Industrial Removal Office records undated, 1899-1922

Contains Board of Directors minutes (1903, 1907), Executive Committee minutes (1907), Removal Committee minutes (1903-1917), Annual Reports (1910, 1913), Monthly Reports (1901-1919), Monthly Bulletins (1914-1915), studies of those removed, Bressler's "The Removal Work, Including Galveston," and several papers relating to the IRO and immigration. Financial papers include a budget (1914), comparative per capita cost figures (1909-1922), audits (1915-1918), receipts and expenditures (1918-1922), investment records, bank balances (1907-1922), removal work cash book (1904-1911), office expenses cash account (1903-1906), and the financial records of other agencies working with the IRO (1906). Includes also removal case records of first the Jewish Agricultural Society (1899-1900), and then of the IRO (1901-1922) when it took over its work, family reunion case records (1901-1904), and the follow-up records of persons removed to various cities (1903-1914). Contains also the correspondence of traveling agents' contacts throughout the U.S. from 1905-1914, among them Stanley Bero, Henry P. Goldstein, Philip Seman, and Morris D. Waldman.

Suomi ja Euroopan kirkollinen jälleenrakentaminen 1945-1948

The international aid that the Evangelical Lutheran Church of Finland received between 1945 and 1948 is the topic of this historical study, in which the process of reconstruction of the Evangelical Lutheran Church of Finland is examined in a European context. The key questions are related not only to the achievements of the reconstruction programs but also to the purposes and objectives of the donating churches. The study pays particular attention to the changes in the ecclesiastical, political and economic fields after the Second World War and asks how the tense political atmosphere of a divided world affected the reconstruction programs of the churches. It is possible to distinguish three periods within the European church reconstruction process. To begin with, the year 1945 was, in general, the year of organization. Many churches had started planning reconstruction work already during the war, but only after the conflict in Europe had ceased did they have a chance to renew contacts, assess the damage and begin operations. The years 1946 and 1947 were the main years of the work. Large reconstruction organizations from American churches donated money, food, clothes and vitamins worth millions of dollars to the European churches. The work started to diminish as early as 1948, partly because Marshall Plan aid and the rising standard of living had reduced the need for material assistance in many countries and partly because other problems overshadowed the reconstruction work of the World Council of Churches: for example, most WCC resources at this time were directed to refugee programs and to Third World churhces. The most important donors from the Evangelical Lutheran Church of Finland's point of view were the American Section of the Lutheran World Federation, the World Council of Churches and the Churches of Denmark, Sweden and England. The amount of money and value of goods received by the Evangelical Lutheran Church of Finland totaled approximately 2.5 million dollars, from which about 60 per cent came from the Lutheran churches of America. The importance of the Lutheran World Federation was even greater because of the productive financial arrangements that increased the American Lutheran funds. In addition the Evangelical Lutheran Church of Finland imported hundreds of tons of tax-free coffee and sold this to Finns. The money gained was used mostly to rebuild destroyed church buildings and to support the work of different ecclesiastical organizations. Smaller amounts were used for scholarship programs, youth work, and supporting sick and disabled church workers.

"Towards 'a real reunion'?" : Archbishop Aleksi Lehtonen's efforts for closer relations with the Church of England 1945–1951

