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- Post3D Bioprinting Meniscus for Future Tissue Replacement in Osteoarthritis Affected Knee Joints(2020) Pettersson, Ida; Olsson Widjaja, Amanda; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Gatenholm, Paul; Simonsson, StinaProgressive cartilage defects in human knee joints are a worldwide health issue that exposes a great burden on society as well as on single individuals. Sudden traumas to the knees can induce tears in the structures of the joints such as the articular cartilage and the meniscus. Lesions in the cartilage tissue of the knee joints have a high probability to eventuate in osteoarthritis (OA). The emerging technique of 3D bioprinting tissue is a novel approach for cartilage repair and regeneration. The choice of bioink and cell type are important factors that considerably impact the resulting cartilage repair potential after the process of 3D bioprinting. Induced pluripotent stem cells possess the ability to differentiate into almost any cell type and their potential in future regenerative medicine is of great interest. The present study has investigated the possibility of 3D bioprinting chondrocytederived human induced pluripotent stem (iPS) cells into meniscus structures with initiated cartilage regeneration. A combination of nanofibrillated cellulose (NFC) and alginate (A) was used as bioink. At first, the bioink was subjected to optimization to augment printing properties and cell viability during and after bioprinting. The composition of nanofibrillated cellulose and alginate originated from a ratio of 60/40 NFC/A (% w/w) that was compared with one ratio of 48/52 NFC/A (% w/w) and one ratio of 80/20 NFC/A (% w/w). The optimization of bioink involved measurements of printability, rheology, and cell viability. The formation of microtissues enabled differentiation of iPS cells toward extracellular matrix (ECM) producing cells prior to 3D bioprinting. 3D bioprinted menisci, as well as cultured microtissues, were analyzed with LIVE/DEAD assay, immunohistochemical analysis, and quantitative reverse transcription polymerase chain reaction (qRT PCR). The work also comprised an in vivo assay of 3D bioprinted constructs transplanted subcutaneously in mice to enable the evaluation of tissue formation in a realistic milieu. Induced pluripotent stem cells further modified to express green fluorescence protein under the aggrecan promotor have been subjected to a screening of a library of small Food and Drug Administration (FDA) approved molecules. Changes in intensity indicated induction or inhibition of the synthesis of aggrecan. Moreover, the screening highlighted molecules included in the induction or inhibition of aggrecan production. These molecules will be subjected to further investigations to evaluate their possible contribution in future OA-treatments. The prevailing work demonstrates the feasibility of utilizing microtissues formed by iPS cells for the 3D bioprinting of cartilage tissue. The culturing of iPS cells as microtissues has proven to induce differentiation and synthesis of components included in the ECM, both in vitro and in vivo. Simultaneously, the study has included a comparison of three bioinks that resulted in an optimized protocol for the production of the bioink composed of 60/40 NFC/A (% w/w). This optimized bioink fulfilled the requirements of being printable while supporting cell viability.
- PostA CRISPR approach to manipulating NK cell receptor ligand expression(2018) Kristensson, Linnea; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringNatural killer (NK) cells are important innate lymphocytes in the immune system and have been considered to be vital in fighting cancer, virus infections as well as regulating immune responses. The activity of NK cells is regulated via a complex signaling between inhibitory and activating receptors. Ligands for most of the ac-tivating receptors have been discovered but no endogenous ligand for one of the important natural cytotoxicity receptors, NKp46 has been identified. Therefore, the long term aim of this thesis project was to define ligand candidates for the acti-vating receptor NKp46 with intermediate aims to manipulate expression of NK cell activating receptor ligands in the K562 cell line using a CRISPR approach. The result from the performed cytotoxicity assays demonstrated the importance of the activating receptors NKp30 and DNAM-1 in elimination of K562 cells and the expression of the complementary ligands NCR3LG1, PVR and NECTIN2 was confirmed using RT-qPCR. The successful creation of a Cas9-expressing K562 cell line was demonstrated, including verification of Cas9 protein expression using both flow cytometry and western blot. Following that, a constructed plasmid with the gRNA for the B7-H6 gene, NCR3LG1, was generated and shown to create a cleavage and mutation within the gene. The engineered cell line was ultimately shown to have a reduced expression of the B7-H6 protein, indicating the successful knockout of the NCR3LG1 gene.
