The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Biomolecular screening for inhibitors of butyrylcholinesterase : identification and characterization using in vitro and in silico tools

Drug discovery is a continuous process where researchers are constantly trying to find new and better drugs for the treatment of various conditions. Alzheimer’s disease, a neurodegenerative disease mostly affecting the elderly, has a complex etiology with several possible drug targets. Some of these targets have been known for years while other new targets and theories have emerged more recently. Cholinesterase inhibitors are the major class of drugs currently used for the symptomatic treatment of Alzheimer’s disease. In the Alzheimer’s disease brain there is a deficit of acetylcholine and an impairment in signal transmission. Acetylcholinesterase has therefore been the main target as this is the main enzyme hydrolysing acetylcholine and ending neurotransmission. It is believed that by inhibiting acetylcholinesterase the cholinergic signalling can be enhanced and the cognitive symptoms that arise in Alzheimer’s disease can be improved. Butyrylcholinesterase, the second enzyme of the cholinesterase family, has more recently attracted interest among researchers. Its function is still not fully known, but it is believed to play a role in several diseases, one of them being Alzheimer’s disease. In this contribution the aim has primarily been to identify butyrylcholinesterase inhibitors to be used as drug molecules or molecular probes in the future. Both synthetic and natural compounds in diverse and targeted screening libraries have been used for this purpose. The active compounds have been further characterized regarding their potencies, cytotoxicity, and furthermore, in two of the publications, the inhibitors ability to also inhibit Aβ aggregation in an attempt to discover bifunctional compounds. Further, in silico methods were used to evaluate the binding position of the active compounds with the enzyme targets. Mostly to differentiate between the selectivity towards acetylcholinesterase and butyrylcholinesterase, but also to assess the structural features required for enzyme inhibition. We also evaluated the compounds, active and non-active, in chemical space using the web-based tool ChemGPS-NP to try and determine the relevant chemical space occupied by cholinesterase inhibitors. In this study, we have succeeded in finding potent butyrylcholinesterase inhibitors with a diverse set of structures, nine chemical classes in total. In addition, some of the compounds are bifunctional as they also inhibit Aβ aggregation. The data gathered from all publications regarding the chemical space occupied by butyrylcholinesterase inhibitors we believe will give an insight into the chemically active space occupied by this type of inhibitors and will hopefully facilitate future screening and result in an even deeper knowledge of butyrylcholinesterase inhibitors.

PHOTOCATALYTIC OXIDATION OF AQUEOUS SOLUTIONS OF JET FUEL AND ICING INHIBITORS

The present paper is devoted to the results of experimental research undertaken into photocatalytical oxidation (PCO) of aqueous solutions of de-icing agents and aqueous extract of jet fuel. The report consists of introduction, literature review, description of materials and methods, discussion of results and conclusions. TiO2 was selected as a photocatalyst for the experiments with synthetic solutions of ethylene glycol, 2-ethoxyethanol and aqueous extract of jet fuel. To explain the PCO mechanisms affecting certain behaviour of de-icing agent under distinctive conditions, the following factors were studied: the impact of initial concentration of pollutant, the role of pH, the presence of tert-butanol as OH·-radicals scavenger and mineral admixtures. PCO under solar radiation performed in two ways: catalysed by irradiated TiO2 slurry or by TiO2 attached to buoyant hollow glass micro-spheres. Special attention was paid to the energy-saving PCO with reduced intensity mixing of the slurry. The effect of PCO was assessed by determination of residual chemical oxygen demand of solution (COD) and by measuring of concentration of glycols. The PCO process efficiency was assumed to be dependent on the TiO2 suspension fractional composition. Thus, the following effects of solutions’ media were viewed: presence of organic admixtures, pH influence, mixing mode during the PCO. The effects of mineral admixtures - Ca2+, Fe3+/2+, Mn2+, SO42- - that are often present in natural and wastewater systems or produced during the degradation of organic pollutants and which can affect the rate of PCO of de-icing agents, were also investigated.

