Induced pluripotent stem cells (iPSCs) can self-renew and differentiate into many other cell types. IPSCs are derived from somatic cells, and upon reprogramming, they share an expression profile similar to embryonic stem cells (ESCs). Among their many applications, iPSCs are an advantageous tool for disease modelling, offering an accurate system to study human molecular networks associated with specific phenotypes. Moreover, progress in genome editing technologies improved the possibilities for investigation of genotype-phenotype relationship for diseases characterized by defined genetic variants. Indeed, CRISPR/Cas9 edited iPSCs lines from healthy donors offer the possibility to investigate molecular networks with comparison to an isogenic control line. Furthermore, the ability of iPSCs to differentiate into neural cells, makes them a good model for studying neurodevelopmental diseases (NDDs). NDDs are characterized by heterogenous genetics and phenotypes. Heterozygous gene variants in the alpha 1 subunit of the sodium-voltage gated channel 1.1 (SCN1A) and in Neurochondrin (NCDN) have been associated with epilepsy. While many variants defining NDDs are associated with genes of transcriptional networks, e.g. the zinc-finger E-box binding homeobox 2 transcription factor (ZEB2) or the RPB1 subunit of RNA polymerase II complex (POLR2A). Although published animal model systems are available, there is a lack of human derived systems to investigate the gene function in disrupted molecular networks in NDDs. In this project, IPSCs deficient for SCN1A, NCDN, ZEB2 and POLR2A were generated using CRISPR/Cas9. To further evaluate the quality of the cell lines as iPSCs model, a POLR2A knock down (K.D.) line carrying a 4 bp insertion and a ZEB2 knock out (K.O.) line carrying a 790 deletion were characterized. Pluripotency and differentiation potential were confirmed by flow cytometry analysis, immunostaining, and qPCR. Both lines maintained genome integrity and editing in the top predicted off targets was excluded with PCR and Sanger sequencing screening. Furthermore, ZEB2 is involved in induction of neural crest cells (NCC); ZEB2 deficient line and the control behave similarly after a week of NCC differentiation. In contrast, POLR2A variants suggest slowing of transcription compared to the wild-type, therefore rate of transcription was measured performing an activity assay. No relevant differences between POLR2A K.D. and control line were observed in transcription rate of early pre-mRNA.

Amyloidosis is a group of diseases where misfolded proteins aggregate in the body. These aggregates are called amyloid and today there are 37 different known amyloid proteins. Diagnosis of amyloidosis is done by Congo Red staining to find amyloid, and typing with immunohistochemistry together with mass spectrometry. An earlier study found an unknown amyloid fibril protein in the Ligamentum flavum of patients suffering from lumbar spinal stenosis (LSS), and another study found specifically amyloid of the precursor protein ApoA-I in the same tissue of patients with LSS. The aim of this project is to type an unknown amyloid fibril protein through immunohistochemistry and mass spectrometry as well as isolating the ApoA-I protein to be able to do further tests on the protein. The unknown amyloid protein was not characterized in this study, but it gave indications on what it is not and how to continue the search in future studies.

<p>Antibodies are a central part of the immune defense system, and a large variability in their specificity is needed in order to be able to react against all possible foreign substances we may encounter during our lives. In this thesis, results are presented from investigations into how an egg-laying mammal, the Australian duck-billed platypus (<i>Ornithorhynchus anatinus</i>) creates antibody variability. Our results show that despite the lack of many V gene families the antibody repertoire in the platypus seems to be well developed. A long and highly variable complementarity-determining region (CDR) 3 compensates for the limited germline diversity. Interestingly, the presence of additional cysteine residues in the CDRs may form stabilizing disulfide bridges in the antigen binding loops and thereby increasing the affinity of the antibody-antigen interaction. </p><p>Although the immune system is necessary for survival, it must be strictly controlled since it may otherwise over-react and cause more harm than benefits. Allergies and autoimmune diseases are examples of such over-reactions by the immune system. Allergies are increasing in the western world and have become one of the main medical issues of the 21^st century. IgE is the central mediator in atopic allergies such as hay fever, eczema and asthma; it is therefore a prime target in the development of allergen-independent preventative treatments. Here we present results from several studies of a novel vaccine strategy aimed at reducing the levels of IgE antibodies. The vaccine results in the induction of anti-IgE antibodies, and the skin reactivity upon allergen challenge was significantly reduced in vaccinated animals. Our results suggest that active immunization against IgE has the potential to become a therapeutic method for humans. In addition, an evaluation of possible adjuvants that could be used as immune stimulators and thus help break self-tolerance at the time of vaccination is presented.</p>

