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.

Enhancing absorptive capacity in a non-research and development. An action research approach to converting individual observations into organisational awareness

The ability to recognize potential knowledge and convert it into business opportunities is one of the key factors of renewal in uncertain environments. This thesis examines absorptive capacity in the context of non-research and development innovation, with a primary focus on the social interaction that facilitates the absorption of knowledge. It proposes that everyone is and should be entitled to take part in the social interaction that shapes individual observations into innovations. Both innovation and absorptive capacity have been traditionally related to research and development departments and institutions. These innovations need to be adopted and adapted by others. This so-called waterfall model of innovations is only one aspect of new knowledge generation and innovation. In addition to this Science–Technology–Innovation perspective, more attention has been recently paid to the Doing–Using–Interacting mode of generating new knowledge and innovations. The amount of literature on absorptive capacity is vast, yet the concept is reified. The greater part of the literature links absorptive capacity to research and development departments. Some publications have focused on the nature of absorptive capacity in practice and the role of social interaction in enhancing it. Recent literature on absorptive capacity calls for studies that shed light on the relationship between individual absorptive capacity and organisational absorptive capacity. There has also been a call to examine absorptive capacity in non-research and development environments. Drawing on the literature on employee-driven innovation and social capital, this thesis looks at how individual observations and ideas are converted into something that an organisation can use. The critical phases of absorptive capacity, during which the ideas of individuals are incorporated into a group context, are assimilation and transformation. These two phases are seen as complementary: whereas assimilation is the application of easy-to-accept knowledge, transformation challenges the current way of thinking. The two require distinct kinds of social interaction and practices. The results of this study can been crystallised thus: “Enhancing absorptive capacity in practicebased non-research and development context is to organise the optimal circumstances for social interaction. Every individual is a potential source of signals leading to innovations. The individual, thus, recognises opportunities and acquires signals. Through the social interaction processes of assimilation and transformation, these signals are processed into the organisation’s reality and language. The conditions of creative social capital facilitate the interplay between assimilation and transformation. An organisation that strives for employee-driven innovation gains the benefits of a broader surface for opportunity recognition and faster absorption.” If organisations and managers become more aware of the benefits of enhancing absorptive capacity in practice, they have reason to assign resources to those practices that facilitate the creation of absorptive capacity. By recognising the underlying social mechanisms and structural features that lead either to assimilation or transformation, it is easier to balance between renewal and effective operations.

Prediction of 3D structure and ligand-binding properties : ABC transporters and flavodiiron proteins from Synechocystis sp. strain PCC 6803

Cyanobacteria are unicellular, non-nitrogen-fixing prokaryotes, which perform photosynthesis similarly as higher plants. The cyanobacterium Synechocystis sp. strain PCC 6803 is used as a model organism in photosynthesis research. My research described herein aims at understanding the function of the photosynthetic machinery and how it responds to changes in the environment. Detailed knowledge of the regulation of photosynthesis in cyanobacteria can be utilized for biotechnological purposes, for example in the harnessing of solar energy for biofuel production. In photosynthesis, iron participates in electron transfer. Here, we focused on iron transport in Synechocystis sp. strain PCC 6803 and particularly on the environmental regulation of the genes encoding the FutA2BC ferric iron transporter, which belongs to the ABC transporter family. A homology model built for the ATP-binding subunit FutC indicates that it has a functional ATPbinding site as well as conserved interactions with the channel-forming subunit FutB in the transporter complex. Polyamines are important for the cell proliferation, differentiation and apoptosis in prokaryotic and eukaryotic cells. In plants, polyamines have special roles in stress response and in plant survival. The polyamine metabolism in cyanobacteria in response to environmental stress is of interest in research on stress tolerance of higher plants. In this thesis, the potd gene encoding an polyamine transporter subunit from Synechocystis sp. strain PCC 6803 was characterized for the first time. A homology model built for PotD protein indicated that it has capability of binding polyamines, with the preference for spermidine. Furthermore, in order to investigate the structural features of the substrate specificity, polyamines were docked into the binding site. Spermidine was positioned very similarly in Synechocystis PotD as in the template structure and had most favorable interactions of the docked polyamines. Based on the homology model, experimental work was conducted, which confirmed the binding preference. Flavodiiron proteins (Flv) are enzymes, which protect the cell against toxicity of oxygen and/or nitric oxide by reduction. In this thesis, we present a novel type of photoprotection mechanism in cyanobacteria by the heterodimer of Flv2/Flv4. The constructed homology model of Flv2/Flv4 suggests a functional heterodimer capable of rapid electron transfer. The unknown protein sll0218, encoded by the flv2-flv4 operon, is assumed to facilitate the interaction of the Flv2/Flv4 heterodimer and energy transfer between the phycobilisome and PSII. Flv2/Flv4 provides an alternative electron transfer pathway and functions as an electron sink in PSII electron transfer.

