Structural studies on members of the picornavirus superfamily

The pathogenic members of the picornavirus superfamily have adverse effects on humans, their crops and their livestock. As structure is related to function, detailed structural studies on these viruses are important not only for fundamental understanding of the viral life cycle, but also for the rational design of vaccines and inhibitors for disease control. These viruses have positive sense, single-stranded RNA genomes enclosed in a protein capsid. X-ray crystallography and cryo-electron microscopy studies have revealed that the isometric members of this group have icosahedrally-symmetric capsids made up of 60 copies of each of the structural proteins. The members that infect animal cells often employ one or more cellular receptors to facilitate cell entry which in some cases is known to initiate the uncoating sequence of the genome. The nature of the interactions between individual viruses and alternative cellular receptors has rarely been probed. The capsid assembly of the members of the picornavirus superfamily is considered to be cooperative and the interactions of RNA and capsid proteins are thought to play an important role in orchestrating virus assembly. The major aims of this thesis were to solve the structures of blackcurrant reversion virus (BRV), human parechovirus 1 (HPEV1) and coxsackievirus A7 (CAV7), as well as the structure of HPEV1 complexed with two of its cellular receptors using cryo-electron microscopy, three-dimensional image reconstruction and homology modeling. Each of the selected viruses represents a taxonomic group where little or no structural data was previously available. The results enabled the detailed comparison of the new structures to those of known picornaviruses, the identification of surface-exposed epitopes potentially important for host interaction, the mapping of RNA-capsid protein interactions and the elucidation of the basis for the specificity of two different receptor molecules for the same capsid. This work will form the basis for further studies on the influence of RNA on parechovirus assembly as a potential target for drug design.

Experimental approach for studying structural changes in axonal membrane upon nerve excitation

The Hodgkin and Huxley (HH) model of action potential has become a central paradigm of neuroscience. Despite its ability to predict action potentials with remarkable accuracy, it fails to explain several biophysical findings related to the initiation and propagation of the nerve impulse. The isentropic heat release and optical phenomena demonstrated by various experiments suggest that action potential is accompanied by a transient phase change in the axonal membrane. In this study a method was developed for preparing a giant axon from the crayfish abdominal cord for studying the molecular mechanisms of action potential simultaneously by electrophysiological and optical methods. Also an alternative setup using a single-cell culture of an Aplysia sensory neuron is presented. In addition to the description of the method, the preliminary results on the effect of phloretin, a dipole potential lowering compound, on the excitability of a crayfish giant axon are presented.

From ‘Afflicted’ to Activists: Structural Violence and Climate Change Discourse in the States of Pernambuco and Paraíba in Northeast Brazil

This study approaches the problem of poverty in the hinterlands of Northeast Brazil through the concept of structural violence, linking the environmental threats posed by climate change, especially those related to droughts, to the broader social struggles in the region. When discussions about potentials and rights are incorporated into the problematic of poverty, a deeper insight is obtained regarding the various factors behind the phenomenon. It is generally believed that climate change is affecting the already marginalized and poor more than those of higher social standing, and will increasingly do so in the future. The data for this study was collected during a three month field work in the states of Pernambuco and Paraíba in Northeast Brazil. The main methods used were semi-structured interviews and participant observation, including attending seminars concerning climate change on the field. The focus of the work is to compare both layman and expert perceptions on what climate change is about, and question the assumptions about its effects in the future, mainly that of increased numbers of ‘climate refugees’ or people forced to migrate due to changes in climate. The focus on droughts, as opposed to other manifestations of climate change, arises from the fact that droughts are not only phenomena that develop over a longer time span than floods or hurricanes, but is also due to the historical persistence of droughts in the region, and both the institutional and cultural linkages that have evolved around it. The instances of structural violence that are highlighted in this study; the drought industry, land use, and the social and power relations present in the region, including those between the civil society, the state and the private agribusiness sector, all work against a backdrop of symbolic and moral realms of value production, where relations between the different actors are being negotiated anew with the rise of the climate change discourse. The main theoretical framework of the study consists of Johan Galtung’s and Paul Farmer’s theory of structural violence, Ulrich Beck’s theory of the risk society, and James Scott’s theory of everyday peasant resistance.

Tandemmassaspektrometrinen menetelmä neutraalien isomeeristen oligosakkaridien rakenteen määrittämisessä

Tutkimuksen tavoitteena oli ottaa käyttöön tandemmassaspektrometrinen (MS/MS) menetelmä, jolla voidaan analysoida polysakkarideista purkautuneiden oligosakkaridien rakenteita. Tavoitteena oli, että menetelmällä voidaan määrittää glykosidisten sidosten eri asemat monosakkaridirakenteiltaan samanlaisista neutraaleista lineaarisista oligosakkarideista. Kirjallisuustutkimuksessa tarkasteltiin oligosakkaridien rakenteiden määrittämiseen käytettyjä MS/MS-menetelmiä ja oligosakkaridien pilkkoutumisreaktioita MS/MS-analyysissa. Kirjallisuuden perusteella MS/MS-analyysissa oligosakkaridien pilkkoutuminen voi tapahtua joko glykosidisen sidoksen katkeamisella tai monosakkaridirenkaan halkeamisella. Monosakkaridirenkaan pilkkoutumisesta muodostuvia tuoteioneja voidaan käyttää glykosidisen sidoksen aseman määrittämiseen. Kokeellisessa tutkimuksessa selvitettiin aluksi monosakkaridirakenteiltaan isomeerisilla disakkaridimalliaineilla glykosidisen sidoksen sijainnin vaikutus disakkaridin pilkkoutumiseen MS/MS-analyysissa. Tämän jälkeen pyrittiin löytämään tunnetuista tri- ja tetrasakkaridimalliaineista näitä eri sidoksille tyypillisiä tuoteionien jakaumia. Tunnettujen tri- ja tetrasakkaridien pilkkoutuminen yhdenmukaisesti disakkaridien pilkkoutumisen kanssa antaisi mahdollisuuden pitkäketjuisempien oligosakkaridien glykosidisten sidosten tunnistamiseen sovelletulla MS/MS-menetelmällä. MS/MS-analyysit tehtiin ioniloukkumassadetektorilaitteistolla käyttäen sähkösumutusionisaatiota (ESI). Oligosakkaridit määritettiin positiivisella ionisaatiolla litium- ja natriumaddukti-ioneina ja negatiivisella ionisaatiolla kloridiaddukti-ioneina. Vertaamalla tri- ja tetrasakkarideista MS/MS-analyyseissa muodostuneita tuoteioneja disakkarideista muodostuneisiin tuoteioneihin, voitiin sekä positiivisella että negatiivisella ionisaatiolla määrittää oligosakkaridin pelkistävän pään sidoksen asema. Negatiivisella ionisaatiolla tri- ja tetrasakkarideista muodostuneista tuoteioneista voitiin määrittää myös muiden kuin pelkistävän pään sidosten asemia. Positiivisella ionisaatiolla muiden sidosten määrittäminen ei ollut mahdollista, koska rengasfragmentti-ioneja muodostui pääosin oligosakkaridin pelkistävästä päästä. Glykosidisen sidoksen katkeamisesta muodostuneet tuoteionit analysoitiin edelleen MS3-analyysilla. MS3-analyysissa muodostuneista tuoteioneista ei voitu tulkita sidosten asemia, koska lähtöionit koostuivat sekä terminaalisen että pelkistävän pään isomeerisista ioneista.