- PostA kinetically-constrained FBA-model of the synthesis of aromatic amino acid-derived products in Saccharomyces cerevisiae(2015) Janasch, Markus; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringDuring the last decades the research interest for creating a more sustainable and environmentallyfriendly society and industry has increased dramatically. The employment of icroorganisms to create valuable compounds such as fuels and chemicals from renewable resources has gained high popularity. With directed modifications in the metabolism of these microorganisms, metabolic engineering seeks to optimize the properties of these organisms for an efficient bioprocess to produce valuable compounds. One characteristic of metabolic engineering is the extensive use of computational models to predict the behavior of the metabolism and thereby identifying suitable targets for genetic interventions in an in silico to in vivo progress. Flux Balance Analysis (FBA) is a popular analysis method for metabolic models, as it only requires the underlying stoichiometric network of the metabolism modelled. With FBA the optimal flux distribution, given a certain objective to be maximized, can be calculated with linear programming algorithms. As FBA only takes the stoichiometry and reaction directionality into account, further physiological constraints have to be included in the framework to increase the predictive strength of the simulations. In this thesis, an expanded form of FBA, that includes kinetic enzyme parameters as additional constraints on the system, was used to analyze a metabolic model of the popular industrially-used organisms Saccharomyces cerevisiae, that included, additionally to the native metabolism, also recombinant enzymatic steps to form the plant secondary metabolites resveratrol and naringenin from the aromatic amino acids phenylalanine and tyrosine. Resveratrol and naringenin have been found to be beneficial to human health by offering anti-inflammatory, anti-carcinogenesis and anti-oxidant properties. The kinetically-constrained model was successfully used to estimate the impact of introducing recombinant pathways on the protein pool in the cell as well as to identify the enzymatic steps offering the highest control over the flux towards these products. This might be used in metabolic engineering to estimate the efficiency of metabolic pathways in the context of the whole metabolism form a protein cost point of view.
- PostA platform for Acetyl-CoA synthesis using xylose as feedstock in Saccharomyces cerevisiae(2015) Englund Örn, Oliver; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringGlobal rise in temperature and diminishing oil reserves has stimulated a market of alternative replacements to traditional petroleum based products. An alternative is the use of a bio refinery capable of converting biomass to the normally petroleum based products. The baker yeast Saccharomyces cerevisiae is an attractive cell factory as its existing large-scale infrastructures for bioethanol production. However, it cannot utilize xylose, an otherwise unusable part of the plant biomass, which represents of utmost importance in the bio-refinery development. To also have a strain capable to produce a wide range of products, it could be used as a platform to base a bio refinery upon. Therefore, the aim of this study is to generate platform strains capable of forming acetyl-CoA, an intermediate metabolite in many of the cells metabolic reactions and also for many other industrially relevant bio-chemicals. With this goal in mind, the metabolism of S. cerevisiae was engineered. The genes encoding an isomerase-based xylose assimilation pathway (RTG, XI, XKS), and a phosphoketolase pathway (XPK, PTA), were cloned into the yeast strain CEN.PK113-5D to enable the yeast to take up and convert xylose into acetyl-CoA. The functionality of this synthetic pathway were evaluated for the production of 3-hydroxypropionic acid via introduction of ACC1** and MCR genes into the engineered strains. By characterisation of all the engineered strains on glucose growth we found increase of acetate production in strains with the phosphoketolase pathway expressed, indicating the in vivo activity of this pathway. However, expression of the xylose assimilation pathway through genome integration did not render the strains able to grow on xylose, suggesting the low efficiency of the assembled xylose assimilation pathway. To overcome this adaptive laboratory evolution is recommended.
