012-02-02 Last updated: 2017-10-17Bibliographically approved
    3. High MAPK p38 activity and low level of IL-10 in intermittent claudication as opposed to stable angina
    Open this publication in new window or tab >>High MAPK p38 activity and low level of IL-10 in intermittent claudication as opposed to stable angina
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    2010 (English)In: International Journal of Angiology, ISSN 0392-9590, E-ISSN 1827-1839, Vol. 29, no 4, p. 331-337Article in journal (Refereed) Published
    Abstract [en]

    AIM:

    The aim of the present pilot study was to relate the activity of MAPK p38 with the levels of pro- and anti-inflammatory cytokines in a small cohort of patients with either stable angina (N=5) or intermittent claudication (N=5) compared to healthy controls (N=10).

    METHODS:

    The activity of MAPK p38 was determined in peripheral blood mononuclear cells, isolated from whole blood by western blot using phospho-specific anti-MAPK p38 antibodies. Cytokine levels of 11 pro- and anti-inflammatory cytokines were determined from the serum using flow cytometry.

    RESULTS:

    We found a significant elevation of the MAPK p38 activity in the intermittent claudication group (P=0.0027) compared with the healthy control group whereas the stable angina group showed similar MAPK p38 activity as the healthy control group. The IL-10 level in serum found in the stable angina group was significantly higher compared with both the healthy control group (P=0.0116) and the intermittent claudication group (P=0.0317).

    CONCLUSION:

    Our results imply that there is a casual relationship between increased levels of the anti-inflammatory cytokines IL-10 and IL-4 and the activity of the MAPK p38. Possibly has IL-10 a protective role that down-regulates the activity of MAPK p38 and thereby further inflammatory processes in stable angina patients.

    Place, publisher, year, edition, pages
    Torino: Minerva Medica, 2010
    Keywords
    p38 mitogen-activated protein kinases, Interleukin-10, Intermittent claudication
    National Category
    Medical and Health Sciences Cardiac and Cardiovascular Systems
    Research subject
    Cardiology
    Identifiers
    urn:nbn:se:oru:diva-12845 (URN)000283516700007 ()20671651 (PubMedID)
    Available from: 2011-01-10 Created: 2011-01-03 Last updated: 2017-12-11Bibliographically approved
    4. Immune cell activation by sewage treatment plant effluents and inland waters in Sweden
    Open this publication in new window or tab >>Immune cell activation by sewage treatment plant effluents and inland waters in Sweden
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-21457 (URN)
    Available from: 2012-02-02 Created: 2012-02-02 Last updated: 2017-10-17Bibliographically approved
  • 3.
    Erdtman, Edvin
    Örebro University, School of Science and Technology.
    5-Aminolevulinic acid and derivatives thereof: properties, lipid permeability and enzymatic reactions2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    5-aminolevulinic acid (5-ALA) and derivatives thereof are widely usedprodrugs in treatment of pre-malignant skin diseases of the cancer treatmentmethod photodynamic therapy (PDT). The target molecule in 5-ALAPDTis protoporphyrin IX (PpIX), which is synthesized endogenously from5-ALA via the heme pathway in the cell. This thesis is focused on 5-ALA,which is studied in different perspectives and with a variety of computationalmethods. The structural and energetic properties of 5-ALA, itsmethyl-, ethyl- and hexyl esters, four different 5-ALA enols, and hydrated5-ALA have been investigated using Quantum Mechanical (QM) first principlesdensity functional theory (DFT) calculations. 5-ALA is found to bemore stable than its isomers and the hydrolysations of the esters are morespontaneous for longer 5-ALA ester chains than shorter. The keto-enoltautomerization mechanism of 5-ALA has been studied, and a self-catalysismechanism has been proposed to be the most probable. Molecular Dynamics(MD) simulations of a lipid bilayer have been performed to study themembrane permeability of 5-ALA and its esters. The methyl ester of 5-ALAwas found to have the highest permeability constant (PMe-5-ALA = 52.8 cm/s).The mechanism of the two heme pathway enzymes; Porphobilinogen synthase(PBGS) and Uroporphyrinogen III decarboxylase (UROD), have beenstudied by DFT calculations and QM/MM methodology. The rate-limitingstep is found to have a barrier of 19.4 kcal/mol for PBGS and 13.7kcal/mol for the first decarboxylation step in UROD. Generally, the resultsare in good agreement with experimental results available to date.

    List of papers
    1. Theoretical study of 5-aminolevulinic acid (5ALA) and some pharmaceutically important derivatives
    Open this publication in new window or tab >>Theoretical study of 5-aminolevulinic acid (5ALA) and some pharmaceutically important derivatives
    2007 (English)In: Chemical Physics Letters, ISSN 0009-2614, E-ISSN 1873-4448, Vol. 434, no 1-3, p. 101-106Article in journal (Refereed) Published
    Abstract [en]

    5-Aminolevulinic acid (5ALA) is the key synthetic building block in protoporphyrin IX (PpIX), the heme chromophore in mitochondria. The addition of extracorporeal 5ALA and its alkyl ester derivatives are in current clinical use in photodynamical diagnostics and photodynamic therapy of tumors and skin disorders. In the current study density functional theory calculations are performed on 5ALA and its methyl, ethyl, and hexyl esters, in order to explore the basic chemical properties of these species. It is concluded that even in aqueous media the zwitterionic form of 5ALA is less stable than the non-zwitterionic one, that the local environment (lipid vs water) affects the energetics of reaction considerably, and that the hexyl species is most prone to hydrolysis of the three alkyl ester derivatives.

    Place, publisher, year, edition, pages
    Amsterdam: North-Holland Publishing Co, 2007
    Keywords
    5-aminolevulinic acid, 5ALA, B3LYP, DFT, Protonation states, Alkyl esters
    National Category
    Theoretical Chemistry Physical Chemistry
    Research subject
    Biochemistry; Physical Chemistry
    Identifiers
    urn:nbn:se:oru:diva-4092 (URN)10.1016/j.cplett.2006.11.084 (DOI)000243820100020 ()2-s2.0-33846018089 (Scopus ID)
    Available from: 2007-06-25 Created: 2007-06-25 Last updated: 2017-10-17Bibliographically approved
    2. Theoretical study of 5-aminolevulinic acid tautomerization: a novel self-catalyzed mechanism
    Open this publication in new window or tab >>Theoretical study of 5-aminolevulinic acid tautomerization: a novel self-catalyzed mechanism
    2008 (English)In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 112, no 18, p. 4367-4374Article in journal (Refereed) Published
    Abstract [en]

    5-Aminolevulinic acid (5ALA) is the key synthetic building block in protoporphyrin IX (PpIX), the heme chromophore in mitochondria. In this study density functional theory calculations were performed on the tautomers of 5ALA and the tautomerization reaction mechanism from its enolic forms (5-amino-4-hydroxypent-3-enoic acid and 5-amino-4-hydroxypent-4-enoic acid) to the more stable 5ALA. The hydrated form 5-amino-4,4-dihydroxypentanoic acid was also studied. The lowest energy pathway of 5ALA tautomerization is by means of autocatalysis, in that an oxygen of the carboxylic group transfers the hydrogen atom as a "crane", with an activation energy of similar to 15 kcal/mol. This should be compared to the barriers of about 35 kcal/mol for water assisted tautomerization, and 60 kcal/mol for direct hydrogen transfer. For hydration of 5ALA, the water catalyzed activation barrier is found to be similar to 35 kcal/mol, approximately 5 kcal/mol lower than direct hydration.

    Place, publisher, year, edition, pages
    Washington DC: American Chemical Society, 2008
    Keywords
    Aminolevulinic Acid/*chemistry, Carboxylic Acids/chemistry, Catalysis, Isomerism, Protons, Quantum Theory, Thermodynamics, Water/chemistry
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Physical Chemistry Theoretical Chemistry
    Research subject
    Biochemistry; Physical Chemistry
    Identifiers
    urn:nbn:se:oru:diva-4625 (URN)10.1021/jp7118197 (DOI)000255486400026 ()18416542 (PubMedID)2-s2.0-43949116597 (Scopus ID)
    Available from: 2008-10-13 Created: 2008-10-13 Last updated: 2017-10-17Bibliographically approved
    3. Modelling the behavior of 5-aminolevulinic acid and its alkyl esters in a lipid bilayer
    Open this publication in new window or tab >>Modelling the behavior of 5-aminolevulinic acid and its alkyl esters in a lipid bilayer
    2008 (English)In: Chemical Physics Letters, ISSN 0009-2614, E-ISSN 1873-4448, Vol. 463, no 1-3, p. 178-182Article in journal (Refereed) Published
    Abstract [en]

    5-Aminolevulinic acid (5ALA) and ester derivates thereof are used as prodrugs in photodynamic therapy (PDT). The behavior of 5ALA and three esters of 5ALA in a DPPC lipid bilayer is investigated. In particular, the methyl ester displays a very different free energy profile, where the highest barrier is located in the region with highest lipid density, while the others have their peak in the middle of the membrane, and also displays a considerably lower permeability coefficient than neutral 5ALA and the ethyl ester. The zwitterion of 5ALA has the highest permeability constant, but a significant free energy minimum in the polar head-group region renders an accumulation in this region.

    Place, publisher, year, edition, pages
    Amsterdam: North-Holland Publishing Co, 2008
    Keywords
    Molecular-dynamics simulations, photodynamic therapy, adenocarcinoma cells, beta transporters, hydrated DPPC, derivates, permeation, protoporphyrin, transition, membranes
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Medicinal Chemistry Physical Chemistry Theoretical Chemistry
    Research subject
    Biochemistry; Physical Chemistry
    Identifiers
    urn:nbn:se:oru:diva-4624 (URN)10.1016/j.cplett.2008.08.021 (DOI)000259150400035 ()2-s2.0-51349091343 (Scopus ID)
    Available from: 2008-10-13 Created: 2008-10-13 Last updated: 2018-01-13Bibliographically approved
    4. Computational Insights into the Mechanism of Substrate Binding in Potphobilinogen Synthase
    Open this publication in new window or tab >>Computational Insights into the Mechanism of Substrate Binding in Potphobilinogen Synthase
    (English)Manuscript (preprint) (Other academic)
    National Category
    Theoretical Chemistry Physical Chemistry
    Research subject
    Physical Chemistry
    Identifiers
    urn:nbn:se:oru:diva-9948 (URN)
    Available from: 2010-03-12 Created: 2010-03-10 Last updated: 2017-10-17Bibliographically approved
    5. Modelling the mechanism of porphobilinogen synthase
    Open this publication in new window or tab >>Modelling the mechanism of porphobilinogen synthase
    (English)Manuscript (preprint) (Other academic)
    National Category
    Physical Chemistry Theoretical Chemistry
    Research subject
    Physical Chemistry; Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-9949 (URN)
    Available from: 2010-03-12 Created: 2010-03-10 Last updated: 2017-10-17Bibliographically approved
    6. Computational insights into the first branching point in porphyrin biosynthesis: decarboxylation of ring D in URO–III by Uroporphyrinogen–III Decarboxylase
    Open this publication in new window or tab >>Computational insights into the first branching point in porphyrin biosynthesis: decarboxylation of ring D in URO–III by Uroporphyrinogen–III Decarboxylase
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Physical Chemistry Theoretical Chemistry
    Research subject
    Physical Chemistry; Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-9950 (URN)
    Available from: 2010-03-12 Created: 2010-03-10 Last updated: 2017-10-17Bibliographically approved
  • 4.
    Kalbina, Irina
    Örebro University, Department of Natural Sciences.
    The molecular mechanisms behind perception and signal transduction of UV-B irradiation in Arabidopsis thaliana2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Elevation of UV-B radiation (280-315 nm), occurring as a result of depletion of the stratospheric ozone, causes a number of physiological and biochemical changes in plants. Damage to the photosynthetic apparatus (including the bleaching of the pigments which trap the sun's energy), to the processes of cell division and growth regulation, and to the composition and replication of genetic material are just some of these changes. The consequences include reduction in growth yield, changes in levels and effects of plant hormones and secondary metabolites, and alteration of interactions between plants and other organisms.

