The Structure and Functions of Hantavirus Nucleocapsid Protein

Hantaviruses, members of the genus Hantavirus in the Bunyaviridae family, are enveloped single-stranded RNA viruses with tri-segmented genome of negative polarity. In humans, hantaviruses cause two diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), which vary in severity depending on the causative agent. Each hantavirus is carried by a specific rodent host and is transmitted to humans through excreta of infected rodents. The genome of hantaviruses encodes four structural proteins: the nucleocapsid protein (N), the glycoproteins (Gn and Gc), and the polymerase (L) and also the nonstructural protein (NSs). This thesis deals with the functional characterization of hantavirus N protein with regard to its structure. Structural studies of the N protein have progressed slowly and the crystal structure of the whole protein is still not available, therefore biochemical assays coupled with bioinformatical modeling proved essential for studying N protein structure and functions. Presumably, during RNA encapsidation, the N protein first forms intermediate trimers and then oligomers. First, we investigated the role of N-terminal domain in the N protein oligomerization. The results suggested that the N-terminal region of the N protein forms a coiled-coil, in which two antiparallel alpha helices interact via their hydrophobic seams. Hydrophobic residues L4, I11, L18, L25 and V32 in the first helix and L44, V51, L58 and L65 in the second helix were crucial for stabilizing the structure. The results were consistent with the head-to-head, tail-to-tail model for hantavirus N protein trimerization. We demonstrated that an intact coiled-coil structure of the N terminus is crucial for the oligomerization capacity of the N protein. We also added new details to the head-to-head, tail-to-tail model of trimerization by suggesting that the initial step is based on interaction(s) between intact intra-molecular coiled-coils of the monomers. We further analyzed the importance of charged aa residues located within the coiled-coil for the N protein oligomerization. To predict the interacting surfaces of the monomers we used an upgraded in silico model of the coiled-coil domain that was docked into a trimer. Next the predicted target residues were mutated. The results obtained using the mammalian two-hybrid assay suggested that conserved charged aa residues within the coiled-coil make a substantial contribution to the N protein oligomerization. This contribution probably involves the formation of interacting surfaces of the N monomers and also stabilization of the coiled-coil via intramolecular ionic bridging. We proposed that the tips of the coiled-coils are the first to come into direct contact and thus initiate tight packing of the three monomers into a compact structure. This was in agreement with the previous results showing that an increase in ionic strength abolished the interaction between N protein molecules. We also showed that residues having the strongest effect on the N protein oligomerization are not scattered randomly throughout the coiled-coil 3D model structure, but form clusters. Next we found evidence for the hantaviral N protein interaction with the cytoplasmic tail of the glycoprotein Gn. In order to study this interaction we used the GST pull-down assay in combination with mutagenesis technique. The results demonstrated that intact, properly folded zinc fingers of the Gn protein cytoplasmic tail as well as the middle domain of the N protein (that includes aa residues 80 248 and supposedly carries the RNA-binding domain) are essential for the interaction. Since hantaviruses do not have a matrix protein that mediates the packaging of the viral RNA in other negatve stranded viruses (NSRV), hantaviral RNPs should be involved in a direct interaction with the intraviral domains of the envelope-embedded glycoproteins. By showing the N-Gn interaction we provided the evidence for one of the crucial steps in the virus replication at which RNPs are directed to the site of the virus assembly. Finally we started analysis of the N protein RNA-binding region, which is supposedly located in the middle domain of the N protein molecule. We developed a model for the initial step of RNA-binding by the hantaviral N protein. We hypothesized that the hantaviral N protein possesses two secondary structure elements that initiate the RNA encapsidation. The results suggest that amino acid residues (172-176) presumably act as a hook to catch vRNA and that the positively charged interaction surface (aa residues 144-160) enhances the initial N-RNA interacation. In conclusion, we elucidated new functions of hantavirus N protein. Using in silico modeling we predicted the domain structure of the protein and using experimental techniques showed that each domain is responsible for executing certain function(s). We showed that intact N terminal coiled-coil domain is crucial for oligomerization and charged residues located on its surface form a interaction surface for the N monomers. The middle domain is essential for interaction with the cytoplasmic tail of the Gn protein and RNA binding.

