Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

The disinfection of wastewater is not something that is currently in use at any wastewater treatmentplant in Sweden. The government has however stated some requirements for some wastewatertreatment plants to have a plan to implement disinfection in their treatment process, if asked.Therefore the goal of the project is to research if disinfection with sodium hypochlorite can beimplemented at Slottshagen wastewater treatment plant. There are several factors that will affect the efficacy of the disinfection of the wastewater. Firstly, thedisinfection process is dependent on the pH of the water solution. This is because the weakhypochlorous acid has better disinfection than the hypochlorite ion, and hypochlorous acid is presentthe most in the solution at pH 3-6. Another factor that is important to consider is the amounts ofnutrients present in the solution, since the chlorine oxidizes the nutrients rather than reacting with theorganisms in the solution. Some of the products from the oxidation of the nutrients are bad for bothhuman health and the environment. Also the temperature of the water and the concentration of thechlorine will affect the disinfection. Lastly, depending on which organism that is sought out to bedisinfected, the chlorine will have more or less effect on that particular organism. To research the chlorination effect, different additions of sodium hypochlorite were added to samplesolutions from the treatment plant and got to react in the solution for 5 minutes. Following thesamples were neutralized with ascorbic acid, and the amount of E. coli , coliform bacteria andintestinal enterococci were analyzed. The disinfection of the water during 1,5 minutes and at a shortertemperature were also analyzed. The results showed that the temperature, the contact time with thesolution and the concentration all are important factors to reach a proper disinfection, but the resultsalso showed that the amount of bacteria in the solution also is an aspect to take into considerationwhen treating wastewater. Finally, the considerations of disinfection of the wastewater with sodium hypochlorite is discussed.The point of addition of the chlorine was concluded to best be directly after the last chemicaltreatment step. When analysing if the disinfection would be possible, the disinfection was from theresults possible, but it was noticed that the results fluctuated a lot. The most probable reason why isbecause of the water quality. The water quality in general fluctuates regularly throughout the day, aswell as in between days of the week. Therefore throughout the disinfection process, the additions ofchlorine would need to be depending on several parameters, making the disinfection difficult toimplement. One solution to this could be to add a lot of chlorine to the bulk water at all times, but itwould cost too much if treating the water for a longer time, as well as it would be a higher risk to theenvironment. A solution to this problem could be to possibly neutralize the water before it is releasedas well, however when analyzing the cost of the ascorbic acid that was used as neutralization in thisproject, the cost to neutralize the water completely would be too high. Lastly it was discussed that theonly time disinfection of the wastewater would give any desirable results, is if the specific organismsthat are being sought out to be treated are bacteria or some viruses. If the particular organism that iswished to be treated is parasites, the disinfection with chlorine would not work.

