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  • 1.
    Agarwal, Prasoon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Regulation of Gene Expression in Multiple Myeloma Cells and Normal Fibroblasts: Integrative Bioinformatic and Experimental Approaches2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The work presented in this thesis applies integrative genomic and experimental approaches to investigate mechanisms involved in regulation of gene expression in the context of disease and normal cell biology.

    In papers I and II, we have explored the role of epigenetic regulation of gene expression in multiple myeloma (MM). By using a bioinformatic approach we identified the Polycomb repressive complex 2 (PRC2) to be a common denominator for the underexpressed gene signature in MM. By using inhibitors of the PRC2 we showed an activation of the genes silenced by H3K27me3 and a reduction in the tumor load and increased overall survival in the in vivo 5TMM model. Using ChIP-sequencing we defined the distribution of H3K27me3 and H3K4me3 marks in MM patients cells. In an integrated bioinformatic approach, the H3K27me3-associated genes significantly correlated to under-expression in patients with less favorable survival. Thus, our data indicates the presence of a common under-expressed gene profile and provides a rationale for implementing new therapies focusing on epigenetic alterations in MM.

    In paper III we address the existence of a small cell population in MM presenting with differential tumorigenic properties in the 5T33MM murine model. We report that the predominant population of CD138+ cells had higher engraftment potential, higher clonogenic growth, whereas the CD138- MM cells presented with less mature phenotype and higher drug resistance. Our findings suggest that while designing treatment regimes for MM, both the cellpopulations must be targeted.

    In paper IV we have studied the general mechanism of differential gene expression regulation by CGGBP1 in response to growth signals in normal human fibroblasts. We found that CGGBP1 binding affects global gene expression by RNA Polymerase II. This is mediated by Alu RNAdependentinhibition of RNA Polymerase II. In presence of growth signals CGGBP1 is retained in the nuclei and exhibits enhanced Alu binding thus inhibiting RNA Polymerase III binding on Alus. Hence we suggest a mechanism by which CGGBP1 orchestrates Alu RNA-mediated regulation of RNA Polymerase II. This thesis provides new insights for using integrative bioinformatic approaches to decipher gene expression regulation mechanisms in MM and in normal cells.

    List of papers
    1. Polycomb target genes are silenced in multiple myeloma
    Open this publication in new window or tab >>Polycomb target genes are silenced in multiple myeloma
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    2010 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 7, p. e11483-Article in journal (Refereed) Published
    Abstract [en]

    Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb-targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies.

    National Category
    Hematology
    Identifiers
    urn:nbn:se:uu:diva-133207 (URN)10.1371/journal.pone.0011483 (DOI)000279715300003 ()20634887 (PubMedID)
    Available from: 2010-11-03 Created: 2010-11-03 Last updated: 2017-12-12Bibliographically approved
    2. The epigenomic map of multiple myeloma reveals the importance of Polycomb gene silencing for the malignancy
    Open this publication in new window or tab >>The epigenomic map of multiple myeloma reveals the importance of Polycomb gene silencing for the malignancy
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Multiple myeloma (MM) is characterized by accumulation of post-germinal center, isotype switched, long-living plasma cells with retained proliferation capacity within the bone marrow. MM is highly heterogeneous and remains fatal. This heterogeneity has hampered identification of a common underlying mechanism for disease establishment and the development of targeted therapy. We recently provided proof-of-principle that gene silencing associated with H3K27me3 contributes to the malignancy of MM. Here we present the first epigenomic map of MM for H3K27me3 and H3K4me3 derived by ChIP- and RNA sequencing from freshly-isolated bone marrow plasma cells from four patients. We compile lists of targets common among the patients as well as unique to MM when compared with PBMCs. Indicating the clinical relevance of our findings, we find increased silencing of H3K27me3 targets with disease progression and in patients presenting with a poor prognosis. Bivalent genes further significantly correlated to under-expressed genes in MM and were unique to MM when compared to PBMCs. Furthermore, bivalent genes, unlike H3K27me3 targets, significantly associated with transcriptional activation upon Polycomb inhibition indicating a potential for drug targeting. Thus, we suggest that gene silencing by Polycomb plays an important role in the development of the malignant phenotype of the MM cell during tumor progression.

    National Category
    Cell and Molecular Biology
    Research subject
    Oncology
    Identifiers
    urn:nbn:se:uu:diva-199492 (URN)
    Available from: 2013-05-06 Created: 2013-05-06 Last updated: 2018-01-11Bibliographically approved
    3. Tumor-initiating capacity of CD138- and CD138+ tumor cells in the 5T33 multiple myeloma model
    Open this publication in new window or tab >>Tumor-initiating capacity of CD138- and CD138+ tumor cells in the 5T33 multiple myeloma model
    Show others...
    2012 (English)In: Leukemia, ISSN 0887-6924, E-ISSN 1476-5551, Vol. 26, no 6, p. 1436-1439Article in journal, Letter (Refereed) Published
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-177948 (URN)10.1038/leu.2011.373 (DOI)000305081000040 ()22289925 (PubMedID)
    Available from: 2012-07-25 Created: 2012-07-20 Last updated: 2017-12-07Bibliographically approved
    4. Growth signals employ CGGBP1 to suppress transcription of Alu-SINEs
    Open this publication in new window or tab >>Growth signals employ CGGBP1 to suppress transcription of Alu-SINEs
    Show others...
    2016 (English)In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 15, no 12, p. 1558-1571Article in journal (Refereed) Published
    Abstract [en]

    CGGBP1 (CGG triplet repeat-binding protein 1) regulates cell proliferation, stress response,cytokinesis, telomeric integrity and transcription. It could affect these processes by modulatingtarget gene expression under different conditions. Identification of CGGBP1-target genes andtheir regulation could reveal how a transcription regulator affects such diverse cellular processes.Here we describe the mechanisms of differential gene expression regulation by CGGBP1 inquiescent or growing cells. By studying global gene expression patterns and genome-wide DNAbindingpatterns of CGGBP1, we show that a possible mechanism through which it affects theexpression of RNA Pol II-transcribed genes in trans depends on Alu RNA. We also show that itregulates Alu transcription in cis by binding to Alu promoter. Our results also indicate thatpotential phosphorylation of CGGBP1 upon growth stimulation facilitates its nuclear retention,Alu-binding and dislodging of RNA Pol III therefrom. These findings provide insights into howAlu transcription is regulated in response to growth signals.

    Keywords
    Alu-SINEs; CGGBP1; ChIP-seq; growth signals; RNA Pol III; transcription; tyrosine phosphorylation
    National Category
    Cell Biology
    Research subject
    Bioinformatics; Biology
    Identifiers
    urn:nbn:se:uu:diva-230959 (URN)10.4161/15384101.2014.967094 (DOI)000379743800011 ()25483050 (PubMedID)
    Funder
    Swedish Cancer SocietySwedish Research Council
    Available from: 2014-09-01 Created: 2014-09-01 Last updated: 2017-12-05Bibliographically approved
  • 2.
    Agarwal, Prasoon
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Kalushkova, Antonia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Enroth, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics.
    Österborg, Anders
    Department of Hematology, Karolinska University Hospital Solna.
    Nilsson, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Öberg, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Jernberg Wiklund, Helena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    The epigenomic map of multiple myeloma reveals the importance of Polycomb gene silencing for the malignancyManuscript (preprint) (Other academic)
    Abstract [en]

    Multiple myeloma (MM) is characterized by accumulation of post-germinal center, isotype switched, long-living plasma cells with retained proliferation capacity within the bone marrow. MM is highly heterogeneous and remains fatal. This heterogeneity has hampered identification of a common underlying mechanism for disease establishment and the development of targeted therapy. We recently provided proof-of-principle that gene silencing associated with H3K27me3 contributes to the malignancy of MM. Here we present the first epigenomic map of MM for H3K27me3 and H3K4me3 derived by ChIP- and RNA sequencing from freshly-isolated bone marrow plasma cells from four patients. We compile lists of targets common among the patients as well as unique to MM when compared with PBMCs. Indicating the clinical relevance of our findings, we find increased silencing of H3K27me3 targets with disease progression and in patients presenting with a poor prognosis. Bivalent genes further significantly correlated to under-expressed genes in MM and were unique to MM when compared to PBMCs. Furthermore, bivalent genes, unlike H3K27me3 targets, significantly associated with transcriptional activation upon Polycomb inhibition indicating a potential for drug targeting. Thus, we suggest that gene silencing by Polycomb plays an important role in the development of the malignant phenotype of the MM cell during tumor progression.

  • 3.
    Agnarsdóttir, Margrét
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Biomarker Discovery in Cutaneous Malignant Melanoma: A Study Based on Tissue Microarrays and Immunohistochemistry2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The incidence of cutaneous malignant melanoma has increased dramatically in Caucasians the last few decades, an increase that is partly explained by altered sun exposure habits. For the individual patient, with a localized disease, the tumor thickness of the excised lesion is the most important prognostic factor. However, there is a need to identify characteristics that can place patients in certain risk groups.

    In this study, the protein expression of multiple proteins in malignant melanoma tumors was studied, with the aim of identifying potential new candidate biomarkers. Representative samples from melanoma tissues were assembled in a tissue microarray format and protein expression was detected using immunohistochemistry. Multiple cohorts were used and for a subset of proteins the expression was also analyzed in melanocytes in normal skin and in benign nevi. The immunohistochemical staining was evaluated manually and for part of the proteins also with an automated algorithm.

    The protein expression of STX7 was described for the first time in tumors of the melanocytic lineage. Stronger expression of STX7 and SOX10 was seen in superficial spreading melanomas compared with nodular malignant melanomas. An inverse relationship between STX7 expression and T-stage was seen and between SOX10 expression and T-stage and Ki-67, respectively. In a population-based cohort the expression of MITF was analyzed and found to be associated with prognosis. Twenty-one potential biomarkers were analyzed using bioinformatics tools and a protein signature was identified which had a prognostic value independent of T-stage. The protein driving this signature was RBM3, a protein not previously described in malignant melanoma. Other markers included in the signature were MITF, SOX10 and Ki-67.

    In conclusion, the protein expression of numerous potential biomarkers was extensively studied and a new prognostic protein panel was identified which can be of value for risk stratification.

    List of papers
    1. Selective expression of Syntaxin-7 protein in benign melanocytes and malignant melanoma
    Open this publication in new window or tab >>Selective expression of Syntaxin-7 protein in benign melanocytes and malignant melanoma
    Show others...
    2009 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 4, p. 1639-1646Article in journal (Refereed) Published
    Abstract [en]

    To search for proteins expressed in human melanocytes and melanoma, we employed an antibody-based proteomics strategy to screen for protein expression in tissue microarrays containing normal tissues, cancer tissues and cell lines. Syntaxin-7 (STX7) was identified as a novel protein, not previously characterized in cells of melanocytic lineage, displaying a cell type-specific protein expression pattern. In tumor tissues, STX7 was expressed in malignant melanoma and lymphoma. The protein was further characterized regarding subcellular localization, specificity, tissue distribution pattern and potential as a diagnostic and prognostic marker using cell lines and tissue microarrays containing normal skin, melanocytic nevi and primary and metastatic melanoma. STX7 was expressed in normal melanocytes, various benign melanocytic nevi, atypical nevi and malignant melanoma. Analysis in two independent melanoma cohorts demonstrated STX7 expression in nearly all investigated tumors, although at varying levels (>90% positive tumors). The expression level of STX7 protein was inversely correlated to tumor stage, suggesting that decreased expression of STX7 is associated with more aggressive tumors. In conclusion, we present protein profiling data for a novel protein showing high sensitivity and specificity for cells of the melanocytic lineage. The presented antibody-based proteomics approach can be used as an effective strategy to identify novel tumor markers and evaluate their potential clinical relevance.

    Keywords
    malignant melanoma, melanocytes, antibody proteomics, tissue microarray, Syntaxin-7
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-105503 (URN)10.1021/pr800745e (DOI)000264928200004 ()19173671 (PubMedID)
    Available from: 2009-06-04 Created: 2009-06-04 Last updated: 2017-12-13Bibliographically approved
    2. SOX10 expression in superficial spreading and nodular malignant melanomas
    Open this publication in new window or tab >>SOX10 expression in superficial spreading and nodular malignant melanomas
    Show others...
    2010 (English)In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 20, no 6, p. 468-478Article in journal (Refereed) Published
    Abstract [en]

    SOX10 is a transcription factor expressed in nerve cells and melanocytes. The aim of this study was to investigate the protein expression pattern of SOX10 in malignant melanoma tumors and to analyze whether the results correlated with clinical parameters and the proliferation marker Ki-67. Furthermore, proliferation and migration were analyzed in three different cell lines employing SOX10 small interfering RNA-mediated silencing. Expression patterns were determined in 106 primary tumors and 39 metastases in addition to 16 normal skin samples and six benign nevi employing immunohistochemistry and tissue microarrays. The immunohistochemical staining was evaluated manually and with an automated algorithm. SOX10 was strongly expressed in the benign tissues, but for the malignant tumors superficial spreading melanomas stained stronger than nodular malignant melanomas (P=0.008). The staining intensity was also inversely correlated with T-stage (Spearman's ρ=-0.261, P=0.008). Overall survival and time to recurrence were significantly correlated with SOX10 intensity, but not in multivariate analysis including T-stage. With the automated algorithm there was an inverse correlation between the SOX10 staining intensity and the proliferation marker, Ki-67 (ρ=-0.173, P=0.02) and a significant difference in the intensity signal between the benign tissues, the primary tumors and the metastases where the metastases stained the weakest (P≤0.001). SOX10 downregulation resulted in variable effects on proliferation and migration rates in the melanoma cell lines. In conclusion, the SOX10 intensity level differed depending on the tissue studied and SOX10 might have a role in survival. No conclusion regarding the role of SOX10 for in-vitro proliferation and migration could be drawn.

    Keywords
    Immunohistochemistry, malignant melanoma, small interfering RNA, SOX10
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-133423 (URN)10.1097/CMR.0b013e3283403ccd (DOI)000283744100005 ()20890226 (PubMedID)
    Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2017-12-12Bibliographically approved
    3. MITF as a Prognostic Marker in Cutaneous Malignant Melanoma
    Open this publication in new window or tab >>MITF as a Prognostic Marker in Cutaneous Malignant Melanoma
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Background: Microphthalmia associated transcription factor (MITF) protein has a central role in the differentiation and survival of melanocytes. The aim of the study was to investigate whether MITF can be employed as a prognostic marker in patients operated on for cutaneous malignant melanoma.

