| 1. |
- Ai, Yuejie, 1982-
(författare)
-
Theoretical studies on photophysics and photochemistry of DNA
- 2010
-
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.
|
|
| 2. |
- Alm, Tove, 1977-
(författare)
-
Interaction engineered three-helix bundle domains for protein recovery and detection
- 2010
-
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.
|
|
| 3. |
- Bodlund, Ida, 1983-
(författare)
-
Coagulant Protein from plant materials: Potential Water Treatment Agent
- 2013
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Access to fresh water is a human right, yet more than 780 million people, especially in rural areas, rely on unimproved sources and the need for finding ways of treating water is crucial. Although the use of natural coagulant protein in drinking water treatment has been discussed for a long time, the method is still not in practice, probably due to availability of material and limited knowledge. In this study, about hundred different crude extracts made from plant materials found in Southern India were screened for coagulation activity. Extracts of three Brassica species (Mustard, Cabbage and Cauliflower) were showing activity comparable to that of Moringa oleifera and were further investigated. Their protein content and profile were compared against each other and with coagulant protein from Moringa. Mustard (large) and Moringa seed proteins were also studied for their effect against clinically isolated bacterial strains. The protein profiles of Brassica extract showed predominant bands around 9kDa and 6.5kDa by SDS-PAGE. The peptide sequence analysis of Mustard large identified the 6.5kDa protein as Moringa coagulant protein (MO2.1) and the 9kDa protein band as seed storage protein napin3. Of thirteen clinical strains analysed, Moringa and Mustard large were proven effective in either aggregation activity or growth kinetic method or both in all thirteen and nine strains respectively. To my knowledge this is the first report on the presence of coagulant protein in Brassica seeds. Owing to the promising results Brassica species could possibly be used as a substitute to Moringa coagulating agent and chemicals in drinking water treatment.
|
|
| 4. |
- Boström, Tove
(författare)
-
High-throughput protein analysis using mass spectrometry-based methods
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers.The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format.
|
|
| 5. |
- Bäcklund, Emma
(författare)
-
Impact of glucose uptake rate on recombinant protein production in Escherichia coli
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures. The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.
|
|
| 6. |
- Cao, Hui, 1969-
(författare)
-
Dynamic Effects on Electron Transport in Molecular Electronic Devices
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- HTML clipboardIn this thesis, dynamic effects on electron transport in molecular electronic devices are presented. Special attention is paid to the dynamics of atomic motions of bridged molecules, thermal motions of surrounding solvents, and many-body electron correlations in molecular junctions. In the framework of single-body Green’s function, the effect of nuclear motions on electron transport in molecular junctions is introduced on the basis of Born-Oppenheimer approximation. Contributions to electron transport from electron-vibration coupling are investigated from the second derivative of current-voltage characteristics, in which each peak is corresponding to a normal mode of the vibration. The inelastic-tunneling spectrum is thus a useful tool in probing the molecular conformations in molecular junctions. By taking account of the many-body interaction between electrons in the scattering region, both time-independent and time-dependent many-body Green’s function formula based on timedependent density functional theory have been developed, in which the concept of state of the system is used to provide insight into the correlation effect on electron transport in molecular devices. An effective approach that combines molecular dynamics simulations and first principles calculations has also been developed to study the statistical behavior of electron transport in electro-chemically gated molecular junctions. The effect of thermal motions of polar water molecules on electron transport at different temperatures has been found to be closely related to the temperature-dependent dynamical hydrogen bond network.
|
|
| 7. |
- Chen, Xing
(författare)
-
Theoretical Studies on Magnetic and Photochemical Properties of Organic Molecules
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The present thesis is concerned with the theoretical studies on magnetic and photochemical properties of organic molecules. The ab initio and first principles theories were employed to investigate the vibrational effects on the isotropic hyperfine coupling constant (HFCC) known as the critical parameter in electron paramagnetic resonance spectrum, the theoretical simulations of the vibronically resolved molecular spectra, the photo-induced reaction mechanism of α-santonin and the spin-forbidden reaction of triplet-state dioxygen with cofactor-free enzyme. The theoretical predictions shed light on the interpretation of experimental observations, the understanding of reaction mechanism, and importantly the guideline and perspective in respect of the popularized applications. We focused on the vibrational corrections to the isotropic HFCCs of hydrogen and carbon atoms in organic radicals. The calculations indicate that the vibrational contributions induce or enhance the effect of spin polarization. A set of rules were stated to guide experimentalist and theoretician in identification of the contributions from the molecular vibrations to HFCCs. And the coupling of spin density with vibrational modes in the backbone is significant and provides the insight into the spin density transfer mechanism in organic π radicals. The spectral characters of the intermediates in solid-state photoarrangement of α-santonin were investigated in order to well understand the underlying experimental spectra. The molecular spectra simulated with Franck-Condon principle show that the positions of the absorption and emission bands of photosantonic acid well match with the experimental observations and the absorption spectrum has a vibrationally resolved character. α-Santonin is the first found organic molecule that has the photoreaction activities. The photorearrangement mechanism is theoretically predicted that the low-lying excited state 1(nπ*) undergoing an intersystem crossing process decays to 3(ππ*) state in the Franck-Condon region. A pathway which is favored in the solid-state reaction requires less space and dynamic advantage on the excited-state potential energy surface (PES). And the other pathway is predominant in the weak polar solvent due to the thermodynamical and dynamical preferences. Lumisantonin is a critical intermediate derived from α-santonin photoreaction. The 3(ππ*) state plays a key role in lumisantonin photolysis. The photolytic pathway is in advantage of dynamics and thermodynamics on the triplet-state PES. In contrast, the other reaction pathway is facile for pyrolysis ascribed to a stable intermediate formed on the ground-state PES. The mechanism of the oxidation reaction involving cofactor-free enzyme and triplet-state dioxygen were studied. The theoretical calculations show that the charge-transfer mechanism is not a sole way to make a spin-forbidden oxidation allowed. It is more likely to take place in the reactant consisting of a non-conjugated substrate. The other mechanism involving the surface hopping between the triplet- and singlet-state PESs via a minimum energy crossing point (MECP) without a significant charge migration. The electronic state of MECP exhibits a mixed characteristic of the singlet and triplet states. The enhanced conjugation of the substrate slows down the spin-flip rate, and this step can in fact control the rate of the reaction that a dioxygen attaches to a substrate.