    This thesis deals with several mechanistic questions related to regulation of responses during UV-B stress in plants. Our results show significant ecotype-specific variability in UV-B response in the model plant Arabidopsis thaliana. Differences at the molecular level (expression of PR-5 and steady-state concentration of H2O2) resulted in statistically significant differences in biomass, rosette size and leaf area. Therefore, it is of great importance to pay attention to the responses of the background ecotypes when for instance studying mechanisms of responses toward ultraviolet-B radiation in mutants.

    Using a DNA microarray approach, we found a number of novel genes to be differentially expressed under UV-B radiation. Two of the genes (PYROA and MEB5.2) were later used as molecular markers for monitoring of UV-B stress. Promoters of PYROA and MEB5.2 were compared with promoters of genes for the phenylpropanoid pathway. The comparisons indicated only few common elements with the UV-B-regulated promoters of CHS, PAL and CHI. In contrast, the genes identified as being UV-B regulated in this study (MEB5.2, PYROA and UBQ3), completely lacked elements required for the UV-B induction of CHS, indicating that these genes are regulated by different transcription factors. In addition, novel unidentified cis-elements are probably also present upstream of the transcription start.

    Reverse and forward genetics were used for searching novel genes responsive to UV-B and for examination of proposed candidates of the UV-B signal transduction chain. Screening of more than 2000 T-DNA mutants for differential response to UV-B resulted in the identification of a mutant displaying insensitivity to UV-B induced inhibition of hypocotyl growth. By using the corresponding knock-out mutants, the involvement of NADPH oxidase and MAPK phosphatase 1 in UV-B signalling was demonstrated.

    For the plant to be able to respond appropriately to UV-B irradiation, UV-B quanta have to be absorbed. There are indirect evidences for the existence of specific UV-B receptor(s), whereas the receptor itself still remains unknown. By the classical approach of action spectroscopy, we undertook an attempt to identify the absorption spectra of the chromophore(s) sensing UV-B radiation in plants. The investigated molecular markers revealed the presence of two potential chromophores absorbing in the UV-B region and peaking at 280-290 and 300 nm, respectively.

    List of papers
    1. Gene regulation by low level UV-B radiation: identification by DNA array analysis
    Open this publication in new window or tab >>Gene regulation by low level UV-B radiation: identification by DNA array analysis
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    2002 (English)In: Photochemical and Photobiological Sciences, ISSN 1474-905X, E-ISSN 1474-9092, Vol. 1, no 9, p. 656-664Article in journal (Refereed) Published
    Abstract [en]

    UV-B radiation alters transcript levels of various defence genes and photosynthetic genes in plants. Utilising a DNA array with 5000 ESTs and cDNAs from Arabidopsis thaliana, 70 genes were found to show a greater than two-fold induction or repression of transcript levels. Six genes (MEB5.2, PyroA, Ubq3, Lhcb6, F5D21.10 and the gene for an RNA polymerase II subunit) were tested for stress specific gene regulation on northern blots with RNA from plants exposed to low dose UV-B radiation, ozone or wounding. Transcript levels for PyroA, Uhq3 and the gene for a RNA polymerase II subunit were all specifically increased by UV-B. MEB5.2 mRNA levels also rose, whereas Lhcb6 and FSD21.10 transcript levels decreased under all stresses. The PyroA gene product in fungi is needed for biosynthesis of pyridoxine, and might have a role in protection against singlet oxygen. The Ubq3 gene encodes the ubiquitin protein that is attached to proteins destined for degradation. MEB5.2 and F5D21.10 represent novel gene products whose function have not yet been identified. Pairwise comparisons between the UV-B inducible promoters have identified a series of elements present in the MEB5.2 and PyroA promoters, absent from promoters of genes for early phenylpropanoid metabolism and that may be responsible for modulating their UV-B responses.

    Keywords
    Arabidopsis/genetics/radiation effects, Base Sequence, DNA; Plant/genetics, Expressed Sequence Tags, Gene Expression Regulation; Enzymologic/radiation effects, Gene Expression Regulation; Plant/*radiation effects, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Plant Proteins/genetics, Promoter Regions (Genetics), RNA Polymerase II/genetics, Ultraviolet Rays
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-2879 (URN)10.1039/B202659G (DOI)12665302 (PubMedID)
    Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2017-12-14Bibliographically approved
    2. Supplementary ultraviolet-B irradiation reveals differences in stress responses between Arabidopsis thaliana ecotypes
    Open this publication in new window or tab >>Supplementary ultraviolet-B irradiation reveals differences in stress responses between Arabidopsis thaliana ecotypes
    2005 (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-2880 (URN)
    Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2017-10-18Bibliographically approved
    3. Wavelength dependence of expression of UV-B-induced molecular markers in Arabidopsis thaliana
    Open this publication in new window or tab >>Wavelength dependence of expression of UV-B-induced molecular markers in Arabidopsis thaliana
    (English)Manuscript (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-2881 (URN)
    Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2017-10-18Bibliographically approved
    4. The role of NADPH oxidase and MAP kinase phosphatase 1 in UV-B-dependent gene expression in Arabidopsis
    Open this publication in new window or tab >>The role of NADPH oxidase and MAP kinase phosphatase 1 in UV-B-dependent gene expression in Arabidopsis
    (English)Manuscript (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-2882 (URN)
    Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2017-10-18Bibliographically approved
    5. An Arabidopsis mutant responsive to UV-B irradiation
    Open this publication in new window or tab >>An Arabidopsis mutant responsive to UV-B irradiation
    (English)Manuscript (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-2883 (URN)
    Available from: 2005-09-14 Created: 2005-09-14 Last updated: 2017-10-18Bibliographically approved
  • 5.
    Karlsson, Mattias
    Örebro University, School of Science and Technology.
    Modulation of cellular innate immune responses by lactobacilli2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Lactobacillus is a genus of lactic acid bacteria frequently used as healthpromoting probiotics. Using probiotics to treat or prevent infections is a novel experimental approach with vast impact on future therapy. Lactobacillus rhamnosus GR-1 is a probiotic investigated for its ability to reduce urogenital disease including urinary tract infections caused by pathogenic Escherichia coli. L. rhamnosus GR-1 has been shown to modulate immunity, thought to influence its probiotic effect. In this thesis, the aim was to study immunomodulation by L. rhamnosus GR-1 and other lactobacilli, with emphasis on elicited immune responses such as nuclear factor-kappaB (NF-κB) activation and cytokine release from human urothelial cells.

    Viable, heat-killed, and isolated released products from L. rhamnosus GR-1 augmented NF-κB activation in E. coli-challenged urothelial cells. Blocking of lipopolysaccharide binding to toll-like receptor 4 completely quelled this augmentation. Size-fractionation, urothelial cell challenge, and two-dimensional gel electrophoresis of L. rhamnosus GR-1 released products presented several candidate proteins with NF-κB modulatory actions including chaperonin GroEL, elongation factur Tu, and a protein from the NLP/P60 protein family. While tumor necrosis factor was correspondingly augmented by L. rhamnosus GR-1, the release of two other cytokines, interleukin (IL)-6 and CXCL8, was reduced. Similar effects were observed in macrophage-like cells stimulated with L. rhamnosus GR-1.

    Many immunomodulatory effects of lactobacilli are believed to be species and strain dependent. Therefore, twelve Lactobacillus strains were used to screen for their effects on CXCL8 release from urothelial cells. A majority of these strains were able to influence CXCL8 release from the cells. Phylogenetic analysis revealed close evolutionary linkage between lactobacilli with similar actions on CXCL8. Increased knowledge on probiotic bacterial products and the mechanism(s) of action could lead to improved future treatments for infections.

    List of papers
    1. Released substances from lactobacilli influence immune responses in human epithelial cells
    Open this publication in new window or tab >>Released substances from lactobacilli influence immune responses in human epithelial cells
    2010 (English)In: Abstracts of the 3rd Swedish-Hellenic Life Sciences Research Conference, Athens, March 25-27, 2010 / [ed] Fragiskos Kolisis, Nikolaos Venizelos, 2010, p. 367-368Conference paper, Oral presentation with published abstract (Refereed)
    National Category
    Medical and Health Sciences Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-12300 (URN)
    Conference
    The 3rd Swedish-Hellenic Life Sciences Research Conference, Athens, March 25-27, 2010
    Note

    in vivo 24 (2010).

    Available from: 2010-10-22 Created: 2010-10-22 Last updated: 2018-04-19Bibliographically approved
    2. Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR4
    Open this publication in new window or tab >>Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR4
    2012 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, p. 15-Article in journal (Refereed) Published
    Abstract [en]

    Background: Epithelial cells of the urinary tract recognize pathogenic bacteria through pattern recognition receptors on their surface, such as toll-like receptors (TLRs), and mount an immune response through the activation of the NF-kappaB pathway. Some uropathogenic bacteria can subvert these cellular responses, creating problems with how the host eliminates pathogens. Lactobacillus is a genus of lactic acid bacteria that are part of the microbiota and consist of many probiotic strains, some specifically for urogenital infections. Immunomodulation has emerged as an important mode of action of probiotic and commensal lactobacilli and given the importance of epithelial cells, we evaluated the effect of the urogenital probiotic Lactobacillus rhamnosus GR-1 on epithelial immune activation.

    Results: Immune activation through the NF-kappaB pathway was initiated by stimulation of T24 urothelial cells with heat-killed Escherichia coli and this was further potentiated when cells were co-cultured with live L. rhamnosus GR-1. Heat-killed lactobacilli were poor activators of NF-kappaB. Concomitant stimulation of bladder cells with E. coli and L. rhamnosus GR 1 increased the levels of the pro-inflammatory cytokine TNF, whereas IL-6 and CXCL8 levels were reduced. Another probiotic, L. rhamnosus GG, was also able to potentiate NF-kappaB in these cells although at a significantly reduced level compared to the GR 1 strain. The transcript numbers and protein levels of the lipopolysaccharide receptor TLR4 were significantly increased after co-stimulation with E. coli and lactobacilli compared to controls. Furthermore, inhibition of TLR4 activation by polymixin B completely blocked the lactobacilli potentiation of NF-kappaB.