    Methods: A cohort study design based on information collected from population-based registers. For included patients tissue microarrays and immunohistochemistry were employed to study the protein expression of MITF in the primary malignant melanoma tumors by estimating the fraction of positive tumor cells and the staining intensity.

    Results: The vast majority of tumors expressed MITF in >25% of the tumor cells with a strong staining intensity and looking at these factors individually these patients had a better prognosis. When cell fraction and intensity were combined a high-risk group dying of malignant melanoma was identified as those with 25% -75% of tumor cells staining with weak intensity and those with <25% of tumor cells staining with strong intensity. However, the majority of the deaths occurred in the lower risk groups.

    Conclusions: Although a high-risk group for death in malignant melanoma was identified we conclude that MITF is not useful as a prognostic marker because of the distribution of that particular expression in the population.

    Impact: Our results indicate a bi-phasic pattern of MITF expression and although not useful as a prognostic marker these results are in line with other experimental studies and are relevant to explore further.

     

    Keywords
    MITF, prognosis, survival, immunohistochemistry, tissue microarray
    National Category
    Cell and Molecular Biology
    Research subject
    Cancer Epidemiology; Pathology
    Identifiers
    urn:nbn:se:uu:diva-143436 (URN)
    Available from: 2011-02-16 Created: 2011-01-20 Last updated: 2018-01-12
    4. Protein Biomarkers in Malignant Melanoma: An Image Analysis-Based Study on Melanoma Markers of Potential Clinical Relevance
    Open this publication in new window or tab >>Protein Biomarkers in Malignant Melanoma: An Image Analysis-Based Study on Melanoma Markers of Potential Clinical Relevance
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The thickness of a primary malignant melanoma tumor is the most important prognostic indicator for a patient with primary cutaneous malignant melanoma. To optimize the management and treatment of melanoma patients there is an unmet need to identify characteristics that can further stratify melanoma patients into high or low risk for progressive disease. Despite numerous studies no single marker has yet been shown to add significant prognostic information. An algorithmic approach, combining data from several markers provides an attractive model to identify patients of increased risk of dying from malignant melanoma. The primary aim of the present study was to analyze the correlation between clinical outcome and protein expression patterns of multiple proteins in malignant melanoma tumors using immunohistochemistry and tissue microarrays. Candidate proteins were identified based on a selective and differential expression pattern in melanoma tumors and tested in a cohort of 143 melanoma patients. Protein expression was analyzed using both manual scoring and automated image analysis-based algorithms. We found no single marker of prognosis that was independent of tumor thickness. When combining potential prognostic markers we could define a prognostic index, based on RBM3, MITF, SOX10 and Ki-67, that was independent of tumor thickness in multivariate analysis. Our findings suggest that a good prognosis signature can be identified in melanoma patients with tumors showing a low fraction of Ki-67 positive tumor cells and a high fraction of RBM3 positive tumor cells combined with low intensity levels of SOX10 and MITF.

     

    Keywords
    malignant melanoma, immunohistochemistry, tissue microarray, protein expression, automated analysis, RBM3, SOX10, MITF, Ki-67
    National Category
    Cell and Molecular Biology
    Research subject
    Pathology; Bioinformatics
    Identifiers
    urn:nbn:se:uu:diva-144108 (URN)
    Available from: 2011-02-16 Created: 2011-01-27 Last updated: 2018-01-12
  • 4.
    Agnarsdóttir, Margrét
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Ponten, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Garmo, Hans
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Wagenius, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Mucci, Lorelei
    Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA, Department of Epidemiology, Harvard School of Public Health, Boston, USA .
    Magnusson, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Holmberg, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Eaker-Fält, Sonja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    MITF as a Prognostic Marker in Cutaneous Malignant MelanomaManuscript (preprint) (Other academic)
    Abstract [en]

    Background: Microphthalmia associated transcription factor (MITF) protein has a central role in the differentiation and survival of melanocytes. The aim of the study was to investigate whether MITF can be employed as a prognostic marker in patients operated on for cutaneous malignant melanoma.

    Methods: A cohort study design based on information collected from population-based registers. For included patients tissue microarrays and immunohistochemistry were employed to study the protein expression of MITF in the primary malignant melanoma tumors by estimating the fraction of positive tumor cells and the staining intensity.

    Results: The vast majority of tumors expressed MITF in >25% of the tumor cells with a strong staining intensity and looking at these factors individually these patients had a better prognosis. When cell fraction and intensity were combined a high-risk group dying of malignant melanoma was identified as those with 25% -75% of tumor cells staining with weak intensity and those with <25% of tumor cells staining with strong intensity. However, the majority of the deaths occurred in the lower risk groups.

    Conclusions: Although a high-risk group for death in malignant melanoma was identified we conclude that MITF is not useful as a prognostic marker because of the distribution of that particular expression in the population.

    Impact: Our results indicate a bi-phasic pattern of MITF expression and although not useful as a prognostic marker these results are in line with other experimental studies and are relevant to explore further.

     

  • 5.
    Agnarsdóttir, Margrét
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Rexhepaj, Elton
    UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Ireland.
    Magnusson, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Patil, Tushar
    Lab Surgpath, Mumbai, India.
    Johansson, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Bergqvist, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Jirström, Karin
    Center for Molecular Pathology, Department of Laboratory Medicine, Skåne University Hospital, Lund University, Malmö, Sweden .
    Uhlen, Mathias
    Department of Proteomics, School of Biotechnology, AlbaNova University Center, KTH-Royal Institute of Technology, SE-10691 Stockholm, Sweden .
    Holmberg, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Gallagher, William
    UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Ireland. .
    Ponten, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular and Morphological Pathology.
    Protein Biomarkers in Malignant Melanoma: An Image Analysis-Based Study on Melanoma Markers of Potential Clinical RelevanceManuscript (preprint) (Other academic)
    Abstract [en]

    The thickness of a primary malignant melanoma tumor is the most important prognostic indicator for a patient with primary cutaneous malignant melanoma. To optimize the management and treatment of melanoma patients there is an unmet need to identify characteristics that can further stratify melanoma patients into high or low risk for progressive disease. Despite numerous studies no single marker has yet been shown to add significant prognostic information. An algorithmic approach, combining data from several markers provides an attractive model to identify patients of increased risk of dying from malignant melanoma. The primary aim of the present study was to analyze the correlation between clinical outcome and protein expression patterns of multiple proteins in malignant melanoma tumors using immunohistochemistry and tissue microarrays. Candidate proteins were identified based on a selective and differential expression pattern in melanoma tumors and tested in a cohort of 143 melanoma patients. Protein expression was analyzed using both manual scoring and automated image analysis-based algorithms. We found no single marker of prognosis that was independent of tumor thickness. When combining potential prognostic markers we could define a prognostic index, based on RBM3, MITF, SOX10 and Ki-67, that was independent of tumor thickness in multivariate analysis. Our findings suggest that a good prognosis signature can be identified in melanoma patients with tumors showing a low fraction of Ki-67 positive tumor cells and a high fraction of RBM3 positive tumor cells combined with low intensity levels of SOX10 and MITF.

     

  • 6.
    Agullo, Luis
    et al.
    Univ Vic Cent Univ Catalonia UVIC UCC, Dept Syst Biol, U Sci Tech, Sagrada Familia 7, Vic 08500, Spain..
    Buch, Ignasi
    Hosp Del Mar Med Res Inst IMIM, Computat Biophys Lab, Barcelona 08003, Spain..
    Gutierrez-de-Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Garcia-Dorado, David
    Vall DHebron Res Inst VHIR, Cardiocirculatory Pathol Grp, Barcelona 08035, Spain..
    Villa-Freixa, Jordi
    Univ Vic Cent Univ Catalonia UVIC UCC, Dept Syst Biol, U Sci Tech, Sagrada Familia 7, Vic 08500, Spain..
    Computational exploration of the binding mode of heme-dependent stimulators into the active catalytic domain of soluble guanylate cyclase2016In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 84, no 10, p. 1534-1548Article in journal (Refereed)
    Abstract [en]

    Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41-2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme-dependent stimulators and heme-independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H-NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an in silico approach. Homology models of the catalytic domain of sGC in inactive or active conformations were constructed using the structure of previously described crystals of the catalytic domains of inactive sGCs (2WZ1, 3ET6) and of active adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 s. Docking of YC-1, a classic heme-dependent stimulator, to all frames of representative trajectories of inactive and active conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high-affinity binding site on the active structure. The site, located between the pseudo-symmetric and the catalytic site just over the loop (2)-(3), does not overlap with the forskolin binding site on adenylate cyclases.

  • 7.
    Ahlford, Annika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Applications of Four-Colour Fluorescent Primer Extension Technology for SNP Analysis and Discovery2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Studies on genetic variation can reveal effects on traits and disease, both in humans and in model organisms. Good technology for the analysis of DNA sequence variations is critical. Currently the development towards assays for large-scale and parallel DNA sequencing and genotyping is progressing rapidly. Single base primer extension (SBE) is a robust reaction principle based on four-colour fluorescent terminating nucleotides to interrogate all four DNA nucleotides in a single reaction. In this thesis, SBE methods were applied to the analysis and discovery of single nucleotide polymorphism (SNP) in the model organism Drosophila melanogaster and in humans.

    The tag-array minisequencing system in a microarray format is convenient for intermediate sized genotyping projects. The system is scalable and flexible to adapt to specialized and novel applications. In Study I of the thesis a tool was established to automate quality control of clustered genotype data. By calculating “Silhouette scores”, the SNP genotype assignment can be evaluated by a single numeric measure. Silhouette scores were then applied in Study I to compare the performance of four DNA polymerases and in Study III to evaluate freeze-dried reagents in the tag-array minisequencing system.

    The characteristics of the tag-array minisequencing system makes it suitable for inexpensive genome-wide gene mapping in the fruit fly. In Study II a high-resolution SNP map, and 293 genotyping assays, were established across the X, 2nd and 3rd chromosomes to distinguish commonly used Drosophila strains. A database of the SNP markers and a program for automatic allele calling and identification of map positions of mutants was also developed. The utility of the system was demonstrated by rapid mapping of 14 genes that disrupt embryonic muscle patterning.

    In Study III the tag-array minisequencing system was adapted to a lab-on-a-chip format for diagnostic testing for mutations in the TP53 gene. Freeze-drying was evaluated for storing reagents, including thermo-sensitive enzymes, on the microchip to reduce the complexity of the integrated test. Correct genotyping results were obtained using freeze-dried reagents in each reaction step of the genotyping protocol, both in test tubes and in single polymer test chambers. The results showed the potential of the approach to be implemented in fully integrated systems.

    The four-colour chemistry of SBE has been developed further to allow massively parallel sequencing (MPS) of short DNA fragments as in the Genome Analyzer system (Solexa/Illumina). In Study IV MPS was used to compare Nimblegen arrays and the SureSelect solution-based system for targeted enrichment of 56 continuous human candidate-gene regions totalling 3.1 Mb in size. Both methods detected known SNPs and discovered novel SNPs in the target regions, demonstrating the feasibility for complexity reduction of sequencing libraries by hybridization methods.

    List of papers
    1.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    2. High-resolution, high-throughput SNP mapping in Drosophila melanogaster
    Open this publication in new window or tab >>High-resolution, high-throughput SNP mapping in Drosophila melanogaster
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    2008 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 5, no 4, p. 323-329Article in journal (Refereed) Published
    Abstract [en]

    Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster. Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1-2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila.

    Keywords
    Animals, Chromosome Mapping, Drosophila melanogaster/embryology/*genetics, Genome; Insect, Muscle Development/*genetics, Mutation, Polymorphism; Single Nucleotide
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-16561 (URN)10.1038/nmeth.1191 (DOI)000254559400019 ()18327265 (PubMedID)
    Available from: 2008-05-28 Created: 2008-05-28 Last updated: 2017-12-08Bibliographically approved
    3. Positional cloning by fast-track SNP-mapping in Drosophila melanogaster
    Open this publication in new window or tab >>Positional cloning by fast-track SNP-mapping in Drosophila melanogaster
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    2008 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 3, no 11, p. 1751-1765Article in journal (Refereed) Published
    Abstract [en]

    Positional cloning of chemically induced mutations is the rate-limiting step in forward genetic screens in Drosophila. Single-nucleotide polymorphisms (SNPs) are useful markers to locate a mutated region in the genome. Here, we provide a protocol for high-throughput, high-resolution SNP mapping that enables rapid and cost-effective positional cloning in Drosophila. In stage 1 of the protocol, we use highly multiplexed tag-array mini-sequencing assays to map mutations to an interval of 1-2 Mb. In these assays, SNPs are genotyped by primer extension using fluorescently labeled dideoxy-nucleotides. Fluorescent primers are captured and detected on a microarray. In stage 2, we selectively isolate recombinants within the identified 1-2 Mb interval for fine mapping of mutations to about 50 kb. We have previously demonstrated the applicability of this protocol by mapping 14 muscle morphogenesis mutants within 4 months, which represents a significant acceleration compared with other commonly used mapping strategies that may take years.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-98390 (URN)10.1038/nprot.2008.175 (DOI)000265781600008 ()18948975 (PubMedID)
    Available from: 2009-02-20 Created: 2009-02-20 Last updated: 2017-12-13Bibliographically approved
    4. Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices
    Open this publication in new window or tab >>Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices
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    2010 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 135, no 9, p. 2377-2385Article in journal (Refereed) Published
    Abstract [en]

    We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-129216 (URN)10.1039/c0an00321b (DOI)000281007300027 ()20668755 (PubMedID)
    Available from: 2010-08-09 Created: 2010-08-09 Last updated: 2017-12-12Bibliographically approved
    5.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
  • 8.
    Ahmed, Meftun
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
    Oscillatory Ca2+ signaling in glucose-stimulated murine pancreatic β-cells: Modulation by amino acids, glucagon, caffeine and ryanodine2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

  • 9. Aho, Leena
    et al.
    Karkola, Kari
    Juusela, Jari
    Alafuzoff, Irina
    Department of Neuroscience and Neurology, University of Kuopio Finland .
    Heavy alcohol consumption and neuropathological lesions: a post-mortem human study2009In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 87, no 12, p. 2786-2792Article in journal (Refereed)
    Abstract [en]

    Epidemiological studies have indicated that excessive alcohol consumption leads to cognitive impairment, but the specific pathological mechanism involved remains unknown. The present study evaluated the association between heavy alcohol intake and the neuropathological hallmark lesions of the three most common neurodegenerative disorders, i.e., Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and vascular cognitive impairment (VCI), in post-mortem human brains. The study cohort was sampled from the subjects who underwent a medicolegal autopsy during a 6-month period in 1999 and it included 54 heavy alcohol consumers and 54 age- and gender-matched control subjects. Immunohistochemical methodology was used to visualize the aggregation of beta-amyloid, hyperphosphorylated tau, and alpha-synuclein and the extent of infarcts. In the present study, no statistically significant influence was observed for alcohol consumption on the extent of neuropathological lesions encountered in the three most common degenerative disorders. Our results indicate that alcohol-related dementia differs from VCI, AD, and DLB; i.e., it has a different etiology and pathogenesis.

