<p>The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins.</p><p>In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells.</p><p>The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-<i>β</i>1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation.</p><p>The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.</p>

<p>We have constructed two adenovirus-based gene expression vector systems permitting regulated protein expression. They are based on the tetracycline-regulated Tet-ON- and the progesterone antagonist RU 486-regulated gene expression systems, which were rescued into E1-deficient adenovirus vectors. The vectors function in a number of cell types representing a broad species-variety and the regulation of protein expression was shown to be tightly controlled in cells not permissive for virus replication. Furthermore, the adenovirus-Tet-ON system was shown to perform in mice after intramuscular administration.</p><p>The novel adenovirus-vector systems were then used to study the effects of overexpression of selected proteins on adenovirus replication during a lytic infection, with focus on regulation of adenovirus alternative splicing. Expression of adenovirus transcription units is to a large extent temporally regulated at the level of alternative pre-mRNA splicing, where viral splice site usage shifts from proximal to distal splice site selection as infection proceeds. This makes adenovirus an appropriate model for mechanistic studies of regulated splicing. We show that overexpression of the essential host cell splicing factor ASF/SF2 inhibits this shift by promoting usage of proximal splice sites. As a consequence, the virus displayed a markedly inhibited growth. Interestingly, mRNA expression from the adenovirus major late promoter was almost completely lost as a consequence of ASF/SF2 overexpression. Collectively, the cellular splicing factor ASF/SF2 prevents adenovirus from entering the late phase of infection. This strongly argues for a need for the virus to block the splicing enhancer activity of ASF/SF2 for establishment of a lytic infection. Further, from analysis of the strict inhibition of late region 1 late pre-mRNA splicing we propose that the temporal regulation of alternative splicing is merely a consequence of fitness rather than profoundly deleterious effects of an unregulated expression. During our studies we noted that in 293 cells, which are used for growth of E1-deficient Ad vectors, an unwanted background reporter gene expression was evident in our vector systems. We therefore introduced an additional regulatory element, functioning as a transcriptional road-block, and showed that this methodological innovation represents a way to overcome the potentially deleterious effects of background reporter gene expression. This modified viral vector system should make it possible to reconstruct recombinant viruses expressing highly toxic proteins.</p><p>In conclusion, this work presents a new <i>in vivo </i>model system to study proteins involved in RNA splicing and other gene regulatory mechanisms.</p>

<p>Fibroblasts continuously strive to organize and compact the surrounding extracellular matrix (ECM). Recent data suggest that this cellular contractility controls interstitial fluid homeostasis in loose connective tissues (CT). The aim of this thesis was to study the effects of prostaglandins on fibroblast contractility and to investigate whether fibroblasts in the interstitial CT surrounding the human endolymphatic duct (ED) can modulate inner ear fluid pressure and endolymph resorption. </p><p>Paper I shows that prostaglandin E1 (PGE₁) and prostacyclin inhibit fibroblast-mediated collagen matrix compaction <i>in vitro</i> and lower the interstitial fluid pressure <i>in vivo</i> in rat dermis. Paper II demonstrates that the inhibition of collagen matrix compaction by PGE₁ is protein kinase A-dependent. Furthermore, PGE₁ induces a complete but reversible actin depolymerization in human dermal fibroblasts by affecting the phosphorylation state of regulatory actin-binding proteins. Paper III describes that the cells of the interstitial CT encompassing the human ED are organized in a network based on intercellular- and cell-ECM contacts. Paper IV shows that two distinct cell phenotypes populate this interstitial CT: one expressing the lymph endothelial marker podoplanin and the other a fibroblast marker. Furthermore, CT cells isolated from human ED tissues exhibited the same tissue compacting properties <i>in vitro</i> as dermal fibroblasts. </p><p>In conclusion, PGE₁ inhibits fibroblast contractility by interfering with the stability and dynamics of the actin cytoskeleton, which leads to a loss of integrin-mediated adhesion to the ECM. These mechanisms are supposedly involved in edema formation in skin during inflammation and might be involved in the formation of endolymphatic hydrops in the inner ear of patients with Ménière’s disease.</p>

<p>Pectinases are a group of enzymes produced by bacteria, fungi, higher plants and animals. Pectinases can modify and degrade pectins, a class of heterogeneous and multifunctional polysaccharides present in middle lamellae and primary cell walls of plants. Pectins have been showed to play diverse roles in cell physiology, growth, adhesion and separation. Pectinases are used technically in the processing of fiber production and fruit juice or wine making. We have studied the mechanisms and applications of pectinases, especially in retting, a microbiological process where bast fibers in flax and other bast fiber cultivars are released from each other and from the woody core.</p><p>A strong correlation was found between the ability to perform retting and the degradation of sparsely esterified pectin, a substrate of polygalacturonase. This led to the conclusion that polygalacturonase plays a key role in the enzymatic retting of flax. We purified and characterized an extracellular polygalacturonase produced by Rhizopus oryzae, a very potent retting organism. The purified enzyme which appeared to be the single active component in retting, has non-methylated polygalacturonan as its preferred substrate. Peptide sequences indicate that the enzyme, like another polygalacturonase (EC. 3.2.1.15), belongs to glycosyl hydrolase family 28. It contains, however, an N-terminal sequence absent from other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium, Ralstonia solanacearum.</p><p>Our finding that removal of calcium ions from the plant material by pre-incubation in dilute acid in enzymatic retting could reduce enzyme consumption by several orders of magnitude, improves the economical feasibility of the enzymatic retting process. Comparisons with different acids showed that the action was mainly pH dependent.</p><p>Pectinases were employed as analytical tools in a study of stored wood discoloration and, together with cellulases, in a mechanical process for making pulp from flax and hemp in paper production. </p>