Tuloverotuksen reaalinen muuttuminen Suomessa : Kuusikymmentä vuotta verojen vuoristorataa

The study seeks to find out whether the real burden of the personal taxation has increased or decreased. In order to determine this, we investigate how the same real income has been taxed in different years. Whenever the taxes for the same real income for a given year are higher than for the base year, the real tax burden has increased. If they are lower, the real tax burden has decreased. The study thus seeks to estimate how changes in the tax regulations affect the real tax burden. It should be kept in mind that the progression in the central government income tax schedule ensures that a real change in income will bring about a change in the tax ration. In case of inflation when the tax schedules are kept nominally the same will also increase the real tax burden. In calculations of the study it is assumed that the real income remains constant, so that we can get an unbiased measure of the effects of governmental actions in real terms. The main factors influencing the amount of income taxes an individual must pay are as follows: - Gross income (income subject to central and local government taxes). - Deductions from gross income and taxes calculated according to tax schedules. - The central government income tax schedule (progressive income taxation). - The rates for the local taxes and for social security payments (proportional taxation). In the study we investigate how much a certain group of taxpayers would have paid in taxes according to the actual tax regulations prevailing indifferent years if the income were kept constant in real terms. Other factors affecting tax liability are kept strictly unchanged (as constants). The resulting taxes, expressed in fixed prices, are then compared to the taxes levied in the base year (hypothetical taxation). The question we are addressing is thus how much taxes a certain group of taxpayers with the same socioeconomic characteristics would have paid on the same real income according to the actual tax regulations prevailing in different years. This has been suggested as the main way to measure real changes in taxation, although there are several alternative measures with essentially the same aim. Next an aggregate indicator of changes in income tax rates is constructed. It is designed to show how much the taxation of income has increased or reduced from one year to next year on average. The main question remains: How aggregation over all income levels should be performed? In order to determine the average real changes in the tax scales the difference functions (difference between actual and hypothetical taxation functions) were aggregated using taxable income as weights. Besides the difference functions, the relative changes in real taxes can be used as indicators of change. In this case the ratio between the taxes computed according to the new and the old situation indicates whether the taxation has become heavier or easier. The relative changes in tax scales can be described in a way similar to that used in describing the cost of living, or by means of price indices. For example, we can use Laspeyres´ price index formula for computing the ratio between taxes determined by the new tax scales and the old tax scales. The formula answers the question: How much more or less will be paid in taxes according to the new tax scales than according to the old ones when the real income situation corresponds to the old situation. In real terms the central government tax burden experienced a steady decline from its high post-war level up until the mid-1950s. The real tax burden then drifted upwards until the mid-1970s. The real level of taxation in 1975 was twice that of 1961. In the 1980s there was a steady phase due to the inflation corrections of tax schedules. In 1989 the tax schedule fell drastically and from the mid-1990s tax schedules have decreased the real tax burden significantly. Local tax rates have risen continuously from 10 percent in 1948 to nearly 19 percent in 2008. Deductions have lowered the real tax burden especially in recent years. Aggregate figures indicate how the tax ratio for the same real income has changed over the years according to the prevailing tax regulations. We call the tax ratio calculated in this manner the real income tax ratio. A change in the real income tax ratio depicts an increase or decrease in the real tax burden. The real income tax ratio declined after the war for some years. In the beginning of the 1960s it nearly doubled to mid-1970. From mid-1990s the real income tax ratio has fallen about 35 %.