    Conclusions: The immunological outcome of E. coli challenge of bladder cells was influenced by probiotic L. rhamnosus GR 1, by enhancing the activation of NF-kappaB and TNF release. Thus the urogenital probiotic L. rhamnosus GR-1 modulated the activation of the NF-kappaB through increased levels of TLR4 on the bladder cells and altered subsequent release of cytokines from urothelial cells. By influencing immunological factors such as TLR4, important in the process of fighting pathogens, lactobacilli could facilitate pathogen recognition and infection clearance.

    Place, publisher, year, edition, pages
    BioMed Central, 2012
    National Category
    Microbiology
    Research subject
    Microbiology; Immunology
    Identifiers
    urn:nbn:se:oru:diva-21288 (URN)10.1186/1471-2180-12-15 (DOI)000301484000001 ()22264349 (PubMedID)2-s2.0-84856001528 (Scopus ID)
    Available from: 2012-02-17 Created: 2012-01-24 Last updated: 2018-05-08Bibliographically approved
    3. Substances released from probiotic Lactobacillus rhamnosus GR-1 potentiate NF-κB activity in Escherichia coli-stimulated urinary bladder cells
    Open this publication in new window or tab >>Substances released from probiotic Lactobacillus rhamnosus GR-1 potentiate NF-κB activity in Escherichia coli-stimulated urinary bladder cells
    Show others...
    2012 (English)In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 66, no 2, p. 147-156Article in journal (Refereed) Published
    Abstract [en]

    Lactobacillus rhamnosus GR-1 is a probiotic bacterium used to maintain urogenital health. The putative mechanism for its probiotic effect is by modulating the host immunity. Urinary tract infections (UTI) are often caused by uropathogenic Escherichia coli that frequently evade or suppress immune responses in the bladder and can target pathways, including nuclear factor-kappaB (NF-κB). We evaluated the role of L. rhamnosus GR-1 on NF-κB activation in E. coli-stimulated bladder cells. Viable L. rhamnosus GR-1 was found to potentiate NF-κB activity in E. coli-stimulated T24 bladder cells, whereas heat-killed lactobacilli demonstrated a marginal increase in NF-κB activity. Surface components released by trypsin- or LiCl treatment, or the resultant heat-killed shaved lactobacilli, had no effect on NF-κB activity. Isolation of released products from L. rhamnosus GR-1 demonstrated that the induction of NF-κB activity was owing to released product(s) with a relatively large native size. Several putative immunomodulatory proteins were identified, namely GroEL, elongation factor Tu and NLP/P60. GroEL and elongation factor Tu have previously been shown to elicit immune responses from human cells. Isolating and using immune-augmenting substances produced by lactobacilli is a novel strategy for the prevention or treatment of UTI caused by immune-evading E. coli.

    Place, publisher, year, edition, pages
    Hoboken, USA: Wiley-Blackwell, 2012
    Keywords
    Immune response, cytokines, lactobacilli, urothelium, uropathogen, secretome
    National Category
    Immunology Microbiology
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-22594 (URN)10.1111/j.1574-695X.2012.00994.x (DOI)000310277300003 ()22620976 (PubMedID)2-s2.0-84867727218 (Scopus ID)
    Note

    Funding Agencies:

    Knowledge Foundation

    Magnus Bergvalls Foundation 

    Sparbanksstiftelsen Nya 

    Carl Tryggers Foundation, Sweden 

    Available from: 2012-04-19 Created: 2012-04-19 Last updated: 2017-12-07Bibliographically approved
    4. Probiotic Lactobacillus rhamnosus alters inflammatory responses of bladder epithelial and macrophage-like cells in co-culture
    Open this publication in new window or tab >>Probiotic Lactobacillus rhamnosus alters inflammatory responses of bladder epithelial and macrophage-like cells in co-culture
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Immunology Microbiology
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-22595 (URN)
    Available from: 2012-04-19 Created: 2012-04-19 Last updated: 2017-10-17Bibliographically approved
    5. Lactobacilli differently regulate expression and secretion of CXCL8 in urothelial cells
    Open this publication in new window or tab >>Lactobacilli differently regulate expression and secretion of CXCL8 in urothelial cells
    2012 (English)In: Beneficial Microbes, ISSN 1876-2883, E-ISSN 1876-2891, Vol. 3, no 3, p. 195-203Article in journal (Refereed) Published
    Abstract [en]

    Modulation of the immune response is an established feature of certain lactobacilli. CXCL8 is an inflammatory chemokine released by the urinary tract mucosa after contact with uropathogenic Escherichia coli during urinary tract infection and is crucial for proper infiltration of immune cells. Nevertheless, persistently high levels of CXCL8 are associated with pathogenicity and malignancy. In this study, we tested twelve Lactobacillus strains for their ability to influence CXCL8 release from urothelial cells. We evaluated how strains from different Lactobacillus species could regulate CXCL8 in human 5637 urothelial cells, either resting cells or cells concomitantly challenged with heat-killed E. coli. A majority of the tested species altered CXCL8 release from the urothelial cells after 24 hours of stimulation. Most species increased CXCL8 release, whereas a few lactobacilli efficiently suppressed CXCL8 secretion from E. coli-challenged cells. While strong CXCL8 modulators such as Lactobacillus reuteri and Lactobacillus delbrueckii were unable to degrade CXCL8 in the extracellular environment, effects on IL8 transcription were evident for selected lactobacilli. Although IL8 transcription was affected by lactobacilli, the influence on mRNA transcript did not correlate to the impact on CXCL8 release. Phylogenetic analysis based on a 16S rRNA dendrogram of the tested lactobacilli and their effect on CXCL8 revealed some linkage to specific Lactobacillus groups. Testing the immunomodulatory nature of lactobacilli can prove important when selecting new probiotic microbes. Moreover, we believe that phylogenetic and phenotypic similarities could be used to analyse the traits governing such modulation.

    Place, publisher, year, edition, pages
    Wageningen Academic Publishers, 2012
    Keywords
    Lactobacillus; immunomodulation; probiotics; chemokines; cytokines
    National Category
    Microbiology Nutrition and Dietetics
    Research subject
    Immunology; Microbiology; Nutrition
    Identifiers
    urn:nbn:se:oru:diva-21701 (URN)10.3920/BM2012.0011 (DOI)000308740900004 ()22835703 (PubMedID)2-s2.0-84869221939 (Scopus ID)
    Funder
    Knowledge Foundation
    Note

    Funding Agencies:

    Magnus Bergvalls Foundation

    Helge Ax:son Johnsons Foundation 

    Available from: 2012-04-19 Created: 2012-02-17 Last updated: 2017-12-07Bibliographically approved
  • 6.
    Khalaf, Hazem
    Örebro University, School of Science and Technology.
    Characterization and environmental influences on inflammatory and physiological responses2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pharmaceuticals are regularly released into the environment, in particular nonsteroidalanti-inflammatory drugs (NSAIDs) and antibiotics. The measuredconcentrations are relatively low and have therefore been considered to be harmless.However, several pharmaceuticals, including naproxen and atenolol, are stable for upto 1 year in the environment, which increases the risk for accumulation. Evaluation ofthe effects of pharmaceuticals on induced inflammatory responses is thereforenecessary for the assessment of potential risks. Since NF-κB and MAPK are the mainpathways known to be critical regulators of inflammatory responses, intracellularsignalling and effects on these systems were examined in vitro using human cell-lines.NSAIDs were shown to significantly reduce NF-κB activity at environmentallyrelevant concentrations. Suppression of immune responses may lead to progressiveinfections since inflammatory responses are controlled by a cooperative activity ofAP-1 and NF-κB. Alterations in the activity of transcription factors and proinflammatorycytokine and chemokine levels such as TNF, IL-6 and CXCL8 areassociated with several human diseases including cystic fibrosis and AIDS. PMAexposure resulted in a rapid NF-κB activation, while extended treatment suppressedNF-κB and activated AP-1. Suppression of NF-κB activity may be due to PKCdependentBcl10 degradation, which decreased in response to PMA and correlatedwith the NF-κB activity. Regulation of cytokine expression revealed that NF-κB wasessential for IL-6 but not CXCL8 expression following specific inhibition of NF-κB,without affecting AP-1 activity. Furthermore, several reports have indicated theimportance of a functional NF-κB complex in zebrafish embryogenesis, whereblockage of NF-κB activation resulted in a deformation of the tail. Our results indicatea suppression of apoptotic pathways following activation of inflammatory mediatorsin response to HK E. coli treatment. These signals acted to direct zebrafish sexdifferentiation towards feminization. NF-κB was shown to regulate zp2 geneexpression, an indicator of oocyte development. Zebrafish sex determination was alsoshown to start early, prior to 16 days post fertilization. The results support thetransition through a juvenile ovary stage and suggests that steriodogenesis is aconsequence of sex differentiation rather than a regulatory mechanism.Control of prescription, use and disposal of pharmaceuticals is therefore importantto preserve human health, biotic processes and to avoid developmental alterations inaquatic organisms. The complexity of regulatory systems involved in inflammationsuggest that there is a need to further evaluate the signalling pathways involved inorder to provide a better understanding of cellular responses to manmade substances,but also to offer an insight into possible development of alternative treatments forhuman diseases with elevated cytokine/chemokine levels.

    List of papers
    1. Differential cytokine regulation by NF-κB and AP-1 in Jurkat T-cells
    Open this publication in new window or tab >>Differential cytokine regulation by NF-κB and AP-1 in Jurkat T-cells
    2010 (English)In: BMC Immunology, ISSN 1471-2172, E-ISSN 1471-2172, Vol. 11, article id 26Article in journal (Refereed) Published
    Abstract [en]

    Background: Activator protein (AP)-1 and nuclear factor (NF)-κB largely control T-cell activation, following binding offoreign antigens to the T-cell receptor leading to cytokine secretion. Elevated levels of pro-inflammatory cytokines andchemokines such as TNF, IL-6 and CXCL8 are associated with several human diseases including cystic fibrosis, pulmonary fibrosis and AIDS. The aim of this study was to investigate the role of the transcription factors, AP-1 and NF-κB, in IL-6 and CXCL8 regulation in Jurkat T-cells.

    Results: Phorbol myristate acetate (PMA) exposure resulted in an up-regulation of AP-1 and down-regulation of NF-κBactivity, however, exposure to heat killed (HK) Escherichia. coli MG1655 resulted in a dose-dependent increase in NF-κBactivity without affecting AP-1. The cytokine profile revealed an up-regulation of the chemokine CXCL8 and the pro-inflammatory cytokines TNF, IL-2 and IL-6 following treatment with both PMA and HK E. coli, while the levels of the anti-inflammatory cytokine IL-10 were not affected by PMA but were significantly down-regulated by HK E. coli. AP-1activation was significantly increased 2 h after PMA exposure and continued to increase thereafter. In contrast, NF-κBresponded to PMA exposure by a rapid up-regulation followed by a subsequent down-regulation. Increased intracellular Ca2+ concentrations countered the down-regulation of NF-κB by PMA, while similar treatment with calcium ionophore resulted in a reduced NF-κB activity following induction with HK E. coli. In order to further study NF-κB activation, we considered two up-stream signalling proteins, PKC and Bcl10. Phosphorylated-PKC levels increased inresponse to PMA and HK E. coli, while Bcl10 levels significantly decreased following PMA treatment. Using an NF-κBactivation inhibitor, we observed complete inhibition of IL-6 expression while CXCL8 levels only decreased by 40% atthe highest concentration. Treatment of Jurkat T-cells with PMA in the presence of JNK-inhibitor suppressed both CXCL8 and IL-6 while PKC-inhibitor primarily decreased CXCL8 expression.

