Rice straw was successfully converted to ethanol by separate enzymatic hydrolysis and fermentation by Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. The hydrolysis temperature and pH of commercial cellulase and beta-glucosidase enzymes were first investigated and their best performance obtained at 45 degrees C and pH 5.0. The pretreatment of the straw with dilute-acid hydrolysis resulted in 0.72 g g (1) sugar yield during 48 h enzymatic hydrolysis, which was higher than steam-pretreated (0.60 g g (1)) and untreated straw (0.46 g g(-1)). Furthermore, increasing the concentration of the dilute-acid pretreated straw from 20 to 50 and 100 g L-1 resulted in 13% and 16% lower sugar yield, respectively. Anaerobic cultivation of the hydrolyzates with M. indicus resulted in 0.36-0.43 g g(-1) ethanol, 0.11-0.17 g g(-1) biomass, and 0.04-0.06 g g(-1) glycerol, which is comparable with the corresponding yields by S. cerevisiae (0.37-0.45 g g(-1) ethanol, 0.04-0.10 g g(-1) biomass and 0.05-0.07 glycerol). These two fungi produced no other major metabolite from the straw and completed the cultivation in less than 25 h. However, R. oryzae produced lactic acid as the major by-product with yield of 0.05-0.09 g g(-1). This fungus had ethanol, biomass and glycerol yields of 0.33-0.41, 0.06-0.12, and 0.03-0.04 g g(-1), respectively. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

In this work two different examples of water absorbtion in polymers are studied by Monte Carlo simulations. Both of them are of large technical and commercial impotance. The first example is the water absorption in polyethylene cables where the water absorption plays a crucial role in the degradation of the cable insulation and thus should be as low as possible. The second example is bio-based superabsorbents made from denatured protein where water absorption capability is the prime desired property. Methods Gibbs Ensemble Monte Carlo simulations [1] were used to study the hydration of polymers. All simulations are performed with two boxes, one of which is filled with water at the start of the simulation, whereas the other contains polymer molecules and possible ions. The polymer molecules are not allowed to swap boxes whereas the water molecules are allowed to do so thus constituting an osmotic Gibbs ensemble [2]. For the polyethylene a connectivity-altering algorithm was used whereas the protein molecules were simulated using a side-chain regrowth model in addition to traditional Monte Carlo moves. For the polyethylene, the TraPPE [3] force field was used and the protein molecules, the Amber force field [4] was used. Water was modelled using simple point charge models [5]. Electrostatic interactions are treated using Ewald summation methods. The protein molecules were of different amino acid compositions and in different conformations, e.g., β-turns and random coils obtained using the amorphous cell method[6]. Studies were made with different degrees of charging on, e.g., lysine side chains mimicking different ionization states. Results The studies of polyethylene revealed the importance of ions left from the polymerisation catalyst for the absorbtion of water and the concomitant degradation of polyethylene cable insulation. Also the absorption properties of the protein molecules is strongly related to the presence of charged groups and fully charged protein molecules absorb large amounts of water. However, neither native nor denatured protein molecules show superabsorbing properties (i.e. absorbing hundreds of times their own mass) as they show in experimental studies and the reasons for this discrepancy will be discussed. References 1. A.Z. Panagiotopoulos, Mol. Phys. 61, 813 (1987). 2. E. Johansson, K. Bolton, D.N. Theodorou, P. Ahlström, J. Chem. Phys., 126, 224902 (2007). 3. M.G. Martin, and J.I. Siepmann, J. Phys. Chem. B, 103, 4508-4517 (1999). 4. W.D. Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K.M. Merz Jr, D.M. Ferguson, D.C. Spellmeyer, T. Fox, J.W. Caldwell, P.A. Kollman (1995). J. Am. Chem. Soc. 117, 5179–5197. 5. H. J. C. Berendsen, J. P. M. Postma and W. F. van Gunsteren, in Intermolecular Forces, B. Pullman, ed. (Reidel, Dordrecht, 1981) p. 331; H. J. C. Berendsen, J. R. Grigera and T. P. Straatsma, J. Phys. Chem. 91, 6269 (1987). 6. D.N. Theodorou, U.W. Suter, Macromolecules, 18, 1467 (1985).

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, Z_basic 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 Z_basic is explored and evaluated. Paper I describes the successful investigation of Z_basic as a purification handle under denaturating conditions. Moreover, Z_basic 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 Z_basic 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.

Stem cell based therapy has the potential to treat several severe diseases; Parkinson’s

disease is one well- known example. Transplantation of stem cell derived cells into

animal models is unfortunately often associated with tumour formation or- uncontrolled

growth of the transplanted cells. One strategy to suppress this tumour formation might be

to induce differentiation of these cells, which in turn would prevent them from dividing.

Neuroblastoma tumors are known to demonstrate the complete transition from an

undifferentiated state to a completely harmful, differentiated appearance and derived cells

can be used as a model for cell differentiation and tumor suppression.

In this Master Thesis’s the conducting polymers PEDOT and PPy, that upon formation

can be doped with biologically active compounds which in- turn can be released in a

controlled manner through electrical stimulation, were formed together with various

drugs (e.g. Methotrexate and Mycophenolic Acid), here shown to have effect on

Neuroblastoma cells. Neuroblastoma- derived cell line SH- SY5Y was used as a model

system for neuronal differentiation and tumour inhibition. Release profiles of

neuroblastoma active drugs following electrical stimulation were evaluated and the

effects from electrochemical processes on simultaneously growing SH- SY5Y cells were

investigated.

The methods to deposit and release the drugs were based on electropolymerization and

electrochemically controlled release, respectively. Controlled release of various drugs

and compounds was monitored using Vis- and UV- spectroscopy and on some occasions

using HPLC.

The electrochemically controlled release of a biologically inactive compound that can be

used as a negative control for electrochemical release in future experiments was shown

and that resulting electrochemical processes have negative effects on neuroblastoma cell

growth.

