Alfaviiruse mittestruktuurne proteaas ja tema liitvalgust substraat: täiuslikult korraldatud kooselu reeglid

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Alfaviirused (sugukond Togaviridae) on artriiti ja entsefaliiti põhjustavad RNA genoomsed viirused. Nende paljunemise strateegia aluseks on viiruse replikaasi valkude süntees ühe nn. mittestruktuurse eelvalgu P1234 kujul ning selle ajaliselt reguleeritud lõikamine valmis valkudeks nsP2 proteaasi abil. Käesoleva väitekirja aluseks olevad uurimistööd viisid nsP2 substraat-spetsiifilisust tagavate mehhanismide väljaselgitamiseni; muu hulgas kirjeldati uudset proteolüütiliste lõikamiste regulatsioonimehhanismi, mis põhineb liitvalgu erinevate regioonide vahelisel „suhtlemisel“ viiruse replikatsiooni kompleksi moodustamise käigus. Sellest saab järeldada, et P1234 lõikamise ajaline regulatsioon sõltub otseselt replikatsioonikompleksi konfiguratsioonidest, millised omakorda on määratud selle komponentide vaheliste interaktsioonide poolt. Seega tõuseb viiruse nsP2 proteaas esile kui keerulise signaalvõrgustiku keskne element, mille roll viirus infektsiooni regulatsioonis seisneb replikatsiooniga kaasnevate sündmuste „jälgimises“ ja nendele reageerimises. Viimane põhineb sellel, et kui viiruse paljunemine jõuab kindla vahe-etapini, siis kaasneb sellega lõikamiskohtade ja/või muude oluliste struktuuride „esitlemine“ proteaasile, mis reageerib toimunud muudatustele lokaalse signaalülekande, mis lõppkokkuvõttes viib replikaasi kompleksi struktuuri järjestikulistele muudatustele, käivitamisega. Kokkuvõttes, tõid läbiviidud uurimised välja asjaolu, et lisaks varem teada olnud lõikamisjärjestuste äratundmisele, omab ka makromolekulaarsete struktuuride moodustamine viiruse valkude poolt olulist (ja mitmel juhul isegi määravat) rolli viiruse proteaasi töö reguleerimisel. Veel enam, eeldati, et seesugune mitmetahuline regulatsioon võib olla paljukomponentsete proteolüütiliste süsteemide üldine omadus. Kirjeldatud avastused ja nende lahtimõtestamine omavad olulist rolli uurimistöödele, mille eesmärgiks on alfaviiruste paljunemist takistavate lähenemiste väljatöötamine. Nii võib saadud tulemuste põhjal järeldada, et lisaks proteaasi aktiivsuse otsesele mõjutamisele võib viiruse replikatsiooni takistada ka mõjutades proteolüüsi regulatsiooni tagavaid molekulide vahelised seoseid.Alphaviruses from the Togaviridae family are RNA viruses that may cause arthritic syndroms and encephalitis. The alphavirus replication strategy relies on the production of replicase proteins initially in the form of non-structural (ns) polyprotein precursor P1234, which during the course of replication becomes proteolytically processed by the virus-encoded nsP2 protease in a temporally regulated manner. The studies that constitute the basis of this thesis led to identification of the requirements for substrate specificity of nsP2 protease and revealed novel mechanism for the regulation of processing based on the specific communication between distant parts of the viral polyprotein brought together during assembly of replication complex. It was concluded that the order of alphaviral ns-polyprotein processing is mostly dependent on the configuration of the replication complex imposed by intermolecular interactions meant to guarantee timely cleavages. The alphaviral protease therefore emerges as an integral part of the sophisticated signaling mechanism, in which the regulatory task of the protease consists of monitoring the succession and completion of the events of viral infection. Once the respective replication status-induced conformational changes within replicase allow the presentation of the scissile bond and/or other essential determinants of substrate recognition like exosites, the local protease signaling is initiated, which apparently leads to further reconfiguration of the viral replication complex. Combined, the studies unveiled the decisive role played by the macromolecular assembly-dependent component of substrate recognition in addition to the sequence-dependent component, the combination of which may be expected to constitute the basis of regulation in multi-site proteolytic systems in general. Described findings and their interpretations are expected to provide with essential grounds and directions for further studies on the restriction of alphaviral replication through affecting the center of viral proteolytic activity or via intervention with its regulation by targeting intramolecular interactions