  • 10. Aho, Leena
    et al.
    Pikkarainen, Maria
    Hiltunen, Mikko
    Leinonen, Ville
    Alafuzoff, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Immunohistochemical Visualization of Amyloid-β Protein Precursor and Amyloid-β in Extra- and Intracellular Compartments in the Human Brain2010In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 20, no 4, p. 1015-1028Article in journal (Refereed)
    Abstract [en]

    Amyloid-beta (Abeta) peptide, a cleavage product of the amyloid-beta protein precursor (AbetaPP), has been reported to be detected in the intracellular compartment. Most studies reporting the presence of intracellular Abeta are based on the use of immunohistochemistry. In this study, the presence of AbetaPP and Abeta was assessed by applying immunohistochemistry in postmortem human brain tissue samples obtained from 10 neurologically intact subjects, the youngest being 2 years of age, one aged with mild cognitive impairment, 14 neurologically diseased, and in one brain biopsy sample obtained from a subject with normal pressure hydrocephalus. Intracellular immunoreactivity was detected in all ages independent of the disease state or existence of extracellular Abeta aggregates with all antibodies directed to AbetaPP, with three Abeta antibodies (4G8, 6E10, and 82E1), clones that are unable to distinguish Abeta from AbetaPP. These results suggest that it is AbetaPP rather than Abeta that is detected intracellularly when using the antibodies listed above. Furthermore, the staining results varied when different pretreatment strategies were applied. Interestingly intracellular Abeta was detected with antibodies directed to the C-terminus of Abeta (neoepitope) in subjects with Alzheimer's disease. The lack of intracellular immunoreactivity in unimpaired subjects, when using antibodies against neoepitopes, may be due to a lack or a low level of the protein that is thus undetectable at light microscopic level by immunohistochemistry method. The staining results and conclusions depended strongly on the chosen antibody and the pretreatment strategy and thus multiple antibodies must be used when assessing the intracellular accumulation of Abeta.

  • 11.
    Ahrén, Anna
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Immunhistokemisk undersökning av paraffinbäddade celler från pleuravätska som kompletterande underlag för diagnos av cancermetastaser2005Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Background. Immunohistochemistry is a useful method in the differential diagnosis between pleural mesotheliomas and metastatic adenocarcinomas in the pleura. Cytokeratin 20 and 7 have been used successfully as markers in studies determining primary location of adenocarcinomas from metastases. The current study is a complementary research of archived paraffininbedded material of cases with cancer origin. This study contributes a bigger statistical material that may facilitate the search for unknown primary site of adenocarcinoma by identification of metastatic cells in the pleura.

    Methods. Cells from the pleura taken from fifteen patients with diagnosed cancer of different types and eleven patients with cancer of unknown origin, were stained with antibodies against the tumour markers: Ber EP 4, calretinin, cytokeratin 20 and 7, estrogen receptor α, thyroid transcription factor, prostate-specific antigen and Cdx2.The staining was conducted in an automated immunohistochemical system. The staining of each kind of antibody was confirmed by a control section staining.

    Results. All control staining ended perfect The whole panel of antibodies used on mammary cancer showed the same pattern for every antibody. Of the patients with cancer of unknown origin there were four that gave the same pattern, two men and two women. The women are deceased. To make a more careful evaluation more information and clinic background is needed. The number of samples is too small to draw any statistical conclusions.

    Comment. Although the control staining was perfect the negative result of CK20 in the cases of diagnosed colon cancer was unexpected. This staining should be performed again to confirm the result. In some cases the number of cells were to few for a certain evaluation. The slides and the results of this work will be archived for further research.

  • 12.
    Alafuzoff, Irina
    et al.
    Department of Neuroscience and Neurology, University of Kuopio Finland .
    Aho, L
    Helisalmi, S
    Mannermaa, A
    Soininen, H
    Beta-amyloid deposition in brains of subjects with diabetes2009In: Neuropathology and Applied Neurobiology, ISSN 0305-1846, E-ISSN 1365-2990, Vol. 35, no 1, p. 60-68Article in journal (Refereed)
    Abstract [en]

    AIM:

    A causative association between diabetes mellitus (DM) and Alzheimer's disease (AD) has been suggested based on clinical and epidemiological studies. One hypothesis is that the link between DM and AD is related to the function of insulin-degrading enzyme (IDE), an enzyme that degrades not only insulin and pancreatic amylin but also beta-amyloid (Abeta). Thus, in diabetics, insulin and Abeta might compete for IDE and this might lead to an increase in Abeta. The objective of this study was to test the hypothesis that hyperinsulinaemia can elevate Abeta levels and thus contribute to AD pathology in humans.

    METHODS:

    Neuropathological examination was carried out employing conventional and immunohistochemical (IHC) methods of the brains obtained post mortem from 701 aged subjects.

    RESULTS:

    The loads of IHC/Abeta, silver stained neuritic plaques (NP) and neurofibrillary tangles (NFT) were significantly higher in subjects carrying the Apolipoprotein E e4 allele. In contrast, the loads of Abeta, NPs and NFT in the brains were not influenced by hyperglycaemia when comparing 134 diabetic with 567 non-diabetic subjects.

    CONCLUSIONS:

    We conclude that the hypothesis that hyperinsulinaemia would significantly elevate the Abeta load and thus increase the extent of AD pathology cannot be supported. Our result challenges the claim that DM is a direct risk factor of developing AD. Thus further studies on pathological lesions in demented diabetics should be conducted.

  • 13.
    Alafuzoff, Irina
    et al.
    Department of Neuroscience and Neurology, University of Kuopio Finland .
    Thal, Dietmar R.
    Arzberger, Thomas
    Bogdanovic, Nenad
    Al-Sarraj, Safa
    Bodi, Istvan
    Boluda, Susan
    Bugiani, Orso
    Duyckaerts, Charles
    Gelpi, Ellen
    Gentleman, Stephen
    Giaccone, Giorgio
    Graeber, Manuel
    Hortobagyi, Tibor
    Höftberger, Romana
    Ince, Paul
    Ironside, James W.
    Kavantzas, Nikolaos
    King, Andrew
    Korkolopoulou, Penelope
    Kovács, Gábor G.
    Meyronet, David
    Monoranu, Camelia
    Nilsson, Tatjana
    Parchi, Piero
    Patsouris, Efstratios
    Pikkarainen, Maria
    Revesz, Tamas
    Rozemuller, Annemieke
    Seilhean, Danielle
    Schulz-Schaeffer, Walter
    Streichenberger, Nathalie
    Wharton, Stephen B.
    Kretzschmar, Hans
    Assessment of beta-amyloid deposits in human brain: a study of the BrainNet Europe Consortium2009In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 117, no 3, p. 309-320Article in journal (Refereed)
    Abstract [en]

    beta-Amyloid (A-beta) related pathology shows a range of lesions which differ both qualitatively and quantitatively. Pathologists, to date, mainly focused on the assessment of both of these aspects but attempts to correlate the findings with clinical phenotypes are not convincing. It has been recently proposed in the same way as iota and alpha synuclein related lesions, also A-beta related pathology may follow a temporal evolution, i.e. distinct phases, characterized by a step-wise involvement of different brain-regions. Twenty-six independent observers reached an 81% absolute agreement while assessing the phase of A-beta, i.e. phase 1 = deposition of A-beta exclusively in neocortex, phase 2 = additionally in allocortex, phase 3 = additionally in diencephalon, phase 4 = additionally in brainstem, and phase 5 = additionally in cerebellum. These high agreement rates were reached when at least six brain regions were evaluated. Likewise, a high agreement (93%) was reached while assessing the absence/presence of cerebral amyloid angiopathy (CAA) and the type of CAA (74%) while examining the six brain regions. Of note, most of observers failed to detect capillary CAA when it was only mild and focal and thus instead of type 1, type 2 CAA was diagnosed. In conclusion, a reliable assessment of A-beta phase and presence/absence of CAA was achieved by a total of 26 observers who examined a standardized set of blocks taken from only six anatomical regions, applying commercially available reagents and by assessing them as instructed. Thus, one may consider rating of A-beta-phases as a diagnostic tool while analyzing subjects with suspected Alzheimer's disease (AD). Because most of these blocks are currently routinely sampled by the majority of laboratories, assessment of the A-beta phase in AD is feasible even in large scale retrospective studies.

  • 14.
    Aldi, S.
    et al.
    KFC Novum Karolinska Inst Huddinge, Dept Mol Med & Surg, S-14186 Stockholm, Sweden..
    Eriksson, L.
    KFC Novum Karolinska Inst Huddinge, Dept Mol Med & Surg, S-14186 Stockholm, Sweden..
    Kronqvist, M.
    KFC Novum Karolinska Inst Huddinge, Dept Mol Med & Surg, S-14186 Stockholm, Sweden..
    Lengquist, M.
    KFC Novum Karolinska Inst Huddinge, Dept Mol Med & Surg, S-14186 Stockholm, Sweden..
    Folkersen, L.
    Tech Univ Denmark, Ctr Biol Sequence Anal, DK-2800 Lyngby, Denmark..
    Perisic, L.
    KFC Novum Karolinska Inst Huddinge, Dept Mol Med & Surg, S-14186 Stockholm, Sweden..
    Grinnemo, K. H.
    KFC Novum Karolinska Inst Huddinge, Dept Mol Med & Surg, S-14186 Stockholm, Sweden..
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hedin, U.
    KFC Novum Karolinska Inst Huddinge, Dept Mol Med & Surg, S-14186 Stockholm, Sweden..
    Osterholm, C.
    KFC Novum Karolinska Inst Huddinge, Dept Mol Med & Surg, S-14186 Stockholm, Sweden..
    Dual roles of heparanase in vascular calcification associated with human carotid atherosclerosis2017In: International journal of experimental pathology (Print), ISSN 0959-9673, E-ISSN 1365-2613, Vol. 98, no 3, p. A5-A5Article in journal (Other academic)
  • 15.
    Alenkvist, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Epac2 signaling at the β-cell plasma membrane2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Secretion of appropriate amounts of insulin from pancreatic β-cells is crucial for glucose homeostasis. The β-cells release insulin in response to glucose and other nutrients, hormones and neurotransmitters, which trigger intracellular signaling cascades, that result in exocytotic fusion of insulin-containing vesicles with the plasma membrane. Increases of the intracellular concentration of calcium ions ([Ca2+]i) trigger exocytosis, whereas the messenger cyclic adenosine monophosphate (cAMP) amplifies various steps of the secretion process. The protein Epac2 mediates some effects of cAMP, but little is known about its regulation in β-cells. In this study, the spatio-temporal dynamics of Epac2 was investigated in insulin-secreting MIN6-cells and primary β-cells using various cell signaling biosensors and live-cell fluorescence microscopy approaches. Increases in the cAMP concentration triggered translocation of Epac2 from the cytoplasm to the plasma membrane. Oscillations of cAMP induced by glucose and the insulin-releasing hormone GLP-1 were associated with cyclic translocation of Epac2. Analyses of Epac2 mutants showed that the high-affinity cyclic nucleotide-binding domain and Ras-association domains were crucial for the translocation, whereas neither the DEP domain, nor the low-affinity cAMP-binding domain were required for membrane binding. However, the latter domain targeted Epac2 to insulin granules at the plasma membrane, which promoted their priming for exocytosis. Depolarization-induced elevations of [Ca2+]i also stimulated Epac2 translocation, but the effects were complex and in the presence of high cAMP concentrations, [Ca2+]i increases often reduced membrane binding. The stimulatory effect of Ca2+ was mediated by increased Ras activity, while the inhibitory effect reflected reduced concentrations of the membrane phospholipid PtdIns(4,5)P2. Anti-diabetic drugs of the sulfonylurea class, suggested to directly activate Epac2, induced translocation indirectly by depolarizing β-cells to increase [Ca2+]i. Epac2 is an activator of Rap GTPases, and its translocation increased Rap activity at the plasma membrane. It is concluded that the subcellular localization of Epac2 is controlled by a complex interplay between cAMP, Ca2+ and PtdIns(4,5)P2 and that the protein controls insulin release by binding to the exocytosis machinery. These results provide new insights into the regulation of β-cell function and may facilitate the development of new anti-diabetic drugs that amplify insulin secretion.

