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.

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.

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 (Inhibitors of Cdk4). The Ink4 family of CKIs consists of four members; p16Ink4a, p15Ink4b, p18Ink4c and p19Ink4d, 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, p16Ink4a is frequently mutated or deleted in human cancer, whereas the other Ink4 genes are only occasionally altered in cancer. The overall aim of this thesis was to study the roles of p18Ink4c and p19Ink4d using in vivo models of cancer and embryonic development. In paper I, we analyzed the tumor spectrum in mice lacking p53, Ink4c and Ink4d. p53 is a tumor suppressor and one of the most frequently mutated genes in human cancer. Mice carrying mutated p53 alleles are highly tumor-prone but develop predominantly lymphomas. However, the combined loss of p53 and Ink4c (but not Ink4d) 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 p18Ink4c. In paper II, loss of Ink4c was evaluated in another tumor-prone mouse model; the Eµ-Myc 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-Myc mice exhibited elevated levels of p18Ink4c mRNA and protein despite high rates of proliferation. Moreover, loss of Ink4c in this model did not affect the rate of cell proliferation or the onset of tumor development. We conclude from these studies that Ink4c is not an important tumor suppressor of Myc-induced lymphomas. To gain insight into the role of Ink4 genes in early vertebrate development, the African clawed frog, Xenopus laevis, was analyzed for the presence of Ink4 homologs. Paper III describes the cloning and characterization of a gene homologous to Ink4d, Xl-Ink4d. This CKI is expressed throughout frog embryo development, making Xl-Ink4d the only CKI present during the cleavage stages of X. laevis. Antisense morpholino oligonucleotides directed against Xl-Ink4d were used to knock down the protein level of Xl-Ink4d during development. This resulted in defects in head tissues and reduced expression of Twist, a gene important for neural crest cell migration. We therefore propose that Xl-Ink4d is important for proper neural crest differentiation in the frog.

<p>Amyloidoses represent a heterogeneous group of diseases characterized by abnormal protein metabolism leading to extracellular deposition of fibrillar, proteinaceous amyloid in various tissues and organs of the body. To date more than 20 different proteins have been linked to diseases with amyloid depositions, of which Alzheimer’s disease and the prion-associated diseases are the most well known. Despite the origin of protein in the amyloid, the fibrils share some common biochemical and biophysical properties such as a diameter of 8-13 nm, a β-pleated sheet secondary structure packed in an ordered crystal-like way, Congo red and thioflavin binding with characteristic spectroscopic patterns and decoration of the fibrils with Serum amyloid P component and glycoseaminoglycans. </p><p>The plasma protein transthyretin (TTR) is associated with familial amyloidosis with polyneuropathy (FAP) and senile systemic amyloidosis (SSA). FAP is a lethal, autosomal inherited disorder caused by point mutations in the TTR-gene. More than 80 different mutations have been associated with amyloid formation and linked to FAP. The interpretation is that amino acid replacements at different sites of the polypeptide lead to reduced stability. Mutant TTR were constructed that have a strong tendency to self-aggregate under physiological conditions. The precipitates were shown to be amyloid by staining with thioflavin T and Congo red. As the mutants were sensitive to trypsin cleavage compared to plasma TTR, we suggest that the mutants represent amyloid precursors or that they may share structural properties with intermediates on a pathway leading to amyloid deposition. Monoclonal antibodies were generated that exclusively recognize the amyloidogenic folding of TTR providing direct biochemical evidence for a structural change in amyloidogenic intermediates. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and is proposed to be displaced at the initial phase of amyloid formation. Amyloidogenic intermediates of TTR were shown to induce a toxic, free radical dependent, response in cultured neuroblastoma cells. Morphological studies revealed a correlation between toxicity (apoptosis) and the presence of immature amyloid suggesting that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.</p><p>Serum amyloid P component (SAP) is a highly conserved plasma glycoprotein universally found associated with amyloid depositions independently of protein origin. SAP’s role in amyloid formation is contradictory since both inhibition and promotion of aggregation have been shown in the case of fibril formation from the Aβ peptide of Alzheimer’s disease. Amyloidogenic prefibrils of TTR were shown to bind SAP and no interference with aggregation was detected. SAP co-localize in patches with mutant TTR on the surface of neuroblastoma cells and prevent apoptosis induced by mutant TTR and Aβ peptide, while several other molecules known to decorate amyloid fibrils were without effect. </p>

