| 1. |
- Arvidson, Nils Gunnar
(författare)
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Disease activity in rheumatoid arthritis : Studies in interleukin-6, tumour necrosis factor alpha, monocyte activity, acute phase markers, glucocorticoids, and disability
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the present studies, aspects of some disease activity measures in rheumatoid arthritis (RA) have been investigated, including the effect of glucocorticoids on this activity. In RA, serum interleukin(IL)-6 levels were elevated and were shown to have a circadian rhythm, with peak levels in the morning, declining towards low or normal levels in the afternoon and evening. In contrast, serum levels of tumour necrosis factor(TNF) alpha were low and stable. In other connective tissue diseases, serum TNF alpha levels were elevated but without circadian variation, while IL-6 levels were low and stable. Nocturnal administration (at 2:00 a.m.) of low-dose prednisolone a few hours before the early morning peak of IL-6 was shown to be significantly more effective in reducing clinical symptoms of disease activity and serum IL-6 levels than the traditional morning administration (at 7:30 a.m.) of the same dose of prednisolone. Circulating monocytes are activated in RA, expressing receptors related to adhesion and phagocytosis. Treatment with glucocorticoids suppressed the expression of these receptors on monocytes, and this may be one mechanism of the beneficial effect of glucocorticoids in RA. Endogenous levels of cortisol seem to play a minor role in expression of monocyte receptors. The different acute phase markers used to assess disease activity in RA showed good corrrelations with each other and with serum IL-6 levels. There were especially strong corrrelations between C-reactive protein (CRP) and Serum amyloid protein A (SAA), and between fibrinogen and erythrocyte sedimentation rate (ESR). Fibrinogen and CRP showed stronger correlation than ESR with the Modified Health Assessment Questionnaire (MHAQ) score and with the neutrophil count. Four simple objective function tests were each compared with the HAQ score a with a radiological joint damage score (Larsen score). The objective function tests correlated with the MHAQ score, and each of these two methods of assessing physical disability correlated with pain, CRP and ESR. In addition, most of the objective function tests correlated significantly with radiological joint damage, while the MHAQ score did not.
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| 2. |
- Blomgren, Bo, 1960-
(författare)
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Morphometrical Methodology in Quantification of Biological Tissue Components
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Objective:To develop and validate computer-assisted morphometrical methods, based on stereological theory, in order to facilitate the analysis and quantitative measurements of biological tissue components.Material and methods:Biopsy specimens from the vaginal wall or from the vestibulum vaginae of healthy women, or from women suffering from incontinence or vestibulitis were used.A number of histochemical methods for light microscopy were used, and modified for the different morphometrical analyses. Electron microscopy was used to reveal collagen fibre diameter.Computer-assisted morphometry, based on image analysis and stereology, was employed to analyse the different tissue components in the biopsies. Computer programs for these purposes were developed and validated.Results:The results show that computer-assisted morphometry is of great value for quantitative measurements of the following tissue components:Epithelium: The epithelial structure, instead of just thickness, was measured in an unbiased way.Collagen: The collagen fibril diameter was determined in electron microscopic specimens, and the collagen content was analysed in light microscopic specimens.Elastic fibres: The amount of elastic fibres in the connective tissue was measured after visualisation by autofluorescence.Vasculature: A stereological method using a cycloid grid was implemented in a computer program. Healthy subjects were compared with patients suffering from vestibulitis. The results were identical in the two groups.Smooth muscle: A stereological method using a point grid was implemented in a computer program. Using the Delesse principle, the fibres were calculated as area fractions. The area fractions were highly variable among the different specimens.Conclusion:Morphometry, used correctly, is an important analysis method in histopathological research. It is important that the methods are as simple and user-friendly as possible. The present studies show that this methodology can be applied for most quantitative histological analyses.