|
|
| 8. |
|
|
| 9. |
- Chen, Zhihui, 1984-
(författare)
-
Light manipulation in micro and nano photonic materials and structures
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Light manipulation is an important method to enhance the light-matter interactions in micro and nano photonic materials and structures by generating usefulelectric field components and increasing time and pathways of light propagationthrough the micro and nano materials and structures. For example, quantum wellinfrared photodetector (QWIP) cannot absorb normal incident radiation so thatthe generation of an electric field component which is parallel to the original incident direction is a necessity for the function of QWIP. Furthermore, the increaseof time and pathways of light propagation in the light-absorbing quantum wellregion will increase the chance of absorbing the photons.The thesis presents the theoretical studies of light manipulation and light-matter interaction in micro and nano photonic materials and structures, aiming atimproving the performance of optical communication devices, photonic integrateddevices and photovoltaic devices.To design efficient micro and nano photonic devices, it is essential to knowthe time evolution of the electromagnetic (EM) field. Two-dimensional and three-dimensional finite-difference time-domain (FDTD) methods have been adopted inthe thesis to numerically solve the Maxwell equations in micro and nano photonicmaterials and structures.Light manipulation in micro and nano material and structures studied in thisthesis includes: (1) light transport in the photonic crystal (PhC) waveguide, (2)light diffraction by the micro-scale dielectric PhC and metallic PhC structures(gratings); and (3) exciton-polaritons of semiconductor quantum dots, (4) surfaceplasmon polaritons at semiconductor-metallic material interface for subwavelengthlight control. All these aspects are found to be useful in optical devices of multiplebeam splitter, quantum well/dot infrared photodetectors, and solar cells.
|
|
| 10. |
- Eklöf, Jens, 1979-
(författare)
-
Plant and microbial xyloglucanases: Function, Structure and Phylogeny
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, enzymes acting on the primary cell wall hemicellulose xyloglucan are studied. Xyloglucans are ubiquitous in land plants which make them an important polysaccharide to utilise for microbes and a potentially interesting raw material for various industries. The function of xyloglucans in plants is mainly to improve primary cell wall characteristics by coating and tethering cellulose microfibrils together. Some plants also utilise xyloglucans as storage polysaccharides in their seeds. In microbes, a variety of different enzymes for degrading xyloglucans have been found. In this thesis, the structure-function relationship of three different microbial endo-xyloglucanases from glycoside hydrolase families 5, 12 and 44 are probed and reveal details of the natural diversity found in xyloglucanases. Hopefully, a better understanding of how xyloglucanases recognise and degrade their substrate can lead to improved saccharification processes of plant matter, finding uses in for example biofuel production. In plants, xyloglucans are modified in muro by the xyloglucan transglycosylase/hydrolase (XTH) gene products. Interestingly, closely related XTH gene products catalyse either transglycosylation (XET activity) or hydrolysis (XEH activity) with dramatically different effects on xyloglucan and on cell wall characteristics. The strict transglycosylases transfer xyloglucan segments between individual xyloglucan molecules while the hydrolases degrade xyloglucan into oligosaccharides. Here, we describe and determine, a major determinant of transglycosylation versus hydrolysis in XTH gene products by solving and comparing the first 3D structure of an XEH, Tm-NXG1 and a XET, PttXET16-34. The XEH activity was hypothesised, and later confirmed to be restricted to subset of the XTH gene products. The in situ localisation of XEH activity in roots and hypocotyls of Arabidopsis was also visualised for the first time. Furthermore, an evolutionary scheme for how XTH gene products developed from bacterial beta-1,3;1,4 glucanases was also presented based on the characterisation of a novel plant endo-glucanase, PtEG16-1. The EG16s are proposed to predate XTH gene products and are with activity on both xyloglucan and beta-1,3;1,4 glucans an “intermediate” in the evolution from beta-1,3;1,4 glucanases to XTH gene products.
|
|
