<p>Immunoglobulin E (IgE) is an important mediator in atopic allergies. This thesis describes the development of a therapeutic vaccine against IgE and its effects in rats and dogs. The development of an assay to determine IgE levels in dogs, and the finding of a chromosome region in rats that affects IgE levels are also reported. </p><p>The vaccine is a chimeric molecule consisting of the constant domains Cε2, Cε3 and Cε4 from IgE. The target domain of the vaccine is the Cε3 domain in the recipient species, which is the domain directly involved in receptor binding, while the flanking regions, Cε2 and Cε4, are from a distantly related mammal. In rats, the vaccine induced an immune response against circulating IgE, which decreased IgE levels by 90% and substantially reduced their allergic symptoms. Further, the effects of adjuvants in rats and dogs were evaluated, and when co-administered with the vaccine certain adjuvants were shown to increase the immune response against IgE. Mineral-oils were the most potent adjuvants in inducing a response against IgE, but metabolizable oils spiked with immunostimulatory substances were also efficient. </p><p>It was also shown that the therapeutic vaccine could induce a decrease in IgE levels in adult dogs, even though their initial levels were exceptionally high compared with humans. The IgE levels in 76 dogs ranged between 1 and 41 μg/ml while humans normally have around 150 ng/ml. However, the high IgE levels did not correlate to any specific breed, nor did they distinguish between dogs that were diagnosed as healthy and those suffering from atopic eczema, autoimmunity or skin parasites. </p><p>Regulation of total IgE levels probably involves many genes. In the final phase of the study, one candidate locus known to be involved in arthritis susceptibility in rats was investigated, and was found also to affect IgE levels.</p>

<p>Protein ubiquitination is an evolutionary conserved mechanism that controls a wide variety of cellular functions. Polyubiquitinated proteins are generally degraded in the proteasome, whereas monoubiquitination controls various other cellular processes, including endocytosis and endosomal sorting.</p><p>Termination of signaling by activated receptor tyrosine kinases (RTKs) largely occurs via their endocytosis and subsequent lysosomal degradation, processes accompanied by receptor ubiquitination. Cbl family proteins are major ubiquitin ligases that promote RTK ubiquitination and downregulation. We showed that epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors are monoubiquitinated at multiple sites following their ligand-induced activation and that a single ubiquitin is sufficient for both receptor internalization and degradation. Cbl also controls EGF receptor (EGFR) downregulation by binding to CIN85, which recruits endophilins to EGFR/Cbl complexes. In the complex with activated EGFRs, Cbl directs monoubiquitination of CIN85, and the entire complex is targeted for degradation in the lysosome. We propose that multiple monoubiquitination of activated receptors and associated protein complexes ensures proper receptor sorting towards the lysosome. Importantly, the functions of Cbl are also negatively controlled in order to maintain cellular homestasis. Sprouty2 blocks EGFR downregulation by sequestering Cbl from activated EGFRs. We showed that Sprouty2 also associates with CIN85 and that this binding is required for efficient inhibition of EGFR ubiquitination and endocytosis. </p><p>Cbl is also implicated in other aspects of RTK signaling, including organization of the actin cytoskeleton. We found that growth factor receptor signals promote lamellipodia formation in neuronal cells via a complex containing Cbl, the adaptor protein ArgBP2 and Pyk2. The lamellipodia formation required intact lipid rafts and the recruitment of Crk and PI(3)K to tyrosine phosphorylated Cbl.</p><p>In conclusion, our findings contribute to a better understanding of monoubiquitin signals in downregulation of RTKs and point at a role of Cbl in the regulation of cytoskeleton dynamics.</p>

<p>TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes.</p><p>The cell cycle inhibitor p21^Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect.</p><p>The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal <i>p21</i> promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as <i>Id2</i> that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene <i>Meox2</i>, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur.</p><p>This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.</p>