<p>Immunoglobulin E (IgE) occurs exclusively in mammals and is one of five immunoglobulin (Ig) classes found in man. Unlike other isotypes, IgE is best known for its pathological effects, whereas its physiological role remains somewhat elusive. </p><p>To trace the emergence of IgE and other post-switch isotypes we have studied Ig expression in two monotreme species, the duck-billed platypus (<i>Ornithorhynchus anatinus</i>) and the short-beaked echidna (<i>Tachyglossus aculeatus</i>), leading to the cloning of IgE, two IgG isotypes in platypus and echidna IgE. The presence of IgE and the conservation of the overall structure in all extant mammalian lineages indicates an early appearance in mammalian evolution and a selective advantage of structural maintenance. Furthermore, both of the two highly divergent platypus γ-chains have three constant domains. Hence, the major evolutionary changes that gave rise to the IgE and IgG isotypes of present day mammals occurred before the separation of monotremes from the marsupial and placental lineages, estimated to have occurred 150-170 million years ago.</p><p>As the central mediator in atopic allergy, IgE is a prime target in the development of preventive treatments. This thesis describes an active immunization strategy that has the potential to reduce IgE to a clinically significant extent. The active vaccine component is a chimeric IgE molecule, Cε2-Cε3-Cε4. The receptor-binding target domain, Cε3, is derived from the recipient species, whereas the flanking domains, acting both as structural support and to break T-cell tolerance, are derived from an evolutionarily distant mammal. Vaccination of ovalbumin-sensitized rats resulted in a substantial reduction in total IgE in three out of four strains, accompanied by a significant reduction in skin-reactivity upon allergen challenge. No cross-linking activity was observed and the response to vaccination was reversible with time. The apparent safety and efficacy of the vaccine suggest that active immunization against IgE has the potential to become a therapeutic method for humans. </p><p>Furthermore, the cloning and expression of the pig (<i>Sus scrufa</i>) ε-chain will facilitate the development of sensitive and specific assays for pig IgE, thus increasing the possibilities of using the pig model in future studies of IgE-mediated reactions.</p>

<p>Tyrosine phosphorylation is a crucial mechanism in cellular signaling and regulates proliferation, differentiation, migration and adhesion. The phosphorylation reaction is reversible and is governed by two families of enzymes: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). This thesis investigates the role of PTPs in regulating receptor protein tyrosine kinases (RTKs), and explores a mechanism for regulation of phosphatase activity.</p><p>Most receptor tyrosine kinases are activated by ligand induced dimerization, which results in an increase in receptor phosphorylation. Preparations of ligand-stimulated dimeric PDGF β-receptors were shown to be less susceptible to dephosphorylation compared with unstimulated receptors. This revealed that reduced receptor dephosphorylation contributes to ligand-induced increase in RTK phosphorylation.</p><p>The receptor-like phosphatase DEP-1 site-selectively dephosphorylates the PDGF β-receptor. One of the most preferred sites is the PLC-γ binding phosphotyrosine pY1021, and the autoregulatory pY857 is one of the least preferred sites. By using chimeric phospho-peptides derived from these two sites as substrate for DEP-1, it was shown that a lysine residue at position +3 acts as a negative determinant for DEP-1 and that an aspartic acid residue at position –1 is a positive determinant.</p><p>The modulatory effect of TC-PTP on PDGF β-receptor signaling was explored by using mouse embryonic fibroblasts derived from TC-PTP knockout mice. PDGF β-receptors derived from knockout cells exhibited a higher level of ligand-induced phosphorylation compared to receptors from wildtype cells. The increase was unevenly distributed between different autophosphorylation sites. The PLC-γ binding site, previously implicated in chemotactic response, displayed the largest increase. Consistently, a cell migration assay revealed hyper-responsiveness to PDGF of TC-PTP knockout cells as compared to wildtype cells.</p><p>Reversible oxidation of the active site cysteine in PTPs is a mechanism, which have been postulated to regulate phosphatase specific activity. An antibody-based generic method for detection of oxidized PTPs was developed. Using this method it was revealed for the first time that UV-induced inactivation of PTPs involves oxidation of the active site cysteine.</p>