    List of papers
    1. Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells
    Open this publication in new window or tab >>Spatial Control of Epac2 Activity by cAMP and Ca2+-Mediated Activation of Ras in Pancreatic beta Cells
    2013 (English)In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 6, no 273, p. ra29-Article in journal (Refereed) Published
    Abstract [en]

    The cAMP (adenosine 3',5'-monophosphate)-activated guanine nucleotide exchange factor (GEF) Epac2 is an important mediator of cAMP-dependent processes in multiple cell types. We used real-time confocal and total internal reflection fluorescence microscopy to examine the spatiotemporal regulation of Epac2, which is a GEF for the guanosine triphosphatase (GTPase) Rap. We demonstrated that increases in the concentration of cAMP triggered the translocation of Epac2 from the cytoplasm to the plasma membrane in insulin-secreting beta cells. Glucose-induced oscillations of the submembrane concentration of cAMP were associated with cyclic translocation of Epac2, and this translocation could be amplified by increases in the cytoplasmic Ca2+ concentration. Analyses of Epac2 mutants identified the high-affinity cAMP-binding and the Ras association domains as crucial for the translocation. Expression of a dominant-negative Ras mutant reduced Epac2 translocation, and Ca2+-dependent oscillations in Ras activity synchronized with Epac2 translocation in single beta cells. The cyclic translocation of Epac2 was accompanied by oscillations of Rap GTPase activity at the plasma membrane, and expression of an inactive Rap1B mutant decreased insulin secretion. Thus, Epac2 localization is dynamically controlled by cAMP as well as by Ca2+-mediated activation of Ras. These results help to explain how oscillating signals can produce pulses of insulin release from pancreatic b cells.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-201253 (URN)10.1126/scisignal.2003932 (DOI)000318350100002 ()
    Available from: 2013-06-10 Created: 2013-06-10 Last updated: 2017-12-06Bibliographically approved
    2. Two-step membrane recruitment of Epac2 primes secretory granules for exocytosis
    Open this publication in new window or tab >>Two-step membrane recruitment of Epac2 primes secretory granules for exocytosis
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-284568 (URN)
    Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2018-01-10
    3. Localization of Epac2 to the plasma membrane is controlled by an interplay between cAMP, Ca2+ and PtdIns(4,5)P2
    Open this publication in new window or tab >>Localization of Epac2 to the plasma membrane is controlled by an interplay between cAMP, Ca2+ and PtdIns(4,5)P2
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-284580 (URN)
    Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2018-01-10
    4. Sulfonylureas trigger Ca2+-dependent Epac2 recruitment and Rap activation at the plasma membrane in β-cells
    Open this publication in new window or tab >>Sulfonylureas trigger Ca2+-dependent Epac2 recruitment and Rap activation at the plasma membrane in β-cells
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-284582 (URN)
    Available from: 2016-04-19 Created: 2016-04-18 Last updated: 2018-01-10
  • 16.
    Alenkvist, Ida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil R
    Barg, Sebastian
    Tengholm, Anders
    Two-step membrane recruitment of Epac2 primes secretory granules for exocytosisManuscript (preprint) (Other academic)
  • 17.
    Alenkvist, Ida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Idevall-Hagren, Olof
    Tengholm, Anders
    Localization of Epac2 to the plasma membrane is controlled by an interplay between cAMP, Ca2+ and PtdIns(4,5)P2Manuscript (preprint) (Other academic)
  • 18.
    Alenkvist, Ida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xu, Yunjian
    Tengholm, Anders
    Sulfonylureas trigger Ca2+-dependent Epac2 recruitment and Rap activation at the plasma membrane in β-cellsManuscript (preprint) (Other academic)
  • 19.
    Alfredsson, Jessica
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Molecular Studies of Mast Cell Migration and Apoptosis: Two Ways of Regulating Mast Cell Numbers at Sites of Inflammation2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Upon activation mast cells release numerous proinflammatory mediators. With this feature, mast cells play an important role in host defense against pathogens, and are involved in tissue remodeling and wound healing. However, in cases of excessive inflammation the effects of mast cells are detrimental. This is observed in allergy, asthma, rheumatoid arthritis, atherosclerosis, certain types of heart failure, and in several other chronic destructive inflammations. Mast cell numbers are typically increased at inflammatory sites. There they act both directly, as effector cells, and in a regulatory manner, secreting agents that recruit and activate other immune cells.

    The studies presented here investigated mechanisms regulating mast cell numbers at sites of inflammation, focusing on cell migration and regulation of survival/apoptosis. We report that SCF-induced mast cell migration requires p38 MAP kinase activity. Moreover, we found that SCF-mediated mast cell survival is regulated through downregulation of the proapoptotic Bcl-2 family member Bim, as well as through phoshorylation of Bim. SCF seems to control Bim protein levels via FOXO transcription factors, and to induce phosphorylation of Bim via the Mek/Erk and the PI3-kinase/Akt signaling pathways. Furthermore, mast cell death triggered by deprivation of SCF and/or IL-3 involves the Bim protein, as demonstrated using bim-/- mast cells. Additional studies revealed that IgE-receptor activation, which occurs in allergy, promotes both prosurvival and proapoptotic signaling events. This includes upregulation of Bim and the prosurvival Bcl-XL and A1, as well as phosphorylation of Akt, FOXO factors, GSK-3β, IκB-α, Bad, and Bim. The simultaneous stimulation of prosurvival and proapoptotic signaling events could be a way to fine-tune the fate of mast cells after IgE-receptor activation and degranulation.

    The new insights about mechanisms involved in mast cell migration and regulation of survival/apoptosis might prove useful for future efforts to design new drugs to be used for mast cell-associated diseases.

    List of papers
    1. Stem Cell Factor-Induced Migration of Mast Cells Requires p38 Mitogen-Activated Protein Kinase Activity
    Open this publication in new window or tab >>Stem Cell Factor-Induced Migration of Mast Cells Requires p38 Mitogen-Activated Protein Kinase Activity
    2001 In: Experimental Cell Research, Vol. 267, no 1, p. 144-151Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-92671 (URN)
    Available from: 2005-03-11 Created: 2005-03-11Bibliographically approved
    2. Stem cell factor promotes mast cell survival via inactivation of FOXO3a mediated transcriptional induction and MEK regulated phosphorylation of the pro-apoptotic protein Bim
    Open this publication in new window or tab >>Stem cell factor promotes mast cell survival via inactivation of FOXO3a mediated transcriptional induction and MEK regulated phosphorylation of the pro-apoptotic protein Bim
    Show others...
    Article in journal (Refereed) Submitted
    Identifiers
    urn:nbn:se:uu:diva-92672 (URN)
    Available from: 2005-03-11 Created: 2005-03-11Bibliographically approved
    3. Porapoptotic Bcl-2 family member Bim is involved in the control of mast cell survival and is induced together with Bcl-XL upon IgE-receptor activation
    Open this publication in new window or tab >>Porapoptotic Bcl-2 family member Bim is involved in the control of mast cell survival and is induced together with Bcl-XL upon IgE-receptor activation
    Show others...
    2005 In: Cell Death and Differentiation, Vol. 12, no 2, p. 136-144Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-92673 (URN)
    Available from: 2005-03-11 Created: 2005-03-11Bibliographically approved
    4. IgE-receptor activation of mast cells regulates phosphorylation and expression of forkhead and Bcl-2 family members
    Open this publication in new window or tab >>IgE-receptor activation of mast cells regulates phosphorylation and expression of forkhead and Bcl-2 family members
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-92674 (URN)
    Available from: 2005-03-11 Created: 2005-03-11 Last updated: 2010-01-13Bibliographically approved
  • 20.
    Alfredsson Timmins, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Functional organisation of the cell nucleus in the fission yeast, Schizosaccharomyces pombe2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In eukaryotes the genome adopts a non-random spatial organisation, which is important for gene regulation. However, very little is known about the driving forces behind nuclear organisation. In the simple model eukaryote fission yeast, Schizosaccharomyces pombe, it has been known for a long time that transcriptionally repressed heterochromatin localise to the nuclear membrane (NM); the centromeres attaches to spindle pole body (SPB), while the telomeres are positioned at the NM on the opposite side of the nucleus compared to the SPB. Studies presented in this thesis aimed at advancing our knowledge of nuclear organisation in Schizosaccharomyces pombe.

    We show that the heterochromatic mating-type region localises to the NM in the vicinity of the SPB. This positioning was completely dependent on Clr4, a histone methyl transferase crucial for the formation of heterochromatin. Additional factors important for localisation were also identified: the chromo domain protein Swi6, and the two boundary elements IR-L and IR-R surrounding this locus. We further identify two other chromo domain proteins; Chp1 and Chp2, as crucial factors for correct subnuclear localisation of this region. From these results we suggest that the boundary elements together with chromodomain proteins in balanced dosage and composition cooperate in organising the mating-type chromatin.

    Gene regulation can affect the subnuclear localisation of genes. Using nitrogen starvation in S. pombe as a model for gene induction we determined the subnuclear localisation of two gene clusters repressed by nitrogen: Chr1 and Tel1. When repressed these loci localise to the NM, and this positioning is dependent on the histone deacetylase Clr3. During induction the gene clusters moved towards the nuclear interior in a transcription dependent manner.

    The knowledge gained from work presented in this thesis, regarding nuclear organisation in the S. pombe model system, can hopefully aid to a better understanding of human nuclear organisation.

    List of papers
    1. The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast
    Open this publication in new window or tab >>The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast
    2007 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 120, no 11, p. 1935-1943Article in journal (Refereed) Published
    Abstract [en]

    The genome has a non-random spatial distribution in the cell nucleus. In Schizosaccharomyces pombe, it has been shown that the centromeres, telomeres and the mating-type region localize to the nuclear membrane (NM), the former by attaching to the spindle pole body (SPB). In addition, reporter genes inserted into these areas are transcriptionally repressed due to the formation of specialized chromatin structures. Performing live cell analysis we found that in a wild-type strain the mating-type region was positioned in the proximity of the SPB, the location where the pericentromeric heterochromatin is also found. In a strain lacking the histone methyltransferase, Clr4, crucial for the formation of heterochromatin, the mating-type region had a random localization in the nucleus. Moreover, in a strain where the two boundary elements IR-L and IR-R had been deleted the mating-type region was displaced from its position at the proximity of the SPB, but remained in the vicinity of the NM. Moreover, in all investigated strains with silencing deficiencies the distance between the mating-type region and the SPB increased. This result indicates a correlation between transcriptional derepression and displacement of the region. Two different models of how the mating-type chromatin is organized in the nucleus are discussed.

    Keywords
    fission yeast, heterochromatin, silencing, subnuclear localization
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-13023 (URN)10.1242/jcs.03457 (DOI)000246665300013 ()17504808 (PubMedID)
    Available from: 2009-08-05 Created: 2009-08-05 Last updated: 2017-12-11Bibliographically approved
    2. Chromo domain proteins in balanced dosage together with boundary elements cooperate in organising the mating-type chromatin in fission yeast
    Open this publication in new window or tab >>Chromo domain proteins in balanced dosage together with boundary elements cooperate in organising the mating-type chromatin in fission yeast
    (English)Manuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    The chromatin in the cell nucleus has a spatial organisation. For example, in the fission yeast, Schizosaccharomyces pombe, transcriptionally repressed heterochromatin is found at the nuclear membrane (NM). The centromeres and the mating-type region localise in the proximity of the spindle pole body (SPB), while the telomeres are found on the opposite side of the nucleus in the proximity of the nucleolus. In a previous study we used the mating-type region as a model to study the driving force behind nuclear organisation. We proposed two mutually exclusive models to explain what determines the localisation of the mating-type region. The first model suggests that solely the amount of heterochromatin in the region affects the localisation, while the other model stipulates that the boundary elements together with heterochromatin formation anchor the mating-type region in the NM in the vicinity of the SPB. Here, we present data that disproves the first model. We found that in a strain expressing tripled amounts of the chromodomain protein Swi6, a structural component of heterochromatin, the mating-type region was delocalised from the proximity of the SPB. A strain deleted of the histone deacetylase clr3+ also had a delocalised mating-type locus. Interestingly, a strain with a point-mutation in clr3-735 producing an enzymatically inactive protein in normal amounts showed an intermediate phenotype. Most importantly, we identify the chromodomain proteins, Chp1 and Chp2, as crucial factors for correct subnuclear localisation of the mating-type region. We suggest that boundary elements together with chromodomain proteins in balanced dosage and composition cooperate in organising the mating-type chromatin.

    Identifiers
    urn:nbn:se:uu:diva-107280 (URN)
    Available from: 2009-08-04 Created: 2009-08-04 Last updated: 2010-01-14Bibliographically approved
    3. Reorganization of chromatin is an early response to nitrogen starvation in Schizosaccharomyces pombe
    Open this publication in new window or tab >>Reorganization of chromatin is an early response to nitrogen starvation in Schizosaccharomyces pombe
    Show others...
    2009 (English)In: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 118, no 1, p. 99-112Article in journal (Refereed) Published
    Abstract [en]

    There are several documented events of changes in subnuclear localization during gene activation. However, there are conflicting data on whether the nuclear periphery is a compartment for gene repression or activation, and whether genes are moved to the pores at the nuclear membrane (NM) or not during gene activation. Nitrogen starvation of fission yeast serves as a good model system for studying gene induction since it causes fast regulation of hundreds of genes. In this study the subnuclear localization of two gene clusters repressed by nitrogen was investigated. During normal growth conditions the gene clusters localized to the nuclear periphery at the opposite side of the nucleus as compared to the spindle pole body (SPB). This constrained localization was dependent on the histone deacetylase Clr3, known to transcriptionally repress genes in these clusters. Already 20 minutes after nitrogen depletion drastic changes in subnuclear localization of the two loci were observed, away from the NM towards the nuclear interior. At least for one of the clusters the movement was clearly transcription dependent. Data presented here illustrates how interconnected events of gene activation and nuclear reorganization are, as well as provides a suggestion of how nuclear organization might be maintained.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-107289 (URN)10.1007/s00412-008-0180-6 (DOI)000262504300009 ()
    Available from: 2009-08-05 Created: 2009-08-05 Last updated: 2017-12-13Bibliographically approved
  • 21.
    Alim, Md. Abdul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Family Medicine and Preventive Medicine. Department of Molecular Medicine and Surgery, Karolinska Institutet.
    Ackermann, Paul W
    Eliasson, Pernilla
    Blomgran, Parmis
    Kristiansson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Family Medicine and Preventive Medicine.
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Peterson, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Family Medicine and Preventive Medicine.
    Increased mast cell degranulation and co-localization of mast cells with the NMDA receptor-1 during healing after Achilles tendon rupture2017In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 370, no 3, p. 451-460Article in journal (Refereed)
    Abstract [en]

    The role of inflammation and the mechanism of tendon healing after rupture has historically been a matter of controversy. The purpose of the present study is to investigate the role of mast cells and their relation to the NMDA receptor-1 (a glutamate receptor) during healing after Achilles tendon rupture. Eight female Sprague Dawley rats had their right Achilles tendon transected. Three weeks after rupture, histological quantification of mast cell numbers and their state of degranulation was assessed by histochemistry. Co-localization of mast cell tryptase (a mast cell marker) and NMDA receptor-1 was determined by immunofluorescence. The intact left Achilles tendon was used as control. An increased number of mast cells and a higher proportion of degranulated mast cells were found in the healing Achilles tendon compared to the intact. In addition, increased co-localization of mast cell tryptase and NMDA receptor-1 was seen in the areas of myotendinous junction, mid-tendon proper and bone tendon junction of the healing versus the intact tendon. These findings introduce a possible role for mast cells in the healing phase after Achilles tendon rupture.

