A major challenge in medical genetics is to identify the mutations underlying heritable diseases. Dogs are excellent genetic models in the search for causative mutations, as they constitute a large library of naturally occurring heritable diseases many of which are analogous to those suffered by man. In addition, these animals have a genome structure well suited to gene mapping. The Shar-Pei dog has two breed-specific features; a strongly selected for wrinkled skin and a high predisposition to an autoinflammatory disease (AID). Abnormalities in the innate immune system cause this type of disease, presenting as spontaneous attacks of inflammation. Persistent inflammation puts an affected Shar-Pei at risk of amyloidosis, organ failure and premature death. In humans, similar AIDs occur and for a majority of cases, no underlying genetic cause has yet been identified. The aim of this thesis was to use the Shar-Pei as a genetic model for autoinflammation in order to find new genes and signalling pathways involved in disease. In paper I, a pleiotropic mutation was identified that could explain both the wrinkled skin and autoinflammation in Shar-Pei. The mutation is associated with an up-regulation of Hyaluronic Acid Synthase 2 (HAS2). Increased expression of HAS2 leads to abnormal depositions of hyaluronic acid (HA) in the skin, resulting in the wrinkled appearance. When fragmented, HA also function as a damage signal sensed by the innate immune system which then responds with inflammation. By selecting for the wrinkled skin, the autoinflammatory disease has inadvertently been enriched in the breed. In paper II, five different inflammatory signs could be associated with the same genetic risk factor, allowing the introduction of a new terminology: Shar-Pei autoinflammatory disease (SPAID) to describe the whole disease complex. In addition, a modifying locus containing several biologically attractive genes was suggested to contribute to varying incidence of amyloidosis in Shar-Pei. In paper III, signs of pathological changes in HA metabolism were investigated in human AID. HA concentration was found to be both higher in subjects with no molecular diagnosis and also associated to disease activity and severity. Taken together, this suggests HA is also involved in human AID.

The purpose of this master thesis project was to investigate the penetration ofbiomolecules into hyaluronic acid (HA) macrogels. The investigations were performedboth in the presence and in the absence of the non-biodegradable surfactantcetylpyridinium chloride (CPC), which earlier has proved to form a micelle-rich shellat the gel surface. In this work investigations were performed to see if properties ofthe biomolecules used, such as size and charge, had any impact on the binding to theHA gels both with and without CPC. The biomolecules used were the proteinscytochrome c, lysozyme, hemoglobin and myoglobin and the polysaccharide dextranof different molecular weights and labeled with fluorescein isothiocyanate.Cetylpyridinium chloride was used as a non-biodegradable model surfactant for abiodegradable betaine ester surfactant. The cetylpyridinium chloride was thereforeserving well when performing control studies for the biodegradable betaine ester,because the two possesses similar properties. Investigations involving the betaineester surfactant was not included in this master thesis project. Also release studiesinvolving the labeled dextran and some of the proteins were performed in thepresence and absence of cetylpyridinium chloride.The binding of CPC to HA was investigated briefly as well as the microstructure ofHA gels saturated with CPC by means of small-angle X-ray scattering, SAXS, atdifferent salt concentrations. The microstructure-investigations indicated that at 10mM NaCl a cubic ordered phase with space group Pm3n was achieved. When the saltconcentration was increased to 40 mM the microstructure was altered to a clearface-centered cubic (FCC) structure. When increasing the NaCl concentrationfurther, to 150 mM, indication of an unordered micellar phase could be seen.Cytochrome c and lysozyme transport into HA gels, to which CPC had bound in anearlier step, could be registered using UV-VIS spectrophotometry. Indications showedthat CPC and cytochrome c was distributed to different parts of the gel frommicroscope pictures taken of the cross-section of gel samples at different time.From release experiments performed with fluorescein isothiocyanate-dextran noconclusions could be drawn on how the different molecular weights of dextranaffected the rate and extent of the amount substance released. Neither could theinfluence of CPC be elucidated. This since the extent of released amounts exceeded100 % for many samples and the duplicate samples investigated showed differentbehavior.The transport of cytochrome c, myoglobin and hemoglobin into HA gels with andwithout beforehand treatment with CPC was evaluated qualitatively. The transportinto gels treated with CPC was successful and complete with cytochrome c andmyoglobin at lower degree of binding but was limited for the bigger proteinhemoglobin. When investigating the release of cytochrome c, myoglobin andhemoglobin from HA gels the extent of released substance was lower with CPCpresent in the solution compared without CPC present. Also some indicationsshowed that the bigger size of hemoglobin affected the rate of release from the gel.

