| 1. |
- Gullfot, Fredrika, 1967-
(författare)
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Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose. Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns. Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.
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| 2. |
- Muzamal, Muhammad, 1986
(författare)
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Steam Explosion of Wood
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rising price of petroleum and environmental concerns regarding CO2 emissions has increased interest in alternative renewable resources. Biomass can be considered as an excellent alternative raw material. A biorefinery uses biomass and produces fuel, energy and value-added chemicals. The biorefinery is an emerging field and requires much development to compete with already established petroleum-based industries. One of the greatest challenges to the biorefinery is that the raw material; biomass, has a complex chemical composition and physical structure. A pretreatment process is necessary to induce physico-chemical changes in the biomass and transform it into easily digestible material. The main factor limiting enzymatic digestion of biomass is accessibility to chemical constituents. Steam Explosion (SE) pretreatment is a promising process that has many potential benefits compared to the alternatives, e.g. it has less hazardous process chemicals and conditions, less environmental impact, fewer energy requirements and lower capital investment.Existing literature on the SE process mainly concerns products obtained after the process. Knowledge about the physical processes that take place during the SE pretreatment is limited. This licentiate thesis is based on experimental and modelling studies performed with the aim of gaining knowledge of the basic mechanisms of this process. The SE is a three-step process that involves; (i) treatment of wood with pressurized steam for a specific period of time, (ii) explosion of wood chips through the rapid release of pressure, and (iii) impact of softened wood chips with other chips and vessel walls. In the experimental part these steps have been carefully isolated and the effects of these steps on internal and external structures of single spruce wood pieces have been studied. The effect of vapour expansion and the creation of stresses during the explosion step on a single cell of spruce wood (with four layers; P, S1, S2 and S3) at high temperature and moisture content have been modelled using the Finite Element Method.The study reveals that all the steps of the SE process contribute to structural changes in the wood material and increase pore size which increases the accessibility of chemical reagents and enzymes. A wood piece disintegrates into smaller pieces during the impact step. The vapour expansion inside cells during the explosion step causes each cell to expand in all directions and creates high stress and strain fields perpendicular to the cell direction. In general, cell wall damage is more likely to occur in cells with thin walls, i.e. earlywood; damaged P, S1 and S3 layers; low MFAs; irregular cross-sections and sharp corners.
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| 3. |
- Kashfi, Pariya, 1980
(författare)
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Towards Usable openEHR-aware Clinical Decision Support: A User-centered Design Approach
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nowadays, the use of computerized approaches to support health care processesin order to improve quality of health care is widespread in the clinical domain.Electronic health records (EHR) and clinical decision support (CDS) are consideredto be two complementary approaches to improve quality of health care.It is shown that EHRs are not able to improve quality of health care withoutbeing supported by other features such as CDS. On the other hand, one of thesuccess factors of CDS is its integration into EHR, and since there are variousinternational EHR standards (such as openEHR) being developed, it is crucialto take these standards into consideration while developing CDS.Various clinical decision support systems (CDSS) are developed but unfortunatelyonly a few of them are being used routinely. Two of the reasons for unacceptability of CDSSs among their users, i.e. clinicians, are shown to be their separation from EHRs and poor usability of the user interfaces. Besides integration into underlying information framework, i.e. EHR systems, consideration of human-computer interaction (HCI) in designing and evaluating CDS isone of the success factors that developers of these systems should keep in mind.This thesis addresses the question of how usable openEHR-aware clinical decision support can be designed and developed in order to improve the quality of health care. To answer this research question, several sub-questions were identified and investigated. This included analyzing \state of the art" in two different aspects of design and development and evaluation of CDS and also investigating application of a customized user-centered design (UCD) process in developing openEHR-based clinical applications.Analysis of state of the art in interplay between HCI and CDS and also the intersection between CDS and EHR revealed that consideration of both HCI and integration of CDS into EHR is more appreciated in theory than in practice and there is still a long way to go before reaching an acceptable level in these two success factors of CDS.Moreover, the experience in designing an openEHR-based clinical application revealed that apart from benefits offered by openEHR approach, such as specifying different roles and involvement of domain experts in defining domain concepts, there are various shortcomings that need to be improved, for instance the limited support for openEHR application developers. Additionally, this study revealed that there are characteristics of the domain, tasks and users in the domain that developers should be informed about while applying UCD methods.
