The Transforming Growth Factor beta (TGFb) superfamily of ligands are highly versatile, functioning throughout development and in adult organisms as diverse as worms and humans to regulate a myriad of biological activities. TGFb family members signal through their cognate serine/threonine kinase receptors to mediate the phosphorylation and activation of receptor-regulated Smads (R-Smads), that then complex with the common Smad (co-Smad/Smad4) to transduce TGFb signals from the membrane into the nucleus. This thesis recounts the first identification of a mammalian Smad-interacting transcription factor, Foxh1. Investigation of the Smad/Foxh1 DNA-binding complex, which mediates TGFb-dependent regulation of transcription from a Gsc enhancer, determined that both Smad and Foxh1 binding sites are required. These studies also defined the first known biological difference between the highly related R-Smads, Smad2 and Smad3. Specifically, it was shown that while both can similarly participate in Smad/Foxh1 DNA-binding complexes, Smad2 activates and Smad3 represses Foxh1-mediated TGFb-dependent transcription. A detailed analysis of the Gsc enhancer element subsequently defined the sequence req irements for a functional Smad/Foxh1 enhancer (SFE). This information was utilized to direct in silico, genome wide searches for genes harbouring evolutionarily conserved SFEs, which successfully expanded the repertoire of Smad/Foxh1 targets. Analysis of these targets revealed novel roles for Smad/Foxh1 signalling in forebrain development and retinoic acid production. Finally, the importance of Foxh1 to heart development was examined. The interaction between Foxh1 and the heart specific factor Nkx2-5 was characterized with respect to TGFb-dependent regulation of Mef2c expression via a compound Foxh/Nkx2-5 enhancer (FNE). Genome-wide searches for similar FNEs identified many potential Foxh1/Nkx2-5 targets, further analysis of which will provide greater insights into how Foxh1 functions in heart development. In summary, the work presented herein expands our understanding of the role of TGFb in development through the identification and characterization of Foxh1 and its genomic targets downstream of TGFb signalling. / PhD