<p>Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for mesenchymal cells and exert its effect by binding to two structurally related receptor tyrosine kinases, denoted α- and β-receptors. PDGF binding induces dimerization of its receptors, both homo-and heterodimerization, leading to their autophosphorylation on tyrosine residues and binding of downstream signaling molecules. This thesis describes autophosphorylation and binding of signal transduction molecules to homo- and heterodimeric PDGF receptor complexes.</p><p> Heterodimeric PDGF receptor complexes have been found to mediate a stronger mitogenic response than homodimeric receptor complexes. It was found that Tyr771 in the PDGF β-receptor was significantly less phosphorylated in the heterodimeric β-receptor compared to the homodimeric receptor, and this correlated with reduced binding of GTPase activating protein (GAP) for Ras and decreased activation of the Ras/Mitogen activated protein kinase pathway. </p><p> The mechanism behind the lowered phosphorylation of Tyr771 in the heterodimeric PDGF β-receptor was investigated. It was found that the SH2 domain-containing tyrosine phosphatase SHP-2 was responsible, at least in part, for the dephosphorylation of Tyr771 in the heterodimeric β-receptor. </p><p> PDGF-induced autophosphorylation of tyrosine residues in the receptors has been proposed to occur <i>in trans </i>between the receptor molecules in the dimers. We demonstrated by phosphopeptide mapping that all major autophosphorylation sites can be phosphorylated <i>in trans, </i>both in the PDGF α- and β-receptors. Analyses of the abilities of heterodimeric receptor complexes of one kinase-active and one kinase-inactive receptor to mediate mitogenicity, chemotaxis and activation of mitogen activated protein kinase revealed that the signaling capacities were retained. This illustrates a functional co-operation between the two receptor molecules in the dimer, where one receptor provides a functional kinase and the other acts as a substrate and provides docking sites for downstream signaling molecules.</p><p> Elucidating the mechanisms behind the unique signaling properties of the heterodimeric PDGF receptor complex, two heterodimer-specific autophosphorylation sites, Tyr692 and Tyr970, were identified and found to interact with the low molecular weight protein tyrosine phosphatase (LMW-PTP). Mutation of Tyr692 or Tyr970 to phenylalanine residues did not affect PDGF-induced mitogenicity, but the Tyr692 to phenylalanine mutation reduced the chemotactic response mediated by the heterodimeric PDGF receptor complex. A mechanism for the lowered chemotactic response was found to involve an increased RasGAP binding and a decreased SHP-2 binding to the heterodimeric β-receptor.</p>

<p>In my thesis, the cell cycle analysis of archaea and hyperthermophilic organisms is presented for the first time. Crenarchaea from the genus <i>Sulfolobus</i> were used as a model system. Plow cytometry and light microscopy were applied to investigate the timing and coordination of different cell cycle events. Furthermore, DNA content, nucleoid structure, and nucleoid distribution at different stages during the cell cycle were studied. The <i>Sulfolobus</i> cell cycle was characterized as having a short pre-replication and a long post-replication period. The presence of a low proportion of cells with segregated genomes in the exponentially growing population suggested 3 considerable time delay between termination of chromosome replication and completion of nucleoid partition, reminiscent of the G₂period in eukaryotic cells.</p><p>The first available collection of conditional-lethal mutants of any archaeon or hyperthemophile was used to elucidate the coordination of cell cycle events. The studies showed that chromosome replication, nucleoid partition and cell division in <i>Sulfolobus</i> <i>acidocaldarius</i>, which are normally tightly coordinated during cellular growth, could be separately inhibited or uncoupled by mutation.</p><p>The <i>ftsZ</i> gene, which is involved in cell division in bacteria and euryarchaea, was isolated from the halophilic archaeon <i>Haloferax mediterranei</i>. Transcriptional start sites were mapped, and putative translation initiation elements were identified. In both the upstream and downstream regions of the <i>ftsZ</i> gene, open reading frames were found to be conserved within the genus <i>Haloferax</i>. Furthermore, at the 3' end of the <i>ftsZ</i> gene, the homologs of the bacterial <i>secE</i> and <i>nusG</i> genes are conserved in almost all euryarchaea analyzed so far. The studies also demonstrated the functional conservation of the FtsZ protein in different archaeal species, as well as between euryarchaea and bacteria.</p>