    Conclusion: The present study shows that NF-κB regulated IL-6 but not CXCL8. This complex regulation of CXCL8suggests that there is a need to further evaluate the signalling pathways in order to develop new treatment fordiseases with elevated CXCL8 levels, such as AIDS and autoimmune diseases.

    Place, publisher, year, edition, pages
    London, United Kingdom: BioMed Central, 2010
    National Category
    Natural Sciences Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-12233 (URN)10.1186/1471-2172-11-26 (DOI)000279893500001 ()20507572 (PubMedID)2-s2.0-77952738203 (Scopus ID)
    Available from: 2010-10-18 Created: 2010-10-18 Last updated: 2018-09-16Bibliographically approved
    2. Influence of growth conditions on in vitro regulation of NF-κB activity in Jurkat T-cells
    Open this publication in new window or tab >>Influence of growth conditions on in vitro regulation of NF-κB activity in Jurkat T-cells
    2010 (English)Manuscript (preprint) (Other academic)
    National Category
    Natural Sciences Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-12234 (URN)
    Available from: 2010-10-18 Created: 2010-10-18 Last updated: 2018-09-06Bibliographically approved
    3. In vitro analysis of inflammatory responses following environmental exposure to pharmaceuticals and inland waters
    Open this publication in new window or tab >>In vitro analysis of inflammatory responses following environmental exposure to pharmaceuticals and inland waters
    Show others...
    2009 (English)In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 407, no 4, p. 1452-1460Article in journal (Refereed) Published
    Abstract [en]

    Pharmaceuticals are regularly released into the environment; in particular non-steroidalanti-inflammatory drugs (NSAIDs) and antibiotics. Erythromycin, naproxen, furosemideand atenolol are reported to be stable for up to 1 year in the environment, which increasesthe risk for accumulation. In the present study we have measured the occurrence andconcentration of pharmaceuticals in river Viskan (Jössabron) downstream of a sewagetreatment plant in Borås, Sweden. Pharmaceuticals and water samples were tested forpotential human risk by evaluating inflammatory responses (NF-κB and AP-1) using humanT24 bladder epithelial cells and Jurkat T-cells. NF-κB activity in T24 cells was significantlyreduced by all NSAIDs analysed (diclofenac, ketoprofen, naproxen, ibuprophen anddextropropoxyphene), but also by trimethoprim, using environmentally relevantconcentrations. NF-κB and AP-1 activation was further analysed in response to watersamples collected from different locations in Sweden. Dose-dependent down-regulation ofAP-1 activity in Jurkat cells was observed at all locations. At two locations (Jössabron andAlmenäs) down-regulation of NF-κB was observed. In contrast, the NF-κB response waspotentiated by exposure to water from both locations following activation of NF-κB bytreatment with heat-killed Escherichia coli. To determine the involvement ofpharmaceuticals in the responses, T24 cells were exposed to the pharmaceutical mixture,based on the determined levels at Jössabron. This resulted in reduction of the NF-κBresponse following exposure to the pharmaceutical mixture alone while no potentiationwas observed when cells were co-exposed to heat killed E. coli and pharmaceuticals. Theobtained results demonstrate that the identified pharmaceuticals affect the inflammatoryresponses and furthermore indicate the presence of unknown substance(s) with the abilityto potentiate inflammatory responses

    Place, publisher, year, edition, pages
    Amsterdam: Elsevier, 2009
    Keywords
    Nuclear factor-κB (NF-κB), Activator protein-1 (AP-1), Pharmaceuticals, Inflammation, Non-steroidal anti-inflammatory drugs
    National Category
    Natural Sciences Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-12235 (URN)10.1016/j.scitotenv.2008.10.016 (DOI)000262573200022 ()19038416 (PubMedID)2-s2.0-57049179481 (Scopus ID)
    Available from: 2010-10-18 Created: 2010-10-18 Last updated: 2017-12-12Bibliographically approved
    4. Zebrafish feminization in response to heat killed bacterial exposure suggests a function for anti-apoptotic genes in oocyte maintenance
    Open this publication in new window or tab >>Zebrafish feminization in response to heat killed bacterial exposure suggests a function for anti-apoptotic genes in oocyte maintenance
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Natural Sciences Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-12238 (URN)
    Available from: 2010-10-18 Created: 2010-10-18 Last updated: 2017-10-17Bibliographically approved
  • 7.
    Larsson, Anders
    Örebro University, School of Science and Technology.
    Androgen receptors and endocrine disrupting substances2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Throughout the animal kingdom, organisms are dependent on substances such as steroid hormones to help them maintain internal balances. Examples of important tasks that are under regulation of steroid hormones are somatic and gonadal development, sexual performance and behavior (both social and sexual) as well as sex differentiation. Balance in the biology of reproduction is important for all organisms, and is sensitive to alterations and disturbances. If the environment is altered in a manner that lead to higher estrogenic or androgenic levels, the sex ratio of organisms that do not rely on genetic differences in the sex differentiation, will be biased towards more females or males in the population. It has been known for some time that there are pollutants in the environment that affect steroid pathways, such as the estrogenic and thyroid systems, but not much has been known about the androgenic systems. Examples of populations being masculinized have been recorded, and estrogenic compounds have been known to act as antiandrogens, but not until recently the first androgen agonist was identified. We used a combination of in vitro and computational modeling to identify the brominated flame retardant, 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane, as a potent androgen agonist to the human androgen receptor.

    In addition to this we cloned and characterized the androgen receptor from, a frequently used model organism, zebrafish (Danio rerio) as a receptor primarily activated by 11-ketotestosterone. This is a feature the zebrafish share with several other teleost fishes, such as the three-spined stickleback. Thus fish androgen receptors differ from most mammalian androgen receptors, where dihydrotestosterone is the most potent activator.

     

    List of papers
    1. Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone
    Open this publication in new window or tab >>Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone
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    2005 (English)In: Reproductive biology and endocrinology, ISSN 1477-7827, Vol. 3:37Article in journal (Refereed) Published
    Abstract [en]

    Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-beta subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.

    National Category
    Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-8835 (URN)10.1186/1477-7827-3-37 (DOI)16107211 (PubMedID)
    Available from: 2009-12-18 Created: 2009-12-18 Last updated: 2017-10-18Bibliographically approved
    2. Zebrafish androgen receptor: isolation, molecular, and biochemical characterization
    Open this publication in new window or tab >>Zebrafish androgen receptor: isolation, molecular, and biochemical characterization
    Show others...
    2008 (English)In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 78, no 2, p. 361-369Article in journal (Refereed) Published
    Abstract [en]

    Androgens play an important role in male sexual differentiation and development. They exert their function by binding to and activating the androgen receptor (Ar), a member of the steroid hormone receptor superfamily. Here, we report on the isolation and characterization of zebrafish Ar. The complete transcript of zebrafish ar is 5.3 kb long encoding a putative polypeptide of 868 amino acids. Our experimental and bioinformatic analysis has found a single ar locus in zebrafish. Phylogenetic analysis using the ligand-binding domain showed that the zebrafish Ar clustered with its cyprinid orthologs to form a separate group, which was closer to the beta clade than to the alpha clade. Tissue-specific expression analysis revealed that the ar mRNA was expressed ubiquitously in all adult tissues tested, with sexually dimorphic expression in the gonad and muscle. While the ar transcript was maternally deposited into the embryo, signs of zygotic expression could be detected as early as 24 h after fertilization, and the expression level increased substantially afterwards. When analyzed during gonad development, the expression level of ar mRNA at 4 wk after fertilization was similar in both developing gonads but later became higher in the transforming testis, suggesting a potential role during male gonad differentiation. We also combined theoretical modeling with in vitro experiments to show that the zebrafish Ar is preferentially activated by 11-ketotestosterone.

    Place, publisher, year, edition, pages
    Champaign, Ill.: Society for the Study of Reproduction, 2008
    Keywords
    Androgens/*metabolism/pharmacology, Animals, Chromosome Mapping, Cloning; Molecular, Computer Simulation, DNA; Complementary/genetics, Female, Gene Expression, Gonads/growth & development, Male, Molecular Sequence Data, Phylogeny, RNA; Messenger/metabolism, Receptors; Androgen/agonists/*classification/*genetics, Sequence Homology; Amino Acid, Testosterone/analogs & derivatives/pharmacology, Transcription; Genetic, Transfection, Zebrafish/genetics/*growth & development, Zebrafish Proteins/*genetics
    National Category
    Natural Sciences Biological Sciences
    Research subject
    biologi
    Identifiers
    urn:nbn:se:oru:diva-3557 (URN)10.1095/​biolreprod.107.062018 (DOI)17942797 (PubMedID)
    Available from: 2008-12-09 Created: 2008-12-09 Last updated: 2017-12-14Bibliographically approved
    3. Identification of the brominated flame retardant 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane as an androgen agonist
    Open this publication in new window or tab >>Identification of the brominated flame retardant 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane as an androgen agonist
    Show others...
    2006 (English)In: Journal of medicinal chemistry, ISSN 0022-2623, Vol. 49, no 25, p. 7366-7372Article in journal (Refereed) Published
    Abstract [en]

    To investigate androgen receptor (AR) activation by exogenous compounds, we used a combination of experimental analysis and theoretical modeling to compare a set of brominated flame retardants (BFRs) to dihydrotestosterone (DHT) with regard to ligand docking, AR binding, and AR activation in human hepatocellular liver carcinoma cells, as well as interacting energy analysis. Modeling of receptor docking was found to be a useful first step in predicting the potential to translocate to the ligand pocket of the receptor, and the computed interaction energy was found to correlate with the observed binding affinity. Flexible alignment studies of the BFR compounds demonstrated that 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (BCH) closely overlap DHT. Combining the theoretical modeling with in vitro ligand-binding and receptor-activation assays, we show that BCH binds to and activates the human AR. The remaining BFRs did not successfully interact with the ligand pocket, were not able to replace a synthetic androgen from the receptor, and failed to activate the receptor.

