| 1. |
- Ahrenstedt, Lage
(författare)
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Surface modification of cellulose materials : from wood pulps to artificial blood vessels
- 2007
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the improvement of two radically different cellulose materials, paper and artificial blood vessels, constructed from two diverse cellulose sources, wood pulp and Acetobacter xylinum. The improvement of both materials was possible due to the natural affinity of the hemicellulose xyloglucan for cellulose. Chemical and mechanical pulps were treated with xyloglucan in the wet-end prior to hand sheet formation or by spray application of dry hand sheets, loading a comparable amount of xyloglucan. The tensile strength increases for the wet-end treatment and spray application were 28% and 71% respectively for bleached soft wood, compared to untreated sheets (20.7 Nm/g). The corresponding strength increases for hand sheets made of thermo-mechanical pulp were 6% and 13% respectively compared to untreated sheets (42.4 Nm/g). The tendency for chemical pulp to be superior to mechanical pulp with respect to strength increase was valid even for tear strength and Scott-Bond. These results suggest, in agreement with other studies, that adhesion of xyloglucan to wood fibres is dependent on their degree of surface lignification. Also, a method was developed to increase the blood compatibility of artificial blood vessels constructed of bacterial cellulose. Xyloglucan was covalently linked to the endothelial cell adhesion motif (Arg-Gly-Asp). To obtain this, new solid-phase coupling chemistry was developed. Xyloglucan oligosaccharides (XGO) were transformed into XGO-succinamic acid via the corresponding XGO--NH2 derivative prior to coupling with the N-terminus of the solid-phase synthesised Gly-Arg-Gly-Asp-Ser peptide. The resin-bound glyco-peptide was then cleaved and enzymatically re-incorporated into high molecular weight xyloglucan. The glyco-peptide was further adsorbed onto bacterial cellulose scaffolds, increasing the adhesion and proliferation of endothelial cells and therefore blood compatibility.
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| 2. |
- Ai, Yuejie, 1982-
(författare)
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Theoretical studies on photophysics and photochemistry of DNA
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Theoretical studies on biological systems like nucleic acid and protein have been widely developed in the past 50 years and will continue to be a topic of interest in forefronts of natural science. In addition to experimental science, computational modeling can give useful information and help us to understand biochemical issues at molecular, atomic and even electronic levels. Deoxyribonucleic acid (DNA), the hereditary basis of life’s genetic identity, has always been major topic of discussions since its structure was built in 1953. However, harmful UV radiation from sunlight can make damage to DNA molecules and eventually give rise to DNA damaging biological consequences, like mutagenesis, carcinogenesis, and cell death. Photostability, photodamage, and photorepair are of vital importance in the photophysics and photochemistry of DNA. In this thesis, we have applied high level computer-aided theoretical methods to explore the underlying mechanisms for these three critical issues of DNA. Special attentions are paid to the following aspects: the properties of the excited states, the design of relevant computational models and the effects of biological environments. We have systematically studied the excited state properties of DNA from single base to base pair and oligonucleotides, where the concerted base pairing and base stacking effects was found to play important roles in DNA photostability. The UV-light induced isomerization mechanism between two photoproducts of DNA photodamage has been revealed in different biological environments. In association with DNA photodamage, the related photorepair processes have been proposed for different lesions in photolyase which is a catalytic enzyme for DNA, and the calculated results well explained the experimental observations. In particular, the internal and external properties of flavin cofactors have been extensively studied by combining the electronic structure and spectroscopic calculations. We have examined the effects of the intramolecular hydrogen bond on spectroscopic properties of flavins. The good agreements with the experimental spectra indicated that the biological self-regulation acted critical role in these biological systems.
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| 3. |
- Akhras, Michael S., 1980-
(författare)
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Nucleic Acid Based Pathogen Diagnostics
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill & Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications.