Mika KT Pajusen väitös "Towards 'a real reunion'?" – Archbishop Aleksi Lehtonen's efforts for closer relations with the Church of England 1945–1951 on yleiseen kirkkohistoriaan lukeutuva tutkimus Englannin kirkon ja Suomen evankelis-luterilaisen kirkon välisistä suhteista Aleksi Lehtosen arkkipiispakaudella 1945–1951. Suhteita on tutkittu kolmesta näkökulmasta: ekumeenisesta, poliittisesta ja kirkkopoliittisesta. Tutkimuskausi alkaa pastori H.M. Waddamsin joulukuussa 1944 Suomeen tekemän vierailun jälkimainingeista ja päättyy arkkipiispa Lehtosen kuolemaan pääsiäisenä 1951. Kirkollisten suhteiden kehitystä rytmittivät lukuisat vierailut, jotka osoittivat Englannin kirkon asenteen muuttumisen sodan aikaisesta neuvostomyönteisyydestä kylmän sodan aikaiseen täysin vastakkaiseen kantaan. Englantilaiset vieraat kohtasivat Suomessa sekä kirkon että yhteiskunnan ylimmän johdon. Molemmat maat olivat valmiita tukemaan hyviä kirkollisia suhteita tilanteen niin salliessa, joskaan eivät kovin suunnitelmallisesti. Suomen evankelis-luterilainen kirkko käytti hyviä suhteita Englannin kirkkoon saadakseen tukea ja ymmärrystä omalle kirkolleen ja yhteiskunnalleen kokemaansa Neuvostoliiton uhkaa vastaan erityisesti vaaran vuosina 1944–1948. Englannin kirkko halusi tukea suomalaista sisarkirkkoaan, mutta varoi, ettei tuottaisi tuellaan enemmän haittaa kuin hyötyä suhteessa Neuvostoliittoon. Sodan jälkeinen ekumeeninen jälleenrakentaminen lähensi kirkkoja toisiinsa. Lehtonen pyrki jatkamaan 1930-luvun kirkkojen välisiä, ehtoollisvieraanvaraisuuden saavuttaneita neuvotteluita kohti täyttä kirkollista yhteyttä. Häntä motivoi sekä evankelis-katolinen teologia että pyrkimys tukea oman maan ja kirkon läntisiä yhteyksiä. Tämä haastoi Englannin kirkon ekumeenisen linjan, joka Suomen kirkon sijasta pyrki jatkamaan neuvotteluja Tanskan, Norjan ja Islannin luterilaisten kirkkojen kanssa, joilla ei vielä ollut virallista ekumeenista sopimusta Englannin kirkon kanssa. Lehtosen pyrkimyksistä huolimatta Englannin kirkko päätyi jättämään Suomen tilanteen hautumaan. Sillä se tarkoitti suhteiden koetinkivenä olleen historiallisen piispuuden leviämistä läpi Suomen kirkon ennen kuin katsoi olevansa valmis jatkamaan kohti täyttä kirkollista yhteyttä. Molemmissa kirkoissa vaikutti pieni, innokkaiden, lähempiä suhteita toivoneiden kirkollisten vaikuttajien ydinjoukko. Englantilaisia Suomen-ystäviä motivoi tarve auttaa Suomea hankalassa poliittisessa tilanteessa. Suomessa arkkipiispa Lehtonen tuki korkeakirkollista liturgista liikettä, jolla oli läheinen yhteys anglikaanisuuteen, mutta joka sai vastaansa vanhoilliset pietistit. Suomen kirkon yleinen mielipide asettui etupäässä pietistiselle kannalle, jolle anglikaanisuus näyttäytyi teologisesti sekä liian katolisena että liian reformoituna. Kirkolliset suhteet tasaantuivat vuoden 1948 Lambeth-konferenssin jälkeen, joka rohkaisi anglikaanisia kirkkoja hyväksymään 1930-luvun neuvottelujen lähempiin kirkollisiin suhteisiin tähtäävät suositukset. Lehtonen näytti tyytyvän tähän. Samaan aikaan lähempää kirkollista kanssakäymistä tukenut ekumeeninen jälleenrakennus tuli tiensä päähän. Lehtonen jatkoi läheisempien suhteiden edistämistä, mutta hänen intonsa hiipui yhdessä heikkenevän terveydentilan kanssa. Osoituksena Lehtosen linjan kapeudesta Suomen evankelis-luterilaisen kirkon piispoista ei löytynyt hänen kuoltuaan ketään, joka olisi jatkanut hänen aktiivista anglikaanimyönteistä linjaansa.

Studies on electrical conductivity of insulator-conductor composites

The variation of electrical resistivity of an insulator-conductor composite, namely, wax-graphite composite, with parameters such as volume fraction, grain size, and temperature has been studied. A model is proposed to explain the observed variations, which assumes that the texture of the composite consists of insulator granules coated with conducting particles. The resistivity of these materials is controlled mainly by the contact resistance between the conducting particles and the number of contacts each particle has with its neighbors. The variation of resistivity with temperature has also been explained with the help of this model and it is attributed to the change in contact area. Journal of Applied Physics is copyrighted by The American Institute of Physics.

