Browsing by Subject "biotekniikka"

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  • Kellock, Miriam (Helsingin yliopisto, 2022)
    Physicochemical lignocellulose pretreatment and subsequent enzymatic conversion of polysaccharides to platform sugars is an important technology in the valorisation of various lignocellulosic biomass streams. This technology is needed in the bio- and circular economy. Lignin, one of the main components in lignocellulose, is known to inhibit enzymatic hydrolysis by non-productively binding enzymes and sterically preventing enzymes to access cellulose. In this work, the aim was to elucidate how lignin in herbaceous plants and softwood is modified during pretreatment and what is the effect of pretreatment severity on lignin-derived inhibition in the enzymatic hydrolysis. Characteristics of cellulases and hemicellulases contributing to binding and inactivation on lignin were investigated. Spruce and wheat straw were hydrothermally pretreated with or without an acid catalyst at increasing severities, followed by isolation of the lignin to explore the inhibitory effects. Lignin inhibition in the enzymatic hydrolysis of microcrystalline cellulose Avicel increased with increasing pretreatment severity. When spruce and wheat straw were pretreated at the same severity, as assessed by the combined severity factor, lignins from both biomasses were equally inhibitory. Furthermore, lignin from mild pretreatment severities did not have a significant effect on the hydrolysis of Avicel. This indicate that the changes in lignin structure during pretreatment are the main reasons for the inhibitory effect of lignin. A decrease in β-O-4 aryl ether bond as well as a change in molecular weight of lignin was observed after pretreatment. The molecular weight of spruce lignin decreased, whereas the molecular weight of wheat straw increased after pretreatment. Degradation and polymerisation reactions competed during pretreatment and the net effect depended on biomass type and pretreatment severity. Lignin-derived inhibition in Avicel hydrolysis strongly correlated with the binding and inactivation of the cellobiohydrolase TrCel7A to lignin. TrCel7A is the main component in the Trichoderma reesei cellulase cocktails. The correlation between enzyme binding to lignin and inhibition in hydrolysis was studied using six purified enzymes common in cellulase cocktails, cellobiohydrolases TrCel7A, TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn2 from T. reesei and a β-glucosidase AnCel3A from Aspergillus niger. The cellobiohydrolases, an endoglucanase and a xylanase were all inhibited by isolated lignin. Interestingly, the most thermostable enzyme AnCel3A exhibited minor binding to lignin and was, in fact, activated by lignin. The activation of AnCel3A was the result of soluble lignin-derived compounds. The enzymes TrCel6A and TrCel7B, which exhibited strong binding to thin lignin films as analysed by quartz crystal microbalance (QCM), were also the enzymes that were most inhibited by lignin in hydrolysis assays. The interactions contributing to enzyme binding to lignin were enzyme-specific, however, some common interactions were identified. Enzymes containing a carbohydrate binding module from family 1 (CBM1), TrCel7A, TrCel6A, TrCel7B and TrCel5A, exhibited greater adsorption to lignin than the enzymes without a CBM. Furthermore, enzymes having a net positive surface charge bound to lignin more than enzymes with a net negative surface charge in the experimental pH. Enzyme surface hydrophobicity was computationally determined. Enzymes containing large uniform hydrophobic patches on the enzyme surface had stronger binding to lignin as only a low amount of enzyme was released from the lignin surface during rinsing with buffer. Thermal stability had a profound effect on lignin tolerance for family GH11 xylanases. Two T. reesei xylanases TrXyn1 and TrXyn2 as well as two forms of a metagenomic xylanase Xyl40 were all inhibited by lignin in hydrolysis assays and bound to lignin after incubation with enzymatically isolated lignin from steam pretreated spruce. Interestingly, the thermostable xylanases Xyl40 produced in Escherichia coli and the catalytic domain of Xyl40 produced in T. reesei remained partially active on the lignin surface, whereas the thermolabile TrXyn1 and TrXyn2 became inactive. N-glycosylation of the catalytic domain of Xyl40 did not affect the hydrolysis yield but had a significant effect on lignin tolerance. The glycosylated xylanase achieved higher hydrolysis yields in the presence of lignin than the deglycosylated xylanase. High thermal stability and structural glycans improved the lignin tolerance of the xylanases studied.
  • Elf, Sonja (Helsingin yliopisto, 2019)
    Despite recent advances in understanding, diagnosis and treatment of cancer, this complex and versatile disease remains one of the leading causes of death worldwide. New and rapid diagnostic methods are needed to detect cancers at their early stages of development, thus enabling earlier prognosis, better risk assessment and more efficient treatment of the disease. There has been an increasing interest in specific molecular biomarkers as the hallmark for cancer research, and the detection of these markers from liquid biopsies using advanced molecular diagnostics methods provides major advantages over the conventional imaging methods currently used in oncology. The aims of this thesis were to examine the applicability of a novel molecular method, SIBA® (Strand Invasion Based Amplification), for the detection of cancer biomarkers, and to develop an assay targeting androgen receptor splice variant 7 (AR-V7) mRNA. The AR-V7 is proposed as a treatment-response biomarker in patients with castration-resistant metastatic prostate cancer (mCRPC). The expression of this variant can indicate resistance to hormonal therapies used for the treatment of advanced prostate cancer. Prostate cancer is the most common cancer after lung cancer in men worldwide and can gradually develop into a highly advanced lethal form, mCRPC, that is not responsive to androgen deprivation therapies. Positive AR-V7 status is suggested to represent the phenotype of this advanced stage of prostate cancer, and its detection can assist in treatment selection for the mCRPC patients. SIBA is a novel isothermal method for the amplification and detection of nucleic acids. The technology offers significant advantages over the more conventional molecular detection method, polymerase chain reaction (PCR), since the amplification reaction occurs at constant temperature and does not require sophisticated laboratory equipment for the thermal cycling. Reverse transcription SIBA (RT-SIBA) enables reverse transcription of RNA to cDNA as well as the simultaneous amplification and detection of the cDNA in one-step reaction under isothermal conditions. The method displays both high analytical sensitivity and specificity to the target nucleic acids. The RT-SIBA technology has not formerly been applied for the detection of human DNA or RNA. The main finding of this thesis was, that the RT-SIBA technology can be applied for rapid detection of specific molecular cancer biomarkers such as the AR-V7 mRNA. In this study, two RT-SIBA assays targeting the full-length androgen receptor (AR-FL) mRNA and the AR splice variant 7 mRNA were developed and optimized. Performance of the assays were evaluated by testing RNA isolates from AR-V7 positive and negative prostate cancer cell lines in the presence of human whole blood and plasma in the reaction. The developed RT-SIBA assays provided high analytical sensitivity and specificity: low copies of the target mRNA were amplified within 20 minutes without the production of non-intended amplicons. The results suggest that the RT-SIBA technology can be utilized for easy and rapid detection of AR-V7 and AR-FL mRNA directly from liquid sample material without a need for time-consuming sample treatment. Further performance evaluation using real AR-V7 positive clinical samples from mCRPC patients is necessary for the reliable validation of the developed assays.