    Keywords
    Binding, Competitive, Cell Line, Tumor, Cyclohexanes/*chemistry/toxicity, Endocrine Disruptors/*chemistry/toxicity, Flame Retardants/*toxicity, Humans, Ligands, Models, Molecular, Receptors, Androgen/*agonists/*chemistry
    National Category
    Natural Sciences Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-3562 (URN)10.1021/jm060713d (DOI)17149866 (PubMedID)
    Available from: 2008-12-09 Created: 2008-12-09 Last updated: 2017-10-18Bibliographically approved
    4. Diastereomers of the Brominated Flame Retardant 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane Induce Androgen Receptor Activation in the HepG2 Hepatocellular Carcinoma Cell Line and the LNCaP Prostate Cancer Cell Line
    Open this publication in new window or tab >>Diastereomers of the Brominated Flame Retardant 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane Induce Androgen Receptor Activation in the HepG2 Hepatocellular Carcinoma Cell Line and the LNCaP Prostate Cancer Cell Line
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    2009 (English)In: Journal of Environmental Health Perspectives, ISSN 0091-6765, E-ISSN 1552-9924, Vol. 117, no 12, p. 1853-1859Article in journal (Refereed) Published
    Abstract [en]

    Background: Reported incidences of prostate cancer and masculinization of animals indicate a release of compounds with androgenic properties into the environment. Large numbers of environmental pollutants have been screened to identify such compounds; however, not until recently was 1,2-dibromo-4-(1,2-dibromoethyl)cyclohex​ane (TBECH) identified as the first potent activator of the human androgen receptor (hAR). TBECH has been found in beluga whales and bird eggs and has also been found to be maternally transferred in zebrafish.

    Objectives: In the present study we investigated interaction energies between TBECH diastereomers (α, β, γ, and δ) and the hAR, and their ability to activate the receptor and induce prostate-specific antigen (PSA) expression in vitro.

    Methods: We performed computational modeling to determine interaction energies between the ligand and the AR ligand-binding site, and measured in vitro competitive binding assays for AR by polarization fluorometry analysis. We used enzyme-linked immunosorbent assays to determine PSA activity in LNCaP and HepG2 cells.

    Results: We found the γ and δ diastereomers to be more potent activators of hAR than the α and β diastereomers, which was confirmed in receptor binding studies. All TBECH diastereomers induced PSA expression in LNCaP cells even though the AR present in these cells is mutated (T877A). Modeling studies of LNCaP AR revealed that TBECH diastereomers bound to the receptor with a closer distance to the key amino acids in the ligand-binding domain, indicating stronger binding to the mutated receptor.

    Conclusions: The present study demonstrates the ability of TBECH to activate the hAR, indicating that it is a potential endocrine disruptor.

    Place, publisher, year, edition, pages
    National Institute of Environmental Health Science, 2009
    Keywords
    androgen, brominated flame retardant, endocrine disruptor
    National Category
    Natural Sciences Chemical Sciences Environmental Sciences Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-9678 (URN)10.1289/ehp.0901065 (DOI)000272474600026 ()20049203 (PubMedID)2-s2.0-75349109314 (Scopus ID)
    Available from: 2010-02-09 Created: 2010-02-09 Last updated: 2017-12-12Bibliographically approved
  • 8.
    Lindh, Ingrid
    Örebro University, School of Science and Technology.
    Plant-produced STI vaccine antigens with special emphasis on HIV-1 p242011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Objective: To establish stable transgenic non-toxic plants as a platform for plant-based vaccine production as well as potential oral delivery system of vaccine antigens for sexually transmitted infections (STIs). The concept is to immunize the mucosal immune system present in the gut-associated lymphoid tissues (GALT). HIV-1 p24 subtype C protein has been used as the main antigen model, in parallel with an engineered unique chimeric MOMP antigen from Chlamydia trachomatis serovar E.

    Methods: Chimeric MOMP and p24 vaccine antigens were successfully inserted into the nuclear genomes of Arabidopsis thaliana and Daucus carota via Agrobacterium-mediated gene transfer. The characteristics of the genetic inserts and corresponding mRNAs and recombinant proteins in planta were described using several methods, including northern, Southern, and western blotting, ELISA, and a commercial HIV Ag/Ab combination assay. Immunogenicity of the antigens was studied in mice models.

    Results: Transgenes of both plant species expressing p24 or chimeric MOMP were successfully generated. Additional HIV-1 vaccine antigen candidates were introduced and the genetic inserts have been confirmed in Arabidopsis thaliana. The Arabidopsis thaliana expressing p24 and chimeric MOMP were demonstrated to be stable over generations and antigenicity analyses showed that plant-derived HIV-1 p24 and chimeric MOMP retained immunological epitopes when they were expressed in planta. Oral administration of transgenic plant material generated a priming effect of the immune competent cells present in the GALT, shown by the presence of antigen-specific-IgG in mice sera after boosting. Mice immunized with plant-derived HIV-1 p24 antigen were also analyzed for antigen-specific faecal IgA as well as cellular immune responses. However, detectable levels of the two latter immune responses were not observed. The Chlamydia trachomatis chimeric MOMP antigen was further evaluated for its potential as a vaccine antigen candidate, with positive results indicating a more rapid clearance of the Chlamydia trachomatis infection post immunization.

    Conclusion: Stable non-toxic transgenic plants expressing either HIV-1 p24 or a novel  Chlamydia trachomatis chimeric MOMP antigens have successfully been developed. The two plant-produced STI vaccine antigens have in initial mice feeding studies provided important proof-of-concept for the oral vaccination approach. Now, immunization studies to expand, en-hance, and improve knowledge of the immune responses generated by the orally delivered transgenic plants are of high priority.

    List of papers
    1. Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses
    Open this publication in new window or tab >>Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses
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    2008 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 116, no 11, p. 985-994Article in journal (Refereed) Published
    Abstract [en]

    Development of transgenic edible plants, to be used as production, storage and delivery systems for recombinant vaccine antigens, is a promising strategy to obtain cost effective vaccines against infectious diseases, not the least for use in developing countries. Therefore, we used Agrobacterium tumefaciens-mediated gene transfer to introduce the p24 gag gene encoding the nucleocapsid protein from HIV-1 subtype C into the Arabidopsis thaliana plant genome. Eighteen plant lines were confirmed positive for the p24 gene by PCR, four of these lines showed an apparent homozygous phenotype when grown on selective medium and these lines also showed transcription of the p24 gene into its corresponding mRNA. The mRNA in all four cases generated the p24 protein in plants, as verified by western blot analysis. The plants were shown to contain between 0.2 µg and 0.5 µg p24 protein per g of fresh tissue. Analysis of the localisation of the p24 protein showed that stem tissue contained the largest amount of protein, more than twice as much as leaf tissue, whereas no p24 protein was detected in roots. By using Southern blotting, we found that 4, 2-3, 2 and 1 T-DNA insertion events took place in the four lines 1, 2, 7, and 10, respectively. The genetic insertions of line 1 were stable from the T1 to the T4 generation and gave rise to the p24 protein in all cases, as verified by western blotting. In mice fed with fresh transgenic A. thaliana (line 10), anti-gag IgG was obtained in serum after a booster injection with recombinant p37Gag. No immune response was observed after equal booster injection of untreated mice or mice fed with A. thaliana WT plants.

    Place, publisher, year, edition, pages
    Oxford: Blackwell, 2008
    National Category
    Medical and Health Sciences Immunology in the medical area
    Research subject
    Immunology
    Identifiers
    urn:nbn:se:oru:diva-4631 (URN)10.1111/j.1600-0463.2008.00900.x (DOI)
    Available from: 2008-10-14 Created: 2008-10-14 Last updated: 2018-01-13Bibliographically approved
    2. Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs
    Open this publication in new window or tab >>Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs
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    2009 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 66, no 1, p. 46-51Article in journal (Refereed) Published
    Abstract [en]

    An optimized gene expression construct was designed in order to increase the accumulation of the HIV-1 subtype C p24 protein in Arabidopsis thaliana and carrot (Daucus carota) plants. An ER retention signal was introduced into the genetic construct generating a p24 protein containing a SEKDEL amino acid sequence at its C-terminus. Mature A. thaliana plants and carrot cells were transformed using Agrobacterium tumefaciens carrying the improved pGreen0229/p24_SEKDEL vector. Several transgenic plant lines were obtained from both plant species by growth on selective medium and confirmed by PCR. Transformed lines were analyzed for p24 protein content by western blotting using anti-p24-specific antibodies and by Southern blotting to establish the number of copies of the insert in the plant nuclear genome. To estimate the accumulation levels of p24 protein in the plants, ELISA was run using soluble plant extracts. By comparing these results with our previous findings, the ER retention signal increased the level of p24 protein 5-fold in the Arabidopsis thaliana plants. In carrot taproot, the content of p24_SEKDEL protein was approximately half of that in Arabidopsis on a fresh weight basis and was stable in planta for several months. However, on a total soluble protein basis, carrots produced considerable higher levels of the p24_SEKDEL protein than Arabidopsis.

    Place, publisher, year, edition, pages
    St. Louis: Academic Press, 2009
    National Category
    Natural Sciences Chemical Sciences Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-5166 (URN)10.1016/j.pep.2008.12.015 (DOI)000265346000008 ()19167502 (PubMedID)2-s2.0-63649152546 (Scopus ID)
    Available from: 2009-11-05 Created: 2009-01-29 Last updated: 2017-12-14Bibliographically approved
    3. Plant-based production of HIV antigens: towards a candidate for an edible vaccine
    Open this publication in new window or tab >>Plant-based production of HIV antigens: towards a candidate for an edible vaccine
    2010 (English)In: In Vivo, ISSN 0258-851X, E-ISSN 1791-7549, Vol. 24, no 3, p. 368-370Article in journal, Meeting abstract (Refereed) Published
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-14224 (URN)
    Projects
    Ätbara vacciner
    Available from: 2012-08-06 Created: 2011-01-25 Last updated: 2018-04-23Bibliographically approved
    4. Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses
    Open this publication in new window or tab >>Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses
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    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4+ T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. Therefore, immediate protection at these surfaces is of high priority for the development of an HIV-1 vaccine. Thus, transgenic plants expressing HIV-1 p24 antigen, which is released by immune competent cells in the GALT during oral administration, are interesting as potential vaccine candidates. In the present study, we used two HIV-1 p24 transgenic plant systems, Arabidopsis thaliana and Daucus carota, in oral immunization experiments. Both transgenic plant systems showed a priming effect in mice and induced humoral immune responses, which could be detected as anti-p24-specific-IgG in sera after an intramuscular p24 protein boost. Initial dose-dependent antigen analyses using transgenic Arabidopsis thaliana indicated that low p24 antigen doses were superior to high p24 antigen doses. No detectable levels of faecal IgA antibodies or cellular immune responses were observed.

    National Category
    Chemical Sciences
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-17314 (URN)
    Available from: 2011-09-22 Created: 2011-09-22 Last updated: 2017-10-17Bibliographically approved
    5. A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate
    Open this publication in new window or tab >>A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate
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    2011 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 80, no 2, p. 194-202Article in journal (Refereed) Published
    Abstract [en]

    The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP – up to 3% of TSP.