Tackling the problem of migrant smuggling into the European Union

Irregular movement on the Mediterranean Sea has been an issue for the European Union already for a few decades. During the last five years the scope of the irregular movement has rapidly expanded and has gained more public attention. The European Union is facing the complicated question of how to retain security and sovereignty while protecting human life. The objective of this thesis is to assess the different approaches to tackle the problem of people smuggling on the external borders of the European Union. This is done by analysing two main proposed policies, one of which focuses on limiting the supply of smuggling services while the other focuses on limiting the demand for smuggling services. The thesis will assess the expected effectiveness and (un)intended consequences of both policy options. The result of this thesis show that the combination of limiting the supply of smuggling services and limiting the demand for smuggling services is the best combination in order to fight human smuggling in the Mediterranean Sea.http://www.ester.ee/record=b4580826*es

People, Places and Pixels: Remote Sensing in the Service of Society

What is the role of Earth remote sensing and other geospatial technologies in our society? Recent global events have brought into focus the role of geospatial science and technology such as remote sensing, GIS, GPS in assisting the professionals who are responsible for operations such as rescue and recovery of sites after a disaster or a terrorist act. This paper reviews the use of recent remote sensing products from satellites such as IKONOS in these efforts. Aerial and satellite imagery used in land mine detection has been evaluated and the results of this evaluation will be discussed. Synopsis of current and future ISS Earth Remote Sensing capabilities will be provided. The role of future missions in humanitarian use of remote sensing will be explored

Assembly of Replication-Incompetent African Horse Sickness Virus Particles: Rational Design of Vaccines for All Serotypes.

UNLABELLED: African horse sickness virus (AHSV), an orbivirus in the Reoviridae family with nine different serotypes, causes devastating disease in equids. The virion particle is composed of seven proteins organized in three concentric layers, an outer layer made of VP2 and VP5, a middle layer made of VP7, and inner layer made of VP3 that encloses a replicase complex of VP1, VP4, and VP6 and a genome of 10 double-stranded RNA segments. In this study, we sought to develop highly efficacious candidate vaccines against all AHSV serotypes, taking into account not only immunogenic and safety properties but also virus productivity and stability parameters, which are essential criteria for vaccine candidates. To achieve this goal, we first established a highly efficient reverse genetics (RG) system for AHSV serotype 1 (AHSV1) and, subsequently, a VP6-defective AHSV1 strain in combination with in trans complementation of VP6. This was then used to generate defective particles of all nine serotypes, which required the exchange of two to five RNA segments to achieve equivalent titers of particles. All reassortant-defective viruses could be amplified and propagated to high titers in cells complemented with VP6 but were totally incompetent in any other cells. Furthermore, these replication-incompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice. Thus, these defective viruses have the potential to be used for the development of safe and stable vaccine candidates. The RG system also provides a powerful tool for the study of the role of individual AHSV proteins in virus assembly, morphogenesis, and pathogenesis. IMPORTANCE: African horse sickness virus is transmitted by biting midges and causes African horse sickness in equids, with mortality reaching up to 95% in naive horses. Therefore, the development of efficient vaccines is extremely important due to major economic losses in the equine industry. Through the establishment of a highly efficient RG system, replication-deficient viruses of all nine AHSV serotypes were generated. These defective viruses achieved high titers in a cell line complemented with VP6 but failed to propagate in wild-type mammalian or insect cells. Importantly, these candidate vaccine strains showed strong protective efficacy against AHSV infection in an IFNAR(-/-) mouse model

