<p>Plants produce a large diverse array of organic compounds that may function in protection against pathogens. Diverse antifungal compounds were reported to exist in barley (Hordeum vulgare L.); the indole alkaloid, gramine, and the pathogenesis-related proteins are some of them. Both the N-methyltransferase that is involved in gramine biosynthesis and PR-17a were studied in barley upon infestation by the bird cherry-oat aphid (Rhopalosiphum padi).</p><p>The effect of infestation by R. padi on induction of PR-17a and N-methyltransferase was investigated in different barley lines, susceptible and resistant.</p><p>The gene expression of PR-17a was down-regulated in the susceptible cv. Golf and to some extent up-regulated at the first days in var. Lina and then down-regulated. The PR-17a was induced by the aphid infestation in the resistant line CI16145; the gene expression was stronger in the infested plants than in the controls. The different responses in resistant and susceptible lines indicate that the induced PR-17a may play a role in the resistance against aphid infestation. PR-17a was up-regulated systemically in the base in barley after infestation by R. padi.</p><p>In the susceptible varieties Lina and Golf, the accumulation of N-methyltransferase did not increase with time from 1 day to 7 days after infestation, as determined by western blots with antibody raised against NMT from barley. The NMT-gene was down-regulated after 7 days infestation in both variety Lina and Golf both locally in the first leaf and in the base. Barley line CI16145 had no accumulation of NMT as was seen by western blotting. There was no induction of NMT in barley upon aphid infestation.</p>

<p>The number of protein-coding genes are likely not sufficient to account for the complexity of higher organisms. It is plausible that the proteome is responsible for the complexity of an organism.</p><p>An important mechanism that increases the protein variability is post-transcriptional modifications that alter the pre-mRNA sequence from that encoded in the genome. In this thesis work I have been focusing on a post-transcriptional process where adenosine (A) is deaminated to inosine (I), A-to-I RNA editing. Inosine is read as a guanosine (G) by the translation machinery, editing within coding regions can therefore give rise to more than one protein isoform from a single gene. A-to-I RNA editing is catalyzed by members of the ADAR enzyme family. ADARs have been found in all metazoans tested and two active ADAR proteins, ADAR1 and ADAR2, have been found in mammals. However, recoding by A-to-I editing is a rarely found event in mammals.</p><p>To detect novel substrates for A-to-I editing we developed an experimental approach to pull down ADAR2 substrates using immunoprecipitations. The captured RNAs were identified by microarray analysis. In this thesis two novel substrates for A-to-I editing are presented that were found using our IP-array approach, in combination with bioinformatic techniques.</p><p>The transcript coding for the GABA_Areceptor subunit α3 (Gabra-3) was found to be selectively edited by both ADAR1 and ADAR2. Editing of Gabra-3 recodes an isoleucine to a methionine and it was found to have a negative effect on the Gabra-3 assembly into the receptor. Moreover, the mouse specific CTN-RNA that codes for the CAT2 Transcribed Nuclear-RNA was shown to be hyper-edited by ADAR2.</p><p>In conclusion, this thesis work has resulted in an experimental method that extracts ADAR substrates. Two novel editing substrates were discovered. Our data adds additional evidence to the fact that RNA editing is of principal significance for a functional brain.</p>

<p>Progression through the G1, S, G2 and M phases of the cell cycle is controlled by cyclin-dependent kinases (Cdks) and cyclins. These proteins form active Cdk:cyclin complexes that phosphorylate specific substrates. The Cdk:cyclin complexes of the G1/S transition regulate the progression of cells into the S phase by phosphorylating the retinoblastoma protein (Rb). This prevents Rb from sequestering E2F, a transcription factor that induces expression of genes required for DNA synthesis. This process is in part regulated by a family of Cdk inhibitors (CKIs) called the Ink4 family (<u>In</u>hibitors of Cd<u>k4</u>). The Ink4 family of CKIs consists of four members; p16^Ink4a, p15^Ink4b, p18^Ink4c and p19^Ink4d, and they bind specifically to Cdk4 and Cdk6, thereby negatively regulating their kinase activities and cell cycle progression. Because of its cell cycle inhibitory role, p16^Ink4a is frequently mutated or deleted in human cancer, whereas the other <i>Ink4</i> genes are only occasionally altered in cancer. The overall aim of this thesis was to study the roles of p18^Ink4c and p19^Ink4d using <i>in vivo</i> models of cancer and embryonic development. In paper I, we analyzed the tumor spectrum in mice lacking <i>p53</i>, <i>Ink4c</i> and <i>Ink4d</i>. p53 is a tumor suppressor and one of the most frequently mutated genes in human cancer. Mice carrying mutated <i>p53</i> alleles are highly tumor-prone but develop predominantly lymphomas. However, the combined loss of <i>p53</i> and <i>Ink4c</i> (but not <i>Ink4d</i>) caused a shift in the tumor spectrum to increased incidences of hemangiomas and hemangiosarcomas, as well as appearance of medulloblastomas, a tumor of the cerebellum. These data, revealed in the absence of p53, suggest a cell-type specific tumor suppressing role for p18^Ink4c. In paper II, loss of <i>Ink4c</i> was evaluated in another tumor-prone mouse model; the Eµ-<i>Myc</i> mouse. This is a transgenic mouse overexpressing c-Myc in B cells causing clonal B cell lymphomas. Surprisingly, precancerous B cells and lymphomas from Eµ-<i>Myc</i> mice exhibited elevated levels of p18^Ink4c mRNA and protein despite high rates of proliferation. Moreover, loss of <i>Ink4c</i> in this model did not affect the rate of cell proliferation or the onset of tumor development. We conclude from these studies that <i>Ink4c</i> is not an important tumor suppressor of Myc-induced lymphomas. To gain insight into the role of <i>Ink4</i> genes in early vertebrate development, the African clawed frog, <i>Xenopus laevis</i>, was analyzed for the presence of <i>Ink4</i> homologs. Paper III describes the cloning and characterization of a gene homologous to <i>Ink4d</i>, <i>Xl-Ink4d</i>. This CKI is expressed throughout frog embryo development, making <i>Xl</i>-Ink4d the only CKI present during the cleavage stages of <i>X. laevis</i>. Antisense morpholino oligonucleotides directed against <i>Xl-Ink4d</i> were used to knock down the protein level of <i>Xl</i>-Ink4d during development. This resulted in defects in head tissues and reduced expression of <i>Twist</i>, a gene important for neural crest cell migration. We therefore propose that <i>Xl</i>-Ink4d is important for proper neural crest differentiation in the frog.</p>