    Place, publisher, year, edition, pages
    Academic Press, 2011
    National Category
    Natural Sciences Chemical Sciences
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-17313 (URN)10.1016/j.pep.2011.08.010 (DOI)000296225800005 ()21903168 (PubMedID)2-s2.0-80052854898 (Scopus ID)
    Projects
    Ätbara vacciner
    Available from: 2012-08-06 Created: 2011-09-22 Last updated: 2018-05-03Bibliographically approved
  • 9.
    Musa, Klefah Abrahem Klefah
    Örebro University, School of Science and Technology.
    Computational studies of photodynamic drugs, phototoxic reactions and drug design2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The most important criterion when designing new drugs or improving old ones in order to prevent side effects or at least diminish them is drug safety. Treatment of all diseases generally needs use of either topical application or systemic medications (transported in the blood) during a certain period of time. These treatments are associated with a number of adverse effects. Photosensitivity is one of those side effects, with phototoxicity as one of the photosensitivity disorders. This adverse side effect arises because of a reaction between UV or visible-light and the drug molecule, its active form or photoproduct(s). Due to phototoxic side effect, unexpected symptoms varying from just a simple rash to severe cutaneous affectations can appear. Furthermore, biomolecular damage occurs once the drug-light interaction takes place persistently and ends with cell death.

    Several drug families, such as over-the-counter drugs in the non-steroidal anti-inflammatory drug family of 2-arylpropionic acid derivatives, or prescription required fluoroquinolone drugs, have the capability to absorb mainly UV light radiation which in turn causes different phototoxic reactions by forming radical derivatives, reactive oxygen species or both. These may effect DNA, protein and lipid cell components leading to photogenotoxicity, photoallergy and lipid peroxidation, respectively. The photodegradation mechanisms of drugs belonging to the above mentioned families including ketoprofen, ibuprofen, flurbiprofen, naproxen, the active form of nabumetone, diclofenac and its main photoproduct, suprofen, tiaprofenic acid, naphazoline, norfloxacin and lomefloxacin are investigated in more detail in this thesis.

    The results obtained by computational density functional theory (DFT) and time-dependent-DFT (TD-DFT) are in line with experimental data available to date. The studies provide detailed insight into the molecular basis and understanding of the full photodegradation mechanisms of drugs mentioned above. This also plays an important role in preventing or at least reducing the phototoxic adverse effects by enabling the development of safe drugs in this area. Hence, new modified non-steroid anti-inflammatory molecules were designed by computational techniques. Obtained results suggest possibility of their future usage as pharmaceuticals with reduced photodegradation and cyclooxygenase 1 induced adverse side effects compared to the parent compounds.

    List of papers
    1. Mechanism of Photoinduced Decomposition of Ketoprofen
    Open this publication in new window or tab >>Mechanism of Photoinduced Decomposition of Ketoprofen
    2007 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 50, no 8, p. 1735-1743Article in journal (Refereed) Published
    Abstract [en]

    UV-induced decarboxylation of the NSAID ketoprofen, followed by activation of molecular oxygen or formation of a decarboxylated peroxide adduct, is explored using computational quantum chemistry. The excited energy surfaces reveal that the neutral species will not decarboxylate, whereas the deprotonated acid decarboxylates spontaneously in the triplet state, and with an associated 3-5 kcal/mol barrier from several low-lying excited singlet states. The observed long lifetimes of the decarboxylated anion is explained in terms of the high stability of the triplet benzoyl ethyl species with protonated carbonylic oxygen, from which there is no obvious decay channel. Mechanisms for the generation of singlet oxygen and superoxide are discussed in detail. Addition of molecular oxygen to give the corresponding peroxyl radical capable of initiating propagating lipid peroxidation reactions is also explored. The computed data explains all features of the observed experimental observations made to date on the photodegradation of ketoprofen.

    Place, publisher, year, edition, pages
    Washington, DC: American Chemical Society, 2007
    National Category
    Theoretical Chemistry Natural Sciences Chemical Sciences
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-4100 (URN)10.1021/jm060697k (DOI)
    Available from: 2007-11-01 Created: 2007-11-01 Last updated: 2017-12-14Bibliographically approved
    2. Theoretical Study of Ibuprofen Phototoxicity
    Open this publication in new window or tab >>Theoretical Study of Ibuprofen Phototoxicity
    2007 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 111, no 46, p. 13345-13352Article in journal (Refereed) Published
    Abstract [en]

    The photochemical properties and degradation of the common nonsteroid anti-inflammatory drug ibuprofen is studied by means of hybrid density functional theory. Computed energies and properties of various species show that the deprotonated form dominates at physiological pH, and that the species will not be able to decarboxylate from a singlet excited state. Instead, decarboxylation will occur, with very high efficiency, provided the deprotonated compound can undergo intersystem crossing from an excited singlet to its excited triplet state. In the triplet state, the C−C bond connecting the carboxyl group is elongated, and the CO2 moiety detaches with a free energy barrier of less than 0.5 kcal/mol. Depending on the local environment, the decarboxylated product can then either be quenched through intersystem crossing (involving the possible formation of singlet oxygen) and protonation, or serve as an efficient source for superoxide anions and the formation of a peroxyl radical that will initiate lipid peroxidation.

    Place, publisher, year, edition, pages
    Washington, DC: American Chemical Society, 2007
    Keywords
    ibuprofen, phototoxicity, degradation, decarboxylation, reactive oxygen species, TD-DFT
    National Category
    Theoretical Chemistry Chemical Sciences
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-4113 (URN)10.1021/jp076553e (DOI)17958415 (PubMedID)
    Available from: 2007-12-17 Created: 2007-12-17 Last updated: 2017-12-14Bibliographically approved
    3. Photochemical and photophysical properties, and photodegradation mechanism, of the non-steroid anti-inflammatory drug Flurbiprofen
    Open this publication in new window or tab >>Photochemical and photophysical properties, and photodegradation mechanism, of the non-steroid anti-inflammatory drug Flurbiprofen
    2009 (English)In: Journal of Photochemistry and Photobiology A: Chemistry, ISSN 1010-6030, E-ISSN 1873-2666, Vol. 202, no 1, p. 48-56Article in journal (Refereed) Published
    Abstract [en]

    The photodegradation mechanism of the widely used non-steroidal anti-inflammatory drug 2-(4-phenyl-3-fluorophenyl) propanoic acid, Flurbiprofen, and its photochemical and photophysical properties have been investigated by means of computational quantum chemistry at the DFT-B3LYP/6-31G(d,p) level. Comparison of computed and experimental singlet and triplet–triplet absorption spectra point to that most experiments, using a range of different solvents, are conducted on the neutral, protonated form of Flurbiprofen. The deprotonated acid, which should dominate at physiological pH, shows no sign of decarboxylation from the lowest singlet excited states, whereas from its first excited triplet state this should readily occur by passing over an energy barrier of <0.5 kcal/mol. Further reactions in the proposed photodegradation mechanism, after decarboxylation, as well as the probability for reactive oxygen species formation are discussed in detail. The generation of the corresponding peroxyl radical from the decarboxylated radical and molecular oxygen is strictly exergonic and occurs without barrier under aerobic conditions. The thus formed peroxyl radical will in turn be capable of initiating propagating lipid peroxidation processes.

    Place, publisher, year, edition, pages
    Amsterdam: Elsevier, 2009
    National Category
    Natural Sciences Chemical Sciences Environmental Sciences
    Research subject
    Environmental Chemistry
    Identifiers
    urn:nbn:se:oru:diva-6319 (URN)10.1016/j.jphotochem.2008.11.010 (DOI)000263500000007 ()2-s2.0-58249098792 (Scopus ID)
    Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2017-12-13Bibliographically approved
    4. Theoretical Study of the Phototoxicity of Naproxen and the Active Form of Nabumetone
    Open this publication in new window or tab >>Theoretical Study of the Phototoxicity of Naproxen and the Active Form of Nabumetone
    2008 (English)In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 112, no 43, p. 10921-10930Article in journal (Refereed) Published
    Place, publisher, year, edition, pages
    Washington, DC: American Chemical Society, 2008
    National Category
    Natural Sciences Chemical Sciences
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-6318 (URN)10.1021/jp805614y (DOI)
    Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2017-12-13Bibliographically approved
    5. Photodegradation mechanism of the common non-steroid anti-inflammatory drug diclofenac and its carbazole photoproduct
    Open this publication in new window or tab >>Photodegradation mechanism of the common non-steroid anti-inflammatory drug diclofenac and its carbazole photoproduct
    2009 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 11, no 22, p. 4601-4610Article in journal (Refereed) Published
    Abstract [en]

    Diclofenac (DF) is a widely used non-steroid anti-inflammatory drug, associated with a range of side effects. The phototoxicity of DF is studied herein employing computational quantum chemistry at the B3LYP/6-31G(d,p) level of theory. The results show that the drug readily absorbs radiation from the UV-region. The deprotonated form spontaneously dechlorinates from its triplet state leading to ring closure and formation of an active photoproduct: chlorocarbazole acetic acid, CCA. The formed CCA is also photodegraded easily from its deprotonated triplet state. Photodegradation routes of deprotonated CCA are decarboxylation (barrier less than 4.5 kcal mol(-1)) and dechlorination (barrier around 6.2 kcal mol(-1)). The energy barrier required for dechlorination to take place from the neutral from is about 20 kcal mol(-1). The differences between the molecular orbitals of the neutral and the deprotonated forms of DF and CCA and spectra obtained using time-dependent density-functional theory (TD-DFT), in addition to the different radical and oxygenated intermediate species formed during the photodegradation mechanism, are discussed in more detail. The theoretical results obtained herein are in line with the experimental results available to date.

    National Category
    Chemical Sciences
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-13296 (URN)10.1039/b900144a (DOI)000266587300022 ()
    Available from: 2011-01-14 Created: 2011-01-11 Last updated: 2017-12-11Bibliographically approved
    6. Photodegradation Mechanism of Nonsteroidal Anti-Inflammatory Drugs Containing Thiophene Moieties: Suprofen and Tiaprofenic Acid
    Open this publication in new window or tab >>Photodegradation Mechanism of Nonsteroidal Anti-Inflammatory Drugs Containing Thiophene Moieties: Suprofen and Tiaprofenic Acid
    2009 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 113, no 32, p. 11306-11313Article in journal (Refereed) Published
    Abstract [en]

    The photodegradation of nonsteroid anti-inflammatory drugs suprofen, 2-[4-(2-thienoyl)phenyl]propionic acid, and tiaprofenic acid, 2-(5-benzoyl-2-thienyl)propanoic acid, is studied by means of density functional theory. Besides the redox properties of the neutral species, we report on absorption spectra and degradation pathways involving excitation, intersystem crossing to the T-1 state. and spontaneous decarboxylation of the deprotonated species of each drug. The energetics and properties of the suprofen and tiaprofenic acid systems are found to be very similar to those of the highly photolabile benzyl analogue ketoprofen. Mechanisms leading to the formation of a closed-shell decarboxylated ethyl species, as well as peroxyl radicals capable of initiating lipid peroxidation reactions, are discussed.