Giloma derive from glial cell, which is the most common malignant and deadly primary tumor that affects the brain and nervous system, and the possible causes are not fully understood. Glioma cells are highly invasive, and can spread to distant area of the brain, this invasive behavior makes complete tumor debulking virtually impossible. Glioma even resists to high dose of radiotherapy and chemotherapy, the prognosis of malignant glioma remains dismal and the estimated median survival time is 12¡Ð15 months. The previous studies showed that the interaction of hyaluronan (HA), the abundant component of the ECM in the adult central nervous system, with cell-surface receptors, CD44 is able to mediate motility, tumor formation and multidrug resistance of glioma. In addition, the interacted between HA and CD44, that could up-regulate glioma HA production. But the effect of hyaluronan synthases (HAS) expression in this regulation mechanism was not described clearly. In this study, the HAS expression was a target gene in the rat glioma cell line¡ÐC6 on the conditions of HA addition or cd44 gene silence, respectively. The results showed that HA addition increased the HAS expression, and cd44 gene silence caused the less expression of HAS, and which could restored by HA addition. Futher, the HA addition could prolong cell proliferation , decrease the expression of the CD44 and GFAP, the astrocyte differentiation marker, and increase brain tumor stem cell marker¡Ðnestin expression, and this result could reappear by the cd44 gene silence alone. However, instead the stemness of cell, the cell toward differentiation and proliferation by HA addition after the cd44 gene silence. From those results, the interaction between HA and CD44 could exist the positive feedback to trigger the HA production, and HA could regulate cells proliferation and differentiation by interaction with CD44 in the glioma cells.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 157-172).

Effective tissue engineering scaffolds should mimic the physical and chemical attributes of native tissue. Native tissues have intricate patterns, a multitude of porosities, and large water contents that are each directly associated with their ability to regulate and support life function. Therefore, the physical architecture of scaffolds intended to mimic these tissues for engineering applications plays an important role in scaffold performance both in vitro and in vivo. Self-assembling molecules organize into intricate patterns with a complexity that resembles that of native tissue. Hyaluronic acid (HA) hydrogels are widely used in tissue engineering for a variety of applications but fail to offer physical architecture beyond the inherent hydrogel porosity. To address this issue, a novel method to impose architecture within thin HA-based films using crystal nucleation was developed in the Schmidt lab [1]. The work described herein extends this method for use in three-dimensional matrices, with the main vii goal being the creation of hydrogels with a complex macroarchitecture. Four in situ self-assembling molecules were used: glycine, guanidine, urea and potassium dihydrogen phosphate. The crystallization of each molecule creates a specific porous network within the hydrogel that is the negative imprint of the crystalline geometry. The novel restriction of aqueous polymer into the molecule interstitial crystalline space allows hydrogels to embody complex geometric lumen architectures. The hydrogels were characterized in terms of their internal architectures, swelling, bulk moduli, biodegradability, cytotoxicity and in vitro cellular response. The unique structure-property relationships displayed by hydrogels templated by each of the crystallizing molecules were characterized in regards to mechanical properties. The need for complex microscopic architecture is conserved over many tissue engineering applications and templated scaffolds were evaluated for two unique applications. Crystal-templated hydrogels were investigated for use as an artificial stem cell niche environment to expand undifferentiated neural progenitor cells. Additionally, the templated hydrogels were evaluated for the in vitro study of myelin expression from Schwann cells. A hydrogel that combines the biocompatible properties of HA and the architectural complexity of native tissue may prove beneficial for biomedical applications. / text

published_or_final_version / Dental Surgery / Master / Master of Dental Surgery