A fundamental process of all living organisms - the transport of molecules across cellular membranes through membrane transport proteins - is investigated.

After a brief review of general properties of biological membranes follows a recollection of the major methods of membrane transport that Nature utilizes (Chapter 1). This is followed by a description of important experimental (Chapter 2) and theoretical methods (Chapter 3) for structural studies of membrane proteins. The findings on membrane protein transport in papers I-IV are then summarized (Chapter 4) and important findings are discussed. The remaining text is a discussion on relevant theoretical and experimental methods.

In orthopaedic practice, fractures are usually stabilised with metal screws or rods. This is done in order to keep the fracture parts in place during the rather slow healing process. The healing time can potentially be reduced by local- or systemic treatment with different bone promoting drugs. In later years, lithium, otherwise used to treat bipolar disease, has shown promise to be such a drug.

The aim of this master thesis was to find a way to coat metal bone screws with lithium and to characterise the coating. The coating was to be designed in such a way that it could release lithium to the surrounding bone tissue.

Lithium chloride was incorporated into a titanate sol-gel and attached to silicon wafers and stainless steel screws by dip coating. Wafers were used for initial in vitro studies of how lithium changed coating characteristics. This was studied using ellipsometry, AFM and SEM. Lithium is most probably physisorbed and not incorporated into the network building up the sol-gel. Coating structure is changed as more lithium is incorporated. For large amounts of lithium, the nanoparticles normally formed when curing the sol-gel are inhibited. One effect of this is reduced bioactivity, seen as a reduced ability for calcium phosphate crystals to nucleate on the coating when immersed in simulated body fluid.

Lithium release was investigated using AAS. Lithium is released from the coating, showing a burst effect. By changing the number of coating layers used, the release profile can be partly altered. The coating was also applied to screws, showing good attachment, and the lithium release profile was similar to the one seen from wafers.

Finally, a screw model was used in rats to assess the effect of local lithium treatment from screws and systemic lithium treatment on fracture healing. In the model, a screw was inserted in tibia, mimicking a fracture. When the bone around the screw was healed, a pullout test was performed, giving information about the strength of the bone surrounding the screw. No significant difference could be found for either local- or systemic lithium treatment compared to control. However, when evaluating the strength of intact bone in a similar way, a positive effect of systemic lithium treatment could be seen. Therefore, it is still likely that lithium has a positive effect on bone and further studies are needed to fully evaluate its role in fracture healing.

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.

Biomolecular interaction analysis to determine the kinetics and affinity between interacting partners is important for the fundamental understanding of biology, as well as for the development of new pharmaceutical substances. A quartz crystal microbalance instrument suitable for kinetics and affinity analyses of interaction events was developed. The functionality of the sensor system was demonstrated by development of an assay for relative affinity determination of lectin-carbohydrate interactions.

Sensor surfaces allowing for effective immobilization of one interacting partner is a key functionality of a biosensor. Here, three different surfaces and immobilization methods were studied. First, optimized preparation conditions for sensor surfaces based on carboxyl-terminated self assembled monolayers were developed and were demonstrated to provide highly functional biosensor surfaces with low non-specific binding. Second, a method allowing for immobilization of very acidic biomolecules based on the use of an electric field was developed and evaluated. The electric field made it possible to immobilize the highly acidic C-peptide on a carboxylated surface. Third, a method for antibody immobilization on a carboxyl surface was optimized and the influence of immobilization pH on the immobilization level and antigen binding capacity was thoroughly assessed. The method showed high reproducibility for a set of antibodies and allowed for antibody immobilization also at low pH.

Three broadly different strategies to increase the sensitivity of electroacoustic sensors were explored. A QCM sensor with small resonator electrodes and reduced flow cell dimensions was demonstrated to improve the mass transport rate to the sensor surface. The use of polymers on QCM sensor surfaces to enhance the sensor response was shown to increase the response of an antibody-antigen model system more than ten-fold. Moreover, the application of high frequency thin film bulk acoustic resonators for biosensing was evaluated with respect to sensing range from the surface. The linear detection range of the thin film resonator was determined to be more than sufficient for biosensor applications involving, for instance, antibody-antigen interactions. Finally, a setup for combined frequency and resistance measurements was developed and was found to provide time resolved data suitable for kinetics determination.

The resonance frequency of acoustic biosensors is today used as a label-free technique for detecting mass changes on sensor surfaces. In combination with an appropriate continuous flow system it has earlier been used for affinity and kinetic rate determination. Here, we assess the potential of a modified acoustic biosensor, monitoring also the real-time dissipation through the resistance of the sensor, to obtain additional kinetic information related to the structure and conformation of the molecules on the surface. Actual interaction studies, including an attempt to determine avidity, are presented along with thorough verification of the experimental setup utilizing true viscous load exposure together with protein and DNA immobilizations. True viscous loads show a linear relationship between resistance and frequency as expected. However, in the interaction studies between antibodies and proteins, as well as in the immobilization of DNA and proteins, higher surface concentrations of interacting molecules led to a decrease (i.e. deviation from the linear trend) in the differential resistance to frequency ratio. This is interpreted as increased surface rigidity at higher surface concentrations of immobilized molecules. Consequently, studies that aim at obtaining biological binding information, such as avidity, from real-time resistance and dissipation data should be conducted at low surface concentrations. In addition, the differential resistance to frequency relationship was found to be highly dependent on the rigidity of the preceding layer(s) of immobilized molecules. This dependence can be utilized to obtain a higher signal-to-noise ratio for resistance measurement by using low surface densities of immobilized interaction partners.

SammanfattningProjektgrupp 14-X5 avser med denna rapport att ge avdelningen Bioreagens på Thermo Fisher Scientific ett underlag för att på sikt kunna byta ut fetalt kalvserum (FCS) mot ett kemiskt definierat suplement vid odling av mushybridomceller för produktion av monoklonala antikroppar. Thermo Fisher Scientific är ett världsomspännande bioteknikföretag som utvecklar blodtestsystem som stöd för klinisk diagnos och uppföljning av allergier, astma och autoimmuna sjukdomar.

