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- Boman, Andrea, 1978-
(författare)
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Lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The pre-symptomatic stage of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) occurs several decades before the clinical onset. Changes in the lysosomal network, i.e. the autophagosomal, endosomal and lysosomal vesicular system, are among the first alterations observed. There are currently no treatments to slow or cure neurodegenerative diseases, and there is a great need for discovery of treatment targets in cellular pathways where pathology pre-dates the neuronal death. It is also crucial to be able to diagnose neurodegenerative diseases earlier, both to enable early intervention treatment and aid in selecting clinical trial populations before the patient has widespread pathology.This thesis aims at investigating the potential of lysosomal network proteins as biomarkers and therapeutic targets in neurodegenerative disease.A targeted search for lysosomal network proteins was performed in cerebrospinal fluid (CSF) from AD patients, and seven proteins: early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), lysozyme, microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were elevated. The levels of EEA1, LAMP-1, LAMP-2, LC3, lysozyme and Rab3 were also measured in CSF from parkinsonian syndrome patients: PD, clinically diagnosed 4-repeat tauopathy, pathologically confirmed corticobasal degeneration (CBD) and pathologically confirmed progressive supranuclear palsy (PSP) patients. LAMP-1 and LAMP-2 were decreased in PD. LC3 and lysozyme levels were increased in 4-repeat tauopathy patients. EEA1 was decreased and lysozyme increased in PSP, and LAMP-1, LAMP-2, LC3 and lysozyme were increased in CBD. The lysosomal network proteins had different CSF protein profiles in all the parkinsonian syndromes, as well as in AD. It should be emphasized that only a select few of the lysosomal network proteins were observed to be changed, rather than a general change in lysosomal network proteins, which implicates the involvement of these seven proteins in specific pathological processes. The most interesting candidates, LAMP-2 and lysozyme, were selected for further study for their involvement in the pathology of AD.Lysozyme was found to co-localise with Aβ plaques in AD patients and overexpression prolonged survival and improved the activity in a Drosophila model of AD. Lysozyme was found to alter the aggregation pathway of Aβ1-42, to counteract the formation of toxic Aβ species and to protect from Aβ1-42 induced cell toxicity. Aβ1-42 in turn was found to increase the expression of lysozyme in both neuronal and glial cells. These data suggest that lysozyme levels rise in AD as a compensatory response which is protective against Aβ associated toxicity.LAMP-2 mRNA and protein were found increased in brain areas relevant for AD pathology and various cellular models showed complex involvement of LAMP-2 in Aβ related pathology, with extensive crosstalk between LAMP-2 and Aβ. Exposure to oligomeric Aβ1-42 caused an upregulation of LAMP-2 and in turn, overexpression of LAMP-2 caused a reduction in secreted levels of Aβ1-42, as well as changing the generation pattern of Aβ and affecting clearance and secretion of Aβ1-42. These data indicate that the increased levels of LAMP-2 in AD could be an attempt to regulate Aβ generation and secretion.In summary, this thesis reports that utilising lysosomal network proteins as biomarkers and novel therapeutic targets for neurodegenerative diseases holds great promise.
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| 2. |
- Karlsson, Annika M., 1972-
(författare)
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Signaling for color change in melanophores : and a biosensor application
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Melanophores are dark brown pigment cells located in the skin of fish, amphibia, reptiles, and many invertebrates. The color of the animal can change via rearrangement of pigment granules, melanosomes, in the cells. The dark melanophores can either hide colorful cells so that the animal appears dark, or let through colors from underneath. The animal regulates its colors and patterns via communicating nerve cells and hormones in the blood stream. It is nowadays well established that melatonin-stimulation of melanophores results in aggregation of melanosomes to the cell center and that the evident outcome is more transparent cells. It has previously been shown that the activity of serine and threonine kinases as well as phosphatases regulates the distribution of melanosomes in the cells.We wanted to study if tyrosine phosphorylations were involved in the regulation of melanosome aggregation. Melatonin-stimulated signaling in the African clawed frog, Xenopus laevis, melanophores was examined. Melansome aggregation was accompanied by tyrosine phosphorylation as shown by immunoblots. Inhibition of tyrosine phosphorylation reduced melanosome aggregation by melatonin, and the phosphorylation most likely regulated pigment aggregation. Tyrosine phosphorylation of the protein was mediated via a Gi/o protein coupled receptor, probably the melatonin receptor Mel1c. The phosphorylation was most likely not a result of the classical Gi/o protein pathway, as Src-kinase and mitogen-activated protein kinase seemed required for phosphorylation and melanosome aggregation. Two candidates for the phosphorylated protein were presented, talin and β-spectrin.The possible involvement of nitric oxide in melanosome aggregation by melatonin was investigated. Nitric oxide appeared to be necessary for melanosome aggregation. The effect of nitric oxide synthase inhibition on melanosome aggregation was not mediated via changes in the tyrosine-phosphorylated protein. We speculated that nitric oxide could affect melanosome distribution via modifications of the actin cytoskeleton. The use of recombinant melanophores as a biosensor has also been examined. A human G protein coupled receptor, opioid receptor 3, was inserted into melanophores by electroporation. The transfected melanophores responded dose-dependently to opioids and an inhibitor of opioid receptors reduced the aggregation response. Future melanophore biosensors migh detect a variety of substances, such as narcotics, pheromones, odors, and tastes.