The aim of this work was to investigate and determine central parameters that can be used to control and increase the solubility, quality and productivity of recombinant proteins. These central parameters should be applicable under the constraints of high-throughput protein production in Escherichia coli. The present investigation shows that alternative methods exist to improve solubility, quality and productivity of the recombinant protein. The hypothesis is that by reducing the synthesis rate of the recombinant protein, a higher quality protein should be produced. The feed rate of glucose can be used to decrease the synthesis rate of the recombinant protein. The influence of feed rate on solubility and proteolysis was investigated using the lacUV5-promoter and two model proteins, Zb-MalE and Zb-MalE31. Zb-MalE31 is a mutated form of Zb-MalE that contains two different amino acids. These altered amino acids greatly affect the solubility of the protein. The soluble fraction is generally twice as high using Zb-MalE compared to Zb-MalE31. Using a low feed rate compared to high benefits the formation of the full-length soluble protein. Furthermore, by using a low feed rate, the proteolysis can be decreased. One other factor that influences the solubility is the amount of inducer used. An increase from 100 µM to 300 µM IPTG only results in more inclusion bodies being formed, the fraction of soluble protein is the same. The quality aspect of protein production was investigated for a secreted version of Zb-MalE using two different feed rates of glucose and the maltose induced promoter PmalK. It was shown that when the protein was secreted to the periplasm, the stringent response as well as the accumulation of acetic acid (even for high feed rates) was reduced. The stringent response and accumulation of acetic acid are factors that are known to affect the quality and quantity of recombinant proteins. Transporting the protein to the periplasm results in this case on a lower burden on the cell, which leads to less degradation products being formed when the protein is secreted to the periplasm. Seeing the feed rate as a critical parameter, the high-throughput production would benefit from a variation in the feed rate. However, since the fed-batch technique is technically complicated for small volumes another approach is needed. E.coli strains that have been mutated to create an internal growth limitation that simulate fed-batch were cultivated in batch and were compared to the parent strain. It was shown that the growth rate and acetic acid formation was comparable to the parent strain in fed-batch. Furthermore it was shown that a higher cell mass was reached using one of the mutants when the cells were cultivated for as long time as possible. The higher cell mass can be used to reach a higher total productivity. / QC 20101112

Systemisk skleros (SSc) är en autoimmun rheumatisk sjukdom som kännetecknas av fibros i huden och/eller de interna organen, vaskulopati och en autoimmun reaktion från immunförsvaret, med eller utan specifika autoantikroppsprofiler. Diagnostisering och sjukdomsbehandling försvåras av sjukdomens heterogena natur. Det finns därför ett behov av pålitliga autoantikroppsbiomarkörer som kan bistå vid diagnostisering av patienter. Autoantikroppar som förekommer i serum kan avslöja sjukdomstillståndet hos patienter samt indikera en prognos om de korreleras till ett specifikt klinisk symtom. Syftet med denna studie var att identifiera autoantikroppar som kanditater till nya biomarkörer inom systemisk skleros i hopp om att dessa ska kunna förbättra stratifiering av patienter, samt att addera kunskap om autoantikroppsreaktivitet inom sjukdomen. Autoantikroppsprofilerna i 107 serumprover, där 55 tillhörde SSc-patienter och 52 tillhörde inflammatoriska kontroller, analyserades i två faser med en planar antigen array för en initial objektiv identifiering av möjliga autoantikroppskandidater, som sedan verifierades i en suspension bead array. Resultaten konfirmerar reaktivitet mot kända autoantigen, så som centromer protein B (CENPB) och DNA topoisomeras I (TOP1), samt identifierar fosfatidylinositol-5-fosfat 4-kinas typ 2 beta (PIP4K2B) som en ny, potentiellt specifik, autoantikroppskandidat för sjukdomen. Tripartite motif containing 21 (TRIM21) som är ett känt autoantigen hos flera systemiska autoimmuna sjukdomar visar potential för att kunna stratifiera sjukdomen. Resultaten i denna studie adderar ny information till kontexten inom autoantikroppsreaktivitet hos patienter med systemisk skleros som efter en mer omfattande dataanalys förhoppningsvis kommer att vara användbar inom diagnostisering av patienter samt för att skräddarsy behandling av sjukdomen. / Systemic sclerosis (SSc) is a highly heterogeneous rheumatic autoimmune disease that is characterized by fibrosis of the skin and/or visceral organs, vasculopathy, and an irregular immune response with or without specific autoantibody profiles. The heterogenic nature of the disease creates a challenge in diagnosis and clinical management of patients. There is thus a need for reliable autoantibody biomarkers that could aid in patient stratification. Serum autoantibodies are indicative of the disease state and may reveal prognosis if correlated to a specific clinical feature. This study aimed to discover novel autoantibody biomarker candidates with the hopes of improving patient stratification and to provide additional knowledge on the autoantibody reactivity in systemic sclerosis. By using a two-phase study design, an autoantibody profiling of 107 serum samples, consisting of 55 SSc samples and 52 inflammatory controls, was performed using the planar antigen array for initial discovery and a suspension bead array for verification. Resulting data confirms reactivity to known targets such as centromere protein B (CENPB) and DNA topoisomerase I (TOP1), while also identifying phosphatidylinositol-5-phosphate 4-kinase type 2 beta (PIP4K2B) as a potential novel target that is specific to the disease. Tripartite motif containing 21 (TRIM21), a known target among several systemic autoimmune diseases, show potential as a target for patient stratification. The results of this study add new information to the context of autoantibody reactivity in systemic sclerosis, which after more extensive data analysis, could be useful in improving the stratification of patients and in tailoring treatment.

