Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents. / QC 20111117 / Development and applications of protein microarrays / The Swedish Human Proteome Resource (HPR) program

>Magister Scientiae - MSc / Saccharomyces cerevisiae is an important host in industrial biotechnology. This yeast is the host of choice for the first and second-generation biofuels for ethanol production. Genome modification in S. cerevisiae has been extremely successful largely due to this yeast’s highly efficient homology-directed DNA repair machinery. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing technology has made multi-gene editing in yeast more accessible. In this study, we aimed at targeting the Cas9 to multiple genomic positions for integrating multiple genes at different sites. We have developed two CRISPR-Cas9 systems, based on published one- and two-plasmid systems, for application in S. cerevisiae strains. In this study, these CRISPR-Cas9 systems were used to transform fungal heterologous genes into yeast using the electroporation transformation method.

Introduction: Clostridium thermocellum is considered a model organism forconsolidated bioprocessing, due to its ability to hydrolyze lignocellulosicbiomass more efficiently than many other organisms and to produce ethanol.In order to meet the industrial requirements of ethanol yield and titer, metabolicengineering efforts have been made resulting in a strain that successfullydisplays increased ethanol yield with reduced amount of some byproducts.However, the ethanol yield in this engineered strain still does not meet theindustrial requirements and significant amounts of amino acids are stillproduced. To attempt to decrease the level of amino acid excretion intended toimprove the ethanol yield in C. thermocellum, it is essential to understand itsmetabolism and how it is affected by different cultivation conditions and mediumcompositions. This study aimed to gain an insight in how carbon- and nitrogenlimitation affect amino acid excretion in C. thermocellum, with the hypothesisthat excess of carbon and nitrogen yields more amino acid excretion. Methods: Mass-balance based calculations of rates and yields were used toanalyze the metabolism of a wild-type of C. thermocellum (DSM 1313) grownanaerobically in carbon- or nitrogen-limiting chemostats. For this, Low-Carbonmedium containing, respectively, cellobiose (5 g/L) and urea (0.15 g/L) as thelimiting nutrient was used. Both cultivations were performed at 55 °C, pH 7.0and 400 RPM shaking at a dilution rate of 0.1 h-1. Conclusion:  Considering yields of total amino acids excreted in bothlimitations, it was hypothesized that C. thermocellum exploited the amino acidexcretion to maintain carbon balance around the pyruvate node caused byexcess of the carbon. Based on yield of valine excreted in particular, it washypothesized that amino acid excretion was used to maintain redox balance inthe metabolism of C. thermocellum, where malate shunt could play a major role.However, results of the Carbon-limitation did not allow any conclusion ofnitrogen excess having an effect on amino acid excretion in C. thermocellum.

Previous small-scale methods for plasmid DNA (pDNA) purification fail to meet theindustry’s demand for sufficient quantities. Greater volumes of bacterial lysates are a consequence of larger volumetric fermentations and traditional large-scale down-stream purification processes have some disadvantages and limitations. The market is believed to continue to expand, hence the need for efficient, cost-effective, andscalable purification processes becomes apparent. A crucial trade-off exists between pDNA yield and purity, necessitating careful consideration in chromatographic pu-rification steps. Each step enhances purity while likely sacrificing yield. In order to achieve a higher degree of pDNA yield, optimal purification entails a single chro-matographic step, specifically anion-exchange chromatography (AEX) in combina-tion with filtration. Alternatively, a two-step purification approach involving AEX followed by hydrophobic interaction chromatography (HIC) is recommended to elim-inate complementary impurities and achieve a high level of purity. Furthermore, the utilization of monolithic chromatographic supports is suggested to facilitate the sug-gested purification strategies. This is due to monoliths promoting higher binding capacities, ensuring robust and consistent results even at high flow rates.

