| 1. |
- Aguda, Adeleke H., 1969-
(författare)
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Structural Study of the WH2 Family and Filamin: Implications for Actin Cytoskeleton Regulation
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular processes like motility, chemotaxis, phagocytosis and morphogenesis are dependent on the dynamic regulation of the actin cytoskeleton. This cytoskeleton system is tightly controlled by a number of diverse actin-binding proteins (ABPs) by various mechanisms described as nucleation, polymerization, capping, severing, depolymerization and sequestration. The ABPs are grouped based on sequence identity as in the Wiskott-Aldrich Syndrome protein homology domain 2 (WH2), and the calponin homology domain (CH) containing proteins.In this work, we elucidate the crystal structures of hybrids of gelsolin domain 1 with thymosin β4, ciboulot domain 2, and the second WH2 domain of N-WASP each bound to actin. We show that the single WH2 motif containing protein thymosin β4 in part sequesters actin by binding its pointed end via a C-terminal helix. This interaction prevents the addition of bound actin protomers to the barbed end of the filament. We propose that sequence variations in some WH2 motifs conferred F-actin binding ability to multiple repeat-containing proteins. These F-actin binding domains interact with the barbed end of a filament and the adjacent WH2 motifs are then freed to add their bound actin to the growing filament end. We demonstrate the binding of ciboulot domains 2 and 3 to both G- and F-actin and that full length ciboulot is capable of binding two actin monomers simultaneously. We have also cloned, expressed, purified and crystallized rod domains 14-16 from the actin crosslinking protein a-filamin. Preliminary X-ray crystallography data gives us hope that we shall be able to solve the structure of this triple domain repeat.
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| 2. |
- Helstad, Klara, 1977-
(författare)
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Managing timber procurement in Nordic purchasing sawmills
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Procurement of sawlogs to purchasing sawmills represents a basic strategic business process. The properties of inbound sawlogs are decisive for the output of sawn products and the cost of raw material contributes substantially to the cost of the final product. Increasing customer orientation and new demands from powerful customers in the building and retail sectors entail new or accentuated demands on management of procurement. Managing raw material procurement and communicating needs to suppliers and logging machine operators are vital issues for sawmills in order to be competitive.The purpose of the thesis is to explore how purchasing sawmills manage procurement of sawlogs. The results are based on 46 in-depth interviews with people involved in the procurement process at seven softwood sawmills in Denmark, Finland and Sweden. The thesis identifies various types of supply uncertainties as well as process improvement and buffer activities that reduce uncertainties. However, the major obstacle in the procurement process is the power/dependence balance in the relationships with suppliers. Beyond doubt, it restricts the manageability of procurement and particularly bucking. The results suggest that there are a number of ways to improve management of procurement, which are currently not fully employed. The thesis provides four key strategic dimensions of the procurement process and suggests a general conceptual model of wood procurement to purchasing sawmills.Further research within the subject can usefully explore the link between procurement management and procurement strategy as well as the relation to other functions' strategies and the corporate strategy. The importance of the identified strategic dimensions of the procurement process needs to be quantified in order to provide normative suggestions.