Neuroblastom är en barncancer som uppstår ur det sympatiska nervsystemet och drabbar omkring 15 barn i Sverige varje år. Högriskvarianten är associerad med mycket hög dödlighet och risk för återfall, vilket tros ha att göra med att tumörernas ovanligt heterogena sammansättning tillåter resistenta subpopulationer att motstå konventionella behandlingsmetoder. Tidigare forskning har identifierat rubbade mekanismer för celldelning som ett tillvägagångssätt för tumörcellerna att överleva DNA-skador som kemoterapeutiska droger orsakar. I detta masterprojekt analyserades fem ultra-högrisk neuroblastomcellinjer i syfte att belysa deras progression genom celldelningen efter behandling med doxorubicin och/eller gemcitabin. Vidare identifierades ataxia telangesia mutated (ATM) serine/threonine kinase som ett essentiellt protein vid inhibering av celldelningen och i samband med reparation av DNA-skador, vilket bekräftades av förhöjt uttryck av fosforylerat ATM i alla fem cellinjer efter behandling med doxorubicin, gemcitabin, och/eller en kombination av båda. Återväxt av tumörcellerna efter inhibering av fosforylerat ATM i kombination med doxorubicin och gemcitabin analyserades sedan, och fördröjd återväxt noterades i en av cellinjerna efter kombinationsbehandling. Sammantaget har nya mekanismer för behandlingsresistens hos tumörceller identifierats och alternativa kombinationsbehandlingar har visat effekt på en av fem testade neuroblastomcellinjer. / Neuroblastoma is a pediatric cancer of the sympathetic nervous system that afflicts around 15 children annually in Sweden. Despite aggressive treatment, high-risk neuroblastoma is associated with a mortality of 50% and relapse rate of up to 60%, emphasizing the need for novel treatment options. In this study, fluoresce activated cell sorting was used to analyze cell cycle progression in five ultra-high-risk neuroblastoma cell lines: BE(2)-C, Kelly, SK-N-AS, SK-N-DZ, and SK-N-FI, post-treatment with doxorubicin and gemcitabine. In line with previous research, doxorubicin primarily induced cell cycle arrest in G2/M-phase and gemcitabine in the S-phase. Combined, the compounds induced varied effects, with accumulation primarily in the G1-and S-phase. Immunoblotting revealed elevated levels of phosphorylated ATM (pATM), a key regulator of cell cycle arrest and DNA damage signaling, across all five cell lines post-treatment to doxorubicin, gemcitabine, and/or the combination, indicating its vital role in their survival. Kelly stood out in both cell cycle progression and ATM phosphorylation, exhibiting minimal to no changes in cell cycle accumulation or pATM expression when exposed to the combined treatment, despite reacting to both monotherapies. These results may indicate that Kelly might implement an alternative mechanism of regulation compared to the remaining cell lines. To explore targeted inhibition of pATM, we employed the ATM kinase inhibitor KU-55933, which in BE(2)-C cells reduced expression levels of pATM when combined with doxorubicin, but not gemcitabine or the combination. Regrowth assays showed increased efficacy of doxorubicin and gemcitabine upon addition of the ATM kinase inhibitor KU-55933 in one of the tested cell lines in comparison to doxorubicin alone. However, longer incubation time is needed before the effect can be fully evaluated. These findings shed light on the differential cell cycle behavior in high-risk neuroblastoma cell lines exposed to combination therapy and suggest a vital role of ATM in the DNA damage response following doxorubicin and gemcitabine treatment. Further investigations are warranted to explore alternative strategies for enhancing the effectiveness of doxorubicin and gemcitabine in the treatment of this aggressive cancer subtype.