Enhancing performance of paper coatings by tailor-made plastic pigments

The paper industry is constantly looking for new ideas for improving paper products while competition and raw material prices are increasing. Many paper products are pigment coated. Coating layer is the top layer of paper, thus by modifying coating pigment also the paper itself can be altered and value added to the final product. In this thesis, synthesis of new plastic and hybrid pigments and their performance in paper and paperboard coating is reported. Two types of plastic pigments were studied: core-shell latexes and solid beads of maleimide copolymers. Core-shell latexes with partially crosslinked hydrophilic polymer core of poly(n-butyl acrylate-co-methacrylic acid) and a hard hydrophobic polystyrene shell were prepared to improve the optical properties of coated paper. In addition, the effect of different crosslinkers was analyzed and the best overall performance was achieved by the use of ethylene glycol dimethacrylate (EGDMA). Furthermore, the possibility to modify core-shell latex was investigated by introducing a new polymerizable optical brightening agent, 1-[(4-vinylphenoxy)methyl]-4-(2-henylethylenyl)benzene which gave promising results. The prepared core-shell latex pigments performed smoothly also in pilot coating and printing trials. The results demonstrated that by optimizing polymer composition, the optical and surface properties of coated paper can be significantly enhanced. The optimal reaction conditions were established for thermal imidization of poly(styrene-co-maleimide) (SMI) and poly(octadecene-co-maleimide) (OMI) from respective maleic anhydride copolymer precursors and ammonia in a solvent free process. The obtained aqueous dispersions of nanoparticle copolymers exhibited glass transition temperatures (Tg) between 140-170ºC and particle sizes from 50-230 nm. Furthermore, the maleimide copolymers were evaluated in paperboard coating as additional pigments. The maleimide copolymer nanoparticles were partly imbedded into the porous coating structure and therefore the full potential of optical property enhancement for paperboard was not achieved by this method. The possibility to modify maleimide copolymers was also studied. Modifications were carried out via N-substitution by replacing part of the ammonia in the imidization reaction with amines, such as triacetonediamine (TAD), aspartic acid (ASP) and fluorinated amines (2,2,2- trifluoroethylamine, TFEA and 2,2,3,3,4,4,4-heptafluorobuthylamine, HFBA). The obtained functional nanoparticles varied in size between 50-217 nm and their Tg from 150-180ºC. During the coating process the produced plastic pigments exhibited good runnability. No significant improvements were achieved in light stability with TAD modified copolymers whereas nanoparticles modified with aspartic acid and those containing fluorinated groups showed the desired changes in surface properties of the coated paperboard. Finally, reports on preliminary studies with organic-inorganic hybrids are presented. The hybrids prepared by an in situ polymerization reaction consisted of 30 wt% poly(styrene- co-maleimide) (SMI) and high levels of 70 wt% inorganic components of kaolin and/or alumina trihydrate. Scanning Electron Microscopy (SEM) images and characterization by Fourier Transform Infrared Spcetroscopy (FTIR) and X-Ray Diffraction (XRD) revealed that the hybrids had conventional composite structure and inorganic components were covered with precipitated SMI nanoparticles attached to the surface via hydrogen bonding. In paper coating, the hybrids had a beneficial effect on increasing gloss levels.

Generation and Characterization of Knockout Mice and Analysis of Patient Material Demonstrates a Role for Tissue Inhibitor of Metalloproteinases 4 (Timp4) in the Cardiovascular System and Tumor Metastasis

This thesis focuses on tissue inhibitor of metalloproteinases 4 (TIMP4) which is the newest member of a small gene and protein family of four closely related endogenous inhibitors of extracellular matrix (ECM) degrading enzymes. Existing data on TIMP4 suggested that it exhibits a more restricted expression pattern than the other TIMPs with high expression levels in heart, brain, ovary and skeletal muscle. These observations and the fact that the ECM is of special importance to provide the cardiovascular system with structural strength combined with elasticity and distensibility, prompted the present molecular biologic investigation on TIMP4. In the first part of the study the murine Timp4 gene was cloned and characterized in detail. The structure of murine Timp4 genomic locus resembles that in other species and of the other Timps. The highest Timp4 expression was detected in heart, ovary and brain. As the expression pattern of Timp4 gives only limited information about its role in physiology and pathology, Timp4 knockout mice were generated next. The analysis of Timp4 knockout mice revealed that Timp4 deficiency has no obvious effect on the development, growth or fertility of mice. Therefore, Timp4 deficient mice were challenged using available cardiovascular models, i.e. experimental cardiac pressure overload and myocardial infarction. In the former model, Timp4 deficiency was found to be compensated by Timp2 overexpression, whereas in the myocardial infarct model, Timp4 deficiency resulted in increased mortality due to increased susceptibility for cardiac rupture. In the wound healing model, Timp4 deficiency was shown to result in transient retardation of re-epithelialization of cutaneous wounds. Melanoma tumor growth was similar in Timp4 deficient and control mice. Despite of this, lung metastasis of melanoma cells was significantly increased in Timp4 null mice. In an attempt to translate the current findings to patient material, TIMP4 expression was studied in human specimens representing different inflammatory cardiovascular pathologies, i.e. giant cell arteritis, atherosclerotic coronary arteries and heart allografts exhibiting signs of chronic rejection. The results showed that cardiovascular expression of TIMP4 is elevated particularly in areas exhibiting inflammation. The results of the present studies suggest that TIMP4 has a special role in the regulation of tissue repair processes in the heart, and also in healing wounds and metastases. Furthermore, evidence is provided suggesting the usefulness of TIMP4 as a novel systemic marker for vascular inflammation.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