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| 3. |
- Larsson, Mårten, 1972-
(författare)
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Megalin, an Endocytotic Receptor with Signalling Potential
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Megalin is an endocytotic receptor belonging to the low-density lipoprotein family. It has often been viewed only as merely a scavenger receptor of absorptive and secretory epithelia. Recent work has revealed that the megalin intracellular domain contains several motifs potentially binding proteins involved in signal transduction.To find potential intracellular proteins binding to megalin, a yeast two-hybrid screening was initiated with the intracellular tail of megalin as the bait. A partial clone encoding the scaffolding protein postsynaptic protein 95 (PSD-95) was found to bind to megalin with its second PDZ-domain. Co-localization experiments in HEK-293 cells and kidney, placenta and parathyroid tissue confirmed this interaction. The PSD-95 related proteins PSD-93 and SAP102 were also confirmed to bind megalin with their PDZ2-domains, but the corresponding domain from SAP97 did not bind. Mutation analysis revealed that an amino acid residue change Ala to Thr was the cause of this.Megalin has within the central nervous system (CNS) been shown to be expressed only in the ependymal cells and choroid plexus. Nothing has been known about megalin expression in the spinal cord. To study spatio-temporal expression of megalin in the spinal cord, extensive staining of prenatal and postnatal mouse spinal cord was undertaken. Megalin expression was found in the dorsal part of the embryonic spinal cord. Most of these cells also expressed vimentin, suggesting that megalin has a role in the normal development of astrocytes. In the postnatal mouse, megalin seems to be expressed in oligodendrocytes only in the spinal cord white matter, and co-incident with myelination. This suggests that megalin is involved in the formation and maintenance of myelin along long spinal pathways. Megalin staining was clearly seen in the nucleus of these cells, indicating that megalin works in a notch-like signalling pathway.Uptake of retinol to the retina pigment epithelium (RPE) has long been thought to be a diffusion process. Staining for megalin in RPE revealed strong expression, and uptake experiments with 3H-retinol bound to retinol-binding protein and blocking with the LDL-receptor family specific antagonist receptor-associated protein (RAP) showed that megalin is a receptor for uptake of retinol to the RPE.
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| 4. |
- Martin, Nathalie, 1972-
(författare)
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Studies on the regulation of the Napin napA promoter by ABI3, bZIP and bHLH transcription factors
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The B3-domain transcription factor ABI3 is a major regulator of gene expression of seed maturation during Arabidopsis embryogenesis. The napA gene encodes for a Brassica napus 2S storage protein specifically expressed in the embryo during the early and mid-maturation phase (MAT program).The napA promoter contains two essential cis-sequences; the B-box, which functions as an Abscisic acid-responsive element (ABRE) and the RY/G cluster. ABI3 is known to target both these cis-sequences. Several bZIP factors expressed during seed maturation, bZIP12, bZIP38 and bZIP66, as well as a heterodimer of ABI5 and bZIP67, can bind the B-box ABRE in a yeast one-hybrid assay. Amongst them ABI3 and bZIP67 are able to activate synergistically the two cis-elements in a transient protoplast assay. We also show that bZIP67 interacts directly with ABI3 in a yeast two-hybrid assay. Therefore, we hypothesize that i)ABI3 is recruited indirectly to napA through molecular interaction with bZIP67 bound to the B-box ABRE, ii) ABI3 binds directly to the RY-element and interacts with bZIP67 targeted to the adjacent G-box found in the napA RY/G-cluster.We also show that the RY/G cluster is responsible for repression of napA expression during the late maturation LEA program, and for repression of ABI3-mediated transactivation during germination. ABI3 from which the A1 activation domain had been removed, can bind to the napA RY-element in a yeast one-hybrid assay, in contrast to full-length ABI3, suggesting that ABI3 DNA-binding abilities are regulated by auto-inhibition. We propose that during late maturation ABI3 loses ability to bind RY, which results in repression of MAT genes but not of LEA genes that contain fewer RY-elements. In parallel, we show that the B3-domain VAL proteins bind to RY-elements and decrease ABI3-mediated transactivation of the napA RY/G and therefore act as active repressors maintaining silencing of MAT genes during vegetative growth.
