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  • Nevalainen, Martti Juha (Helsingin yliopisto, 2004)
  • Dahlman, Tuula (Helsingin yliopisto, 2003)
  • Lantto, Raija (Helsingin yliopisto, 2007)
    The effects of tyrosinase, laccase and transglutaminase (TG) were studied in different meat protein systems. The study was focused on the effects of the enzymes on the gel formation properties of myofibrils, and on the textural and water-holding properties of the heated meat systems. The cross-linking efficiency of a novel Trichoderma reesei tyrosinase was compared to that of the commercial Agaricus bisporus tyrosinase. Trichoderma tyrosinase was found to be superior compared to the Agaricus enzyme in its protein cross-linking efficiency and in the incorporation of a small molecule into a complex proteinaceous substrate. Tyrosinase, laccase and TG all polymerised myofibrillar proteins, but laccase was also found to cause protein fragmentation. A positive connection between covalent cross-link and gel formation was observed with tyrosinase and TG. Laccase was able to increase the gel formation only slightly. With an excessive laccase dosage the gel formation declined due to protein fragmentation. Tyrosinase, laccase and TG had different effects on the texture and water-holding of the heated chicken breast meat homogenates. Tyrosinase improved the firmness of the homogenate gels free of phosphate and with a low amount of meat. TG improved the firmness of all studied homogenates. Laccase weakened the gel firmness of the low-meat, low-salt and low-salt/phosphate homogenates and maintained the firmness on the control level in the homogenate free of phosphate. Tyrosinase was the only enzyme capable of reducing the weight loss in the homogenates containing a low amount of meat and a low amount of NaCl. TG was the only enzyme that could positively affect the firmness of the homogenate gel containing both low NaCl and phosphate amounts. In pilot scale the test products were made of coarsely ground chicken breast fillet with a moderate amount of salt. Increasining the amount of meat, salt and TG contents favoured the development of firmness of the test products. The evaporation loss decreased slightly along with increasing TG and NaCl amounts in the experimental conditions used, indicating a positive interaction between these two factors. In this work it was shown that tyrosinase, laccase and TG affected the same myofibrillar proteins, i.e. myosin and troponin T. However, these enzymes had distinguishable effects on the gel formation of a myofibril system as well as on the textural and water-holding properties of the finely ground meat homogenates, reflecting distinctions at least in the reaction mechanisms and target amino acid availability in the protein substrates for these enzymes.
  • Boije af Gennäs, Gustav (Helsingin yliopisto, 2011)
    The protein kinases (PKs) belong to the largest single family of enzymes, phosphotransferases, which catalyze the phosphorylation of other enzymes and proteins and function primarily in signal transduction. Consequently, PKs regulate cell mechanisms such as growth, differentiation, and proliferation. Dysfunction of these cellular mechanisms may lead to cancer, a major predicament in health care. Even though there is a range of clinically available cancer-fighting drugs, increasing number of cancer cases and setbacks such as drug resistance, constantly keep cancer research active. At the commencement of this study an isophthalic acid derivative had been suggested to bind to the regulatory domain of protein kinase C (PKC). In order to investigate the biological effects and structure-activity relationships (SARs) of this new chemical entity, a library of compounds was synthesized. The best compounds induced apoptosis in human leukemia HL-60 cells and were not cytotoxic in Swiss 3T3 fibroblasts. In addition, the best apoptosis inducers were neither cytotoxic nor mutagenic. Furthermore, results from binding affinity assays of PKC isoforms revealed the pharmacophores of these isophthalic acid derivatives. The best inhibition constants of the tested compounds were measured to 210 nM for PKCα and to 530 nM for PKCδ. Among natural compounds targeting the regulatory domain of PKC, the target of bistramide A has been a matter of debate. It was initially found to activate PKCδ; however, actin was recently reported as the main target. In order to clarify and to further study the biological effects of bistramide