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| 4. |
- Alm, Tove, 1977-
(författare)
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Interaction engineered three-helix bundle domains for protein recovery and detection
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- HTML clipboard The great advances in DNA technology, e.g. sequencing and recombinant DNA techniques, have given us the genetic information and the tools needed to effectively produce recombinant proteins. Recombinant proteins are valuable means in biotechnological applications and are also emerging as alternatives in therapeutic applications. Traditionally, monoclonal antibodies have been the natural choice for biotechnological and therapeutic applications due to their ability to bind a huge range of different molecules and their natural good affinity. However, the large size of antibodies (150 kDa) limits tissue penetration and the recombinant expression is complicated. Therefore, alternative binders with smaller sizes have been derived from antibodies and alternative scaffolds.In this thesis, two structurally similar domains, Zbasic and ABDz1, have been used as purification tags in different contexts. They are both three-helical bundles and derived from bacterial surface domains, but share no sequence homology. Furthermore, by redesign of the scaffold used for ABDz1, a molecule intended for drug targeting with extended in-vivo half-life has been engineered. In Papers I and II, the poly-cationic tag Zbasic is explored and evaluated. Paper I describes the successful investigation of Zbasic as a purification handle under denaturating conditions. Moreover, Zbasic is evaluated as an interaction domain in matrixassisted refolding. Two different proteins were successfully refolded using the same setup without individual optimization. In Paper II, Zbasic is further explored as a purification handle under non-native conditions in a multi-parallel setup. In total, 22 proteins with varying characteristics are successfully purified using a multi-parallel protein purification protocol and a robotic system. Without modifications, the system can purify up to 60 proteins without manual handling. Paper I and II clearly demonstrate that Zbasic can be used as an interaction domain in matrix-assisted refolding and that it offers a good alternative to the commonly used His6-tag under denaturating conditions. In paper III, the small bifunctional ABDz1 is selected from a phage display library. Endowed with two different binding interfaces, ABDz1 is capable of binding both the HSA-sepharose and the protein A-derived MabSelect SuRe-matrix. The bifunctionality of the domain is exploited in an orthogonal affinity setup. Three target proteins are successfully purified using the HSA-matrix and the MabSelect SuRe-matrix. Furthermore, the purity of the target proteins is effectively improved by combining the two chromatographic steps. Thus, paper III shows that the small ABDz1 can be used as an effective purification handle and dual affinity tag without target specific optimization. Paper IV describes the selection and affinity maturation of small bispecific drug-targeting molecules. First generation binders against tumor necrosis factor-α are selected using phage display. Thereafter on-cell surface display and flow cytometry is used to select second-generation binders. The binding to tumor necrosis factor-α is improved up to 30 times as compared to the best first generation binder, and a 6-fold improvement of the binding strength was possible with retained HSA affinity. Paper III and IV clearly demonstrate that dual interaction surfaces can successfully be grafted on a small proteinaceous domain, and that the strategy in paper IV can be used for dual selection of bifunctional binders.
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| 5. |
- Andersen, Malin, 1977-
(författare)
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Computational and experimental approaches to regulatory genetic variation
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Genetic variation is a strong risk factor for many human diseases, including diabetes, cancer, cardiovascular disease, depression, autoimmunity and asthma. Most of the disease genes identified so far alter the amino acid sequences of encoded proteins. However, a significant number of genetic variants affecting complex diseases may alter the regulation of gene transcription. The map of the regulatory elements in the human genome is still to a large extent unknown, and it remains a challenge to separate the functional regulatory genetic variations from linked neutral variations. The objective of this thesis was to develop methods for the identification of genetic variation with a potential to affect the transcriptional regulation of human genes, and to analyze potential regulatory polymorphisms in the CD36 glycoprotein, a candidate gene for cardiovascular disease. An in silico tool for the prediction of regulatory polymorphisms in human genes was implemented and is available at www.cisreg.ca/RAVEN. The tool was evaluated using experimentally verified regulatory single nucleotide polymorphisms (SNPs) collected from the scientific literature, and tested in combination with experimental detection of allele specific expression of target genes (allelic imbalance). Regulatory SNPs were shown to be located in evolutionary conserved regions more often than background SNPs, but predicted transcription factor binding sites were unable to enrich for regulatory SNPs unless additional information linking transcription factors with the target genes were available. The in silico tool was applied to the CD36 glycoprotein, a candidate gene for cardiovascular disease. Potential regulatory SNPs in the alternative promoters of this gene were identified and evaluated in vitro and in vivo using a clinical study for coronary artery disease. We observed association to the plasma concentrations of inflammation markers (serum amyloid A protein and C-reactive protein) in myocardial infarction patients, which highlights the need for further analyses of potential regulatory polymorphisms in this gene. Taken together, this thesis describes an in silico approach to identify putative regulatory polymorphisms which can be useful for directing limited laboratory resources to the polymorphisms most likely to have a phenotypic effect.