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p>

<p>The goal of this project is to express both the normal CYP26 B1 and the spliced CYP26 B1 from human in <em>Escherichia coli </em>(E.coli) cells for further crystallization. This will be achieved by cloning in the DNA fragments into the Champion pET SUMO vector that is later transformed into <em>E.coli </em>cells. The CYP26 B1 contains a hydrophobic helix at the N-terminal of the protein, making both protein expression and crystallization difficult. Two variants of both full-length CYP26 B1 and the spliced variant will therefore be made, one with the trans-membrane helix present and one without the helix. The SUMO-vector will produce a fusion protein that will make CYP26 B1 more hydrophilic and improve the purification of the two proteins.</p>

<p>Flaviviruses, family flaviviridae, are often associated with severe diseases and many of the viruses have been shown to affect the immune response. Interferons confer important ways of defending the host against viral infections because they provide a link between the innate and the adaptive immunity. Langat virus (LGTV), belonging to the Tick-Borne Encephalitis (TBE) complex of viruses, has recently been shown to provide interactions with interferon receptors and inhibit the interferon-mediated response. Similarly, it has been demonstrated that the TBE virus NS5 protein affects type 1 interferon signaling. In this study we have analyzed the interaction between the TBE NS5 protein and interferon receptors by using the yeast two-hybrid system. Our results support the idea that the inhibition of interferon signaling does not involve a direct interaction between TBEV NS5 and the interferon receptors IFNAR2, IFNGR1 and IFNGR2.</p>

<p>Infection by the parasite Bonamia ostreae has infected and caused major mortality of the flat oyster, Ostrea edulis, over the last 25 years throughout the coasts of Europe and the United States of America. The conventional techniques for the diagnosis of infection with Bonamia ostreae are typically by histology and cytology. Both have a low sensitivity and Bonamia ostreae in weekly infected oysters can remain undetected when analyzed by such techniques. Molecular methods like the Polymerase Chain Reaction have recently been applied for a more reliable and sensitive detection of Bonamia ostreae.</p><p>The aim of this project was to optimize a PCR for the specific detection of the 18S Small Ribosomal subunit rDNA gene of Bonamia ostreae in formalin fixed Ostrea edulis tissues. While the PCR was successfully optimized for purified oyster DNA from fresh tissue it was difficult to apply on formalin fixed oyster tissues due to poor quality DNA from the fixed tissues. Ethanol fixed tissues were also tested for Bonamia ostreae, however, the primers were not specific for Bonamia ostreae and uninfected oysters also tested positive which led to the conclusion that the PCR could not be used as a reliable detection method for Bonamia ostreae in oysters. Despite using alternative primers which were designed to amplify other components of the Bonamia ostreae genome no consistent results were achieved to reliably use the PCR method for the accurate detection of Bonamia ostreae in oysters. The conclusion of this project is that other genomic sites in Bonamia ostreae must be identified as a target for PCR for this test to be specific.</p>

