Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In Drosophila, one major defense mechanism is the production of a battery of antimicrobial peptides (AMPs). The expression of AMPs is primarily regulated at the level of transcription and constitutes both constitutive expression in a tissue-specific manner and inducible systemic expression in response to infection. The aim of my thesis has been to investigate the regulation of AMP gene expression at different levels. I have studied a novel cis-regulatory element, Region 1 (R1) found in the proximal promoter of all Cecropin genes in Drosophila melanogaster, as well as in other species of Drosophila. We found that the R1 element was important for the expression of CecropinA1 (CecA1) both in vitro and in vivo. A signaling-dependent R1-binding activity (RBA) was identified in nuclear extracts from Drosophila cells and flies. The molecular nature of the RBA, has despite considerable effort, not yet been identified. I also have studied the role of the JNK pathway in transcriptional regulation of AMP genes. The role of the JNK pathway in the regulation of AMP genes has long been elusive, however, in this study we showed that the pathway is directly involved in the expression of AMP genes. Analysis of cells mutant for JNK pathway components showed severely reduced AMP gene expression. Fur-thermore, over-expression of a JNK pathway-inhibitor also inhibited AMP gene expression. Lastly, I have studied transcription factors that have not previously been implicated in transcriptional regulation of AMP genes. In a yeast screen, three members of the POU family of transcription factors were identified as regulators of CecA1. Two of them, Drifter (Dfr) and POU do-main protein 1 (Pdm1) were further characterized. We showed that Dfr was able to promote AMP gene expression in the absence of infection, suggest-ing it to play a role in constitutive expression of AMP genes. Indeed, down-regulation of Dfr expression using RNAi severely reduced the constitutive expression of AMP genes in the male ejaculatory duct. We also identified an enhancer element important for Dfr-mediated expression of CecA1. Pdm1, on the other hand, was shown to be important for the systemic expression of AMP genes. In Pdm1 mutant flies, several AMP genes are systemically expressed even in the absence of infection, suggesting that Pdm1 works as a repressor of those genes. However, at least on AMP gene, AttacinA (AttA) requires Pdm1 for its expression, suggesting that Pdm1 works as an activator for this gene. Upon infection, Pdm1 was rapidly degraded, but, regenerated shortly after infection. We propose that the degradation of Pdm1 is important for the activation of the Pdm1-repressed genes and that regeneration sup-ports the expression of AttA.

Adenosine (A) to inosine (I) RNA editing is a widespread post-transcriptional mechanism in mammals that contributes to increase the protein diversity. Adenosine deaminases that act on RNA (ADARs) are the enzymes catalyzing RNA editing. ADARs are particularly active within the brain where they act on transcripts involved in neurotransmission. In this work the editing efficiency of all known site-selectively edited substrates have been analyzed during development of the mouse brain. We show that there is a global regulation of RNA editing, where editing levels of sites increase as the brain matures. This increase in editing efficiency cannot be explained by an increase in ADAR protein expression. During differentiation of primary cells from the mouse brain, editing levels increases similar to what we observe in vivo. Interestingly, the subcellular localization of the ADAR enzymes of cultured neurons show a different distribution in immature compared mature neurons. An accumulation of the ADAR enzymes in the nucleus may explain elevated A-to-I editing during brain development. Furthermore, we find that certain adenosines work as principal sites where editing of the transcript is initiated. Presumably, these sites are kinetically favored and are hypothesized to recruit the ADAR enzymes to the RNA substrate. Editing is then coupled to sites located in multiples of 12 nucleotides from each other. Interestingly, these sites reside on the same side in the 3D helix structure. The Gabra-3 transcript is site-selectively edited at a single position changing an isoleucine codon for a methionine upon editing. Gabra-3 encodes the a3 subunit of the GABAA receptor. We show that receptors assembled with edited a3 are less stable at the cell surface than the non-edited a3. We propose that the amino acid change upon editing, could affect protein interactions important for trafficking and stability of the GABAA receptors. Further, the editing event in a3 may have the function to reduce the number of a3 subunits in favor of other a subunits. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Myc is a transcription factor frequently found deregulated in human cancer. Cells with deregulated expression of Myc carry a selective advantage against its neighbours due to the fact that Myc-mediated transcription governs crucial cellular events such as proliferation and growth. In addition, Myc has been implicated in several other aspects of tumour biology like cellular immortality, the formation of new blood vessels and the colonization of distant tissues through the process of metastasis. Therapy aimed at disrupting essential pathways regulated by Myc is important because of the many different types of cancers that depend on continued signalling along these pathways.  This thesis describes new treatment opportunities for cancers with a high Myc signature. In Paper Ι, we describe a new role for the DNA methyltransferase inhibitor Decitabine in the treatment of Myc transformed tumours cells. We show that the therapeutic potential of Decitabine in the treatment of Burkitt Lymphoma relies not only on its ability to cause reactivation of silenced genes such as pro-apoptotic PUMA, but also on the DNA damage that this drug induces. In vivo, Decitabine delays disease progression of transplanted lymphoma cells. In Paper ΙΙ, we identify the DNA damage checkpoint kinase Chk1 as a therapeutic target in Myc overexpressing cancers. We show that targeting Chk1 with shRNA or with a novel small molecule inhibitor cause a delay in disease progression of transplanted lymphoma cells in vivo. In Paper ΙΙΙ, the Chk1-related kinase Chk2 is evaluated as a therapeutic target in Myc overexpressing cancers. Myc overexpressing cells are not dependent on Chk2 but we show that Chk2 abrogation using shRNA causes polyploidization and protection against DNA damage. However, Chk2-targeted therapy elicits a synergistic lethal response in combination with inhibition of the DNA repair associated protein PARP. In conclusion, this thesis shows the potential of targeting the DNA damage machinery and the functional hubs important for maintenance of genomic stability in tumours with a deregulated expression of Myc.