A, the total syntheses of the natural compound and two isomers were performed. Biological assays of the compounds revealed accumulation of 4n polyploid cells as the primary mode of action and the compounds showed similar overall antiproliferative activities. However, each compound showed a distinct distribution of antimitotic effect presumably via actin binding, proapoptotic effect presumably via PKCδ, and pro-differentiation effect as evidenced by CD11b expression. Furthermore, it was shown that the antimitotic and proapoptotic effects of bistramide A were not secondary effects of actin binding but independent effects. The third aim in this study was to synthesize a library of a new class of urea-based type II inhibitors targeted at the kinase domain of anaplastic lymphoma kinase (ALK). The best compounds in this library showed IC50 values as low as 390 nM for ALK while the initial low cellular activities were successfully increased even by more than 70 times for NPM-ALK- positive BaF3 cells. More importantly, selective antiproliferative activity on ALK-positive cell lines was achieved; while the best compound affected the BaF3 and SU-DHL-1 cells with IC50 values of 0.5 and 0.8 μM, respectively, they were less toxic to the NPM-ALK-negative human leukemic cells U937 (IC50 = 3.2 μM) and BaF3 parental cells (IC50 = 5.4 μM). Furthermore, SAR studies of the synthesized compounds revealed functional groups and positions of the scaffold, which enhanced the enzymatic and cellular activities.
  • Vulli, Jaana (Helsingin yliopisto, 2013)
    A small set of highly conserved signaling molecules performs a great number of tasks in different animals and developmental contexts. Among them, the bone morphogenetic proteins (BMPs) constitute a group of growth and differentiation factors that are involved in numerous developmental processes affecting cell proliferation, apoptosis and differentiation. In the fruit fly, Drosophila melanogaster, three BMP type proteins have been identified, each of which has a homolog in mammals. Decapentaplegic (Dpp) is a BMP2/4 type protein which plays a major role in dorsal-ventral patterning of the early embryo. It participates in midgut development, patterning and growth of imaginal tissues, wing vein formation and maintenance of germline stem cells in the germarium. Dpp is a morphogen which requires a second BMP type protein, Screw (Scw) or Glass bottom boat (Gbb) to be able to form proper concentration gradients in developing tissues. Scw and Gbb belong to the BMP5/6/7/8 subfamily and their expression domains are different; Scw is specifically expressed during the early events of embryogenesis, while Gbb has more functional roles during later stages of fly development, like wing morphogenesis. BMP type proteins are produced as large proproteins that require proteolytic cleavage prior to secretion and extracellular gradient formation. This study concentrated on the cleavage of Dpp and Scw to reveal the meaning of post-translational modifications in concentration gradient formation and BMP signaling. Three furin recognition sites were identified in the Dpp proprotein. Mutational analyses indicate that the upstream optimal furin site of the prodomain (furin site (FS) II) is critical for producing ligands and creating a long range concentration gradient in a wing imaginal disc. Cleavage of the other two FSs produce the differently sized Dpp ligands that contribute to BMP gradient formation in the early embryo and wing imaginal disc. It was noted that the cleavage requirements of BMP2/4 type proteins in different species vary to establish species-specific regulation of BMP signaling. Discovery of the scwE1 allele, that causes dominant negative effect in embryos heterozygous for a hypomorphic dpp allele, gave more information about how the cleavage patterns of prodomains can contribute to creating diversity in the regulation of signaling. The mutation responsible for the dominant negative function in scwE1 was located in the cleavage site that is in the prodomain of Scw. Mutational analyses showed that the mature ligand of ScwE1 is produced in lower amounts and in complex with an N-terminal prodomain peptide. ScwE1 preferentially binds Dpp and disrupts normal gradient formation possibly through interactions with molecules within the extracellular matrix. Phylogenetic analyses and functional studies of BMP cleavage mutants propose a mechanism by which post-translational regulation of proproteins modulates BMP signaling.
