Structure and function of GDNF receptor alpha splice variants

The growth factors of the glial cell line-derived neurotrophic factor (GDNF) family consisting of GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), are involved in the development, differentiation and maintenance of many types of neurons. They also have important functions outside the nervous system in the development of kidney, testis and thyroid gland. Each of these GFLs preferentially binds to one of the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptors α (GFRα). GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The GFLs in the complex with their cognate GFRα receptors all bind to and signal through the receptor tyrosine kinase RET. Alternative splicing of the mouse GFRα4 gene yields three splice isoforms. These had been described as putative GPI-anchored, transmembrane and soluble forms. My goal was to characterise the function of the different forms of mouse GFRα4. I firstly found that the putative GPI-anchored GFRα4 (GFRα4-GPI) is glycosylated, membrane-bound, GPI-anchored and interacts with PSPN and RET. We also showed that mouse GFRα4-GPI mediates PSPN-induced phosphorylation of RET, promotes PSPN-dependent neuronal differentiation of the rat pheochromocytoma cell line PC6-3 and PSPN-dependent survival of cerebellar granule neurons (CGN). However, although this receptor can mediate PSPN-signalling and activate RET, GFRα4-GPI does not recruit RET into lipid rafts. The recruitment of RET into lipid rafts has previously been thought to be a crucial event for GDNF- and GFL-mediated signalling via RET. I secondly demonstrated that the putative transmembrane GFRα4 (GFRα4-TM) is indeed a real transmembrane GFRα4 protein. Although it has a weak binding capacity for PSPN, it can not mediate PSPN-dependent phosphorylation of RET, neuronal differentiation or survival. These data show that GFRα4-TM is inactive as a receptor for PSPN. Surprisingly, GFRα4-TM can negatively regulate PSPN-mediated signalling via GFRα4-GPI. GFRα4-TM interacts with GFRα4-GPI and blocks PSPN-induced phosphorylation of RET, neuronal differentiation as well as survival. Taken together, our data show that GFRα4-TM may act as a dominant negative inhibitor of PSPN-mediated signaling. The most exciting part of my work was the finding that the putative soluble GFRα4 (GFRα4-sol) can form homodimers and function as an agonist of the RET receptor. In the absence of PSPN, GFRα4-sol can promote the phosphorylation of RET, trigger the activation of the PI-3K/AKT pathway, induce neuronal differentiation and support the survival of CGN. Our findings are in line with a recent publication showing the GFRα4-sol might contribute to the inherited cancer syndrome multiple endocrine neoplasia type 2. Our data provide an explanation to how GFRα4-sol may cause or modify the disease. Mammalian GFRα4 receptors all lack the first Cys-rich domain which is present in other GFRα receptors. In the final part of my work I have studied the function of this particular domain. I created a truncated GFRα1 construct lacking the first Cys-rich domain. Using binding assays in both cellular and cell-free systems, phosphorylation assays with RET, as well as neurite outgrowth assays, we found that the first Cys-rich domain contributes to an optimal function of GFRα1, by stabilizing the interaction between GDNF and GFRα1.