Tiedon jakaminen tiimityössä

Knowledge-sharing in a teamwork The study examines the link between knowledge-sharing that takes place in a team and the dimensions and objectives of the team s activities. The question the study poses is: How does knowledge-sharing in a team relate to the team s activities? The exchange of knowledge is examined using knowledge-sharing networks and the conversion model, which describes the process of knowledge formation. The answer to the question is sought through four empirical articles describing the activities of a team from the viewpoint of quality, fairness, power related to knowledge management, and performance. One of the articles used in the study describes the role of networks in work life more generally. It attempts to shed light on the manner in which team-related networks operate as part of a more extensive structure of organizational networks. Finland is one of the most eager users of teamwork, if numbers are used as a yardstick. About half of all Finnish wage earners worked in teams in 2009, and comparisons show that the use of teams in Finland is above the EU average. This study focuses on so-called semi-autonomous teams, which carry out permanent work tasks. In such teams, tasks are interdependent, and teams are jointly responsible for ensuring that the work is done. Team members may also, at least to some extent, agree between themselves on how the tasks are carried out and are able to take part in the decision-making process. Such teamwork makes knowledge-sharing an important element for the team s activities. Knowledge and knowledge-sharing have become a major resource, allowing organizations to operate and even compete in today s increasingly competitive markets. A single team or a single organization cannot, however, possess all the knowledge required for carrying out the tasks assigned to it. Although it is difficult to copy the knowledge generated in an organization, it is important to share the knowledge within and between organizations. External links supply teams and organizations with important knowledge that allows them to keep their operations up-to-date and their structures well-functioning. In fact, knowledge provides teams and organizations with an intangible resource that improves their capacity to interact with their environment and to adjust to it. For this reason, it is important to examine both the internal and external knowledge-sharing taking place in a team. The findings of the study show that in terms of quality, fairness, performance and the knowledge management issues concerning a team, its social network structure is both internally and externally connected with its activities. A team structure that is internally coherent and at the same time open to external contacts, is, with certain restrictions, connected with the quality, fairness, and performance of the team. The restrictions concern differences between procedural and interactional justice, public and private sectors, and the team leaders and ordinary team members. The role of the team leader is closely connected with the management of networks that are considered valuable. The results of the study indicate that teamwork is supervisor-dominated. Thus, teamwork does not substantially strengthen the influence of individual employees as players in knowledge-transfer networks. However, ordinary team members possess important peer contacts inside the organization. Teamwork clearly allows employees to interact in a democratic manner, and here the transfer of tacit knowledge plays an important role. Keywords: teamwork, knowledge-sharing, social networks, organization

Food intake, growth and social interactions of signal crayfish, Pacifastacus leniusculus (Dana)

The knowledge about the optimal rearing conditions, such as water temperature and quality, photoperiod and density, with the understanding of animal nutritional requirements forms the basis of economically stable aquaculture for freshwater crayfish. However, the shift from a natural environment to effective culture conditions induces several changes, not only at the population level, but also at the individual level. The social contacts between conspecifics increase with increasing animal density. The competition for limited resources (e.g. food, shelter, mates) is more severe with the presence of agonistic behaviour and may lead to unequal distribution of these. The objectives of this study were to: 1) study the distribution of a common food resource between communally reared signal crayfish (Pacifastacus leniusculus) and to assign potential feeding hierarchy on the basis of individual food intake measurements, 2) explore the possibilities of size distribution manipulations to affect population dynamics and food intake to improve growth and survival in culture and 3) study the effect of food ration and spatial distribution on food intake and to explore the effect of temperature and food ration on growth and body composition of freshwater crayfish. The feeding ranks between animals were assigned with a new method for individual food intake measurement of communally reared crayfish. This technique has a high feasibility and a great potential to be applied in crayfish aquaculture studies. In this study, signal crayfish showed high size-related variability in food consumption both among individuals within a group (inter-individual) and within individual day-to-day variation (intra-individual). Increased competition for food led to an unequal distribution of this resource and this may be a reason for large growth differences between animals. The consumption was significantly higher when reared individually in comparison with communal housing. These results suggest that communally housed crayfish form a feeding hierarchy and that the animal size is the major factor controlling the position in this hierarchy. The optimisation of the social environment ( social conditions ) was evaluated in this study as a new approach to crayfish aquaculture. The results showed that the absence of conspecifics (individual rearing vs. communal housing) affects growth rate, food intake and the proportion of injured animals, whereas size variation between animals influences the number and duration of agonistic encounters. In addition, animal size had a strong influence on the fighting success of signal crayfish reared in a social milieu with a wide size variation of conspecifics. Larger individuals initiated and won most of the competitions, which suggests size-based social hierarchy of P. leniusculus. This is further supported by the fact that the length and weight gain of smaller animals increased after size grading, maybe because of a better access to the food resource due to diminished social pressure. However, the high dominance index was not based on size under conditions of limited size variation, e.g. those characteristic of restocked natural populations and aquaculture, indicating the important role of behaviour on social hierarchy.