Functions of Alphavirus Macrodomain-containing protein nsP3

Alphaviruses are positive strand RNA viruses that replicate in association with cellular membranes. The viral RNA replication complex consists of four non-structural proteins nsP1-nsP4 which are essential for viral replication. The functions of nsP1, nsP2 and nsP4 are well established, but the roles of nsP3 are mainly unknown. In this work I have clarified some of the functions of nsP3 in order to better understand the importance of this protein in virus replication. Semliki Forest virus (SFV) has been mostly used as a model alphavirus during this work, but some experiments have also been conducted with Sindbis and Chikungunya viruses. NsP3 is composed of three different protein domains. The N-terminus of nsP3 contains an evolutionarily conserved macrodomain, the central part of nsP3 contains a domain that is only found in alphaviruses, and the C-terminus of the protein is hypervariable and predicted to be unstructured. In this work I have analyzed the functions of nsP3 macrodomain, and shown that viral macrodomains bind poly(ADP-ribose) and that they do not resemble cellular macrodomains in their properties. Furthermore, I have shown that some macrodomains, including viral macrodomains of SFV and hepatitis E virus, also bind poly(A). Mutations in the ligand binding pocket of SFV macrodomain hamper virus replication but do not confer lethality, indicating that macrodomain function is beneficial but not mandatory for virus replication. The hypervariable C-terminus of nsP3 is heavily phosphorylated and is enriched in proline residues. In this work it is shown that this region harbors an SH3 domain binding motif (Sh3BM) PxRxPR through which cellular amphiphysin is recruited to viral replication sites and to nsP3 containing cytoplasmic aggregate structures. The function of Sh3BM was destroyed by a single point mutation, which led to impaired viral RNA replication in HeLa cells, pointing out the functional importance of amphiphysin recruitment by the Sh3BM. In addition, evidence is provided tho show that the endosomal localization of alphavirus replication is mediated by nsP3 and that the phosphorylation of hypervariable region might be important for the endosomal targeting. Together these findings demonstrate that nsP3 contains multiple important host interaction motifs and domains, which facilitate successful viral propagation in host cells.

Hantavirus glycoprotein GN in virion assembly and regulation of interferon response

Hantaviruses have a tri-segmented negative-stranded RNA genome. The S segment encodes the nucleocapsid protein (N), M segment two glycoproteins, Gn and Gc, and the L segment the RNA polymerase. Gn and Gc are co-translationally cleaved from a precursor and targeted to the cis-Golgi compartment. The Gn glycoprotein consists of an external domain, a transmembrane domain and a C-terminal cytoplasmic domain. In addition, the S segment of some hantaviruses, including Tula and Puumala virus, have an open reading frame (ORF) encoding a nonstructural potein NSs that can function as a weak interferon antagonist. The mechanisms of hantavirus-induced pathogenesis are not fully understood but it is known that both hemorrhagic fever with renal syndrome (HFRS) and hantavirus (cardio) pulmonary syndrome (HCPS) share various features such as increased capillary permeability, thrombocytopenia and upregulation of TNF-. Several hantaviruses have been reported to induce programmed cell death (apoptosis), such as TULV-infected Vero E6 cells which is known to be defective in interferon signaling. Recently reports describing properties of the hantavirus Gn cytoplasmic tail (Gn-CT) have appeared. The Gn-CT of hantaviruses contains animmunoreceptor tyrosine-based activation motif (ITAM) which directs receptor signaling in immune and endothelial cells; and contain highly conserved classical zinc finger domains which may have a role in the interaction with N protein. More functions of Gn protein have been discovered, but much still remains unknown. Our aim was to study the functions of Gn protein from several aspects: synthesis, degradation and interaction with N protein. Gn protein was reported to inhibit interferon induction and amplication. For this reason, we also carried out projects studying the mechanisms of IFN induction and evasion by hantavirus. We first showed degradation and aggresome formation of the Gn-CT of the apathogenic TULV. It was reported earlier that the degradation of Gn-CT is related to the pathogenicity of hantavirus. We found that the Gn-CT of the apathogenic hantaviruses (TULV, Prospect Hill virus) was degraded through the ubiquitin-proteasome pathway, and TULV Gn-CT formed aggresomes upon treatment with proteasomal inhibitor. Thus the results suggest that degradation and aggregation of the Gn-CT may be a general property of most hantaviruses, unrelated to pathogenicity. Second, we investigated the interaction of TULV N protein and the TULV Gn-CT. The Gn protein is located on the Golgi membrane and its interaction with N protein has been thought to determine the cargo of the hantaviral ribonucleoprotein which is an important step in virus assembly, but direct evidence has not been reported. We found that TULV Gn-CT fused with GST tag expressed in bacteria can pull-down the N protein expressed in mammalian cells; a mutagenesis assay was carried out, in which we found that the zinc finger motif in Gn-CT and RNA-binding motif in N protein are indispensable for the interaction. For the study of mechanisms of IFN induction and evasion by Old World hantavirus, we found that Old World hantaviruses do not produce detectable amounts of dsRNA in infected cells and the 5 -termini of their genomic RNAs are monophosphorylated. DsRNA and tri-phosphorylated RNA are considered to be critical activators of innate immnity response by interacting with PRRs (pattern recognition receptors). We examined systematically the 5´-termini of hantavirus genomic RNAs and the dsRNA production by different species of hantaviruses. We found that no detectable dsRNA was produced in cells infected by the two groups of the old world hantaviruses: Seoul, Dobrava, Saaremaa, Puumala and Tula. We also found that the genomic RNAs of these Old World hantaviruses carry 5´-monophosphate and are unable to trigger interferon induction. The antiviral response is mainly mediated by alpha/beta interferon. Recently the glycoproteins of the pathogenic hantaviruses Sin Nombre and New York-1 viruses were reported to regulate cellular interferon. We found that Gn-CT can inhibit the induction of IFN activation through Toll-like receptor (TLR) and retinoic acid-inducible gene I-like RNA helicases (RLH) pathway and that the inhibition target lies at the level of TANK-binding kinase 1 (TBK-1)/ IKK epislon complex and myeloid differentiation primary response gene (88) (MyD88) / interferon regulatory factor 7 (IRF-7) complex.