The Structure and Function of αI Domains in Collagen Receptor and Leukocyte Integrins

Integrins are heterodimeric, signaling transmembrane adhesion receptors that connect the intracellular actin microfilaments to the extracellular matrix composed of collagens and other matrix molecules. Bidirectional signaling is mediated via drastic conformational changes in integrins. These changes also occur in the integrin αI domains, which are responsible for ligand binding by collagen receptor and leukocyte specific integrins. Like intact integrins, soluble αI domains exist in the closed, low affinity form and in the open, high affinity form, and so it is possible to use isolated αI domains to study the factors and mechanisms involved in integrin activation/deactivation. Integrins are found in all mammalian tissues and cells, where they play crucial roles in growth, migration, defense mechanisms and apoptosis. Integrins are involved in many human diseases, such as inflammatory, cardiovascular and metastatic diseases, and so plenty of effort has been invested into developing integrin specific drugs. Humans have 24 different integrins, four of which are collagen receptor (α1β1, α2β1, α10β1, α11β1) and five leukocyte specific integrins (αLβ2, αMβ2, αXβ2, αDβ2, αEβ7). These two integrin groups are quite unselective having both primary and secondary ligands. This work presents the first systematic studies performed on these integrin groups to find out how integrin activation affects ligand binding and selectivity. These kinds of studies are important not only for understanding the partially overlapping functions of integrins, but also for drug development. In general, our results indicated that selectivity in ligand recognition is greatly reduced upon integrin activation. Interestingly, in some cases the ligand binding properties of integrins have been shown to be cell type specific. The reason for this is not known, but our observations suggest that cell types with a higher integrin activation state have lower ligand selectivity, and vice versa. Furthermore, we solved the three-dimensional structure for the activated form of the collagen receptor α1I domain. This structure revealed a novel intermediate conformation not previously seen with any other integrin αI domain. This is the first 3D structure for an activated collagen receptor αI domain without ligand. Based on the differences between the open and closed conformation of the αI domain we set structural criteria for a search for effective collagen receptor drugs. By docking a large number of molecules into the closed conformation of the α2I domain we discovered two polyketides, which best fulfilled the set structural criteria, and by cell adhesion studies we showed them to be specific inhibitors of the collagen receptor integrins.

Facilitating corporate entrepreneurial behavior: A study of corporate entrepreneurial behavior in corporate banking