<p>Integrins are heterodimeric cell surface receptors, composed of an α- and a β-subunit, which mediate cell-extracellular matrix (ECM) interactions. Integrins mediate intracellular signals in response to extracellular stimuli, and cooperate with growth factor and other cytokine receptors. Cells execute their differentiated functions anchored to an ECM. In this thesis functional properties of the two collagen-binding integrins α2β1 and α11β1 were studied. In addition, the impact of β1 cytoplasmic tyrosines in collagen-induced signalling was analyzed.</p><p>The integrin α11β1 is the latest identified collagen-binding integrin. In this study, tissue distribution of α11 mRNA and protein during embryonal development was explored, and the first α11β1-mediated cellular functions were established. Both α11 protein and mRNA were present in mesenchymal cells in intervertebral discs and around the cartilage of the developing skeleton. α11 protein was also detected in cornea keratinocytes. α11β1 mediated cation-dependent adhesion to collagen types I and IV and localized to focal adhesions. In addition, α11β1 mediated contraction of a collagen lattice and supported cell migration through a collagen substrate. PDGF-BB and FBS both stimulated α11β1-mediated contraction and directed migration.</p><p>Expression of β1Y783,795F in β1-null cells, prevents activation of FAK in response to fibronectin, and decreases cell migration. In this study, we investigated how this mutation affected α2β1-mediated functions in response to collagen. The β1 mutation impaired collagen gel contraction and prevented activation of FAK, Cas and Src on planar collagen, but not in collagen gels. PDGF-BB stimulated contraction via αvβ3, which also induced activation of Cas in collagen gels. The YY-FF mutation also abolished β1A-dependent downregulation of β3.</p><p>In the final study integrin-crosstalk during collagen gel contraction was investigated. In cells lacking collagen-binding integrins αvβ3 mediated contraction. Clustering of β1-integrins by antibodies and PDGF-BB stimulated αvβ3-mediated contraction in an ERK-dependent way. Expression of α2β1, but not α11β1, prevented αvβ3-mediated contraction. Contraction by α2β1 and α11β1 was ERK-independent.</p>

<p>Transforming growth factor-β (TGF-β) superfamily members are multi-functional regulators of cell fate. These factors signal by binding to a limited number of highly conserved transmembrane type I and type II serine/threonine kinase receptors. These receptors initiate signals into the cell via the Smad proteins. Up to date, 8 different mammalian Smads are reported and are divided into three subgroups; receptor regulated Smads (R-Smads), common mediator Smads (Co-Smads) and inhibitory Smads (I-Smads). This thesis investigates the function and regulation of TGF-β/Smad signaling through identification and characterization of Smad interacting proteins.</p><p>I-Smads, i.e. Smad6 and Smad7, are potent antagonists of the TGF-β superfamily signaling. We found that Smad7, but not Smad6, inhibits TGF-β1-induced growth inhibition and expression of immediate early response genes. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-β receptor activation. Moreover, we found that the MH2 domain is important for nuclear export.</p><p>To investigate further the role of inhibitory Smads, we have identified AMSH as a Smad6 interacting protein using a yeast two-hybrid screening method. AMSH was previously discovered as the associated molecule with the SH3 domain of STAM. AMSH interacts with I-Smads, but not with R- and Co-Smads upon receptor activation and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. AMSH was found to prevent Smad6 from binding to activated type I receptors and/or activated R-Smads. Smad anchor for receptor activation (SARA) is critical for Smad2 and Smad3 activation by TGF-β receptors. The present studies show that the localization of SARA in early endosomes is regulated through its FYVE domain. We have found that the FYVE domain of SARA is sufficient and necessary for the early endosomal localization, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF-β/Smad signaling.</p><p>Both Notch and BMP signaling pathways are important for vascular development. We have found that Herp2, which is originally known as one of the Notch target genes, is synergistically induced upon activation of Notch and BMP signaling pathways in endothelial cells (ECs). The critical elements for synergistical activation of Herp2 gene by BMP and Notch pathway were identified. Furthermore, the Notch intracellular domain interacts with Smad5 upon BMP receptor and this interaction becomes stronger in the presence of pCAF. Interestingly, Herp2 was found to antagonize BMP receptor- or Id-mediated EC migration.</p>