In recent years, significant success has been made in the field of regenerative medicine. Tissue engineering scaffolds have been developed to repair and replace different types of tissues. The overall goal of the current work was to develop scaffolds of native extracellular matrix components for soft tissue regeneration, more specifically, neural tissue engineering. To date, much research has been focused on developing a nerve guidance scaffold for its ability to fill and heal the gap between the damaged nerve ends. Such scaffolds are marked by several intrinsic properties including: (1) a biodegradable scaffold or conduit, consisting of native ECM components, with controlled internal microarchitecture; (2) support cells (such as Schwann cells) embedded in a soft support matrix; and (3) sustained release of bioactive factors. In the current dissertation, we have developed such scaffolds of native biomaterials including hyaluronic acid (HA) and collagen. HA is a nonsulphated, unbranched, high-molecular weight glycosaminoglycan which is ubiquitously secreted by cells in vivo and is a major component of extracellular matrix (ECM). High concentrations of HA are found in cartilage tissue, skin, vitreous humor, synovial fluid of joints and umbilical cord. HA is nonimmunogenic, enzymatically degradable, non-cell adhesive which makes HA an attractive material for biomedical research. Here we developed new photopolymerizable HA based materials for soft tissue repair application. First, we developed interpenetrating polymer networks (IPN) of HA and collagen with controlled structural and mechanical properties. The IPN hydrogels were enzymatically degradable, porous, viscoelastic and cytocompatible. These properties were dependent on the presence of crosslinked networks of collagen and GMHA and can be controlled by fine tuning the polymer ratio. We further developed these hydrogel constructs as three dimensional cellular constructs by encapsulating Schwann cells in IPN hydrogels. The hydrogel constructs supported cell viability, spreading, proliferation, and growth factor release from the encapsulated cells. Finally, we fabricated scaffolds of photopolymerizable HA with controlled microarchitecture and developed designer scaffolds for neural repair using layer-by-layer fabrication technique. Lastly, we developed HA hydrogels with unique anisotropic swelling behavior. We developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked regions. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions. / text

Cell behavior is influenced to a large extent by the surrounding microenvironment. Therefore, in the body, the cellular microenvironment is highly controlled with cells growing within well-defined tissue architectures. However, traditional culture techniques allow only for the random placement of cells onto culture dishes and biomaterials. Cell micropatterning strategies aim to control the spatial localization of cells on their underlying material and in relation to other cells. Developing such strategies provides us with tools necessary to eventually fabricate the highly-controlled microenvironments found in multicellular organisms. Employing natural extracellular matrix (ECM) materials in patterning techniques can increase biocompatibility. In the future, with such technologies, we can hope to conduct novel studies in cell biology or optimize cell behavior and function towards the development of new cell-based devices and tissue engineering constructs. Herein, a novel cell patterning platform was developed on a hydrogel base of crosslinked hyaluronic acid (HA). Hydrogels are often employed in tissue engineering due to their ability to mimic the physicochemical properties of natural tissues. HA is a polymer present in all connective tissues. Cell-adhesive regions on the hydrogel were created using the RGDS peptide sequence, found within the cell-adhesive ECM protein, fibronectin. The peptide was bound to a 2-nitrobenzyl “caging group” via a photolabile bond to render the peptide light-responsive. Finally, this “caged” peptide was covalently bound to the hydrogel to form a novel HA hydrogel with a cell non-adhesive surface which could be activated with near-UV light to become adhesive. In this way, we successfully formed chemically patterned cell-adhesive regions on a HA hydrogel using light as a stimulus to form controlled cell patterns. While the majority of cell patterning strategies to date are limited to patterning one cell population and cannot be changed with time, our strategy was novel in using small, adhesive, caged peptides combined with multiple, aligned light exposure steps to allow for dynamic chemical cell patterning on a hydrogel. Multiple cell populations, even held apart from one another, were successfully patterned on the same hydrogel. Furthermore, cell patterns were deliberately modified with time to direct cell growth and/or migration on the hydrogel base.