  • 22.
    Alimohammadi, Mohammad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Molecular Targets in Autoimmune Polyendocrine Syndrome Type1 and Their Clinical Implications2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Autoimmune diseases occur when the immune system attacks and destroys healthy body tissue. Autoimmunity is known to cause a wide range of disorders, and is suspected to be responsible for many more. Most autoimmune disorders are chronic and cause severe morbidity for the patients, and are also costly for society. A majority of these disorders are today considered as complex diseases with incompletely known etiology. Hence, model systems for studying the pathogenesis of autoimmunity are important to unravel its causes.

    Autoimmune Polyendocrine Syndrome Type 1 (APS-1), (OMIM 240300), is a rare autoimmune disorder. Patients with APS-1 progressively develop multiple organ-specific autoimmune lesions involving both endocrine and non endocrine tissues. Typical autoimmune disease components in APS-1 are hypoparathyroidism, Addison’s disease, vitiligo, alopecia and type 1 diabetes. The gene preventing APS-1 has been identified and designated Autoimmune Regulator (AIRE). It has been shown that mutations of AIRE cause loss of tolerance to self-structures, resulting in organ-specific autoimmunity.

    Although APS-1 is a rare syndrome occurring mainly in genetically isolated populations, the disease components of APS-1 are, in isolated forms, not unusual in the general population and affect many patients. Hence, APS-1 is an attractive model disease for studies of molecular mechanisms underlying organ-specific autoimmunity.

    This thesis concerns investigations in which two novel autoantigens are identified in APS-1 and used in serological diagnosis of the disease. NALP5, is identified as a parathyroid autoantigen - an important finding since autoimmune hypoparathyroidism is one of the cardinal symptoms of APS-1. Additionally, KCNRG is identified as a bronchial autoantigen in APS-1 patients with respiratory symptoms. Finally, studies that compare the immune response in APS-1 patients and the mouse model for APS-1 are presented.

    List of papers
    1. Aire deficient mice do not develop the same profile of tissue-specific autoantibodies as APECED patients
    Open this publication in new window or tab >>Aire deficient mice do not develop the same profile of tissue-specific autoantibodies as APECED patients
    Show others...
    2006 (English)In: Journal of Autoimmunity, ISSN 0896-8411, E-ISSN 1095-9157, Vol. 27, no 2, p. 96-104Article in journal (Refereed) Published
    Abstract [en]

    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, or APS1), is a monogenic autoimmune disease caused by mutations in the autoimmune regulator (AIRE) gene. The three main components of APECED are chronic mucocuteaneous candidiasis, hypoparathyroidism and adrenocortical insufficiency. However, several additional endocrine or other autoimmune disease components, or ectodermal dystrophies form the individually variable clinical picture of APECED. An important feature of APECED is a spectrum of well-characterized circulating autoantibodies, reacting against tissue-specific autoantigens. Aire deficient mice develop some characteristics of APECED phenotype. In order to investigate whether the Aire deficient mice produce autoantibodies similar to human APECED, we studied the reactivity of Aire mouse sera against mouse homologues of 11 human APECED antigens. None of the APECED antigens indicated elevated reactivity in the Aire knock-out mouse sera, implying the absence of APECED associated autoantibodies in Aire deficient mice. These findings were supported by the failure of the autoantigens to activate mouse T-cells. Furthermore, Aire knock-out mice did not express increased levels of anti-nuclear antibodies compared to wt mice. This study indicates that spontaneous induction of tissue-specific autoantibodies similar to APECED does not occur in the rodent model suggesting differences in the immunopathogenic mechanisms between mice and men.

    Keywords
    APS1, Autoantigen, Autoimmune regulator, Knock-out mouse
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-98037 (URN)10.1016/j.jaut.2006.06.001 (DOI)000241661500004 ()16820279 (PubMedID)
    Available from: 2009-02-11 Created: 2009-02-11 Last updated: 2017-12-14Bibliographically approved
    2. Autoimmune Polyendocrine Syndrome Type 1: NALP5 in Autoimmune Polyendocrine Syndrome Type 1
    Open this publication in new window or tab >>Autoimmune Polyendocrine Syndrome Type 1: NALP5 in Autoimmune Polyendocrine Syndrome Type 1
    Show others...
    2006 (English)In: The New England Journal of Medicine, Vol. 358, no 10, p. 1018-28Article in journal (Refereed) Published
    Abstract [en]

    Background Autoimmune polyendocrine syndrome type 1 (APS-1) is a multiorgan autoimmune disorder caused by mutations in AIRE, the autoimmune regulator gene. Though recent studies concerning AIRE deficiency have begun to elucidate the molecular pathogenesis of organ-specific autoimmunity in patients with APS-1, the autoantigen responsible for hypoparathyroidism, a hallmark of APS-1 and its most common autoimmune endocrinopathy, has not yet been identified.

    Methods We performed immunoscreening of a human parathyroid complementary DNA library, using serum samples from patients with APS-1 and hypoparathyroidism, to identify patients with reactivity to the NACHT leucine-rich-repeat protein 5 (NALP5). Subsequently, serum samples from 87 patients with APS-1 and 293 controls, including patients with other autoimmune disorders, were used to determine the frequency and specificity of autoantibodies against NALP5. In addition, the expression of NALP5 was investigated in various tissues.

    Results NALP5-specific autoantibodies were detected in 49% of the patients with APS-1 and hypoparathyroidism but were absent in all patients with APS-1 but without hypoparathyroidism, in all patients with other autoimmune endocrine disorders, and in all healthy controls. NALP5 was predominantly expressed in the cytoplasm of parathyroid chief cells.

    Conclusions NALP5 appears to be a tissue-specific autoantigen involved in hypoparathyroidism in patients with APS-1. Autoantibodies against NALP5 appear to be highly specific and may be diagnostic for this prominent component of APS-1.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-98038 (URN)
    Available from: 2009-02-11 Created: 2009-02-11 Last updated: 2009-09-29Bibliographically approved
    3. NALP5 - a Target for Autoantibodies in AIRE Deficient Mice Reflecting the Autoimmune Status
    Open this publication in new window or tab >>NALP5 - a Target for Autoantibodies in AIRE Deficient Mice Reflecting the Autoimmune Status
    Show others...
    (English)Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-98039 (URN)
    Available from: 2009-02-11 Created: 2009-02-11 Last updated: 2010-05-06Bibliographically approved
    4. Pulmonary Autoimmunity as a Feature of Autoimmune Polyendocrine Syndrome Type 1 and Identification of KCNRG as a Bronchial Autoantigen
    Open this publication in new window or tab >>Pulmonary Autoimmunity as a Feature of Autoimmune Polyendocrine Syndrome Type 1 and Identification of KCNRG as a Bronchial Autoantigen
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    2009 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 11, p. 4396-4401Article in journal (Refereed) Published
    Abstract [en]

    Patients with autoimmune polyendocrine syndrome type 1 (APS-1) suffer from multiple organ-specific autoimmunity with autoantibodies against target tissue-specific autoantigens. Endocrine and nonendocrine organs such as skin, hair follicles, and liver are targeted by the immune system. Despite sporadic observations of pulmonary symptoms among APS-1 patients, an autoimmune mechanism for pulmonary involvement has not been elucidated. We report here on a subset of APS-1 patients with respiratory symptoms. Eight patients with pulmonary involvement were identified. Severe airway obstruction was found in 4 patients, leading to death in 2. Immunoscreening of a cDNA library using serum samples from a patient with APS-1 and obstructive respiratory symptoms identified a putative potassium channel regulator (KCNRG) as a pulmonary autoantigen. Reactivity to recombinant KCNRG was assessed in 110 APS-1 patients by using immunoprecipitation. Autoantibodies to KCNRG were present in 7 of the 8 patients with respiratory symptoms, but in only 1 of 102 APS-1 patients without respiratory symptoms. Expression of KCNRG messenger RNA and protein was found to be predominantly restricted to the epithelial cells of terminal bronchioles. Autoantibodies to KCNRG, a protein mainly expressed in bronchial epithelium, are strongly associated with pulmonary involvement in APS-1. These findings may facilitate the recognition, diagnosis, characterization, and understanding of the pulmonary manifestations of APS-1.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-98040 (URN)10.1073/pnas.0809986106 (DOI)000264278800062 ()19251657 (PubMedID)
    Available from: 2009-02-11 Created: 2009-02-11 Last updated: 2017-12-14Bibliographically approved
  • 23.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bjerke, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Lab Med, SE-14186 Stockholm, Sweden..
    Edlund, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
    Nelander, Sven
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Westermark, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Origin of the U87MG glioma cell line: Good news and bad news2016In: Science Translational Medicine, ISSN 1946-6234, E-ISSN 1946-6242, Vol. 8, no 354, article id 354re3Article in journal (Refereed)
    Abstract [en]

    Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.

  • 24.
    Allkja, Jontana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The influence of copper on mast cell during bacterial infections2017Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    Mast cells are innate immune cells generally known for their harmful effects, particularly their role in asthma and autoimmune diseases. However, they can also recognize pathogens and mount a response to them. Copper is essential for proper cell function and homeostasis. Research has shown that changes in copper levels can affect mast cell morphology and gene expression of many of its immune mediators. It has also been shown that the copper transporter Ctr2 plays an important role in mast cell physiology. In this project, we investigated whether the same effects can be seen when mast cells are stimulated with bacteria, namely Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). For this purpose, wild type and Ctr2 knockout mast cells were treated with different concentrations of copper for 72 hours and later co-cultured with either S. aureus or E. coli. Wild type cells were also treated with a copper chelator to investigate the effect of copper starvation. Release of IL-6 and CCL2, two common mediators released in response to bacteria, were measured by ELISA, while their gene expression was measured by qPCR. Results showed that either copper starvation or, 10 μM copper cause an increase in IL-6 and CCL2 protein levels, but have no effect on gene expression. The 10 μM copper effect was not observed in the Ctr2 knockout cells. In conclusion, not much is known about the mechanisms involved in the mast cell response to bacteria. However, it appears that copper can affect either different stages of the same pathway, or different pathways in a similar manner.

  • 25.
    Amandusson, Åsa
    Klin o experimentell medicin, Linköpings universitet.
    Estrogen receptor expression in relation to pain modulation and transmission: experimental studies in rats2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [sv]

    Estrogens have a remarkably wide range of actions in the mammalian brain. They not only play a pivotal role in reproductive behavior and sexual differentiation, but also contribute to e.g. thermoregulation, feeding, memory, neuronal survival and the perception of somatosensory stimuli. A multitude of studies on both animals and human subjects has demonstrated potential effects of gonadal hormones, such as estrogens, on pain transmission. These effects most likely involve multiple neuroanatomical circuits as well as diverse neurochemical systems and therefore need to be evaluated specifically in relation to the localization and intrinsic characteristics of the neurons engaged. The overall aim of this thesis is to gain specific knowledge of the possible cellular mechanisms by which estrogens may influence the transmission of nociceptive stimuli at the level of the spinal cord.

    The estrogen receptors, by which estrogens regulate non-genomic as well as genomic mechanisms, are crucial to estrogen signaling in general and essential to the estrogen-induced effects in the brain. In Paper I, we use immunohistochemistry to label neurons containing estrogen receptor-! (ERα) in the medullary and spinal dorsal horn of female rats. Large numbers of ER!-expressing neurons were found in lamina I and lamina II, i.e. in the areas involved in the processing of primary afferent nociceptive information. This distribution in part overlaps that of enkephalin, a potent pain-inhibiting endogenous opioid. The effects of gonadal hormones on pain modulation may, to a great extent, be blocked by the opioid antagonist naloxone, suggesting an involvement of the endogenous opioid system in the prosecution of hormonal pain regulation. By combining immunohistochemical labeling of ERα with in situ hybridization of preproenkephalin mRNA (Paper II), we demonstrate that the majority of enkephalinergic neurons in the superficial laminae of the spinal and medullary dorsal horn express ER!. This co-localization and the fact that the preproenkephalin gene contains a sequence that binds ERs, suggest that estrogens may potentially regulate enkephalin expression in these cells. This is further supported by the findings in Paper III in which we show that a single subcutaneous injection of estradiol induces a significant increase (on average 68%) in preproenkephalin mRNA content in the spinal cord after 4 hours. The expression of the enkephalin gene in the spinal cord is thus sensitive to fluctuating estradiol levels. In Paper IV, a noxious injection of formalin is used to induce activation of a neuronal population involved in nociceptive transmission from the face. By using a dual-labeling immunohistochemistry protocol, we were able to identify ER!-expressing cells within this neuronal population suggesting that nociceptive-responsive neurons in the medullary dorsal horn express ER!. In all, our findings provide morphological as well as biochemical evidence in support for an estrogen-dependent modulation of nociceptive processing at the level of the dorsal horn.

    List of papers
    1. Estrogen receptor-α expression in nociceptive-responsive neurons in the medullary dorsal horn of the female rat
    Open this publication in new window or tab >>Estrogen receptor-α expression in nociceptive-responsive neurons in the medullary dorsal horn of the female rat
    2010 (English)In: European Journal of Pain, ISSN 1090-3801, E-ISSN 1532-2149, Vol. 14, no 3, p. 245-248Article in journal (Refereed) Published
    Abstract [en]

    Estrogens exert a substantial influence on the transmission of nociceptive stimuli and the susceptibility to pain disorders as made evident by studies in both animals and human subjects. The estrogen receptor (ER) seems to be of crucial importance to the cellular mechanisms underlying such an influence. However, it has not been clarified whether nociceptive neurons activated by pain express ERs. In this study, a noxious injection of formalin was given into the lower lip of female rats, thereby activating nociceptive neurons in the trigeminal subnucleus caudalis as demonstrated by immunohistochemical labeling of Fos. Using a dual-label immunohistochemistry protocol ERalpha-containing cells were visualized in the same sections. In the superficial layers of the medullary dorsal horn, 12% of ERalpha-labeled cells, mainly located in lamina II, also expressed noxious-induced Fos. These findings show that nociceptive-responsive neurons in the medullary dorsal horn express ERalpha, thus providing a possible morphological basis for the hypothesis that estrogens directly regulate pain transmission at this level.

