<p>Heparan sulfate proteoglycans are proteins with long, unbranched heparan sulfate (HS) polysaccharide chains attached to them. They are found on cell surfaces and in basement membranes where they exert their action by interacting with a wide range of enzymes and signaling molecules and are thereby involved in a range of various processes both during embryonic development and in adult physiology.</p><p>A great part of the biological functionality of proteoglycans can be directly related to the polysaccharide part. HS chains display very variable sulfation patterns where highly sulfated regions are responsible for a large part of the biological activity. The biosynthesis of HS is a complex process in which a number of enzymes are involved. Better comprehension of how this process is regulated could reveal clues to how formation of HS sulfation patterns occurs, and thereby how HS functionality is controlled.</p><p>This thesis is focusing on regulation of one of the enzymes responsible for HS sulfation, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), in an attempt to understand these mechanisms better. Different aspects of NDST regulation were studied in three projects:</p><p>I) “Heparin/heparan sulfate biosynthesis: Processive formation of N-sulfated domains”, where the sulfate donor PAPS is shown to influence the manner in which NDST modifies the substrate, affecting the domain structure of the polysaccharide.</p><p>II) “Heparan sulfate biosynthesis: Characterization of an NDST1 splice variant”, where a splice variant of NDST1 which appears to influence NDST1 protein levels and affect HS structure is described.</p><p>III) “Heparan sulfate biosynthesis in zebrafish: Five NDST genes with distinct expression patterns during embryonic development”, in which five zebrafish NDSTs were cloned and shown to be expressed in a temporally and spatially regulated manner.</p>

<p>Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition.</p><p>We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing.</p><p>By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.</p>

<p>It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of <i>Chironomus tentans</i> provide a suitable model system for studying gene expression<i> in situ</i>, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in <i>C. tentans</i> named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of <i>C. tentans</i> salivary gland cells.</p><p>Hrp59 is a <i>C. tentans</i> homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in <i>Drosophila</i> cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences.</p><p>Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1.</p><p>Hrp65 binds to HEL/UAP56 in <i>C. tentans</i> cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.</p>

<p>Gene expression in the eukaryotic cell is a fundamental cellular process, which consists of several distinct steps but extensively coupled to each other. From site of transcription in the nucleus to the cytoplasm, both mRNA and rRNA are associated with a proper set of proteins. These proteins influence RNA processing, transport as well as ribosome maturation. We have tried to take advantage of different model systems to understand the process of eukaryotic gene expression at the post-transcription level. To this end, we have focused on identification and characterization of several specific proteins in the context of mRNP and rRNP particles.</p><p>We have characterized a novel yeast gene MRD1, which encodes a protein with five RNA-binding domains (RBDs) and is essential for viability. Mrd1p is present in the nucleolus and the nucleoplasm. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and 40S ribosomal subunits. Mrd1p associates with the 35S prerRNA and the U3 snoRNA and is required for the initial processing of pre-rRNA at the A₀-A₂ sites. The presence of five RBDs in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage.</p><p>Meanwhile, an MRD1 homologue, Ct-RBD-1 with six RBDs, has also been identified and shown to involve in ribosome biogenesis in Chironomus tentans. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In the cytoplasm, Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that Ct-RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.</p><p>We have characterized a novel abundant nucleolar protein, p100 in C. tentans. The p100 protein is located in the fibrillar compartment of the nucleolus, and remains in the nucleolus after digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. These results suggest that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus.</p><p>We have characterized a Dbp5 homologue in C. tentans, Ct-Dbp5. The protein becomes associated with nascent pre-mRNAs at a large number of active genes, including the Balbiani ring (BR) genes. Ct-Dbp5 is bound to nascent BR pre-mRNP particles and accompanies them through the nucleoplasm and the nuclear pore into the cytoplasm. Nuclear accumulation of Ct-Dbp5 takes place when synthesis and/or export of mRNA are inhibited. Our results indicate that most or all of the shuttling Ct-Dbp5 exiting from the nucleus associated with mRNP. Furthermore, Ct-Dbp5 is present along the mRNP fibril extending into the cytoplasm, supporting the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation.</p><p>We have shown that the export receptor CRM1 in C. tentans is associated with BR pre-mRNP while transcription takes place. We have also shown that the GTPase Ran binds to BR pre-mRNP, but its binding mainly in the interchromatin. Although both CRM1 and Ran accompany BR pre-mRNP through the nuclear pore, Leptomycin B treatment reveals that a NES-CRM1-RanGTP complex is not essential for export of the BR mRNP. Our results suggest that several export receptors associate with BR mRNP and that these receptors might have redundant functions in the nuclear export of BR mRNP.</p><p>We have analyzed four SR proteins, SC35, ASF/SF2, 9G8 and hrp45, in C. tentans. All four SR proteins genes are expressed in salivary gland cells and in several other tissues in a tissue specific pattern. We found that about 90% of all nascent pre-mRNAs bind all four SR proteins, and that approximately 10% of the pre-mRNAs associate with different subsets of the four SR proteins, suggesting that not all of four SR proteins are needed for processing of pre-mRNA. None of three examined SR proteins leave BR pre-mRNP as splicing is completed. Instead, 9G8 accompanies the mRNP to the cytoplasm, while SC35 and hrp45 leave the BR mRNP at the nuclear side of the nuclear pore complex.</p>

The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.

