Characterization of the pore structure is a crucial part in themanufacturing of porous media used for purification of biologicalpharmaceuticals. This project took place at Cytiva in Uppsala and aimedat optimizing a newly developed method in pore structurecharacterization called size-exclusion quantification NMR (SEQ-NMR). Bymeasuring with diffusion NMR on a polymer solution before and afterequilibration with a material of interest the pore structure of thematerial can be determined. This project aimed at reducing the durationof a SEQ-NMR experiment while examining the performance of the methodduring different conditions with the goal of making the methodapplicable for quality control procedures. The method was optimizedboth by simulations and by experimental diffusion NMR measurements. Itwas discovered that the performance of the method could be improved byhaving an optimal mixture of the polymer solution and duringexperiments distributing ten measurement points with linear spacing.With these parameters optimized the duration of the method could bereduced with 22 hours landing on a total duration of 8 hours. Theduration combined with the complexity of the method still makes themethod unsuitable for use in quality control of porous media. Despitethe small possibility of SEQ-NMR being a quality control method thisproject has proven the method to be both reproducible and sensitive.

Antibodies are the gold standard molecular recognition elements and a cornerstone of molecular biology. They are used in immunoassays to precisely measure a specific analyte, but certain targets are especially challenging. Difficult targets include small molecules and molecules that do not induce an immune response. Aptamers are short oligonucleotides that can form 3-dimensional structures and bind targets with high specificity. Aptamers are smaller and more flexible than antibodies and could therefore solve this problem. In contrast to antibodies, aptamers are synthetically produced, so they can have affinity for molecules that do not induce an immune response. This also makes them cheaper, faster and more ethical to produce. They are also easily modified and have the ability to renature and can therefore be reused.  Our conclusions are that aptamers can outperform antibodies, especially for small molecule targets, and that the synthetic production of aptamers gives them a further advantage over antibodies. Our report compares several different types of detection methods that use aptamers and we conclude that fluorescence-based methods are the most easy to use with basic lab equipment, can be made similar to the ELISA kits in addition to giving highly sensitive detection. We describe a variety of fluorescence-based detection strategies but the optimal method will depend on the specific aptamer and target. The report also includes an ethical analysis where antibodies and aptamers are compared. This report is commissioned by Mercodia AB, a company that develops, manufactures and distributes immunoassays for biomarkers within the field of metabolic disorders. They commissioned this report in order to give an overview of how aptamers interact with their target, and also to compare aptamer-based detection strategies with sensitivity prioritized over selectivity.  This was done by literature research.

The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system. CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction. Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures. / QC 20101222

Food waste and fashion pollution are two of the significant global environmental issues throughout the recent past. In this research, it was investigated the feasibility of making a leather-like material from bread waste using biotechnology as the bridging mechanism. The waste bread collected from the supermarkets were used as the substrate to grow filamentous fungi species Rhizopus Delemar and Fusarium Venenatum. Tanning of fungal protein fibres was successfully performed using vegetable tanning, confirmed using FTIR and SEM images. Furthermore, glycerol and a biobased binder treatment was performed for the wet-laid fungal microfibre sheets produced. Overall, three potential materials were able to produce with tensile strengths ranging from 7.74 ± 0.55 MPa to 6.92 ± 0.51 MPa and the elongation% from 16.81 ± 1.61 to 4.82 ± 0.36. The binder treatment enhanced the hydrophobicity even after the glycerol treatment, an added functional advantage for retaining flexibility even after contact with moisture. The fungal functional material produced with bread waste can be tailored successfully into leather substitutes using an environmentally benign procedure.

Microscopy imaging based techniques, such as the Cell Painting assay, could be used to generate images that visualize the Mechanism of Action (MoA) of a drug, which could be of great use in drug development. In order to extract information and predict the MoA of a new compound from these images we need powerful image analysis tools. The purpose with this project is to further develop a Deep Learning model to predict the MoA of different drugs from Cell Painting images using Convolutional Neural Networks (CNNs) and Conformal Prediction. The specific task was to compare the accuracy of different CNN architectures and to compare the efficiency of different nonconformity functions.  During the project the CNN architectures ResNet50, ResNet101 and DenseNet121 were compared as well as the nonconformity functions Inverse Probability, Margin and a combination of them both. No significant difference in accuracy between the CNNs and no difference in efficiency between the nonconformity functions was measured. The results showed that the model could predict the MoA of a compound with high accuracy when all compounds were used both in training, validation and test of the model, which validates the implementations. However, it is desirable for the model to be able to predict the MoA of a new compound if the model has been trained on other compounds with the same MoA. This could not be confirmed through this project and the model needs to be further investigated and tested with another dataset in order to be used for that purpose.