Fetalt kalvserum är en tillsats i många odlingsmedier som ofta är nödvändig för att cellerna ska växa. Det finns dock många problem med FCS. Det är en biprodukt av köttindustrin och produceras på ett etiskt tveksamt sätt, variation mellan olika batcher förekommer och då det är en animalisk produkt finns en risk för kontamination av bland annat bakterier, virus och prioner. Av dessa anledningar vill man byta ut FCS mot ett kemiskt definierat, serumfritt supplement. Vi har utrett vilka ämnen eller grupper av ämnen som har störst potential att vara bra substitut för FCS, samt rangordnat dessa. Genom våra artikelstudier har vi kommit fram till att man kan dela in alternativen i tre grupper: lipider, tillväxtfaktorer och små biomolekyler. Bland lipiderna är det linol- och oljesyra som i flera artiklar har visats ha god effekt på både celltillväxt och antikroppsproduktion. Kolesterol har även visats ha positiva effekter. Tillväxtfaktorerna som har valts är epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukin-2 (IL-2) och interleukin-6 (IL-6). Dessa har främst en positiv effekt på cellernas antikroppsproduktion. Bland de små biomolekyler som har valts ut finns en mängd olika ämnen som på olika sätt kan bidra till att skapa en bra miljö för hybridomceller i ett serumfritt medium.

För att få en klarare bild av de olika förslagen har de jämförts. Vi menar att de ämnen som är komponenter i det basala mediet Ham F-12 bör prioriteras då det används som standard vid odling av en bred grupp av celltyper. Denna grupp inkluderar linolsyra, putrescin, tymidin, hypoxantin samt liponsyra. Därefter anser vi att återstående lipider (oljesyra och kolesterol) ska prioriteras då de har visats kunna öka både hybridomcellers tillväxt och antikroppsproduktion i ett serumfritt medium. Tillväxtfaktorer, som främst ökar antikroppsproduktionen hos cellerna  och i vissa fall även deras livslängd placerar vi på tredje plats i vår rangordning. Övriga ämnen i gruppen “Små biomolekyler” (paraaminobensoesyra och glutation) prioriterar vi sist då de inte är komponenter i Ham F-12 och de inte explicit visats påverka celltillväxten ellerantikroppsproduktionen, men har identifierats i det serumfria mediet IBL media III.

Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and sugar residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D) while phylogenetic analysis has resulted in classification into thirteen subfamilies (SF1-13). Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters (donor) with alcohols or sugars (acceptor) resulting in compounds with modified lipophilicity, having a great potential for use in the tailor-made modification of natural antioxidants for cosmetic, cosmeceutical and pharmaceutical industries. The work described in this thesis focused on the selection,characterization and application of FAEs for the synthesis of bioactive esters with antioxidant activity in non-conventional media. The basis of the current classification systems was investigated in relation with the enzymes’ synthetic and hydrolytic abilities while the developed processes were evaluated for their efficiency and sustainability.

Paper I was dedicated to the screening and evaluation of the synthetic abilities of 28 fungal FAEs using acceptors of different lipophilicity at fixed conditions in detergentless microemulsions. It was revealed that FAEs classified in phylogenetic subfamilies related to acetyl xylan esterases (SF5 and 6) showed increased transesterification rates and selectivity. In general, FAEs showed preference on more hydrophilic alcohol acceptors and in descending order to glycerol > 1-butanol > prenol. Homology modeling and small molecule docking simulations were employed as tools for the identification of a potential relationship between the predicted surface and active site properties of selected FAEs and the transesterification selectivity.

Papers II- IV focused on the characterization of eight promising FAEs and the optimization of reaction conditions for the synthesis of two bioactive esters (prenyl ferulate and L-arabinose ferulate) in detergentless microemulsions. The effect of the medium composition, the donor and acceptor concentration, the enzyme load, the pH, the temperature and the agitation on the transesterification yield and selectivity were investigated. It was observed that the acceptor concentration and enzyme load were crucial parameters for selectivity. Fae125 (Type A, SF5) iiexhibited highest prenyl ferulate yield (81.1%) and selectivity (4.685) converting 98.5% of VFA to products after optimization at 60 mM VFA, 1.5 M prenol, 0.04 mg FAE mL-1, 40oC, 24 h, 53.4:43.4:3.2 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. On the other hand, FaeA1 (Type A, SF5) showed highest L-arabinose ferulate yield (52.2 %) and selectivity (1.120) at 80 mM VFA, 55 mM L-arabinose, 0.02 mg FAE mL-1, 50oC, 8 h, 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0.

In paper V, the effect of reaction media on the enzyme stability and transesterification yield and selectivity was studied in different solvents for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate. The best performing enzyme (Fae125) was used in the optimization of reaction conditions in the best solvent (n-hexane) via response surface methodology. Both bioconversions were best described by a two-factor interaction model while optimal conditions were determined as the ones resulting in highest yield and selectivity.Highest prenyl ferulate yield (87.5%) and selectivity (7.616) were observed at 18.56 mM prenol mM-1VFA, 0.04 mg FAE mL-1, 24.5 oC, 24.5 h, 91.8: 8.2 v/v n-hexane: 100 mM sodium acetate pH 4.7. Highest L-arabinose ferulate yield (56.2%) and selectivity (1.284) were observed at 2.96 mM L-arabinose mM-1VFA, 0.02 mg FAE mL-1, 38.9 oC, 12 h, 90.5: 5.0: 4.5 v/v/v n-hexane: dimethyl sulfoxide: 100 mM sodium acetate pH 4.7. The enzyme could be reused for six consecutive reaction cycles maintaining 66.6% of its initial synthetic activity. The developed bioconversions showed exceptional biocatalyst productivities (> 300 g product g-1FAE) and the waste production was within the range of pharmaceutical processes.