  • Häggman, Hely (Suomen metsätieteellinen seura, 1991)
  • Hyvärinen, Kati Susanna (2001)
    Gene technology is a rapidly growing area in the field of biotechnology. People's lives are pervaded by a flow of biomedical knowledge showing the associations between biology and identity. Gene testing has already improved lives of a number of people. Some reliable, low cost tests are used to clarify a diagnosis and direct a physician toward appropriate treatments, while others allow families to avoid having children with devastating diseases or identify people at high risk for conditions that may be preventable. The goal of this paper is to study the kind of attitudes Finnish general population has toward gene tests. Rapidly developing gene technology gives people the reason to think of how to act in a situation where the hereditary make up of the future generations can easily be altered to please ones ideals. People were asked to describe in their own words what benefits and drawbacks do they find in prenatal gene tests. The basic finding of my study is that people people are concerned about the new innovations, yet raising hopes for the new discoveries. Respondents show more positive attitudes toward prenatal gene tests than negative. The most crucial finding, however, is that the tests are able to provide information about the health of a baby. Everyone is wishing for healthy offspring and hopes to avoid unnecessary pain and worries. The most active respondents seem to be females in the most acute child bearing age. Marital status, nor ones education level do not seem to explain my findings. Income level, however, does indicate that people with more money seem to have stronger attitudes toward the issue. Prenatal gene tests raise a lot of feelings because they clearly have two different sides to an issue. The ones against the testing are afraid of its possible effects to eugenics, the ones in favor of the test plead to the decrease of human suffering. The question is difficult do to the fact that it has no either or answer. Public needs more information about the issue as well as clear laws and regulations to follow the correct use of those tests.
  • Ollikainen, Pia (Helsingin yliopisto, 2013)
    Bovine milk is a rich source of nutrients, such as protein, fat, carbohydrate, minerals and vitamins. In addition to the major protein components of casein and whey proteins, milk has low concentrations of compounds with bioactivity, growth factors and insulin. Insulin-like growth factor I (IGF-I) and transforming growth factor-β2 (TGF-β2) are the most studied because of their various growth-promoting activities and the possibility to use them as bioactive supplements in foodstuffs. In the present work heat-stability and separation of IGF-I, TGF-β2 and insulin were studied using ELISA immunoassay. Milk from a dairy farm was heat-treated at 65, 72, 90 or 135⁰C for 15 s using an indirect pilot heating processor. Both IGF-I and TGF-β2 showed good heat stability up to 90⁰C, but at 135⁰C both growth factors had lost most of their immunochemical activity. Heating also activated the latent forms of IGF-I and TGF-β2 to immunoreactive form, which was possible to analyse without any separate pre-treatment. Heat-activation of TGF-β2 was temperature-dependent and at 90⁰C all the TGF-β2 concentration was activated by heat. IGF-I was less sensitive, but at 90⁰C it showed a sharp increase in immunoreactivity. Heating as a pre-treatment method was introduced as a new method to analyse and compare IGF-I concentrations in samples with no or low whey protein concentrations. When heat-treated milks were separated to casein and whey fractions by acid, ultracentrifugation or membrane filtration, the heat-activated immunoreactive form of the growth factor followed the casein fraction and the latent form remained in whey. Bovine insulin was heat stable during pasteurization at 65 and 72⁰C, but lost some of its immunochemical activity at higher temperatures. During membrane filtration a higher share of insulin was concentrated in MF retentate than in UF retentate when compared to IGF-I. An in vitro digestion experiment was performed in order to compare the digestibility of the latent and active forms of TGF-β2. In the adult model TGF-β2 was hydrolysed in gastric phase, but in the infant model a small share of TGF-β2 survived the gastric and duodenal phases. There was no difference in survival or digestibility between the latent or active forms of the growth factor. The digestibility of IGF-I was tested in the infant model and this growth factor was hydrolysed during duodenal phase. The results obtained in this study have great importance when separating and concentrating the growth factors from milk or colostrum. If the separation and concentration is performed from whey, these results show that the heating history of milk has a great effect on the yields. The results also demonstrate that heating as a pre-treatment method and membrane filtration can be used to lead growth factors to different fractions, depending on the intended usage.