Racial Segregation in Indianapolis, 1990–2010: A Spatial Perspective

The index of dissimilarity is the most widely used method for measuring racial segregation. When applied to Indianapolis, this index has returned results showing the city to be among the most segregated in the country. The resulting measure, however, suffers from two shortcomings. First, the index of dissimilarity is sensitive to the census-defined geographic unit chosen for the analysis; thus, this index returns different (though proportionate) results depending on whether the population data are aggregated to larger or smaller enumeration units. Second, the index of dissimilarity cannot account for the influence of spatial proximity; adjacent census blocks interact regardless of administrative boundaries. In place of the index of dissimilarity, we apply the segregation index in order to treat the phenomena as a surface that is simultaneously smooth and continuous. In this article, we calculate the segregation index for Indianapolis from 1990 to 2010 using the kernel density estimation method. The results of the analysis are presented in three pairs of decennial maps. These maps add to the understanding of residential segregation by resolving in a statistically reliable manner the phenomenon’s geographic component. Our visualization of segregation confirms its presence in distinct clusters, its growth over time, and a strong bias of this growth to be contiguous. In a manner akin to examinations of residential segregation’s impact on education attainment and health outcomes, careful description of segregation’s spatial aspect leads to a more nuanced understanding of phenomenon’s pervasiveness across social life

Measurement and Modeling of Ground-Level Ozone Concentration in Catania, Italy using Biophysical Remote Sensing and GIS

This experimental study examined spatial variation of ground level ozone (O3) in the city of Catania, Italy using thirty passive samplers deployed in a 500-m grid pattern. Significant spatial variation in ground level O3 concentrations (ranging from 12.8 to 41.7 g/m3) was detected across Catania’s urban core and periphery. Biophysical measures derived from satellite imagery and built environment characteristics from GIS were evaluated as correlates of O3 concentrations. A land use regression model based on four variables (land surface temperature, building area, residential street length, and distance to the coast) explained 74% of the variance (adjusted R2) in measured O3. The results of the study suggest that biophysical remote sensing variables are worth further investigation as predictors of ground level O3 (and potentially other air pollutants) because they provide objective measurements that can be tested across multiple locations and over time

Mutation of CD2AP and SH3KBP1 binding motif in alphavirus nsP3 hypervariable domain results in attenuated virus

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3

Climate Change Adaptation Science Activities at NASA Johnson Space Center

The Johnson Space Center (JSC), located in the southeast metropolitan region of Houston, TX is the prime NASA center for human spaceflight operations and astronaut training, but it also houses the unique collection of returned extraterrestrial samples, including lunar samples from the Apollo missions. The Center's location adjacent to Clear Lake and the Clear Creek watershed, an estuary of Galveston Bay, puts it at direct annual risk from hurricanes, but also from a number of other climate-related hazards including drought, floods, sea level rise, heat waves, and high wind events all assigned Threat Levels of 2 or 3 in the most recent NASA Center Disaster/Risk Matrix produced by the Climate Adaptation Science Investigator Working Group. Based on prior CASI workshops at other NASA centers, it is recognized that JSC is highly vulnerable to climate-change related hazards and has a need for adaptation strategies. We will present an overview of prior CASI-related work at JSC, including publication of a climate change and adaptation informational data brochure, and a Resilience and Adaptation to Climate Risks Workshop that was held at JSC in early March 2012. Major outcomes of that workshop that form a basis for work going forward are 1) a realization that JSC is embedded in a regional environmental and social context, and that potential climate change effects and adaptation strategies will not, and should not, be constrained by the Center fence line; 2) a desire to coordinate data collection and adaptation planning activities with interested stakeholders to form a regional climate change adaptation center that could facilitate interaction with CASI; 3) recognition that there is a wide array of basic data (remotely sensed, in situ, GIS/mapping, and historical) available through JSC and other stakeholders, but this data is not yet centrally accessible for planning purposes