Paper VI focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger(AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamic acid esters. For this purpose, L-arabinose ferulateand caffeate were synthesized enzymatically. kcat/Kmratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification describing more adequately the enzyme specificitiesin the natural environment.

With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides. Combined together, these two versions of antigen arrays offer a versatile approach for multiplexed characterization of antibody binding selectivity, off-target interactions, as well as mapping for the amino acids of epitopes involved in antibody binding.

A new material model is proposed for the description of stress-softening observed in cyclic tension tests performed on soft biological tissues. The modeling framework is based on the concept of internal variables introducing a scalar-valued variable for the representation of fiber damage. Remanent strains in fiber direction can be represented as a result of microscopic damage of the fiber crosslinks. Particular internal variables are defined able to capture the nature of soft biological tissues that no damage occurs in the physiological loading domain. A specific model is adjusted to experimental data taking into account the supra-physiological loading regime. For the description of the physiological domain polyconvex functions are used which also take into account fiber dispersion in a phenomenological approach. The applicability of the model in numerical simulations is shown by a representative example where the damage distribution in an arterial cross-section is analyzed.

The extent of devastation caused by a biological warfare attack is highly correlated to the time from release to detection. As a step towards lowering the detection time the international project TWOBIAS was launched. Here, the main goal is to develop an automated, specific and sensitive combined detection and identification instrument capable of identifying a biological threat within an hour. The identification unit is comprised of a sample preparation module, an amplification module and a detection module and utilizes a proximity ligation assay in combination with circle-to-circle amplification in order to detect a biological threat. This thesis describes the automation of the sample preparation steps of the assay and the integration with the downstream units. The functionality of the sample preparation module was verified by subjecting it to biological samples in a laboratory and at a real-life location. The results showed that the sample preparation module was capable of preparing a sample collected in a complex environment with the same results as a sample prepared in a laboratory. 

A superabsorbent polymer (SAP) from chitosan was provided via carboxymethylation of chitosan, followed by cross-linking with glutaraldehyde and freeze-drying. This work was focused on an investigation of the effects of monochloroacetic acid (MCAA), sodium hydroxide, and reaction time on preparation of carboxymethylchitosan (CMCS). The CMCS products were characterized using FTIR spectroscopy, and their degrees of substitution (DS) were measured using conductimetry and FTIR analysis. The highest DS value was obtained when the carboxymethylation reaction was carried out using 1.75 g MCAA and 1.75 g NaOH per g of chitosan in 4 h. The water solubilities of the CMCS products at various pHs were also evaluated, and the results indicated a significant impact of the reaction parameters on the solubility of CMCS. The CMCSs with the highest DS value resulted in SAPs having the highest water-binding capacity (WBC). TheWBCof the best SAP measured after 10 minexposure in distilled water, 0.9% NaCl solution, synthetic urine, and artificial blood was 104, 33, 30, and 57 g/g, respectively. The WBC of this SAP at pH 2–9 passed a maximum at pH 6.

Using a human-capital perspective and the similarity-attraction paradigm, we examine the role of general and specific human capital in the decision policies of 114 Swedish loan officers in their assessments of small-business loan requests. We found that human capital characteristics had marginal impact on decision policy contingencies and that specific human capital had no significant influence on the probability of loan approval. However, we did find that the similarity between the loan officers’ human capital and the pplicants’ human capital was a significant indicator of loan approval. The findings offer interesting insight into the heterogeneity of loan decision processes and outcomes and future research opportunities are suggested.

Using a human-capital perspective and the similarity-attraction paradigm, we examine the role of general and specific human capital in the decision policies of 114 Swedish loan officers in their assessments of small-business loan requests. We found that human capital characteristics had marginal impact on decision policy contingencies and that specific human capital had no significant influence on the probability of loan approval. However, we did find that the similarity between the loan officers’ human capital and the pplicants’ human capital was a significant indicator of loan approval. The findings offer interesting insight into the heterogeneity of loan decision processes and outcomes and future research opportunities are suggested.

Functional surface display of small affinity proteins, namely, affibodies (6.5 kDa), was evaluated for the model cyanobacterium Synechocystis sp. strain PCC 6803 through anchoring to native surface structures. These structures included confirmed or putative subunits of the type IV pili, the S-layer protein, and the heterologous Escherichia coli autotransporter antigen 43 system. The most stable display system was determined to be through C-terminal fusion to PilA1, the major type IV pilus subunit in Synechocystis, in a strain unable to retract these pili (Delta pilT1). Type IV pilus synthesis was upheld, albeit reduced, when fusion proteins were incorporated. However, pilus-mediated functions, such as motility and transformational competency, were negatively affected. Display of affibodies on Synechocystis and the complementary anti-idiotypic affibodies on E. coli or Staphylococcus carnosus was able to mediate interspecies cell-cell binding by affibody complex formation. The same strategy, however, was not able to drive cell-cell binding and aggregation of Synechocystis-only mixtures. Successful affibody tagging of the putative minor pilin PilA4 showed that it locates to the type IV pili in Synechocystis and that its extracellular availability depends on PilA1. In addition, affibody tagging of the S-layer protein indicated that the domains responsible for the anchoring and secretion of this protein are located at the N and C termini, respectively. This study can serve as a basis for future surface display of proteins on Synechocystis for biotechnological applications. IMPORTANCE Cyanobacteria are gaining interest for their potential as autotrophic cell factories. Development of efficient surface display strategies could improve their suitability for large-scale applications by providing options for designed microbial consortia, cell immobilization, and biomass harvesting. Here, surface display of small affinity proteins was realized by fusing them to the major subunit of the native type IV pili in Synechocystis sp. strain PCC 6803. The display of complementary affinity proteins allowed specific cell-cell binding between Synechocystis and Escherichia coli or Staphylococcus carnosus. Additionally, successful tagging of the putative pilin PilA4 helped determine its localization to the type IV pili. Analogous tagging of the S-layer protein shed light on the regions involved in its secretion and surface anchoring.