<p>Initiation of protein synthesis in <i>E.coli </i>involves several steps, which lead to the formation of the first peptide bond. This process requires three initiation factors: IF1, IF2 and IF3. Using a novel technique of combined light scattering and stopped-flow, we elucidated the importance of IF2•GTP conformation for the recruitment of 50S to 30S pre-initiation complex. Moreover, GTP hydrolysis is essential for IF2 release and later binding of ternary complex. Interestingly, a switch in IF2 affinity to G-nucleotides is induced during 30S pre-initiation complexes formation. </p><p>We found that IF1, previously with unknown functions in vitro, increases the rate of naked 70S dissociation by a factor 80 and acts as a fidelity factor in preventing 70S formation containing elongator tRNA instead of fMet-tRNA^fMet. We showed that RRF/EFG/IF3 split both naked and post-termination complexes while IF1/IF3 split only naked ribosomes. The mechanisms of action of RRF/ EFG, the order of their binding to 70S, as well as, the three different conformation of EF-G on the ribosomes are emphasized. Interestingly, 70S formation rate is dependent on the concentration of IF3 and not linear with 50S subunits concentration. We demonstrated that the rate-limiting step in 70S formation is IF3 dissociation from 30S complexes.</p><p>The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNA^fMet (initiator tRNA), Met-tRNA^fMet(non-formylated initiator tRNA) and Phe-tRNA^Phe (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNA^fMet, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes.</p><p>Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNA^fMet binding to 30S subunits, subsequently; the stabilization of fMet-tRNA^fMet by IF2 increases the rate of IF3 dissociation. Later, IF2 in GTP conformation allows 50S docking to 30S pre-initiation complex free of IF3 followed by GTP hydrolysis allowing IF2 release for ternary complex to bind and start elongation of protein synthesis. </p>

<p>Prostate cancer is common in the Western society and current treatments are often associated with side effects, therefore improved therapeutic strategies are desired. 2-methoxyestradiol (2-ME), an endogenous metabolite of estradiol-17β inhibits tumor growth <i>in vivo</i> as it prevents angiogenesis. 2-ME has also direct cytotoxic effects on tumor cells. In this study, we have investigated the potential use of PET to record effects 2-ME on prostate cancer cell (PC3) aggregates. The anti-proliferative and pro-apoptotic effects of 2-ME on PC3 cell aggregates <i>in vitro</i> were correlated with the uptake of deoxy-D-glucose, FMAU and choline labeled with ¹⁸F, ¹¹C or ³H. 2-ME clearly reduced growth of PC3 aggregates and induced apoptosis in a dose-dependent manner. However, the PET tracers failed to record the cytotoxicity of 2-ME on PC3 aggregates. </p><p>Further, the signaling events responsible for 2-ME induced prostate cancer cell death were investigated. We found that Smad7, previously implicated in TGF-β-induced responses, is required for 2-ME-induced p38 MAPK activation and subsequent apoptosis in PC-3U cells, as shown by the use of antisense or siRNA techniques and a specific inhibitor of p38 MAPK (SB203580). Interestingly, Smad7 also regulated the expression of the pro-apoptotic Bim protein. </p><p>Shb is a Src Homology 2 domain adapter protein with pro-apoptotic effects. PC3 clones overexpressing Shb exhibited increased rates of apoptosis, both in the presence or absence of 2-ME, as they failed to activate survival mechanisms through ERK and Akt in response to 2-ME. Notably, Shb cells displayed increased activity of the pro-apoptotic kinase c-Abl. Pre-treatment with SB203580 or c-Abl (STI-571) inhibitors completely blocked the apoptotic response to 2-ME. </p><p>In conclusion, Smad7 and Shb appear to be crucial for 2-ME-induced PC3 cell apoptosis via their activation of p38 MAPK and c-Abl. Future therapies exploring these pathways can be envisaged as treatment of prostate cancer.</p>