<p>In about half of all newly diagnosed cancer cases, conventional treatment is not adequately curative, mainly due to the failure of conventional techniques to find and kill residual cells and metastases, which might consist of only a few malignant cells, without causing unacceptable complications to healthy tissue. To solve the problem a more selective delivery of cytotoxic substances to tumour cells is needed. The approach applied here is called ‘tumour targeting’ and implies the use of biomolecules that recognise specific molecular structures on the malignant cell surface. Such molecules are then used for a selective transport of toxic agents to the cancer cells. </p><p>The use of radionuclides as cytotoxic substances has a number of advantages: 1) radiation does not cause severe resistance; 2) there is a cross-fire effect and 3) smaller amounts of nuclides are required than other cytotoxic substances to cause the same damage. Such an approach is called radionuclide tumour therapy. Several factors are important for the success of radionuclide therapy, such as the pharmacokinetics of the radiolabelled substance and its radiocatabolites, as well as the physical and chemical properties of the radiolabel used.</p><p>Nuclear properties of the label should be consistent with the problem to be solved: primary diagnostics; quantification of pharmacokinetics and dose planning; or therapy. From this point of view, radiohalogens are an attractive group of radiolabels. Halogens have nuclides with a variety of physical properties while the chemical and biological properties of halogens are very similar. The same labelling procedures can be used for all heavy halogens, i.e. bromine, iodine and astatine. It has been demonstrated that the biodistribution of proteins labelled with different heavy halogens is quite similar. </p><p>The main goal of the study was to develop protein radiohalogenation methods that provide a stable halogen-protein bond, convenient labelling chemistry that preserves the binding properties of proteins, long intracellular retention of radioactivity in targeted cells and quick release of radiohalogenated catabolites from the blood circulation. Radiohalogenation of proteins using indirect methods was studied, including optimisation of labelling chemistry and biological characterisation of some labelled conjugates. Two groups for indirect radiohalogenation were used, representing two different labelling principles: activated ester of benzoic acid (1) and the derivative of <i>closo</i>-dodecaborate anion (2). The non-phenolic linker (1) as well as the borate-halogen moiety (2) probably prevent dehalogenation. The negative charge of the potential catabolic products of (2) might trap radiohalogens intracellularly. </p>

<p>Proteins belonging to the Cbl family are multidomain scaffolds that participate in numerous processes, assembling signaling complexes and mediating attachment of ubiquitin to receptor and non-receptor tyrosine kinases.</p><p>We characterized a novel role for Cbl and Cbl-b in ligand-dependent internalization of growth factor receptors. Upon stimulation with epidermal growth factor (EGF), Cbl proteins associate with EGF receptor, become phosphorylated, and bind to the three SH3 domains of CIN85, which brings endophilins to the complex with active receptors. Endophilins can induce internalization of the plasma membrane, contributing to formation of clathrin-coated pits. We identified a minimal binding domain for CIN85 in the carboxyl termini of Cbl/Cbl-b and observed constitutive association between CIN85, Cbl/Cbl-b and oncogenically stimulated receptor tyrosine kinases. In addition to functioning as a ubiquitin ligase, Cbl forms a complex with CIN85 and endophilin, which is required for efficient endocytosis and downregulation of membrane receptors.</p><p>In EGF stimulated cells, we observed inducible modification of CIN85 and related CMS proteins by attachment of a single ubiquitin molecule. Monoubiquitination of CIN85 was mediated by the RING finger and dependent on the carboxyl terminal part of Cbl/Cbl-b, and demanded an intact carboxyl terminus of CIN85. Prolonged stimulation with EGF induced concomitant degradation of EGF receptors, Cbl, and monoubiquitinated forms of CIN85 in lysosomes.</p><p>Cbl regulates cytoskeletal processes in a variety of cell systems. We identified SH3P2, a protein with SH3 domain and ankyrin repeats, as a Cbl partner and described its phosphorylation by Src and its distribution in fibroblasts and osteoclasts. SH3P2 formed inducible complexes with Cbl and actin in spread cells and colocalized with dynamic actin structures.</p><p>Our data contribute to better understanding of the role of Cbl in downregulation of receptor tyrosine kinases as well as in controlling actin rearrangement.</p>

