| 1. |
- Eliasson, Pernilla, 1979-
(författare)
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Live and Let Die : Critical regulation of survival in normal and malignant hematopoietic stem and progenitor cells
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and produce all mature blood cells throughout the life of an organism. This is tightly regulated to maintain a balance between survival, proliferation, and differentiation. The HSCs are located in specialized niches in the bone marrow thought to be low in oxygen, which is suggested to be involved in the regulation of HSC maintenance, proliferation, and migration. However, the importance of hypoxia in the stem cell niche and the molecular mechanisms involved remain fairly undefined. Another important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3, which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause constitutively active signaling. This leads to uncontrolled survival and proliferation, which can result in development of acute myeloid leukemia (AML). One of the purposes with this thesis is to investigate how survival, proliferation and self-renewal in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse bone marrow. We found that hypoxia maintained an immature phenotype. In addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the signature of HSCs with long term multipotential capacity. A dormant state of HSCs is suggested to be critical for protecting and preventing depletion of the stem cell pool. Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced cell death, implicating that hypoxia is important in the bone marrow niche to limit reactive oxidative species (ROS) production and give life-long protection of HSCs. Another focus in this thesis is to investigate downstream pathways involved in tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic cells expressing mutated FLT3.
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| 2. |
- Gallwitz, Maike, 1977-
(författare)
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Sculpted through Time : Evolution and Function of Serine Proteases from the Mast Cell Chymase Locus
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of granule serine proteases, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an in vivo substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.
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| 3. |
- Jerström Skarman, Petra
(författare)
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The role of calcium and calcium-regulated proteins in neutrophil phagocytosis
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neutrophil phagocytosis is an essential component of the innate immunity against invading pathogens. The two types of phagocytosis that are investigated in detail are IgG- and C3bi-mediated phagocytosis. Although the two types are controlled differently, they share the same driving force - reorganisation of the actin cytoskeleton. Subcellular elevations of intracellular free calcium concentration ([Ca2+]1), are critical for this kind of functional response within the neutrophils.The aim of this study was to try to understand how calcium and certain calciumregulated proteins control phagocytosis in neutrophils, especially the remodelling of the actin cytoskeleton during pseudopod fonnation and the regulation of phagolysosome fusion.By immunofluorescence staining (IF) and confocal microscopy, we analysed the distribution of Ca2+ stores using antibodies against Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase (SERCA2) and calreticulin, during phagocytosis. The results showed a distinct accumulation of Ca2+ stores around phagosomes and pseudopods. This accumulation is coherent with a local Ca2+ rise seen in the area of phagocytosis and provides a model for how this localised [Ca2+]i is regulated in neutrophils. To further investigate if inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores are involved, we analysed the subcellular distribution of IP3-receptors (IP3Rs), which are located on the ci+ stores. The IP3Rs translocated in a similar manner as did SERCA2 and calreticulin, indicating that the IP3-sensitive Ca2+ stores are involved.During phagocytosis, an accumulation of phospholipid- and calcium-binding proteins, annexins, can be seen in the periphagosomal area. Several studies have demonstrated that certain annexins promote Ca2+-dependent contact between phospholipid vesicles and/or isolated neutrophil-specific granule membranes. This suggests that annexins, apart from being involved in vesicle aggregation and fusion, participate together with filamentous actin (F-actin) in phagolysosome formation, by establishing a connection between the phagosomal membrane and granule membranes prior to fusion. A prerequisite for phagolysosome fusion is the elimination of F-actin around the phagosomes to facilitate the membrane contact between lysosomes and phagosomes. We have, therefore, investigated the role of gelsolin, which is a protein that severs Factin by binding to the barbed ends, and thereby inhibits further polymerisation. The results show that both annexin I and Ill, and gelsolin translocates to the area of phagocytosis, in a Ca2+ -independent manner, where they eo-localise with F-actin.
