Neurofilament proteins in the postnatal rat hippocampus. Developmental expression and changes in experimental epilepsy

Neurofilament proteins (NFs) are the major components of the intermediate filaments of the neuronal cytoskeleton. The three different NF proteins; the low (NF-L), medium (NF-M),and dendrites.NF proteins play an important role in neuronal development, and plasticity,and seem to contribute to the pathophysiology of several diseases. However, the detailed expression patterns of NF proteins in the course of postnatal aturation, and in response to seizures in the rat have remained unknown. In this work, I have studied the developmental expression and cellular distribution of the three NF proteins in the rat hippocampus during the postnatal development. The reactivity of NF proteins in response to kainic acid (KA)-induced status epilepticus (SE)was studied in the hippocampus of 9-day-old rats, and using in vitro organotypic hippocampal slices cultures prepared from P6-7 rats. The results showed that NF-L and NF-M proteins are expressed already at the postnatal day 1, while the expression of NF-H mainly occurred during the second postnatal week. The immunoreactivity of NF proteins varied depending on the cell type and sub-cellular location in the hippocampus. In adult rats, KA-induced SE typically results in severe and permanent NF degradation. However, in our P9 rats KA-induced SE resulted in a transient increase in the expression of NF proteins during the first few hours but not degradation. No neuronal death or mossy fiber sprouting was observed at any time after SE. The in vitro studies with OHCs, which mimick the in vivo developing models where a local injection of KA is applied(e.g. intrahippocampal), indicated that NF proteins were rapidly degraded in response to KA treatment, this effect being effectively inhibited by the treatment with the AMPA receptor antagonist CNQX, and calpain inhibitor MDL-28170. These compounds also significantly ameliorated the KA-induced region-specific neuronal damage. The NMDA receptor antagonist and the L-type Ca2+ channel blocker did not have any significant effect. In conclusion, the results indicate that the developmental expression of NF in the rat hippocampus is differentially regulated and targeted in the different hippocampal cell types during the postnatal development. Furthermore, despite SE, the mechanisms leading to NF degradation and neuronal death are not activated in P9 rats unlike in adults. The reason for this remains unknown. The results in organotypic hippocampal cultures confirm the validity of this in vitro model to study development processes, and to perform pharmacological studies. The results also suggest that calpain proteases as interesting pharmacological targets to reduce neuronal damage after acute excitotoxic insults.

On Undecidable Dynamical Properties of Reversible One-Dimensional Cellular Automata

Cellular automata are models for massively parallel computation. A cellular automaton consists of cells which are arranged in some kind of regular lattice and a local update rule which updates the state of each cell according to the states of the cell's neighbors on each step of the computation. This work focuses on reversible one-dimensional cellular automata in which the cells are arranged in a two-way in_nite line and the computation is reversible, that is, the previous states of the cells can be derived from the current ones. In this work it is shown that several properties of reversible one-dimensional cellular automata are algorithmically undecidable, that is, there exists no algorithm that would tell whether a given cellular automaton has the property or not. It is shown that the tiling problem of Wang tiles remains undecidable even in some very restricted special cases. It follows that it is undecidable whether some given states will always appear in computations by the given cellular automaton. It also follows that a weaker form of expansivity, which is a concept of dynamical systems, is an undecidable property for reversible one-dimensional cellular automata. It is shown that several properties of dynamical systems are undecidable for reversible one-dimensional cellular automata. It shown that sensitivity to initial conditions and topological mixing are undecidable properties. Furthermore, non-sensitive and mixing cellular automata are recursively inseparable. It follows that also chaotic behavior is an undecidable property for reversible one-dimensional cellular automata.

Kainic acid-induced seizures: inflammation and excitotoxic neuronal damage in the developing rat hippocampus

Epileptic seizures are harmful to the developing brain. During epileptic seizures, overactivation of glutamate receptors (GluR) leads to neuronal degeneration, defined as excitotoxicity. The hippocampus is especially vulnerable to excitotoxic neuronal death, but its mechanism has remained incompletely known in the developing brain. Recently, signs of activation of inflammatory processes after epileptic seizures have been detected in the hippocampus. The purpose of this thesis was to study the inflammatory reaction and death mechanisms in excitoxic neurodegeneration induced by the glutamate analogue kainic acid (KA) in the developing hippocampus. Organotypic hippocampal slice cultures (OHCs), prepared from 6-7-day-old rats (P6-7) and treated with KA, served as an in vitro model. KA-induced status epilepticus in P9 and P21 rats was used as an in vivo model. The results showed that the pyramidal cell layers of the hippocampus were the most susceptible to irreversible and age-specific neurodegeneration, which occurred in the juvenile (P21), but not in the immature (P9), rat hippocampus. The primary death mechanism was necrosis as there were no significant changes in the expression of selected apoptosis markers and morphological cellular features of necrosis were found. Inflammatory response was similarly age-dependent after KA treatment as a rapid, fulminant and wide response was detected in the juvenile, but not in the immature, rat brain. An anti-inflammatory drug treatment, given before KA, was not neuroprotective in OHCs, possibly because of the timing of the treatment. In summary, the results suggest that KA induces an age-dependent inflammatory response and necrotic neurodegeneration, which may cause disturbances in hippocampal connectivity and promote epileptogenesis.