  • Sund, Jukka (Helsingin yliopisto, 2014)
    Nanotechnology has been one of the major success stories of the early 21st century. The foundation for this success rests on the discovery that a small size confers completely new properties on materials. Nowadays, engineered nanomaterials (ENMs) are used in a plethora of applications such as paints, cosmetics, food products and electronics. These new properties, however, potentially make ENMs more reactive in biological systems than their large-scale counterparts. Already, asbestos-like effects have been described in mice after exposure to certain forms of carbon nanotubes (CNTs), while nano-sized titanium dioxide (nTiO2) has been shown to evoke inflammation in mouse lung. Therefore, extensive nanosafety studies have to be performed to ensure that no adverse effects are suffered by either workers or end-users of ENM products. This thesis has investigated the biological effects of ENMs by proteomic methods, first by evaluating the uptake and interactions of ENMs with plasma and cellular proteins followed by an analysis of the effects of ENM exposure on the intracellular proteome and secretome of human primary macrophages. The results revealed that ENM interactions with cellular proteins were governed by the surface reactivity of ENMs, whereas interactions with plasma proteins seemed to depend on the combination of both surface reactivity and active recognition, namely tagging of ENMs by opsonin proteins. The binding of cellular proteins to ENMs and subsequent interference with cellular processes might represent a novel cause of ENM toxicity, especially since transmission electron microscopy (TEM) micrographs indicated that several ENM species could be visualized free in the cytoplasm. The cytoplasmic protein expression changes after exposure to silica coated and uncoated nTiO2 revealed that silica coated TiO2 induced stronger protein expression changes in the macrophages. Most of the proteins with altered expression were related to phagocytosis, oxidative stress and inflammation. These proteome changes indicate that macrophages are actively engulfing ENMs and processing them. Moreover, the up-regulation of oxidative stress related proteins might be an indication of oxidative burst. Finally, nTiO2 treatment evoked acetylation of cytoplasmic proteins, a previously uncharacterized phenomenon in cells exposed to ENMs. The results from the macrophage secretome analysis showed that asbestos and long rigid carbon nanotubes (R CNTs) produced a similar response, while protein secretion profile of macrophages exposed to long tangled carbon nanotubes (T CNTs) exhibited a distinct profile. Bioinformatic analysis revealed that R CNTs evoked secretion of inflammation and apoptosis related proteins, possibly because of lysosomal damage. Functional assay confirmed that R CNT exposure triggered apoptosis in macrophages, while T CNTs and asbestos did not. This thesis offers new knowledge concerning the biological effects of engineered nanomaterials. Proteomic methods proved to be useful in the ENM-protein interaction studies revealing that it would be beneficial to include the ENM-protein interaction experiments as part of the routine ENM characterization when assessing the health effects of ENMs. By employing quantitative proteomics, we obtained a global view of both cytoplasmic and secreted proteome changes of macrophages exposed to different ENMs.