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| 3. |
- Dauksaite, Vita, 1966-
(författare)
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The Modular Domain Structure of ASF/SF2: Significance for its Function as a Regulator of RNA Splicing
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- ASF/SF2 is an essential splicing factor, required for constitutive splicing, and functioning as a regulator of alternative splicing. ASF/SF2 is modular in structure and contains two amino-terminal RNA binding domains (RBD1 and RBD2), and a carboxy-terminal RS domain. The results from my studies show that the different activities of ASF/SF2 as a regulator of alternative 5’ and 3’ splice site selection can be attributed to distinct domains of ASF/SF2.I show that ASF/SF2-RBD2 is both necessary and sufficient to reproduce the splicing repressor function of ASF/SF2. A SWQDLKD motif was shown to be essential for the splicing repressor activity of ASF/SF2. In conclusion, this study demonstrated that ASF/SF2 encodes for distinct domains responsible for its function as a splicing enhancer (the RS domain) or a splicing repressor (the RBD2) protein. Using a model transcript containing two competing 3’ splice sites it was further demonstrated that the activity of ASF/SF2 as a regulator of alternative 3’ splice site selection was directional: i.e. resulting in RS or RBD1 mediated activation of upstream 3’ splice site selection while simultaneously causing an RBD2 mediated repression of downstream 3’ splice site usage.In alternative 5’ splice site selection, the RBD2 alone was sufficient to reproduce the activity of the full-length protein as an inducer of proximal 5’ splice site usage, while RBD1 had the opposite effect and induced distal 5’ splice site selection. The conserved SWQDLKD motif and the RNP-1 type RNA recognition motif in ASF/SF2-RBD2 were both essential for this induction. The activity of the ASF/SF2-RBD2 domain as a regulator of alternative 5’ splice site was shown to correlate with the RNA binding capacity of the domain.Collectively, my results suggest that the RBD2 domain in ASF/SF2 plays the most decisive role in the alternative 5’ and 3’ splice site regulatory activities of ASF/SF2.
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| 4. |
- Edlund, Sofia
(författare)
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Mechanisms for TGF-β-Mediated Regulation of the Actin Filament System and Apoptosis
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transforming growth factor-β (TGF-β) is a member of a large superfamily of cytokines which participate in many different types of cellular processes, such as growth inhibition, cell migration, differentiation, cell adhesion, wound healing and immunosuppression. Alterations of TGF-β superfamily signalling results in several different disorders, including bone disease, vascular disease and cancer. The TGF-β signalling pathways involve several different proteins, such as the Smad proteins, which upon receptor activation are translocated to the nucleus, where they affect transcriptional responses. The actin cytoskeleton is an organised network of filaments with a highly dynamic structure, which is under a continuous reconstruction to control the morphology, survival, growth and motility of eukaryotic cells. The members of the family of small GTP-binding proteins have been shown to be important regulators of the actin cytoskeleton.TGF-β was found to induce short term as well as long term actin reorganisation in prostate cancer cells. The short term response included membrane ruffling, and required signalling by the small GTPases Cdc42 and Rho as well as, the involvement of the mitogen-activated protein kinases p38 (p38 MAPK). The long term response included formation of stress fibers and required a cooperation between Smad and Rho GTPase signalling pathways involving the Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). The TGF-β-induced activation of Cdc42 was, furthermore, shown to require the inhibitory Smad7 and p38 MAP kinase, via a PI3K-dependent pathway. Mixed lineage kinase 3 (MLK3), a mediator downstream of Cdc42, was necessary for the Cdc42-dependent actin filament reorganisation.Apoptosis is an important and carefully regulated process in human development and disease, which allows the multicellular organisms to remove cells that are in excess or potentially dangerous. TGF-β family members can induce apoptosis in many different cell types, in the presence or absence of other growth factors. Smad7 had previously been shown to be necessary for TGF-β-induced apoptosis of epithelial cells. We could show that Smad7 is required for TGF-β-induced activation of the TGF-β activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK pathway, which subsequently leads to apoptosis in prostate cancer cells.Members of the lymphoid enhancer factor-1/T-cell factor (LEF1/TCF) family of transcription factors have, together with β-catenin, been shown to be nuclear effectors in the Wnt-signalling pathway. We investigated a possible cross-talk between the TGF-β and Wnt signalling pathways. We found that TGF-β, in a Smad7-dependent manner induced a nuclear accumulation of β-catenin and enhanced the transcriptional activity of β-catenin and the induction of the downstream target gene c-myc. Since β-catenin and c-Myc has been shown to promote apoptosis, our results suggests the possibility that β-catenin contributes to TGF-β-induced apoptosis