  • Koivistoinen, Outi (Helsingin yliopisto, 2013)
    The use of metabolic engineering as a tool for production of biochemicals and biofuels requires profound understanding of cell metabolism. The pathways for the most abundant and most important hexoses have already been studied quite extensively but it is also important to get a more complete picture of sugar catabolism. In this thesis, catabolic pathways of L-rhamnose and D-galactose were studied in fungi. Both of these hexoses are present in plant biomass, such as in hemicellulose and pectin. Galactoglucomannan, a type of hemicellulose that is especially rich in softwood, is an abundant source of D-galactose. As biotechnology is moving from the usage of edible and easily metabolisable carbon sources towards the increased use of lignocellulosic biomass, it is important to understand how the different sugars can be efficiently turned into valuable biobased products. Identification of the first fungal L-rhamnose 1-dehydrogenase gene, which codes for the first enzyme of the fungal catabolic L-rhamnose pathway, showed that the protein belongs to a protein family of short-chain alcohol dehydrogenases. Sugar dehydrogenases oxidising a sugar to a sugar acid are not very common in fungi and thus the identification of the L-rhamnose dehydrogenase gene provides more understanding of oxidative sugar catabolism in eukaryotic microbes. Further studies characterising the L-rhamnose cluster in the yeast Scheffersomyces stipitis including the expression of the L-rhamnonate dehydratase in Saccharomyces cerevisiae finalised the biochemical characterisation of the enzymes acting on the pathway. In addition, more understanding of the regulation and evolution of the pathway was gained. D-Galactose catabolism was studied in the filamentous fungus Aspergillus niger. Two genes coding for the enzymes of the oxido-reductive pathway were identified. Galactitol dehydrogenase is the second enzyme of the pathway converting galactitol to L-xylo-3-hexulose. The galactitol dehydrogenase encoding gene ladB was identified and the deletion of the gene resulted in growth arrest on galactitol indicating that the enzyme is an essential part of the oxido-reductive galactose pathway in fungi. The last step of this pathway converts D-sorbitol to D-fructose by sorbitol dehydrogenase encoded by sdhA gene. Sorbitol dehydrogenase was found to be a medium chain dehydrogenase and transcription analysis suggested that the enzyme is involved in D-galactose and D-sorbitol catabolism.   The thesis also demonstrates how the understanding of cell metabolism can be used to engineer yeast to produce glycolic acid. Glycolic acid is a chemical, which can be used for example in the cosmetic industry and as a precursor for biopolymers. Currently, glycolic acid is produced by chemical synthesis in a process requiring toxic formaldehyde and fossil fuels. Thus, a biochemical production route would be preferable from a sustainability point of view. Yeasts do not produce glycolic acid under normal conditions but it is a desired production host for acid production because of its natural tolerance to low pH conditions. As a proof of concept, pure model substrates, e.g. D-xylose and ethanol, were used as starting materials for glycolic acid production but the knowledge can be further applied to an expanded substrate range such as biomass derived sugars. Already the introduction of a heterologous glyoxylate reductase gene resulted in glycolic acid production in the yeasts S. cerevisiae and Kluyveromyces lactis. Further modifications of the glyoxylate cycle increased the production of glycolic acid and it was successfully produced in bioreactor cultivation. The challenge of biotechnology is to produce high value products from cheap raw materials in an economically feasible way. This thesis gives more basic understanding to the topic in the form of new information regarding L-rhamnose and D-galactose metabolism in eukaryotic microbes as well as provides an example on how cell metabolism can be engineered in order to turn the cell into a cell factory that is able to produce a useful chemical.
  • Rahikainen, Jenni (Helsingin yliopisto, 2013)
    Lignin, a major non-carbohydrate polymer in lignocellulosic plant biomass, restricts the action of hydrolytic enzymes in the enzymatic hydrolysis of lignocellulosic feedstocks. Non-productive enzyme adsorption onto lignin is a major inhibitory mechanism, which results in decreased hydrolysis rates and yields and difficulties in enzyme recycling. The mechanisms of non-productive binding are poorly understood; therefore, in this thesis, enzyme-lignin interactions were studied using isolated lignins from steam pretreated and non-treated spruce and wheat straw as well as monocomponent cellulases with different modular structures and temperature stabilities. The origin of the isolated lignin had an undisputable effect on non-productive binding. Ultrathin lignin films, prepared from steam pretreated and non-treated lignin preparations, were employed in QCM adsorption studies in which Trichoderma reesei Cel7A (TrCel7A) was found to bind more onto lignin isolated from steam pretreated biomass than onto lignin isolated from non-treated lignocellulosic biomass. Botanical differences in lignin chemistry had only a minor effect on nonproductive binding when enzyme binding to non-treated wheat straw and spruce lignin was compared. Increase in temperature was found to increase the inhibitory effect arising from non-productive enzyme binding to lignin. Different enzymes were shown to have a characteristic temperature at which the inhibition emerged. Thermostable enzymes were the most lignin-tolerant at high temperatures, suggesting that in addition to the surface properties of an enzyme, non-productive binding onto lignin may be influenced by stability of the enzyme structure. In addition, for lignin-bound T. reesei cellulases, increase in temperature resulted in loss of catalytic activity and tighter binding, suggesting that at high temperature enzyme binding to lignin was probably coupled to conformational changes in the protein folding. With TrCel7A, carbohydrate-binding module (CBM) was found to increase nonproductive adsorption to lignin. The Talaromyces emersonii Cel7A catalytic module was linked to a CBM from TrCel7A, giving rise to a fusion enzyme TeCel7A-CBM1. Despite a similar CBM, TeCel7A-CBM adsorbed significantly less to lignin than TrCel7A, indicating that the catalytic module (TeCel7A) had a strong contribution to the low binding. Probably, the contribution of CBM or catalytic core module in non-productive binding varies between different enzymes, and the role of the CBM is not always dominant. To date, very little attention has been paid to the role of electrostatic interactions in lignin-binding. In this work, binding of Melanocarpus albomyces Cel45A endoglucanase onto lignin was found to be very dependent on pH, suggesting that electrostatic interactions were involved in the binding. At high pH, significantly less non-productive binding occurred, probably due to increasing electrostatic repulsion between negatively charged enzymes and lignin. Modification of the charged chemical groups in enzymes or lignin may be a viable strategy to reduce nonproductive enzyme binding in the hydrolysis of lignocellulosic substrates.