Aspiration-based utility functions in a planning model for timber flow management.

The study presents a theory of utility models based on aspiration levels, as well as the application of this theory to the planning of timber flow economics. The first part of the study comprises a derivation of the utility-theoretic basis for the application of aspiration levels. Two basic models are dealt with: the additive and the multiplicative. Applied here solely for partial utility functions, aspiration and reservation levels are interpreted as defining piecewisely linear functions. The standpoint of the choices of the decision-maker is emphasized by the use of indifference curves. The second part of the study introduces a model for the management of timber flows. The model is based on the assumption that the decision-maker is willing to specify a shape of income flow which is different from that of the capital-theoretic optimum. The utility model comprises four aspiration-based compound utility functions. The theory and the flow model are tested numerically by computations covering three forest holdings. The results show that the additive model is sensitive even to slight changes in relative importances and aspiration levels. This applies particularly to nearly linear production possibility boundaries of monetary variables. The multiplicative model, on the other hand, is stable because it generates strictly convex indifference curves. Due to a higher marginal rate of substitution, the multiplicative model implies a stronger dependence on forest management than the additive function. For income trajectory optimization, a method utilizing an income trajectory index is more efficient than one based on the use of aspiration levels per management period. Smooth trajectories can be attained by squaring the deviations of the feasible trajectories from the desired one.

Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.

Monetary policy rules and estimated reaction functions for the European Central Bank

