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.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 3 uppsatser.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Hyaluronan is a polymer that among other things is used in fillers. Products containing Hyaluronan is sometimes discoloured over time and the mechanism behind this discolouration is still unknown. However, it was suspected that discolouration occurs during the degradation due to high pH values or with a phosphate buffer. The discolouration of Hyaluronan that occurs with phosphate buffer was studied in more detail in this project. The samples of Hyaluronan with different concentrations of phosphate buffer were left at 90 °C in an oven to speed up the discolouration. These samples were then analyzed byusing UV/Vis spectrophotometry to measure the absorption and capillary viscometry to measure the molecular weight. The results showed that the discolouration increased with time and that the samples with the higher concentration of buffer got more discoloured faster. The molecular weight showed a decreasing trend with time. It also suggested that the phosphate buffer had an impact on the molecular weight. The samples with the highest concentration of phosphate buffer had a lower molecular weight compared to samples with no phosphate buffer. The main conclusion from this study is that the phosphate buffer had an effect on the discolouration of Hyaluronan.

O propósito deste trabalho é avaliar as características e classificação de compostos orgânicos, presentes em frações de petróleo, em saturados, aromáticos, resinas e asfaltenos (SARA) e identificar a classe homóloga (compostos de enxofre, nitrogênio, oxigênio, enxofre-oxigênio, nitrogênio-oxigênio ou apenas hidrogênio e carbono), construindo um modelo, com base no peso molecular, e utilizando um banco de dados em Excel. No modelo, os compostos orgânicos são organizados em uma matriz atômica de séries homólogas. Essas são séries de compostos com propriedades químicas similares, que diferem por um peso molecular constante (CH2). Esses compostos são classificados por relações heurísticas de Hidrogênio/ Carbono (H/C) e Ligação Dupla Equivalente (DBE). DBE é o número de anéis ou ligações ? envolvendo o átomo de carbono, porque cada anel ou ligações ? resultam na perda de um átomo de hidrogênio. O banco de dados foi desenvolvido em Excel, usando programação em VBA (Visual Basic for Applications). Os dados experimentais foram obtidos utilizando a técnica analítica de espectrometria de massa por MALDI-TOF. Os resultados obtidos mostram que o algoritmo em VBA é capaz de identificar os compostos de uma amostra, dentro da faixa de erro definida e de acordo com a calibração do espectrômetro de massa. / The purpose of this study is to evaluate the characteristics and classification of organic compounds in petroleum fractions as saturates, aromatics, resins and asphaltenes (SARA) and identify the possible homologous class (compounds of sulfur, nitrogen, oxygen, sulfur-oxygen, nitrogen-oxygen or only hydrogen and carbon), building a model and using a database in Excel, based on molecular weight. In the modeling, organic compounds are organized in an atomic matrix of homologous series. These are series of similar chemical properties, which differ by a constant molecular weight (CH2). Then, these compounds are classified through heuristics of hydrogen/ carbon ratio (H/C) and DBE (Double Bond Equivalent) relations. DBE is the number of rings or ? bonds involving carbon, because each ring or ? bonds results in a hydrogen atoms loss. The database was developed in Excel using VBA (Visual Basic for Applications) programming. The experimental data were obtained by analytical technique of MALDI-TOF mass spectrometry. The results show that the VBA algorithm is able to identify compounds from a given sample, within an error marge defined according to the mass spectrometer calibration.

Os modelos moleculares são construídos com base na caracterização da composição do petróleo, que consiste em conhecer informações moleculares detalhadas com o objetivo de identificar os compostos presentes. Este trabalho tem como objetivo construir um banco de dados para a classificação de componentes constituintes de asfaltenos em grupos moleculares. A classificação é feita através da semelhança entre suas massas moleculares em grupos de séries homólogas. Tal classificação é feita por meio da utilização dos dados provenientes da análise de espectrometria de massa e de um algoritmo de busca em linguagem VBA (Visual Basic for Applications), com o objetivo de identificar uma molécula específica em um conjunto de moléculas conhecidas (banco de dados). O método proposto nesse trabalho é baseado na classificação dos compostos através de suas massas moleculares, isto é, os compostos presentes em uma mistura são separados em classes por uma diferença de massa tão pequena quanto se queira. Pode-se, assim, observar a que grupo de moléculas um composto de petróleo pertence por meio de sua massa molecular. Como o banco de dados foi gerado por compostos cuja composição é conhecida, sabe-se que composição um dado composto terá entre as possíveis composições. Para gerar moléculas baseado no conhecimento da composição de petróleo, usam-se as fórmulas gerais dos compostos orgânicos que são formados por séries homólogas. Séries homólogas são séries de compostos orgânicos com propriedades químicas similares cujos componentes diferem entre si por uma massa molecular relativa constante (CH2). Tais séries são geradas e seus pesos moleculares são calculados e armazenados juntamente com suas fórmulas moleculares, em ordem crescente de massa, podendo assim, gerar os grupos moleculares com o auxílio de uma rotina computacional em linguagem VBA. / Molecular models are built based on the characterization of the composition of oil, which consists of detailed molecular information to know in order to identify the compounds present. This work aims to build a database for classifying constituent components of asphaltenes in molecular groups. The classification is done by the similarity of their molecular weights with the use of groups of homologous series. This classification is made by using data from the analysis of mass spectrometry and a search algorithm in language VBA (Visual Basic for Applications) with the aim of identifying a molecule in a specific set of molecules known (database data). The method proposed in that work is based on the classification of the compounds through their molecular weights, i.e., the compounds in a mixture are separated into classes by a mass difference as small as one likes. One can therefore see that the group of molecules from petroleum belongs through its molecular weight. Because the database is generated from compounds whose composition is known, it is known that a given compound composition will have all the possible compositions. To generate molecules based on knowledge of the oil composition, it is used the general formulas of organic compounds which are formed by homologous series. Homologous series are series of organic compounds with similar chemical properties whose components differ by a constant relative molecular mass (CH2). Such series are generated and their molecular weights are calculated and stored together with their molecular formulas, in ascending order of mass, being able to generate the molecular groups with the help of a computing routine in VBA language.

Statement of the problem The primary objective of this thesis was to determine the best study design to evaluate the safety and effectiveness of an intermediate dose of low molecular weight heparin for secondary prevention of pregnancy associated VTE (PAVTE). An RCT was deemed unfeasible,so the use of a single arm study with prior evaluation of feasibility with a pilot study is proposed. // Methods - A systematic review was conducted to evaluate the efficacy of current strategies used for secondary prevention of PAVTE.A survey was used to elicit the non-inferiority margin. // Results - The pooled proportion of recurrent VTE in patients treated with full dose LMWH was 0.012(95% CI 0.006 to 0.02) and the rate of major bleeding was 0.025(95% CI=0.01 to 0.041). The non-inferiority margin was elicited at 2.5%. // Conclusions - Although a randomized controlled trial should be conducted whenever possible, in certain scenarios they are unfeasible. Therefore, an alternative study design should perhaps be used to evaluate the safety and efficacy of therapeutic strategies.