    Place, publisher, year, edition, pages
    Elsevier, 2010
    Keywords
    Estrogen receptor, Spinal trigeminal nucleus, Gonadal hormone, Pain, Fos
    National Category
    Physiology
    Research subject
    Clinical Neurophysiology
    Identifiers
    urn:nbn:se:uu:diva-120674 (URN)10.1016/j.ejpain.2009.05.008 (DOI)19525133 (PubMedID)
    Available from: 2010-03-15 Created: 2010-03-15 Last updated: 2018-01-12Bibliographically approved
    2. Estrogen-induced alterations of spinal cord enkephalin gene expression
    Open this publication in new window or tab >>Estrogen-induced alterations of spinal cord enkephalin gene expression
    Show others...
    1999 (English)In: Pain, ISSN 0304-3959, E-ISSN 1872-6623, Vol. 83, no 2, p. 243-248Article in journal (Refereed) Published
    Abstract [en]

    Enkephalin-synthesizing neurons in the superficial laminae of the spinal and trigeminal dorsal horn are critical components of the endogenous pain-modulatory system. We have previously demonstrated that these neurons display intracellular estrogen receptors, suggesting that estrogen can potentially influence their enkephalin expression. By using Northern blot, we now show that a bolus injection of estrogen results in a rapid increase in spinal cord enkephalin mRNA levels in ovariectomized female rats. Thus, 4 h after estrogen administration the enkephalin mRNA-expression in the lumbar spinal cord was on average 68% higher (P<0.05) than in control animals injected with vehicle only. A small increase in the amount of enkephalin mRNA was also seen after 8 h (P<0.05), whereas no difference between estrogen-injected and control animals was found after 24 h or at time periods shorter than 4 h. Taken together with the previous anatomical data, the present findings imply that estrogen has an acute effect on spinal opioid levels in areas involved in the transmission of nociceptive information.

    National Category
    Cell and Molecular Biology
    Research subject
    Medical Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-125109 (URN)10.1016/S0304-3959(99)00109-8 (DOI)10534596 (PubMedID)
    Available from: 2010-05-07 Created: 2010-05-07 Last updated: 2018-01-12Bibliographically approved
    3. Estrogen receptor-like immunoreactivity in the medullary and spinal dorsal horn of the female rat
    Open this publication in new window or tab >>Estrogen receptor-like immunoreactivity in the medullary and spinal dorsal horn of the female rat
    1995 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 196, no 1-2, p. 25-28Article in journal (Refereed) Published
    Abstract [en]

    Using an immunohistochemical technique, we demonstrate that large numbers of neurons in the laminar spinal trigeminal nucleus and spinal gray matter of the female rat express estrogen receptors (ER). Densely packed ER-immunoreactive neurons were present in lamina II, but labeled neurons were also present in lamina I, the neck of the dorsal horn, and in lamina X. Labeling was present throughout the length of the spinal cord, with the exception of segments caudal to S1, which were unlabeled. The distribution of ER-containing neurons to areas that are involved in processing of primary afferent nociceptive information suggests that the pain modulatory effects of estrogen may be exerted at the spinal level.

    National Category
    Physiology
    Research subject
    Clinical Neurophysiology
    Identifiers
    urn:nbn:se:uu:diva-125104 (URN)7501248 (PubMedID)
    Available from: 2010-05-07 Created: 2010-05-07 Last updated: 2018-01-12Bibliographically approved
    4. Colocalization of oestrogen receptor immunoreactivity and preproenkephalin mRNA expression fo neurons in the superficial laminae of the spinal and medullary dorsal horn of rats
    Open this publication in new window or tab >>Colocalization of oestrogen receptor immunoreactivity and preproenkephalin mRNA expression fo neurons in the superficial laminae of the spinal and medullary dorsal horn of rats
    1996 (English)In: Eur J Neurosci, Vol. 8, no 11, p. 2240-2245Article in journal (Refereed) Published
    Abstract [en]

    A double-labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor-like immunoreactivity and preproenkephalin-A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double-labelled, displaying both oestrogen receptor-like-immunoreactivity and preproenkephalin-A mRNA; cell counts showed that 40-60% of the of the oestrogen receptor-like-immunoreactive cells in the superficial laminae also were labelled for preproenkephalin-A mRNA, and that 60-70% of the preproenkephalin-A mRNA-labelled neurons in the same laminae displayed oestrogen receptor-like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin-A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.

    National Category
    Cell and Molecular Biology
    Research subject
    Medical Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-125106 (URN)8950107 (PubMedID)
    Available from: 2010-05-07 Created: 2010-05-07 Last updated: 2018-01-12Bibliographically approved
  • 26.
    Amandusson, Åsa
    et al.
    Klin o experimentell medicin.
    Hallbeck, M
    Hallbeck, AL
    Hermansson, O
    Blomqvist, A
    Estrogen-induced alterations of spinal cord enkephalin gene expression1999In: Pain, ISSN 0304-3959, E-ISSN 1872-6623, Vol. 83, no 2, p. 243-248Article in journal (Refereed)
    Abstract [en]

    Enkephalin-synthesizing neurons in the superficial laminae of the spinal and trigeminal dorsal horn are critical components of the endogenous pain-modulatory system. We have previously demonstrated that these neurons display intracellular estrogen receptors, suggesting that estrogen can potentially influence their enkephalin expression. By using Northern blot, we now show that a bolus injection of estrogen results in a rapid increase in spinal cord enkephalin mRNA levels in ovariectomized female rats. Thus, 4 h after estrogen administration the enkephalin mRNA-expression in the lumbar spinal cord was on average 68% higher (P<0.05) than in control animals injected with vehicle only. A small increase in the amount of enkephalin mRNA was also seen after 8 h (P<0.05), whereas no difference between estrogen-injected and control animals was found after 24 h or at time periods shorter than 4 h. Taken together with the previous anatomical data, the present findings imply that estrogen has an acute effect on spinal opioid levels in areas involved in the transmission of nociceptive information.

  • 27.
    Amandusson, Åsa
    et al.
    Klin o experimentell medicin.
    Hermansson, O
    Blomqvist, A
    Colocalization of oestrogen receptor immunoreactivity and preproenkephalin mRNA expression fo neurons in the superficial laminae of the spinal and medullary dorsal horn of rats1996In: Eur J Neurosci, Vol. 8, no 11, p. 2240-2245Article in journal (Refereed)
    Abstract [en]

    A double-labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor-like immunoreactivity and preproenkephalin-A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double-labelled, displaying both oestrogen receptor-like-immunoreactivity and preproenkephalin-A mRNA; cell counts showed that 40-60% of the of the oestrogen receptor-like-immunoreactive cells in the superficial laminae also were labelled for preproenkephalin-A mRNA, and that 60-70% of the preproenkephalin-A mRNA-labelled neurons in the same laminae displayed oestrogen receptor-like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin-A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.

  • 28.
    Amin, Kawa
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
    The relationship between inflammation and structural changes in the airways of the lower and upper respiratory tract: Studies in patients with asthma, Sjögren's syndrome, rhinitis and children with otitis media with effusion2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The pathophysiology of asthma, Sjögrens syndrome (SS), rhinitis, and otitis media with effusion (OME) in children has been extensively investigated in upper and lower respiratory tract, respectively, and shown to comprise structural changes in the airways and involvement of inflammatory cells. By comparing diseases that have bronchial hyperresponsivenses or mucosal inflammation as a common denominator, it may be possible to learn more about the mechanisms underlying inflammation in the upper and lower respiratory tract.

    With immunohistochemical techniques and a panel of monoclonal antibodies, inflammatory cells were identified and structural changes of the airway were quantitatively studied in the bronchial, nasal and middle ear mucosa and submucosa.

    The highest number of eosinophils and mast ceils in bronchial, nasal or middle ear biopsies was found in patients with atopic asthma (AA), perennial non-allergic rhinitis (PNAR) and children with OME. The number of the neutrophils was highest in SS, non-atopic asthma (NAA) and children with OME. The number of T lymphocytes in SS and AA was significantly higher than in NAA and healthy controls (HC). The degree of epithelial damage was higher in the AA group andin patients with perennial allergic rhinitis where the biopsy was taken within the pollen season (PARSEASON) group compared to the other patient groups. The tenascin- and Iaminin-positive layers in AA and SS were thicker than other groups. In AA, and PARSEASON a significant negative correlation was found between epithelial integrity and the count for eosinophils or neutrophils. The most pronounced epithelial damage was observed in patients with allergic rhinitis in areas characterized by an increased number of inflammatory cells. Eosinophils in asthmatic and PARSEASON patients and neutrophils in SS were found in the area of epithelial damage.

    This work has demonstrated a quantitatively different inflammatory profile in AA, NAA and SS, different profiles in perennial allergic and non-allergic rhinitis and a specific inflammatory profile in the middle ear of children with OME suggesting differences in the pathogenesis of respiratory tract diseases.

  • 29.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Analysis of somatropin by double-injection capillary-zone electrophoresis in polybrene/chondroitin sulfate a double-coated capillaries2016In: Capillary Electrophoresis of Proteins and Peptides: Methods and Protocols / [ed] Nguyet Thuy Tran, Myriam Taverna, New York: Springer-Verlag New York, 2016, p. 93-105Chapter in book (Refereed)
    Abstract [en]

    Purity determination of somatropin as a recombinant protein is important to ensure its safety and quality. This is carried out by capillary zone electrophoresis in double-injection mode using polybrene/chondroitin sulfate A double-coated capillaries. Modification of the capillary wall eliminates protein-wall interactions which results in improved accuracy and precision of the determinations. In the double-injection mode two somatropin samples are analyzed within a single electrophoretic run. Prior to the second injection, the first injected plug is electrophoresed for a predetermined time period in order to adjust the inter- plug distance. Here, the principle for the separation of somatropin charge variants is described.

  • 30.
    Andersson, Annika K.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
    Role of Inducible Nitric Oxide Synthase and Melatonin in Regulation of β-cell Sensitivity to Cytokines2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The mechanisms of β-cell destruction leading to type 1 diabetes are complex and not yet fully understood, but infiltration of the islets of Langerhans by autoreactive immune cells is believed to be important. Activated macrophages and T-cells may then secrete cytokines and free radicals, which could selectively damage the β-cells. Among the cytokines, IL-1β, IFN-γ and TNF-α can induce expression of inducible nitric synthase (iNOS) and cyclooxygenase-2. Subsequent nitric oxide (NO) and prostaglandin E2 (PGE2) formation may impair islet function.

    In the present study, the ability of melatonin (an antioxidative and immunoregulatory hormone) to protect against β-cell damage induced by streptozotocin (STZ; a diabetogenic and free radical generating substance) or IL-1β exposure was examined. In vitro, melatonin counteracted STZ- but not IL-1β-induced islet suppression, indicating that the protective effect of melatonin is related to interference with free radical generation and DNA damage, rather than NO synthesis. In vivo, non-immune mediated diabetes induced by a single dose of STZ was prevented by melatonin.

    Furthermore, the effects of proinflammatory cytokines were examined in islets obtained from mice with a targeted deletion of the iNOS gene (iNOS -/- mice) and wild-type controls. The in vitro data obtained show that exposure to IL-1β or (IL-1β + IFN-γ) induce disturbances in the insulin secretory pathway, which were independent of NO or PGE2 production and cell death. Initially after addition, in particular IL-1β seems to be stimulatory for the insulin secretory machinery of iNOS –/- islets, whereas IL-1β acts inhibitory after a prolonged period. Separate experiments suggest that the stimulatory effect of IL-1β involves an increased gene expression of phospholipase D1a/b. In addition, the formation of new insulin molecules appears to be affected, since IL-1β and (IL-1β + IFN-γ) suppressed mRNA expression of both insulin convertase enzymes and insulin itself.

    List of papers
    1. Melatonin protects against streptozotocin, but not Interleukin-1β-induced damage of rodent pancreatic β-cells
    Open this publication in new window or tab >>Melatonin protects against streptozotocin, but not Interleukin-1β-induced damage of rodent pancreatic β-cells
    2001 In: J. Pineal Res., ISSN 0742-3098, Vol. 30, no 3, p. 157-165Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-90711 (URN)
    Available from: 2003-09-03 Created: 2003-09-03Bibliographically approved
    2. Cytokine-induced inhibition of insulin release from mouse pancreatic β-cells deficient in inducible nitric oxide synthase
    Open this publication in new window or tab >>Cytokine-induced inhibition of insulin release from mouse pancreatic β-cells deficient in inducible nitric oxide synthase
    2001 In: Biochem. Biophys. Res. Commun., ISSN 0006-291, Vol. 281, no 2, p. 396-403Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-90712 (URN)
    Available from: 2003-09-03 Created: 2003-09-03Bibliographically approved
    3. Cytokine-induced prostaglandin E2 formation is reduced from mouse pancreatic islets deficient in inducible nitric oxide synthase
    Open this publication in new window or tab >>Cytokine-induced prostaglandin E2 formation is reduced from mouse pancreatic islets deficient in inducible nitric oxide synthase
    (English)Manuscript (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-90713 (URN)
    Available from: 2003-09-03 Created: 2003-09-03 Last updated: 2018-01-13
    4. Role of phospholipase D, insulin and proinsulin convertase gene expression in altered insulin secretion from β-cells deficient in inducible nitric oxide synthase following IL-1β and IFN-γ exposure
    Open this publication in new window or tab >>Role of phospholipase D, insulin and proinsulin convertase gene expression in altered insulin secretion from β-cells deficient in inducible nitric oxide synthase following IL-1β and IFN-γ exposure
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-90714 (URN)
    Available from: 2003-09-03 Created: 2003-09-03 Last updated: 2010-01-13Bibliographically approved
  • 31.
    Andersson, Annika K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Thorvaldson, Lina
    Carlsson, Carina
    Sandler, Stellan
    Cytokine-induced prostaglandin E2 formation is reduced from mouse pancreatic islets deficient in inducible nitric oxide synthaseManuscript (Other academic)
  • 32.
    Andersson, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Department of Medical Cell Biology: Annual Report 20072008Collection (editor) (Other (popular science, discussion, etc.))
  • 33.
    Andersson, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Department of Medical Cell Biology: Annual Report 20082009Collection (editor) (Other (popular science, discussion, etc.))
  • 34. Andersson, Cecilia
    et al.
    Henriksson, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy.
    Mirazimi, Ali
    Different localization of CCHFV vRNA compared cRNA during infection as determined by in situ padlock probe detectionManuscript (preprint) (Other academic)
  • 35.
    Andersson, Charlotte
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
    Towards Pharmacological Treatment of Cystic Fibrosis2002Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated.

    Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated.

    Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate in vitro evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease.

    Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.

    List of papers
    1. Activation of deltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX
    Open this publication in new window or tab >>Activation of deltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX
    2000 In: Eur Respir J., Vol. 15, no 5, p. 937-41Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-89966 (URN)
    Available from: 2002-10-11 Created: 2002-10-11Bibliographically approved
    2. Activation of CFTR by genistein in human airway epithelial cell lines
    Open this publication in new window or tab >>Activation of CFTR by genistein in human airway epithelial cell lines
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-89967 (URN)
    Available from: 2002-10-11 Created: 2002-10-11 Last updated: 2010-01-13Bibliographically approved
    3. S-Nitrosoglutathione induces functional ?F508-CFTR in airway epithelial cells
    Open this publication in new window or tab >>S-Nitrosoglutathione induces functional ?F508-CFTR in airway epithelial cells
    2002 In: Biochem Biophys Res Commun., Vol. 297, p. 552-557Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-89968 (URN)
    Available from: 2002-10-11 Created: 2002-10-11Bibliographically approved
    4. Determination of chloride efflux by X-ray microanalysis versus MQAE-fluorescence
    Open this publication in new window or tab >>Determination of chloride efflux by X-ray microanalysis versus MQAE-fluorescence
    2002 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 59, no 6, p. 531-355Article in journal (Refereed) Published
    Abstract [en]

    The importance of chloride channels for the cell is demonstrated by a number of serious human diseases that are due to mutations in chloride channels. The most well-known of these diseases is cystic fibrosis. Investigations into the mechanisms of the disease and possible treatments require the study of chloride fluxes at the level of individual cells. The present study compares two methods for studies of chloride transport: X-ray microanalysis and MQAE fluorescence with image analysis. As an experimental system, the cAMP-activated chloride channel in cultured respiratory epithelial cells was chosen. Both methods showed that stimulation with the cAMP-elevating agents forskolin and IBMX decreased the chloride content of the cells by about 20-27%. Inducing a driving force for chloride by replacing extracellular chloride by nitrate resulted in a chloride efflux that was significantly increased in the presence of forskolin and IBMX. This study shows that X-ray microanalysis and MQAE fluorescence are adequate and comparable methods for measuring cAMP-dependent chloride transport in individual cells.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89969 (URN)10.1002/jemt.10234 (DOI)12467030 (PubMedID)
    Available from: 2002-10-11 Created: 2002-10-11 Last updated: 2017-12-14Bibliographically approved
    5. Assessment of chloride secretion in human nasal epithelial cells by X-ray microanalysis
    Open this publication in new window or tab >>Assessment of chloride secretion in human nasal epithelial cells by X-ray microanalysis
    2001 In: J Microsc., Vol. 203, no 3, p. 277-84Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-89970 (URN)
    Available from: 2002-10-11 Created: 2002-10-11Bibliographically approved
    6. CFTR) activity in nasal epithelial cells from cystic fibrosis patients with severe genotypes
    Open this publication in new window or tab >>CFTR) activity in nasal epithelial cells from cystic fibrosis patients with severe genotypes
    2002 In: Clin Sci., Vol. 103, p. 417-424Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-89971 (URN)
    Available from: 2002-10-11 Created: 2002-10-11Bibliographically approved
  • 36.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gene amplification and adaptive evolution in bacteria2009In: Annual Review of Genetics, ISSN 0066-4197, E-ISSN 1545-2948, Vol. 43, p. 167-195Article, review/survey (Refereed)
    Abstract [en]

    Gene duplication-amplification (GDA) processes are highly relevant biologically because they generate extensive and reversible genetic variation on which adaptive evolution can act. Whenever cellular growth is restricted, escape from these growth restrictions often occurs by GDA events that resolve the selective problem. In addition, GDA may facilitate subsequent genetic change by allowing a population to grow and increase in number, thereby increasing the probability for subsequent adaptive mutations to occur in the amplified genes or in unrelated genes. Mathematical modeling of the effect of GDA on the rate of adaptive evolution shows that GDA will facilitate adaptation, especially when the supply of mutations in the population is rate-limiting. GDA can form via several mechanisms, both RecA-dependent and RecA-independent, including rolling-circle amplification and nonequal crossing over between sister chromatids. Due to the high intrinsic instability and fitness costs associated with GDAs, they are generally transient in nature, and consequently their evolutionary and medical importance is often underestimated.

  • 37.
    Andersson, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
    Bringing time into molecular and cellular biology2013In: Journal of Analytical Oncology, ISSN 1927-7229, Vol. 2, no 2, p. 65-68Article in journal (Refereed)
    Abstract [en]

    In conjunction with the defense of a doctoral thesis on the deciphering of complex protein interactions on living cells, six scientists shared their view on time in molecular and cellular biology. This brief review takes the form of a conference report and summarizes the contributions of the speakers and the defense. Opportunities and challenges for time resolved assays in molecular and cellular biology were vividly discussed during two days with a pan-European audience. Awareness of biological timeframes and understanding the temporal aspects were claimed critical for analytical applications in biology.

  • 38.
    Andersson, Ki
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Earth Sciences, Department of Earth Sciences, Palaeontology group.
    Aspects of locomotor evolution in the Carnivora (Mammalia)2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In this thesis, the shape of the distal humerus trochlea is analysed using landmark-based morphometrics and multivariate methods, with the aim of exploring locomotor evolution in carnivorans. Elbow joint morphology is used together with body size and craniodental morphology to characterize past and present carnivorans. Evolutionary implications are studied at the ordinal, familial, and species levels, testing specific hypotheses about scaling, morphological constraints, evolutionary trajectories, and potential for social pack-hunting behaviour. The circumference of the distal humerus trochlea is found to be highly correlated with body mass, and appears to scale similarly throughout the order Carnivora. A general predictive model for carnivoran bodymass is presented (a=0.601; b= 2.552; r2=0.952, SEE=0.136, p<0001, n=92), which removes the need for the investigator to actively choose between the diverging estimates that different predictors and their equations often produce. At the elbow joint, manual manipulation and locomotion appear to be conflicting functions, thus suggesting mutually exclusive lifestyles involving either forelimb grappling or pursuit. At large body sizes, carnivorans are distributed over a strongly dichotomised pattern (grappling or locomotion), a pattern coinciding with the postulated threshold in predator-prey size ratio at 21.5-25 kg. This pattern is compared to that of two carnivoran faunas from the Tertiary. In the Oligocene (33.7-23.8 Myr BP), the overall pattern is remarkably similar to that observed for extant Carnivora. In the Miocene (23.8-11.2 Myr BP) carnivores show a similarly dichotomised pattern as the Oligocene and Recent, although the whole pattern is shifted towards larger body sizes. This difference is suggested to be a reflection of the extraordinary species richness of browsing ungulates in the early Miocene of North America. Such an increase in prey spectrum would create a unique situation, in which large carnivores need not commit to a cursorial habitus in order to fill their nutritional requirements. Finally, the elbow joints and craniodental morphology (14 measurements) of fossil canids were examined with the aim of assessing the potential for pack-hunting in fossil canids. It is clear that small and large members of the Recent Caninae share similar craniodental morphologies. However, this pattern is not present in Borophaginae and Hesperocyoninae. In the latter, large representatives are characterized by being short-faced, with reduced anterior premolars and enlarged posterior premolars, thus approaching a “pantherine-like” craniodental configuration. These traits are interpreted as an adaptation for killing prey with canine bites. It is similarly determined that, unlike recent Caninae, all analyzed species of borophagines and hesperocyonines have retained the ability to supinate their forearms. It is therefore likely that manual manipulation was part of their hunting behaviour, thus removing an essential part of the argument for social pack-hunting in these forms, as the benefits of such a strategy become less obvious.

    List of papers
    1. Predicting carnivoran body mass from a weight bearing joint
    Open this publication in new window or tab >>Predicting carnivoran body mass from a weight bearing joint
    2004 (English)In: Journal of Zoology, ISSN 0952-8369, E-ISSN 1469-7998, Vol. 262, no 2, p. 161-172Article in journal (Refereed) Published
    Abstract [en]

    Predictors used to calculate the body mass of extinct carnivorans often scale differently between different taxa, thus yielding body mass estimates that diverge considerably depending on which predictive equation is used. This requires the investigator to choose the ones most suitable, a procedure that is best avoided if possible. The carnivoran elbow joint is here explored with the aim of producing a single general body mass predictor that can be used over a broad range of terrestrial and arboreal carnivorans. The circumference of the distal humerus trochlea is found to be highly correlated with body mass, and trochlea circumference seems to scale similarly throughout the order Carnivora. This scaling is not as theoretically predicted by elastic similarity and is slightly higher than that predicted by geometric similarity, indicating a slight positive allometry for the latter. Some degree of differential scaling between carnivoran families and between animals of large and small size cannot be ruled out, but this result is inconclusive. A predictive model that allows mass estimations for a broad range of carnivorans is presented (a=0.601; b=2.552; r2=0.952, SEE=0.136, P<0001, n=92). Body mass for eight extinct carnivoran species are calculated and these generally conform to earlier mass predictions.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-90745 (URN)10.1017/S0952836903004564 (DOI)
    Available from: 2003-09-01 Created: 2003-09-01 Last updated: 2017-12-14Bibliographically approved
    2. Elbow-joint morphology as a guide to forearm function and foraging behaviour in mammalian carnivores
    Open this publication in new window or tab >>Elbow-joint morphology as a guide to forearm function and foraging behaviour in mammalian carnivores
    2004 (English)In: Zoological Journal of the Linnean Society, ISSN 0024-4082, E-ISSN 1096-3642, Vol. 142, no 1, p. 91-104Article in journal (Refereed) Published
    Abstract [en]

    Among the hunting strategies employed by members of the order Carnivora (Mammalia), two, stalk and ambush and sustained pursuit, are particularly prevalent among larger species of the order. It has been difficult to identify morphological traits that support this distinction and ecological observations have shown that most carnivorans adopt a continuum of strategies, depending on available habitat and prey. In this paper, the shape of the distal humerus articulation is analysed, with the aim of exploring the use of the forelimb in prey procurement, and as a guide to such behaviour among fossil carnivorans. The results suggest that manual manipulation and locomotion are conflicting functions. Elbow-joint morphology supports a division between grapplers (i.e. ambushers) and nongrapplers (i.e. pursuers). Joints of the former are characterized by being relatively wide and the latter, by being relatively narrow and box-like with pronounced stabilizing features. At intermediate and large body sizes, carnivorans show a pattern suggesting mutually exclusive feeding strategies that involve either grappling with prey or sustained pursuit. The former allows for large body sizes, such as pantherine felids and ursids; the latter includes species of only moderate size, such as hyenids and canids. Elbow-joint morphology is closely linked to phylogeny, but the morphology of the cheetah converges with that of nongrapplers, showing that strong selective forces may override the phylogenetic component. Two taxa of giant mustelids from the Miocene were analysed to test whether this sort of analysis is applicable to carnivorans of the past. The African Late Miocene species Ekorus ekakeran has a joint morphology comparable to that of modern-day nongrapplers. Two joint morphologies were found in the North American Late Oligocene-Early Miocene Megalictis ferox. The first morphology is comparable to that of modern pantherine cats and the second forms  an  intermediate  between  grapplers  and  nongrapplers  that  is  not  present  in  the  modern  carnivoran  fauna.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-90746 (URN)10.1111/j.1096-3642.2004.00129.x (DOI)
    Available from: 2003-09-01 Created: 2003-09-01 Last updated: 2017-12-14Bibliographically approved
    3. The evolution of cursorial carnivores in the Tertiary: implications of elbow-joint morphology
    Open this publication in new window or tab >>The evolution of cursorial carnivores in the Tertiary: implications of elbow-joint morphology
    2003 In: Biology letters, Vol. Published online 6 AugustArticle in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-90747 (URN)
    Available from: 2003-09-01 Created: 2003-09-01Bibliographically approved
    4. Potential for pack-hunting in Tertiary canids (Canidae, Carnivora)
    Open this publication in new window or tab >>Potential for pack-hunting in Tertiary canids (Canidae, Carnivora)
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-90748 (URN)
    Available from: 2003-09-01 Created: 2003-09-01 Last updated: 2010-01-13Bibliographically approved
  • 39.
    Andrén, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Induction of HPV-16 Late Gene Expression Through Use of Small Molecule Drugs2016Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Cervical cancer is the second most common cancer in women worldwide. The principal cause of cervical cancer is infection with human papillomavirus (HPV). HPV-16 is a high-risk virus and it is responsible for a high portion of all HPV-caused cancers. The HPV-16 genome consists of early and late genes. The virus initially infects basal cells of the cervix epithelium and in these cells early genes are expressed, whilst late genes, L1 and L2, are only expressed in the upper cell layers of the epithelium. Proteins encoded by the late genes are highly immunogenic, thus it is speculated that expression of the late genes earlier in the virus life cycle could lead to clearance of the virus due to interference of the immune system.

        The aim of this study was to treat reporter cell lines with three different small molecule drugs to see if they had the ability to induce HPV-16 late gene expression. The reporter cell lines used in this study had been previously created by transfecting HeLa-cells with plasmids representing the HPV-16 genome. In these plasmids, L1 is replaced with a CAT reporter gene that encodes the CAT protein, which can be easily quantified using a sandwich ELISA.

        Upon treating the reporter cell lines with TPA, a significant induction of late gene expression was detected. Furthermore, treatment with valproic acid showed some induction of late gene expression. In conclusion, TPA and valproic acid was deemed to have potential to act as a candidate drugs for treatment of HPV infections. 