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| 5. |
- Ekman, Simon
(författare)
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Specific signaling through heteromeric PDGF receptor complexes
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for mesenchymal cells and exert its effect by binding to two structurally related receptor tyrosine kinases, denoted α- and β-receptors. PDGF binding induces dimerization of its receptors, both homo-and heterodimerization, leading to their autophosphorylation on tyrosine residues and binding of downstream signaling molecules. This thesis describes autophosphorylation and binding of signal transduction molecules to homo- and heterodimeric PDGF receptor complexes. Heterodimeric PDGF receptor complexes have been found to mediate a stronger mitogenic response than homodimeric receptor complexes. It was found that Tyr771 in the PDGF β-receptor was significantly less phosphorylated in the heterodimeric β-receptor compared to the homodimeric receptor, and this correlated with reduced binding of GTPase activating protein (GAP) for Ras and decreased activation of the Ras/Mitogen activated protein kinase pathway. The mechanism behind the lowered phosphorylation of Tyr771 in the heterodimeric PDGF β-receptor was investigated. It was found that the SH2 domain-containing tyrosine phosphatase SHP-2 was responsible, at least in part, for the dephosphorylation of Tyr771 in the heterodimeric β-receptor. PDGF-induced autophosphorylation of tyrosine residues in the receptors has been proposed to occur in trans between the receptor molecules in the dimers. We demonstrated by phosphopeptide mapping that all major autophosphorylation sites can be phosphorylated in trans, both in the PDGF α- and β-receptors. Analyses of the abilities of heterodimeric receptor complexes of one kinase-active and one kinase-inactive receptor to mediate mitogenicity, chemotaxis and activation of mitogen activated protein kinase revealed that the signaling capacities were retained. This illustrates a functional co-operation between the two receptor molecules in the dimer, where one receptor provides a functional kinase and the other acts as a substrate and provides docking sites for downstream signaling molecules. Elucidating the mechanisms behind the unique signaling properties of the heterodimeric PDGF receptor complex, two heterodimer-specific autophosphorylation sites, Tyr692 and Tyr970, were identified and found to interact with the low molecular weight protein tyrosine phosphatase (LMW-PTP). Mutation of Tyr692 or Tyr970 to phenylalanine residues did not affect PDGF-induced mitogenicity, but the Tyr692 to phenylalanine mutation reduced the chemotactic response mediated by the heterodimeric PDGF receptor complex. A mechanism for the lowered chemotactic response was found to involve an increased RasGAP binding and a decreased SHP-2 binding to the heterodimeric β-receptor.
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| 6. |
- Enarsson, Mia, 1974-
(författare)
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Roles of PDGF for Neural Stem Cells
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Stem cells are endowed with unique qualities: they can both self-renew and give rise to new mature cell types. Central nervous system (CNS) stem cells can give rise to neurons and glia. What factors regulate stem cell fate decisions? Identifying signals that are involved in the regulation of CNS stem cell proliferation, survival, differentiation and migration is fundamental to the understanding of CNS development. In addition, this knowledge hopefully will contribute to more efficient therapies of CNS damages and diseases.The focus of this thesis was to investigate mechanisms of CNS stem cell proliferation and differentiation. We have studied the role for platelet-derived growth factor (PDGF) in these cellular events both in vitro and in vivo. Previous reports have shown that PDGF are implicated in brain tumorigenesis and also supports neuronal differentiation of CNS stem cells. We have found that PDGF promotes survival and proliferation of immature neurons, thereby supporting neuronal differentiation. The intracellular Ras/ERK signaling pathway probably mediates the mitogenic activity of PDGF. In contrast, neuronal differentiation is not dependent on the Ras/ERK pathway. A genetic expression profile of stem cells during their differentiation was obtained. This microarray analysis suggests that PDGF-treated stem cells are at an intermediate stage between proliferation and differentiation. Furthermore, we generated transgenic mice that overexpress Pdgf-b in neural stem cells. Preliminary data indicate no signs of enhanced proliferation of immature neurons. Instead, increased apoptosis was detected in the developing striatum.The results presented in this thesis show how CNS stem cells are regulated by PDGF. PDGFs are widely expressed in the developing CNS and also in some brain tumors, which are thought to arise from CNS stem cells. Thus, this knowledge may contribute to an increased understanding of brain tumorigenesis in addition to normal CNS development.