People are, in their daily lives, exposed to a number of airborne foreign compounds that do not normally affect the body. However, depending on the nature of these compounds, dose and duration of exposure, various airway symptoms may arise. Early symptoms are often manifested as upper airway mucosal inflammation which generates changes in protein composition in the airway lining fluid. This thesis aims at identifying, understanding mechanisms and characterizing protein alterations in the upper airway mucosa that can be used as potential new biomarkers for inflammation in the mucosa. The protein composition in the mucosa was studied by sampling of nasal lavage fluid that was further analyzed with a proteomic approach using twodimensional gel electrophoresis and mass spectrometry. Additionally, by studying factors on site through environmental examination, health questionnaires and biological analyses, we have tried to understand the background to these protein alterations and their impact on health. Respiratory symptoms from the upper airways are common among people who are exposed to irritative and microbial agents. This thesis have focused on personnel in swimming pool facilities exposed to trichloramine, metal industry workers exposed to metalworking fluids, employees working in damp and moldy buildings and infants diagnosed with respiratory syncytial virus infection. The common denominator in these four studies is that the subjects experience upper airway mucosal inflammation, which is manifested as cough, rhinitis, phlegm etc. In the three occupational studies, the symptoms were work related. Notably, a high prevalence of perceived mucosal symptoms was shown despite the relatively low levels of airborne irritants revealed by the environmental examination. Protein profiling verified an ongoing inflammatory response by identification of several proteins that displayed altered levels. Interestingly, innate immune proteins dominated and four protein alterations occurred in most of the studies; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin. Similarly, these proteins were also found in nasal fluid from children with virus infection and in addition a truncated form of SPLUNC1 and two other S100 proteins (S100A7-like 2 and S100A16), not previously found in nasal secretion, were identified. Altogether, the results indicate the potential use of a proteomic approach for identifying new biomarkers for the upper respiratory tract at an early stage in the disease process after exposure to irritant and microbial agents. The results indicate an effect on the innate immunity system and the proteins; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin are especially promising new biomarkers. Moreover, further studies of these proteins may help us to understand the molecular mechanisms involved in irritant-induced airway inflammation.

The transcriptome of the mammalian brain is extensively modified by adenosine to inosine (A-to-I) nucleotide conversion by two adenosine deaminases (ADAR1 and ADAR2). As adenosine and inosine have different base pairing properties, A-to-I RNA editing shapes the functional output of both coding and non-coding RNAs (ncRNAs) in the brain. The aim of this thesis was to identify editing events in small regulatory ncRNAs (miRNAs) and to determine their temporal and spatial editing status in the developing and adult mouse brain. To do this, we initially analyzed the editing status of miRNAs from different developmental time points of the mouse brain. We detected novel miRNA substrates subjected to A-to-I editing and found a general increase in miRNA editing during brain development, implicating a more stringent control of miRNAs as the brain matures. Most of the edited miRNAs were found to be transcribed as a single long consecutive transcript from a large gene cluster. However, maturation from this primary miRNA (pri-miRNA) transcript into functional forms of miRNAs is regulated individually, and might be influenced by the ADAR proteins in an editing independent matter. We also found that edited miRNAs were highly expressed at the synapse, implicating a role as local regulators of synaptic translation. We further show that the increase in editing during development is explained by a gradual accumulation of the ADAR enzymes in the nucleus. Specifically for ADAR2, we found a developmentally increasing interaction with two factors, importin-α4 and Pin1, that facilitate nuclear localization of the editing enzyme. We have also found that selectively edited stem loops often are flanked by other long stem loop structures that induce editing in cis. This may explain why multiple pri-miRNAs are edited within the same cluster. In conclusion, this thesis has significantly increased the understanding of the dynamics of both editing substrates and enzymes in the developing and mature brain. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Viral proteases are important targets for anti-viral drugs. Discovery of protease inhibitors as anti-viral drugs is aided by an understanding of the interactions between viral protease and inhibitors. This thesis addresses the characterization of protease-inhibitor interactions for application to drug discovery and model system development. The choice of a relevant target is essential to molecular interaction studies. Therefore, full-length NS3 protein of hepatitis C virus (HCV) was obtained, providing a more relevant target and a better model for the development of HCV protease inhibitors. In addition, resistance to anti-viral drugs, a serious problem in the treatment of AIDS, prompted the investigation of resistant variants of human immunodeficiency virus (HIV) protease. Drug resistance was initially explored by characterization of the interactions between a series of closely related inhibitors and resistant variants of HIV protease, using an inhibition assay to determine the inhibition dissociation constants (Ki). The relationship between structure, activity and resistance profiles was not clarified, indicating that the effect of structural changes in the inhibitors and the protease are not predictable and must be analyzed case wise. It was proposed that additional kinetic characterization of the interactions was required and a biosensor-based method allowing for determination of affinity, KD, and interaction rate constants, kon and koff, was adopted. The increased physiological relevance of this method was confirmed, and the affinity data have better correlation with cell culture data. In addition, interactions between clinical inhibitors of HIV protease and enzyme variants indicate that increased dissociation rates (koff) are associated with the development of resistance. Thermodynamic characterization of the interactions between HIV-1 protease and clinically relevant inhibitors revealed distinct energetic characteristics for inhibitors. The resolution of the energetics of association and dissociation identified an inhibitor with unique interaction characteristics and confirmed the validity of using this method for further characterization of molecular interactions. This work resulted in the development of model systems for the analysis of kinetics, resistance and thermodynamic characteristics of protein-inhibitor interactions. The results give increased understanding of the biomolecular interactions and can be applied to drug discovery.