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| 4. |
- Karlson, Ulrika, 1975-
(författare)
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Cutting Edge – Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- It is well established that mast cells (MC) are key players in airway pathologies such as allergic asthma, but they are also known to contribute to host defense and tissue remodeling. MC serine proteases are the major protein components of mast cell granules and accordingly, are most likely involved in many aspects of MC function. Two major groups of MC serine proteases have been described; chymases, which cleave a target preferentially after aromatic amino acids, and tryptases, which cleave preferentially after positively charged residues. Biochemical characterization of these proteases is a first step towards understanding their contribution to MC function. One of the issues addressed in this thesis is the target specificity of two rodent MC chymases, rat mast cell protease (rMCP)-4 and rMCP-5. The substrate specificity was analyzed using a substrate phage display technique, in which a large library of peptide substrates is screened simultaneously in a single reaction. The substrate analysis revealed that rMCP-4 displays very stringent substrate specificity, with striking preference for two subsequent aromatic amino acids N-terminal of the cleavage site. This chymase therefore holds a substrate recognition profile clearly distinct from other chymases. Database searches using the generated peptide sequence identified several interesting potential targets for rMCP-4, such as the FcγRIII and the TGFβ receptor. The phage display technique was also used to analyze the substrate specificity of rMCP-5. rMCP-5 is the rat chymase most closely related in sequence to human chymase. Interestingly, rMCP-5, unlike human chymase, was shown to hydrolyze substrates after small aliphatic amino acids, but not after aromatic residues. rMCP-5 and human chymase might therefore have different biological functions. Thus, studies of cleavage specificity can be a successful approach both to elucidate subtle differences in specificity of closely related proteases, as well as to identify new biological targets for a protease.The MC tryptases contribute to the pro-inflammatory activities of the MC. To assess the requirements for activation and stability of a mouse tryptase, mMCP-6, recombinant mMCP-6 protein was produced in mammalian cells. A low pH (<6.5), as well as a negatively charged proteoglycan, e.g. heparin, were shown to be necessary both to obtain and maintain activity. With this in mind, heparin antagonists were studied for their potential to inhibit mMCP-6 and human tryptase. Indeed, the heparin antagonists were shown to be highly efficient tryptase inhibitors. Thus, heparin antagonists might be promising candidates to attenuate inflammatory disorders, such as allergic asthma.
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| 5. |
- Thorpe, Michael
(författare)
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Haematopoietic Serine Proteases : A Cleavage Specificity Analysis
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mast cells are innate immune cells, historically involved in allergy responses involving IgE. Through this, they have earned a reputation as a fairly detrimental cell type. Their beneficial roles remain somewhat enigmatic although they clearly have the ability to modulate the immune system. This is due to their ability to synthesise many cytokines and chemokines as well as immediately release potent granule-stored mediators. One such mediator is a serine protease, chymase, which has been targeted by pharmaceutical companies developing inhibitors for use in inflammatory conditions.In order to address roles of the proteases, information regarding their cleavage specificity using substrate phage display can help find potential in vivo substrates. The human chymase cleaves substrates with aromatic amino acids in the P1 position and has a preference for negatively charged amino acids in the P2’ position. The molecular interactions mediating this P2’ preference was investigated by site-directed mutagenesis, where Arg143 and Lys192 had a clear effect in this selectivity.As humans express one chymase and rodents express multiple chymases, extrapolating data between species is difficult. Here, the crab-eating macaque was characterised, which showed many similarities to the human chymase including a near identical extended cleavage specificity and effects of human chymase inhibitors. Appropriate models are needed when developing human inhibitors for therapeutic use in inflammatory conditions.The effects of five specific chymase inhibitors in development were also tested. The selectivity of inhibitors was dependent on both Arg143 and Lys192, with a greater effect of Lys192. Identification of residues involved in specific inhibitor interactions is important for selective inhibitor development.Another innate cell type, the NK cell, is important in virus and tumour defence. In the channel catfish, a serine protease from an NK-like cell, granzyme-like I, was characterised. A strict preference for Met in the P1 position was seen, and caspase 6 was identified as a potential in vivo target. This may highlight a novel apoptosis-inducing mechanism from a similar cell type has been conserved for approximately 400 myr.Here, important residues mediating chymases’ specificity and interactions with inhibitors has been addressed, as well as finding a new animal model for providing ways to combat their roles in pathological settings.
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