Tjock-och ändtarmscancer (kolorektalcancer) är den tredje vanligaste cancertypen och den näst vanligaste cancer-relaterade dödsorsaken i världen. Östrogen har visat sig ha en skyddande roll mot kolorektalcancer och östrogenreceptor beta är den dominerande östrogenreceptorn i normalt kolonepitel. Immunceller påverkar utvecklingen hos tumörer och forskning på samspelet mellan cancerceller och immunceller kan vara viktig för framtida cancerforskning. Den här studien har för avsikt att undersöka hur koloncancerceller påverkar makrofager och vice versa genom att utföra samkultursexperiment och analysera genuttryck med RT-qPCR. Tumör-associerade makrofager (TAM) polariserades från THP-1 celler och odlades tillsammans med SW480-kolorektalceller med eller utan uttryck av ERβ. En immunfluorescens-analys gjordes på en musmodell av kolit (tarminflammation) för att undersöka andelen av olika immunceller i tjocktarmen. Analysen gjordes i QuPath och beräkningarna mellan en erfaren och en oerfaren användare jämfördes. Denna studie visade att bildandet av TAM:s med hjälp av konditionerat medium från SW480-celler ändrade genuttrycket mot en pro-inflammatorisk fenotyp. Samkultur-experimenten uppvisade motstridiga resultat men antyder att genuttrycket för TAM:s ändras av att vara i samkultur med SW480-celler och att genuttrycket för SW480-celler ändras av att vara i samkultur med THP- 1-celler. Vidare visade resultatet från samkulturen att ERβ-uttrycket i SW480 cellerna påverkade deras genuttryck. Immunofluorescence-analysen av mustarmen demonstrerade att immuncellen med den högsta andelen var dendritiska celler medan celltypen med minst andel var cytotoxiska T-celler, som till antalet var ungefär hälften så många som T-hjälparceller. Analysen visade att skillnaden mellan en erfaren och en oerfaren användare var signifikant för två av tio immunceller. Slutsatserna från denna studie var att SW480-celler har en inverkan på genuttrycket av TAM:s och att genuttrycket i SW480 påverkades av att vara i samodling med THP-1-celler polariserade till makrofagliknande celler (av PMA) eller TAM:s (genom konditionerat medium från SW480-celler). Vidare tycks ERβ påverka uttrycket av ICAM1 och IL-1β i SW480 celler under samkultur med makrofagliknande- eller TAM-THP-1 celler. Genom att fortsätta undersöka förhållandet mellan makrofager och kolorektalcancerceller kan forskningen breddas vilket kan leda till nya behandlingsmetoder i framtiden. / Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer-related mortality in the world. Estrogen has been found to have a protective role in the development of colorectal cancer and estrogen receptor beta is the predominant estrogen receptor in normal colonic epithelium. Immune cells influence tumor progression and research on the crosstalk between cancer cells and immune cells could be important in future therapies. This study aims to investigate how colorectal cancer cells influence macrophages and vice versa by conducting co-culture experiments and analyzing gene expression using RT-qPCR. Tumor-associated macrophages (TAMs) were polarized from THP-1 cells and cultured together with SW480 colorectal cancer cells with or without the expression of ERβ. Immunofluorescence analysis was performed on colon tissue samples from a colitis-induced mice model to investigate the percentage of different immune cells in the colon. The analysis was done in QuPath and the calculations between an inexperienced user and an experienced user were compared to investigate how the results differ. We found that the formation of TAMs using SW480 conditioned media changed gene expression toward a pro-inflammatory phenotype. The co-culture experiments showed conflicting results but suggest the gene expression of TAMs is altered by being cultured