The production of olive oil has seen an increase in recent years due to a broader understanding of the health benefits of the Mediterranean Aliment Culture. With this expanding industry we also see an increase in the waste products associated with olive oil production. Given the high polluting content of the waste streams and the economic costs associated with its removal and processing, waste remediation and disposal has become a significant point of interest for both producers and local bodies. In this project, wastes of the olive oil production industry are examined for their use as the raw material for a novel product used in the control of horticulturally important diseases, examining the effect of extraction protocols on the activity of the final product. Active fractions of the olive oil wastes were identified from literature and protocols for their extraction and recovery developed; incorporating both standard solvent extraction and novel ultrasound-assisted extraction. Criteria for the analysis of extract quality were outlined and potential target applications identified. The biophenolic compounds of olive wastes were identified as providing the majority of the active fraction, so protocols were developed for the recovery of these compounds. Standard solvent extraction and ultrasound-assisted extraction were examined for their effectiveness of biophenolic recovery and their effect on product quality. Certain horticulturally important diseases were identified as potential targets, and bioassays undertaken to determine the ability of a crude extract to inhibit and control these diseases. It was found that the action of ultrasound during extraction provides a greater degree of recovery of biophenolic compounds, with minimal loss of product quality; as determined by bioassays and total biophenol determination. This increase in recovery is due primarily to the destruction of cellular material resulting in higher rates and absolute yields of recovery. This work provides evidence of the occurrence of some interesting phenomenon in the recovery of biophenols from olive wastes that deserves further examination. The crude olive leaf extract was shown to have an inhibitory effect on bacteria and effectively no inhibitory effect on fungal species in the total biophenol ranges tested. Erwinia amylovora and Staphylococcus aureus both showed a large susceptibility to the olive leaf extract. Results showed a higher degree of susceptibility of Gram positive bacteria and a potential resistance in soil microbes. For bacterial species, total biophenol concentrations of 0.15 to 3.50 mg GAE/ml provided inhibitory effects, while with the fungal species tested, no inhibitory effects were found at total biophenol concentrations of up to 2.50 mg GAE/ml. Some evidence exists that there is an opportunity for the economic recovery of olive biophenols for use as a novel product, but more work is required to determine specific applications and/or targets of use, as well as optimisation of the extraction and purification protocol. A sample removed from interfering compounds will allow the examination of activity of particular compounds and hence a better understanding of the action of the olive waste extract.

The aim of this study was to observe biofilm formation by Enterobacter sakazakii (E. Sakazakii) from different clinical, dairy and environmental origins on three infant feeding tubes made of different materials. Infant formula milk was selected as the medium for E. sakazakii growth. Seventeen isolates from different origins were retrieved and tested for purity, using a plating method and biochemical tests to eliminate the non E. sakazakii strains from this study. A method to rapidly and accurately detect viable cells of E. sakazakii on infant feeding tube surfaces using of the BacTrac® 4000 microbiological growth analyser was developed. The sources of errors such as from cleaning, operation and handling procedures were assessed prior to experimental runs. The strength of biofilm formation by different isolates of E. sakazakii on plastic surfaces was scrutinised using a microtiter plate assay. The results from the microtitre plate assay were based on the absorbance at 550 nm of crystal violet stained films and showed that all the clinical isolates were able to attach and form strong biofilms on the plate. Some environmental isolates formed strong or weak biofilms and some did not produce biofilm at all. However, dairy isolates formed both strong and weak biofilms in the microtitre plate when incubated in 10% reconstituted infant formula milk. The further studies were to quantify biofilm formation by three isolates of different origin on three different materials of infant feeding tubes using a batch system. Tubing pieces were incubated with infant formula milk inoculated with E. sakazakii cells at approximately 8 log CFU mL-1 and the biofilm formation was assessed at three time intervals: 4, 12 and 24 hours. Biofilm formation on the tubing by clinical isolates was also observed using epifluorescence microscopy and the scanning electron microscope. E. sakazakii from clinical, dairy and environmental isolates were able to form biofilm on three different materials of infant feeding tubes. The results showed that the initial attachment at 4 h on silicone tubing was low compared with the other two tubes. The scanning electron micrographs showed the surface characteristics of each tubing and the biofilm formation by E. sakazakii clinical isolates after 4, 12 and 24 hours. Silicone tubing appeared to be the best choice for premature babies that need feeding using feeding tubes, as it was slow to become colonised compared with the PVC and polyurethane tubing.