- PostA Rolling Circle Amplification-Based Methodology for Making Long, SequenceRepeating, DNA Duplexes(2018) Dekoning, Nora; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringIn vitro studies of sequence-specific DNA-protein interactions using techniques where long DNA molecules is needed are currently limited by the size of synthesized DNA molecules, or is restricted to the commercially available DNA. For single molecule DNA imaging techniques using for instance nanochannels confinement, the small sized molecules make the interactions difficult to detect. The commercially available DNA, on the other hand, does not allow any freedom in choosing or changing the DNA sequence. Therefore, a method for producing long DNA molecules containing a sequence of choice would alleviate these limitations and greatly improve the possibility to study DNA-protein interactions. The general concept of this paper was to create long, double-stranded DNA molecules with a sequence that is specifically designed to interact with the protein internal host factor. To make such molecules, three experimental procedures were developed, building on the single-stranded DNA products from a rolling circle amplification (RCA) reaction. The first experimental procedure was based on the presumed perfect annealing of smaller complementary strands to the single-stranded RCA product. The second experiment assumed that single-stranded gaps would be present in the duplex after annealing, hence the addition of a polymerisation reaction. In the third experiment, the RCA was run for 24 hours, to allow double-stranded product to be formed in the RCA. The DNA strands were visualized using fluorescence microscopy, with the goal of studying them and their protein interactions in nanochannels. To be able to use this detection method, an external fluorescent dye, YOYO, is used in the main aim of the project. However, as these types of dyes change the native structure of DNA, an extra aim was to use the fluorescent base analogue tC incorporated into one of the duplex strands, leaving the native structure intact. All three experimental procedures were shown to be capable of producing apparently double-stranded DNA molecules, that were larger than 100 kilo-base pairs albeit with a broad size distribution. This shows that the main aim in terms of procuing DNA molecules has been completed. The tC-containing DNA molecules were not visible under the microscope with the settings used, but appears to be promising.
- PostA Step Toward Personalized Cancer Treatment Simultaneous Detection of Multiple Types of Chemotherapyinduced DNA Damage Using Single Molecule Imaging(2022) Foss, Ebba; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering; Westerlund, Fredrik; Akwasi Aning, ObedChemotherapy is commonly used to treat cancer today, either alone or more commonly as part of combination therapy. Response to a certain chemotherapeutic agent is highly individual, both in terms of treatment efficacy and the extent to which healthy cells are affected. For several drugs, induced DNA damage provides the main cytotoxic effect, and a method for evaluating this damage could therefore prove a powerful tool in treatment planning. In this thesis, a single molecule imaging approach is used to assess chemotherapy-induced DNA damage, allowing visualisation of damage sites on individual DNA strands. While previous studies have focused on one damage type, or collective damage without distinction between types, a novel modification to pre-existing techniques that allows for this distinction has recently been demonstrated. In this thesis, the alkylating agent temozolomide was used to illustrate how different damage types can be distinguished with a single molecule imaging approach. This is done using repair enzymes associated with different DNA repair pathways. The repair enzymes sequentially incorporate spectrally distinct fluorescent nucleotides at the damage site which are then visualized as fluorescent spots of two different colours on individual DNA molecules. This distinction could be shown with high repeatability in terms of colour ratio. While both enzymes used separately clearly repaired the treated DNA, there appeared to be an overlap when applying them sequentially. This could suggest a problem with enzyme specificity. Further exploration of this issue is needed to verify the feasibility of single molecule imaging for the purpose of simultaneous detection of chemotherapy-induced DNA damage types.