Hyaluronic acid (HA) is a high value biopolymer that has numerous biomedical and cosmetic applications. It is currently derived from two sources, namely animal tissues and bacterial fermentation (Fong Chong et al. 2005). The molecular weight (Mw) of HA can vary from several hundred thousand dalton (Da) to approximately 8 MDa (Widner et al. 2005). High Mw HA has surgical applications, and therefore constitutes a major component of the lucrative HA market. The current need is largely met by extraction from animal tissues, e.g., rooster comb and bovine vitreous humor (Shiedlin et al. 2004). However, the potential of contamination with adventitious agents (e.g., viruses) have raised regulatory concerns regarding the use of animal extracts in pharmaceutical products. Moreover, with recent reports of zoonotic diseases (e.g., bovine spongiform encephalitis and avian influenza virus), pharmaceutical companies are moving towards microbial HA sources. Although HA obtained from bacterial fermentation does not have the problem of viral contamination, this approach has not yet resulted in a process where HA of sufficiently high Mw for surgical applications can be derived. While attempts have been made to produce higher Mw HA through cross-linking, cross-linked HA is undesirable for certain medical procedures (e.g., ophthalmic applications) which requires a natural polymer with a short half-life. Nevertheless, due to its availability and the relative ease of purification, bacterial fermentation has the potential of replacing extraction from animal tissues as a preferred commercial source of HA. This thesis presents a good example of a metabolic engineering study where modern techniques (e.g., molecular biology, fermentation and omics technologies) are used to explain complex cell metabolism. The hypothesis for this study was that the precursors to HA, i.e., UDP-glucuronic acid and UDP-N-acetylglucosamine and consequently the genes involved in precursor generation, are important for HA Mw. However, environmental manipulation, e.g., anaerobic versus aerobic or glucose versus maltose, often results in large global changes in metabolite concentration and enzyme activities. This makes it impossible to resolve issues related to Mw control. Classical statistical methods do not provide a meaningful inference as the number of explanatory variables always exceeds the number of independent observations. Hence, it is difficult to distinguish between causative and accidental correlation. This work first examined the influence of manipulation of metabolite concentrations in the hyaluronan pathway to find an explanation for the mechanism of Mw control. To achieve this, the five essential genes of the hyaluronan synthesis (has) operon in Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) were first examined. These genes are involved in two pathways which lead to the production of either UDP-glucuronic acid or UDP-N-acetylglucosamine. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased HA Mw, while overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased HA Mw. The Mw variation generated provided a stepping stone for further understanding of Mw control of HA. The highest Mw observed was achieved with combined overexpression of pgi and glmU. This study proved that there is a positive correlation between UDP-N-acetylglucosamine and Mw. The first model for HA Mw control based on the concentration of activated sugar precursors is described in this study (Chapter 3). This correlation observed led to the hypothesis that high Mw HA can be achieved when an appropriate balance of the two HA precursor is maintained. Three genes in the two precursor pathways are not found in the has operon of S. zooepidemicus. To obtain a complete overview of all genes in the HA pathway, these genes were also examined using overexpression studies. Individual overexpression of these genes had negligible effects on HA Mw and production. Despite the positive correlation previously observed between UDP-N-acetylglucosamine and Mw, sequential overexpression of genes involved in the UDP-N-acetylglucosamine precursor pathway did not increase Mw of HA produced. This is surprising since the highest pool of UDP-N-acetylglucosamine was achieved in this case. This suggests that a threshold effect is present in the correlation between UDP-N-acetylglucosamine and Mw. This threshold effect may be defined by a balance between the two precursors. To investigate this phenomenon further, the precursor ratio was also manipulated by co-metabolising glucose and N-acetylglucosamine. Similar to the previous experiment, a significant increase in UDP-N-acetylglucosamine levels was observed despite only a marginal increase in Mw (Chapter 4). Surprisingly, an increase in Mw was observed with the introduction of a plasmid in S. zooepidemicus. This plasmid effect was studied on a global scale using transcriptome and proteome analysis to understand the changes occurring in the system. The increase in Mw due to the plasmid effect is independent of the functions, i.e., nisin promoter or antibiotic resistance, encoded in the plasmid. A gene involved in UDP-N-acetylglucosamine production, UDP-N-acetylglucosamide 1-carboxivinyltransferase (murA), was significantly down-regulated in both the plasmid bearing strain and the high Mw strain (pgi). In addition, overexpression of murA decreased both the concentration of activated sugar precursors and HA Mw. There was however no evidence of down-regulation of murA in the plasmid containing strain from transcriptomics data. This suggests that control is exerted either at the translation level or by protein degradation (Chapter 5). This thesis contributes and represents an ongoing effort to understand the elusive mechanism of Mw control of HA.