    National Category
    Chemical Sciences
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-13254 (URN)10.1021/jp904171p (DOI)000268661100033 ()
    Available from: 2011-01-17 Created: 2011-01-11 Last updated: 2017-12-11Bibliographically approved
    7. Theoretical assessment of naphazoline redoxchemistry and photochemistry
    Open this publication in new window or tab >>Theoretical assessment of naphazoline redoxchemistry and photochemistry
    2007 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 111, no 15, p. 3977-3981Article in journal (Refereed) Published
    Abstract [en]

    The imidazoline derivative naphazoline (2-(1-naphtylmethyl)-2-imidazoline) is an α2-adrenergic agonist used as non-prescription eye and nasal preparations. Besides its functionality in generating vascoconstriction and decongestion in the patient, the toxicity, ROS generating capability, and recently also possible antioxidant capacity of the compound have been reported in the literature. In the current work the structural and electronic features of the drug are explored, using computational chemical tools. Electron affinities, ionization potentials, and excitation energies are reported, as well as charge and spin distributions of various forms of the drug. The difference in photochemical behavior between the protonated and unprotonated (basic) species is explained by the molecular orbital distributions, allowing for efficient excitation quenching in the basic structure but clear naphthalene to imidazolene charge transfer upon HOMO→ LUMO excitation in the protonated form, enabling larger intersystem crossing capability to the imidazole localized excited triplet and a resulting higher singlet oxygen quantum yield.

    Place, publisher, year, edition, pages
    Washington, DC: American Chemical Society, 2007
    Keywords
    Molecular Structure, Naphazoline, Oxidation-Reduction, Photochemistry, Density functional theory
    National Category
    Theoretical Chemistry Natural Sciences Chemical Sciences
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-4105 (URN)10.1021/jp070207f (DOI)17388561 (PubMedID)
    Available from: 2007-11-02 Created: 2007-11-02 Last updated: 2017-12-14Bibliographically approved
    8. Theoretical assessment of norfloxacin redox and photochemistry
    Open this publication in new window or tab >>Theoretical assessment of norfloxacin redox and photochemistry
    2009 (English)In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 113, no 40, p. 10803-10810Article in journal (Refereed) Published
    Abstract [en]

    Norfloxacin, 1-ethyl-6-fluoro-1,4-dihydo-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxyli c acid, NOR, is an antibiotic drug from the fluoroquinoline family. The different protonation states of this drug formed throughout the pH range is studied by means of density functional theory (DFT) and the spectra of the NOR species computed using time-dependent DFT. Details about their photochemistry are obtained from investigating the highest occupied and lowest unoccupied molecular orbitals. The predominant species under physiological pH, the zwitterion, is the most photoliable one, capable of producing singlet oxygen or/and superoxide radical anions from its triplet state. In addition, the main photodegradation step, defluorination, occurs more easily from this species compared with the other forms. The defluorination from the excited triplet state requires passing a barrier of 16.3 kcal/mol in the case of the zwitterion. The neutral and cationic forms display higher transition barriers, whereas the reaction path of defluorination is completely endothermic for the anionic species. The theoretical results obtained herein are in line with previous experimental data.

    National Category
    Chemical Sciences
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-13209 (URN)10.1021/jp904671s (DOI)000270362900026 ()
    Available from: 2011-01-17 Created: 2011-01-11 Last updated: 2017-12-11Bibliographically approved
    9. Photophysical and photochemical properties and photodehalogenation of antibiotic drug lomefloxacin
    Open this publication in new window or tab >>Photophysical and photochemical properties and photodehalogenation of antibiotic drug lomefloxacin
    (English)Manuscript (preprint) (Other academic)
    National Category
    Physical Chemistry
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-15445 (URN)
    Available from: 2011-05-02 Created: 2011-05-02 Last updated: 2017-10-17Bibliographically approved
    10. New non-steroidal anti-inflammatory molecules with reduced photodegradation side effects and enhanced COX-2 selectivity
    Open this publication in new window or tab >>New non-steroidal anti-inflammatory molecules with reduced photodegradation side effects and enhanced COX-2 selectivity
    (English)Manuscript (preprint) (Other academic)
    National Category
    Physical Chemistry
    Research subject
    Chemistry
    Identifiers
    urn:nbn:se:oru:diva-15446 (URN)
    Available from: 2011-05-02 Created: 2011-05-02 Last updated: 2017-10-17Bibliographically approved
  • 10.
    Pradhan, Ajay
    Örebro University, School of Science and Technology.
    Molecular mechanisms of zebrafish sex differentiation and sexual behavior2015Doctoral thesis, comprehensive summary (Other academic)
    List of papers
    1. Activation of NF-kappa B Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish
    Open this publication in new window or tab >>Activation of NF-kappa B Protein Prevents the Transition from Juvenile Ovary to Testis and Promotes Ovarian Development in Zebrafish
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    2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 45, p. 37926-37938Article in journal (Refereed) Published
    Abstract [en]

    Testis differentiation in zebrafish involves juvenile ovary to testis transformation initiated by an apoptotic wave. The molecular regulation of this transformation process is not fully understood. NF-kappa B is activated at an early stage of development and has been shown to interact with steroidogenic factor-1 in mammals, leading to the suppression of anti-Mullerian hormone (Amh) gene expression. Because steroidogenic factor-1 and Amh are important for proper testis development, NF-kappa B-mediated induction of anti-apoptotic genes could, therefore, also play a role in zebrafish gonad differentiation. The aim of this study was to examine the potential role of NF-kappa B in zebrafish gonad differentiation. Exposure of juvenile zebrafish to heat-killed Escherichia coli activated the NF-kappa B pathways and resulted in an increased ratio of females from 30 to 85%. Microarray and quantitative real-time-PCR analysis of gonads showed elevated expression of NF-kappa B-regulated genes. To confirm the involvement of NF-kappa B-induced anti-apoptotic effects, zebrafish were treated with sodium deoxycholate, a known inducer of NF-kappa B or NF-kappa B activation inhibitor (NAI). Sodium deoxycholate treatment mimicked the effect of heat-killed bacteria and resulted in an increased proportion of females from 25 to 45%, whereas the inhibition of NF-kappa B using NAI resulted in a decrease in females from 45 to 20%. This study provides proof for an essential role of NF-kappa B in gonadal differentiation of zebrafish and represents an important step toward the complete understanding of the complicated process of sex differentiation in this species and possibly other cyprinid teleosts as well.

    Place, publisher, year, edition, pages
    The American Society for Biochemistry and Molecular Biology, 2012
    National Category
    Developmental Biology
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-32903 (URN)10.1074/jbc.M112.386284 (DOI)000310642200028 ()2-s2.0-84868315208 (Scopus ID)
    Funder
    Swedish Research CouncilKnowledge Foundation
    Note

    Funding Agency: Orebro University; Agri-Food and Veterinary Authority; Temasek Life Sciences Laboratory, Singapore (se även Forskningsfinansiärer)

    Available from: 2014-01-02 Created: 2014-01-02 Last updated: 2018-11-29Bibliographically approved
    2. Juvenile Ovary to Testis Transition in Zebrafish Involves Inhibition of Ptges
    Open this publication in new window or tab >>Juvenile Ovary to Testis Transition in Zebrafish Involves Inhibition of Ptges
    2014 (English)In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 91, no 2, p. 1-15Article in journal (Refereed) Published
    Abstract [en]

    The sex differentiation mechanisms in zebrafish (Danio rerio) remains elusive, partly due to the absence of sex chromosomes but also the process appears to depend on the synchrony of multiple genes and possibly environmental factors. Zebrafish gonadal development is initiated through the development of immature oocytes. Depending on multiple signaling cues, in about half of the individuals, the juvenile ovaries degenerate or undergo apoptosis to initiate testes development while the other half maintains the oogenic pathway. We have previously shown that activation of NFkappaB and prostaglandin synthase 2 (ptgs2) results in female biased sex ratios. Prostaglandin synthase and prostaglandins are involved in multiple physiological functions including cell survival and apoptosis. In the present study we show that inhibition of ptgs2 by meloxicam result in male biased sex ratios. On further evaluation, we observed that exposure with the prostaglandin D2 (PGD2) analogue BW-245C induced SRY-box containing gene 9a (sox9a) and resulted in male biased sex ratios. On the other hand, prostaglandin E2 (PGE2) treatment resulted in female biased sex ratios and involved activation of NFkappaB and the beta-catenin pathway as well as inhibition of sox9. Exposure to the beta-catenin inhibitor, PNU-74654, resulted in up-regulation of ptgds and male biased sex ratios which further confirmed the involvement of beta-catenin in the female differentiation pathway. In this study we show that PGD2 and PGE2 can program the gonads to either the testis or ovary differentiation pathways, indicating that prostaglandins are involved in the regulation of zebrafish gonadal differentiation.

    Keywords
    fish reproduction; gene expression; prostaglandins; sex determination; sex differentiation
    National Category
    Developmental Biology
    Research subject
    Molecular Biology
    Identifiers
    urn:nbn:se:oru:diva-35473 (URN)10.1095/biolreprod.114.119016 (DOI)000341300400009 ()24920039 (PubMedID)
    Funder
    Knowledge Foundation
    Note

    Funding Agency:

    Örebro University

    Available from: 2014-06-24 Created: 2014-06-24 Last updated: 2017-12-05Bibliographically approved
    3. Inhibition of Retinoic Acid Synthesis Disrupts Spermatogenesis and Fecundity in Zebrafish
    Open this publication in new window or tab >>Inhibition of Retinoic Acid Synthesis Disrupts Spermatogenesis and Fecundity in Zebrafish
    (English)Manuscript (preprint) (Other academic)
    National Category
    Developmental Biology
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-43321 (URN)
    Available from: 2015-03-05 Created: 2015-03-05 Last updated: 2017-10-17Bibliographically approved
    4. Zebrafish Sexual Behavior: role of sex steroid hormones and prostaglandins
    Open this publication in new window or tab >>Zebrafish Sexual Behavior: role of sex steroid hormones and prostaglandins
    (English)Manuscript (preprint) (Other academic)
    National Category
    Developmental Biology
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-43322 (URN)
    Available from: 2015-03-05 Created: 2015-03-05 Last updated: 2017-10-17Bibliographically approved
  • 11.
    Rahman, Aminur
    Örebro University, School of Science and Technology.
    Bioremediation of Toxic Metals for Protecting Human Health and the Ecosystem2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Heavy metal pollutants, discharged into the ecosystem as waste by anthropogenic activities, contaminate drinking water for millions of people and animals in many regions of the world. Long term exposure to these metals, leads to several lethal diseases like cancer, keratosis, gangrene, diabetes, cardio- vascular disorders, etc. Therefore, removal of these pollutants from soil, water and environment is of great importance for human welfare. One of the possible eco-friendly solutions to this problem is the use of microorganisms that can accumulate the heavy metals from the contaminated sources, hence reducing the pollutant contents to a safe level.