  • 40.
    Annerén, Cecilia
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
    The Tyrosine Kinase GTK: Signal Transduction and Biological Function2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Protein tyrosine kinases play an important role in the regulation of various cellular processes such as

    growth, differentiation and survival. GTK, a novel SRC-like cytoplasmic tyrosine kinase, was recently cloned from a mouse insulinoma cell line and the present work was conducted in order to find a biological function of GTK in insulin producing and neuronal cells. It was observed that kinase active GTK-mutants, expressed in RINm5F cells, transferred to the cell nucleus and increased the levels of the cell cycle regulatory protein p27KIP1, reduced cell growth and stimulated glucagon mRNA expression. Furthermore, wild type GTK induces neurite outgrowth in the rat adrenal pheochromocytoma PC12 cell line, through activation of the RAP1-pathway, suggesting a role of GTK for cell differentiation. Studies using transgenic mice, expressing GTK under the control of the rat insulin 1 promoter, demonstrated a dual role of GTK for β-cell growth: Whereas GTK increases the β-cell mass and causes enhanced β-cell proliferation in response to partial pancreatectomy it also induced β-cell death in response to proinflammatory cytokines and impaired the glucose tolerance in mice treated with the β-cell toxin streptozotocin suggesting a possible role of GTK for β-cell destruction in Type 1 diabetes. We have also observed that GTK-transgenic islets and GTK-expressing RINm5F cells exhibit a reduced insulininduced activation of the insulin receptor substrate (IRS-1 and IRS-2)-pathways, partly due to an increased basal activity of these. GTK was found to associate with and phosphorylate the SH2 domain adapter protein SHB, which could explain many of the GTK-dependent effects both in vitro and in vivo. In summary, the present work suggests that the novel tyrosine kinase GTK is involved in various signal transduction pathways, regulating different cellular responses, such as proliferation, differentiation and survival.

    List of papers
    1.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    2. Increased cytokine-induced cytotoxicity of pancreatic islet cells from transgenic mice expressing the Src-like tyrosine kinase GTK
    Open this publication in new window or tab >>Increased cytokine-induced cytotoxicity of pancreatic islet cells from transgenic mice expressing the Src-like tyrosine kinase GTK
    2001 (English)In: Mol Med., Vol. 7, p. 301-310Article in journal (Refereed) Published
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89441 (URN)
    Available from: 2001-09-25 Created: 2001-09-25 Last updated: 2012-12-17
    3. Dual role of the tyrosine kinase GTK and the adaptor protein SHB in β-cell growth: enhanced β-cell replication after 60% pancreatectomy and increased sensitivity to streptozotocin
    Open this publication in new window or tab >>Dual role of the tyrosine kinase GTK and the adaptor protein SHB in β-cell growth: enhanced β-cell replication after 60% pancreatectomy and increased sensitivity to streptozotocin
    2002 (English)In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 172, no 1, p. 145-153Article in journal (Refereed) Published
    Abstract [en]

    Transgenic CBA mice expressing either the tyrosine kinase GTK (gut tyrosine kinase) or the adaptor protein SHB (Src homology 2 protein of beta-cells) under the control of the rat insulin promoter exhibited an increased beta-cell mass, but also elevated cytokine-induced islet cell death compared with control mice. To further investigate the importance of GTK and SHB for beta-cell death and proliferation, these mice were subjected to a 60% partial pancreatectomy (Px) or a sham-operation and beta-cell replication was determined by autoradiographic detection of [(3)H]thymidine incorporation into islet cells positively stained for insulin. The Px-operated control mice exhibited a moderate and insignificant increase in beta-cell replication 4 days after Px compared with the sham-operated mice (0.27+/-0.08% vs 0.08+/-0.02%). In contrast, the Px-induced beta-cell proliferation was significantly increased in both the GTK- and SHB-transgenic mice compared with the corresponding sham-treated animals (0.64+/-0.12% vs 0.11+/-0.04% and 0.44+/-0.11% vs 0.09+/-0.04% respectively). This effect was dependent on intracellular signal transduction pathways activated or enhanced by GTK and SHB overexpression, since the proliferation of acinar cells, located in the vicinity of the islets, was equal in the transgenic and control mice. GTK- and SHB-transgenic mice, treated with a sub-diabetogenic dose of the beta-cell toxin streptozotocin (STZ) on day 0 and subjected to a glucose tolerance test on day 3, exhibited an impaired glucose tolerance in comparison with the STZ-treated control mice. Pretreatment with STZ blunted the regenerative response to Px in the transgenic mice. Furthermore, the SHB-transgenic islets were significantly more damaged with respect to beta-cell loss, compared with the islets of the control mice. Previous and present data suggest a dual role of GTK and SHB for beta-cell growth: whereas these proteins increase the beta-cell mass and induce beta-cell proliferation after 60% Px, SHB and GTK also enhance beta-cell death under certain stressful conditions.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89442 (URN)10.1677/joe.0.1720145 (DOI)000173471200013 ()11786382 (PubMedID)
    Available from: 2001-09-25 Created: 2001-09-25 Last updated: 2017-12-14Bibliographically approved
    4.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    5. GTK, a Src-related tyrosine kinase, induces nerve growth factor-independent neurite outgrowth in PC12 cells through activation of the Rap1 pathway: Relationship to Shb tyrosine phosphorylation and elevated levels of focal adhesion kinase
    Open this publication in new window or tab >>GTK, a Src-related tyrosine kinase, induces nerve growth factor-independent neurite outgrowth in PC12 cells through activation of the Rap1 pathway: Relationship to Shb tyrosine phosphorylation and elevated levels of focal adhesion kinase
    2000 (English)In: J Biol Chem, Vol. 275, p. 29153-29161Article in journal (Refereed) Published
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-89444 (URN)
    Available from: 2001-09-25 Created: 2001-09-25 Last updated: 2012-12-17
  • 41.
    Annerén, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tyrosine kinase signalling in embryonic stem cells2008In: Clinical Science, ISSN 0143-5221, E-ISSN 1470-8736, Vol. 115, no 1-2, p. 43-55Article, review/survey (Refereed)
    Abstract [en]

    Pluripotent ES (embryonic stem) cells can be expanded in culture and induced to differentiate into a wide range of cell types. Self-renewal of ES cells involves proliferation with concomitant suppression of differentiation. Some critical and conserved pathways regulating self-renewal in both human and mouse ES cells have been identified, but there is also evidence suggesting significant species differences. Cytoplasmic and receptor tyrosine kinases play important roles in proliferation, survival, self-renewal and differentiation in stem, progenitor and adult cells. The present review focuses on the role of tyrosine kinase signalling for maintenance of the undifferentiated state, proliferation, survival and early differentiation of ES cells.

  • 42.
    Annerén, Cecilia
    et al.
    Harvard University, Department of Molecular and Cellular Biology.
    Cowan, Chad A
    Harvard University, Department of Molecular and Cellular Biology.
    Melton, Douglas A
    Harvard University, Department of Molecular and Cellular Biology.
    The Src family of tyrosine kinases is important for embryonic stem cell self-renewal.2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 30, p. 31590-8Article in journal (Refereed)
    Abstract [en]

    cYes, a member of the Src family of non-receptor tyrosine kinases, is highly expressed in mouse and human embryonic stem (ES) cells. We demonstrate that cYes kinase activity is regulated by leukemia inhibitory factor (LIF) and serum and is down-regulated when cells differentiate. Moreover, selective chemical inhibition of Src family kinases decreases growth and expression of stem cell genes that mark the undifferentiated state, including Oct3/4, alkaline phosphatase, fibroblast growth factor 4, and Nanog. A synergistic effect on differentiation is observed when ES cells are cultured with an Src family inhibitor and low levels of retinoic acid. Src family kinase inhibition does not interfere with LIF-induced JAK/STAT3 (Janus-associated tyrosine kinases/signal transducer and activator of transcription 3) or p42/p44 MAPK (mitogen-activated protein kinase) phosphorylation. Together the results suggest that the activation of the Src family is important for maintaining mouse and human ES in an undifferentiated state and may represent a third, independent pathway, downstream of LIF in mouse ES cells.

  • 43.
    Annerén, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lindholm, Cecilia K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kriz, Vitezslav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    The FRK/RAK-SHB signaling cascade: a versatile signal-transduction pathway that regulates cell survival, differentiation and profileration2003In: Current molecular medicine, ISSN 1566-5240, E-ISSN 1875-5666, Vol. 3, no 4, p. 313-324Article in journal (Refereed)
    Abstract [en]

    Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK / RAK (also named BSK / IYK or GTK). FRK / RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and β-cells, where they both induce PC12 cell differentiation and β-cell proliferation. Furthermore, β-cell apoptosis is augmented by these proteins under conditions that cause β-cell degeneration. The FRK / RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2.

    Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development.

    In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express this kinase.

  • 44.
    Annerén, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    GTK Tyrosine Kinase-induced Alteration of IRS-protein Signalling in Insulin Producing Cells2002In: Molecular medicine (Cambridge, Mass. Print), ISSN 1076-1551, E-ISSN 1528-3658, Vol. 8, no 11, p. 705-713Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Insulin receptor substrate proteins (IRS) mediate various effects of insulin, including regulation of glucose homeostasis, cell growth and survival. To understand the underlying mechanisms explaining the effects of the Src-related tyrosine kinase GTK on beta-cell proliferation and survival, insulin-signalling pathways involving IRS-1 and IRS-2 were studied in islet cells and RINm5F cells overexpressing wild-type and two different mutants of the SRC-related tyrosine kinase GTK. MATERIALS AND METHODS: Islets isolated from transgenic mice and RINm5F cells overexpressing wild-type and mutant GTK were analysed for IRS-1, IRS-2, SHB, AKT and ERK phosphorylation/activity by Western blot analysis. RESULTS: RINm5F cells expressing the kinase active mutant Y504F-GTK and islet cells from GTK(Y504F) -transgenic mice exhibited reduced insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2. In RINm5F cells, the diminished IRS-phosphorylation was accompanied by a reduced insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3K), AKT and Extracellular Signal-Regulated Kinase, partly due to an increased basal activity. In addition, increased tyrosine phosphorylation of the SHB SH2 domain-adaptor protein and its association with IRS-2, IRS-1 and focal adhesion kinase was observed in these cells. RINm5F cells overexpressing wild-type GTK also exhibited reduced activation of IRS-2, PI3K and AKT, whereas cells expressing a GTK mutant with lower kinase activity (GTK(Y394F)) exhibited insignificantly altered responses to insulin compared to the mock transfected cells. Moreover, GTK was shown to associate with and phosphorylate SHB in transiently transfected COS-7 cells, indicating that SHB is a specific substrate for GTK. CONCLUSIONS: The results suggest that GTK signals via SHB to modulate insulin-stimulated pathways in beta cells and this may explain previous results showing an increased beta-cell mass in GTK-transgenic mice.

  • 45.
    Anvari, Ebrahim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wikström, Per
    Walum, Erik
    Welsh, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice2015In: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 49, no 11, p. 1308-1318Article in journal (Refereed)
    Abstract [en]

    In type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) overactivity. Five different NOX enzymes (NOX1-5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet or HFD-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1-3 days), high-glucose-induced human islet cell reactive oxygen species (ROS) production and death were prevented by GLX351322. We propose that while short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4 activity, for example, during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX inhibition may be a therapeutic strategy in type 2 diabetes.

  • 46.
    Aqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Exceptionally large entropy contributions enable the high rates of GTP hydrolysis on the ribosome2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 15817Article in journal (Refereed)
    Abstract [en]

    Protein synthesis on the ribosome involves hydrolysis of GTP in several key steps of the mRNA translation cycle. These steps are catalyzed by the translational GTPases of which elongation factor Tu (EF-Tu) is the fastest GTPase known. Here, we use extensive computer simulations to explore the origin of its remarkably high catalytic rate on the ribosome and show that it is made possible by a very large positive activation entropy. This entropy term (T Delta S-double dagger) amounts to more than 7 kcal/mol at 25 degrees C. It is further found to be characteristic of the reaction mechanism utilized by the translational, but not other, GTPases and it enables these enzymes to attain hydrolysis rates exceeding 500 s(-1). This entropy driven mechanism likely reflects the very high selection pressure on the speed of protein synthesis, which drives the rate of each individual GTPase towards maximal turnover rate of the whole translation cycle.

  • 47.
    Ashrafzadeh, Parham
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Exploring Cellular Dynamics: From Vesicle Tethering to Cell Migration2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cells in the body communicate with each other in order to cooperate efficiently. This communication is in part achieved by regulated secretion of signaling molecules, which when released from a cell may activate receptors present at the plasma membrane of an adjacent cell. Such signals affect both cell fate and behavior. Dysregulated signaling may lead to disease, including cancer. This thesis is focused on how exocytosis and subsequent activation and trafficking of receptors can be regulated, and what the consequences of this regulation may be for cell migration.

    Actin filaments are important transport structures for secretory vesicle trafficking. In Paper 1, actin polymerization was shown to induce formation of ordered lipid domains in the plasma membrane. Accordingly, actin filaments may thus create and stabilize specific membrane domains that enable docking of vesicles containing secretory cargo.

    The RhoGEF FGD5 regulates Cdc42 which can result in cytoskeletal rearrangements. In Paper II, FGD5 was shown to be selectively expressed in blood vessels and required for normal VEGFR2 signaling. FGD5 protected VEGFR2 from proteasome-mediated degradation and was essential for endothelial cells to efficiently respond to chemotactic gradients of VEGFA.

    The exocyst component EXOC7 is essential for tethering secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. In Paper III, EXOC7 was required for trafficking of VEGFR2-containing vesicles to the inner plasma membrane and VEGFR2 presentation at the cell surface.

    The ability of tumor cells to escape the primary tumor and establish metastasis is in part dependent on their capacity to migrate. In Paper IV, a method based on time-lapse microscopy and fluorescent dyes was created to analyze single cancer cell migration in mixed cancer cell cultures, and in particular the influence of different types on neighboring cells was assessed.

    In conclusion, these studies have enhanced our understanding of the mechanisms behind cellular trafficking, and may be applied in the future to develop more specific therapeutics to treat cancer and other diseases associated with abnormal angiogenesis and cellular migration.

    List of papers
    1. Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains
    Open this publication in new window or tab >>Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains
    2013 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 3, p. 1102-1111Article in journal (Refereed) Published
    Abstract [en]

    The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37°C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerisation decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of 4-phosphate-inositides lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, lead to the formation of ordered domains. The increase in ordered domains was correlated with an increase in actin filaments just beneath the plasma membrane. In live cell plasma membrane blebs, which are detached from the underlying actin filaments, the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form when actin filaments attach to the plasma membrane. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions.

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-196480 (URN)10.1016/j.bbamem.2012.12.004 (DOI)000315473400021 ()23246974 (PubMedID)
    Available from: 2013-03-10 Created: 2013-03-10 Last updated: 2018-01-11Bibliographically approved
    2. FGD5 sustains vascular endothelial growth factor A (VEGFA) Signaling through inhibition of proteasome-mediated VEGF-receptor 2 degradation
    Open this publication in new window or tab >>FGD5 sustains vascular endothelial growth factor A (VEGFA) Signaling through inhibition of proteasome-mediated VEGF-receptor 2 degradation