Regulation of cell growth, death, and polarization by ERBB4

Regulation of cell growth, death, and polarization by ERBB4 ErbB4 is a member of the epidermal growth factor receptor (EGFR, ErbB) family. The other members are EGFR, ErbB2 and ErbB3. ErbB receptors are important regulators for example in cardiovascular, neural and breast development but control key cellular functions also in many adult tissues. Abnormal ErbB signaling has been shown to be involved in various illnesses such as cancers and heart diseases. Among the ErbBs, ErbB4 has been shown to have unique signaling characteristics. ErbB4 exists in four alternatively spliced isoforms that are expressed in a tissue-specific manner. Two of the isoforms can be cleaved by membrane proteases, resulting in release of soluble intracellular domains (ICD). Once released into the cytosol, the ICD is capable of translocating into the nucleus and participating in regulation of transcription. The functional differences and the tissue-specific expression patterns suggest isoformspecific roles for ErbB4 isoforms. However, the abilities of ErbB4 isoforms to differently regulate cellular functions were discovered only recently and are not well understood. This study aimed to determine the expression patterns of ErbB4 in normal and diseased tissue, and to define whether the cleavable and non-cleavable isoforms could regulate different target genes and therefore, cellular functions. In this study, a comprehensive ErbB4 expression pattern in several normal tissues, various cancers and non-neoplastic diseases was determined. In addition, the data demonstrated that the cleavable and non-cleavable ErbB4 isoforms could regulate different cellular functions and target genes. Finally, this study defined the cellular responses regulated by ErbB4 during kidney development.

Integration in project business : mechanisms for integrating customers and the project network during the life-cycle of industrial projects

This thesis focuses on integration in project business, i.e. how projectbased companies organize their product and process structures when they deliver industrial solutions to their customers. The customers that invest in these solutions run their businesses in different geographical, political and economical environments, which should be acknowledged by the supplier when providing solutions comprising of larger and more complex scopes than previously supplied to these customers. This means that the suppliers are increasing their supply range by taking over some of the activities in the value chain that have traditionally been handled by the customer. In order to be able to provide the functioning solutions, including more engineering hours, technical equipment and a wider project network, a change is needed in the mindset in order to be able to carry out and take the required responsibility that these new approaches bring. For the supplier it is important to be able to integrate technical products, systems and services, but the supplier also needs to have the capabilities to integrate the cross-functional organizations and departments in the project network, the knowledge and information between and within these organizations and departments, along with inputs from the customer into the product and process structures during the lifecycle of the project under development. Hence, the main objective of this thesis is to explore the challenges of integration that industrial projects meet, and based on that, to suggest a concept of how to manage integration in project business by making use of integration mechanisms. Integration is considered the essential process for accomplishing an industrial project, whereas the accomplishment of the industrial project is considered to be the result of the integration. The thesis consists of an extended summary and four papers, that are based on three studies in which integration mechanisms for value creation in industrial project networks and the management of integration in project business have been explored. The research is based on an inductive approach where in particular the design, commissioning and operations functions of industrial projects have been studied, addressing entire project life-cycles. The studies have been conducted in the shipbuilding and power generation industries where the scopes of supply consist of stand-alone equipment, equipment and engineering, and turnkey solutions. These industrial solutions include demanding efforts in engineering and organization. Addressing the calls for more studies on the evolving value chains of integrated solutions, mechanisms for inter- and intra-organizational integration and subsequent value creation in project networks have been explored. The research results in thirteen integration mechanisms and a typology for integration is proposed. Managing integration consists of integrating the project network (the supplier and the sub-suppliers) and the customer (the customer’s business purpose, operations environment and the end-user) into the project by making use of integration mechanisms. The findings bring new insight into research on industrial project business by proposing integration of technology and engineering related elements with elements related to customer oriented business performance in contemporary project environments. Thirteen mechanisms for combining products and the processes needed to deliver projects are described and categorized according to the impact that they have on the management of knowledge and information. These mechanisms directly relate to the performance of the supplier, and consequently to the functioning of the solution that the project provides. This thesis offers ways to promote integration of knowledge and information during the lifecycle of industrial projects, enhancing the development towards innovative solutions in project business.