  • Zaki, Urfa (Helsingin yliopisto, 2019)
    Cerebral dopamine neurotrophic factor (CDNF) belongs to the the family of neurotrophic factors that are evolutionary conserved, having a unique structure, with two domains: C-terminal domain and the N-terminal domain, and a cysteine bridge. It is known to be involved in the repair of the dopaminergic neurons when studied in the animal models of PD, which shows their different mode of action as compared to other neurotrophic factors, highlighting their therapeutic potential. Analysis of the crystal structure shows that CDNF and MANF consist of two domains: the saposin-like N-terminal domain with five α-helices stabilized by three disulphide bridges, and presumably unstructured C-terminal domain with a disulphide bridge. Characteristic feature of saposin-like proteins is their ability to interact with membranes or lipids. The lipid interaction may be crucial for the activity of CDNF and MANF proteins. In the first part of this project, the binding of CDNF was tested with several oxidized lipids, using two methods; Co-sedementation assay and lipid fluorescence assay;with two different types of probes. According to the results, CDNF seemed to show binding with POVPC. The second part of the project involved testing the binding and internalization of CDNF to mouse myoblast cells in the presence of oxidized lipid; POVPC. It was observed that CDNF seemed to show binding to the cell surface of the mouse myoblast cells (C2C12) and is also observed to be internalized to the cells as well. However, as these are the preliminary results, so we need to further test the binding between the protein and other lipids and devise more precise protocols for the testing the internalization to the cells.
  • Banerjee, Rishi (Helsingin yliopisto, 2019)
    After birth, stem cells act as the source of reparative and regenerative potential in various tissues. Among different tissues and organs in human body, tooth is one of the organs which does not undergo continuous regeneration. Therefore, tooth regeneration must be studied in a different animal, which possesses continuously growing teeth. In mouse, the incisor undergoes continuous growth which is fueled by the interaction between epithelial and mesenchymal stem cell compartments located at its apical end. The inferior alveolar nerve, which supports mandibular dentition, and its surrounding blood vessels (combinedly known as neurovascular bundle or NVB) were previously shown to act as a source of the mesenchymal stem cells during incisor growth and regeneration. However, the regulation of the cells in the NVB is not well understood. The primary aim of my master’s thesis was to characterize the effect of the Hh pathway modification on cellular properties of the NVB and the MSCs within it. The Ptch2 KO mouse model used in this study demonstrated increase in the number of blood vessel in the NVB. Additionally, analysis of the structure of skin in the mouse model was the second aim of my project, which showed significant increase in the thickness of the dermis at the postnatal day 1. Collectively, the change in structure of skin and NVB showed that Ptch2 might regulates the cellular properties of tooth mesenchyme and dermis by modulating the structural components of the NVB of continuously growing mice incisor and skin, respectively.
  • Sultana, Nasrin (Helsingin yliopisto, 2020)
    Tiivistelmä – Referat – Abstract Plant lives and grows in variable environment and climate conditions. Everyday plants can be confronted with a variety of abiotic (temperature, light, salt, water availability) and biotic stress (pathogens, insects etc). These abiotic and biotic stress can halt plant growth and influence crop productivity. Plant has evolved signaling mechanism and different responses to adapt or respond with these unfavorable environmental conditions. Our group’s previous research identified a new mutant in the model plant Arabidopsis thaliana with a striking phenotype – when the plants ages it progressively becomes yellow and eventually the entire plant is white. The mutant was named “white” after its striking appearance. The phenotype is associated with increased accumulation of mRNA transcript for stress and senescence regulated genes. Mapping of the mutation identified a 4 bp deletion in a gene EGY1 that encodes a metalloprotease located in the chloroplast. To identify molecular mechanisms that regulate this unusual type of premature senescence, a suppressor mutants screen was performed in the white mutant, and three suppressors that restore normal appearance to the plant was identified. Mapping of one of these suppressors, identified a mutation in STAY GREEN1 (SGR1) as a likely candidate. SGR1 encodes the protein that catalyze the first step in chlorophyll breakdown, removal of Mg2+ from chlorophyll. The overall aim of my master thesis was to understand the molecular mechanisms behind the development of the age and chlorophyll related phenotypes in the white mutant and its two suppressors S1 and S2. Furthermore, with gene expression analysis, plant stress and senescence responses were studied in white, S1 and S2. By complementation method I proved that mutations in SGR1 gene caused the development of suppressor mutant phenotype and restoration of wild type allele of SGR1 gene restore white phenotype in suppressor mutant. Measurements of chlorophyll concentration provided further evidence that the mutation in SGR1 stabilizes the suppressor mutant phenotype, stops chlorophyll breakdown and keep the leaves green. Gene expression study using qPCR with marker genes provided insight of molecular changes within these phenotypes.