Hepatitis C is a serious disease that affects about 200 million people worldwide. No anti-HCV vaccine or specific anti-viral drugs are available today. Non-structural protein 3 (NS3) of HCV is a bifunctional serine protease/helicase, and the protease has become a prime target in the search for anti-HCV drugs. In this work, the complete HCV NS3 gene has been cloned and expressed, and the protein has been purified using affinity chromatography. An assay for measuring the protease activity of full-length NS3 protease has been developed and used for inhibition studies. A series of peptide-based inhibitors of NS3 protease varying in length, the composition of the side-chain and the N- and C-terminal groups have been studied. Potent tetra-, penta- and hexapeptide inhibitors of the NS3 protease were discovered. Hexapeptides with an acyl sulfonamide C-terminal residue were the most potent inhibitors of the NS3 protease, having nanomolar Ki-values. The selectivity of the inhibitors was assessed using other serine and cysteine proteases. NS3 protease inhibitors with electrophilic C-terminal groups were non-selective while those comprising a C-terminal carboxylate or acyl sulfonamide group were selective. All inhibitors with a small hydrophobic P1 side-chain residue were non-selective for the NS3 protease, being good inhibitors of human leukocyte elastase. This result highlights the importance of the P1 residue for inhibitor selectivity, which stems from the major role of this residue in determining substrate specificity of serine proteases. Electrophilic inhibitors often cause slow-binding inhibition of serine and cysteine proteases. This was observed with other proteases used in our work but not with NS3 protease, which indicates that mechanism of inhibition of NS3 protease by electrophilic inhibitors may not involve formation of a covalent bond. The structure-activity relationships obtained in this work can be used for improvement of peptide-based inhibitors of HCV NS3 protease towards higher inhibitory potency and selectivity.

A variety of HIV-1 protease inhibitors and their interactions with the enzyme have been characterized in order to identify novel and improved drugs against AIDS. The investigated inhibitors were represented by clinical and non-clinical inhibitors, active site and allosteric inhibitors, transition-state analogues and metal-ions. In addition, different enzyme variants were used to investigate the contribution of different amino acid residues to the interaction with different ligands. The problem of resistance has been addressed by exploring novel types of inhibitors, and resistant mutants of HIV-1 protease. A study resolving the inhibition of HIV-1 protease by Cu2+ showed that the enzyme can be allosterically inhibited and that copper inhibition is a result of an interaction with His-69 and a subsequent conformational change. Several types of transition-state analogues were analyzed with respect to their inhibition of wild-type and resistant mutants of HIV-1 protease. Unfortunately cyclic compounds were not found to be better than linear compounds. Moreover, it was not possible to identify structure-activity relationships that clearly correlated with efficacy towards mutants and a biosensor based method for more detailed kinetic studies was therefore adopted. By cross-linking the immobilized enzyme on the biosensor matrix, a stable surface was obtained and kinetic rate constants could be determined for the interaction between the enzyme and inhibitors. Additional improvements in the methodology involved identification of a more representative interaction model, allowing more detailed studies of interactions with resistant mutants and varying conditions. Finally, absorption to lipid membranes and interaction with human serum albumin and α1-glycoprotein by clinical drugs were studied in a simplified ADME model system for improvement of the earlier stages of drug development. These studies have revealed important characteristics of these drugs that can potentially be modeled into new compounds that have improved efficacy of both wild-type and resistant mutants of HIV-1 protease.