Research framework for the connection between environmental collaboration and firm performance

This thesis focuses on collaborative activities with regard to environmental issues both within the firm and outside the firm with the key suppliers and customers, i.e. internal and external environmental collaboration. Integrating environmental thinking into supply chain management has received increasing interest in recent years. The relational view and the natural resource-based-view together suggest that environmental capabilities can be built jointly with supply chain partners and used to gain sustained competitive advantage. Several studies have been undertaken to analyse the connection between environmental activities and firm performance but most studies have taken only economic performance into account. This study pays attention also to two other dimensions of firm performance, intra-firm supply chain performance and environmental performance, and aims at presenting the linkages between them and environmental collaboration. This thesis creates a research framework for the connections between environmental collaboration and firm performance and suggests approaches to analyse these. In order to find out the key concepts and their relationship, an extensive literature review is conducted. The research framework proposes a positive connection between internal and external environmental collaboration and all three dimensions of firm performance. In addition, environmental performance and intra-firm supply chain performance are expected to contribute positively to economic performance. Hence, firms are suggested to benefit from environmental collaboration both within the firm and outside the firm. Empirical testing of the developed research framework is out of the scope of this study. However, this thesis proposes using a mixed methods research approach, including survey research and multiple case studies. Finland State of Logistics 2012 survey commissioned by the Finnish Ministry of Transport and Communications and conducted by Turku School of Economics is used as an example of data for the quantitative phase. The applicability of these two methods is discussed at a general level and with regard to analysing the research framework developed in the thesis. Future research will aim at the development of the research framework and the methods in order to confirm the connection between environmental collaboration and firm performance.

The effects of maternal hyperglycemia on human umbilical vascular gene expression and neonatal rat lung development

Background: Maternal diabetes affects many fetal organ systems, including the vasculature and the lungs. The offspring of diabetic mothers have respiratory adaptation problems after birth. The mechanisms are multifactorial and the effects are prolonged during the postnatal period. An increasing incidence of diabetic pregnancies accentuates the importance of identifying the pathological mechanisms, which cause the metabolic and genetic changes that occur in offspring, born to diabetic mothers. Aims and methods: The aim of this thesis was to determine changes both in human umbilical cord exposed to maternal type 1 diabetes and in neonatal rat lungs after streptozotocin-induced maternal hyperglycemia, during pregnancy. Rat lungs were used as a model for the potential disease mechanisms. Gene expression alterations were determined in human umbilical cords at birth and in rat pup lungs at two week of age. During the first two postnatal weeks, rat lung development was studied morphologically and histologically. Further, the effect of postnatal hyperoxia on hyperglycemia-primed rat lungs was investigated at one week of age to mimic the clinical situation of supplemental oxygen treatment. Results: In the umbilical cord, maternal diabetes had a major negative effect on the expression of genes involved in blood vessel development. The genes regulating vascular tone were also affected. In neonatal rat lungs, intrauterine hyperglycemia had a prolonged effect on gene expression during late alveolarization. The most affected pathway was the upregulation of extracellular matrix proteins. Newborn rat lungs exposed to intrauterine hyperglycemia had thinner saccular walls without changes in airspace size, a smaller relative lung weight and lung total tissue area, and increased cellular apoptosis and proliferation compared to control lungs, possibly reflecting an aberrant maturational adaptation. At one and two weeks of age, cell proliferation and secondary crest formation were accelerated in hyperglycemia-exposed lungs. Postnatal hyperoxic exposure, alone caused arrested alveolarization with thin-walled and enlarged alveoli. In contrast, the dual exposure of intrauterine hyperglycemia and postnatal hyperoxia resulted in the phenotype of thick septa together with arrested alveolarization and decreased number of small pulmonary arteries. Conclusions: Maternal diabetic environment seems to alter the umbilical cord gene expression profile of the regulation of vascular development and function. Fetal hyperglycemia may additionally affect the genetic regulation of the postnatal lung development and may actually induce prolonged structural alterations in neonatal lungs together with a modifying effect on the deleterious pulmonary exposure of postnatal hyperoxia. This, combined with the novel human umbilical cord gene data could serve as stepping stones for future therapies to curb developmental aberrations.