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

<b>PAPER 1</b> In the first paper we show that reducing the average molecular weight from ~350 kDa to &lt;6kDa by acid hydrolysis diminished the cell-stimulating activity of poly-M, measured as TNFproduction from human monocytes. However, the activity of the resulting oligomers (M-blocks) was greatly enhanced when covalently attached to particles (plastic beads or biodegradable albumin particles). Similar results were obtained with detoxified/deacylated LPS (DLPS) and glucuronic acid polymers (C6OXY), but not with G-blocks that by themselves are not active. These results suggest that the supramolecular structure affects the potency of polysaccharide stimuli, and that M-blocks attached to biodegradable albumin particles could possibly be exploited as an immunostimulant for protection against various diseases. <b>PAPER 2</b> In paper 2, according to the reviewers suggestion, the designation M-polymers of different molecular size was used in place of poly-M (~350 kDa) and M-blocks (~3 kDa). In this study we demonstrated that M-blocks and DLPS attached to particles engaged different receptors than soluble poly-M and DLPS in activation of monocytes. By using blocking mAbs to CD14, CD11b and CD18, we found that particulate stimuli employed the β2- integrin CD11b/CD18 in addition to the shared CD14 for signaling TNF-production. Moreover, whereas poly-M only bound to CD14-expressing CHO-cells, M-particles preferentially bound to CHO-cells expressing β2-integrins. However, the DLPS- and M-particles failed to activate NF-κB-translocation in CHO-cells co-transfected with CD14 and β2-integrins, suggesting that additional molecules are required for activation of CHO-cells. The major conclusion drawn from this work is that the supramolecular structure, in addition to influence the potency, affects the cellular receptor engagement by carbohydrates like poly-M and DLPS. This points to the importance of comparing the mechanisms involved in activation of immune cells by soluble bacterial components and whole bacteria to achieve a better understanding of inflammatory diseases like sepsis. <b>PAPER 3</b> Poly-M activates cells in a CD14-dependent manner, but CD14 is linked to the membrane with a GPI-anchor and mediates activation by interaction with other, signal-transducing molecules, like the TLRs. By using blocking mAbs to TLR2 (generated in our lab, paper 5) and TLR4, we found that both receptors were involved in mediating TNF-production from human monocytes in response to poly-M. Furthermore, TLR4 mutant (C3H/HeJ) and knockout (TLR4-/-) murine macrophages were completely non-responsive to poly-M, whereas TLR2-deficient macrophages showed reduced TNF-responses. These findings indicate that CD14, TLR2 and TLR4 on primary cells all participate in cytokine-induction by poly-M, and that TLR4 may be necessary for activation. <b>PAPER 4</b> In addition to CD14, β2-integrins have been implicated in LPS-induced cellular activation, and in this study we compared the involvement of CD14 and β2-integrins in TNF-production and NF-κB-activation induced by LPS and GBS cell wall fragments. With blocking mAbs to CD14 and CD18 we found that LPS and GBS cell walls shared CD14, but in addition the cell walls employed CD11/CD18 in mediating TNF-production from human monocytes. Both stimuli specifically induced NF-κB-translocation in CD14-transfected CHO-cells, but only LPS could activate cells transfected with CD11/CD18. The lack of response to GBS cell walls in CD11/CD18-transfected CHO-cells indicated that the cell walls need CD14 for cell activation. Further in paper 4 we demonstrate the ability of GBS cell walls to activate LPS-hyporesponsiv C3H/HeJ mouse macrophages, suggesting that LPS and GBS cell walls employ different receptors/signaling mechanisms in murine macrophages. <b>PAPER 5</b> When it was discovered that human TLR2 and TLR4 are involved in microbial recognition, we started to generate a mouse mAb to human TLR2, and in paper 5 we report the production and characterization of the mAb TL2.1. We subsequently used this mAb to evaluate the role of TLR2 in mediating activation by heat-killed GBS and L monocytogenes. L. monocytogenes, but not GBS, activated TLR2-transfected CHO-cells to IL-6-production, and the response was inhibited by TL2.1. A CD14 mAb and TL2.1 both inhibited TNF-production from monocytes induced by L. monocytogenes, but neither mAb affected the TNF-response triggered by GBS. Our results suggest that CD14 and TLR2 are engaged in cell activation by L. monocytogenes, but that neither receptor seem to be involved in activation by GBS. This study was the first to show that human TLR2 can discriminate between two G+ bacteria. <b>PAPER 6</b> In paper 6 we report the generation of a new TLR2 mAb, TL2.3, that stained with the same specificity as TL2.1 (anti-TLR2, paper 5). We used these mAbs to investigate the expression of TLR2 protein in human cells. We found that TLR2 was highly expressed in blood monocytes, less in granulocytes, and not present in lymphocytes. The protein level was measured on quiescent and activated cells by extra- and intracellular flow cytometry, and by immunoprecipitation of TLR2 from metabolic S35-labeled cells. Surprisingly, TLR2 protein was detected in activated B-cells located in lymphoid germinal centers, indicating that subsets of lymphocytes may express TLR2. We further show that TLR2 protein was differentially regulated on monocytes and granulocytes after exposure to LPS, pro- or anti-inflammatory cytokines. However, we could not correlate the regulation of TLR2 to cellular responses, as for instance the three anti-inflammatory cytokines TGFβ, IL-4 and IL-10 all inhibited lipopeptideinduced TNF-production, but either did not affect, reduced, or increased the level of surface TLR2, respectively. Thus, the biological significance of TLR2-regulation remains to be found.

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.

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.

No description available.

Trypanosomes are unicellular protozoan parasites responsible for several human diseases that affect millions of people and cause thousands of casualties every year. They also represent a primitive eukaryotic model system harboring unique processes and basic regulatory mechanisms such as RNA-editing, polycistronic transcription and trans-splicing, first described in these organisms. Unlike most eukaryotes where levels of gene expression are controlled at initiation of transcription, trypanosomes use post-transcriptional events as the main regulators. This thesis explores the epigenetic mechanisms involved in gene expression control in trypanosomes, providing the first evidences for a functional “histone-code” as well as the existence and location of DNA methylation in Trypanosoma cruzi. Chromatin immunoprecipitation (ChIP) was used for the profiling of acetylated and methylated histones in T. cruzi, showing that the modified histones were exclusively localized at bidirectional transcription start sites. In addition, promoters from highly transcribed genes were found depleted of nucleosomes, while DNA regions expected to be silent were not enriched in the investigated modified histones. Furthermore, we showed that the histone patterns were developmentally regulated. The first in depth characterization of the DNA methylation patterns in T. cruzi was presented in this work. We detected m5C in regions of transcriptional initiation and termination, retrotransposons, pseudogenes and the kinetoplast minicircle. We also showed that the amount of methylation changes during development, with an increase in non-replicative forms. We also characterized the DNA-interacting properties of a T. cruzi polypyrimidine-tract binding protein (TcPTB), and its potential role as a transcription factor. TcPTB was found to interact with single-stranded DNA present in promoters bound by its mammalian homologue as well as to the region of transcriptional initiation in Leishmania major. We also demonstrated that T. cruzi polypyrimidine stretches were able to confer ssDNA conformations. Overall, these results provide new insights into the biology of ancient pathogenic parasites, which might be exploited for drug development.