<p>In this thesis I have studied the mechanism by which vitamin B12 regulates the expression of the <i>cob</i> operon and the <i>btuB</i> gene in <i>Salmonella typhimurium</i>. The <i>cob</i> operon encodes most of the 25 genes required for the <i>de novo</i> synthesis of vitamin B12, and the <i>butB</i> gene encodes the outer membrane protein needed for transport of exogenous vitamin B12 into the cell. Vitamin B12 is used as a cofactor in four enzymatic reactions in <i>Salmonella typhimurium</i>. The regulation by vitamin B12 of the <i>cob</i> operon and the <i>btuB</i> gene requires sequences in the long leader regions of the respective mRNAs. Proper folding of the reader mRNA is essential for normal repression, in particular a hairpin structure that sequesters the ribosomal binding site (RBS). The upstream leader region contains two conserved sequence elements that are required for the vitamin B12 regulation; the translational enhancer (TE) element element and the B12 box. The TE element confers its enhancer function by resolving the downstream inhibitory RBS hairpin through basepairing with nucleotides in the stem. In the presence of vitamin B12, either B12 itself, or a B12 regulatory factor binds to the upstream reader region and prevents the enhancer function. This will inhibit unfolding of the RBS hairpin and repress translation.</p>

In this thesis I have studied the mechanism by which vitamin B12 regulates the expression of the cob operon and the btuB gene in Salmonella typhimurium. The cob operon encodes most of the 25 genes required for the de novo synthesis of vitamin B12, and the butB gene encodes the outer membrane protein needed for transport of exogenous vitamin B12 into the cell. Vitamin B12 is used as a cofactor in four enzymatic reactions in Salmonella typhimurium. The regulation by vitamin B12 of the cob operon and the btuB gene requires sequences in the long leader regions of the respective mRNAs. Proper folding of the reader mRNA is essential for normal repression, in particular a hairpin structure that sequesters the ribosomal binding site (RBS). The upstream leader region contains two conserved sequence elements that are required for the vitamin B12 regulation; the translational enhancer (TE) element element and the B12 box. The TE element confers its enhancer function by resolving the downstream inhibitory RBS hairpin through basepairing with nucleotides in the stem. In the presence of vitamin B12, either B12 itself, or a B12 regulatory factor binds to the upstream reader region and prevents the enhancer function. This will inhibit unfolding of the RBS hairpin and repress translation.

Antibodies are a central part of the immune defense system, and a large variability in their specificity is needed in order to be able to react against all possible foreign substances we may encounter during our lives. In this thesis, results are presented from investigations into how an egg-laying mammal, the Australian duck-billed platypus (Ornithorhynchus anatinus) creates antibody variability. Our results show that despite the lack of many V gene families the antibody repertoire in the platypus seems to be well developed. A long and highly variable complementarity-determining region (CDR) 3 compensates for the limited germline diversity. Interestingly, the presence of additional cysteine residues in the CDRs may form stabilizing disulfide bridges in the antigen binding loops and thereby increasing the affinity of the antibody-antigen interaction. Although the immune system is necessary for survival, it must be strictly controlled since it may otherwise over-react and cause more harm than benefits. Allergies and autoimmune diseases are examples of such over-reactions by the immune system. Allergies are increasing in the western world and have become one of the main medical issues of the 21st century. IgE is the central mediator in atopic allergies such as hay fever, eczema and asthma; it is therefore a prime target in the development of allergen-independent preventative treatments. Here we present results from several studies of a novel vaccine strategy aimed at reducing the levels of IgE antibodies. The vaccine results in the induction of anti-IgE antibodies, and the skin reactivity upon allergen challenge was significantly reduced in vaccinated animals. Our results suggest that active immunization against IgE has the potential to become a therapeutic method for humans. In addition, an evaluation of possible adjuvants that could be used as immune stimulators and thus help break self-tolerance at the time of vaccination is presented.