Fibroblast growth factors (FGFs) constitute a family (currently FGF-1 to FGF-23) of polypeptides that are essential in embryonal development and adult physiology, in animals from nematodes to humans. FGFs bind to four receptor tyrosine kinases, denoted FGFR-1 to FGFR-4. For proper function, the FGFs and their receptors depend on specific polysaccharide co-receptors, denoted heparan sulfate (HS). This thesis describes HS regulation of FGFR-1 signal transduction using blood vessel endothelial cells as a model. We have determined HS structural features, necessary for FGF-2 induced FGFR-1 activation, using chemically modified heparin, which is structurally related to HS. Modified heparin, lacking sulfation at the 6-O position was inhibitory for FGFR-1 kinase activation and FGF-2 induced angiogenesis. Inhibition of blood vessel formation using modified heparin could be useful in treatment of diseases characterized by excess blood vessel formation. The critical role of HS sulfation for proper growth factor function was further underscored using an embryonal stem (ES) cell model. ES cells lacking expression of two isoforms of N-deacetyl N-sulfotransferase, NDST-1 and –2, failed to undergo embryonal development and to establish a vascular system. Exogenous heparin could not support development, but HS delivered from other ES cells allowed formation of primitive vessels and subsequent sprouting angiogenesis. We have, furthermore, shown that the mechanism whereby HS supports FGF receptor activation is qualitative, as well as quantitative. Kinase activity could be induced by FGF-2 in the absence of HS, but this allowed only selected phosphorylation. In the presence of HS, the kinase activity was stabilized, allowing a broader spectrum of phosphorylation of sites on the FGF receptor itself as well as on cytoplasmic substrates. Finally, using selected microarrays, we have examined the potential regulation of enzymes in the HS biosynthesis pathway and of different proteoglycans to which HS is attached. Overall, we found no evidence for dramatic regulation on the transcriptional level, but could identify specific upregulation of HS proteoglycan syndecan-2, during blood vessel formation in vitro. In conclusion, our studies demonstrate selective and complex regulation of HS synthesis and structure, essential in guiding growth factor function during health and disease.

Over 20 different plasma proteins have been shown to have the capacity to undergo conformational changes and self-assemble into highly stable and insoluble amyloid fibrils. One, transthyretin (TTR), consists of 127 amino acid residues arranged in eight β-strands (named A to H) and is involved in two different clinical forms of amyloidosis. In familial amyloidotic polyneuropathy (FAP), mutated TTR is found in the amyloid deposits while in senile systemic amyloidosis (SSA) only wild type TTR is present in the amyloid deposits. In this study, we have identified a novel form of amyloidosis that is caused by the deposition of an N-terminal fragment of apolipoprotein A-IV (apoA-IV). Interestingly, apoA-IV amyloid was found deposited in a patient that also suffered from SSA. Thus, this patient had two biochemically distinct and concurrent forms of amyloidosis that were derived from apoA-IV and TTR. We have also discovered that two different morphological deposition patterns (identified as patterns A and B) exist in TTR-derived amyloidosis. Pattern A, observed in all SSA patients studied and in half of the FAP patients examined contained large homogenous deposits that were composed of short randomly oriented fibrils. In contrast, pattern B was observed in the remaining FAP patients and was represented by smaller-sized deposits that consisted of longer fibrils that were arranged in parallel bundles. The predominant TTR component deposited also differed between the two amyloid patterns. Amyloid pattern A contained mainly C-terminal TTR fragments while pattern B amyloid consisted of full-length TTR. Our findings suggest that two different mechanisms of fibril formation may exist in TTR-derived amyloidosis. We have found two epitopes, corresponding to strand C and H that are surface-exposed in TTR-derived amyloid fibrils but hidden and part of the hydrophobic core in the native molecular structure. This indicates that TTR undergoes partial unfolding during fibril formation.