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| 4. |
- Angleby, Helen
(författare)
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Analysis of domestic dog mitochondrial DNA sequence variation for forensic investigations
- 2005
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The first method for DNA analysis in forensics was presented in 1985. Since then, the introduction of the polymerase chain reaction (PCR) has rendered possible the analysis of small amounts of DNA and automated sequencing and fragment analysis techniques have facilitated the analyses. In most cases short tandemly repeated regions (STRs) of nuclear DNA are analysed in forensic investigations, but all samples cannot be successfully analysed using this method. For samples containing minute amounts of DNA or degraded DNA, such as shed hairs, analysis of mitochondrial DNA (mtDNA) is generally more successful due to the presence of thousands of copies of mtDNA molecules per cell. In Sweden, ~40 % of all households have cats or dogs. With ~9 million humans shedding ~100 scalp hairs per day, and ~1.6 million cats and ~1 million dogs shedding hairs it is not surprising that shed hairs are one of the most common biological evidence found at crime scenes. However, the match probability for domestic dog mtDNA analysis has only been investigated in a few minor studies. Furthermore, although breed –sequence correlations of the noncoding mtDNA control region (CR) have been analysed in a few studies, showing limited correlations, no largescale studies have been performed previously. Thus, there have not been any comprehensive studies of forensic informativity of dog mtDNA. In the two papers presented in this thesis we have tried to lay a foundation for forensic use of analysis of domestic dog mtDNA. In the first paper, CR sequences were analysed and the exclusion capacity was investigated for a number of different populations. This is also the first comprehensive study of the correlation between mtDNA CR type and breed, type, and geographic origin of domestic dogs. Since the exclusion capacity for analysis of domestic dog CR sequences is relatively low, it was investigated in the second paper to what extent the discrimination power is improved by analysis of coding sequence. The exclusion capacity improved considerably when 3,000 base pairs of coding sequences where analysed in addition to CR sequences. This study will hopefully work as a basis for future development of analysis of dog mtDNA for forensic purposes.
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| 5. |
- Bondesson, Laban
(författare)
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Microscopic views of drug solubility
- 2006
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The development of computational models for predicting drug solubility has increased drastically during the last decades. Nevertheless these models still have diffculties to estimate the aqueous solubility as accurate as desired. In this thesis di erent aspects that are known to have a large impact on the aqueous solubility of a molecule have been studied in detail using various theoretical methods with intension to provide microscopic view on drug solubility. The rst aspect studied is the hydrogen bond energies. Eight drug molecules have been calculated using density functional theory and the validity of additive model that has often been used in solubility models is examined. The impact of hydrogen bonds in Infrared and Raman spectra of three commonly used drug molecules has also been demonstrated. The calculated spectra are found to be in good agreement with the experimental data. Another aspect that is important in solubility models is the volume that a molecule occupies when it is dissolved in water. The volume term and its impact on the solvation energy has therefore also been calculated using three di erent methods. It was shown that the calculated volume di ered signi cantly dependent on which method that had been used, especially for larger molecules. Most of the solubility models assume the solute molecule to be in the bulk of the solvent. The molecular behavior at the water/gas interface has been investigated to see how it di ers from bulk. It was seen that the concentration close to the interface was almost three times higher than in the bulk. The increase in concentration close to the surface depends on the larger gap between the interface energy and the gas phase energy than between the bulk energy and the gas phase energy.
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| 6. |
- Bäcklund, Emma
(författare)
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Growth rate control of periplasmic product retention in Escherichia coli
- 2008
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The recombinant product is secreted to the periplasm in many processes where E. coli is used as host. One drawback with secretion is the undesired leakage of the periplasmic products to the medium. The aim of this work was to find strategies to influence the periplasmic retention of recombinant products. We have focused on the role of the specific growth rate, a parameter that is usually controlled in industrial bioprocesses. The hypothesis was that the stability of the outer membrane in E. coli is gained from a certain combination of specific phospholipids and fatty acids on one side and the amount and specificity of the outer membrane proteins on the other side, and that the specific growth rate influences this structure and therefore can be used to control the periplasmic retention. We found that is possible to control the periplasmic retention by the growth rate. The leakage of the product increased as the growth rate increased. It was however also found that a higher growth rate resulted in increased productivity. This resulted in equal amounts of product inside the cells regardless of growth rate. We also showed that the growth rate influenced the outer membrane composition with respect to OmpF and LamB while OmpA was largely unaffected. The total amount of outer membrane proteins decreased as the growth rate increased. There were further reductions in outer membrane protein accumulation when the recombinant product was secreted to the periplasm. The lowered amount of outer membrane proteins may have contributed to the reduced ability for the cell to retain the product in the periplasm. The traditional way to control the growth rate is through a feed of substrate in a fed-batch process. In this work we used strains with a set of mutations in the phosphotransferase system (PTS) with a reduced uptake rate of glucose to investigate if these strains could be used for growth rate control in batch cultivations without the use of fed-batch control equipment. The hypothesis was that the lowering of the growth rate on cell level would result in the establishment of fed-batch similar conditions. This study showed that it is possible to control the growth rate in batch cultivations by using mutant strains with a decreased level of substrate uptake rate. The mutants also produced equivalent amounts of acetic acid as the wild type did in fed-batch cultivation with the same growth rate. The oxygen consumption rates were also comparable. A higher cell density was reached with one of the mutants than with the wild type in batch cultivations. It is possible to control the growth rate by the use of the mutants in small-scale batch cultivations without fed-batch control equipment.