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| 7. |
- Grundström, Gunilla, 1968-
(författare)
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Functional Studies of Collagen-Binding Integrins α2β1 and α11β1 : Interplay between Integrins and Platelet-Derived Growth Factor Receptors
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Integrins are heterodimeric cell surface receptors, composed of an α- and a β-subunit, which mediate cell-extracellular matrix (ECM) interactions. Integrins mediate intracellular signals in response to extracellular stimuli, and cooperate with growth factor and other cytokine receptors. Cells execute their differentiated functions anchored to an ECM. In this thesis functional properties of the two collagen-binding integrins α2β1 and α11β1 were studied. In addition, the impact of β1 cytoplasmic tyrosines in collagen-induced signalling was analyzed.The integrin α11β1 is the latest identified collagen-binding integrin. In this study, tissue distribution of α11 mRNA and protein during embryonal development was explored, and the first α11β1-mediated cellular functions were established. Both α11 protein and mRNA were present in mesenchymal cells in intervertebral discs and around the cartilage of the developing skeleton. α11 protein was also detected in cornea keratinocytes. α11β1 mediated cation-dependent adhesion to collagen types I and IV and localized to focal adhesions. In addition, α11β1 mediated contraction of a collagen lattice and supported cell migration through a collagen substrate. PDGF-BB and FBS both stimulated α11β1-mediated contraction and directed migration.Expression of β1Y783,795F in β1-null cells, prevents activation of FAK in response to fibronectin, and decreases cell migration. In this study, we investigated how this mutation affected α2β1-mediated functions in response to collagen. The β1 mutation impaired collagen gel contraction and prevented activation of FAK, Cas and Src on planar collagen, but not in collagen gels. PDGF-BB stimulated contraction via αvβ3, which also induced activation of Cas in collagen gels. The YY-FF mutation also abolished β1A-dependent downregulation of β3.In the final study integrin-crosstalk during collagen gel contraction was investigated. In cells lacking collagen-binding integrins αvβ3 mediated contraction. Clustering of β1-integrins by antibodies and PDGF-BB stimulated αvβ3-mediated contraction in an ERK-dependent way. Expression of α2β1, but not α11β1, prevented αvβ3-mediated contraction. Contraction by α2β1 and α11β1 was ERK-independent.
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| 8. |
- Gustafsson, Ingegerd
(författare)
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Biological and Pharmacological Factor that Influence the Selection of Antibiotic Resistance
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic treatment causes an ecological disturbance on the human microflora. Four commensal bacteria: E. coli, enterococci, a-streptococci and coagulase-negative staphylococci, from patients with extensive, high antibiotic usage were investigated with regard to resistance pattern and mutation frequency. Among 193 investigated strains it was found that high antibiotic usage selected for resistant bacteria and enriched for bacteria with a small but significantly increased mutation frequency.The relative biological fitness cost of resistance in Staphylococcus epidermidis was assessed in a human in vivo model where the indigenous flora was present. In vitro data of the bacterial growth rate correlated well to in vivo fitness assayed in the competition experiments on skin.An in vitro kinetic model was shown to be a useful tool to establish the pharmacokinetic and pharmacodynamic (PK/PD) indices for efficacy of antibiotics. It was confirmed that the time, when the concentration exceeds the minimal inhibitory concentration (MIC), correlates with efficacy for b-lactam antibiotics. To achieve maximal killing for penicillin-resistant pneumococci, with an MIC of 2 mg/L, the peak concentration was also of importance.Suboptimal dosing regimen facilitates selection of resistance. Penicillin-resistant pneumococci were easily selected in a mixed population with penicillin-sensitive, -intermediate and -resistant pneumococci in an in vitro kinetic model. The selection of the resistant strain was prevented when the benzylpenicillin concentration exceeded the MIC for approximately 50% of 24 h.