  • Kiviniemi, Eero A. (Helsingin yliopisto, 2018)
    Microbial cellulases, e.g. cellobiohydrolases, are able to degrade cellulose and lignocellulosic biomass to smaller glucose-containing monomers and oligomers. Cellulases are often multi-domain enzymes comprised of different protein domains (i.e. modules), which have different functions. The main two components, which often appear in cellulases, are the cellulose-binding module (CBM) and the catalytic domain. The CBMs bind to cellulose, bringing the catalytic domains close to their substrate and increasing the amount of enzymes on the substrate surface. The catalytic domain performs the cleavage of the substrate, e.g. in the case of cellobiohydrolases hydrolyses or “cuts” the crystalline cellulose chain into smaller soluble saccharides, mainly cellobiose. Unlike aerobic fungi, which utilize free extracellular enzymes to break down cellulose, anaerobic microbes often use a different kind of strategy. Their cellulases are organized and bound to the cell surface in a macromolecular protein complex, the cellulosome. The core of the cellulosome is formed of a scaffolding protein (the scaffoldin) consisting mainly of multiple consecutive cohesin domains, into which the catalytic subunits of enzymes attach via a dockerin domain. This creates a protein complex with multiple different catalytic domains and activities arranged in close proximity to each other. Dockerins and cohesins are known to bind each other with one of the strongest receptor-ligand -pair forces known to nature. Dockerin containing fusion proteins have also been successfully combined in vitro with proteins containing their natural counterparts, cohesins, to create functional multiprotein complexes. In this Master’s thesis work the goal was to 1) produce fusion proteins in which different CBMs were connected to dockerin domains, 2) combine these fusions with cohesin-catalytic domain fusion proteins to create stable CBM and catalytic domain containing enzyme complexes, 3) to characterize these enzyme complexes in respect of their thermostability and cellulose hydrolysis capacity and 4) to ultimately create a robust and fast domain shuffling method for multi-domain cellobiohydrolases (CBH) to facilitate their faster screening. The hypothesis of the experiments was that different CBMs fused with a dockerin domain and the cellobiohydrolase catalytic domain fused with a cohesin domain could be produced separately and then be combined to produce a functional two-domain enzyme with a dockerin-cohesin “linker” in between. In this way time and work could be saved because not every different CBM- catalytic domain -pair would have to be cloned and produced separately. Several CBM-dockerin fusion proteins (in which the CBM were of fungal or bacterial origin) were tested for expression in heterologous hosts, either in Saccharomyces cerevisiae or Escherichia coli. The purified proteins were combined with a fungal glycoside hydrolase family 7 (GH7) cellobiohydrolase-cohesin fusion protein produced in S. cerevisiae. The characterization of the catalytic domain-CBM -complexes formed through cohesin-dockerin interaction included thermostability measurements using circular dichroism and activity assays using soluble and insoluble cellulosic substrate. The results were compared to enzyme controls comprising of the same CBM and catalytic domain connected by a simple peptide linker. The results showed that the cohesin-dockerin –linked cellobiohydrolase complex performed in the cellulose hydrolysis studies in a similar manner as the directly linked enzyme controls at temperature of 50˚C and 60 ˚C. At temperatures of 70 ˚C the complex did not perform as well as the control enzymes, apparently due to the instability of the dockerin-cohesin interaction. The thermostability measurements of the enzymes, together with the previously published data supported the hydrolysis results and this hypothesis. The future work should be aimed at enhancing the thermostability of the cohesin-dockerin interaction as well as on verifying the results on different cellulase fusion complexes.
  • Oja, Sofia (Helsingin yliopisto, 2020)
    Mesenchymal stromal cells (MSC) are a potential tool for cell-based therapies subject to intensive investigation. MSCs display immunomodulatory functions and a broad differentiation capacity, and thus have several potential therapeutic applications, such as treatment of immunological disorders and correction of tissue defects. MSCs have been widely studied and utilized in treatment of Graft-versus-Host disease, a severe complication of stem cell transplantation, and have also been evaluated in treatment of Crohn’s disease, multiple sclerosis, and chronic inflammation. The repair of bone and cartilage defects is another application of significant interest. MSCs are classified as advanced therapy medicinal products (ATMPs), which are manufactured under supervision by regulatory authorities. The aim of regulation is to produce safe and effective medicinal products, and therefore safety risks and sources of alterations or impairment of functionality should be evaluated carefully. MSCs are somatic cells, which can be isolated from various sources, such as from bone marrow, adipose tissue, or umbilical cord. For production of medicinal products, MSCs are usually expanded extensively in cell cultures. Culture conditions are known to affect to the characteristic and functionality of MSCs. Animal-derived components during culture such as bovine serum are to be avoided if possible, due to risks of immune reactions and zoonotic infections. Therefore, animal serum-free culture medium is considered safe option for MSC cultures. This thesis focuses on determining optimal expansion and differentiation conditions for clinical-grade bone marrow-derived MSCs by using platelet-derived culture medium supplements to replace bovine serum. This study also aimed to improve quality of MSCs products by introducing an imaging-based screening method to detect aging-related morphological changes in MSC cultures. Finally, we investigated how additional freezing steps during the manufacturing process affect the basic manufacturing parameters and alter the cellular aging process. We found in this study that platelet lysate with two freeze-thaw cycles effectively supported MSC expansion and maintained their functionality in ambient oxygen concentration. Platelet lysate also promoted osteogenic differentiation at least equally with bovine serum in two-dimensional plate culture and slightly better if a three-dimensional matrix was used. We were able to detect and quantify aging-related morphological changes from MSCs cultures and found that a rapid increase in cell size reflects the expression of aging markers. We found that freezing at early phases of cell cultures did not alter the characteristics or functionality of the MSC. This study has yielded insights into the establishment and scaling up of animal-serum free MSC cultures. In addition, our screening method for aged cells could be implemented into the clinical-grade manufacturing of MSCs to monitor cell quality during processing.