In this thesis, enzymes acting on the primary cell wall hemicellulose xyloglucan are studied.  Xyloglucans are ubiquitous in land plants which make them an important polysaccharide to utilise for microbes and a potentially interesting raw material for various industries.  The function of xyloglucans in plants is mainly to improve primary cell wall characteristics by coating and tethering cellulose microfibrils together.  Some plants also utilise xyloglucans as storage polysaccharides in their seeds. In microbes, a variety of different enzymes for degrading xyloglucans have been found.  In this thesis, the structure-function relationship of three different microbial endo-xyloglucanases from glycoside hydrolase families 5, 12 and 44 are probed and reveal details of the natural diversity found in xyloglucanases.  Hopefully, a better understanding of how xyloglucanases recognise and degrade their substrate can lead to improved saccharification processes of plant matter, finding uses in for example biofuel production. In plants, xyloglucans are modified in muro by the xyloglucan transglycosylase/hydrolase (XTH) gene products.  Interestingly, closely related XTH gene products catalyse either transglycosylation (XET activity) or hydrolysis (XEH activity) with dramatically different effects on xyloglucan and on cell wall characteristics.  The strict transglycosylases transfer xyloglucan segments between individual xyloglucan molecules while the hydrolases degrade xyloglucan into oligosaccharides.  Here, we describe and determine, a major determinant of transglycosylation versus hydrolysis in XTH gene products by solving and comparing the first 3D structure of an XEH, Tm-NXG1 and a XET, PttXET16-34.  The XEH activity was hypothesised, and later confirmed to be restricted to subset of the XTH gene products.  The in situ localisation of XEH activity in roots and hypocotyls of Arabidopsis was also visualised for the first time.  Furthermore, an evolutionary scheme for how XTH gene products developed from bacterial beta-1,3;1,4 glucanases was also presented based on the characterisation of a novel plant endo-glucanase, PtEG16-1. The EG16s are proposed to predate XTH gene products and are with activity on both xyloglucan and beta-1,3;1,4 glucans an “intermediate” in the evolution from beta-1,3;1,4 glucanases to XTH gene products. / QC 20110401

In the eukaryotic cell nucleus, spatial organization and dynamics of the genome is important in the regulation of gene expression. This thesis describes the use of the fission yeast, Schizosaccharomyces pombe, to study chromatin regulation and dynamics. We used nitrogen starvation to induce transcription of genes in fission yeast cells. In induced genes, nucleosomes get evicted in both the promoter and in the open reading frame (ORF). In the genes with the highest expression more nucleosomes get evicted from the ORF than from the promoter. This indicates that large rearrangements of the chromatin are occurring during a drastic gene induction. Many of the genes that become expressed early after nitrogen starvation are located together in clusters. In a cell where nitrogen is present in the surrounding media the gene clusters locate close to the nuclear periphery. When the nitrogen source is removed from the media, the clusters move to a more internal position. Thus rearrangement of chromatin due to gene induction, described in the first study, is accompanied by subnuclear changes of localization. Another type of regulation is the silencing of genes. We have studied a factor necessary for correct repression of genes located in silent chromatin, in S. pombe. The protein, Clr2, is part of the SHREC complex containing a remodeler (Mit1) and a histone deacetylase (Clr3). By bioinformatic analysis of Clr2 and newly sequenced fungi genomes, three motifs were identified. To gather more information about important parts of the Clr2 protein, deletions were made. When removing from about 20 to 100 amino acids in the middle of the protein, silencing of a reporter gene inserted at the mating-type region, inner repeats of centromere 1 and at the central core of centromere 2, failed. This indicates that Clr2 has an important role in establishing silent chromatin.