  • Lietzén, Niina (Helsingin yliopisto, 2012)
    Proteomics is defined as large-scale study of proteins, and with current proteomic methods thousands of proteins can be characterized in a single experiment. Mass spectrometry (MS) has an important role in the characterization of complex protein samples. In addition, various bioinformatics tools have become increasingly important in the interpretation of complex proteomic data. The combination of proteomics and bioinformatics is nowadays an important tool to study cellular signaling mechanisms. When host cell recognizes the invading virus, multiple cell signaling cascades are activated resulting in antiviral immune responses, inflammation and programmed cell death of the infected cell. The detailed mechanisms of host cell defense responses activated upon viral infection are still partially unknown. The aim of this project was to develop and utilize proteomic and bioinformatic methods to characterize host responses to viral infection. Three different proteomic approaches were used in this project to study virus-induced changes in human epithelial cells and macrophages. Viral RNA-induced responses in HaCaT keratinocytes were studied using two-dimensional gel electrophoresis and MS. Influenza A virus-induced changes in the intracellular compartments and secretomes of human primary macrophages were characterized using iTRAQ labeling-based quantitative proteomics. Finally, viral RNA-triggered protein secretion from human primary macrophages was studied using SDS-PAGE, liquid chromatography and MS. In addition, two computational tools, Compid and Pripper, were developed to simplify the analysis of our proteomic data. Our studies showed that both influenza A virus and viral RNA trigger significant changes in the proteomes of macrophages and HaCaT keratinocytes. Virus-induced changes in the expression of 14-3-3 signaling proteins as well as rearrangement of host cell cytoskeleton were detected in HaCaT keratinocytes. Caspase-3-dependent apoptosis was detected in HaCaT keratinocytes and macrophages after viral stimulation. Our studies with macrophages also showed that several inflammatory pathways, and especially the NLRP3 inflammasome, are activated as a result of viral RNA and influenza A virus infection. Additionally, we showed that cathepsins, src tyrosine kinase and P2X7 receptor were involved in the inflammasome activation. Finally, we showed that viral stimulation triggered extensive protein secretion from macrophages. In conclusion, our proteomic experiments have given an in-depth view of cellular events activated in human primary macrophages and HaCaT keratinocytes after viral infection.
  • Siljamäki, Pia (Helsingin yliopisto, 2014)
    Staphylococcus epidermidis (SE) is an opportunistic pathogen capable of infecting humans and animals. It is a frequent cause of hospital-acquired infections in humans and of intramammary infections (IMIs) in dairy cows. The protein expression profiles and the genomes of three SE strains, one associated with bovine mastitis (PM221), one representing a commensal/low-virulent human strain (ATCC12228 isolated from a healthy human host) and the third being a virulent human strain (RP62A isolated from a catheter-associated sepsis) were compared. At the genome and total proteome levels, the bovine strain PM221 was found to be more similar to ATCC12228 than to RP62A. The strain-specific differences found between PM221 and ATCC12228 are believed to have roles in adaptation and virulence. These findings may explain why ATCC12228 was able to cause persistent mastitis in an experimental infection study albeit with a milder clinical outcome than encountered with PM221. These findings strengthen the hypothesis that humans could represent an important source of SE-mediated infections in dairy cows. The exoproteome analysis revealed that the exoproteome of PM221 resembled more that of RP62A than the exoproteome of ATCC12228. The major part of the identified exoproteins was predicted to be cytoplasmic, indicating that these proteins might be surface-displayed moonlighting proteins or that they had been embedded in membrane vesicles. PM221 and RP62A were shown to be more efficient in non-classical protein secretion. These findings may explain the higher virulence capacity of PM221 than that of ATCC12228. Our studies also indicated that PM221 uses less aggressive strategies than RP62A when establishing an infection in vivo. Phenotypic tests indicated that SE may use several strategies to improve its adaptation to different environments; the virulent bovine and human strains (PM221 and RP62A) were able to use biofilm formation for colonization and adaptation, and PM221 and ATCC12228 could downregulate their tricarboxylic acid cycle activity and increase the formation of small colony variants to improve bacterial survival during the stationary phase. In addition, the bovine PM221 strain may possess an advantage since it has higher tributyrin activity in the milk environment, whereas the human strains may benefit from their higher urease and/or catalase activities helping these strains to survive in their ecological niches and indwelling medical devices. In conclusion, the present studies demonstrate that the SE species may exploit diverse strategies involving specific changes in their late stationary phase metabolism as well as in protein export strategies accompanied by increased production of certain enzymes in order to ensure better adaptation and/or successful infection.