Mast cell (MC) accumulation around tumours is an old observation gaining new relevance due to the multifaceted nature of MCs and their many roles in immunity, beyond allergy. Knowledge about tumour specific recruitment of, and interactions with, MCs is needed to unravel the function of their presence. This study investigates the participation of mast cells in the tumourigenesis of Hodgkin lymphoma (HL), a tumour with many inflammatory features. We report that MC recruitment into HL lymphomatous tissue is possibly due to the production of CCL5/RANTES by malignant Hodgkin and Reed-Sternberg (HRS) cells. In addition, increased levels of IL-9, a cytokine implicated in mast cell heterogeneity and as an autocrine growth factor for HRS cells, were found in HL patient sera and correlate with negative prognostic factors. The ubiquitous expression of CD30 by HRS cells has been implicated in HL tumour development. In HL tissue MCs were found to be the predominant CD30 ligand (CD30L) expressing cells, and through CD30L/CD30 engagement they induced a proliferative response in HRS cells. This interaction proved to be bi-directional as it induced a degranulation-independent de novo synthesis of a specific set of chemokines in MCs, including IL-8. This novel trigger of MC activation is suggested to be of importance also in atopic dermatitis (AD) and psoriasis since increased numbers of CD30L and IL-8 positive MCs were detected along with increased expression of CD30. Data presented in this study supports a specific recruitment of MCs into HL tumours and co-operative interactions between HRS cells and MCs. Our identification of reversed signalling via CD30L as a novel MC trigger provides a mechanism behind leukocyte infiltration and chronic development in diseases associated with CD30 and MCs, such as HL, AD and psoriasis.

Mast cells are long-lived effector cells of importance for both acute and chronic inflammations. Mast cells can be activated in many different ways, leading to the release of inflammatory mediators. In contrast to most other inflammatory cells, activated mast cells have the capacity to recover, regranulate and thereby be activated again. In this thesis I have investigated the mechanisms involved in regulating activation-induced mast cell survival. We have found that cross-linking of FcεRI-bound IgE with an antigen (IgER-CL) induces a survival program in mast cells. Upon IgER-CL, mouse and human mast cells upregulate the pro-survival Bcl-2 family gene A1/Bfl-1. A1-/- mast cells degranulate upon FcεRI activation but they cannot recover most likely due to the lack of A1. Sensitized and provoked A1-/- mice exhibit lower amounts of mast cells compared to littermate controls. In contrast to mast cells, no Bfl-1 expression or survival promotion can be detected in basophils after IgER-CL. Another mast cell secretagogue, an adenosine receptor agonist, neither promoted upregulation of A1 nor survival. Although it is well established that mast cell survival is dependent on stem cell factor (SCF), it has not been described how this process is regulated. We have found that SCF promotes survival through Akt-mediated inhibition of the forkhead transcription factor FOXO3a and its transcriptional target Bim, a BH3-only pro-apoptotic protein. SCF-treatment prevents upregulation of Bim protein expression and leads to an upregulation of Bim phosphorylation through PI3-kinase and MEK-dependent pathways. Overexpression of FOXO3a causes an upregulation of Bim and induces mast cell apoptosis, even in the presence of SCF. Taken together, the work in this thesis demonstrates that A1/Bfl-1 and Bim play key roles in mast cell survival. These findings might be of importance in understanding the mechanisms of mast cell longevity and hence for possible new therapeutics used for mast cell-associated inflammations.

Upon activation mast cells release numerous proinflammatory mediators. With this feature, mast cells play an important role in host defense against pathogens, and are involved in tissue remodeling and wound healing. However, in cases of excessive inflammation the effects of mast cells are detrimental. This is observed in allergy, asthma, rheumatoid arthritis, atherosclerosis, certain types of heart failure, and in several other chronic destructive inflammations. Mast cell numbers are typically increased at inflammatory sites. There they act both directly, as effector cells, and in a regulatory manner, secreting agents that recruit and activate other immune cells. The studies presented here investigated mechanisms regulating mast cell numbers at sites of inflammation, focusing on cell migration and regulation of survival/apoptosis. We report that SCF-induced mast cell migration requires p38 MAP kinase activity. Moreover, we found that SCF-mediated mast cell survival is regulated through downregulation of the proapoptotic Bcl-2 family member Bim, as well as through phoshorylation of Bim. SCF seems to control Bim protein levels via FOXO transcription factors, and to induce phosphorylation of Bim via the Mek/Erk and the PI3-kinase/Akt signaling pathways. Furthermore, mast cell death triggered by deprivation of SCF and/or IL-3 involves the Bim protein, as demonstrated using bim-/- mast cells. Additional studies revealed that IgE-receptor activation, which occurs in allergy, promotes both prosurvival and proapoptotic signaling events. This includes upregulation of Bim and the prosurvival Bcl-XL and A1, as well as phosphorylation of Akt, FOXO factors, GSK-3β, IκB-α, Bad, and Bim. The simultaneous stimulation of prosurvival and proapoptotic signaling events could be a way to fine-tune the fate of mast cells after IgE-receptor activation and degranulation. The new insights about mechanisms involved in mast cell migration and regulation of survival/apoptosis might prove useful for future efforts to design new drugs to be used for mast cell-associated diseases.