The community of medium-sized carnivores: the interactions between species, habitats and rabies

The composition of the carnivore community influences the different forms of inter-specific interactions. Furthermore, inter-specific interactions of carnivores have important implications for intra-guild competition, epidemiology and strategies of species-specific population management. Zoonooses, such as rabies, are diseases that can be transmitted from wildlife to people. Knowing the ecological characteristics of the species helps us to choose the right preventive actions and to time them accurately. In this thesis, I have studied how raccoon dogs Nyctereutes procyonoides, European badgers Meles meles, red foxes Vulpes vulpes and domestic cats Felis silvestris catus act as members of carnivore community, and how these interactions relate to the transmission risk of rabies. In the study area, these species form a community of medium-sized and rather generalist predators. They live in the same areas, in spatially and temporally overlapping home ranges and use the same habitats and dens and even have similar diets. However, there is no direct evidence of competition. Shared dens point to good tolerance of other species. Numerous observations of animals moving in each other’s proximity give similar clues. However, overlapping home ranges and similar habitat preferences lead to frequent inter-specific contacts, which increase the risk of possible rabies transmission. Also, the new insight of habitat use gained by this study illustrates the similar favouring of deciduous forests and fields by these sympatric medium-sized carnivores, creating a basis for contact zones, i.e. risky habitats for rabies transmission and spread. This study is so far the only simultaneous radio tracking study of raccoon dogs, badgers, foxes and cats. These results give new insight of the interactions in the carnivore community, as well as of the behaviour of each individual species. Also, these results have significant implications for the planning of rabies control. In order to reach viable management decisions, not only one or two species should be taken into consideration, but the whole community. In particular, this changes the perspective to inter-specific contacts, animal densities, densities of individuals susceptible to diseases and the magnitude of preventive actions. Rabies should be considered as a multi-vector disease, at least in Finland and the Baltic states. It is of interest for disease management to be able to model an epizootic with local parameters to reflect the real situation and also to suite best the local management needs.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

The hydrogen bond: a molecular beam microwave spectroscopist's view with a universal appeal

In this manuscript, we propose a criterion for a weakly bound complex formed in a supersonic beam to be characterized as a `hydrogen bonded complex'. For a `hydrogen bonded complex', the zero point energy along any large amplitude vibrational coordinate that destroys the orientational preference for the hydrogen bond should be significantly below the barrier along that coordinate so that there is at least one bound level. These are vibrational modes that do not lead to the breakdown of the complex as a whole. If the zero point level is higher than the barrier, the `hydrogen bond' would not be able to stabilize the orientation which favors it and it is no longer sensible to characterize a complex as hydrogen bonded. Four complexes, Ar-2-H2O, Ar-2-H2S, C2H4-H2O and C2H4-H2S, were chosen for investigations. Zero point energies and barriers for large amplitude motions were calculated at a reasonable level of calculation, MP2(full)/aug-cc-pVTZ, for all these complexes. Atoms in molecules (AIM) theoretical analyses of these complexes were carried out as well. All these complexes would be considered hydrogen bonded according to the AIM theoretical criteria suggested by Koch and Popelier for C-H center dot center dot center dot O hydrogen bonds (U. Koch and P. L. A. Popelier, J. Phys. Chem., 1995, 99, 9747), which has been widely and, at times, incorrectly used for all types of contacts involving H. It is shown that, according to the criterion proposed here, the Ar-2-H2O/H2S complexes are not hydrogen bonded even at zero kelvin and C2H4-H2O/H2S complexes are. This analysis can naturally be extended to all temperatures. It can explain the recent experimental observations on crystal structures of H2S at various conditions and the crossed beam scattering studies on rare gases with H2O and H2S.