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| 5. |
- Mårtensson, Mattias
(författare)
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Evaluation of Errors and Limitations in Ultrasound Imaging Systems
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- There are binding regulations requiring safety and efficacy aspects of medical devices. The requirements ask for documentation that the devices are safe and effective for their intended use, i.e. if a device has a measuring function it must be correct. In addition to this there are demands for quality systems describing development, manufacturing, labelling, and manufacturing of a device. The requirements are established to guarantee that non-defective medical devices are used in the routine clinical practice. The fast rates in which the imaging modalities have evolved during the last decades have resulted in numerous new diagnostic tools, such as velocity and deformation imaging in ultrasound imaging. However, it seems as if the development of evaluation methods and test routines has not been able to keep up the same pace. Two of the studies in this thesis, Study I and IV, showed that computed tomography-based and ultrasound based volume measurements can yield very disparate measurements, and that tissue Doppler imaging-based ultrasound measurements can be unreliable. Furthermore, the new ultrasound modalities impose higher demands on the ultrasound transducers. Transducers are known to be fragile, but defective transducers were less of a problem earlier when the ultrasound systems to a lesser extent were used for measurements. The two other studies, Study II and III, showed that serious transducer errors are very common, and that annual testing of the transducers is not sufficient to guarantee an error free function. The studies in the thesis indicate that the system with Notified Bodies, in accordance with the EU’s Medical Device Directive, checking the function and manufacturing of medical devices does not work entirely satisfactory. They also show that the evaluation of new methods have led to the undesirable situation, where new measuring tools, such as volume rendering from imaging systems, and tissue Doppler-based velocity and deformation imaging in echocardiography are available for clinicians without proven knowledge about their accuracy.
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| 6. |
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| 7. |
- Sakwe, Amos M., 1962-
(författare)
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The Role of Protein Kinase C in the Extracellular Ca2+-regulated Secretion of Parathyroid Hormone
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca2+ concentration ([Ca2+]o) in the body. The secretion of this hormone is suppressed at high [Ca2+]o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca2+ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca2+]o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca2+]o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca2+]o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca2+]o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca2+-independent. The magnitude of neomycin-induced release of Ca2+ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca2+ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca2+]o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca2+ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.
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| 8. |
- Valsecchi, Isabel, 1973-
(författare)
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AtZDP, a Plant 3' DNA Phosphatase, Involved in DNA Repair
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- DNA bases can be modified by endogenous agents (e.g. oxidized by products of respiration and photosynthesis or methylated by gene silencing processes) as well as by environmental agents (e.g. oxidized by UV light). In the process of removing modified bases, a 3’-phosphate group is sometimes left in the resulting gap, and has to be removed since it blocks the incorporation of a new nucleotide by DNA polymerase. The aim of this thesis was the characterization of AtZDP, a plant enzyme with a DNA 3’-phosphatase activity.By homologous modeling, the existence of four domains was predicted in AtZDP, three independent zinc-finger and one DNA 3’-phosphatase domains. AtZDP was found to be localized in the nucleus by bimolecular fluorescence complementation. Western blotting analysis showed that the enzyme was ubiquitously expressed in plant tissues.AtZDP was found in a 600,000 molecular-weight protein complex by gel chromatography and glycerol gradient sedimentation centrifugation. The fractions containing AtZDP in the complex displayed 3’-DNA phosphatase activity as shown by desphosphorylation of a DNA oligonucleotide with a 3’-phosphate terminus. Also fractions of the gel chromatography corresponding to lower molecular weight showed 3’-DNA phosphatase activity, but antibodies against AtZDP did not recognize this fraction inferring that in plants, at least another protein with similar activity exists.In mammals, polynucleotide kinase, an enzyme with the same activity phosphatase activity as AtZDP, is involved in single-strand and double-strand repair pathways. To elucidate if AtZDP could be part of similar pathways, different double strand and single-strand oligonucleotides with 3’-phosphate termini were separately incubated with AtZDP. All substrates were dephosphorylated by AtZDP, assuming that this enzyme could potentially be involved in double-strand DNA repair. A double-strand oligonucleotide containing a one-bp gap with a 3’-phosphate terminus was repaired by a leaf protein extract. The activities of a 3’-DNA phosphatase, a flap 5’ to 3’ endonuclease-like, a DNA polymerase and a DNA ligase were observed. The presence of these enzymes revealed that these damages are in plants predominantly repaired by long-patch base excision repair.
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