<p>Abstract</p><p>G-protein coupled receptors (GPCRs) are having the high medical importance since almost half of the medicinal drugs are designed as modulators of receptor molecules. Crystal structure or NMR structures of GPCRs are very difficult to determine because all GPCRs are typically bound to the cell membrane and thus their molecular activation mechanism is still unclear. The recent publication of the crystal structure of the 2-adrenoreceptor will provide new insights in the field of GPCR research.</p><p>Ghrelin is a peptide growth hormone which binds to the growth hormone secretagogue receptor (GHS-R) and stimulates the release of growth hormone. Based on the known ghrelin receptor binding core sequences wFwLL (upper letter and lower letter representative for L-form and D-form of the amino acids respectively), we prepared two novel peptide analogs with terminal S-(2-aminoethylsulfenyl) cysteine residues. These peptides were tested for their ability to suppress the binding of ghrelin to transfected COS7 cell-line (Kidney fibroblast line from the green African monkey) cells expressing the ghrelin wild-type receptor or certain mutants thereof. As a result we observed a significant reduction of the total number of binding sites accessible for ghrelin, which increased with the time the cells were incubated with our test compounds. This observations support our hypothesis that the peptides we tested form a covalent bond with free thiols located closely to the ligand binding-site of the receptor protein by disulfide thiol exchange which is an interesting target for development of anti-obesity drugs.</p>

Ebolavirus (EBOV) is a single stranded RNA virus that causes haemorrhagic fever in humans and other mammals. The EBOV encodes 7 proteins, NP, L, VP30, VP35, VP40, GP and VP24. VP24 is believed to be one of the EBOV proteins that causes the extreme virulence of the pathogen. The protein blocks the interaction between PY-STAT1 and KPNA, a protein that is involved in the import of PY-STAT1 into the nucleus. The nuclear import of PYSTAT1 is therefore blocked. This leads to the inhibition of IFN signalling. The purpose of this study was to express and purify VP24 and KPNA5. The proteins recombinantly expressed as a fusion tag in E. coli in lysogeny broth. Purification of VP24 was done using immobilized metal ion affinity chromatography, size exclusion chromatography and ion exchange chromatography. Characterization of the protein was analysed using circular dichroism. The results obtained from this study showed that VP24 could be purified in pH 10 buffers with little loss of protein due to aggregation and the protein was folded with an alphahelical structure. The expression and purification of KPNA5 was more complicated and further evaluation is left for future studies. The established protocol for expression and purification of VP24 and the initial work on KPNA5 will go a long way to aiding future studies on the system, thus the answer to the question regarding the extreme virulence of EBOV will be closer.

Zona pellucida glycoproteins (ZP) are important proteins for maturation of the oocytes in eukaryotes, these proteins are encoded by cluster of zp genes. zp2.3 and zp3.5 genes are expressed during the developing and maturation of the oocytes in zebrafish ovaries. Both of the gens have a CCAAT box in their promoter regions, playing a big role in the expression of the both genes in zebrafish oocytes. The transcription of the genes in the eukaryotes requires transcription factors to initiate and promote the transcription, the transcription factors can bind to the promoter region and initiate the transcription process. The nuclear factor y (NFY) regulates the genes by binding to the CCAAT boxes in their promoter regions, it consist from many subunit such as NF-YA and NF-YB. Here in this study we characterize the expression pattern of NF-YA and NF-YB by screening these genes expression in several organs and tissues, also to determine its roles in the expression of the zp2.3 and zp3.5 genes in the adultzebrafish ovary.

Different mutations within the NALP3 gene are thought to be associated with development of several types of hereditary auto inflammatory disorders such as neonatal onset multisystem inflammatory disorder (NOMID) and muckle-wells syndrome (MWS). In this work two separate mutations E688K and G569R were supposed to be constructed by site-directed mutagenesis in the cloned wild type NALP3 genes and further expressed in bacterial and mammalian host cells for functional studies in protein -protein interaction models.

De senaste åren har molekylärbiologisk data växt explosionsartat. Ny teknik gör att information kan härledas ur data snabbare och mer än tidigare. Faktum är att det tillkommer så mycket ny data att de nya resultaten inte längre kan publiceras i artiklar. Dessa sekvenser av data finns istället bara åtkomliga i speciella databaser. Dessa databaser är tillgängliga för allmänheten dels genom att ladda ned dem eller att använda en Internet browser. Det har, för ett par år sedan, kommit en ny standard för att strukturera upp och lagra data på. Detta nya sätt är XML. Det har föreslagits att XML skall ersätta eller ta över delar av de databaser som finns då XML skall vara ett bättre sätt att strukturera upp och lagra molekylärbiologisk data. Det finns däremot inga forskningar som direkt har undersökt hur bra XML är jämte andra databaser när det gäller Molekylärbiologisk data. Detta arbete skall ge en närmare inblick i hur bra databaser och XML kan hantera representation och lagring av sekvensdata som DNA och proteiner, då data av den sorten innehåller väldigt mycket information och är svår att representera. Resultaten kommer att visa att XML inte är riktigt tillräckligt bra för att ersätta de äldre databaserna. Resultaten kommer dessutom också att visa att de äldre databaserna, även om de är de bästa för tillfället, inte är perfekta för ändamålet att lagra sekvensdata.