During development of the mammalian embryo, spatial and temporal expression of fibroblast growth factors (FGFs) and their cognate receptors are vital in the regulation of a number of patterning processes. Inappropriate or decreased expression leads to severe malformations and even embryonic death. The objectives of this thesis have been to evaluate the usefulness of differentiating embryonic stem (ES) cells as a model to study FGF and FGF receptors in endothelial and hematopoietic cell function in vitro and in vivo, and the effect of an activating mutation in the platelet-derived growth factor receptor-β (PDGFR-β) on endothelial cells and vessel formation. Aggregates of differentiating ES cells, denoted embryoid bodies, faithfully recapitulate many developmental processes. Embryoid bodies cultured in fetal calf serum spontaneously develop cardiomyocytes and endothelial cells. The endothelial cells organize into lumen-containing vessels carrying erythroblasts. Administration of FGF or vascular endothelial growth factor (VEGF)-A promotes development of specific vascular phenotypes. About 20% of endothelial cells in embryoid bodies and teratomas express FGFR-1, and these FGFR-1-expressing endothelial cells are mitogenically active in the absence of exogenous stimuli and respond to VEGF-A to the same extent as endothelial cells lacking FGFR-1 expression. FGFR-1 deficiency leads to arrest in hematopoietic differentiation, whereas endothelial cell development is enhanced. As a consequence, teratomas derived from ES cells lacking FGFR-1 expression display vessels composed of a double layer of endothelial cells. The hyperactivity of endothelial cells derived from FGFR-1-deficient ES cells is suggested to be due to hyperactivity of VEGF receptor-2, as well as to loss of negative regulators of angiogenesis, such as interleukin-4. Mutation of platelet-derived factor receptor-β (PDGFR-β) to replace D849 in the activating loop in the kinase domain with V leads to ligand-independent kinase activity, increased basal signal transduction, and enhanced expression of VEGF-A as well as VEGFR-2. As a result, endothelial cell sprouts covered with pericyte-like cells are formed in a VEGF-A/VEGFR-2 dependent manner in ES cells expressing the mutated PDGFR-β. In conclusion, embryoid bodies represent a high-quality model for the study of growth factor-regulated vascular development and sprouting angiogenesis.

Amyloid is a form of abnormal protein aggregation within the living body. Massive deposits can lead to organ failure. There is also increasing evidence that smaller pre-amyloid aggregates exert direct toxic effects to cells. To date 25 different proteins are known to occur as amyloid deposition in human tissues, although not all of these conditions are known to be associated with clinical diseases. This thesis deals with the very common form of amyloid localized to the arterial media. The fibril protein called ‘medin’ was identified in 1999. Medin is a 50 amino acid residue internal fragment of the precursor protein lactadherin. Lactadherin, first found in human milk, is expressed in various tissues such as breast epithelium (including carcinomas), macrophages and aorta. The function of the protein is not known but it has several functional domains. There is an EFG like domain, including an RGD-sequence, in the N-terminal part of the molecule. The C-terminal part consists of C1 and C2 coagulation factor V and VIII like domains. Medin is from within the C2 domain. This region is suggested to be involved in phosphatidyl serine binding, important in phagocytosis of apoptotic cells. Medin amyloid was originally described from studies of the aorta. It is shown here that deposits are more widely spread and can be found in many large arteries, particularly within the upper part of the body. The prevalence of medin amyloid increases with age and deposits are found, to a certain degree, in virtually everyone above the age of 60 years. The amyloid is not only found extracellularly but intracellular deposits may also occur. Amyloid is usually associated with elastic lamina or lamellae which often show signs of fragmentation. Given the localization of amyloid to elastic structures of the arterial media, three different vascular diseases were studied: temporal (giant cell) arteritis, thoracic aortic aneurysm and thoracic aortic dissection. Medin amyloid was found in temporal arteries with and without inflammation. In inflamed arteries, amyloid was mainly located along the broken internal elastic lamina. Medin was also demonstrated within giant cells. It is suggested that medin may be an antigen triggering autoimmune giant cell arteritis. In the study of thoracic aortic aneurysms and dissections, we found significant less medin amyloid in diseased aortic tissues compared with a control material. On the other hand, immunoreactive medin, probably in the state of oligomeric aggregates, was regularly found in association with aneurysms and dissections but not in the control material. It is suggested that medin oligomers exert toxic effects on smooth muscle cells which may lead to weakening of the arterial wall with aneurysm or dissection as a consequence.