with SW480 and that the gene expression of SW480 cells was affected by being cultured with THP-1 cells. Further, the ERβ expression in SW480 cells affected the gene expression of the cells during co-culture with macrophage-like or TAM THP-1 cells. In the immunofluorescence analysis of mouse colon, the immune cell type with the highest abundance was dendritic cells and the lowest seem to be cytotoxic T-cells, which was around half of the number of T-helper cells. There was a significant difference between the analysis of experienced and inexperienced annotators for two out of ten markers. The conclusions from this study were that SW480 cells have an impact on the gene expression of TAMs and that the gene expression in SW480 was influenced by being in co-culture with THP-1 cells polarized into macrophage-like cells (by PMA) or TAMs (by conditioned media from SW480 cells). Further, ERβ impacted the expression of ICAM1 and IL-1β in SW480 cells during co-culture with macrophage-like or TAM THP-1 cells. By further studying the correlation between macrophages and CRC cells, the research can be broadened which can lead to new approaches to CRC therapies in the future.

The development of a biologic protein involves different stages and becomes a highly complex process which can be costly and time consuming to scale up for industrial production. Therefore, optimization is a necessary part of the production process development to lower the production expenses.An on-going project is working on upscaling the production of a protein derived from mussel adhesive proteins (MAPs) which has great properties to be used as a pharmaceutical drug or in medical devices. The protein is expressed in a bacterial host cell and the necessary post translational modifications (PTMs) are done in-vitro using enzymes. The work presented in this report was done to optimize both the protein production in lab scale bioreactors and the enzymatic reaction using an immobilized prolyl-4-hydroxylase (P4H) which does a post translational modification on prolyl-residues. Additionally, an interaction study was conducted to better understand the hydroxylation using the prolyl-4-hydroxylase.For the bioreactor optimization four initial trials were performed testing different growth and induction temperatures and also comparing exponential to linear feeding. From these trials it appeared that having 30 ℃ growth overnight and induction at the same temperature in combination with an exponential feeding rate gave the best results. The modifications done by the prolyl-4-hydroxylase were analysed by LC-MS and suggest that longer incubation time and more immobilized protein gives more modifications in the tested ranges and the possibilities of reusing the immobilized proteins looks promising. No conclusive data was discovered for the optimal substrate concentration. The interaction study revealed the importance of reagents used for catalysis with the enzyme to be present for interaction to occur, however more work needs to be done to discover an accurate KD for the interaction.

G-protein coupled receptors (GPCRs) play a key role in a broad range of biological processes by binding to a wide variety of signaling molecules, which have resulted in 34% of all FDA-approved drugs which target GPCRs. The human genome encodes for approximately 800 GPCR members of which about 140 non-olfactory receptors remain orphans with an unknown function and endogenous ligand. Despite prolonged efforts to deorphanize the unresolved receptors, they remain orphans until this day. By studying scientific publications, this thesis has clarified the challenges with the deorphanization of GPCRs to explain why there are still so many orphan GPCRs when they have confirmed involvement in so many human disorders.