<p>Glycosidases hydrolyse the glycosidic bond in carbohydrates. Structural studies of three glycosidases with different substrate specificities are presented in this work.</p><p>Dextranase catalyzes the hydrolysis of <i>α</i>-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from <i>Penicillium minioluteum</i> was solved in the apo-enzyme (1.8 Å resolution) and product-bound (1.65 Å resolution) forms. The main domain of the enzyme is a right-handed β-helix, which is connected to a β-sandwich domain at the N-terminus. Using NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon. A new clan is suggested that links glycoside hydrolase (GH) families 28 and 49.</p><p>Endo-<i>β</i>-1,4-D-mannanase catalyzes the depolymerization of <i>β</i>-1,4-mannan polymers. The structure of endo-1,4-<i>β</i>-mannanase Man5A from blue mussel <i>Mytilus edulis</i> has been determined at 1.6 Å resolution. Kinetic analysis of Man5A revealed that the enzyme requires at least 6 subsites for efficient hydrolysis. The architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that Man5A represents a new subfamily in GH 5. </p><p>Both the Dex49A and the Man5A structures were determined by multiple-wavelength anomalous diffraction using the selenium <i>K</i>-edge with selenomethionyl enzymes expressed in the yeast <i>Pichia pastoris</i>.</p><p>Endoglucanase Cel6A from <i>Thermobifida fusca</i> hydrolyzes the <i>β</i>-1,4 linkages in cellulose. The structure of the catalytic domain of Cel6A from <i>T. fusca</i> in complex with a non-hydrolysable substrate analogue has been determined to 1.5 Å resolution. The glycosyl unit in subsite –1 was sterically hindered by Tyr73 and forced into a distorted ²S_O conformation. In the enzyme where Tyr73 was mutated to a serine residue the hindrance was removed and the glycosyl unit in subsite –1 had a relaxed ⁴C₁ chair conformation.</p>

<p>Adenovirus E1A is needed to activate early viral genes and induce cell cycle progression to optimise the conditions for viral replication. This is mostly achieved through interactions between the first exon of E1A and cellular transcriptional regulatory proteins. The carboxy terminus of E1A binds the cellular corepressor of transcription C-terminal Binding Protein (CtBP), resulting in derepression of CtBP target genes. </p><p>Inducible stable U2OS cell lines were established, expressing wild type E1A (E1Awt) and a mutant unable to bind CtBP (E1A∆CID). Low inducible levels and loss of protein expression after prolonged induction together with induction of apoptosis were consistent with the fact that wild type E1A is a cytotoxic protein and correlated with the ability of CtBP to repress proapoptotic genes. E1A∆CID did not induce apoptosis and could be expressed at high levels for prolonged time periods. Moreover, the binding of CtBP contributed to E1A-induced activation of viral E1B and E4 genes, through possible targeting of Sp1 and ATF transcription factors.</p><p>In a micorarray study on mRNA levels in E1A-expressing cells, several genes consistent with the tumour suppressive and apoptotic properties of E1Awt were identified as differentially expressed. Furthermore, the differences between the two cell lines correlated with the presence of binding sites for CtBP-interacting transcription factors in the promoters of regulated genes, enabling the possible identification of new CtBP target genes. </p><p>Finally, a molecular characterisation of the CtBP mechanism of repression revealed that positioning proximal to the basal promoter element was required for efficient repression, suggesting that CtBP interferes with the basal transcriptional machinery. Two separate domains were identified in CtBP, conferring transcriptional repression and activation when expressed alone, achieved through their interaction with HDACs and HATs, respectively. However, together they cooperate to ensure maximal repression through recruitment of histone deacetylase and inhibition of histone acetyl transferase activity.</p><p>Together, these data shows important modulation of E1A activities by the binding of CtBP and suggests the involvement of acetylation/deacetylation complexes for the regulation of E1A function.</p>

Basement membranes are thin extracellular sheets that surround muscle, fat and peripheral nerve cells and underlay epithelial and endothelial cells. Laminins are one of the main protein families of these matrices. Integrins and dystroglycan are receptors for laminins, connecting cells to basement membranes. Each laminin consists of three different chains, (α, β, γ). Laminin-1 (α1β1γ1) was the first laminin to be found and is the most frequently studied. Despite this, it was unclear where its α1 chain was expressed. A restricted distribution of the α1 chain in the adult epithelial basement membranes was demonstrated in the present study. In contrast, dystroglycan was found to have a much broader distribution. Dystroglycan is an important receptor for α2-laminins in muscle, but binds also α1-laminins. The more ubiquitous α5-laminins were also shown to bind dystroglycan, but with distinctly lower affinity than α1- and α2- laminins. The biological roles of different laminin isoforms have been investigated. Differences were found in the capacity of various tested laminins to promote epithelial cell adhesion. The α5-laminins were potent adhesive substrates, a property shown to be dependent on α3 and α6 integrins. Each receptor alone could promote efficient epithelial cell adhesion to α5-laminins. Yet, only α6 integrin and in particular the α6A cytoplasmic splice variant could be linked to laminin-mediated activation of a mitogen-activated protein kinase (MAP kinase) pathway. Attachment to either α1- or α5-laminins activated extracellular-signal regulated kinase (ERK) in cells expressing the integrin α6A variant, but not in cells expressing α6B. A new role for dystroglycan as a suppressor of this activation was demonstrated. Dystroglycan antibodies, or recombinant fragments with high affinity for dystroglycan, decreased ERK activation induced by integrin α6 antibodies. Integrin αvβ3 was identified as a novel co-receptor for α5-laminin trimers. Cell attachment to α5-laminins was found to facilitate growth factor induced cell proliferation. This proliferation could be blocked by antibodies against integrin αvβ3 or by an inhibitor of the MEK/ERK pathway. Therefore, integrin αvβ3 binding to α5-laminins could be of biological significance.