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| 9. |
- Haglund, Kaisa, 1978-
(författare)
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Ubiquitination and Receptor Endocytosis
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein ubiquitination is an evolutionary conserved mechanism that controls a wide variety of cellular functions. Polyubiquitinated proteins are generally degraded in the proteasome, whereas monoubiquitination controls various other cellular processes, including endocytosis and endosomal sorting.Termination of signaling by activated receptor tyrosine kinases (RTKs) largely occurs via their endocytosis and subsequent lysosomal degradation, processes accompanied by receptor ubiquitination. Cbl family proteins are major ubiquitin ligases that promote RTK ubiquitination and downregulation. We showed that epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors are monoubiquitinated at multiple sites following their ligand-induced activation and that a single ubiquitin is sufficient for both receptor internalization and degradation. Cbl also controls EGF receptor (EGFR) downregulation by binding to CIN85, which recruits endophilins to EGFR/Cbl complexes. In the complex with activated EGFRs, Cbl directs monoubiquitination of CIN85, and the entire complex is targeted for degradation in the lysosome. We propose that multiple monoubiquitination of activated receptors and associated protein complexes ensures proper receptor sorting towards the lysosome. Importantly, the functions of Cbl are also negatively controlled in order to maintain cellular homestasis. Sprouty2 blocks EGFR downregulation by sequestering Cbl from activated EGFRs. We showed that Sprouty2 also associates with CIN85 and that this binding is required for efficient inhibition of EGFR ubiquitination and endocytosis. Cbl is also implicated in other aspects of RTK signaling, including organization of the actin cytoskeleton. We found that growth factor receptor signals promote lamellipodia formation in neuronal cells via a complex containing Cbl, the adaptor protein ArgBP2 and Pyk2. The lamellipodia formation required intact lipid rafts and the recruitment of Crk and PI(3)K to tyrosine phosphorylated Cbl.In conclusion, our findings contribute to a better understanding of monoubiquitin signals in downregulation of RTKs and point at a role of Cbl in the regulation of cytoskeleton dynamics.
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| 10. |
- Itoh, Fumiko, 1972-
(författare)
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Regulation of TGF-β/Smad Signaling Through Smad Interacting Proteins
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transforming growth factor-β (TGF-β) superfamily members are multi-functional regulators of cell fate. These factors signal by binding to a limited number of highly conserved transmembrane type I and type II serine/threonine kinase receptors. These receptors initiate signals into the cell via the Smad proteins. Up to date, 8 different mammalian Smads are reported and are divided into three subgroups; receptor regulated Smads (R-Smads), common mediator Smads (Co-Smads) and inhibitory Smads (I-Smads). This thesis investigates the function and regulation of TGF-β/Smad signaling through identification and characterization of Smad interacting proteins.I-Smads, i.e. Smad6 and Smad7, are potent antagonists of the TGF-β superfamily signaling. We found that Smad7, but not Smad6, inhibits TGF-β1-induced growth inhibition and expression of immediate early response genes. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-β receptor activation. Moreover, we found that the MH2 domain is important for nuclear export.To investigate further the role of inhibitory Smads, we have identified AMSH as a Smad6 interacting protein using a yeast two-hybrid screening method. AMSH was previously discovered as the associated molecule with the SH3 domain of STAM. AMSH interacts with I-Smads, but not with R- and Co-Smads upon receptor activation and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. AMSH was found to prevent Smad6 from binding to activated type I receptors and/or activated R-Smads. Smad anchor for receptor activation (SARA) is critical for Smad2 and Smad3 activation by TGF-β receptors. The present studies show that the localization of SARA in early endosomes is regulated through its FYVE domain. We have found that the FYVE domain of SARA is sufficient and necessary for the early endosomal localization, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF-β/Smad signaling.Both Notch and BMP signaling pathways are important for vascular development. We have found that Herp2, which is originally known as one of the Notch target genes, is synergistically induced upon activation of Notch and BMP signaling pathways in endothelial cells (ECs). The critical elements for synergistical activation of Herp2 gene by BMP and Notch pathway were identified. Furthermore, the Notch intracellular domain interacts with Smad5 upon BMP receptor and this interaction becomes stronger in the presence of pCAF. Interestingly, Herp2 was found to antagonize BMP receptor- or Id-mediated EC migration.
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