<p>In this thesis, methods to study gene and protein expression in cells and tissues were developed and utilized in combination with protein-specific antibodies, with the overall objective to attain greater understanding of protein function.</p><p>To analyze protein expression in <i>in vitro</i> cultured cell lines, a cell microarray (CMA) was developed, facilitating antibody-based protein profiling of cell lines using immunohistochemistry (IHC). Staining patterns in cell lines were analyzed using image analysis, developed to automatically identify cells and immunohistochemical staining, providing qualitative and quantitative measurements of protein expression. Quantitative IHC data from CMAs stained with nearly 3000 antibodies was used to evaluate the adequacy of using cell lines as models for cancer tissue. We found that cell lines are homogenous with respect to protein expression profiles, and generally more alike each other, than corresponding cancer cells <i>in vivo</i>. However, we found variability between cell lines in regards to the level of retained tumor phenotypic traits, and identified cell lines with a preserved link to corresponding cancer, suggesting that some cell lines are appropriate model systems for specific tumor types. </p><p>Specific gene expression patterns were analyzed in vitiligo vulgaris and malignant melanoma. Transcriptional profiling of vitiligo melanocytes revealed dysregulation of genes involved in melanin biosynthesis and melanosome function, thus highlighting some mechanisms possibly involved in the pathogenesis of vitiligo. Two new potential markers for infiltrating malignant melanoma, Syntaxin-7 and Discs large homolog 5, were identified using antibody-based protein profiling of melanoma in a tissue microarray format. Both proteins were expressed with high specificity in melanocytic lesions, and loss of Syntaxin-7 expression was associated with more high-grade malignant melanomas.</p><p>In conclusion, the combination of antibody-based proteomics and microarray technology provided valuable information of expression patterns in cells and tissues, which can be used to better understand associations between protein signatures and disease.</p>