  • Timonen, Marjut (Helsingin yliopisto, 2010)
    Boron neutron capture therapy (BNCT) is a radiotherapy that has mainly been used to treat malignant brain tumours, melanomas, and head and neck cancer. In BNCT, the patient receives an intravenous infusion of a 10B-carrier, which accumulates in the tumour area. The tumour is irradiated with epithermal or thermal neutrons, which result in a boron neutron capture reaction that generates heavy particles to damage tumour cells. In Finland, boronophenylalanine fructose (BPA-F) is used as the 10B-carrier. Currently, the drifting of boron from blood to tumour as well as the spatial and temporal accumulation of boron in the brain, are not precisely known. Proton magnetic resonance spectroscopy (1H MRS) could be used for selective BPA-F detection and quantification as aromatic protons of BPA resonate in the spectrum region, which is clear of brain metabolite signals. This study, which included both phantom and in vivo studies, examined the validity of 1H MRS as a tool for BPA detection. In the phantom study, BPA quantification was studied at 1.5 and 3.0 T with single voxel 1H MRS, and at 1.5 T with magnetic resonance imaging (MRSI). The detection limit of BPA was determined in phantom conditions at 1.5 T and 3.0 T using single voxel 1H MRS, and at 1.5 T using MRSI. In phantom conditions, BPA quantification accuracy of ± 5% and ± 15% were achieved with single voxel MRS using external or internal (internal water signal) concentration references, respectively. For MRSI, a quantification accuracy of <5% was obtained using an internal concentration reference (creatine). The detection limits of BPA in phantom conditions for the PRESS sequence were 0.7 (3.0 T) and 1.4 mM (1.5 T) mM with 20 × 20 × 20 mm3 single voxel MRS, and 1.0 mM with acquisition-weighted MRSI (nominal voxel volume 10(RL) × 10(AP) × 7.5(SI) mm3), respectively. In the in vivo study, an MRSI or single voxel MRS or both was performed for ten patients (patients 1-10) on the day of BNCT. Three patients had glioblastoma multiforme (GBM), and five patients had a recurrent or progressing GBM or anaplastic astrocytoma gradus III, and two patients had head and neck cancer. For nine patients (patients 1-9), MRS/MRSI was performed 70-140 min after the second irradiation field, and for one patient (patient 10), the MRSI study began 11 min before the end of the BPA-F infusion and ended 6 min after the end of the infusion. In comparison, single voxel MRS was performed before BNCT, for two patients (patients 3 and 9), and for one patient (patient 9), MRSI was performed one month after treatment. For one patient (patient 10), MRSI was performed four days before infusion. Signals from the tumour spectrum aromatic region were detected on the day of BNCT in three patients, indicating that in favourable cases, it is possible to detect BPA in vivo in the patient’s brain after BNCT treatment or at the end of BPA-F infusion. However, because the shape and position of the detected signals did not exactly match the BPA spectrum detected in the in vitro conditions, assignment of BPA is difficult. The opportunity to perform MRS immediately after the end of BPA-F infusion for more patients is necessary to evaluate the suitability of 1H MRS for BPA detection or quantification for treatment planning purposes. However, it could be possible to use MRSI as criteria in selecting patients for BNCT.