Polyhydroxyalkanoates (PHAs) are polyesters composed of (R)-3-hydroxy-fatty acids. A variety of gram-positive as well as gram-negative bacteria and some archaea are able to produce these biopolymers as energy and carbon storage materials. In times of unbalanced growth, when carbon is available in excess but other nutrients are limited, PHA inclusions are formed. These granules are water-insoluble, stored intracellularly and can be maintained outside the cell as beads. The key enzyme for the formation of PHA inclusions is the PHA synthase PhaC, which catalyses the polymerization of (R)- 3-hydroxyacyl-CoA to PHA with the concomitant release of CoA. The PHA synthase from Ralstonia eutropha (currently Cupriavidus necator), which is covalently bound to the PHA granule surface, tolerates fusions to its N terminus without loss of activity. In this study it was investigated if it would also tolerate translational fusions to its C terminus. A specially designed linker was employed, aiming at maintaining the hydrophobic surroundings of the R. eutropha synthase C terminus to allow proper folding and activity. Two reporter proteins were tested as fusion partners, the maltose binding protein MalE and the green fluorescent protein GFP. As GFP is a hydrophobic protein itself, no additional linker between the PHA synthase and the reporter protein was necessary to produce PHA granules displaying the functional fusion protein on the surface. Principally, the PHA synthase PhaC tolerates translational fusions to its C terminus but the nature of the fusion partner influences the functionality. Recently, PHA granules have often been acknowledged as bio-beads. A one-step production allows the formation of functionalised beads without the need for further cross-linking to impart desired surface properties. PHA beads displaying a gold- or silica-binding peptide at the N terminus of PhaC were constructed and tested for their applicability. Additionally, these beads were able to bind IgG due to the ZZ domain of the IgG binding protein A, which was employed as a linker sequence. These functionalised beads can be used as molecular tools in bioimaging and biomedicine, combining organic core with inorganic-binding shell structures. In a different biomimetic approach, the display of ten lysine residues at the granule surface was achieved using the phasin protein PhaP as the anchoring matrix. Extensive work was performed in an attempt to also employ the synthase protein, but was unsuccessful. These positively charged bio-beads can be used for dispersion or crosslinking experiments as well as silica binding.

This work is a comparison of a well-established and a novel, "green" and efficient technique to separate peptides of pharmaceutical interest. An attempt is made to derive the chromatographic retention behaviour from these techniques to a number of property descriptors derived from the linear sequence of amino acids. A set of therapeutic peptides were carefully chosen to be experimentally evaluated using in silico-based descriptor calculations. A principle component analysis was performed to assess the distribution of calculated descriptors for including peptides with variable properties. A diluent optimization study was also included to find the optimal diluent for peptides with minimal diluent effects and peak splitting phenomena. The results showed that the solvents tert-butanol and methanol performed best between 20-30 and 50 volumetric percent water as additive in SFC and HPLC, respectively. These diluents were then used for the peptides within the set to evaluate the retention and selectivity in HPLC and SFC. SFC performed well in terms of resolving power. Inparticular, SFC was able to separate Leuprolide and Triptorelin while HPLC was not. A comparison was also made in between the two stationary phases CN and XT, where a global selectivity was shown to be higher for CN. This work does also assess a novel method for determining solubility of analytes in supercritical fluid. The method was evaluated using the pharmaceutical compounds caffeine and aspirin and then used to determine solubility of Leu-Enkephalin in 20% (v/v%) methanol. The solubility of caffeine was determined to be 0.45 mg ml-1 in pure SF-CO2 under 140 bar pressure and 3.9 mg ml-1 for aspirin in 2.4% methanol. Both values correlated well with measurements from four acknowledged papers within this field. Leu-Enkephalin was found to have a solubility of 1.90 mg ml-1 using a solvent corresponding to the initial phase condition of the gradient used for peptide analysis in SFC. Further experimental work is required before the method can be implemented as a useful tool in preparative chromatography, however the results presented here show the compatibility of assessing biomolecules in both pure SF-CO2 and mixed with modifier. The possibility to determine solubility with additional modifier infers an important step of including and evaluating these compounds creating a solid support to subsequent large scale separation.