Plastics have a wide application field, where cosmetic products are one of them. Polymers which are building blocks to plastics exists in many variants, overall they can be categorized into two groups; microplastics and water soluble polymers. Both polymer types are important to study and understand since polymers in general are not covered by any legislation. To gain a more profound understanding of their impact on environment this study was conducted. In collaboration with SSNC (Naturskyddsföreningen), a database containing hundreds cosmetic products was processed. The most occurring polymers were quantified and prevalent ingredients having “poly” in their name were selected for further investigation namely Nylon 12-20 (microplast) and Acrylates C/10-30 Alkyl-crosspolymer (water soluble). A standardized analysis method OECD 301 F was performed to test the polymers biodegrading ability. Results from biodegradation method showed that, neither of the two polymers is readily biodegradable in aqueous environment, despite their different properties. In connection with the obtained results, a filtration analysis was performed, with the purpose to determine the possibility to capture the polymers using microfilters. Results mainly showed flowthrough of both polymers. Relating the results to reality implies that these polymers are not captured in waste water treatment plant due to inefficient filtration and thereby spread to the environment. In addition more research should be devoted to water-soluble polymers and their impact on nature. Based on all compiled results, it is proposed that legislation addressing microplastics should be edited and revised in such a way that water soluble polymers are included in future prohibitions (against microplastics). / Plast har många användningsområden varav kosmetiska produkter är ett av dem. I kosmetika används exempelvis naturliga ämnen, sådana som förekommer i naturen och således kan brytas ned. Sedan finns syntetiska polymerer, sådana som syntetiseras och tillverkas av människor. Det finns oerhört många varianter av syntetiska polymerer som används inom kosmetika, generellt kan de kategoriseras i två grupper; mikroplaster och vattenlösliga polymerer. Med hjälp av Naturskyddsföreningens databas som innehåller hundratals kosmetikaingredienser, söktes de komponenter som hade ”poly” i sitt namn eftersom de inte täcks av lagstiftning. Bland dessa, valdes två mest förekommande polymerer för att studeras vidare, nämligen Nylon 12-20 (mikroplast) och Acrylates C/10-30 alkyl crosspolymer (vattenlöslig). En standardiserad analysmetod OECD 301 F tillämpades för att testa deras biologiska nedbrytbarhet. Resultatet från nedbrytbarhetstestet visade att ingen av polymertyperna, trots deras åtskiljande egenskaper, är lättnedbrytbar i vattenmiljöer. I följd av resultatet från analysen genomfördes en filtreringsanalys på KTH, för att avgöra om respektive polymer går att fånga upp i olika storlekar av mikrofilter. Resultatet visade att ingen av dem fångades upp (med den utrustning som var tillgänglig på KTH). Baserat på resultaten föreslås det att lagstiftningar som innefattar mikroplaster bör redigeras och revideras på ett sådant sätt att vattenlösliga polymerer är inkluderade i framtida förbud (mot mikroplaster). Utöver detta bör mer forskning ägnas åt vattenlösliga polymerer och deras påverkan på naturen.