Stability and properties of cellulose-sodium hydroxide solution systems

Since cellulose is a linear macromolecule it can be used as a material for regenerated cellulose fiber products e.g. in textile fibers or film manufacturing. Cellulose is not thermoformable, thus the manufacturing of these regenerated fibers is mainly possible through dissolution processes preceding the regeneration process. However, the dissolution of cellulose in common solvents is hindered due to inter- and intra-molecular hydrogen bonds in the cellulose chains, and relatively high crystallinity. Interestingly at subzero temperatures relatively dilute sodium hydroxide solutions can be used to dissolve cellulose to a certain extent. The objective of this work was to investigate the possible factors that govern the solubility of cellulose in aqueous NaOH and the solution stability. Cellulose-NaOH solutions have the tendency to form a gel over time and at elevated temperature, which creates challenges for further processing. The main target of this work was to achieve high solubility of cellulose in aqueous NaOH without excessively compromising the solution stability. In the literature survey an overview of the cellulose dissolution is given and possible factors contributing to the solubility and solution properties of cellulose in aqueous NaOH are reviewed. Furthermore, the concept of solution rheology is discussed. In the experimental part the focus was on the characterization of the used materials and properties of the prepared solutions mainly concentrating on cellulose solubility and solution stability.

Segmental Durations of Speech

This dissertation considers the segmental durations of speech from the viewpoint of speech technology, especially speech synthesis. The idea is that better models of segmental durations lead to higher naturalness and better intelligibility. These features are the key factors for better usability and generality of synthesized speech technology. Even though the studies are based on a Finnish corpus the approaches apply to all other languages as well. This is possibly due to the fact that most of the studies included in this dissertation are about universal effects taking place on utterance boundaries. Also the methods invented and used here are suitable for any other study of another language. This study is based on two corpora of news reading speech and sentences read aloud. The other corpus is read aloud by a 39-year-old male, whilst the other consists of several speakers in various situations. The use of two corpora is twofold: it involves a comparison of the corpora and a broader view on the matters of interest. The dissertation begins with an overview to the phonemes and the quantity system in the Finnish language. Especially, we are covering the intrinsic durations of phonemes and phoneme categories, as well as the difference of duration between short and long phonemes. The phoneme categories are presented to facilitate the problem of variability of speech segments. In this dissertation we cover the boundary-adjacent effects on segmental durations. In initial positions of utterances we find that there seems to be initial shortening in Finnish, but the result depends on the level of detail and on the individual phoneme. On the phoneme level we find that the shortening or lengthening only affects the very first ones at the beginning of an utterance. However, on average, the effect seems to shorten the whole first word on the word level. We establish the effect of final lengthening in Finnish. The effect in Finnish has been an open question for a long time, whilst Finnish has been the last missing piece for it to be a universal phenomenon. Final lengthening is studied from various angles and it is also shown that it is not a mere effect of prominence or an effect of speech corpus with high inter- and intra-speaker variation. The effect of final lengthening seems to extend from the final to the penultimate word. On a phoneme level it reaches a much wider area than the initial effect. We also present a normalization method suitable for corpus studies on segmental durations. The method uses an utterance-level normalization approach to capture the pattern of segmental durations within each utterance. This prevents the impact of various problematic variations within the corpora. The normalization is used in a study on final lengthening to show that the results on the effect are not caused by variation in the material. The dissertation shows an implementation and prowess of speech synthesis on a mobile platform. We find that the rule-based method of speech synthesis is a real-time software solution, but the signal generation process slows down the system beyond real time. Future aspects of speech synthesis on limited platforms are discussed. The dissertation considers ethical issues on the development of speech technology. The main focus is on the development of speech synthesis with high naturalness, but the problems and solutions are applicable to any other speech technology approaches.