Inom dagens tillverkande företag pågår en allt större konkurrens om att vinna marknadens kunder. En av de främsta konkurrenskrafterna är korta ledtider. Som ett led i detta har vetenskapliga filosofier och metoder inom Lean blivit allt mer förekommande inom verksamheterna. Teorierna handlar om att skapa effektiva processer och att producera rätt produkt, till rätt kvalitet inom rätt tid. Under de senaste åren har en ökad medvetenhet för inköp-och orderhanteringens betydelse trätt fram. Enligt tidigare forskning kan en effektiv inköps-och orderhanteringsprocess bidra till att den totala tillverkningsprocessens ledtid effektiviseras. Sekventiella arbetsprocesser är vanligt förekommande trots att administrativa handläggningsaktiviteter ofta kan utföras parallellt, detta medför en onödig fördröjning av ledtiden. Studiens syfte är undersöka hur orderhanteringsprocessen ledtid kan reduceras. En ledtidsreducering baserat på att finna icke värdeskapande aktiviteter och problemområden inom och åtgärda dessa med hjälp av lämpliga åtgärdsmetoder.   Arbetet är en fallstudie som utförts på teknikutvecklingsföretaget Syntronic. Arbetets empiriska studie gjordes genom intervjuer, observationer och dokument. För att underlätta analysarbetet användes ett fiskbensdiagram för att finna orsakerna till problemområden och icke värdeskapande aktiviteter i processen. För att förtydliga processflödet och vart i processen de identifierade problemområdena uppstod kartlades observationen genom en värdeflödesanalys. Arbetets litteraturstudie bestod av teorier kring orderhantering, effektivisering, ledtider och Lean.   Analysarbetet gav upphov till att bristande kommunikation, avsaknad av standardiserat arbetssätt, bristande planering, bristande affärssystem samt bristande helhetssyn var orsakerna till den ineffektiva orderhanteringsprocessen. Studien resulterade i att lämpliga förbättringsåtgärder och arbetsmetoder kunde urskiljas. Metoder inom Lean som 5S, Kaizen och TQM (Total Quality Management) ansågs kunna lämpa sig för fallstudien. Analysarbetet inspirerade även till att utveckla förslag till en ny arbetsmetod: 5S Administration Plus. Arbetsmodellen talar för vikten att standardisera arbetsrutiner, följa upp och utvärdera för att bibehålla förbättringsarbetet.   Studiens teoretiska bidrag talar för hur kombinationen av metoder inom Lean kan tillämpas som effektiviseringsverktyg i en administrativ orderhanteringsprocess. Det praktiska bidraget svarar på studiens syfte och frågeställning angående hur orderhanteringsprocessen kan effektiviseras.

This thesis relates the physicochemical properties of phenolic compounds to their antioxidant activities. It focuses on the partitioning of phenolic compounds between hydrophilic and lipophilic environments and the relevance this has to their in vivo health effects. Data in the literature was lacking so the phase partition coefficients (log P) of 53 phenolic antioxidants were measured by reversed-phase HPLC and calculated by log P prediction software. There was a very strong linear correlation between measured and calculated values (r=0.91). The importance of log P in determining antioxidant assay values was then tested by developing an assay system capable of measuring activities of both hydrophilic and lipophilic antioxidants. This Lipid Peroxidation Inhibition Capacity Assay (LPIC), based on using liposomes to simulate a cell membrane environment, was then used to measure the activity of antioxidants with a broad range of structures. The activities were correlated against log p, the difference of heat of formation (∆Hf) and half-wave potential (Ep/2) and used to derive a predictive model to calculate the LPIC activity. There was a highly significant linear correlation between the calculated and measured values. The LPIC activities also correlated well to published LDL inhibition activities but not to measured ORAC activities. These findings suggested that behaviours of antioxidants in the small unilamellar vesicles of the LPIC assay were similar to that in the LDL assay but not to the aqueous phase based ORAC assay. The LPIC assay may therefore be a better indicator of potential health benefits of antioxidants in the human body than the ORAC assay. The possible mechanistic reasons are that it may better reflect ability to prevent the oxidation of LDL blood stream particles that leads to cardiovascular disease and also takes into account the importance of membrane solubility which can raise the cellular concentration and thus potential to protect cells from oxidative damage. KEYWORDS: LPIC, LDL; Antioxidant; Phytochemical; Polyphenolic; Phenolic acid; Flavonoids; log P; Partition Coefficient; Liposome; Lipid bilayer; Lipid Membrane; ORAC; Comet assay; Flow Cytometry. / Whole document restricted, but available by request, use the feedback form to request access.