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| 6. |
- Anderson, Mattias
(författare)
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Amine Transaminases in Multi-Step One-Pot Reactions
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amine transaminases are enzymes that catalyze the mild and selective formation of primary amines, which are useful building blocks for biologically active compounds and natural products. In order to make the production of these kinds of compounds more efficient from both a practical and an environmental point of view, amine transaminases were incorporated into multi-step one-pot reactions. With this kind of methodology there is no need for isolation of intermediates, and thus unnecessary work-up steps can be omitted and formation of waste is prevented. Amine transaminases were successfully combined with other enzymes for multi-step synthesis of valuable products: With ketoreductases all four diastereomers of a 1,3-amino alcohol could be obtained, and the use of a lipase allowed for the synthesis of natural products in the form of capsaicinoids. Amine transaminases were also successfully combined with metal catalysts based on palladium or copper. This methodology allowed for the amination of alcohols and the synthesis of chiral amines such as the pharmaceutical compound Rivastigmine. These examples show that the use of amine transaminases in multi-step one-pot reactions is possible, and hopefully this concept can be further developed and applied to make industrial processes more sustainable and efficient in the future.
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| 7. |
- Andersson, Ken G., 1987-
(författare)
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Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Directed evolution is today an established strategy for generation of new affinity proteins. This thesis describes the development of a cell-display method using Escherichia coli for directed evolution of Affibody molecules. Further, the thesis describes rational design of Affibody-based tracers, intended for future patient stratification using medical imaging. Fusing recombinant proteins to various autotransporters is a promising approach for efficient surface display on the surface of E. coli, as well as for construction of high-complexity libraries. In paper I, we successfully engineered an expression vector for display of Affibody molecules using the autotransporter AIDA-I. In paper II, a large Affibody library of 2.3x109 variants was constructed and screening using FACS resulted in new specific binders in the nanomolar range. In paper III, we demonstrated Sortase-mediated secretion and conjugation of binders directly from the E. coli surface. The three following studies describe rational design of Affibody-based tracers against two cancer-associated targets for molecular imaging. First, anti-HER3 Affibody molecules were labelled with 111In, and SPECT imaging showed that the conjugates specifically targeted HER3-expressing xenografts. Furthermore, labeling with 68Ga for PET imaging showed that tumor uptake correlated with HER3 expression, suggesting that the tracers have potential for patient stratification. The last study describes the development and investigation of anti-EGFR Affibody-based imaging agents. Labeled with 89Zr, the Affibody tracer demonstrated higher tumor uptake at 3 h post injection than the anti-EGFR antibody cetuximab at 48 h post injection. In conclusion, this thesis describes new tools and knowledge that will hopefully contribute to the development of affinity proteins for biotechnology, therapy and medical imaging in the future.
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| 8. |
- Andersson, Sofia, 1978-
(författare)
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Characterization of Bacterial Biofilms for Wastewater Treatment
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Research performed at the Division of Environmental Microbiology has over the last years resulted in the isolation of possible bacterial key-organisms with efficient nutrient removal properties (Comamonas denitrificans, Brachymonas denitrificans, Aeromonas hydrophila). Effective use of these organisms for enhanced nutrient removal in wastewater treatment applications requires the strains to be retained, to proliferate and to maintain biological activity within theprocess. This can be achieved by immobilization of the organisms using an appropriate system.Two putative immobilization systems, agar entrapment and biofilm formation, wereassessed. Surface attached biofilm growth provided better results with respect to cell retention,proliferation and microbial activity than immobilization in agar beads. Thus, biofilm physiology was further characterized using simplified systems of single, dual or multi strain bacterial consortia containing the key-organisms as well as other wastewater treatment isolates. Mechanisms for initial adherence, biofilm formation over time, dynamics and characteristics of extracellular polymeric substances (EPS) and exopolysaccharides, nutrient removal activity as well as the effect of bacterial interactions were investigated. The results showed that all theassessed bacterial strains could form single strain biofilm providing that a suitable nutrientsupply was given. Production of EPS was found to be critical for biofilm development and both EPS and polysaccharide residue composition varied with bacterial strain, culture conditions and biofilm age. Denitrification and phosphorus removal activity of the keyorganisms was maintained in biofilm growth. Co-culturing of two or more strains resulted in both synergistic and antagonistic effects on biofilm formation as well as the microbial activitywithin the biofilm. Bacterial interactions also induced the synthesis of new polysaccharideswhich were not produced in pure strain biofilms.The complexity of single and mixed strain biofilm development and the implications of interactions on biofilm performance were underlined in this study. The data presented can be useful for modeling of biofilm systems, serve as a tool for selection of bacterial strain combinations to use for bioaugmentation/bioremediation or provide a base for further experiment design.