- PostAccelerated shelf life tests of wheat tortillas A study of microbial and textural deterioration in wheat tortilla(2018) Nilsson, Fredrik; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThis study tried to shorten the time required for shelf life studies in product development of wheat tortillas, by developing accelerated shelf life test (ASLT) models for microbial and textural shelf life. ASLTs speed up product degradation by exposure to extreme environmental conditions and in this case were temperature and relative humidity (RH) used. The samples were divided into four storage groups: 3°C and 70% RH, 20°C and 30% RH, 27°C and 80% RH as well as 40°C and 80% RH. Texture was determined with a fold/roll-method, an instrument and by a sensory panel. Microbial concentrations were determined by a laboratory company. Gas composition inside the bags, tortilla pH, water content and water activity were measured to determine possible links to the shelf life. It was not possible to calculate an ASLT model for the microbial growth because of unknown starting number of microbes. The microbial tests however showed that a high temperature and humidity caused the number of bacteria to rapidly increase above what is considered acceptable. Microbial ASLTs thus seem possible, but more tests with a lower detection limit are needed to create a model. To avoid bad taste from nonpathogenic bacterial growth, consumers are recommended to avoid storing the tortillas in high temperature and/or humidity. Textural ASLTs also seem possible in regards of rollability and foldability, but more tests are needed to precisely determine the accelerating factor. Stickiness and translucency seems to deteriorate much slower than rollability and foldability under normal storage conditions. It is therefore suggested to be enough to check that freshly baked tortillas meet the quality requirements of stickiness and translucency. Tortillas seemed to lose textural quality at the same rate, or slower, in refrigerator compared to room conditions. Longer shelf life studies are therefore suggested, to verify if this is the case. If so, storage in a refrigerator could possibly prolong shelf life of wheat tortillas. Changes in the modified atmosphere inside the bags seems to correlate with textural degradation. A study with atmospheric air instead would therefore be interesting as an attempt to determine if it retards textural degradation in wheat tortillas.
- PostAdaptive evolution of Yarrowia lipolytica(2017) Hellgren, John; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringWe need renewable resources to allow sustainable production of fuels. By using lipid accu-mulating yeast, biodiesel can be produced in a sustainable way from resources that were previously not used, for example lignocellulose-based sources such as agricultural waste. However, for this process to be proﬁtable, the tolerance of the yeast needs to be improved. This project aims to improve the tolerance of the oleaginous yeast Yarrowia lipolytica to-wards osmotic and saline stress by using the method adaptive laboratory evolution. This method has previously been shown to be eﬃcient in constructing strains to tolerate new conditions without the need of prior knowledge. After evolving Y. lipolytica for 220 gen-eration in minimal medium containing 1.4 M NaCl, an improved performance in the same medium was observed, along with evolved cross-tolerance towards low pH. This indicates that this adaptive evolution of Y. lipolytica resulted in improved ionic tolerance rather than pure osmotic tolerance. The evolved strains were sent for whole genomic sequencing to ﬁnd out which mutations that caused this phenotype. During this project, two previous CRISPR/Cas9 strategies were combined and adapted for eﬃcient markerless reverse en-gineering. When genome data arrives, this strategy will be used for reconstruction of can-didate mutations to ﬁnd out which mutations are important for the observed phenotype. The gained knowledge from this evolution experiment can later be used for constructing a robust industrial strain that eﬃciently converts lignocellulose-based material to biodiesel, allowing sustainable production of fuels.
- PostAdaptive laboratory evolution of Saccharomyces cerevisiae for increased tolerance towards compounds of industrial interest(2016) Malina, Carl; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringOver the last decades, microbial production of fuels and chemicals has become an increas-ingly attractive alternative to petroleum-based production. This has created a demand cell factories able to produce a wide range of compounds. The yeast Saccharomyces cere-visiae is widely used in biotechnology with successful applications in the production of both bulk and fine chemicals. However, in order to reach the full potential of yeast as a cell factory challanges still remain. These include creating strains tolerant to stress conditions, such as inhibition at the high product titers required in industrial production. Traits conferring these properties are often complex and encoded by several genes. In order to obtain strains with improved tolerance, adaptive laboratory evolution (ALE) is often used. This thesis was part of an ongoing project of ALE for increased tolerance towards compounds with potential industrial applications. The work in this thesis can be divided into two main parts. The first part was screening for tolerance of S. cerevisiae towards four diols and two diamines by microplate cultivation. For both the diols and diamines, clear trends of decreasing fitness as compound concentration was increased was seen. For the diols tested, it seems increasing toxicity correlates with increasing chain length and branching. The second part was characterization of pimelic acid tolerant strains from ALE, by shake flask cultivation and HPLC analysis of metabolites. In addi-tion, the genomes of 21 strains were resequenced. Results from the shake flask cultures showed that, in presence of pimelic acid, most strains have an impaired growth on non-fermentable carbon sources. Furthermore, HPLC analysis of metabolites revealed that glycerol and acetate accumulated during cultivation while ethanol was slowly consumed, implicating a defective respiratory system. Through genome resequencing, in total 47 genes were found to be mutated across all of the evolved strains.