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| 7. |
- Calles, Karin, 1976-
(författare)
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Regulation of productivity in Trichoplusia ni and Spodoptera frugiperda Sf9 serum-free cultures
- 2005
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this work has been to characterize the effects of conditioned medium (CM) on insect cell productivity and physiology in order to get a better understanding about the mechanisms that regulate productivity in serum-free media. Two cell lines have been investigated, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T. ni, BTI-Tn-5B1-4). The baculovirus expression vector system (BEVS) was used for protein expression, using the ligand-binding domain of the human glucocorticoid receptor as a model protein. Addition of CM at inoculation led to a shorter lag phase and that the cells reached the maximum cell density faster than cells in fresh medium for both Sf9 and T. ni cells. Sf9 cells passed a switch in growth kinetics after 30-40 passages. At this point, CM lost its stimulating effect on proliferation. CM also affected the cell size and cell cycle progression. Sf9 and T. ni cells became smaller when CM was added at inoculation because they had a minor arrest in the cell cycle after inoculation and therefore started to divide earlier than cells in fresh medium. For Sf9 cells, this was illustrated by a smaller arrest in G2/M in the beginning of culture and the cells were consequently less synchronized. For T. ni cells, the initial decrease in the S phase population was followed by an earlier increase of the S phase population for the cells with CM than for the cells in fresh medium. Addition of 20 % CM or CM filtrated with a 10 kDa cut-off filter to Sf9 cultures had a negative effect on the specific productivity. However, addition of CM to Sf9 cells that had passed the switch in growth kinetics had no negative effect on productivity. This indicates that CM not affects the protein production per se, but rather through its effects on cell physiology. Instead, the degree of cells synchronized in G2/M is important for high productivity and the gradually decreasing degree of synchronization during the course of a culture might be the explanation behind the cell density dependent decrease in productivity for Sf9 cells. This was further supported by the positive effects on productivity achieved by synchronizing Sf9 cells in G2/M by yeastolate limitation, which counteracted the cell density-dependent drop in productivity and hence a higher volumetric yield was achieved. Addition of 20 % CM to T. ni cultures had a positive effect on productivity. The specific productivity was maintained at a high level longer than for cells in 100 % fresh medium. The product concentration was 34 % higher and the maximum product concentration was obtained 24 hours earlier for the cells with the addition of CM. These results show that the effects of CM on productivity are not the same for the two cell lines and that the mechanism regulating productivity are quite complex.
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| 8. |
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| 9. |
- Eriksson, Ulrika, 1974-
(författare)
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Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free cultures
- 2005
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells.Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation.Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics.In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity.Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well.
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| 10. |
- Guldevall, Karolin
(författare)
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Development of Microchip-based Assays to Study Immune Cell Interactions at the Single Cell Level
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measures at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity. Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach. To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. One design is aimed towards screening applications, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated; this program is also described and evaluated. We here show that our silicon microwell platform allows long-term studies (up to several days), with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using time-lapse imaging we confirmed immune cell heterogeneity in NK cell populations regarding both cytotoxicity and migrational behavior. The automatic counting program was tested and showed similar results compared to both manual counting and FACS. In addition, the large numbers of wells that can be simultaneously imaged, provide new statistical information that will lead to a better understanding of the function and regulation of the immune system at the single cell level. Altogether, our technique enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications.
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