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- Vijayaraghavan, Balaje
(författare)
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Identification and characterization of nuclear envelope protein interactions
- 2015
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Nuclear envelope which surrounds the chromatin of eukaryotic cells contains more than a hundred transmembrane proteins. Mutations in some genes encoding nuclear envelope proteins give rise to human diseases including neurological disorders. The function of many nuclear envelope proteins is not well established. This is partly because nuclear envelope proteins and their interactions are difficult to study due to the inherent resistance to extraction of nuclear envelope proteins. We have developed a novel method called MCLIP, to identify interacting partners of nuclear envelope proteins in live cells. Using MCLIP, we found three new binding partners of the inner nuclear membrane protein Samp1: the intermediate filament protein Lamin B1, the LINC complex protein Sun1 and the G-protein Ran. Furthermore, using in vitro studies, we show that Samp1 binds both Emerin and Ran directly. We have also studied the interaction between Samp1 and Ran in detail.
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| 3. |
- Karlsson, Sofia, 1974-
(författare)
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Studies of prostaglandin E2 formationin human monocytes
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Prostaglandin (PG) E2 is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE2 has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE2 is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A2 (PLA2), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE2 synthesising enzymes are not completely established. PGE2 was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA2a (cPLA2a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE2. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH2. The conversion of PGH2 to the final product PGE2 was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA2α was markedly advantageous for the formation of PGE2. Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE2 in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA2a.
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- Muthukrishnan, Uma, 1984-
(författare)
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The release of histone proteins from cells via extracellular vesicles
- 2018
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes.In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting.In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway.Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent.The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion.In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients. In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.
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- Ungerbäck, Jonas, 1982-
(författare)
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Notch signalling in carcinogenesis : With special emphasis on T-cell lymphoma and colorectal cancer
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Notch signalling pathway is an evolutionary conserved pathway, named after the Notch receptors, Notch1-4 in mammals, which upon cell-cell contact and ligand binding releases the intracellular domain (NICD). NICD translocates into the nucleus where it binds the transcriptional repressor RBP-Jk, which together with co-activators belonging to the Mastermind-like family of proteins form a transcriptional activation complex. This complex activates genes controlling cell fate decision, embryonic development, proliferation, differentiation, adult homeostasis and stem cell maintenance. On the other hand, disrupted Notch signalling may result in pathological conditions like cancer, although the mechanisms behind the disruption are often complex and in many cases largely unknown.Notch1 drives the lymphocyte differentiation towards a T-cell fate and activating mutations in the gene have been suggested to be involved in T-cell lymphoma. In paper I, genetic alterations in Notch1 and the Notch1 regulating gene CDC4 were investigated in tumours from murine T-cell lymphoma induced with phenolphthalein, 1,3-butadiene or 2’,3’-dideoxycytidine. We identified activating Notch1 mutations in 39% of the lymphomas, suggesting that Notch1 is an important target gene for mutations in chemically induced lymphomas.While it is known that constitutively activated Notch signalling has a clear oncogenic function in several solid malignancies as well, the molecular mechanisms are less known in this context. Unpublished data of our lab, together with other recent studies, suggest that mutations of Notch and Notch-related genes per se are uncommon in solid malignancies including colorectal cancer, while a growing body of evidence indicates that aberrant Wnt/b-catenin signalling may result in pro-tumoural Notch activation in these contexts. In paper II, we therefore investigated potential transcriptional interactions between the Notch and Wnt signalling pathways in colorectal cancer cell lines. The proximal Notch and Wnt pathway gene promoters were bioinformatically identified and screened for putative TCF/LEF1 and RBP-Jk sites. In canonical Wnt signalling, Apc negatively regulates b-catenin leading to repression of TCF/LEF1 target genes. Upon repression of the Wnt pathway we observed that several genes in the Notch pathway, including Notch2, were transcriptionally downregulated. We also confirmed binding of Lef1 to Notch2 as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for at least one LEF1/TCF-site in the Notch2 promoter upon co-transfection of HT29 or HCT116 cells with mutated b-catenin. HT29 cell lines were also treated with the g-secretase inhibitor DAPT, leading to inactivation of the Notch pathway by preventing release of NICD. However, results showed no effects on Apc, b-catenin or their target cyclin D1. Taken together, these results indicate that the Wnt pathway may function as a regulator of the Notch pathway through the TCF/LEF1 target gene program in colon cancer cell lines.In summary, Notch pathway deregulation is of importance in both murine T-cell lymphoma and human colorectal cancer, although the mechanisms differ. The current results give new insights in Notch pathway alterations as well as the signalling networks in which the Notch pathway interacts, and thus increase the understanding of Notch’s involvement in malignant diseases.