- PostAltered immune phenotype in patients with cirrhosis results in impaired vaccine-induced immunity following COVID-19 vaccination(2022) Blick, Elin; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Nygård, Yvonne; Martner, Anna
- PostAntimicrobial silk(2016) Floderus Savonen, Lotta; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringSpider silk has shown potential for use as a biomaterial. If fused to antimicrobial peptides (AMPs), recombinant spider silk could be used for medical applications and reduce the need to use conventional antibiotics to battle infections. Recombinant spider silk 4RepCT was fused to the AMPs Magainin I, Lactoferricin and a synthetically derived AMP referred to as WGR. Polystyrene disks were coated with the AMP-silk fusion proteins and the disks were incubated with cultures of Staphylococcus aureus and Escherichia coli, to test the AMP-silks antimicrobial activity. All AMP-silk fusion proteins significantly decreased bacterial adhesion of S. aureus to the disks after 48 hours of incubation compared to uncoated disks. The Mag- and WGR-silks were e↵ective already after 24 hours. The recombinant silk itself seemed to have an antimicrobial e↵ect by reducing bacterial adhesion of both bacterial strains to the polystyrene disks. Results indicated that addition of the AMPs improved this e↵ect on S. aurues, but not on E.coli.
- PostAssessment of fermentability of steam-pretreated spruce tips, needles and branches for bioethanol applications(2018) Asp, Tobias; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThe production of bioethanol from fermentation-based bioprocesses utilizing ligno-celluosic feedstocks is an option for replacing fossil based fuels. By using genetically modified yeasts that co-consume glucose and xylose, it is possible to ferment lingo-cellulosic materials such as spruce for bioethanol production. In this project the fermentability of spruce tips, needles and branches, pretreated by acid-catalyzed steam explosion according to a design of experiments plan, was evaluated in terms of ethanol titer, rate and yield as well as cell viability. In order to ferment the spruce tips, needles and branches, suÿcient cell concentrations are needed. A preculture method was developed where enough cells were produced and harvested in the same physiological state in di˙erent batches for simultaneous saccharification and co-fermentation. A anaerobic shake flask system was used to ferment the spruce tips, needles and branches. Final ethanol titers of up to 10 g l-1, average volumetric ethanol production rates of up to 0.35 g l-1 h-1 and final yields of ethanol on available glucose and on available glucose together with xylose of up to 0.19 gEthanol gGlucose-1 and 0.16 gEthanol gGlucose+Xylose-1 respectively were observed. The final cell concentrations, colony forming units and growth rates observed were all fairly low values of up to 3.00 x 105 cells ml-1, 1.19 x 107 CFU ml-1 and 2.36 x 10-2 h-1 respectively. Even though there may be some indications on preferable pretreatment conditions in terms of fermentability, more testing and experiments are required to make statisti-cally significant recommendations.However, trends observed in this project points to either high temperature and short time or low temperature and long time as prefer-able pretreatment process conditions. Materials pretreated under these conditions showed the highest final titers and yields.