Structure and Dynamics of Coil-like Molecules by Residual Dipolar Couplings

Nuclear magnetic resonance (NMR) spectroscopy provides us with many means to study biological macromolecules in solution. Proteins in particular are the most intriguing targets for NMR studies. Protein functions are usually ascribed to specific three-dimensional structures but more recently tails, long loops and non-structural polypeptides have also been shown to be biologically active. Examples include prions, -synuclein, amylin and the NEF HIV-protein. However, conformational preferences in coil-like molecules are difficult to study by traditional methods. Residual dipolar couplings (RDCs) have opened up new opportunities; however their analysis is not trivial. Here we show how to interpret RDCs from these weakly structured molecules. The most notable residual dipolar couplings arise from steric obstruction effects. In dilute liquid crystalline media as well as in anisotropic gels polypeptides encounter nematogens. The shape of a polypeptide conformation limits the encounter with the nematogen. The most elongated conformations may come closest whereas the most compact remain furthest away. As a result there is slightly more room in the solution for the extended than for the compact conformations. This conformation-dependent concentration effect leads to a bias in the measured data. The measured values are not arithmetic averages but essentially weighted averages over conformations. The overall effect can be calculated for random flight chains and simulated for more realistic molecular models. Earlier there was an implicit thought that weakly structured or non-structural molecules would not yield to any observable residual dipolar couplings. However, in the pioneering study by Shortle and Ackerman RDCs were clearly observed. We repeated the study for urea-denatured protein at high temperature and also observed indisputably RDCs. This was very convincing to us but we could not possibly accept the proposed reason for the non-zero RDCs, namely that there would be some residual structure left in the protein that to our understanding was fully denatured. We proceeded to gain understanding via simulations and elementary experiments. In measurements we used simple homopolymers with only two labelled residues and we simulated the data to learn more about the origin of RDCs. We realized that RDCs depend on the position of the residue as well as on the length of the polypeptide. Investigations resulted in a theoretical model for RDCs from coil-like molecules. Later we extended the studies by molecular dynamics. Somewhat surprisingly the effects are small for non-structured molecules whereas the bias may be large for a small compact protein. All in all the work gave clear and unambiguous results on how to interpret RDCs as structural and dynamic parameters of weakly structured proteins.

Occupational Rhinitis : Diagnosis and Health-related Quality of Life

Occupational rhinitis is mainly caused by work environment and not by stimuli encountered outside the workplace. It differs from rhinitis that is worsened by, but not mainly caused by, workplace exposures. Occupational rhinitis can develop in response to allergens, inhaled irritants, or corrosive gases. The thesis evaluated the use of challenge tests in occupational rhinitis diagnostics, studied the long-term health-related quality of life among allergic occupational rhinitis patients, and the allergens of wheat grain among occupational respiratory allergy patients. The diagnosed occupational rhinitis was mainly allergic rhinitis, which was caused by occupational agents, most commonly flours and animal allergens. The non-IgE-mediated rhinitis reactions were less frequent and caused more often asthma than rhinitis. Both nasal challenges and inhalation challenges were found to be safe tests. The inhalation challenge tests had considerably resource-intensive methodology. However, the evaluation of nasal symptoms and signs together with bronchial reactions saved time and expense compared with the organization of multiple individual challenges. The scoring criteria used matched well with the weighted amount of discharge ≥ 0.2 g and in most cases gave comparable results. The challenge tests are valuable tools when there is uncertainty whether the patient's exposure should be reduced or discontinued. It was found that continuing exposure decreases health-related quality of life among patients with allergic occupational rhinitis despite of rhinitis medications, still approximately ten years after the diagnosis. Health-related quality of life among occupational rhinitis patients without any longer occupational exposure was mainly similar than that of the healthy controls. This highlights the importance of the reduction and cessation of occupational exposure. To achieve this, 17% of occupational rhinitis patients had been re-educated. Alpha-amylase inhibitors, lipid transfer protein 2G, thaumatin -like protein, and peroxidase I were found to be relevant allergens in Finnish patients with occupational respiratory wheat allergy. Of these allergens, thaumatin-like protein and lipid transfer protein 2G were found as new allergens associated with baker's rhinitis and asthma. The knowledge of the new clinically relevant proteins can be used in the future in the development of better standardized diagnostic preparations.