We are glad to announce the Special Issue “Nanostructured Solar Cells”, published in Nanomaterials. This issue consists of eight articles, two communications, and one review paper, covering major important aspects of nanostructured solar cells of varying types. From fundamental physicochemical investigations to technological advances, and from single junction solar cells (silicon solar cell, dye sensitized solar cell, quantum dots sensitized solar cell, and small molecule organic solar cell) to tandem multi-junction solar cells, all aspects are included and discussed in this issue to advance the use of nanotechnology to improve the performance of solar cells with reduced fabrication costs.

Utilization of bacterial nanocellulose (BNC) for large-scale applications is restricted by low productivity in static cultures and by the high cost of the medium. Fiber sludge, a waste stream from pulp and paper mills, was enzymatically hydrolyzed to sugar, which was used for the production of BNC by the submerged cultivation of Komagataeibacter xylinus. Compared with a synthetic glucose-based medium, the productivity of purified BNC from the fiber sludge hydrolysate using shake-flasks was enhanced from 0.11 to 0.17 g/(L x d), although the average viscometric degree of polymerization (DPv) decreased from 6760 to 6050. The cultivation conditions used in stirred-tank reactors (STRs), including the stirring speed, the airflow, and the pH, were also investigated. Using STRs, the BNC productivity in fiber-sludge medium was increased to 0.32 g/(L x d) and the DPv was increased to 6650. BNC produced from the fiber sludge hydrolysate was used as an additive in papermaking based on the chemithermomechanical pulp (CTMP) of birch. The introduction of BNC resulted in a significant enhancement of the mechanical strength of the paper sheets. With 10% (w/w) BNC in the CTMP/BNC mixture, the tear resistance was enhanced by 140%. SEM images showed that the BNC cross-linked and covered the surface of the CTMP fibers, resulting in enhanced mechanical strength.

Prostate cancer is one leading cause of cancer-related death among men in Western countries. The standard therapies for localized prostate cancer include radical prostatectomy and radiation therapy. Such measures are relatively effective in the short term, but many patients ultimately relapse. These patients may benefit from a combination of standard therapy and oncolytic virus therapy. My work aimed to develop viruses for this purpose.

TARP is a protein that in males is specifically expressed in prostate epithelial and cancer cells. In my thesis, I characterized the TARP promoter and showed that TARP expression is regulated at the transcriptional level by testosterone through binding of the androgen receptor in the proximal TARP promoter. I further developed TARP promoter-based regulatory sequences for prostate-specific gene expression. A sequence comprising a PSA enhancer, a PSMA enhancer and the TARP promoter was constructed and designated PPT. An adenoviral vector containing the PPT sequence shielded from transcriptional interference by an H19 insulator showed high prostate-specific transcriptional activity in human cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer the PPT sequence is not active. Therefore, a two-step transcriptional amplification (TSTA) system was used together with the PPT sequence to develop an adenovirus that confers prostate-specific transgene expression also in murine cells.

I constructed a conditionally replicating adenovirus where the E1A gene expression is controlled by an H19 insulator-shielded PPT regulatory sequence, Ad[I/PPT-E1A]. This virus exhibited absolute prostate specificity in terms of E1A expression, viral replication and cytolysis in vitro and in vivo. Importantly, our virus is active both in the presence and absence of testosterone, which may prove beneficial for patients treated by hormonal withdrawal.

Hopefully, my work will improve existing gene therapy strategies for prostate cancer and in the long term improve the prognosis for patients with prostate cancer.

A majority of methods for identifying sequences in the human genome involve target sequence amplification through PCR. This work presents novel methods for amplifying circularized DNA and presents solutions for some major limitations of PCR.

We have developed a novel method to amplify circularized DNA molecules based on a serial rolling-circle replication reaction, called circle to circle amplification (C2CA). Amplified DNA circles can be detected in array-based analyses or in real-time using molecular beacons. The amplification mechanism allows higher precision in quantification than in exponential amplification methods like PCR, and more products can be generated than in PCR.

A major limitation of PCR is that amplification artifacts arise when large numbers of specific primer pairs are simultaneously added to a reaction. We have developed a solution to this problem that enables multiplex PCR amplification of specific target sequences without producing amplification artifacts. The procedure is based on oligonucleotide constructs, called selectors. The selectors identify defined target nucleic acid sequences, and they act as ligation templates to direct circularization of these targets. The selectors contain a general primer-pair motif that allows the circularized targets to be amplified in multiplex using a universal PCR primer pair. We also developed a computer program, PieceMaker, that finds an optimal design of selector probes for a given selector application. We demonstrate the method by performing a 96-plex PCR of specific DNA sequences with high success-rate and reproducibility.

Comparison of vacuum and high pressure evaporated wood hydrolyzate for ethanol production by repeated fed-batch using flocculating Saccharomyces cerevisiae

We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA-1) affibody and it's Z domain binding partner as a model. Free Z(SPA-1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA-1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA-1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA-1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA-1) complex is associated with a large change in conformational entropy that opposes binding.

The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA).

In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity.

A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation.

A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization.

Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K-d = 0.1 mu M. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T-M similar to 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with Delta H degrees(bind) = -9.0(+/- 0.1) kcal mol(-1). The heat capacity change Delta C-P degrees,(bind) = -0.43(+/- 0.01) kcal mol(-1) K-1 is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-T Delta S degrees(bind) = -0.6(+/- 0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.