The recombinant product is secreted to the periplasm in many processes where E. coli is used as host. One drawback with secretion is the undesired leakage of the periplasmic products to the medium. The aim of this work was to find strategies to influence the periplasmic retention of recombinant products. We have focused on the role of the specific growth rate, a parameter that is usually controlled in industrial bioprocesses. The hypothesis was that the stability of the outer membrane in E. coli is gained from a certain combination of specific phospholipids and fatty acids on one side and the amount and specificity of the outer membrane proteins on the other side, and that the specific growth rate influences this structure and therefore can be used to control the periplasmic retention. We found that is possible to control the periplasmic retention by the growth rate. The leakage of the product increased as the growth rate increased. It was however also found that a higher growth rate resulted in increased productivity. This resulted in equal amounts of product inside the cells regardless of growth rate. We also showed that the growth rate influenced the outer membrane composition with respect to OmpF and LamB while OmpA was largely unaffected. The total amount of outer membrane proteins decreased as the growth rate increased. There were further reductions in outer membrane protein accumulation when the recombinant product was secreted to the periplasm. The lowered amount of outer membrane proteins may have contributed to the reduced ability for the cell to retain the product in the periplasm. The traditional way to control the growth rate is through a feed of substrate in a fed-batch process. In this work we used strains with a set of mutations in the phosphotransferase system (PTS) with a reduced uptake rate of glucose to investigate if these strains could be used for growth rate control in batch cultivations without the use of fed-batch control equipment. The hypothesis was that the lowering of the growth rate on cell level would result in the establishment of fed-batch similar conditions. This study showed that it is possible to control the growth rate in batch cultivations by using mutant strains with a decreased level of substrate uptake rate. The mutants also produced equivalent amounts of acetic acid as the wild type did in fed-batch cultivation with the same growth rate. The oxygen consumption rates were also comparable. A higher cell density was reached with one of the mutants than with the wild type in batch cultivations. It is possible to control the growth rate by the use of the mutants in small-scale batch cultivations without fed-batch control equipment. / QC 20101108

Syftet med projektet är att undersöka effekten av att leda över vattenverksslam från Norrvattens vattenverk, Görvälnverket, till Käppalaverkets reningsverk som ett steg i en mer hållbar slamhantering. Detta slamhanterings alternativ jämförs med en framtida lokal slamhantering vid Görvälnverket. Studien undersöker möjligheten att leda vattenverksslam över till Käppalaverket utifrån fyra huvudaspekter, vattenrening, drift, kostnad och miljöpåverkan. En litteraturstudie genomfördes med syfte att utvärdera hur vattenverksslammet kan påverka reningsprocesserna vid Käppalaverket. En Livscykelanalys genomfördes med syfte att utvärdera miljöpåverkan av att leda över vattenverksslam till Käppalaverket. Genomförbarheten utvärderades med hjälp av en multikriterieanalys, där tekniska, miljömässiga och ekonomiska aspekter utvärderades.  Resultat från studien visade att den framtida lokala slamhanteringen är mer fördelaktig från ett tekniskt och ekonomiskt perspektiv, än överledning av vattenverksslammet till Käppalaverket. En nackdel med överledning av vattenverksslam till Käppalaverket är att vattenverksslammet sannolikt kommer påverka avvattningen av avloppsslammet, vilket resulterar i en högre polymerförbrukning och en ökad hydraulisk belastning på centrifuger och rötkammare. Överledningen av vattenverksslam antas däremot inte ha en negativ påverka på kvaliteten av reningen vid Käppalaverket. Att leda vattenverksslam över till Käppalaverket ger en lägre miljöpåverkan med avseende på kemikalieutsläpp till vattenmiljön men en högre miljöpåverkan med avseende på transporter och energiförbrukning.  Överledning av vattenverksslam till Käppalaverket bedöms som genomförbart, men denna studie visar att den framtida lokala slamhanteringen på Görvälnverket är ett mer fördelaktigt alternativ. Om överledning av vattenverksslam till Käppalaverket fortsatt är ett aktuellt alternativ rekommenderas det att en mer djupgående studie utförs, där vattenverksslammet tillsätts till Käppalaverket för att utvärdera dess påverkan. / The aim with this project is to investigate the impact of leading the produced waterworks sludge (WWS) from Norrvattens drinking water treatment plant (DWTP), Görvälnverket, over to Käppalaverkets wastewater treatment plant (WWTP) as a step in a more sustainable sludge management. This alternative is compared to a future sludge management at Görvälnverket. The study, investigating the feasibility of leading the WWS over to Käppalaverket, is based on four main aspects, water treatment, operation, cost, and environmental impact. A literature study was performed to evaluate the effect of WWS on Käppalaverket. A life cycle assessment (LCA) analysis was performed to evaluate the environmental impact of leading the WWS over to Käppalaverket. The feasibility was evaluated using a multi-criteria decision analysis (MCDA), where technical, environmental, and economic aspects were considered. The study shows that the future sludge management is more favourable than leading the WWS over to Käppalaverket from a technical and economic aspect. The main drawback with leading the WWS over to Käppalaverket is that the WWS will likely impact the dewatering of the sewage sludge, resulting in a higher polymer consumption and an increased hydraulic load on centrifuges and digesters. However, the addition of WWS at Käppalaverket is not assumed to negatively impact the quality of the treatment at Käppalaverket. Leading the WWS over to Käppalaverket results in a lower environmental impact regarding chemical emissions but results in a higher environmental impact regarding transportation and energy consumption. Leading the WWS over to Käppalaverket was found to be feasible, although the future sludge management at Görvälnverket was found to be more favourably in this study. A more in depth study on the feasibility of leading the WWS over to Käppalaverkets is required to fully assess this aspect. A trial where the WWS is added to Käppalaverket is recommended to further evaluate the impact of the WWS.