- PostBio-electrochemical activity of Clostridium ljungdahlii at different cathodic potentials(2021) Hadi, Fahim; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Olsson, Lisbeth; Im, ChaehoHumans are responsible for a large portion of the emissions of CO2 gas since the industrial revolution. These emissions must decrease so that the increase of temperature should not exceed an increase of 2 °C by the end of this century. Gas fermentation is a promising technology that could reduce the emissions of CO2 into the atmosphere. Clostridium ljungdahlii is an interesting anaerobic microorganism capable of CO2 fixation and produce valuable chemicals such as ethanol. C. ljungdahlii is also known for the capability of accepting electrons from an electrode in a bio-electrochemical system. The bio electrochemical activity of C. ljungdahlii was thus investigated at the cathodic potentials - 400, -600, -800 and -1000 mV. This was done by running biotic and abiotic bio electrochemical system experiments at the mentioned potentials, as well as serum flask control experiments. The results were analysed by investigating product formation, growth, pH, current consumption, and the mechanism of electron transfer in the bio-electrochemical system reactors. No planktonic cell growth was observed in the optical density measurements, but CO2 fixation by C. ljungdahlii in a bio-electrochemical system was observed. C. ljungdahlii could in a bio-electrochemical system produce 2,3-butanediol at - 800 mV, and though not reproducible, also ethanol at -600 mV. C. ljungdahlii seem on the other hand to consume formate at all potentials applied. The current consumption of C. ljungdahlii was very low indicating that H2 could have been produced but was not consumed by C. ljungdahlii.
- PostBiofilm Reduction Modelling in a Drip-flow Reactor. Developing a Method for Growing Biofilms in a Laboratory Setting, to Evaluate the Effects of an Anti-fouling Product Used in Paper Machines.(2020) Frithiofson, Emil; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Norbeck, Joakim; W. Janco, EmmaBacterial biofilms naturally form in paper machines, causing problems in production. Reducing the amount of biofilm formed is therefore of interest to the paper industry. To model biofilm reduction, a method was developed for growing biofilms in a laboratory setting at BIM Kemi Sweden AB. The goal was for the method to be used to evaluate effect of the anti-fouling product Bimogard produced by the company, a non-biocidal agent that reduces formation of process disrupting biofilms in paper machines. Initial experiments were carried out using petri dishes for cultivation, but the main part of the work was carried out using a drip-flow biofilm reactor. The biofilms were quantified by staining with safranin and measuring absorbance. Different process parameters for running the reactor were examined and improved, including what medium concentration to use and whether to inoculate with a mono-culutre of Pseudomonas fluorescens or a co-culture where Bacillus subtilis was also added. The final method, using the mono-culture and 7.5% of standard medium concentration, was used to evaluate the effects of adding Bimogard to the nutrient medium. The addition of Bimogard significantly reduced the amount of biofilm formed, but only at low concentrations.
- PostBiophysical approaches in a structure-guided SMYD3 ligand discovery(2019) Talu, Martin Johannes; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Norbeck, Joakim; Talibov, Vladimir O.In eukaryotic cells, DNA is wrapped around nucleosomal cores formed of protein heterooctamers, which consist of core histones. Nucleosomes are the main units of chromatin organization. Chromatin exist in two states - either as eu- or heterochromatin, which either promotes or silences gene expression as a consequence of its packing states. Eukaryotic cells have developed epigenetic regulation to control the chromatin state, and to guarantee a high level of differentiation. The basis of epigenetic regulation are patterns of post-translational modifications of the nucleosomal proteins. These modifications are performed by epigenetic enzymes. This thesis focuses on one of these enzymes - the human lysine methyltransferase SMYD3. SMYD3 is also capable of interacting with certain cytosolic proteins, such as the molecular chaperone HSP90 - the human Heat Shock Protein 90. Both proteins are of high interest in the drug research and development landscape, as a drastic change in their activity and expression levels have both been shown to be related to several cancers or neurodevelopmental diseases. In this work, various truncated forms of HSP90 were produced and probed for their interactions with SMYD3 using surface plasmon resonance-based biosensor technology. A previously reported interaction of SMYD3 with the C-terminal domain of HSP90 was confirmed, with an affinity discovered to be KD = 1.3 × 10−5M. Additionally, the biosensor-based assay was used to test potential ligands of SMYD3, including low affinity fragment-like organic molecules. To complement the study, extensive crystallization and co-crystallization trials were carried out with SMYD3. As a result, conditions for the formation of various crystal forms of SMYD3 were mapped, with the best crystal form found to have high stability and good diffraction properties. A set of experiments presented herein develops expertise in the tools one can use for an efficient and rational ligand discovery campaign targeting SMYD3 histone methyltransferase.