Degradation characteristics in response to electron beam sterilization of designed and biodegradable aliphatic polyester scaffolds are relevant for clinically successful synthetic graft tissue regeneration Scaffold degradation in vitro and in vivo were documented and correlated to the macroscopic structure and chemical design of the original polymer The materials tested were of inherently diverse hydrophobicity and crystallinity poly(L-lactide) (poly(LLA)) and random copolymers from L-lactide and epsilon-caprolactone or 1.5-dioxepan-2-one, fabricated into porous and non-porous scaffolds After sterilization, the samples underwent hydrolysis in vitro for up to a year In vivo, scaffolds were surgically implanted into rat calvarial defects and retrieved for analysis after 28 and 91 days In vitro, poly(L-lactide-co-1, 5-dioxepan-2-one) (poly(LLA-co-DXO)) samples degraded most rapidly during hydrolysis, due to the pronounced chain-shortening reaction caused by the sterilization. This was indicated by the rapid decrease in both mass and molecular weight of poly(LLA-co-DXO). Poly(L-lactide-co-epsilon-caprolactone) (poly(LLA-co-CL)) samples were also strongly affected by sterilization, but mass loss was more gradual; molecular weight decreased rapidly during hydrolysis Least affected by sterilization were the poly(LLA) samples, which subsequently showed low mass loss rate and molecular weight decrease during hydrolysis. Mechanical stability varied greatly. poly(LLA-co-CL) withstood mechanical testing for up to 182 days, while poly(LLA) and poly(LLA-co-DXO) samples quickly became too brittle Poly(LLA-co-DXO) samples unexpectedly degraded more rapidly in vitro than in vivo. After sterilization by electron beam irradiation, the three biodegradable polymers present widely diverse degradation profiles, both in vitro and in vivo. Each exhibits the potential to be tailored to meet diverse clinical tissue engineering requirements

Vaginitis is a vaginal infection which affects many women all over the world. The disorder is characterized by an infection of the vaginal area which can cause problems like abnormal vaginal discharge, itching and redness.

The two most common causes of vaginitis are bacterial vaginosis and Candida vaginitis. The prevalence of bacterial vaginosis in Sweden is around 10-20 % and approximately 75 % of all women will once in their lifetime suffer from vaginal yeast infection.

The clinical symptoms of vaginal infections are not specific and the diagnosis methods of bacterial vaginosis and Candida vaginitis are subjective and depended on the acuity of the clinician. Due to the lack of standardized and objective diagnostic tools, misdiagnosis and consequently incorrect treatment may occur.

As vaginal infections and symptoms impact greatly of women´s quality of life and vaginitis have been associated with serious public health consequences, it is essential to diagnose and treat the conditions correctly. Hence, there is a great need of better methods of diagnosing these conditions.

The aim of this master thesis was to develop quantitative species-specific real-time PCR assays to use in diagnosing the two most common causes of vaginitis i.e. bacterial vaginosis and Candida vaginitis.

Potential markers for bacterial vaginosis (Atopobium vaginae, BVAB2, Gardnerella vaginalis, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Megasphaera type 1, Megasphaera type 2, Mobiluncus curtisii, Mobiluncus mulieris and Leptotrichia/Sneathia species) and Candida vaginitis (Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis) were chosen. Primers and probes were designed and tested on reference strains and vaginal samples. Single- and multiplex PCR reactions were successfully optimized with the designed oligonucleotides. Furthermore, standard curves with excellent linearity were created and covered more than five orders of magnitude. These developed quantitative species-specific real-time PCR assays will, in a prospective medical validation, quantify 300 vaginal samples from women visiting the RFSU Clinic in Stockholm.

Hydrogels are used for various applications, for example as transporters in drug delivery, in control lenses, and as superabsorbent material in diapers.[1] Most synthetic produced hydrogels are based on synthetic polymers. Even though they are efficient and cheap, they are not biodegradable and sometimes even toxic. To produce more environmental friendly and biodegradable superabsorbent polymers (Bio- SAPs), other building blocks can be used, such as polysaccharides[2] and various protein structures, for example fish shells[3], collagen[4], soy protein[5] and egg protein[6]. Experimental studies at the University of Boras show that it is possible to produce Bio-SAPs from by-products of ethanol production from ligno-cellulose.[2, 6, 7] 2. Method We have studied the absorption properties of protein structures in silico as a comparison to experimental studies. The NPT Gibbs Ensemble Monte Carlo (GEMC) simulation scheme with two phases is used in order to calculate the absorption capacity of the protein. Pure water was simulated in the first GEMC-phase and the peptide in the second phase. The simulations were made with SPC/E water model [8] and the AMBER99 atomistic force field for the peptides [9]. Furthermore, mesoscopic studies with coarse grained force fields have been done. To facilitate faster computations, we used cell lists for the atom-atom interactions, configurational bias algorithm to build the water molecules and the peptide side-chains, and the cavity bias algorithm [10] for molecule insertions. Model peptides have been studied with varying secondary structure, temperature and protonation (pH). We also plan to study how cross-links affect the absorption. One of the peptides we study is a 20 amino acid long peptide called SSP1.[11] This peptide is designed to form a fibrous structure a hydrogel, and its structure is well defined. We have also studied a peptide which changes secondary structure when changing the pH, and concentration.[12] This makes it possible to compare absorption properties with respect to the secondary structure. 3. Conclusion We have simulated peptides with the Gibbs Ensemble Monte Carlo scheme in order to study the water absorption rate dependent of structure, charge, pH and temperature. This information is useful when developing new biodegradable superabsorbent materials.

With the availability of many completely sequenced genomes, scientific research has shifted from genes to the products of the genes, the proteins. Structural genomics groups have been established worldwide, with the objective of determining protein structures on a genome-wide scale. New methods for protein production and structural determination have become necessary.

Two methods for high throughput analysis of proteins are presented in the first part of this thesis. The first method is the thermofluor method, which presents a fast way to identify stabilizing conditions for a particular protein. It was shown that the addition of a stabilizing additive, identified with the thermofluor method, significantly increased the likelihood of growing protein crystals. The second method presented in this thesis provides a fast and robust way to detect metal containing proteins.

The second part of this thesis describes the crystal structures of two RNA modifying enzymes, the pseudouridine synthase TruD and the RNA m5C methyltransferase YebU. The catalytic domain of TruD was shown to bear remarkable structural similarity to the other pseudouridine families despite a lack of sequence similarity. In addition to the catalytic domain, the structure of TruD also contained an insertion domain with a novel fold.