This thesis studies the interest-rate policy of the ECB by estimating monetary policy rules using real-time data and central bank forecasts. The aim of the estimations is to try to characterize a decade of common monetary policy and to look at how different models perform at this task.The estimated rules include: contemporary Taylor rules, forward-looking Taylor rules, nonlinearrules and forecast-based rules. The nonlinear models allow for the possibility of zone-like preferences and an asymmetric response to key variables. The models therefore encompass the most popular sub-group of simple models used for policy analysis as well as the more unusual non-linear approach. In addition to the empirical work, this thesis also contains a more general discussion of monetary policy rules mostly from a New Keynesian perspective. This discussion includes an overview of some notable related studies, optimal policy, policy gradualism and several other related subjects. The regression estimations are performed with either least squares or the generalized method of moments depending on the requirements of the estimations. The estimations use data from both the Euro Area Real-Time Database and the central bank forecasts published in ECB Monthly Bulletins. These data sources represent some of the best data that is available for this kind of analysis. The main results of this thesis are that forward-looking behavior appears highly prevalent, but that standard forward-looking Taylor rules offer only ambivalent results with regard to inflation. Nonlinear models are shown to work, but on the other hand do not have a strong rationale over a simpler linear formulation. However, the forecasts appear to be highly useful in characterizing policy and may offer the most accurate depiction of a predominantly forward-looking central bank. In particular the inflation response appears much stronger while the output response becomes highly forward-looking as well.

A maximal operator, Carleson's embedding, and tent spaces for vector-valued functions

This thesis is concerned with the area of vector-valued Harmonic Analysis, where the central theme is to determine how results from classical Harmonic Analysis generalize to functions with values in an infinite dimensional Banach space. The work consists of three articles and an introduction. The first article studies the Rademacher maximal function that was originally defined by T. Hytönen, A. McIntosh and P. Portal in 2008 in order to prove a vector-valued version of Carleson's embedding theorem. The boundedness of the corresponding maximal operator on Lebesgue-(Bochner) -spaces defines the RMF-property of the range space. It is shown that the RMF-property is equivalent to a weak type inequality, which does not depend for instance on the integrability exponent, hence providing more flexibility for the RMF-property. The second article, which is written in collaboration with T. Hytönen, studies a vector-valued Carleson's embedding theorem with respect to filtrations. An earlier proof of the dyadic version assumed that the range space satisfies a certain geometric type condition, which this article shows to be also necessary. The third article deals with a vector-valued generalizations of tent spaces, originally defined by R. R. Coifman, Y. Meyer and E. M. Stein in the 80's, and concerns especially the ones related to square functions. A natural assumption on the range space is then the UMD-property. The main result is an atomic decomposition for tent spaces with integrability exponent one. In order to suit the stochastic integrals appearing in the vector-valued formulation, the proof is based on a geometric lemma for cones and differs essentially from the classical proof. Vector-valued tent spaces have also found applications in functional calculi for bisectorial operators. In the introduction these three themes come together when studying paraproduct operators for vector-valued functions. The Rademacher maximal function and Carleson's embedding theorem were applied already by Hytönen, McIntosh and Portal in order to prove boundedness for the dyadic paraproduct operator on Lebesgue-Bochner -spaces assuming that the range space satisfies both UMD- and RMF-properties. Whether UMD implies RMF is thus an interesting question. Tent spaces, on the other hand, provide a method to study continuous time paraproduct operators, although the RMF-property is not yet understood in the framework of tent spaces.

The effect of structure on the dilute solution properties of branched polysaccharides studied with SEC and AsFlFFF