    In this thesis an arsenic resistant bacterium Lysinibacillus sphaericus B1-CDA, a chromium resistant bacterium Enterobacter cloacae B2-DHA and a nickel resistant bacterium Lysinibacillus sp. BA2 were isolated and studied. The minimum inhibitory concentration values of these isolates are 500 mM sodium arsenate, 5.5 mM potassium chromate and 9 mM nickel chloride, respectively. The time of flight-secondary ion mass spectrometry and inductively coupled plasma-mass spectroscopy analyses revealed that after 120 h of exposure, the intracellular accumulation of arsenic in B1-CDA and chromium in B2-DHA were 5.0 mg/g dwt and 320 μg/g dwt of cell biomass, respectively. However, the arsenic and chromium contents in the liquid medium were reduced to 50% and 81%, respectively. The adsorption values of BA2 when exposed to nickel for 6 h were 238.04 mg of Ni(II) per gram of dead biomass indicating BA2 can reduce nickel content in the solution to 53.89%. Scanning electron micrograph depicted the effect of these metals on cellular morphology of the isolates. The genetic composition of B1-CDA and B2-DHA were studied in detail by sequencing of whole genomes. All genes of B1-CDA and B2-DHA predicted to be associated with resistance to heavy metals were annotated.

    The findings in this study accentuate the significance of these bacteria in removing toxic metals from the contaminated sources. The genetic mechanisms of these isolates in absorbing and thus removing toxic metals could be used as vehicles to cope with metal toxicity of the contaminated effluents discharged to the nature by industries and other human activities.

    List of papers
    1. Isolation and characterization of a Lysinibacillus strain B1-CDA showing potential for bioremediation of arsenics from contaminated water
    Open this publication in new window or tab >>Isolation and characterization of a Lysinibacillus strain B1-CDA showing potential for bioremediation of arsenics from contaminated water
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    2014 (English)In: Journal of Environmental Science and Health. Part A: Toxic/Hazardous Substances and Environmental Engineering, ISSN 1093-4529, E-ISSN 1532-4117, Vol. 49, no 12, p. 1349-1360Article in journal (Refereed) Published
    Abstract [en]

    The main objective of this study was to identify and isolate arsenic resistant bacteria that can be used for removing arsenic from the contaminated environment. Here we report a soil borne bacterium, B1-CDA that can serve this purpose. B1-CDA was isolated from the soil of a cultivated land in Chuadanga district located in the southwest region of Bangladesh. The morphological, biochemical and 16S rRNA analysis suggested that the isolate belongs to Lysinibacillus sphaericus. The minimum inhibitory concentration (MIC) value of the isolate is 500mM (As) as arsenate. TOF-SIMS and ICP-MS analysis confirmed intracellular accumulation and removal of arsenics. Arsenic accumulation in cells amounted to 5.0mg g(-1) of the cells dry biomass and thus reduced the arsenic concentration in the contaminated liquid medium by as much as 50%. These results indicate that B1-CDA has the potential for remediation of arsenic from the contaminated water. We believe the benefits of implementing this bacterium to efficiently reduce arsenic exposure will not only help to remove one aspect of human arsenic poisoning but will also benefit livestock and native animal species. Therefore, the outcome of this research will be highly significant for people in the affected area and also for human populations in other countries that have credible health concerns as a consequence of arsenic-contaminated water.

    Keywords
    Pollution, toxic metals, arsenics, bioremediation, bacteria, bioaccumulation
    National Category
    Environmental Sciences
    Research subject
    Enviromental Science
    Identifiers
    urn:nbn:se:oru:diva-36537 (URN)10.1080/10934529.2014.928247 (DOI)000340370000002 ()25072766 (PubMedID)2-s2.0-84905275614 (Scopus ID)
    Funder
    Sida - Swedish International Development Cooperation AgencySwedish Research Council Formas
    Note

    Funding Agency:

    Nilsson-Ehle (The Royal Physiographic Society in Lund) foundation in Sweden

    Available from: 2014-09-16 Created: 2014-09-15 Last updated: 2018-12-07Bibliographically approved
    2. Bioremediation of hexavalent chromium (VI) by a soil-borne bacterium, Enterobacter cloacae B2-DHA
    Open this publication in new window or tab >>Bioremediation of hexavalent chromium (VI) by a soil-borne bacterium, Enterobacter cloacae B2-DHA
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    2015 (English)In: Journal of Environmental Science and Health. Part A: Toxic/Hazardous Substances and Environmental Engineering, ISSN 1093-4529, E-ISSN 1532-4117, Vol. 50, no 11, p. 1136-1147Article in journal (Refereed) Published
    Abstract [en]

    Chromium and chromium containing compounds are discharged into the nature as waste from anthropogenic activities, such as industries, agriculture, forest farming, mining and metallurgy. Continued disposal of these compounds to the environment leads to development of various lethal diseases in both humans and animals. In this paper, we report a soil borne bacterium, B2-DHA that can be used as a vehicle to effectively remove chromium from the contaminated sources. B2-DHA is resistant to chromium with a MIC value of 1000 mu g mL(-1) potassium chromate. The bacterium has been identified as a Gram negative, Enterobacter cloacae based on biochemical characteristics and 16S rRNA gene analysis. TOF-SIMS and ICP-MS analyses confirmed intracellular accumulation of chromium and thus its removal from the contaminated liquid medium. Chromium accumulation in cells was 320 mu g/g of cells dry biomass after 120-h exposure, and thus it reduced the chromium concentration in the liquid medium by as much as 81%. Environmental scanning electron micrograph revealed the effect of metals on cellular morphology of the isolates. Altogether, our results indicate that B2-DHA has the potential to reduce chromium significantly to safe levels from the contaminated environments and suggest the potential use of this bacterium in reducing human exposure to chromium, hence avoiding poisoning.

    Keywords
    Bioremediation, chromium, Enterobacter cloacae, human health, soil borne bacterium, tannery effluents
    National Category
    Environmental Sciences
    Research subject
    Enviromental Science
    Identifiers
    urn:nbn:se:oru:diva-45753 (URN)10.1080/10934529.2015.1047670 (DOI)000359339900006 ()26191988 (PubMedID)2-s2.0-84937800926 (Scopus ID)
    Funder
    Sida - Swedish International Development Cooperation Agency, AKT-2010-018Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, 229-2007-217
    Note

    Funding Agency:

    Nilsson-Ehle (The Royal Physiographic Society in Lund) Foundation in Sweden

    Available from: 2015-09-09 Created: 2015-09-09 Last updated: 2018-12-07Bibliographically approved
    3. Biosorption of nickel by Lysinibacillus sp BA2 native to bauxite mine
    Open this publication in new window or tab >>Biosorption of nickel by Lysinibacillus sp BA2 native to bauxite mine
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    2014 (English)In: Ecotoxicology and Environmental Safety, ISSN 0147-6513, E-ISSN 1090-2414, Vol. 107, p. 260-268Article in journal (Refereed) Published
    Abstract [en]

    The current scenario of environmental pollution urges the need for an effective solution for toxic heavy metal removal from industrial wastewater. Bioremediation is the most cost effective process employed by the use of microbes especially bacteria resistant to toxic metals. In this study, Lysinibacillus sp. BA2, a nickel tolerant strain isolated from bauxite mine was used for the biosorption of Ni(II). Lysinibacillus sp. BA2 biomass had isoelectric point (pI) of 3.3. The maximum negative zeta potential value (-39.45) was obtained at pH 6.0 which was highly favourable for Ni(II) biosorption. 238.04 mg of Ni(II) adsorbed on one gram of dead biomass and 196.32 mg adsorbed on one gram of live biomass. The adsorption of Ni(II) on biomass increased with time and attained saturation after 180 mm with rapid biosorption in initial 30 min. The Langmuir and Freundlich isotherms could fit well for biosorption of Ni(II) by dead biomass while Langmuir isotherm provided a better fit for live biomass based on correlation coefficient values. The kinetic studies of Ni(II) removal, using dead and live biomass was well explained by second-order kinetic model. Ni(II) adsorption on live biomass was confirrned by SEM-EDX where cell aggregation and increasing irregularity of cell morphology was observed even though cells were in non-growing state. The FTIR analysis of biomass revealed the presence of carboxyl, hydroxyl and amino groups, which seem responsible for biosorption of Ni(II). The beads made using dead biomass of Lysinibacillus sp. BA2 could efficiently remove Ni(II) from effluent solutions. These microbial cells can substitute expensive methods for treating nickel contaminated industrial wastewaters.

    Place, publisher, year, edition, pages
    Elsevier, 2014
    Keywords
    Lysinibacillus sp BA2, Heavy metals, Biosorption, Adsorption isotherm
    National Category
    Other Biological Topics
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-51857 (URN)10.1016/j.ecoenv.2014.06.009 (DOI)000342122000036 ()25011123 (PubMedID)2-s2.0-84903900011 (Scopus ID)
    Available from: 2016-08-29 Created: 2016-08-29 Last updated: 2018-12-07Bibliographically approved
    4. Comparative genome analysis of Lysinibacillus B1-CDA, a bacterium that accumulates arsenics
    Open this publication in new window or tab >>Comparative genome analysis of Lysinibacillus B1-CDA, a bacterium that accumulates arsenics
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    2015 (English)In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 106, no 6, p. 384-392Article in journal (Refereed) Published
    Abstract [en]

    Previously, we reported an arsenic resistant bacterium Lysinibacillus sphaericus B1-CDA, isolated from an arsenic contaminated lands. Here, we have investigated its genetic composition and evolutionary history by using massively parallel sequencing and comparative analysis with other known Lysinibacillus genomes. Assembly of the sequencing reads revealed a genome of similar to 4.5 Mb in size encompassing similar to 80% of the chromosomal DNA. We found that the set of ordered contigs contains abundant regions of similarity with other Lysinibacillus genomes and clearly identifiable genome rearrangements. Furthermore, all genes of B1-CDA that were predicted be involved in its resistance to arsenic and/or other heavy metals were annotated. The presence of arsenic responsive genes was verified by PCR in vitro conditions. The findings of this study highlight the significance of this bacterium in removing arsenics and other toxic metals from the contaminated sources. The genetic mechanisms of the isolate could be used to cope with arsenic toxicity.

    Place, publisher, year, edition, pages
    Academic Press, 2015
    Keywords
    Toxic metals, Bioremediation, Lysinibacillus sphaericus B1-CDA, Genome sequencing, de novo assembly, Gene prediction
    National Category
    Environmental Sciences Environmental Biotechnology
    Research subject
    Enviromental Science
    Identifiers
    urn:nbn:se:oru:diva-47292 (URN)10.1016/j.ygeno.2015.09.006 (DOI)000365613100010 ()26387925 (PubMedID)2-s2.0-84948102629 (Scopus ID)
    Note

    Funding Agencies:

    Swedish International Development Cooperation Agency (SIDA) AKT-2010-018

    Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) 229-2007-217

    Nilsson-Ehle (The Royal Physio-graphic Society in Lund) foundation in Sweden

    Available from: 2016-01-05 Created: 2016-01-04 Last updated: 2018-12-07Bibliographically approved
    5. Genome analysis of Enterobacter cloacae B2-DHA: A bacterium resistant to chromium and/or other heavy metals
    Open this publication in new window or tab >>Genome analysis of Enterobacter cloacae B2-DHA: A bacterium resistant to chromium and/or other heavy metals
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Other Biological Topics
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-51858 (URN)
    Available from: 2016-08-31 Created: 2016-08-29 Last updated: 2018-12-06Bibliographically approved