YebU was also found have two structurally distinct domains. The N-terminal catalytic domain has a high structural similarity to other RNA m5C methyltransferases. The C-terminal domain was revealed to be a so-called PUA domain, which had not been predicted by previous sequence alignments.

Human cancer cell lines grown in vitro are frequently used to decipher basic cell biological phenomena but also to specifically study different forms of cancer. Here we present the first large-scale study of protein expression patterns in cell lines using an antibody-based proteomics approach. We analyzed the expression pattern of 5436 proteins in 45 different cell lines using hierarchical clustering, principal component analysis and two-group comparisons for the identification of differentially expressed proteins. The results show that protein profiles of cell lines, as determined using immunohistochemistry, allow for a hierarchical clustering that overall reflects tumor tissues of origin. Hematological cell lines appear to retain their protein profiles to a higher degree than cell lines established from solid tumors, resulting in a clustering that well reflects progenitor cell types. The discrepancy may reflect different levels of in vitro induced alterations in adherent and suspension grown cell lines, respectively. In addition, multiple myeloma cells and cells of myeloid origin were found to share a protein profile, relative the protein profile of lymphoid leukemia and lymphoma cells, possibly reflecting their common dependency of bone marrow microenvironment.

Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus Phytophthora are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus Saprolegnia, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen Phytophthora infestans and the fish pathogen Saprolegnia monoica.

A new Oomycete CesA gene family was identified. It contains four homologues designated as CesA1, CesA2, CesA3 and CesA4. The gene products of CesA1, 2 and 4 contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete CesA genes. CesA3 is the dominantly expressed CesA homologue in the mycelium of both S. monoica and P. infestans, while CesA1 and CesA2 are up-regulated in virulent life stages of P. infestans. CesA4 was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole CesA gene family in P. infestans lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on SmCesA gene expression, which was accompanied by an increased b-glucan synthase activity in vitro. In addition, these inhibitors slowed down the growth of the mycelium from S. monoica. Zoospores from P. infestans treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the CesA gene family.

Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by P. infestans. 

A method for extraction of therapeutic proteins from dried blood spots (DBS) followed by quantification on Gyrolab(TM) has been developed. The method makes it possible to measure the concentration of the analyte in the range 100-6000 ng/mL. The procedure can generate full analytical information from 15 μL blood originally sampled from a subject. The modest sample requirements allows for sampling a full pre-clinical pharmacokinetic profile from a single mouse. This may allow for reduced usage of animals during preclinical development of new therapeutic proteins in accordance with the 3R’s, replace, refine and reduce.

Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway.

In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (Z_MAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted Z_RAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the Z_RAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of Z_RAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target.

Development of molecules with the ability to selectively inhibit particular protein-protein interactions is important in providing tools for understanding cell biology In this work, we describe efforts to select small Ras- and Raf-specific three-helix bundle affibody binding proteins capable of inhibiting the interaction between H-Ras and Raf-1, from a combinatorial library displayed on bacteriophage Target-specific variants with typically high nanomolar or low micromolar affinities (K-D) could be selected successfully against both proteins, as shown by dot blot, ELISA and real-time biospecific interaction analyses Affibody molecule variants selected against H-Ras were shown to bind epitopes overlapping each other at a site that differed from that at which H-Ras interacts with Raf-1 In contrast, an affibody molecule isolated during selection against Raf-1 was shown to effectively inhibit the interaction between H-Ras and Raf-1 in a dose-dependent manner Possible intracellular applications of the selected affibody molecules are discussed

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.

Small populations are facing a particular risk of extinction due to a lack of appropriate genetic diversity and associated negative effects, factors dealt with in the discipline of conservation genetics. Many orchid species exhibit characteristics that make them a perfect study object in the scope of conservation genetics. The aim with this thesis was to investigate genetic structure at different levels in two orchid species Gymnadenia conopsea, geographically widespread, although diminishing and G. odoratissima with a long history of being rare. Microsatellite markers, developed in and used in studies of G. conopsea were also used in the study of G. odoratissima.

Populations of G. conopsea expressed high levels of genetic variation and a certain amount of gene flow, although investigated mating pattern in a small population indicated non-random mating among individuals, with the majority of pollen exchange between near neighbours, and noticeable levels of geitonogamous pollinations. Further a pronounced year to year variation in flowering frequency among individuals was found.

It was also discovered that flowering time variants (early and late) within the species G. conopsea were highly differentiated and seem to have had a more ancient historical separation than the separation between the two different species, G. conopsea and G. odoratissima.

Levels of genetic variation in the rare congener, G. odoratissima differed between island and mainland populations where the more numerous island populations expressed larger levels of genetic variation and were less differentiated compared to the few remaining and genetically depauperated mainland populations.

Uppsala University Library, Box 510, 75120, Uppsala, Sweden

The number of gene products available for structural and functional study is increasing at an unprecedented rate as a result of the successful whole genome sequencing projects. Systematic structure determination of proteins on a genomic scale, called structural genomics, can significantly contribute to the field of protein science and to functional annotation of newly identified genes.

This thesis covers different aspects of protein production in Eschericiha coli for structural studies in the context of structural genomics. Protocols have been downscaled and standardized to allow for a rapid assessment of the production characteristics for multiple proteins in parallel under a number of different conditions. Foremost, the ability of different proteins and peptide tags to affect the solubility of the recombinant protein when produced as fusion proteins has been systematically studied. Large differences in the success-rate for production of soluble protein in E. coli were found depending on the fusion partner used, with a more than two-fold increase in the number of proteins produced as soluble when comparing the best and the poorest fusion tags. For different constructs with a histidine tag, commonly used to facilitate protein purification, large differences in yield depending on the design of the expression vector were found. When comparing different fusion proteins produced from identical expression vectors, fusions to the GB1 domain were found to result in the highest yield of purified target protein, on average 25 % higher than any of the other fusions.