Hyaluronic acid (HA) is a biopolymer with valuable applications in the pharmaceutical and cosmetic industries. The molecular weight of HA is important for its rheological, biological and commercial properties. Currently, high molecular weight HA is extracted from animal sources. Recent pandemic outbreaks of viruses H5N1 and H1N1 (avian and swine influenza) have raised concerns regarding the safety of animal-derived pharmaceuticals; making fermentation the preferred source of HA. Throughout this study, the mechanism of molecular weight control of HA polymerisation in S. zooepidemicus was investigated. While several aspects are still unknown, it was found that levels of activated monomers, (UDP-sugars) have a fundamental role in molecular weight control. High levels of activated sugars strongly correlated with high molecular weight. Throughout rational strain engineering, several strains harbouring a collection of genes were engineered using S. zooepidemicus as a host microorganism. Five genes of the so-called has operon were cloned into a nisin inducible plasmid and transformed into S. zooepidemicus. Several significant changes were observed in HA molecular weight. In order to understand those changes as a complex system, rather than isolated parts of the cell, a systems approach was undertaken. The main goal of this approach was to examine the structure and dynamics of cellular functions using global measurements on changes in proteins or metabolite concentrations, in response to genetic perturbations. As part of the systems biotechnology approach, methods for proteins and metabolites harvesting were optimised. Harvesting metabolites and proteins in microorganisms is a non-trivial process (Chapter 4 and 5). Encapsulated bacteria presents additional challenges since capsular polysaccharides interfere with extraction and downstream analysis. In terms of metabolite harvesting, several studies have reported rapid turnovers in low abundant intracellular sugar metabolites. Four different protocols for cellular harvesting were tested. The best method found was centrifugation, which allowed for efficient medium removal and enabled quantification of the broadest range of sugar metabolites. Unlike observations for other microbes, changes in metabolite pools due to a delay of extraction by centrifugation were not observed. Our hypothesis was that the capsule itself isolates the cells from their surroundings and still supports them with nutrients during cellular harvesting (Chapter 3). Protein harvesting also proved to be technically challenging (Chapter 5), especially when fluorescent dyes were employed for protein visualisation(Chapter 6). Despite this, using hyaluronidase to remove the HA capsule the first reference map for S. zooepidemicus was completed. Besides giving insight into the most abundantly expressed proteins, as well as facilitating the design of better diagnostics and treatments for streptococcal infections, our reference map can be used to engineer superior production strains (Chapter 5). In Chapter 6, proteins involved in MW control were separated using 2D differential in gel electrophoresis (DIGE) and identified by mass spectrometry. Moreover, the wild-type was compared with both the empty vector control strain and a high molecular-weight producing strain harbouring phosphoglucoisomerase (pgi). An enzyme involved in controlling the levels of the intracellular pool of one of the activated sugars, UDP-N-acetylglucosamide-1-carboxivinyltransferase, was down-regulated in the control line. Overexpression of UDP-N-Acetylglucosamide 1-carboxivinyltransferase, decreased both the concentration of activated precursors and HA molecular weight. Gene knockout of one of the copies of that particular gene could potentially guide us to further improvement of the strain. By overexpressing all the genes involved in the HA pathway (Appendix A) we have pushed gene expression as far as possible and the next option was to look at process optimisation. Several studies have found that culture conditions affect HA molecular weight: higher molecular weight is produced under aerobic conditions and when using maltose as the carbon source. To overcome the lower sugar uptake and growth rate observed under maltose, a two stage batch fermentation process was conducted. By feeding glucose when cell growth was stopped through amino acid auxotrophy, we achieved high molecular weight HA production in stationary phase. Using engineered strains, HA >5 MDa was obtained (Chapter 7). This thesis represents a good example of systems biotechnology for strain improvement and is a step forward in understanding the mechanism of molecular weight control of HA production by bacterial fermentation.