  • Deb, Debashish (Helsingin yliopisto, 2019)
    There is significant reduction in number of approved drugs for acute myeloid leukemia in recent years. Partially it may be due to the failure of discovery and validation approach to new drugs as well as the complexity of the disease. Ex vivo functional drug testing is a promising approach to identify novel treatment strategies for acute myeloid leukemia (AML). In ideal condition, an effective drug should eradicate the immature AML blasts, but spare non-malignant hematopoietic cells. However, current strategies like conventional cell viability assay fail to measure cell population-specific drug responses. Hence, development of more advanced approaches is needed. Using multiparameter, high-content flow cytometry (FC), we simultaneously evaluated the ex vivo sensitivity of different cell populations in multiple (10) primary AML samples to 7 FDA/EMA-approved drugs and 8 drug combinations. Amongst the 7 tested drugs, venetoclax, cytarabine and dasatinib were very cytotoxic with venetoclax had the highest blast-specific toxicity, and combining cytarabine with JAK inhibitor ruxolitinib effectively targeted all leukemic blasts but spared non-malignant hematopoietic cells. Taken together, we show that the ex vivo efficacy of targeted agents for specific AML cell population can be assessed with a cell phenotype, FC-based approach. Furthermore, we put an effort to analyze the potential of this assay and biomarkers to predict the clinical outcome of individual patients and future perspectives.
  • Mäntylahti, Sampo (Helsingin yliopisto, 2014)
    In the field of bioscience there is an ongoing explosive growth in discovery and information. Novel means in biotechnology as well as in medicines are introduced at an unseen rate. One of the aspects contributing to this development is the increased understanding of protein function and structure. Proteins have a role in almost every biological process. The function and structure of proteins are linked. Recent studies have discovered that the understanding of the protein structure has been biased. Namely, the studies have unearthed a previously dismissed protein structure state: intrinsically disordered proteins (IDPs). In this highly dynamic state a protein is without a globular fold, but does not meet the requirements of a random coil either. Rapid transition between folds renders most of the established research techniques to be poor methods to study the IDPs. Nuclear magnetic resonance (NMR) is a spectroscopy method, which enables the study of molecules at atomic resolution. The technique is based upon manipulation of the nuclear spins in specifically produced sample under strong magnetic field. In this method, spins of the system generate quantum coherence state(s), which is utilized to obtain information about the system. NMR is suitable for studying samples in solid and liquid mediums, but in case of biomolecules, water solution is preferable as it resembles in vivo environment. Highly mobile structure and chemical composition of IDPs cause many established NMR experiments to fail. Development of NMR pulse sequences is an obvious approach to solve the problem. This thesis presents a number of NMR pulse sequences, which are designed to improve acquisition of information from highly mobile sections of proteins. The key aspect is to utilize H atom instead of HN in coherence transfer. Additional improvements include limited residue specific identification and novel coherence transfer pathways. Articles I, II, and III present triple resonance experiments, which correlate protein backbone atoms. Combination of the spectra enables full sequential assignment. Article IV introduces an improved pulse sequence for measuring J couplings between nitrogen and amide proton. The experiments were subjected to experimental verification. Comparisons were drawn between established pulse sequences. In both globular proteins and IDPs the results show improvement over established pulse sequences. The proposed sequences yielded improved assignment coverage, resolution and sensitivity enhancement.
  • Auvinen, Pauliina (Helsingin yliopisto, 2018)
    Assisted reproductive technology (ART) refers to treatments used for infertile couples to achieve pregnancy in vitro. The main technology of ART is in vitro fertilization (IVF), which may also include intracytoplasmic sperm injection (ICSI) and/or embryo cryopreservation and frozen embryo transfer (FET). ART treatments are well-accepted in Western countries and there is an increasing number of children being conceived in that way. Even though, majority of ART derived newborns appear healthy, they have been associated with increased risks of adverse perinatal outcomes, especially, alterations in birth size as well as higher frequencies of imprinting disorders and alterations in epigenetic modifications, such as in DNA methylation, of imprinted genes. Epigenetically regulated imprinted genes have crucial roles in fetal and placental growth during development and they are known to be affected by environmental factors. Since ART takes place in the early embryo in vulnerable time-period of epigenetic reprogramming, ART has been suggested to impact on epigenetic profiles of the embryo, consequently, affecting the phenotype of newborns, and therefore potentially causing long-term health effects. This thesis aimed to study whether ART has effects on DNA methylation in the placenta and whether ART has effects on the phenotype of newborns. To study these effects, this thesis focused on the sixth binding sequence of CTCF (CTCF6) of H19 ICR1 of the growth-related imprinted IGF2/H19 gene locus. The aim was also to study whether the possible changes associate with the rs10732516 G/A polymorphism locating at CTCF6 of H19 ICR1. DNA methylation levels of placental tissue as well as white blood cells in umbilical cord blood of ART derived, and spontaneously conceived newborns were explored by mass spectrometry-based Sequenom MassARRAY® EpiTYPER® method and traditional bisulfite sequencing. To study the effects of ART on the phenotype of newborns, the birth weight, length and head circumference of ART and control newborns were explored using international growth standards. Moreover, placental weights were compared. The results of this thesis showed slightly, but consistently decreased DNA methylation levels at H19 ICR1 in the paternal allele of ART derived placentas in rs10732516 patA/matG genotype, but not in patG/matA genotype. Thus, the results suggest that the changes in DNA methylation at IGF2/H19 in the placenta are genotype-specific and associate with the rs10732516 polymorphism. Similar decreased methylation levels in the paternal allele of patA/matG genotype was not detected in white blood cells suggesting that the effects on DNA methylation levels are also cell type-specific. The effects of ART on the phenotype also associated with the rs10732516 polymorphism. Fresh embryo transfer derived newborns with A/A genotype were seen to have smaller birth weight than newborns with G/G genotype. Moreover, in A/A genotype, frozen embryo transfer derived newborns were demonstrated to be heavier and to have heavier placentas than fresh embryo transfer derived newborns. The findings of this thesis suggest that ART has effects on DNA methylation in the placenta and on the phenotype of newborns, and the effects associate with the rs10732516 G/A polymorphism. This underlines the significance of the polymorphism when studying the effects of ART. However, further investigations are needed to confirm these findings and to discern whether the changes are due to the ART procedures or underlying infertility.