- PostBioprospecting for novel laminarin-degrading enzymes in marine microorganisms - A step towards the use of macroalgae in bioprocesses(2015) Olsson, Joakim; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringCurrently, efforts are being made to find alternative biomasses for biofuel production, which are not competing with food production. Several kelp species have potential for large-scale cultivation, harvest and biorefinery processes. On the Swedish west coast, Laminaria digitata and Saccharina latissima are two such species. These kelps can contain up to 33% of the storage carbohydrate laminarin (dry weight), which can be hydrolysed to glucose and subsequently converted into bioethanol by for instance yeast. Enzymes able to digest the β-1,3 or β-1,6 bonds of the laminarin, to release glucose, are in large unknown and this project has been about finding microorganisms expressing such enzymes, known as laminarases. Samples from partly decomposed L. digitata and S. latissima specimens were streaked on nutrient agar plates for proliferation and isolation of surface microorganisms. Isolated organisms were subsequently screened for growth on laminarin and promising strains were identified through 16s/18s rRNA sequencing. After growth experiments on liquid medium with different carbon sources, two bacterial strains, Pseudoalteromonas ssp., were selected for further characterisation. The two strains were grown in algae extract and the sugar composition was monitored over time. In the extract, mainly mannitol and laminarin were present but glucose was formed during the cultivation, indicating the presence of hydrolytic enzymes, after which the hydrolytic activity on pure laminarin was investigated. The supernatants of one of the strains showed in vitro activity, further indicating the presence of extracellular hydrolytic enzymes. More investigations are needed on whether the enzymes can be used in any type of process. This thesis work has been successful in isolating marine organisms with laminarin-degrading activity.
- PostBiotransformation of brewer’s spent grain and application as a food ingredient in extruded breakfast cereals(2020) Thorvaldsson, Mira; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Alminger, Marie; Krona, AnnikaBrewers' spent grain (BSG) is a beer brewing by-product, that mainly consists of fibers (more than 50%) and proteins (up to 30%). BSG has, due to its content, a big potential to be used in the food industry but it is currently used as animal feed or discarded. To improve BSG's usability as a food ingredient bioprocessing with slime producing lactic acid bacteria has been used in this project. Incorporation of fermented BSG as a food ingredient in breakfast cereals has been performed with extrusion method. A process and recipe have been developed to produce breakfast cereal prototypes. Evaluations of the taste and texture of the breakfast cereal prototypes have been performed with texture measurements but mainly with consumer panels (focus groups). The final breakfast cereal prototypes produced for the last focus group were all with 30% fermented BSG. The BSG were from three different breweries, Dugges, Senson and Peroni, and the fermentation was performed with and without slime production by the lactic acid bacteria. The most preferred breakfast cereal prototype was with BSG from the brewery Dugges and fermented with slime producing lactic acid bacteria. The conclusion of this master thesis project was that it is possible to make breakfast cereal products with a content of 30% fermented BSG with extrusion method with a pleasant taste and texture.