Studies on the Cell Wall Structure and on the Mechanical Properties of Norway Spruce

The structure and the mechanical properties of wood of Norway spruce (Picea abies [L.] Karst.) were studied using small samples from Finland and Sweden. X-ray diffraction (XRD) was used to determine the orientation of cellulose microfibrils (microfibril angle, MFA), the dimensions of cellulose crystallites and the average shape of the cell cross-section. X-ray attenuation and x-ray fluorescence measurements were used to study the chemical composition and the trace element content. Tensile testing with in situ XRD was used to characterise the mechanical properties of wood and the deformation of crystalline cellulose within the wood cell walls. Cellulose crystallites were found to be 192 284 Å long and 28.9 33.4 Å wide in chemically untreated wood and they were longer and wider in mature wood than in juvenile wood. The MFA distribution of individual Norway spruce tracheids and larger samples was asymmetric. In individual cell walls, the mean MFA was 19 30 degrees, while the mode of the MFA distribution was 7 21 degrees. Both the mean MFA and the mode of the MFA distribution decreased as a function of the annual ring. Tangential cell walls exhibited smaller mean MFA and mode of the MFA distribution than radial cell walls. Maceration of wood material caused narrowing of the MFA distribution and removed contributions observed at around 90 degrees. In wood of both untreated and fertilised trees, the average shape of the cell cross-section changed from circular via ambiguous to rectangular as the cambial age increased. The average shape of the cell cross-section and the MFA distribution did not change as a result of fertilisation. The mass absorption coefficient for x-rays was higher in wood of fertilised trees than in that of untreated trees and wood of fertilised trees contained more of the elements S, Cl, and K, but a smaller amount of Mn. Cellulose crystallites were longer in wood of fertilised trees than in that of untreated trees. Kraft cooking caused widening and shortening of the cellulose crystallites. Tensile tests parallel to the cells showed that if the mean MFA is initially around 10 degrees or smaller, no systematic changes occur in the MFA distribution due to strain. The role of mean MFA in defining the tensile strength or the modulus of elasticity of wood was not as dominant as that reported earlier. Crystalline cellulose elongated much less than the entire samples. The Poisson ratio νca of crystalline cellulose in Norway spruce wood was shown to be largely dependent on the surroundings of crystalline cellulose in the cell wall, varying between -1.2 and 0.8. The Poisson ratio was negative in kraft cooked wood and positive in chemically untreated wood. In chemically untreated wood, νca was larger in mature wood and in latewood compared to juvenile wood and earlywood.

Lung structure and function studied by synchrotron radiation

A novel method for functional lung imaging was introduced by adapting the K-edge subtraction method (KES) to in vivo studies of small animals. In this method two synchrotron radiation energies, which bracket the K-edge of the contrast agent, are used for simultaneous recording of absorption-contrast images. Stable xenon gas is used as the contrast agent, and imaging is performed in projection or computed tomography (CT) mode. Subtraction of the two images yields the distribution of xenon, while removing practically all features due to other structures, and the xenon density can be calculated quantitatively. Because the images are recorded simultaneously, there are no movement artifacts in the subtraction image. Time resolution for a series of CT images is one image/s, which allows functional studies. Voxel size is 0.1mm3, which is an order better than in traditional lung imaging methods. KES imaging technique was used in studies of ventilation distribution and the effects of histamine-induced airway narrowing in healthy, mechanically ventilated, and anaesthetized rabbits. First, the effect of tidal volume on ventilation was studied, and the results show that an increase in tidal volume without an increase in minute ventilation results a proportional increase in regional ventilation. Second, spiral CT was used to quantify the airspace volumes in lungs in normal conditions and after histamine aerosol inhalation, and the results showed large patchy filling defects in peripheral lungs following histamine provocation. Third, the kinetics of proximal and distal airway response to histamine aerosol were examined, and the findings show that the distal airways react immediately to histamine and start to recover, while the reaction and the recovery in proximal airways is slower. Fourth, the fractal dimensions of lungs was studied, and it was found that the fractal dimension is higher at the apical part of the lungs compared to the basal part, indicating structural differences between apical and basal lung level. These results provide new insights to lung function and the effects of drug challenge studies. Nowadays the technique is available at synchrotron radiation facilities, but the compact synchrotron radiation sources are being developed, and in relatively near future the method may be used at hospitals.