  • Belevich, Ilya (Helsingin yliopisto, 2007)
    Terminal oxidases are the final proteins of the respiratory chain in eukaryotes and some bacteria. They catalyze most of the biological oxygen consumption on Earth done by aerobic organisms. During the catalytic reaction terminal oxidases reduce dioxygen to water and use the energy released in this process to maintain the electrochemical proton gradient by functioning as a redox-driven proton pump. This membrane gradient of protons is extremely important for cells as it is used for many cellular processes, such as transportation of substrates and ATP synthesis. Even though the structures of several terminal oxidases are known, they are not sufficient in themselves to explain the molecular mechanism of proton pumping. In this work we have applied a complex approach using a variety of different techniques to address the properties and the mechanism of proton translocation by the terminal oxidases. The combination of direct measurements of pH changes during catalytic turnover, time-resolved potentiometric electrometry and optical spectroscopy, made it possible to obtain valuable information about various aspects of oxidase functioning. We compared oxygen binding properties of terminal oxidases from the distinct heme-copper (CcO) and cytochrome bd families and found that cytochrome bd has a high affinity for oxygen, which is 3 orders of magnitude higher than that of CcO. Interestingly, the difference between CcO and cytochrome bd is not only in higher affinity of the latter to oxygen, but also in the way that each of these enzymes traps oxygen during catalysis. CcO traps oxygen kinetically - the molecule of bound dioxygen is rapidly reduced before it can dissociate. Alternatively, cytochrome bd employs an alternative mechanism of oxygen trapping - part of the redox energy is invested into tight oxygen binding, and the price paid for this is the lack of proton pumping. A single cycle of oxygen reduction to water is characterized by translocation of four protons across the membrane. Our results make it possible to assign the pumping steps to discrete transitions of the catalytic cycle and indicate that during in vivo turnover of the oxidase these four protons are transferred, one at a time, during the P→F, F→OH, Oh→Eh, and Eh→R transitions. At the same time, each individual proton translocation step in the catalytic cycle is not just a single reaction catalyzed by CcO, but rather a complicated sequence of interdependent electron and proton transfers. We assume that each single proton translocation cycle of CcO is assured by internal proton transfer from the conserved Glu-278 to an as yet unidentified pump site above the hemes. Delivery of a proton to the pump site serves as a driving reaction that forces the proton translocation cycle to continue.
  • Manner, Jukka (Helsingin yliopisto, 2003)
  • Kolehmainen, Leena (Helsingin yliopisto, 2005)
  • Meinander, Kristian (Helsingin yliopisto, 2014)
    The highly prostate specific serine protease kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen, PSA) is widely used as a biomarker for prostate cancer and it has also been postulated that it may play a part in tumour growth. Especially interestesting is the antiangiogenic effect exerted by proteolytically active KLK3 in cell line models. In order to stimulate the proteolytic activity of KLK3, a series of peptides have been developed by phage display methodology. Even though the peptides are quite potent KLK3 stimulators, they are not directly suitable for in vivo studies or use as drugs. Even though there are many natural and unnatural biologically active peptides, they suffer from rapid clearance via the liver and kidneys and proteolytic degradation of the compounds both in the gastrointestinal tract and other parts of the body. This gives peptides a poor oral bioavailability meaning that they are usually administered as intravenous or intramuscular injections. Several different strategies have been developed in order to access compounds with improved bioavailability including modifications of the peptide structure, development of pseudopeptides and development of small molecular weight peptidomimetics. This thesis concentrates on the further development of the two most potent peptides known to stimulate KLK3, i.e. B-2 and C-4. The main part of the work was concentrated on the replacement of disulphide bridges in the peptides in order to both gain more information on which residues are necessary for obtaining the biological activity and at the same time also gain information on how changes to the geometry of the disulphide bridge affects the activity. A series of different disulphide bridge mimicking building blocks were designed and synthesised with the intention of using them in a protocol for solid-phase synthesis of KLK3 stimulating peptides. Unfortunately, the use of these building blocks in the synthesis of pseudopeptides based on C-4 turned out to be an unsurmountable challenge and the synthesis had to be completed using a different strategy in which the key step was the use of ring-closing metathesis (RCM) for the cyclisation of the partly completed pseudopeptide. Pleasingly, the synthesis of pseudopeptide analogues of the B-2 peptide using the building blocks was more successful. In total three pseudopeptide analogues of C-4 and four of B-2 were synthesised and shown to retain the biological activity of the parent peptides. Based on the information from the synthesised pseudopeptides and a molecular modelling study, a 4-quinolone based peptidomimetic was designed to mimic the C-4 peptide and a synthetic protocol was devised to access this compound. Even though the synthesis of the desired target compound has so far not been successful, the synthetic protocol that was designed has given access to a number of 1,2,8-trisubstituted 4-quinolone derivatives.