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.

Understanding the bioactivity of plant tannins: developments in analysis methods and structure-activity studies

Tannins, typically segregated into two major groups, the hydrolyzable tannins (HTs) and the proanthocyanidins (PAs), are plant polyphenolic secondary metabolites found throughout the plant kingdom. On one hand, tannins may cause harmful nutritional effects on herbivores, for example insects, and hence they work as plants’ defense against plant-eating animals. On the other hand, they may affect positively some herbivores, such as mammals, for example by their antioxidant, antimicrobial, anti-inflammatory or anticarcinogenic activities. This thesis focuses on understanding the bioactivity of plant tannins, their anthelmintic properties and the tools used for the qualitative and quantitative analysis of this endless source of structural diversity. The first part of the experimental work focused on the development of ultra-high performance liquid chromatography−tandem mass spectrometry (UHPLC-MS/MS) based methods for the rapid fingerprint analysis of bioactive polyphenols, especially tannins. In the second part of the experimental work the in vitro activity of isolated and purified HTs and their hydrolysis product, gallic acid, was tested against egg hatching and larval motility of two larval developmental stages, L1 and L2, of a common ruminant gastrointestinal parasite, Haemonchus contortus. The results indicated clear relationships between the HT structure and the anthelmintic activity. The activity of the studied compounds depended on many structural features, including size, functional groups present in the structure, and the structural rigidness. To further understand tannin bioactivity on a molecular level, the interaction between bovine serum albumin (BSA), and seven HTs and epigallocatechin gallate was examined. The objective was to define the effect of pH on the formation on tannin–protein complexes and to evaluate the stability of the formed complexes by gel electrophoresis and MALDI-TOF-MS. The results indicated that more basic pH values had a stabilizing effect on the tannin–protein complexes and that the tannin oxidative activity was directly linked with their tendency to form covalently stabilized complexes with BSA at increased pH.

A modified structural stress method for fatigue assessment of welded structures

Fatigue life assessment of weldedstructures is commonly based on the nominal stress method, but more flexible and accurate methods have been introduced. In general, the assessment accuracy is improved as more localized information about the weld is incorporated. The structural hot spot stress method includes the influence of macro geometric effects and structural discontinuities on the design stress but excludes the local features of the weld. In this thesis, the limitations of the structural hot spot stress method are discussed and a modified structural stress method with improved accuracy is developed and verified for selected welded details. The fatigue life of structures in the as-welded state consists mainly of crack growth from pre-existing cracks or defects. Crack growth rate depends on crack geometry and the stress state on the crack face plane. This means that the stress level and shape of the stress distribution in the assumed crack path governs thetotal fatigue life. In many structural details the stress distribution is similar and adequate fatigue life estimates can be obtained just by adjusting the stress level based on a single stress value, i.e., the structural hot spot stress. There are, however, cases for which the structural stress approach is less appropriate because the stress distribution differs significantly from the more common cases. Plate edge attachments and plates on elastic foundations are some examples of structures with this type of stress distribution. The importance of fillet weld size and weld load variation on the stress distribution is another central topic in this thesis. Structural hot spot stress determination is generally based on a procedure that involves extrapolation of plate surface stresses. Other possibilities for determining the structural hot spot stress is to extrapolate stresses through the thickness at the weld toe or to use Dong's method which includes through-thickness extrapolation at some distance from the weld toe. Both of these latter methods are less sensitive to the FE mesh used. Structural stress based on surface extrapolation is sensitive to the extrapolation points selected and to the FE mesh used near these points. Rules for proper meshing, however, are well defined and not difficult to apply. To improve the accuracy of the traditional structural hot spot stress, a multi-linear stress distribution is introduced. The magnitude of the weld toe stress after linearization is dependent on the weld size, weld load and plate thickness. Simple equations have been derived by comparing assessment results based on the local linear stress distribution and LEFM based calculations. The proposed method is called the modified structural stress method (MSHS) since the structural hot spot stress (SHS) value is corrected using information on weld size andweld load. The correction procedure is verified using fatigue test results found in the literature. Also, a test case was conducted comparing the proposed method with other local fatigue assessment methods.

Structural development of the harvester head saw unit

The main goal of the thesis was to further develop harvester head saw device to the Finnish forest machine manufacturer. The work was done from the basis of the manufacturer´s current production model and the earlier study from this same subject called: “Development of chain saw for harvester” Tero Kaatrasalo, 2004. The work was focused to improving the serviceability and reliability of the saw device, but design also included adding few beforehand determined new features into the saw unit. This was done to give some added value for the end customer. The work includes analysis of the earlier saw devices and ideations of the improvements for the structure.