Balancing Social Citizenship and New Paternalism : Finnish activation policy and street-level practice in a comparative perspective

The study examines the origin and development of the Finnish activation policy since the mid-1990s by using the 2001 activation reform as a benchmark. The notion behind activation is to link work obligations to welfare benefits for the unemployed. The focus of the thesis is policy learning and the impact of ideas on the reform of the welfare state. The broader research interests of the thesis are summarized by two groups of questions. First, how was the Finnish activation policy developed and what specific form did it receive in the 2001 activation reform? Second, how does the Finnish activation policy compare to the welfare reforms in the EU and in the US? What kinds of ideas and instruments informed the Finnish policy? To what extent can we talk about a restructuring or transformation of the Nordic welfare policy? Theoretically, the thesis is embedded in the comparative welfare state research and the concepts used in the contemporary welfare state discourse. Activation policy is analysed against the backdrop of the theories about the welfare state, welfare state governance and citizenship. Activation policies are also analysed in the context of the overall modernization and individualization of lifestyles and its implications for the individual citizen. Further, the different perspectives of the policy analysis are applied to determine the role of implementation and street-level practice within the whole. Empirically, the policy design, its implementation and the experiences of the welfare staff and recipients in Finland are examined. The policy development, goals and instruments of the activation policies have followed astonishingly similar paths in the different welfare states and regimes over the last two decades. In Finland, the policy change has been manifested through several successive reforms that have been introduced since the mid-1990s. The 2001 activation reform the Act on Rehabilitative Work Experience illustrates the broader trend towards stricter work requirements and draws its inspiration from the ideas of new paternalism. The ideas, goals and instruments of the international activation trend are clearly visible in the reform. Similarly, the reform has implications for the traditional Nordic social policies, which incorporate institutionalised social rights and the provision of services.

The Relational Self, the Social Bond and the Dynamics of Personal Relationships : A Sociological Analysis

This study analyses personal relationships linking research to sociological theory on the questions of the social bond and on the self as social. From the viewpoint of disruptive life events and experiences, such as loss, divorce and illness, it aims at understanding how selves are bound to their significant others as those specific people ‘close or otherwise important’ to them. Who form the configurations of significant others? How do different bonds respond in disruptions and how do relational processes unfold? How is the embeddedness of selves manifested in the processes of bonding, on the one hand, and in the relational formation of the self, on the other? The bonds are analyzed from an anti-categorical viewpoint based on personal citations of significance as opposed to given relationship categories, such as ‘family’ or ‘friendship’ – the two kinds of relationships that in fact are most frequently significant. The study draws from analysis of the personal narratives of 37 Finnish women and men (in all 80 interviews) and their entire configurations of those specific people who they cite as ‘close or otherwise important’. The analysis stresses the subjective experiences, while also investigating the actualized relational processes and configurations of all personal relationships with certain relationship histories embedded in micro-level structures. The research is based on four empirical sub-studies of personal relationships and a summary discussing the questions of the self and social bond. Discussion draws from G. H. Mead, C. Cooley, N. Elias, T. Scheff, G. Simmel and the contributors of ‘relational sociology’. Sub-studies analyse bonds to others from the viewpoint of biographical disruption and re-configuration of significant others, estranged family bonds, peer support and the formation of the most intimate relationships into exclusive and inclusive configurations. All analyses examine the dialectics of the social and the personal, asking how different structuring mechanisms and personal experiences and negotiations together contribute to the unfolding of the bonds. The summary elaborates personal relationships as social bonds embedded in wider webs of interdependent people and social settings that are laden with cultural expectations. Regarding the question of the relational self, the study proposes both bonding and individuality as significant. They are seen as interdependent phases of the relationality of the self. Bonding anchors the self to its significant relationships, in which individuality is manifested, for example, in contrasting and differentiating dynamics, but also in active attempts to connect with others. Individuality is not a fixed quality of the self, but a fluid and interdependent phase of the relational self. More specifically, it appears in three formats in the flux of relational processes: as a sense of unique self (via cultivation of subjective experiences), as agency and as (a search for) relative autonomy. The study includes an epilogue addressing the ambivalence between the social expectation of individuality in society and the bonded reality of selves.

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.