Injury to epithelial barriers puts metazoans at risk of loss of body fluid and contamination of their body by foreign particles. This risk is even exacerbated in insects, which have an open circulatory system and as a result, quickly need to seal wounds in order to keep a fairly constant internal milieu. Due to paucity of information on biochemical and molecular basis of insects’ clot, we studied how hemolymph of Drosophila melanogaster forms a clot, leading to a better understanding of responses after injury or infection in flies. By comparing hemolymph of Drosophila after bleeding with that described for an earlier model Galleria mellonella, we showed that a bona fide clot forms in Drosophila. The Drosophila clot is a fibrous network of crosslinked hemolymph proteins, which incorporates blood cells (plasmatocytes) extending shorter cellular processes of filopodia compared to cells outside the clot. Also, some plasmatocytes in the clot show features of apoptotic death while other blood cells (crystal cells) quickly rupture. The clot sequesters bacteria, as bacteria tethered to clot did not move. Clotting factors isolated include, Hemolectin (Hml) previously implicated in clotting, the immune induced protein Fondue and hemolymph proteins such as apolipophorin 2, fat body protein 1 and larval serum protein 1 γ. Hml mutants were more susceptible to infections when tested in a genetically sensitized background, suggesting that the clot may contribute to innate immunity. Clot also formed in hemolymph without phenoloxidase, an enzyme required for melanization and previously thought to be important for clot formation. However, we found that PO activity strengthens the clot to form a more solid plug. We found PO activity in clot to be induced in a transcription independent manner by inner membrane phospholipids: phosphatidylserine (PS) and phosphatidylinositol (PI) exposed on dead plasmatocytes and ruptured crystal cells. This is in contrast to induction of the enzyme during infection, which requires microbial components and transcriptional induction. However, both activation of PO in the clot and activation after infection appear to depend on proteases. Surprisingly, neither PS nor PI induced PO activity in the lepidopteran Galleria mellonella, in which the enzyme activity was instead induced by the microbial components peptidoglycan. This result may caution against generalizations of findings from using only one particular insect species. Finally, we found that the rupture of crystal cell during clot formation requires the Drosophila TNF homologue Eiger, JNK homologue Basket and small GTPases. This work therefore adds hemolymph clotting to the responses after injury or infection in flies and largely establishes Drosophila as a model to study coagulation of insect hemolymph. This will lead to a more comprehensive picture of Drosophila immunity with implications for other innate immune systems including our own. / At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper 5: Manuscript

The number of protein-coding genes are likely not sufficient to account for the complexity of higher organisms. It is plausible that the proteome is responsible for the complexity of an organism. An important mechanism that increases the protein variability is post-transcriptional modifications that alter the pre-mRNA sequence from that encoded in the genome. In this thesis work I have been focusing on a post-transcriptional process where adenosine (A) is deaminated to inosine (I), A-to-I RNA editing. Inosine is read as a guanosine (G) by the translation machinery, editing within coding regions can therefore give rise to more than one protein isoform from a single gene. A-to-I RNA editing is catalyzed by members of the ADAR enzyme family. ADARs have been found in all metazoans tested and two active ADAR proteins, ADAR1 and ADAR2, have been found in mammals. However, recoding by A-to-I editing is a rarely found event in mammals. To detect novel substrates for A-to-I editing we developed an experimental approach to pull down ADAR2 substrates using immunoprecipitations. The captured RNAs were identified by microarray analysis. In this thesis two novel substrates for A-to-I editing are presented that were found using our IP-array approach, in combination with bioinformatic techniques. The transcript coding for the GABAA receptor subunit α3 (Gabra-3) was found to be selectively edited by both ADAR1 and ADAR2. Editing of Gabra-3 recodes an isoleucine to a methionine and it was found to have a negative effect on the Gabra-3 assembly into the receptor. Moreover, the mouse specific CTN-RNA that codes for the CAT2 Transcribed Nuclear-RNA was shown to be hyper-edited by ADAR2. In conclusion, this thesis work has resulted in an experimental method that extracts ADAR substrates. Two novel editing substrates were discovered. Our data adds additional evidence to the fact that RNA editing is of principal significance for a functional brain.

Infection by the parasite Bonamia ostreae has infected and caused major mortality of the flat oyster, Ostrea edulis, over the last 25 years throughout the coasts of Europe and the United States of America. The conventional techniques for the diagnosis of infection with Bonamia ostreae are typically by histology and cytology. Both have a low sensitivity and Bonamia ostreae in weekly infected oysters can remain undetected when analyzed by such techniques. Molecular methods like the Polymerase Chain Reaction have recently been applied for a more reliable and sensitive detection of Bonamia ostreae. The aim of this project was to optimize a PCR for the specific detection of the 18S Small Ribosomal subunit rDNA gene of Bonamia ostreae in formalin fixed Ostrea edulis tissues. While the PCR was successfully optimized for purified oyster DNA from fresh tissue it was difficult to apply on formalin fixed oyster tissues due to poor quality DNA from the fixed tissues. Ethanol fixed tissues were also tested for Bonamia ostreae, however, the primers were not specific for Bonamia ostreae and uninfected oysters also tested positive which led to the conclusion that the PCR could not be used as a reliable detection method for Bonamia ostreae in oysters. Despite using alternative primers which were designed to amplify other components of the Bonamia ostreae genome no consistent results were achieved to reliably use the PCR method for the accurate detection of Bonamia ostreae in oysters. The conclusion of this project is that other genomic sites in Bonamia ostreae must be identified as a target for PCR for this test to be specific.