Kardiovaskulära sjukdomar är fortfarande en av de främsta dödsorsakerna i dag. Den främsta orsaken till sjukdomen är skador på hjärtcellerna, som kanske inte kan repareras av sig själva, så att utveckla en ny terapi för att regenerera eller reparera hjärtcellerna är ett viktigt mål inom forskningen om hjärtsjukdomar. Tidigare forskning har visat att vissa kardiogena parakrina faktorer som tillväxtfaktorer eller utsöndrade proteiner kan påverka hjärtats utveckling och regenerering. I den här studien valdes en av de parakrina faktorerna, placentaväxtfaktor (PLGF), ut för att verifiera dess inflytande på kardiomyogenes och vaskulogenes genom in vivo- och in vitro-experiment. Genom in vivo-experiment injicerades humana embryonala stamceller (hESC) som innehöll kemiskt modifierat PLGF mRNA i njurkapselskiktet hos musen, och en månad senare samlades njurarna in och färgades genom sektionering för att bestämma differentieringen av hESC. Genom in vitro-experiment inducerades hESC som avlägsnats PLGF-genen genom CRISPR-Cas9 till tre olika celler (kardiomyocyter, glatta muskelceller och endotelceller) enligt ett etablerat protokoll, och sedan analyserades cellerna med fluorescensaktiverad cellsortering (FACS) för att bestämma differentieringsnivån. Enligt experimenten krävs PLGF för induktion av kärlceller in vitro (endotelceller och glatta muskelceller) och skapandet av in vivo-kärl som genererats från hESCs, och det kan också påverka induktionen av kardiomyocyter i viss utsträckning, vilket krävde ytterligare forskning. Denna studie ger information om PLGF:s kritiska funktioner i kardiomyogenes och vaskulogenes, vilket kan bana väg för utvecklingen av nya terapier för regenerering av hjärtat. / Cardiovascular disease is still a leading cause of mortality nowadays. The main cause of the disease is the damage to heart cells, which may not be repaired by itself so developing a new therapy to regenerate or repair the heart cells is a major goal in cardiac disease research. Through previous research, some cardiogenic paracrine factors like growth factors or secreted proteins may influence heart development and regeneration. In this study, one of the paracrine factors, the placental growth factor (PLGF), was selected to verify its influence on cardiomyogenesis and vasculogenesis through in vivo and in vitro experiments. Through in vivo experiments, human embryonic stem cells(hESCs) containing PLGF mRNA which was modified chemically were injected into the mouse kidney capsule layer, and one month later the kidneys were collected and stained by sectioning to determine the differentiation of hESCs. Through in vitro experiments, hESC which were removed PLGF gene by CRISPR-Cas9 were induced into three different cells (cardiomyocyte, smooth muscle cell, and endothelial cell) followed an established protocol, and then the cells were analyzed by fluorescence-activated cell sorting (FACS) to determine the differentiation level. According to experiments, PLGF is required for the induction of in vitro vascular cells (endothelial cell and smooth muscle cell) and the creation of in vivo vasculature generated from hESCs, and it may also influence cardiomyocyte induction to some extent which needed further research. This study offers information on the critical functions of PLGF in cardiomyogenesis and vasculogenesis, potentially paving the way for the development of new heart regeneration therapies.

Microalgae are gaining more attention for several reasons such as being potential producers of sustainable fuel, for use as health supplements and in skincare. Simris Alg is a Swedish company that produces Omega 3 supplements from a primary producer of these fatty acids - the algal diatom Phaeodactylum tricornutum, which is a sustainable alternative to Omega 3 derived from fish. Omega 3 fatty acids constitute a small fraction of the total algal biomass, and to increase profitability and utilize all of the biomass, the purpose of this thesis project is to present potential applications for the residual material that is left after oil extraction. A general composition study was made of Simris Alg algal residue material, and results are compared to those found in previous studies of P. tricornutum biomass. An optimization of the fractionation is needed to separate the storage carbohydrate chrysolaminarin and cell wall component glucuronomannan, followed by analysis for confirmation. Also, it would be interesting to separate chitin, if there is any, since despite the presence of chitin synthases, it is unclear whether the diatom actually produces chitin. When gathering information, no actual experimental characterization of carbohydrate active enzymes involved in synthesis of the main carbohydrates investigated were found. Such information would be useful to increase production of the carbohydrate of interest, if valuable applications are found. Potential applications of various cell components, such as carbohydrates, in skincare would be interesting to investigate, as well as optimizing fucoxanthin extraction for use as an additional high value product next to Omega 3.