Novel CO2 Regulated Proteins in Synechocystis PCC 6803

The large biodiversity of cyanobacteria together with the increasing genomics and proteomics metadata provide novel information for finding new commercially valuable metabolites. With the advent of global warming, there is growing interest in the processes that results in efficient CO2 capture through the use of photosynthetic microorganisms such as cyanobacteria. This requires a detailed knowledge of how cyanobacteria respond to the ambient CO2. My study was aimed at understanding the changes in the protein profile of the model organism, Synechocystis PCC 6803 towards the varying CO₂ level. In order to achieve this goal I have employed modern proteomics tools such as iTRAQ and DIGE, recombinant DNA techniques to construct different mutants in cyanobacteria and biophysical methods to study the photosynthetic properties. The proteomics study revealed several novel proteins, apart from the well characterized proteins involved in carbon concentrating mechanisms (CCMs), that were upregulated upon shift of the cells from high CO₂ concentration (3%) to that in air level (0.039%). The unknown proteins, Slr0006 and flavodiiron proteins (FDPs) Sll0217-Flv4 and Sll0219-Flv2, were selected for further characterization. Although slr0006 was substantially upregulated under C_i limiting conditions, inactivation of the gene did not result in any visual phenotype under various environmental conditions indicating that this protein is not essential for cell survival. However, quantitative proteomics showed the induction of novel plasmid and chromosome encoded proteins in deltaslr0006 under air level CO₂ conditions. The expression of the slr0006 gene was found to be strictly dependent on active photosynthetic electron transfer. Slr0006 contains conserved dsRNA binding domain that belongs to the Sua5/YrdC/YciO protein family. Structural modelling of Slr0006 showed an alpha/beta twisted open-sheet structure and a positively charged cavity, indicating a possible binding site for RNA. The 3D model and the co-localization of Slr0006 with ribosomal subunits suggest that it might play a role in translation or ribosome biogenesis. On the other hand, deletions in the sll0217-sll218- sll0219 operon resulted in enhanced photodamage of PSII and distorted energy transfer from phycobilisome (PBS) to PSII, suggesting a dynamic photoprotection role of the operon. Constructed homology models also suggest efficient electron transfer in heterodimeric Flv2/Flv4, apparently involved in PSII photoprotection. Both Slr0006 and FDPs exhibited several common features, including negative regulation by NdhR and ambiguous cellular localization when subjected to different concentrations of divalent ions. This strong association with the membranes remained undisturbed even in the presence of detergent or high salt. My finding brings ample information on three novel proteins and their functions towards carbon limitation. Nevertheless, many pathways and related proteins remain unexplored. The comprehensive understanding of the acclimation processes in cyanobacteria towards varying environmental CO₂ levels will help to uncover adaptive mechanisms in other organisms, including higher plants.

Interdependence of Emerging Eastern European stock markets

One of the main developments in the global economy during the past decades has been the growth of emerging economies. Projections for their long-term growth, changes in the investment climate, corporate transparency and demography point to an increasing role for these emerging economies in the global economy. Today, emerging economies are usually considered as financial markets offering opportunities for high returns, good risk diversification and improved return-to-risk ratios. However, researchers have noted that these advantages may be in decline because of the increasing market integration. Nevertheless, it is likely that certain financial markets and specific sectors will remain partially segmented and somewhat insulated from the global economy for the year to come. This doctoral dissertation investigates several stock markets in Emerging Eastern Europe (EEE), including the ones in Russia, Poland, Hungary, the Czech Republic, Bulgaria and Slovenia. The objective is to analyze the returns and financial risks in these emerging markets from international investor’s point of view. This study also examines the segmentation/integration of these financial markets and the possibilities to diversify and hedge financial risk. The dissertation is divided into two parts. The first includes a review of the theoretical background for the articles and a review of the literature on EEE stock markets. It includes an overview of the methodology and research design applied in the analysis and a summary of articles from the second part of this dissertation and their main findings. The second part consists of four research publications. This work contributes to studies on emerging stock markets in four ways. First, it adds to the body of research on the pricing of risk, providing new empirical evidence about partial stock market segmentation in EEE. The results suggest that the aggregate emerging market risk is a relevant driver for stock market returns and that this market risk can be used to price financial instruments and forecast their performance. Second, it contributes to the empirical research on the integration of stock markets, asset prices and exchange rates by identifying the relationships between these markets through volatility and asset pricing. The results show that certain sectors of stock markets in EEE are not as integrated as others. For example, the Polish consumer goods sector, the Hungarian telecommunications sector, and the Czech financial sector are somewhat isolated from their counterparts elsewhere in Europe. Nevertheless, an analysis of the impact of EU accession in 2004 on stock markets suggests that most of the EEE markets are becoming increasingly integrated with the global markets. Third, this thesis complements the scientific literature in the field of shock and volatility spillovers by examining the mechanism of spillover distribution among the EU and EEE countries. The results illustrate that spillovers in emerging markets are mostly from a foreign exchange to the stock markets. Moreover, the results show that the effects of external shocks on stock markets have increased after the enlargement of the EU in 2004. Finally, this study is unique because it analyzes the effects of foreign macroeconomic news on geographically closely related countries. The results suggest that the effects of macroeconomic announcements on volatility are significant and have effect that varies across markets and their sectors. Moreover, the results show that the foreign macroeconomic news releases, somewhat surprisingly, have a greater effect on the EEE markets than the local macroeconomic news. This dissertation has a number of implications for the industry and for practitioners. It analyses financial risk associated with investing in Emerging Eastern Europe. Investors may use this information to construct and optimize investment portfolios. Moreover, this dissertation provides insights for investors and portfolio managers considering asset allocation to protect value or obtain higher returns. The results have also implications for asset pricing and portfolio selection in light of macroeconomic news releases.