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| 3. |
- Nilsson, Ulrika K., 1969-
(författare)
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Lysophosphatidic acid : Physiological effects and structure-activity relationships
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lipids havepreviously been considered primarily as building blocks of the cell membrane, but are now also recognized as important cell signaling molecules. Lysophosphatidic acid (LPA) is a glycerophospholipid consisting of a phosphate head group, a linker region, and a lipophilic tail. LPA has earlier been shown to exert a diversity of cellular effects such as aggregation, apoptosis, contraction, migration, and proliferation. The effects of LPA are elicited by activation of its cognate G protein-coupled receptors LPA1, LPA2, and LPA3. In the present study we have used cultures of human smooth muscle cells (SMCs) and erythroleukemia cells (HEL), and isolated human platelets to characterize physiological effects of LPA compared with adrenaline and noradrenaline as well as structure-activity relationships of LPA. SMCs were isolated from biopsies of human myometrium obtained at cesarean sections. We show that cultured myometrial SMCs express multiple LPA and α2-adrenergic receptor subtypes. Treatment of SMCs with LPA and noradrenaline resulted in increases in proliferation. However, LPA elicits a much more pronounced stimulatory effect than noradrenaline. The ability to increase calcium might be one explanation why LPA is more effective. Further studies indicated that several pathways mediate the growth stimulatory effect of LPA where transactivation of epidermal growth factor receptors through matrix metalloproteinases as well as calcium/calmodulin-dependent protein kinases appears to be important. LPA enantiomers and LPA analogues were synthesized and characterized due to their capacity to increase calcium in HEL cells. Our study is the first to show that both natural (R) and unnatural (S) LPA enantiomers are capable of stimulating cells, suggesting LPA receptors are not stereoselective. Moreover, we have synthesized a LPA analogue with higher maximal effect than LPA by reducing the hydrocarbon chain length. In platelets we demonstrated that LPA is a weak calciumelevating compound which failed to stimulate aggregation. However, in combination with adrenaline, another weak platelet agonist, a complete aggregatory response was obtained in blood from some healthy individuals. These results are important since platelet activation is a key step in distinguishing normal from pathological hemostasis. Since LPA is present at high concentrations in atherosclerotic lesions, the synergistic effect of LPA and adrenaline might be a new risk factor for arterial thrombosis.
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| 4. |
- Svensson, Samuel, 1962-
(författare)
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Melanophore a2-Adrenoceptors.
- 1993
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The present study provides new insight into the pharmacology, the signal transduction mechanisms and the molecular biology of a2-adrenoceptors (a2-ARs) in black pigment cells (melanophores) of a teleost fish, the cuckoo wrasse (Labrus ossifagus L.). Stimulation of these receptors leads to an aggregation of intracellular pigment granules, which is the cellular mechanism underlying the ability of fishes to change color.The pharmacology of the melanophore a2-AR is very similar to its mammalian counterparts. However, the melanophore a2-AR is unique in one respect, UK 14,304, a potent agonist at mammalian a2-ARs, is an antagonist in melanophores.Noradrenaline and the selective a2-AR agonist B-HT 920 are pharmacologically heterogeneous regarding their ability to induce pigment aggregation. This may reflect that B-HT 920 and noradrenaline interact with different a2-AR sites.Pigment aggregation induced by a2-ARs seems to involve multiplesignaling pathways. Attenuation of intracellular cAMP production and a subsequent reduction of protein kinase A activity may serve as one mechanism, and an additional signal mechanism may involve activation of a phosphatase.An a2-adrenoceptor (a2p) from L. ossifagus skin mRNA was cloned. The deduced amino acid sequence showed significant homology with the human a2-ARs. When expressed in a mammalian cell line, the pharmacology of the Melanin-concentrating hormone (MCH) induces pigment aggregationthrough a unique receptor. However, MCH receptors and a2-ARs might share a common signal transduction mechanism, namely attenuation of cAMP production.When melanophores are maintained in tissue culture media, the sensitivity to noradrenaline is increased and the sensitivity to MCH is decreased. This reciprocal change in sensitivity may be due to an increase in a2-AR coupling at the expense of MCH receptor couplingand/or to a down regulation of MCH receptors.
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