- PostBreaking intrinsic antibiotic resistance. Outer membrane-permeabilizing peptides from innate bacterial proteomes.(2022) Persson, Jonatan; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Westerlund, Fredrik; Schäfer, Ann-Britt; Wenzel, MichaelaIntrinsic antibiotic resistance is the innate ability of bacteria to resist antimicrobial agents. The most defining structure that confers intrinsic antibiotic resistance, is the outer membrane of Gram-negative bacteria. It is a highly impermeable barrier that impedes various antibiotics from reaching their targets. A lot of research focuses on finding and developing novel drug candidates with increased antibacterial activity, but less research has been done on selective outer membrane permeabilization compounds. Such compounds could render Gram-negative bacteria susceptible to almost all known antimicrobials. It has been shown that the bacterial proteome is host to proteins containing a membrane-anchoring motif, which exerts selectivity towards the outer membrane. The aim of this Master thesis is to investigate the outer membrane permeabilizing properties of peptides derived from membrane-binding domains of innate peptides. This is realized through development and optimization of assays to assess the peptides outer and inner membrane activity against Escherichia coli. In addition, antibacterial activity was tested against both E. coli and the Gram-positive Bacillus subtilis. Here I show that 12 out of 14 innate peptides derived from E. coli and B. subtilis are active against the outer membrane of E. coli. Furthermore, FtsA10, MreB9, MurG12, FtsY11 from E. coli and MinD11 from B. subtilis are selective towards the outer membrane well below their growth inhibitory concentrations.
- PostCell wall stress response proteins in B. subtilis and their potential as new antibiotic targets: YtrB and YtrE, subunits of a putative ABC transporter(2022) Hammer úr Skúoy, Pauline; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Wenzel, Michaela; Wenzel, MichaelaMulti-resistant bacteria exist worldwide and are a serious problem with 25 000 deaths per year only in the EU. The effort of finding new antibiotics has also reduced over the past decades due to resistance being established even before the drug reaches the market. A relatively new idea is to design antibiotics with multiple targets and/or multiple effects. One of these targets with multiple effects is the cell wall stress response. Previous investigations used proteomic studies of the cell wall stress response in Bacillus subtilis (B. subtilis) and found different marker proteins for different classes of antibiotics. In this study, we focused on the cell wall synthesis marker proteins YtrB and YtrE, which are annotated as nucleotide-binding subunits of a putative ABC transporter. The aim was to study their localisation, importance for growth at different temperatures, and their impact on antibiotic sensitivity to evaluate the transporter’s potential as a novel drug target. GFP fusions and deletion mutants were constructed and used in growth curve experiments, bacterial cytological profiling (BCP), and minimal inhibitory concentration (MIC) assays. Neither YtrB nor YtrE were of high importance for the growth in B. subtilis at different temperatures. They were, however, important for the resistance against the lantibiotic nisin and the beta lactams ampicillin and ertapenem. By connecting the localisation of YtrB in both rigid and fluid regions of the membrane to previous studies and to our MIC results, it can be hypothesised that the ABC transporter may have multiple functions. Among these are the function as an importer of precursor molecules for cell wall synthesis and/or the function as a membrane-bound remover or inhibitor of beta lactams. The ABC transporter has, as such, the potential of being a target for new potentiators: sensitizers to decrease the cell wall thickness or increase the activity of beta lactams. However, more studies need to be done with additional antibiotics and strains (a reporter fusion of the promoter of the ytr operon, whole ytr operon deletion, and N-terminal GFP fusion to YtrE) to determine their potential use within healthcare.
- PostCharacterization and differentiation of MAFA reporter hiPS cells into pancreatic beta cells(2019) Sjölin, Jacob; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Esbjörner Winters, Elin; Gupta, ShaileshStem cell research is a wide field within science. One of the main research areas with the field is the differentiation of various cell types. However one of the main difficulties within this research area is to achieve a high yield of target cells from the differentiation process. This project aims at improving the yield of pancreatic beta cell differentiation by utilizing a reporter cell line for V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA). This reporter line has genetically tagged MAFA with green fluorescent protein (GFP). As MAFA is only expressed in beta cells, this cell line can be used in order to sort beta cells, which are expressing the GFP-tagged MAFA, from non-beta cells using fluorescent activated cell sorting (FACS). The cells were differentiated from human pluripotent stem cells (hiPSCs) into beta cells and were characterized at different stages using flow cytometry and immunofluorescence. In this project six differentiation experiments were performed, showing that MAFA-GFP is successfully expressed by beta cells at the end stages of the differentiation process. However, additional research is required to perform the cell sorting and test to the insulin response to glucose of these cells.