Gallstas är ett patologiskt tillstånd där antingen produktionen eller flödet av galla är hämmat. Orsaken kan vara mekaniskt stopp och kallas då för extrahepatisk eller annan underliggande kronisk leversjukdom och kallas då för intrahepatisk. Vid gallstas samlas kopparassocierat protein (KAP) i levern. Dessa proteiner kan synliggöras med orcein. Orcein är en histokemisk färg som färgar KAP, och elastiska fibrer. Syftet med arbetet är att utifrån etablerade orceinprotokoll utföra avvikelser för att se hur dessa påverkar orceins förmåga att synliggöra elastiska fibrer. Metoden ska valideras med positivt kontrollmaterial för att på sikt användas inom diagnostik för att påvisa KAP i levervävnad vid avdelningen för Klinisk patologi i Region Kalmar Län. Som kontrollmaterial användes tunntarm och extransplanterad cirrotisk lever. Initialt testades etablerade protokoll och utifrån resultaten gjordes fortsatta tester. Fortsatta tester inkluderade (i) varierande inkubationstider i orcein vid rumstemperatur samt vid 60°C, (ii) varierande koncentration av surgjord kaliumpermanganat samt varierande inkubationstid i orcein i rumstemperatur, (iii) jämförelse mellan orcein från två olika tillverkare (HistoLab och RAL-diagnostic), samt (iv) validering av metod med positivt kontrollmaterial, vid varierande inkubationstid i värme (60°C) och med orcein från annan tillverkare (HistoLab). Resultaten visade att elastiska fibrer och bakgrund färgades mer intensivt vid längre inkubationstid samt att värme minskar bakgrunden i tunntarm. Högre koncentrationer kaliumpermanganat påverkade inte resultatet märkbart förutom vid längre inkubationstid där bakgrunden blev svagare. Stor skillnad upptäcktes mellan olika tillverkare av orcein. Vid infärgning av lever syntes KAP först efter infärgning över natt med RAL-diagnostics orcein och efter enbart 30 minuter med HistoLabs orcein. Bakgrunden i lever är allmänt högre än i tunntarm. Den slutsats som drogs av arbetet är att elastiska fibrer lämpligast synliggörs vid lång inkubationstid i 60°C. KAP synliggörs med båda varianterna av orcein, vidare tester är däremot nödvändigt. / Cholestasis is a pathological condition where bile flow from the liver to the intestines is somehow obstructed. Obstruction can result from mechanical blockage of the ducts (called extrahepatic) or metabolic inhibition (called intrahepatic). During cholestasis copper associated protein accumulate in the liver. These proteins can be made visible with the use of orcein. Orcein is a histochemical stain that stain copper associated proteins and elastic fibres. The purpose of this thesis was to use established protocols to stain elastic fibres and to make know deviations from these protocols to studied orceins ability to stain these structures. Validation of the method shall be performed with known copper associated protein material, so that the method can be used in the long run for diagnostic use at the department of Clinical Pathology, Region Kalmar County. Initial tests were conducted to compare the different protocols. Based on the results further testing was done. These tests included variation in temperature, variation in incubation time in orcein, variation in potassium permanganate concentration and testing the outcome of orcein from different manufacturers (RAL-diagnostic and HistoLab).  A validation was also conducted. The results showed that elastic fibres gained increased visibility with longer incubation time, higher temperatures increased visibility of elastic fibres even further and reduced background. Higher concentrations of potassium permanganate had limited effect, though it seemed to reduce background staining at longer incubations. Orcein from different manufacturers was also showed to preform very differently, with HistoLab giving best results. Copper associated proteins was visualised in liver with both types of orcein. The results showed a faster staning with orcein from HistoLab (30 min) than orcein from RAL-diagnostic (overnight). The conclusion is that elastic fibres is best visualised with orcein in 60°C and with longer incubation time. Visualisation of copper associated proteins can be done with orcein from both manufacturers, although further testing is required.

Hjärt-kärlsjukdomar är de vanligaste dödsorsakerna i Sverige och under 2016 stod de för 35 % av alla dödsfall i Sverige. Vid behandling av hjärtinfarkt är målet att återställa blodflödet till hjärtat. Trots att återställandet av blodflödet (reperfusion) till hjärtat leder till förbättrat kort- och långsiktigt hälsotillstånd så kan reperfusionen leda till irreversibla skador såsom kardiell kontraktionsbandsnekros. Luxol-fast-blue är en specialfärgning som används för att undersöka myelin samt visats färga in myofibrilla degenereringar såsom kontraktionsbandsnekros. Syftet med detta examensarbete var att göra det möjligt att påvisa akut hjärtinfarkt med luxor-fast-blue, en mer specifik färgmetod istället för rutindiagnostisk klassisk hematoxylin och eosin-färgning. Arbetet utfördes genom att vävnad från hjärta med känd hjärtinfarkt förbereddes och färgades med luxor-fast-blue. Metoden för infärgning med luxor-fast-blue modifierades för att uppnå optimal infärgning av kontraktionsbandsnekros vid hjärtinfarkt. De olika modifikationerna som gjordes var ändrad tid vid infärgning, differentiering och kärninfärgning. Resultatet av arbetet visade att vävnaden, med hjärtinfarkt, som färgades med luxor-fast-blue över natt, differentieringen under 50 sekunder och kärninfärgning med nuclear-fast-red under en minut var den mest optimala infärgningen av kontraktionsbandsnekros vid hjärtinfarkt. Där kunde blå infärgade kontraktionsband visualiseras i hjärtinfarktsbiopsin medan resterande celler var infärgade rött.