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- Tapani, Sofia, 1982
(författare)
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Three dimensional mathematical modelling of pronuclei migration for the mouse
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The main question addressed in this thesis is what happens between the moment when the sperm enters the egg and the fusion of the male and the female pronuclei. Orientation of the apposing pronuclei most likely plays a decisive role in the polarity of the developing embryo. The migration and the dependence between the pronuclei have been investigated through three different measures of correlation. It was concluded that a measure based on the projection of the movement onto the axis between the pronuclei’s centres was preferred. Two mathematical models that describe the pronuclei dynamics have been constructed in the form of stochastic differential equations. The models concern pronuclei migration from the time of the sperm entry to the fusion and spatial orientation of this fusion. First, a basic model was created. This was then developed into a refined model. The methodology consists of using stacks of confocal microscopy time-lapse images of the pronuclei migration together with statistical methods to identify realistic parameters in the models. Given different angles between the sperm entry and the position of the second polar body, the final models are then used to produce distributions of orientations of the meeting positions between the pronuclei. However, the main result is the suggested models themselves which describe the main features of the migration. The basic fitted model is based on two forces of attraction. Migration is directed towards the centre but also towards the other pronucleus. Parameter values corresponding to the size of these forces are estimated from data of both eggs treated with a microtubule inhibitor and untreated eggs. The refined model is also based on centring and attraction to the other pronucleus, but the centring is modelled into two mechanisms of pushing and pulling of the microtubule exerted forces. Simulated distributions from the models could for instance be used as initial value distributions for future models of egg cleavage.
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| 9. |
- Olausson, Johan, 1980-
(författare)
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Studies of recombinant forms of Aleuria aurantia lectin
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The presented work describes construction and analysis of recombinantly produced forms of Aleuria aurantia lectin (AAL). The binding properties of the produced AAL forms were studied using techniques such as tryptophan fluorescence, hemagglutination analysis, ELISA and surface plasmon resonance analysis.Lectins are proteins that are ubiquitous in nature with the ability to bind specifically to different types of carbohydrates. The physiological function of different lectins is not always known, but they are involved in many recognition events at molecular and cellular levels. In research, lectins are widely used for structural and functional studies of complex carbohydrates, and they are also used to detect changes in the carbohydrate pattern on glycoproteins in different diseases.With the use of recombinant technology it is now possible to refine properties of lectins such as decreasing the valency and alter specificity and affinity. This may be a way of constructing more suitable reagents for use in diagnostic glycosylation analysis assays.AAL has been extensively used in different types of research for its ability to bind the monosaccharide fucose and to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. AAL was expressed recombinantly (rAAL) and its properties was investigated. These studies reveled that one of the binding sites in rAAL had unusually high affinities towards fucose and fucosecontaining oligosaccharides with Kd-values in the nanomolar range. This binding site is not detected in AAL that have been exposed to fucose during its purification, and therefore we proposed that this site may be blocked with free fucose in commercial preparations of AAL.Normally lectin-oligosaccharide interactions are considered to be of weak affinity, so the finding of a high affinity site was interesting for the future study of recombinant forms of AAL. The next step was to produce recombinant AAL forms with decreased valency. This was done using site-directed mutagenesis. First a monomeric form of AAL (mAAL) was constructed and then a monovalent form of AAL, containing only one fucose-binding site (S2-AAL) was constructed. Both of these forms had retained ability to bind fucose. The binding characteristics of mAAL were similar to that of rAAL, but mAAL showed decreased hemagglutinating activity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and did not bind to sialylated fuco-oligosaccharides such as sialyl-LewisX. This study shows that molecular engineering techniques could be important tools for development of reliable and specific diagnostic and biological assays for carbohydrate analysis.
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| 10. |
- Molin, Jesper, 1987
(författare)
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Designing a digital pathology workstation for routine practice
- 2015
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The role of the pathology lab is important in the future of cancer care. In orderto further personalize the care for cancer patients, more precise review of tumorspecimens is needed in order to guide clinicians between different treatmentstrategies.New digital imaging technologies is one promising possibility that might allowpathologists performing more and better work with the same amount ofresources. Early scanning systems and workstations have been shown to beinefficient and have not met the pathologists’ needs, who still perform most oftheir diagnostic work with mechanical microscopes.In this thesis, we analyze the pathologist's work with early digital workstationsand present a set of new solutions in order to increase the performance of theinteraction with these systems.First, we review the implementation process of two current digital systems intwo pathology labs in Sweden (Paper I), followed by study of the navigationbehavior that is performed by pathologists when they explore large digital slidesof cancer specimens (Paper II).With a specific focus on design solutions that work within medical routinepractice, three different input devices for navigation in large images wascompared with pathologists as participants (Paper III), as well as a visualizationtechnique, inspired by semantic zoom in order to facilitate certain tasks forpathologists (Paper IV).The results provided in this thesis points towards the same conclusion that havemade in other domains: When good usability engineering is combined withtechnological advances, this can make novel technology become useful for real.For a Human-Computer Interaction (HCI) researcher, the pathologist caserepresents an especially demanding use of zoomable user interfaces. This hasdriven us to enhance efficiency of such interfaces further in order for them tobecome useful. The research findings offered within this thesis are particularlyimportant to the field of digital pathology. However, our findings could alsohave a bearing on the design of zoomable user interfaces.
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