Cereal arabinoxylans, guar galactomannans, and dextrans produced by lactic acid bacteria(LAB) are a structurally diverse group of branched polysaccharides with nutritional and industrial functions. In this thesis, the effect of the chemical structure on the dilute solution properties of these polysaccharides was investigated using size-exclusion chromatography(SEC) and asymmetric flow field-flow fractionation (AsFlFFF) with multiple-detection. The chemical structures of arabinoxylans were determined, whereas galactomannan and dextran structures were studied in previous investigations. Characterization of arabinoxylans revealed differences in the chemical structures of cereal arabinoxylans. Although arabinoxylans from wheat, rye, and barley fiber contained similar amounts of arabinose side units, the substitution pattern of arabinoxylans from different cereals varied. Arabinoxylans from barley husks and commercial low-viscosity wheat arabinoxylan contained a lower number of arabinose side units. Structurally different dextrans were obtained from different LAB. The structural effects on the solution properties could be studied in detail by modifying pure wheat and rye arabinoxylans and guar galactomannan with specific enzymes. The solution characterization of arabinoxylans, enzymatically modified galactomannans, and dextrans revealed the presence of aggregates in aqueous polysaccharide solutions. In the case of arabinoxylans and dextrans, the comparison of molar mass data from aqueous and organic SEC analyses was essential in confirming aggregation, which could not be observed only from the peak or molar mass distribution shapes obtained with aqueous SEC. The AsFlFFF analyses gave further evidence of aggregation. Comparison of molar mass and intrinsic viscosity data of unmodified and partially debranched guar galactomannan, on the other hand, revealed the aggregation of native galactomannan. The arabinoxylan and galactomannan samples with low or enzymatically extensively decreased side unit content behaved similarly in aqueous solution: lower molar mass samples stayed in solution but formed large aggregates, whereas the water solubility of the higher-molar-mass samples decreased significantly. Due to the restricted solubility of galactomannans in organic solvents, only aqueous galactomannan solutions were studied. The SEC and AsFlFFF results differed for the wheat arabinoxylan and dextran samples. Column matrix effects and possible differences in the separation parameters are discussed, and a problem related to the non-established relationship between the separation parameters of the two separation techniques is highlighted. This thesis shows that complementary approaches in the solution characterization of chemically heterogeneous polysaccharides are needed to comprehensively investigate macromolecular behavior in solution. These results may also be valuable when characterizing other branched polysaccharides.

The Reception of the Maccabean Martyrs : Their Historiographical and Paradigmatic Functions in Antique and Late-antique Jewish and Christian Sources

The study attempts a reception-historical analysis of the Maccabean martyrs. The concept of reception has fundamentally to do with the re-use and interpretation of a text within new texts. In a religious tradition, certain elements become re-circulated and thus their reception may reflect the development of that particular tradition. The Maccabean martyrs first appear in 2 Maccabees. In my study, it is the Maccabean martyr figures who count as the received text; the focus is shifted from the interrelations between texts onto how the figures have been exploited in early Christian and Rabbinic sources. I have divided my sources into two categories and my analysis is in two parts. First, I analyze the reception of the Maccabean martyrs within Jewish and Christian historiographical sources, focusing on the role given to them in the depictions of the Maccabean Revolt (Chapter 3). I conclude that, within Jewish historiography, the martyrs are given roles, which vary between ultimate efficacy and marginal position with regard to making a historical difference. In Christian historiographical sources, the martyrs role grows in importance by time: however, it is not before a Christian cult of the Maccabean martyrs has been established, that the Christian historiographies consider them historically effective. After the first part, I move on to analyze the reception in sources, which make use of the Maccabean martyrs as paradigmatic figures (Chapter 4). I have suggested that the martyrs are paradigmatic in the context of martyrdom, persecution and destruction, on one hand, and in a homiletic context, inspiring religious celebration, on the other. I conclude that, as the figures are considered pre-Christian and biblical martyrs, they function well in terms of Christian martyrdom and have contributed to the development of its ideals. Furthermore, the presentation of the martyr figures in Rabbinic sources demonstrates how the notion of Jewish martyrdom arises from experiences of destruction and despair, not so much from heroic confession of faith in the face of persecution. Before the emergence of a Christian cult of the Maccabean martyrs, their identity is derived namely from their biblical position. Later on, in the homiletic context, their Jewish identity is debated and sometimes reconstructed as fundamentally Christian , despite of their Jewish origins. Similar debate about their identity is not found in the Rabbinic versions of their martyrdom and nothing there indicates a mutual debate between early Christians and Jews. A thematic comparison shows that the Rabbinic and Christian cases of reception are non-reliant on each other but also that they link to one another. Especially the scriptural connections, often made to the Maccabean mother, reveal the similarities. The results of the analyses confirm that the early history of Christianity and Rabbinic Judaism share, at least partly, the same religious environment and intertwining traditions, not only during the first century or two but until Late Antiquity and beyond. More likely, the reception of the Maccabean martyrs demonstrates that these religious traditions never ceased to influence one another.