Bread waste represents a significant portion of global food waste, necessitating innovative approaches for its valorization. This research project explores the utilization of bread waste through fermentation with Aspergillus oryzae to produce fungal film which could be used for textile applications. While previous studies have examined various applications of food waste, this project specifically targets the textile industry, aiming to mitigate pollution associated with conventional textile manufacturing. The objective of this project was to investigate the feasibility of creating fungal films derived from bread waste. Additionally, to analyze the material's properties through assessments of tensile strength, microscopic analysis, and the identification of an appropriate methodology for this investigation. The biomass suspension was prepared using an ultrafine grinder, and a kitchen blender was subsequently employed to minimize the presence of remaining solids from the grinding process. Additionally, a range of strategies for film casting and wet laying were implemented and evaluated. Wet laying involved combining fungal biomass with tannin to mimic the characteristics of leather. On the other hand, casting utilized pre-treated biomass suspension to assess the formation and quality of the films. As the research progressed and different tannins were used to treat the biomass, a methodology was developed, and glycerol was introduced as a plasticizer. Furthermore, nanocellulose was later incorporated exclusively for the casting of the films to serve as a binder. The films that were produced gave interesting results are observed in casted sheets containing nanocellulose and glycerol-infused biomass (3% BM + 2% Cellulose + 0.13 g Glycerol), exhibiting exceptional tensile strength (35.1 ± 3.42 MPa) and elongation (16.7 ± 5.98%). Wet laid biomass sheets treated with Tara and glycerol display tensile strength (19.9 ± 3.55 MPa) and elongation (6.66 ± 3.02%). These findings signify the potential for developing fungal films from bread waste, necessitating further research to refine methodologies. Overall, this research project paves the way for future advancements in fungal films derived from bread waste. By investigating the use of Aspergillus oryzae and employing wet laying and casting techniques, the project establishes a foundation for sustainable textile production. The successful utilization of bread waste not only addresses the issue of food waste but also contributes to reducing pollution in the textile industry.

To diagnose patients suffering from blood clotting disorders latex immununoassays (LIA) can be used. A time consuming manual tangential flow filtration (TFF) process suggests the implementation of an automatic TFF system to improve the efficiency, profitability, and expandability of the production facility of LIA at Nordic Biomarker. Tests were made of the automatic TFF system's ability to perform the desired steps of concentration, dilution and diafiltration, both with purified water and mimicked product. The mimicked product of micro particles (MP) mixed with monoclonal antibodies (mAb) was also used to further test the system's pressure control, safety alarms and stops, and to determine a permeate flux by a critical flux experiment. The results imply a functional TFF system able to automatically concentrate the process fluid and maintain a stable volume during diafiltration, although an additional permeate pump was ordered to be able to attain a fully functional performance of the automatic TFF process. The final part of the implementation was to initiate a validation draft including a risk assessment, OQ plan and PQ plan that resulted in a plan of the main tests to be performed. To conclude, the essential part of the implementation of a high quality and efficient automatic TFF process was conducted to facilitate future expansion of the production of LIA.