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| 9. |
- Andrade, Jorge, 1969-
(författare)
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Grid and High-Performance Computing for Applied Bioinformatics
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The beginning of the twenty-first century has been characterized by an explosion of biological information. The avalanche of data grows daily and arises as a consequence of advances in the fields of molecular biology and genomics and proteomics. The challenge for nowadays biologist lies in the de-codification of this huge and complex data, in order to achieve a better understanding of how our genes shape who we are, how our genome evolved, and how we function. Without the annotation and data mining, the information provided by for example high throughput genomic sequencing projects is not very useful. Bioinformatics is the application of computer science and technology to the management and analysis of biological data, in an effort to address biological questions. The work presented in this thesis has focused on the use of Grid and High Performance Computing for solving computationally expensive bioinformatics tasks, where, due to the very large amount of available data and the complexity of the tasks, new solutions are required for efficient data analysis and interpretation. Three major research topics are addressed; First, the use of grids for distributing the execution of sequence based proteomic analysis, its application in optimal epitope selection and in a proteome-wide effort to map the linear epitopes in the human proteome. Second, the application of grid technology in genetic association studies, which enabled the analysis of thousand of simulated genotypes, and finally the development and application of a economic based model for grid-job scheduling and resource administration. The applications of the grid based technology developed in the present investigation, results in successfully tagging and linking chromosomes regions in Alzheimer disease, proteome-wide mapping of the linear epitopes, and the development of a Market-Based Resource Allocation in Grid for Scientific Applications.
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| 10. |
- Berglund, Lisa, 1978-
(författare)
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Selection of antigens for antibody-based proteomics
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human genome is predicted to contain ~20,500 protein-coding genes. The encoded proteins are the key players in the body, but the functions and localizations of most proteins are still unknown. Antibody-based proteomics has great potential for exploration of the protein complement of the human genome, but there are antibodies only to a very limited set of proteins. The Human Proteome Resource (HPR) project was launched in August 2003, with the aim to generate high-quality specific antibodies towards the human proteome, and to use these antibodies for large-scale protein profiling in human tissues and cells. The goal of the work presented in this thesis was to evaluate if antigens can be selected, in a high-throughput manner, to enable generation of specific antibodies towards one protein from every human gene. A computationally intensive analysis of potential epitopes in the human proteome was performed and showed that it should be possible to find unique epitopes for most human proteins. The result from this analysis was implemented in a new web-based visualization tool for antigen selection. Predicted protein features important for antigen selection, such as transmembrane regions and signal peptides, are also displayed in the tool. The antigens used in HPR are named protein epitope signature tags (PrESTs). A genome-wide analysis combining different protein features revealed that it should be possible to select unique, 50 amino acids long PrESTs for ~80% of the human protein-coding genes. The PrESTs are transferred from the computer to the laboratory by design of PrEST-specific PCR primers. A study of the success rate in PCR cloning of the selected fragments demonstrated the importance of controlled GC-content in the primers for specific amplification. The PrEST protein is produced in bacteria and used for immunization and subsequent affinity purification of the resulting sera to generate mono-specific antibodies. The antibodies are tested for specificity and approved antibodies are used for tissue profiling in normal and cancer tissues. A large-scale analysis of the success rates for different PrESTs in the experimental pipeline of the HPR project showed that the total success rate from PrEST selection to an approved antibody is 31%, and that this rate is dependent on PrEST length. A second PrEST on a target protein is somewhat less likely to succeed in the HPR pipeline if the first PrEST is unsuccessful, but the analysis shows that it is valuable to select several PrESTs for each protein, to enable generation of at least two antibodies, which can be used to validate each other.
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