The suitability for further structural studies was tested at an intermediate scale for proteins that were identified as soluble in the expression screening. For this purpose, protocols for rapid purification and biophysical characterization using nuclear magnetic resonance and circular dichroism spectroscopy were developed and tested on 19 proteins, of which four were structured.

We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.

Over the last few years, genome-wide association studies (GWAS) have been used to identify numerous obesity associated SNPs in the human genome. By using linkage studies, candidate obesity genes have been identified. When SNPs in the first intron of FTO were found to be associated to BMI, it became the first gene to be linked to common obesity. In order to look for causative explanations behind the associated SNPs, a re-sequencing of FTO had been performed on the SOLiD sequencing platform. In-house candidate gene, SLCX, was also sequenced in order to evaluate a potential obesity association. The purpose of this project was to analyse the sequences and also to evaluate the quality of the SOLiD sequencing. A part of the project consisted in performing PCRs and selecting genomic regions for future sequencing projects. I developed and implemented a sequence analysis strategy to identify obesity associated SNPs. I found 39 obesity-linked SNPs in FTO, a majority of which were located in introns 1 and 8. I also identified 3 associated intronic SNPs in SLCX. I found that the SOLiD sequencing coverage varies between non-repetitive and repetitive genomic regions, and that it is highest near amplicon ends. Interestingly, coverage varies significantly between different amplicons even after repetitive sequences have been removed, which indicates that it is affected by features inherent to the sequence. Still, the observed allele frequencies for known SNPs were highly correlated with the SNP frequencies documented in HapMap. In conclusion, I verify that SNPs in FTO are associated with obesity and also identify a previously unassociated gene, SLCX, as a potential obesity gene. Re-sequencing of genomic regions on the SOLiD platform was proven to be successful for SNP identification, although the difference in sequencing coverage might be problematic.

Biofuels that are renewable and environmentally benign constitute an important area of research, as the supply of fossil fuels decreases and the amount of green house gases in the atmosphere increases. Biohydrogen is not as well explored as other biofuels, but its properties render it a promising complement, as it is clean and can be used directly in fuel cells to generate electricity, the only waste products being water and heat. Hydrogenproducing microorganisms have the potential to be used to recycle industrial waste, such as carbohydrates from food manufacturing. Hence the cost of waste disposal could be reduced whilst biofuel is being produced through microbial processes.

Escherichia coli is a well-known microorganism that produces hydrogen under fermentative conditions, through the conversion of formate to hydrogen gas and carbon dioxide, via an enzyme complex called formate hydrogenlyase (FHL). The complex is anchored to the inner cell membrane and consists of seven subunits: a formate dehydrogenase, a [Ni-Fe] hydrogenase, three electron carrier proteins, which together make up a large ‘hydrophilic domain’, and two integral membrane proteins (the ‘membrane domain’).

Even though the entire bacterial genome is known, the FHL complex remains little understood and has proven difficult to isolate and characterise. During this project, a genetically modified strain producing only the hydrophilic domain of FHL was constructed, and the resultant sub-complex was purified. It was hoped that, if a stable and homogenous core complex could be isolated, it might be subjected to further analysis, such as elucidating the subunit stoichiometry and solving the structure.

Furthermore, FHL is notoriously oxygen labile, which hampers its study and technological development. However, oxygen tolerance is a natural feature found in some other [Ni-Fe] hydrogenases, and recent research shows that this property is likely dependent on the presence of extra cysteine residues near an important metal cluster in the enzyme. These cysteines are not present in FHL and a complex that could be active in both aerobic and anaerobic conditions may be a useful tool in optimising microbial biohydrogen processes. Thus, three strains that each expressed a modified FHL variant carrying single Cysteine-for-Glycine substitutions were constructed. The modified FHL complexes proved to remain active in vivo, and can serve as the basis of genetically engineering oxygen tolerance into this important enzyme.

Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine.

Mutagenesis libraries are the heart of combinatorial protein engineering where proteins such as antibodies are evolved for improved functionality. Despite recent improvements in gene synthesis and selection methodologies, current methods still fail to provide practical means for synthesis of complete antibody scFv and screening of theoretical diversities, hence forcing the user to focused diversity screening and assembly of shorter oligos to avoid synthesis errors and maximize library functionality. Here we demonstrate a way to generate highly functional tailored mutagenesis libraries for efficient antibody affinity maturation using a rapid cell-free solid phase cloning method with single strand diversity oligonucleotides. For this we are utilizing a combination of a high-fidelity polymerase for PCR-based incorporation of Uracil into a wild-type template, bead-based solid-phase technology for elution of single strand DNA, oligonucleotide annealing, extension and automation, and an uracil excision enzyme cocktail for in vitro degradation of template DNA to minimize background. Our method allowed for fast (8 hours) mutagenesis and automated cloning of a complete set of 50 position specific alanine-mutations for mapping of the paratope of a scFv antibody in a single robot run. We further exemplify our method by generating and stratifying a set of antibody scFv affinity maturation libraries with targeted diversity into critical or nonessential paratope positions, as well as by a complete randomization in all positions. The libraries were subjected to bacterial surface display selections and output was followed by Illumina deep sequencing and binding analysis by SPR. The functional quality of our libraries were high, with a yield of >99% functional diversity in the case for two of our libraries. We were further able to target all positions in all loops with diversity, and we could show the ability to target all six loops with diversity at the same time. The comparison of different library focus showed us that scFv libraries with diversity targeted to non-essential enhancing paratope positions more quickly rendered enrichment of improved binders compared to random diversity or paratope-targeted libraries. Surprisingly several of the improved binders from the random library had beneficial mutations in the same positions targeted by the smaller focused non-essential enhancing residue focused library indicating a possible benefit of focusing diversity to these spots. We believe our method for construction of libraries with site directed mutagenesis to be a viable way for generation of functional and diverse genetic libraries, particularly suitable for affinity maturation and paratope mapping of antibodies.