Escherichia coli strains typical of UPEC (uropathogenic E. coli) and NBM (newborn meningitis) isolates carry chromosomally located PAIs (pathogenicity islands) that are absent in non-pathogenic E. coli strains. The PAIs include genes for virulence factors such as toxins and genes coding for specific adhesins and pili/fimbriae formation. Commonly, the gene clusters for such fimbriae in E. coli consist of a set of genes for biogenesis of the actual fimbriae organelles: a chaperone, an usher, the fimbrial subunits, and an adhesin, as well as some regulatory genes. Genetic studies of the fimbrial gene clusters in PAIs containing the pap genes, the prs genes, or the sfa genes led to the discovery of nearby open reading frames coding for putative cytoplasmic 17 kDa proteins — the X genes. Molecular genetic studies of the sfaXII locus in the clinical NMEC isolate IHE3034 have been performed. The results suggested that expression of the sfaXII gene had regulatory functions affecting both type 1 fimbriae expression and the flagella-mediated motility. Type 1 fimbriae expression was found to be affected at the level of fim operon transcription and a major reason was SfaXII-mediated modulation of expression from the fimB and fimE recombinase genes. Quantification of SfaII-fimbriated bacteria in a comparison between wild type and SfaXII mutant strains gave no indication that the sfaXII gene product also would be affecting expression and/or biogenesis of SfaII fimbriae. Biomechanical properties of the SfaII fimbriae produced by wild type and the sfaXII mutant IHE3034 were studied using force measuring optical tweezers (FMOT) and compared to other PAI-encoded fimbriae as well as to the type 1 fimbriae encoded on the core chromosome. The FMOT methodology assesses unfolding and refolding properties and we found that S fimbriae had weaker layer-to-layer interactions than both P and type 1 fimbriae, however the unfolding kinetics was slightly faster. The expression profile and regulation of the sfaXII gene were determined by use of reporter gene fusions and it was found that expression was affected by environmental cues such as pH, osmolarity and temperature. It was also discovered that the nucleoid structuring protein H-NS and the sigma factor RpoS had strong direct or indirect repressive effects on sfaXII gene expression. Further genomic analysis of the PAI fimbrial operons revealed that in some cases an additional ORF was found between the X genes and the fimbrial adhesion genes. Examination of the sfaII operon in IHE3034 indicated that this new gene, denoted sfaYII, coded for a protein that had the EAL domain motif and thereby could be considered a putative phosphodiesterase involved in controlling the level of cyclic-di-GMP in the bacterial cells.

This dissertation presents a generalized quantum chemical approach for electron transport in molecular electronic devices based on Green's function scattering theory. It allows to describe both elastic and inelastic electron transport processes at first principles levels of theory, and to treat devices with metal electrodes either chemically or physically bonded to the molecules on equal footing. Special attention has been paid to understand the molecular length dependence of current-voltage characteristics of molecular junctions. Effects of external electric fields have been taken into account non-perturbatively, allowing to treat electrochemical gate-controlled single molecular field effect transistors for the first time. Inelastic electron tunneling spectroscopy of molecular junctions has been simulated by including electron-vibration couplings. The calculated spectra are often in excellent agreement with experiment, revealing detailed structure information about the molecule and the bonding between molecule and metal electrodes that are not accessible in the experiment. An effective central insertion scheme (CIS) has been introduced to study electronic structures of nanomaterials at first principles levels. It takes advantage of the partial periodicity of a system and uses the fact that long range interaction in a big system dies out quickly. CIS method can save significant computational time without loss of accuracy and has been successfully applied to calculate electronic structures of one- , two- , and three-dimensional nanomaterials, such as sub-116 nm long conjugated polymers, sub-200nm long single-walled carbon nanotubes, sub-60 base pairs DNA segments, nanodiamondoids of sub-7.3nm in diameter and Si-nanoparticles of sub-6.5nm in diameter at the hybrid density functional theory level. The largest system under investigation consists of 100,000 electrons. The formation of energy bands and quantum confinement effects in these nanostructures have been revealed. Electron transport properties of polymers, SWCNTs and DNA have also been calculated. / QC 20100729. Ändrat felaktig titel "Theoretical Chemistry, Molecular and Nano-electronics" 20100729.