<p>This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation.</p><p>The interstitial fluid pressure (P_IF) is of importance for control of tissue fluid balance. A lowering of P_IF <i>in vivo</i> will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling P_IF.</p><p>Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of P_IF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of P_IF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored P_IF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered P_IF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered P_IF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from <i>Streptococcus equi</i> modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction <i>in vitro</i> and control of P_IF <i>in vivo</i>, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence P_IF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.</p>

<p>Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4)disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of <i>N</i>-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified <i>N</i>-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs).</p><p>Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn²⁺-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups.</p><p>The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.</p>

The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate. The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, qp, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins. By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation, In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied. In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the PmalK production system. A novel fed-batch process based on the promoters for the universal stress proteins A and B (PuspA, PuspB) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects. / <p>QC 20101008</p>

Integrins are heterodimeric cell surface receptors, composed of an α and a β subunit, mainly binding for extracellular matrix proteins. lntegrin subunit β1 can combine with at least 12 a subunits and thus form the biggest subfamily within the integrin family. In this thesis, functional properties of the splice variant β1Β, and the effects of several mutations in the cytoplasmic tail of integrin subunit β1Α were studied. In addition, the border between the transmembrane and cytoplasmic domains of several integrin subunits was determined. The β1Β splice variant has been reported to have a dominant negative effect on functions of β1Α integrins. In this study, it was studied if the expression of β1Β had similar negative effects on the αvβ3 integrin functions since the β3 subunit is structurally similar to β1Α. The β1Β subunit was expressed in an integrin β1-deficient cell line and it was found that the presence of β1Β does not interfere with adhesion or signaling of endogenous αvβ3 The border between the cytoplasmic domain and the C-terminal end of the transmembrane domain of integrin α and β subunits has been unclear. This question was experimentally addressed for integrin subunits β1, β2, α2 and α5. It was found that integrin subunits contain a positively charged lysine, which is embedded in the membrane in the absence of interacting proteins. The functional importance of the lysine in integrin transmembrane domains was investigated by mutating this amino acid to leucine in β1Α. The mutation affected cell spreading and tyrosine phosphorylation of the adapter protein CAS. The activation of focal adhesion kinase and tyrosine phosphorylation of paxillin was not affected. Furthermore, the mutation of two tyrosines to phenylalanines in the β1Α cytoplasmic tail was found to reduce the capability of β1Α integrins to mediate cell spreading and migration. Activation of focal adhesion kinase in response to the later β1Α mutant was shown to be impaired as well as tyrosine phosphorylation of adapter proteins paxillin and tensin whereas overall tyrosine phosphorylation of CAS was unaffected. These data suggests the presence of focal adhesion kinase-dependent and -independent pathways for tyrosine phosphorylation of CAS after integrin β1Α-mediated adhesion.

The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins. In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells. The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-β1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation. The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.

We have constructed two adenovirus-based gene expression vector systems permitting regulated protein expression. They are based on the tetracycline-regulated Tet-ON- and the progesterone antagonist RU 486-regulated gene expression systems, which were rescued into E1-deficient adenovirus vectors. The vectors function in a number of cell types representing a broad species-variety and the regulation of protein expression was shown to be tightly controlled in cells not permissive for virus replication. Furthermore, the adenovirus-Tet-ON system was shown to perform in mice after intramuscular administration. The novel adenovirus-vector systems were then used to study the effects of overexpression of selected proteins on adenovirus replication during a lytic infection, with focus on regulation of adenovirus alternative splicing. Expression of adenovirus transcription units is to a large extent temporally regulated at the level of alternative pre-mRNA splicing, where viral splice site usage shifts from proximal to distal splice site selection as infection proceeds. This makes adenovirus an appropriate model for mechanistic studies of regulated splicing. We show that overexpression of the essential host cell splicing factor ASF/SF2 inhibits this shift by promoting usage of proximal splice sites. As a consequence, the virus displayed a markedly inhibited growth. Interestingly, mRNA expression from the adenovirus major late promoter was almost completely lost as a consequence of ASF/SF2 overexpression. Collectively, the cellular splicing factor ASF/SF2 prevents adenovirus from entering the late phase of infection. This strongly argues for a need for the virus to block the splicing enhancer activity of ASF/SF2 for establishment of a lytic infection. Further, from analysis of the strict inhibition of late region 1 late pre-mRNA splicing we propose that the temporal regulation of alternative splicing is merely a consequence of fitness rather than profoundly deleterious effects of an unregulated expression. During our studies we noted that in 293 cells, which are used for growth of E1-deficient Ad vectors, an unwanted background reporter gene expression was evident in our vector systems. We therefore introduced an additional regulatory element, functioning as a transcriptional road-block, and showed that this methodological innovation represents a way to overcome the potentially deleterious effects of background reporter gene expression. This modified viral vector system should make it possible to reconstruct recombinant viruses expressing highly toxic proteins. In conclusion, this work presents a new in vivo model system to study proteins involved in RNA splicing and other gene regulatory mechanisms.