  • Kaya, Meryem Ecem (Helsingin yliopisto, 2019)
    Synthetic biology is an emerging interdisciplinary field of biology that aims to system-atically design artificial biological systems. As synthetic biologists seek increasingly complex control over cellular processes to achieve robust and predictable systems. A new frontier in synthetic biology is engineering synthetic microbial consortia. This ap-proach employs the concept of division of labor, instead of introducing large genetic cir-cuitry to homogenous cell populations. In this approach, different cell types are assigned to execute a portion of the overall circuit. Each cell type communicates with their co-worker subpopulations to complete the circuit. The main advantage of this strategy is the reduced metabolic burden on each cell type. Thus, leading to more reliable and stable overall performance. In this work, to simplify cellular communication between the mem-bers of the consortium, we used the simple architecture of quorum sensing machinery. We constructed a toolbox that contains promoter, receptor and quorum sensing signal synthase genes along with fluorescent reporters. Using this toolbox, we constructed dif-ferent cell types that can be used in synthetic consortia forming various communication topologies. We characterized the constructed cell types individually and in co-cultures.
  • Rytioja, Johanna (Helsingin yliopisto, 2016)
    Basidiomycete white rot fungi are wood-rotting species and their impact to the global carbon cycle is significant. White rot fungi are capable of degrading all the polymeric cell wall components of the plant biomass from polysaccharides, cellulose, hemicelluloses and pectin, to the aromatic heteropolymer lignin. This is due to their ability to produce diverse set of extracellular enzymes that degrade or modify the plant cell wall concomitantly releasing carbon. Research on plant-biomass-degrading fungi has concentrated on isolation and characterization of enzymes especially from the ascomycete fungi for biotechnological applications, such as bioenergy, food processing and waste treatment. More recently genomic studies have opened the reservoir of the plant-biomass-degrading potential of basidiomycete fungi including wood-rotting, litter-decomposing, plant-pathogenic and ectomycorrhizal species. Dichomitus squalens is a white-rot fungus, which colonises softwood and is able to efficiently degrade lignin and cellulose. Previously, intensive studies on white rot fungi have been focused on lignin degradation by oxidative enzymes. The aim of this study was to analyse the potential of the plant-cell-wall-modifying enzymes of D. squalens. Plant biomass degradation by D. squalens was studied at different levels from gene expression to enzyme production. The focus was to dissect the overall degradation of plant biomass polymers, especially cellulose degrading enzymes of D. squalens. The cellulose degradation by D. squalens was studied at the transcript level during growth on spruce wood sticks and in microcrystalline cellulose-containing liquid medium. Selected cellulases and oxidoreductases, which putatively act on cellulose were expressed simultaneously on spruce, the natural substrate of the fungus, and microcrystalline cellulose in time- and substrate-dependent manner. To clarify the adaptation of D. squalens to different plant biomass, the transcriptome and secretome of the fungus were studied in different wood and non-woody substrates. The study confirmed that lignin degradation occurs at the initial stage of growth and D. squalens has retained the diverse enzyme set both for the degradation of wood and non-woody plant biomass. The cellobiohydrolases (CBHs) and cellobiose dehydrogenase of D. squalens were biochemically characterized. In hydrolysis of different plant-derived biomasses, CBHs released reducing sugars alone and in combination with oxidative laccase enzyme. The study shows that D. squalens encodes a complete enzymatic repertoire for plant biomass degradation. In addition, the data emphasise the role of oxidoreductases in the white rot fungal degradation of cellulose and other plant cell wall polymers. Results suggest that white rot fungal plant cell wall converting enzymes are promising candidates in the biotechnological applications using plant biomass.
  • Rommi, Katariina (VTT Technical Research Centre of Finland Ltd, 2016)
    New protein sources are needed to fulfil the growing global demand for food protein. The co-stream from cold pressing of rapeseed oil, press cake, is a rich source of protein (32 26%) with good nutritional value. Several technologies based on alkaline or saline extraction have been developed for enrichment of rapeseed protein, but high energy and water consumption due to dilute conditions and multiple processing steps limit their sustainability and profitability. In the present study, enzyme-aided methods for extraction of protein from rapeseed cold-press cake were developed, and factors influencing protein extractability and properties of the obtained protein-rich fractions were elucidated. Pretreatments and carbohydrate-hydrolyzing enzyme treatment were used to facilitate extraction at reduced water content and without chemicals such as alkali or salt. Particularly a pectinase preparation with β-glucanase side activity enabled effective hydrolysis of rapeseed cell wall polysaccharides, increasing protein recovery by aqueous extraction. Similar improvement of protein extractability was, however, not achieved by mechanical cell wall disruption, suggesting that protein release was not to a major extent physically hindered by the residual cell structures in the press cake. Instead, enzyme treatment reduced the water holding capacity of the press cake and released hydrolysis products which are suggested to have enhanced the solubility of rapeseed protein during water extraction. At 10% solid content, enzyme-aided water extraction produced protein-enriched fractions with a similar yield and estimated production costs as conventional alkaline extraction (pH 10) and isoelectric precipitation. Techno-economic evaluation of different extraction schemes also suggested substantial reduction of energy costs when the extraction was carried out at 20% solid content. Due to the co-extraction of carbohydrates, enzyme-aided protein extraction was less selective than non-enzymatic alkaline extraction and isoelectric precipitation. On the other hand, carbohydrates are suggested to have improved the solubility and dispersion stability of protein extracts by electrostatic interactions. The results indicate that enzyme-aided fractionation methods are suitable for rapeseed protein production and may offer a techno-economically feasible alternative to alkaline or saline extraction. Additionally, protein hydrolyzates were extracted from dry-fractionated rapeseed press cake by proteolytic enzyme treatment. The hydrolyzates showed high inhibition of radical-induced oxidation in vitro, and their observed antioxidativity correlated with their protein and sinapic acid concentration. The hydrolyzates were also able to inhibit the myeloperoxidase and elastase enzymes involved in inflammation and skin aging, therefore having novel application potential as active ingredients or natural preservatives in skin care products.