The formation and maintenance of tissues in multicellular animals are crucially dependent on cellular interactions with the extracellular matrix (ECM). Two different studies on such interactions are presented herein. Studies on expression of laminins in normal and dystrophic skeletal muscle, clarified a much debated issue regarding discrepancies seen for laminin α1-chain expression between human and mouse tissues. Lack of laminin α1-chain expression was verified in both mouse and human skeletal muscle. Furthermore, the earlier discrepancies seen for laminin α1-chain expression was explained by showing that an antibody-reagent, commonly used in human studies, recognised the laminin α5-chain rather than the laminin α1-chain The integrin α11-chain (forming α11β1 integrin) is the latest addition to the integrin receptor family, and belongs to the I domain-containing group of integrin α-chains. Previous studies had shown that α11β1 is a collagen receptor. In the present study, the in vitro and in vivo functions of the α11-chain were further characterised. Distribution studies on embryonic human and mouse tissues showed that the α11-chain was expressed on mesenchymal cells in the developing tendon, perichondrium, intervertebral disc, and cornea. The interactions of α11β1 integrin with collagen type I and IV were studied in vitro. The α11β1 bound to these collagens in a manner similar to integrin α2β1 (with collagen type I being the preferred ligand for α11β1). Furthermore, α11β1 was shown to mediate migration on collagen type I coated surfaces, and to mediate contraction of collagen type I gels. The in vivo functions of the α11-chain were investigated by the generation of integrin α11-chain null-mice, using gene targeted disruption of the itga11 in embryonic stem cells. Two independent lines of mice lacking α11 protein were generated. Phenotypic analysis of these mice indicated a role for α11β1 in the formation of the musculoskeletal system.

RNase P is an essential ribonuclease responsible for removal of the 5’ leader of tRNA precursors. Bacterial RNase P consists of an RNA subunit and a small basic protein. The catalytic activity is associated with the RNA subunit, i.e. bacterial RNase P RNA is a ribozyme. The protein subunit is, however, essential for activity in vivo. RNase P RNA, as well as the holoenzyme, requires the presence of divalent metal ions for activity. The aim of this thesis was to increase our understanding of the catalytic mechanism of RNase P RNA mediated cleavage. The importance of the nucleotides close to the cleavage site and the roles of divalent metal ions in RNase P RNA-catalyzed reaction were investigated. Escherichia coli RNase P RNA (M1 RNA) was used as a model system. It was shown that different metal ions have differential effects on cleavage site recognition. Cleavage activity was rescued by mixing metal ions that do not promote cleavage activity by themselves. This suggests that efficient and correct cleavage is the result of metal ion cooperativity in the RNase P RNA-mediated cleavage reaction. The results suggested that one of the metal ions involved in this cooperativity is positioned in the vicinity of a well-known interaction between RNase P RNA and its substrate. Based on my studies on how different metal ions bind to RNA and influence its activity we raise the interesting possibility that the activity of biocatalysts that depend on RNA for activity are up- or downregulated depending on the intracellular concentrations of the bulk biological metal ions Mg2+ and Ca2+. The nucleotides upstream of the cleavage site in the substrate were found to influence the cleavage efficiency. This was not exclusively due to intermolecular base pairing within the substrate but also dependent on the identities of the nucleotides at position –2 and –1. The strength of the base pair at position –1/+73 was demonstrated to affect cleavage efficiency. These observations are in keeping with previous suggestion that the nucleotides close to the cleavage site are important for RNase P cleavage. We conclude that the residue at -1 is a positive determinant for cleavage by RNase P. Hence, my studies extend our understanding of the RNase P cleavage site recognition process.