This study discusses the pronounced importance of corporate entrepreneurial behavior, CEB, facilitation and enablement as a key dimension in the rapidly changing business environment within companies. The research target is a large finance company in Finland, where regulations, compliance and processes restrict and refine extensively business approach. The purpose of this study is to foster the understanding of corporate entrepreneurial behavior and requirements and identify the supporters and inhibitors of facilitation of it. Furthermore, this study examines who should be driving the implementation and offer concrete outcome for the company to get the facilitation started and berth it as part of the organizational culture and values. The theoretical background is constructed from literature related to concept of corporate entrepreneurial behavior, factors supporting and hindering the facilitation based on previous studies and innovation management. Furthermore theoretical framework of middle managers entrepreneurial behavior in facilitation process was researched. Additionally top down and bottom up approach of conversational space building within the organization in order to foster innovation and involving mindset and behavior was in the core literature. The empirical research conducted for the study consists three parts; innovation audit questionnaire, semi-structured interviews and secondary data from previously made research within the case company. Questionnaire and interviews were targeted to eight middle managers within the company, the head of branch regions in corporate segment. The secondary data was collected from over 300 employees in the case company by an external company. Research results were analyzed mainly by themes and source division in adaption with the theoretical framework. The study finds that facilitation of CEB should be a strategic choice and requires strong management support and examples. Behavior should be involved with organizational culture, values, structures and processes. The companies´ willingness to take risks and encourage employees at all levels to participate and be involved by taking ownership and responsibility is in the core. CEB is found to be a key dimension in increasing employee satisfaction and engagement, competitive advantages and economic growth of companies. There is increased interest towards CEB in the case company but there is lack in the mutual consensus of it. CEB is not in the strategy although the mindset and support from management is in place. There is no concrete enablement and space for innovation and CEB although the platform would be receptive. Further research is needed to build shared vision of CEB and how to make it a part of the organizational culture and values in addition to building the conversational space.

Finnish perceptions on management accounting systems: towards an upgraded sophistication model

This study is based on a large survey study of over 1500 Finnish companies’ usage, needs and implementation difficulties of management accounting systems. The study uses quantitative, qualitative and mixed methods to answer the research questions. The empirical data used in the study was gathered through structured interviews with randomly selected companies of varying sizes and industries. The study answers the three research questions by analyzing the characteristics and behaviors of companies working in Finland. The study found five distinctive groups of companies according to the characteristics of their cost information and management accounting system use. The study also showed that the state of cost information and management accounting systems depends on the industry and size of the companies. It was found that over 50% of the companies either did not know how their systems could be updated or saw systems as inadequate. The qualitative side also highlighted the needs for tailored and integrated management accounting systems for creating more value to the managers of companies. The major inhibitors of new system implementation were the lack of both monetary and human resources. Through the use of mixed methods and design science a new and improved sophistication model is created based on previous research results combined with the information gathered from previous literature. The sophistication model shows the different stages of management accounting systems in use and what companies can achieve with the implementation and upgrading of their systems.

VAP-1 as drug target in inflammation : structural features determining ligand interactions

Vascular adhesion protein-1 (VAP-1), which belongs to the copper amine oxidases (CAOs), is a validated drug target in inflammatory diseases. Inhibition of VAP-1 blocks the leukocyte trafficking to sites of inflammation and alleviates inflammatory reactions. In this study, a novel set of potent pyridazinone inhibitors is presented together with their X-ray structure complexes with VAP-1. The crystal structure of serum VAP-1 (sVAP-1) revealed an imidazole binding site in the active site channel and, analogously, the pyridazinone inhibitors were designed to bind into the channel. This is the first time human VAP-1 has been crystallized with a reversible inhibitor and the structures reveal detailed information of the binding mode on the atomic level. Similarly to some earlier studied inhibitors of human VAP-1, the designed pyridazinone inhibitors bind rodent VAP-1 with a lower affinity than human VAP-1. Therefore, we made homology models of rodent VAP-1 and compared human and rodent enzymes to determine differences that might affect the inhibitor binding. The comparison of the crystal structures of the human VAP-1 and the mouse VAP-1 homology model revealed key differences important for the species specific binding properties. In general, the channel in mouse VAP-1 is more narrow and polar than the channel in human VAP-1, which is wider and more hydrophobic. The differences are located in the channel leading to the active site, as well as, in the entrance to the active site channel. The information obtained from these studies is of great importance for the development and design of drugs blocking the activity of human VAP-1, as rodents are often used for in vivo testing of candidate drugs. In order to gain more insight into the selective binding properties of the different CAOs in one species a comprehensive evolutionary study of mammalian CAOs was performed. We found that CAOs can be classified into sub-families according to the residues X1 and X2 of the Thr/Ser-X1-X2-Asn-Tyr-Asp active site motif. In the phylogenetic tree, CAOs group into diamine oxidase, retina specific amine oxidase and VAP-1/serum amine oxidase clades based on the residue in the position X2. We also found that VAP-1 and SAO can be further differentiated based on the residue in the position X1. This is the first large-scale comparison of CAO sequences, which explains some of the reasons for the unique substrate specificities within the CAO family.