  • Partti, Edvard (Helsingin yliopisto, 2018)
    Kaurapohjaiset elintarvikkeet ovat terveellisiä. Monet niiden terveyshyödyt johtuvat kauran liukoisen ravintokuidun suuresta β-glukaanipitoisuudesta. β-glukaanin terveysvaikutukset ovat riippuvaisia sen molekyylipainosta ja viskositeetista. Viskositeetilla on myös muuta merkitystä kaurapohjaisissa elintarvikkeissa kuten kaurajugurteissa ja kauramaidoissa. Aiemmassa Folafibre-tutkimusprojektissa oli tutkittu kaurakuidun folaattipitoisuuden (B9 vitamiini) kasvattamista fermentoimalla sitä eri mikrobeilla. Hyvin folaattia tuottaneet mikrobit myös alensivat kaurakuituvalmisteen viskositeettia, ja erittivät glykosyylihydrolaaseja ja/tai proteaasia. Oli kuitenkin jäänyt epäselväksi, olivatko entsyymit ainut syy viskositeetin alenemiseen, ja kuinka paljon kullakin niistä oli vaikutusta viskositeettiin. Lisäksi tämän alan kirjallisuudessa on pidetty epäselvänä, onko esim. tärkkelyksen ja β-glukaanin välillä jotain interaktioita jotka mm. nostavat niiden viskositeettia yli yksittäisten polymeerien viskositeetin summan. Näitä voitaisiin selvittää puhdistamalla viskositeettia alentaneista mikrobikannoista kyseiset entsyymit ja tutkimalla niiden vaikutusta kaurakuidun viskositeettiin. Yhdeksi mikrobiksi valittiin Exiguobacterium sp. RB3 kanta, koska em. syiden lisäksi tässä bakteerisuvussa esiintyy psykrofiilejä ja mikrobeja jotka voivat kasvaa korkeassa pH:ssa, jolloin sen erittämät entsyymit saattaisivat olla aktiivisia matalissa lämpötiloissa ja korkeassa pH:ssa, ja olla siten teollisesti kiinnostavia. Toiseksi mikrobiksi valittiin Bacillus sp. ABM5119, koska sitä oli käytetty monissa Folafibre-projektin tutkimuksissa. Työn tavoitteena oli puhdistaa Bacillus sp. ABM5119:n endo-β-1,4-glukanaasi, ja Exiguobacterium sp. RB3:n α-amylaasi ainakin siinä määrin, että ne ovat muista endoaktiivisista glykosyylihydrolaaseista ja proteaaseista puhtaita, ja sitten mitata puhdistettuja entsyymejä ja proteaasia käyttäen niiden vaikutus yhdessä ja erikseen keitetyn kaurakuituvalmisteen viskositeettiin. Lisäksi tavoitteena oli karakterisoida Exiguobacterium sp. RB3 amylaasin olennaisimmat biokemialliset ominaisuudet, sekvensoida sen geenin amylaasia koodaava alue, ja selittää sen ominaisuuksia myös sekvenssistä johdettavissa olevan tiedon perusteella. Keitetyn kaurakuitupreparaatin viskositeettia alentavat eniten α-amylaasi ja endo-β-1,4-glukanaasi. Proteaasi ei vaikuta kaurakuitupreparaatin viskositeettiin, kun kuitupreparaatti on keitetty. Endo-β-1,4-glukanaasi alentaa viskositeettia yhtä hyvin kuin β-1,3-1,4-glukanaasi. Synergiaa viskositeetin alentamisessa α-amylaasilla ja endo-β-1,4-glukanaasilla ei havaittu, mutta havaittiin että β-glukaani saattaa estää tärkkelyksen retrogradaatiota. Hyvin pienetkin entsyymiaktiivisuudet vaikuttavat viskositeettiin. Jos halutaan β-glukaanin viskositeetin ja siten terveysvaikutusten säilyvän, täytyy esim. kauramaidon valmistuksessa käytettävien entsyymivalmisteiden olla β-glukanaaseista hyvin puhtaita. Mikrobifermentaatioissa tapahtuvat viskositeetin alenemat johtuvat lähinnä mikrobien erittämistä glykosyylihydrolaaseista, ei niinkään esim. niiden metabolian sivutuotteista kuten happiradikaaleista. Exiguobacterium sp. RB3 α-amylaasi on rakenteeltaan Bacillus licheniformis α-amylaasin kaltainen glykosyylihydrolaasiryhmän 13 entsyymi. Se sitoo rakenteeseensa kolme kalsiumiatomia, ja kalsiumpitoisuus vaikuttaa sen aktiivisuuteen. Se on aktiivisimmillan pH alueella 5,0 – 7,5. Se sietää detergenttejä, toisin kuin eräs aiemmin karakterisoitu Exiguobacterium-α-amylaasi. RB3 α-amylaasin turnover number oli korkea, 29000 1/s. Exiguobacterium-suvun psykrofiilisessä haarassa esiintyy kahta eri α-amylaasia, joista yksi on tässä karakterisoitu, ja toinen on selvästi erilainen rakenteeltaan ja biokemiallisilta ominaisuuksiltaan.