In situ cryocrystallization of low melting chloro and bromo substituted anilines

In situ cryocrystallographic Studies of chloro and bromo substituted anilines have been performed to evaluate the role of halogen...halogen interactions and the subsequent formation of supramolecular assemblies in the solid state. Ortho Cl/Br substituted anilines are isostructural and belong to the trigonal P3(1) space group. Halogen...halogen intermolecular contacts along with stronger N-H center dot center dot center dot N hydrogen bonds generate helical motifs along the crystallographic c-axis. (C) 2009 Elsevier B.V. All rights reserved.

N-{1-[(3-Bromopropyl)aminocarbonyl]ethyl}-2-(2-nitrobenzenesulfonamido)propionamide

In the title compound, C15H21BrN4O6S, all three NH groups are involved in intermolecular N-H center dot center dot center dot O interactions which, together with two intermolecular C-H center dot center dot center dot O contacts, lead to a continuous antiparallel beta-sheet structure. There are no pi-pi interactions between molecules, and two C-H center dot center dot center dot pi interactions primarily govern the linkage between sheets.

4-Chloro-N-(3-chlorophenyl)benzamide

The title compound, C13H9Cl2N, has an intramolecular C-H center dot center dot center dot O close contact, and presents the NH group syn to the meta-chloro group in the aniline ring and trans to the C=O group. The crystal packing is formed by infinite chains of N-H center dot center dot center dot O hydrogen bonds along the c axis. Cl center dot center dot center dot Cl [3.474 (1) angstrom] contacts link chains. The crystal used for data collection was a twin, the domains related by the twin law 0.948 (1)/0.052 (1).

Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.

Molecular details of phage phi6 RNA-dependent RNA synthesis

For most RNA viruses RNA-dependent RNA polymerases (RdRPs) encoded by the virus are responsible for the entire RNA metabolism. Thus, RdRPs are critical components in the viral life cycle. However, it is not fully understood how these important enzymes function during viral replication. Double-stranded RNA (dsRNA) viruses perform the synthesis of their RNA genome within a proteinacous viral particle containing an RdRP as a minor constituent. The phi6 bacteriophage is the best-studied dsRNA virus, providing an excellent background for studies of its RNA synthesis. The purified recombinant phi6 RdRP is highly active in vitro and it possesses both RNA replication and transcription activities. The crystal structure of the phi6 polymerase, solved in complex with a number of ligands, provides a working model for detailed in vitro studies of RNA-dependent RNA polymerization. In this thesis, the primer-independent initiation of the phi6 RdRP was studied in vitro using biochemical and structural methods. A C-terminal, four-amino-acid-long loop protruding into the central cavity of the phi6 RdRP has been suggested to stabilize the incoming nucleotides of the initiation complex formation through stacking interactions. A similar structural element has been found from several other viral RdRPs. In this thesis, this so-called initiation platform loop was subjected to site-directed mutagenesis to address its role in the initiation. It was found that the initiation mode of the mutants is primer-dependent, requiring either an oligonucleotide primer or a back-priming initiation mechanism for the RNA synthesis. The crystal structure of a mutant RdRP with altered initiation platform revealed a set of contacts important for primer-independent initiation. Since phi6 RdRP is structurally and functionally homologous to several viral RdRPs, among them the hepatitis C virus RdRP, these results provide further general insight to understand primer-independent initiation. In this study it is demonstrated that manganese phasing could be used as a practical tool for solving structures of large proteins with a bound manganese ion. The phi6 RdRP was used as a case study to obtain phases for crystallographic analysis. Manganese ions are naturally bound to the phi6 RdRP at the palm domain of the enzyme. In a crystallographic experiment, X-ray diffraction data from a phi6 RdRP crystal were collected at a wavelength of 1.89 Å, which is the K edge of manganese. With this data an automatically built model of the core region of the protein could be obtained. Finally, in this work terminal nucleotidyl transferase (TNTase) activity of the phi6 RdRP was documented in the isolated polymerase as well as in the viral particle. This is the first time that such an activity has been reported in a polymerase of a dsRNA virus. The phi6 RdRP used uridine triphosphates as the sole substrate in a TNTase reaction but could accept several heterologous templates. The RdRP was able to add one or a few non-templated nucleotides to the 3' end of the single- or double-stranded RNA substrate. Based on the results on particle-mediated TNTase activity and previous structural information of the polymerase, a model for termination of the RNA-dependent RNA synthesis is suggested in this thesis.

Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (Mstopol) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage m the absence of Mg2+ but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim. (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+, in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike Mstopol and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.